Mark J. Czaja

The intracellular storage and utilization of lipids are critical to maintain cellular energy homeostasis. During nutrient deprivation, cellular lipids stored as triglycerides in lipid droplets are hydrolysed into fatty acids for energy. A second cellular response to starvation is the induction of autophagy, which delivers intracellular proteins and organelles sequestered in double-membrane vesicles (autophagosomes) to lysosomes for degradation and use as an energy source. Lipolysis and autophagy share similarities in regulation and function but are not known to be interrelated. Here we show a previously unknown function for autophagy in regulating intracellular lipid stores (macrolipophagy). Lipid droplets and autophagic components associated during nutrient deprivation, and inhibition of autophagy in cultured hepatocytes and mouse liver increased triglyceride storage in lipid droplets. This study identifies a critical function for autophagy in lipid metabolism that could have important implications for human diseases with lipid over-accumulation such as those that comprise the metabolic syndrome.

<h3>Background & Aims</h3> The pathogenesis of liver fibrosis involves activation of hepatic stellate cells, which is associated with depletion of intracellular lipid droplets. When hepatocytes undergo autophagy, intracellular lipids are degraded in lysosomes. We investigated whether autophagy also promotes loss of lipids in hepatic stellate cells to provide energy for their activation and extended these findings to other fibrogenic cells. <h3>Methods</h3> We analyzed hepatic stellate cells from C57BL/6 wild-type, <i>Atg7^F/F</i>, and <i>Atg7^F/F-GFAP-Cre</i> mice, as well as the mouse stellate cell line JS1. Fibrosis was induced in mice using CCl₄ or thioacetamide (TAA); liver tissues and stellate cells were analyzed. Autophagy was blocked in fibrogenic cells from liver and other tissues using small interfering RNAs against <i>Atg5</i> or <i>Atg</i>7 and chemical antagonists. Human pulmonary fibroblasts were isolated from samples of lung tissue from patients with idiopathic pulmonary fibrosis or from healthy donors. <h3>Results</h3> In mice, induction of liver injury with CCl₄ or TAA increased levels of autophagy. We also observed features of autophagy in activated stellate cells within injured human liver tissue. Loss of autophagic function in cultured mouse stellate cells and in mice following injury reduced fibrogenesis and matrix accumulation; this effect was partially overcome by providing oleic acid as an energy substrate. Autophagy also regulated expression of fibrogenic genes in embryonic, lung, and renal fibroblasts. <h3>Conclusions</h3> Autophagy of activated stellate cells is required for hepatic fibrogenesis in mice. Selective reduction of autophagic activity in fibrogenic cells in liver and other tissues might be used to treat patients with fibrotic diseases.

The relative balance between the quantity of white and brown adipose tissue can profoundly affect lipid storage and whole-body energy homeostasis. However, the mechanisms regulating the formation, expansion, and interconversion of these 2 distinct types of fat remain unknown. Recently, the lysosomal degradative pathway of macroautophagy has been identified as a regulator of cellular differentiation, suggesting that autophagy may modulate this process in adipocytes. The function of autophagy in adipose differentiation was therefore examined in the current study by genetic inhibition of the critical macroautophagy gene autophagy-related 7 (Atg7). Knockdown of Atg7 in 3T3-L1 preadipocytes inhibited lipid accumulation and decreased protein levels of adipocyte differentiation factors. Knockdown of Atg5 or pharmacological inhibition of autophagy or lysosome function also had similar effects. An adipocyte-specific mouse knockout of Atg7 generated lean mice with decreased white adipose mass and enhanced insulin sensitivity. White adipose tissue in knockout mice had increased features of brown adipocytes, which, along with an increase in normal brown adipose tissue, led to an elevated rate of fatty acid, beta-oxidation, and a lean body mass. Autophagy therefore functions to regulate body lipid accumulation by controlling adipocyte differentiation and determining the balance between white and brown fat.

Intracellular lipids are stored in lipid droplets (LDs) and metabolized by cytoplasmic neutral hydrolases to supply lipids for cell use. Recently, an alternative pathway of lipid metabolism through the lysosomal degradative pathway of autophagy has been described and termed lipophagy. In this form of lipid metabolism, LD triglycerides (TGs) and cholesterol are taken up by autophagosomes and delivered to lysosomes for degradation by acidic hydrolases. Free fatty acids generated by lipophagy from the breakdown of TGs fuel cellular rates of mitochondrial β-oxidation. Lipophagy therefore functions to regulate intracellular lipid stores, cellular levels of free lipids such as fatty acids and energy homeostasis. The amount of lipid metabolized by lipophagy varies in response to the extracellular supply of nutrients. The ability of the cell to alter the amount of lipid targeted for autophagic degradation depending on nutritional status demonstrates that this process is selective. Intracellular lipids themselves regulate levels of autophagy by unclear mechanisms. Impaired lipophagy can lead to excessive tissue lipid accumulation such as hepatic steatosis, alter hypothalamic neuropeptide release to affect body mass, block cellular transdifferentiation and sensitize cells to death stimuli. Future studies will likely identify additional mechanisms by which lipophagy regulates cellular physiology, making this pathway a potential therapeutic target in a variety of diseases.

Despite extensive efforts, little progress has been made in identifying the factors that induce hepatic fibrosis. Transforming growth factor-beta (TGF-beta) has been shown to enhance collagen production, therefore its role in hepatic fibrosis was investigated. Treatment of cultured hepatic cells with TGF-beta 1 increased type I procollagen mRNA levels 13-fold due to post-transcriptional gene regulation. When two animal models of hepatic fibrosis, murine schistosomiasis and CCl4-treated rats, were examined, they both exhibited increased levels of TGF-beta 1 gene expression at times that somewhat preceded the increase in collagen synthesis. In contrast, in murine schistosomiasis, mRNA levels of tumor necrosis factor and interleukin-1 peaked early in the fibrogenic process. Immunohistochemical analysis showed TGF-beta 1 to be present in normal mouse liver and to be markedly increased in mice infected with schistosomiasis. TGF-beta 1 appeared in the hepatic parenchyma, primarily in hepatocytes. These findings strongly suggest a role for TGF-beta 1 in a pathophysiological state.

Autophagy has emerged as a critical lysosomal pathway that maintains cell function and survival through the degradation of cellular components such as organelles and proteins. Investigations specifically employing the liver or hepatocytes as experimental models have contributed significantly to our current knowledge of autophagic regulation and function. The diverse cellular functions of autophagy, along with unique features of the liver and its principal cell type the hepatocyte, suggest that the liver is highly dependent on autophagy for both normal function and to prevent the development of disease states. However, instances have also been identified in which autophagy promotes pathological changes such as the development of hepatic fibrosis. Considerable evidence has accumulated that alterations in autophagy are an underlying mechanism of a number of common hepatic diseases including toxin-, drug- and ischemia/reperfusion-induced liver injury, fatty liver, viral hepatitis and hepatocellular carcinoma. This review summarizes recent advances in understanding the roles that autophagy plays in normal hepatic physiology and pathophysiology with the intent of furthering the development of autophagy-based therapies for human liver diseases.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis and varying degrees of necroinflammation. Although chronic oxidative stress, inflammatory cytokines, and insulin resistance have been implicated in the pathogenesis of NAFLD, the mechanisms that underlie the initiation and progression of this disease remain unknown. c-Jun N-terminal kinase (JNK) is activated by oxidants and cytokines and regulates hepatocellular injury and insulin resistance, suggesting that this kinase may mediate the development of steatohepatitis. The presence and function of JNK activation were therefore examined in the murine methionine- and choline-deficient (MCD) diet model of steatohepatitis. Activation of hepatic JNK, c-Jun, and AP-1 signaling occurred in parallel with the development of steatohepatitis in MCD diet–fed mice. Investigations in jnk1 and jnk2 knockout mice demonstrated that jnk1, but not jnk2, was critical for MCD diet–induced JNK activation. JNK promoted the development of steatohepatitis as MCD diet–fed jnk1 null mice had significantly reduced levels of hepatic triglyceride accumulation, inflammation, lipid peroxidation, liver injury, and apoptosis compared with wild-type and jnk2 −/− mice. Ablation of jnk1 led to an increase in serum adiponectin but had no effect on serum levels of tumor necrosis factor-α. In conclusion, JNK1 is responsible for JNK activation that promotes the development of steatohepatitis in the MCD diet model. These findings also provide additional support for the critical mechanistic involvement of JNK1 overactivation in conditions associated with insulin resistance and the metabolic syndrome. (HEPATOLOGY 2006;43:163–172.)

Recent evidence that excessive lipid accumulation can decrease cellular levels of autophagy and that autophagy regulates immune responsiveness suggested that impaired macrophage autophagy may promote the increased innate immune activation that underlies obesity. Primary bone marrow-derived macrophages (BMDM) and peritoneal macrophages from high-fat diet (HFD)-fed mice had decreased levels of autophagic flux indicating a generalized impairment of macrophage autophagy in obese mice. To assess the effects of decreased macrophage autophagy on inflammation, mice with a Lyz2-Cre-mediated knockout of Atg5 in macrophages were fed a HFD and treated with low-dose lipopolysaccharide (LPS). Knockout mice developed systemic and hepatic inflammation with HFD feeding and LPS. This effect was liver specific as knockout mice did not have increased adipose tissue inflammation. The mechanism by which the loss of autophagy promoted inflammation was through the regulation of macrophage polarization. BMDM and Kupffer cells from knockout mice exhibited abnormalities in polarization with both increased proinflammatory M1 and decreased anti-inflammatory M2 polarization as determined by measures of genes and proteins. The heightened hepatic inflammatory response in HFD-fed, LPS-treated knockout mice led to liver injury without affecting steatosis. These findings demonstrate that autophagy has a critical regulatory function in macrophage polarization that downregulates inflammation. Defects in macrophage autophagy may underlie inflammatory disease states such as the decrease in macrophage autophagy with obesity that leads to hepatic inflammation and the progression to liver injury.

Patients with chronic kidney disease (CKD) develop increased levels of the phosphate-regulating hormone, fibroblast growth factor (FGF) 23, that are associated with a higher risk of mortality. Increases in inflammatory markers are another common feature that predicts poor clinical outcomes. Elevated FGF23 is associated with higher circulating levels of inflammatory cytokines in CKD, which can stimulate osteocyte production of FGF23. Here, we studied whether FGF23 can directly stimulate hepatic production of inflammatory cytokines in the absence of α-klotho, an FGF23 coreceptor in the kidney that is not expressed by hepatocytes. By activating FGF receptor isoform 4 (FGFR4), FGF23 stimulated calcineurin signaling in cultured hepatocytes, which increased the expression and secretion of inflammatory cytokines, including C-reactive protein. Elevating serum FGF23 levels increased hepatic and circulating levels of C-reactive protein in wild-type mice, but not in FGFR4 knockout mice. Administration of an isoform-specific FGFR4 blocking antibody reduced hepatic and circulating levels of C-reactive protein in the 5/6 nephrectomy rat model of CKD. Thus, FGF23 can directly stimulate hepatic secretion of inflammatory cytokines. Our findings indicate a novel mechanism of chronic inflammation in patients with CKD and suggest that FGFR4 blockade might have therapeutic anti-inflammatory effects in CKD.

Autophagy is a critical pathway for the degradation of intracellular components by lysosomes. Established functions for both macroautophagy and chaperone-mediated autophagy in hepatic lipid metabolism, insulin sensitivity and cellular injury suggest a number of potential mechanistic roles for autophagy in nonalcoholic steatohepatitis (NASH). Decreased autophagic function in particular may promote the initial development of hepatic steatosis and progression of steatosis to liver injury. Additional functions of autophagy in immune responses and carcinogenesis may also contribute to the development of NASH and its complications. The impairment in autophagy that occurs with cellular lipid accumulation, obesity and aging may therefore have an important impact on this disease, and agents to augment hepatic autophagy have therapeutic potential in NASH.

Autophagy is a lysosomal degradative pathway that functions to promote cell survival by supplying energy in times of stress or by removing damaged organelles and proteins after injury. The involvement of autophagy in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) was first suggested by the finding that this pathway mediates the breakdown of intracellular lipids in hepatocytes and therefore may regulate the development of hepatic steatosis. Subsequent studies have demonstrated additional critical functions for autophagy in hepatocytes and other hepatic cell types such as macrophages and stellate cells that regulate insulin sensitivity, hepatocellular injury, innate immunity, fibrosis, and carcinogenesis. These findings suggest a number of possible mechanistic roles for autophagy in the development of NAFLD and progression to NASH and its complications. The functions of autophagy in the liver, together with findings of decreased hepatic autophagy in association with conditions that predispose to NAFLD such as obesity and aging, suggest that autophagy may be a novel therapeutic target in this disease.

Collagen is the predominant component of the extracellular matrix of the heart, where it is organized in a hierarchy of structures. To establish the cellular origin of the various collagen types, type I-procollagen alpha 2 chain and types III and IV collagen mRNAs were examined in preparations of myocytes and non-myocyte heart cells freshly isolated from rats 1 to 6 months old. The cardiomyocytes appeared morphologically intact and functionally competent. Fibroblast-like cells predominated in the non-myocyte cell fractions but endothelial and smooth muscle cells were also present. RNA from whole ventricular tissue served as a control. Northern and dot blot analyses were used to establish the presence or absence of mRNAs. In RNA prepared from whole ventricular tissue, the mRNAs for alpha-, beta-, and gamma-actin isotypes were detected whereas mRNA for alpha-actin was found in myocytes and those for beta- and gamma-actins were found in non-myocyte cells, confirming further the nature of the cell populations. Procollagen types I and III mRNAs were not detected in the total RNA of cardiomyocytes but mRNA for type IV collagen was present. The mRNAs for all three collagen types were present in the non-myocyte cells. These results suggest that in the rat heart the non-myocyte cells, probably fibroblasts, are responsible for interstitial collagen production. Both cell populations may engage in the formation of basement membrane collagen type IV.

<i>In vitro</i> studies of hepatocytes have implicated over-activation of c-Jun N-terminal kinase (JNK) signaling as a mechanism of tumor necrosis factor-α (TNF)-induced apoptosis. However, the functional significance of JNK activation and the role of specific JNK isoforms in TNF-induced hepatic apoptosis <i>in vivo</i> remain unclear. JNK1 and JNK2 function was, therefore, investigated in the TNF-dependent, galactosamine/lipopolysaccharide (GalN/LPS) model of liver injury. The toxin GalN converted LPS-induced JNK signaling from a transient to prolonged activation. Liver injury and mortality from GalN/LPS was equivalent in wild-type and <i>jnk1^–/–</i> mice but markedly decreased in <i>jnk2^–/–</i> mice. This effect was not secondary to down-regulation of TNF receptor 1 expression or TNF production. In the absence of <i>jnk2</i>, the caspase-dependent, TNF death pathway was blocked, as reflected by the failure of caspase-3 and -7 and poly(ADP-ribose) polymerase cleavage to occur. JNK2 was critical for activation of the mitochondrial death pathway, as in <i>jnk2^–/–</i> mice Bid cleavage and mitochondrial translocation and cytochrome <i>c</i> release were markedly decreased. This effect was secondary to the failure of <i>jnk2^–/–</i> mice to activate caspase-8. Liver injury and caspase activation were similarly decreased in <i>jnk2</i> null mice after GalN/TNF treatment. Ablation of <i>jnk2</i> did not inhibit GalN/LPS-induced c-Jun kinase activity, although activity was completely blocked in <i>jnk1^–/–</i> mice. Toxic liver injury is, therefore, associated with JNK over-activation and mediated by JNK2 promotion of caspase-8 activation and the TNF mitochondrial death pathway through a mechanism independent of c-Jun kinase activity.

Hepatocyte resistance to tumor necrosis factor α (TNF)-induced apoptosis is dependent on activation of the transcription factor nuclear factor κB (NF-κB). To determine the mechanism by which NF-κB protects against TNF toxicity, the effect of NF-κB inactivation on the proapoptotic c-Jun NH2-terminal kinase (JNK) signaling pathway was examined in the rat hepatocyte cell line RALA255-10G. Adenovirus-mediated NF-κB inactivation led to a prolonged activation of JNK and increased activating protein-1 (AP-1) transcriptional activity in response to TNF treatment. Inhibition of the function of the JNK substrate and AP-1 subunit c-Jun blocked cell death from NF-κB inactivation and TNF as determined by measures of cell survival, numbers of apoptotic and necrotic cells, and DNA hypoploidy. Inhibition of c-Jun function blocked mitochondrial cytochrome c release and activation of caspase-3 and -7. NF-κB therefore blocks the TNF death pathway through down-regulation of JNK and c-Jun/AP-1. In conclusion, sustained JNK activation that occurs in the absence of NF-κB initiates apoptosis through a c-Jun–dependent induction of the mitochondrial death pathway.

Activation of c-Jun N-terminal kinase (JNK) has been implicated as a mechanism in the development of steatohepatitis. This finding, together with the reported role of JNK signaling in the development of obesity and insulin resistance, two components of the metabolic syndrome and predisposing factors for fatty liver disease, suggests that JNK may be a central mediator of the metabolic syndrome and an important therapeutic target in steatohepatitis. To define the isoform-specific functions of JNK in steatohepatitis associated with obesity and insulin resistance, the effects of JNK1 or JNK2 ablation were determined in developing and established steatohepatitis induced by a high-fat diet (HFD). HFD-fed jnk1 null mice failed to develop excessive weight gain, insulin resistance, or steatohepatitis. In contrast, jnk2(-/-) mice fed a HFD were obese and insulin-resistant, similar to wild-type mice, and had increased liver injury. In mice with established steatohepatitis, an antisense oligonucleotide knockdown of jnk1 decreased the amount of steatohepatitis in concert with a normalization of insulin sensitivity. Knockdown of jnk2 improved insulin sensitivity but had no effect on hepatic steatosis and markedly increased liver injury. A jnk2 knockdown increased hepatic expression of the proapoptotic Bcl-2 family members Bim and Bax and the increase in liver injury resulted in part from a Bim-dependent activation of the mitochondrial death pathway.JNK1 and JNK2 both mediate insulin resistance in HFD-fed mice, but the JNK isoforms have distinct effects on steatohepatitis, with JNK1 promoting steatosis and hepatitis and JNK2 inhibiting hepatocyte cell death by blocking the mitochondrial death pathway.

Since interferons have been shown to affect the synthesis of matrix proteins such as collagen in several in vitro systems, the potential role of gamma-interferon in inhibiting hepatic fibrosis was investigated. Hepatic cells, consisting primarily of hepatocytes, were treated with recombinant gamma-interferon for 24 hr. Northern blot hybridization showed that gamma-interferon treatment caused a profound decrease in pro-alpha 2(I)collagen mRNA levels but an increase in beta-actin mRNA content. The effects of gamma-interferon were then studied in an in vivo model of hepatic fibrogenesis, murine schistosomiasis. Schistosoma-infected mice were treated with daily i.m. injections of gamma-interferon for a 4-week period starting 4 weeks after the initial infection. gamma-Interferon treatment decreased collagen deposition as determined by histologic evaluation and measurement of total liver collagen content. Northern blots showed Types I and III procollagen mRNA levels for treated, infected animals to be only 32 and 29% that of infected controls, but beta-actin mRNA levels were significantly elevated. These results indicate a potential role for gamma-interferon as an antifibrogenic agent in vivo.

Autophagy is a lysosomal pathway by which intracellular organelles and proteins are degraded to supply the cell with energy and to maintain cellular homeostasis. Recently, lipid droplets (LDs) have been identified as a substrate for macroautophagy. In addition to the classic pathway of lipid metabolism by cytosolic lipases, LDs are sequestered in autophagosomes that fuse with lysosomes for the breakdown of LD components by lysosomal enzymes. The ability of autophagy to respond to changes in nutrient supply allows the cell to alter LD metabolism to meet the cell's energy demands. Pathophysiological changes in autophagic function can alter cellular lipid metabolism and promote disease states. Autophagy therefore represents a new cellular target for abnormalities in lipid metabolism and accumulation.

Insulin resistance and increased cytochrome P450 2E1 (CYP2E1) expression are both associated with and mechanistically implicated in the development of nonalcoholic fatty liver disease. Although currently viewed as distinct factors, insulin resistance and CYP2E1 expression may be interrelated through the ability of CYP2E1-induced oxidant stress to impair hepatic insulin signaling. To test this possibility, the effects of <i>in vitro</i> and <i>in vivo</i> CYP2E1 overexpression on hepatocyte insulin signaling were examined. CYP2E1 overexpression in a hepatocyte cell line decreased tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 in response to insulin. CYP2E1 overexpression was also associated with increased inhibitory serine 307 and 636/639 IRS-1 phosphorylation. In parallel, the effects of insulin on Akt activation, glycogen synthase kinase 3, and FoxO1a phosphorylation, and glucose secretion were all significantly decreased in CYP2E1 overexpressing cells. This inhibition of insulin signaling by CYP2E1 overexpression was partially c-Jun N-terminal kinase dependent. In the methionine- and choline-deficient diet mouse model of steatohepatitis with CYP2E1 overexpression, insulin-induced IRS-1, IRS-2, and Akt phosphorylation were similarly decreased. These findings indicate that increased hepatocyte CYP2E1 expression and the presence of steatohepatitis result in the down-regulation of insulin signaling, potentially contributing to the insulin resistance associated with nonalcoholic fatty liver disease.

Ito cells are perisinusoidal cells thought to be a major source of collagen in normal and fibrotic livers. These cells appear to have features similar to several cell types but when cultured assume a fibroblast-like morphology. In this study we evaluated the phenotype of both freshly isolated and cultured Ito cells by examining their gene expression. To better define the modulators of Ito-cell collagen synthesis, we also examined the effect of transforming growth factor-β1, tumor necrosis factor-α and dexamethasone on collagen synthesis by these cells. Northern hybridization analysis revealed that cultured Ito cells expressed different types of procollagen mRNAs than did freshly isolated cells. Cultured cells contained large amounts of type I procollagen mRNA and lesser amounts of types III and IV, whereas freshly isolated cells contained more type IV procollagen mRNA than types I and III. Treatment of cultured cells with either transforming growth factor-β1 or tumor necrosis factor-α resulted in a greater than threefold increase in total collagen content, and the effects of these cytokines on Ito-cell collagen synthesis involved different levels of gene regulation. Transforming growth factor-β1-treated cells had an approximately threefold increase in their type I procollagen mRNA levels, whereas no increase in this mRNA level was found in tumor necrosis factor-α-treated cells. Transforming growth factor-β1 treatment induced a twofold increase in transforming growth factor-β1 mRNA content in cultured cells. In contrast to transforming growth factor-β1, dexamethasone inhibited type I procollagen and transforming growth factor-β1 mRNA content by at least twofold in cultured cells. These results suggest that cultured Ito cells alter their collagen gene expression such that they become phenotypically more fibroblast-like. Furthermore, transforming growth factor-β1's induction of its own mRNA in Ito cells suggests that these cells are capable of amplifying their collagen synthesis. Finally, the inhibition of transforming growth factor-β1 and procollagen type I gene expression by dexamethasone suggests another way steroids may be beneficial in the treatment of certain forms of chronic liver disease.

Considerable indirect evidence suggests that cytokine tumor necrosis factor alpha contributes to the hepatocellular damage caused by toxic liver injury. The effects of tumor necrosis factor alpha neutralization on liver cell injury were determined in an in vivo model of toxic liver injury.The in vivo effects of tumor necrosis factor alpha were examined in carbon tetrachloride liver injury through the administration of a soluble tumor necrosis factor receptor to neutralize the effects of this cytokine.Soluble tumor necrosis factor receptor treatment decreased the degree of liver injury as measured by reduced levels of serum liver enzymes and improved histology. Soluble tumor necrosis factor receptor administration also lowered the mortality from a lethal dose of carbon tetrachloride from 60% to 16%. Tumor necrosis factor alpha neutralization had no detrimental effect on liver regeneration as determined by the timing of histone gene expression and postinjury liver weight.These data provide direct evidence for a role of tumor necrosis factor alpha in toxin-induced liver cell injury. In addition, these investigations suggest that soluble tumor necrosis factor receptor therapy may be of benefit in the treatment of human liver disease.

Toxins convert the hepatocellular response to tumor necrosis factor-α (TNF-α) stimulation from proliferation to cell death, suggesting that hepatotoxins somehow sensitize hepatocytes to TNF-α toxicity. Because nuclear factor-κB (NF-κB) activation confers resistance to TNF-α cytotoxicity in nonhepatic cells, the possibility that toxin-induced sensitization to TNF-α killing results from inhibition of NF-κB-dependent gene expression was examined in the RALA rat hepatocyte cell line sensitized to TNF-α cytotoxicity by actinomycin D (ActD). ActD did not affect TNF-α-induced hepatocyte NF-κB activation but decreased NF-κB-dependent gene expression. Expression of an IκB superrepressor rendered RALA hepatocytes sensitive to TNF-α-induced apoptosis in the absence of ActD. Apoptosis was blocked by caspase inhibitors, and TNF-α treatment led to activation of caspase-2, caspase-3, and caspase-8 only when NF-κB activation was blocked. Although apoptosis was blocked by the NF-κB-dependent factor nitric oxide (NO), inhibition of endogenous NO production did not sensitize cells to TNF-α-induced cytotoxicity. Thus NF-κB activation is the critical intracellular signal that determines whether TNF-α stimulates hepatocyte proliferation or apoptosis. Although exogenous NO blocks RALA hepatocyte TNF-α cytotoxicity, endogenous production of NO is not the mechanism by which NF-κB activation inhibits this death pathway.

Autophagy is a lysosomal pathway that degrades and recycles intracellular organelles and proteins to maintain energy homeostasis during times of nutrient deprivation and to remove damaged cell components. Recent studies have identified new functions for autophagy under basal and stressed conditions. In the liver and pancreas, autophagy performs the standard functions of degrading mitochondria and aggregated proteins and regulating cell death. In addition, autophagy functions in these organs to regulate lipid accumulation in hepatic steatosis, trypsinogen activation in pancreatitis, and hepatitis virus replication. This review discusses the effects of autophagy on hepatic and pancreatic physiology and the contribution of this degradative process to diseases of these organs. The discovery of novel functions for this lysosomal pathway has increased our understanding of the pathophysiology of diseases in the liver and pancreas and suggested new possibilities for their treatment.

The interferons are a group of endogenous proteins that exhibit a variety of biological functions in addition to their ability to induce resistance to viruses. In order to evaluate the anti-fibrogenic actions of interferon, we have delineated the level of regulation responsible for gamma-interferon-induced changes in collagen and fibronectin gene expression in cultured fibroblasts. Confluent human skin fibroblasts were exposed to 500 anti-viral units/ml of gamma-interferon. RNA was then extracted from the cells, and steady-state mRNA levels were determined by Northern and dot blot hybridization studies. Cells exposed to interferon had type I procollagen mRNA levels that were 23% of control and type III procollagen mRNA levels only 7% of control. The interferon-treated cells also had beta-actin mRNA levels that were decreased to 51% that of untreated cells but had fibronectin steady-state mRNA levels that were 560% of control levels. Nuclear run-on assays revealed that interferon did not affect the transcriptional rates of types I and III collagen or beta-actin, but it did increase the transcriptional rate of fibronectin to 670% of control levels. These findings demonstrate that gamma-interferon causes a marked decrease in types I and III procollagen mRNA levels in vitro by a post-transcriptional mechanism while inducing fibronectin expression at a transcriptional level.

Oxidative stress is a common mechanism of liver injury. Recent investigations have demonstrated that oxidant-induced liver injury is mediated by the direct effects of reactive oxygen species on signal transduction pathways. Although the function of cell signaling in this form of injury is complex and likely variable depending on the type and duration of oxidative stress, common regulatory pathways of hepatocyte oxidant injury have been identified that include the mitogen-activated protein kinases extracellular signal-regulated kinase 1/2 (ERK1/2) c-Jun N-terminal kinase (JNK), and the nuclear factor κB (NF-κB) pathway. Studies in cultured hepatocyte and rodent models of oxidative stress have demonstrated that ERK1/2 typically induces resistance to oxidant stress, whereas JNK promotes cell death. The effects of NF-κB activation are more complex and cell-type specific. A further understanding of the signaling pathways that regulate oxidant-induced liver injury may suggest new therapies for hepatic diseases resulting from oxidative stress.

Recent investigations have demonstrated a complex interrelationship between autophagy and cell death. A common mechanism of cell death in liver injury is tumor necrosis factor (TNF) cytotoxicity. To better delineate the in vivo function of autophagy in cell death, we examined the role of autophagy in TNF-induced hepatic injury. Atg7Δhep mice with a hepatocyte-specific knockout of the autophagy gene atg7 were generated and cotreated with D-galactosamine (GalN) and lipopolysaccharide (LPS). GalN/LPS-treated Atg7Δhep mice had increased serum alanine aminotransferase levels, histological injury, numbers of TUNEL (terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling)-positive cells and mortality as compared with littermate controls. Loss of hepatocyte autophagy similarly sensitized to GalN/TNF liver injury. GalN/LPS injury in knockout animals did not result from altered production of TNF or other cytokines. Atg7Δhep mice had accelerated activation of the mitochondrial death pathway and caspase-3 and -7 cleavage. Increased cell death did not occur from direct mitochondrial toxicity or a lack of mitophagy, but rather from increased activation of initiator caspase-8 causing Bid cleavage. GalN blocked LPS induction of hepatic autophagy, and increased autophagy from beclin 1 overexpression prevented GalN/LPS injury. Autophagy, therefore, mediates cellular resistance to TNF toxicity in vivo by blocking activation of caspase-8 and the mitochondrial death pathway, suggesting that autophagy is a therapeutic target in TNF-dependent tissue injury.

Autophagy is a lysosomal degradative pathway critical for the removal and breakdown of cellular components such as organelles and proteins. Despite striking similarities in the regulation and function of autophagy and lipid metabolism, the two processes have only recently been shown to be interrelated. This review details new findings of critical functions for autophagy in lipid metabolism and storage. Studies in hepatocytes and liver have demonstrated that macroautophagy mediates the breakdown of lipids stored in lipid droplets and that an inhibition of autophagy leads to the development of a fatty liver. In contrast, in adipocytes the loss of macroautophagy decreases the amount of lipid stored in adipose tissue through effects on white and brown adipocyte differentiation. Other investigations have indicated that the relationship between autophagy and lipids is bidirectional, with changes in cellular lipid content altering autophagic function. These newly described links between autophagy and lipid metabolism and storage have provided new insights into the mechanisms of both processes. The findings also suggest possible new therapeutic approaches to the problems of lipid overaccumulation and impaired autophagy that occur with aging and the metabolic syndrome.

Overactivation of the innate immune response underlies many forms of liver injury including that caused by hepatotoxins. Recent studies have demonstrated that macrophage autophagy regulates innate immunity and resultant tissue inflammation. Although hepatocyte autophagy has been shown to modulate hepatic injury, little is known about the role of autophagy in hepatic macrophages during the inflammatory response to acute toxic liver injury. Our aim therefore was to determine whether macrophage autophagy functions to down regulate hepatic inflammation.Mice with a LysM-CRE-mediated macrophage knockout of the autophagy gene ATG5 were examined for their response to toxin-induced liver injury from D-galactosamine/lipopolysaccharide (GalN/LPS).Knockout mice had increased liver injury from GalN/LPS as determined by significant increases in serum alanine aminotransferase, histological evidence of liver injury, positive terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling, caspase activation and mortality as compared to littermate controls. Levels of proinflammatory tumor necrosis factor and interleukin (IL)-6 hepatic mRNA and serum protein were unchanged, but serum IL-1β was significantly increased in knockout mice. The increase in serum IL-1β was secondary to elevated hepatic caspase 1 activation and inflammasome-mediated cleavage of pro-IL-1β to its active form. Cultured hepatic macrophages from GalN/LPS-treated knockout mice had similarly increased IL-1β production. Dysregulation of IL-1β was the mechanism of increased liver injury as an IL-1 receptor antagonist prevented injury in knockout mice in concert with decreased neutrophil activation.Macrophage autophagy functions to limit acute toxin-induced liver injury and death by inhibiting the generation of inflammasome-dependent IL-1β.

Activation of the Hippo pathway effector Yap underlies many liver cancers, however no germline or somatic mutations have been identified. Autophagy maintains essential metabolic functions of the liver, and autophagy-deficient murine models develop benign adenomas and hepatomegaly, which have been attributed to activation of the p62/Sqstm1-Nrf2 axis. Here, we show that Yap is an autophagy substrate and mediator of tissue remodeling and hepatocarcinogenesis independent of the p62/Sqstm1-Nrf2 axis. Hepatocyte-specific deletion of Atg7 promotes liver size, fibrosis, progenitor cell expansion, and hepatocarcinogenesis, which is rescued by concurrent deletion of Yap. Our results shed new light on mechanisms of Yap degradation and the sequence of events that follow disruption of autophagy, which is impaired in chronic liver disease.

Abstract The function of the lysosomal degradative pathway of autophagy in cellular injury is unclear, because findings in nonhepatic cells have implicated autophagy as both a mediator of cell death and as a survival response. Autophagic function is impaired in steatotic and aged hepatocytes, suggesting that in these settings hepatocellular injury may be altered by the decrease in autophagy. To delineate the specific function of autophagy in the hepatocyte injury response, the effects of menadione-induced oxidative stress were examined in the RALA255-10G rat hepatocyte line when macroautophagy was inhibited by a short hairpin RNA (shRNA)-mediated knockdown of the autophagy gene atg5. Loss of macroautophagy sensitized cells to apoptotic and necrotic death from normally nontoxic concentrations of menadione. Loss of macroautophagy led to overactivation of the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway that induced cell death. Death occurred from activation of the mitochondrial death pathway with cellular adenosine triphosphate (ATP) depletion, mitochondrial cytochrome c release, and caspase activation. Sensitization to death from menadione occurred despite up-regulation of other forms of autophagy in compensation for the loss of macroautophagy. Chaperone-mediated autophagy (CMA) also mediated resistance to menadione. CMA inhibition sensitized cells to death from menadione through a mechanism different from that of a loss of macroautophagy, because death occurred in the absence of JNK/c-Jun overactivation or ATP depletion. Conclusion: Hepatocyte resistance to injury from menadione-induced oxidative stress is mediated by distinct functions of both macroautophagy and CMA, indicating that impaired function of either form of autophagy may promote oxidant-induced liver injury. HEPATOLOGY 2010

Cellular lipid breakdown from LD stores depends on the direct actions of cytosolic neutral lipases on LDs and on acidic lysosomal lipases that act on LDs delivered to lysosomes by macroautophagy. Lipophagy (a selective form of macroautophagy) relies on the sensing of LD-associated proteins such as Rab7. This selectivity allows autophagy to specifically break down LD lipid stores in times of prolonged nutrient deprivation. Lipolysis by neutral lipases and lipophagy are not independent pathways but have significant crosstalk, such as the degradation of perilipins by autophagy to facilitate the actions of ATGL, or the ability of neutral lipases to supply lipids for autophagosome biogenesis. The functions of lipophagy in diverse processes ranging from steatosis to macrophage activation, and the fact that autophagy levels are often altered in disease, suggest that lipophagy is important in the pathogenesis of metabolic diseases. The selective breakdown by autophagy of lipid droplet (LD)-stored lipids, termed lipophagy, is a lysosomal lipolytic pathway that complements the actions of cytosolic neutral lipases. The physiological importance of lipophagy has been demonstrated in multiple mammalian cell types, as well as in lower organisms, and this pathway has many functions in addition to supplying free fatty acids to maintain cellular energy stores. Recent studies have begun to delineate the molecular mechanisms of the selective recognition of LDs by the autophagic machinery, as well as the intricate crosstalk between the different forms of autophagy and neutral lipases. These studies have led to increased interest in the role of lipophagy in both human disease pathogenesis and therapy. The selective breakdown by autophagy of lipid droplet (LD)-stored lipids, termed lipophagy, is a lysosomal lipolytic pathway that complements the actions of cytosolic neutral lipases. The physiological importance of lipophagy has been demonstrated in multiple mammalian cell types, as well as in lower organisms, and this pathway has many functions in addition to supplying free fatty acids to maintain cellular energy stores. Recent studies have begun to delineate the molecular mechanisms of the selective recognition of LDs by the autophagic machinery, as well as the intricate crosstalk between the different forms of autophagy and neutral lipases. These studies have led to increased interest in the role of lipophagy in both human disease pathogenesis and therapy. an autophagic vacuole formed by the fusion of an autophagosome and an endosome to deliver autophagic cargo to the lysosome as an alternative to direct autophagosome–lysosome fusion. cellular organelle resulting from the fusion of an autophagosome and a lysosome. In this structure, the cargo from the autophagosome mixes with the hydrolytic enzymes of the lysosome for degradation and release into the cytosol. a double-membrane vesicle in the cytosol of the cell that forms around cellular components for translocation to a lysosome as part of the process of macroautophagy. The origin of the membrane remains unclear with the ER, mitochondria, Golgi apparatus, mitochondrial and plasma membranes, and LDs all being implicated as sources. a type of autophagy that targets cytosolic proteins containing a pentapeptide motif that is recognized by the chaperone Hsc70. This complex is translocated to the lysosomal membrane where binding to the Lamp-2A receptor leads to internalization and degradation. cellular organelle in which TGs and cholesterol are stored in a central core surrounded by a phospholipid monolayer and a variety of proteins. a selective form of macroautophagy that specifically targets LD-contained lipids for degradation by lysosomal acid lipases. the major type of autophagy in which cytosolic organelles and proteins are sequestered in a double-membrane structure, termed the autophagosome, which fuses with a lysosome to form an autolysosome in which the cellular components are degraded and the products released into the cytosol. fat-laden macrophages that accumulate in the walls of arterial blood vessels as part of the process of atherosclerosis. a liver disorder with histological similarity to alcoholic liver disease that occurs in the absence of excessive alcohol intake. NAFLD is a continuum from simple lipid overaccumulation or steatosis to steatosis together with hepatocyte injury, inflammation, and eventual fibrosis. The mechanisms underlying this liver disease are unclear but the condition is strongly associated with insulin insensitivity and is a component of the metabolic syndrome. the more serious form of NAFLD in which steatosis is accompanied by hepatocyte injury and inflammation. Whereas simple steatosis is considered benign, NASH can progress to chronic liver disease and its complications. a member of a family of proteins that coat LDs and function to regulate the breakdown of lipids by both cytosolic lipases and lipophagy, in part by controlling the exposure of proteins in these pathways to the lipid core of the LD.

The prevalence of the metabolic syndrome and nonalcoholic fatty liver disease (NAFLD) in humans increases with age. It is unknown whether this association is secondary to the increased incidence of risk factors for NAFLD that occurs with aging, reflects the culmination of years of exposure to lifestyle factors such as a high-fat diet (HFD), or results from physiological changes that characterize aging. To examine this question, the development of NAFLD in response to a fixed period of HFD feeding was examined in mice of different ages. Mice aged 2, 8, and 18 months were fed 16 weeks of a low-fat diet or HFD. Increased body mass and insulin insensitivity occurred in response to HFD feeding irrespective of the age of the mice. The amount of HFD-induced hepatic steatosis as determined biochemically and histologically was also equivalent among the three ages. Liver injury occurred exclusively in the two older ages as reflected by increased serum alanine aminotransferase levels, positive terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling, and caspase activation. Older mice also had an elevated innate immune response with a more pronounced polarization of liver and adipose tissue macrophages into an M1 phenotype. Studies of cultured hepatocytes from young and old mice revealed that aged cells were selectively sensitized to the Fas death pathway.Aging does not promote the development of hepatic steatosis but leads to increased hepatocellular injury and inflammation that may be due in part to sensitization to the Fas death pathway and increased M1 macrophage polarization.

Background & Aims Acid sphingomyelinase (ASMase) is activated in non-alcoholic steatohepatitis (NASH). However, the contribution of ASMase to NASH is poorly understood and limited to hepatic steatosis and glucose metabolism. Here we examined the role of ASMase in high fat diet (HFD)-induced NASH. Methods Autophagy, endoplasmic reticulum (ER) stress and lysosomal membrane permeabilization (LMP) were determined in ASMase−/− mice fed a HFD. The impact of pharmacological ASMase inhibition on NASH was analyzed in wild type mice fed a HFD. Results ASMase deficiency determined resistance to hepatic steatosis mediated by a HFD or methionine-choline deficient diet. ASMase−/− mice were resistant to HFD-induced hepatic ER stress, but sensitive to tunicamycin-mediated ER stress, indicating selectivity in the resistance of ASMase−/− mice to ER stress and steatosis. Autophagic flux, determined in the presence of rapamycin and/or chloroquine, was lower in primary mouse hepatocytes (PMH) from ASMase−/− mice and accompanied by increased p62 levels, suggesting autophagic impairment. Moreover, autophagy suppression by chloroquine and brefeldin A caused ER stress in PMH from ASMase+/+ mice but not in ASMase−/− mice. ASMase−/− PMH exhibited increased lysosomal cholesterol loading, decreased LMP and apoptosis resistance induced by O-methyl-serine dodecylamide hydrochloride or palmitic acid, effects that were reversed by decreasing cholesterol levels by oxysterol 25-hydroxycholesterol. In vivo pharmacological ASMase inhibition by amitriptyline, a widely used tricyclic antidepressant, protected wild type mice against HFD-induced hepatic steatosis, fibrosis, and liver damage, effects indicative of early-stage NASH. Conclusions These findings underscore a critical role for ASMase in diet-induced NASH and suggest the potential of amitriptyline as a treatment for patients with NASH. Acid sphingomyelinase (ASMase) is activated in non-alcoholic steatohepatitis (NASH). However, the contribution of ASMase to NASH is poorly understood and limited to hepatic steatosis and glucose metabolism. Here we examined the role of ASMase in high fat diet (HFD)-induced NASH. Autophagy, endoplasmic reticulum (ER) stress and lysosomal membrane permeabilization (LMP) were determined in ASMase−/− mice fed a HFD. The impact of pharmacological ASMase inhibition on NASH was analyzed in wild type mice fed a HFD. ASMase deficiency determined resistance to hepatic steatosis mediated by a HFD or methionine-choline deficient diet. ASMase−/− mice were resistant to HFD-induced hepatic ER stress, but sensitive to tunicamycin-mediated ER stress, indicating selectivity in the resistance of ASMase−/− mice to ER stress and steatosis. Autophagic flux, determined in the presence of rapamycin and/or chloroquine, was lower in primary mouse hepatocytes (PMH) from ASMase−/− mice and accompanied by increased p62 levels, suggesting autophagic impairment. Moreover, autophagy suppression by chloroquine and brefeldin A caused ER stress in PMH from ASMase+/+ mice but not in ASMase−/− mice. ASMase−/− PMH exhibited increased lysosomal cholesterol loading, decreased LMP and apoptosis resistance induced by O-methyl-serine dodecylamide hydrochloride or palmitic acid, effects that were reversed by decreasing cholesterol levels by oxysterol 25-hydroxycholesterol. In vivo pharmacological ASMase inhibition by amitriptyline, a widely used tricyclic antidepressant, protected wild type mice against HFD-induced hepatic steatosis, fibrosis, and liver damage, effects indicative of early-stage NASH. These findings underscore a critical role for ASMase in diet-induced NASH and suggest the potential of amitriptyline as a treatment for patients with NASH.

Considerable evidence suggests that monocytes/macrophages play a crucial role in the process of liver injury and repair. Recent investigations have focused on the function of various macrophage-produced cytokines in liver disease. Much is still unknown, however, about the mechanism of macrophage recruitment and activation during liver disease. To further define this process, the gene expression of the monocyte chemoattractant monocyte chemoattractant protein 1 (MCP-1) was examined in animal and human liver disease. MCP-1 mRNA was not found in normal rat liver by Northern blot analysis. After single-dose treatments with the hepatotoxins carbon tetrachloride and galactosamine, MCP-1 mRNA was detectable beginning at 2 and 4 h after treatment, respectively, and was expressed continuously until 60-72 h. During chronic carbon tetrachloride administration, MCP-1 mRNA levels were elevated for the entire 10 weeks of treatment with peak levels of expression occurring early (weeks 1-3) and late (weeks 8-10) in this model. Isolated liver cell fractions from rats treated for 3 weeks with carbon tetrachloride revealed the major cellular source of MCP-1 mRNA to be fat-storing or Ito cells, with some expression occurring in the endothelial cell fraction. Studies of potential inducers of hepatic MCP-1 expression showed that lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-1 alpha and beta treatments all led to MCP-1 expression. Finally, studies of human liver samples revealed MCP-1 gene expression in nondiseased liver and greatly increased levels in livers from patients with fulminant hepatic failure. These data implicate MCP-1 from fat-storing cells as a modulator of the process of liver injury and further support a role for MCP-1 in the pathogenesis of human disease.

Oxidative stress has been implicated as a mechanism for a variety of forms of liver injury. Although reactive oxygen species (ROS) may damage cellular macromolecules directly, oxidant-induced cell death may result from redox effects on signal transduction pathways. To understand the mechanisms of hepatocyte death from oxidative stress, the functions of the mitogen-activated protein kinases (MAPKs) were determined during oxidant-induced hepatocyte injury from menadione. Low, nontoxic, and high toxic concentrations of the superoxide generator menadione were established in the RALA255-10G rat hepatocyte cell line. Death from menadione was blocked by catalase and ebselen, indicating that death was secondary to oxidant generation and not arylation. Treatment with a nontoxic menadione concentration resulted in a brief activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). In contrast, treatment with a toxic menadione concentration induced a prolonged activation of both ERK and JNK. Chemical inhibition of ERK function sensitized RALA hepatocytes to death from previously nontoxic menadione concentrations in association with sustained JNK activation. Adenoviral expression of a dominant-negative protein for c-Jun, a downstream substrate for JNK, blocked death from menadione. The pro-apoptotic effect of c-Jun was not mediated through the mitochondrial death pathway. In conclusion, RALA hepatocyte resistance to oxidant-induced death from menadione is dependent on ERK, whereas cell death is mediated by AP-1 activation. These findings identify signaling pathways that may be therapeutic targets in the prevention or treatment of oxidant-induced liver injury.

Glucocorticoids have been shown to be useful in the treatment of certain types of chronic liver disease both by inhibiting fibrosis and by improving liver function. We have previously demonstrated in an in vivo model of hepatic fibrogenesis that dexamethasone inhibits the synthesis of types I and IV collagen. In the present study we have evaluated the level of regulation responsible for the dexamethasone-induced changes in collagen gene expression in a defined in vitro system. Primary cultures of adult rat hepatocytes treated with and without dexamethasone under classical cell culture conditions or using defined media were evaluated for synthesis and abundance of procollagen and beta-actin mRNAs. Cells treated with dexamethasone had decreased types I and IV procollagen mRNA steady state levels due in part to diminished transcription rates of the genes. On the other hand, beta-actin mRNA levels were unaffected by dexamethasone. Transient expression experiments were performed to more precisely define the mechanism whereby dexamethasone affects type I procollagen gene transcription. The recombinant plasmid, pAZ1009, containing the mouse alpha 2(I) procollagen gene promoter linked to the chloramphenicol acetyltransferase gene, was transfected into mouse fibroblast cell lines. Cells transfected with the pAZ1009 plasmid in the presence of dexamethasone had a significant decrease in chloramphenicol acetyltransferase activity when compared to cells not exposed to dexamethasone. These data suggest that dexamethasone inhibits collagen synthesis through a direct effect on the collagen gene promoter and appears also to have a post-transcriptional effect on procollagen mRNA content.

Macroautophagy has been implicated as a mechanism of cell death. However, the relationship between this degradative pathway and cell death is unclear as macroautophagy has been shown recently to protect against apoptosis. To better define the interplay between these two critical cellular processes, we determined whether inhibition of macroautophagy could have both pro-apoptotic and anti-apoptotic effects in the same cell. Embryonic fibroblasts from mice with a knock-out of the essential macroautophagy gene atg5 were treated with activators of the extrinsic and intrinsic death pathways. Loss of macroautophagy sensitized these cells to caspase-dependent apoptosis from the death receptor ligands Fas and tumor necrosis factor-alpha (TNF-alpha). Atg5-/- mouse embryonic fibroblasts had increased activation of the mitochondrial death pathway in response to Fas/TNF-alpha in concert with decreased ATP levels. Fas/TNF-alpha treatment failed to up-regulate macroautophagy, and in fact, decreased activity at late time points. In contrast to their sensitization to Fas/TNF-alpha, Atg5-/- cells were resistant to death from menadione and UV light. In the absence of macroautophagy, an up-regulation of chaperone-mediated autophagy induced resistance to these stressors. These results demonstrate that inhibition of macroautophagy can promote or prevent apoptosis in the same cell and that the response is governed by the nature of the death stimulus and compensatory changes in other forms of autophagy. Experimental findings that an inhibition of macroautophagy blocks apoptosis do not prove that autophagy mediates cell death as this effect may result from the protective up-regulation of other autophagic pathways such as chaperone-mediated autophagy.

Nonalcoholic fatty liver disease (NAFLD) is now recognized as both an important component of the metabolic syndrome and the most prevalent liver disease in the United States. Although the mechanisms for development of steatosis and chronic liver injury in NAFLD remain unclear, recent investigations have indicated that overactivation of c-Jun N-terminal kinase (JNK) is crucial to this process. These findings, together with evidence for the involvement of JNK signaling in other manifestations of the metabolic syndrome such as obesity and insulin resistance, have suggested that JNK could be a novel therapeutic target in this disorder. This review details findings that JNK mediates lipid accumulation and cell injury in fatty liver disease and discusses the possible cellular mechanisms of JNK actions.

Abstract Oxidative stress has been implicated as the mechanism of hepatocyte injury from numerous agents. Although reactive oxygen species injure cells by the modification of critical cellular macromolecules, recent studies have demonstrated the mechanistic involvement of oxidant stress‐induced alterations in signal transduction cascades. Studies in menadione‐treated hepatocytes have demonstrated differential effects of mitogen‐activated protein kinase activation on hepatocyte death from acute oxidative stress. Activation of the extracellular signal‐regulated kinase pathway 1/2 (ERK1/2) confers hepatocyte resistance to death whereas sustained c‐Jun N‐terminal kinase (JNK)/c‐Jun/AP‐1 activation promotes apoptosis. Redundant protective signals such as the protein kinase C/protein kinase D pathways also downregulate the JNK/c‐Jun/AP‐1 cascade and provide resistance to cell death. Although ERK1/2 overactivation also acts as a protective response to chronic oxidative stress, enhanced activation of this kinase sensitizes hepatocytes to death from free fatty acids in this setting. The outcome from challenge with an oxidative stress, therefore, depends on the integration of a series of signaling cascades that both protect against and promote hepatocyte apoptosis.

In vitro studies have demonstrated a critical role for high-mobility group box 1 (HMGB1) in autophagy and the autophagic clearance of dysfunctional mitochondria, resulting in severe mitochondrial fragmentation and profound disturbances of mitochondrial respiration in HMGB1-deficient cells. Here, we investigated the effects of HMGB1 deficiency on autophagy and mitochondrial function in vivo, using conditional Hmgb1 ablation in the liver and heart. Unexpectedly, deletion of Hmgb1 in hepatocytes or cardiomyocytes, two cell types with abundant mitochondria, did not alter mitochondrial structure or function, organ function, or long-term survival. Moreover, hepatic autophagy and mitophagy occurred normally in the absence of Hmgb1, and absence of Hmgb1 did not significantly affect baseline and glucocorticoid-induced hepatic gene expression. Collectively, our findings suggest that HMGB1 is dispensable for autophagy, mitochondrial quality control, the regulation of gene expression, and organ function in the adult organism.

The generation of excessive amounts of reactive oxygen species (ROS) leads to cellular oxidative stress that underlies a variety of forms of hepatocyte injury and death including that from alcohol. Although ROS can induce cell damage through direct effects on cellular macromolecules, the injurious effects of ROS are mediated largely through changes in signal transduction pathways such as the mitogen-activated protein kinase c-Jun N-terminal kinase (JNK). In response to alcohol, hepatocytes have increased levels of the enzyme cytochrome P450 2E1 (CYP2E1) which generates an oxidant stress that promotes the development of alcoholic steatosis and liver injury. These effects are mediated in large part through overactivation of JNK that alters cell death pathways. Targeting the JNK pathway or its downstream effectors may be a useful therapeutic approach to the oxidative stress generated by CYP2E1 in alcoholic liver disease.

Background and Aims The proinflammatory cytokine IL‐1β has been implicated in the pathophysiology of nonalcoholic and alcoholic steatohepatitis. How IL‐1β promotes liver injury in these diseases is unclear, as no IL‐1β receptor‐linked death pathway has been identified. Autophagy functions in hepatocyte resistance to injury and death, and findings of decreased hepatic autophagy in many liver diseases suggest a role for impaired autophagy in disease pathogenesis. Recent findings that autophagy blocks mouse liver injury from lipopolysaccharide led to an examination of autophagy’s function in hepatotoxicity from proinflammatory cytokines. Approach and Results AML12 cells with decreased autophagy from a lentiviral autophagy‐related 5 ( Atg5 ) knockdown were resistant to toxicity from TNF, but sensitized to death from IL‐1β, which was markedly amplified by TNF co‐treatment. IL‐1β/TNF death was necrosis by trypan blue and propidium iodide positivity, absence of mitochondrial death pathway and caspase activation, and failure of a caspase inhibitor or necrostatin‐1s to prevent death. IL‐1β/TNF depleted autophagy‐deficient cells of ATP, and ATP depletion and cell death were prevented by supplementation with the energy substrate pyruvate or oleate. Pharmacological inhibitors and genetic knockdown studies demonstrated that IL‐1β/TNF‐induced necrosis resulted from lysosomal permeabilization and release of cathepsins B and L in autophagy‐deficient cells. Mice with a tamoxifen‐inducible, hepatocyte‐specific Atg5 knockout were similarly sensitized to cathepsin‐dependent hepatocellular injury and death from IL‐1β/TNF in combination, but neither IL‐1β nor TNF alone. Knockout mice had increased hepatic inflammation, and IL‐1β/TNF‐treated, autophagy‐deficient AML12 cells secreted exosomes with proinflammatory damage–associated molecular patterns. Conclusions The findings delineate mechanisms by which decreased hepatocyte autophagy promotes IL‐1β/TNF‐induced necrosis from impaired energy homeostasis and lysosomal permeabilization and inflammation through the secretion of exosomal damage–associated molecular patterns.

The heterodimeric integrin receptor α4β7 regulates CD4 T cell recruitment to inflamed tissues, but its role in the pathogenesis of non-alcoholic steatohepatitis (NASH) is unknown. Herein, we examined the role of α4β7-mediated recruitment of CD4 T cells to the intestine and liver in NASH.Male littermate F11r+/+ (control) and junctional adhesion molecule A knockout F11r-/- mice were fed a normal diet or a western diet (WD) for 8 weeks. Liver and intestinal tissues were analyzed by histology, quantitative reverse transcription PCR (qRT-PCR), 16s rRNA sequencing and flow cytometry. Colonic mucosa-associated microbiota were analyzed using 16s rRNA sequencing. Liver biopsies from patients with NASH were analyzed by confocal imaging and qRT-PCR.WD-fed knockout mice developed NASH and had increased hepatic and intestinal α4β7+ CD4 T cells relative to control mice who developed mild hepatic steatosis. The increase in α4β7+ CD4 T cells was associated with markedly higher expression of the α4β7 ligand mucosal addressin cell adhesion molecule 1 (MAdCAM-1) in the colonic mucosa and livers of WD-fed knockout mice. Elevated MAdCAM-1 expression correlated with increased mucosa-associated Proteobacteria in the WD-fed knockout mice. Antibiotics reduced MAdCAM-1 expression indicating that the diet-altered microbiota promoted colonic and hepatic MAdCAM-1 expression. α4β7 blockade in WD-fed knockout mice significantly decreased α4β7+ CD4 T cell recruitment to the intestine and liver, attenuated hepatic inflammation and fibrosis, and improved metabolic indices. MAdCAM-1 blockade also reduced hepatic inflammation and fibrosis in WD-fed knockout mice. Hepatic MAdCAM-1 expression was elevated in patients with NASH and correlated with higher expression of α4 and β7 integrins.These findings establish α4β7/MAdCAM-1 as a critical axis regulating NASH development through colonic and hepatic CD4 T cell recruitment.Non-alcoholic steatohepatitis (NASH) is an advanced and progressive form of non-alcoholic fatty liver disease (NAFLD), and despite its growing incidence no therapies currently exist to halt NAFLD progression. Herein, we show that blocking integrin receptor α4β7-mediated recruitment of CD4 T cells to the intestine and liver not only attenuates hepatic inflammation and fibrosis, but also improves metabolic derangements associated with NASH. These findings provide evidence for the potential therapeutic application of α4β7 antibody in the treatment of human NASH.

Iron is a mineral essential for blood production and a variety of critical cellular functions. Altered iron metabolism has been increasingly observed in many diseases and disorders, but a comprehensive and mechanistic understanding of the cellular impact of impaired iron metabolism is still lacking. We examined the effects of iron overload or iron deficiency on cellular stress responses and autophagy which collectively regulate cell homeostasis and survival. Acute iron loading led to increased mitochondrial ROS (mtROS) production and damage, lipid peroxidation, impaired autophagic flux, and ferroptosis. Iron-induced mtROS overproduction is the mechanism of increased lipid peroxidation, impaired autophagy, and the induction of ferroptosis. Iron excess-induced ferroptosis was cell-type dependent and regulated by activating transcription factor 4 (ATF4). Upregulation of ATF4 mitigated iron-induced autophagic dysfunction and ferroptosis, whereas silencing of ATF4 expression impaired autophagy and resulted in increased mtROS production and ferroptosis. Employing autophagy-deficient hepatocytes and different autophagy inhibitors, we further showed that autophagic impairment sensitized cells to iron-induced ferroptosis. In contrast, iron deficiency activated the endoplasmic reticulum (ER) stress response, decreased autophagy, and induced apoptosis. Decreased autophagy associated with iron deficiency was due to ER stress, as reduction of ER stress by 4-phenylbutyric acid (4-PBA) improved autophagic flux. The mechanism of decreased autophagy in iron deficiency is a disruption in lysosomal biogenesis due to impaired posttranslational maturation of lysosomal membrane proteins. In conclusion, iron excess and iron deficiency cause different forms of cell stress and death in part through the common mechanism of impaired autophagic function.

Reactive oxygen intermediates (ROI) have been implicated in the induction of hepatocyte apoptosis that results from a variety of forms of liver injury. Exogenous oxidants induce hepatocyte apoptosis and may mediate death during inflammatory liver injury. Lethal levels of intracellularly generated ROI resulting from hepatotoxin metabolism, or the induction of enzymes in the cytochrome P450 family, are also important inducers of apoptosis. In addition, ROI production may mediate death from a number of diverse factors, including tumor necrosis factor-alpha, bile acids, ischemia, and transforming growth factor-beta1. Oxidants alter many redox-sensitive cellular signaling pathways, including mitogen-activated protein kinases and transcription factors such as activator protein-1 and nuclear factor-kappaB. The mechanisms of oxidant-induced hepatocyte apoptosis remain unclear, but probably involve effects on cell signaling, as well as direct chemical interactions. The delineation of stimulus-specific mechanisms of oxidant-dependent hepatocyte apoptosis is important to the design of effective therapies for a number of forms of liver injury.

Activation of the JNK/activator protein-1 (AP-1)-signaling pathway is a common mediator of hepatocyte death from a variety of stimuli. Although the mechanism by which JNK or AP-1 promotes death is unknown, it results when activation of this signaling pathway is unusually prolonged. Although JNK/AP-1 mediates TNF-induced cell death at or above the level of the mitochondria, the ability of JNK/AP-1 to promote death from necrosis as well as apoptosis suggests that JNK/AP-1 may induce death by several mechanisms. Recognition of JNK/AP-1 signaling as a critical promoter of hepatocyte death raises the possibility that the therapeutic manipulation of this pathway may be effective in the treatment of human liver disease.

Hepatocytes can be sensitized to tumor necrosis factor (TNF)-α toxicity by repression of NF-κB activation or inhibition of RNA synthesis. To determine whether both forms of sensitization lead to TNF-α cytotoxicity by similar mechanisms, TNF-α-induced cell death in RALA255-10G hepatocytes was examined following infection with an adenovirus, Ad5IκB, that blocks NF-κB activation or following cotreatment with actinomycin D (ActD). TNF-α treatment of Ad5IκB-infected cells resulted in 44% cell death within 6 h. ActD/TNF-α induced no death within 6 h but did lead to 37% cell death by 24 h. In both instances, cell death occurred by apoptosis and was associated with caspase activation, although caspase activation in ActD-sensitized cells was delayed. CrmA and chemical caspase inhibitors blocked Ad5IκB/TNF-α-induced cell death but did not inhibit ActD/TNF-α-induced apoptosis. A Fas-associated protein with death domain (FADD) dominant negative decreased Ad5IκB/TNF-α- and ActD/TNF-α-induced cell death by 81 and 47%, respectively. However, downstream events differed, since Ad5IκB/TNF-α but not ActD/TNF-α treatment caused mitochondrial cytochrome crelease. These results suggest that NF-κB inactivation and inhibition of RNA synthesis sensitize RALA255-10G hepatocytes to TNF-α toxicity through distinct cell death pathways that diverge below the level of FADD. ActD-induced hepatocyte sensitization to TNF-α cytotoxicity occurs through a FADD-dependent, caspase-independent pathway of apoptosis. Hepatocytes can be sensitized to tumor necrosis factor (TNF)-α toxicity by repression of NF-κB activation or inhibition of RNA synthesis. To determine whether both forms of sensitization lead to TNF-α cytotoxicity by similar mechanisms, TNF-α-induced cell death in RALA255-10G hepatocytes was examined following infection with an adenovirus, Ad5IκB, that blocks NF-κB activation or following cotreatment with actinomycin D (ActD). TNF-α treatment of Ad5IκB-infected cells resulted in 44% cell death within 6 h. ActD/TNF-α induced no death within 6 h but did lead to 37% cell death by 24 h. In both instances, cell death occurred by apoptosis and was associated with caspase activation, although caspase activation in ActD-sensitized cells was delayed. CrmA and chemical caspase inhibitors blocked Ad5IκB/TNF-α-induced cell death but did not inhibit ActD/TNF-α-induced apoptosis. A Fas-associated protein with death domain (FADD) dominant negative decreased Ad5IκB/TNF-α- and ActD/TNF-α-induced cell death by 81 and 47%, respectively. However, downstream events differed, since Ad5IκB/TNF-α but not ActD/TNF-α treatment caused mitochondrial cytochrome crelease. These results suggest that NF-κB inactivation and inhibition of RNA synthesis sensitize RALA255-10G hepatocytes to TNF-α toxicity through distinct cell death pathways that diverge below the level of FADD. ActD-induced hepatocyte sensitization to TNF-α cytotoxicity occurs through a FADD-dependent, caspase-independent pathway of apoptosis. tumor necrosis factor-α RALA255-10G actinomycin D 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tumor necrosis factor receptor 1 Fas-associated protein with death domain N-[(indole-2-carbonlyl)alaninyl]-3-amino-4-oxo-5-fluoropentanoic acid N-[(1,3-dimethylindole-2-carbonyl)-valinyl]-3-amino-4-oxo-5-fluoropentanoic acid polyacrylamide gel electrophoresis fluorescence-activated cell sorting Prominent among the varied physiological effects of the cytokine tumor necrosis factor-α (TNF-α)1 is its ability to act as a cytotoxin and induce apoptotic or necrotic cell death (1Baker S.J. Reddy E.P. Oncogene. 1998; 17: 3261-3270Crossref PubMed Scopus (479) Google Scholar). Although TNF-α cytotoxicity has been widely investigated in the context of its potential as an antineoplastic agent, recent studies have demonstrated that TNF-α may also induce death in cells in normal tissue undergoing injury or inflammation. TNF-α toxicity is particularly important to the pathophysiology of liver disease, and TNF-α has been implicated as a mediator of hepatocyte death following injury from toxins, ischemia/reperfusion, and hepatitis virus (for a review, see Ref. 2Bradham C.A. Plumpe J. Manns M.P. Brenner D.A. Trautwein C. Am. J. Physiol. 1998; 275: G387-G392PubMed Google Scholar). In toxin-induced liver injury, endogenously produced TNF-α induces a significant proportion of the subsequent liver cell death as evidenced by the ability of TNF-α neutralization to dramatically reduce liver injury from toxins such as carbon tetrachloride (3Czaja M.J. Xu J. Alt E. Gastroenterology. 1995; 108: 1849-1854Abstract Full Text PDF PubMed Scopus (191) Google Scholar), actinomycin D (ActD) (4Leist M. Gantner F. Naumann H. Bluethmann H. Vogt K. Brigelius-Flohe R. Nicotera P. Volk H.-D. Wendel A. Gastroenterology. 1997; 112: 923-934Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), and ethanol (5Iimuro Y. Gallucci R.M. Luster M.I. Kono H. Thurman R.G. Hepatology. 1997; 26: 1530-1537Crossref PubMed Scopus (447) Google Scholar). Hepatocytes are normally resistant to TNF-α cytotoxicity (6Leist M. Gantner F. Bohlinger I. Germann P.G. Tiegs G. Wendel A. J. Immunol. 1994; 153: 1778-1788PubMed Google Scholar, 7Xu Y. Jones B.E. Neufeld D. Czaja M. Gastroenterology. 1998; 115: 1229-1237Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar); therefore, these toxins sensitize hepatocytes to cell death from TNF-α by an as yet unknown mechanism. In vitroinvestigations into the mechanisms of TNF-α cytotoxicity in nonhepatic cells have demonstrated that binding of TNF-α to tumor necrosis factor receptor 1 (TNFR-1) results in receptor trimerization and the recruitment of a series of intracellular proteins (1Baker S.J. Reddy E.P. Oncogene. 1998; 17: 3261-3270Crossref PubMed Scopus (479) Google Scholar). Initially, TNFR-associated death domain protein binds to the TNFR-1. TNFR-associated death domain protein then recruits TNFR-associated factor 2, Fas-associated protein with death domain (FADD), and receptor-interacting protein (1Baker S.J. Reddy E.P. Oncogene. 1998; 17: 3261-3270Crossref PubMed Scopus (479) Google Scholar, 8Hsu H. Shu H.-B. Pan M.-G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1726) Google Scholar). Binding of TNFR-associated death domain protein and FADD to the TNFR-1 leads to the recruitment, oligomerization, and activation of caspase-8 (8Hsu H. Shu H.-B. Pan M.-G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1726) Google Scholar, 9Boldin M.P. Goncharov T.M. Golster Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2100) Google Scholar). Activated caspase-8 subsequently initiates a proteolytic cascade involving other caspase family members, ultimately leading to apoptosis (10Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4105) Google Scholar, 11Sun X.-M. MacFarlane M. Zhuang J. Wolf B.B. Green D.R. Cohen G.M. J. Biol. Chem. 1999; 274: 5053-5060Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar). Activation of these downstream caspases may be amplified by factors released from mitochondria such as cytochrome c (12Bradham C.A. Gian T. Streetz K. Trautwein C. Brenner D.A. Lemasters J.J. Mol. Cell. Biol. 1998; 18: 6353-6364Crossref PubMed Scopus (367) Google Scholar, 13Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). Alternative caspase-8-independent mechanisms by which TNF-α receptor binding initiates downstream caspase activation may also exist. Investigations have demonstrated a FADD-independent pathway of TNF-α-induced caspase activation involving RAIDD (14Duan H. Dixit V.M. Nature. 1997; 385: 86-89Crossref PubMed Scopus (468) Google Scholar). Despite their differences, these pathways all ultimately transduce the TNF-α death signal through the activation of caspases.The resistance of nontransformed cells to TNF-α-induced cytotoxicity is thought to depend on the ability of TNF-α signaling to up-regulate a protective cellular gene(s). This conclusion is based on the finding that inhibition of RNA synthesis by ActD or of protein synthesis by cycloheximide sensitizes nonhepatic cells (15Kull F.C. Cuatrecasas P. Cancer Res. 1981; 41: 4885-4890PubMed Google Scholar) and hepatocytes (4Leist M. Gantner F. Naumann H. Bluethmann H. Vogt K. Brigelius-Flohe R. Nicotera P. Volk H.-D. Wendel A. Gastroenterology. 1997; 112: 923-934Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 7Xu Y. Jones B.E. Neufeld D. Czaja M. Gastroenterology. 1998; 115: 1229-1237Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) to TNF-α-induced cell death. Recent investigations have demonstrated that cellular activation of the transcription factor NF-κB is critical for the induction of resistance to TNF-α toxicity (16Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2441) Google Scholar, 17Wang C.-Y. Mayo M.W. Baldwin A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2500) Google Scholar, 18Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2926) Google Scholar, 19Liu Z.G. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1778) Google Scholar). Blocking NF-κB activation in cultured hepatocytes (12Bradham C.A. Gian T. Streetz K. Trautwein C. Brenner D.A. Lemasters J.J. Mol. Cell. Biol. 1998; 18: 6353-6364Crossref PubMed Scopus (367) Google Scholar, 20Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar) or in the liver in vivo (21Iimuro Y. Nishiura T. Hellerbrand C. Behrns K.E. Schoonhoven R. Grisham J.W. Brenner D.A. J. Clin. Invest. 1998; 101: 802-811Crossref PubMed Scopus (418) Google Scholar), converts the hepatocellular TNF-α response from one of proliferation to one of apoptosis. These results have suggested that toxins such as ActD or carbon tetrachloride may sensitize hepatocytes to TNF-α toxicity by blocking up-regulation of an NF-κB-dependent protective gene. However, because of differences in the amount of cell death caused by these two forms of TNF-α toxicity (7Xu Y. Jones B.E. Neufeld D. Czaja M. Gastroenterology. 1998; 115: 1229-1237Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 20Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar), we hypothesized that ActD and NF-κB inhibition sensitize hepatocytes to TNF-α-induced death by different mechanisms. By contrasting the involvement of caspases, FADD, and cytochrome c in these two forms of sensitization to TNF-α-induced cytotoxicity, the present studies demonstrate that two distinct TNF-α cell death pathways exist in hepatocytes.DISCUSSIONTNF-α has multiple biological effects on hepatocytes that include either the stimulation of cellular proliferation or the initiation of cell death. In rat liver, TNF-α induces both responses under different physiological circumstances, stimulating hepatocyte proliferation after partial hepatectomy (29Akerman P. Cote P. Yang S.Q. McClain C. Nelson S. Bagby G.J. Diehl A.M. Am. J. Physiol. 1992; 263: G9579-G9585Google Scholar, 30Yamada Y. Kirillova I. Peschon J.J. Fausto N. Proc. Natl. Acad. Sci. 1997; 94: 1441-1446Crossref PubMed Scopus (827) Google Scholar) and inducing cell death during hepatotoxic injury (3Czaja M.J. Xu J. Alt E. Gastroenterology. 1995; 108: 1849-1854Abstract Full Text PDF PubMed Scopus (191) Google Scholar). Our previous investigations have demonstrated that hepatocyte NF-κB activation is essential to preventing TNF-α cytotoxicity (20Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar), similar to findings in some nonhepatic cells (16Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2441) Google Scholar, 17Wang C.-Y. Mayo M.W. Baldwin A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2500) Google Scholar, 18Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2926) Google Scholar, 19Liu Z.G. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1778) Google Scholar). Despite the complex and diverse signaling cascades initiated by TNF-α, inhibition of activation of the single transcription factor NF-κB is sufficient to convert the TNF-α response from proliferation to cell death both in cultured RALA hepatocytes (20Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar) and in hepatocytes in vivo following partial hepatectomy (21Iimuro Y. Nishiura T. Hellerbrand C. Behrns K.E. Schoonhoven R. Grisham J.W. Brenner D.A. J. Clin. Invest. 1998; 101: 802-811Crossref PubMed Scopus (418) Google Scholar). These results suggest that NF-κB is the transcriptional regulator of a cellular gene(s) that when up-regulated by TNF-α blocks the cellular death response to TNF-α and allows hepatocellular proliferation to occur.The concept that NF-κB up-regulates a protective cellular gene is consistent with findings that inhibition of RNA or protein synthesis sensitizes resistant cells including hepatocytes to cytotoxicity from TNF-α (4Leist M. Gantner F. Naumann H. Bluethmann H. Vogt K. Brigelius-Flohe R. Nicotera P. Volk H.-D. Wendel A. Gastroenterology. 1997; 112: 923-934Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 7Xu Y. Jones B.E. Neufeld D. Czaja M. Gastroenterology. 1998; 115: 1229-1237Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 15Kull F.C. Cuatrecasas P. Cancer Res. 1981; 41: 4885-4890PubMed Google Scholar). Previous studies in RALA hepatocytes demonstrated that ActD did not prevent TNF-α-induced activation of NF-κB as measured by DNA binding activity, but ActD did partially inhibit NF-κB-dependent gene expression (20Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar). NF-κB inactivation or RNA/protein synthesis inhibition may therefore act at different levels to ultimately block expression of the same NF-κB-dependent cellular gene. To examine this question, TNF-α-induced cell death in RALA hepatocytes was compared following NF-κB inactivation or ActD treatment. The present results indicate that the two forms of TNF-α-induced cell death differ significantly in several respects. The first difference is in the timing of cell death. NF-κB inactivation resulted in immediate cell death with increased numbers of apoptotic cells within only 1 h of TNF-α treatment (20Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar) and the majority of cell loss occurring in 6 h. In contrast, ActD/TNF-α treatment led to a delayed induction of cell death. No death occurred within 6 h after treatment, and a small increase in the number of apoptotic cells was detected only after 8 h of treatment. This disparate timing of cell death in the two models suggests that the two forms of sensitization activate distinct cell death pathways.Despite the time differential, both forms of TNF-α toxicity resulted in apoptotic cell death. Consistent with prior findings that RALA hepatocyte death from Ad5IκB/TNF-α occurred by apoptosis (20Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar), ActD/TNF-α-induced cell death was also apoptotic as determined by fluorescent microscopic studies, PARP cleavage, and FACS analysis. All of these measures of apoptosis confirmed MTT and cell count data indicating that cell death did not commence until 8 h after ActD/TNF-α treatment. An additional hallmark of apoptosis in ActD/TNF-α-treated cells was the presence of caspase activation. Western immunoblotting and caspase-3-like enzyme activity assays demonstrated that caspase activation occurred following TNF-α stimulation in cells sensitized by either Ad5IκB infection or ActD treatment. In keeping with the divergent time courses of apoptosis in the two models, caspase activation following ActD sensitization occurred much later than that seen following inhibition of NF-κB. Caspase activation was not evident until 24 h, in contrast to the rapid caspase activation previously detected within 2 h after Ad5IκB/TNF-α treatment (20Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar). Although both models of TNF-α sensitization resulted in caspase activation at times appropriate to the occurrence of apoptosis, viral and chemical caspase inhibitors only blocked death in Ad5IκB/TNF-α-treated cells. The failure of caspase inhibitors to prevent ActD/TNF-α-induced apoptosis was not because of insufficient caspase inhibition, because the chemical inhibitor IDN-1529 markedly suppressed caspase-3-like activity in these cells and completely abrogated the degradation of cellular DNA. These data are consistent with reports of a caspase-activated DNAase responsible for DNA fragmentation in apoptosis (31Enari M. Sakakura H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2795) Google Scholar) and the fact that DNA fragmentation is a late phenomenon not essential for the occurrence of apoptotic cell death (32Deas O. Dumont C. MacFarlane M. Rouleau M. Hebib C. Harper F. Hirsch F. Charpentier B. Cohen G.M. Senik A. J. Immunol. 1998; 161: 3375-3383PubMed Google Scholar).RALA hepatocytes still exhibited morphological features of apoptosis on fluorescent microscopy despite effective caspase inhibition. The dissociation between the caspase-dependent apoptotic parameters of PARP and internucleosomal DNA cleavage and the caspase-independent morphological changes demonstrated in the present study are consistent with recent reports identifying the ability of a caspase-independent factor to induce an apoptotic nuclear morphology (33Samejima K. Tone S. Kottke T.J. Enari M. Sakahira H. Cooke C.A. Durrieu F. Martins L.M. Nagata S. Kaufmann S.H. Eurnshow W.C. J. Cell Biol. 1998; 143: 225-239Crossref PubMed Scopus (110) Google Scholar, 34Marzo I. Susin S.A. Petit P.X. Ravagnan L. Brenner C. Larochette N. Zamzami N. Kroemer G. FEBS Lett. 1998; 427: 198-202Crossref PubMed Scopus (125) Google Scholar). Although caspase activation occurred with ActD/TNF-α treatment and was associated with caspase-dependent biochemical hallmarks of apoptosis, caspase activation did not mediate apoptosis from ActD/TNF-α treatment. Results in ActD/TNF-α-treated cells are in marked contrast to those in Ad5IκB/TNF-α-treated cells in which caspase activation was critical to the induction of apoptosis. These data demonstrate that NF-κB inactivation and ActD sensitize RALA hepatocytes to TNF-α cytotoxicity by distinct mechanisms. In the absence of NF-κB, a critical inhibitor of the TNFR-1-triggered caspase cascade may not be up-regulated, leading to a rapid caspase activation that commits the cell to apoptosis. ActD sensitized hepatocytes to TNF-α-induced death by a slower, and as yet undetermined mechanism in which caspase activation was not critical for the commitment to cell death. To our knowledge, these findings represent the first report of TNF-α-induced apoptosis occurring by a caspase-independent mechanism and add to the other recent examples of caspase-independent apoptosis (32Deas O. Dumont C. MacFarlane M. Rouleau M. Hebib C. Harper F. Hirsch F. Charpentier B. Cohen G.M. Senik A. J. Immunol. 1998; 161: 3375-3383PubMed Google Scholar, 35Trapani J.A. Jans D.A. Jans P.J. Smyth M.J. Browne K.A. Sutton V.R. J. Biol. Chem. 1998; 273: 27934-27938Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar).These results differ from a recent report by Li et al. (36Li J. Bombeck C.A. Yang S. Kim Y.-M. Billiar T.R. J. Biol. Chem. 1999; 274: 17325-17333Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar), who demonstrated that in primary rat hepatocyte cultures ActD/TNF-α caused a rapid, caspase-dependent apoptosis associated with cytochrome c release. A potential explanation for these divergent findings is that Li et al. employed a much higher ActD concentration (200 ng/ml), which by itself causes a significant amount of hepatocyte apoptosis. Unlike our lower ActD dose, their concentration may have more effectively inhibited NF-κB-dependent gene transcription, making their model equivalent to our Ad5IκB/TNF-α treatment. In addition, chemotherapeutic drugs that interfere with macromolecular synthesis induce apoptosis through the Fas death pathway (37Müller M. Strand S. Hug H. Heinemann E.M. Walczak H. Hofmann W.J. Stremmel W. Krammer P.H. Galle P.R. J. Clin. Invest. 1997; 99: 403-413Crossref PubMed Scopus (715) Google Scholar, 38Micheau O. Solary E. Hammann A. Dimanche-Boitrel M.T. J. Biol. Chem. 1999; 274: 7987-7992Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar), and high dose ActD may also activate this caspase-dependent pathway in hepatocytes. Alternatively, primary hepatocyte cultures exist in a nonproliferative, proapoptotic state that may alter cell death responses as compared with RALA hepatocytes or hepatocytes in vivo.To determine the level at which the two TNF-α death pathways diverge in RALA hepatocytes, the function of the TNFR-1-binding protein FADD was blocked. Expression of a dominant negative FADD significantly reduced cell death following either Ad5IκB/TNF-α or ActD/TNF-α treatment, indicating that these pathways diverge below the level of FADD. These data demonstrate that in RALA hepatocytes FADD can transduce the TNF-α death signal via a caspase-independent pathway, in addition to the pathway involving caspase-8 activation previously described in nonhepatic cells (9Boldin M.P. Goncharov T.M. Golster Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2100) Google Scholar). This finding, together with the recent demonstration of FADD-dependent, caspase-independent induction of necrosis in Jurkat cells by Fas ligand (39Kawahara A. Ohsawa Y. Matsumura H. Uchiyama Y. Nagata S. J. Cell Biol. 1998; 143: 1353-1360Crossref PubMed Scopus (274) Google Scholar), points to the ability of FADD to initiate cell death through caspase-independent mechanisms. Subsequent to the engagement of FADD, the two pathways of TNF-α-induced cell death in hepatocytes diverge, with mitochondrial cytochrome c release occurring with Ad5IκB/TNF-α- but not ActD/TNF-α-induced apoptosis. Mitochondrial release of cytochromec is not essential for many forms of death receptor-induced apoptosis but has been proposed to serve as an accelerator of this process (40Stennicke H.R. Jürgensmeier J.M. Shin H. Deveraux Q. Wolf B.B. Yang X. Zhou Q. Ellerby H.M. Ellerby L.M. Bredesen D. Green D.R. Reed J.C. Froelich C.J. Salvesen G.S. J. Biol. Chem. 1998; 273: 27084-27090Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar). Receptor-mediated apoptosis has been demonstrated to result from cytochrome c-independent caspase activation through the direct actions of autoactivated caspase-8 on caspase-3 and -7 (40Stennicke H.R. Jürgensmeier J.M. Shin H. Deveraux Q. Wolf B.B. Yang X. Zhou Q. Ellerby H.M. Ellerby L.M. Bredesen D. Green D.R. Reed J.C. Froelich C.J. Salvesen G.S. J. Biol. Chem. 1998; 273: 27084-27090Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar). In RALA hepatocytes, cytochrome c release may be required for the induction of a rapid, caspase-dependent apoptotic pathway. In the absence of cytochrome c release, the engagement of FADD triggers an alternative, caspase-independent death pathway. In nonhepatic cells, FADD mediates activation of acid sphingomyelinase, leading to ceramide generation (41Schwandner R. Wiegmann K. Bernardo K. Kreder D. Krönke M. J. Biol. Chem. 1998; 273: 5916-5922Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). We have previously demonstrated that RALA hepatocytes sensitized to ceramide toxicity by ActD undergo caspase-independent apoptosis (42Jones B.E. Lo C.R. Srinivasan A. Valentino K.L. Czaja M.J. Hepatology. 1999; 30: 215-222Crossref PubMed Scopus (45) Google Scholar). It is therefore possible that FADD-dependent ceramide signaling induces cell death in ActD/TNF-α-treated RALA hepatocytes. Cell death may occur from mitochondrial damage with resultant toxic generation of reactive oxygen species and depletion of ATP. Further studies must now identify both the mechanism of this caspase-independent death and the factors that regulate whether sensitized hepatocytes enter a rapid, caspase-dependent or slower, caspase-independent pathway of TNF-α-induced apoptosis. Prominent among the varied physiological effects of the cytokine tumor necrosis factor-α (TNF-α)1 is its ability to act as a cytotoxin and induce apoptotic or necrotic cell death (1Baker S.J. Reddy E.P. Oncogene. 1998; 17: 3261-3270Crossref PubMed Scopus (479) Google Scholar). Although TNF-α cytotoxicity has been widely investigated in the context of its potential as an antineoplastic agent, recent studies have demonstrated that TNF-α may also induce death in cells in normal tissue undergoing injury or inflammation. TNF-α toxicity is particularly important to the pathophysiology of liver disease, and TNF-α has been implicated as a mediator of hepatocyte death following injury from toxins, ischemia/reperfusion, and hepatitis virus (for a review, see Ref. 2Bradham C.A. Plumpe J. Manns M.P. Brenner D.A. Trautwein C. Am. J. Physiol. 1998; 275: G387-G392PubMed Google Scholar). In toxin-induced liver injury, endogenously produced TNF-α induces a significant proportion of the subsequent liver cell death as evidenced by the ability of TNF-α neutralization to dramatically reduce liver injury from toxins such as carbon tetrachloride (3Czaja M.J. Xu J. Alt E. Gastroenterology. 1995; 108: 1849-1854Abstract Full Text PDF PubMed Scopus (191) Google Scholar), actinomycin D (ActD) (4Leist M. Gantner F. Naumann H. Bluethmann H. Vogt K. Brigelius-Flohe R. Nicotera P. Volk H.-D. Wendel A. Gastroenterology. 1997; 112: 923-934Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), and ethanol (5Iimuro Y. Gallucci R.M. Luster M.I. Kono H. Thurman R.G. Hepatology. 1997; 26: 1530-1537Crossref PubMed Scopus (447) Google Scholar). Hepatocytes are normally resistant to TNF-α cytotoxicity (6Leist M. Gantner F. Bohlinger I. Germann P.G. Tiegs G. Wendel A. J. Immunol. 1994; 153: 1778-1788PubMed Google Scholar, 7Xu Y. Jones B.E. Neufeld D. Czaja M. Gastroenterology. 1998; 115: 1229-1237Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar); therefore, these toxins sensitize hepatocytes to cell death from TNF-α by an as yet unknown mechanism. In vitroinvestigations into the mechanisms of TNF-α cytotoxicity in nonhepatic cells have demonstrated that binding of TNF-α to tumor necrosis factor receptor 1 (TNFR-1) results in receptor trimerization and the recruitment of a series of intracellular proteins (1Baker S.J. Reddy E.P. Oncogene. 1998; 17: 3261-3270Crossref PubMed Scopus (479) Google Scholar). Initially, TNFR-associated death domain protein binds to the TNFR-1. TNFR-associated death domain protein then recruits TNFR-associated factor 2, Fas-associated protein with death domain (FADD), and receptor-interacting protein (1Baker S.J. Reddy E.P. Oncogene. 1998; 17: 3261-3270Crossref PubMed Scopus (479) Google Scholar, 8Hsu H. Shu H.-B. Pan M.-G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1726) Google Scholar). Binding of TNFR-associated death domain protein and FADD to the TNFR-1 leads to the recruitment, oligomerization, and activation of caspase-8 (8Hsu H. Shu H.-B. Pan M.-G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1726) Google Scholar, 9Boldin M.P. Goncharov T.M. Golster Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2100) Google Scholar). Activated caspase-8 subsequently initiates a proteolytic cascade involving other caspase family members, ultimately leading to apoptosis (10Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4105) Google Scholar, 11Sun X.-M. MacFarlane M. Zhuang J. Wolf B.B. Green D.R. Cohen G.M. J. Biol. Chem. 1999; 274: 5053-5060Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar). Activation of these downstream caspases may be amplified by factors released from mitochondria such as cytochrome c (12Bradham C.A. Gian T. Streetz K. Trautwein C. Brenner D.A. Lemasters J.J. Mol. Cell. Biol. 1998; 18: 6353-6364Crossref PubMed Scopus (367) Google Scholar, 13Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). Alternative caspase-8-independent mechanisms by which TNF-α receptor binding initiates downstream caspase activation may also exist. Investigations have demonstrated a FADD-independent pathway of TNF-α-induced caspase activation involving RAIDD (14Duan H. Dixit V.M. Nature. 1997; 385: 86-89Crossref PubMed Scopus (468) Google Scholar). Despite their differences, these pathways all ultimately transduce the TNF-α death signal through the activation of caspases. The resistance of nontransformed cells to TNF-α-induced cytotoxicity is thought to depend on the ability of TNF-α signaling to up-regulate a protective cellular gene(s). This conclusio

We studied the mechanisms by which excess copper exerts, and zinc mitigates, toxic effects on HepG2 cells. Survival and cell growth were reduced in media containing greater than 500 microM copper chloride for 48 h; LD50 was 750 microM. At 1,000 microM copper for 1 h, there was a general reduction of protein synthesis, and no recognizable changes in cellular ultrastructure. Incubation of cells with 200 microM zinc acetate before exposure to copper, raised the LD50 for confluent cells to 1,250 microM copper chloride, improved protein synthesis, and increased synthesis of a 10-kD protein, apparently metallothionein. The mitigation, by zinc, of copper's toxicity may in part be mediated through induction of this protein in the hepatocyte.

Tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) have a number of in vitro functions that could be important in vivo in acute liver injury and repair. Therefore, we investigated these two cytokines in acute liver damage. Northern blots of RNA isolated from rats sacrificed at various time intervals after a single oral dose of CCl4 revealed that TNF-α mRNA levels were elevated within 6 hr of CCl4 administration and returned to control values by 24–32 hr. In contrast, TGF-β1 mRNA levels started to rise significantly at 24 hr, peaked at 48 hr, and approached baseline levels by 72 hr. Identical changes in TNF-α and TGF-β1 mRNA levels were also seen with D-galactosamine-induced hepatotoxicity. Immunohistochemical analysis using a TGF-β1 antibody demonstrated increased hepatic staining in CCl4-treated rats, at times corresponding to the increases in TGF-β1 gene expression. Therefore, there is a differential expression of these cytokines in acute CCl4 and galactosamine hepatotoxicity with an early rise in TNF-α, suggesting that this cytokine may affect inflammation and cell toxicity, while TGF-β1 peaks later, when it may regulate hepatocyte proliferation and extracellular matrix repair.

Sustained activation of the c-Jun NH 2 -terminal kinase (JNK) signaling pathway mediates the development and progression of experimental diet-induced nonalcoholic fatty liver disease (NAFLD). Delineating the mechanism of JNK overactivation in the setting of a fatty liver is therefore essential to understanding the pathophysiology of NAFLD. Both human and experimental NAFLD are associated with oxidative stress and resultant lipid peroxidation, which have been proposed to mediate the progression of this disease from simple steatosis to steatohepatitis. The ability of oxidants and the lipid peroxidation product 4-hydroxynonenal (HNE) to activate JNK signaling suggested that these two factors may act synergistically to trigger JNK overactivation. The effect of HNE on hepatocyte injury and JNK activation was therefore examined in cells under chronic oxidant stress from overexpression of the prooxidant enzyme cytochrome P450 2E1 (CYP2E1), which occurs in NAFLD. CYP2E1-generated oxidant stress sensitized a rat hepatocyte cell line to death from normally nontoxic concentrations of HNE. CYP2E1-overexpressing cells underwent a more profound depletion of glutathione (GSH) in response to HNE secondary to decreased γ-glutamylcysteine synthetase activity. GSH depletion led to overactivation of JNK/c-Jun signaling at the level of mitogen-activated protein kinase kinase 4 that induced cell death. Oxidant stress and the lipid peroxidation product HNE cause synergistic overactivation of the JNK/c-Jun signaling pathway in hepatocytes, demonstrating that HNE may not be just a passive biomarker of hepatic oxidant stress but rather an active mediator of hepatocellular injury through effects on JNK signaling.

During sepsis, bacterial products, particularly LPS, trigger injury in organs such as the liver. This common condition remains largely untreatable, in part due to a lack of understanding of how high concentrations of LPS cause cellular injury. In the liver, the lysosomal degradative pathway of autophagy performs essential hepatoprotective functions and is induced by LPS. We, therefore, examined whether hepatocyte autophagy protects against liver injury from septic levels of LPS. Mice with an inducible hepatocyte-specific knockout of the critical autophagy gene Atg7 were examined for their sensitivity to high-dose LPS. Increased liver injury occurred in knockout mice, as determined by significantly increased serum alanine aminotransferase levels, histological evidence of liver injury, terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling, and effector caspase-3 and -7 activation. Hepatic inflammation and proinflammatory cytokine induction were unaffected by the decrease in hepatocyte autophagy. Although knockout mice had normal NF-κB signaling, hepatic levels of Akt1 and Akt2 phosphorylation in response to LPS were decreased. Cultured hepatocytes from knockout mice displayed a generalized defect in Akt signaling in response to multiple stimuli, including LPS, TNF, and IL-1β. Akt activation mediates hepatocyte resistance to TNF cytotoxicity, and anti-TNF antibodies significantly decreased LPS-induced liver injury in knockout mice, indicating that the loss of autophagy sensitized to TNF-dependent liver damage. Hepatocyte autophagy, therefore, protects against LPS-induced liver injury. Conditions such as aging and steatosis that impair hepatic autophagy may predispose to poor outcomes from sepsis through this mechanism.

Reactive oxygen intermediates (ROI) have been implicated as mediators of hepatocyte death resulting from a variety of forms of liver injury. To delineate the mechanisms that underlie ROI-induced apoptosis, the roles of caspase activation and nuclear factor-kappaB (NF-kappaB) signaling were determined in the rat hepatocyte cell line RALA255-10G after treatment with H(2)O(2) or the superoxide generator menadione. By 8 h, H(2)O(2) and menadione caused 26% and 33% cell death, respectively. Death from both ROI occurred by apoptosis as indicated by morphology under fluorescence microscopy, the induction of caspase activation and DNA fragmentation, and the cleavage of poly(ADP-ribose) polymerase. Despite the presence of caspase activation in both forms of apoptosis, caspase inhibition blocked H(2)O(2)- but not menadione-induced apoptosis. In contrast, inhibition of NF-kappaB activation decreased cell death from both ROI. Different ROI, therefore, induce distinct apoptotic pathways in RALA hepatocytes that are both caspase dependent and independent. In contrast to the known protective effect of NF-kappaB activation in tumor necrosis factor-alpha-induced hepatocyte apoptosis, NF-kappaB promotes hepatocellular death from ROI in these cells.

The mechanisms underlying hepatocyte sensitization to tumor necrosis factor-alpha (TNF-alpha)-mediated cell death remain unclear. Increases in hepatocellular oxidant stress such as those that occur with hepatic overexpression of cytochrome P-450 2E1 (CYP2E1) may promote TNF-alpha death. TNF-alpha treatment of hepatocyte cell lines with differential CYP2E1 expression demonstrated that overexpression of CYP2E1 converted the hepatocyte TNF-alpha response from proliferation to apoptotic and necrotic cell death. Death occurred despite the presence of increased levels of nuclear factor-kappaB transcriptional activity and was associated with increased lipid peroxidation and GSH depletion. CYP2E1-overexpressing hepatocytes had increased basal and TNF-alpha-induced levels of c-Jun NH(2)-terminal kinase (JNK) activity, as well as prolonged JNK activation after TNF-alpha stimulation. Sensitization to TNF-alpha-induced cell death by CYP2E1 overexpression was inhibited by antioxidants or adenoviral expression of a dominant-negative c-Jun. Increased CYP2E1 expression sensitized hepatocytes to TNF-alpha toxicity mediated by c-Jun and overwhelming oxidative stress. The chronic increase in intracellular oxidant stress created by CYP2E1 overexpression may serve as a mechanism by which hepatocytes are sensitized to TNF-alpha toxicity in liver disease.

The prevention of injury from reactive oxygen species is critical for cellular resistance to many death stimuli. Resistance to death from the superoxide generator menadione in the hepatocyte cell line RALA255–10G is dependent on down-regulation of the c-Jun N-terminal kinase (JNK)/AP-1 signaling pathway by extracellular signal-regulated kinase 1/2 (ERK1/2). Because protein kinase C (PKC) regulates both oxidant stress and JNK signaling, the ability of PKC to modulate hepatocyte death from menadione through effects on AP-1 was examined. PKC inhibition with Ro-31-8425 or bisindolylmaleimide I sensitized this cell line to death from menadione. Menadione treatment led to activation of PKCμ, or protein kinase D (PKD), but not PKCα/β, PKCζ/λ, or PKCδ/θ. Menadione induced phosphorylation of PKD at Ser-744/748, but not Ser-916, and translocation of PKD to the nucleus. PKC inhibition blocked menadione-induced phosphorylation of PKD, and expression of a constitutively active PKD prevented death from Ro-31-8425/menadione. PKC inhibition led to a sustained overactivation of JNK and c-Jun in response to menadione as determined by in vitro kinase assay and immunoblotting for the phosphorylated forms of both proteins. Cell death from PKC inhibition and menadione treatment resulted from c-Jun activation, since death was blocked by adenoviral expression of the c-Jun dominant negative TAM67. PKC and ERK1/2 independently down-regulated JNK/c-Jun, since inhibition of either kinase failed to affect activation of the other kinase, and simultaneous inhibition of both pathways caused additive JNK/c-Jun activation and cell death. Resistance to death from superoxide therefore requires both PKC/PKD and ERK1/2 activation in order to down-regulate proapoptotic JNK/c-Jun signaling. The prevention of injury from reactive oxygen species is critical for cellular resistance to many death stimuli. Resistance to death from the superoxide generator menadione in the hepatocyte cell line RALA255–10G is dependent on down-regulation of the c-Jun N-terminal kinase (JNK)/AP-1 signaling pathway by extracellular signal-regulated kinase 1/2 (ERK1/2). Because protein kinase C (PKC) regulates both oxidant stress and JNK signaling, the ability of PKC to modulate hepatocyte death from menadione through effects on AP-1 was examined. PKC inhibition with Ro-31-8425 or bisindolylmaleimide I sensitized this cell line to death from menadione. Menadione treatment led to activation of PKCμ, or protein kinase D (PKD), but not PKCα/β, PKCζ/λ, or PKCδ/θ. Menadione induced phosphorylation of PKD at Ser-744/748, but not Ser-916, and translocation of PKD to the nucleus. PKC inhibition blocked menadione-induced phosphorylation of PKD, and expression of a constitutively active PKD prevented death from Ro-31-8425/menadione. PKC inhibition led to a sustained overactivation of JNK and c-Jun in response to menadione as determined by in vitro kinase assay and immunoblotting for the phosphorylated forms of both proteins. Cell death from PKC inhibition and menadione treatment resulted from c-Jun activation, since death was blocked by adenoviral expression of the c-Jun dominant negative TAM67. PKC and ERK1/2 independently down-regulated JNK/c-Jun, since inhibition of either kinase failed to affect activation of the other kinase, and simultaneous inhibition of both pathways caused additive JNK/c-Jun activation and cell death. Resistance to death from superoxide therefore requires both PKC/PKD and ERK1/2 activation in order to down-regulate proapoptotic JNK/c-Jun signaling. The ability of the cell to resist injury from excessive levels of reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; Bis I, bisindolylmaleimide I; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; PKC, protein kinase C; PKD, protein kinase D; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; HA, hemagglutinin. 1The abbreviations used are: ROS, reactive oxygen species; Bis I, bisindolylmaleimide I; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; PKC, protein kinase C; PKD, protein kinase D; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; HA, hemagglutinin. is a critical survival mechanism in response to a variety of environmental stresses. Until recently, oxidative stress was thought to trigger cell death through the adverse effects of biochemical reactions between oxidants and cellular macromolecules. However, it is now known that oxidant-induced death pathways are far more complex, with death also resulting from the effects of oxidants on signal transduction pathways (1Czaja M.J. Antioxid. Redox Signal. 2002; 4: 759-767Google Scholar, 2Gabbita S.P. Robinson K.A. Stewart C.A. Floyd R.A. Hensley K. Arch. Biochem. Biophys. 2000; 376: 1-13Google Scholar). Central among these signal transducers of oxidant-induced death are the mitogen-activated protein kinases (MAPKs). In the hepatocyte cell line RALA255-10G, resistance to toxicity from the ROS superoxide depends on activation of the MAPK extracellular signal-regulated kinase 1/2 (ERK1/2). Treatment of these cells with the superoxide generator menadione induces ERK1/2 activation (3Czaja M.J. Liu H. Wang Y. Hepatology. 2003; 37: 1405-1413Google Scholar). Inhibition of ERK1/2 signaling causes sustained activation of the c-Jun N-terminal kinase (JNK)/c-Jun/AP-1 pathway, resulting in cell death from normally nontoxic concentrations of menadione (3Czaja M.J. Liu H. Wang Y. Hepatology. 2003; 37: 1405-1413Google Scholar). Overactivation of JNK/AP-1 signaling is known to mediate cell death from a number of stimuli in both hepatocytes and nonhepatic cells (4Shaulian E. Karin M. Nat. Cell Biol. 2002; 4: E131-E136Google Scholar, 5Czaja M.J. Am. J. Physiol. Gastrointest. Liver Physiol. 2003; 284: G875-G879Google Scholar). Restricting the duration of this proapoptotic AP-1 activation following superoxide-generated cellular stress is required for hepatocyte resistance to oxidative stress.The critical nature of cellular resistance to oxidant stress suggests the likelihood that redundant or complementary signaling pathways exist in order to protect hepatocytes against oxidant injury. However, upstream inhibitors of AP-1 activation other than ERK1/2 have not been identified after oxidative stress in hepatocytes. In addition to their effects on MAPK signaling, oxidants have been demonstrated to phosphorylate and thereby activate protein kinase C (PKC) isoforms. Multiple PKC isoforms are phosphorylated in response to oxidative stress induced by hydrogen peroxide (6Konishi H. Tanaka M. Takemura Y. Matsuzaki H. Ono Y. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11233-11237Google Scholar, 7Konishi H. Yamauchi E. Taniguchi H. Yamamoto T. Matsuzaki H. Takemura Y. Ohmae K. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6587-6592Google Scholar), including PKCμ or protein kinase D (PKD) (8Storz P. Toker A. EMBO J. 2003; 22: 109-120Google Scholar, 9Waldron R.T. Rozengurt E. J. Biol. Chem. 2000; 275: 17114-17121Google Scholar). Although originally described as a PKC family member, PKD has distinct features that make it part of a separate kinase family that also includes PKD2 and PKD3 (10Van Lint J. Rykx A. Maeda Y. Vantus T. Sturany S. Malhotra V. Vandenheede J.R. Seufferlein T. Trends Cell Biol. 2002; 12: 193-200Google Scholar). Both serine and tyrosine phosphorylation of PKD have been reported to result from hydrogen peroxide treatment (8Storz P. Toker A. EMBO J. 2003; 22: 109-120Google Scholar, 9Waldron R.T. Rozengurt E. J. Biol. Chem. 2000; 275: 17114-17121Google Scholar). Hydrogen peroxide-induced phosphorylation of Ser-744/748 within the PKD activation loop occurs by a PKC-dependent mechanism (11Waldron R.T. Rey O. Iglesias T. Tugal T. Cantrell D. Rozengurt E. J. Biol. Chem. 2001; 276: 32606-32615Google Scholar, 12Waldron R.T. Rozengurt E. J. Biol. Chem. 2003; 278: 154-163Google Scholar). In addition to phosphorylation, PKD activation involves translocation from the cytoplasm to other cellular compartments, including the nucleus and mitochondria (13Rey O. Sinnett-Smith J. Zhukova E. Rozengurt E. J. Biol. Chem. 2001; 276: 49228-49235Google Scholar, 14Storz P. Hausser A. Link G. Dedio J. Ghebrehiwet B. Pfizenmaier K. Johannes F.J. J. Biol. Chem. 2000; 275: 24601-24607Google Scholar). PKD activation has been reported to up-regulate NF-κB signaling, and the protective effects of PKD activation against death from hydrogen peroxide were associated with PKD-dependent NF-κB activation (8Storz P. Toker A. EMBO J. 2003; 22: 109-120Google Scholar). Interestingly, PKD has also been reported to regulate JNK/c-Jun signaling (15Brandlin I. Eiseler T. Salowsky R. Johannes F.J. J. Biol. Chem. 2002; 277: 45451-45457Google Scholar, 16Brandlin I. Hubner S. Eiseler T. Martinez-Moya M. Horschinek A. Hausser A. Link G. Rupp S. Storz P. Pfizenmaier K. Johannes F.J. J. Biol. Chem. 2002; 277: 6490-6496Google Scholar, 17Hurd C. Rozengurt E. Biochem. Biophys. Res. Commun. 2001; 282: 404-408Google Scholar, 18Hurd C. Waldron R.T. Rozengurt E. Oncogene. 2002; 21: 2154-2160Google Scholar), suggesting the possibility that PKD activation induced by oxidative stress may also regulate the AP-1 pathway.The objective of the present study was to examine whether PKC is an upstream regulator of the AP-1 death pathway in a hepatocyte cell line exposed to the superoxide generator menadione. The studies demonstrate that menadione causes a PKC-dependent activation of PKD. Inhibition of PKC/PKD activation leads to increased toxicity from menadione associated with sustained activation of the JNK/AP-1 pathway. Death resulting from PKC/PKD inhibition is blocked by the c-Jun dominant negative TAM67, suggesting that PKD-dependent resistance to menadione toxicity is the result of down-regulation of AP-1 signaling. These data therefore demonstrate for the first time a critical physiologic role for PKC/PKD in the regulation of AP-1 signaling.EXPERIMENTAL PROCEDURESCells and Culture Conditions—All studies were performed in the adult rat hepatocyte line RALA255–10G (RALA hepatocytes). These cells are conditionally immortalized with a mutant SV40 virus expressing a temperature-sensitive T antigen (19Chou J.Y. Mol. Cell. Biol. 1983; 3: 1013-1020Google Scholar). Cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 4% fetal bovine serum (Gemini, Woodland, CA) and antibiotics (Invitrogen) at the permissive temperature of 33 °C. For experiments, cells were plated and cultured at 33 °C for 24 h and then cultured in Dulbecco's modified Eagle's medium, 2% fetal bovine serum, antibiotics, and 1 μm dexamethasone at the restrictive temperature of 37 °C, as previously described (20Jones B.E. Lo C.R. Liu H. Srinivasan A. Streetz K. Valentino K.L. Czaja M.J. J. Biol. Chem. 2000; 275: 705-712Google Scholar). Under these conditions, T antigen expression is suppressed, the cells are nontransformed, and they display a differentiated hepatocyte phenotype (19Chou J.Y. Mol. Cell. Biol. 1983; 3: 1013-1020Google Scholar, 21Chou J.Y. Yeoh G.C. Cancer Res. 1987; 47: 5415-5420Google Scholar). Cells were then placed in serum-free medium containing dexamethasone for 18 h prior to the start of an experiment.Cells were pretreated for 1 h with the PKC inhibitors 10 μm Ro-31-8425, 10 μm bisindolylmaleimide I (Bis I), or 10 μm chelerythrine chloride (Calbiochem) dissolved in Me2SO. Cells were treated with menadione (Sigma) at the concentrations indicated. Some cells were pretreated for 1 h prior to the addition of Ro-31-8425 with 10 μm ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one) (Biomol, Plymouth Meeting, PA), 1000 units of catalase polyethylene glycol (Sigma), or 50 μm Val-Ala-Asp-fluoromethylketone (Calbiochem). Ebselen and Val-Ala-Asp-fluoromethylketone were dissolved in Me2SO. In experiments with inhibitors dissolved in Me2SO, untreated control cells received equivalent amounts of Me2SO.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay—Cell death was determined by the MTT assay (22Mosmann T. J. Immunol. Methods. 1983; 65: 55-63Google Scholar). At 24 h after treatment, the cell culture medium was aspirated, and an equal volume of a 1 mg/ml MTT solution, pH 7.4, in Dulbecco's modified Eagle's medium was added to the cells. After incubation at 37 °C for 1 h, the MTT solution was removed, and 1.5 ml of N-propyl alcohol was added to solubilize the formazan product. The absorbance of this compound was measured at 560 nm in a spectrophotometer. The percentage of cell death was calculated by dividing the optical density of a treatment group by the optical density for untreated, control cells, multiplying by 100, and subtracting that number from 100.Fluorescence Microscopy—The numbers of apoptotic and necrotic cells were quantified by fluorescence microscopy after costaining with acridine orange and ethidium bromide (23Duke R.C. Cohen J.J. Coligan J.E. Kruisbeek A.M. Marguiles D.H. Shevack E.M. Strober W. Current Protocols in Immunology. John Wiley & Sons, Inc., New York1992: 1-16Google Scholar), as previously described (24Liu H. Lo C.R. Jones B.E. Pradhan Z. Srinivasan A. Valentino K.L. Stockert R.J. Czaja M.J. J. Biol. Chem. 2000; 275: 40155-40162Google Scholar). Cells with a shrunken cytoplasm and a condensed or fragmented nucleus as determined by acridine orange staining were considered apoptotic. Necrotic cells were detected by positive staining with ethidium bromide. A minimum of 400 cells per dish were examined, and the numbers of apoptotic, and necrotic cells are expressed as a percentage of the total number of cells counted.Protein Isolation, Immunoprecipitation, and Western Blotting—For the isolation of total cellular protein, cells were harvested in phosphate-buffered saline, centrifuged, and resuspended in cell lysis buffer containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, pH 7.6, 1% Nonidet P-40, 1 μg/ml leupeptin, 2 μg/ml pepstatin, 1 mm phenylmethylsulfonyl fluoride, 2 mm β-glycerophosphate, 5 mm sodium pyrophosphate, and 2 mm sodium orthovanadate. Protein concentrations were determined using the Bio-Rad protein assay according to the manufacturer's instructions.To isolate nuclear protein, cells were scraped into hypotonic lysis buffer containing 10 mm Hepes, pH 7.4, 10 mm NaCl, 0.1 mm EDTA, pH 7.6, 0.4% Nonidet P-40, 1 μg/ml leupeptin, 2 μg/ml pepstatin, 1 mm phenylmethylsulfonyl fluoride, 2 mm β-glycerophosphate, 5 mm sodium pyrophosphate, and 2 mm sodium orthovanadate. Lysates were pipetted vigorously and centrifuged at 800 × g, at 4 °C for 8 min. Supernatants were transferred into new tubes and ultracentrifuged at 100,000 × g, at 4 °C for 60 min. The supernatants were saved as cytosolic fractions. The pellets were lysed in hypertonic lysis buffer (20 mm Hepes, pH 7.4, 400 mm NaCl, 1 mm EDTA, pH 7.6, 1% Nonidet P-40, 1 μg/ml leupeptin, 2 μg/ml pepstatin, 1 mm phenylmethylsulfonyl fluoride, 2 mm β-glycerophosphate, 5 mm sodium pyrophosphate, 2 mm sodium orthovanadate) and centrifuged at 20,000 × g at 4 °C for 20 min. The supernatants were used as nuclear fractions. Protein concentrations were determined using the Bio-Rad protein assay as above.For immunoprecipitations, cells were lysed in a buffer containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, pH 7.6, 1% Triton X-100, 1 μg/ml leupeptin, 2 μg/ml pepstatin, and 1 mm phenylmethylsulfonyl fluoride. Protein determination was performed as above, and 350 μg of protein were immunoprecipitated by a 1-h incubation with 2 μg of anti-hemagglutinin (anti-HA) antibody purified from the 12CA5 hybridoma. Samples were then incubated with protein A/G-agarose (Sigma) for 30 min. The immune complexes were washed five times with 20 mm Tris, pH 7.5, 500 mm sodium chloride and resolved on Western blots as described subsequently.For Western blotting, 50 μg of protein were denaturated at 100 °C for 5 min in Laemmli sample buffer containing 62.5 mm Tris-HCl, pH 6.8, 2% SDS, 25% glycerol, 0.01% bromphenol blue, and 5% β-mercaptoethanol. Samples were applied to 8–10% SDS-polyacrylamide gels and resolved at 100 V over 3 h. Proteins were transferred to nitrocellulose membranes (Schleicher & Schuell) in transfer buffer containing 25 mm Tris, pH 8.3, 192 mm glycine, 0.01% SDS, and 15% methanol using a Bio-Rad Trans-blot SD semidry transfer cell to which 150 mA were applied for 90 min. Membranes were blocked in 5% nonfat dry milk in 20 mm Tris, pH 7.5, 500 mm sodium chloride, and 0.5% Tween 20 (TBS-T) for 1 h. Membranes were exposed to antibodies against PKD; PKD phosphorylated at Ser-744/748 or Ser-916; phosphorylated PKCα/β; phosphorylated PKCδ/θ; phosphorylated PKCζ/λ; phosphorylated and total ERK1/2 (Cell Signaling, Beverly, MA); JNK1 and JNK2 and c-Jun (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); protein-disulfide isomerase (a kind gift from R. J. Stockert) (25Terada K. Manchikalapudi P. Noiva R. Jauregui H.O. Stockert R.J. Schilsky M.L. J. Biol. Chem. 1995; 270: 20410-20416Google Scholar); and Nopp140 (kindly provided by U. T. Meier) (26Meier U.T. J. Biol. Chem. 1996; 271: 19376-19384Google Scholar). Primary antibodies were used at 1:1000 to 1:6000 dilutions in 5% nonfat milk or bovine serum albumin in TBS-T for 18 h at 4 °C. Goat anti-rabbit and anti-mouse IgG antibodies conjugated with horseradish peroxidase (KPL, Gaithersburg, MD) were used as secondary antibodies at a dilution of 1:10,000 in 5% nonfat milk TBS-T for 1 h. Signals were detected by chemiluminescence (Western Lightning Chemiluminescence Plus; PerkinElmer Life Sciences) and exposure to x-ray film.Transient Transfections for PKD Overexpression—RALA hepatocytes were transiently transfected with an expression vector containing the Escherichia coli β-galactosidase gene, CMV-βGal, or with PKD.SS738/742EE. PKD.SS738/742EE expresses an HA-tagged mutant PKD in which the activation loop residues have been replaced with negatively charged Glu, resulting in a constitutively active PKD (27Storz P. Doppler H. Toker A. Mol. Cell. Biol. 2004; 24: 2614-2626Google Scholar). Cells were plated at a lower density and cultured at 37 °C for a shorter period of time than in the other experiments to allow for a less confluent culture necessary for optimal transfection efficiency. Transfections were performed with FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. Transfection efficiency was determined by β-galactosidase staining of CMV-βGal-transfected cells using a commercial kit (Invitrogen). Twenty-four hours after transfection, cells were treated with Ro-31-8425 and/or 20 μm menadione as described previously. The percentage cell survival was determined by an MTT assay 18 h later.JNK Assay—JNK activity was measured in cell lysates using a stress-activated protein kinase/JNK assay kit (Cell Signaling), according to the manufacturer's instructions. An N-terminal c-Jun-(1–89) fusion protein bound to glutathione-Sepharose beads was used to immobilize JNK from cell lysates containing 250 μg of total protein. After washing, the kinase reaction was performed in the presence of cold ATP using the c-Jun fusion protein as a substrate. Samples were resolved on 10% SDS-polyacrylamide gels, and the amount of phosphorylated c-Jun was detected with an antibody specific for c-Jun phosphorylated at serine 63. As a control for the loading of equivalent amounts of protein among samples, total c-Jun levels were analyzed by immunoblotting with a rabbit phosphorylation-independent c-Jun antibody (Santa Cruz Biotechnology). Proteins were visualized using a secondary antibody and chemiluminescent substrate as described above.Luciferase Assay—RALA hepatocytes were cultured as previously described and transiently transfected with reporter genes using LipofectAMINE Plus (Invitrogen) 18 h prior to Ro-31-8425/menadione treatments. Cells were transfected with the AP-1-regulated firefly luciferase reporter gene Coll73-Luc (28Webb P. Lopez G.N. Uht R.M. Kushner P.J. Mol. Endocrinol. 1995; 9: 443-456Google Scholar) and the constitutive Renilla luciferase vector pRL-TK (Promega, Madison, WI). Luciferase activities were assayed as previously described (24Liu H. Lo C.R. Jones B.E. Pradhan Z. Srinivasan A. Valentino K.L. Stockert R.J. Czaja M.J. J. Biol. Chem. 2000; 275: 40155-40162Google Scholar), and firefly luciferase activity was normalized to Renilla luciferase activity.Adenoviruses—The adenoviruses Ad5LacZ, containing the β-galactosidase gene (29Iimuro Y. Nishiura T. Hellerbrand C. Behrns K.E. Schoonhoven R. Grisham J.W. Brenner D.A. J. Clin. Invest. 1998; 101: 802-811Google Scholar), and Ad5TAM, which expresses TAM-67, a dominant negative c-Jun (30Bradham C.A. Hatano E. Brenner D.A. Am. J. Physiol. 2001; 281: G1279-G1289Google Scholar), were employed. The adenoviruses were grown in 293 cells, purified by banding twice on CsCl gradients, and titered by plaque assay as previously described (31Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Google Scholar). RALA hepatocytes were infected at a multiplicity of infection of 20 as previously described (24Liu H. Lo C.R. Jones B.E. Pradhan Z. Srinivasan A. Valentino K.L. Stockert R.J. Czaja M.J. J. Biol. Chem. 2000; 275: 40155-40162Google Scholar).Statistical Analysis—All numerical results are expressed as mean ± S.E. and represent data from three independent experiments with duplicate dishes in each treatment group. Statistical significance was determined by Student's t test. Calculations were made with Sigma Plot 2000 (SPSS Science, Chicago, IL).RESULTSPKC Inhibitors Sensitize RALA Hepatocytes to Death from Menadione—Menadione is a quinone compound that undergoes redox cycling resulting in the formation of superoxide (32Monks T.J. Hanzlik R.P. Cohen G.M. Ross D. Graham D.G. Toxicol. Appl. Pharmacol. 1992; 112: 2-16Google Scholar, 33Thor H. Smith M.T. Hartzell P. Bellomo G. Jewell S.A. Orrenius S. J. Biol. Chem. 1982; 257: 12419-12425Google Scholar). Recent investigations have demonstrated that RALA hepatocyte death from menadione-induced oxidative stress is regulated by both ERK1/2 and JNK MAPKs (3Czaja M.J. Liu H. Wang Y. Hepatology. 2003; 37: 1405-1413Google Scholar). However, it remains unclear how oxidative stress triggers activation of these MAPKs. To delineate upstream signals that regulate MAPK-dependent oxidant-induced death in RALA hepatocytes, the effect of PKC inhibition on menadione toxicity was examined. Cells were pretreated with vehicle or the PKC inhibitor Ro-31-8425 (34Merritt J.E. Sullivan J.A. Tse J. Wilkinson S. Nixon J.S. Cell Signal. 1997; 9: 53-57Google Scholar) and nontoxic and toxic concentrations of menadione that have been previously established (3Czaja M.J. Liu H. Wang Y. Hepatology. 2003; 37: 1405-1413Google Scholar). By 24 h of MTT assay, Ro-31-8425 sensitized RALA hepatocytes to 22 and 41% cell death, respectively, from the usually nontoxic 20 and 25 μm concentrations of menadione (Fig. 1A). This PKC inhibitor also further increased death from a toxic 30 μm menadione concentration by almost 2-fold (Fig. 1A), indicating that PKC-dependent protective mechanisms were still operative even at toxic levels of oxidative stress. No toxicity occurred from Ro-31-8425 treatment alone (data not shown).To ensure that death was secondary to PKC inhibition, the effect of a second PKC inhibitor, Bis I (35Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. J. Biol. Chem. 1991; 266: 15771-15781Google Scholar), on menadione toxicity was investigated. Bis I alone was nontoxic (data not shown), but sensitization of RALA hepatocytes to death from menadione occurred with Bis I pretreatment. Death from Bis I/menadione cotreatment was 28% for 20 μm menadione and 47% for 25 μm menadione at 24 h (Fig. 1B), similar to findings for Ro-31-8425/menadione. In contrast, chelerythrine, another purported chemical PKC inhibitor (36Herbert J.M. Augereau J.M. Gleye J. Maffrand J.P. Biochem. Biophys. Res. Commun. 1990; 172: 993-999Google Scholar), failed to sensitize RALA hepatocytes to death from menadione (data not shown).Death from Ro-31-8425/Menadione Cotreatment Results from Caspase-independent Apoptosis—The induction of cell death from combined Ro-31-8425/menadione treatment was additionally confirmed by fluorescence microscopy of acridine orange/ethidium bromide-costained cells. At 24 h, there was a marked increase in the percentage of apoptotic cells after Ro-31-8425/menadione cotreatment but only a slight increase in the number of necrotic cells (Fig. 2A). Apoptosis was secondary to oxidative stress as demonstrated by significant inhibition of death by the antioxidants ebselen and catalase (Fig. 2B). Death was not prevented by the caspase inhibitor Val-Ala-Asp-fluoromethylketone (Fig. 2B). Thus, similar to findings of caspase-independent apoptosis in RALA hepatocytes and nonhepatic cell types from toxic concentrations of menadione (3Czaja M.J. Liu H. Wang Y. Hepatology. 2003; 37: 1405-1413Google Scholar, 37Joza N. Susin S.A. Daugas E. Stanford W.L. Cho S.K. Li C.Y. Sasaki T. Elia A.J. Cheng H.Y. Ravagnan L. Ferri K.F. Zamzami N. Wakeham A. Hakem R. Yoshida H. Kong Y.Y. Mak T.W. Zuniga-Pflucker J.C. Kroemer G. Penninger J.M. Nature. 2001; 410: 549-554Google Scholar), Ro-31-8425/menadione treatment resulted in an oxidant-induced, caspase-independent apoptosis.Fig. 2PKC inhibition leads to caspase-independent apoptosis from menadione. A, the numbers of apoptotic and necrotic cells were determined in acridine orange and ethidium bromide-costained cells by fluorescence microscopy as described under “Experimental Procedures.” Cells were untreated (Con) or treated with 25 μm menadione (Men), Ro-31-8425 (Ro), or a combination of the two (Ro/Men). The percentages of cells that were apoptotic or necrotic were determined at 12 h after menadione treatment. B, percentage of cell death at 24 h in RALA hepatocytes treated with Ro-31-8425 and 25 μm menadione alone (ϕ), or together with pretreatment with ebselen (Eb), catalase (Cat), or Val-Ala-Asp-fluoromethylketone (ZVAD). The data are from three independent experiments performed in duplicate (*, p < 0.0001).View Large Image Figure ViewerDownload (PPT)Resistance to Menadione Toxicity Requires Early PKC Signaling—To delineate the temporal involvement of protective PKC signaling in the menadione death pathway, RALA hepatocytes were examined for menadione-induced cell death after different times of Ro-31-8425 treatment. Conversion of the Ro-31-8425 1 h pretreatment to 1 h post-treatment still sensitized the cells to significant toxicity from menadione but reduced death from Ro-31-8425/menadione treatment by 36% (Fig. 3). When Ro-31-8425 treatment was delayed to 2 h after menadione administration, the amount of cell death was not significantly different from that of 25 μm menadione alone (Fig. 3). These data indicate that PKC-dependent signaling mediates an immediate protective response against menadione-induced oxidative stress.Fig. 3Early inhibition of PKC signaling is required to sensitize cells to death from menadione. RALA hepatocytes were treated with 25 μm menadione alone (Men) or with Ro-31-8425 as a 1-h pretreatment (–1h) or as a treatment 1, 2, or 4 h after the menadione. The percentage of cell death was determined at 24 h by an MTT assay. The results represent data from three independent experiments performed in duplicate.View Large Image Figure ViewerDownload (PPT)Menadione Causes Selective PKD Ser-744/748 Phosphorylation and Nuclear Translocation—To identify the PKC isoform mediating RALA hepatocyte resistance to menadione toxicity, levels of active, phosphorylated PKC were examined after menadione treatment. Menadione induced an increase in phospho-PKCμ or phospho-PKD within 1 h after menadione treatment (Fig. 4A). Menadione-induced phosphorylation was specific for Ser-744/748, since no change was detected in the levels of phosphorylation at the Ser-916 residue. Levels of total PKD were also unaffected by menadione treatment. Menadione had no effect on the levels of phosphorylated PKCα/β, PKCζ/λ, or PKCδ/θ (Fig. 4A). Selective PKD Ser-744/748 phosphorylation was induced by both nontoxic and toxic concentrations of menadione (Fig. 4B).Fig. 4Menadione induces selective activation of PKD Ser-744/748. A, protein was isolated from untreated RALA hepatocytes, and cells were treated with 25 μm menadione for the indicated number of hours. Aliquots of protein were immunoblotted with antibodies against PKD phosphorylated at Ser-744/748 (P-PKD(Ser744/8)) or Ser-916 (P-PKD(Ser916)), total PKD, and phosphorylated forms of PKCα/β (P-PKCα/β), PKCζ/λ (P-PKCζ/λ), and PKCδ/θ (P-PKCδ/θ). B, immunoblots performed with the same antibodies on cells untreated or treated with the indicated micromolar concentrations of menadione for 2 h. C, RALA hepatocytes were untreated or treated with 25 μm menadione for the indicted number of minutes. Nuclear and cytosolic protein fractions were obtained as detailed under “Experimental Procedures.” Protein aliquots were immunoblotted with the PKD antibodies plus antibodies to Nopp140 and protein-disulfide isomerase (PDI). The results are representative of three independent experiments. Numerical results under the PKD Western blots represent the relative signal intensity among samples from densitometry scanning of the three experiments.View Large Image Figure ViewerDownload (PPT)Once

Deficiency of serum ceruloplasmin is a characteristic biochemical abnormality of Wilson's disease, although the mechanism of this finding is unknown. Ceruloplasmin messenger RNA (mRNA) levels were therefore examined in five patients with Wilson's disease and five controls with other types of hepatic disease. Northern and dot blot hybridizations showed that detectable ceruloplasmin mRNA was present in all of the patients with Wilson's disease, including one patient with no detectable serum ceruloplasmin. However, the ceruloplasmin mRNA levels in the Wilson's disease patients were only 33% that of controls (P less than 0.001). In contrast, albumin mRNA levels in the Wilson's disease patients averaged 161% that of controls. In an attempt to better delineate the level of gene expression responsible for this decrease in ceruloplasmin mRNA, the nuclear run-on assay was used to analyze transcriptional rates. The amount of ceruloplasmin gene transcription in four Wilson's patients was decreased to 44% that of three controls. These results indicate that the diminished serum ceruloplasmin levels in patients with Wilson's disease are due at least in part to a decrease in ceruloplasmin gene transcription.

Expression of c-myc regulates apoptotic cell death in the human hepatoma cell line HuH-7 during culture in serum-free medium (SFM) plus zinc. To understand the mechanism of this c-myc effect, the ability of various serum-contained factors to prevent apoptosis was determined. Apoptosis was not inhibited by growth factors and was even accelerated by supplementation with insulin-like growth factor I or insulin. Cell death was prevented by SFM supplementation with the amino acid glutamine but not serine or asparagine. Improved cell survival with glutamine was associated with increased levels of glutathione (GSH). In HuH-7 cells cultured in SFM plus zinc, c-myc expression led to decreased levels of GSH, and elevated intracellular levels of hydrogen peroxide (H2O2). Cell death induced by c-myc expression was inhibited by the addition of catalase or dimethyl sulfoxide, a hydroxyl radical scavenger, or by increased intracellular expression of catalase. In contrast to findings in fibroblasts, c-myc-dependent apoptosis during serum deprivation in HuH-7 hepatoma cells was unrelated to a loss of growth factors. Apoptosis resulted from H2O2-mediated oxidative stress with associated glutamine dependent intracellular GSH depletion.

Chronic oxidative stress induced by overexpression of the cytochrome P450 isoform 2E1 (CYP2E1) has been implicated in hepatocyte injury and death. However, the mechanism by which CYP2E1 overexpression may promote cell death is unknown. Acute oxidative stress activates mitogen-activated protein kinases (MAPK), suggesting that chronic oxidant generation by CYP2E1 may regulate cellular responses through these signaling pathways. The effect of CYP2E1 overexpression on MAPK activation and their function in altering death responses of CYP2E1-overexpressing hepatocytes were investigated. Chronic CYP2E1 overexpression led to increased extracellular signal-regulated kinase 1/2 (ERK1/2) activation constitutively and in response to oxidant stress from the superoxide generator menadione. CYP2E1-overexpressing cells were resistant to menadione toxicity through an ERK1/2-dependent mechanism. Similar to menadione, the polyunsaturated fatty acid (PUFA) arachidonic acid (AA) induced an increased activation of ERK1/2 in hepatocytes that overexpressed CYP2E1. However, CYP2E1-overexpressing cells were sensitized to necrotic death from AA and the PUFA gamma-linolenic acid, but not from saturated or monounsaturated fatty acids. Death from PUFA resulted from oxidative stress and was blocked by inhibition of ERK1/2, but not p38 MAPK or activator protein-1 signaling. CYP2E1 expression induced ERK1/2 activation through increased epidermal growth factor receptor (EGFR)/c-Raf signaling. Inhibition of EGFR signaling reversed CYP2E1-induced resistance to menadione and sensitization to AA toxicity. In conclusion, chronic CYP2E1 overexpression leads to sustained ERK1/2 activation mediated by EGFR/c-Raf signaling. This adaptive response in hepatocytes exposed to chronic oxidative stress confers differential effects on cellular survival, protecting against menadione-induced apoptosis, but sensitizing to necrotic death from PUFA.

Central to the mechanisms underlying a variety of forms of liver injury are factors produced from the accompanying inflammatory response. Liver injury triggers the recruitment and activation of macrophages and neutrophils, a process mediated in part by lipopolysaccharide.1 These activated cells produce a number of potentially injurious factors that may promote hepatocyte injury. The most critical of these is the cytokine tumor necrosis factor (TNF)-α. Through either direct toxic or proinflammatory effects, TNF may promote injury from toxins,2 ischemia/reperfusion,3 and hepatitis viruses.4 Strategies to block the production, activity, or death pathway signaling of TNF therefore represent potential therapies for a variety of liver diseases. However, while progress has been made in understanding the mechanisms of hepatocyte sensitization to TNF cytotoxicity, effective means of suppressing this effect in vivo are still lacking. BMI, body mass index; GalN, galactosamine; LPS, lipopolysaccharide; PPAR, peroxisome proliferator-activated receptor; TNF, tumor necrosis factor; TNFR, TNF receptor. Crucial to understanding liver injury in nonalcoholic fatty liver disease (NAFLD) is the delineation of mechanisms of progression from benign fatty liver to hepatocyte injury and steatohepatitis. Several facts suggest that the inflammatory response and TNF in particular may promote this liver injury. First, the disease lacks an apparent direct death stimulus such as a hepatotoxin or hypoxia, suggesting that indirect factors must cause cell injury. Second, steatohepatitis frequently occurs in the setting of obesity and insulin resistance or diabetes which are increasingly recognized as proinflammatory states with increased oxidative stress, cytokine production, and cellular stress pathway signaling.5, 6 Obesity in particular is associated with increased TNF production from fat stores. The ability of TNF to act as a hepatotoxin, and the presence of increased levels of this cytokine in conditions associated with NAFLD, make TNF a prime candidate to promote progression to steatohepatitis. This possibility is supported by studies in leptin deficient ob/ob mice with obesity and fatty liver in which TNF inhibition reduced steatosis and liver injury.7, 8 However, studies of ob/ob mice lacking type I and II TNF receptors (TNFR1 and TNFR2) have suggested that TNF is not involved in their liver disease.9 How TNF could cause steatotic cell injury is unclear because TNF is normally a mitogen unless hepatocellular resistance mechanisms to the toxic effects of TNF are somehow circumvented. One possibility is that hepatocyte sensitization to TNF injury in the setting of NAFLD results from the cellular effects of overexpression of the pro-oxidant enzyme cytochrome P450 2E1 in this disease.10 An additional factor in the regulation of liver injury occurring in the setting of obesity and insulin resistance is the influence of adipocyte produced proteins.11 Adipocytes not only store excess energy, but also respond to metabolic signals by secreting proteins that exert local, central, and peripheral effects. Principal among these adipocyte specific or enriched factors, or adipokines, are leptin, resistin, and adiponectin. Leptin has multiple actions that include decreasing food intake and increasing energy expenditure. Serum leptin levels increase in proportion with body mass index (BMI). Whether leptin levels are altered in NAFLD is controversial, with some studies demonstrating increased levels in this disease,12 but others finding no correlation between serum leptin and the development of steatohepatitis.13 Resistin is an adipocyte produced protein whose main effect is to increase hepatic glucose production.14 Serum resistin levels are increased in human obesity,15 but levels in NAFLD have not yet been examined. Thus, no clear evidence yet implicates either leptin or resistin in the development of NASH. Adiponectin is present in significant concentrations in human serum (5–30 nM), and circulates in several forms including dimers, trimers, and a high-molecular weight complex consisting of up to six trimers.16 Two adiponectin receptors, AdipoR1 and AdipoR2, have been cloned.17 Liver expresses both receptor genes and has the highest expression of AdipoR2 among organs. Adiponection is secreted by adipocytes in inverse proportion to BMI.18 Serum adiponectin levels are also reduced with insulin resistance and diabetes.19 Metabolically adiponectin acts to reduce body fat,20 improve hepatic and peripheral insulin sensitivity,21 and decrease serum fatty acid levels in association with increased fatty acid oxidation in muscle.22 However, adiponectin has significant anti-inflammatory as well as metabolic effects. Adiponectin blocks macrophage phagocytosis and lipopolysaccharide-induced TNF release in vitro, possibly through inhibition of NF-κB activation.23-25 These dual metabolic and anti-inflammatory beneficial effects of adiponectin have been utilized to effectively treat atherosclerosis.26 Recent studies have now suggested that adiponectin can also prevent liver disease. Initially adiponectin null mice were reported to develop more extensive carbon tetrachloride-induced hepatic fibrosis than wild-type mice.27 A direct antifibrotic effect of adiponectin was suggested by findings of adiponectin receptor gene expression in hepatic stellate cells, and the inhibition of stellate cell proliferation, migration, and transforming growth factorβ1 expression by adiponectin treatment.27 A second study examined the effects of adiponectin administration on both ob/ob mice and mice fed a high fat, ethanol-containing diet.28 In both models, adiponectin significantly decreased levels of steatosis, liver injury and serum TNF. Ethanol-induced steatohepatitis was associated with a reduction in serum adiponectin levels, suggesting that a relative deficiency of this adipokine may have promoted liver disease. The article by Masaki et al. in this issue of HEPATOLOGY further expands the spectrum of adiponectin's protective effects during liver injury.29 The authors examined the effect of adiponectin administration on liver injury in KK-Ay obese mice sensitized to acute hepatotoxicity from LPS or TNF by cotreatment with the toxin galactosamine (GalN). Obese mice were markedly more sensitive to both forms of injury than lean controls, consistent with previous findings of LPS sensitivity in obese mice and rats.30 As expected, the obese mice had a 30% decrease in serum and adipose levels of adiponectin. Pretreatment with adiponectin reduced mortality, transaminase elevations and the amount of apoptosis induced by GalN/LPS by approximately 50%. This reduction in liver injury was associated with marked decreases in serum and hepatic TNF levels that rose with GalN/LPS treatment, leading the authors to conclude that the protective effect of adiponectin was mediated by its inhibition of TNF synthesis or release. However, adiponectin also significantly decreased liver injury from GalN/TNF administration in this study, suggesting that the major mechanism of adiponectin's action was downstream of TNF activation. The reduction in TNF could have been a secondary manifestation of the decrease in liver injury and therefore the stimulus for inflammation. It would be interesting to determine in this model whether adiponectin inhibits NF-κB activation, and therefore the production of other cytokines that may modulate liver injury. An alternative mechanism of adiponectin's effect may have been the ability of this adipokine to prevent the GalN/LPS-induced reduction in peroxisome proliferator-activated receptor-α (PPAR-α) expression, because PPAR-α-mediated signaling has been previously demonstrated to block liver injury in experimental NAFLD.31 While the mechanism of adiponectin's protective effect requires further study, these investigations provide the first demonstration that adiponectin can prevent acute hepatic injury from LPS/TNF in a steatotic liver. The exciting evidence of protective effects of adiponectin in liver injury in obese mice raises the possibility that adiponectin may be a treatment for human NAFLD. Hui and colleagues provide additional support for this concept in a second article in this month's Journal.32 In a study of over 100 patients with NAFLD, multivariate analysis revealed that decreased serum adiponectin levels and increased TNF and soluble TNFR2 levels correlated with the presence of NASH independent of the presence of insulin resistance. Levels of adiponectin were lower in NASH than in simple steatosis, and correlated with the degree of hepatic necroinflammation. This important study provides both concrete evidence for the involvement of TNF and adiponectin in human NAFLD, and suggests that adiponectin may be a critical factor in the progression of this disease. These two studies along with previous investigations suggest an emerging concept of the interrelationship of adiponectin with the liver (Fig. 1). In addition, they provide three forms of cogent evidence that support further study of adiponectin as a therapy for NAFLD: (1) this molecule has metabolic and anti-inflammatory properties that could alleviate both the fat accumulation and liver injury that mark this disease; (2) adiponectin has been effective in inhibiting liver injury in two animal models of hepatic steatosis; and (3) NASH may occur in the setting of relative adiponectin “deficiency.” A possible mechanistic scenario for NAFLD is that conditions such as insulin resistance and obesity lead to increased levels of fatty acids and the development of hepatic steatosis. However, these states also suppress adiponectin levels leading to a proinflammatory condition and the generation of injurious factors such as TNF. Individual variation in the degree of adiponectin suppression, the amount of inflammatory cell activation, and/or the hepatic susceptibility to injury from inflammatory mediators may determine which individuals progress to NASH. However, the actions of adiponectin are likely to be more complex than simple TNF suppression, and may include direct protective effects on hepatocytes and anti-fibrotic effects on hepatic stellate cells. Thus, adiponectin therapy is potentially applicable to liver diseases other than NAFLD. The role of adiponectin in other steatotic diseases such as alcoholic steatohepatitis, and any liver disease dependent on inflammatory mediators, requires further study as well. Effects of adiponectin on hepatic steatosis and injury. Adiponectin produced by adipose tissue has both physiological effects on lipid homeostasis (blue arrows) and anti-inflammatory effects (red lines) that may modulate hepatic steatosis and injury. Local effects of adiponectin on adipose tissue include a reduction in body fat and tumor necrosis factor (TNF) production. Adiponectin's peripheral effects on muscle lead to increased fatty acid oxidation and insulin sensitivity. The net result of these effects on adipose and muscle tissue is a decrease in serum fatty acid levels that may prevent hepatic fat accumulation. In the setting of hepatic injury, adiponectin may additionally act to inhibit both liver injury and fibrosis. This adipokine may inhibit Kupffer cell (KC) activation and release of injurious substances such as the cytokine TNF. This effect together with reduced adipose production of TNF may prevent hepatocyte injury. Adiponectin may also have direct effects on the hepatic stellate cell (HSC), blocking its proliferation and secretion of the profibrogenic cytokine transforming growth factor-β1 (TGF-β1). Finally, adiponectin also has as yet poorly described central effects on the brain that may impact on liver steatosis, injury or fibrosis.

Although it has been suggested that retinoids regulate Ito cell proliferation and collagen synthesis, little is known about the ability of Ito cells to respond to retinoids in vivo. Because retinoids may mediate their molecular effects through nuclear receptors, Ito cells were examined for the presence of one of these receptors, nuclear retinoic acid receptor-β. The modulation of nuclear retinoic acid receptor-β expression was also studied during cell culture and hepatic fibrogenesis. Northern hybridization analysis revealed that Ito cells freshly isolated from normal rat liver contained nuclear retinoic acid receptor-β messenger RNA at levels significantly higher than those found in other hepatic cell types. Ito cells also contained messenger RNA for two other nuclear retinoic acid receptors, nuclear retinoic acid receptor-α and nuclear retinoic acid receptor-γ. Using an antibody to human nuclear retinoic acid receptor-β, the nuclear presence of this receptor was demonstrated in normal Ito cells. In contrast, Ito cells cultured for at least 7 days had no detectable messenger RNA or nuclear staining for nuclear retinoic acid receptor-β despite a 20 ± 5-fold increase in the messenger RNA level of another retinoid binding protein, cellular retinol binding protein. Analysis of Ito cells isolated from rats with carbon tetrachloride—induced hepatic fibrosis revealed an 81% ± 3% decrease in nuclear retinoic acid receptor-β messenger RNA levels in these cells when compared with normal Ito cells. No difference in the messenger RNA levels of cellular retinol binding protein was found in Ito cells isolated from either normal or fibrotic liver. The effect of retinoid treatment on Ito cell nuclear retinoic acid receptor-β messenger RNA level was also studied because retinoic acid treatment of other cells has been shown to induce nuclear retinoic acid receptor-β gene expression. Treatment for 48 hr with either 10−6 mol/L retinoic acid or 10−5 mol/L retinyl acetate induced the expression of nuclear retinoic acid receptor-β messenger RNA in primary cultured Ito cells. These data demonstrate that Ito cells possess a nuclear retinoic acid receptor allowing them to respond to retinoic acid in vivo. Increased Ito cell proliferation and collagen synthesis during cell culture or hepatic fibrosis may result in part from decreased Ito cell retinoid responsiveness as reflected by the loss or decrease in nuclear retinoic acid receptor-β gene expression observed under these two conditions. Furthermore, the ability of retinoids to induce Ito cell nuclear retinoic acid receptor-β messenger RNA expression suggests that Ito cell retinoid unresponsiveness during cell culture and hepatic fibrogenesis is reversible. (HEPATOLOGY) 1992;15:336–342.

Endogenous lipopolysaccharide has been implicated as a cofactor in the hepatocellular injury and death resulting from toxic liver injury. To prevent this lipopolysaccharide-induced injury and to further understand the mechanism of this effect, an anti-lipopolysaccharide antibody was administered to rats in which toxic hepatocellular injury was induced. Rats were given the hepatotoxin galactosamine together with an isotypic control antibody B55 or the anti-lipopolysaccharide antibody E5. E5 treatment resulted in reductions of serum AST levels of 43% at 36 hr (p < 0.02) and 60% at 48 hr (NS) after galactosamine administration. These decreases in AST values were accompanied by diminished histological evidence of injury and inflammation. In carbon tetrachloride-induced liver injury, E5 similarly reduced serum AST levels at 36 and 48 hr by 47% (p < 0.04) and 54% (p < 0.03), respectively. E5 treatment was equally effective in reducing AST levels 48 hr after administration of carbon tetrachloride, whether the initial dose of antibody was given 1 hr before or 3 or 6 hr after the administration of this toxin. To understand the mechanism of this E5 effect, the activation of the toxic cytokine tumor necrosis factor-alpha and the chemotactic cytokine monocyte chemoattractant protein 1 was examined by Northern-blot analysis of RNA from rat livers after galactosamine-induced injury and treatment with B55 or E5. Despite E5's efficacy in reducing hepatocellular damage, E5 treatment did not affect the timing or magnitude of tumor necrosis factor-alpha or monocyte chemoattractant protein 1 activation during galactosamine-induced injury.(ABSTRACT TRUNCATED AT 250 WORDS)

We have previously shown that dexamethasone increases albumin mRNA and decreases procollagen steady-state mRNA levels in rat hepatocyte cultures. These studies were extended by evaluating an in vivo model of fibrogenesis (murine schistosomiasis) and by determining a more precise level of gene expression responsible for these changes. Control mice and litter mates infected with Schistosomiasis mansoni were evaluated at 8 weeks postinfection when the livers of the infected mice had become fibrotic and their serum albumin levels significantly decreased. The addition of 4 micrograms/mL dexamethasone to the drinking water of half of the infected mice led to a 75% decrease in the liver collagen content as determined by high-performance liquid chromatography. RNA was extracted from the livers of mice under three conditions: control and infected +/- dexamethasone. This RNA was then hybridized with cDNA probes to determine steady-state levels of specific mRNAs. In the infected mice, albumin mRNA levels were decreased compared to control; however, infected mice treated with dexamethasone increased their albumin mRNA content by 3-fold at 8 weeks. Types I and IV procollagen steady-state mRNA levels in infected mice were increased compared to control while dexamethasone suppressed the mRNA level of collagen in infected mice by 50%. The level of gene expression responsible for these steady-state changes was evaluated by nuclear run-on analysis. While the effect of schistosomiasis on these genes was primarily at a transcriptional level, dexamethasone exerted its effect on different genes in the injured liver by diverse mechanisms, i.e., decreasing collagen synthesis at a transcriptional level and increasing albumin by posttranscriptional mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Autophagy is a cellular catabolic process that relies on the cooperation of autophagosomes and lysosomes. During starvation, the cell expands both compartments to enhance degradation processes. We found that starvation activates a transcriptional program that controls major steps of the autophagic pathway, including autophagosome formation, autophagosome-lysosome fusion, and substrate degradation. The transcription factor EB (TFEB), a master gene for lysosomal biogenesis, coordinated this program by driving expression of autophagy and lysosomal genes. Nuclear localization and activity of TFEB were regulated by serine phosphorylation mediated by the extracellular signal-regulated kinase 2, whose activity was tuned by the levels of extracellular nutrients. Thus, a mitogen-activated protein kinase-dependent mechanism regulates autophagy by controlling the biogenesis and partnership of two distinct cellular organelles.

Tumor necrosis factor (TNF)-alpha causes much of the hepatocellular injury and cell death that follows toxin-induced liver damage. The mechanism by which toxic liver injury sensitizes hepatocytes to TNF-alpha cytotoxicity is unknown. The aim of this study was to determine the role of the antioxidant glutathione in this process.A rat hepatocyte cell line and primary hepatocytes sensitized to TNF-alpha toxicity by the addition of actinomycin D were examined for changes in glutathione levels and for the effects of glutathione depletion or supplementation on cell death. The in vivo effects of glutathione depletion were determined in mice treated with galactosamine plus lipopolysaccharide.Treatment of hepatocytes with actinomycin D and TNF-alpha induced apoptotic cell death without affecting cellular glutathione levels or production of the reactive oxygen intermediate H2O2. Glutathione depletion induced by diethyl maleic acid significantly increased TNF-alpha-induced cell death even when this agent was administered 2 hours after TNF-alpha treatment. Hepatocyte cell death was not affected by glutathione supplementation. In mice treated with galactosamine plus lipopolysaccharide, glutathione depletion increased mortality from liver injury from 32% to 72%.TNF-alpha-induced cytotoxicity in hepatocytes occurs in the absence of glutathione depletion. However, a preexisting reduction in glutathione levels can significantly increase cell death from TNF-alpha.

Hepatic expression of the protooncogenes c-fos and c-myc occurs within 2 h after partial hepatectomy, and these immediate early genes are thought to prime the hepatocytes for subsequent proliferation. To examine whether such gene activation occurred in the setting of hepatocyte proliferation after toxic liver injury, protooncogene expression was examined during the regenerative response following liver injury from carbon tetrachloride (CCl4) or galactosamine (GalN). The pattern of protooncogene expression after CCl4 mirrored that seen after partial hepatectomy, with rises in c-fos and c-myc mRNA content within 2 h, and then a rapid return to baseline levels. In contrast, early c-fos and c-myc expression did not occur after GalN injury. Instead GalN-induced regeneration led to a delayed, and prolonged c-fos and c-myc activation which peaked 24-48 h after injury. Increases in c-jun, jun-B, and jun-D mRNA levels also occurred in both models at times similar to the rises of c-fos and c-myc expression. Although the timing of DNA synthesis was identical after GalN or CCl4 treatment, the proliferative response after GalN injury was significantly less than that of CCl4, and marked by the histologic appearance of oval cells. The coadministration of 2-acetylaminofluorene, an inhibitor of differentiated hepatocyte proliferation, together with CCl4 altered the usual pattern of post-CCl4 protooncogene expression to one resembling that seen after GalN injury. Thus, the timing of protooncogene expression during liver regeneration may vary considerably. These variations may influence the nature of the proliferative response in terms of which cell type(s) proliferates, and the amount of regeneration that ensues.

Acetaminophen (APAP)-induced liver injury is the most common cause of acute liver failure (ALF) in the Western world. APAP toxicity progresses to multiorgan dysfunction and thus has broader whole-body implications. Importantly, greater 30-day mortality has been observed in liver transplant recipients following ALF due to APAP-related versus non-APAP-related causes. Reasons for this discrepancy have yet to be determined. Extrahepatic toxicities of APAP overdose may represent underappreciated and unaddressed comorbidities within this patient population. In the present study, rapid induction of apoptosis following APAP overdose was observed in the intestine, an organ that greatly influences the physiology of the liver. Strikingly, apoptotic cells appeared to be strictly restricted to the intestinal crypts. The use of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) reporter mice confirmed that the LGR5-positive (+) crypt base stem cells were disproportionately affected by APAP-induced cell death. Although the apoptotic cells were cleared within 24 hours after APAP treatment, potentially long-lived consequences on the intestine due to APAP exposure were indicated by prolonged deficits in gut barrier function. Moreover, small intestinal cell death was found to be independent of tumor necrosis factor receptor signaling and may represent a direct toxic insult to the intestine by exposure to high concentrations of APAP. Conclusion: APAP induces intestinal injury through a regulated process of apoptotic cell death that disproportionately affects LGR5+ stem cells. This work advances our understanding of the consequences of APAP toxicity in a novel organ that was not previously considered as a significant site of injury and thus presents potential new considerations for patient management.

One mechanism underlying the development of alcoholic liver disease is overactivation of the innate immune response. Recent investigations indicate that the lysosomal pathway of autophagy down-regulates the inflammatory state of hepatic macrophages, suggesting that macrophage autophagy may regulate innate immunity in alcoholic liver disease. The function of macrophage autophagy in the development of alcoholic liver disease was examined in studies employing mice with a myeloid-specific decrease in autophagy.Littermate control and Atg5Δmye mice lacking Atg5-dependent myeloid autophagy were administered a Lieber-DeCarli control (CD) or ethanol diet (ED) alone or together with lipopolysaccharide (LPS) and examined for the degree of liver injury and inflammation.Knockout mice with decreased macrophage autophagy had equivalent steatosis but increased mortality and liver injury from ED alone. Increased liver injury and hepatocyte death also occurred in Atg5Δmye mice administered ED and LPS in association with systemic inflammation as indicated by elevated serum levels of proinflammatory cytokines. Hepatic macrophage and neutrophil infiltration were unaffected by decreased autophagy, but levels of proinflammatory cytokine gene induction were significantly increased in the livers but not adipose tissue of knockout mice treated with ED and LPS. Inflammasome activation was increased in ED/LPS-treated knockout mice resulting in elevated interleukin (IL)-1β production. Increased IL-1β promoted alcoholic liver disease as liver injury was decreased by the administration of an IL-1 receptor antagonist.Macrophage autophagy functions to prevent liver injury from alcohol. This protection is mediated in part by down-regulation of inflammasome-dependent and inflammasome-independent hepatic inflammation. Therapies to increase autophagy may be effective in this disease through anti-inflammatory effects on macrophages.

Fibroblast growth factor 23 (FGF23) is a bone marrow cell produced hormone that functions in the intestine and kidney to regulate phosphate homeostasis. Increased serum FGF23 is a well-established predictor of mortality in renal disease, but recent findings linking increased levels to hepatic and cardiac diseases have suggested that other organs are sources of FGF23 or targets of its effects. The potential ability of the liver to produce FGF23 in response to hepatocellular injury was therefore examined. Very low levels of Fgf23 mRNA and FGF23 protein were detected in normal mouse liver, but the amounts increased markedly during acute liver injury from the hepatotoxin carbon tetrachloride. Serum levels of intact FGF23 were elevated during liver injury from carbon tetrachloride. Chronic liver injury induced by a high fat diet or elevated bile acids also increased hepatic FGF23 levels. Stimulation of toll-like receptor (TLR) 4-driven inflammation by gut-derived lipopolysaccharide (LPS) underlies many forms of liver injury, and LPS induced Fgf23 in the liver as well as in other organs. The LPS-inducible cytokines IL-1β and TNF increased hepatic Fgf23 expression as did a TLR2 agonist Pam2CSK3. Analysis of Fgf23 expression and FGF23 secretion in different hepatic cell types involved in liver injury identified the resident liver macrophage or Kupffer cell as a source of hepatic FGF23. LPS and cytokines selectively induced the hormone in these cells but not in hepatocytes or hepatic stellate cells. FGF23 failed to exert any autocrine effect on the inflammatory state of Kupffer cells but did trigger proinflammatory activation of hepatocytes. During liver injury inflammatory factors induce Kupffer cell production of FGF23 that may have a paracrine proinflammatory effect on hepatocytes. Liver-produced FGF23 may have systemic hormonal effects as well that influence diseases in in other organs.

Ceramide has been implicated as a second messenger in intracellular signaling pathways leading to apoptosis in nonhepatic cells. To determine whether ceramide can mediate hepatocyte apoptosis, the cytotoxicity of ceramide was determined in rat hepatocytes. The rat hepatocyte cell line, RALA255-10G, and primary rat hepatocytes were completely resistant to toxicity from 10 to 100 micromol/L C2 ceramide. Resistance was not the result of a failure to take up ceramide, because ceramide treatment did cause nuclear factor-kappaB (NF-kappaB) activation. Because ceramide may mediate cell death from tumor necrosis factor alpha (TNF-alpha), the ability of RNA synthesis inhibition and NF-kappaB inactivation to sensitize hepatocytes to ceramide toxicity was examined. RALA hepatocytes were sensitized to ceramide toxicity by coadministration of actinomycin D (ActD). Cell death occurred by apoptosis as determined by the presence of morphological evidence of apoptosis, caspase activation, poly(ADP-ribose) polymerase (PARP) degradation, and DNA hypoploidy. Despite the induction of apoptosis associated with caspase activation, cell death from ActD/ceramide was not blocked by caspase inhibition. Inhibition of NF-kappaB activation also sensitized RALA hepatocytes to ceramide toxicity, but to a lesser extent than for TNF-alpha. Thus, unlike many nonhepatic cell types, rat hepatocytes are resistant to cell death from ceramide because of the transcriptionally dependent up-regulation of a protective gene(s). The ability of ActD and NF-kappaB inactivation to sensitize RALA hepatocytes to ceramide toxicity suggests that ceramide may act as a downstream mediator of TNF-alpha toxicity.

Monocyte chemoattractant protein 1 (MCP-1) is a potent monocyte/macrophage chemoattractant expressed by fat-storing cells (FSCs) in rat models of liver injury. This study investigated the mechanism of this activation of hepatic MCP-1 expression.The regulation of MCP-1 messenger RNA (mRNA) expression and protein synthesis was examined in FSC lines derived from CCl4-induced cirrhotic rat liver (cirrhotic FSCs) and normal rat liver (normal FSCs).Northern blot hybridization analysis revealed low levels of MCP-1 mRNA in cultured cirrhotic FSCs that increased markedly after treatment with tumor necrosis factor alpha, interleukin 1 alpha, or transforming growth factor beta 1. All three cytokines increased the synthesis and secretion of MCP-1 protein. Oxygen free radical production also increased MCP-1 mRNA levels. These increases in MCP-1 mRNA were blocked by dexamethasone. In normal FSCs, levels of MCP-1 mRNA and secreted protein were increased in response to cytokines or oxygen free radical production, but the magnitude and duration of this increase was less than in cirrhotic FSCs.In liver injury, monocyte/macrophage recruitment and activation from FSC production of MCP-1 may be stimulated by cytokines and oxygen free radicals. During chronic liver injury leading to cirrhosis, FSCs may become hypersensitive to these stimuli, further fueling the inflammatory response.

We have all learned in textbooks that “lysosomes contain hydrolases able to degrade all types of intracellular molecules which include proteases, glycosidases, nucleotidases and lipases.” To date, the only logical explanation for the presence of lipases inside of lysosomes was for the degradation of lipoproteins internalized by endocytosis, and for the breakdown of intralysosomal vesicles derived from fusion with autophagosomes or multivesicular bodies. However, in our recent work we found a novel role for ysosomal lipases in the basic cellular process that regulates intracellular lipid stores that we have named “macrolipophagy”.

Overactivation of c-Jun N-terminal kinase (JNK)/c-Jun signaling is a central mechanism of hepatocyte injury and death including that from oxidative stress. However, the functions of JNK and c-Jun are still unclear, and this pathway also inhibits hepatocyte death. Previous studies of menadione-induced oxidant stress demonstrated that toxicity resulted from sustained JNK/c-Jun activation as death was blocked by the c-Jun dominant negative TAM67. To further delineate the function of JNK/c-Jun signaling in hepatocyte injury from oxidant stress, the effects of direct JNK inhibition on menadione-induced death were examined. In contrast to the inhibitory effect of TAM67, pharmacological JNK inhibition by SP600125 sensitized the rat hepatocyte cell line RALA255-10G to death from menadione. SP600125 similarly sensitized mouse primary hepatocytes to menadione toxicity. Death from SP600125/menadione was c-Jun dependent as it was blocked by TAM67, but independent of c-Jun phosphorylation. Death occurred by apoptosis and necrosis and activation of the mitochondrial death pathway. Short hairpin RNA knockdowns of total JNK or JNK2 sensitized to death from menadione, whereas a jnk1 knockdown was protective. Jnk2 null mouse primary hepatocytes were also sensitized to menadione death. JNK inhibition magnified decreases in cellular ATP content and β-oxidation induced by menadione. This effect mediated cell death as chemical inhibition of β-oxidation also sensitized cells to death from menadione, and supplementation with the β-oxidation substrate oleate blocked death. Components of the JNK/c-Jun signaling pathway have opposing functions in hepatocyte oxidant stress with JNK2 mediating resistance to cell death and c-Jun promoting death.

Glial cell line–derived neurotrophic factor (GDNF) is a protein that is required for the development and survival of enteric, sympathetic, and catecholaminergic neurons. We previously reported that GDNF is protective against high fat diet (HFD)‐induced hepatic steatosis in mice through suppression of hepatic expression of peroxisome proliferator activated receptor‐γ and genes encoding enzymes involved in de novo lipogenesis. We also reported that transgenic overexpression of GDNF in mice prevented the HFD‐induced liver accumulation of the autophagy cargo‐associated protein p62/sequestosome 1 characteristic of impaired autophagy. Here we investigated the effects of GDNF on hepatic autophagy in response to increased fat load, and on hepatocyte mitochondrial fatty acid β‐oxidation and cell survival. GDNF not only prevented the reductions in the liver levels of some key autophagy‐related proteins, including Atg5, Atg7, Beclin‐1 and LC3A/B‐II, seen in HFD‐fed control mice, but enhanced their levels after 12 weeks of HFD feeding. In vitro , GDNF accelerated autophagic cargo clearance in primary mouse hepatocytes and a rat hepatocyte cell line, and reduced the phosphorylation of the mechanistic target of rapamycin complex downstream‐target p70S6 kinase similar to the autophagy activator rapamycin. GDNF also enhanced mitochondrial fatty acid β‐oxidation in primary mouse and rat hepatocytes, and protected against palmitate‐induced lipotoxicity. Conclusion : We demonstrate a role for GDNF in enhancing hepatic autophagy and in potentiating mitochondrial function and fatty acid oxidation. Our studies show that GDNF and its receptor agonists could be useful for enhancing hepatocyte survival and protecting against fatty acid–induced hepatic lipotoxicity.

Activation of extracellular signal–regulated kinase (ERK) 1/2 promotes hepatocyte proliferation in response to growth stimuli, but whether constitutive hepatocyte ERK1/2 signaling functions in liver physiology is unknown. To examine the role of ERK1/2 in hepatic homeostasis, the effects of a knockout of Erk1 and/or Erk2 in mouse liver were examined. The livers of mice with a global Erk1 knockout or a tamoxifen‐inducible, hepatocyte‐specific Erk2 knockout were normal. In contrast, Erk1/2 double‐knockout mice developed hepatomegaly and hepatitis by serum transaminases, histology, terminal deoxynucleotide transferase‐mediated deoxyuridine triphosphate nick end‐labeling, and assays of hepatic inflammation. Liver injury was associated with biochemical evidence of cholestasis with increased serum and hepatic bile acids and led to hepatic fibrosis and mortality. RNA sequencing and polymerase chain reaction analysis of double‐knockout mouse livers revealed that the rate‐limiting bile acid synthesis gene Cyp7a1 ( cholesterol 7α‐hydroxylase ) was up‐regulated in concert with decreased expression of the transcriptional repressor short heterodimer partner . Elevated bile acids were the mechanism of liver injury, as bile acid reduction by SC‐435, an inhibitor of the ileal apical sodium–dependent bile acid transporter, prevented liver injury. Conclusion: Constitutive ERK1 and ERK2 signaling has a redundant but critical physiological function in the down‐regulation of hepatic bile acid synthesis to maintain normal liver homeostasis.

The typical proliferative response of hepatocytes to tumor necrosis factor (TNF) can be converted to a cytotoxic one by transcriptional arrest. Although NF-κB activation is critical for hepatocyte resistance to TNF toxicity, the contribution of other TNF-inducible transcription factors remains unknown. To determine the function of c-Myc in hepatocyte sensitivity to TNF, stable transfectants of the rat hepatocyte cell line RALA255-10G containing sense and antisense c-myc expression vectors were isolated with increased (S-Myc cells) and decreased (AN-Myc cells) c-Myc transcriptional activity. While S-Myc cells proliferated in response to TNF treatment, AN-Myc cells underwent 32% cell death within 6 h. Fluorescent microscopic studies indicated that TNF induced apoptosis and necrosis in AN-Myc cells. Cell death was associated with DNA hypoploidy and poly(ADP-ribose) polymerase cleavage but occurred in the absence of detectable caspase-3, -7, or -8 activation. TNF-induced, AN-Myc cell death was dependent on Fas-associated protein with death domain and partially blocked by caspase inhibitors. AN-Myc cells had decreased levels of NF-κB transcriptional activity, but S-Myc cells maintained resistance to TNF despite NF-κB inactivation, suggesting that c-Myc and NF-κB independently mediate TNF resistance. Thus, in the absence of sufficient c-Myc expression, hepatocytes are sensitized to TNF-induced apoptosis and necrosis. These findings demonstrate that hepatocyte resistance to TNF is regulated by multiple transcriptional activators. The typical proliferative response of hepatocytes to tumor necrosis factor (TNF) can be converted to a cytotoxic one by transcriptional arrest. Although NF-κB activation is critical for hepatocyte resistance to TNF toxicity, the contribution of other TNF-inducible transcription factors remains unknown. To determine the function of c-Myc in hepatocyte sensitivity to TNF, stable transfectants of the rat hepatocyte cell line RALA255-10G containing sense and antisense c-myc expression vectors were isolated with increased (S-Myc cells) and decreased (AN-Myc cells) c-Myc transcriptional activity. While S-Myc cells proliferated in response to TNF treatment, AN-Myc cells underwent 32% cell death within 6 h. Fluorescent microscopic studies indicated that TNF induced apoptosis and necrosis in AN-Myc cells. Cell death was associated with DNA hypoploidy and poly(ADP-ribose) polymerase cleavage but occurred in the absence of detectable caspase-3, -7, or -8 activation. TNF-induced, AN-Myc cell death was dependent on Fas-associated protein with death domain and partially blocked by caspase inhibitors. AN-Myc cells had decreased levels of NF-κB transcriptional activity, but S-Myc cells maintained resistance to TNF despite NF-κB inactivation, suggesting that c-Myc and NF-κB independently mediate TNF resistance. Thus, in the absence of sufficient c-Myc expression, hepatocytes are sensitized to TNF-induced apoptosis and necrosis. These findings demonstrate that hepatocyte resistance to TNF is regulated by multiple transcriptional activators. tumor necrosis factor TNF receptor Fas-associated protein with death domain 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide fluorescence-activated cell sorting poly(ADP-ribose) polymerase inhibitor of apoptosis polyacrylamide gel electrophoresis Tumor necrosis factor (TNF)1 is a pleiotrophic cytokine that can induce either proliferative or cytotoxic responses in a variety of cultured cells including hepatocytes (1Tracy K.J. Remick D.G. Friedland J.S. Cytokines in Health and Disease. Marcel Dekker, New York1997: 223-240Google Scholar). The biological effects of TNF in cultured hepatocytes are relevant to the liverin vivo, since TNF also acts as a hepatic mitogen (2Akerman P. Cote Y. Yang S.Q. McClain C. Nelson S. Bagby G.J. Diehl A.M. Am. J. Physiol. 1992; 263: G579-G585Crossref PubMed Google Scholar, 3Yamada Y. Kirillova I. Peschon J.J. Fausto N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1441-1446Crossref PubMed Scopus (840) Google Scholar) or cytotoxin (4Blazka M.E. Wilmer J.L. Holladay S.D. Wilson R.E. Luster M.I. Toxicol. Appl. Pharmacol. 1995; 133: 43-52Crossref PubMed Scopus (298) Google Scholar, 5Czaja M.J. Xu J. Alt E. Gastroenterology. 1995; 108: 1849-1854Abstract Full Text PDF PubMed Scopus (191) Google Scholar, 6Hishinuma I. Nagakawa J.I. Hirota K. Miyamoto K. Tsukidate L. Yamanaka T. Katayama K.I. Yamatsu I. Hepatology. 1990; 12: 1187-1191Crossref PubMed Scopus (169) Google Scholar) in vivo, depending on the pathophysiological setting. TNF has been implicated as a mediator of hepatocyte death following injury from toxins, ischemia/reperfusion, and hepatitis virus (for a review, see Ref. 7Bradham C.A. Plümpe J. Manns M.P. Brenner D.A. Trautwein C. Am. J. Physiol. 1998; 275: G387-G392PubMed Google Scholar). In the absence of an injurious cofactor such as a toxin, hepatocytes are resistant to TNF cytotoxicity, and the mechanism by which they become sensitized to TNF-induced death in the setting of cell injury remains unknown. The pathway from TNF stimulation to cell death has been well described (for a review, see Ref. 8Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5154) Google Scholar). TNF binding to the type 1 TNF receptor (TNFR-1) causes receptor trimerization and the recruitment and binding of a series of intracellular proteins including TNFR-associated death domain protein and Fas-associated protein with death domain (FADD). FADD binding leads initially to activation of caspase-8, and subsequently to activation of caspase-3, resulting in apoptosis (8Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5154) Google Scholar). While the steps in the TNF death pathway leading to apoptosis are known, the mechanism by which cells inactivate this caspase cascade and maintain resistance to TNF toxicity is unclear. A recent advance in our understanding of cellular TNF resistance has come from the demonstration that activation of the transcription factor NF-κB is critical for the induction of cellular resistance to TNF toxicity (9Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2935) Google Scholar, 10Liu Z. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1783) Google Scholar, 11Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2449) Google Scholar, 12Wang C.-Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2512) Google Scholar). Inhibition of NF-κB activation in cultured hepatocytes (13Bradham C.A. Qian T. Streetz K. Trautwein C. Brenner D.A. LeMasters J. Mol. Cell. Biol. 1998; 18: 6353-6364Crossref PubMed Scopus (367) Google Scholar, 14Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar) or in the liver in vivo (15Iimuro Y. Nishiura T. Hellerbrand C. Behrns K. Schoonhoven R. Grisham J.W. Brenner D.A. J. Clin. Invest. 1998; 101: 802-811Crossref PubMed Scopus (424) Google Scholar) converts the hepatocellular TNF response from one of proliferation to one of apoptosis. This finding fits well with the fact that in vitro resistance to TNF-induced cytotoxicity requires RNA and protein synthesis (16Kull F.C. Cuatrecasas P. Cancer Res. 1981; 41: 4885-4890PubMed Google Scholar), suggesting that TNF signaling up-regulates a protective cellular gene(s). NF-κB inactivation may sensitize cells to TNF toxicity by preventing the transcriptional up-regulation of an NF-κB-dependent protective gene(s). However, TNF activates other transcriptional activators, including c-Myc and AP-1, and their potential contribution to the transcriptional regulation of hepatocyte resistance to TNF toxicity is unknown. c-Myc is a transcription factor that regulates cell proliferation, differentiation, and apoptosis (for a review, see Ref. 17Dang C.V. Mol. Cell. Biol. 1999; 19: 1-11Crossref PubMed Scopus (1384) Google Scholar). c-Myc expression not only promotes proliferation but also can induce or sensitize cells to apoptosis (18Hoffman B. Lieberman D.A. Oncogene. 1998; 17: 3351-3357Crossref PubMed Scopus (134) Google Scholar, 19Packham G. Cleveland J.L. Biochim. Biophys. Acta. 1995; 1242: 11-28PubMed Google Scholar). Overexpression of c-myc under circumstances in which this gene is usually down regulated such as serum deprivation, results in apoptotic cell death in nonhepatic cells (20Evan G.I. Wyllie A.H. Gilbert C.S. Littlewood T.D. Land H. Brooks M. Waters C.M. Penn L.Z. Hancock D.C. Cell. 1992; 69: 119-128Abstract Full Text PDF PubMed Scopus (2773) Google Scholar) and in a hepatoma cell line (21Xu J. Xu Y. Nguyen Q. Novikoff P.M. Czaja M.J. Am. J. Physiol. 1996; 270: G60-G70Crossref PubMed Google Scholar). c-Myc expression has been reported to be induced by TNF alone (22Manchester K.M. Heston W.D.W. Donner D.B. Biochem. J. 1993; 290: 185-190Crossref PubMed Scopus (32) Google Scholar, 23Ninomiya-Tsuji J. Torti F.M. Ringold G.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9611-9615Crossref PubMed Scopus (34) Google Scholar) or in combination with cycloheximide (24Janicke R.U. Lee F.H.H. Porter A.G. Mol. Cell. Biol. 1994; 14: 5661-5670Crossref PubMed Scopus (94) Google Scholar). Previous investigations in nonhepatic cells have consistently reported that increased c-Myc expression initiates or promotes TNF-induced apoptosis (24Janicke R.U. Lee F.H.H. Porter A.G. Mol. Cell. Biol. 1994; 14: 5661-5670Crossref PubMed Scopus (94) Google Scholar, 25Janicke R.U. Lin X.Y. Lee F.H.H. Porter A.G. Mol. Cell. Biol. 1996; 16: 5245-5253Crossref PubMed Scopus (55) Google Scholar, 26Klefstrom J. Arighi E. Littlewood T. Jäättelä M. Saksela E. Evan G.I. Alitalo K. EMBO J. 1997; 16: 7382-7392Crossref PubMed Scopus (107) Google Scholar, 27Klefstrom J. Västrik I. Saksela E. Valle J. Eilers M. Alitalo K. EMBO J. 1994; 13: 5442-5450Crossref PubMed Scopus (134) Google Scholar). However, in TNF-dependent liver injury in vivoinduced by the toxin galactosamine (6Hishinuma I. Nagakawa J.I. Hirota K. Miyamoto K. Tsukidate L. Yamanaka T. Katayama K.I. Yamatsu I. Hepatology. 1990; 12: 1187-1191Crossref PubMed Scopus (169) Google Scholar), TNF induces hepatocyte injury and death associated with a block in the up-regulation ofc-myc mRNA expression that normally occurs during a hepatic proliferative response (28Schmiedeberg P. Biempica L. Czaja M.J. J. Cell. Physiol. 1993; 154: 294-300Crossref PubMed Scopus (51) Google Scholar). These findings suggested that hepatocytes may undergo TNF-induced death in the absence of c-Myc expression or even become sensitized to TNF toxicity by a failure to up-regulate c-Myc. We therefore tested the hypothesis that c-Myc expression promotes hepatocyte resistance to TNF toxicity by examining the sensitivity of rat hepatocyte cell lines with differential c-Myc expression to TNF toxicity. Cells lines with differential c-Myc expression were derived from the wild-type RALA255–10G rat hepatocyte cell line (29Chou J.Y. Mol. Cell. Biol. 1983; 3: 1013-1020Crossref PubMed Scopus (47) Google Scholar). These cells are conditionally transformed with a temperature-sensitive T antigen. Cells were grown at the permissive temperature of 33 °C and then maintained at 37 °C to allow suppression of T antigen expression and development of a differentiated hepatocyte phenotype as described previously (29Chou J.Y. Mol. Cell. Biol. 1983; 3: 1013-1020Crossref PubMed Scopus (47) Google Scholar). All experiments were performed in cells cultured at 37 °C. The c-myc cDNA subcloned into the expression vector pMEP4 (Invitrogen, San Diego, CA) as described previously (21Xu J. Xu Y. Nguyen Q. Novikoff P.M. Czaja M.J. Am. J. Physiol. 1996; 270: G60-G70Crossref PubMed Google Scholar) was transfected into RALA hepatocytes using LipofectAMINE Plus (Life Technologies, Inc.) according to the manufacturer's instructions. Stable transfectants were selected by resistance to 200 μg/ml hygromycin (Calbiochem). The subsequent experiments employed pooled transfectants expressing sense (S-Myc cells) and antisense (AN-Myc cells) c-myc constructs. All cells were cultured in 50 μm zinc for 4 days prior to the start of experiments in order to induce transgene expression from pMEP4, which contains a zinc-inducible human MT IIa promoter. In some experiments, cells were treated with rat recombinant TNF (TNF-α, R & D Systems, Minneapolis, MN) at a concentration of 10 ng/ml, 50 μm C2 ceramide (Biomol, Plymouth Meeting, PA), or 1.25 μmol/106 cells of hydrogen peroxide (H2O2) (Sigma). To inhibit caspase activity, cells were pretreated for 1 h before the addition of TNF with the following caspase inhibitors dissolved in dimethyl sulfoxide: 100 μm Val-Ala-Asp-fluoromethylketone (BACHEM, Torrance, CA), 50 μm N-[(indole-2-carbonyl)-alaninyl]-3-amino-4-oxo-5-fluoropentanoic acid (IDN-1529), orN-[(1,3-dimethylindol-2-carbonyl)-valinyl]-3-amino-4-oxo-5-fluoropentanoic acid (IDN-1965) (IDUN Pharmaceuticals, La Jolla, CA). IDN-1529 and IDN-1965 have broad anti-caspase activity, inhibiting caspase-1, -3, -6, and -8. 2J. Wu, personal communication. RALA hepatocytes were transiently transfected with luciferase reporter genes using LipofectAMINE Plus. Cells were transfected with NF-κB-Luc (30Galang C.K. Der C.J. Hauser C.A. Oncogene. 1994; 9: 2913-2921PubMed Google Scholar), which contains three NF-κB binding sites, or pMyc 3E1b-Luc (31Gupta S. Seth A. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3216-3220Crossref PubMed Scopus (140) Google Scholar), which contains three c-Myc binding sites, driving firefly luciferase reporter genes. Cells were cotransfected with pRL-TK (Promega, Madison, WI) a Renilla luciferase vector driven by a Herpes simplex virus thymidine kinase promoter, which served as a control for transfection efficiency. To assay luciferase activity, cells were washed in phosphate-buffered saline and lysed in 1% Triton X-100, and the cell extract was assayed for firefly luciferase activity in a luminometer. Renilla luciferase was assayed in the same sample according to the manufacturer's instructions. Firefly luciferase activity was then normalized to Renillaluciferase activity. RNA was extracted from cells as described previously (32Czaja M.J. Weiner F.R. Freedman J.H. J. Cell. Physiol. 1991; 147: 434-438Crossref PubMed Scopus (25) Google Scholar). Steady-state mRNA levels were determined by Northern blot hybridizations using samples of 20 μg of total RNA (32Czaja M.J. Weiner F.R. Freedman J.H. J. Cell. Physiol. 1991; 147: 434-438Crossref PubMed Scopus (25) Google Scholar). The membranes were hybridized with [32P]dCTP (PerkinElmer Life Sciences)-labeled cDNA clones for lactate dehydrogenase A (33Shim H. Chun Y.S. Lewis B.C. Dang C.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1511-1516Crossref PubMed Scopus (254) Google Scholar) and glyceraldehyde-3-phosphate dehydrogenase (34Tso J.Y. Sun X.-H. Kao T.-H. Reece K.S. Wu R. Nucleic Acids Res. 1985; 13: 2485-2502Crossref PubMed Scopus (1760) Google Scholar). The hybridized filters were washed under stringent conditions (32Czaja M.J. Weiner F.R. Freedman J.H. J. Cell. Physiol. 1991; 147: 434-438Crossref PubMed Scopus (25) Google Scholar). Relative cell number was determined by the MTT assay, as described previously (14Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar). Cell survival was calculated as a percentage of control cells by taking the optical density reading of cells given a particular treatment, dividing that number by the optical density reading for the untreated, control cells, and then multiplying by 100. The numbers of apoptotic and necrotic cells were determined by examining cells under fluorescence microscopy following costaining with acridine orange and ethidium bromide (14Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar). The percentage of cells with apoptotic morphology (nuclear and cytoplasmic condensation, nuclear fragmentation, membrane blebbing, and apoptotic body formation) under acridine orange staining was determined by examining >400 cells/dish. Necrosis was determined by the presence of ethidium bromide staining in the same cell population. The identification of hypoploid cells by FACS detection of DNA loss after controlled extraction of low molecular weight DNA was performed as described previously (35Jones B.E. Lo C.R. Liu H. Srinivasan A. Streetz K. Valentino K.L. Czaja M.J. J. Biol. Chem. 2000; 275: 705-712Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Cells were trypsinized and centrifuged, and the cell pellets were fixed in 70% ethanol and placed at −20 °C for a minimum of 17 h. The cells were washed and resuspended in Hanks' buffered saline solution and incubated in phosphate-citric acid buffer (0.2 m Na2HPO4, 0.1 mcitric acid, pH 7.8) for 5 min. The cells were then centrifuged, and the pellet was resuspended in Hanks' buffered saline solution containing propidium iodide (20 μg/μl) and RNase (100 μg/ml). Following a 30-min incubation at room temperature, the cells were analyzed on a FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA) at an excitation of 488 nm. DNA fluorescence pulse processing was used to discriminate between single cells and aggregates of cells (Doublet Discrimination) by evaluating the FL2-Width versusFL2-Area scatter plot. Light scatter gating was used to eliminate smaller debris from analysis. An analysis gate was set to limit the measurement of hypoploidy to an area of 10-fold loss of DNA content. For protein isolation for Western immunoblots of c-Myc and protein-disulfide isomerase, cells were washed in phosphate-buffered saline, centrifuged, and resuspended in lysis buffer composed of 50 mm Tris, pH 7.5, 150 mm sodium chloride, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mm dithiothreitol, 1 mm EDTA, 1 mm EGTA, 1 mmphenylmethylsulfonyl fluoride, and 2 μg/ml of pepstatin A, leupeptin, and aprotinin. Cells were then mixed at 4 °C for 30 min. After centrifugation, the supernatant was collected, and the protein concentration was determined by the Bio-Rad protein assay. Fifty micrograms of protein were resolved on 10% SDS-PAGE as described previously (14Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar). Membranes were stained with Ponceau red to ensure equivalent amounts of protein loading and electrophoretic transfer among samples. Membranes were exposed to a rabbit anti-c-Myc polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or protein-disulfide isomerase rabbit antiserum (36Terada K. Manchikalapudi P. Noiva R. Jauregui H.O. Stockert R.J. Schilsky M.L. J. Biol. Chem. 1995; 270: 20410-20416Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), at 1:1000 dilutions followed by a goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (Life Technologies) at a 1:20,000 dilution. Proteins were visualized by chemiluminescence (SuperSignal West Dura Extended; Pierce). For poly(ADP-ribose) polymerase (PARP) immunoblots, cells were washed in phosphate-buffered saline, centrifuged, and resuspended in lysis buffer containing 20 mm Tris, pH 7.5, 1% SDS, 2 mm EDTA, 2 mm EGTA, 6 mmβ-mercaptoethanol, and the protease inhibitors as above. After a 10-min incubation on ice, the cell suspension was sonicated. Fifty micrograms of protein were resolved on 8% SDS-PAGE and immunoblotted with a rabbit anti-PARP polyclonal antibody (Santa Cruz Biotechnology) at a 1:1000 dilution followed by a goat anti-rabbit antibody at a 1:20,000 dilution. For caspase immunoblots, cells were scraped in medium; centrifuged; resuspended in lysis buffer containing 10 mm HEPES, pH 7.4, 42 mm MgCl2, 1% Triton X-100, and the protease inhibitors listed previously; and mixed at 4 °C for 30 min. Fifty micrograms of protein were resolved on 10% SDS-PAGE and immunoblotted with rabbit polyclonal anti-caspase-3, -7, and -8 antibodies (IDUN Pharmaceuticals) at 1:2000, 1:1000, and 1:4000 dilutions, respectively, followed by a goat anti-rabbit secondary antibody at a 1:10,000 dilution. To examine mitochondrial cytochrome c release, mitochondrial fractions were prepared by differential centrifugation in sucrose as described previously (35Jones B.E. Lo C.R. Liu H. Srinivasan A. Streetz K. Valentino K.L. Czaja M.J. J. Biol. Chem. 2000; 275: 705-712Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Fifty micrograms of mitochondrial protein were subjected to 15% SDS-PAGE as described above. A mouse anti-cytochrome c monoclonal IgG (Pharmingen, San Diego, CA) and a mouse anti-cytochrome oxidase subunit IV monoclonal IgG (Molecular Probes, Inc., Eugene, OR) were used at 1:1000 dilutions together with a goat anti-mouse IgG conjugated to horseradish peroxidase (Life Technologies). The following adenoviruses were employed: a control virus Ad5LacZ that expresses theEscherichia coli β-galactosidase gene; NFD-4 containing a dominant negative FADD; a CrmA-expressing adenovirus; and Ad5IκB, which expresses a mutated IκB that irreversibly binds NF-κB, preventing its activation (13Bradham C.A. Qian T. Streetz K. Trautwein C. Brenner D.A. LeMasters J. Mol. Cell. Biol. 1998; 18: 6353-6364Crossref PubMed Scopus (367) Google Scholar). Viruses were grown in 293 cells; purified by banding twice on CsCl gradients; dialyzed against 5 mm Tris, pH 8.0, 50 mm MgCl2, 3% glycerol, and 0.05% bovine serum albumin; and stored at −80 °C. Cells were infected with 5 × 109 particles of the appropriate virus per 35-mm culture dish (∼1.5 × 103 particles/cell or 5–15 plaque-forming units/cell) as described previously (14Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar). Nuclear proteins were isolated by the method of Schreiber et al. (37Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3917) Google Scholar), modified as described previously (21Xu J. Xu Y. Nguyen Q. Novikoff P.M. Czaja M.J. Am. J. Physiol. 1996; 270: G60-G70Crossref PubMed Google Scholar). Electrophoretic mobility shift assays were performed on 5 μg of protein with a 32P-end-labeled oligonucleotide for the NF-κB consensus sequence (Santa Cruz Biotechnology). The DNA binding reaction was performed as described previously (21Xu J. Xu Y. Nguyen Q. Novikoff P.M. Czaja M.J. Am. J. Physiol. 1996; 270: G60-G70Crossref PubMed Google Scholar); the samples were resolved on a 4% polyacrylamide gel, dried, and subjected to autoradiography. All numerical results are reported as mean ± S.E. and represent data from a minimum of three independent experiments performed in duplicate. RALA hepatocytes were transfected with the pMEP4 expression vector containing the c-myc cDNA in either a sense or antisense orientation. Stable transfectants were selected in hygromycin and initially screened for c-myc expression by Northern blot analysis. Two polyclonal cell lines were selected in which expression of sense c-myc (S-Myc cells) and antisense c-myc(AN-Myc cells) constructs resulted in maximally increased and decreasedc-myc levels, respectively. Western immunoblotting confirmed that S-Myc cells had increased c-Myc levels compared with AN-Myc cells, while the two cell lines had equivalent levels of the constitutively expressed protein-disulfide isomerase (Fig. 1 A). The relative amounts of c-Myc transcriptional activity in the two cells lines were measured with a transiently transfected c-Myc firefly luciferase reporter, and the results were normalized to a cotransfected Renillaluciferase reporter under the control of a minimal reporter. c-Myc transcriptional activity in untreated cells was increased over 14-fold in S-Myc cells as compared with AN-Myc cells (Fig. 1 B). Although c-Myc-dependent transcriptional activity increased in both cell lines following TNF treatment, the activity in AN-Myc cells was still less than 10% of the activity in S-Myc cells (Fig. 1 B). As additional evidence of differential c-Myc transcriptional activity in the two cell lines, mRNA levels for the c-Myc-dependent lactate dehydrogenase A gene (33Shim H. Chun Y.S. Lewis B.C. Dang C.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1511-1516Crossref PubMed Scopus (254) Google Scholar), were determined by Northern blot analysis. S-Myc cells had significantly increased expression of lactate dehydrogenase A relative to AN-Myc cells, while RNA levels of the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase gene were equivalent in the two cell lines (Fig. 1 C). Thus, as assessed by protein levels, transcriptional activity, and c-Myc-dependent gene expression, c-Myc levels were increased in S-Myc cells relative to AN-Myc cells. TNF treatment of RALA hepatocytes results in a proliferative response (14Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar), similar to the known mitogenic effect of TNF on the liver in vivo following partial hepatectomy (2Akerman P. Cote Y. Yang S.Q. McClain C. Nelson S. Bagby G.J. Diehl A.M. Am. J. Physiol. 1992; 263: G579-G585Crossref PubMed Google Scholar, 3Yamada Y. Kirillova I. Peschon J.J. Fausto N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1441-1446Crossref PubMed Scopus (840) Google Scholar). To convert the TNF response from proliferation to apoptosis requires either cotreatment with the RNA synthesis inhibitor actinomycin D or inhibition of activation of the transcription factor NF-κB (14Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar, 35Jones B.E. Lo C.R. Liu H. Srinivasan A. Streetz K. Valentino K.L. Czaja M.J. J. Biol. Chem. 2000; 275: 705-712Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Similar to wild-type RALA hepatocytes, S-Myc cells were resistant to TNF toxicity as determined by MTT assays 6 and 24 h after TNF treatment (Fig. 2 A). Despite the use of highly confluent cultures, S-Myc cell number increased 11% at 24 h, indicating that these cells underwent a proliferative response to TNF, a result identical to previous findings in wild-type cells (14Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar). In contrast, TNF treatment of AN-Myc cells resulted in a 32% decrease in cell number within only 6 h and only a slight further decrease in cell number by 24 h (Fig. 2 A). In keeping with previously published results (14Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar), TNF at 10 ng/ml resulted in a maximal death response because no further decrease in cell number occurred when AN-Myc cells were treated with a higher TNF concentration of 30 ng/ml (data not shown). To examine whether AN-Myc cell sensitivity to TNF toxicity represented a nonspecific sensitization to any cell death stimulus, cell survival was determined following treatment with C2 ceramide and H2O2. Ceramide is a known apoptotic stimulus that has been implicated as a downstream mediator of TNF-induced cell death (38Kolesnick R.N. Krönke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (730) Google Scholar). The oxidant H2O2 triggers apoptosis in many cell types, including RALA cells (39Jones B.E. Lo C.R. Liu H. Pradhan Z. Garcia L. Srinivasan A. Valentino K.L. Czaja M.J. Am. J. Physiol. 2000; 278: G693-G699Crossref PubMed Google Scholar), and oxidative stress has been implicated as a mechanism of TNF toxicity (1Tracy K.J. Remick D.G. Friedland J.S. Cytokines in Health and Disease. Marcel Dekker, New York1997: 223-240Google Scholar). Identical to previous reports in wild-type RALA hepatocytes (40Jones B.E. Lo C.R. Srinivasan A. Valentino K.L. Czaja M.J. Hepatology. 1999; 30: 215-222Crossref PubMed Scopus (45) Google Scholar), both S-Myc and AN-Myc cells were resistant to ceramide toxicity at the 6-h time point, at which sensitization to TNF toxicity had occurred (Fig. 2 A). Both S-Myc and AN-Myc cells underwent significant cell death 24 h after H2O2 treatment (Fig. 2 A), indicating no significant alteration in sensitivity to this toxin between the two cell lines. Inhibition of c-Myc expression did not sensitize RALA hepatocytes indiscriminately to any form of cell death but specifically modulated resistance to TNF-induced cell death. Cell death from TNF may result from apoptosis or necrosis depending on the cell type. To determine which type of cell death occurred in TNF-treated AN-Myc cells, cells were examined for morphological and biochemical evidence of apoptosis. AN-Myc cells were examined under fluorescence microscopy following costaining with acridine orange and ethidium bromide to quantitate the numbers of apoptotic and necrotic cells. Over the 6 h after TNF treatment, AN-Myc cells had marked increases in both the numbers of apoptotic and necrotic cells (Fig. 2 B). As additional evidence that inhibition of c-Myc expression sensitized cells to death at least in part from apoptosis, FACS analysis was performed to quantitate the numbers of hypoploid cells as a measure of the presence of DNA fragmentation. Despite 24 h of culture in serum-free medium, S-Myc cells had a low level of hypoploidy that decreased slightly with TNF t

While Ito cells appear to be a major source of increased matrix synthesis during hepatic fibrogenesis, the cellular changes that occur in these cells during liver fibrosis have not been well delineated. In this study we examined Ito cell gene expression in isolated cells from normal rats, and rats with carbon tetrachloride-induced fibrosis, in order to better define the changes occurring in these cells during this pathologic process. Specifically, we addressed three questions: (1) which matrix genes are over expressed in Ito cells in fibrotic liver; (2) do these cells increase their expression of the fibrogenic cytokine transforming growth factor-β1 (TGF-β1); and (3) do Ito cells change their phenotype during hepatic fibrogenesis as reflected by alterations in the expression of their intermediate filament genes? Northern hybridization analysis revealed that Ito cells isolated from fibrotic livers had significant increases in mRNA levels of types I, III and IV procollagen compared to normal cells, while no increases were found in hepatocytes, and Kupffer/endothelial cells had only an increase in type I procollagen mRNA. Analysis of other matrix proteins which increase during hepatic fibrogenesis revealed elevations in laminin B and fibronectin mRNA levels only in Ito cells. Increased Ito cell matrix gene expression was also associated with a 4-fold increase in TGF-β1 levels in these cells. No increase in TGF-β1 mRNA was found in hepatocytes, and less than a 2-fold increase was found in Kupffer/endothelial cells isolated from fibrotic livers. Ito cells isolated from both normal and fibrotic livers expressed vimentin, desmin and β-actin mRNA, but no α-actin mRNA, implying that Ito cells in these fibrotic livers retained a normal Ito cell phenotype. These studies suggest that increases in Ito cell mRNA levels of matrix proteins contribute to the development of hepatic fibrosis. Furthermore, enhanced Ito cell expression of TGF-β1 mRNA may further augment this cell's matrix synthesis. Finally, the increases in the mRNA of these matrix proteins appear to occur in Ito cells which are not phenotypically altered by this pathologic process.

Increased expression of cytochrome P450 2E1 (CYP2E1) occurs in alcoholic liver disease, and leads to the hepatocellular generation of toxic reactive oxygen intermediates (ROI). Oxidative stress created by CYP2E1 overexpression may promote liver cell injury by sensitizing hepatocytes to oxidant-induced damage from Kupffer cell-produced ROI or cytokines. To determine the effect of CYP2E1 expression on the hepatocellular response to injury, stably transfected hepatocytes expressing increased (S-CYP15) and decreased (AN-CYP10) levels of CYP2E1 were generated from the rat hepatocyte line RALA255-10G. S-CYP15 cells had increased levels of CYP2E1 as demonstrated by Northern blot analysis, immunoblotting, catalytic activity, and increased cell sensitivity to death from acetaminophen. Death in S-CYP15 cells was significantly decreased relative to that in AN-CYP10 cells following treatment with hydrogen peroxide and the superoxide generator menadione. S-CYP15 cells underwent apoptosis in response to these ROI, whereas AN-CYP10 cells died by necrosis. This differential sensitivity to ROI-induced cell death was partly explained by markedly decreased levels of glutathione (GSH) in AN-CYP10 cells. However, chemically induced GSH depletion triggered cell death in S-CYP15 but not AN-CYP10 cells. Increased expression of CYP2E1 conferred hepatocyte resistance to ROI-induced cytotoxicity, which was mediated in part by GSH. However, CYP2E1 overexpression left cells vulnerable to death from GSH depletion.

Common features of chronic alcoholic liver disease are progressive hypoalbuminemia and a spectrum of liver fibrosis. The molecular mechanisms that account for these effects are still the subject of controversy. Therefore, in the present study we evaluated albumin and collagen gene expression in livers of alcohol abusers and patients with virus-induced liver disease. Albumin and pro alpha 1(I) collagen messenger RNA levels were determined in 30 patients who underwent diagnostic liver biopsy. Of 14 alcoholics, 7 had alcoholic hepatitis alone and the other 7 had cirrhosis plus alcoholic hepatitis. Of 16 nonalcoholic patients with chronic viral infection, 6 had chronic active hepatitis and 10 had cirrhosis plus chronic active hepatitis. Total RNA was extracted from a portion of each biopsy specimen, hybridized with a human albumin or collagen complementary DNA clone, and compared with 2 normal surgical specimens, which served as controls. The Northern hybridization studies showed that (a) despite the presence of inflammation and fibrosis, the albumin messenger RNA levels of alcoholics were similar to those of the controls; (b) these alcoholics had significantly higher levels of albumin messenger RNA than did patients with similar histological levels of disease due to viral infection; and (c) all the categories of patients had markedly increased procollagen messenger RNA levels compared with controls. Given these results it is tempting to speculate that alcohol may actually increase albumin messenger RNA content in humans as it does in animals. Furthermore, the increased procollagen messenger RNA levels in fibrotic livers suggest that an increase in collagen syntheses may be a significant factor in the pathogenesis of hepatic fibrosis.

Toxin‐induced liver diseases lack effective therapies despite increased understanding of the role factors such as an overactive innate immune response play in the pathogenesis of this form of hepatic injury. Pentamidine is an effective antimicrobial agent against several human pathogens, but studies have also suggested that this drug inhibits inflammation. This potential anti‐inflammatory mechanism of action, together with the development of a new oral form of pentamidine isethionate VLX103, led to investigations of the effectiveness of this drug in the prevention and treatment of hepatotoxic liver injury. Pretreatment with a single injection of VLX103 in the d ‐galactosamine (GalN) and lipopolysaccharide (LPS) model of acute, fulminant liver injury dramatically decreased serum alanine aminotransferase levels, histological injury, the number of terminal deoxynucleotide transferase–mediated deoxyuridine triphosphate nick end‐labeling (TUNEL)‐positive cells and mortality compared with vehicle‐injected controls. VLX103 decreased GalN/LPS induction of tumor necrosis factor (TNF) but had no effect on other proinflammatory cytokines. VLX103 prevented the proinflammatory activation of cultured hepatic macrophages and partially blocked liver injury from GalN/TNF. In GalN/LPS‐treated mice, VLX103 decreased activation of both the mitochondrial death pathway and downstream effector caspases 3 and 7, which resulted from reduced c‐Jun N‐terminal kinase activation and initiator caspase 8 cleavage. Delaying VLX103 treatment for up to 3 hours after GalN/LPS administration was still remarkably effective in blocking liver injury in this model. Oral administration of VLX103 also decreased hepatotoxic injury in a second more chronic model of alcohol‐induced liver injury, as demonstrated by decreased serum alanine and aspartate aminotransferase levels and numbers of TUNEL‐positive cells. Conclusion : VLX103 effectively decreases toxin‐induced liver injury in mice and may be an effective therapy for this and other forms of human liver disease. (H epatology 2017;66:922–935).

Stathmin 1 performs a critical function in cell proliferation by regulating microtubule polymerization. This proliferative function is thought to explain the frequent overexpression of stathmin in human cancer and its correlation with a bad prognosis. Whether stathmin also functions in cell death pathways is unclear. Stathmin regulates microtubules in part by binding free tubulin, a process inhibited by stathmin phosphorylation from kinases including c-Jun N-terminal kinase (JNK). The involvement of JNK activation both in stathmin phosphorylation, and in hepatocellular resistance to oxidative stress, led to an examination of the role of stathmin/JNK crosstalk in oxidant-induced hepatocyte death. Oxidative stress from menadione-generated superoxide induced JNK-dependent stathmin phosphorylation at Ser-16, Ser-25 and Ser-38 in hepatocytes. A stathmin knockdown sensitized hepatocytes to both apoptotic and necrotic cell death from menadione without altering levels of oxidant generation. The absence of stathmin during oxidative stress led to JNK overactivation that was the mechanism of cell death as a concomitant knockdown of JNK1 or JNK2 blocked death. Hepatocyte death from JNK overactivation was mediated by the effects of JNK on mitochondria. Mitochondrial outer membrane permeabilization occurred in stathmin knockdown cells at low concentrations of menadione that triggered apoptosis, whereas mitochondrial β-oxidation and ATP homeostasis were compromised at higher, necrotic menadione concentrations. Stathmin therefore mediates hepatocyte resistance to death from oxidative stress by down regulating JNK and maintaining mitochondrial integrity. These findings demonstrate a new mechanism by which stathmin promotes cell survival and potentially tumor growth.

Potential conflict of interest: Nothing to report. Supported by National Institutes of Health grants R01DK044234 and R01AA022601 (to M.J.C.) and T32DK108735 (to M.A.). See Article On Page 736 Significant advances have been made over the past several years in understanding the importance of the intracellular proinflammatory structure termed the inflammasome in promoting inflammatory, autoimmune, and metabolic diseases. Sterile inflammation is triggered when cells sense danger from exposure to damage‐associated molecular patterns (DAMPs) released by dying cells rather than pathogen‐associated molecular patterns (PAMPs) from microorganisms. Inflammation in the liver is regulated by Kupffer cell recognition of DAMPs and PAMPs, and the resulting activation of these resident macrophages leads to the recruitment of circulating monocytes and neutrophils that together comprise the hepatic innate immune response. DAMPs identified to induce sterile inflammation include extracellular adenosine triphosphate, high mobility group box protein 1 (HMGB1), heat shock protein 70, hyaluronic acid, nuclear DNA, and uric acid. DAMPs are sensed by extracellular pattern recognition receptors (PRRs) which include nucleotide‐binding oligomerization domain‐like receptors and toll‐like receptors (TLRs). After DAMP stimulation, two steps lead to inflammasome activation. First, transcription of cytokine and inflammasome component genes is increased. Second, inflammasome assembly occurs that allows activation of caspase 1, which cleaves pro–interleukin (IL)‐1β into its active secreted form. Secreted IL‐1β regulates the recruitment and activation of other immune cells to amplify the inflammatory response.1 Overactivation of the sterile inflammatory response has been implicated in the pathogenesis of a number of liver diseases, including drug‐induced liver injury, alcoholic and nonalcoholic steatohepatitis, and ischemia/reperfusion injury.1 In the mouse model of acetaminophen hepatotoxicity, DAMPs including HMGB1, hyaluronic acid, DNA, keratin, and heat shock protein 70 are released. With antibody neutralization of HMGB1, or in mice lacking TLR4 or TLR9 receptors, liver injury from acetaminophen is significantly reduced.1 The PRRs TLR2, TLR4, and TLR9 have been shown to mediate steatosis and hepatitis in murine nonalcoholic fatty liver disease driven by inflammasome‐produced IL‐1β.2 TLR4 and TLR9 receptor signaling also underlies mouse models of liver injury and inflammation from alcohol and ischemia/reperfusion.1 Together, these studies demonstrate a central role for the DAMP–PRR–inflammasome cascade in liver injury. The specific inflammasome NLRP3 (NOD, LRR, and pyrin domain‐containing 3) has emerged as an important mediator of sterile inflammation including in the liver. In murine liver injury components of the NLRP3 inflammasome pathway are up‐regulated and their deletion reduces liver disease.4 Wree et al.6 previously described the ability of the NLRP3 inflammasome to generate spontaneous liver inflammation, hepatocyte death by pyroptosis, and fibrosis in studies employing transgenic mice with constitutive NLRP3 activation globally or conditionally in myeloid cells. In a continuation of this work in this issue of Hepatology, they further investigated the mechanism by which NLRP3 drives hepatic inflammation and fibrosis.7 They hypothesized that NLRP3 inflammasome effects are mediated not only by inflammasome‐produced IL‐1β, but also by the inflammasome‐independent cytokines tumor necrosis factor (TNF) and IL‐17, which have known roles in liver injury and fibrosis. To examine this question, the authors crossed Nlrp3A350V mice with constitutive myeloid cell NLRP3 activation with global TNF or IL‐17 knockout mice. Inflammation in Nlrp3A350V mice was associated with elevated IL‐1β, TNF, and IL‐17 levels and increased chemokine (C‐X‐C motif) ligand 1 and 2 resulting in hepatic neutrophil translocation. The knockout mice had increased numbers of hepatic macrophages with a proinflammatory phenotype. Increased hepatic stellate cell (HSC) activation and hepatic fibrosis were also found in Nlrp3A350V mice. All of these NLRP3‐induced effects were completely ameliorated by knockout of TNF, indicating that TNF is a critical effector molecule for NLRP3. Knockout of IL‐17 reduced some of the changes caused by NLRP3 inflammasome activation but had little effect on the development of inflammation and fibrosis compared with TNF. With high‐dose lipopolysaccharide administration, liver inflammation was amplified in Nlrp3A350V mice as reflected by increased neutrophil and macrophage infiltration. Inflammation was abrogated in Nlrp3A350V mice with the TNF but not IL‐17 knockout. These findings delineate a new mechanism by which TNF acts as a central mediator of liver disease.8 The study establishes TNF as the principal cytokine responsible for the recruitment of proinflammatory cells and activation of HSCs after macrophage NLRP3 activation (Fig. 1). This role of TNF could be due in part to enhanced IL‐1β signaling, because the NLRP3‐TNF double knockout mice had reduced IL‐1β levels, indicating a positive feedback of TNF on IL‐1β. TNF can induce hepatocyte apoptosis and necrosis, which serve as a stimulus for HSC activation, and liver fibrosis often correlates with the extent of hepatocyte death. Alternatively, DAMPs can directly cause HSC activation. In experimental liver fibrosis, hepatic expression of the NLRP1, NLRP3, and absent in melanoma 2 inflammasomes is increased in HSCs as well as in Kupffer cells, suggesting that HSC inflammasome activation may be functionally important.9 IL‐1β and TNF may also cause liver fibrosis by increasing HSC survival.10 Thus, TNF may promote inflammasome‐dependent liver fibrosis through multiple mechanisms.Figure 1: Effects of the NLRP3 inflammasome on liver injury and fibrosis. DAMPs produced by dying hepatocytes are sensed by PRRs on Kupffer cells leading to the assembly and activation of the NLRP3 inflammasome and increased pro–IL‐1β production. Caspase 1 is activated and cleaves pro–IL‐1β into its active secreted form. IL‐1β can directly promote hepatocyte cell death, the recruitment of monocytes and neutrophils, and HSC survival and activation. IL‐1β also induces TNF synthesis and secretion from Kupffer cells, which further increases IL‐1β production. TNF functions as the principal effector cytokine in mediating the effects of inflammasome‐dependent IL‐1β on liver injury, inflammation, and fibrosis.The findings of this study are important in light of the failure of anti‐TNF therapy in human trials of alcoholic hepatitis due largely to increased mortality from infections. IL‐1β lacks the antimicrobial functions of TNF, suggesting that IL‐1β might be a safer biological target than TNF in liver disease patients who are susceptible to infections. If TNF production is secondary to IL‐1β, then there is a further rationale for the current efforts to test anti–IL‐1β therapy in human liver disease. However, prior findings that an anti‐IL‐1 agent in Nlrp3A350V mice led to a reduction in inflammation but not fibrosis,6 suggest that mono‐therapy directed at IL‐1β may not be effective. Therapeutics to specifically block TNF effects on injury and inflammation may therefore be unavoidable for anticytokine therapy to succeed in preventing fibrosis in some liver diseases. Wree et al.6 have shown previously that global NLRP3 activation produces a more severe phenotype than the myeloid‐specific activation of NLRP3, suggesting the importance of this inflammasome in cells other than macrophages. Further studies are needed to examine the role of inflammasomes and their downstream cytokines individually in hepatocytes, Kupffer cells, and HSCs to further clarify the interrelationship of these cells to the development of hepatic sterile inflammation and fibrosis. Specific cell‐targeted therapies can then be designed to dampen inflammation and fibrosis. These studies are important in light of the current lack of any hepatic anticytokine therapy despite the long‐established role of proinflammatory cytokines in liver disease. Author names in bold designate shared co‐first authorship.

To determine whether intracellular signaling events involved in apoptosis may also mediate necrosis, the role of the transcription factor AP-1 was investigated in a hepatoma cell model of cellular necrosis induced by oxidant stress. Treatment of the human hepatoma cell line HuH-7 with H 2 O 2 caused dose-dependent necrosis as determined by light microscopy, fluorescent staining, and an absence of DNA fragmentation. H 2 O 2 treatment led to increases in c- fosand c- jun mRNA levels, Jun nuclear kinase activity, and AP-1 DNA binding. AP-1 transcriptional activity measured with an AP-1-driven luciferase reporter gene was also increased. To determine whether this AP-1 activation contributed to H 2 O 2 -induced cell necrosis, HuH-7 cells were stably transfected with an antisense c- jun expression vector. Cells expressing antisense c- jun had decreased levels of AP-1 activation and significantly increased survival after H 2 O 2 exposure. These data indicate that AP-1 activation occurs during oxidant-induced cell necrosis and contributes to cell death. Necrosis is therefore not always a passive process but may involve the activation of intracellular signaling pathways similar to those that mediate apoptosis.

HepatologyVolume 52, Issue 6 p. 2118-2126 Liver Injury/RegenerationFree Access Nuclear factor κB up-regulation of CCAAT/enhancer-binding protein β mediates hepatocyte resistance to tumor necrosis factor α toxicity† Yongjun Wang, Yongjun Wang Department of Medicine and Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NYSearch for more papers by this authorRajat Singh, Rajat Singh Department of Medicine and Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NYSearch for more papers by this authorYouqing Xiang, Youqing Xiang Department of Medicine and Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NYSearch for more papers by this authorLinda E. Greenbaum, Linda E. Greenbaum Departments of Cancer Biology and Medicine, Jefferson Medical College, Philadelphia, PASearch for more papers by this authorMark J. Czaja, Corresponding Author Mark J. Czaja mark.czaja@einstein.yu.edu Department of Medicine and Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY fax: 718-430-8975Marion Bessin Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461===Search for more papers by this author Yongjun Wang, Yongjun Wang Department of Medicine and Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NYSearch for more papers by this authorRajat Singh, Rajat Singh Department of Medicine and Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NYSearch for more papers by this authorYouqing Xiang, Youqing Xiang Department of Medicine and Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NYSearch for more papers by this authorLinda E. Greenbaum, Linda E. Greenbaum Departments of Cancer Biology and Medicine, Jefferson Medical College, Philadelphia, PASearch for more papers by this authorMark J. Czaja, Corresponding Author Mark J. Czaja mark.czaja@einstein.yu.edu Department of Medicine and Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY fax: 718-430-8975Marion Bessin Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461===Search for more papers by this author First published: 20 August 2010 https://doi.org/10.1002/hep.23929Citations: 13 † Potential conflict of interest: Nothing to report. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract The sensitization of hepatocytes to cell death from tumor necrosis factor α (TNFα) underlies many forms of hepatic injury, including that from toxins. Critical for hepatocyte resistance to TNFα toxicity is activation of nuclear factor κB (NF-κB) signaling, which prevents TNFα-induced death by the up-regulation of protective proteins. To further define the mechanisms of hepatocyte sensitization to TNFα killing, immunoblot analysis comparing livers from mice treated with lipopolysaccharide (LPS) alone or LPS together with the hepatotoxin galactosamine (GalN) was performed to identify TNFα-induced protective proteins blocked by GalN. Levels of CCAAT/enhancer-binding protein β (C/EBPβ) were increased after LPS treatment but not GalN/LPS treatment. In a nontransformed rat hepatocyte cell line, TNFα-induced increases in C/EBPβ protein levels were dependent on NF-κB–mediated inhibition of proteasomal degradation. Pharmacological inhibition of c-Jun N-terminal kinase (JNK) did not affect C/EBPβ degradation, indicating that the process was JNK-independent. C/EBPβ functioned to prevent cell death as adenoviral C/EBPβ overexpression blocked TNFα-induced apoptosis in cells sensitized to TNFα toxicity by NF-κB inhibition. C/EBPβ inhibited TNFα-induced caspase 8 activation and downstream mitochondrial cytochrome c release and caspase 3 and caspase 7 activation. Studies in primary hepatocytes from c/ebpβ−/− mice confirmed that loss of C/EBPβ increased death from TNFα. c/ebpβ−/− mice were also sensitized to liver injury from a nontoxic dose of LPS or TNFα. The absence of jnk2 failed to reverse the GalN-induced block in C/EBPβ induction by LPS, again demonstrating that C/EBPβ degradation was JNK-independent. Conclusion: C/EBPβ is up-regulated by TNFα and mediates hepatocyte resistance to TNFα toxicity by inhibiting caspase-dependent apoptosis. In the absence of NF-κB signaling, proteasomal degradation of C/EBPβ is increased by a JNK-independent mechanism and promotes death from TNFα. (HEPATOLOGY 2010;.) Tumor necrosis factor α (TNFα) is a critical mediator of multiple forms of liver injury, including that resulting from toxins,1, 2 ischemia/reperfusion,3, 4 viral hepatitis,5, 6 and cholestasis.7, 8 Central to TNFα's role as a hepatotoxic factor is its ability under certain pathophysiological conditions to induce apoptotic cell death. TNFα binding to the type 1 TNFα receptor recruits a series of intracellular proteins that ultimately activate initiator caspase 8.9 Caspase 8 activation triggers the sequential release of lysosomal cathepsin B,10 cleavage of the proapoptotic Bcl-2 family member Bid,11 initiation of the mitochondrial death pathway with release of cytochrome c, and activation of downstream effector caspases that induce apoptosis.12 Hepatocytes are normally resistant to TNFα cytotoxicity through TNFα-induced activation of the transcription factor nuclear factor κB (NF-κB).13, 14 Loss of NF-κB activity in hepatocytes in culture,14 or in vivo,15 sensitizes the cells to death through this apoptotic pathway.10, 13, 14 TNFα-dependent liver injury from hepatotoxins such as carbon tetrachloride, galactosamine, and alcohol may result from a block in protective NF-κB signaling. A mechanism of NF-κB–mediated resistance to TNFα toxicity is down-regulation of the mitogen-activated protein kinase c-Jun N-terminal kinase (JNK).16-18 In the absence of NF-κB signaling, TNFα-induced JNK activation is converted from a transient to a prolonged response that triggers cell death. Although the central function of JNK is to increase gene transcription through the phosphorylation and activation of the activator protein-1 component c-Jun, JNK regulates TNFα toxicity through nontranscriptional effects on protein degradation. The induction of cell death by JNK overactivation occurs in part from alterations in the half-lives of two proteins that mediate hepatocyte TNFα resistance: cFLIP and Mcl-1.19, 20 Loss of the transcription factor NF-κB therefore sensitizes hepatocytes to TNFα cytotoxicity in part through JNK-dependent effects on protein degradation. CCAAT/enhancer-binding protein β (C/EBPβ) is a member of a family of transcription factors that regulate several critical cellular functions, including apoptosis.21 C/EBPβ has three protein forms that in rodents are termed LAP1 (38 kDa), LAP2 (34 kDa), and LIP (20 kDa).21 LAP1 and LAP2 act as transcriptional activators and LIP as a dominant negative. C/EBPβ promotes cell survival in several nonhepatic cell types after DNA damage.22-24 In addition, a novel nontranscriptional function of C/EBPβ as a caspase inhibitor has been described in hepatic stellate cells.22 Whether C/EBPβ performs this function in other cell types is unknown. In hepatocytes, C/EBPβ promotes apoptosis from the death receptor Fas.25 C/EBPβ regulation by TNFα has been shown to occur in hepatocytes at the level of subcellular localization as TNFα induces C/EBPβ translocation to the nucleus, where it regulates hepatocyte differentiation and proliferation through effects on gene transcription.26-28 It is unknown whether C/EBPβ functions in hepatocyte death from TNFα. Galactosamine/lipopolysaccharide (GalN/LPS)-induced liver injury is a well-established experimental model of TNFα-dependent hepatic injury.29, 30 GalN is a hepatocyte-specific transcriptional inhibitor that at subtoxic doses sensitizes hepatocytes to death from LPS-induced TNFα. A feature of this model is that protein changes induced by LPS alone can be contrasted with those from combined GalN/LPS treatment to identify protective proteins whose induction by TNFα is blocked by GalN. Using this approach, we identified C/EBPβ as a factor whose induction by LPS was blocked by the hepatotoxin GalN. In vitro and in vivo studies demonstrated that C/EBPβ blocks TNFα-induced apoptosis in hepatocytes at the level of caspase 8 activation. These findings identify C/EBPβ as an NF-κB–regulated antiapoptotic factor that mediates hepatocyte resistance to TNFα toxicity. Abbreviations C/EBPβ, CCAAT/enhancer-binding protein β; GalN, galactosamine; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; mRNA, messenger RNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-κB, nuclear factor κB; TNFα, tumor necrosis factor α; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling. Materials and Methods Materials and Methods are available in the Supporting Information online. Results GalN Blocks LPS-Induced Increase in C/EBPβ in Mouse Liver. As a strategy to identify novel factors mediating hepatocyte resistance to hepatotoxin-induced, TNFα-dependent liver injury, immunoblot analysis was performed on hepatic proteins isolated from LPS- and GalN/LPS-treated mice. An increase in hepatic levels of a specific protein by LPS alone that is blocked by GalN cotreatment identifies that protein as a potential TNFα-inducible protective factor. C/EBPβ levels were examined because of the known function of this protein as a caspase inhibitor. LPS administration markedly increased both the LAP1 and LAP2 forms of C/EBPβ protein in mouse liver within 4 hours (Fig. 1A). However, cotreatment with GalN blocked this LPS-induced increase in C/EBPβ (Fig. 1A). In contrast, levels of C/EBPα were unaffected by LPS or GalN treatment and served as a loading control (Fig. 1A). These findings suggested that a hepatotoxin-mediated inhibition of C/EBPβ induction may sensitize hepatocytes to death from TNFα. Figure 1Open in figure viewerPowerPoint LPS induction of C/EBPβ protein is blocked by GalN cotreatment. (A) Total hepatic protein was isolated from the livers of an untreated mouse and mice treated for the hours indicated with LPS alone, or LPS together with GalN, and immunoblotted with antibodies against C/EBPβ or C/EBPα. Results are representative of three independent experiments. (B) Fold increase in hepatic C/EBPβ mRNA levels as determined by real-time polymerase chain reaction at the times indicated after LPS or GalN/LPS treatment. C/EBPβ mRNA levels were normalized to those for TATA-box binding protein (n = 3). GalN inhibits hepatocyte transcription, suggesting that this hepatotoxin may block C/EBPβ induction by this mechanism. To determine whether GalN blocked C/EBPβ up-regulation at the messenger RNA (mRNA) level, hepatic levels of C/EBPβ mRNA after LPS or GalN/LPS treatment were determined by real-time polymerase chain reaction. Levels of C/EBPβ mRNA increased three- to eight-fold at 1-6 hours after treatment with LPS alone (Fig. 1B). GalN did block the increase in C/EBPβ mRNA at 2 hours, but at the other time points C/EBPβ mRNA levels increased two- to four-fold despite GalN cotreatment (Fig. 1B). Thus, GalN reduced but did not block completely the LPS-induced increase in C/EBPβ mRNA. These findings are in contrast to the complete absence of any increase in C/EBPβ protein in GalN/LPS-treated mice, suggesting that the lack of C/EBPβ protein induction in these mice was mediated at least in part through changes in protein translation or degradation. TNFα and LPS Induce an NF-κB–Dependent Increase in C/EBPβ in RALA Hepatocytes. To determine whether TNFα and LPS regulate C/EBPβ specifically in hepatocytes, the effects of TNFα and LPS on C/EBPβ levels were examined in RALA hepatocytes cultured under nontransformed conditions. TNFα treatment increased cellular C/EBPβ protein levels within 2 hours (Fig. 2A). The increase in C/EBPβ was further augmented by cotreatment with LPS (Fig. 2A), indicating that TNFα and LPS both up-regulate C/EBPβ protein content in hepatocytes. The normal up-regulation of C/EBPβ by TNFα was dependent in part on protein synthesis as the induction was partially blocked by the protein synthesis inhibitor cycloheximide (Fig. 2B). Figure 2Open in figure viewerPowerPoint TNFα induction of C/EBPβ is mediated by NF-κB–dependent inhibition of proteasomal degradation. (A) RALA hepatocytes were untreated or treated with TNFα alone or together with LPS for the indicated number of hours. Total protein was isolated and aliquots immunoblotted with antibodies for C/EBPβ or β-actin. (B) RALA hepatocytes received no pretreatment or were pretreated with cycloheximide (CHX) for 1 hour, then treated with TNFα for the number of hours shown. Total protein was immunoblotted for C/EBPβ and β-actin. (C) Following infection with Ad5LacZ or Ad5IκB, cells were left untreated or treated with TNFα in the absence or presence of 1 hour of MG132 (MG) pretreatment. Total protein was isolated 4 hours after TNFα administration and immunoblotted with C/EBPβ or β-actin antibodies. Levels of β-actin demonstrated equal loading among protein samples. Immunoblots are representative of four independent experiments. Numerical data under the immunoblots represents the relative signal intensity by densitometry scanning of four experiments. TNFα-induced activation of the transcription factor NF-κB is a critical protective response for hepatocyte resistance to TNFα toxicity.14 To investigate the role of NF-κB in TNFα up-regulation of C/EBPβ, NF-κB activation was inhibited with the adenovirus Ad5IκB which expresses a mutant IκB that irreversibly binds and inactivates NF-κB.15 The TNFα-mediated increase in C/EBPβ was abrogated in Ad5IκB-infected cells, but not in control Ad5LacZ-infected hepatocytes (Fig. 2C), indicating that NF-κB activation mediated the TNFα-induced increase in C/EBPβ. The total block in induction of C/EBPβ protein in GalN/LPS-treated mice, despite an increase in C/EBPβ mRNA, suggested that NF-κB signaling regulates C/EBPβ in vivo at the level of protein degradation. To test this possibility, cells were treated with TNFα in the absence or presence of the proteasomal inhibitor MG132.31 MG132 treatment alone in Ad5LacZ- or Ad5IκB-infected cells increased cellular C/EBPβ protein content to a level equivalent to that in TNFα-treated, Ad5LacZ-infected cells (Fig. 2C), demonstrating constitutive regulation of C/EBPβ levels by proteasomal degradation. Cotreatment with MG132 had no effect on C/EBPβ levels in TNFα-treated, Ad5LacZ-infected cells (Fig. 2C), indicating that C/EBPβ was not regulated by proteasomal degradation in these cells. In contrast, MG132 had a marked effect on C/EBPβ levels in cells lacking NF-κB. Inhibition of proteasomal function in Ad5IκB-infected cells increased TNFα-induced C/EBPβ content to levels equivalent to those in TNFα-treated, Ad5LacZ-infected cells (Fig. 2C). Thus, despite the fact that the TNFα-induced increase in C/EBPβ depended in part on protein synthesis (Fig. 2B), the up-regulation of C/EBPβ levels by TNFα treatment was largely dependent on an NF-κB–dependent inhibition of C/EBPβ protein degradation. As previous studies have demonstrated that JNK overactivation resulting from a block in NF-κB signaling alters protein degradation,19, 20 the possible involvement of JNK in the increased degradation of C/EBPβ with NF-κB inhibition was examined. Pretreatment of cells with the pharmacological JNK inhibitor SP60012532 failed to reverse the block in C/EBPβ up-regulation that occurred in the absence of NF-κB signaling (data not shown). Taken together, these findings demonstrate that the up-regulation of hepatocyte levels of C/EBPβ in response to TNFα is dependent on NF-κB-mediated inhibition of proteasomal degradation by a JNK-independent mechanism. C/EBPβ Regulates Hepatocyte Death from TNFα. Studies in nonhepatic cells have demonstrated an antiapoptotic function for C/EBPβ.22-24 The ability of proteasomal inhibition to increase levels of C/EBPβ led us to investigate whether MG132 was able to block hepatocyte death from TNFα. Despite mild toxicity from MG132 treatment alone, proteasomal inhibition significantly decreased the amount of cell death in Ad5IκB-infected cells treated with TNFα (Fig. 3A). To determine whether C/EBPβ functioned to prevent RALA hepatocyte death from TNFα, the effect of C/EBPβ overexpression on TNFα-induced apoptosis in RALA hepatocytes with an inhibition of NF-κB activation was assessed. Cells infected with the C/EBPβ-expressing adenovirus WT-C/EBPβ alone or coinfected with WT-C/EBPβ and either Ad5LacZ or Ad5IκB expressed increased levels of C/EBPβ compared with cells infected with Ad5LacZ alone (Fig. 3B). Cells were coinfected with Ad5IκB and either Ad5LacZ as a control or WT-C/EBPβ and treated with TNFα. When compared with Ad5IκB/Ad5LacZ-coinfected cells, the amount of cell death after TNFα treatment was significantly decreased in Ad5IκB/WT-C/EBPβ–coinfected cells at 6 and 12 hours by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Fig. 3C). The ability of C/EBPβ expression to block cell death from TNFα was confirmed by fluorescence microscopic studies of cells costained with acridine orange/ethidium bromide to quantify the numbers of apoptotic and necrotic cells. As previously established, death from NF-κB inactivation and TNFα was predominantly apoptotic, and no significant increase occurred in the numbers of necrotic cells (data not shown). The marked increase in apoptotic cells with TNFα administration was significantly reduced by adenoviral expression of C/EBPβ (Fig. 3D). Thus, the NF-κB–dependent increase in C/EBPβ in TNFα-treated RALA hepatocytes is a mechanism of cellular resistance to TNFα-induced apoptosis. Figure 3Open in figure viewerPowerPoint C/EBPβ blocks RALA hepatocyte death from TNFα. (A) Percentage cell death at 6 hours by MTT assay in Ad5IκB-infected cells treated with TNFα or MG132 (MG) alone or in combination. *P < 0.00001 versus cells treated with TNFα alone (n = 8). (B) Immunoblots of total protein from RALA hepatocytes infected with WT-C/EBPβ, Ad5LacZ, and Ad5IκB as indicated and probed for C/EBPβ or β-actin. (C) Percentage cell death by MTT assay at 6 and 12 hours after TNFα treatment in cells coinfected with Ad5IκB and either Ad5LacZ or WT-C/EBPβ *P < 0.01 versus TNFα-treated Ad5IκB/Ad5LacZ-coinfected cells (n = 3-4). (D) Percentage of apoptotic cells in Ad5IκB/Ad5LacZ (LacZ) and Ad5IκB/WT-C/EBPβ (C/EBP) coinfected cells untreated or TNFα-treated for 6 or 12 hours. *P < 0.01 versus TNFα-treated Ad5IκB/Ad5LacZ-infected cells (n = 3-4). C/EBPβ Inhibits TNFα-Induced Caspase Activation. The sensitization of hepatocytes to TNFα toxicity by NF-κB inhibition occurs through caspase-dependent apoptosis.17, 33 The ability of C/EBPβ to function as a caspase inhibitor suggested that the mechanism of C/EBPβ's inhibition of TNFα-induced apoptosis may be through blocking caspase activation. Adenoviral expression of C/EBPβ significantly decreased levels of activity of the initiator caspase 8 in both untreated and TNFα-treated cells in which NF-κB was inhibited by Ad5IκB (Fig. 4A). Inhibition of caspase 8 by C/EBPβ prevented TNFα-induced activation of the mitochondrial death pathway as WT-C/EBPβ decreased the amount of truncated Bid that translocated to the mitochondria and blocked the cytochrome c release from mitochondria into cytoplasm that occurred in Ad5IκB/Ad5LacZ-coinfected cells (Fig. 4B). In contrast, levels of cytochrome oxidase, a mitochondrial protein not released during apoptosis, were equivalent in Ad5LacZ- and WT-C/EBPβ–infected cells after TNFα treatment and indicated equal protein loading (Fig. 4B). As a result of the inhibition of cytochrome c release, downstream effector caspase 3 and caspase 7 activation was blocked in cells overexpressing C/EBPβ as detected by decreases in the active, cleaved caspase forms on immunoblots (Fig. 4C). The block in caspase 3 activation was confirmed through measurement of caspase 3 activity, which was significantly decreased by C/EBPβ overexpression in both untreated and TNFα-treated cells (Fig. 4D). These results indicate that C/EBPβ blocks TNFα-induced apoptosis by the inhibition of caspase activation. Figure 4Open in figure viewerPowerPoint C/EBPβ blocks TNFα-induced caspase activation. (A) Relative levels of caspase 8 activity in untreated control (Con) and 4-hour TNFα-treated cells. Cells were coinfected with Ad5IκB and either Ad5LacZ or WT-C/EBPβ as indicated. *P < 0.0002 versus Ad5IκB/Ad5LacZ-coinfected cells with the same treatment (n = 7). (B) Immunoblots of mitochondrial (Mit) or cytosolic (Cyt) proteins from coinfected cells untreated or treated with TNFα and probed with antibodies for truncated Bid (tBid), cytochrome c (Cyt c), cytochrome oxidase (Cyt ox) or β-actin. (C) Immunoblots of total protein from cells coinfected with Ad5IκB and Ad5LacZ or WT-C/EBPβ as indicated. Cells were untreated or treated with TNFα for the indicated number of hours. Proteins were immunoblotted with antibodies for caspase 3 (Casp 3), caspase 7 (Casp 7) or β-actin. Arrows indicate the cleaved forms of caspase 3 and caspase 7. Immunoblots are representative of results from three independent experiments. (D) Relative levels of caspase 3 activity in untreated and 6-hour TNFα-treated cells coinfected with Ad5IκB and either Ad5LacZ or WT-C/EBPβ. *P < 0.0002 versus Ad5IκB/Ad5LacZ-coinfected cells with the same treatment (n = 4). Absence of C/EBPβ Sensitizes Primary Hepatocytes to Death from TNFα. Our findings suggested that the loss of C/EBPβ would result in an increase in hepatocyte sensitivity to TNFα. To investigate this possibility, primary hepatocytes were isolated from littermate control wild-type mice and c/ebpβ knockout mice, placed in culture, and examined for their sensitivity to TNFα-induced death. TNFα treatment alone was not sufficient to induce death in either wild-type or C/EBPβ null hepatocytes (data not shown). When the hepatocytes were sensitized to TNFα by infection with Ad5IκB, however, cell death at 10 and 24 hours in the knockout cells was two-fold greater than in wild-type cells (Fig. 5A). Knockout cells had greater levels of the cleaved active forms of caspase 3 and caspase 7 that resulted in increased caspase activity as indicated by cleavage of the caspase substrate poly(ADP-ribose) polymerase (Fig. 5B). We have therefore been able to demonstrate with both overexpression and loss-of-function approaches that C/EBPβ mediates hepatocyte resistance to TNFα cytotoxicity. Figure 5Open in figure viewerPowerPoint C/EBPβ knockout hepatocytes undergo increased cell death from TNFα. (A) Percentage cell death by MTT assay at the indicated times after TNFα treatment in Ad5IκB-infected wild-type and c/ebpβ knockout hepatocytes. *P < 0.00005 versus untreated cells (n = 12). (B) Total protein from untreated or TNFα-treated Ad5IκB-infected primary mouse hepatocytes from wild-type (WT) and c/ebpβ knockout (KO) mice were immunoblotted for C/EBPβ, caspase 3 (Casp 3), caspase 7 (Casp 7), poly(ADP-ribose) polymerase (PARP), or β-actin. The cleaved forms of caspase 3 and caspase 7 and poly(ADP-ribose) polymerase are indicated by arrows. Data are representative of three independent experiments. Sensitization to LPS- and TNFα-Induced Liver Injury Occurs in the Absence of C/EBPβ. The in vivo function of C/EBPβ in LPS-induced liver injury was determined. The ability of C/EBPβ to block TNFα-dependent liver injury in vivo was examined by comparing the degree of liver injury in wild-type and c/ebpβ−/− mice after the administration of a usually nontoxic dose of LPS. Wild-type mice had normal ALT levels after treatment with low-dose LPS, but ALT levels were increased in knockout mice (Fig. 6A). Reflective of the predominantly apoptotic nature of TNFα-induced hepatocyte death, a much greater increase occurred in the numbers of terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL)-positive cells in LPS-treated c/ebpβ−/− mice compared with littermate controls (Fig. 6B). The steady-state numbers of TUNEL-positive cells in the liver were increased eight-fold at 6 hours and four-fold at 24 hours in null mice compared with control mice. To ensure that injury from LPS represented toxicity from TNFα, C/EBPβ null mice were examined for sensitivity to injury from TNFα. An injection of TNFα led to liver injury in knockout but not wild-type mice as demonstrated by increased serum ALT levels (Fig. 6C) and numbers of apoptotic cells (Fig. 6D) at 6 hours. C/EBPβ therefore mediates hepatocyte resistance to TNFα toxicity in vivo as well as in vitro. Figure 6Open in figure viewerPowerPoint C/EBPβ knockout mice are sensitized to liver injury from LPS and TNFα. (A) Serum ALT levels in control wild-type (WT) and c/ebpβ knockout (KO) mice 6 and 24 hours after LPS injection. *P < 0.03, **P < 0.001 versus wild-type mice (n = 10-12). (B) Numbers of apoptotic cells by TUNEL staining in the livers of the same animals. *P < 0.0001 versus wild-type mice (n = 5-7). (C) Serum ALT levels in wild-type mice and c/ebpβ knockout mice 6 hours after injection with TNFα. *P < 0.05 versus wild-type mice (n = 4). (D) Numbers of apoptotic cells in the livers of the same animals. *P < 0.01 versus wild-type mice (n = 4). (E) Immunoblots of total hepatic protein from untreated and GalN/LPS-treated wild-type and jnk2 null mice for C/EBPβ and β-actin. The last lane contains protein from a wild-type mouse injected with LPS alone to demonstrate normal levels of C/EBPβ induction by LPS. Blots are representative of three experiments. In the absence of NF-κB signaling, TNFα-induced JNK activation is converted from a transient to prolonged response that triggers cell death in part through altered protein degradation of antiapoptotic proteins. To examine whether the proapoptotic effects of JNK during TNFα-dependent injury in vivo are mediated via degradation of C/EBPβ, we investigated the effect of loss of jnk2 on C/EBPβ induction after GalN/LPS treatment. Our previous investigations have demonstrated that the jnk2 gene expresses the JNK isoforms that promote liver injury from GalN/LPS.34 Western blots of total liver protein from GalN/LPS-treated wild-type and jnk2 null mice for C/EBPβ revealed that the absence of jnk2 failed to reverse the GalN-induced inhibition of C/EBPβ induction by LPS (Fig. 6E). Mice null for jnk1 are not protected from GalN/LPS toxicity34 and also failed to up-regulate C/EBPβ (data not shown). Thus, consistent with the in vitro findings in cells with NF-κB inhibition, C/EBPβ degradation that occurred in vivo during GalN/LPS-induced liver injury was not mediated by JNK. Discussion Significant progress has been made in defining the mechanisms by which hepatocytes lose resistance to TNFα toxicity and undergo TNFα-induced cell death. Critical for resistance to TNFα-induced apoptosis is the ability of the hepatocyte to activate the NF-κβ signaling pathway in response to TNFα stimulation.13-15 Prominent among the forms of hepatic injury mediated by sensitization to TNFα toxicity are those induced by hepatotoxins.1, 2 Hepatotoxins invariably impair macromolecular synthesis, suggesting that they may sensitize hepatocytes to TNFα-dependent injury from a toxin-induced block in the transcriptional or translational induction of protective signals by NF-κB. The identification of the protective protein effectors of NF-κB signaling may therefore increase our understanding of the mechanisms of toxic liver injury and suggest new therapies for its prevention. These studies identify for the first time that C/EBPβ is an NF-κB-regulated mediator of hepatocellular resistance to TNFα toxicity. C/EBPβ is one member of a family of leucine-zipper transcription factors that regulate cell proliferation, differentiation, and metabolism through effects on gene expression. In addition to its role in transcription, Buck et al.22 have demonstrated a novel nontranscriptional function of C/EBPβ as a caspase inhibitor. In the present studies, C/EBPβ was up-regulated by LPS/TNFα in vitro and in vivo, which suggested that this protein may have an antiapoptotic function in TNFα-induced liver injury. Although TNFα has been shown to alter the subcellular localization of C/EBPβ,26-28 TNFα regulation of C/EBPβ protein levels has not been reported previously in hepatocytes. Consistent with a function for C/EBPβ as a protective factor against TNFα-induced cell apoptosis was that C/EBPβ up-regulation was NF-κB–dependent. Although LPS/TNFα increased C/EBPβ mRNA levels and protein synthesis, the primary mechanism by which NF-κB regulated cellular C/EBPβ content was through a decrease in proteasomal degradation of C/EBPβ. Findings from both gain-of-function studies in RALA hepatocytes and loss-of-function studies in primary mouse hepatocytes demonstrated that C/EBPβ mediates hepatocyte resistance to TNFα toxicity. The absence of C/EBPβ alone was insufficient to sensitize mouse hepatocytes to death from TNFα; however, the significance of this finding is unclear, because NF-κB activation occurs in primary hepatocytes in response to the stress of the liver perfusion and cell isolation.35 Other NF-κB–dependent protective factors may have been up-regulated by this perfusion-induced NF-κB activation that compensated for the loss of C/EBPβ. Alternatively, the null mice may have up-regulated other compensatory protective factors that negated the loss of C/EBPβ. Nonetheless, the findings identify C/EBPβ as a new antiapoptotic protein regulated by NF-κB at the level of protein degradation. Confirmatory of the in vitro hepatocyte data were findings that C/EBPβ was up-regulated and functioned in hepatotoxic liver injury in vivo. Identical to results in RALA hepatocytes, hepatic C/EBPβ protein levels were markedly increased by the TNFα inducer LPS. Consistent with the ability of GalN to block the up-regulation of NF-κB–induced protective signaling, mice cotreated with GalN and LPS failed to up-regulate C/EBPβ. C/EBPβ was protective against TNFα cytotoxicity, because null mice but not wild-type mice developed liver injury from low-dose LPS or TNFα alone. Injury in C/EBPβ null mice was far less than that elicited by the combination of GalN and LPS in wild-type mice. These results suggest that C/EBPβ functions as one of a redundant set of NF-κB–regulated antiapoptotic factors in the hepatocyte. Alternatively, as with the studies in cultured hepatocytes from these mice, the null mice may have responded to the knockout of C/EBPβ by up-regulating other antiapoptotic factors in compensation for the loss of C/EBPβ that in part masked the true importance of C/EBPβ as an antiapoptotic factor in vivo. The mechanism of the antiapoptotic effect of C/EBPβ was at least in part at the level of initiator caspase 8 activation, because C/EBPβ blocked the activation of this caspase and therefore the downstream mitochondrial death pathway and effector caspase cleavage. However, further studies must be performed to delineate the mechanism by which C/EBPβ blocks caspase 8 activation to confirm this possibility. Our finding is consistent with that of Buck et al.,22 who similarly found that C/EBPβ inhibited caspase 8 activation in hepatic stellate cells. This effect in hepatocytes appears to be specific for the TNFα death pathway. In contrast to the present finding of an antiapoptotic function for C/EBPβ in TNFα-mediated hepatocyte injury, studies in C/EBPβ null mice demonstrated that C/EBPβ promotes hepatocyte apoptosis from the Fas death receptor.25 Fas-mediated cell death is also caspase 8 mediated, yet C/EBPβ promoted this form of apoptosis. The mechanism of the differential effect of C/EBPβ on the TNFα and Fas death receptor pathways remains to be determined, but the current study suggests the interesting possibility that TNFα, through induction of C/EBPβ, may potentiate Fas toxicity. A protective mechanism of NF-κB signaling is its inhibition of proapoptotic JNK overactivation.16-18 JNK signaling alters the half-lives of proteins that mediate hepatocyte resistance to TNFα toxicity. JNK1 has been reported to promote TNFα-induced death by mediating degradation of the antiapoptotic factor cFLIP.19 Conversely, other studies have suggested an antiapoptotic effect of JNK1 through an increase in the half-life of Mcl-1.20 NF-κβ is therefore known to regulate death from TNFα through JNK-dependent effects on protein degradation. Levels of C/EBPβ were similarly regulated through NF-κβ–dependent effects on the rate of C/EBPβ protein degradation. However, this effect was JNK-independent, because it was not blocked in vitro by pharmacological JNK inhibition. The absence of jnk2 in vivo, which prevented GalN/LPS-induced liver injury,34 also failed to restore the LPS-induced increase in C/EBPβ, indicating that jnk2 potentiation of liver injury does not occur through degradation of C/EBPβ. This study is the first to demonstrate a JNK-independent effect of NF-κB on protein degradation that modulates hepatocyte resistance to death from TNFα. The new identification of C/EBPβ as an NF-κB–regulated antiapoptotic factor in the TNFα death pathway adds to the mechanistic complexity of TNFα-induced hepatocyte injury. This complexity results in part from the presence of both JNK-dependent and JNK-independent effects of NF-κB on proteasomal degradation. The existence of multiple mechanisms of resistance against the TNFα-activated apoptotic death pathway attests to the importance of hepatic resistance to TNFα toxicity in maintaining normal liver function. Acknowledgements The authors thank David Brenner for providing the Ad5LacZ and Ad5IκB adenoviruses and Xiao-Ming Yin for providing the anti-Bid antibody. Supporting Information Additional Supporting Information may be found in the online version of this article. Filename Description HEP_23929_sm_SuppInfoFigure1.tif11.2 MB Supporting Fig. S1. β-galactosidase staining of Ad5LacZ-infected cells. Representative image of β-galactosidase-stained Ad5LacZ-infected (A) or uninfected (B) RALA hepatocytes (100×). 23929Supplemental_Materials_and_Methods.doc87 KB Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. References 1 Czaja MJ, Xu J, Alt E. Prevention of carbon tetrachloride-induced rat liver injury by soluble tumor necrosis factor receptor. Gastroenterology 1995; 108: 1849- 1854. 2 Yin M, Wheeler MD, Kono H, Bradford BU, Gallucci RM, Luster MI et al. Essential role of tumor necrosis factor α in alcohol-induced liver injury in mice. Gastroenterology 1999; 117: 942- 952. 3 Teoh N, Field J, Sutton J, Farrell G. Dual role of tumor necrosis factor-α in hepatic ischemia-reperfusion injury: studies in tumor necrosis factor-α gene knockout mice. HEPATOLOGY 2004; 39: 412- 421. 4 Zhou W, Zhang Y, Hosch MS, Lang A, Zwacka RM, Engelhardt JF. Subcellular site of superoxide dismutase expression differentially controls AP-1 activity and injury in mouse liver following ischemia/reperfusion. HEPATOLOGY 2001; 33: 902- 914. 5 Su F, Schneider RJ. Hepatitis B virus HBx protein sensitizes cells to apoptotic killing by tumor necrosis factor α. Proc Natl Acad Sci U S A 1997; 94: 8744- 8749. 6 Kallinowski B, Haseroth K, Marinos G, Hanck C, Stremmel W, Theilmann L, et al. Induction of tumour necrosis factor (TNF) receptor type p55 and p75 in patients with chronic hepatitis C virus (HCV) infection. Clin Exp Immunol 1998; 111: 269- 277. 7 Lesage G, Glaser S, Ueno Y, Alvaro D, Baiocchi L, Kanno N, et al. Regression of cholangiocyte proliferation after cessation of ANIT feeding is coupled with increased apoptosis. Am J Physiol Gastrointest Liver Physiol 2001; 281: G182- G190. 8 Yerushalmi B, Dahl R, Devereaux MW, Gumpricht E, Sokol RJ. Bile acid-induced rat hepatocyte apoptosis is inhibited by antioxidants and blockers of the mitochondrial permeability transition. HEPATOLOGY 2001; 33: 616- 626. 9 Schattenberg JM, Czaja MJ. TNF and TNF receptors. In: JF Dufour, P-A Clavien, eds. Signaling Pathways in Liver Diseases. 2nd ed. Berlin, Germany: Springer- Verlag; 2010: 161- 179. 10 Guicciardi ME, Miyoshi H, Bronk SF, Gores GJ. Cathepsin B knockout mice are resistant to tumor necrosis factor-α-mediated hepatocyte apoptosis and liver injury: implications for therapeutic applications. Am J Pathol 2001; 159: 2045- 2054. 11 Yin XM. Signal transduction mediated by Bid, a pro-death Bcl-2 family proteins, connects the death receptor and mitochondria apoptosis pathways. Cell Res 2000; 10: 161- 167. 12 Zhao Y, Li S, Childs EE, Kuharsky DK, Yin XM. Activation of pro-death Bcl-2 family proteins and mitochondria apoptosis pathway in tumor necrosis factor-α-induced liver injury. J Biol Chem 2001; 276: 27432- 27440. 13 Bradham CA, Qian T, Streetz K, Trautwein C, Brenner DA, Lemasters JJ. The mitochondrial permeability transition is required for tumor necrosis factor α-mediated apoptosis and cytochrome c release. Mol Cell Biol 1998; 18: 6353- 6364. 14 Xu Y, Bialik S, Jones BE, Iimuro Y, Kitsis RN, Srinivasan A, et al. NF-κB inactivation converts a hepatocyte cell line TNF-α response from proliferation to apoptosis. Am J Physiol 1998; 275: C1058- C1066. 15 Iimuro Y, Nishiura T, Hellerbrand C, Behrns KE, Schoonhoven R, Grisham JW, et al. NFκB prevents apoptosis and liver dysfunction during liver regeneration. J Clin Invest 1998; 101: 802- 811. 16 De Smaele E, Zazzeroni F, Papa S, Nguyen DU, Jin R, Jones J, et al. Induction of gadd45β by NF-κB downregulates pro-apoptotic JNK signalling. Nature 2001; 414: 308- 313. 17 Liu H, Lo CR, Czaja MJ. NF-κB inhibition sensitizes hepatocytes to TNF-induced apoptosis through a sustained activation of JNK and c-Jun. HEPATOLOGY 2002; 35: 772- 778. 18 Tang G, Minemoto Y, Dibling B, Purcell NH, Li Z, Karin M, et al. Inhibition of JNK activation through NF-κB target genes. Nature 2001; 414: 313- 317. 19 Chang L, Kamata H, Solinas G, Luo JL, Maeda S, Venuprasad K, et al. The E3 ubiquitin ligase itch couples JNK activation to TNFα-induced cell death by inducing c-FLIPL turnover. Cell 2006; 124: 601- 613. 20 Kodama Y, Taura K, Miura K, Schnabl B, Osawa Y, Brenner DA. Antiapoptotic effect of c-Jun N-terminal kinase-1 through Mcl-1 stabilization in TNF-induced hepatocyte apoptosis. Gastroenterology 2009; 136: 1423- 1434. 21 Ramji DP, Foka P. CCAAT/enhancer-binding proteins: structure, function and regulation. Biochem J 2002; 365: 561- 575. 22 Buck M, Poli V, Hunter T, Chojkier M. C/EBPβ phosphorylation by RSK creates a functional XEXD caspase inhibitory box critical for cell survival. Mol Cell 2001; 8: 807- 816. 23 Ewing SJ, Zhu S, Zhu F, House JS, Smart RC. C/EBPβ represses p53 to promote cell survival downstream of DNA damage independent of oncogenic Ras and p19(Arf). Cell Death Differ 2008; 15: 1734- 1744. 24 Yoon K, Zhu S, Ewing SJ, Smart RC. Decreased survival of C/EBP β-deficient keratinocytes is due to aberrant regulation of p53 levels and function. Oncogene 2007; 26: 360- 367. 25 Mukherjee D, Kaestner KH, Kovalovich KK, Greenbaum LE. Fas-induced apoptosis in mouse hepatocytes is dependent on C/EBPβ. HEPATOLOGY 2001; 33: 1166- 1172. 26 Diehl AM, Yang SQ, Yin M, Lin HZ, Nelson S, Bagby G. Tumor necrosis factor-α modulates CCAAT/enhancer binding proteins-DNA binding activities and promotes hepatocyte-specific gene expression during liver regeneration. HEPATOLOGY 1995; 22: 252- 261. 27 Trautwein C, Rakemann T, Malek NP, Plumpe J, Tiegs G, Manns MP. Concanavalin A-induced liver injury triggers hepatocyte proliferation. J Clin Invest 1998; 101: 1960- 1969. 28 Yin M, Yang SQ, Lin HZ, Lane MD, Chatterjee S, Diehl AM. Tumor necrosis factor α promotes nuclear localization of cytokine-inducible CCAAT/enhancer binding protein isoforms in hepatocytes. J Biol Chem 1996; 271: 17974- 17978. 29 Leist M, Gantner F, Bohlinger I, Tiegs G, Germann PG, Wendel A. Tumor necrosis factor-induced hepatocyte apoptosis precedes liver failure in experimental murine shock models. Am J Pathol 1995; 146: 1220- 1234. 30 Nowak M, Gaines GC, Rosenberg J, Minter R, Bahjat FR, Rectenwald J, et al. LPS-induced liver injury in D-galactosamine-sensitized mice requires secreted TNF-α and the TNF-p55 receptor. Am J Physiol Regul Integr Comp Physiol 2000; 278: R1202- R1209. 31 Rock KL, Gramm C, Rothstein L, Clark K, Stein R, Dick L, et al. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 1994; 78: 761- 771. 32 Bennett BL, Sasaki DT, Murray BW, O'Leary EC, Sakata ST, Xu W, et al. SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci U S A 2001; 98: 13681- 13686. 33 Schwabe RF, Uchinami H, Qian T, Bennett BL, Lemasters JJ, Brenner DA. Differential requirement for c-Jun NH2-terminal kinase in TNFα- and Fas-mediated apoptosis in hepatocytes. FASEB J 2004; 18: 720- 722. 34 Wang Y, Singh R, Lefkowitch JH, Rigoli RM, Czaja MJ. TNF-induced toxic liver injury results from JNK2-dependent activation of caspase-8 and the mitochondrial death pathway. J Biol Chem 2006; 281: 15258- 15267. 35 Wang H, Gao X, Fukumoto S, Tademoto S, Sato K, Hirai K. Differential expression and regulation of chemokines JE, KC, and IP-10 gene in primary cultured murine hepatocytes. J Cell Physiol 1999; 181: 361- 370. Citing Literature Volume52, Issue6December 2010Pages 2118-2126 FiguresReferencesRelatedInformation

Toxin-induced liver injury was formerly considered a passive biochemical event, but recent evidence has demonstrated that signal transduction pathways actively modulate the hepatocyte’s response to this form of injury. Investigations have examined the effects of a variety of toxins on the activation of receptor-coupled signal transduction, mitogen-activated protein kinases, and Fas signaling, as well as the generation of second messengers such as ceramide and nitric oxide. Many of these pathways culminate in the activation of transcription factors such as activator protein-1, c-Myc, or nuclear factor-κB. This Themes article discusses the effects of toxic injury on these signaling pathways and their known functions in regulating hepatocyte death and proliferation following injury.

Abstract The molecular basis for increased metallothionein concentrations in copperresistant hepatoma cells was examined. The copper‐resistant cell line HAC 600 , which is maintained in 600 μm copper, had increased steady‐state mRNA levels for both the metallothionein‐1 (MT‐1) and the metallothionein‐2 (MT‐2) genes. Levels of mRNA were increased 11‐fold for MT‐1 and 15‐fold for MT‐2, with no significant change in α‐tubulin mRNA content. HAC 600 NM cells, which are copper‐resistant cells kept in a normal copper concentration for over 1 year, also had eight‐ and tenfold increases in MT‐1 and MT‐2 mRNA levels. Nuclear run‐on assays showed that MT‐1 and MT‐2 gene transcription was increased nine‐ and eightfold in HAC 600 cells and seven‐ and tenfold in HAC 600 NM cells, respectively. Southern blot analysis showed amplification of both metallothionein genes in HAC 600 and HAC 600 NM cells. Thus the molecular basis of increased metallothionein in these hepatoma cells involved a stable gene amplification of both MT genes. The greater increase in metallothionein mRNA levels in HAC 600 cells relative to the changes in transcription suggests that posttranscriptional mechanisms of gene regulation may also be acting in these cells.

The relationship between the degradative process of autophagy and cellular death pathways remains unclear. Macroautophagy may potentially function to prevent or promote cell death, and both effects have been reported in studies of cells with a block in macroautophagy. To better delineate the function of macroautophagy in cell death, we contrasted the responses to death stimuli in wild-type and atg5-/- murine embryonic fibroblasts. We have reported that a knockout of the critical macroautophagy gene ATG5 sensitizes cells to death receptor ligand-induced death from Fas and tumor necrosis factor-α. Death occurs by caspase-dependent apoptosis resulting from activation of the mitochondrial death pathway. In contrast, atg5-/- cells are more resistant to death induced by oxidative stress from menadione or UV light. This resistance was associated with an up-regulation of chaperone-mediated autophagy. Inhibition of this form of autophagy sensitizes cells to death from menadione, suggesting that the compensatory up-regulation of chaperone-mediated autophagy, and not the loss of macroautophagy, prevents death from menadione. These findings demonstrate that the effects of a loss of macroautophagy on the cellular death response differ depending on the mechanism of cellular injury and the compensatory changes in other forms of autophagy.Addendum to: Wang Y, Singh R, Massey AC, Kane SS, Kaushik S, Grant T, Xiang Y, Cuervo AM, Czaja MJ. Loss of macroautophagy promotes or prevents fibroblast apoptosis depending on the death stimulus. J Biol Chem 2008; 283:4766-77.

Autophagy is a critical survival mechanism that underlies the cellular response to a variety of death stimuli including that of oxidative stress. Known functions of autophagy that may promote cell survival from oxidants include the supply of substrates to maintain energy homeostasis during injury, the removal of damaged organelles that may otherwise trigger apoptosis and the degradation of oxidized proteins that may compromise cellular function. However, whether autophagy prevents cell death from oxidants by any of these mechanisms remains unclear and other novel unidentified functions of autophagy may mediate cell survival. Our recent work examined the function of autophagy in the resistance of hepatocytes to death from oxidative stress. The function of autophagy was investigated in the hepatocyte model of oxidant stress induced by the superoxide generator menadione. Inhibition of macroautophagy in hepatocytes by a lentiviral genetic knockdown of ATG5 sensitized these cells to death from a normally nontoxic concentration of menadione. Death occurred through a sequence of events that included ATP depletion, mitochondrial death pathway activation leading to cytochrome c release, effector caspase 3 and 7 activation and resultant apoptosis. These findings suggest that macroautophagy has a protective mitochondrial effect either through the removal of damaged mitochondria by mitophagy or by the generation of substrates essential for the maintenance of ATP levels. However, the menadione-induced changes in mitochondria occur downstream of activation of the mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) and its effector substrate c-Jun, a signaling pathway whose overactivation we had previously demonstrated mediates hepatocyte death from menadione. Menadione-induced JNK/c-Jun activation is converted from a transient to a prolonged activation in the absence of macroautophagy. JNK/c-Jun signaling is an upstream initiator of cell death as JNK/c-Jun inhibition blocks mitochondrial ATP depletion, subsequent downstream events and cell death. The mechanism of JNK overactivation in the absence of macroautophagy is unclear and potentially involves known functions of macroautophagy such as the removal of limited numbers of damaged mitochondria that otherwise would trigger JNK activation. Alternatively, the loss of macroautophagy may have modulated MAPK signaling more directly by altering levels of upstream activating kinases, scaffolding proteins or deactivating phosphatases that determine levels of JNK activation. The effects were not specific for JNK, as activation of the protective MAPK extracellular signal-regulated kinase (ERK) 1/2 is also increased by menadione treatment in the absence of macroautophagy. Although further studies must delineate the molecular mechanism, the findings do demonstrate for the first time that macroautophagy functions to downregulate a pro-apoptotic MAPK signaling pathway. Our investigations examined the function of chaperone-mediated autophagy (CMA) as well as macroautophagy in hepatocyte resistance to oxidant stress. Inhibition of CMA by a genetic knockdown of LAMP-2A also sensitizes cells to death from menadione. Although a simultaneous genetic knockdown of both forms of autophagy could not be established due to lethality, the combination of a pharmacological inhibitor of macroautophagy in cells with a genetic knockdown of CMA leads to a greater amount of menadione-induced cell death than does the inhibition of either form of autophagy alone. The two types of autophagy mediate hepatocyte resistance to oxidant stress by distinct mechanisms. In contrast to findings in cells lacking macroautophagy, inhibition of CMA sensitizes cells to death from menadione without inducing JNK/c-Jun hyperactivation or cellular ATP depletion. CMA limits the chronic accumulation of oxidized proteins in the liver during aging. CMA may promote hepatocyte survival from menadione by removing oxidized proteins that initiate cell death pathways directly or indirectly through the promotion of cellular dysfunction. Aggregated proteins that accumulate in neurodegenerative disorders associated with oxidant stress are likely CMA targets in the brain, but specific protein targets in acute and more generalized oxidant injuries in the liver remain to be delineated. The synergistic increase in cell death from a loss of both macroautophagy and CMA, and their differential effects on JNK activity and cellular ATP levels, demonstrate distinct, non-overlapping functions for the two forms of autophagy in hepatocellular resistance to oxidative stress that need further elucidation. The findings highlight important differences in autophagic function between hepatocytes and fibroblasts. Our prior investigations in ATG5 knockout murine embryonic fibroblasts (MEFs) revealed that macroautophagy and CMA have redundant functions in these cells during oxidant stress from menadione. MEFs lacking Atg5 and macroautophagy have increased resistance to menadione toxicity because the upregulation of CMA in compensation for the loss of macroautophagy protects the cells from oxidative stress. Thus, CMA is able to duplicate the functions of macroautophagy in MEFs and mediate survival from a menadione challenge. In contrast, although similar crosstalk occurs in hepatocytes that results in increased CMA in ATG5 knockdown cells, hepatocytes are sensitized to death from menadione. These findings indicate that CMA cannot replace the protective function of macroautophagy in hepatocytes as occurs in MEFs. Our study demonstrates that although crosstalk between these two autophagic pathways occurs in hepatocytes, the resultant upregulation of one form of autophagy cannot always replace the function of the impaired pathway. Functional redundancy for protection from oxidant stress does not exist for the pathways of macroautophagy and CMA in hepatocytes. This difference from MEFs may reflect an increased vulnerability of hepatocytes to oxidant stress, a greater dependency of hepatocytes on autophagy for resistance to oxidant stress or fundamental differences in the functions of autophagy in the two cell types. The present study expands the mechanisms by which autophagy may protect against oxidative stress to include the regulation of MAPK signaling and establishes that macroautophagy and CMA perform distinct functions during hepatocellular oxidative stress (Fig. 1). Hepatic diseases mediated by oxidant stress may be promoted by factors that impair hepatocyte autophagic function such as lipid accumulation or aging. These findings highlight the need to develop additional compounds to increase autophagic function that may serve as therapeutic agents in the prevention or treatment of liver injury. Figure 1 Mechanisms by which autophagy promotes hepatocyte resistance to oxidative stress. In response to oxidative stress, macroautophagy can serve a protective function through the breakdown of cellular constituents such as lipid droplets and mitochondria to ...

The endogenous cellular signals that initiate the transition of hepatocytes from quiescence to proliferation remain unclear. The protein stathmin 1 (STMN1) is highly expressed in dividing cells, including hepatocytes, and functions to promote cell mitosis through physical interactions with tubulin and microtubules that regulate mitotic spindle formation. The recent finding that STMN1 mediates the resistance of cultured hepatocytes to oxidant stress led to an examination of the expression and function of this protein in the liver in vivo. STMN1 messenger RNA (mRNA) and protein were essentially undetectable in normal mouse liver but increased markedly in response to oxidant injury from carbon tetrachloride. Similarly, levels of STMN1 mRNA and protein were increased in human livers from patients with acute fulminant hepatic failure. To determine STMN1 function in the liver in vivo, mice were infected with a control or Stmn1-expressing adenovirus. Stmn1 expression induced spontaneous liver enlargement with a doubling of the liver to body weight ratio. The increase in liver mass resulted, in part, from hepatocellular hypertrophy but mainly from an induction of hepatocyte proliferation. STMN1 expression led to marked increases in the numbers of 5-bromo-2'-deoxyuridine-positive and mitotic hepatocytes and hepatic nuclear levels of cyclins and cyclin-dependent kinases. STMN1-induced hepatocyte proliferation was followed by an apoptotic response and a return of the liver to its normal mass. Conclusion: STMN1 promotes entry of quiescent hepatocytes into the cell cycle. STMN1 expression by itself in the absence of any reduction in liver mass is sufficient to stimulate a hepatic proliferative response that significantly increases liver mass.

Interferon-γ (IFNγ) is a central activator of immune responses in the liver and other organs. IFNγ triggers tissue injury and inflammation in immune diseases, which occur predominantly in females for unknown reasons. Recent findings that autophagy regulates hepatotoxicity from proinflammatory cytokines led to an examination of whether defective hepatocyte autophagy underlies sex-specific liver injury and inflammation induced by IFNγ.A lentiviral autophagy-related 5 (Atg5) knockdown was performed to decrease autophagy-sensitized alpha mouse liver (AML 12) hepatocytes to death from IFNγ in combination with IL-1β or TNF. Death was necrosis attributable to impaired energy homeostasis and adenosine triphosphate depletion. Male mice with decreased autophagy from a tamoxifen-inducible, hepatocyte-specific Atg5 knockout were resistant to IFNγ hepatotoxicity whereas female knockout mice developed liver injury and inflammation. Female mice had increased IFNγ-induced signal transducer and activator of transcription 1 (STAT1) levels compared to males. Blocking STAT1, but not interferon regulatory factor 1, signaling prevented IFNγ-induced hepatocyte death in autophagy-deficient AML12 cells and female mice. The mechanism of death is STAT1-induced overexpression of nitric oxide synthase 2 (NOS2) as in vitro hepatocyte death and in vivo liver injury were blocked by NOS2 inhibition.Decreased hepatocyte autophagy sensitizes mice to IFNγ-induced liver injury and inflammation through overactivation of STAT1 signaling that causes NOS2 overexpression. Hepatotoxicity is restricted to female mice, suggesting that sex-specific effects of defective autophagy may underlie the increased susceptibility of females to IFNγ-mediated immune diseases.

Mutant human hepatoblastoma cell lines resistant to copper toxicity were isolated from mutagenized HuH7. Two copper resistant cell lines (CuR), CuR 23 and CuR 27, had reduced basal expression of metallothionein (MT) messenger RNA (mRNA) and exhibited minimal or no increase in resistance to cadmium or zinc toxicity. Copper uptake, efflux of newly transported copper, glutathione content, and efflux rate were comparable with HuH7, whereas holoceruloplasmin synthesis and secretion were slightly decreased. Subcellular distribution of copper at steady-state showed an increase in organelle and membrane fractions with a reduction in cytosol. Expression of ATP7B mRNA was fivefold increased, and ATP7B protein approximately threefold increased in both CuR 23 and 27. Another cell line, CuR 41, showed increased basal expression of MT and ATP7B mRNA but not ATP7B protein, and resistance to cadmium and zinc toxicity. Copper uptake in CuR 41 was comparable with HuH7, but initial rates of efflux of copper and glutathione were reduced. The synthesis of holoceruloplasmin but not ceruloplasmin peptide was markedly diminished in CuR 41. Subcellular distribution of copper showed an increase in cytosolic and decreased organelle and membrane-associated copper. These data suggest that cellular resistance to copper toxicity was achieved in two independent cell lines without MT induction and that the induction of ATP7B may lead to the enhanced intracellular sequestration of copper by organelles.

Protein kinase CK2 (formerly casein kinase II) is a tetrameric enzyme constitutively expressed in all eurakyotic tissues that plays a significant role in the regulation of cell proliferation, malignant transformation, and apoptosis. The catalytic alpha-subunit of the enzyme is known to exist in three isoforms CK2alpha, CK2alpha' and CK2alpha". CK2alpha" is highly expressed in liver compared with other tissues and is required for the normal trafficking of several hepatocellular membrane proteins. Initial studies of dengue virus infection indicated that the CK2alpha"-deficient membrane trafficking mutant cell line (Trf1) was resistant to virus-induced cell death compared with the parental human hepatoma (HuH)-7 hepatoma line. Expression of recombinant CK2alpha" in Trf1 was capable of reverting this resistant phenotype. This study was extended to TNF-alpha in addition to other stimuli of cell death in an attempt to uncover common death pathways that might be modulated by CK2alpha". Evaluation of different pathways involved in death signaling suggest that the regulation of a critical proapoptotic step in HuH-7 cells by CK2alpha" is mediated by a JNK signaling cascade.

Since c-myc expression is increased during apoptosis in toxin-induced liver injury in vivo, the role of c-myc in liver cell apoptosis was investigated. The human hepatoma cell line HuH-7, which constitutively expresses c-myc, was stably transfected with sense and antisense c-myc expression vectors under the control of the zinc-inducible metallothionein promoter. None of the three cell types (wild-type, sense c-myc, or antisense c-myc) underwent apoptosis when cultured in serum-free medium (SFM). With the addition of SFM plus 37.5 microM zinc, wild-type and sense c-myc-expressing cells underwent rapid cell death, whereas antisense c-myc-expressing cells exhibited increased survival. This cell death had the light, fluorescent, and electron microscopic appearance of apoptosis, but did not result in DNA fragmentation. This apoptosis could be terminated by the addition of medium containing 2% fetal calf serum or the overexpression of bcl-2 but not by supplementation with specific growth factors. Altering c-myc expression did not affect cellular metallothionein mRNA levels or the rate of cell death from copper or cadmium. The requirement for zinc and absence of DNA fragmentation in c-myc-induced hepatoma cell apoptosis under serum-free conditions provides further evidence of the complex regulation of apoptosis in different cell types.

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disease in this country and a major global health problem that is likely to worsen due to the epidemic of obesity and diabetes.1 No established therapy exists for NAFLD, but one experimental approach has been to treat with agents that increase insulin sensitivity. This strategy is based on the concept that the mechanism underlying the development of NAFLD and other components of the metabolic syndrome is insulin resistance.1 Prominent among the insulin-sensitizing agents being examined as NAFLD treatments has been the thiazolidinedione class of antidiabetic drugs which includes pioglitazone. A randomized, placebo-controlled short-term study of pioglitazone in nondiabetic patients with NAFLD recently demonstrated significant metabolic and histologic improvement with a reduction in liver injury and fibrosis in the pioglitazone-treated patients.2 IL-6, interleukin-6; NAFLD, nonalcoholic fatty liver disease; PPARγ, peroxisome proliferator-activated receptor γ; STAT3, signal transducer and activator of transcription 3; TNFα, tumor necrosis factor-α. The thiazolidinediones are ligands for the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) which mediates the effects of these drugs.3 PPARγ is a transcription factor that regulates critical cellular processes such as metabolism, proliferation, differentiation, and inflammation. Thiazolidinediones decrease insulin resistance, because PPARγ activation increases insulin sensitivity through a complex series of effects involving multiple organs but centered primarily on the adipocyte. The highest cellular levels of PPARγ are in adipocytes in which activation of this nuclear receptor induces white adipocyte differentiation and lipid storage. Increased sequestration of lipid in adipose tissue lowers serum free fatty acid levels,4 decreasing lipid delivery to other organs such as the liver and skeletal muscle where deleterious lipid deposition may occur and promote insulin resistance. PPARγ activation also up-regulates adipocyte production of adiponectin which can act to increase hepatic glucose uptake and suppress glucose production.5, 6 Finally, stimulation of the PPARγ pathway inhibits adipocyte production of tumor necrosis factor-α (TNFα) and resistin, which mediate insulin resistance.7 Although the levels are much lower than in adipose tissue, PPARγ is expressed in normal liver, and levels increase in rodent models of steatosis. Gavrilova et al.8 delineated the relative contributions of adipocyte versus hepatocyte PPARγ effects in response to the thiazolidinedione rosiglitazone by examining its differential effects in wild-type and lipoatrophic mice with normal or a knockout of hepatocyte PPARγ expression. In the absence of fat tissue (lipoatrophic mice), rosiglitazone induced hepatic steatosis that was PPARγ-mediated because fat accumulation failed to occur in mice with a hepatocyte-specific PPARγ knockout. In mice with normal fat tissue, the effect of rosiglitazone was to decrease hepatic lipid content through a mechanism independent of hepatocyte PPARγ because an equivalent effect occurred in hepatocyte PPARγ knockout mice. Thus, the effect of isolated PPARγ activation in the liver was to promote lipid accumulation, but the effect of PPARγ on fat tissue superseded the direct hepatic effects and led to a decrease in hepatic steatosis. The macrophage is another cell in which PPARγ activation may mediate the therapeutic effects of thiazolidinediones in NAFLD.9 PPARγ inhibits macrophage activation and proinflammatory cytokine production,9 and therefore acts in both adipocytes and macrophages to decrease the levels of cytokines that mediate peripheral and hepatic insulin resistance and liver inflammation in NAFLD. The critical function of macrophage PPARγ signaling in the maintenance of insulin sensitivity is evident because a macrophage-specific knockout of PPARγ is sufficient to induce insulin resistance and hepatic steatosis.10 Thus, PPARγ-dependent effects of thiazolidinediones on macrophages as well as adipocytes likely mediate the potential benefits these drugs have on insulin responsiveness and hepatic lipid accumulation. In this issue of HEPATOLOGY, novel studies by Aoyama and colleagues11 examine the physiological effects of pioglitazone on partial hepatectomy–induced liver regeneration in the KK-Ay mouse model of NAFLD. Partial hepatectomy is associated with transient hepatic lipid accumulation that has been proposed to provide the energy required for cell proliferation.12 Controversy exists on whether hepatic steatosis impairs regenerative potential. Obese mice with a genetic loss of leptin signaling have a defective regenerative response to partial hepatectomy,13 although the inability of exogenous leptin to reverse this defect despite resolution of the hepatic steatosis suggests that increased hepatic lipid content may not be the mechanism of this impairment.14 Both normal15 and defective16 hepatic regeneration has been demonstrated in mice with diet-induced NAFLD. Finally, recent studies by Newberry et al.17 have suggested that regeneration after partial hepatectomy is unaffected by hepatic lipid content, because mouse models with a wide range of hepatic triglyceride levels failed to exhibit any differences in proliferation. The studies by Aoyama et al.11 were conducted in mutant KK-Ay mice, which develop obesity, insulin resistance, and hyperleptinemia with leptin resistance in the absence of a genetic defect in leptin signaling. In theory, the mice therefore represent a good rodent model of human NAFLD. These mice exhibited a profound regenerative defect, because for 48 hours after partial hepatectomy they had absolutely no increase in hepatocyte bromodeoxyuridine staining or cyclin D1 expression. There was also significant mortality in the KK-Ay mice which was attributed to impaired regeneration. It seems unlikely, however, that failed regeneration was the only mechanism as some mice died within 12-24 hours, well before regeneration occurs normally. Pioglitazone pretreatment augmented the hepatic proliferative response in KK-Ay mice in response to partial hepatectomy. Bromodeoxyuridine and proliferating cell nuclear antigen staining was markedly increased with pioglitazone treatment, albeit to levels still well below those in wild-type mice. This effect was achieved with a short 5-day course of pioglitazone. Mortality from partial hepatectomy was also blocked by pioglitazone. Interestingly, after partial hepatectomy, the previously hyperglycemic KK-Ay mice developed hypoglycemia and marked hyperinsulinemia that was blocked by pioglitazone treatment. The paradoxical decrease in serum glucose in the diabetic animals along with the sudden increase in insulin production in response to partial hepatectomy is difficult to explain. These findings do suggest that the prosurvival effect of pioglitazone may have been mediated by its metabolic effects that stabilized insulin responsiveness and gluconeogenesis. The marked increase in adiponectin levels in the pioglitazone-treated animals may have mediated this effect, as well as having a direct effect on hepatocellular proliferation.18 The hypoglycemia in the previously hyperglycemic mutant mice suggests that they might have become acutely more insulin sensitive, and hepatic insulin hypersensitivity has been reported in diabetic rats after partial hepatectomy.19 Energy homeostasis may have been severely disrupted in the liver and other organs in KK-Ay mice after partial hepatectomy, which could have led to both impaired regeneration and death. To address this possibility, it would be interesting to examine hepatic adenosine triphosphate levels in wild-type and untreated and pioglitazone-treated animals as well as the effects of glucose supplementation on mortality and the regenerative defect in mutant mice. In keeping with the known ability of PPARγ to inhibit inflammatory responses, pioglitazone treatment blunted increases in TNFα and interleukin-6 (IL-6) after partial hepatectomy. These two cytokines have beneficial proliferative and hepatoprotective effects, so it seems counterintuitive that pioglitazone-induced decreases in these factors were beneficial. However, the mutant mice had abnormal levels of these cytokines after partial hepatectomy with a markedly increased early and a second later peak in TNFα messenger RNA expression, and a normal early rise but an additional second peak in serum IL-6. Hyperproduction of these cytokines may have been deleterious, and although the excess TNFα did not lead to apoptotic liver injury as indicated by the absence of cytokeratin-18 cleavage, TNFα may have inhibited proliferation. In addition, a more careful assessment for liver injury, including that resulting in necrosis, should be performed. KK-Ay mice had a normal early induction of signal transducer and activator of transcription 3 (STAT3) phosphorylation, but STAT3 phosphorylation was sustained, an effect that was prevented by pioglitazone. Depending on the hepatic cell type undergoing STAT3 activation, prolonged STAT3 signaling may have had beneficial or detrimental hepatic effects.20 Assuming that pioglitazone's effects were PPARγ-mediated, studies should be performed in selective cellular PPARγ knockouts to determine whether the drug's proliferative effects are mediated by hepatocytes, nonparenchymal cells, and/or extrahepatic cells. Of note is that prior studies in partially hepatectomized mice have demonstrated that thiazolidinediones, including pioglitazone, decreased regeneration.21, 22 However, in contrast to the investigations by Aoyama et al., the studies were conducted in normal animals. The current finding of the opposite effect in a fatty liver further suggests that steatosis may alter the regenerative response. The data available from these studies are not sufficient to suggest a mechanism for this differential effect. If direct hepatic effects of pioglitazone are involved, one simple explanation may be differences in receptor number. PPARγ levels are low in normal rodent and human liver and decrease further in response to partial hepatectomy.22 In contrast, obese and diabetic animals, including KK-Ay mice, have increased PPARγ levels. More detailed studies contrasting the signaling responses of normal versus steatotic animals to PPARγ activation may provide a better understanding of whether proliferative responses are altered in fat-laden hepatocytes. The novel findings in the study by Aoyama et al. add to the potential beneficial effects of pioglitazone on the steatotic liver (Fig. 1). It is impressive that significant metabolic and regenerative improvements were achieved with a short course of therapy. Whether these effects can translate to humans, in whom PPAR signaling often differs from rodents, remains to be determined. First, the effects of pioglitazone after partial hepatectomy or on a more modest proliferative response need to be examined in other rodent NAFLD models. However, while awaiting further experimental evidence that pioglitazone may in fact promote hepatocyte proliferation in the setting of a fatty liver, it is interesting to speculate how a proliferative effect of pioglitazone may expand its use. Pioglitazone may particularly benefit older individuals with NAFLD in whom liver regenerative capacity may also be impaired by aging. Patients with steatotic livers without frank steatohepatitis may benefit from a preoperative course of pioglitazone before undergoing resective hepatic surgery. Steatotic donor livers for transplant might be preconditioned with pioglitazone to reduce primary nonfunction that may result in part from impaired regeneration.23 Although these therapeutic uses are highly speculative and their value must be weighed against potential side effects, the fact that thiazolidinediones may have additional beneficial effects on a steatotic liver beyond insulin sensitization does provide further hope that these agents will prove efficacious in the treatment of human NAFLD. Biological effects of pioglitazone treatment. The primary effects of pioglitazone (dotted blue lines) are on white adipocytes and macrophages. In white adipose tissue, pioglitazone: (1) increases adipose mass and triglyceride (TG) storage leading to decreased release of free fatty acids (FFA); (2) inhibits production of proinflammatory cytokines including TNFα, IL-6, and monocyte chemoattractant protein 1 (MCP-1); and (3) stimulates adiponectin production. Pioglitazone also directly inhibits macrophage cytokine production. Effects on the liver and skeletal muscle are secondary to those on adipocytes and macrophages. The reduction in serum FFA decreases lipid accumulation in both liver and muscle. Decreased lipid accumulation, reduced TNFα levels, and increased adiponectin all act to reduce liver and skeletal muscle insulin resistance. Increased insulin sensitivity results in decreased gluconeogenesis in the liver and increased glucose uptake in muscle. Changes in cytokines, adiponectin levels and/or insulin sensitivity, or an as-yet undefined direct hepatic effect of pioglitazone may increase the liver's proliferative capacity. Serum factors are shown in green.

Investigations into the mechanism of impaired autophagy in fatty liver disease have identified increased intracellular calcium as a mediator of this defect. Park et al.1 have demonstrated that saturated free fatty acids (FFAs) induce increased cytosolic calcium in hepatocytes that inhibits autophagic function. The decrease in autophagy can be reversed in obese mice by administration of a calcium-channel blocker, which results in a reduction in steatohepatitis. These findings suggest the possibility that a common class of medications extensively employed in humans for cardiovascular disease (CVD) may be efficacious in the therapy of nonalcoholic steatohepatitis (NASH). The finding that hepatocyte lipids are metabolized by the lysosomal degradative pathway of macroautophagy2 has generated growing interest in the mechanistic involvement of autophagy in NASH.3 Potential mechanisms by which hepatocyte autophagy may prevent NASH development and/or progression include not only the limitation of excessive hepatic lipid accumulation by increasing lipid metabolism, but also promotion of hepatocyte survival postinjury from oxidant stress or tumor necrosis factor.3 Macrophage autophagy may protect against NASH by down-regulating the innate immune response.4 These potentially beneficial effects of autophagy, together with the fact that autophagic function is decreased in hepatocytes and macrophages in steatotic livers,2, 4 suggest that a therapeutic approach directed at increasing hepatic autophagy may be an effective treatment for NASH. A better understanding of the mechanism(s) that underlies the defect in autophagy in hepatocytes and other cells in hepatic steatosis may lead to the identification of novel compounds to reverse this problem. Initial findings of a decrease in levels of critical autophagy proteins in the liver, such as autophagy-related protein 7, as a mechanism for the defect have not been reproduced.3 Subsequent studies have pointed to a problem not in autophagosome formation, but in the process of fusion between autophagosome and -lysosome or in lysosomal function and therefore degradation of the contents of the autolysosome.5 The mechanism of the fusion defect has been attributed to altered membrane lipid content secondary to the dyslipidemia associated with insulin resistance (IR) and obesity. One report implicated both the unsaturated fatty acid, oleate, and the saturated fatty acid, palmitate, in decreasing autophagy through defective fusion.6 Other studies have shown differential effects with oleate actually increasing autophagy. This finding would be consistent with the general concept that saturated, but not unsaturated, FFAs are toxic in NASH.7 The importance of the finding that decreased autophagy in steatotic hepatocytes is a failure of fusion is that agents that only target the autophagic pathway upstream of this block may be ineffective in increasing hepatic autophagy. One potential mechanism of saturated FFA toxicity in hepatocytes is endoplasmic reticulum (ER) stress.7 Palmitate-induced ER stress in hepatoma cells decreases activity of sarco-ER calcium ATPase (SERCA), which normally maintains cellular calcium homeostasis by sequestering calcium in the ER.8 Park et al. reasoned that saturated FFAs may impair autophagy through ER stress-induced SERCA inhibition given that the ER stress inducer, thapsigargin, which inhibits SERCA, also blocks autophagosome-lysosome fusion. Studying HepG2 cells, they found that the saturated FFAs, palmitate and stearate, but not oleate, induced cellular accumulation of ubiquitinated proteins and the autophagy-degraded protein, p62, along with autophagosomes. By numerous techniques, they demonstrated that saturated FFAs inhibit autophagic function primarily by blocking the fusion step. One limitation of this study is that the investigations were all performed in HepG2 cells, and it would be important to know whether the findings are reproducible in primary hepatocytes. However, these results, together with the previous study by Koga et al.,6 strongly suggest that FFAs inhibit hepatocyte autophagy at the level of fusion. To prove that saturated FFAs inhibit autophagy through SERCA and not nonspecific ER stress, Park et al. first showed that only ER stress inducers that also inhibit SERCA decrease autophagy. In addition, they demonstrated that genetic silencing of SERCA increased, whereas SERCA overexpression decreased, protein accumulation from saturated FFAs. Palmitate elevated cytosolic calcium levels, an effect prevented by the calcium-channel blockers, verapamil and nicardipine, which prevent extracellular calcium entry into the cytoplasm. Both agents also reversed inhibition of autophagy by palmitate. Genetic inhibition of the primary calcium channel present in HepG2 cells also partially blunted the palmitate-induced decrease in autophagy. Although these findings do not conclusively prove that saturated FFAs act directly on SERCA, the data do clearly indicate that the effect of palmitate on autophagy is mediated by increased cytosolic calcium. Other agents that increase hepatocyte calcium through the inositol triphosphate receptor, such as glucagon and vasopressin, also increased HepG2 protein accumulation, although not to the extent of saturated FFAs. The efficacy of verapamil in obese mice is also demonstrated by the study. Mice fed 2 months of high-fat diet and treated with verapamil for 10 days had marked decreases in hepatic steatosis (HS) and macrophage infiltration. Unfortunately, the effects on liver injury are not discussed. There was indirect evidence of reduced hepatic cytosolic calcium in the verapamil-treated mice, and the drug suppressed the increase in cytosolic calcium from palmitate in cultured mouse hepatocytes. Autophagic function, including autophagosome-lysosome fusion, was restored by verapamil in obese mice. The effects of verapamil were somewhat liver specific given that white adipose tissue mass and inflammatory cytokine production were unaffected. Whether decreased HS and inflammation resulted completely from direct hepatic effects is unclear, given that these mice also had slightly reduced body weights as well an almost complete reversal of their glucose intolerance and IR. The mechanism by which increased calcium inhibits fusion is not addressed, and, interestingly, calcium is essential for fusion to occur.6 The possibility exists that calcium-channel blockers may affect autophagy through mechanisms other than an effect on fusion. However, the findings do significantly expand our understanding of how saturated FFAs are potentially toxic in NASH by establishing a novel mechanism by which ER stress and calcium disequilibrium can inhibit autophagy (Fig. 1). The study raises the question of whether calcium-channel blockers, with a proven safety profile from extensive human use in CVD, can treat human NASH and, possibly, alcoholic fatty liver disease as well. It should be noted that whether autophagy is defective in human NASH remains unknown. Autophagic function is almost impossible to assess in human tissue, with the possible exception of studies of autophagic flux in tissue explants. Published reports of levels of autophagy in human liver disease typically only provide assessments of steady-state levels of autophagosomes in which increases or decreases may both reflect either elevated or reduced autophagic function. A number of studies have examined calcium-channel blockers in metabolic syndrome with mixed conclusions on whether these agents have beneficial effects on glucose tolerance, hyperinsulinemia, and hyperlipidemia. Studies specifically examining NASH are lacking, although one study demonstrated no effect of amiodipine.9 The findings of Park et al. provide a scientific basis for a closer examination of these drugs in NASH. The lack of data on autophagic function in human NASH makes it possible that only a subset of patients with this disease have impaired autophagy and will therefore benefit from autophagy-directed therapy. Only by identifying these patients, and through the use of new agents developed specifically to target autophagy, will the role of autophagy in human NASH be determined. Mark J. Czaja, M.D. Department of Medicine and Marion Bessin Liver Research Center Albert Einstein College of Medicine Bronx, NY Author names in bold designate shared co-first authorship.

We have developed the methodology for evaluating the effects of pathophysiological conditions on the molecular mechanisms of hepatic protein synthesis and fibrogenesis in baboons and man. Total RNA was extracted from percutaneous liver biopsies of five baboons who were chronically fed an ethanol-rich liquid diet, their pair-fed controls and from humans with a variety of liver abnormalities. Chronic alcohol administration in baboons with liver fibrosis and normal serum albumin levels increased in vitro protein synthesis as measured by [35S]methionine incorporation, albumin mRNA content and Type I procollagen mRNA content. There was no difference in the beta-actin (a constitutive protein) mRNA content. In humans, serum albumin levels correlated with albumin mRNA content as indicated by the intensity of dot blot hybridization and Type I procollagen mRNA levels correlated with the activity of liver fibrosis. The use of RNA-DNA hybridization to investigate procollagen mRNA from human biopsies appears to be a valuable tool for evaluating the potential for collagen synthesis and the future course of liver disease. Besides the use of RNA-DNA hybridization, we describe other methodologies which are useful in delineating the levels of gene expression responsible for hepatic mRNA regulation in normal liver and disease states in man. The use of molecular techniques to evaluate human liver disease provides an opportunity to develop clinically relevant information while at the same time offering the additional advantage of providing fundamental knowledge about fibrogenesis.

FundingSupported by the National Institutes of Health (R01DK044234, R01AA022601, and R01DK111678) (to M.J.C.). See Article on page 1937 Overactivation of the innate immune response amplifies hepatic injury and promotes the progression to fibrosis and cirrhosis in many liver diseases. Hepatic and systemic inflammation are triggered in part by factors released from dying hepatocytes, but recent evidence demonstrates that increased inflammation occurs in end‐stage cirrhotic liver disease, which typically has low levels of hepatocyte injury.1 Clinical decompensation in patients with cirrhosis is associated with increased inflammation, which is even greater in patients with acute‐on‐chronic liver failure (ACLF).2 These recent findings suggest the possibility that cirrhosis may be a target for anti‐inflammatory drug therapy. Inflammation in cirrhosis is driven partly by the increased intestinal translocation of bacterial products (pathogen‐associated molecular patterns) that activate systemic monocytes and resident liver macrophages. Factors released by dying hepatocytes (damage‐associated molecular patterns) are presumably decreased in end‐stage cirrhosis lacking a significant component of active liver injury, suggesting that other monocyte/macrophage activators fuel the increased inflammation in decompensated cirrhosis. A better understanding of the mechanisms of macrophage activation in cirrhosis may facilitate the therapeutic targeting of this pathway in cirrhotic patients. Hepatocyte‐synthesized albumin is the most abundant plasma protein and performs beneficial functions including maintaining intravascular oncotic pressure and acts as a molecular carrier for different endogenous and exogenous molecules such as fatty acids, metals, hormones, and drugs.3 A free cysteine‐34 amino acid residue characterizes the reduced form of human mercaptoalbumin (HMA) and is responsible in part for its antioxidant activity.3 Reversible or irreversible oxidation of the cysteine‐34 thiol group generates human non‐mercaptoalbumin 1 (HNA1) and non‐mercaptoalbumin 2 (HNA2), respectively. These oxidized forms of albumin have impaired scavenger activity for reactive oxygen and nitrogen species and accumulate in the serum of cirrhotic patients serving as a biomarker of oxidative stress in this disease.4 In this issue of Hepatology, Alcaraz‐Quiles et al. 6 delineate a new role for oxidized albumin as a contributor to the increased systemic inflammation in advanced liver disease. Consistent with previous studies, they demonstrate that in patients with stages of chronic liver disease ranging from compensated and decompensated cirrhosis to ACLF, the serum HNA1 and HNA2 levels increase with worsening severity of liver disease. Levels of serum proinflammatory cytokines interleukin (IL)‐6, IL‐1β, tumor necrosis factor‐α, and IL‐8 were increased in decompensated cirrhosis and ACLF patients compared with patients with compensated cirrhosis and correlated with HNA1 and/or HNA2 levels, suggesting a direct relationship between elevated oxidized albumin and systemic inflammation. To further confirm causality, serum white blood cells from healthy donors and cirrhotic patients were treated in vitro with ex vivo oxidized HNA1 and HNA2. Cytokine production increased in the cells from both groups in response to HNA1 but not HNA2. HNA1 also upregulated levels of cyclooxygenase‐2 and PGE2‐synthase and eicosanoid release. Subpopulation analysis revealed that monocytes and not neutrophils were responsive to the proinflammatory effects of HNA1. A rationale for the differential effects of HNA1 and HNA2 was not supplied, except that it did not result from a difference in their levels of reactive oxygen or nitrogen species. A phospho‐kinase array analysis identified increased phosphorylation and therefore activation of the mitogen‐activated protein kinase (MAPK) p38α by HNA1. The mechanistic involvement of p38 MAPK was confirmed by the ability of a p38 MAPK inhibitor, but not other MAPK inhibitors, to block the proinflammatory effect of HNA1. Although confirmatory evidence from cells with a genetic knockout/knockdown of p38 MAPK would be important, the data indicate that oxidized albumin is an activator of monocyte inflammation in cirrhosis through a p38 MAPK‐mediated effect. The novelty of these findings is that they identify albumin as a Trojan horse in cirrhosis. Although the maintenance of adequate serum HMA levels is important for the many beneficial functions of albumin, measures of total albumin in the clinic do not include HNA1 or HNA2, which require a more sophisticated analysis to detect. This hidden oxidized fraction of albumin can trigger an injurious proinflammatory macrophage response that promotes cirrhotic decompensation or ACLF. Whether albumin oxidation occurs exclusively systemically, or within the liver as well to activate hepatic resident macrophages, is an unanswered question. Infusion of human albumin is a common therapy in cirrhotic liver disease. An increased supply of functional HMA should benefit the patient, but oxidation of the exogenous albumin could possibly worsen the clinical condition by increasing inflammation. Therefore, the situation in cirrhosis is more complex than the simple need to replenish HMA and ideally albumin‐directed therapies should remove harmful HNA1 as well. Contributing to the lack of efficacy for liver support devices could be that they fail to reduce levels of oxidized albumin, whereas plasma transfusion is more effective because it removes harmful oxidized albumin as well.3 MAPKs are known to be important modulators of liver pathophysiology that are upregulated by oxidative stress.7 Although studies such as the current one point to multiple mechanisms by which oxidative stress promotes liver disease, antioxidant therapies have been unsuccessful for diseases of the liver as well as other organs.8 MAPKs as the downstream effectors of oxidant stress therefore represent a potentially more efficacious target than global oxidant neutralization. The present findings are novel in that they involve the p38 MAPK pathway, whereas previous studies have largely implicated the MAPKs c‐Jun N‐terminal kinase (JNK) and to a lesser extent extracellular signal‐regulated kinase in liver pathophysiology. The finding of p38 MAPK as a regulatory element in inflammation defines this kinase as a new candidate for inhibiting systemic and possibly hepatic inflammation as well. Unexamined in the present study is the receptor by which HNA1 activates the monocyte p38 MAPK pathway. Receptor identification would be interesting as it might identify an alternative, more selective approach to the inhibition of this proinflammatory pathway. Phosphorylation of p38 MAPK results from activation of a cascade of upstream kinases including apoptosis signal‐regulating kinase 1 (ASK1). ASK1 has been identified as a potential therapeutic target in a number of diseases because of its involvement in apoptosis, inflammation and fibrosis, and human ASK1 inhibitors have been developed.9 A randomized phase II trial recently suggested that the ASK1 inhibitor selonsertib may be effective in reducing fibrosis in patients with nonalcoholic steatohepatitis and stage 2 or 3 fibrosis.10 This benefit could have been secondary to inhibition of downstream p38 MAPK activation and/or JNK inhibition. An important question becomes whether HNA1‐induced p38 MAPK activation in monocytes results from ASK1 activation. If activation is ASK1‐dependent, the findings of the present study suggest that an ASK1 inhibitor already in human clinical trials for liver disease may prevent decompensation in end‐stage cirrhosis. Alternatively, direct p38 MAPK inhibitors could be used as a therapy (Fig. 1). Such new molecular approaches are desperately needed for a highly prevalent disease with significant mortality and a lack of effective therapies.Figure 1: The oxidized albumin form HNA1 promotes systemic inflammation and decompensation in cirrhosis. With liver cirrhosis induced by viral infections, alcohol or obesity, within the liver or systemically, HMA is reversibly (HNA1) and irreversibly (HNA2) oxidized. Serum HNA1 is taken up by an unknown mechanism into circulating monocytes to phosphorylate (P) and activate p38α MAPK, which induces the production and secretion into the blood of proinflammatory cytokines such as tumor necrosis factor‐α and IL‐1β. The mechanism of p38 MAPK activation is unknown but may be through effects on ASK1, which phosphorylates downstream MAPK kinases (MKK4/7 and MKK3/6) that then phosphorylate JNK and p38 MAPK, respectively. Proinflammatory cytokines released as the result of increased macrophage activation have deleterious effects on the liver and other organs such as the kidney, to promote clinical decompensation or ACLF in cirrhotic patients. The ASK1 inhibitor selonsertib or other direct p38 MAPK inhibitors may be a prevention or treatment of decompensated cirrhosis by downregulating this inflammatory pathway. Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; TNF‐α, tumor necrosis factor‐α.Potential conflict of interest Nothing to report.

HepatologyVolume 69, Issue 1 p. 446-448 Hepatology Elsewhere A Novel Mechanism of Starvation-Stimulated Hepatic Autophagy: Calcium-Induced O-GlcNAc-Dependent Signaling Yang Shen Ph.D., Department of Medicine, Division of Digestive Diseases, Emory University School of Medicine, Atlanta, GeorgiaSearch for more papers by this authorMark J. Czaja M.D., orcid.org/0000-0003-2306-7219 Department of Medicine, Division of Digestive Diseases, Emory University School of Medicine, Atlanta, GeorgiaSearch for more papers by this author Yang Shen Ph.D., Department of Medicine, Division of Digestive Diseases, Emory University School of Medicine, Atlanta, GeorgiaSearch for more papers by this authorMark J. Czaja M.D., orcid.org/0000-0003-2306-7219 Department of Medicine, Division of Digestive Diseases, Emory University School of Medicine, Atlanta, GeorgiaSearch for more papers by this author First published: 02 August 2018 https://doi.org/10.1002/hep.30118Citations: 1 Supported by NIH grants R01DK044234, R01AA022601, and R01DK111678. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume69, Issue1January 2019Pages 446-448 RelatedInformation

Commentary to: Cyclic AMP Inhibition of Proliferation of Hepatocellular Carcinoma Cells Is Mediated by Akt Lunhua Liu, Yili Xie and Liguang LouVol: 4 | Issue: 11 | pgs: 1240-1247

The model of toxic liver injury was used to examine the role of manganese superoxide dismutase (MnSOD) expression in cellular resistance to tumor necrosis factor (TNF)-alpha toxicity. The effects of the hepatotoxin D-galactosamine (GalN) and lipopolysaccharide (LPS) on hepatic and splenic TNF-alpha and MnSOD expression were studied. Treatment with GalN and LPS alone or in combination led to equivalent increases in hepatic and splenic TNF-alpha gene expression. Hepatic MnSOD mRNA levels were not affected by GalN or GalN with LPS but were increased 13-fold by LPS alone. Splenic MnSOD mRNA levels were increased twofold by GalN and 12-fold by either LPS alone or GalN plus LPS. The determination of MnSOD protein content, however, revealed no changes in hepatic or splenic steady-state levels of the protein with any of the treatments, despite the marked increases in MnSOD gene expression. Hepatic MnSOD enzyme activity was also unchanged by LPS or GalN plus LPS administration. Biosynthesis of MnSOD protein in rat hepatocytes isolated from an in vivo LPS-treated rat was unchanged compared with control. MnSOD mRNA levels were increased when GalN treatment was combined with uridine rescue, but again no change in protein was seen. The lack of any increase in MnSOD protein after GalN or LPS administration indicates that MnSOD upregulation is not involved in cellular resistance against TNF-alpha cytotoxicity in the liver in vivo.

Toxin-induced liver injury was formerly considered a passive biochemical event, but recent evidence has demonstrated that signal transduction pathways actively modulate the hepatocyte's response to this form of injury. Investigations have examined the effects of a variety of toxins on the activation of receptor-coupled signal transduction, mitogen-activated protein kinases, and Fas signaling, as well as the generation of second messengers such as ceramide and nitric oxide. Many of these pathways culminate in the activation of transcription factors such as activator protein-1, c-Myc, or nuclear factor-KB. This Themes article discusses the effects of toxic injury on these signaling pathways and their known functions in regulating hepatocyte death and proliferation following injury.