Ken‐ichi Isobe

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70-90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.

Abstract We recently identified CD8+CD122+ regulatory T cells that directly control CD8+ and CD4+ cells without intervention of APCs. In this study, we investigated the effector mechanism of CD8+CD122+ regulatory T cells by using an in vitro regulation system. The profile of cytokine expression revealed that IL-10 was predominantly produced by CD8+CD122+ cells, whereas other cytokines were similarly expressed in CD8+CD122+ cells and CD8+CD122− cells. Suppression of both proliferation and IFN-γ production by CD8+CD122− cells by CD8+CD122+ cells was blocked by adding anti-IL-10 Ab to the culture but not by adding anti-TGF-β Ab. When IL-10 was removed from the conditioned medium from CD8+CD122+ cells, the conditioned medium no longer showed regulatory activity. Finally, CD8+CD122+ cells from IL-10-deficient mice had no regulatory activity in vitro and reduced regulatory activity in vivo. Our results clearly indicate that IL-10 is produced by CD8+CD122+ cells and mediates the regulatory activity of these cells.

GADD34 is a protein that is induced by stresses such as DNA damage. The function of mammalian GADD34 has been proposed by in vitro transfection, but its function in vivo has not yet been elucidated. Here we generated and analyzed GADD34 knockout mice. Despite their embryonic stage- and tissue-specific expressions, GADD34 knockout mice showed no abnormalities at fetal development and in early adult life. However, in GADD34-/- mouse embryonic fibroblasts (MEFs), recovery from a shutoff of protein synthesis was delayed when MEFs were exposed to endoplasmic reticulum (ER) stress. The phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2alpha) at Ser51 induced by thapsigargin or DTT was prolonged in GADD34-/- MEF, although following treatment with tunicamycin, the eIF2alpha phosphorylation level did not change in either GADD34+/+ or GADD34-/- cells. ER stress stimuli induced expressions of Bip (binding Ig protein) and CHOP (C/EBP homologous protein) in MEF of wild-type mice. These expressions were strongly reduced in GADD34-/- MEF, which suggests that GADD34 up-regulates Bip and CHOP. These results indicate that GADD34 works as a sensor of ER stress stimuli and recovers cells from shutoff of protein synthesis.

Forkhead transcription factor, DAF-16, regulates genes that contribute both to longevity and resistance to various stresses in C. elegans. We and others have reported that members of the FOXO, mammalian homologs of DAF-16, also regulate genes related to stress resistance, such as GADD45. The NAD-dependent protein deacetylase, SIR2, is required for life span extension in yeast induced by caloric restriction, which also increases longevity in a wide variety of other organisms, including mammals. Sir2.1, a homolog of yeast SIR2, also extends life span by acting in a DAF-16 signaling pathway in C. elegans. We demonstrate that mammalian SIRT1 (Sir2α) physiologically interacts with FOXO. Acetylation of FOXO4, by the transcriptional coactivator p300, counteracted transcriptional activation of FOXO4 by p300. In contrast, mammalian SIRT1 was found to bind to FOXO4, catalyze its deacetylation in an NAD-dependent manner, and thereby increase its transactivation activity. The activity of FOXO4 is suppressed or enhanced by SIRT1 inhibitor, nicotinamide, or its activator, resveratrol, respectively. In response to oxidative stress, FOXO accumulates within the nucleus and induces GADD45 expression. FOXO-mediated GADD45 induction is markedly impaired in the cell, which depleted SIRT1 expression by RNA-interference. These results indicate that mammalian SIRT1 plays a pivotal role for FOXO function via NAD-dependent deacetylation in response to oxidative stress, and thereby may contribute to cellular stress resistance and longevity.

Alteration of tryptophan (TRP) metabolism elicited by proinflammatory cytokines has gained attention as a new concept to explain the etiological and pathophysiological mechanisms of major depression. The kynurenine (KYN) pathway, which is initiated by indoleamine 2,3-dioxygenase (IDO), is the main TRP metabolic pathway. It shares TRP with the serotonin (5-HT) pathway. Proinflammatory cytokines induce IDO under stress, promote the KYN pathway, deprive the 5-HT pathway of TRP, and reduce 5-HT synthesis. The resultant decrease in 5-HT production may relate to the monoamine hypothesis of major depression. Furthermore, metabolites of the KYN pathway have neurotoxic/neuroprotective activities; 3-hydroxykynurenine and quinolinic acid are neurotoxic, whereas kynurenic acid is neuroprotective. The hippocampal atrophy that appears in chronic depression may be associated with imbalances in neurotoxic/neuroprotective activities. Because proinflammatory cytokines also activate the hypothalamo-pituitary-adrenal (HPA) axis, these imbalances may inhibit the hippocampal negative feedback system. Thus, changes in the TRP metabolism may also relate to the HPA axis-hyperactivity hypothesis of major depression. In this article, we review the changes in TRP metabolism by proinflammatory cytokines under stress, which is assumed to be a risk factor for major depression, and the relationship between physiological risk factors for major depression and proinflammatory cytokines.

Imaging of Ancient SchwannomaKenichi Isobe1, Tominaga Shimizu, Tsutomu Akahane and Hiroyuki KatoAudio Available | Share

Abstract Experimental autoimmune encephalomyelitis (EAE) is one of the best-documented animal models of autoimmune disease. We examined the role of CD8+CD122+ regulatory T cells, which we previously identified as naturally occurring regulatory T cells that effectively regulate CD8+ T cells, in EAE. Depletion of CD8+CD122+ regulatory T cells by in vivo administration of anti-CD122 mAb resulted in persistent EAE symptoms. Transfer of CD8+CD122+ regulatory T cells into EAE mice at the peak EAE score clearly improved symptoms, indicating an important role of CD8+CD122+ regulatory T cells in the recovery phase of EAE. This was further confirmed by an increase and a decrease in the number of infiltrating T cells in the CNS and T cell cytokine production in mice that were depleted of or complemented with CD8+CD122+ cells. Furthermore, transfer of preactivated CD8+CD122+ regulatory T cells resulted in diminished EAE symptoms, especially in the recovery phase of EAE. These results elucidate the essential role of CD8+CD122+ regulatory T cells in the recovery phase of EAE and suggest the preventive effect of preactivated CD8+CD122+ regulatory T cells for EAE.

We have reported that histone acetylation induced by trichostatin A (TSA) promotes p21 waf1/cip1 (p21) expression, the GC-box located just upstream of TATA box was responsible for TSA-induced promoter activation, and both Sp1 and Sp3 were the working activator of this GC-box. To understand the molecular pathway from histone acetylation to this Sp1 family factors-mediated promoter activation, we investigated the function of p300, one of the histone acetyltransferase, in the present work. The evidence supporting the linkage between p300 and TSA-induced p21 promoter activation were realized from the following findings: 1) cotransfection of p300 elevated p21 promoter activity, and this elevation was dependent on TSA-responsive GC-box; 2) TSA-induced promoter activation was blocked by the introduction of p300 dominant-negative mutant into cells; and 3) Sp1- or Sp3-mediated activation was also suppressed by this p300 dominant-negative mutant. Our data also suggested that p300 collaborates with Sp1 in a way which is different from that when p300 collaborates with p53 in p21 transcription. We have reported that histone acetylation induced by trichostatin A (TSA) promotes p21 waf1/cip1 (p21) expression, the GC-box located just upstream of TATA box was responsible for TSA-induced promoter activation, and both Sp1 and Sp3 were the working activator of this GC-box. To understand the molecular pathway from histone acetylation to this Sp1 family factors-mediated promoter activation, we investigated the function of p300, one of the histone acetyltransferase, in the present work. The evidence supporting the linkage between p300 and TSA-induced p21 promoter activation were realized from the following findings: 1) cotransfection of p300 elevated p21 promoter activity, and this elevation was dependent on TSA-responsive GC-box; 2) TSA-induced promoter activation was blocked by the introduction of p300 dominant-negative mutant into cells; and 3) Sp1- or Sp3-mediated activation was also suppressed by this p300 dominant-negative mutant. Our data also suggested that p300 collaborates with Sp1 in a way which is different from that when p300 collaborates with p53 in p21 transcription. p21 waf1/cip1 trichostatin A DNA binding domain activation domain base pair phenylmethylsulfonyl fluoride p21 waf1/cip1 (p21)1 is a gene functioning as a cell cycle blocker, and its expression is usually regulated at transcription level. p21 was first cloned and characterized as an important effector that acts to inhibit cyclin-dependent kinase activity in p53-mediated cell cycle arrest induced by DNA damage (1.el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7936) Google Scholar, 2.Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5239) Google Scholar, 3.El-Deiry W.S. Harper J.W. O'Connor P.M. Velculescu V.E. Canman C.E. Jackman J. 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During the study of cellular senescence, we found that the inhibitors of histone deacetylase, either sodium butyrate or TSA, can promote p21 transcription and induce growth arrest and senescence-like state in NIH 3T3 cells (9.Xiao H. Hasegawa T. Miyaishi O. Ohkusu K. Isobe K. Biochem. Biophys. Res. Commun. 1997; 237: 457-460Crossref PubMed Scopus (99) Google Scholar). 2H. Xiao, T. Hasegawa, and K-i. Isobe, unpublished data.The minimal region of the mouse p21 promoter, containing from −60 to +40 bp relative to the TATA box, is essential and sufficient for the induction of p21 promoter by TSA. We reported also that a GC-box in this region is critical for both basal and TSA-induced promoter activity and that Sp1 and Sp3 are the functional activators of this GC-box (10.Xiao H. Hasegawa T. Isobe K. J. Cell. Biochem. 1999; 73: 291-302Crossref PubMed Scopus (132) Google Scholar). However, one question remained, how does histone acetylation affect Sp1-mediated transcription? Recently, the study of transcriptional regulation has been moving its focus to chromatin level. The molecules involved in chromatin transcription include DNA (promoter, enhancer, or silencer), histones, and non-histone proteins. It has become increasingly apparent that the equilibrium of histone acetylation and deacetylation plays an important role in transcriptional regulation (11.Wade PA. Pruss D. Wolffe A.P. Trends Biochem. Sci. 1997; 22: 128-132Abstract Full Text PDF PubMed Scopus (410) Google Scholar, 12.Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2401) Google Scholar). Several mammalian histone acetyltransferases and histone deacetylases have been cloned in recent years (13.Wang L. Mizzen C. Ying C. Candau R. Barlev N. Brownell J. Allis C.D. Berger S.L. Mol. Cell. Biol. 1997; 17: 519-527Crossref PubMed Scopus (145) Google Scholar, 14.Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1533) Google Scholar, 15.Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2402) Google Scholar, 16.Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar, 17.Mizzen C.A. Yang X.J. Kokubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J. Wang L. Berger S.L. Kouzarides T. Nakatani Y. Allis C.D. Cell. 1996; 87: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar, 18.Taunton J. Hassig C.A. Schreiber S.L. Science. 1996; 272: 408-411Crossref PubMed Scopus (1534) Google Scholar, 19.Emiliani S. Fischle W. Van L.C. Al-Abed Y. Verdin E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2795-2800Crossref PubMed Scopus (277) Google Scholar). p300 was first cloned as an E1A associated protein with properties of a transcriptional adapter (20.Eckner R. Ewen M.E. Newsome D. Gerdes M. DeCaprio J.A. Lawrence J.B. Livingston D.M. Genes Dev. 1994; 8: 869-884Crossref PubMed Scopus (925) Google Scholar). This protein was found later to possess intrinsic histone acetyltransferase activity and works as a coactivator in MyoD-, p53-, and SRC-1-mediated transcription (21.Yuan W. Condorelli G. Caruso M. Felsani A. Giordano A. J. Biol. Chem. 1996; 271: 9009-9013Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 22.Somasundaram K. El-Deiry W.S. Oncogene. 1997; 14: 1047-1057Crossref PubMed Scopus (162) Google Scholar, 23.Smith C.L. Onate S.A. Tsai M.J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8884-8888Crossref PubMed Scopus (368) Google Scholar). Indirect evidence has implicated p300 in cell cycle control and differentiation (24.Puri P.L. Avantaggiati M.L. Balsano C. Sang N. Graessmann A. Giordano A. Levrero M. EMBO J. 1997; 16: 369-383Crossref PubMed Scopus (236) Google Scholar, 25.Billon N. van Grunsven L.A. Rudkin B.B. Oncogene. 1996; 13: 2047-2054PubMed Google Scholar). Although p300 has been found to be required for induction of p21 expression in keratinocyte differentiation (26.Missero C. Calautti E. Eckner R. Chin J. Tsai L.H. Livingston D.M. Dotto G.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5451-5455Crossref PubMed Scopus (328) Google Scholar), the cis-element in the p21 promoter and the sequence-specific transcriptional activator remained unknown. The present work is part of our effort to understand the linkage between histone acetylation and Sp1-mediated transcription. Here we show evidence that p300 is required for TSA-induced, Sp1-mediated p21 transcription and discuss the possible mechanism of the functional interaction between Sp1 and p300 in p21 transcriptional activation. HeLa cells from ATCC and COS-1 cells from RIKEN (Wako, Japan) were cultured in a 37 °C humidified atmosphere containing 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Trichostatin A (Wako, Osaka) was dissolved with ethanol at a concentration of 1 mg/ml and stored at −20 °C. The working solution was diluted with distilled phosphate-buffered saline to 10 μg/ml concentration and stored at 4 °C for less than 1 week. The full-length (pGL3b-4542), the wild type minimal region (pGL3b-60 or pRL-60), and the GC-box mutated minimal region (pGL3b-#4 or pRL-#4) of the p21 promoter inserted in two types of luciferase reporter containing vectors have been described elsewhere (10.Xiao H. Hasegawa T. Isobe K. J. Cell. Biochem. 1999; 73: 291-302Crossref PubMed Scopus (132) Google Scholar). All expression vectors employed in this study were driven by the CMV promoter. Sp1 cDNA expressing constructs (pCGN-Sp1) and its backbone vector pCGN were kind gifts from Thomas Shenk (Princeton University) (27.Parks C.L. Shenk T. J. Biol. Chem. 1996; 271: 4417-4430Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Sp3 cDNA expressing construct (pCGN-Sp3) was generated by subcloning human Sp3 cDNA from pCMV4-Sp3flu into pCGN vector (10.Xiao H. Hasegawa T. Isobe K. J. Cell. Biochem. 1999; 73: 291-302Crossref PubMed Scopus (132) Google Scholar). p300 cDNA expressing construct(pGAL4p300) and its dominant-negative mutant (pGAL4p300/1514–1922) were provided by Antonio Giordano (Thomas Jefferson University) (28.Dennig J. Beato M. Suske G. EMBO J. 1996; 15: 5659-5667Crossref PubMed Scopus (203) Google Scholar). pCAF cDNA expression vector (pCI-pCAF) was from Yoshihiro Nakatani (National Institutes of Health) (16.Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar). The Sp1 expression construct used forin vitro transcription and translation experiments was made by introducing the 2.0-kilobase XbaI/SmaI fragment of pERV2/Sp1 (21.Yuan W. Condorelli G. Caruso M. Felsani A. Giordano A. J. Biol. Chem. 1996; 271: 9009-9013Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar) into a T7 promoter containing expression vector, pcDNA3.1/Myc.HisC (Invitrogen), at XbaI andEcoRV sites. pRL-TK (Promega), a Renillaluciferase reporter vector, was used for transfection efficiency control. For mammalian two-hybrid experiments, the following constructs were used. 1) pG5luc was a 5-GAL4 DNA binding sites-containing firefly luciferase vector (Promega). 2) pGAL4p300 was the same construct as mentioned above, which is a chimeric fusion of GAL4 DNA binding domain (DBD) and full-length p300 cDNA. 3) pVP16pCAF was created by inserting the EagI/KpnI fragment of pCAF cDNA from pCI-pCAF into pACT vector (Promega), which has a VP16 activation domain (AD) sequence, at NotI and KpnI sites. 4) pVP16Sp1N was made by two steps. The 1.5-kilobase BamHI fragment of Sp1 cDNA from pCGN-Sp1 was inserted into pcDNA3.1/Myc.HisC vector at the BamHI site first, and then the EcoRV and KpnI insert from correctly directed clone was subcloned into pACT vector at the EcoRV and KpnI sites. 5) pVP16Sp1C was made by the same strategy as that for pVP16Sp1N except the 0.5-kilobase BamHI fragment of Sp1 cDNA was used, and 6) pRL-TK as mentioned above. Cells were seeded into 48-well plate at a density of 30,000 cells/well the day before transfection. In the transfection experiments with reporter plasmid only, HeLa cells were transfected with 100 ng of promoter-reporter construct by standard calcium phosphate procedure. Treatments of TSA were started after 24 h and continued for another 24 h generally, except where noted in the figure legends. In cDNA cotransfection experiments, transfection was performed by using FugeneTM6 (Roche Molecular Biochemicals). The DNA mixture contained 20 ng of reporter and 20–100 ng of cDNA and either pCGN or pcDNA3.1 to adjust a total amount of DNA to 120 ng per well. For mammalian two-hybrid assays, COS-1 cells were transfected by standard calcium phosphate procedure with the combination of pG5luc, pGAL4, and pVP16 (or their corresponding fusions) in 1:1:1 molar ratio. pGEM-5Zf(+) vector was also added as carrier and for making the concentration of DNA to 0.3 μg/well. In all of these experiments, after 2 days of transfection, 5 of 50 μl of total cell lysate was used, and its luciferase activity was assayed by luminometer and dual luciferase assay kit (Promega). The luciferase activities were normalized against the activity of pRL-TK control vector (Promega) and protein concentration. Every experiment was performed at least three times. Labeled proteins were produced from pGAL4p300, pCI-pCAF, and pcDNA3.1/Myc.HisCSp1 plasmids by in vitro transcription and translation system (TNT kit, Promega) in the presence of [35S]methionine. Two μl of in vitro translated 35S protein and 2 μg of specific antibody were incubated in 50 μl of binding buffer (25 mm Tris-HCl (pH 8.0), 10% glycerol, 75 mmKCl, 5 mm MgCl2, 0.1 mm EDTA, 0.1% Nonidet P-40, 1 mm dithiothreitol, 0.5 mm PMSF) at 4 °C for 1 h and with 10 μl of 3% bovine serum albumin-blocked protein G-Sepharose (Amersham Pharmacia Biotech, 50% slurry) for an additional 1 h. After washing with binding buffer three times, the beads were resuspended in 50 μl of the same buffer and incubated with 2 μl of labeled pCAF or Sp1 reaction mixture at room temperature for 30 min. Then the beads were washed again and boiled for 2 min in the presence of 20 μl of Laemmli buffer. Finally, the supernatants were separated on 8% SDS-polyacrylamide electrophoresis gel. For the immunoprecipitation of unlabeled cellular p300 protein, HeLa cell lysates in radioimmune precipitation buffer (25 mm Tris-HCl (pH 8.0), 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 2.5 mm EDTA, 1 mm dithiothreitol, 0.5 mm PMSF) were immunoprecipitated with anti-p300 antibody and protein G-Sepharose. The beads were washed by radioimmune precipitation buffer three times and binding buffer once. Thereafter, the beads were used for pull-down assay as described above. The experimental procedure was the same as that described previously (10.Xiao H. Hasegawa T. Isobe K. J. Cell. Biochem. 1999; 73: 291-302Crossref PubMed Scopus (132) Google Scholar) except that the nuclear extract was from HeLa cells, and only the32P-labeled oligonucleotides probe with the wild type p21 promoter sequence (−40 to −10 bp from TATA box) was used. The polyclonal antibodies directed against Sp1 (catalogue number sc-59), Sp3 (sc-644), p300 N-terminal (p300-N, sc-584), p300 C-terminal (p300-C, sc-585) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The monoclonal antibody for p300 (p300-mono, 05–267) was from Upstate Biotechnology (Lake Placid, NY). We have reported that TSA greatly induced the p21 promoter activity and determined the cis-element responsive to TSA in NIH 3T3 cells (10.Xiao H. Hasegawa T. Isobe K. J. Cell. Biochem. 1999; 73: 291-302Crossref PubMed Scopus (132) Google Scholar). We found that these results are not cellular-specific. In HeLa cells, the morphological differentiation and elevated p21 expression were also noticed after TSA treatment, and the minimal promoter region and TSA-responsive GC-box were the same as those in NIH 3T3 cells (results not shown). To confirm the functional effects of Sp1 family transcription factors on p21 promoter, especially on the TSA response of this promoter, we cotransfected wild type or the TSA-responsive GC-box-mutated promoter-luciferase constructs with Sp1 or Sp3 cDNA expression construct into HeLa cells (Fig.1). While Sp1 and Sp3 can obviously activate wild type minimal promoter (pRL21–60) in a dose-dependent manner, they poorly activate the GC-box mutated minimal promoter (pRL21-#4) and empty vector (pRL-null). These results suggest that both Sp1 and Sp3 are the transactivators of the p21 promoter in HeLa cells, and they function through the TSA-responsive GC-box in vivo. Therefore, TSA, GC-box, and Sp1/Sp3 are tightly interrelated in p21 promoter activity. Because no changes in Sp1/Sp3 protein expression or in their DNA binding activity were found after TSA treatment (data not shown), we considered that TSA-induced p21 promoter activity may result from some kinds of interaction between Sp1/Sp3 and histone acetyltransferase or deacetylase. As the first step to issue this question, we examined the effect of p300 on p21 promoter. As shown in Fig. 2, transfection of wild type p300 expression construct, pGAL4p300, into cells enhanced either full-length (pGL3b-4542) or minimal promoter (pGL3b-60) activity. The experiments performed using the intact wild-type p300 (pCMVβp300 provided from Prof. David M. Livingston) gave results similar to those using the GAL4 derivative (data not shown).This suggested the positive effect of p300 on p21 transcription. Different from p21 wild type minimal promoter, the activity of the GC-box mutated promoter (pGL3b-#4) was not affected by p300 cotransfection. Thus the TSA-responsive GC-box is also required for the function of p300 to the p21 promoter, as it is required for the function of Sp1/Sp3 on p21 promoter. The interrelationship of p300 and TSA-induced promoter activation was further evidenced by cotransfection of a dominant-negative mutant of p300 expression construct (pGAL4p300/1514–1922) with p21 promoter construct, followed by TSA treatment. The product of pGAL4p300/1514–1922 encodes only a small portion of p300 protein, corresponding to the CH3 domain and its surroundings, and has been reported to be capable of interfering with the activity of endogenous protein (28.Dennig J. Beato M. Suske G. EMBO J. 1996; 15: 5659-5667Crossref PubMed Scopus (203) Google Scholar, 30.Avantaggiati M.L. Ogryzko V. Gardner K. Giordano A. Levine A.S. Kell K. Cell. 1997; 89: 1175-1184Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). In our experiment, this mutant lost the up-regulating effect of wild type p300 on the p21 promoter (Fig.3 A). Meanwhile, in the cells expressing this mutant p300, the p21 minimal promoter activity induced by TSA was obviously dose-dependent suppressed (Fig.3 B). These results suggest that the p300 molecule is also involved in TSA-induced p21 promoter activation. Considering the facts that both Sp1/Sp3 and p300 can up-regulate p21 promoter and function through the same GC-box in TSA-induced p21 promoter activation, the cooperative relationship between them might be present. To expose the interrelationship of Sp1/Sp3 and p300, we investigated the requirement of p300 for Sp1/Sp3-mediated transactivation. When Sp1 or Sp3 was cotransfected with various amounts of dominant negative mutant p300, Sp1/Sp3-mediated promoter activation was dose-dependent blocked (Fig.4). This result suggests that p300 is required for Sp1/Sp3-mediated p21 transcription and that some kind of collaboration between p300 and Sp1/Sp3 was present during this process. The cooperation of p300 and Sp1/Sp3 shown in functional assays described above may be from direct or indirect interaction of p300 with Sp1/Sp3. As p300 has been shown to physically interact with various transcription factors, we examined the physical relationship between p300 and Sp1 in vitro and in vivo. Fig.5 showed the results from two kinds ofin vitro protein binding assays. In immunoprecipitation pull-down assay (Fig. 5 A), the positive control, recombinant pCAF protein, can associate with recombinant p300 (lane 4) or native p300 (lane 7), whereas recombinant Sp1 cannot (lanes 5 and 8). That Sp1 does not associate with p300 in vitro was also implied from super shift assay (Fig.5 B). In this experiment, three kinds of antibodies for p300 failed to produce any shift or the loss of Sp1 and Sp3 complexes which were binding to the TSA-responsive GC-box-containing oligonucleotides (lanes 4–6). To examine whether there is the physical interaction between p300 and Sp1 in cellular situation, mammalian two-hybrid assay was performed. Fig.6 A shows the structures of fusion proteins produced in transfected cells and the schematic representation of this two-hybrid system. Briefly, after cotransfecting the GAL4 binding sites-containing reporter construct with GAL4DBD-fused vector as well as VP16AD-fused vector, the luciferase activity induced was detected. As shown in Fig. 6 B, when the GAL4DBD-fused p300 protein expressed together with the VP16AD-fused pCAF protein, which is a known p300 association protein, the luciferase activity in cells increased more than 2-fold. However, when the GAL4DBD-fused p300 expressed with VP16AD-fused Sp1, neither its N-terminal nor C-terminal portion, the luciferase activity was not affected compared with that with VP16AD only. This means that the exogenous p300 and Sp1 did not interact directly in this system. In vivoco-immunoprecipitation was also tried to detect the interaction of endogenous p300 and Sp1, but no positive clue was found (data not shown).Figure 6p300 does not directly interact with Sp1 in mammalian two-hybrid system. A, schematic representation of the two-hybrid system. The full-length p300 was cloned into a GAL4DBD-containing vector; the almost full-length of pCAF or indicated regions of Sp1 were inserted into a VP16AD-containing vector. Both of them were cotransfected with a GAL4 DNA binding sites-containing luciferase reporter vector. B, luciferase activity induced by different combinations of cotransfection. COS-1 cells seeded in a 48-well plate were transfected with the DNA mixture composed of the same molar ratio of pG5luc, GAL4p300, and VP16pCAF, VP16Sp1N, or VP16Sp1C. 30 ng of pRL-TK and a different amount of pGEM-5Zf(+) were used as internal control and carrier DNA, respectively. The total amount of DNA is 0.3 μg/well. Every point was triplicate, and the data are representative of three experiments. +, present; −, absent.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The original purpose of this work is to elucidate how TSA affected Sp1-mediated p21 transcription. In view of recent progress in transcriptional regulation, we assumed that some kinds of histone acetyltransferase and/or deacetylase may be altered physically or functionally during TSA treatment and thus influence function of Sp1 family transcription factors. In working to try to identify these involved histone acetyltransferase and/or deacetylase, we found that p300, one of the histone acetyltransferases, up-regulates p21 promoter and functions through the GC-box responsible to TSA (Figs. 2 and 3). The dominant negative mutant of p300 can clearly block TSA-induced p21 activation, further supporting the opinion that p300 functionally contributes to TSA-induced p21 activation. It has been reported that p300 works as a coactivator for various transcription activators, such as MyoD, p53, RAR, CREB, c-Jun, and for basal transcription factors, for example TBP (21.Yuan W. Condorelli G. Caruso M. Felsani A. Giordano A. J. Biol. Chem. 1996; 271: 9009-9013Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 22.Somasundaram K. El-Deiry W.S. Oncogene. 1997; 14: 1047-1057Crossref PubMed Scopus (162) Google Scholar, 23.Smith C.L. Onate S.A. Tsai M.J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8884-8888Crossref PubMed Scopus (368) Google Scholar). There is no report up to now implying the interaction of p300 and Sp1 family transcription factors. The same GC-box dependence of Sp1/Sp3 and p300 in p21 promoter activation reported in our present work, thereby, raises the possibility that p300 may cooperate with Sp1 family transcription factors as well. To address this question, the dominant negative mutant of p300 was cotransfected with Sp1 or Sp3 based on the idea that if the cooperative relationship is present, cellular knockout of p300 will abolish the function of Sp1/Sp3. Our results support this idea (Figs. 3 and 4). Owen, et al. (31.Owen G.I. Ricker J.K. Tung L. Takimoto G. Horowitz K.B. J. Biol. Chem. 1998; 273: 10696-10701Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar) reported recently that progesterone regulates transcription of p21 gene through Sp1 and CBP/p300. They showed data indicating that progesterone regulates p21 promoter activity through a Sp1 site and E1A can abolish the effect of progesterone on p21 promoter containing this Sp1 site. Given that other proteins except CBP/p300 also associate with E1A (32.Sandmoller A. Meents H. Arnold H.H. Mol. Cell. Biol. 1996; 16: 5846-5856Crossref PubMed Scopus (18) Google Scholar, 33.Mazzarelli J.M. Mengus G. Davidson I. Ricciardi R.P. J. Virol. 1997; 71: 7978-7983Crossref PubMed Google Scholar, 34.Sundqvist A. Sollerbrant K. Svensson C. FEBS Lett. 1998; 429: 183-188Crossref PubMed Scopus (89) Google Scholar), we think the result from cotransfecting with E1A may not be enough for supporting the involvement of p300 in this Sp1 site-mediated transcription. By comparing the different effect of p300 on wild type or GC-box-mutated promoter and utilizing its dominant negative mutant to suppress TSA- and Sp1/Sp3-induced transactivation, we provide direct evidence here that p300 can regulate the activity of the p21 promoter through Sp1-mediated transcription. p300 is also involved in p21 transcription through a p53-mediated way (22.Somasundaram K. El-Deiry W.S. Oncogene. 1997; 14: 1047-1057Crossref PubMed Scopus (162) Google Scholar, 30.Avantaggiati M.L. Ogryzko V. Gardner K. Giordano A. Levine A.S. Kell K. Cell. 1997; 89: 1175-1184Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). In that case, p300 physically interacts with p53 and is recruited by p53 to the basal transcription machinery. However, the molecular basis of the collaboration of p300 and Sp1 in p21 promoter regulation seems different. As the results from in vitro andin vivo experiments failed to provide any evidence that p300 directly interact with Sp1 (Figs. 5, 6), we considered that p300 and Sp1 might indirectly interact through a common multi-protein complex which is important for p21 transcription. Given that either p300 or Sp1 can interact with TBP and other basal transcription factors (35.Dallas P.B. Yaciuk P. Moran E. J. Virol. 1997; 71: 1726-1731Crossref PubMed Google Scholar, 36.Abraham S.E. Lobo S. Yaciuk P. Wang H.G. Moran E. Oncogene. 1993; 8: 1639-1647PubMed Google Scholar, 37.Emili A. Greenblatt J. Ingles C.J. Mol. Cell. Biol. 1994; 14: 1582-1593Crossref PubMed Scopus (226) Google Scholar), and that the Sp1 binding site in p21 promoter is so close to TATA box, this multi-protein complex might be the transcription initiation complex. Thus, we propose one hypothesis here, that the functional collaboration of p300 and Sp1 in p21 expression may rely on the effect of p300 on the activity of the initiation complex or the interaction of this complex with Sp1, which is the base for Sp1-mediated transcription in p21 activation. In fact, a multi-protein transcriptional complex containing TATA-binding protein, Sp1, and cAMP-response element-binding protein (CBP/p300) has been noticed recently (29.Shetty S. Takahashi T. Matsui H. Ayengar R. Raghow R. Biochem. J. 1999; 339: 751-758Crossref PubMed Scopus (42) Google Scholar). We prefer to say, based on the results presented here, that p300 is an indispensable factor for the p21 promoter activation, not only in a p53-mediated, DNA damage-responsible way but also in Sp1-mediated, basal and histone acetylation-effected way. Further work remains to be done to identify more details about the cooperation of p300 and Sp1. We thank Prof. Jonathan M. Horowitz (University of North Carolina), Prof. Thomas Shenk (Princeton University), Prof. Antonio Giordano (Thomas Jefferson University), Prof. David M. Livingston (Dana-Faber Cancer Institute), and Prof. Yoshihiro Nakatani (National Institutes of Health) for providing plasmids.

Plasminogen activator inhibitor-1 (PAI-1) is one of the primary inhibitors of the fibrinolytic system and has been implicated in a variety of thrombotic disorders. In this report, stress-induced changes in murine PAI-1 gene expression were investigated to study the role of this inhibitor in the development of stress-induced hypercoagulability. Restraint stress led to a dramatic induction of plasma PAI-1 antigen and of tissue PAI-1 mRNA with maximum induction in adipose tissues. In situ hybridization analysis of the stressed mice revealed that strong signals for PAI-1 mRNA were localized to hepatocytes, renal tubular epithelial cells, adrenomedullar chromaffin cells, neural cells in the paraaortic sympathetic ganglion, vascular smooth muscle cells, and adipocytes, but not to endothelial cells. These observations indicate that the stress induces the PAI-1 gene expression in a tissue-specific and cell type-specific manner. The induction of PAI-1 mRNA by restraint stress was greater than that observed for heat shock protein, a typical stress protein, suggesting that PAI-1 is one of the most highly induced stress proteins. Importantly, the magnitude of induction of PAI-1 mRNA by stress increased markedly with age, and this increase in PAI-1 correlated with tissue thrombosis in the older stressed mice. Moreover, much less tissue thrombosis was induced by restraint stress in young and aged PAI-1-deficient mice compared with age-matched wild-type mice. These results suggest that the large induction of PAI-1 by stress increases the risk for thrombosis in the older populations, and that the adipose tissue may be involved.

In Parkinson's disease, characteristic pathological features are the cell death of nigrostriatal dopamine neurons and the formation of Lewy bodies composed of oxidized proteins. Mitochondrial dysfunction and aggregation of abnormal proteins have been proposed to cause the pathological changes. However, the relation between these two factors remains to be clarified. In this study, the effects of mitochondrial dysfunction on the oxidative modification and accumulation of proteins were analyzed using an inhibitor of mitochondrial complex I, rotenone, and antibodies against acrolein- and dityrosine-modified proteins. Under conditions inducing mainly apoptosis in neuroblastoma SH-SY5Y cells, rotenone markedly increased oxidized proteins, especially those modified with acrolein, even though the increase in intracellular reactive oxygen and nitrogen species was only transient and was not so marked. In addition, the activity of the proteasome system degrading oxidized proteins was reduced profoundly after treatment with rotenone. The 20S beta subunit of proteasome was modified with acrolein, to which other acrolein-modified proteins were found to bind, as shown by coprecipitation with the antibody against 20S beta subunit. These results suggest that mitochondrial dysfunction, especially decreased activity of complex I, may reduce proteasome activity through oxidative modification of proteasome itself and aggregation with other oxidized proteins. This mechanism might account for the accumulation of modified protein and, at least partially, for cell death of the dopamine neurons in Parkinson's disease.

Histone deacetylase inhibitor-induced expression of p21WAF1 is p53 independent. In the present study, we provide evidence that trichostatin A (TSA), a specific inhibitor of histone deacetylase, can elevate H3 and H4 acetylation and p21WAF1 expression in NIH3T3 cells at first. To identify the transcription factor which is responsible for histone deacetylase inhibitor-induced expression of p21WAF1 and understand the potential events occurred during this process, we analyze the response of the mouse p21WAF1 promoter to TSA in detail. The region responsive to TSA treatment in the p21 promoter is located -100 bp upstream from transcription initiation site and contains a GC-box. The mutation introduced into this GC-box decreases most of the basal and TSA-induced promoter activity. The results from gel-shift assay show that Sp1 and Sp3 bind to this GC-rich region. Cotransfection with Sp1 and/or Sp3 expression constructs elevate both basal and induced promoter activity, and this elevation is dependent on the present of the GC-box. By contrast, cotransfection with reverse oriented Sp1 or Sp3 cDNA decreased basal and induced-promoter activity, as well as GC-box dependency. These findings provide physical and functional evidence which strongly indicated that both Sp1 and Sp3 are responsible for TSA-induced transactivation of the murine p21WAF1 promoter in NIH3T3 cells.

Peroxynitrite, a product of nitric oxide and superoxide, is one of the most potent oxidants and it has been suggested to be involved in many neurodegenerative disorders. The mechanism of the cytotoxicity by peroxynitrite was examined using 3-morpholinosydonimine (SIN-1) as a peroxynitrite donor and SH-SY5Y cells as a model of dopamine neurons. SIN-1 was found to induce apoptotic cell death with typical nucleosomal DNA fragmentation with activation of caspase 3-like proteases. The signal transduction of apoptosis was studied in concern to mitogen-activated protein kinases (MAPKs). After SIN-1 treatment, phosphorylation of p38 was detected, followed by that of Erk. SB202190, an inhibitor of p38, suppressed Erk phosphorylation to the basal level and partially reduced the activation of caspase 3-like proteases and also the cell death. These results suggest that peroxynitrite may activate p38 MAPK pathway to induce apoptosis in dopamine cells via activation of caspase 3-like proteases.

Peroxynitrite, one of the most reactive radicals, is produced from superoxide anion and nitric oxide. A peroxynitrite generator, 3-morpholinosydonimine (SIN-1), was found to induce the expression of three different growth arrest and DNA damage-inducible (GADD) mRNA, GADD34, GADD45, and GADD153, at the early phase during cell death in human neuroblastoma SH-SY5Y cells. In addition, peroxynitrite activated p38 MAPK just before induction of three GADD mRNA. A specific inhibitor of p38 MAPK, SB202190, markedly suppressed peroxynitrite-induced expression of three GADD mRNA in SH-SY5Y cells. The expression of three GADD genes and also p38 MAPK phosphorylation were suppressed by treatment with radical scavengers, superoxide dismutase plus catalase and glutathione. Glutathione depletion by L-buthionine-S, R-sulfoximine (BSO), increased the vulnerability of the cells to peroxynitrite. These findings indicate that peroxynitrite-mediated oxidative stress activated p38 MAPK to induce three GADD genes.

Research Article1 August 1985free access Structure of the murine Thy-1 gene. V. Giguére V. Giguére Search for more papers by this author K. Isobe K. Isobe Search for more papers by this author F. Grosveld F. Grosveld Search for more papers by this author V. Giguére V. Giguére Search for more papers by this author K. Isobe K. Isobe Search for more papers by this author F. Grosveld F. Grosveld Search for more papers by this author Author Information V. Giguére, K. Isobe and F. Grosveld The EMBO Journal (1985)4:2017-2024https://doi.org/10.1002/j.1460-2075.1985.tb03886.x PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have cloned the murine Thy-1.1 (AKR) and Thy-1.2 (Balb/c) genes. The complete exon/intron structure and the nucleotide sequence of the Thy-1.2 gene was determined. The gene contains four exons and three intervening sequences. The complete transcriptional unit gives rise to a tissue and developmental stage-specific mRNA of 1850 bp. The 5′ end of the gene has multiple initiation sites and a non-TATA box promoter. The 3′ end shows a single polyadenylation site after a very long untranslated region. Previous ArticleNext Article Volume 4Issue 81 August 1985In this issue RelatedDetailsLoading ...

One of the most important clinical problems in caring for elderly patients is treatment of pressure ulcers. One component of normal wound healing is the generation of an inflammatory reaction, which is characterized by the sequential infiltration of neutrophils, macrophages and lymphocytes. Neutrophils migrate early in the wound healing process. In aged C57BL/6 mice, wound healing is relatively inefficient. We examined the effects of neutrophil numbers on wound healing in both young and aged mice. We found that the depletion of neutrophils by anti-Gr-1 antibody dramatically delayed wound healing in aged mice. The depletion of neutrophils in young mice had less effect on the kinetics of wound healing. Intravenous G-CSF injection increased the migration of neutrophils to the wound site. While the rate of wound repair did not change significantly in young mice following G-CSF injection, it increased significantly in old mice.

CD8(+)CD122(+) regulatory T cells (CD8(+)CD122(+) Treg) are naturally occurring Treg that effectively suppress the proliferation and IFN-gamma production of both CD8(+) and CD4(+) target cells. This study investigated the molecular mechanisms of the recognition of target cells by CD8(+)CD122(+) Treg using an in vitro culture system that reconstitutes the regulatory action of these cells. Naive CD8(+)CD122(+) Treg co-cultured with pre-activated T cells became active Treg that produced IL-10 and suppressed IFN-gamma production from the target T cells. CD8(+)CD122(+) Treg effectively suppressed the IFN-gamma production of the target cells of syngeneic mouse strains but not of allogeneic mouse strains with incompatible MHC. By using MHC-congeneic mouse strains, MHC-restricted suppression by CD8(+)CD122(+) Treg was further confirmed. The blockade of cell surface molecules either on the Treg or on the target cells by specific blocking antibodies indicated that H-2K, H-2D, alphabetaTCR and CD8 were involved in the regulatory action but I-A and Qa-1 were not. These results indicate that CD8(+)CD122(+) Treg recognize already-activated T cells via the interaction of conventional MHC class I-alphabetaTCR and become active regulatory cells that produce IL-10 and suppress the target cells.

Abstract The importance of CD8 + CD122 + Treg in the maintenance of immune homeostasis has been previously demonstrated in mice. Because the expression pattern of CD8 and CD122 in humans is different from that in mice, human CD8 + Treg that correspond to the murine CD8 + CD122 + Treg have not been identified. In this study, we performed DNA microarray analyses to compare the gene expression profiles of CD8 + CD122 + cells and CD8 + CD122 − cells in mice and found that CXCR3 was preferentially expressed in CD8 + CD122 + cells. When we analyzed the expression of CD122 and CXCR3 in murine CD8 + cells, we observed a definite population of CD122 + CXCR3 + cells. CD8 + CXCR3 + cells in mice showed similar regulatory activities to CD8 + CD122 + cells by in vivo and in vitro assays. While CD8 + CD122 + CXCR3 + cells are present in mice, CD8 + CXCR3 + cells, but not CD8 + CD122 + cells, are present in humans. In the in vitro assay, human CD8 + CXCR3 + cells showed the regulatory activity of producing IL‐10 and suppressing IFN‐γ production from CD8 + CXCR3 − cells. These results suggest that human CD8 + CXCR3 + T cells are the counterparts of murine CD8 + CD122 + Treg.

Abstract We identified CD8+CD122+ regulatory T cells (Tregs) and demonstrated their importance in the maintenance of immune homeostasis and in the recovery from experimental autoimmune encephalomyelitis. In this paper, we show that CD8+CD122+ Tregs effectively prevent and cure colitis in a mouse model. In our experiments, colitis was induced in lymphocyte-deficient RAG-2−/− mice by transferring CD4+CD45RBhigh cells that were excluded with CD4+ Tregs. Cotransfer of CD8+CD122+ cells clearly suppressed the development of colitis, and this suppressive effect was similar to that of CD4+CD45RBlow cells that were mostly CD4+ Tregs. CD8+CD122+ cells obtained from IL-10−/− mice were unable to suppress colitis, indicating that IL-10 is an important effect-transmitting factor in the suppression of colitis. CD8+CD122+ cells showed a suppressive effect when they were transferred 4 wk after CD4+CD45RBhigh cells, indicating the therapeutic potential of CD8+CD122+ cells. A mixture of CD8+CD122+ cells and CD4+CD45RBlow cells was far more effective than single Tregs, indicating the synergistic effect of these Tregs. These overall findings demonstrate the potential role of CD8+ Tregs, and possibly together with CD4+ Tregs, in the medical care of inflammatory bowel disease patients.

Apigenin, a naturally occurring, non-mutagenic flavonoid, has been shown to inhibit UV-induced skin tumorigenesis in mice when topically applied. In this report we have used the mouse keratinocyte 308 cell line, which contains a wild-type p53 gene, to study the effect of apigenin treatment on p53 protein levels and the expression of its downstream partner, p21/waf1. Cells were treated with 70 microM apigenin for various times and levels of p53 and p21/waf1 protein were assessed by western blot analysis. The level of p53 protein was induced 27-fold after 4 h of apigenin treatment and levels remained elevated through 10 h of exposure. After 24 h of exposure to 70 microM apigenin, p53 protein levels returned to control levels. p21/waf1 protein levels increased approximately 1. 5-2-fold after 4 h and remained elevated at 24 h. To investigate the mechanism of p53 protein accumulation, we compared the half-life of p53 protein in vehicle- and apigenin-treated cells. Cells were incubated for 4 h in the presence of apigenin, then cycloheximide was added to inhibit further protein synthesis and p53 protein levels were measured by western blot. The half-life of p53 protein was found to be increased an average of 8-fold in apigenin-treated cells compared with vehicle-treated cells (t(1/2) = 131 min versus 16 min in apigenin- versus vehicle-treated cells, respectively). The mechanism of p53 protein stabilization is currently being investigated. To determine whether p53 was transcriptionally active, we also performed gel mobility shift assays and transient transfection studies using a luciferase plasmid under the control of the p21/waf1 promoter. Both p53 DNA-binding activity and transcriptional activation peaked after 24 h of exposure to apigenin. These studies suggest that apigenin may exert anti-tumorigenic activity by stimulating the p53-p21/waf1 response pathway.

Sodium butyrate, a histone deacetylase inhibitor, has been shown to induce differentiation of many cancer cells and senescence-like state of human fibroblasts. Here we show that sodium butyrate also induces senescence-like state of NIH3T3 cells. The treated cells were blocked at G1 phase and featured morphologically like senescent cells with enlarged cytoplasm and multiple nuclei. The expression of p21(WAF/CIP1) (p21) increased after sodium butyrate treatment at transcriptional level. To analyze the induction of promoter activity, we isolated 4.6 kb of murine p21 promoter and inserted it upstream of a luciferase reporter gene. When this construct was transiently transfected into NIH3T3 cells, sodium butyrate enhanced the luciferase activity. p53 independency of sodium butyrate-inducible p21 promoter activity was confirmed by using the deletion mutants lacking p53 binding sites and p53 deficient cells in transfection experiments.

Alzheimer’s disease is characterized by the presence of senile plaques in the brain composed primarily of amyloid-β peptide. Microglia have been reported to surround these Aβ plaques, which have opposite roles, provoking a microglia-mediated inflammatory response that contributes to neuronal cell loss or the removal of Aβ and damaged neurons. We herein analyzed the process of expression of Matrix metalloproteinases induced by Aβ stimulation. We found that Aβ1-42 induces a high level of MMP3, MMP12 and MMP13 in the microglia. The signal transduction pathway for the expression of these MMPs mRNA induced by Aβ1-42 depends on PI3K/Akt.

Impaired or deficient autophagy is believed to cause or contribute to aging, as well as several age-related pathologies. Thymic epithelial cells had a high constitutive level of autophagy. The autophagic process may play a supporting role or even a crucial role in the presentation of self-Ags in the thymus to shape the T-cell repertoires. Autophagic activity in the liver is important for the balance of energy and nutrients for basic cell functions. The abundance of autophagic structure in both cortical and medullary thymic epithelial cells and liver with mouse age has not been examined in detail. Here, we demonstrated that the architecture of mouse thymus and liver markedly changed with age. We found that the expression of LC3 detected by immunofluorescence and Western blot analysis was greatly decreased in thymus and liver of 12-month-old mice. The same level of reduction was observed in thymus and liver of 24-month-old mice. Ultrastructure analysis by an electron microscope revealed that the number of autophagic structure/vacuole in total thymic epithelial cells and hepatocytes decrease with age. The age-related decrease of autophagic structure in thymic epithelial cells may cause the reduction of immunocompetent T-cell pool in aged mice. The age-related decrease of autophagy in liver may induce accumulation of cellular materials in liver of aged mice.

Acrolein, a compound found in cigarette smoke, is a major risk factor for respiratory diseases. Previous research determined that both acrolein and cigarette smoke produced reactive oxygen species (ROS). As many types of pulmonary injuries are associated with inflammation, this study sought to ascertain the extent to which exposure to acrolein advanced inflammatory state in the lungs. Our results showed that intranasal exposure of mice to acrolein increased CD11c+F4/80high macrophages in the lungs and increased ROS formation via induction of NF-κB signaling. Treatment with acrolein activated macrophages and led to their increased production of ROS and expression of several key pro-inflammatory cytokines. In in vitro studies, acrolein treatment of bone marrow-derived GM-CSF-dependent immature macrophages (GM-IMs), activated the cells and led to their increased production of ROS and expression of several key pro-inflammatory cytokines. Acrolein treatment of macrophages induced apoptosis of lung epithelial cells. Inclusion of an inhibitor of ROS formation markedly decreased acrolein-mediated macrophage activation and reduced the extent of epithelial cell death. These results indicate that acrolein can cause lung damage, in great part by mediating the increased release of pro-inflammatory cytokines/factors by macrophages.

Angiogenic cell therapy represents a novel strategy for ischemic diseases, but some patients show poor responses. We investigated the therapeutic potential of an induced pluripotent stem (iPS) cell sheet created by a novel magnetite tissue engineering technology (Mag-TE) for reparative angiogenesis. Mouse iPS cell-derived Flk-1(+) cells were incubated with magnetic nanoparticle-containing liposomes (MCLs). MCL-labeled Flk-1(+) cells were mixed with diluted extracellular matrix (ECM) precursor and a magnet was placed on the reverse side. Magnetized Flk-1(+) cells formed multi-layered cell sheets according to magnetic force. Implantation of the Flk-1(+) cell sheet accelerated revascularization of ischemic hindlimbs relative to the contralateral limbs in nude mice as measured by laser Doppler blood flow and capillary density analyses. The Flk-1(+) cell sheet also increased the expressions of VEGF and bFGF in ischemic tissue. iPS cell-derived Flk-1(+) cell sheets created by this novel Mag-TE method represent a promising new modality for therapeutic angiogenesis.

Although older subjects are susceptible to thrombosis under septic conditions, the underlying molecular mechanisms have not been fully elucidated. Since elevated plasminogen activator inhibitor-1 (PAI-1) primarily contributes to endotoxin-induced thrombosis, we first compared the induction of PAI-1 by lipopolysaccharide (LPS) between young and aged mice. The higher induction of PAI-1 antigen and mRNA with increased renal glomerular fibrin deposition was observed in LPS-treated aged mice compared to young mice. In situ hybridization analysis showed that the aging-associated induction of PAI-1 mRNA by LPS was pronounced in hepatocytes and in renal glomerular cells. The increased magnitude of the response of aged mice to lower doses of LPS was observed in terms of renal glomerular fibrin deposition and PAI-1 mRNA induction in the tissues. Furthermore, older PAI-1 deficient mice treated with LPS developed much less fibrin deposition in kidneys. Importantly, a larger induction of receptor molecules for LPS (eg, CD14 and Toll-like receptor 4) was demonstrated in LPS-treated aged mice as compared with young mice. The enhanced LPS signaling in aged mice was also demonstrated by the marked induction of nuclear factor-kappaB in the tissues after endotoxin treatment. As a consequence, increases in an inflammatory cytokine, tumor necrosis factor-alpha, were pronounced in plasma and tissues of LPS-treated aged mice. These results emphasize the key role played by PAI-1 in aging-associated deterioration in this thrombosis model, and suggest that the hyperresponse of PAI-1 gene to LPS results from the enhanced LPS signaling and the subsequent inflammatory response in aged mice.

We have used the yeast one-hybrid system to clone transcription factors that bind to specific sequences in the proximal promoters of the type I collagen genes. We utilized as bait the sequence between −180 and −136 in the pro-α2(I) collagen promoter because it acts as a functional promoter element and binds several DNA-binding proteins. Three cDNA clones were isolated that encoded portions of the mouse SPR2 transcription factor, whereas a fourth cDNA contained a potential open reading frame for a polypeptide of 775 amino acids and was designated BFCOL1. Recombinant BFCOL1 was shown to bind to the −180 to −152 segment of the mouse pro-α2(I) collagen proximal promoter and to two discrete sites in the proximal promoter of the mouse pro-α1(I) gene. The N-terminal portion of BFCOL1 contains its DNA-binding domain. DNA transfection experiments using fusion polypeptides with the yeast GAL4 DNA-binding segment indicated that the C-terminal part of BFCOL1 contained a potential transcriptional activation domain. We speculate that BFCOL1 participates in the transcriptional control of the two type I collagen genes. We have used the yeast one-hybrid system to clone transcription factors that bind to specific sequences in the proximal promoters of the type I collagen genes. We utilized as bait the sequence between −180 and −136 in the pro-α2(I) collagen promoter because it acts as a functional promoter element and binds several DNA-binding proteins. Three cDNA clones were isolated that encoded portions of the mouse SPR2 transcription factor, whereas a fourth cDNA contained a potential open reading frame for a polypeptide of 775 amino acids and was designated BFCOL1. Recombinant BFCOL1 was shown to bind to the −180 to −152 segment of the mouse pro-α2(I) collagen proximal promoter and to two discrete sites in the proximal promoter of the mouse pro-α1(I) gene. The N-terminal portion of BFCOL1 contains its DNA-binding domain. DNA transfection experiments using fusion polypeptides with the yeast GAL4 DNA-binding segment indicated that the C-terminal part of BFCOL1 contained a potential transcriptional activation domain. We speculate that BFCOL1 participates in the transcriptional control of the two type I collagen genes.

Abstract We have established a novel injury model in the central nervous system by a stereotaxic injection of ethanol into rat striatum to induce necrosis. With this model, we clarify a function of inducible nitric oxide synthase (iNOS) in a healing mechanism around a necrotic lesion. A semiquantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) revealed that the iNOS mRNA arose at 6 h, peaked at 24 h, and declined to a lower level 48 h after an intrastriatal 5‐μL ethanol injection. From in situ hybridization, this iNOS mRNA was expressed in the area surrounding the injury. By immunohistochemistry, mononuclear cells at this boundary area of necrosis were stained with anti‐iNOS antibody on the first day after the injury. These cells turned out to be reactive microglia from the positive staining of GSA‐I‐B 4 , ED‐1 and OX‐42. Haematoxylin‐eosin (HE) staining showed that neurons in this boundary area gradually disappear up to 5 days after the injury with an increment of microglial cells, and this area became cavernous. Nuclei of neurons in this area were stained positive by the terminal deoxynucleotidyl‐transferase‐mediated dUTP‐biotin nick end‐labelling (TUNEL) assay on the first day after the injury. These TUNEL‐positive neurons gradually disappeared toward the third day, while microglial cells increased. L‐Ng‐nitro‐arginine methylester (L‐NAME), a competitive NOS inhibitor, administration diminished the elimination of neurons by microglia in this boundary area surrounding necrosis. Microglial NO may act as a neurotoxic agent to eliminate damaged neurons near the necrosis in the form of delayed neuronal death, and may reintegrate the neuronal circuits with functionally intact neurons.

In spite of its reputation as a tumour marker, little is known about the function of carcinoembryonic antigen (CEA). We examined the mRNA expression of CEA and NCA in 26 gastric and 14 colorectal cancers together with adjacent morphologically normal mucosae. There was no significant difference between the CEA mRNA levels of colorectal cancer and adjacent mucosa, whereas the CEA mRNA levels were significantly elevated in gastric cancer compared with normal gastric mucosa. The expression of NCA, on the other hand, was more cancer-specific and was significantly up-regulated in both gastric and colorectal cancers compared with the corresponding normal mucosae. No correlation was found between the mRNA level and plasma CEA value. No significant up-regulation was recognised in the node positive cancer, cancer with distant metastasis, or the metastatic tissues themselves. Marked diversity in the degree of differentiation in gastric cancer tissues, however, resulted in varied expression of the CEA gene family, compared with the constantly high expression found in colorectal cancer. Further analysis revealed significant up-regulation of NCA in well and moderately differentiated gastric cancers over poorly differentiated cancers, suggesting that NCA might have roles in differentiation.

Abstract Pluripotent embryonic stem (ES) cells hold great potential for cell-based therapies. Although several recent studies have reported the potential of ES cell-derived progenitors for skeletal muscle regeneration, how the cells contribute to reconstitution of the damaged myofibers has remained elusive. Here, we demonstrated the process of injured muscle regeneration by the engraftment of ES cell-derived mesodermal progenitors. Mesodermal progenitor cells were induced by a conventional differentiation system and isolated by flow cytometer of platelet-derived growth factor receptor-α (PDGFR-α), a marker of paraxial mesoderm, and vascular endothelial growth factor receptor-2 (VEGFR-2), a marker of lateral mesoderm. The PDGFR-α+ population that represented the paraxial mesodermal character demonstrated significant engraftment when transplanted into the injured muscle of immunodeficient mouse. Moreover, the PDGFR-α+ population could differentiate into the muscle satellite cells that were the stem cells of adult muscle and characterized by the expression of Pax7 and CD34. These ES cell-derived satellite cells could form functional mature myofibers in vitro and generate myofibers fused with the damaged host myofibers in vivo. On the other hand, the PDGFR-α−VEGFR-2+ population that showed lateral mesodermal character exhibited restricted potential to differentiate into the satellite cells in injured muscle. Our results show the potential of ES cell-derived paraxial mesodermal progenitor cells to generate functional muscle stem cells in vivo without inducing or suppressing gene manipulation. This knowledge could be used to form the foundation of the development of stem cell therapies to repair diseased and damaged muscles. Disclosure of potential conflicts of interest is found at the end of this article.

Systemic sclerosis (SSc) is an autoimmune disease characterized by the excessive deposition of collagen in the skin or other organs and the production of specific antinuclear antibodies (ANAs). Recently, bleomycin (BLM)-induced experimental scleroderma was reported in a murine model. Here, we present further development of this model and suggest that it is appropriate for the analysis of human diffuse type SSc. BLM was injected into the shaved backs of C3H or BALB/c mice (100 microg/mouse) 5 days per week for 3 weeks. Skin fibrosis was confirmed and pathological changes were seen in the lower part of the esophagus and stomach similar to those seen in SSc. The sera from these mice had autoantibodies specific to the damaged tissues and ANAs. Transfer of CD4(+) T cells from BLM-treated BALB/c mice induced the same pathological changes and antibody production in untreated-BALB/c nude mice. Hence, tissue fibrosis and the production of ANAs are probably associated with CD4(+) T-cell activity in this model. In conclusion, this model will be valuable for investigating the relationship between tissue fibrosis and abnormalities of the immune system.

While Gr1(+)CD11b(+) cells are known to regulate immune responses and accumulate in most cancer tissues, the function of Gr1(+)CD11b(+) cells in inflammation is poorly understood. We investigated the role of Gr1(+)CD11b(+) cells in a dextran sulphate sodium (DSS)-treated mouse model of ulcerative colitis (UC). C57BL/6 mice were treated with 2% DSS in drinking water for 5 days. Disease progression and recovery were assessed by body weight, disease activity index score (DAI) score and colon length. Splenic Gr1(+)CD11b(+) cell number was greatly increased during the recovery phase of DSS-induced colitis. DSS-derived splenic Gr1(+)CD11b(+) cells were administered intravenously to recipient (C57BL/6) mice during the early phase of DSS treatment. The transplanted splenic DSS-induced Gr1(+)CD11b(+) cells improved DSS-induced colitis and promoted efficient colonic mucosal healing. We found that the CD11b(+) single positive cells increased in the course of DSS-induced colitis in lamina propria. The transplantation of splenic Gr1(+)CD11b(+) cells induced feedback suppression of myeloid-lineage cell development. Namely, the transplantation of splenic Gr1(+)CD11b(+) cells greatly suppressed the migration of CD11b(+) single positive cells to the lamina propria. Further, transplantation of Gr-1(+)CD11b(+) cells greatly suppressed the increase of the same population, especially during the late phase of DSS colitis both in spleen and bone marrow.

If induced pluripotent stem (iPS) cells are to be used to treat damaged tissues or repair organs in elderly patients, it will be necessary to establish iPS cells from their tissues. To determine the feasibility of using this technology with elderly patients, we asked if it was indeed possible to establish iPS cells from the bone marrow (BM) of aged mice. BM cells from aged C57BL/6 mice carrying the green fluorescence protein (GFP) gene were cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) for 4 days. Four factors (Oct3/4, Sox2, Klf4 and c-Myc) were introduced into the BM-derived myeloid (BM-M) cells. The efficiency of generating iPS cells from aged BM cultured in GM-CSF was low. However, we succeeded in obtaining BM-M-iPS cells from aged C57BL/6 mice, which carried GFP. Our BM-M-iPS cells expressed SSEA-1 and Pou5f1 and were positive for alkaline phosphatase staining. The iPS cells did make teratoma with three germ layers following injection into syngeneic C57BL/6 mice, and can be differentiated to three germ layers in vitro. By co-culturing with OP9, the BM-M-iPS cells can be differentiated to the myeloid lineage. The differentiated BM-M-iPS cells proliferated well in the presence of GM-CSF, and lost expression of Nanog and Pou5f1, at least in part, due to methylation of their promoters. On the contrary, Tnf and Il1b gene expression was upregulated and their promoters were hypomethylated.

Dextran sulfate sodium (DSS) is commonly used in rodent IBD models to chemically induce acute intestinal inflammation. The acute course of colitis includes colon tissue damages and recovery from wounded tissues. As skin wound repair was delayed by splenectomy, we asked whether splenectomy would induce the delay of colonic wound healing. In splenectomized mice, body weight recovery, disease score and colon length were delayed. Surprisingly we found a great increase of Gr1+CD11b+ cells in spleen and bone marrow of DSS-administered mice. Anti-Gr-1 antibody treatment worsened the DSS- administered colitis. These results indicate that Gr1+CD11b+ cells induced by DSS worked to repair colon wound healing and repair colitis. Keywords: Ulcerative colitis, DSS, Gr-1, CD11b, myeloid-lineage, Inflammatory bowel disease (IBD), gastrointestinal, Crohn's disease, atherosclerosis, hypercholesterolemic, splenectomy, secreting antibodies, Dextran sulfate sodium (DSS), bloody diarrhea, epithelial cell damage, cell infiltration, oral administration, Biomedical, Colon Histology, epithelium, Disease Activity Index, gross bleeding, stool consistency, Immune Cells, myeloid cell, Lamina propria cells, Biosciences, thymus, bone marrow, Colon lengths, parasites, fungus, Trypanosoma cruzi infection, autoimmune, encephalomyelitis, high inflammatory monocytes

The wound repair process is a highly ordered sequence of events that encompasses haemostasis, inflammatory cell infiltration, tissue regrowth and remodelling. Wound healing follows tissue destruction so we hypothesized that antibodies might bind to wounded tissues, which would facilitate the engulfment of damaged tissues by macrophages. Here, we show that B cells, which produce antibodies to damaged tissues, are engaged in the process of wound healing. Splenectomy delayed wound healing, and transfer of spleen cells into splenectomized mice recovered the delay in wound healing. Furthermore, wound healing in splenectomized nude mice was also delayed. Transfer of enriched B220(+) cells by magnetic beads accelerated wound healing in splenectomized mice. We detected immunoglobulin G1 (IgG1) binding to wounded tissues by using fluorescein isothiocyanate-labelled anti-IgG1 6-24 hr after wounding. Splenectomy reduced the amount of IgG1 binding to wounded tissues. Immunoblotting studies revealed several bands, which were reduced by splenectomy. Using immunoprecipitation with anti-IgG bound to protein G we found that the intensity of several bands was lower in the serum from splenectomized mice than in that from sham-operated mice. These bands were matched to myosin IIA, carbamoyl-phosphate synthase, argininosuccinate synthase, actin and alpha-actinin-4 by liquid chromatography tandem mass spectrometry analysis.

An incorrect file for Movie S2 was uploaded with this manuscript. Please view the correct Movie S2 here: http://www.plosone.org/corrections/pone.0061540.001.cn.wmv

In Japan, the number of patients with streptococcal toxic shock syndrome is reported to be increasing. mef(A) gene-positive macrolide-resistant emm1 strains are thought to possibly contribute to the rise in the frequency of STSS. Although analyses of macrolide-resistant mechanisms, including mef(A) resistance, have been performed mainly in Streptococcus pneumoniae, the role of this gene in Streptococcus pyogenes has not been completely investigated. Therefore, to the best of our knowledge, we established the first mef(A)-knockout strain using an emm1-type S. pyogenes strain, and tested its susceptibility to erythromycin, clarithromycin and azithromycin. We found that the antimicrobial susceptibilities were almost identical to those of the parental strain. Hence, we established a knockout strain for another gene, msr(D), that is located immediately downstream of mef(A). The macrolide resistances of the resulting strain significantly decreased, and were further altered when both mef(A) and msr(D) were knocked out. The introduction of the msr(D) gene into a macrolide-sensitive strain conferred more resistance than the introduction of the mef(A) gene. The erythromycin susceptibilities of knockout strains were further dissected using two additional emm4- and emm75-type S. pyogenes strains. We found almost identical results for both strains except for the mef(A) knockout emm4 type, whose susceptibility was altered, although the change was less than that for the msr(D) knockout. These results suggest that both mef(A) and msr(D) are involved in macrolide resistance in S. pyogenes, and that the msr(D) gene plays a more predominant role in macrolide resistance than mef(A).

(−)Deprenyl, a monoamine oxidase B (MAO B) inhibitor is known to upregulate activities of anti-oxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) in brain dopaminergic regions. The drug is also the sole chemical which has been repeatedly shown to increase life spans of several animal species including rats, mice, hamsters and dogs. Further, the drug was recently found to enhance anti-oxidant enzyme activities not only in brain dopaminergic regions but also in extra-brain tissues such as the heart, kidneys, adrenal glands and the spleen. We and others have also observed mobilization of many humoral factors (interferone (INF)-γ, tumor necrosis factor (TNF)-α, interleukine (IL)-1β,2,6, trophic factors, etc.) and enhancement of natural killer (NK) cell functions by (−)deprenyl administration. An apparent extension of life spans of experimental animals reported in the past may be better explained by these new observations that (−)deprenyl upregulate SOD and CAT activities not only in the brain but also in extra-brain vital organs and involve anti-tumorigenic as well as immunomodulatory effect as well. These combined drug effects may lead to the protection of the homeostatic regulations of the neuro-immuno-endocrine axis of an organism against aging.

Background. Gene expression of nm23 has been investigated in number of tumors, including breast cancer, colon cancer, malignant melanoma, and hepatocellular carcinoma. Its down-regulation has been shown to be associated with metastasis or disease progression in some of the tumors. Methods. nm23 H-1 mRNA levels were investigated in 31 surgically resected specimens of gastric cancer by Northern blot analysis, and association with clinical stages was determined. Results. There was no significant difference in nm23 expression between tumor and matching normal mucosa. There was a significant down-regulation of the nm23 gene in gastric cancer with serosal invasion (T3 and T4 by the TNM classification) and nodal metastasis (pN1 and pN2), although the association of the down-regulation with clinically manifest hepatic or peritoneal metastasis was not significant. The tumors with low nm23 expression were associated with a poor prognosis. Conclusions. It was suggested that the down-regulation of nm23 gene might have a role in metastasis and invasion in gastric cancer, possibly leading to a poor prognosis.

In this study, we investigated the expression of genes for inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6) of Kupffer cells in the presence of lipopolysaccharide (LPS), and the tissue expression of iNOS in a rat liver after LPS injection at various time intervals. The effects of L-NG-nitroarginine-methyl-esther HCI (L-NAMF), a NO inhibitor, also were examined. The mRNA transcripts of TNF-α IL-1β and IL-6 were rapidly detected no more than at 1 h after LPS stimulation, whereas the iNOS transcript was detectable from 3 h after LPS stimulation and maximally increased at 12 h. This fact suggested that these early induced cytokines were related to expression of iNOS. Using an anti-iNOS antiserum raised against recombinant iNOS protein, immunohistochemical analysis was made to reveal kinetics of NO producing cells. The cells immunoreactive for iNOS appeared at 6 h post-LPS injection in the sinusoids of the liver. By structural and immunohistochemical studies, almost all iNOS positive cells were identified as Kupffer cells and endothelial cells. The number of cells immunoreactive for iNOS increased until 12 h post-LPS injection. At 24 h after LPS injection, iNOS positive cells were restricted to the foci of spotty necrosis. Hepatic injury measured by released enzymes was increased by pretreatment of L-NAME before LPS injection. J. Cell. Biochem. 65:349–358. © 1997 Wiley-Liss, Inc.

A novel superfamily of guanine nucleotide exchange factors for Rho GTPases includes DOCK180 and zizimin1. The zizimin subfamily includes three genes of which only zizimin1 has been cloned. We report here the cloning of zizimin2, identified in a screen for genes enriched in germinal center B cells. Zizimin2 and zizimin1 have similar primary structures and both proteins bound and activated Cdc42 but not the Cdc42-related proteins TC10 or TCL. Their tissue distributions are distinct, however, with zizimin2 expressed predominantly in lymphocytes and an opposite pattern for zizimin1. Zizimin3 was also analyzed and showed distinct GTPase specificity and tissue distribution.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that induces immune tolerance in mice. Our prior study showed that high tumoral IDO expression in endometrial cancer tissues correlates with disease progression and impaired patient survival. The purpose of the present study was to clarify the functional role of IDO in human endometrial cancer cells and to investigate the therapeutic potential of IDO inhibitors.IDO cDNA was transfected into the human endometrial carcinoma cell line AMEC, resulting in the establishment of stable clones of IDO-overexpressing AMEC cells (AMEC-IDO). AMEC-IDO cells were characterized in vitro as well as in vivo using a mouse xenograft model.There was no significant difference in in vitro cell proliferation, migration, or chemosensitivity to paclitaxel between AMEC-IDO and control vector-transfected cells (AMEC-pcDNA). However, in vivo tumor growth was markedly enhanced in AMEC-IDO-xenografted nude mice when compared with AMEC-pcDNA-xenografted mice. Splenic natural killer (NK) cell counts in AMEC-IDO-xenografted mice were significantly decreased when compared with control mice. Furthermore, conditioned medium obtained from AMEC-IDO cell cultures markedly reduced the NK lysis activity of nude mice. Finally, oral administration of the IDO inhibitor 1-methyl-D-tryptophan in combination with paclitaxel in AMEC-IDO-xenografted mice strongly potentiated the antitumor effect of paclitaxel, resulting in significantly prolonged survival.This is the first evidence showing that IDO overexpression in human cancer cells contributes to tumor progression in vivo with suppression of NK cells. Our data suggest that targeting IDO may be a novel therapeutic strategy for endometrial cancer.

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism. IDO is immunosuppressive and is induced by inflammation in macrophages and dendritic cells (DCs). Previous studies have shown the serum Kyn/Trp levels in patients with hemolytic anemia to be notably high. In the present study, we demonstrated that hemoglobin (Hb), but not hemin or heme-free globin (Apo Hb), induced IDO expression in bone marrow-derived myeloid DCs (BMDCs). Hb induced the phosphorylation and degradation of I kappaB alpha. Hb-induced IDO expression was inhibited by inhibitors of PI3-kinase (PI3K), PKC and nuclear factor (NF)-kappaB. Hb translocated both RelA and p52 from the cytosol to the nucleus and induced the intracellular generation of reactive oxygen species (ROS). Hb-induced IDO expression was inhibited by anti-oxidant N-acetyl-L-cysteine (NAC) or mixtures of SOD and catalase, however, IDO expression was enhanced by 3-amino-1,2,4-triazole, an inhibitor of catalase, suggesting that the generation of ROS such as O(2) (-), H(2)O(2), and hydroxyl radical is required for the induction of IDO expression. The generation of ROS was inhibited by a PKC inhibitor, and this action was further enhanced by addition of a PI3K inhibitor. Hb induced Akt phosphorylation, which was inhibited by a PI3K inhibitor and enhanced by a PKC inhibitor. These results suggest that the activation of NF-kappaB through the PI3K-PKC-ROS and PI3K-Akt pathways is required for the Hb-induced IDO expression in BMDCs.

Thymic epithelial cells (TECs) are present in both cortical and medullary thymic areas, and have crucial roles in functional T-cell development. In this study, we studied the differentiation of induced pluripotent stem cells (iPSCs) to TEC. When iPSC were cultured for 4 days in collagen IV-coated dishes in the presence of both activin A and lithium chloride (LiCl), the cells differentiated to definitive endoderm through mesendoderm. Further treatment with Fgf8 followed by Fgf7, Fgf10 and BMP4 differentiated iPSC to thymic epithelial progenitor cells (TEPCs) by phenotype. Gene expression of Hoxa3, Pax1 and Pax9 was observed and cell surface proteins EpCAM1 and MTS24 were detected at day 14 of iPSC differentiation. TEPCs differentiated to medullary TECs (mTECs) by phenotype following the addition of receptor activator nuclear factor B ligand with LiCl. Thus, we successfully induced efficient differentiation from mouse iPSC to TEPCs and mTEC by phenotype using chemically defined conditions.

Induced pluripotent stem (iPS) cells are the novel stem cell population induced from somatic cells. It is anticipated that iPS will be used in the expanding field of regenerative medicine. Here, we investigated whether implantation of fetal liver kinase-1 positive (Flk-1+) cells derived from iPS cells could improve angiogenesis in a mouse hind limb model of ischemia.Flk-1+ cells were induced from iPS cells after four to five days of culture. Hind limb ischemia was surgically induced and sorted Flk-1+ cells were directly injected into ischemic hind limbs of athymic nude mice. Revascularization of the ischemic hind limb was accelerated in mice that were transplanted with Flk-1+ cells compared with control mice, which were transplanted with vehicle, as evaluated by laser Doppler blood flowmetry. Transplantation of Flk-1+ cells also increased expression of VEGF mRNA in ischemic tissue compared to controls.Direct local implantation of iPS cell-derived Flk-1+ cells would salvage tissues from ischemia. These data indicate that iPS cells could be valuable in the therapeutic induction of angiogenesis.

Several types of cellular stress induce expression of growth arrest and DNA damage protein 34 (Gadd34). Autophagy occurs under both basal conditions and conditions of stress, such as starvation. Gadd34 and autophagy are both induced under starvation conditions. In this study we found that starvation induced the expression of Gadd34, reduced mTOR activity, and induced autophagy in wild type mice, but not Gadd34 KO mice. Gadd34 bound to and dephosphorylated pTSC2 at Thr1462. Dephosphorylation of TSC2 during the starvation time period leads to the suppression of mTOR, which is a potent inhibitor of autophagy. We concluded that starvation-induced Gadd34 suppresses mTOR and, thereby, induces autophagy.

There is no consensus on the best surgical treatment for deep-seated atypical lipomatous tumor (ALT) of the extremities; furthermore, the appropriate duration for follow-up observation remains unclear. We investigated clinical and functional median-term outcomes in the primary operations for ALT of the extremities in order to find its best treatment methods and observation periods.From 1996 to 2009, we diagnosed 41 patients with deep-seated ALT of the extremities. Wide resection was performed on 11 patients and marginal resection was performed on 30 patients. The minimum follow-up was 5 years (median, 8.5; range, 5-17.4). Patients were evaluated for their local recurrence, dedifferentiation, and post-operative function using the ISOLS/MSTS scoring system.Recurrence and dedifferentiation rates were both 0% for the wide resection group, while the rates were 23% (7/30) and 3% (1/30) for the marginal resection group, respectively. Median duration before recurrence was 7.2 years (range, 4.0-14.2). Local recurrence-free survival rate was significantly higher in the wide resection group (P = 0.013). In the marginal resection group, 10% (3/30) of the cases showed residual tumor. The localization of these tumors was all intermuscular. The ISOLS/MSTS scores were 98% (range, 90-100) for wide resection and 99% (range, 93-100) for marginal resection, with no statistical difference (P = 0.694). No ALT-related deaths occurred during the observation period.In addition to long-term (at least 8 years) of continuous observation, a wide resection is necessary in order to prevent recurrence, dedifferentiation, and residual tumor.

Chronic obstructive pulmonary disease (COPD) is characterized by lung destruction and inflammation. As a major compound of cigarette smoke, acrolein plays a critical role in the induction of respiratory diseases. GADD34 is known as a growth arrest and DNA damage-related gene, which can be overexpressed in adverse environmental conditions. Here we investigated the effects of GADD34 on acrolein-induced lung injury. The intranasal exposure of acrolein induced the expression of GADD34, developing the pulmonary damage with inflammation and increase of reactive oxygen species (ROS). Conversely, the integrality of pulmonary structure was preserved and the generation of ROS was reduced in GADD34-knockout mice. Acrolein-induced phosphorylation of eIF2α in GADD34-knockout epithelial cells by shRNA protected cell death by reducing misfolded protein-caused oxidative stress. These data indicate that GADD34 participates in the development of acrolein-induced lung injury.

Inflammatory bowel disease confers an increased risk of developing colitis-associated colon cancer (CAC). During the active colitis or developing tumor stage, commensal bacteria show dynamic translocation. However, whether alteration of the bacterial composition in the gut causes CAC is still unclear. To clarify the effect of commensal bacteria on CAC development, we employed an azoxymethane (AOM) and dextran sodium sulfate (DSS)-induced murine CAC model treated with or without antibiotics. In addition, we analyzed the effects of antibiotics on infiltration of myeloid cells, colonic inflammatory responses, and colorectal cancer formation. We found that vancomycin treatment dramatically suppressed tumor development. In addition, AOM/DSS treatment greatly induced the infiltration of Gr-1(high)/CD11b(high) neutrophils to the colon, which led to the production of tumor necrosis factor α and inducible nitric oxide synthase. Vancomycin treatment suppressed the infiltration of neutrophils induced by AOM/DSS. Moreover, vancomycin treatment greatly reduced the colon injury and DNA damage caused by AOM/DSS-induced NO radicals. Our results indicate that vancomycin-sensitive bacteria induced colon inflammation and DNA damage by attracting neutrophils into damaged colon tissue, thus promoting tumor formation.

Abstract The role of glial cell line‐derived neurotrophic factor (GDNF) in the survival of dopaminergic neurons has been well documented, but its effect on dopamine biosynthesis remains to be elucidated. In this study, the effect of GDNF on the gene expression of tyrosine hydroxylase (TH), the rate‐limiting enzyme of dopamine biosynthesis, was investigated. We found that GDNF elevated the expression of the TH gene at both mRNA and protein levels in TGW cells, a human neuroblastoma cell line. GDNF significantly enhances the transcription rate of the TH gene as actinomycin D prevented the induction of TH mRNA and GDNF increased the activity of the TH promoter. In addition, GDNF exerts a relatively weak but significant effect on the stability of TH mRNA, because GDNF delayed the degradation of TH mRNA and strengthened a special TH mRNA/protein interaction known to be relevant with TH mRNA stability. By comparing several human neurogenic cell lines, we found that GDNF‐induced TH expression was only observed in the cells possessing Ret protein and coincided with the expression levels. Taken together, these results indicate that GDNF up‐regulates the expression of the TH gene by promoting the transcription of the TH gene and the stability of TH mRNA with the Ret receptor dependency in some neuroblastoma cell lines. Thus, GDNF exerts its neurotrophic role not only in promoting cells survival, but also in affecting dopamine biosynthesis.

Recently, others and we have shown that one of the functions of GADD34 is a recovery from a shutoff of protein synthesis induced by endoplasmic reticulum stress. GADD34 has been shown to induce growth arrest and apoptosis. Main protein of apoptosis is p53, especially phosphorylation of p53. And one of the main proteins of growth arrest is p21/WAF1. Here we analyzed the effects of GADD34 on p53 phosphorylation and p21/WAF1 transcription. Transfected Myc-tagged p53 was dose-dependently phosphorylated at Ser15 by increasing the amount of GADD34. Transfection of GADD34 also induced the endogenous phosphorylation of p53 and enhanced p21 protein expression. Transfection of GADD34 induced p21/WAF1 promoter activity. This activity was dependent on p53, because GADD34 transfection to p53-deficient cells produced only a slight increase of p21/WAF1 promoter activity. The p21/WAF1 promoter activity was greatly enhanced by the transfection of p53. Both GADD34 and p53 transfection induced much higher p21/WAF1 promoter activity. The promoter activity of p21/WAF1 was very low in GADD34 deficient MEF. The transfection of GADD34 increased the p21/WAF1 promoter activity in GADD34 deficient MEF.

The immunoregulatory action of saikosaponin-d (SSd), which was isolated from the root of Bupleurum falcatum L. and has a steroid-like structure, was examined on splenic T lymphocytes of C57BL/6 mice. SSd displayed a definite action in vitro to bidirectionally control the growth response of T lymphocytes stimulated by concanavalin A, anti-CD3 monoclonal antibody, and calcium ionophore A23187 plus phorbol 12-myristate 13-acetate. Low concentrations (I-3 μg/ml) of SSd upregulated the responses to suboptimum stimuli of agonists, particularly during the relatively late stage of the responses, whereas it downregulated the responses to supraoptimal stimuli. Under appropriate experimental conditions, SSd promoted interleukin-2 (IL-2) production and IL-2 receptor expression. It also accelerated c-fos gene transcription, but it did not modulate the level of tyrosine phosphorylation of cellular proteins. We concluded from these results that SSd uniquely modulates T lymphocyte function and that at least one target of the action of SSd is located at or before the step of c-fos gene transcription and after T-cell receptor/CD3-mediated protein tyrosine kinase activation.

The relationship was analyzed between drug resistance and MDR1 (with MDR signifying multiple drug resistance) and glutathione S transferase-pi (GST-pi) gene expression in four stomach and four colon cancer cell lines. Northern blot analysis by pmdr1 probe showed that stomach cancer cell lines had no detectable level of MDR1 mRNA expression. By contrast, some levels of MDR1 mRNA expression were found in two colon cancer cell lines, indicating doxorubicin resistance. To examine the MDR1 mRNA in each cell level, in situ hybridization was used. It was found that all colon cell lines and two stomach cell lines had more silver grains per cell than KB cells (a human KB kidney epidermoid carcinoma cell line). However, the number of silver grains in each cell was heterogeneous in the colon and stomach cell lines. Low-level MDR1 mRNA expression could be detected even in cell lines without MDR1 mRNA expression by northern blot hybridization. These results suggest the possibility that all gastrointestinal cell lines can acquire multiple drug resistance. In addition, all examined gastrointestinal cell lines had high GST-pi mRNA expression. This GST-pi gene expression shows cisplatin resistance in the examined cell lines. Heterogeneity of GST-pi mRNA expression also was shown at the cellular level.

We evaluated the medium and long-term outcomes on the basis of patients' function and general quality of life after three different surgical procedures for osteosarcoma around the knee joints, that is, amputation, prosthetic reconstruction and rotationplasty. Twenty-six procedures in 22 patients who survived for at least 1 year after surgery were assessed for functional analysis (scores of the Musculoskeletal Tumor Society), and health-related quality of life assessment (SF-36) was applied to 17 patients who are alive without the disease. The patients treated with rotationplasty showed significantly high functional scores in two of six categories as compared with those undergoing the other two procedures. The scores of SF-36 also showed higher values for seven of eight subscales, however, no significant differences were observed for any subscale. We demonstrated that despite no statistical difference in patient self-assessment of outcome between the treatment modalities, there were functional benefits of rotationplasty over prosthetic reconstruction and amputation.

Alzheimer's disease (AD) is characterized by the presence of senile plaques composed primarily of amyloid-β peptide (Aβ) in the brain. Microglia have been reported to surround these Aβ plaques, which have opposite roles, provoking a microglia-mediated inflammatory response that contributes to neuronal cell loss or the removal of Aβ and damaged neurons. To perform these tasks microglia migrate to the sites of Aβ secretion. We herein analyzed the process of chemokine expression induced by Aβ stimulation in primary murine microglia and Ra2 microglial cell line. We found that Aβ1-42 induced the expressions of CCL7, CCL2, CCL3, CCL4 and CXCL2 in the microglia. The signal transduction pathway for the expression of CCL2 and CCL7 mRNA induced by Aβ1-42 was found to depend on phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK), whereas the pathway for CCL4 depended only on PI3K/Akt. These inflammatory chemokine expressions by Aβ stimulation emphasize the contribution of neuroinflammatory mechanisms to the pathogenesis of AD.

3-Hydroxyanthranilic acid (3HAA) is one of the tryptophan metabolites along the kynurenine pathway and induces apoptosis in T cells. We investigated the mechanism of 3HAA-induced apoptosis in mouse thymocytes. The optimal concentration of 3HAA for apoptosis induction was 300-500 microM. The induction of apoptosis by a suboptimal concentration (100 microM) of 3HAA was enhanced by superoxide dismutase (SOD) as well as MnCl2 and further promoted in the presence of catalase. The 3HAA-mediated generation of intracellular reactive oxygen species (ROS) was enhanced by SOD or MnCl2 and inhibited by catalase. Corresponding to apoptosis induction, the generation of cinnabarinic acid (CA) through the oxidation of 3HAA was enhanced by SOD or MnCl2 in the presence of catalase. The synthesized CA possessed more than 10 times higher apoptosis-inducing activity than 3HAA. The intracellular ROS generation was induced by CA within 15 min and decreased to the control levels within 4 h, whereas the 3HAA-induced ROS generation increased gradually up to 4 h. Corresponding to ROS generation, the mitochondrial membrane potential was downregulated within 15 min and retained by the CA treatment. Apoptosis induction by 3HAA or CA was dependent on caspases, and caspase-3 was much more strongly activated by CA than 3HAA. In conclusion, the CA generated from 3HAA possesses a strong apoptosis-inducing activity in thymocytes through ROS generation, the loss of mitochondrial membrane potential, and caspase activation.

CD8+CD122+ regulatory T cells are a newly identified, naturally occurring type of regulatory T cell that produce interleukin-10 (IL-10) and effectively suppress interferon-gamma (IFN-gamma) production from both CD8+ and CD4+ target cells. Molecular mechanisms responsible for the recognition of target cells by CD8+CD122+ regulatory T cells were investigated in this study by using an in vitro culture system that reconstitutes the regulatory action of these cells. CD8+CD122( regulatory T cells did not produce IL-10 and did not suppress the IFN-gamma production of allogeneic target T cells when they were stimulated by immobilized anti-CD3 antibody alone, but they clearly produced IL-10 and suppressed the IFN-gamma production of target cells when stimulated by anti-CD3 plus anti-CD28-coated beads. IFN-gamma production by major histocompatibility complex-class I-deficient T cells was also suppressed by CD8+CD122+ regulatory T cells stimulated with anti-CD3 plus anti-CD28 antibody but was not suppressed by cells stimulated by anti-CD3 alone. Experiments examining the blockade of cell surface molecules expressed on either the regulatory cells or the target cells by adding specific neutralizing antibodies in the culture indicated that CD80, CD86, and CD28 molecules were involved in the regulatory action, but cytotoxic T lymphocyte antigen-4, inducible costimulatory molecule (ICOS) and programmed death-1 (PD-1) molecules were not. Finally, CD8+CD122+ cells isolated from CD28-knockout (CD28-/-) mice showed no regulatory activity. These results indicate that CD8+CD122(+) regulatory T cells recognize target T cells via the interaction of CD80/CD86-CD28 molecules to become active regulatory cells that produce suppressive factors such as IL-10.

Embryonic stem cells (ESCs) are a renewable cell source of tissue for regenerative therapies. The addition of bone morphogenetic protein 4 (BMP4) to serum-free ESC cultures can induce primitive streak-like mesodermal cells. In differentiated mouse ESCs, platelet-derived growth factor receptor-alpha (PDGFR-alpha) and E-cadherin (ECD) are useful markers to distinguish between paraxial mesodermal progenitor cells and undifferentiated and endodermal cells, respectively. Here, we demonstrate methods for BMP4-mediated induction of paraxial mesodermal progenitors using PDGFR-alpha and ECD as markers for purification and characterization. Serum-free monolayers of ESCs cultured with BMP4 could efficiently promote paraxial mesodermal differentiation akin to embryonic mesodermal development. BMP4 treatment alone induced paraxial mesodermal progenitors that could differentiate into osteochondrogenic cells in vitro and in vivo. Furthermore, early removal of BMP4 followed by lithium chloride (LiCl) promoted the differentiation to myogenic progenitor cells. These myogenic progenitors were able to differentiate further in vitro into mature skeletal muscle cells. Thus, we successfully induced the efficient bidirectional differentiation of mouse ESCs toward osteochondrogenic and myogenic cell types using chemically defined conditions.

We identified CD8+CD122+ regulatory T cells in the mouse. Some immunologists consider CD8+CD122+ cells to be memory T cells despite our report of their regulatory function. Here, we propose a dual phenotype of these cells. Murine CD8+CD122+ T cells demonstrate both memory and regulatory features in their functional profiles. Human CD8+CXCR3+ T cells, which are thought to be the human counterpart of murine CD8+CD122+ regulatory T cells, do not match human central memory T cells of the CD8+CD45RA−CCR7+ phenotype. Thus, we must consider human CD8+ regulatory T cells and murine CD8+ regulatory T cells separately. Of human CD8+ regulatory T cells, CD8+CXCR3+ regulatory T cells can be divided into further subsets and we may be able to distinguish memory T cells and regulatory T cells. Of murine CD8+CD122+ regulatory T cells, it seems to be impossible to divide CD8+CD122+CD44+CD62L+ regulatory T cells into further subsets at present, indicating that this single population of cells has activities of both regulatory T cells and memory T cells.

The kynurenine (KYN) pathway, which is initiated by indoleamine 2,3-dioxygenase, is the main tryptophan (TRP) metabolic pathway. It shares TRP with the serotonin (5-HT) pathway. We investigated the influence of inescapable-predator (rat) stress on behavior and brain TRP metabolism in mice. Male ICR mice (4W) were exposed to 20-min inescapable-predator stress. Behavior on an elevated plus-maze, and TRP, KYN, and 5-HT levels in the prefrontal cortex, hippocampus, amygdala, and dorsal raphe nuclei were measured 1 and 4 weeks after stress exposure. Predator stress increased the number of open-arm entries (NOA) 4 weeks after stress exposure without altering the number of closed-arm entries (NCA). Thus, the open/closed-arm entry ratio (NOA/NCA) increased after stress exposure. Predator stress increased KYN levels in the prefrontal cortex (until 4 weeks after stress exposure) and dorsal raphe nuclei (for 1 week after stress exposure), decreased 5-HT levels in all brain regions (until 4 weeks after stress exposure). Thus, predator stress increased the KYN/5-HT ratio in all regions, in particular in the prefrontal cortex and hippocampus until 4 weeks after stress exposure. Predator stress shifted the balance between the KYN and 5-HT pathways to the KYN pathway, and induced behavioral disinhibition.

The specific processes that cause aging of the cardiac tissue remain elusive. C57BL/6 (B6) mice are commonly used for investigating age-related diseases in mammals. We thus sought to evaluate the cardiac aging process in B6 mice. Cardiac tissues from the newborn (B6 NB), 2 month-old (B6 2M) and 21–27 month-old B6 mice (B6 aged) were used for the investigation. Several age-related cellular processes were evaluated, including telomere shortening, changes in p53 and p16 expression, changes in mitochondria DNA expression and DNA deletion, and alteration of mitochondria. We found that the aging of the B6 mice cardiac tissue is associated with the maintenance of telomere length, increased expression of p53 and p16, mild changes in mitochondrial DNA expression but widespread DNA deletion, and significant alterations of the mitochondrial ultrastructure within the cardiac tissue. The results of our studies suggest that mitochondrial DNA deletions, which affect the mitochondrial ultrastructure, cytochrome C oxidase activity, and p53 expression, are significantly associated with cardiac aging and may be a source of age-related heart failure.

Abstract The prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing in parallel with the prevalence of obesity. DNA damage-inducible protein 34 (GADD34/Ppp1r15a), originally isolated from UV-inducible transcripts in Chinese hamster ovary (CHO) cells, dephosphorylates several kinases that function in important signaling cascades, including dephosphorylation of eIF2α. We examined the effects of GADD34 on natural life span by using GADD34-deficient mice. Here we observed for the first time that with age GADD34-deficient mice become obese, developing fatty liver followed by liver cirrhosis, hepatocellular carcinoma and insulin resistance. We found that myofibroblasts and immune cells infiltrated the portal veins of aged GADD34-deficient mouse livers. A high-fat diet (HFD) induced a higher level of steatosis in young GADD34-deficient mice compared with WT mice. Differentiation into fat is dependent on insulin signaling. Insulin signaling in young GADD34-deficient mice was higher than that in WT mice, which explained the higher fat differentiation of mouse embryonic fibroblasts (MEFs) observed in GADD34-deficient mice. Through aging or a HFD, insulin signaling in GADD34-deficient liver converted to be down regulated compared with WT mice. We found that a HFD or palmitate treatment converted insulin signaling by up-regulating TNF-α and JNK.

Expression of the manganese superoxide dismutase (Mn-SOD) is induced by tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and lipopolysaccharide (LPS). Recently, a TNF-responsive element (TNFRE) was identified within the second intron of the murine Mn-SOD gene. The 5′ CCAAT/enhancer binding protein (C/EBP)-related region within the TNFRE was responsive to TNF, whereas the 3′ NF-κB-related region alone was not. This report describes the minimal promoter region of the Mn-SOD gene and investigates the cis-acting elements and trans-acting factors responsible for TNF-α-induced Mn-SOD gene expression. Reporter plasmid transfection studies demonstrated that inducible transcription factors enhanced the transcriptional activity of the Mn-SOD gene through the intronic enhancer region. Electrophoretic mobility shift assays demonstrated that after TNF-α stimulation, p50 and p65 NF-κB subunits bound specifically to the newly identified NF-κB transcription factor-binding site, distinct from the previously described NF-κB site, within the intronic enhancer region. In addition, site-directed mutagenesis and cotransfection studies demonstrated that the NF-κB p65 subunit enhanced the transcriptional activity of the Mn-SOD gene through the newly identified NF-κB site. These results show that a NF-κB p65 subunit is mainly involved in the molecular mechanisms controlling TNF-α-mediated Mn-SOD gene transcription. J. Cell. Biochem. 77:474–486, 2000. © 2000 Wiley-Liss, Inc.

ABSTRACT C methylation at genomic CpG dinucleotides has been implicated in the regulation of a number of genetic activities during vertebrate cell differentiation and embryo development. The methylated CpG could induce chromatin condensation through the recruitment of histone deacetylase (HDAC)-containing complexes by methyl-CpG-binding proteins. These proteins consist of the methylated-DNA binding domain (MBD). Unexpectedly, however, several studies have identified MBD-containing proteins encoded by genes of Drosophila melanogaster , an invertebrate species supposed to be void of detectable m 5 CpG. We now report the genomic structure of a Drosophila gene, dMBD2/3 , that codes for two MBD-containing, alternatively spliced, and developmentally regulated isoforms of proteins, dMBD2/3 and dMBD2/3Δ. Interestingly, in vitro binding experiments showed that as was the case for vertebrate MBD proteins, dMBD2/3Δ could preferentially recognize m 5 CpG-containing DNA through its MBD. Furthermore, dMBD2/3Δ as well as one of its orthologs in mouse, MBD2b, could function in human cells as a transcriptional corepressor or repressor. The activities of HDACs appeared to be dispensable for transcriptional repression by dMBD2/3Δ. Finally, dMBD2/3Δ also could repress transcription effectively in transfected Drosophila cells. The surprisingly similar structures and characteristics of the MBD proteins as well as DNA cytosine (C-5) methyltransferase-related proteins in Drosophila and vertebrates suggest interesting scenarios for their roles in eukaryotic cellular functions.

Age-dependent changes in noradrenergic innervations of the hippocampal dentate gyrus (DG) and the frontal cortex (FC) have been studied in male F344 rats. The projections from the nucleus locus coeruleus (LC) to DG or FC with advancing age (from 7 to 27 months) in rats have been quantified by electrophysiological and immunohistochemical methods. In the electrophysiological study, we observed that the percentage of LC neurons activated antidromically by electrical stimulation (P-index) of DG or FC decreased with age. We found that the percentage of LC neurons showing multiple antidromic latencies (M-index), which suggests axonal branching of individual LC neurons, increased markedly between 15 and 17 months in DG or FC. In DG, the M-index increased steadily between 15 and 24 months. In contrast, the increased M-index in FC was maintained until 24 months. The increased M-index in both targets declined at 27 months. These results suggest that LC neurons give rise to axonal branching following the loss of projections to DG or FC with age. In the immunohistochemical study, the density of dopamine-beta-hydroxylase-positive axonal varicosities was measured in molecular, granule cell and polymorphic layers of DG. The density in the polymorphic layer significantly decreased in the earlier stage of ageing (7-19 months), whilst the density in the molecular and granule cell layers decreased in the later stage (27 months). These findings suggested that a layer-specific decline occurred with age in the noradrenergic axon terminals in DG.

Development and aging of the immune system lead to an accumulation of memory T cells over the long term. The predominance of T cells of the memory phenotype in the T cell population induces an age-related decline in protective immune responses. We found that development and aging of the immune system were accelerated in p53-deficient (p53-/-) mice; the accumulation of memory T cells was spontaneously accelerated, and a strong T cell-dependent Ab response and Th2 cytokine expression (IL-4, IL-6, and IL-10) were induced by Ag stimulation in young p53-/- mice in the developmental stage. The high T cell proliferative response in the young mice rapidly progressed to a depressed proliferative response in adult mice. It was suggested that the loss of regulation of the cell cycle, DNA repair, and apoptosis by p53 deficiency potentially leads to immunosenescence with the accumulation of memory T cells.

CBP, a protein that binds to cyclic adenosine monophosphate-regulated enhancer-binding protein, and homologue protein, p300, have histone acetyltransferase (HAT) activity and are important in gene transcription, although their physiological functions in vivo remain to be further elucidated. By using immunoprecipitation and HAT activity assay we have found that p300 and CBP have similar tissue patterns of HAT activities, with the highest level in the brain, a relatively high level in the lung, spleen, and heart, an intermediate level in testes and muscle, and a lower level in liver and kidney; that HAT activities of p300 and CBP are relatively stable with advancing age in most examined tissues, but in liver, muscle, and testes, the activities are attenuated with aging; and that HAT activities of p300 and CBP are high in the brain and liver of E14 fetal and newborn mice. These data suggest that the HAT activities of p300 and CBP are important for gene transcription involved in tissue-specific expression, aging, and developing processes.

In a previous study, we have demonstrated that damaged neurons within a boundary area around necrosis fall into delayed neuronal death owing to the cytotoxic effect of microglial nitric oxide (NO), and these neurons are finally eliminated by activated microglia. In this process, microglia are activated to release NO, increase in number, and accumulate toward the damaged area. In this study, we investigated the expression of macrophage colony-stimulating factor (M-CSF, also called colony stimulating factor-1; CSF-1) and other cytokines, which are reported to relate to activation, proliferation, or migration of microglia. The mRNA of M-CSF arose biphasically from 30 min to 1 hr and from 6 to 72 hr after the injury, as demonstrated by semiquantitative RT-PCR. However, another cytokine of granulocyte-macrophage CSF (GM-CSF) or interleukin-3 (IL-3), which causes proliferation of microglia in vitro, was not detected. From immunohistochemical studies, positive staining of M-CSF was observed mainly in neuron-specific enolase (NSE)-positive cells from 1 to 12 hr after the injury, and after that M-CSF became positive in Griffonia simplicifolia isolectin-B4 (GSA-I-B4)-positive cells from 24 to 72 hr in the boundary area around necrosis. These results suggest that neurons around the damaged area express M-CSF in the early phase after injury, which may initially activate microglia, and these activated microglia also express M-CSF later, causing further proliferation or migration of microglia themselves to eliminate damaged neurons or necrotic brain tissue.

Okadaic acid (OA) is a protein phosphatase (PP) inhibitor and induces hyperphosphorylation of p53. We investigated whether the inhibition of PP1 by OA promotes the phosphorylation of the serine 15 of p53. In vitro dephosphorylation assay showed that PP1 dephosphorylated ultraviolet C (UVC)‐induced phospho‐ser15 of p53, and that OA treatment inhibited it. One of the PP1 regulators, growth arrest and DNA damage 34 (GADD34), disturbed PP1 binding with p53, interfered with the dephosphorylation of p53 and increased the amount of phospho‐p53 after UVC‐treatment. This report provides the first evidence that PP1, but not PP2A, dephosphorylates phospho‐serine 15 of p53.

We previously found that an aminopeptidase inhibitor, ubenimex (bestatin), had a growth‐suppressive effect on Choriocarcinoma cell lines in vitro. To clarify the mechanism of this action, we investigated the expression of aminopeptidase N (AP‐N/CD13) on Choriocarcinoma cells and other human tumor cells. Two Choriocarcinoma cell lines, NaUCC‐4 and Be Wo, had higher AP‐N activity than other cell lines (358.8 and 340.2 nmol/h/10 6 cells, respectively), as did the human myeloid leukemia cell line, HL‐60 (373.8 nmol/h/10 6 cells). These Choriocarcinoma and leukemia cell lines with abundant AP‐N activity showed much higher sensitivity to bestatin (IC50 = 0.5, 2.1 and l.0μg/ml, respectively) than the other cell lines. By imniuiioblotting and immunocytochemical staining, AP‐N was detected as an approximately 165‐kDa protein and localized on the cell membrane in Choriocarcinoma cells. We also examined the effects of two other aminopeptidase inhibitors and three anti‐CD13 monoclonal antibodies (MAbs) (WM15, MCS2 and MY7) on the growth of NaUCC‐4 cells. Cell growth was markedly suppressed by the AP‐N inhibitor actinonin as well as bestatin, but not by the AP‐B inhibitor arphamenine. Of the three MAbs, only WM15, which is able to inhibit AP‐N activity, suppressed cell growth in a dose‐dependent manner. These results indicate that AP‐N inhibitors show a growth‐suppressive effect, presumably through inhibition of the enzymatic activity of AP‐N on tumor cells, and suggest that AP‐N may play important roles in the growth of certain tumors, such as Choriocarcinoma and leukemia.

To better understand the link between chromatin modification and manganese superoxide dismutase (Mn-SOD) gene expression, we have investigated the level of histone acetylation at Mn-SOD proximal promoter. TSA induced the expression of Mn-SOD mRNA and its transcriptional activity in C2C12 cells. Sp1 binding sites in the proximal promoter region of Mn-SOD were transcriptionally responsive to TSA by transfection studies. We have detected a localized acetylation of histones H3 and H4, in vivo occupation by Sp1, early growth responsive-1 (Egr-1), and histone deacetylase-1 (HDAC1) in the proximal promoter region of Mn-SOD gene using chromatin immunoprecipitation assays. Our findings indicate that Mn-SOD gene expression is repressed by Sp1-HDAC1 complex. This repression is released by a localized histone acetylation and at least in parts a displacement by Egr-1 in response to TSA.

Alveolar macrophages (AM) from normal rats had immunosuppressive activity to mitogen-induced proliferative responses of splenic lymphocytes. We studied the mechanism and the implication of the nitric oxide synthetase pathway in AM-mediated suppression of concanavalin A (Con A)-induced lymphocyte proliferation. The culture supernatant from AM cultures alone did not have immunosuppressive activity to Con A-induced proliferative responses of non-adherent spleen cells (n-ad SC), but the culture supernatant from co-culture of AM and autologous n-ad SC had this activity. Con A-pulsed AM also liberated the immunosuppressive factor. When AM and autologous n-ad SC were cultured separately under the condition that medium could freely communicate, the culture supernatant did not suppress the Con A-induced proliferative response of n-ad SC. This indicated that the immunosuppressive factor was liberated when AM was activated by cell-to-cell contact with n-ad SC. Further, we examined the immunosuppressive activity of the culture supernatant of co-culture of AM and autologous n-ad SC to Con A-induced responses of allogeneic n-ad SC and xenogeneic murine n-ad SC, and allogeneic mixed leucocyte reaction, and found that this culture supernatant could suppress all these proliferative responses. Nitrate (NO2-) synthesis was markedly augmented in the culture supernatants of Con A-pulsed AM and co-culture of AM and n-ad SC. NG-monomethyl-L-arginine (MMA), a specific competitive inhibitor of the nitric oxide synthetase pathway (NOSP), extinguished both NO2- synthesis by AM and AM-mediated immunosuppressive activity. These data suggest that NOSP was important in AM-mediated suppression of Con A-induced lymphocyte proliferation.

GADD34 is a protein that is induced by a variety of stressors, including DNA damage, heat shock, nutrient deprivation, energy depletion, and endoplasmic reticulum stress. Here, we demonstrated that GADD34 induced by vesicular stomatitis virus (VSV) infection suppressed viral replication in wild-type (WT) mouse embryo fibroblasts (MEFs), whereas replication was enhanced in GADD34-deficient (GADD34-KO) MEFs. Enhanced viral replication in GADD34-KO MEFs was reduced by retroviral gene rescue of GADD34. The level of VSV protein expression in GADD34-KO MEFs was significantly higher than that in WT MEFs. Neither phosphorylation of eIF2alpha nor cellular protein synthesis was correlated with viral replication in GADD34-KO MEFs. On the other hand, phosphorylation of S6 and 4EBP1, proteins downstream of mTOR, was suppressed by VSV infection in WT MEFs but not in GADD34-KO MEFs. GADD34 was able to associate with TSC1/2 and dephosphorylate TSC2 at Thr1462. VSV replication was higher in TSC2-null cells than in TSC2-expressing cells, and constitutively active Akt enhanced VSV replication. On the other hand, rapamycin, an mTOR inhibitor, significantly suppressed VSV replication in GADD34-KO MEFs. These findings demonstrate that GADD34 induced by VSV infection suppresses viral replication via mTOR pathway inhibition, indicating that cross talk between stress-inducible GADD34 and the mTOR signaling pathway plays a critical role in antiviral defense.

The kynurenine (KYN) pathway, which is initiated by indoleamine 2,3-dioxygenase (IDO), is a tryptophan (TRP) metabolic pathway. It shares TRP with the serotonin (5-hydroxytryptamine, 5-HT) pathway. In major depression, activation of the KYN pathway may deplete 5-HT. In the present study we investigated the influence of various risk factors for depression, such as ageing, social isolation and psychological stress, on TRP metabolism. Male ICR mice (postnatal day, PND, 21) were divided into two housing conditions, isolation and group housing, reared for 4 weeks (young adult) or 5 months (adult) and exposed to novelty stress. We measured TRP, KYN and 5-HT contents in the prefrontal cortex, hippocampus, amygdala and dorsal raphe nuclei to investigate the balance between the KYN and 5-HT pathways. Ageing decreased TRP and KYN and increased 5-HT. Thus, ageing shifted the balance to the latter. In the younger group, social isolation decreased TRP and KYN and increased the 5-HT/TRP ratio, whereas novelty stress increased TRP and KYN and decreased the 5-HT/TRP ratio. Thus, social isolation shifted the balance to the latter, whereas novelty stress shifted it to the former. In the older group, these effects were restricted to specific brain regions. Ageing and social isolation counteracted novelty stress effects on TRP metabolism.

Human histocompatibility antigens are quite heterogeneous and promote the rejection of transplanted tissue. Recent advances in stem cell research that enable the use of a patient's own stem cells for transplantation are very important because rejection could be avoided. In particular, Yamanaka's group in Japan gave new hope to patients with incurable diseases when they developed induced murine pluripotent stem cells (iPSCs) in 2006 and human iPSCs in 2007. Whereas embryonic stem cells (ESCs) are derived from the inner cell mass and are supported in culture by LIF, iPSCs are derived from fetal or adult somatic cells. Through the application of iPSC technology, adult somatic cells can develop a pluripotent state. One advantage of using iPSCs instead of ESCs in regenerative medicine is that (theoretically) immune rejection could be avoided, although there is some debate about immune rejection of a patient's own iPSCs. Many diseases occur in elderly patients. In order to use regenerative medicine with the elderly, it is important to demonstrate that iPSCs can indeed be generated from older patients. Recent findings have shown that iPSCs can be established from aged mice and aged humans. These iPSCs can differentiate to cells from all three germ layers. However, it is not known whether iPSCs from aged mice or humans show early senescence. Before clinical use of iPSCs, issues related to copy number variation, tumorigenicity and immunogenicity must be resolved. It is particularly important that researchers have succeeded in generating iPSCs that have differentiated to somatic cells related to specific diseases of the elderly, including atherosclerosis, diabetes, Alzheimer's disease and Parkinson's disease. These efforts will facilitate the use of personalized stem cell transplantation therapy for currently incurable diseases.

Growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a) is a family of GADD proteins that are induced by DNA damage. GADD34 protein has been suggested to regulate inflammation or host defense systems. However, the in vivo function of GADD34 in inflammation is still unclear. Long lasting inflammation, such as that seen in Crohn's disease and ulcerative colitis, is associated with a higher incidence of colorectal cancer (CRC).Using a colitis-associated cancer model, we analysed GADD34-deficient (KO) mice to study the effect of GADD34 on colitis and colorectal tumorigenesis.We found a higher incidence of CRC in wild-type (WT) mice than in GADD34KO mice. Moreover, dextran sodium sulfate (DSS)-induced inflammatory responses were downregulated by GADD34 deficiency. The expression of pro-inflammatory mediators such as TNFα, IL-6, and iNOS/NOS2 was higher in the colons of WT mice than GADD34KO mice. IL-6 is known to activate STAT3 signalling in colonic epithelial cells and subsequently induced epithelial proliferation. We found that IL-6-STAT3 signalling and epithelial proliferation were higher in WT mice compared with GADD34KO mice.These results indicated that GADD34 upregulated pro-inflammatory mediator production leading to a higher tumour burden following azoxymethane (AOM)/DSS treatment.

The glycosyltransferases chondroitin sulfate synthase 1 (CHSY1) and exostoses-like 3 (EXTL3) specifically function in biosynthesis of the glycans chondroitin sulfate and heparan sulfate, respectively. Although these glycans play important roles in pathogenesis of various tumors, their significance in soft tissue sarcoma remains unknown. Here, we asked whether CHSY1 or EXTL3 expression correlates with malignant potential of soft tissue sarcomas with myxoid substance. To do so, we examined 40 samples representing specific types, including 12 cases of myxoid liposarcoma, 14 of myxofibrosarcoma, 12 of malignant peripheral nerve sheath tumor, and 2 of low-grade fibromyxoid sarcoma. We performed immunohistochemistry with anti-CHSY1 and anti-EXTL3 antibodies and compared enzyme expression levels with tumor histologic grade as assessed by the Fédération Nationale des Centres de Lutte Contre le Cancer classification and with patient 5-year survival rate. CHSY1 and EXTL3 were expressed in 72.5% and 32.5% of all tumors, respectively. Notably, CHSY1 was strongly expressed in myxofibrosarcoma and malignant peripheral nerve sheath tumor compared with other tumors and significantly associated with higher- rather than lower-grade tumors (P < .01). High expression of CHSY1 was also significantly associated with poorer patient outcomes (P = .031) and higher stages assessed by American Joint Committee on Cancer staging system (P = .004). By contrast, EXTL3 expression was not correlated with histologic grade or patient prognosis. We conclude that CHSY1 expression is closely associated with malignant potential of soft tissue sarcomas with myxoid substance.

Expression of the manganese superoxide dismutase (Mn‐SOD) is induced by pro‐inflammatory cytokines. We investigated the cis ‐acting elements within a tumor necrosis factor‐responsive element (TNFRE) which was identified in the second intron of the murine Mn‐SOD gene. Site‐directed mutagenesis, reporter plasmid transfection studies and electrophoretic mobility shift assays demonstrated that inducible transcription factors enhanced the transcriptional activity of the Mn‐SOD gene through the TNFRE. The cooperation between proteins binding to the newly identified NF‐κB and C/EBP sites led to synergistic gene transcription. This report provides the first evidence that cooperation between two distinct cis ‐acting elements may be required for induction of Mn‐SOD gene expression mediated by lipopolysaccharide and interferon‐γ.

A histone deacetylase inhibitor has been shown to induce differentiation of many cancer cells and senescence-like state of human fibroblasts. Previously, our data suggested that the region responsive to trichostatin A (TSA), a specific inhibitor of histone deacetylase, treatment in the p21WAF1promoter is located −100 bp upstream from transcription initiation site and contains a GC-box where both Sp1 and Sp3 are responsible. Here we show that another zinc-finger transcription factor, BFCOL1, which binds to the proximal proα2(I) collagen promoter, could also bind to this GC-box of the p21 promoter. In addition, we cloned a gene whose product interacts with this factor by yeast two-hybrid method. The cloned gene was a variant of GADD34 and lacking one PEST region. We found that this cDNA product decreased the DNA binding activity of BFCOL1 to the GC-rich region of p21 minimal promoter.

The incidence of all cancer increases with age through most of the human life span, but its real incidence at very old ages has not been well elucidated to date. Clarification of the real incidence of cancer in old age, especially among centenarians, may well provide pivotal information to understand the characteristics of humankind. In this study, autopsy records of the Annual of the Pathological Autopsy Cases in Japan, 1991-1996, vols. 34-39 (Japanese Society of Pathology, Tokyo) were used. Cases over 90 years old were studied individually for accurate analysis. The incidence of cancer peaked in the 6th decade and that of multiple cases in the 8th decade. In groups over 90 years of age, the incidence at 5-year intervals did not show any significant decrement. Moreover, the metastatic rate and rate of death due to cancer among centenarians was about three-fourths and two-thirds, respectively, of that of cases aged 90-94 years. The decrease in the metastatic ratio and less mortality due to cancer occurring at the oldest ages are considered due to the nature of cancer itself. The fact that the incidence of cancer does not increase would suggest that certain people among those of advanced age have a special resistance to it.

Manganese superoxide dismutase Mn-SOD plays a major role in protecting mitochondria from oxidative damage. Overexpression of Mn-SOD maintains cell survival under conditions that lead to apoptotic death. In addition to the antioxidative enzyme, platelet-derived growth factor (PDGF) is a principal survival factor that inhibits apoptosis and promotes proliferation by activating survival signaling pathways in various cells. Here we show that PDGF induced the expression of the Mn-SOD gene in NIH3T3 cells, and its induction was associated with early growth response-1 (Egr-1), a transcription factor. An electrophoretic mobility shift assay demonstrated that Egr-1 bound to the proximal promoter of the Mn-SOD gene in response to PDGF. The proximal promoter region of Mn-SOD was shown to be transcriptionally responsive to both basal and PDGF stimulation by transfection studies. Forced expression of Egr-1 in the cells activated Mn-SOD transcription in a dose-dependent manner. The pathway by which PDGF induced Egr-1 involved the mitogen-activated protein kinase kinase-1 (MEK1) and extracellular signal-regulated kinases 1 and 2 (ERK1/2), because the effect of PDGF on the induction of Egr-1 was blocked by U0126, a specific MEK1 inhibitor. These findings indicate that the induction of Mn-SOD is part of the anti-apoptotic properties mediated by PDGF.

Staphylococcal enterotoxin A (SEA)- or SEB-stimulated T-lymphocyte proliferation was suppressed by the addition of high numbers of murine peritoneal macrophages or rat peritoneal or alveolar macrophages, whereas lower numbers of murine peritoneal macrophages enhanced the T-lymphocyte response. Suppression was associated with the increase of accumulation of nitrite, a product of nitric oxide, in the culture supernatants. This macrophage-mediated suppression was totally reversed by the addition of NG-monomethyl-L-arginine, a homolog of L-arginine, indicating that macrophage-mediated suppression of T-lymphocyte proliferation was mediated through the nitric oxide-synthesizing pathway activity. Macrophages in large numbers spontaneously produced nitric oxide in culture supernatant fluids. By the addition of autologous or allogeneic spleen cells but not thymocytes to SEA- or SEB-stimulated macrophage culture, nitric oxide production was greatly increased. When T lymphocytes in spleen cells were killed by antibody before addition to macrophage culture, nitric oxide production was diminished to the basal level. These results suggest that in addition to the action to support the process of T-lymphocyte activation by SEA or SEB, macrophages display a feedback regulatory action on the SEA- or SEB-stimulated T-cell proliferative response by releasing nitric oxide through interaction between macrophages and activated T lymphocytes.

Hypercoagulability and thrombotic tendency are frequently induced by a variety of stressors. Clinically, aged subjects and obese patients are more susceptible to thrombotic diseases associated with stress, but the underlying mechanisms are unknown. We investigated the expression of a procoagulant gene, tissue factor (TF), in a mouse model of restraint stress. Twenty hours of restraint stress to mice caused a substantial induction of TF mRNA in several tissues. Importantly, the magnitude of induction of TF mRNA by restraint stress was larger in aged mice compared with young mice. In situ hybridization analysis of the stressed aged mice revealed that strong signals for TF mRNA were localized to renal epithelial cells, smooth muscle cells, adventitial cells, and adipocytes but not to vascular endothelial cells. These observations suggest that restraint stress induces the TF expression in a tissue-specific and cell type–specific manner. Genetically obese mice were also hyperresponsive to restraint stress in the induction of TF gene, especially in their livers and adipose tissues. Stress-induced microthrombi formation was pronounced in renal glomeruli and within the vasculature in adipose tissues of aged mice. Tumor necrosis factor-α (TNF-α) antigen in plasma was elevated by stress in aged mice and obese mice, and pretreatment of mice with anti–TNF-α antibody partially attenuated the stress-mediated induction of TF gene in adipose tissues in these mice. These results suggest that the induction of TF gene may increase the risk of stress-associated thrombosis in older and obese subjects and that TNF-α may be involved.

Telomerase, the reverse transcriptase that maintains telomere DNA, is usually undetectable in adult human tissues, but is positive in embryonic tissues and in cancers. However, in rodents, several organs of normal adult animals express substantial amounts of telomerase activity. To elucidate relevant control mechanisms operating on the tissue-specific expression of telomerase in rodents, we examined the transcriptional regulation of telomerase reverse transcriptase (mTERT) gene in muscle cell differentiation. Reverse transcriptase-polymerase chain reaction analysis showed that the reduction of telomerase activity was caused by the decrease of mTERT mRNA level during myogenesis. Transfections of mTERT promoter showed that the proximal 225-base pair region is the core promoter responsible for basal transcriptional activity and also participates in the reduced transcription after muscle differentiation. Electrophoretic mobility shift assays showed that this region contained the GC-boxes, which bind to Sp1 family proteins, and the E-box, which binds to c-Myc. Furthermore, DNA binding activities of Sp1, Sp3, and c-Myc were down-regulated during myogenesis. These data suggest that Sp1, Sp3, and c-Myc have critical roles of TERT transactivation in mouse, and the lack of these transcription factors cause down-regulation of mTERT gene expression in muscle cells differentiation. Telomerase, the reverse transcriptase that maintains telomere DNA, is usually undetectable in adult human tissues, but is positive in embryonic tissues and in cancers. However, in rodents, several organs of normal adult animals express substantial amounts of telomerase activity. To elucidate relevant control mechanisms operating on the tissue-specific expression of telomerase in rodents, we examined the transcriptional regulation of telomerase reverse transcriptase (mTERT) gene in muscle cell differentiation. Reverse transcriptase-polymerase chain reaction analysis showed that the reduction of telomerase activity was caused by the decrease of mTERT mRNA level during myogenesis. Transfections of mTERT promoter showed that the proximal 225-base pair region is the core promoter responsible for basal transcriptional activity and also participates in the reduced transcription after muscle differentiation. Electrophoretic mobility shift assays showed that this region contained the GC-boxes, which bind to Sp1 family proteins, and the E-box, which binds to c-Myc. Furthermore, DNA binding activities of Sp1, Sp3, and c-Myc were down-regulated during myogenesis. These data suggest that Sp1, Sp3, and c-Myc have critical roles of TERT transactivation in mouse, and the lack of these transcription factors cause down-regulation of mTERT gene expression in muscle cells differentiation. telomerase reverse transcriptase human TERT mouse TERT base pair(s) horseradish peroxidase reverse transcriptase-polymerase chain reaction polyacrylamide gel electrophoresis 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid telomere repeat amplification protocol electrophoretic mobility shift assay kilobase(s) basic-helix-loop-helix Telomeres are specialized structures at the ends of chromosomes composed of DNA and proteins that are essential for maintaining the stability of the eukaryote genome (1Blackburn E.H. Nature. 1991; 350: 569-573Crossref PubMed Scopus (3006) Google Scholar). In vertebrates, they consist of tandem hexanucleotide repeats, (TTAGGG)n, maintained by a specialized ribonucleoprotein enzyme, called telomerase, which adds motif-specific nucleotides using its RNA subunit as a template. Recently, three major telomerase subunits have been identified. The telomerase RNA component provides the template for telomere repeat synthesis (2Blasco M.A. Funk W. Villeponteau B. Greider C.W. Science. 1995; 269: 1267-1270Crossref PubMed Scopus (356) Google Scholar, 3Feng J. Funk W.D. Wang S.S. Weinrich S.L. Avilion A.A. Chiu C.P. Adams R.R. Chang E. Allsopp R.C., Yu, J. Le S. West M.D. Harley C.B. Andrews W.H. Greider C.W. Villeponteau B. Science. 1995; 269: 1236-1241Crossref PubMed Scopus (2054) Google Scholar), the telomerase-associated protein (TEP1) binds to telomerase RNA and coordinates assembly of telomerase holoenzyme (4Nakayama J.I. Saito M. Nakamura H. Matsuura A. Ishikawa F. 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Allsopp R.C. Chin L. Morin G.B. DePinho R.A. Oncogene. 1998; 16: 1723-1730Crossref PubMed Scopus (288) Google Scholar, 9Takakura M. Kyo S. Kanaya T. Tanaka M. Inoue M. Cancer Res. 1998; 58: 1558-1561PubMed Google Scholar). These findings indicate that TERT is a rate-limiting determinant of catalytic activity of telomerase. Analysis of the human and mouse TERT promoters reveals that they are regulated by a number of inducible transcription factors, including c-Myc and NF-κB (10Takakura M. Kyo S. Kanaya T. Hirano H. Takeda J. Yutsudo M. Inoue M. Cancer Res. 1999; 59: 551-557PubMed Google Scholar, 11Greenberg R.A. O'Hagan R.C. Deng H. Xiao Q. Hann S.R. Adams R.R. Lichtsteiner S. Chin L. Morin G.B. DePinho R.A. Oncogene. 1999; 18: 1219-1226Crossref PubMed Scopus (356) Google Scholar, 12Horikawa I. Cable P.L. Afshari C. Barrett J.C. Cancer Res. 1999; 59: 826-830PubMed Google Scholar, 13Kyo S. Takakura M. Taira T. Kanaya T. Itoh H. Yutsudo M. Ariga H. Inoue M. Nucleic Acids Res. 2000; 28: 669-677Crossref PubMed Scopus (427) Google Scholar, 14Yin L. Hubbard A.K. Giardina C. J. Biol. Chem. 2000; 275: 36671-36675Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). These transcription factors are likely to contribute to the observed instances of TERT gene activation. Most telomerase-positive cells are highly regenerative or immortal. Among normal tissues of adult humans, telomerase activity was almost undetectable except germ line cells, although very small amounts of activity are detectable in normal bone marrow, peripheral blood leukocytes, lymphoid cells, and skin epidermis (15Broccoli D. Young J.W. de Lange T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9082-9086Crossref PubMed Scopus (718) Google Scholar, 16Hiyama K. Hirai Y. Kyoizumi S. Akiyama M. Hiyama E. Piatyszek M.A. Shay J.W. Ishioka S. Yamakido M. J. Immunol. 1995; 155: 3711-3715PubMed Google Scholar, 17Taylor R.S. Ramirez R.D. Ogoshi M. Chaffins M. Piatyszek M.A. Shay J.W. J. Invest. Dermatol. 1996; 106: 759-765Abstract Full Text PDF PubMed Scopus (273) Google Scholar, 18Wright W.E. Piatyszek M.A. Rainey W.E. Byrd W. Shay J.W. Dev. Genet. 1996; 18: 173-179Crossref PubMed Scopus (1122) Google Scholar). In contrast, highly regenerative tissues of normal rodents express modest levels of telomerase even in adult animals (19Yamaguchi Y. Nozawa K. Savoysky E. Hayakawa N. Nimura Y. Yoshida S. Exp. Cell Res. 1998; 242: 120-127Crossref PubMed Scopus (52) Google Scholar, 20Nozawa K. Kurumiya Y. Yamamoto A. Isobe Y. Suzuki M. Yoshida S. J. Biochem. (Tokyo). 1999; 126: 361-367Crossref PubMed Scopus (22) Google Scholar). In the normal mouse, telomerase activity exists in colon, liver, ovary, and testis but not in brain, heart, stomach, and muscle (21Prowse K.R. Greider C.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4818-4822Crossref PubMed Scopus (611) Google Scholar, 22Chadeneau C. Siegel P. Harley C.B. Muller W.J. Bacchetti S. Oncogene. 1995; 11: 893-898PubMed Google Scholar). Moreover, cell differentiation causes reduced telomerase activity in some kinds of cell types such as murine F9 teratocarcinoma and C2C12 myoblast cells (23Bestilny L.J. Brown C.B. Miura Y. Robertson L.D. Riabowol K.T. Cancer Res. 1996; 56: 3796-3802PubMed Google Scholar, 24Holt S.E. Wright W.E. Shay J.W. Mol. Cell. Biol. 1996; 16: 2932-2939Crossref PubMed Scopus (362) Google Scholar). These results suggest that this remarkable difference among various tissues in mouse may reflect the different regulation mechanisms of telomerase expression, which may cause the tissue-specific features of differentiation and proliferation. However, the detailed mechanisms that contribute to the telomerase expression during development and cell differentiation in mouse are largely unknown. In this study, to understand the mechanisms that reduce mTERT gene expression during muscle cell differentiation, we constructed a series of reporter plasmids containing the 5′-franking sequence of mTERT gene and transfected these constructs into mouse cell lines, including C2C12 myoblasts. Transcriptional activity of mTERT was dependent on the proximal 225-bp region of promoter, and this promoter region contained E-boxes and GC-boxes, which bind to bHLH proteins and Sp1 family proteins, respectively. We attempted to identify the transcription factors directing mTERT expression and found that Sp1, Sp3, and c-Myc, but not MyoD, play crucial roles in the regulation of mTERT transcription during muscle cell differentiation. Cell culture reagents and fetal bovine serum were obtained from Sigma-Aldrich (St. Louis, MO). Antibodies were from the following sources: anti-c-Myc, anti-MyoD, anti-Sp1, and anti-Sp3 from Santa Cruz Biotechnology (Santa Cruz, CA); horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G and HRP-conjugated anti-mouse immunoglobulin G from New England BioLabs (Beverly, MA). Sp1 and c-Myc consensus oligonucleotides were obtained from Santa Cruz Biotechnology. Mouse NIH3T3 fibroblasts, C3H10T1/2 fibroblasts, and C2C12 myoblasts were obtained from Riken Cell Bank (Tukuba, Japan) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (standard medium). In some experiments, subconfluent C2C12 cells were induced to differentiate by lowering fetal bovine serum to final concentration of 0.5% (differentiation medium). Total cytoplasmic RNA was isolated using the guanidine isothiocyanate method from the cells that were cultured in standard medium or differentiation medium for 1, 4, 7, or 10 days. Samples of 20 μg of total RNA were denatured, separated by electrophoresis in a 1% agarose gel containing formaldehyde, and transferred to GeneScreen membranes (PerkinElmer Life Sciences). The membranes were prehybridized and then hybridized with cDNA probes labeled with [α-32P]dCTP using a random primer labeling system (Amersham Pharmacia Biotech, Buckinghamshire, UK). After hybridization, the membranes were washed and exposed to x-ray film. All blots were rehybridized with a glyceraldehyde-3-phosphate dehydrogenase cDNA probe to normalize for mRNA loading differences. To quantify the contents of mRNA in the cells, the membranes were exposed to imaging plates and radioactivities were measured with a bioimage analyzer, Fijix BAS 1500 (Fuji Film, Tokyo, Japan). The RT-PCR primers, 5′-AGACTSCGCTTCATCCCCAAG-3′ (sense) and 5′-GTCTGGAGGCTGTTCACCTGC-3′ (antisense), were constructed according to the mouse TERT cDNA (GenBank™ accession numberAF073311) (7Martin-Rivera L. Herrera E. Albar J.P. Blasco M.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10471-10476Crossref PubMed Scopus (202) Google Scholar). Total RNA was isolated from C2C12 cells as described above. First-strand cDNA was synthesized with 1 μg of total RNA using a First Strand cDNA synthesis kit (Life Technologies, Inc., Gaithersburg, MD) in the presence of 1.6 μg of oligo-(dT)2 primer in a final volume of 25 μl. After denaturation for 5 min at 94 °C, 4 μl of reaction product was amplified by PCR for 29 cycles (94 °C for 30 s; 55 °C for 30 s; 72 °C for 1 min). The amplified products were separated by electrophoresis on either a 2% agarose gel or 8% polyacrylamide gel electrophoresis (PAGE) and visualized with ethidium bromide. The DNA sequences of RT-PCR products were confirmed at least once by DNA sequencing and were found to be identical to the corresponding sequence of mouse TERT cDNA (nucleotides 1857–2975, data not shown). Each RT-PCR was performed three times with independent preparations of RNA, and typical results are shown in Fig. 1. As an internal control, RT-PCR of glyceraldehyde-3-phosphate dehydrogenase was performed for all RNA samples, using the PCR primers, 5′-ACCACAGTCCATGCCATCAC-3′ (sense) and 5′-TCCACCACCCTGTTGCTGTA-3′ (antisense). The linearity of RT-PCR with respect to RNA amount was determined, and the estimation of mRNA for mTERT was done within a linear range. C2C12 cells were washed twice with ice-cold phosphate-buffered saline, scraped off, and transferred to 1.5-ml microtubes. After centrifugation for 3 min at 1000 ×g at 4 °C, pellets were suspended in 500 μl of the wash buffer (pH 7.5) containing 10 mm HEPES, 1.5 mmMgCl2, 10 mm KCl, 1 mmdithiothreitol, and centrifuged again for 3 min at 1000 ×g at 4 °C. Whole cell extracts were prepared by suspending cells in 30 μl of CHAPS lysis buffer, containing 10 mm Tris-HCl, pH 7.5, 1 mm MgCl2, 1 mm EGTA, 0.1 mm phenylmethylsulfonyl fluoride, 5 mm 2-mercaptoethanol, 0.5% CHAPS, and 10% glycerol. Cell suspensions were subjected to three cycles of freezing and thawing using liquid nitrogen. After being placed on ice for 30 min, they were centrifuged for 30 min at 10,000 × g and the supernatant was collected (CHAPS extracts). Three independent experiments were performed to measure telomerase activity of the cells cultured with differentiation medium. The TRAP (telomere repeat amplification protocol) assay was employed with minor modifications described by Kimet al. (25Kim N.W. Wu F. Nucleic Acids Res. 1997; 25: 2595-2597Crossref PubMed Scopus (621) Google Scholar). The cell extract (representing 1 × 105 cells) was incubated for 30 min at 37 °C in a mixture containing 20 mm Tris-HCl (pH 8.3), 1.5 mm MgCl2, 63 mm KCl, 0.005% Tween 20, 1 mm EGTA, 50 μm each of dATP, dGTP, dTTP, 10 μm dCTP, 2 μCi of [α-32P]dCTP, 0.1 μg/μl bovine serum albumin, 2 units of Taq DNA polymerase, 0.1 μg of TS primer (5′-AATCCGTCGAGCAGAGTT-3′), and 0.1 μg of ACX primer (5′-GCGCGGCTTACCCTTACCCTTACCCTAACC-3′). After the mixtures were heated at 94 °C for 90 s, they were subjected to 30 cycles of PCR amplification (one cycle consisting of 94 °C for 30 s and 58 °C for 45 s). After the reaction, PCR products were analyzed by 12.5% non-denaturing PAGE (1-mm thick). The gels were autoradiographed with the x-ray film (Hyperfilm MP, Amersham Pharmacia Biotech, Buckinghamshire, UK) at −80 °C for 2 h, and telomerase activity was assessed on incorporated radioactive substrate in ladders of product DNA, multiplies of 6 bases corresponding to the telomere repeat unit. The mouse TERT promoter-luciferase reporter plasmids were constructed by pGL3-basic vector (Promega, WI) and the DNA, which was obtained by PCR amplification using mouse genomic DNA according to a published sequence (GenBank™ accession number AF121949) (11Greenberg R.A. O'Hagan R.C. Deng H. Xiao Q. Hann S.R. Adams R.R. Lichtsteiner S. Chin L. Morin G.B. DePinho R.A. Oncogene. 1999; 18: 1219-1226Crossref PubMed Scopus (356) Google Scholar). Various lengths of DNA fragments upstream of the initiating ATG codon of the mTERT gene were amplified and inserted into the pGL3-basic vector. For the construction of reporter plasmids containing substitution mutations in transcription factor binding sites, site-specific mutagenesis was performed by a PCR-based protocol. Expression vector for c-Myc protein (pcDNA3-cMyc) encoding the full-length of c-Myc was a kind gift from Rónán C. O'Hagan and Ronald A. DePinho, Harvard Medical School (26O'Hagan R.C. Schreiber-Agus N. Chen K. David G. Engelman J.A. Schwab R. Alland L. Thomson C. Ronning D.R. Sacchettini J.C. Meltzer P. DePinho R.A. Nat. Genet. 2000; 24: 113-119Crossref PubMed Scopus (120) Google Scholar). Expression vector pCGN-Sp1, encoding the full-length of human Sp1, and its backbone vector (pCGN) were kind gifts from Thomas Shenk, Princeton University (27Parks C.L. Shenk T. J. Biol. Chem. 1996; 271: 4417-4430Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). pCGN-Sp3, encoding the full-length of Sp3, was described previously (28Xiao H. Hasegawa T. Isobe K. J. Cell. Biochem. 1999; 73: 291-302Crossref PubMed Scopus (132) Google Scholar). One microgram of mTERT promoter-luciferase expression plasmid and 0.5 μg of pSV2neo vector were cotransfected into C2C12 cells grown in a 12-well plate using SuperFect reagent (Qiagen, Hilden, Germany) as previously described (29Maehara K. Hasegawa T. Isobe K.I. J. Cell. Biochem. 2000; 77: 474-486Crossref PubMed Scopus (43) Google Scholar). The transfected colonies were selected by 400 μg/ml G418 (Life Technologies, Inc.) for 3 weeks. More than 50 colonies were pooled. Cell extracts were from the transfectants that were grown in the presence of 200 μg/ml G418 and differentiated in Dulbecco's modified Eagle's medium containing 0.5% (v/v) fetal bovine serum with antibiotics and G418 for 10 days. Luciferase activity of these transfectants was normalized against the number of cells. A series of culture experiments were repeated at least three times. Transient transfection of luciferase reporter plasmids was carried out using SuperFect reagent (Qiagen) as previously described (29Maehara K. Hasegawa T. Isobe K.I. J. Cell. Biochem. 2000; 77: 474-486Crossref PubMed Scopus (43) Google Scholar). In general, the day before transfection, cells were plated onto 12-well tissue culture plates at a density of 20,000 cells/well, supplemented with fresh medium before transfection. A total of 0.825 μg of DNA consisting of 0.75 μg of the indicated luciferase reporter plasmid and 0.075 μg of the pRL-thymidine kinase control vector (pRL-TK) (Promega, WI) per well was used for transfection studies. For cotransfection studies, cells grown in 12-well plates were transfected with 0.75 μg of luciferase reporter plasmid, 0.075 μg of pRL-TK, and 0.75 or 0.25 μg of the indicated expression plasmids. PCGN or pcDNA3 was used to adjust the total amount of expression plasmid DNA. After harvest, the cells were assayed by the Dual-Luciferase Reporter Assay system (Promega, WI), using a luminometer (EG&G Berthold, Germany). Protein concentrations of the cell lysates were determined by the method of Bradford with the Bio-Rad protein assay dye reagent (Bio-Rad, CA). Promoter activities were expressed as relative activities, normalized against the concentration of the protein. All transfection experiments were repeated at least three times. Nuclear extracts were prepared from the C2C12 cells that were cultured in differentiation medium for 1, 4, 7, or 10 days using the rapid preparation as previously described (29Maehara K. Hasegawa T. Isobe K.I. J. Cell. Biochem. 2000; 77: 474-486Crossref PubMed Scopus (43) Google Scholar). The protein concentrations of the nuclear fractions were determined by the Bradford assay, and all extracts were stored at −70 °C. Electrophoretic mobility shift assays (EMSAs) were performed using 5 μg of nuclear extract from either untreated or differentiation medium-treated cells. Synthetic complementary oligonucleotides with a G overhang were annealed and labeled with [α-32P]dCTP, using the Klenow fragment. DNA binding reactions were performed as previously described (29Maehara K. Hasegawa T. Isobe K.I. J. Cell. Biochem. 2000; 77: 474-486Crossref PubMed Scopus (43) Google Scholar). Molar excess (5-, 20-, 100-fold) of c-Myc, Sp1 consensus oligonucleotides, MyoD binding element, or oligonucleotides containing mutated sequences were used as an unlabeled competitor. The following pairs of oligonucleotides derived from the muscle creatine kinase promoter were used as MyoD binding oligonucleotides; 5′-GCCCCAACACCTGCTGCCTGA-3′ and 5′-GTCAGGCAGCAGGTGTTGGGG-3′ (30Puri P.L. Avantaggiati M.L. Balsano C. Sang N. Graessmann A. Giordano A. Levrero M. EMBO J. 1997; 16: 369-383Crossref PubMed Scopus (229) Google Scholar). For EMSAs using antibodies, nuclear extracts were preincubated with antibodies (0.2–0.4 μg per reaction) for 20 min at 4 °C. The reactions were separated on 7% or 6% polyacrylamide gels. Gels were dried and subjected to autoradiography. The following pairs of oligonucleotides were used: E-201 containing the nucleotide −211 to −186; 5′-CCGGGGAACACACCTGGTCCTCATGC-3′, 5′-GCATGAGGACCAGGTGTGTTCCCCGG-3′, GC-88 containing the nucleotide −97 to −68; 5′-TTCCTCCGTTCCCAGCCTCATCTTTTTCGT-3′, 5′-ACGAAAAAGATGAGGCTGGGAACGGAGGAA-3′, GC-105 containing the nucleotide −116 to −88; 5′-TCCGCCTGAATCCCGCCCCTTCCTCCGTT-3′, 5′-AACGGAGGAAGGGGCGGGATTCAGGCGGA-3′, and GC-143 containing the nucleotide −153 to −125; 5′-ATTGCTGCGACCCCGCCCCTTCCGCTACA-3′, 5′-TGTAGCGGAAGGGGCGGGGTCGCAGCAAT-3′. The positions of each oligonucleotide are shown in Fig. 4. Cells cultured in differentiation medium for 1, 4, 7, and 10 days were lysed in a buffer consisting of 20 mm Tris-HCl (pH 8.0), 137 mm NaCl, 2 mm EDTA, 10% (v/v) glycerol, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm sodium vanadate, and 1% (v/v) Triton X-100. Each cell lysate containing 25 μg of protein was run under reducing conditions in 8% or 10% sodium dodecyl sulfate-polyacrylamide electrophoresis gels (SDS-PAGE), transferred onto polyvinylidene difluoride membranes (Millipore, MA), and reacted with antibodies as described. The primary antibody was detected by counterstaining with an HRP-linked antibody and visualized by enhanced chemiluminescence. Nuclear extracts (20 μg) were subjected to SDS-PAGE and Western blotting. C2C12 cells in culture were induced to differentiate by lowering serum concentration. Differentiation of C2C12 myoblasts into myotubes was associated with a diminished activity of telomerase (Fig. 1 A), as observed previously (23Bestilny L.J. Brown C.B. Miura Y. Robertson L.D. Riabowol K.T. Cancer Res. 1996; 56: 3796-3802PubMed Google Scholar, 24Holt S.E. Wright W.E. Shay J.W. Mol. Cell. Biol. 1996; 16: 2932-2939Crossref PubMed Scopus (362) Google Scholar). During the culture in differentiation medium, mTERT mRNA levels were also gradually decreased without changing the expression of glyceraldehyde-3-phosphate dehydrogenase mRNA level (Fig. 1 B). However, the culture in differentiation medium resulted in up-regulation of muscle-specific genes, MyoD or myogenin (Fig. 1 B), in agreement with previous reports (31Olson E.N. Klein W.H. Genes Dev. 1994; 8: 1-8Crossref PubMed Scopus (606) Google Scholar, 32Davis R.L. Weintraub H. Lassar A.B. Cell. 1987; 51: 987-1000Abstract Full Text PDF PubMed Scopus (2422) Google Scholar, 33Wright W.E. Sassoon D.A. Lin V.K. Cell. 1989; 56: 607-617Abstract Full Text PDF PubMed Scopus (924) Google Scholar, 34Edmondson D.G. Olson E.N. Genes Dev. 1989; 3: 628-640Crossref PubMed Scopus (597) Google Scholar). Thus, the decrease of mTERT mRNA level was negatively correlated with the expressions of muscle-specific genes. These results indicate that the reduction of telomerase activity during myogenesis is regulated by the reduction of mTERT gene expression in C2C12. To understand the mechanisms of the mTERT expression in cell differentiation, analysis of the mTERT promoter is indispensable. As the first step, luciferase assays were performed using mouse NIH3T3 and C3H10T1/2 as well as C2C12 cell lines. These cell lines were transiently transfected with a series of 5′ terminus-truncated mutants of the mTERT promoter linked to the luciferase reporter gene. As shown in Fig.2, a 1.6-kb region of mTERT promoter (−1561/+53-Luc) demonstrated the significant transcriptional activities in all these cell lines. NIH3T3 and C3H10T1/2 conferred higher transcriptional activity than C2C12. C2C12 exhibited modest transcriptional activity equivalent to 30–40% of NIH3T3, but this activity was still significant, because it was 250-fold the activity in promoterless reporter plasmid (pGL3-basic). Although deletions from −1561 to −225 of mTERT gene promoter resulted in no significant alterations in luciferase activity, truncation of a further 166 base pairs (from −225 to −59) led to a remarkable reduction of the transcriptional activity in these three cell lines (Fig. 2). These findings suggest that the proximal 225 bp is the core promoter essential for basal transcriptional activation of mTERT. Subsequently, C2C12 cells were stably transfected with reporter plasmids and induced to differentiate by the culture in differentiation medium for 10 days. Transfectants with −1561/+53-Luc, −414/+53-Luc, or −225/+53-Luc reporter plasmids exhibited the reduction of transcriptional activities (Fig. 3), being consistent with the decrease of mTERT mRNA level in muscle cell differentiation (Fig. 1 B). These results suggest that the core promoter region (−225/+53) is also responsible for reduction of mTERT transcription in muscle cell differentiation. From these observations, we defined a 225-bp fragment as the mTERT minimal promoter and assumed that myogenesis-responsive elements are located in this region. According to data base analysis, we noticed that three GC clusters and two E-boxes are located in this region, which are known to bind with Sp1 family proteins and bHLH proteins, respectively (Fig. 4). To examine the role of these cis-elements more precisely, luciferase assays were performed with reporter plasmids with site-specific mutants, which were introduced into the mTERT minimal promoter (Fig.5). One E-box is located at the 5′-end of the minimal promoter, and the deletion of this site resulted in a 50% reduction in transcriptional activity in C2C12 cells, as shown in Fig.2 (−225/+53-Luc and −185/+53-Luc). Abrogation of this E-box (E-201) by substitution mutations also led to a 50% reduction of transcriptional activity (Fig. 5, Ebox-mt #1), suggesting that this E-box is essential for transactivation. In contrast, the mutation of another E-box (E-4) that is adjacent to the transcription start site led to no significant alteration of transcriptional activity (Ebox-mt #2). Although a mutation in the GC-box (GC-88) at −88 bp of minimal promoter slightly increased transcriptional activity (GC-mt #3), the mutations in the other two GC-boxes (GC-143 and GC-105) reduced the transcriptional activity (GC-mt #1, #2). Especially, abrogation of the GC-box located at −143 bp (GC-143) of the minimal promoter led to a remarkable reduction (>95%) of transcriptional activity (GC-mt #1). Abrogation of the GC-box located at −105 bp (GC-105) also caused a >50% reduction of transcriptional activity (GC-mt #5). Mutations of all of two E-boxes and three GC-boxes led to a marked loss of transcriptional activity (99.9 ± 0.2% reduction, GC-E-mt), and mutations of only two GC-boxes (GC-143 andGC-105) except GC-88 also caused a >98% reduction of transcription activity (GC-mt #4). These results indicate that several cis-elements, including GC-143 and -105, as well as E-201, are important for basal transcriptional activity of mTERT gene. In particular, two GC-boxes (GC-143 and-105) are the most essential cis-elements for basal transactivation of mTERT gene in C2C12 cells. As shown in Fig. 3, a core promoter region (−225/+53) is responsible for reduction of mTERT transcription during myogenesis. To identify the role of cis-elements in this core promoter, C2C12 cells were stably transfected with reporter plasmids containing mutations in five protein binding sites and induced to differentiate with differentiation medium for 10 days (Fig.6). Transfectants with GC-mt #1, GC-mt #2, and Ebox-mt #1 reporter plasmids, which have single mutation among these cis-elements, reduced transcriptional activity during myogenesis (75–85% reduction), as well as transfectants with either −1561/+53-Luc or −225/+53-Luc reporter plasmid. Mutation of two E-boxes (Ebox-mt #2) also caused a decrease of luciferase activity (75% reduction). This result indicates that GC-boxes in the mTERT core promoter are responsible for differentiational down-regulation of mTERT gene in C2C12 cells. Abrogation of two GC-boxes (GC-143 and GC-105; GC-mt #4) also caused a slight decrease of transcription activity during myogenesis (15% reduction, Fig. 6). However, we could not clearly ascertain whether the rest of the E-boxes in the mTERT core promoter are indispensable for muscle cell differentiation or not, because there was not enough gene expression activity to be lost further with GC-mt #4 reporter plasmid, as shown in Fig. 5. In establishing these stable transfectants, several clones were shown to have different basal transcriptional activities from those of the transient transfectants (Fig. 5), because these results might represent the difference of the number of reporter gene copies, which were integrated into host chromosomal DNA. To identify the proteins that bind to these GC-boxes (GC-143 and GC-105) in mTERT gene core promoter, EMSA was performed with 32P-labeled oligonucleotides corresponding to each GC-box and nuclear extracts from C2C12 cells cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Two major DNA·protein complexes were observed by using either GC-143 or GC-105 for probes (Fig.7 A, lane 1 or5). The specificity of these complexes was confirmed by competiti

In the axon terminals of the locus coeruleus (LC) neurons, a high level of axonal branching was occurred in the middle-aged brain, and the increased branching was maintained in the aged brain. In the present study, we hypothesized that neurotrophic support is necessary for the morphological age-related changes seen in the noradrenergic innervations from the LC to frontal cortex. Through immunohistochemical and quantitative image analyses, we examined the age-dependent effects of brain-derived neurotrophic factor (BDNF) on the noradrenergic axon terminals in the frontal cortex of F344 rats. We continuously infused BDNF into the frontal cortex of young (6-months-old), middle-aged (13-months-old), or aged (25-months-old) rats. Exogenous BDNF infusion caused a marked increase in the density of noradrenergic axons in the aged brain, but no trophic action of BDNF was observed in the young and middle-aged brain. Neutralization of endogenous BDNF with a specific function-blocking antibody to BDNF led to a reduction in noradrenergic axons in the frontal cortex of 19-month-old rats. The present results suggest that BDNF is not involved in the augmentation of noradrenergic innervations in the aging brain, but it is necessary for the maintenance of noradrenergic innervations in the aged brain.

Electrochemotherapy (ECT) delivers nonpermeable anticancer drugs to cell interiors by temporally increasing the permeability of the cytoplasmic membrane under locally applied pulsating electrical stimuli. This treatment results in consistent and enhanced pharmacological effects of drugs on the targeted tissue. ECT has been used for surface skin cancer but never for musculoskeletal tumors. This report describes a clinical trial of ECT for digital chondrosarcoma. A 74-year-old woman with a digital chondrosarcoma was administered electric stimulation with two surface electrodes 10 min after intratumoral multiple injection of bleomycin sulfate and 15 s after intraarterial perfusion of bleomycin sulfate. Biopsy performed after ECT showed 90% tumor necrosis. Marginal resection of the tumor was followed by autologous bone grafting to fill the bone defect. Although the follow-up period was short (3 years), the patient remained disease-free after ECT and was satisfied that amputation of the affected finger could be avoided. This preliminary result suggests that ECT is a viable modality for limb-preserving treatment of patients with sarcoma of the extremities.

In the brain of a patient with Alzheimer's disease, beta amyloid peptide (Aβ) is thought to be taken up by glial cells such as astrocyte and microglia to be degraded. However, it is unclear whether the Aβ is absorbed by astrocyte or microglia. The purpose of our study is to determine which type of glial cell, astrocyte or microglia, can take up Aβ. Beta amyloid 1-40 (Aβ1-40) was directly infused into the frontal cortex or hippocampus for 14 days. Dual-labeling immunohistochemistry for Aβ1-40 with an astrocytic (GFAP) or microglial (CD11b) marker was performed to examine co-localization of Aβ1-40 and glial markers. In the Aβ1-40 infused site, immunoreactivity of Aβ1-40 was observed only in astrocytes, not in microglia. In addition, Aβ40-1, a reverse peptide of Aβ1-40, was not taken up by astrocytes. These results suggested that the astrocyte-specific uptake of Aβ occurred in the rat brain.

Ig-like transcripts (ILT/leukocyte Ig-like receptor/monocyte/macrophage Ig-like receptor or CD85) are encoded on human chromosome 19q13.4, designated the human leukocyte receptor complex, and are predominantly expressed on myeloid lineage cells. We investigated the transcriptional regulation of ILT1, ILT2, and ILT4 genes to elucidate control mechanisms operating on the specific expression of ILT receptors. Inhibitory ILT2 and ILT4 both have a similar genomic structure, in which the approximately 160-bp 5'-flanking regions function as core promoters with critically important PU.1 binding sites. However, an Sp1 family-binding GC-box is more influential in trans-activation of ILT2 than ILT4. Additionally, ILT4 transcription is tightly regulated by chromatin modifications accompanied by histone acetylation, which strictly controls expression within myeloid lineage cells. Activating ILT1 carries a core promoter corresponding to the intronic region of ILT2 and ILT4, where PU.1 and Runx1 binding sites are essential, but a downstream heat shock element also augments promoter activity. Thus, each ILT is regulated by a distinct transcriptional mechanism, although PU.1 acts as a common trans-acting factor. We also found that human CMV infection strongly trans-activates inhibitory ILT2 and ILT4 genes through the expression of immediate-early proteins.

Cells regulate the rate of protein synthesis during conditions of cell stress to adapt to environmental changes. However, the molecular interactions between signaling pathways controlling translation and the cellular response to stress remain to be elucidated. Here, we show that the expression of growth arrest and DNA damage protein 34 (GADD34) is induced by energy depletion and that the expression of this protein protects cells from apoptotic cell death. During conditions of cell stress, GADD34 forms a stable complex with tuberous sclerosis complex (TSC) 1/2, causes TSC2 dephosphorylation, and inhibits signaling by mammalian target of the rapamycin (mTOR). These findings demonstrate that crosstalk between GADD34 and the mTOR signaling pathways contributes to the response of the protein synthetic machinery to environmental stress. GADD34 may find clinical potential as a target drug for the treatment of mTOR-associated diseases.

Continuous low-dose injection of d-galactose induces changes in mice that resemble accelerated aging. As such, these mice have been used as models to study mechanisms of aging. Here, we examined whether repeated (daily, for 60 days) subcutaneous injections (at 50 mg d-galactose/kg) into young adult (i.e., 2-month-old) mice induced changes in key immune system organs that were on par with those associated with aging. The results showed that galactose-treated mice develop histologic changes in their thymic cortical and medullary regions; immunohistochemical analysis revealed unorganized distributions of keratin-5 and keratin-8 proteins in the thymus of these hosts. These histological changes in the thymus of d-galactose-treated mice were also observed in the organs of aged (i.e., 24-month-old control mice); however, in this latter group, these changes were accompanied by a strong infiltration of adipose cells. Galactose-treated mice also evinced alterations within their splenic white and red pulp. Further, ultrastructural analyses of the thymus and spleen of the treated mice revealed increases in irregularly shaped lymphocytes bearing visible pyknosis. It was also seen that levels of autophagy within thymic epithelial cells were greatly decreased in the tissues of the galactose-treated mice, an outcome also seen in aged mice. Lastly, the level of memory T-lymphocytes and percentage of IgM-B220-B-lymphocytes in spleens of the galactose-treated mice were both increased (albeit insignificantly so) relative to values among splenocytes of age-matched control; however, these levels were not clealy as elevated as would be expected in “elderly” mice. Taken together, our results strongly suggest that d-galactose treatment can induce structural changes in the thymus and spleen, and some changes in organ-associated cell phenotypes, that are similar to several effects seen with aging. However, the fact that many endpoints do not appear to be truly reflective of what should be seen in immune system organs/cells of “elderly” mice now calls into question the appropriateness of the use of d-galactose (i.e., is it histologically/immunotoxicologically-proper?) to create age-mimicry in mice.

MRL-lpr mice are severely impaired in the Fas pathway of apoptosis induction. We here evaluate another pathway of apoptosis induction in MRL-lpr mice which is protein kinase C (PKC) dependent. Despite the defect of the Fas pathway, apoptosis developed during culture in vitro in splenic T lymphocytes from MRL-lpr mice more extensively than in T lymphocytes from MRL-(+/+) mice. Apoptosis induction in the former cells was then found to be greatly promoted by PKC inhibitor H-7, and partially prevented by PKC activator phorbol 12-myristate 13-acetate (PMA). High sensitivity to H-7, but not to PKA inhibitor HA 1004, of these cells for apoptosis induction was confirmed by detailed time course and dose-dependency experiments of the drug effect. Population analysis showed that both CD4+ T lymphocytes and CD8+ T lymphocytes from MRL-lpr mice were highly sensitive to H-7, whereas CD8+ T lymphocytes, but not CD4+ T lymphocytes, from MRL-(+/+) mice were susceptible to the reagent. Interestingly, B220+ Thy-1+ CD4-CD8- T lymphocytes from MRL-lpr mice were most sensitive to H-7 for apoptosis induction. Correspondingly, the membrane-translocated activated PKC-alpha level in splenic T lymphocytes from MRL-lpr was more extensively up-regulated by PMA than in splenic T lymphocytes from MRL-(+/+). These results suggest that some signal consistently activates PKC in MRL-lpr T lymphocytes, and this event is needed for survival of these cells. On the other hand, CD4+ CD8+ thymocytes were deleted by apoptosis in culture with PMA, whether these thymocytes were from MRL-lpr mice or MRL-(+/+) mice. This finding suggested that the apoptosis induction pathway linked to PKC activation is intact in CD4+ CD8+ thymocytes from the Fas-defective MRL-lpr mice. We conclude from these results that the PKC-dependent signal pathways for either cell death or cell activation are intact or even accelerated in lpr mice, which could both compensate for the loss of the Fas pathway and promote the generation of autoreactive T lymphocytes.

We have used the yeast two-hybrid system to clone the protein that interacts with the BFCOL1 (binding factor of a type-I collagen promoter) zinc-finger transcription factor that was cloned previously as the factor that binds to the two mouse proximal promoters of the type-I collagen genes. We utilized as bait the N-terminal domain of BFCOL1 that includes the zinc-finger DNA-binding domain. One cDNA contained a potential open reading frame for a polypeptide of 392 amino acids and was identical to PTRF (polymerase I and transcript-release factor), which is involved in transcription termination of the RNA polymerase I reaction. Northern-blot analysis revealed that the pattern of mRNA expression was similar to that of the type-I collagen gene. In addition, we detected the mRNA expression only in a fibroblast cell line and two bone cell lines, but not in other blood and neuronal cell lines. Recombinant protein was shown to enhance the binding of BFCOL1 to its binding site in the mouse proalpha2(I) collagen proximal promoter in vitro. The transient-transfection experiment showed that PTRF had a suppressive effect on the mouse proalpha2(I) collagen proximal promoter activity. We speculate that PTRF might play a role in the RNA polymerase II reaction as well as that of RNA polymerase I.

Exposure of lymphocytes to 0.2–2 mM HgCl 2 , a thiol‐reactive heavy metal, induced extensive tyrosine phosphorylation of multiple cellular proteins. The phosphorylation started as quickly as 5 s after exposure to HgCl 2 , and was irreversible. Another 3 thiol‐reactive chemicals also displayed similar, though less marked, actions, whereas dithiothreitol, a reducing agent, antagonized the HgCl 2 action. The demonstrated new action of HgCl 2 indispensably required membrane‐intact cells as a target. Whereas exposure of lymphocytes to &gt; 0.2 mM HgCl 2 caused rapid cell death, 0.01–0.1 mM HgCl 2 affected the cells so as to accelerate their c‐ fos transcription. These results suggest a novel redox‐linked mechanism of cell surface triggering of intracellular protein kinase activity, which is independent of receptor‐ligand interactions.

Alzheimer's disease is characterized by numerous amyloid‐β peptide (Aβ) plaques surrounded by microglia. Here we report that Aβ induces the proliferation of the mouse microglial cell line Ra2 by increasing the expression of macrophage colony‐stimulating factor (M‐CSF). We examined signal cascades for Aβ‐induced M‐CSF mRNA expression. The induction of M‐CSF was blocked by a phosphatidylinositol 3 kinase (PI3‐kinase) inhibitor (LY294002), a Src family tyrosine kinase inhibitor (PP1) and an Akt inhibitor. Electrophoretic mobility shift assays showed that Aβ enhanced NF‐κB binding activity to the NF‐κB site of the mouse M‐CSF promoter, which was blocked by LY294002. These results indicate that Aβ induces M‐CSF mRNA expression via the PI3‐kinase/Akt/NF‐κB pathway.

Indoleamine 2,3-dioxygenase (IDO)-initiated tryptophan metabolism along the kynurenine (Kyn) pathway in some dendritic cells (DC) such as plasmacytoid DC (pDC) regulates T-cell responses. It is unclear whether bone marrow-derived myeloid DC (BMDC) express functional IDO. The IDO expression was examined in CD11c+CD11b+ BMDC differentiated from mouse bone marrow cells using GM-CSF. CpG oligodeoxynucleotides (CpG) induced the expression of IDO protein with the production of nitric oxide (NO) in BMDC in cultures for 24 h. In the enzyme assay using cellular extracts of BMDC, the IDO activity of BMDC stimulated with CpG was enhanced by the addition of a NO synthase (NOS) inhibitor, suggesting that IDO activity was suppressed by NO production. On the other hand, the concentration of Kyn in the culture supernatant of BMDC was not increased by stimulation with CpG. Exogenously added Kyn was taken up by BMDC independently of CpG stimulation and NO production, and the uptake of Kyn was inhibited by a transport system L-specific inhibitor or high concentrations of tryptophan. The uptake of tryptophan by BMDC was markedly lower than that of Kyn. In conclusion, IDO activity in BMDC is down-regulated by NO production, whereas BMDC strongly take up exogenous Kyn.

Recent studies have shown that cell transplantation therapies, such as endothelial precursor cells, bone marrow-derived mononuclear cells (BM-MNCs) and mesenchymal stem cells, are effective on diabetic polyneuropathy through ameliorating impaired nerve blood flow in diabetic rats. Here, we investigated the effects of BM-MNCs transplantation in diabetic polyneuropathy using BM-MNCs derived from adult (16-week-old) diabetic (AD), adult non-diabetic (AN) or young (8-week-old) non-diabetic (YN) rats.BM-MNCs of AD and AN were isolated after an 8-week diabetes duration. The BM-MNCs were characterized using flow cytometry analysis of cell surface markers and reverse transcription polymerase chain reaction of several cytokines. BM-MNCs or saline were injected into hind limb muscles. Four weeks later, the thermal plantar test, nerve conduction velocity, blood flow of the sciatic nerve and capillary-to-muscle fiber ratio were evaluated.The number of CD29(+)/CD90(+) cells that host mesenchymal stem cells in BM-MNCs decreased in AD compared with AN or YN, and transcript expressions of basic fibroblast growth factor and nerve growth factor in BM-MNCs decreased in AD compared with AN or YN. Impaired thermal sensation, decreased blood flow of the sciatic nerve and delayed nerve conduction velocity in 8-week-diabetic rats were significantly ameliorated by BM-MNCs derived from YN, whereas BM-MNCs from AD or AN rats did not show any beneficial effect in these functional tests.These results show that cytokine production abilities and the mesenchymal stem cell population of BM-MNCs would be modified by aging and metabolic changes in diabetes, and that these differences could explain the disparity of the therapeutic efficacy of BM-MNCs between young and adult or diabetic and non-diabetic patients in diabetic polyneuropathy.

The thymus is mainly composed of two types of epithelial cells, medullary thymic epithelial cells and cortex thymic epithelial cells (mTECs and cTECs). The tissue structure and mechanism for T cell development are complicated, with generation of the thymus regulated by complex molecular and cellular interactions of the thymic microenvironment during embryogenesis. Since the development of organ regeneration techniques became available, complete in vitro regeneration of the thymus has been attempted. Steric induction systems are thought to be optimal for tissue regeneration, but three-dimensional (3-D) induction of TECs from induced pluripotent stem cells (iPSCs) has not yet been reported. Here, we demonstrate the induction of functional TECs from iPSCs by a 3-D spheroid culture system with recruitment of robust numbers of T cells into the peripheral blood. Purified iPSC-derived TECs showed a sufficient expression level of FoxN1 comparable to TECs, and phenotypic analysis revealed that iPSC-derived TECs were expressing K5. Moreover, transplants of cell aggregations into recipient mice were not rejected and there was normal support of T cell development. Functional analysis revealed that T cells showed immune tolerance to both donor and recipient MHCs and could reject an allogeneic third party's skin graft without tumorigenesis. Taken together, these findings raised the possibility of using iPSC-derived TECs induced by 3-D spheroid culture in future regenerative therapy for patients with immunodeficiency.