Masaki Noda

Matricellular proteinsOsteopontin (OPN) is a phosphorylated acidic glycoprotein that has been implicated in a number of physiological and pathological events, including maintenance or reconfiguration of tissue integrity during inflammatory processes.As such, it is required for stress-induced bone remodeling and certain types of cell-mediated immunity.It also acts in dystrophic calcification, coronary restenosis, and tumor cell metastasis.An RGD-containing protein, OPN exists both as an immobilized ECM molecule in mineralized tissues and as a cytokine in body fluids; it is not a significant part of typical nonmineralized ECM.OPN can engage a number of receptors, including the integrins α v (β 1 , β 3 , or β 5 ) and (α 4 , α 5 , α 8 , or α 9 )β 1 , and it may also be a ligand for certain variant forms of CD44, specifically v6 and/or v7, but possibly only in conjunction with a β 1 integrin (1).These receptors directly or indirectly activate cellular signaling pathways, allowing OPN to mediate cell-matrix, and possibly cell-cell, interactions.Several studies have demonstrated that OPN delivers a prosurvival, antiapoptotic signal to the cell.Here, we argue that OPN influences cellular functions in a unique manner, by mimicking key aspects of an ECM signal outside the confines of the ECM.We will explore this idea by reviewing recent data concerning OPN signaling and the consequences of OPN deficiency in several settings, notably inflammatory processes involving immune cells and bone cells.

The effect of transforming growth factor-β (TGFβ) on bone in vivo was examined. Twelve daily injections of 1 μg TGFβ directly onto the periostea of parietal bones of neonatal rats stimulated the formation of periosteal woven bone. The thickness of the treated parietal bones increased at least 2- fold in a dose-dependent manner. This TGFβ effect was localized at the sites of injection, and no change was observed in contralateral parietal bones and tibiae. The body weight in these growing rats was not affected by TGFβ1. TGFβ2 had effects similar to those of TGFβ1 on the parietal bones in vivo. These results reveal for the first time that TGFβ stimulates bone formation in vivo and indicate its anabolic role in local bone metabolism. (Endocrinology124: 2991-2994, 1989)

SOX9 is a transcription factor that plays a key role in chondrogenesis.Aggrecan is one of the major structural components in cartilage; however, the molecular mechanism of aggrecan gene regulation has not yet been fully elucidated.TC6 is a clonal chondrocytic cell line derived from articular cartilage.The purpose of this study was to examine whether SOX9 modulates aggrecan gene expression and to further identify molecules that regulate Sox9 expression in TC6 cells.SOX9 overexpression in TC6 cells enhanced by ϳ3-fold the transcriptional activity of the AgCAT-8 construct containing 8-kilobase (kb) promoter/ first exon/first intron fragments of the aggrecan gene.SOX9 enhancement of aggrecan promoter activity was lost when we deleted a 4.5-kb fragment from the 3-end of the 8-kb fragment corresponding to the region including the first intron.In TC6 cells, SOX9 enhanced the transcriptional activity of a reporter construct containing the Sry/Sox consensus sequence >10-fold.SOX9 enhancement of aggrecan gene promoter activity and SOX9 transactivation through the Sry/Sox consensus sequence were not observed in osteoblastic osteosarcoma cells (ROS17/2.8),indicating the dependence on the cellular background.Northern blot analysis indicated that TC6 cells constitutively express Sox9 mRNA at relatively low levels.To examine regulation of Sox9 gene expression, we investigated the effects of calciotropic hormones and cytokines.Among these, retinoic acid (RA) specifically enhanced Sox9 mRNA expression in TC6 cells.The basal levels of Sox9 expression and its enhancement by RA were observed similarly at both permissive (33 °C) and nonpermissive (39 °C) temperatures.Furthermore, RA treatment enhanced the transcriptional activity of a reporter construct containing the Sry/Sox consensus sequence in TC6 cells.Moreover, RA treatment also enhanced the transcriptional activity of another reporter construct containing the enhancer region of the type II procollagen gene in TC6 cells.These observations indicate that SOX9 enhances aggrecan promoter activity and that its expression is up-regulated by RA in TC6 cells.

Secreted phosphoprotein 1 (Spp-1; osteopontin) is one of the abundant noncollagenous proteins in bone matrix and is produced by osteoblasts. We examined the promoter region of the mouse Spp-1 gene and identified a sequence responsible for 1,25-dihydroxyvitamin D3 enhancement of the Spp-1 gene expression. This 24-base-pair (bp) sequence (vitamin D response element) is located 761 bp upstream of the transcription start site and consists of two direct repeats of a unique 9-bp motif, AGGTTCACG. The vitamin D response element confers responsiveness of a heterologous promoter to 1,25-dihydroxyvitamin D3 in a position- and orientation-independent and copy-number-dependent manner. The basal level of expression of the reporter constructs containing this sequence and its response to 1,25-dihydroxyvitamin D3 were not affected by cotreatment with transforming growth factor beta or the tumor promoter phorbol 12-myristate 13-acetate or by cotransfection with a JUN expression vector. The vitamin D response element forms DNA-protein complexes, as indicated by gel-retardation assays. The addition of a monoclonal antibody raised against the vitamin D receptor further retarded the mobility of the DNA-protein complex. Another antibody that recognizes the DNA binding region of the vitamin D receptor attenuated its binding to the sequence. These results indicate that this 24-bp sequence containing two 9-bp motifs binds to the vitamin D receptor and mediates the vitamin D3 enhancement of murine Spp-1 gene expression.

We have used homologous recombination in embryonic stem cells to generate mice with a targeted disruption of the osteopontin (Opn, or Spp1, for secreted phosphoprotein 1) gene. Mice homozygous for this disruption fail to express osteopontin (OPN) as assessed at both the mRNA and protein level, although an N-terminal fragment of OPN is detectable at extremely low levels in the bones of -/- animals. The Opn -/- mice are fertile, their litter size is normal, and they develop normally. The bones and teeth of animals not expressing OPN are morphologically normal at the level of light and electron microscopy, and the skeletal structure of young animals is normal as assessed by radiography. Ultrastructurally, proteinaceous structures normally rich in OPN, such as cement lines, persist in the bones of the Opn-/- animals. Osteoclastogenesis was assessed in vitro in cocultures with a feeder layer of calvarial osteoblast cells from wild-type mice. Spleen cells from Opn-/- mice cells formed osteoclasts 3- to 13-fold more frequently than did control Opn+/+ cells, while the extent of osteoclast development from Opn -/- bone marrow cells was about 2- to 4-fold more than from the corresponding wild-type cells. Osteoclast development occurred when Opn-/- spleen cells were differentiated in the presence of Opn-/-osteoblasts, indicating that endogenous OPN is not required for this process. These results suggest that OPN is not essential for normal mouse development and osteogenesis, but can modulate osteoclast differentiation.

Postmenopausal osteoporosis and rheumatoid joint destruction result from increased osteoclast formation and bone resorption induced by receptor activator of NF-κB ligand (RANKL) and tumor necrosis factor (TNF). Osteoclast formation induced by these cytokines requires NF-κB p50 and p52, c-Fos, and NFATc1 expression in osteoclast precursors. c-Fos induces NFATc1, but the relationship between NF-κB and these other transcription factors in osteoclastogenesis remains poorly understood. We report that RANKL and TNF can induce osteoclast formation directly from NF-κB p50/p52 double knockout (dKO) osteoclast precursors when either c-Fos or NFATc1 is expressed. RANKL- or TNF-induced c-Fos up-regulation and activation are abolished in dKO cells and in wild-type cells treated with an NF-κB inhibitor. c-Fos expression requires concomitant RANKL or TNF treatment to induce NFATc1 activation in the dKO cells. Furthermore, c-Fos expression increases the number and resorptive capacity of wild-type osteoclasts induced by TNF in vitro. We conclude that NF-κB controls early osteoclast differentiation from precursors induced directly by RANKL and TNF, leading to activation of c-Fos followed by NFATc1. Inhibition of NF-κB should prevent RANKL- and TNF-induced bone resorption. Postmenopausal osteoporosis and rheumatoid joint destruction result from increased osteoclast formation and bone resorption induced by receptor activator of NF-κB ligand (RANKL) and tumor necrosis factor (TNF). Osteoclast formation induced by these cytokines requires NF-κB p50 and p52, c-Fos, and NFATc1 expression in osteoclast precursors. c-Fos induces NFATc1, but the relationship between NF-κB and these other transcription factors in osteoclastogenesis remains poorly understood. We report that RANKL and TNF can induce osteoclast formation directly from NF-κB p50/p52 double knockout (dKO) osteoclast precursors when either c-Fos or NFATc1 is expressed. RANKL- or TNF-induced c-Fos up-regulation and activation are abolished in dKO cells and in wild-type cells treated with an NF-κB inhibitor. c-Fos expression requires concomitant RANKL or TNF treatment to induce NFATc1 activation in the dKO cells. Furthermore, c-Fos expression increases the number and resorptive capacity of wild-type osteoclasts induced by TNF in vitro. We conclude that NF-κB controls early osteoclast differentiation from precursors induced directly by RANKL and TNF, leading to activation of c-Fos followed by NFATc1. Inhibition of NF-κB should prevent RANKL- and TNF-induced bone resorption. Osteoclasts are specialized bone-resorbing cells derived from multipotent myeloid progenitor cells. They play a crucial homeostatic role in skeletal modeling and remodeling and destroy bone in many pathologic conditions (1Boyle W.J. Simonet W.S. Lacey D.L. Nature. 2003; 423: 337-342Crossref PubMed Scopus (4851) Google Scholar, 2Teitelbaum S.L. J. Bone Miner. Metab. 2000; 18: 344-349Crossref PubMed Scopus (100) Google Scholar). Understanding of the regulation of osteoclast formation, activation, and survival has increased dramatically in recent years following the identification of osteoprotegerin and of receptor activator of NF-κB (RANK) 3The abbreviations used are: RANK, receptor activator of NF-κB; RANKL, RANK ligand; TNF, tumor necrosis factor; dKO, double knock-out; WT, wild type; M-CSF, macrophage colony-stimulating factor; MDS, M-CSF-dependent splenocytes; GFP, green fluorescent protein; RT, reverse transcription; NFAT, nuclear factor of activated T cells; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay. 3The abbreviations used are: RANK, receptor activator of NF-κB; RANKL, RANK ligand; TNF, tumor necrosis factor; dKO, double knock-out; WT, wild type; M-CSF, macrophage colony-stimulating factor; MDS, M-CSF-dependent splenocytes; GFP, green fluorescent protein; RT, reverse transcription; NFAT, nuclear factor of activated T cells; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay. and its ligand, RANKL (3Lacey D.L. 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In addition, TNF increases expression of RANK and c-Fms on the surface of osteoclast precursors to amplify RANKL and M-CSF signaling (20Yao Z. Li P. Zhang Q. Schwarz E.M. Keng P. Arbini A. Boyce B.F. Xing L. J. Biol. Chem. 2006; 281: 11846-11855Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 21Zhang Y.H. Heulsmann A. Tondravi M.M. Mukherjee A. Abu-Amer Y. J. Biol. Chem. 2001; 276: 563-568Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar). TNF also induces activation of the transcription factors NF-κB, AP-1, and nuclear factor of activated T cells c1 (NFATc1, also known as NFAT2) in osteoclast precursors (reviewed in Refs. 1Boyle W.J. Simonet W.S. Lacey D.L. Nature. 2003; 423: 337-342Crossref PubMed Scopus (4851) Google Scholar and 22Boyce B.F. Yamashita T. Yao Z. Zhang Q. Li F. Xing L. J. Bone Miner. Metab. 2005; 23: 11-15Crossref PubMed Scopus (74) Google Scholar, 23Karsenty G. Wagner E.F. Dev. 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Chem. 2004; 279: 26475-26480Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). NFATc1 is a member of the NFAT transcription factor family of five proteins that regulate the expression of cytokines and immunoregulatory genes in several cell types (34Crabtree G.R. Olson E.N. Cell. 2002; 109: S67-S79Abstract Full Text Full Text PDF PubMed Scopus (1087) Google Scholar, 35Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2212) Google Scholar, 36Macian F. Nat. Rev. Immunol. 2005; 5: 472-484Crossref PubMed Scopus (1101) Google Scholar). NFATc1 rescues osteoclastogenesis in cells lacking c-Fos (29Takayanagi H. Kim S. Koga T. Nishina H. Isshiki M. Yoshida H. Saiura A. Isobe M. Yokochi T. Inoue J. Wagner E.F. Mak T.W. Kodama T. Taniguchi T. Dev. Cell. 2002; 3: 889-901Abstract Full Text Full Text PDF PubMed Scopus (1965) Google Scholar, 33Matsuo K. Galson D.L. Zhao C. Peng L. Laplace C. Wang K.Z. Bachler M.A. Amano H. Aburatani H. Ishikawa H. Wagner E.F. J. Biol. Chem. 2004; 279: 26475-26480Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 37Koga T. Inui M. Inoue K. Kim S. Suematsu A. Kobayashi E. Iwata T. Ohnishi H. Matozaki T. Kodama T. Taniguchi T. Takayanagi H. Takai T. Nature. 2004; 428: 758-763Crossref PubMed Scopus (678) Google Scholar, 38Ishida N. Hayashi K. Hoshijima M. Ogawa T. Koga S. Miyatake Y. Kumegawa M. Kimura T. Takeya T. J. Biol. Chem. 2002; 277: 41147-41156Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 39Hirotani H. Tuohy N.A. Woo J.T. Stern P.H. Clipstone N.A. J. Biol. Chem. 2004; 279: 13984-13992Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar), indicating that c-Fos is upstream of NFATc1. NF-κB comprises a family of five transcription factors, and expression of both p50 and p52 is required for osteoclast formation (30Franzoso G. Carlson L. Xing L. Poljak L. Shores E.W. Brown K.D. Leonardi A. Tran T. Boyce B.F. Siebenlist U. Genes Dev. 1997; 11: 3482-3496Crossref PubMed Scopus (863) Google Scholar, 31Iotsova V. Caamano J. Loy J. Lewin A. Bravo R. Nat. Med. 1997; 3: 1285-1289Crossref PubMed Scopus (877) Google Scholar). NF-κB p50/p52 double knock-out (dKO) mice do not form osteoclasts, whereas osteoclast formation in p50 or p52 single knock-out mice is normal. The dKO mice have increased numbers of CD11b+/RANK+ osteoclast precursors in their spleens (40Xing L. Bushnell T.P. Carlson L. Tai Z. Tondravi M. Siebenlist U. Young F. Boyce B.F. J. Bone Miner. Res. 2002; 17: 1200-1210Crossref PubMed Scopus (134) Google Scholar), indicating that p50/p52 expression is required for progression of these cells along the osteoclast differentiation pathway. The defect cannot be rescued by treatment of dKO splenocytes with RANKL or TNF (40Xing L. Bushnell T.P. Carlson L. Tai Z. Tondravi M. Siebenlist U. Young F. Boyce B.F. J. Bone Miner. Res. 2002; 17: 1200-1210Crossref PubMed Scopus (134) Google Scholar), each of which activates NF-κB in wild-type (WT) osteoclasts (25Kong Y.Y. Yoshida H. Sarosi I. Tan H.L. Timms E. Capparelli C. Morony S. Oliveira-dos-Santos A.J. Van G. Itie A. Khoo W. Wakeham A. Dunstan C.R. Lacey D.L. Mak T.W. Boyle W.J. Penninger J.M. Nature. 1999; 397: 315-323Crossref PubMed Scopus (2846) Google Scholar, 26Abu-Amer Y. Ross F.P. McHugh K.P. Livolsi A. Peyron J.F. Teitelbaum S.L. J. Biol. Chem. 1998; 273: 29417-29423Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). This is important because TNF can induce osteoclast formation in vitro from WT, RANKL–/–, or RANK–/– osteoclast precursors (41Yamashita T. Xing L. Li P. Schwarz E.M. Dougall W.C. Boyce B.F. J. Bone Miner. Res. 2002; 17: S131Google Scholar, 42Armstrong A.P. Tometsko M.E. Glaccum M. Sutherland C.L. Cosman D. Dougall W.C. J. Biol. Chem. 2002; 277: 44347-44356Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 43Kim N. Kadono Y. Takami M. Lee J. Lee S.H. Okada F. Kim J.H. Kobayashi T. Odgren P.R. Nakano H. Yeh W.C. Lee S.K. Lorenzo J.A. Choi Y. J. Exp. Med. 2005; 202: 589-595Crossref PubMed Scopus (301) Google Scholar), indicating that p50 and p52 expression is required for this in vitro effect of TNF. Despite these observations, the mechanism whereby TNF induces osteoclast formation in vitro in the absence of RANKL/RANK signaling remains unknown. Although it is established that RANKL activates c-Fos and NFATc1 in osteoclasts or their precursors (29Takayanagi H. Kim S. Koga T. Nishina H. Isshiki M. Yoshida H. Saiura A. Isobe M. Yokochi T. Inoue J. Wagner E.F. Mak T.W. Kodama T. Taniguchi T. Dev. Cell. 2002; 3: 889-901Abstract Full Text Full Text PDF PubMed Scopus (1965) Google Scholar, 33Matsuo K. Galson D.L. Zhao C. Peng L. Laplace C. Wang K.Z. Bachler M.A. Amano H. Aburatani H. Ishikawa H. Wagner E.F. J. Biol. Chem. 2004; 279: 26475-26480Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar), there are conflicting data on its effects on NF-κB activation (44Zhu L.L. Zaidi S. Moonga B.S. Troen B.R. Sun L. Biochem. Biophys. Res. Commun. 2005; 326: 131-135Crossref PubMed Scopus (21) Google Scholar). Recent studies have indicated that NF-κB p65 and to a lesser extent p50 proteins are recruited along with NFATc2 to the NFATc1 promoter within 1 h of treatment of osteoclast precursors with RANKL (45Asagiri M. Sato K. Usami T. Ochi S. Nishina H. Yoshida H. Morita I. Wagner E.F. Mak T.W. Serfling E. Takayanagi H. J. Exp. Med. 2005; 202: 1261-1269Crossref PubMed Scopus (655) Google Scholar). c-Fos is not recruited to the NFATc1 promoter until much later (at 24 h), by which time NFATc2 and NF-κB p65 and p50 are no longer detectable. Interestingly, by this time, NFATc1 has been recruited to its own promoter. These investigators suggested that RANKL induces cooperation of NFATc2 pre-existing in precursors with other transcription factors, such as NF-κB to activate initial induction of NFATc1, followed by a later auto-amplification phase of NFATc1 to induce osteoclast formation. Despite these demonstrations of transient NF-κB p65 and p50 association with NFATc2, but not NFATc1, on the NFATc1 promoter, it is still not clear what the relationship is among NF-κB, c-Fos, and NFATc1 during the early events that mediate RANKL or TNF-induced osteoclast formation. This is exemplified by the pathways illustrated in recent review papers of signaling downstream from RANK (46Takayanagi H. Nat. Rev. Immunol. 2007; 7: 292-304Crossref PubMed Scopus (1321) Google Scholar, 47Asagiri M. Takayanagi H. Bone. 2007; 40: 251-264Crossref PubMed Scopus (1024) Google Scholar). Thus, it is still not clear whether the essential role of NF-κB p50 and p52 in osteoclast formation is up- or downstream of c-Fos given that expression of both is required for osteoclastogenesis in vitro and in vivo, nor is it clear whether it is necessary for NF-κB p50 and p52 to interact with NFATc1 in osteoclast precursors for their differentiation into osteoclasts. Here, we report that RANKL or TNF can induce osteoclast formation from NF-κB dKO osteoclast precursors when c-Fos is expressed, indicating that NF-κB is upstream of c-Fos. RANKL or TNF treatment and c-Fos expression in dKO cells also induces NFATc1 expression, and retroviral expression of NFATc1 plus treatment with these cytokines rescue the defect in osteoclast formation. These findings indicate that interaction between NFATc1 and NF-κB p50 or p52 is not required for NFATc1 to execute its osteoclastogenic effect. Animals—Generation of NF-κB p50/p52 dKO mice (C57Bl/6 × 129) was described previously (30Franzoso G. Carlson L. Xing L. Poljak L. Shores E.W. Brown K.D. Leonardi A. Tran T. Boyce B.F. Siebenlist U. Genes Dev. 1997; 11: 3482-3496Crossref PubMed Scopus (863) Google Scholar), and mice were used when they were 3–6 weeks old. Littermates of dKO mice that have normal teeth eruption and skeletal development were used as WT controls. The Institutional Animal Care and Use Committee approved all animal studies. Reagents—Recombinant human M-CSF, murine RANKL, interleukin-1β, and TNFα were purchased from R&D Systems, Inc. (Minneapolis, MN). Polybrene and puromycin were obtained from Sigma. NF-κB activation inhibitor was purchased from Calbiochem. Constructs and Transfection—The coding regions of genes were amplified by PCR from cDNAs and cloned into the pMX-puro retroviral vector (33Matsuo K. Galson D.L. Zhao C. Peng L. Laplace C. Wang K.Z. Bachler M.A. Amano H. Aburatani H. Ishikawa H. Wagner E.F. J. Biol. Chem. 2004; 279: 26475-26480Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 48Kitamura T. Onishi M. Kinoshita S. Shibuya A. Miyajima A. Nolan G.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9146-9150Crossref PubMed Scopus (223) Google Scholar). Each 5′ primer contains a Kozak sequence following the start codon. c-Fos was murine, and NF-κB p50 and p52 were of human origin. The pMSCV-caNFATc1 construct was a gift from Dr. N. Clipstone (Northwestern University, Chicago, IL) (49Neal J.W. Clipstone N.A. J. Biol. Chem. 2001; 276: 3666-3673Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The pMX-GFP-puro vector was used as a control for infection efficiency. These retrovirus vectors were transiently transfected into the Plat-E retroviral packaging cell line (50Morita S. Kojima T. Kitamura T. Gene Ther. 2000; 7: 1063-1066Crossref PubMed Scopus (1348) Google Scholar), and viral supernatant was collected 48 h later. Osteoclastogenesis and Viral Infection—Splenocytes were extracted from spleens through a fine wire mesh and cultured with conditioned medium from a M-CSF-producing cell line (51Takeshita S. Kaji K. Kudo A. J. Bone Miner. Res. 2000; 15: 1477-1488Crossref PubMed Scopus (478) Google Scholar) (1:20 dilution) for 3 days in α-modified essential medium with 10% fetal calf serum (Hyclone Laboratories, Logan, UT) to enrich for osteoclast precursors, which we named M-CSF-dependent splenocytes (MDS). Then the cells were infected with the retroviral supernatants in the presence of M-CSF (10 ng/ml) and Polybrene (8 μg/ml). On day 2 after infection, puromycin (2 μg/ml) was added to the cultures to select for geneintegrated cells. ∼50–80% of MDS were GFP-positive under fluorescence microscopy 2 days after infection, and this increased to >90% following puromycin treatment. The cells were cultured with M-CSF (10 ng/ml) for 3–7 days. RANKL (10 ng/ml) and TNF (20 ng/ml) (these doses for RANKL and TNF effectively induce osteoclast formation from WT spleen cells) were added every other day. The experiments were stopped during this time based on a visual assessment of multinucleated cell formation using an inverted microscope. Optimal osteoclast formation occurs in WT cell preparations 1–2 days after those from the dKO mice. The cells were fixed, stained for tartrate-resistant acid phosphatase (TRAP) activity to identify osteoclasts as TRAP+ cells containing ≥3 nuclei, and counted, as described previously (52Hughes D.E. Dai A. Tiffee J.C. Li H.H. Mundy G.R. Boyce B.F. Nat. Med. 1996; 2: 1132-1136Crossref PubMed Scopus (696) Google Scholar). For functional studies, infected cells were cultured on bone slices for 10 days under the same conditions as described above. Osteoclasts were then removed, resorption pits were visualized after 0.1% toluidine blue staining, and the mean pit area was measured, as described previously (53Li P. Schwarz E.M. O'Keefe R.J. Ma L. Looney R.J. Ritchlin C.T. Boyce B.F. Xing L. Arthritis Rheum. 2004; 50: 265-276Crossref PubMed Scopus (181) Google Scholar). Quantitative Real-time RT-PCR—RNA from MDS or infected cells was extracted using the RNeasy kit and the QiaShredder from Qiagen (Valencia, CA). cDNA synthesis was performed as described previously (40Xing L. Bushnell T.P. Carlson L. Tai Z. Tondravi M. Siebenlist U. Young F. Boyce B.F. J. Bone Miner. Res. 2002; 17: 1200-1210Crossref PubMed Scopus (134) Google Scholar). Quantitative PCR amplification was performed with gene-specific primers using an iCycler real-time PCR machine using iQ SYBR Green super-mix (both from Bio-Rad Laboratories) according to the manufacturer's instructions. The primer sequences are as follows: NFATc1, forward, 5′-CACATTCTGGTCCATACGA-3′, and reverse, 5′-CGTGTAGCTGCACAATGG-3′; c-fos, forward, 5′-CTGTCAACACACAGGACTTTT-3′, and reverse, 5′-AGGAGATAGCTGCTCTACTTTG-3′; β-actin, forward, 5′-ACCCAGATCATGTTTGAGAC-3′, and reverse, 5′-GTCAGGATCTTCATGAGGTAGT-3′. The relative standard curve method was used to calculate the amplification difference for each primer set (54Johnson M.R. Wang K. Smith J.B. Heslin M.J. Diasio R.B. Anal. Biochem. 2000; 278: 175-184Crossref PubMed Scopus (323) Google Scholar). The standard curve was made from four points corresponding to 10-fold cDNA serial dilution for each gene. For each sample, the relative amount was calculated from its respective standard curve. The quantity of c-fos or NFATc1 mRNA was then obtained by division of each value by the actin value. Standards and samples were run in triplicate. Western Blot Analysis—Infected cells were lysed with radioimmunoprecipitation buffer (50 mm HEPES at pH 7, 1% Triton X-100, 1 mm EDTA, 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate, 150 mm NaCl with protease inhibitors, and sodium orthovanadate). The lysates (20 μg of protein) were resolved by 10% SDS-PAGE and immunoblotted with a rabbit anti-c-Fos, mouse anti-NFATc1 (both from Santa Cruz Biotechnology, Santa Cruz, CA), or a mouse anti-actin (Sigma) antibody. NFATc1 Nuclear Translocation—c-Fos or GFP virus-infected MDS were cultured with M-CSF and RANKL in 96-well culture plates to generate osteoclasts. After mature osteoclasts were observed, cells were fixed with 10% neutral buffered formalin and permeabilized using 0.1% Triton X-100. Immunofluorescent staining was performed using mouse anti-NFATc1 antibody followed by Cy3-conjugated anti-murine immunoglobulin (Jackson ImmunoResearch, West Grove, PA). Subcellular localization of Cy3-labeled NFATc1 was observed using fluorescence microscopy. Electrophoretic Mobility Shift Assay (EMSA)—To assess c-Fos activation, EMSA was performed as described previously (55Feng J.Q. Xing L. Zhang J.H. Zhao M. Horn D. Chan J. Boyce B.F. Harris S.E. Mundy G.R. Chen D. J. Biol. Chem. 2003; 278: 29130-29135Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Briefly, 5 μg of nuclear extracts prepared from untreated MDS or RANKL- or TNF-treated MDS were incubated with 32P-end-labeled 21-mer double-stranded oligonucleotide containing the consensus AP-1 site (5′-CGCTTGATGACTCAGCCGGAA-3′) (Santa Cruz Biotechnology) for 30 min at room temperature. The DNA-protein complex formed was then separated from free oligonucleotide on 5% native polyacrylamide gels. Binding specificity was examined by competition with 30-fold excess unlabeled AP-1 oligonucleotide. SP-1 binding sequence 5′-CGAGCCGGCCCCGCCCATC-3′ (Invitrogen) was used as a loading control. Statistics—All experiments were performed more than once with similar results. Results are given as mean ± S.E. Comparisons were made by analysis of variance and Mann-Whitney's U test for unpaired data. p values <0.05 were considered statistically significant. NF-κB Is Upstream from c-Fos in RANKL and TNF-induced Osteoclast Formation—c-Fos, like NF-κB, is activated by RANKL and TNF to induce osteoclast formation (15Takayanagi H. Kim S. Matsuo K. Suzuki H. Suzuki T. Sato K. Yokochi T. Oda H. Nakamura K. Ida N. Wagner E.F. Taniguchi T. Nature. 2002; 416: 744-749Crossref PubMed Scopus (589) Google Scholar), but it is not known whether c-Fos can substitute for NF-κB expression in osteoclast precursors. To examine this question, we overexpressed c-Fos in NF-κB p50/p52 dKO MDS. MDS were used as osteoclast precursors in our study because NF-κB dKO mice have severe osteopetrosis, which prevents harvesting of bone marrow cells. We first examined whether NF-κB p50, p52, and p50+p52 retroviral expression would rescue the defect in osteoclast formation in dKO MDS when the cells were treated with RANKL. Overexpression of p50+p52 in dKO cells rescued osteoclast formation, inducing ∼150 osteoclasts/96 wells (Fig. 1A). In comparison, RANKL-treated GFP-infected WT cells typically formed ∼300 TRAP+ osteoclasts (data not shown), indicating that the maximal rescue efficiency of our system is ∼50% of that of WT cells. c-Fos expression alone, like p50, p52, or p50+p52, did not induce osteoclasts in the absence of RANKL (Fig. 1, A and B). However, when RANKL was added to c-Fos-expressing dKO cells, the combination rescued the osteoclast formation defect, inducing ∼150 osteoclasts (Fig. 1, B and C). The TRAP+ cells that formed on plastic dishes and bone slices from dKO cells overexpressing c-Fos appeared similar to those of WT MDS treated with M-CSF and RANKL, and they formed numerous resorption pits on bone slices, typical of mature WT osteoclasts (Fig. 1D). These data indicate that c-Fos activated by RANKL can efficiently substitute for the lack of p50 and p52 in dKO cells in these culture conditions and that the resulting c-Fos-overexpressing osteoclasts have a bone resorptive capacity similar to that of WT cells. TNF plays important roles in osteoclast formation, activation, and survival in a number of pathologi

The cytokine and cell attachment protein osteopontin (OPN) is not necessary for the development and survival of mice in a clean animal facility. The primary role of OPN appears to be that of facilitating recovery of the organism after injury or infection, which generally causes an increase in its expression. It also is essential for some forms of bone remodeling. OPN stimulates cellular signaling pathways via various receptors found on most cell types and can encourage cell migration. OPN modulates immune and inflammatory responses and possibly negatively regulates Ras signaling pathways. Its apparent ability to enhance cell survival by inhibiting apoptosis may explain why the metastatic proficiency of tumor cells increases with increased OPN expression.

Osteopontin is one of the major noncollagenous bone matrix proteins produced by osteoblasts and osteoclasts, bone cells that are uniquely responsible for the remodeling of mineralized tissues. Osteoclasts express the αvβ3 integrin, which is one of the receptors for osteopontin. Recent knockout studies revealed that noncollagenous bone matrix proteins are functionally important in regulation of bone metabolism. However, the significance of the presence of osteopontin in in vivo has not been known. We report here that osteopontin knockout mice are resistant to ovariectomy-induced bone resorption compared with wild-type mice. Microcomputed tomography analysis indicated about 60% reduction in bone volume by ovariectomy in wild-type mice, whereas the osteopontin-deficient mice exhibited only about 10% reduction in trabecular bone volume after ovariectomy. Reduction in uterine weight was observed similarly in both wild-type and osteopontin-deficient mice, indicating the specificity of the effect of osteopontin deficiency on bone metabolism. We propose that osteopontin is essential for postmenopausal osteoporosis in women. Strategies to counteract osteopontin’s action may prove effective in suppressing osteoporosis.

Lymphocyte recirculation through secondary lymphoid organs is essential for immunosurveillance and lymphocyte effector functions. Here, we show that signals through β2-adrenergic receptors (β2ARs) expressed on lymphocytes are involved in the control of lymphocyte dynamics by altering the responsiveness of chemoattractant receptors. Agonist stimulation of lymphocyte β2ARs inhibited egress of lymphocytes from lymph nodes (LNs) and rapidly produced lymphopenia in mice. Physiological inputs from adrenergic nerves contributed to retention of lymphocytes within LNs and homeostasis of their distribution among lymphoid tissues. β2ARs physically interacted with CCR7 and CXCR4, chemokine receptors promoting lymphocyte retention in LNs. Activation of β2ARs enhanced retention-promoting signals through CCR7 and CXCR4, and consequently inhibited lymphocyte egress from LNs. In models of T cell–mediated inflammatory diseases, β2AR-mediated signals inhibited LN egress of antigen-primed T cells and reduced their recruitment into peripheral tissues. Thus, this study reveals a novel mechanism for controlling lymphocyte trafficking and provides additional insights into immune regulation by the nervous system.

Various aspects of the immune system display circadian rhythms. Although lymphocyte trafficking has been suggested to show diurnal variations, the mechanisms and influences on immune responses are unclear. Here, we show in mice that inputs from adrenergic nerves contribute to the diurnal variation of lymphocyte recirculation through lymph nodes (LNs), which is reflected in the magnitude of the adaptive immune response. Neural inputs to β2-adrenergic receptors (β2ARs) expressed on lymphocytes reduced the frequency of lymphocyte egress from LNs at night, which was accompanied by an increase of lymphocyte numbers in LNs. Immunization during the period of lymphocyte accumulation in LNs enhanced antibody responses. The diurnal variation of the humoral immune response was dependent on β2AR-mediated neural signals and was diminished when lymphocyte recirculation through LNs was stopped. This study reveals the physiological role of adrenergic control of lymphocyte trafficking in adaptive immunity and establishes a novel mechanism that generates diurnal rhythmicity in the immune system.

<h2>Abstract</h2> Bone morphogenetic protein (BMP) controls osteoblast proliferation and differentiation through Smad proteins. Here we show that Tob, a member of the emerging family of antiproliferative proteins, is a negative regulator of BMP/Smad signaling in osteoblasts. Mice carrying a targeted deletion of the <i>tob</i> gene have a greater bone mass resulting from increased numbers of osteoblasts. Orthotopic bone formation in response to BMP2 is elevated in <i>tob</i>-deficient mice. Overproduction of Tob represses BMP2-induced, Smad-mediated transcriptional activation. Finally, Tob associates with receptor-regulated Smads (Smad1, 5, and 8) and colocalizes with these Smads in the nuclear bodies upon BMP2 stimulation. The results indicate that Tob negatively regulates osteoblast proliferation and differentiation by suppressing the activity of the receptor-regulated Smad proteins.

The combinatorial action of separate <i>cis</i>-acting elements controls the cell-specific expression of type I collagen genes. In particular, we have shown that two short elements located between -3.2 and -2.3 kb and named TSE1 and TSE2 are needed for expression of the mouse <i>COL1a1</i> gene in tendon fibroblasts. In this study, we analyzed the <i>trans</i>-acting factors binding to TSE1 and TSE2. Gel shift experiments showed that scleraxis (SCX), which is a basic helix-loop-helix transcription factor that is expressed selectively in tendon fibroblasts, binds TSE2, preferentially as a SCX/E47 heterodimer. In transfection experiments, overexpression of SCX and E47 strongly enhanced the activity of reporter constructs harboring either four copies of TSE2 cloned upstream of the <i>COL1a1</i> minimal promoter or a 3.2-kb segment of the <i>COL1a1</i> proximal promoter. Analysis of TSE1 showed that it contains a consensus binding site for NFATc transcription factors. This led us to show that the <i>NFATc4</i> gene is expressed in tendons of developing mouse limbs and in TT-D6 cells, a cell line that has characteristics of tendon fibroblasts. In gel shift assays, TSE1 bound NFATc proteins present in nuclear extracts from TT-D6 cells. In transfection experiments, overexpression of NFATc transactivated a reporter construct harboring four copies of TSE1 cloned upstream of the <i>COL1a1</i> minimal promoter. By contrast, inhibition of the nuclear translocation of NFATc proteins in TT-D6 cells strongly inhibited the expression of the <i>COL1a1</i> gene. Taken together, these results suggest that SCX and NFATc4 cooperate to activate the <i>COL1a1</i> gene specifically in tendon fibroblasts.

Reduced mechanical stress to bone in bedridden patients and astronauts leads to bone loss and increase in fracture risk which is one of the major medical and health issues in modern aging society and space medicine. However, no molecule involved in the mechanisms underlying this phenomenon has been identified to date. Osteopontin (OPN) is one of the major noncollagenous proteins in bone matrix, but its function in mediating physical-force effects on bone in vivo has not been known. To investigate the possible requirement for OPN in the transduction of mechanical signaling in bone metabolism in vivo, we examined the effect of unloading on the bones of OPN(-/-) mice using a tail suspension model. In contrast to the tail suspension-induced bone loss in wild-type mice, OPN(-/-) mice did not lose bone. Elevation of urinary deoxypyridinoline levels due to unloading was observed in wild-type but not in OPN(-/-) mice. Analysis of the mechanisms of OPN deficiency-dependent reduction in bone on the cellular basis resulted in two unexpected findings. First, osteoclasts, which were increased by unloading in wild-type mice, were not increased by tail suspension in OPN(-/-) mice. Second, measures of osteoblastic bone formation, which were decreased in wild-type mice by unloading, were not altered in OPN(-/-) mice. These observations indicate that the presence of OPN is a prerequisite for the activation of osteoclastic bone resorption and for the reduction in osteoblastic bone formation in unloaded mice. Thus, OPN is a molecule required for the bone loss induced by mechanical stress that regulates the functions of osteoblasts and osteoclasts.

Development of the musculoskeletal system requires coordinated formation of distinct types of tissues, including bone, cartilage, muscle, and tendon. Compared to muscle, cartilage, and bone, cellular and molecular bases of tendon development have not been well understood due to the lack of tendon cell lines. The purpose of this study was to establish and characterize tendon cell lines. Three clonal tendon cell lines (TT-E4, TT-G11, and TT-D6) were established using transgenic mice harboring a temperature-sensitive mutant of SV40 large T antigen. Proliferation of these cells was significantly enhanced by treatment with bFGF and TGF-beta but not BMP2. Tendon phenotype-related genes such as those encoding scleraxis, Six1, EphA4, COMP, and type I collagen were expressed in these tendon cell clones. In addition to tendon phenotype-related genes, expression of osteopontin and Cbfal was observed. These clonal cell lines formed hard fibrous connective tissue when implanted onto chorioallantoic membrane in ovo. Furthermore, these cells also formed tendon-like tissues when they were implanted into defects made in patella tendon in mice. As these tendon cell lines also produced fibrocartilaginous tissues in tendon defect implantation experiments, mesenchymal stem cell properties were examined. Interestingly, these cells expressed genes related to osteogenic, chondrogenic, and adipogenic lineages at low levels when examined by RT-PCR. TT-G11 and TT-E4 cells differentiated into either osteoblasts or adipocytes, respectively, when they were cultured in cognate differentiation medium. These observations indicated that the established tendon cell line possesses mesenchymal stem cell-like properties, suggesting the existence of mesenchymal stem cell in tendon tissue.

Type beta transforming growth factor (TGF beta) was shown to regulate the production of several extracellular matrix proteins. Osteopontin (OP) is a recently discovered bone matrix protein which was shown to promote the attachment of osteoblastic rat osteosarcoma ROS 17/2.8 cells to their substrate. We examined the effects of TGF beta on OP production and OP mRNA in ROS 17/2.8 cells. Four-day treatment with 4 ng/ml TGF beta 1 increased substantially the level of osteopontin in the cell culture media, as estimated by immunoblotting. Metabolic labeling showed that this effect was associated with a 3-4-fold increase in OP biosynthesis. TGF beta 1 also increased, in a dose-dependent manner starting at 0.4 ng/ml, the steady-state level of OP mRNA. The increase in OP mRNA was first detected 48 h after the addition of TGF beta 1 and lasted at least until 120 h. The half-life of OP mRNA, estimated in the presence of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, was about 10 h and was not altered by TGF beta 1. On the other hand, the increase in OP mRNA was blocked by actinomycin D. Nuclear run-on assays indicated that TGF beta 1 increased the rate of transcription of the OP gene. Examination of hormonal interactions showed that TGF beta 1 opposed or compensated for the reduction in OP mRNA produced by dexamethasone and that TGF beta 1 did not further augment OP mRNA levels which had been increased by 1,25-dihydroxyvitamin D3 treatment. TGF beta 2 had similar effects on OP gene expression as TGF beta 1. In conclusion, TGF beta promotes the production of osteopontin in the osteoblastic osteosarcoma cells through a pathway which is at least in part mediated by transcriptional events.

Two flat revertants have been isolated from mutagen-treated populations of Kirsten murine sarcoma virus (Ki-MuSV)-transformed NIH/3T3 cells. These revertants, which appear to be cellular variants resistant to transformation by the Ki-MuSV oncogene v-Ki-ras, contain Ki-MuSV-specific DNA, elevated levels of the v-Ki-ras gene product p21, and rescuable transforming virus. Cell hybridization studies indicated that the revertant phenotype is dominant in hybrids between revertant cells and cells transformed by Ki-MuSV or the closely related Harvey MuSV and BALB MuSV. Analysis of hybrid cells resulting from the fusion of these revertants to cell lines transformed by other retroviruses showed that the action of certain oncogenes structurally unrelated to v-Ki-ras also could be suppressed. Thus, there appear to be functional relationships and diversities among transforming genes (oncogenes) not readily apparent from their structural characteristics.

Rheumatoid arthritis is one of the most critical diseases that impair the quality of life of patients, but its pathogenesis has not yet been fully understood. Osteopontin (OPN) is an extracellular matrix protein containing Arg-Gly-Asp (RGD) sequence, which interacts with alpha(v)beta3 integrins, promotes cell attachment, and cell migration and is expressed in both synovial cells and chondrocytes in rheumatoid arthritis; however, its functional relationship to arthritis has not been known. Therefore, we investigated the roles of OPN in the pathogenesis of inflammatory process in a rheumatoid arthritis model induced by a mixture of anti-type II collagen mAbs and lipopolysaccharide (mAbs/LPS). mAbs/LPS injection induced OPN expression in synovia as well as cartilage, and this expression was associated with joint swelling, destruction of the surface structures of the joint based on scanning electron microscopy, and loss of toluidine blue-positive proteoglycan content in the articular cartilage in wild-type mice. In contrast, OPN deficiency prevented the mice from such surface destruction, loss of proteoglycan in the articular joint cartilage, and swelling of the joints even when the mice were subjected to mAbs/LPS injection. Furthermore, mAbs/LPS injection in wild-type mice enhanced the levels of CD31-positive vessels in synovia and terminal deoxynucleotidyltransferase-mediated UTP end labeling-positive chondrocytes in the articular cartilage, whereas such angiogenesis as well as chondrocyte apoptosis was suppressed significantly in OPN-deficient mice. These results indicated that OPN plays a critical role in the destruction of joint cartilage in the rheumatoid arthritis model in mice via promotion of angiogenesis and induction of chondrocyte apoptosis.

Abstract TGFβ1 from porcine platelets increased alkaline phosphatase (AP) activity in the rat osteoblastic cell line ROS 17/2.8 about three‐fold. This effect was dose‐dependent with an ED 50 of about ∼0.2 ng/ml and was larger during logarithmic growth than at confluence. TGFβ1 inhibited cell growth by about 30% with similar dose dependence. Thirty min exposure to TGFβ1 was sufficient to increase AP activity 3 days later by about two‐fold but did not affect cell growth, suggesting dissociation between effects on proliferation and differentiation. The rise in AP activity started 6 h after TGFβ1 addition and was blocked by cycloheximide and actinomycin D. TGFβ1 also increased AP mRNA by two‐ to three‐fold and this effect was not blocked by cycloheximide. The half‐life of AP mRNA, estimated following the addition of 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole was about ten h in both control and TGFβ1‐treated cells. The mRNAs for type I procollagen and osteonectin were also increased by TGFβ1 but fibronectin mRNA was decreased. TGFβ2 effects on AP and cell growth were similar to those of TGFβ1, except for lack of activity following transient exposure. At saturating concentrations, TGFβ2 (2 ng/ml) or dexamethasone (10 −7 M), which has similar effects on these cells, did not further augment the effects of TGFβ1 (at 2 ng/ml). Above findings suggest that TGFβ promotes osteoblatic differentiation in rat osteosarcoma cells at least in part by acting at the pretranslational level.

Osteoporosis is one of the major health problems in our modern world. Especially, disuse (unloading) osteoporosis occurs commonly in bedridden patients, a population that is rapidly increasing due to aging-associated diseases. However, the mechanisms underlying such unloading-induced pathological bone loss have not yet been fully understood. Since sympathetic nervous system could control bone mass, we examined whether unloading-induced bone loss is controlled by sympathetic nervous tone. Treatment with β-blocker, propranolol, suppressed the unloading-induced reduction in bone mass. Conversely, β-agonist, isoproterenol, reduced bone mass in loaded mice, and under such conditions, unloading no longer further reduced bone mass. Analyses on the cellular bases indicated that unloading-induced reduction in the levels of osteoblastic cell activities, including mineral apposition rate, mineralizing surface, and bone formation rate, was suppressed by propranolol treatment and that isoproterenol-induced reduction in these levels of bone formation parameters was no longer suppressed by unloading. Unloading-induced reduction in the levels of mineralized nodule formation in bone marrow cell cultures was suppressed by propranolol treatment in vivo. In addition, loss of a half-dosage in the dopamine β-hydroxylase gene suppressed the unloading-induced bone loss and reduction in mineralized nodule formation. Unloading-induced increase in the levels of osteoclastic activities such as osteoclast number and surface as well as urinary deoxypyridinoline was all suppressed by the treatment with propranolol. These observations indicated that sympathetic nervous tone mediates unloading-induced bone loss through suppression of bone formation by osteoblasts and enhancement of resorption by osteoclasts. Osteoporosis is one of the major health problems in our modern world. Especially, disuse (unloading) osteoporosis occurs commonly in bedridden patients, a population that is rapidly increasing due to aging-associated diseases. However, the mechanisms underlying such unloading-induced pathological bone loss have not yet been fully understood. Since sympathetic nervous system could control bone mass, we examined whether unloading-induced bone loss is controlled by sympathetic nervous tone. Treatment with β-blocker, propranolol, suppressed the unloading-induced reduction in bone mass. Conversely, β-agonist, isoproterenol, reduced bone mass in loaded mice, and under such conditions, unloading no longer further reduced bone mass. Analyses on the cellular bases indicated that unloading-induced reduction in the levels of osteoblastic cell activities, including mineral apposition rate, mineralizing surface, and bone formation rate, was suppressed by propranolol treatment and that isoproterenol-induced reduction in these levels of bone formation parameters was no longer suppressed by unloading. Unloading-induced reduction in the levels of mineralized nodule formation in bone marrow cell cultures was suppressed by propranolol treatment in vivo. In addition, loss of a half-dosage in the dopamine β-hydroxylase gene suppressed the unloading-induced bone loss and reduction in mineralized nodule formation. Unloading-induced increase in the levels of osteoclastic activities such as osteoclast number and surface as well as urinary deoxypyridinoline was all suppressed by the treatment with propranolol. These observations indicated that sympathetic nervous tone mediates unloading-induced bone loss through suppression of bone formation by osteoblasts and enhancement of resorption by osteoclasts. Osteoporosis is one of the major age-related diseases in our modern world (1Riggs B.L. Hartmann L.C. N. Engl. J. Med. 2003; 348: 618-629Crossref PubMed Scopus (829) Google Scholar, 2Raisz L.G. Rodan G.A. Endocrinol. Metab. Clin. N. Am. 2003; 32: 15-24Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 3Riggs B.L. J. Cell. Biochem. 2003; 88: 209-215Crossref PubMed Scopus (40) Google Scholar, 4Riggs B.L. J. Cell. Biochem. 2003; 88: 209-215Crossref PubMed Scopus (43) Google Scholar). Especially, high fracture risk in osteoporosis patients results in not only loss of quality of life but also loss of life in a certain fraction of aged patients. The number of osteoporosis patients is estimated to be close to 10% of the whole population in many advanced countries. Among this patient population, a significant number of patients have disuse osteoporosis based on bedridden conditions caused by aging-related cardiovasucular as well as cerebrovascular diseases (5Ehrlich P.J. Lanyon L.E. Osteoporosis Int. 2002; 13: 688-700Crossref PubMed Scopus (387) Google Scholar). Bone has been known to be lost upon the removal of the mechanical stimuli, and prolonged lack of mechanical stimuli leads to disuse osteoporosis (5Ehrlich P.J. Lanyon L.E. Osteoporosis Int. 2002; 13: 688-700Crossref PubMed Scopus (387) Google Scholar, 6Lanyon L. Skerry T. J. Bone Miner. Res. 2001; 16: 1937-1947Crossref PubMed Scopus (201) Google Scholar, 7Bikle D.D. Sakata T. Halloran B.P. Gravit. Space Biol. Bull. 2003; 16: 45-54PubMed Google Scholar, 8Bikle D.D. Halloran B.P. J. Bone Miner. Res. 1991; 6: 527-530PubMed Google Scholar, 9Serhan C.N. N. Engl. J. Med. 2004; 350: 1902-1903Crossref PubMed Scopus (6) Google Scholar). However, the mechanisms underlying such disuse osteoporosis are largely unknown (2Raisz L.G. Rodan G.A. Endocrinol. Metab. Clin. N. Am. 2003; 32: 15-24Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 5Ehrlich P.J. Lanyon L.E. Osteoporosis Int. 2002; 13: 688-700Crossref PubMed Scopus (387) Google Scholar, 6Lanyon L. Skerry T. J. Bone Miner. Res. 2001; 16: 1937-1947Crossref PubMed Scopus (201) Google Scholar). Bone mass is determined by the actions of osteoblasts, which make bone, and those of osteoclasts, which resorb bone (2Raisz L.G. Rodan G.A. Endocrinol. Metab. Clin. N. Am. 2003; 32: 15-24Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 12Augat P. Simon U. Liedert A. Claes L. Osteoporosis Int. 2005; 16: S36-S43Crossref PubMed Scopus (220) Google Scholar, 13Strewler G.J. N. Engl. J. Med. 2004; 350: 1172-1174Crossref PubMed Scopus (38) Google Scholar, 14Mohamed A.M. N. Engl. J. Med. 2003; 349: 1671Crossref PubMed Scopus (3) Google Scholar, 15Fuller K.E. N. Engl. J. Med. 2004; 350: 189-192Crossref PubMed Scopus (18) Google Scholar). The balance between the two activities is under the control of hormones and cytokines. Usually, simultaneous changes in the two activities are considered to be coupled to compensate bone loss. However, in the case of disuse osteoporosis, bone formation activities are not enhanced even in the presence of enhanced bone resorption. Rather, bone formation is significantly suppressed in disuse osteoporosis (16Ishijima M. Tsuji K. Rittling S.R. Yamashita T. Kurosawa H. Denhardt D.T. Nifuji A. Noda M. J. Exp. Med. 2001; 193: 399-404Crossref PubMed Scopus (200) Google Scholar, 17Harada S. Rodan G.A. Nature. 2003; 423: 349-355Crossref PubMed Scopus (1139) Google Scholar). Therefore, disuse osteoporosis is a critical pathological situation where bone mass is continuously lost without having any compensatory activity against the reduction of bone. However, how such a critical reduction in bone formation occurs in unloading-induced pathological bone loss is not yet known. Bone formation and bone resorption are under the control of the systemic hormones and local cytokines (2Raisz L.G. Rodan G.A. Endocrinol. Metab. Clin. N. Am. 2003; 32: 15-24Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 6Lanyon L. Skerry T. J. Bone Miner. Res. 2001; 16: 1937-1947Crossref PubMed Scopus (201) Google Scholar). However, none of these factors have been proven to be the major cause of the disuse osteoporosis. In addition to the bedridden patients, astronauts under gravity conditions also lose bone due to the loss of mechanical stress. Analysis of such astronauts returning from space indicated that sympathetic nervous tone is enhanced in their muscle (18Fu Q Levine B.D. Pawelczyk J.A. Ertl A.C. Diedrich A. Cox J.F. Zuckerman J.H. Ray C.A. Smith M.L. Iwase S. Saito M. Sugiyama Y. Mano T. Zhang R. Iwasaki K. Lane L.D. Buckey Jr., J.C. Cooke W.H. Robertson R.M. Baisch F.J. Blomqvist C.G. Eckberg D.L. Robertson D. Biaggioni I. J. Physiol. 2002; 544: 653-664Crossref PubMed Scopus (76) Google Scholar, 19Cox J.F. Tahvanainen K.U. Kuusela T.A. Levine B.D. Cooke W.H. Mano T. Iwase S. Saito M. Sugiyama Y. Ertl A.C. Biaggioni I. Diedrich A. Robertson R.M. Zuckerman J.H. Lane L.D Ray C.A White R.J. Pawelczyk J.A. Buckey Jr., J.C. Baisch F.J. Blomqvist C.G. Robertson D. Eckberg D.L. J. Physiol. 2002; 538: 309-320Crossref PubMed Scopus (73) Google Scholar). Sympathetic nervous system would regulate bone mass via bone formation by osteoblasts systemically (20Takeda S. Elefteriou F. Levasseur R. Liu X. Zhao L. Parker K.L. Armstrong D. Ducy P. Karsenty G. Cell. 2002; 111: 305-317Abstract Full Text Full Text PDF PubMed Scopus (1232) Google Scholar). However, nothing has been known about how this system is related to pathophysiology in bone metabolism in the body. Therefore, we examined whether sympathetic nervous tone is involved in reduction of bone mass in a disuse osteoporosis model via osteoblastic and osteoclastic cells using hind limb unloading. Animals—Male 129 or C57BL/6J mice (10–14 weeks old) were used for the experiments. Mice were housed for at least 1 week prior to the study. The mice were subjected to either intraperitoneal injections of propranolol (20 μg/g of body weight/day) (21Commins S.P. Watson P.M. Levin N. Beiler R.J. Gettys T.W. J. Biol. Chem. 2000; 275: 33059-33067Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), continuous administration of guanethidine via an osmotic minipump (20 μg/g of body weight/day) (22Sherman B.E. Chole R.A. J. Bone Miner. Res. 2000; 15: 1354-1360Crossref PubMed Scopus (19) Google Scholar), or intraperitoneal injections of isoproterenol (6 μg/g of body weight/day) (23Takeda S. Elefteriou F. Levasseur R. Liu X. Zhao L. Parker K.L. Armstrong D. Ducy P. Karsenty G. Cell. 2002; 111: 305-317Abstract Full Text Full Text PDF PubMed Scopus (1373) Google Scholar). In the case of osmotic minipump implantation, the pump was implanted 12 h before the start of hind limb unloading. Osmotic minipumps were implanted into the subcutaneous tissue in the back of the animals according to the manufacturer's instruction. For dopamine β-hydroxylase (DBH) 1The abbreviations used are: DBH, dopamine β-hydroxylase; BV/TV, bone volume/tissue volume; BFR, bone formation rate; TRAP, tartrate-resistant acid phosphatase; MAR, bone mineral apposition rate; MS, mineralizing surface; CT, computerized tomography. gene deletion experiments, heterozygous knockout mice with a C57BL6/129sv F2 background and wild type litter mate mice were used (13-week-old females) (24Thomas S.A. Matsumoto A.M. Palmiter R.D. Nature. 1995; 374: 643-646Crossref PubMed Scopus (468) Google Scholar). All of the mice were injected intraperitoneally with calcein at 4 mg/kg at 4 and 2 days before sacrifice. After treatment for 10 or 14 days, mice were anesthetized with tribromoethanol at 200 mg/kg and were sacrificed by cervical dislocation. Hind Limb Unloading Model—Hind limb unloading was conducted by applying a tape to the surface of the hind limb to set a metal clip (10Ishijima M. Tsuji K. Rittling S.R. Yamashita T. Kurosawa H. J. Bone Miner. Res. 2002; 17: 661-667Crossref PubMed Scopus (88) Google Scholar, 16Ishijima M. Tsuji K. Rittling S.R. Yamashita T. Kurosawa H. Denhardt D.T. Nifuji A. Noda M. J. Exp. Med. 2001; 193: 399-404Crossref PubMed Scopus (200) Google Scholar). The end of the clip was fixed to an overhead bar. The height of the bar was adjusted to maintain the mice at an ∼30° head down tilt with the hind limbs elevated above the floor of the cage. The mice were subjected to hind limb unloading for 10 or 14 days. Loaded control mice were also housed individually under the same conditions except for hind limb unloading for the same duration. Body Weight—The body weight of the mice was monitored during the experimental period. There were no significant changes in body weight in any of the groups during the course of the study. This confirmed that stress could be considered minimal in our experiments as previously described (16Ishijima M. Tsuji K. Rittling S.R. Yamashita T. Kurosawa H. Denhardt D.T. Nifuji A. Noda M. J. Exp. Med. 2001; 193: 399-404Crossref PubMed Scopus (200) Google Scholar, 21Commins S.P. Watson P.M. Levin N. Beiler R.J. Gettys T.W. J. Biol. Chem. 2000; 275: 33059-33067Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Two-dimensional Micro-CT Analysis of Bone—Bone volume/tissue volume (BV/TV) was determined based on two-dimensional micro-CT analyses using a micro-CT apparatus (Musashi, Nittetsu-ELEX Co., Kita-Kyushu City, Japan). The data were quantified by using automated image analysis system (Luzex-F, Nireco). The fractional bone volume (BV/TV) was obtained in an area of 0.47 mm2 with its closest and furthest edges at 0.28 and 0.84 mm, respectively, distal to the growth plate of the proximal ends of the tibiae. The threshold level for the measurements was set at 110 for the analyses (16Ishijima M. Tsuji K. Rittling S.R. Yamashita T. Kurosawa H. Denhardt D.T. Nifuji A. Noda M. J. Exp. Med. 2001; 193: 399-404Crossref PubMed Scopus (200) Google Scholar, 21Commins S.P. Watson P.M. Levin N. Beiler R.J. Gettys T.W. J. Biol. Chem. 2000; 275: 33059-33067Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Histomorphometric Analysis of Bone—At the end of the experiments, the right femora of each mouse were removed and fixed in 70% ethanol. Serial 3-μm-thick sagittal sections were made as undecalcified sections (right femora). For bone formation rate (BFR), metaphyseal cancellous bone in the femora was used to obtain bone fraction in a rectangular area of 0.34 mm2 (0.5 × 0.67 mm) with its closest and furthest edges at 0.3 and 0.8 mm distal to the growth plate, respectively. For decalcified sections, the left tibiae of the mice were removed at the end of the experiments and fixed in 4% paraformaldehyde and then decalcified in 20% EDTA. Serial 5-mm-thick sagittal sections were made using a microtome and stained for tartrate-resistant acid phosphatase (TRAP). TRAP-positive multinucleated cells attached to bone were scored as osteoclasts. Measurements were made within an area of 0.24 mm2 (0.6 × 0.4 mm), with its closest and furthest edges at 0.3 and 0.7 mm distal to the growth plate of the proximal ends of the tibiae. Histomorphometry was conducted to quantify the number of osteoclasts (Oc.N/BS) and osteoclast surface (Oc.S/BS) as defined by Parfitt et al. (11Parfitt A.M. Drezner M.K. Glorieux F.H. Kanis J.A. Malluche H. Meunier P.J. Ott S.M. Recker R.R. J. Bone Miner. Res. 1987; 2: 595-610Crossref PubMed Scopus (4920) Google Scholar). Nodule Formation Analysis—We cultured the cells obtained from the bone marrow of the animals that were subjected to hind limb unloading (12Augat P. Simon U. Liedert A. Claes L. Osteoporosis Int. 2005; 16: S36-S43Crossref PubMed Scopus (220) Google Scholar) or loading in combination with the treatment with adrenergic modulators. The cells were cultured in the presence of ascorbic acid (50 μg/ml) and β-glycerophosphate (10 mm) in α-minimal essential medium supplemented with 10% FBS, 1% antibiotics. After 3 weeks in culture, alizarin red staining was conducted. This staining was used to visualize the calcified materials formed in vitro. Briefly, after the cultures were terminated, the cells were fixed in 100% ethanol and then were stained in alizarin red solution (1%) for 1 min. The cultures were then rinsed several times with water. The area of alizarin red positive nodules was measured by using an image analyzer. Urinary Deoxypyridinoline—Deoxypyridinoline levels in urine at the end of the hind limb unloading were measured by enzyme-linked immunosorbent assay (Metra Biosystems) (6Lanyon L. Skerry T. J. Bone Miner. Res. 2001; 16: 1937-1947Crossref PubMed Scopus (201) Google Scholar). Urine samples were collected from five mice per group, which were housed in a metabolic cage during the last 24 h and analyzed. Statistical Analysis—Data were expressed as means ± S.D., and statistical evaluation was performed based on analysis of variance, using a statistical software package for Windows, Statview version 5.0 (SAS Institute). A p value less than 0.05 was considered to be statistically significant. Hind limb unloading reduced bone volume in vehicle-treated control mice as reported previously (16Ishijima M. Tsuji K. Rittling S.R. Yamashita T. Kurosawa H. Denhardt D.T. Nifuji A. Noda M. J. Exp. Med. 2001; 193: 399-404Crossref PubMed Scopus (200) Google Scholar) (Fig. 1, a and b, column 1 versus column 2). In contrast, treatment with propranolol, a β-adrenergic blocker acting at the receptor levels, suppressed hind limb unloading-induced reduction in bone mass expressed as BV/TV (Fig. 1, a and b; no significant difference between columns 3 and 4). With regard to the drug effect in unloaded mice, propranolol treatment resumed the bone loss induced by unloading (Fig. 1b, column 2 versus column 4). Control-loaded mice treated with propranolol revealed significant difference in bone mass compared with unloaded vehicle-treated mice (Fig. 1b, column 2 versus column 3). If hind limb unloading suppressed the levels of bone mass and bone formation through sympathetic tone, not only the actions of the blockers for sympathetic signaling, which act at the receptor levels, but also the depletion of the presynaptic transmitter reservoir should affect the unloading-induced reduction in bone mass. Therefore, guanethidine sulfate was administrated to deplete norepinephrine at the presynaptic nerve ending levels in the animals that were subjected to hind limb unloading. As shown in Fig. 2, guanethidine treatment suppressed unloading-induced bone loss (Fig. 2, a and b). The pattern of the levels in BV/TV was similar to that in the case of propranolol (Fig. 1). Body weight was not altered significantly due to unloading or drug treatment in all of our experiments (Fig. 2, c–e).Fig. 2Guanethidine treatment suppressed unloading-induced pathological bone loss. During hind limb unloading of mice (129 strain) for 14 days, guanethidine treatment or vehicle was administered using an osmotic minipump (model 1002). The number of the mice used for the experiments represented in each bar is indicated as N. During the 14-day hind limb unloading of 129 mice, guanethidine treatment was carried out.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further test whether unloading signaling is exerted in the line of sympathetic tone, we examined the effects of activation by isoproterenol of sympathetic tone. This was in order to determine whether isoproterenol may mask the unloading-induced signals. Isoproterenol treatment reduced bone mass in control-loaded mice as reported previously (Fig. 3, a and b, column 1 versus column 3), and under this condition, hind limb unloading no longer reduced bone mass (Fig. 3, a and b; compare columns 3 and 4). Notably, the levels of bone mass reduction by isoproterenol treatment were similar to those in unloaded mice (Fig. 3b, column 3 versus column 2). Thus, the data of these three sets of experiments are in accordance with the notion that unloading signaling is suppressed by blockers for sympathetic signaling, and such signaling is no longer active in the presence of the action of β-adrenergic agonist. In order to examine the mechanisms of β-adrenergic actions in unloading-induced bone loss, we conducted dynamic histomorphometry. Hind limb unloading induced reduction in the levels of bone mineral apposition rate (MAR)), mineralizing surface (MS), and BFR (Fig. 4, a–d) after 10 days of hind limb unloading and propranolol treatment suppressed the unloading-induced decrease in MAR, MS, and BFR (Fig. 4, a–d). The patterns of the columns were similar to those in the case of bone mass (Fig. 1). Guanethidine treatment also suppressed the hind limb unloading-induced reduction in MAR, MS, and BFR (Fig. 5, a–d) in the mice subjected to hind limb unloading for 2 weeks. Again, the patterns of the columns were similar to those in the case of bone mass (Fig. 1). Thus, depletion of sympathetic neurotransmitter has effects similar to the block of β-adrenergic receptor with respect to bone formation activity in vivo. Isoproterenol treatment alone suppressed the levels of mineral apposition rate, mineralizing surface, and bone formation rate, whereas in the presence of isoproterenol treatment, 10 days of hind limb unloading failed to suppress all of these parameters (Fig. 6, a–d). It is again notable that the patterns of the columns in Fig. 6 were similar to those observed in the effects of isoproterenol on bone mass (Fig. 3). These three lines of evidence based on dynamic histomorphometry indicated that bone formation is the target of sympathetic tone in mice subjected to hind limb unloading.Fig. 5Guanethidine treatment suppressed unloading-induced reduction in bone formation in vivo. Mice were treated with guanethidine as described in the legend to Fig. 3. The number of mice in each group is indicated as N. a, calcein double-labeled surfaces of the bones at the ends of the femora after hind limb unloading (Unload) or loading (Load) in vehicle- or guanethidine-treated mice. The arrows indicate the lines of calcein labeling (light green) used to obtain data shown in b–d. b–d, in the undecalcified sections of the distal ends of the femora, MAR (b), MS (c), and BFR (d) were measured in all areas at 0.3–0.8 mm distal to the growth plate in the metaphyseal region as described under “Materials and Methods.” The mice were injected intraperitoneally with calcein at 4 mg/kg 4 and 2 days before sacrifice at 2 weeks. Data are expressed as means and S.D. for five bones from each of the vehicle- and guanethidine-treated mice groups. *, statistically significant difference (p < 0.05). #, statistically significant difference between the vehicle group and drug-treated group (p < 0.05) (either control-loaded or unloaded groups). §, statistically significant difference between vehicle-treated unloaded group and drug-treated control-loaded group (p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Isoproterenol treatment suppressed the level of bone formation parameters, and unloading did not further suppress these parameters in vivo. Mice were treated with isoproterenol as described in the legend to Fig. 3. The number of mice in each group is indicated as N. a, calcein double-labeled surfaces of the bones at the ends of the femora after hind limb unloading (Unload) or loading (Load) in vehicle or isoproterenol-treated mice. The arrows indicate the lines of calcein labeling (light green) used to obtain data shown in b–d. b–d, in the undecalcified sections of the distal ends of the femora, MAR (b), MS (c), and BFR (d) were measured in all areas at 0.3–0.8 mm distal to the growth plate in the metaphyseal region as described under “Materials and Methods.” The mice were injected intraperitoneally with calcein at 4 mg/kg 4 and 2 days before sacrifice at 10 days. Data are expressed as means and S.D. for five bones from each of the vehicle- and isoproterenol-treated mouse groups. *, statistically significant difference (p < 0.05). #, statistically significant difference between the vehicle group and drug-treated group (p < 0.05) (either control-loaded or unloaded groups).View Large Image Figure ViewerDownload Hi-res image Download (PPT) We further examined whether our observations can be detected at cell levels in culture. For this purpose, a nodule formation assay was conducted by using bone marrow cells obtained from the bone of the mice after they were subjected to hind limb unloading or control loading in the presence or the absence of the treatment with pharmacological agents. Hind limb unloading reduced nodule formation in the cultures of cells obtained from the animals (Fig. 7, a and b, column 1 versus column 2). Propranolol treatment in vivo suppressed the unloading-induced reduction in the mineralized nodule formation in culture (Fig. 7, a and b, column 3 versus column 4). Isoproterenol treatment suppressed the levels of nodule formation in loaded control mice (Fig. 8, a and b, column 1 versus column 3), and in the presence of isoproterenol treatment in vivo, hind limb unloading failed to further reduce the levels of nodule formation in bone marrow cells in culture (Fig. 8, a and b, column 3 versus column 4). Thus, these in vitro experiments indicated that β-adrenergic sympathetic tone mediates unloading-induced reduction in mineralization of bone marrow cell cultures.Fig. 8Isoproterenol treatment in vivo suppressed the levels of nodule formation, and unloading conditions failed to further reduce the mineralization levels in vitro. Mice were treated with isoproterenol as described in legend to Fig. 6. The number of mice in each group is indicated as N. The cells obtained from the bone marrow of the animals subjected to hind limb unloading with the isoproterenol or vehicle treatment were cultured in the presence of ascorbic acid and β-glycelophosphare. After 3 weeks, alizarin red staining was conducted, and the area of the alizarin red-positive nodule was quantified. *, statistically significant difference (p < 0.05). #, statistically significant difference between the vehicle group and drug-treated group (p < 0.05) (either control-loaded or unloaded groups).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In order to examine the effects of the sympathetic nervous tone on unloading-induced reduction in bone mass and bone formation in a genetic model rather than pharmacological modulation, DBH (dopamine β-hydroxylase) knockout mice were subjected to hind limb unloading. Hind limb unloading reduced bone mass by about 68.6% in wild type littermate mice (Fig. 9a, column 1 versus column 2). Heterozygous loss for the dopamine β-hydroxylase gene attenuated the reduction in bone loss by about 29.7% after hind limb unloading (Fig. 9a, column 3 versus column 4). The rate of bone loss due to unloading (calculated as (control load – unload)/(control load × 100%) was significantly reduced from 68 ± 7% in DBH+/+ (wild type) to 30 ± 24% in DBH+/– (Fig. 9c)(p < 0.05). The nodule formation in cultures of bone marrow cells of the wild type littermate was reduced by unloading (Fig. 9b, column 1 versus column 2). The marrow cells obtained from DBH gene heterozygous knockout mice indicated suppression of hind limb unloading-induced reduction in nodule formation (Fig. 9b, column 3 versus column 4). During the course of unloading-induced bone loss, bone resorption also occurs as critical events to reduce bone mass. Unloading in tail-suspended mice caused an increase in osteoclast number (Oc.N/BS) and osteoclast surface (Oc.S/BS) based on histomorphometry in vivo as reported previously (Fig. 10, b–g, column 1 versus column 2). In contrast, inhibition of sympathetic tone by treatment with propranolol or guanethidine, suppressed such unloading-induced increase in osteoclast number (Oc.N/BS) (Fig. 10, a and b for propranolol, d for guanethidine; column 3 versus column 4) and osteoclast surface (Oc.S/BS) (Fig. 10, c for propranolol, e for guanethidine; column 3 versus column 4). The levels of Oc.N/BS and Oc.S/BS were enhanced by either unloading or isoproterenol treatment alone to similar levels (Fig. 10, f and g, column 2 versus column 3). The simultaneous presence of unloading conditions and isoproterenol treatment resulted in similar levels in the increase in osteoclast number (Oc.N/BS) and surface (Oc.S/BS) to those in mice subjected to either one of the two conditions alone (Fig. 10, f and g, column 4 versus columns 2 and 3). Furthermore, unloading-induced increase in deoxypyridinoline excretion into urine (Fig. 11, column 1 versus column 2) was also suppressed by guanethidine treatment (Fig. 11, column 3 versus column 4).Fig. 11Pharmacological inhibition of sympathetic tone suppresses unloading-induced systemic bone loss assessed by the levels of urinary deoxypyridinoline excretion. Mice were subjected to hind limb unloading and guanethidine treatment as described under “Materials and Methods.” After 14 days, urine samples of the mice were collected, and the amount of deoxypyridinoline in the urine was measured as described under “Materials and Methods.” *, statistically significant difference (p < 0.05). The number of mice in each group is indicated as N. #, statistically significant difference between the vehicle group and drug-treated group (p < 0.05) (either control-loaded or unloaded groups). §, statistically significant difference between vehicle-treated unloaded group and drug-treated control-loaded group (p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our data reveal that sympathetic nervous tone is mediating unloading-induced bone loss via reduction in osteoblastic cell activity as well as enhancement in osteoclastic cell activity. This is the first report that sympathetic control of the bone mass is involved in the unloading-induced bone loss by controlling osteoblasts. Unloading-induced bone loss was suppressed by the treatment of the animals with propranolol, a receptor antagonist, suggesting that β-adrenergic receptors in the sympathetic nervous system are the target of unloading-induced bone loss. The peripheral sympathetic nervous targets receive signals from the proximal upper nervous ending, which releases noradrenalin as a neurotransmitter into the synaptic gap. We observed that unloading-induced bone loss was again suppressed by the treatment of the animals with guanethidine. These data indicate that the depletion of noradrenalin in the proximal ending of the synaptic gap could suppress the unloading-induced osteoporosis. Furthermore, unloading failed to further suppress the bone volume that was reduced by a β-adrenergic agonist, isoproterenol. These three series of observations further indicate that sympathetic nervous tone is involved in unloading-induced pathological bone loss. We also examined the effects of heterozygous deletion of the DBH gene. DBH is required for the sympathetic nervous tone. Therefore, we subjected the heterozygous knockout mice to hind limb unloading. Unloading-induced bone loss was attenuated by the absence of the half-dosage of dopamine β-hydroxylase gene, indicating that the presence of a full dosage of DBH gene in the animals is necessary for the complete effects of the unloading-induced bone loss. Furthermore, DBH data excluded the possibility that pharmacological experiments might be influenced by possible artifacts due to the systemic drug administration. Thus, both pharmacological and genetic interventions of sympathetic signals supported the idea that the sympathetic nervous system causes pathological loss of bone in unloading-induced osteopenia. Since bone formation is the critical activity to determine the levels of bone loss due to unloading, it is the major target to elucidate the mechanisms required for unloading-induced loss of bone mass. Dynamic histomorphometric analyses on osteoblastic cell activity in vivo revealed that the reduction in bone formation activity in vivo due to unloading was suppressed by a series of pharmacological agents including propranolol and guanethidine. Furthermore, bone cell culture experiments using the bone marrow cells taken from the animals subjected to either pharmacological or genetic interventions of the sympathetic nervous tone indicate that these interventions suppressed unloading-induced reduction in mineralized nodule formation. The interpretation of the reduction in the formation of osteoblastic bone nodules after 3 weeks in culture would be that progenitor cell populations for osteoblastic cell lineage could be reduced at the point of harvesting the cells from animals at the end of unloading. This suppression was blocked by the treatment with propranolol. We also carried out 3-week culture experiments to form bone nodules in the presence or absence of isoproterenol or propranolol and guanethidine in culture using bone marrow cells that were taken from wild type (untreated) C57Bl6 mice. There was no effect of these agents in culture to modulate the nodule formation in the bone marrow cells from untreated mice (data not shown). These data support the idea that the progenitor population in bone marrow in vivo would be reduced by the unloading condition or by treatment with drugs such as propranolol, guanethidine, and isoproterenol during the periods of the tail suspension experiments. Therefore, the action of sympathetic nervous tone during unloading-induced bone loss renders cell level effects on osteoblastic cell activity. It is known that propranolol at doses of 10 μg/g body weight could reduce blood pressure in mice. It is also known that such blood pressure change could be enhanced by the treatment with isoproterenol. However, there has yet been no clear evidence in clinical or experimental settings in terms of a direct relationship between blood pressure and bone mass. However, at this point, we cannot fully exclude the possibility that propranolol or isoproterenol used in our experiments may have affected bone mass via regulation of blood pressure, and these points have to be elucidated in the future. It is intriguing to consider the differences in the time courses of the β-adrenergic receptor modulators. Propranolol has been known to reduce blood pressure in days, whereas isoproterenol and guanethidine alter blood pressure immediately. It is not known whether such time course differences seen in their effects on blood pressure may also affect their modulation of the bone mass. However, both propranolol and guanethidine blocked unloading-induced bone loss similarly in our experiments. This may partially be due to the nature of bone where the biological read out (i.e. bone mass) could be detected in a relatively slow manner compared with blood pressure. If these types of drugs could be proven to be effective in the treatment of unloading-induced osteoporosis, it has to be considered that side effects might occur in the treatment of the patients. In the future, we may have to identify certain windows of the dosages by which bone effects may be obtained without affecting the blood pressure, or these drugs may be used only for those patients whose side effects could be predicted to be less based on the individual genomic data. Our data indicated that sympathetic tones regulate unloading-induced enhancement in bone resorption. This was evidenced by the observations that inhibition of sympathetic tone by propranolol and guanethidine, suppressed unloading-induced bone resorption, and this leads to suppression in bone loss. Histomorphometric analyses indicated that unloading activates bone resorption through the increase in osteoclast number and osteoclast surface. These increases in the bone resorption parameters due to unloading were suppressed by treatment with propranolol and guanethidine. Such observations regarding the inhibitors for sympathetic tone effects on unloading-induced bone resorption were not limited to particular bone, since suppression of sympathetic tone by the treatment with guanethidine also suppressed unloading-induced increase in deoxypyridinoline excretion into urine, which is a systemic bone resorption maker. These data revealed that sympathetic tone regulates unloading-induced bone resorption as well. Involvement of sympathetic tone in the induction of bone resorption after unloading was further supported by the analysis on the mice subjected to simultaneous unloading and isoproterenol treatment. Either isoproterenol treatment or unloading alone could cause an equivalent increase in the levels of bone loss as well as an increase in the levels of bone resorption parameters (osteoclast number and osteoclast surface). The simultaneous presence of unloading conditions and isoproterenol treatment resulted in an increase in bone loss as well as an increase in osteoclast number and osteoclast surface to levels equivalent to those in the cases of either one of the two conditions alone. These data further support the notion that sympathetic nervous tone and the unloading condition would share signaling pathways. Unloading induces rapid bone loss and increases fracture risk significantly, especially in elderly bedridden patients. In fact, deoxypyridinoline excretion into urine was reported to increase in astronauts within a few hours after they are exposed to microgravity conditions in space. Such rapid bone loss is caused by an immediate response of osteoclastic activity when the body is subjected to unloading conditions. Although this phenomenon is so clearly observed in human (bedridden patients and astronauts) and various animal models, the mechanism for such a response of bone resorbing activity has not been identified. Since the nervous system could elicit signals at a relatively fast speed, our identification of the sympathetic tone as responsible for the unloading-induced increase in bone resorption and loss of bone mass would explain the rapid response of osteoclasts to unloading. Although we have used DBH knockout mice to see the effects of such a deletion of the gene on the bone loss due to tail suspension, this enzyme is also required to produce epinephrine in the adrenals as well as norepinephrine. The enzyme required to produce epinephrine from norepinephrine, phenylethanolamine-N-methyltransferase, has been suggested in several reports to be subjected to induction by immobilization-induced stress. As a result, the DBH heterozygous knock-out mice results may not represent conclusive proof of a prominent role for the sympathetic nervous system here. However, our data on the effects of unloading on bone in DBH knockout mice is at least in part in accordance with the idea that sympathetic tone is involved in the bone loss due to unloading. In conclusion, our data indicate that sympathetic tone is in charge of the pathological reduction in bone mass upon unloading by suppressing osteoblastic cell actions and enhancing osteoclastic actions. These data predict that identification of the involvement of systemic modulation in the bone loss in unloading conditions could give a clue to appropriate measures to treat patients with disuse osteoporosis (9Serhan C.N. N. Engl. J. 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Extracellular matrix production and degradation by bone cells are critical steps in bone metabolism. Mutations of the gene encoding MMP-2, an extracellular matrix-degrading enzyme, are associated with a human genetic disorder characterized by subcutaneous nodules, arthropathy, and focal osteolysis. It is not known how the loss of MMP-2 function results in the pathology. Here, we show that Mmp2-/- mice exhibited opposing bone phenotypes caused by an impaired osteocytic canalicular network. Mmp2-/- mice showed decreased bone mineral density in the limb and trunk bones but increased bone volume in the calvariae. In the long bones, there was moderate disruption of the osteocytic networks and reduced bone density throughout life, whereas osteoblast and osteoclast function was normal. In contrast, aged but not young Mmp2-/- mice had calvarial sclerosis with osteocyte death. Severe disruption of the osteocytic networks preceded osteocyte loss in Mmp2-/- calvariae. Successful transplantation of wild-type periosteum restored the osteocytic canalicular networks in the Mmp2-/- calvariae, suggesting local roles of MMP-2 in determining bone phenotypes. Our results indicate that MMP-2 plays a crucial role in forming and maintaining the osteocytic canalicular network, and we propose that osteocytic network formation is a determinant of bone remodeling and mineralization. Extracellular matrix production and degradation by bone cells are critical steps in bone metabolism. Mutations of the gene encoding MMP-2, an extracellular matrix-degrading enzyme, are associated with a human genetic disorder characterized by subcutaneous nodules, arthropathy, and focal osteolysis. It is not known how the loss of MMP-2 function results in the pathology. Here, we show that Mmp2-/- mice exhibited opposing bone phenotypes caused by an impaired osteocytic canalicular network. Mmp2-/- mice showed decreased bone mineral density in the limb and trunk bones but increased bone volume in the calvariae. In the long bones, there was moderate disruption of the osteocytic networks and reduced bone density throughout life, whereas osteoblast and osteoclast function was normal. In contrast, aged but not young Mmp2-/- mice had calvarial sclerosis with osteocyte death. Severe disruption of the osteocytic networks preceded osteocyte loss in Mmp2-/- calvariae. Successful transplantation of wild-type periosteum restored the osteocytic canalicular networks in the Mmp2-/- calvariae, suggesting local roles of MMP-2 in determining bone phenotypes. Our results indicate that MMP-2 plays a crucial role in forming and maintaining the osteocytic canalicular network, and we propose that osteocytic network formation is a determinant of bone remodeling and mineralization. Bone is continuously remodeled to adopt a volume appropriate for the local environment; the amount of bone deposited depends on the balance between bone formation and resorption by bone cells, osteoblasts, osteoclasts, and osteocytes (1Bilezikian J. Raisz L. Rodan G. Principles of Bone Biology. Academic Press, San Diego, CA2002: 3-126Google Scholar). Osteoblasts are bone-forming cells that differentiate from mesenchymal stem cells and secrete extracellular matrix (ECM) 4The abbreviations used are: ECM, extracellular matrix; DMP-1, dentin matrix protein 1; DMEM, Dulbecco's modified Eagle's medium; MMP, matrix metalloproteinase; NAO, nodulosis, arthropathy, and osteolysis; BMD, bone mineral density; pQCT, peripheral quantitative computed tomography; MAR, mineral apposition rate; BFR/BS, ratio of bone formation rate to bone surface. proteins, which are subsequently mineralized. Osteoclasts are bone-resorbing cells that differentiate from hematopoietic stem cells and degrade bone ECM proteins after demineralization in the extracellular space (Howship's lacunae) adjacent to the ruffled borders. In contrast to osteoblasts and osteoclasts, which act at bone surfaces, osteocytes, cells of osteoblastic lineage, are embedded in bone and are terminally differentiated. Osteocytes extend their dendritic processes into the bone matrix to constitute a well developed canalicular network with other cells. Although osteocytes are the most abundant cell type in bone tissue, their role in bone metabolism is not firmly established. ECM production and degradation by bone cells are critical steps in bone metabolism (1Bilezikian J. Raisz L. Rodan G. Principles of Bone Biology. Academic Press, San Diego, CA2002: 3-126Google Scholar), and disturbed ECM turnover leads to bone disease. Type I collagen is a major ECM component. Secreted type I collagen molecules are processed by propeptidases and cross-linked by lysyl oxidases into mature collagen. Mutations of genes encoding type I collagen cause the bone disease osteogenesis imperfecta (2Byers P.H. Steiner R.D. Annu. Rev. Med. 1992; 43: 269-282Crossref PubMed Scopus (216) Google Scholar). Type I collagens are mainly degraded by matrix metalloproteinases (MMPs), which exert their enzymatic activity at a neutral pH in a zinc ion-dependent manner (3Nagase H. Woessner Jr., J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3902) Google Scholar, 4Andersen T.L. del Carmen Ovejeroqq M. Kirkegaard T. Lenhard T. Foged N.T. Delaisse J.M. Bone. 2004; 35: 1107-1119Crossref PubMed Scopus (122) Google Scholar). Several MMPs are expressed in bone tissue (5Tezuka K. Nemoto K. Tezuka Y. Sato T. Ikeda Y. Kobori M. Kawashima H. Eguchi H. Hakeda Y. Kumegawa M. J. Biol. Chem. 1994; 269: 15006-15009Abstract Full Text PDF PubMed Google Scholar, 6Breckon J.J. Papaioannou S. Kon L.W. Tumber A. Hembry R.M. Murphy G. Reynolds J.J. Meikle M.C. J. Bone Miner. Res. 1999; 14: 1880-1890Crossref PubMed Scopus (47) Google Scholar, 7Sasano Y. Zhu J.X. Tsubota M. Takahashi I. Onodera K. Mizoguchi I. Kagayama M. J. Histochem. Cytochem. 2002; 50: 325-332Crossref PubMed Scopus (106) Google Scholar, 8Nakamura H. Sato G. Hirata A. Yamamoto T. Bone. 2004; 34: 48-56Crossref PubMed Scopus (63) Google Scholar, 9Hatori K. Sasano Y. Takahashi I. Kamakura S. Kagayama M. Sasaki K. Anat. Rec. A. Discov. Mol. Cell. Evol. Bio. 2004; 277: 262-271Crossref PubMed Scopus (43) Google Scholar). MMPs may play a role in osteoclastic bone resorption (4Andersen T.L. del Carmen Ovejeroqq M. Kirkegaard T. Lenhard T. Foged N.T. Delaisse J.M. Bone. 2004; 35: 1107-1119Crossref PubMed Scopus (122) Google Scholar, 5Tezuka K. Nemoto K. Tezuka Y. Sato T. Ikeda Y. Kobori M. Kawashima H. Eguchi H. Hakeda Y. Kumegawa M. J. Biol. Chem. 1994; 269: 15006-15009Abstract Full Text PDF PubMed Google Scholar). Osteoblasts and osteocytes also produce MMPs such as MMP-2 and MMP-13 (7Sasano Y. Zhu J.X. Tsubota M. Takahashi I. Onodera K. Mizoguchi I. Kagayama M. J. Histochem. Cytochem. 2002; 50: 325-332Crossref PubMed Scopus (106) Google Scholar, 8Nakamura H. Sato G. Hirata A. Yamamoto T. Bone. 2004; 34: 48-56Crossref PubMed Scopus (63) Google Scholar, 9Hatori K. Sasano Y. Takahashi I. Kamakura S. Kagayama M. Sasaki K. Anat. Rec. A. Discov. Mol. Cell. Evol. Bio. 2004; 277: 262-271Crossref PubMed Scopus (43) Google Scholar). Recent linkage analysis suggests that a loss of function mutation of MMP2 causes a human autosomal recessive disorder with multicentric nodulosis, arthropathy with joint erosion, and osteolysis, termed NAO syndrome (10Martignetti J.A. Aqeel A.A. Sewairi W.A. Boumah C.E. Kambouris M. Mayouf S.A. Sheth K.V. Eid W.A. Dowling O. Harris J. Glucksman M.J. Bahabri S. Meyer B.F. Desnick R.J. Nat. Genet. 2001; 28: 261-265Crossref PubMed Scopus (251) Google Scholar, 11Vu T.H. Nat. Genet. 2001; 28: 202-203Crossref PubMed Scopus (43) Google Scholar). This syndrome also includes facial abnormalities and generalized osteoporosis (12Al Aqeel A. Al Sewairi W. Edress B. Gorlin R.J. Desnick R.J. Martignetti J.A. Am. J. Med. Genet. 2000; 93: 11-18Crossref PubMed Scopus (48) Google Scholar, 13Al-Mayouf S.M. Majeed M. Hugosson C. Bahabri S. Am. J. Med. Genet. 2000; 93: 5-10Crossref PubMed Scopus (47) Google Scholar, 14Al-Otaibi L. Al-Mayouf S.M. Majeed M. Al-Eid W. Bahabri S. Hugosson C.O. Pediatr. Radiol. 2002; 32: 523-528Crossref PubMed Scopus (9) Google Scholar). The finding that NAO syndrome is caused by a loss of MMP-2 activity raises questions about how an ECM-degrading enzyme affects bone volume (10Martignetti J.A. Aqeel A.A. Sewairi W.A. Boumah C.E. Kambouris M. Mayouf S.A. Sheth K.V. Eid W.A. Dowling O. Harris J. Glucksman M.J. Bahabri S. Meyer B.F. Desnick R.J. Nat. Genet. 2001; 28: 261-265Crossref PubMed Scopus (251) Google Scholar, 11Vu T.H. Nat. Genet. 2001; 28: 202-203Crossref PubMed Scopus (43) Google Scholar). We previously generated mice devoid of MMP-2 by gene targeting and observed a subtle retardation in their growth rate (15Itoh T. Ikeda T. Gomi H. Nakao S. Suzuki T. Itohara S. J. Biol. Chem. 1997; 272: 22389-22392Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). In the present study, we found marked skeletal abnormalities in the Mmp2-/- mice with decreased mineralization of long bones but increased bone deposition with osteocytic death in the calvariae. The data suggest that mild impairment in canalicular formation affects bone mineralization, but complete disruption of the osteocytic networks enhances osteoblastic activity and increases bone formation. We propose that osteocytes, through their canalicular networks, control osteoblast function and possibly modulate secondary mineralization. Animals—The MMP-2-deficient mice (15Itoh T. Ikeda T. Gomi H. Nakao S. Suzuki T. Itohara S. J. Biol. Chem. 1997; 272: 22389-22392Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar) used in this study were generated previously and backcrossed 9-11 times to C57BL/6J mice. Homozygous mutants were obtained by crossing heterozygotes. The Mmp2-/- mice were maintained in the animal facility of the RIKEN Brain Science Institute, with dry food pellets and water available ad libitum. The Col1a1r/r mice (16Liu X. Wu H. Byrne M. Jeffrey J. Krane S. Jaenisch R. J. Cell Biol. 1995; 130: 227-237Crossref PubMed Scopus (244) Google Scholar, 17Zhao W. Byrne M.H. Wang Y. Krane S.M. J. Clin. Investig. 2000; 106: 941-949Crossref PubMed Scopus (119) Google Scholar) were maintained in the animal facility at Massachusetts General Hospital. All of the experimental protocols were approved by the Institutional Animal Care and Use Committees. Radiographic Measurement—For soft x-ray radiograms, the mice were subjected to X-irradiation at 25 mA for 2 s. For bone mineral density (BMD) measurement, whole bodies and isolated bones were scanned with a Lunar PIXImus2 densitometer (GE Yokogawa Medical Systems, Tokyo, Japan). Peripheral Quantitative Computed Tomography (pQCT) Analysis—For pQCT analyses, isolated bones were subjected to XCT Research SA+ (Stratec Medizintechnik GmbH, Pforzheim, Germany). Femoral bones were scanned in two 0.46-mm-thick slices with a 0.08-mm voxel size. The slice 1.4 mm from the distal growth plate was used for cancellous BMD, and the slice 5.5 mm from the distal growth plate was used for cortical BMD. Calvarial bones were scanned at a 0.46-mm slice thickness and 0.05-mm voxel size. The coronal slice at the middle of parietal bones was used for calvarial BMD. Bone Histomorphometry—Tibial and femoral bones were fixed in 70% ethanol, and the nondecalcified bones were embedded in methylmethacrylate. For cancellous bone, longitudinal sections of tibial bone (3 μm thick) were stained with toluidine blue. Measurements were made at 400× magnification on a minimum of 20 optical fields between 450 and 1650 μm from the epiphyseal growth plate and 150 μm from the lateral cortices of the secondary spongiosa. For cortical bone, cross-sections (25 μm thick) of the femoral midshafts were stained with toluidine blue. The measurements were made at 400× magnification on a minimum of 30 optical fields. Calvarial bones were fixed in 70% ethanol, and the nondecalcified bones were embedded in glycomethacrylate. Coronal sections (3 μm thick) were cut and stained with toluidine blue. The measurements were made at 400× magnification on a minimum of 12 optical fields at 0.3 mm from the calvarial midline to both lateral sides. The mineral apposition rate was calculated by measuring the interval between two calcein-labeled lines following two intraperitoneal injections of calcein (4.0 mg/kg each). The nomenclature, symbols, and units used are those recommended by the Nomenclature Committee of the American Society of Bone and Mineral Research (18Parfitt A.M. Drezner M.K. Glorieux F.H. Kanis J.A. Malluche H. Meunier P.J. Ott S.M. Recker R.R. J. Bone Miner. Res. 1987; 2: 595-610Crossref PubMed Scopus (4920) Google Scholar). Histology—The bones were isolated and fixed in 4% paraformaldehyde, decalcified in 25% formic acid for 4-14 days at room temperature, and embedded in paraffin. For MMP-2 staining, anti-human MMP-2 monoclonal antibody 42-5D11 (Daiichi, Takaoka, Japan) was used at 10 μg/ml. For dentin matrix protein 1 (DMP-1) and sclerostin (the SOST gene product) staining, anti-rat DMP-1 polyclonal antibody M176 (Takara Bio, Ohtsu, Japan) and anti-mouse SOST polyclonal antibody MAB1589 (R & D Systems, Minneapolis, MN) were used at 2 and 50 μg/ml, respectively, after 0.25% trypsin/EDTA treatment at 37 °C for 15 min. Staining of Bone Canaliculi—Bone canaliculi were stained using the previously described Bodian method with minor modifications (19Kusuzaki K. Kageyama N. Shinjo H. Murata H. Takeshita H. Ashihara T. Hirasawa Y. Acta Orthop. Scand. 1995; 66: 166-168Crossref PubMed Scopus (10) Google Scholar, 20Kusuzaki K. Kageyama N. Shinjo H. Takeshita H. Murata H. Hashiguchi S. Ashihara T. Hirasawa Y. Bone. 2000; 27: 655-659Crossref PubMed Scopus (45) Google Scholar). Deparaffinized sections were stained with protein-silver solution containing copper balls at 37 °C for 24 h and reduced in 1% hydroquinone and 5% formalin for 30 min. The sections were then treated with 0.5% gold chloride solution for 50 min and 2% oxalic acid solution for 60 min. Finally, they were dehydrated and embedded for microscopic observation. Cell Culture—Bone marrow was flushed from the femora, and the cells were plated at 1 × 106 cells/well. For mineralized nodule formation, the cells were cultured for 25 days in Dulbecco's modified Eagle's medium (DMEM) containing 50 μg/ml ascorbic acid and 10 mm β-glycerophosphate. The mineralized nodules were stained with alizarin red. For osteoclast formation, cells were cultured in DMEM containing 10 nm 1, 25 (OH)2 vitamin D3, and 100 nm dexamethasone for 10 days and visualized by tartrate-resistant acid phosphatase staining. Calvarial primary cells were obtained from the outgrowth cultures of bone fragments of calvariae taken from 55-week-old Mmp2+/+ and Mmp2-/- mice. The cells were cultured in DMEM. After reaching confluence, the cells were reseeded at 3 × 104 cells/well into 96-well plates and cultured in DMEM containing 0.5 or 5% fetal bovine serum. Three days after plating, the cells were subjected to 3-(4,5-dimethylthioazol-2yl)-2,5-diphenyltetrazolium bromide assay and alkaline phosphatase assay, for measuring proliferation and osteoblastic differentiation, respectively. Transplantation—Calvarial periosteal tissues were stripped from 6-week-old Mmp2+/- and heads of 10-day-old Mmp2-/- mice, and the skins were stitched. Four Mmp2+/- and three Mmp2-/- donor mice and seven Mmp2-/- recipient mice were used. Two Mmp2+/- mice, which were littermates of Mmp2-/- recipient mice, were used as positive controls. The calvarial tissues of the recipient mice were harvested 2 weeks after transplantation and subjected to fixation, decalcification, and paraffin embedding. Deparaffinized sections were subjected to MMP-2 staining and subsequently to the Bodian method. Statistical Analysis—All of the comparisons were made with the Mann-Whitney U test, and the values are expressed as the means ± S.E. A p value of less than 0.05 was considered significant. n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Mmp2-/- Mice Have Facial Abnormalities and Other Bone Alterations—Mmp2-/- mice had facial abnormalities such as short snouts, hypertelorism, and dome-shaped heads (Fig. 1A). The skulls of 45-week-old Mmp2-/- mice also differed from those of Mmp2+/+ (wild-type) mice, with deformed parietal bones and sutures (Fig. 1B). The jaws of Mmp2-/- mice were ∼20% smaller than those of Mmp2+/+ mice (data not shown), and Mmp2-/- mice had a smaller body size (Fig. 1C) (15Itoh T. Ikeda T. Gomi H. Nakao S. Suzuki T. Itohara S. J. Biol. Chem. 1997; 272: 22389-22392Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). Radiographic analyses also revealed osteopenia in Mmp2-/- mice (Fig. 1C). There was medullary widening with cortical thinning of the femoral bones. Spontaneous fractures often occurred in the tibiae in mice 3 months of age and older. Dual energy x-ray absorptiometry revealed reduced the BMD/unit area in several bones of both 7- and 35-week-old Mmp2-/- mice (Fig. 1D;7 weeks, 55.1 ± 0.6 (+/+) versus 52.3 ± 0.7 (-/-) mg/cm2, p < 0.01; 35 weeks, 67.4 ± 0.7 (+/+) versus 62.0 ± 0.6 (-/-) mg/cm2, p < 0.01). Osteopenia and facial abnormalities are features of NAO syndrome (12Al Aqeel A. Al Sewairi W. Edress B. Gorlin R.J. Desnick R.J. Martignetti J.A. Am. J. Med. Genet. 2000; 93: 11-18Crossref PubMed Scopus (48) Google Scholar, 13Al-Mayouf S.M. Majeed M. Hugosson C. Bahabri S. Am. J. Med. Genet. 2000; 93: 5-10Crossref PubMed Scopus (47) Google Scholar, 14Al-Otaibi L. Al-Mayouf S.M. Majeed M. Al-Eid W. Bahabri S. Hugosson C.O. Pediatr. Radiol. 2002; 32: 523-528Crossref PubMed Scopus (9) Google Scholar). Although the skulls of 45- and 60-week-old Mmp2-/- mice had higher BMD (Fig. 1E; 85.3 ± 1.4 (+/+) versus 92.1 ± 1.8 (-/-) mg/cm2, p < 0.01), the skulls of 7-week-old Mmp2-/- mice had reduced BMD (Fig. 1E; 62.1 ± 0.5 (+/+) versus 55.8 ± 1.6 (-/-) mg/cm2, p < 0.01). The calvarial bones of 55-week-old Mmp2-/- mice were thicker than those of Mmp2+/+ mice (see Fig. 5, A and B). Sclerotic changes occurred only in the calvarial bones. Thus, we observed a novel result of the Mmp2-null mutation, increased calvarial BMD. Some patients with NAO do have sclerotic sutures (13Al-Mayouf S.M. Majeed M. Hugosson C. Bahabri S. Am. J. Med. Genet. 2000; 93: 5-10Crossref PubMed Scopus (47) Google Scholar). Heterozygous (Mmp2+/-) mice do not have skeletal abnormalities (not shown), consistent with the inheritance pattern in human NAO syndrome. Gender effects were not observed in the knock-out mice, nor are they reported in NAO patients. Arthropathy, characteristic of human NAO syndrome (12Al Aqeel A. Al Sewairi W. Edress B. Gorlin R.J. Desnick R.J. Martignetti J.A. Am. J. Med. Genet. 2000; 93: 11-18Crossref PubMed Scopus (48) Google Scholar, 13Al-Mayouf S.M. Majeed M. Hugosson C. Bahabri S. Am. J. Med. Genet. 2000; 93: 5-10Crossref PubMed Scopus (47) Google Scholar, 14Al-Otaibi L. Al-Mayouf S.M. Majeed M. Al-Eid W. Bahabri S. Hugosson C.O. Pediatr. Radiol. 2002; 32: 523-528Crossref PubMed Scopus (9) Google Scholar), was not observed in Mmp2-/- mice at any age (data not shown). The Mmp2-/- mice, however, are more susceptible to antibody-induced arthritis (21Itoh T. Matsuda H. Tanioka M. Kuwabara K. Itohara S. Suzuki R. J. Immunol. 2002; 169: 2643-2647Crossref PubMed Scopus (242) Google Scholar), suggesting that some additional factors, environmental or genetic, could be involved in the pathogenesis of arthritis in human NAO syndrome. In addition, the focal osteolysis of NAO syndrome was not observed in the Mmp2-/- mice.FIGURE 5Sclerotic changes of calvarial bones of Mmp2-/- mice. A, representative calvarial sections indicating the increased thickness of Mmp2-/- calvariae at 55 weeks-old. Left end, midline suture. B, quantification of the thickness (+/+, n = 6; -/-, n = 6; at each age) and BMD by pQCT (n = 5 for each genotype at 7 weeks of age, n = 3 for each genotype at 55 weeks of age). C, diagram of calvarial bone. D, upper row, MAR and BFR/BS of calvarial outer membrane. Lower row, MAR and BFR/BS of calvarial inner membrane. These parameters were significantly higher in Mmp2-/- calvaria at 55 weeks of age. *, p < 0.05; **, p < 0.01; ***, p < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Decreased Matrix Mineralization in the Long Bones of Mmp2-/- Mice with No Evidence of Osteoblastic and Osteoclastic Dysfunction—To analyze the bone alterations in Mmp2-/- mice in detail, we performed pQCT analyses and histomorphometric analyses on long bones in 7-week-old Mmp2-/- mice. Long bone is composed of two structure types, cancellous and cortical bone (Fig. 2A) (1Bilezikian J. Raisz L. Rodan G. Principles of Bone Biology. Academic Press, San Diego, CA2002: 3-126Google Scholar). There was no significant difference in cancellous bone matrix volume between genotypes (Fig. 2F). The pQCT analyses revealed that the BMD/unit of volume was significantly reduced in both cancellous and cortical bone (Fig. 2, B-E; cancellous bone (D), 235.5 ± 3.8 mg/cm3 (+/+) versus 197.3 ± 3.8 mg/cm3 (-/-), p < 0.001; cortical bone (E), 928.0 ± 6.6 mg/cm3 (+/+) versus 822.2 ± 9.5 mg/cm3 (-/-), p < 0.001). In cortical bone, there were more insufficiently mineralized regions (<690 mg/cm3; Fig. 2, B and C). Histomorphometric analyses revealed that the mineral apposition rate (MAR) was unchanged in Mmp2-/- mice (Fig. 3, A, B, and D). These morphometric data suggested that the timing and rate of matrix mineralization were normal in Mmp2-/- mice. None of the parameters reflecting the in vivo properties of osteoblasts and osteoclasts was significantly different (Fig. 3, A, B, and D). Furthermore, cultures of bone marrow cells obtained from Mmp2-/- mice developed normally mineralized nodules and tartrate-resistant acid phosphatase-positive osteoclasts in vitro (Fig. 4A). These in vivo and in vitro observations suggest that osteoblast and osteoclast functions are not appreciably altered in Mmp2-/- mice. Thus, the results suggest that “secondary” mineralization (22Meunier P.J. Boivin G. Bone. 1997; 21: 373-377Crossref PubMed Scopus (251) Google Scholar, 23Follet H. Boivin G. Rumelhart C. Meunier P.J. Bone. 2004; 34: 783-789Crossref PubMed Scopus (231) Google Scholar) is decreased in the limb bones of Mmp2-/- mice. Secondary mineralization, a slow and gradual maturation of the bone mineral component, is measured by quantitative microradiography and was not performed here. Decreases in secondary mineralization are usually due to high rates of bone turnover and a shortening of the life span of basic structural units; high rates of bone turnover, however, were not found in Mmp2-/- mice.FIGURE 4Unaltered properties of osteoblastic and osteoclastic cells from Mmp2-/- mice in vitro. A, in vitro bone marrow cell cultures. Osteoblastic nodule formation and osteoclast formation rates were not different between genotypes. (n = 6 for each genotype and each assay). B, in vitro calvarial cell culture. Proliferation and osteoblastic differentiation were measured by 3-(4,5-dimethylthioazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay and alkaline phosphatase (ALP) assay, respectively. Proliferation and osteoblastic differentiation of cells from Mmp2-/- mice remained unchanged (n = 6 for each genotype).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Bone Formation Is Enhanced in the Calvariae of Mmp2-/- Mice—Calvarial thickness and BMD were normal at 7 weeks of age (Fig. 5, A and B). Histomorphometric analyses of the calvariae demonstrated normal bone formation in Mmp2-/- calvariae. The matrix was fully mineralized in Mmp2-/- calvariae at this age. The thickness and BMD of Mmp2-/- calvaria, however, were significantly increased by 55 weeks of age (Fig. 5, A and B; thickness, 254 ± 7 μm (+/+) versus 376 ± 12 μm (-/-); p < 0.001, BMD, 851.7 ± 4.3 mg/cm3 (+/+) versus 1051.5 ± 3.0 mg/cm3 (-/-), p < 0.0001). Consistent with these findings, osteoblastic activities, represented by MAR and the ratio of bone formation rate to bone surface (BFR/BS), were significantly increased at 55 weeks of age (Fig. 5D), suggesting that the augmented bone formation resulted in the sclerosis of aged Mmp2-/- calvariae. Osteoblastic cells derived from Mmp2-/- calvariae, however, had proliferation and differentiation properties identical to those of cells derived from Mmp2+/+ calvariae in vitro (Fig. 4B). Thus, in vivo specific mechanisms likely induce calvarial osteoblast hyperactivity in aged Mmp2-/- mice. Osteocytic Cell Death Is Increased in the Calvariae of Mmp2-/- Mice, Not in the Long Bones—Further exploration of bone sections revealed that the ratio of empty lacunae increased with age only in Mmp2-/- calvariae (Fig. 6, A and B; 3 weeks, 2.8 ± 0.3% (+/+) versus 2.3 ± 1.0% (-/-), p > 0.05; 11 weeks, 17.1 ± 1.9% (+/+) versus 30.0 ± 3.2% (-/-), p < 0.001; 55 weeks, 20.0 ± 1.8% (+/+) versus 52.9 ± 1.6% (-/-), p < 0.001). A large number of osteocytes in Mmp2-/- calvariae were positive for TdT-mediated dUTP-biotin nick end labeling at 9 weeks of age, suggesting that the empty lacunae resulted from apoptotic cell death (data not shown). In contrast, the ratios of empty lacunae in Mmp2-/- femora were comparable with those in Mmp2+/+ femora at any age examined (Fig. 6, A and B). There were no differences between genotypes in other bones (Fig. 6C). Thus, the calvaria-specific bone phenotype is closely associated with osteocytic cell death and is caused by the loss of the osteocytic canalicular network. MMP-2 Deficiency Affects Development of the Canalicular Network in Both Long Bones and Calvariae—The above data demonstrated alterations in structure and deposition of bone in long bones and calvariae in Mmp2-/- mice, despite the normal intrinsic properties of the osteoblasts and osteoclasts. To assess the MMP-2 sites of action in bone, immunohistochemistry was performed in sections of femora and calvariae. MMP-2 was detected in the vicinity of osteocytes in both bone types (Fig. 7, A and C). One characteristic feature of bone matrix is a well developed cellular network (1Bilezikian J. Raisz L. Rodan G. Principles of Bone Biology. Academic Press, San Diego, CA2002: 3-126Google Scholar) comprised of osteocytes, which have their cell bodies in the lacunae and extensive dendritic processes in the canalicular channels throughout bone. At a higher magnification, MMP-2 was observed in and around the osteocytic lacunae as well as along the canalicular channels in femora and calvaria (Fig. 7, A and C). There were strong MMP-2 signals close to the bone surface, which represented newly formed osteocytic canaliculi (Fig. 7A, inset). The bone surface mature osteoblasts extended their processes after being embedded in the bone matrix and differentiating into mature osteocytes. These findings suggest that MMP-2 contributes to form and/or maintain the osteocytic network. To examine the role of MMP-2 in the osteocytic network, we stained bone sections using the Bodian method to visualize osteocytic canaliculi (19Kusuzaki K. Kageyama N. Shinjo H. Murata H. Takeshita H. Ashihara T. Hirasawa Y. Acta Orthop. Scand. 1995; 66: 166-168Crossref PubMed Scopus (10) Google Scholar, 20Kusuzaki K. Kageyama N. Shinjo H. Takeshita H. Murata H. Hashiguchi S. Ashihara T. Hirasawa Y. Bone. 2000; 27: 655-659Crossref PubMed Scopus (45) Google Scholar). Bodian staining revealed a significant impairment of the fine, slender structures of the osteoblastic/osteocytic network in Mmp2-/- femora (Fig. 7, E and G; number of connections between pairs of adjacent lacunae per section, 10.9 ± 0.6 (+/+) versus 3.7 ± 0.3 (-/-), p < 0.001; number of processes protruding from a lacuna per section, 25.9 ± 1.0 (+/+) versus 17.1 ± 0.7 (-/-), p < 0.001). This network was disrupted in Mmp2-/- calvariae (Fig. 7, F and G; number of connections, 6.4 ± 0.3 (+/+) versus 0.3 ± 0.2 (-/-), p < 0.001; number of processes, 13.0 ± 0.5 (+/+) versus 8.3 ± 0.6 (-/-), p < 0.001). Thus, Mmp2-/- mice had a moderately disrupted osteocytic canalicular network in the femora and a severely disrupted osteocytic canalicular network in the calvariae. Restored Canalicular Formation by Wild-type Periosteal Transplantation—To determine whether MMP-2 activity is required locally for osteocytic canalicular formation, we transplanted the periosteal tissues of Mmp2+/- or Mmp2-/- mice onto the calvariae of 10-day-old Mmp2-/- mice. Two weeks after the transplantation, we examined osteocytic canalicular networks in the recipient mice. Osteocytic canalicular networks were successfully formed in one of four Mmp2-/- mice transplanted with Mmp2+/- periosteum (Fig. 8A). To avoid nonspecific osteocyte damage in the recipients, we did not irradiate the recipients before transplantation. Thus, the low rate of canalicular formation may be explained in part by immunological rejection of some of the periosteal transplants. The newly formed osteocytic canaliculi were located in areas adjacent to the transplanted periosteum; MMP-2 was also identified in these areas by immunohistochemistry. We did not observe any formation of osteocytic canaliculi in the sections from the recipients transplanted with Mmp2-/- periosteum. These results suggest that MMP-2 acts locally to form osteocytic canaliculi. Differential Localizations of DMP-1 and Sclerostin in Mmp2-/- Long Bones and Calvarial Bones—The results just described indicated differences in osteocyte canalicular networks in the long bones and calvariae of Mmp2-/- mice. To study

TGF-beta modulates growth and differentiation in many cell types. MC3T3E1 is a clonal non-transformed murine bone cell line which differentiates in culture. We tested the effect of porcine TGF-beta on the proliferation and differentiation of MC3T3E1 cells in monolayer cultures by following cell number, and alkaline phosphatase activity. TGF-beta treatment (2 ng/ml) altered the shape of MC3T3E1 cells from cuboidal to elongated/spindle-shape. TGF-beta inhibited the growth of MC3T3E1 by up to 40% (P less than 0.02) in a dose-dependent manner with half maximal inhibition at 1 ng/ml. Growth inhibition depended on serum concentration, maximal inhibition occurring at 2% serum. Expression of alkaline phosphatase, which peaks in vitro when the cells reach confluence, was strongly inhibited by TGF-beta, in a dose-dependent manner with half maximal inhibition at around 0.05 ng/ml and complete inhibition at 2 ng/ml. Alkaline phosphatase inhibition was irreversible after 24 hours exposure to TGF-beta.

Genetic and cell biological studies have indicated that Indian hedgehog (Ihh) plays an important role in bone development and osteoblast differentiation. However, the molecular mechanism by which Ihh regulates osteoblast differentiation is complex and remains to be fully elucidated. In this study, we investigated the role of Ihh signaling in osteoblast differentiation using mesenchymal cells and primary osteoblasts. We observed that Ihh stimulated alkaline phosphatase (ALP) activity, osteocalcin expression, and calcification. Overexpression of Gli2- but not Gli3-induced ALP, osteocalcin expression, and calcification of these cells. In contrast, dominant-negative Gli2 markedly inhibited Ihh-dependent osteoblast differentiation. Ihh treatment or Gli2 overexpression also up-regulated the expression of Runx2, an essential transcription factor for osteoblastogenesis, and enhanced the transcriptional activity and osteogenic action of Runx2. Coimmunoprecipitation analysis demonstrated a physical interaction between Gli2 and Runx2. Moreover, Ihh or Gli2 overexpression failed to increase ALP activity in Runx2-deficient mesenchymal cells. Collectively, these results suggest that Ihh regulates osteoblast differentiation of mesenchymal cells through up-regulation of the expression and function of Runx2 by Gli2.

Bone morphogenetic protein 2 (BMP-2) is a potent inducer of differentiation of osteoblasts both in vivo and in vitro. We examined the action of BMP-2 on expression of a helix-loop-helix-type transcription factor, Id (inhibitor of differentiation), in osteoblast-like cells, as well as in osteoblast-enriched cells and possible precursor cells. To our surprise, BMP-2 enhanced Id gene expression in the cell types of osteoblastic lineage we examined. The maximal BMP-2 enhancement was observed within 24 hr in early proliferating cultures and the enhancement lasted up to 96 hr. The BMP-2 effect was not blocked by actinomycin D, while it was blocked by cycloheximide, suggesting that BMP-2 regulates Id gene expression at least in part via posttranscriptional events, which require protein synthesis. Other experiments indicated that BMP-2 did not further enhance Id mRNA levels promoted by dexamethasone, while BMP-2 did not resume the Id mRNA levels suppressed by 1,25-dihydroxyvitamin D3. Similar BMP-2 enhancement of Id message expression was also observed in osteoblast-enriched fetal rat calvaria cells as well as C3H10T1/2 cells. These results indicate that BMP-2 enhances expression of Id in early cultures of osteoblastic cells and suggest that enhancement of Id expression may somehow be involved in the promotion of differentiation by this cytokine in these osteoblastic cells and in their precursor cells.

Abstract To clarify the role of OPN in bone formation under mechanical stress, we examined the expression and the function of OPN in bone using an expansion force-induced osteogenesis model. Our results indicated that OPN expression was enhanced during the bone formation and that OPN would be one of the positive factors for the bone formation under mechanical stress. Introduction: Bone formation is known to be stimulated by mechanical stress; however, molecules involved in stress-dependent regulation of bone formation have not yet been fully characterized. Extracellular matrix proteins such as osteopontin (OPN) could play a role in mediation of the mechanical stress signal to osteoblasts. However, the function of OPN in bone formation under mechanical force is not known. Therefore, we examined the expression and the role of OPN in bone formation in vivo under tensile mechanical stress. Materials and Methods: Sagittal sutures of mice were subjected to expansion mechanical stress by setting orthodontic spring wires, and OPN expression during bone formation within the suture gap was examined. Results: Expansion of the sutures resulted in bone formation at the edges of the parietal bones within the sagittal suture. Immunohistochemical analysis revealed abundant accumulation of OPN protein in the matrix of newly formed bone on the inner edge of the parietal bone within the mechanically expanded sutures. Osteoblasts forming bone within the suture subjected to tensile stress also exhibited high levels of OPN protein expression. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that OPN mRNA expression was enhanced in wild-type calvariae subjected to expansion force compared with the control calvariae where dead spring wires were set without expansion stress. In addition, type I collagen mRNA was also expressed in the calvariae under the mechanical stimuli. To understand the function of OPN, sagittal sutures in OPN-deficient mice were subjected the expansion stress, and bone formation within the suture to fill the expanded gap was compared with that observed in wild-type mice. OPN deficiency reduced bone formation at the edge of the parietal bone in contact with the expanded suture gap. Conclusions: These observations revealed that OPN plays a pivotal role in bone formation under tensile mechanical stress.

Abstract Osteoclasts are unique cells that resorb bone, and are involved in not only bone remodeling but also pathological bone loss such as osteoporosis and rheumatoid arthritis. The regulation of osteoclasts is based on a number of molecules but full details of these molecules have not yet been understood. MicroRNAs are produced by Dicer cleavage an emerging regulatory system for cell and tissue function. Here, we examine the effects of Dicer deficiency in osteoclasts on osteoclastic activity and bone mass in vivo. We specifically knocked out Dicer in osteoclasts by crossing Dicer flox mice with cathepsin K‐Cre knock‐in mice. Dicer deficiency in osteoclasts decreased the number of osteoclasts (N.Oc/BS) and osteoclast surface (Oc.S/BS) in vivo. Intrinsically, Dicer deficiency in osteoclasts suppressed the levels of TRAP positive multinucleated cell development in culture and also reduced NFATc1 and TRAP gene expression. MicroRNA analysis indicated that expression of miR‐155 was suppressed by RANKL treatment in Dicer deficient cells. Dicer deficiency in osteoclasts suppressed osteoblastic activity in vivo including mineral apposition rate (MAR) and bone formation rate (BFR) and also suppressed expression of genes encoding type I collagen, osteocalcin, Runx2, and Efnb2 in vivo. Dicer deficiency in osteoclasts increased the levels of bone mass indicating that the Dicer deficiency‐induced osteoclastic suppression was dominant over Dicer deficiency‐induced osteoblastic suppression. On the other hand, conditional Dicer deletion in osteoblasts by using 2.3 kb type I collagen‐Cre did not affect bone mass. These results indicate that Dicer in osteoclasts controls activity of bone resorption in vivo. J. Cell. Biochem. 109: 866–875, 2010. © 2009 Wiley‐Liss, Inc.

Renin angiotensin system (RAS) regulates circulating blood volume and blood pressure systemically, whereas RAS also plays a role in the local milieu. Previous in vitro studies suggested that RAS may be involved in the regulation of bone cells. However, it was not known whether molecules involved in RAS are present in bone in vivo. In this study, we examined the presence of RAS components in adult bone and the effects of angiotensin II type 2 (AT2) receptor blocker on bone mass. Immunohistochemistry revealed that AT2 receptor protein was expressed in both osteoblasts and osteoclasts. In addition, renin and angiotensin II-converting enzyme were expressed in bone cells in vivo. Treatment with AT2 receptor blocker significantly enhanced the levels of bone mass, and this effect was based on the enhancement of osteoblastic activity as well as the suppression of osteoclastic activity in vivo. These results indicate that RAS components are present in adult bone and that blockade of AT2 receptor results in alteration in bone mass.

Osteoclastic bone resorption requires a number of complex steps that are under the control of local regulatory molecules. Osteopontin is expressed in osteoclasts and is also present in bone matrix; however, its biological function has not been fully understood. To elucidate the role of osteopontin in the process of osteoclastic bone resorption, we conducted ectopic bone implantation experiments using wild-type and osteopontin knockout mouse. In the wild-type group, bone discs from calvariae implanted ectopically in muscle were resorbed, and their mass was reduced by 25% within 4 weeks. In contrast, the mass of the bone discs from calvariae of osteopontin knockout mice was reduced by only 5% when implanted in osteopontin knockout mice. Histological analyses indicated that the number of osteoclasts associated with the implanted bones was reduced in the osteopontin knockout mice. As osteopontin deficiency does not suppress osteoclastogenesis per se, we further examined vascularization immunohistologically and found that the number of vessels containing CD31-positive endothelial cells around the bone discs implanted in muscle was reduced in the osteopontin knockout mice. Furthermore, sc implantation assays indicated that the length and branching points of the newly formed vasculatures associated with the bone discs were also reduced in the absence of osteopontin. In this assay, tartrate-resistant acid phosphatase-positive area of the bone discs was also reduced in the osteopontin knockout mice, indicating further the link between the osteopontin-dependent vascularization and osteoclast accumulation. The bone resorption defect could be rescued by topical administration of recombinant osteopontin to the bones implanted in muscle. These observations indicate that osteopontin is required for efficient vascularization by the hemangiogenic endothelial cells and subsequent osteoclastic resorption of bones.

Osteoblasts, the bone-forming cells, synthesize the macromolecules of the bone matrix including: type I collagen; osteocalcin; osteonectin; osteopontin; proteoglycan I and II; bone sialoprotein; matrix gla-protein; bone glycoprotein 75; several other proteins, which have not been extensively characterized; growth factors, including transforming growth factor beta and fibroblast growth factor. Osteoblasts also have high levels of the membrane-bound enzyme, alkaline phosphatase, which plays a role in matrix mineralization, and receptors for tissue-specific hormones, such as parathyroid hormone, as well as many other hormones, cytokines and growth factors, which regulate bone growth, differentiation and metabolism. The expression of these various proteins, most of which are not unique to bone but which together characterize the bone phenotype, is induced during osteoblastic differentiation in a stepwise fashion, suggestive of multiple regulatory factors. The detailed sequence of the expression of osteoblastic genes in situ has not been fully characterized. It appears that type I collagen and alkaline phosphatase are expressed early during the commitment to the osteoblastic phenotype, whereas osteopontin and osteocalcin appear late during osteoblastic differentiation. Diversity among "osteoblastic" cells is also apparent, probably not all osteoblastic cells express all the features. A large number of osteoblastic models are currently available to study the expression of osteoblast-related genes in vitro. These include primary cultures from calvaria or trabecular bone from several species, including humans, osteosarcoma-derived cell lines, and experimentally immortalized cells. Some of these in vitro models, especially the calvaria-derived cultures, undergo changes which mimic osteoblastic differentiation in vivo. The study of these and other cell models started providing insights into the regulation of gene expression in osteoblastic cells. In addition to a vast body of information on the conditions required for the expression of various proteins in culture and their regulation by hormones and growth factors, more detailed information on specific genes has recently been obtained. For example, regulation of type I collagen gene expression has been studied in osteosarcoma cell lines where 1,25(OH)2 vitamin D3 was shown to act via specific DNA segment(s) in the 5' flanking region of the gene, while parathyroid hormone affected gene expression by altering the stability of the transcripts. TGF beta 1, which stimulates osteogenesis, was shown to promote the transcription of osteopontin and type I collagen, the latter effect requiring the binding site for the transactivating protein, nuclear factor I.(ABSTRACT TRUNCATED AT 400 WORDS)

CLC-3 is a member of the CLC chloride channel family and is widely expressed in mammalian tissues. To determine the physiological role of CLC-3, we generated CLC-3-deficient mice (Clcn3-/- ) by targeted gene disruption.Together with developmental retardation and higher mortality, the Clcn3-/- mice showed neurological manifestations such as blindness, motor coordination deficit, and spontaneous hyperlocomotion. In histological analysis, the Clcn3-/- mice showed a pattern of progressive degeneration of the retina, hippocampus and ileal mucosa, which resembled the phenotype observed in cathepsin D knockout mice. The defect of cathepsin D results in a lysosomal accumulation of ceroid lipofuscin containing the mitochondrial F1F0 ATPase subunit c. In immunohistochemistry and Western blot analysis, we found that the subunit c was heavily accumulated in the lysosome of Clcn3-/- mice. Furthermore, we detected an elevation in the endosomal pH of the Clcn3-/- mice.These results indicated that the neurodegeneration observed in the Clcn3-/- mice was caused by an abnormality in the machinery which degrades the cellular protein and was associated with the phenotype of neuronal ceroid lipofuscinosis (NCL). The elevated endosomal pH could be an important factor in the pathogenesis of NCL.

Fibroblast growth factor (FGF) and type beta transforming growth factor (TGF beta) are potent modulators of proliferation and differentiation in many types of cells. TGF beta acts in an autocrine manner, and the regulation of TGF beta gene expression is one of the crucial events in the control of cellular functions. This study examines FGF regulation of TGF beta 1 gene expression in osteoblast-like cells. Bovine basic FGF (bFGF) increased the steady-state level of 2.5-kb TGF beta 1 mRNA two- to threefold in rat osteosarcoma (ROS17/2.8) cells in a dose-dependent manner, starting at 0.1 ng/ml. The increase of the message was detectable within 3 h after the addition of bFGF, peaked at 6 h, and lasted at least up to 48 h. This effect was blocked by a protein kinase inhibitor, K252a, indicating the involvement of phosphorylation. bFGF increased the rate of TGF beta 1 gene transcription estimated by nuclear run-on assay, while the stability of TGF beta 1 mRNA was not altered. bFGF increased the TGF beta activity in the conditioned media, estimated by DNA synthesis inhibition assay using mink lung epithelial (CCL-64) cells. Parathyroid hormone reduced the abundance of TGF beta 1 mRNA in ROsS17/2.8 cells and opposed the bFGF effect on TGF beta 1 mRNA. bFGF also increased the steady-state level of TGF beta 1 mRNA in mouse calvaria-derived MC3T3E1 and human osteosarcoma SaOS-2 cells. These findings indicate that FGF enhances the expression of TGF beta 1 gene in osteoblast-like cells and point to the tight relationship of the two growth factors involved in the control of cellular functions.

Osteopontin (OP) or bone sialoprotein is a recently characterized extracellular matrix protein which is abundant in bone and is produced by osteoblasts. Parathyroid hormone (PTH) is a potent calcitropic hormone which regulates osteoblastic function including the synthesis of extracellular matrix proteins. This study examines the effect of human PTH (hPTH-[1-34]) on the expression of this novel protein in rat osteoblast-like cells. hPTH(1-34) significantly decreased the amount of OP in culture media of the rat osteoblastic osteosarcoma cell line, ROS 17/2.8, detected by Western immunoblot analysis. hPTH(1-34) also suppressed the steady-state level of OP mRNA two- to threefold with an ED50 of approximately 3 X 10(-10) M. This inhibition was detectable at 24 h, reached its nadir at 48 h, and lasted at least up to 96 h. The hPTH(1-34) effects were mimicked by isobutylmethylxanthine, cholera toxin, 8-bromo-cAMP, forskolin, and isoproterenol. hPTH(1-34) suppressed by two- to threefold the rate of OP gene transcription, estimated by nuclear run-on assays. The suppression of OP mRNA levels by hPTH(1-34) was also seen when basal levels were increased by transforming growth factor type beta, or 1,25-dihydroxyvitamin D3, or were decreased by dexamethasone. A similar decrease in the steady-state level of OP mRNA by hPTH(1-34) was also observed in primary cultures of osteoblast-enriched cells from fetal rat calvaria. These findings indicate that hPTH(1-34) suppresses the production of the novel extracellular matrix protein, OP, in osteoblasts at least in part through transcriptional control.

The lateral mobility of alkaline phosphatase (AP) in the plasma membrane of osteoblastic and nonosteoblastic cells was estimated by fluorescence redistribution after photobleaching in embryonic and in tumor cells, in cells that express AP naturally, and in cells transfected with an expression vector containing AP cDNA. The diffusion coefficient (D) and the mobile fraction, estimated from the percent recovery (%R), were found to be cell-type dependent ranging from (0.58 +/- 0.16) X 10(-9) cm2s-1 and 73.3 +/- 10.5 in rat osteosarcoma cells ROS 17/2.8 to (1.77 +/- 0.51) X 10(-9) cm2s-1 and 82.8 +/- 2.5 in rat osteosarcoma cells UMR106. Similar values of D greater than or equal to 10(-9) cm2s-1 with approximately 80% recovery were also found in fetal rat calvaria cells, transfected skin fibroblasts, and transfected AP-negative osteosarcoma cells ROS 25/1. These values of D are many times greater than "typical" values for membrane proteins, coming close to those of membrane lipid in fetal rat calvaria and ROS 17/2.8 cells (D = [4(-5)] X 10(-9) cm2s-1 with 75-80% recovery), estimated with the hexadecanoyl aminofluorescein probe. In all cell types, phosphatidylinositol (PI)-specific phospholipase C released 60-90% of native and transfection-expressed AP, demonstrating that, as in other tissue types, AP in these cells is anchored in the membrane via a linkage to PI. These results indicate that the transfected cells used in this study possess the machinery for AP insertion into the membrane and its binding to PI. The fast AP mobility appears to be an intrinsic property of the way the protein is anchored in the membrane, a conclusion with general implications for the understanding of the slow diffusion of other membrane proteins.

PEBP2αA/AML3/CBFA1 is one of the transcription regulators that belong to the PEBP2/AML family. The knockout mice, where the gene encoding PEBP2αA/AML3/CBFA1 was inactivated, showed no osteogenesis, indicating the critical role of this transcription factor in osteoblastic differentiation (Komori, Y. et al. Cell 89:755–764; 1997). The aim of this study is to examine the regulation of PEBP2αA/AML3/CBFA1 expression in skeletal (MC3T3E1, ROS17/2.8) and nonskeletal (C3H10T1/2, C2C12, NIH3T3) cell lines. The basal levels of PEBP2αA/AML3/CBFA1 were time dependent and were increased during culture in ROS17/2.8 by day 2, remaining similar during cultures in other types of cells. Treatment with a 100-ng/mL BMP4/7 heterodimer enhanced the expression of PEBP2αA/AML3/CBFA1 mRNA levels in MC3T3E1 and C2C12 cells, whereas BMP2 did not significantly alter PEBP2αA/AML3/CBFA1 mRNA levels in both skeletal and nonskeletal cells. The PEBP2αA/AML3/CBFA1 mRNA level in ROS17/2.8 cells was relatively high on day 2, and was not further enhanced by treatment with BMP4/7. In contrast to the reported type I collagen gene upregulation by the overexpression of Osf2/CBFA1, which differs from PEBP2αA/AML3/CBFA1 by containing a unique 87 amino acid sequence at its amino terminal end, overexpression of PEBP2αA/AML3/CBFA1 suppressed type I collagen mRNA levels in MC3T3E1, C2C12, and C3H10T1/2 cells and suppressed osteocalcin mRNA levels in ROS17/2.8 cells. The osteopontin mRNA level was enhanced by overexpression of PEBP2αA/AML3/CBFA1 in MC3T3E1, while the level was similar in ROS17/2.8 cells and was suppressed in C2C12 cells. These data indicate that PEBP2αA/AML3/CBFA1 is one of the targets of BMP4/7 and participates in the regulation of the expression of genes related to osteoblast phenotype. The overexpression study suggests that PEBP2αA/AML3/CBFA1 and Osf2/CBFA1 may have a different function in the regulation of the expression of the genes related to the osteoblast phenotype.

Osteocalcin (OC) is one of the abundant noncollagenous bone matrix proteins produced exclusively by osteoblasts, and its serum level is used as an indicator of bone metabolism in patients. Transforming growth factor-β (TGFβ) is abundant in bones and platelets, promotes wound healing in vivo, and is a potent stimulator of the production of extracellular matrix proteins in fibroblasts and osteoblasts. The effects of TGFβ on OC gene expression were examined in rat osteoblastlike cells, ROS17/2.8. TGFjSl decreased OC levels in the culture media 2- to 3-fold. TGFβ1 also decreased the level of osteocalcin mRNA about 3-fold in a dose-dependent manner. TGFβ2 and TGFβ1,2, a heterodimeric form, showed similar effects on OC mRNA levels as TGFβ1. The suppression of the OC message level was detectable at 24 h and lasted for up to 72 h. This effect on OC mRNA was blocked by cycloheximide. The stability of OC mRNA was not changed by TGFjSl. On the other hand, the rate of OC gene transcription was reduced 4- to 5-fold, as estimated by in vitro nuclear transcription (run-on) assay. TGFβ1 blocked the increase in the OC mRNA level induced by PTH or 1,25-dihydroxyvitamin D3. These results indicate that TGFβ inhibits osteocalcin gene expression at least in part through transcriptional control. (Endocrinology124: 612–617, 1989)

Abstract Increased levels of ePP i in mice deficient in TNALP (i.e., Akp2 −/− ) lead to elevated OPN concentrations. We examined the skeletal phenotype of mice lacking both OPN and TNALP and concluded that the increased OPN levels contribute to the hypophosphatasia phenotype characteristic of Akp2 −/− mice. We also found that extracellular OPN regulates the PP i output by osteoblasts. Introduction: Akp2 −/− display mineralization deficiencies characterized by rickets/osteomalacia. This defect has been attributed to the increased levels of extracellular inorganic pyrophosphate (ePP i ), a substrate of tissue‐nonspecific alkaline phosphatase (TNALP) and a potent inhibitor of mineral deposition. Because elevated levels of ePP i induce Opn gene expression, the Akp2 −/− mice also display increased levels of osteopontin (OPN), another inhibitor of mineralization. Materials and Methods: Akp2 −/− mice were bred into the Opn −/− line. The resulting double knockout mice were analyzed for skeletal abnormalities by histology and μCT. Calvarial osteoblasts were assayed for their ability to mineralize in vitro and were probed for changes in gene expression. Results: Mice lacking both Akp2 and Opn showed partial normalization at the histological level with regard to mineral deposition and BMD. However, high ePP i levels remained in Akp2 −/− mice. We found that Opn −/− mice have themselves elevated levels of ePP i attributable to an increase in Enpp1 and Ank expression and a concomitant downregulation of Akp2 expression in Opn −/− osteoblasts, but that Opn −/− mice have more mineralized osteoid than wildtype (WT) controls despite their elevated ePP i levels. Addition of exogenous OPN to Opn −/− osteoblasts results in downregulation of Enpp1 and Ank gene expression and a reduction of the PP i output by these cells. Conclusions: Deletion of both Akp2 and Opn can partially rescue the hypomineralized phenotype of Akp2 −/− mice. However, these double knockout mice do not display corrected ePP i levels, and we conclude that regulation of hydroxyapatite deposition requires the coordinated actions of both PP i and OPN and that the hypophosphatasia phenotype in Akp2 −/− mice results from the combined inhibitory action of increased levels of both ePP i and OPN. Our data also suggest that the ePP i ‐mediated regulation of OPN and the OPN‐mediated regulation of ePP i are linked counterregulatory mechanisms that control the concentrations of these two important mineralization inhibitors, OPN and ePP i .

Mechanosensing is one of the crucial components of the biological events. In bone, as observed in unloading-induced osteoporosis in bed ridden patients, mechanical stress determines the levels of bone mass. Many molecules have been suggested to be involved in sensing mechanical stress in bone, while the full pathways for this event has not yet been identified. We examined the role of TRPV4 in unloading-induced bone loss. Hind limb unloading induced osteopenia in wild-type mice. In contrast, TRPV4 deficiency suppressed such unloading-induced bone loss. As underlying mechanism for such effects, TRPV4 deficiency suppressed unloading-induced reduction in the levels of mineral apposition rate and bone formation rate. In these mice, unloading-induced increase in the number of osteoclasts in the primary trabecular bone was suppressed by TRPV4 deficiency. Unloading-induced reduction in the longitudinal length of primary trabecular bone was also suppressed by TRPV4 deficiency. TRPV4 protein is expressed in both osteoblasts and osteoclasts. These results indicated that TRPV4 plays a critical role in unloading-induced bone loss.

Alkaline phosphatase [ALP; orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] is a ubiquitous enzyme of unknown function expressed at high levels in cells of mineralizing tissues. To study the structure, function, and expression of ALP, a full-length cDNA of rat ALP (2415 bases) was isolated from a ROS 17/2.8 osteosarcoma cell lambda gt10 cDNA library. The predicted amino acid sequence spans 524 residues and includes an N-terminal signal peptide of 17 amino acids, the phosphohydrolase active site, a rather hydrophilic backbone with five potential N-glycosylation sites, and a short hydrophobic C-terminal sequence. ALP negative CHO cells transfected with an expression vector containing the ALP coding sequences express ALP. The rat bone, liver, and kidney ALP shows remarkable 90% homology with the corresponding human enzyme, the most divergent region being the C-terminal hydrophobic domain through which the enzyme may be anchored to the plasma membrane. The rat ALP also shows 50% homology with the human placental and intestinal ALP and 25% homology with the Escherichia coli ALP. The amino acids involved in catalysis show nearly complete homology among all known ALP sequences, suggesting that these enzymes evolved from a common ancestral gene. The rat ALP cDNA pRAP 54, used as a hybridization probe in RNA blot analysis of several tissues that express ALP, revealed the presence of an ALP mRNA of approximately equal to 2500 bases. Furthermore, hybridization patterns derived from Southern blot analysis of rat chromosomal DNA offered molecular evidence that the ALP expressed in ROS 17/2.8 osteosarcoma and various rat tissues, excluding the intestine, is the product of the same single copy gene.

Two cDNA clones of rat alkaline phosphatase (AP) were isolated from a rat osteosarcoma lambda gt 11 cDNA library (ROS 17/2.8) utilizing a human bone-liver-kidney (BLK) type AP cDNA. These clones contain overlapping DNA sequences of 597 and 520 bp, respectively, corresponding to the 3' noncoding region of AP mRNA. The sequence homology with the human BLK AP cDNA is 61%. In Northern blot analysis the rat cDNA hybridizes to a single band of 2.5 kb mRNA from ROS 17/2.8 and rat liver, under highly stringent conditions. Steady state levels of AP mRNAs measured in several rat osteosarcoma cell lines (ROS 17/2.8, ROS 2/3, ROS 25/1, UMR 106) correlate with the level of AP enzymatic activity in these cells. Dexamethasone, which stimulates AP enzymatic activity in ROS 17/2.8 cells, increases the relative abundance of AP mRNA in a dose-dependent manner. This probe can be used to study AP expression in rat tissues and cells.

Osteocalcin (bone gamma-carboxyglutamic acid-containing protein) is exclusively produced by osteoblasts, which are the major target cells of parathyroid hormone (PTH) in bone. This study examined the effect of human (h) PTH(1-34) on osteocalcin gene expression in the rat osteoblast-like osteosarcoma cells ROS17/2.8. hPTH(1-34) increased in a dose-dependent manner the steady state levels of osteocalcin mRNA 2- to 3-fold with an ED50 of about 5 X 10(-10) M. This effect was detectable at 12 h, peaked at 24 h, and lasted at least up to 48 h. Forskolin, cyclic 8-bromo-AMP, isobutylmethylxanthine, cholera toxin, and (-)-isoproterenol similarly elevated osteocalcin mRNA. hPTH(1-34) did not alter the transcriptional rate of the osteocalcin gene, estimated by nuclear run-on assays, but increased the stability of osteocalcin mRNA. hPTH(1-34) also increased 2- to 3-fold the osteocalcin level in the culture media determined by radioimmunoassay. PTH, thus, promoted osteocalcin gene expression in these cells at least in part through mRNA stabilization via cyclic AMP mediation, a mechanism known only in few systems.

Abstract Bone regeneration for the defects in revision surgery of joint replacement is an increasingly important issue. To repair bone defects, bone cell activation by growth factors using synthetic resorbable scaffold is a useful and safe option. We examine the efficiency of nanogel‐crosslinking hydrogel as a novel synthetic scaffold for BMP to stimulate osteoblasts and to induce bone formation. Cholesterol‐bearing pullulan nanogel‐crosslinking hydrogel (CHPA/Hydrogel) was used to deliver BMP. The CHPA hydrogel pellets were implanted in vivo. Single implantation of CHPA/hydrogel containing low amounts of BMP induced osteoblastic activation and new bone formation in vivo. Furthermore, nanogel in a disc shape established recruitment of osteoblastic cells that vigorously formed bone to heal the calvarial defects, which did not heal spontaneously without it. In conclusion, CHPA/hydrogel serves as an efficient and versatile scaffold for the stimulation of osteoblasts to form bone and to repair defects via delivery of BMP. J. Cell. Physiol. 220: 1–7, 2009. © 2009 Wiley‐Liss, Inc.

The sympathetic nervous system suppresses bone mass by mechanisms that remain incompletely elucidated. Using cell-based and murine genetics approaches, we show that this activity of the sympathetic nervous system requires osteopontin (OPN), a cytokine and one of the major members of the noncollagenous extracellular matrix proteins of bone. In this work, we found that the stimulation of the sympathetic tone by isoproterenol increased the level of OPN expression in the plasma and bone and that mice lacking OPN (OPN-KO) suppressed the isoproterenol-induced bone loss by preventing reduced osteoblastic and enhanced osteoclastic activities. In addition, we found that OPN is necessary for changes in the expression of genes related to bone resorption and bone formation that are induced by activation of the sympathetic tone. At the cellular level, we showed that intracellular OPN modulated the capacity of the β2-adrenergic receptor to generate cAMP with a corresponding modulation of cAMP-response element binding (CREB) phosphorylation and associated transcriptional events inside the cell. Our results indicate that OPN plays a critical role in sympathetic tone regulation of bone mass and that this OPN regulation is taking place through modulation of the β2-adrenergic receptor/cAMP signaling system.

The synergistic effect of the GPCR β2AR on signaling through another GPCR, PTHR, is explained by the release of Gβγ from the heterotrimeric Gαiβγ protein, activating adenylate cyclase AC2 and subsequent prolonged cAMP signaling in internal compartments Cells express several G-protein-coupled receptors (GPCRs) at their surfaces, transmitting simultaneous extracellular hormonal and chemical signals into cells. A comprehensive understanding of mechanisms underlying the integrated signaling response induced by distinct GPCRs is thus required. Here we found that the β2-adrenergic receptor, which induces a short cAMP response, prolongs nuclear cAMP and protein kinase A (PKA) activation by promoting endosomal cAMP production in parathyroid hormone (PTH) receptor signaling through the stimulatory action of G protein Gβγ subunits on adenylate cyclase type 2.

This paper proposes a multicomponent topology optimization method that considers assemblability. Generally, it is difficult to consider assemblability in topology optimization; however, in this study, we achieve it by introducing a fictitious physical model. To perform multicomponent topology optimization, the extended level set method is used to represent multiple components. First, the assembly constraints are formulated using a fictitious physical model limited to two components. Then, by considering stepwise assembly, the constraint is extended to three or more components. In addition, topology optimization algorithms are constructed using the finite element method. Several numerical examples demonstrate that the proposed method can obtain structures with assemblability and has low initial structure dependence.

Osteopontin is an RGDS-containing protein that acts as a ligand for the α_vβ₃ integrin, which is abundantly expressed in osteoclasts, cells responsible for bone resorption in osteopenic diseases such as osteoporosis and hyperparathyroidism. However, the role of osteopontin in the process of bone resorption has not yet been fully understood. Therefore, we investigated the direct function of osteopontin in bone resorption using an organ culture system. The amount of ⁴⁵Ca released from the osteopontin-deficient bones was not significantly different from the basal release from wild type bones. However, in contrast to the parathyroid hormone (PTH) enhancement of the ⁴⁵Ca release from wild type bones, PTH had no effect on ⁴⁵Ca release from organ cultures of osteopontin-deficient bones. Because PTH is located upstream of receptor activator of NF-κB ligand (RANKL), that directly promotes bone resorption, we also examined the effect of RANKL. Soluble RANKL with macrophage-colony stimulating factor enhanced⁴⁵Ca release from the bones of wild type fetal mice but not from the bones of osteopontin-deficient mice. To obtain insight into the cellular mechanism underlying the phenomena observed in osteopontin-deficient bone, we investigated the number of tartrate-resistant acid phosphatase (TRAP)-positive cells in the bones subjected to PTH treatment in cultures. The number of TRAP-positive cells was increased significantly by PTH in wild type bone; however, no such PTH-induced increase in TRAP-positive cells was observed in osteopontin-deficient bones. These results indicate that the absence of osteopontin suppressed PTH-induced increase in bone resorption via preventing the increase in the number of osteoclasts in the local milieu of bone.

A series of Al, Ti, Zn and Zr compounds, i.e., their metal alkoxides, organic acid and enolate salts, halid and oxide, were evaluated as intramolecular transesterification catalysts for the thermal depolymerization reaction of poly(L-lactic acid) oligomer resulting in LL-lactide, meso-lactide and DD-lactide by gas chromatography using a β-cyclodexstrin chiral stationary phase capillary column. The activity of the intramolecular transesterification compared with that of stannous 2-ethylhexanoate was in the following order : Sn>Zn>Zr>Ti>Al.

Osteopontin has been implicated in the metastasis of tumors, and human tumors with high metastatic activity often express osteopontin at high levels. Osteopontin contains an arginine-glycine-aspartate (RGD) motif that is recognized by integrin family members to promote various cell activities including attachment to substrate and it is abundant in bone, to which certain tumors preferentially metastasize. Therefore, we investigated the role of osteopontin in the experimental metastasis of tumor cells using recently established osteopontin-deficient mice. B16 melanoma cells, which produce little osteopontin, were injected into the left ventricle of osteopontin-deficient mice or wild-type mice. Animals were killed 2 weeks after injection. The number of tumors was reduced in the bones of osteopontin-deficient mice compared with the bones in wild-type mice. The number of tumors in the adrenal gland also was reduced. To investigate the osteopontin effect on metastases via a different route, we injected B16 melanoma cells into the femoral vein. Through this route, the number of lung tumors formed was higher than in the intracardiac route and was again less in osteopontin-deficient mice compared with wild-type mice. In conclusion, in an experimental metastasis assay, the number of tumors found in bone (after intracardiac injection) and lung (after left femoral vein injection) was significantly reduced in osteopontin-deficient mice compared with wild-type mice. Tumor numbers in other organs examined were small and not significantly different in the two situations.

Embryonic development of tendons is in close association with that of cartilage and bone. Although these tissues are derived from mesenchymal progenitor cells which also give rise to muscle and fat, their fates clearly diverse in early embryonic stages. Transcription factors may play pivotal roles in the process of determination and differentiation of tendon cells as well as other cells in the skeletal system. Scleraxis, a basic helix-loop-helix (bHLH) type transcription factor, is expressed in mesenchymal progenitors that later form connective tissues including tendons. Sox9 is an HMG-box containing transcription factor, which is expressed at high levels in chondrocytes. We hypothesized that the two transcription factors regulate the fate of cells that interact with each other at the interface between the two tissues during divergence of their differentiation pathways. To address this point, we investigated scleraxis and Sox9 mRNA expression during mouse embyogenesis focusing on the coordinated development of tendons and skeletons. In the early stage of mesenchymal tissue development at 10.5 d.p.c. scleraxis and Sox9 transcripts were expressed in the mesenchymal progenitor cells in the appendicular and axial mesenchyme. At 11.5 d.p.c., scleraxis transcripts were observed in the mesenchymal tissue surrounding skeletal primordia which express Sox9. From this stage. seleraxis expression was closely associated with, but distinct from, formation of skeletal primordia. At 13.5 d.p.c., scleraxis was expressed broadly in the interface between muscle and skeletal primordia while Sox9 expression is confined within the early skeletal primordia. Then, at 15.5 d.p.c., scleraxis transcripts were more restricted to tendons. These observations revealed the presence of temporal and spatial association of scleraxis expression during embryonic development of tendon precursor cells in close association with that of Sox9 expression in chondrogenic cells in skeletal tissues.

Recent development in three-dimensional (3D) imaging of cancellous bone has made possible true 3D quantification of trabecular architecture. This provides a significant improvement in the measures available to study and understand the mechanical functions of cancellous bone. We recently reported that the presence of osteopontin (OPN) was required for the effects of mechanical stress on bone as OPN-null (OPN-/-) mice showed neither enhancement of bone resorption nor suppression of bone formation when they were subjected to unloading by tail suspension. However, in this previous study, morphological analyses were limited to two-dimensional (2D) evaluation. Although bone structure is 3D and thus stress effect should be evaluated based on 3D parameters, no such 3D morphological features underlying the phenomenon have been known. To elucidate the role of OPN in mediating mechanical stress effect based on true quantitative examination of bone, we evaluated 3D trabecular structures of hindlimb bones of OPN-/- mice after tail suspension. Tail suspension significantly reduced 3D parameters of bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and anisotropy and increased 3D parameters on trabecular separation (Tb.Sp) in wild-type mice. In contrast, these 3D parameters were not altered after tail suspension in OPN-/- mice. These data provided evidence that OPN is required for unloading-induced 3D bone loss.

To elucidate regulatory mechanism(s) underlying differentiation of osteoblasts, we examined involvement of helix-loop-helix (HLH)-type transcription factors in osteoblast-specific expression of a phenotypic marker gene which encodes osteocalcin, a major noncollagenous bone matrix protein, exclusively expressed in osteoblasts. Overexpression of a dominant negative HLH protein, Id-1, decreased the activity of the 1.1-kb osteocalcin gene promoter cotransfected into rat osteoblastic osteosarcoma ROS17/2.8 cells. Analysis of deletion mutants revealed that a 264-bp fragment of osteocalcin promoter (-198 to +66) was sufficient for the Id-1-dependent suppression. Furthermore, the activity of the same promoter fragment (-198 to +66) was enhanced when antisense Id-1 expression vector was cotransfected. This osteocalcin gene promoter region contains two sites of an E-box motif, a consensus binding site for HLH proteins, which we refer to as OCE1 (CACATG, at -102) and OCE2 (CAGCTG, at -149), respectively. Mutagenesis in OCE1 but not OCE2 led to greater than 50% reduction in transcriptional activity of the osteocalcin gene promoter. Electrophoresis mobility shift assay indicated that factors in nuclear extracts prepared from ROS17/2.8 cells bound to the 30-bp oligonucleotide probe containing the E-box motif of OCE1. This binding was competed out by OCE1 oligonucleotide but neither by OCmE1 oligonucleotide in which E-box motif was mutated nor by OCE2. The OCE1-binding activity in the nuclear extracts of ROS17/2.8 cells was reduced by 70% when bacterially expressed Id-1 protein was added to the reaction mixture, suggesting the involvement of HLH proteins in the DNA/protein complex formation. In contrast to the osteoblast-like cells, OCE1-binding activity in the nuclear extracts of C3H10T1/2 fibroblasts was very low. However, when these fibroblasts were treated with recombinant human bone morphogenetic protein-2 which induced expression of osteocalcin as well as other phenotypic markers of osteoblasts, OCE1-binding activity was increased approximately 40-fold, indicating that OCE1 would be involved in the tissue-specific expression of the osteocalcin gene. These findings indicated for the first time that osteoblast-specific gene transcription is regulated via the interaction between certain E-box binding transcription factor(s) in osteoblasts and the OCE1 sequence in the promoter region of the osteocalcin gene.

Abstract Objective Human synovium–derived mesenchymal stem cells (MSCs) can efficiently differentiate into mature chondrocytes. It has been suggested that DNA methylation is one mechanism that regulates human chondrogenesis; however, the methylation status of genes related to chondrogenic differentiation is not known. The purpose of this study was to investigate the CpG methylation status in human synovium–derived MSCs during experimental chondrogenesis, with a view toward potential therapeutic use in osteoarthritis. Methods Human synovium–derived MSCs were subjected to chondrogenic pellet culture for 3 weeks. The methylation status of 12 regions in the promoters of 10 candidate genes ( SOX9 , RUNX2 , CHM1 , FGFR3 , CHAD , MATN4 , SOX4 , GREM1 , GPR39 , and SDF1 ) was analyzed by bisulfite sequencing before and after differentiation. The expression levels of these genes were analyzed by real‐time reverse transcription–polymerase chain reaction. Methylation status was also examined in human articular cartilage. Results Bisulfite sequencing analysis indicated that 10 of the 11 CpG‐rich regions analyzed were hypomethylated in human progenitor cells before and after 3 weeks of pellet culture, regardless of the expression levels of the genes. The methylation status was consistently low in SOX9 , RUNX2 , CHM1 , CHAD , and FGFR3 following an increase in expression upon differentiation and was low in GREM1 and GPR39 following a decrease in expression upon chondrogenesis. One exceptional instance of a differentially methylated CpG‐rich region was in a 1‐kb upstream sequence of SDF1 , the expression of which decreased upon differentiation. Paradoxically, the hypermethylation status of this region was reduced after 3 weeks of pellet culture. Conclusion The DNA methylation levels of CpG‐rich promoters of genes related to chondrocyte phenotypes are largely kept low during chondrogenesis in human synovium–derived MSCs.

Parathyroid hormone (PTH), the major calcium-regulating hormone, and norepinephrine (NE), the principal neurotransmitter of sympathetic nerves, regulate bone remodeling by activating distinct cell-surface G protein-coupled receptors in osteoblasts: the parathyroid hormone type 1 receptor (PTHR) and the β(2)-adrenergic receptor (β(2)AR), respectively. These receptors activate a common cAMP/PKA signal transduction pathway mediated through the stimulatory heterotrimeric G protein. Activation of β(2)AR via the sympathetic nervous system decreases bone formation and increases bone resorption. Conversely, daily injection of PTH (1-34), a regimen known as intermittent (i)PTH treatment, increases bone mass through the stimulation of trabecular and cortical bone formation and decreases fracture incidences in severe cases of osteoporosis. Here, we show that iPTH has no osteoanabolic activity in mice lacking the β(2)AR. β(2)AR deficiency suppressed both iPTH-induced increase in bone formation and resorption. We showed that the lack of β(2)AR blocks expression of iPTH-target genes involved in bone formation and resorption that are regulated by the cAMP/PKA pathway. These data implicate an unexpected functional interaction between PTHR and β(2)AR, two G protein-coupled receptors from distinct families, which control bone formation and PTH anabolism.

Mechanical stress is known to alter bone mass and the loss of force stimuli leads to reduction of bone mass. However, molecules involved in this phenomenon are incompletely understood. As mechanical force would affect signaling events in cells, we focused on a calcium channel, TRPV4 regarding its role in the effects of force stimuli on calcium in osteoblasts. TRPV4 expression levels were enhanced upon differentiation of osteoblasts in culture. We found that BMP-2 treatment enhanced TRPV4 gene expression in a dose dependent manner. BMP-2 effects on TRPV4 expression were suppressed by inhibitors for transcription and new protein synthesis. In these osteoblasts, a TRPV4-selective agonist, 4α-PDD, enhanced calcium signaling and the effects of 4α-PDD were enhanced in differentiated osteoblasts compared to the control cells. Fluid flow, as a mechanical stimulation, induced intracellular calcium oscillation in wild type osteoblasts. In contrast, TRPV4 deficiency suppressed calcium oscillation significantly even when the cells were subjected to fluid flow. These data suggest that TRPV4 is involved in the flow-induced calcium signaling in osteoblasts.

Bone formation is precisely regulated by cell-cell communication in osteoblasts. We have previously demonstrated that genetic deletion of Col6a1 or Col12a1 impairs osteoblast connections and/or communication in mice, resulting in bone mass reduction and bone fragility. Mutations of the genes encoding collagen VI cause Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM), which have overlapping phenotypes involving connective tissue and muscle. Recent studies have identified COL12A1 gene mutations in patients with UCMD- and BM-like disorders harboring no COL6 mutations, indicating the shared functions of these collagens in connective tissue homeostasis. The purpose of this investigation has been to test the hypothesis that collagens VI and XII have coordinate regulatory role(s) during bone formation. We analyzed the localization of collagens VI and XII relative to primary osteoblasts during osteogenesis. Immunofluorescence analysis demonstrated that collagens VI and XII colocalized in matrix bridges between adjacent cells during periods when osteoblasts were establishing cell-cell connections. Quantification of cells harboring collagen bridges demonstrated that matrix bridges were composed of collagens VI and XII but not collagen I. Interestingly, matrix bridge formation was impaired in osteoblasts deficient in either Col6a1 or Col12a1, suggesting that both collagens were indispensable for matrix bridge formation. These data demonstrate, for the first time, a functional relationship between collagens VI and XII during osteogenesis and indicate that a complex containing collagens VI and XII is essential for the formation of a communicating cellular network during bone formation.

This paper provides an extended level set (X-LS) based topology optimiza- tion method for multi material design. In the proposed method, each zero level set of a level set function {\phi}ij represents the boundary between materials i and j. Each increase or decrease of {\phi}ij corresponds to a material change between the two materials. This approach reduces the dependence of the initial configuration in the optimization calculation and simplifies the sensitivity analysis. First, the topology optimization problem is formulated in the X-LS representation. Next, the reaction-diffusion equation that updates the level set function is introduced, and an optimization algorithm that solves the equilibrium equations and the reaction-diffusion equation using the fi- nite element method is constructed. Finally, the validity and utility of the proposed topology optimization method are confirmed using two- and three- dimensional numerical examples.

Recent advances in additive manufacturing have enabled the fabrication of structures in which members made of multiple materials are placed in appropriate positions. However, dimensional constraints must be considered in such a process because of the size limitations of the 3D printer. This paper proposes a topology optimization method that considers the dimensional constraints of each material component. An extended level-set method is used to represent multiple material phases in the structural design. Constraint functions are formulated to control the size of each component. The weighted covariance matrix is used for the bounding box constraint. The sensitivity is based on the topological derivative and adjoint variable method. The proposed method was applied to solving the minimum compliance problem for two- and three-dimensional numerical examples to demonstrate its effectiveness and its potential contribution to enriching designs in additive manufacturing.

Microphthalmia-associated transcription factor (Mitf) regulates the differentiation of melanocytes, optic cup-derived retinal pigment epithelium (RPE), and some types of bone marrow-derived cells. Mitf consists of at least five isoforms with different N-termini, each of which is encoded by a separate exon 1. Here we identified a novel isoform, termed mouse Mitf-D/human MITF-D, that is expressed in RPE, macrophages, and osteoclasts affected by the Mitf mutations, but not expressed in other Mitf target cells, including melanocyte-lineage cells and natural killer cells. The initiation Met of MITF-D is located in the downstream domain (B1b domain) that is shared by other MITF isoforms. The 5'-untranslated region of MITF-D mRNA is encoded by the newly identified first exon of the MITF gene, termed exon 1D, which is located 3 kb upstream of the exon encoding the B1b domain. Thus, the MITF gene generates multiple isoforms with different expression patterns by using the alternative promoters in a cell-dependent manner, thereby providing the molecular basis for the phenotypic variability seen in the MITF/Mitf mutants.

Bone morphogenetic protein (BMP) signaling regulates body axis determination, apoptosis, and differentiation of various types of cells including neuron, gut, and bone cells. However, the molecules involved in such BMP regulation of biological events have not been fully understood. Here, we examined the involvement of Cas-interacting zinc finger protein (CIZ) in the modulation of BMP2-induced osteoblastic cell differentiation. CIZ overexpression in osteoblastic MC3T3E1 cells suppressed BMP2-enhanced expression of alkaline phosphatase, osteocalcin, and type I collagen genes. Upstream analyses revealed that CIZ overexpression also suppressed BMP2-induced enhancement of the mRNA expression of Cbfa1, which is a critical transcription factor for osteoblastic differentiation. BMP-induced Smad1 and Smad5 activation of GCCG-mediated transcription was blocked in the presence of CIZ overexpression. CIZ overexpression alone in the absence of BMP2 moderately enhanced basal levels of Cbfa1 mRNA expression. CIZ overexpression also enhanced 1.8-kb Cbfa1 promoter activity in the absence of BMP2, whereas it suppressed the promoter activity in the presence of BMP2. Finally, CIZ overexpression suppressed the formation of mineralized nodules in osteoblastic cell cultures. These data indicate that CIZ is a novel type inhibitor of BMP/Smad signaling.

The effect of leukemia inhibitory factor (LIF) on proliferation and phenotypic expression in murine osteoblast-like (MC3T3E1) cells was examined. LIF inhibited the proliferation of these cells by up to 20% and DNA synthesis was inhibited in a dose-dependent manner with an ED50 of about 0.2 ng/ml. The effect of LIF relative to matched controls increased with decreasing serum concentration, reaching 30% inhibition at 0.2% serum. LIF also reduced the stimulatory effects of platelet-derived growth factor and insulin-like growth factor I on DNA synthesis. The inhibition of the DNA synthesis by saturating concentration of transforming growth factor beta was further enhanced by the addition of LIF, suggesting independent pathways for the action of the two growth inhibitors. In addition, LIF reduced alkaline phosphatase activity and the abundance of type I collagen messenger RNA, but increased the level of osteopontin messenger RNA. These findings suggest that LIF may play a role in regulating the function of osteoblasts.

An accurate assessment of the cervical lymph node metastasis status in oral cavity cancer not only helps predict the prognosis of patients, but also helps surgeons to perform the appropriate treatment. We investigated the utilization of microarray technology focusing on the differences in gene expression profiles between primary tumors of oral squamous cell carcinoma that had metastasized to cervical lymph nodes and those that had not metastasized in the hope of finding new biomarkers to serve for diagnosis and treatment of oral cavity cancer. To design this experiment, we prepared two groups: the learning case group with 30 patients and the test case group with 13 patients. All tissue samples were performed using laser captured microdissection to yield cancer cells, and RNA was isolated from purified cancer cells. To identify a predictive gene expression signature, the different gene expressions between the two groups with and without metastasis in the learning case ( n = 30) were analyzed, and the 85 genes expressed differentially were selected. Subsequently, to construct a more accurate prediction model, we further selected the genes with a high power for prediction from the 85 genes using the AdaBoost algorithm. The eight candidate genes, DCTD , IL‐15 , THBD , GSDML , SH3GL3 , PTHLH , RP5‐1022P6 and C9orf46 , were selected to achieve the minimum error rate. Quantitative reverse transcription–polymerase chain reaction was carried out to validate the selected genes. From these statistical methods, the prediction model was constructed including the eight genes and this model was evaluated by using the test case group. The results in 12 of 13 cases (∼92.3%) were predicted correctly. ( Cancer Sci 2007; 98: 740–746)

A tumor-derived factor believed to cause hypercalcemia by acting on the parathyroid hormone (PTH) receptor was recently purified, cloned, and found to have NH2-terminal sequence homology with PTH. The 1-34 region of this protein was synthesized, evaluated for its postreceptor effects on the ROS 17/2.8 cell line, and its properties were compared to 1-34 PTH. Both 1-34 human humoral hypercalcemia factor (HCF) and 1-34 PTH stimulated adenylate cyclase with an effective concentration (EC)50 of approximately 1 nM. The extent of stimulation by both peptides was equally enhanced by dexamethasone. They both had a pronounced inhibitory effect on growth in the presence of dexamethasone, with an EC50 of approximately 0.1 nM, reduced alkaline phosphatase (AP) activity by approximately 70% in the absence of dexamethasone and by approximately 80% in the presence of dexamethasone with an EC50 of 0.03 nM, and when present at a concentration of 10 nM, reduced AP mRNA levels (estimated by Northern analysis) by approximately 80% in the presence or absence of dexamethasone. Thus, in addition to similar dose-response curves for adenylate cyclase stimulation, both HCF and PTH produced identical postreceptor effects in ROS 17/2.8 cells. These effects of HCF are probably mediated by the interaction of the tumor-derived factor with the PTH receptor.

Sclerostin (SOST), a member of the cystine-knot superfamily, is essential for proper skeletogenesis because a loss-of-function mutation in the SOST gene results in sclerosteosis featured with massive bone growth in humans. To understand the function of SOST in developmental skeletal tissue formation, we examined SOST gene expression in embryonic osteogenesis in vitro and in vivo. During osteoblastic differentiation in primary calvarial cells, the levels of SOST expression were increased along with those of alkaline phosphatase activity and nodule formation. In situ hybridization study revealed that SOST mRNA expression was observed in the digits in embryonic 13-d limb buds, and SOST expression was observed in osteogenic front in embryonic 16.5-d postcoitus embryonic calvariae, and this expression persisted in the peripheral area of cranial bone in the later developmental stage (embryonic 18.5-d post coitum). These temporal and spacial expression patterns in vivo and in vitro were in parallel to those of osterix (Osx), which is a critical transcriptional factor for bone formation. Similar coexpression of SOST and Osx mRNA was observed when the primary osteoblastic calvarial cells were cultured in the presence of bone morphogenetic protein (BMP)2 in vitro. Moreover, endogenous expression of SOST and Osx mRNA was inhibited by infection of noggin-expression adenovirus into the primary osteoblastic calvarial cells, suggesting that endogenous BMPs are required for these cells to express SOST and Osx mRNA. Thus, expression and regulation of SOST under the control of BMP were closely associated with those of Osx in vivo and in vitro.

Osteoporosis is a major health problem; however, the mechanisms regulating adult bone mass are poorly understood. Cas-interacting zinc finger protein (CIZ) is a nucleocytoplasmic shuttling protein that localizes at cell adhesion plaques that form where osteoblasts attach to substrate. To investigate the potential role of CIZ in regulating adult bone mass, we examined the bones in CIZ-deficient mice. Bone volume was increased and the rates of bone formation were increased in CIZ-deficient mice, whereas bone resorption was not altered. CIZ deficiency enhanced the levels of mRNA expression of genes encoding proteins related to osteoblastic phenotypes, such as alkaline phosphatase (ALP) as well as osterix mRNA expression in whole long bones. Bone marrow cells obtained from the femora of CIZ-deficient mice revealed higher ALP activity in culture and formed more mineralized nodules than wild-type cells. CIZ deficiency enhanced bone morphogenetic protein (BMP)-induced osteoblastic differentiation in bone marrow cells in cultures, indicating that BMP is the target of CIZ action. CIZ deficiency increased newly formed bone mass after femoral bone marrow ablation in vivo. Finally, BMP-2-induced bone formation on adult mouse calvariae in vivo was enhanced by CIZ deficiency. These results establish that CIZ suppresses the levels of adult bone mass through inhibition of BMP-induced activation of osteoblasts.

TGFβ modulates the growth and differentiation of various cell types, in part by regulating the production of extracellular matrix proteins. In rat osteoblast - like cells TGFβ stimulates the production of collagen, osteopontin and osteonectin. On the other hand, TGFβ inhibits the production of osteocalcin, one of the most abundant non-collagenous bone matrix proteins, which is only expressed in osteoblasts. Inhibition of osteocalcin expression by TGFβ in the rat osteoblatic osteosarcoma, ROS 17/2.8 cells, occurs at least in part through transcriptional control.

Abstract Dendritic cells (DCs) are professional APCs that can control immune responses against self and altered self, typically foreign, determinants. DCs can be divided into several subsets, including CD8α+ and CD8α− DCs. These subsets possess specific functions. For example, mouse splenic CD8α+, but not CD8α− DCs selectively take up dying cells and cross-present cell-associated Ags to naive T cells. In this study, we identified genes that were more expressed in CD8α+ than CD8α− DCs by microarray analysis. Only one of these genes, when the extracellular domains were linked to human IgG Fc domain, could bind to late apoptotic or necrotic cells. This gene was a new member of the triggering receptor expressed on myeloid cells (Trem) family, Trem-like 4 (Treml4). Treml4 mRNA and protein, the latter detected with a new mAb, were predominantly expressed in spleen. Treml4, like other Trem family members, could associate with the adaptor molecule DNAX activation protein 12 kDa, but neither DNAX activation protein 10 kDa nor FcRγ. Consistent with the microarray data, we confirmed that Treml4 protein was more expressed on CD8α+ than CD8α− DCs, and we also found that Treml4 was expressed at high levels on splenic macrophages in spleen, particularly red pulp and marginal metallophilic macrophages. In addition, Treml4 expression on DCs was not changed after maturation induced by TLR ligands. Thus, Treml4 is a new Trem family molecule that is abundantly expressed on CD8α+ DCs and subsets of splenic resident macrophages, and can recognize dead cells by different types of phagocytes in spleen.

Regeneration of bone requires the combination of appropriate drugs and an appropriate delivery system to control cell behavior. However, the delivery of multiple drugs to heal bone is complicated by the availability of carriers. The aim of this study was to explore a new system for delivery of a selective EP4 receptor agonist (EP4A) in combination with low-dose bone morphogenetic protein 2 (BMP-2).Combined delivery of EP4A and BMP-2 was carried out with a nanogel-based scaffold in the shape of a disc, to repair critical-size circle-shaped bone defects in calvariae that otherwise did not heal spontaneously.Combination treatment with EP4A and low-dose BMP-2 in nanogel efficiently activated bone cells to regenerate calvarial bone by forming both outer and inner cortical plates as well as bone marrow tissue to regenerate a structure similar to that of intact calvaria. EP4A enhanced low-dose BMP-2-induced cell differentiation and activation of transcription events in osteoblasts.These data indicate that combined delivery of EP4A and low-dose BMP-2 via nanogel-based hydrogel provides a new system for bone repair.

SOX9 is a transcription factor that activates type II procollagen (Col2a1) gene expression during chondrocyte differentiation. Glucocorticoids are also known to promote chondrocyte differentiation via unknown molecular mechanisms. We therefore investigated the effects of a synthetic glucocorticoid, dexamethasone (DEX), on Sox9 gene expression in chondrocytes prepared from rib cartilage of newborn mice. Sox9 mRNA was expressed at high levels in these chondrocytes. Treatment with DEX enhanced Sox9 mRNA expression within 24 h and this effect was observed at least up to 48 h. The effect of DEX was dose dependent, starting at 0.1 nM and maximal at 10 nM. The half life of Sox9 mRNA was approximately 45 min in the presence or absence of DEX. Western blot analysis revealed that DEX also enhanced the levels of SOX9 protein expression. Treatment with DEX enhanced Col2a1 mRNA expression in these chondrocytes and furthermore, DEX enhanced the activity of Col2-CAT (chloramphenicol acetyltransferase) construct containing a 1.6 kb intron fragment where chondrocyte-specific Sry/Sox- consensus sequence is located. The enhancing effect of DEX was specific to SOX9, as DEX did not alter the levels of Sox6 mRNA expression. These data suggest that DEX promotes chondrocyte differentiation through enhancement of SOX9.

Inactivation mutation of the recently discovered klotho gene in mice causes a syndrome resembling aging. Manifestations include short life span, atherosclerosis, gonadal atropy, skin atropy, emphysema, ataxia and ectopic calcification. These mice also exhibit abnormally high bone density in the epiphyses of their tibiae based on X-ray and histological analyses. However, micro-structures of the trabecular bones in arbitrary two-dimensional planes or three-dimensional regions are difficult to analyze by these techniques. Therefore, we applied high resolution micro-computed tomography (microCT) to characterize the micro-structural abnormality in the trabecular bone in long bones as well as in vertebrae of four- to six-week-old klotho mutant mice. Two-dimensional microCT analyses in the mid-sagittal plane as well as three-dimensional microCT analyses indicated that the trabecular bone volume fraction measured in the proximal metaphyses of the tibiae was increased more than twofold in klotho mutant mice compared with the wild-type mice. Similarly, the trabecular bone area fraction in the mid-sagittal plane of the lumbar vertebral bodies was also increased by about 80% at the proximal and distal ends. No significant difference was observed with regard to the cortical thickness in the mid-shaft of femora between klotho mutant and wild-type mice. Three-dimensional microCT analyses also indicated that the trabecular number and thickness of the proximal metaphyses of the tibiae were increased by about 80% and 300% respectively in the klotho mutant mice, while trabecular separation was 60% less in klotho mutant mice compared with the wild-type mice. These quantitative microCT analyses indicate that the inactivation of klotho gene expression results in an increase in three-dimensional bone volume fraction, number and thickness of the trabecular bones in these mice.

Abstract Osterix is a recently identified zinc‐finger‐containing transcription factor, which is required for skeletogenesis as no bone formation was observed in osterix‐deficient mice. Osterix was first cloned as a gene whose expression was enhanced by BMP in C2C12 cells. As BMP induces ectopic bone formation in vivo via a pathway reminiscent to endochondral bone formation, BMP may also regulate osterix gene expression in chondrocytes. However, no information was available regarding the BMP actions on osterix gene expression in chondrocytes. We therefore examined the effects of BMP‐2 on osterix gene expression in chondrocytes in culture. RT‐PCR analysis indicated that osterix mRNA was expressed in the primary cultures of chondrocytes derived from mouse rib cartilage. The treatment with BMP‐2 enhanced the levels of osterix transcripts within 24 h and the enhancement was still observed at 48 h based on RT‐PCR analysis. This BMP effect was specific to this cytokine, as TGF‐β did not alter osterix gene expression. BMP effects on the osterix mRNA levels were also confirmed by Northern blot analysis. The enhancing effect of BMP on osterix gene expression was observed in a dose‐dependent manner starting at 200 ng/ml. The BMP enhancement of the osterix gene expression in chondrocytes was blocked in the presence of a protein synthesis inhibitor, cycloheximide, while it was still observed in the presence of 5,6‐dichloro‐1‐β D‐ribofuranosylbenzimidazol (DRB) suggesting the involvement of post‐transcriptional events, which require new protein synthesis. These results indicated that osterix gene is expressed in the primary cultures of chondrocytes and its expression is under the control of BMP‐2. J. Cell. Biochem. 88: 1077–1083, 2003. © 2003 Wiley‐Liss, Inc.

Coordinated regulation of the activities of bone morphogenetic protein (BMP) and its inhibitors is essential for skeletal development since loss-of-function experiments show that both BMPs and BMP inhibitory signals, such as noggin, are required to establish proper formation of skeletal tissues. In this paper, we asked how and when noggin would be functional to interact with BMPs during skeletogenesis in mammals. For this purpose, we first analyzed the spatial and temporal patterns of noggin, BMP-2, BMP-4, and BMP-7 expression during early skeletogenesis in mouse embryos. In situ hybridization study revealed that noggin expression was detected at a low level in limb mesenchyme, whereas BMP-7 was expressed at a high level throughout limb mesenchyme 10.5 days postcoitum (dpc) in mouse embryos. One day later, noggin mRNA was expressed at a high level in the prechondrogenic condensations in appendicular and axial skeletal primordia, where sox9 transcripts were also expressed. At this stage, noggin-expressing cells were surrounded by those expressing BMP-7. The chondrogenic cell condensation continued to express noggin transcripts in 12.5 dpc and 13.5 dpc embryos, and again the noggin-expressing cells within the cartilaginous tissue were surrounded by those expressing BMP-7. We further examined interaction of noggin and BMPs by using organ cultures of 11.5 dpc mouse forelimbs and found that implantation of carriers containing BMP-7 protein into the forelimb explants induced noggin expression in the limb mesenchyme. BMP-7 also induced type II collagen and sox9 mRNAs in the same cell population, indicating that noggin induction occurred in the chondrogenic precursor cells. BMP-7 effects on noggin expression were observed in a dose-dependent manner within a dose range of 10-100 ng/microliter. These results suggest that BMP-7 induced expression of noggin transcripts within skeletal cell condensation and that this noggin expression in turn could act antagonistically to attenuate BMP action in the early skeletogenesis.

Cell differentiation is determined by a certain set of transcription factors such as MyoD in myogenesis. However, transcription factors that play a positive role in phenotypic gene expression in skeletal cells are largely unknown, except the recently identified CBFA1. Scleraxis is a helix-loop-helix-type transcription factor whose transcripts are expressed in sclerotome and in a certain set of skeletal cells; however, nothing is known about its function with regard to the regulation of cell function. To examine possible roles of scleraxis, we overexpressed scleraxis in osteoblastic ROS17/2.8 cells, which express low levels of scleraxis. Scleraxis overexpression enhanced expression of the aggrecan gene, which is not normally expressed at high levels in these osteoblastic cells. Overexpression of scleraxis also increased mRNA levels of type II collagen and osteopontin while suppressing expression of osteoblast phenotype-related genes encoding type I collagen and alkaline phosphatase. Transient transfection experiments indicated that scleraxis enhanced the chloramphenicol acetyltransferase activity of the reporter construct AgCAT-8, which contained an 8-kilobase pair (kb) fragment of the aggrecan gene including both the promoter and its first intron. Deletion analysis identified a 1-kb region that is responsive to scleraxis within the aggrecan gene. This region contains two adjacent E-box sequences. A 29-base pair DNA fragment (AgE) containing these E-box sequences bound to proteins in the ROS17/2.8 cell nuclear extracts as well as to <i>in vitro</i> translated scleraxis. This binding was competed with unlabeled AgE, but not with a mutated E-box DNA sequence (mAgE), indicating the specificity of the binding activity. The AgE binding activity in the ROS17/2.8 cell nuclear extracts was enhanced in the cells overexpressing scleraxis and was supershifted by the antiserum raised against scleraxis. Furthermore, AgE, but not mAgE, conferred responsiveness to scleraxis overexpression to a heterologous promoter. Finally, replacement mutation of the AgE sequence within the 2.5-kb AgCAT-1 construct significantly reduced its responsiveness to scleraxis. These results indicate that overexpression of a single helix-loop-helix-type transcription factor, scleraxis, enhances aggrecan gene expression via binding to the E-box-containing AgE sequence in ROS17/2.8 cells.

Id is one of the helix-loop-helix family of proteins that regulate differentiation in several types of cells, including myoblasts. We found that Id mRNA was constitutively expressed in ROS17/2.8 rat osteoblastic osteosarcoma cells and that the level of Id message in these cells was suppressed by 1,25-dihydroxyvitamin D3 (vitamin D), a calcitropic hormone known to enhance expression of differentiation-related phenotypes in these cells. The vitamin D suppression was dose-dependent, starting at 10(-11) M and saturating at 10(-8) M. This vitamin D effect was seen within 6 hr after the initiation of the treatment and lasted at least 48 hr. Cycloheximide did not block the vitamin D suppression of Id message level. Vitamin D reduced the rate of Id gene transcription by approximately 80% as estimated by nuclear run-on assay. Electrophoretic mobility-shift assay indicated the specific binding of nuclear factors from ROS17/2.8 cells to an E-box DNA sequence, and the binding signal was enhanced in nuclear extracts of the cells treated with vitamin D. The suppressive effect was specific to vitamin D, since another calcitropic hormone, the synthetic compound dexamethasone, did not suppress Id message expression. These observations indicate the presence of helix-loop-helix proteins in osteoblastic cells and indicate that vitamin D, a potent regulator of differentiation in several types of cells, controls the expression of the helix-loop-helix molecules.

Abstract Bone formation is under the control of a set of transcription factors. Ids are inhibitory helix‐loop‐helix (HLH) transcription factors and expression of Id genes in osteoblasts is under the control of calciotropic agents such as BMP and vitamin D. However, the function of Ids during bone formation in vivo has not yet been elucidated. We, therefore, examined the role of Id1 and Id3 in the regulation of bone metabolism in vivo. Using wild type and Id1/Id3 heterozygous knockout mice, we analyzed calvarial bone formation in the suture by X‐ray picture, proliferation, and mineralization activities of primary calvarial osteoblasts by MTT assay and alizarin red staining and onthotopic in vivo bone formation by BMP injection onto calvaria and micro CT analysis. The width of calvarial sutures was reduced by more than 50% in Id1/Id3 heterozygous knock out mice. Analyses on the cellular basis for the mechanism underlying the defects in the mutant mice revealed suppression of proliferation and mineralization in osteoblasts derived from Id1/Id3 heterozygous knock out mice. Furthermore, Id1/Id3 heterozygous knock out mice suppressed BMP‐induced bone formation in vivo. These results indicated that Id1 and Id3 are positive factors to promote bone formation in vivo. © 2004 Wiley‐Liss, Inc.

Endothelins (ETs) are vasoconstrictive peptides produced mainly by endothelial cells. The ET receptors are expressed in many types of cells including osteoblast-like cells. The purpose of this study was to examine the effects of endothelin on the expression of osteoblastic phenotype-related genes. We found that endothelin-1 (ET-1) enhanced approximately two-fold the mRNA expression of both osteopontin and osteocalcin genes in rat osteoblastic osteosarcoma ROS17/2.8 cells. These effects were dose-dependent, peaking at 10(-7) M. The ET-1 enhancement of the abundance of osteopontin and osteocalcin mRNAs was time-dependent, with a maximal effect at 24 h. ET-1 modulation of the expression of the two phenotype-related gene products of osteoblasts suggests that endothelin is one of the cytokines which modulate osteoblastic functions and that this molecule may play a role in the regulation of bone metabolism.

Mechanical stress to bone plays a crucial role in the maintenance of bone homeostasis. It causes the deformation of bone matrix and generates strain force, which could initiate the mechano-transduction pathway. The presence of osteopontin (OPN), which is one of the abundant proteins in bone matrix, is required for the effects of mechanical stress on bone, as we have reported that OPN-null (OPN-/-) mice showed resistance to unloading-induced bone loss. However, cellular mechanisms underlying the phenomenon have not been completely elucidated. To obtain further insight into the role of OPN in mediating mechanical stress effect on bone, we examined in vitro mineralization and osteoclast-like cell formation in bone marrow cells obtained from hind limb bones of OPN-/- mice after tail suspension. The levels of mineralized nodule formation of bone marrow cells derived from the femora subjected to unloading were decreased compared with that from loaded control in wild-type mice. However, these were not decreased in cells from OPN-/- mice after tail suspension compared with that from loaded OPN-/- mice. Moreover, while spreading of osteoclast-like cells derived from bone marrow cells of the femora subjected to unloading was enhanced compared with that from loaded control in wild-type mice, this enhancement of spreading of these cells derived from the femora subjected to unloading was not recognized compared with those from loaded control in OPN-/- mice. These data provided cellular bases for the effect of the OPN deficiency on in vitro reduced mineralized nodule formation by osteoblasts and on enhancement of osteoclast spreading in vitro induced by the absence of mechanical stress. These in vitro results correlate well with the resistance to unloading-induced bone loss in OPN-/- mice in vivo, suggesting that OPN has an important role in the effects of unloading-induced alterations of differentiation of bone marrow into osteoblasts and osteoclasts.

Abstract Recovery of bone loss is one of the active research issues in bone medicine due to the need for efficient measures for bone gain. We examined here a novel drug delivery system using a nanogel of cholesterol‐bearing pullulan (CHP) in combination with prostaglandin E 2 (PGE 2 ). PGE 2 or PGE 2 /CHP, vehicle (saline containing 0.06% ethanol and 0.02% Tween 80) or CHP were injected on to the calvariae of mice once every day for 5 days per week for 4 weeks. Low dosage of PGE 2 (0.6 µg) alone or CHP alone did not induce new bone formation in this system. In contrast, PGE 2 (0.6 µg)/CHP induced new bone formation. Bone formation activities of PGE 2 was enhanced by CHP nanogels only at the site of injection (calvaria) but not in the distant sites of the skeleton, showing that PGE 2 /CHP could avoid systemic effects. In spite of the fact that previously reported animal models of bone formation by PGE 2 were associated with loss of body weight, bone formation based on PGE 2 /CHP did not associate with loss of body weight. Furthermore, only a single application of PGE 2 in combination with nanogel cross‐linking hydrogel sphere (PGE 2 /CHP‐PEO) induced new bone formation. Thus, nanogel‐based delivery system is an efficient delivery system of bone anabolic agent, PGE 2 . J. Cell. Biochem. 101:1063–1070, 2007. © 2007 Wiley‐Liss, Inc.

Abstract Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Since tumor metastasis shortens patients' lifetime, establishment of therapy for anti‐metastasis is very important. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, extracellular matrix (ECM) invasion and cell proliferation via interaction with its receptor, that is, αvβ3 integrin. OPN is believed to be a positive regulator of tumor metastasis in vivo. However, how OPN regulates metastasis is largely unknown. Here, we explore the role of OPN in cell migration. Serum from wild‐type mice induced cell migration of B16 melanoma cells, while serum from OPN‐deficient mouse suppressed this event. The presence of recombinant OPN significantly enhanced cell migration compared to albumin containing medium. OPN‐induced cell migration was suppressed by inhibiting the ERK/MAPK pathway indicating that OPN‐induced cell migration depends on this pathway. Overexpression of OPN in these cancer cells per se promoted cell proliferation and tended to increase B16 cell migration suggesting that OPN promotes bone metastasis by playing dual roles both in host microenvironment and in tumor cell itself. In conclusion, the elevated OPN expression in host tissue and tumor cell itself promotes tumor cell migration reading to tumor metastasis, suggesting that neutralization of OPN‐induced signal might be effective in suppression of tumor metastasis. J. Cell. Biochem. 101: 979–986, 2007. © 2007 Wiley‐Liss, Inc.

Bisphosphonates (BPs) are a major class of antiresorptive drug, and their molecular mechanisms of antiresorptive action have been extensively studied. Recent studies have suggested that BPs target bone-forming cells as well as bone-resorbing cells. We previously demonstrated that local application of a nitrogen-containing BP (N-BP), alendronate (ALN), for a short period of time increased bone tissue in a rat tooth replantation model. Here, we investigated cellular mechanisms of bone formation by ALN. Bone histomorphometry confirmed that bone formation was increased by local application of ALN. ALN increased proliferation of bone-forming cells residing on the bone surface, whereas it suppressed the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in vivo. Moreover, ALN treatment induced more alkaline phosphatase-positive and osteocalcin-positive cells on the bone surface than PBS treatment. In vitro studies revealed that pulse treatment with ALN promoted osteocalcin expression. To track the target cells of N-BPs, we applied fluorescence-labeled ALN (F-ALN) in vivo and in vitro. F-ALN was taken into bone-forming cells both in vivo and in vitro. This intracellular uptake was inhibited by endocytosis inhibitors. Furthermore, the endocytosis inhibitor dansylcadaverine (DC) suppressed ALN-stimulated osteoblastic differentiation in vitro and it suppressed the increase in alkaline phosphatase-positive bone-forming cells and subsequent bone formation in vivo. DC also blocked the inhibition of Rap1A prenylation by ALN in the osteoblastic cells. These data suggest that local application of ALN promotes bone formation by stimulating proliferation and differentiation of bone-forming cells as well as inhibiting osteoclast function. These effects may occur through endocytic incorporation of ALN and subsequent inhibition of protein prenylation.

Abstract Members of the triggering expressed on myeloid cells (Trem) receptor family fine-tune inflammatory responses. We previously identified one of these receptors, called Treml4, expressed mainly in the spleen, as well as at high levels by CD8α+ dendritic cells and macrophages. Like other Trem family members, Treml4 has an Ig-like extracellular domain and a short cytoplasmic tail that associates with the adaptor DAP12. To follow up on our initial results that Treml4-Fc fusion proteins bind necrotic cells, we generated a knockout mouse to assess the role of Treml4 in the uptake and presentation of dying cells in vivo. Loss of Treml4 expression did not impair uptake of dying cells by CD8α+ dendritic cells or cross-presentation of cell-associated Ag to CD8+ T cells, suggesting overlapping function between Treml4 and other receptors in vivo. To further investigate Treml4 function, we took advantage of a newly generated mAb against Treml4 and engineered its H chain to express three different Ags (i.e., OVA, HIV GAGp24, and the extracellular domain of the breast cancer protein HER2). OVA directed to Treml4 was efficiently presented to CD8+ and CD4+ T cells in vivo. Anti–Treml4-GAGp24 mAbs, given along with a maturation stimulus, induced Th1 Ag-specific responses that were not observed in Treml4 knockout mice. Also, HER2 targeting using anti-Treml4 mAbs elicited combined CD4+ and CD8+ T cell immunity, and both T cells participated in resistance to a transplantable tumor. Therefore, Treml4 participates in Ag presentation in vivo, and targeting Ags with anti-Treml4 Abs enhances immunization of otherwise naive mice.

We have established a novel mouse mutant, klotho (kl), by insertional mutation of a transgene and identified the structural gene. The mouse homozygous for the mutation exhibits multiple pathological conditions resembling age-related disorders in humans and can be regarded as a model of human premature aging syndromes. However, the pathophysiological role of Klotho protein has not been clarified.A replication-deficient adenoviral vector expressing the membrane form of the mouse klotho gene was constructed and we examined Klotho expression in vitro. The recombinant adenoviral vector was then administered intravenously into klotho mice at 4-5 weeks of age and its therapeutic potential was examined.Expression of Klotho protein was observed in the adenoviral vector-infected CHO cells. The klotho mice infused with the recombinant adenovirus showed a significant extension of life span and gain in body weight at 1 week after treatment. Macroscopic and histological analyses demonstrated the improvement of multiple pathological findings such as restoration from atrophy and cell formation and differentiation in the gonadal cells, immune tissues and subcutaneous fat.We showed that local expression of the klotho gene retards or partially improves pathological abnormalities in several organs of klotho mice after onset of the phenotypes. Therefore, the recombinant adenovirus vector will provide an important tool for investigating the molecular mechanism of the Klotho protein and give clues to understanding the individual disease mechanisms.

Tob (transducer of erbB2) is a member of antiproliferative family proteins and acts as a bone morphogenic protein inhibitor as well as a suppressor of proliferation in T cells, which have been implicated in postmenopausal bone loss. To determine the effect of Tob deficiency on estrogen deficiency-induced bone loss, we analyzed bone metabolism after ovariectomy or sham operation in Tob-deficient mice. Ovariectomy in WT mice decreased trabecular bone volume and bone mineral density (BMD) as expected. In Tob-deficient mice, ovariectomy reduced bone volume and BMD. However, even after ovariectomy, both trabecular bone volume and BMD levels in Tob-deficient bone were comparable to those in sham-operated WT bones. Bone formation parameters (mineral apposition rate and bone formation rate) in the ovariectomized Tob-deficient mice were significantly higher than those in the ovariectomized WT mice. In contrast, the ovariectomy-induced increase in the bone resorption parameters, osteoclast surface, and osteoclast number was similar between Tob-deficient mice and WT mice. Furthermore, in ex vivo nodule formation assay, ovariectomy-induced enhancement of nodule formation was significantly higher in the bone marrow cells from Tob-deficient mice than in the bone marrow cells from ovariectomized WT mice. Both Tob and estrogen signalings converge at bone morphogenic protein activation of alkaline phosphatase and GCCG-reporter gene expression in osteoblasts, revealing interaction between the two signals. These data indicate that Tob deficiency prevents ovariectomy-induced bone loss through the superenhancement of osteoblastic activities in bone and that this results in further augmentation in the bone formation rate and the mineral apposition rate after ovariectomy in vivo .

Intermittent PTH treatment increases cancellous bone mass in osteoporosis patients; however, it reveals diverse effects on cortical bone mass. Underlying molecular mechanisms for anabolic PTH actions are largely unknown. Because PTH regulates expression of osteopontin (OPN) in osteoblasts, OPN could be one of the targets of PTH in bone. Therefore, we examined the role of OPN in the PTH actions in bone. Intermittent PTH treatment neither altered whole long-bone bone mineral density nor changed cortical bone mass in wild-type 129 mice, although it enhanced cancellous bone volume as reported previously. In contrast, OPN deficiency induced PTH enhancement of whole-bone bone mineral density as well as cortical bone mass. Strikingly, although PTH suppressed periosteal bone formation rate (BFR) and mineral apposition rate (MAR) in cortical bone in wild type, OPN deficiency induced PTH activation of periosteal BFR and MAR. In cancellous bone, OPN deficiency further enhanced PTH increase in BFR and MAR. Analysis on the cellular bases for these phenomena indicated that OPN deficiency augmented PTH enhancement in the increase in mineralized nodule formation in vitro. OPN deficiency did not alter the levels of PTH enhancement of the excretion of deoxypyridinoline in urine, the osteoclast number in vivo, and tartrate-resistant acid phosphatase-positive cell development in vitro. These observations indicated that OPN deficiency specifically induces PTH activation of periosteal bone formation in the cortical bone envelope.

Osteopontin (OPN) is a major non-collagenous bone matrix protein implicated in the regulation of cell function. Although OPN is rich in the cementum of the tooth, the significance of OPN in this tissue is not understood. Tooth root resorption is the most frequent complication of orthodontic tooth movement (TM). The objective of this study was to examine the pathophysiological role of OPN in cementum of the tooth root. For this purpose, the upper right first molar (M1) in OPN-deficient and wild-type (WT) mice was subjected to mechanical force via 10 gf NiTi coil spring while the left side molar was kept intact to serve as an internal control. Micro-CT section and the level of tartrate resistant acid phosphatase (TRAP)-positive cells on the tooth root surface defined as odontoclasts were quantified at the end of the force application. In WT mice, force application to the tooth caused appearance of odontoclasts around the mesial surface of the tooth root resulting in tooth root resorption. In contrast, OPN deficiency significantly suppressed the force-induced increase in the number of odontoclasts and suppressed root resorption. This force application also induced increase in the number of TRAP-positive cells in the alveolar bone on the pressure side defined as osteoclasts, while the levels of the increase in osteoclastic cell number in such alveolar bone were similar between the OPN-deficient and WT mice. These observations indicate that OPN deficiency suppresses specifically tooth root resorption in case of experimental force application.

Molecular mechanisms underlying unloading-induced reduction of bone formation have not yet been fully understood. In vitro, Runx2 has been suggested to be involved in mechanical signaling in osteoblasts. However, the roles of Runx2 in vivo during the bone response to mechanical stimuli have not yet been known. The purpose of this paper was to examine the roles of Runx2 in unloading-induced bone loss in vivo. Tail suspension was conducted for 2 wk using 9- to 11-wk-old Runx2 heterozygous knockout mice (Runx2(+/-)) and wild-type (Wt) littermates. Bones were subjected to two-dimensional micro-x-ray computed tomography, bone histomorphometry and RT-PCR analyses. Loss of half Runx2 gene dosage-exacerbated unloading-induced bone loss in trabecular and cortical envelopes. Unloading-induced reduction in mineral apposition rate and bone formation rate in cortical bone as well as trabecular bone was exacerbated in Runx2(+/-) mice, compared with Wt mice. Bone resorption parameters were not significantly affected by unloading or Runx2(+/-) genotype. Basal Runx2 and osterix mRNA levels in bone were reduced by 50% in Wt, whereas unloading in Runx2(+/-) mice did not further alter Runx2 and osterix mRNA levels. In contrast, osteocalcin mRNA levels were reduced by unloading, regardless of Runx2 gene dosage. These data demonstrated that full Runx2 gene dosage is required for maintaining normal function of osteoblasts in mechanical unloading or nonphysiological condition. Finally, we propose Runx2 as a critical target gene in unloading to alter osteoblastic activity and bone formation in vivo.

Abstract Objective To investigate the molecular mechanisms underlying particle‐induced osteolysis, we focused on osteopontin (OPN), a cytokine and cell‐attachment protein that is associated with macrophage chemoattractant and osteoclast activation. Methods We compared OPN protein levels in human periprosthetic osteolysis tissues with those in osteoarthritis (OA) synovial tissues. To investigate the functions of OPN during particle‐induced osteolysis in vivo, titanium particles were implanted onto the calvaria of OPN‐deficient mice and their wild‐type (WT) littermates. Mice were killed on day 10 and evaluated immunohistologically. The effects of OPN deficiency on the secretion of inflammatory cytokines were examined using cultured bone marrow–derived macrophages (BMMs). BMMs from OPN‐deficient and WT mice were cultured with titanium particles for 12 hours, and the concentrations of inflammatory cytokines in the conditioned media were measured by enzyme‐linked immunosorbent assay. Results Expression of OPN protein was enhanced in human periprosthetic osteolysis tissues as compared with OA synovial tissues. In the particle‐induced model of osteolysis of the calvaria, bone resorption was significantly suppressed by OPN deficiency via inhibition of osteoclastogenesis, whereas an inflammatory reaction was observed regardless of the genotype. Results of immunostaining indicated that OPN protein was highly expressed in the membrane and bone surface at the area of bone resorption in WT mice. When BMMs were exposed to titanium particles, the concentration of proinflammatory cytokines, such as tumor necrosis factor α, interleukin‐1α (IL‐1α), IL‐1β, and IL‐6, as well as chemotactic factors, such as monocyte chemoattractant protein 1 and macrophage inflammatory protein 1α, in the conditioned medium were significantly reduced by OPN deficiency. Whereas phagocytic activity of BMMs was not attenuated by OPN deficiency, phagocytosis‐mediated NF‐κB activation was impaired in OPN‐deficient BMMs. These data indicated that OPN was implicated in the development of particle‐induced osteolysis via the orchestration of pro‐/antiinflammatory cytokines secreted from macrophages. Conclusion OPN plays critical roles in wear debris–induced osteolysis, suggesting that OPN is a candidate therapeutic target for periprosthetic osteolysis.

In this letter, we developed, for the first time, 10.3-Gb/s burst-mode 3R receiver incorporating a full automatic gain control optical receiver and 82.5-GS/s over-sampling clock and data recovery (CDR) for 10G-EPON. Burst-mode preamplifier, limiting amplifier, and CDR were newly fabricated on 0.13-¿m SiGe bipolar complementary metal-oxide-semiconductor process to completely meet the IEEE802.3av 10G-EPON standards. The 10.3-Gb/s burst-mode 3R receiver successfully achieves the burst receiver sensitivity of -30.1 dBm (at bit error rate (BER) =1 ×10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-3</sup> ) and the dynamic rage more than 24.1 dB (at BER=1 ×10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-3</sup> ) with the assistance of FEC RS(255, 223).

Osteoclastogenesis is under the control of posttranscriptional and transcriptional events. However, posttranscriptional regulation of osteoclastogenesis is incompletely understood. CNOT3 is a component of the CCR4 family that regulates mRNA stability, but its function in bone is not known. Here, we show that Cnot3 deficiency by deletion of a single allele induces osteoporosis. Cnot3 deficiency causes an enhancement in bone resorption in association with an elevation in bone formation, resulting in high-turnover type bone loss. At the cellular level, Cnot3 deficiency enhances receptor activator of NF-κB ligand (RANKL) effects on osteoclastogenesis in a cell-autonomous manner. Conversely, Cnot3 deficiency does not affect osteoblasts directly. Cnot3 deficiency does not alter RANKL expression but enhances receptor activator of NF-κB (RANK) mRNA expression in bone in vivo. Cnot3 deficiency promotes RANK mRNA stability about twofold in bone marrow cells of mice. Cnot3 knockdown also increases RANK mRNA expression in the precursor cell line for osteoclasts. Anti-CNOT3 antibody immunoprecipitates RANK mRNA. Cnot3 deficiency stabilizes luciferase reporter expression linked to the 3'-UTR fragment of RANK mRNA. In contrast, Cnot3 overexpression destabilizes the luciferase reporter linked to RANK 3'-UTR. In aged mice that exhibit severe osteoporosis, Cnot3 expression levels in bone are reduced about threefold in vivo. Surprisingly, Cnot3 deficiency in these aged mice further exacerbates osteoporosis, which also occurs via enhancement of osteoclastic activity. Our results reveal that CNOT3 is a critical regulator of bone mass acting on bone resorption through posttranscriptional down-regulation of RANK mRNA stability, at least in part, even in aging-induced osteoporosis.

Mothers againstdecapentaplegic-related proteins (Smads) are essential intracellular components for the signal transduction of transforming growth factor-β (TGF-β) family members. Smad1 mediates bone morphogenetic protein (BMP) signals, whereas Smad2 functions downstream of TGF-β. TGF-β is expressed in osteoblastic cells and acts as an autocrine and/or paracrine factor in regulation of osteoblastic functions. In this study, we examined the levels and functions of Smad2 in osteoblastic cells. Smad2 mRNA expression was hardly detectable by Northern blot analysis in an osteoblast-like cell line, ROS17/2.8, as well as in primary rat calvaria (PRC) cells. Overexpression of Smad2 gene enhanced endogenous Smad4 gene expression in both ROS17/2.8 and PRC cells, while Smad3 levels were not altered. Smad2 overexpression suppressed osteocalcin mRNA expression in ROS17/2.8 cells. Furthermore, Smad2 overexpression also suppressed transcriptional activity of the 1-kilobase pair osteocalcin gene promoter, which was linked to chloramphenicol acetyltransferase reporter gene in both ROS and PRC cells. Since core binding factor A1 (CBFA1) is involved in osteocalcin gene expression, we further examined CBFA1 expression in the Smad2-overexpressing ROS17/2.8 and PRC cells. The levels of CBFA1 mRNA were suppressed by the overexpression of Smad2 by about 50% in both ROS17/2.8 and PRC cells. TGF-β treatment enhanced Smad4 expression in PRC cells, and this TGF-β effect was blocked by the cotreatment with BMP, indicating that TGF-β signaling pathway is interfered by BMP. These data indicate that Smad2 regulates Smad4 specifically and that CBFA1 gene is one of the downstream targets of Smad2.

Bone diseases such as osteoporosis and osteoarthritis are regarded as age-associated diseases, and occur in a significantly increasing number of patients, but the underlying mechanisms of these age-associated bone diseases are not yet clear. We have established a transgenic mouse line by an insertion mutation. These mice exhibit many features related to precocious aging. Homozygote mutant mice, which lack expression of the newly identified targeted gene,klotho (kl), exhibit atherosclerosis, emphysema, hypogonadism and calcification of soft tissues, and die within 3-4 months. We describe here the radiological and histological characteristics of the skeletal abnormalities in the bones of the mice with a mutation in the kl gene locus. In heterozygous mice (+/kl), the skeletal patterns and structures remain normal and most features are similar to those in the wild-type, whereas histological examinations of homozygous mice (kl/kl) show abnormal elongation of the trabecular bone(s) in the epiphyses of long bones. As with their long bones, on radiographic examination the mid parts of the vertebral bones of these mice show less radiopacity compared with the wild-type, again resembling human vertebrae of osteoporotic patients. The elongation of the trabecular bones results in high radiopacity on both ends of each of the vertebrae, and in the epiphyses of the long bones. Cancellous bone volume in the epiphyses of the homozygote mice is three times that of the wild-type mice. The kl/kl mice are smaller than the wild-type litter mates and hence the size of their long bones is less than that of the wild-type litter mates. These observations, and the osteopenia in the vertebrae and long bones in these mice, suggest the presence of abnormality in bone metabolism, the elongation of the trabecular bone apparently resulting from the relatively low levels of bone resorption. Therefore, thekl/kl mutant mice could serve as an interesting tool to study the effects of the lack of the product of the new gene,klotho, on bone metabolism.

Osteoclasts regulate homeostasis of bone development. A defect in osteoclast development results in osteopetrosis. Recently, the involvement of several molecules in osteoclast development has been found. Osteoclast-associated receptor (OSCAR) is one of such molecules critical for osteoclast differentiation. However, it remains unclear how OSCAR transduces signals for osteoclast differentiation. Here, we found that the FcRgamma chain, a signal transducing adaptor molecule for Fc receptors, is associated with OSCAR and is involved in the cell surface expression of OSCAR. Furthermore, FcRgamma is required for signal transduction by OSCAR. These findings suggest that the FcRgamma-mediated signal transduction by OSCAR is involved in osteoclast differentiation.

Mice homozygous for klotho gene deletion are well established aging models as they mimic certain aspects of human senescence e.g. osteoporosis. Induced senescence may affect cellular functions and alter the histological properties of the extracellular matrices. The present study examined the histological and ultrastructural features of osteocytes and the surrounding bone matrix in klotho-deficient mice. As expected, osteoblasts showed a flattened shape with a weak immunoreactivity for alkaline phosphatase, and the bone matrix contained many empty osteocytic lacunae. The walls of both normal and empty lacunae were intensely immunopositive for osteopontin and dentin matrix protein-1, but featured an inconsistent immunoreactivity for osteocalcin and type I collagen. Not surprisingly, TUNEL-positivity, indicative of apoptosis, was found in many osteoblasts, osteocytes, and bone marrow cells of the klotho-deficient mice. In transmission electron microscopy, an amorphous matrix containing non-collagenous organic materials was recognizable around osteoblasts and in the osteocytic lacunae. Some osteoblasts on the bone surface featured these amorphous materials in vacuoles associated with their trans-Golgi network, indicating that, under klotho-deficient conditions, they synthesize and secrete the non-collagenous structures. Some osteocytes displayed pyknosis or degenerative traits. Thus, our findings provide histological evidence that klotho gene deletion influences the spatial distribution of osteocytes and the synthesis of bone matrix proteins in addition to the accelerated aging of bone cells.

Osteoporosis due to unloading-induced bone loss is a critical issue in the modern aging society. Although the mechanisms underlying this phenomenon are largely unknown, osteopontin (OPN) is one of the critical mediators required for unloading-induced bone loss [M. Ishijima, S.R. Rittling, T. Yamashita, K. Tsuji, H. Kurosawa, A. Nifuji, D.T. Denhardt, and M. Noda, Enhancement of osteoclastic bone resorption and suppression of osteoblastic bone formation in response to reduced mechanical stress do not occur in the absence of osteopontin, J Exp Med, 193 (2001) 399-404]. To clarify the molecular bases for OPN actions, we carried out microarray analyses on the genes expressed in the femoral bone marrow cells in wild type and OPN−/− mice. The removal of the mechanical load induced bone loss in wild type, but not in OPN−/− mice, as previously reported. Expression analysis of 9586 cDNAs on a microarray system revealed that OPN deficiency blocked tail-suspension-induced expression of ten genes (group A). This observation was confirmed based on semi-quantitative RT-PCR analyses. On the other hand, expression of four genes (group B) was not altered by tail suspension in wild type but was enhanced in OPN-deficient mice. NF-κB p105 subunit gene (Nfkb1) was found in group A and Bax in group B. p53 gene expression was upregulated by tail suspension in wild type mice, but it was no longer observed in OPN−/− mice. These data indicate that OPN acts to mediate mechanical stress signaling upstream to the genes encoding apoptosis-related molecules, and its action is associated with alteration of the genes.

The message of Id (for "inhibitor of DNA binding") was expressed in an osteoblastic cell line, MC3T3E1, when the cells were in early cultures, while undetectable after the cells became confluent. The abundance of Id message in MC3T3E1 cells in early cultures was increased when the cells were treated with dexamethasone. This effect was time and dose dependent in a range between 10(-11)M and 10(-7)M. Id message expression was not enhanced by TGF-beta, 1,25-dihydroxyvitamin D3, retinoic acid, interleukin 1-alpha, interleukin 6, tumor necrosis factor-alpha or parathyroid hormone. These observations indicate for the first time the presence of HLH protein family member, Id, in osteoblast-like cells and its regulation by dexamethasone.

Studies of bone cells in culture have raised two salient questions: are the findings representative of the in vivo situation and can the conflicting data from different cell models be reconciled? Review of the literature indicates that all osteoblastic cells, defined by their origin or by their ability to produce mineralized matrix, have a few common properties: production of type I collagen; increased alkaline phosphatase activity; and parathyroid hormone-stimulated adenylate cyclase. Other features, such as osteocalcin and prostaglandin E production and the response to prostaglandin E, are selectively expressed by certain cell types. Pilot studies on mRNA levels of ‘bone proteins’ in developing calvaria suggest that such differences may reflect stages in osteoblastic differentiation. Immortalization of calvaria-derived cells using a SV40 large T antigen vector, which may freeze the cells in their particular state of differentiation (as proposed for leukaemia cells), yields phenotypes consistent with that hypothesis. Immortal cell lines may thus help to characterize osteoblastic differentiation. The diversity of osteoblast responses in culture to hormones and growth factors could be due to these phenotype differences but could also represent a subspecialization of differentiated cells. In addition, in the organism regulatory agents act in concert on a heterogeneous interactive cell population. Nonetheless cell cultures can be useful in screening for and predicting in vivo responses, as was shown by the 1,25-(OH)2D3 stimulation of osteocalcin, and for studying the molecular mechanisms of regulatory effects. Cell lines are also convenient for the production of specific proteins and cDNA libraries, and for the expression of specific genes.

Regulation of bone remodeling determines the levels of bone mass and its imbalance causes major skeletal diseases such as osteoporosis. A zinc finger protein, Schnurri-2 (SHN-2), was recently demonstrated to regulate bone morphogenetic protein-dependent adipogenesis and lymphogenesis. However, the role of SHN-2 in bone is not known. Here, we investigated the effects of Shn-2 deficiency on bone metabolism and cell function in Shn-2-null mice. Lack of SHN-2 expression reduced bone remodeling by suppressing both osteoblastic bone formation and osteoclastic bone resorption activities in vivo. Shn-2 deficiency suppressed osterix and osteocalcin expression as well as in vitro mineralization. Conversely, Shn-2 overexpression enhanced osteocalcin promoter activity and bone morphogenetic protein-dependent osteoblastic differentiation. Shn-2 deficiency suppressed Nfatc1 and c-fos expression leading to reduction of tartrate-resistant acid phosphatase-positive cell development in vivo as well as in the cultures of bone marrow cells. These studies demonstrate that SHN-2 regulates the activities of critical transcription factors required for normal bone remodeling. Regulation of bone remodeling determines the levels of bone mass and its imbalance causes major skeletal diseases such as osteoporosis. A zinc finger protein, Schnurri-2 (SHN-2), was recently demonstrated to regulate bone morphogenetic protein-dependent adipogenesis and lymphogenesis. However, the role of SHN-2 in bone is not known. Here, we investigated the effects of Shn-2 deficiency on bone metabolism and cell function in Shn-2-null mice. Lack of SHN-2 expression reduced bone remodeling by suppressing both osteoblastic bone formation and osteoclastic bone resorption activities in vivo. Shn-2 deficiency suppressed osterix and osteocalcin expression as well as in vitro mineralization. Conversely, Shn-2 overexpression enhanced osteocalcin promoter activity and bone morphogenetic protein-dependent osteoblastic differentiation. Shn-2 deficiency suppressed Nfatc1 and c-fos expression leading to reduction of tartrate-resistant acid phosphatase-positive cell development in vivo as well as in the cultures of bone marrow cells. These studies demonstrate that SHN-2 regulates the activities of critical transcription factors required for normal bone remodeling. Adult bone is remodeled to meet continuously changing structural and metabolic requirements. To maintain bone mass, resorption by osteoclasts must be balanced by the formation of bone by osteoblasts. In bone diseases such as osteoporosis the balance between formation and resorption is lost, leading to reduced bone mass, which increases the risk of fractures (1Raisz L.G. J. Clin. Investig. 2005; 115: 3318-3325Crossref PubMed Scopus (1290) Google Scholar, 2Martin T.J. Sims N.A. Trends Mol. Med. 2005; 11: 76-81Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar). Identification of molecules involved in adult bone mass determination is critical to better understand the function of such molecules and may render clues for treatment of bone diseases such as osteoporosis. Several molecules have been reported to modulate adult bone mass in mouse models. Genetic analyses of high bone mass trait in the C3H strain and Balb strain identified a mutation in ALOX15 that may be responsible for increase in adult bone mass (3Klein R.F. Allard J. Avnur Z. Nikolcheva T. Rotstein D. Carlos A.S. Shea M. Waters R.V. Belknap J.K. Peltz G. Orwoll E.S. Science. 2004; 303: 229-232Crossref PubMed Scopus (251) Google Scholar). Activating transcription factor 4 positively determines adult bone mass (4Yang X. Matsuda K. Bialek P. Jacquot S. Masuoka H.C. Schinke T. Li L. 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Loss of half the Runt-related transcription factor-2 (Runx2) 4The abbreviations used are: Runx2, Runt-related transcription factor-2; Shn, Schnurri; NFAT, nuclear factor of activated T cell; BMD, bone mineral density; BMP, bone morphogenetic protein; ALP, alkaline phosphatase; BFR, bone formation rate; M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of NF-κB ligand; MS/BS, mineralizing surface/bone surface; OPG, osteoprotegerin; WT, wild type; CT, computed tomography. gene dosage causes failure in the recovery of trabecular bone mass after bone marrow ablation specifically in aged adult mice (7Tsuji K. Komori T. Noda M. J. Bone Miner. Res. 2004; 19: 1481-1489Crossref PubMed Scopus (32) Google Scholar). In human, suppression of Wnt inhibitor signaling molecules such as Dickkopf increases adult bone mass (8Morvan F. Boulukos K. Clement-Lacroix P. Roman S. Suc-Royer I. Vayssiere B. Ammann P. Martin P. Pinho S. Pognonec P. Mollat P. Niehrs C. Baron R. Rawadi G. J. Bone Miner. Res. 2006; 21: 934-945Crossref PubMed Scopus (474) Google Scholar), and loss of function mutation in sclerostin enhances adult bone volume in patients with sclerostosis (9Gardner J.C. van Bezooijen R.L. Mervis B. Hamdy N.A. Lowik C.W. Hamersma H. Beighton P. Papapoulos S.E. J. Clin. Endocrinol. Metab. 2005; 90: 6392-6395Crossref PubMed Scopus (145) Google Scholar). These molecules mostly act on osteoblasts to modulate only one of the two arms of bone remodeling. Drosophila Schnurri (Shn) and mammalian Shn homologues Shn-1, Shn-2, and Shn-3 are zinc finger-type molecules implicated in cell regulation of these species (10Grieder N.C. Nellen D. Burke R. Basler K. Affolter M. Cell. 1995; 81: 791-800Abstract Full Text PDF PubMed Scopus (174) Google Scholar, 11Arora K. Dai H. Kazuko S.G. Jamal J. O'Connor M.B. Letsou A. Warrior R. Cell. 1995; 81: 781-790Abstract Full Text PDF PubMed Scopus (194) Google Scholar, 12Staehling-Hampton K. Laughon A.S. Hoffmann F.M. 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SHN-2 was recently identified to regulate adipogenesis and lymphogenesis (20Jin W. Takagi T. Kanesashi S.N. Kurahashi T. Nomura T. Harada J. Ishii S. Dev. Cell. 2006; 10: 461-471Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 21Takagi T. Harada J. Ishii S. Nat. Immunol. 2001; 2: 1048-1053Crossref PubMed Scopus (66) Google Scholar, 22Kimura M.Y. Hosokawa H. Yamashita M. Hasegawa A. Iwamura C. Watarai H. Taniguchi M. Takagi T. Ishii S. Nakayama T. J. Exp. Med. 2005; 201: 397-408Crossref PubMed Scopus (54) Google Scholar). SHN-2 binds to Smad proteins to up-regulate peroxisome proliferator-activated receptor γ gene expression (20Jin W. Takagi T. Kanesashi S.N. Kurahashi T. Nomura T. Harada J. Ishii S. Dev. Cell. 2006; 10: 461-471Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), and it is also required for positive selection of thymocytes (21Takagi T. Harada J. Ishii S. Nat. Immunol. 2001; 2: 1048-1053Crossref PubMed Scopus (66) Google Scholar). SHN-2 competitively inhibits NF-κB binding and leads to the suppression of type 2 helper T cell differentiation (22Kimura M.Y. Hosokawa H. Yamashita M. Hasegawa A. Iwamura C. Watarai H. Taniguchi M. Takagi T. Ishii S. Nakayama T. J. Exp. Med. 2005; 201: 397-408Crossref PubMed Scopus (54) Google Scholar). Recently, SHN-3, another member of the Schnurri family, was reported to suppress adult bone mass levels through RUNX2 protein degradation (23Jones D.C. Wein M.N. Oukka M. Hofstaetter J.G. Glimcher M.J. Glimcher L.H. Science. 2006; 312: 1223-1227Crossref PubMed Scopus (189) Google Scholar). However, SHN-3 shares only limited homology with SHN-2 (17Wu L.C. Gene Expr. 2002; 10: 137-152Crossref PubMed Scopus (60) Google Scholar). Here, we report on Shn-2 deficiency-suppressed bone formation as well as bone resorption. Shn-2 deficiency reduced the expression levels of osteoblastic transcription factor osterix as well as osteoclastic transcriptional factors, nuclear factor of activated T cells (Nfat) c1 and c-fos. Shn-2 overexpression enhanced promoter activity of osteocalcin, which is downstream of master regulators in osteoblasts. Furthermore, Shn-2 overexpression activated bone morphogenetic protein (BMP)-induced osteoblastic differentiation. Thus, Shn-2 is a novel regulator of adult bone remodeling. Animals—Shn-2 knock-out mice were described previously (21Takagi T. Harada J. Ishii S. Nat. Immunol. 2001; 2: 1048-1053Crossref PubMed Scopus (66) Google Scholar). Male BALB/c background 12-week-old Shn-2 knock-out mice and wild type littermates (n = 7) were used for the studies of bone mineral density (BMD), two-dimensional micro-computed tomography (μCT), bone histomorphometric analysis, bone marrow cell cultures, and measurement of BMP-induced alkaline phosphatase (ALP) activity. For experiments on the evaluations of time course study of two-dimensional micro-CT, we used 1-, 4-, and 8-week-old Shn-2-deficient mice and wild type littermates (n = 3). All the animal experiments were approved by the Animal Welfare Committee of Tokyo Medical and Dental University. Measurement of BMD and μCT Analysis of Bone—BMD was measured based on dual-energy x-ray absorptiometry (DEXA) as described previously (24Kitahara K. Ishijima M. Rittling S.R. Tsuji K. Kurosawa H. Nifuji A. Denhardt D.T. Noda M. Endocrinology. 2003; 144: 2132-2140Crossref PubMed Scopus (52) Google Scholar). Briefly, BMD (g/cm2) of the femora and tibia were subjected to dual x-ray absorptiometry using a device specifically designed for small animals (PIXI; GE Luner, Madison, WI). Two-dimensional μCT data were collected using the Musashi system (Nittetsu-ELEX, Osaka, Japan). For the evaluation of cortical bone volume, total cortical bone area as well as cortical thickness in the midshaft was measured. Calvarial thickness and bone area were measured in the coronal section of parietal bone. For cancellous bone, bone volume/tissue volume values were evaluated using μCT slices at the sagittal section of femora as well as coronal section of spine (L4) as described previously (24Kitahara K. Ishijima M. Rittling S.R. Tsuji K. Kurosawa H. Nifuji A. Denhardt D.T. Noda M. Endocrinology. 2003; 144: 2132-2140Crossref PubMed Scopus (52) Google Scholar). Histomorphometric Analysis and Skeletal Preparation—Bone formation rate (BFR) in cortical and cancellous regions in femora was measured in the undecalcified section as previously described (24Kitahara K. Ishijima M. Rittling S.R. Tsuji K. Kurosawa H. Nifuji A. Denhardt D.T. Noda M. Endocrinology. 2003; 144: 2132-2140Crossref PubMed Scopus (52) Google Scholar). In the decalcified section, tartrate-resistant acid phosphatase-positive multinucleated cells were counted as osteoclasts to evaluate osteoclast number/bone surface and osteoclast surface/bone surface. Skeletal preparation was performed as described previously (25Hogan B. Beddington R. Costantini F. Lacy E. Manipulating the Mouse Embryo. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 379-381Google Scholar). Briefly, day 3 mice skins were removed and fixed in 95% EtOH overnight and then stained Alcian blue (15 mg/100 ml in 80% EtOH) followed by alizarin red (5 mg/100 ml in 2% KOH). RNA Extraction, cDNA Synthesis, and PCR—RNA was extracted from mouse femur, MC3T3E1 cells, and RAW264.7 cells based on the acid guanidinium thiocyanate-phenol-chloroform method. First-strand cDNA was synthesized using 1 μg of the total RNA and Molony murine leukemia virus reverse transcriptase. Amplification was performed as described previously (26Pfaffl M.W. Nucleic Acids Res. 2001; 29: 2002-2007Crossref Scopus (25832) Google Scholar). Quantitative real-time PCR was performed using iQ SYBR Green Supermix (Bio-Rad). The quantification was done as described previously (27Salingcarnboriboon R. Yoshitake H. Tsuji K. Obinata M. Amagasa T. Nifuji A. Noda M. Exp. Cell Res. 2003; 287: 289-300Crossref PubMed Scopus (224) Google Scholar). Expression values were normalized to glyceraldehyde-3-phosphate dehydrogenase. The primers for real-time reverse transcription PCR were as follows: Shn-2, forward, 5′-GCC GCC CAC CCT GAC TTA C-3′, reverse, 5′-CTT CTC ATC CAC ACT GCT CTT GC-3′; Col1a1, forward, 5′-CTG ACT GGA AGA GCG GAG AG-3′, reverse, 5′-GCA CAG ACG GCT GAG TAG G-3′; Runx2, forward, 5′-TGG CTT GGG TTT CAG GTT AGG G-3′, reverse, 5′-TCG GTT TCT TAG GGT CTT GGA GTG-3′; osterix, forward, 5′-AGC GAC CAC TTG AGC AAA CAT C-3′, reverse 5′-CGG CTG ATT GGC TTC TTC TTC C-3′; Alp, forward, 5′-GCT ATC TGC CTT GCC TGT ATC TG-3′, reverse, 5′-AGG TGC TTT GGG AAT CTG TGC-3′; Rankl, forward, 5′-CCT GAG GCC CAG CCA TTT-3′, reverse, 5′-CTT GGC CCA GCC TCG AT-3′; osteoprotegerin (Opg), forward, 5′-TAC CTG GAG ATC GAA TTC TGC TT-3, reverse, 5′-CCA TCT GGA CAT TTT TTG CAA A-3′;c-fos, forward, 5′-CCG TGT CAG GAG GCA GAG C-3′, reverse 5′-GCA GCC ATC TTA TTC CGT TCC C-3′; Nfatc1, forward, 5′-AGC CCA AGT CTC ACC ACA GG-3′, reverse, 5′-CAG CCG TCC CAA TGA ACA GC-3′; and glyceraldehyde-3-phosphate dehydrogenase, forward, 5′-AGA AGG TGG TGA AGC AGG CAT C-3′, reverse, 5′-CGA AGG TGG AAG AGT GGG AGT TG-3′. Cells—Primary osteoblasts were obtained from the outgrowth cultures of bone fragments of calvariae taken from wild type and Shn-2-deficient mice. These cells were used for measurement of BMP-induced ALP activity. Bone marrow cells were flushed out after the proximal epiphyseal ends were removed from right tibiae of wild type and Shn-2-deficient mice and used for mineralized nodule formation assay and osteoclast development. Osteoclas-togenesis was examined both in bone marrow cells and splenocytes obtained from wild type and Shn-2-deficient mice. The MC3T3E1 osteoblastic cell line was obtained from RIKEN (Saitama, Japan). These cells were used for experiments of real-time PCR, overexpression of Shn-2, and luciferase assays. The RAW264.7 cell line was purchased from the Dainippon Pharmaceutical Co., Ltd. and maintained with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. These cells were used for experiment of RANKL (100 ng/ml)-induced Shn-2 mRNA expression. Bone Marrow Cultures—Marrow cells were plated in 24-well plates (2.0 cm2/well) at a density of 2 × 106 cells/well. Tartrateresistant acid phosphatase-positive osteoclast-like multinucleated cells were formed in α-minimal essential medium supplemented with 10% fetal bovine serum, 100 μg/ml antibiotics-antimycotics mixture, 10 nm 1.25(OH)2 vitamin D3, and 100 nm dexamethasone. The medium was changed every 3-4 days. For mineralized nodule formation, bone marrow cells were cultured in a standard growth medium containing 50 μg/ml ascorbic acid and 10 mm sodium β-glycerophosphate. The medium was changed every 3-4 days. The cultures were stained in a saturated solution of alizarin red on day 21. The area of mineralized nodules/total dish surface was measured by using the Luzex-F automated image analyzer (Nireco). Measurement of ALP Activity—ALP activity measurement was made in primary osteoblasts by using p-nitrophenyl phosphate as substrate (28Maeda Y. Tsuji K. Nifuji A. Noda M. J. Cell Biochem. 2004; 93: 337-344Crossref PubMed Scopus (57) Google Scholar). These cells were maintained in α-minimal essential medium. Cell differentiation was induced by treatment with 100 ng/ml rhBMP-2 in a standard growth medium. Transfections and Reporter Assays—MC3T3E1 cells (1 × 105 cells/well in six-well tissue culture plates) were transfected with various combinations of the following plasmids using the FuGENE 6 transfection reagent: reporter constructs (12×GCCG/luciferase, 1.1 kb osteocalcin-gene-2/luciferase), Shn-2 expression vector, and empty vector as a control. After 24-72 h of incubation, cell extracts were prepared and used to measure luciferase activity based on the Dual Luciferase™ reporter assay system (Promega), and the values were normalized against the efficiency of transfection using the same system. Both firefly and Renilla luciferase activities were measured by AutoLumat (LB953; EG & G Brethhold). Osteoclastogenesis in Stromal Cell-free Condition—Lymphocytes and monocytes were obtained from spleen by using Lympholite-M (Cedarlane) according to the manufacturer's protocol. These cells were cultured for 12 h with 5 ng/ml macrophage colony-stimulating factor (M-CSF), and non-adherent cells were further cultured in the presence of 30 ng/ml M-CSF for 3 days. RANKL (100 ng/ml) was added with 30 ng/ml M-CSF, and tartrate-resistant acid phosphatase-positive multinucleated cells were counted as osteoclasts. Statistical Evaluations—The results were presented as mean values ± S.D. Statistical analysis was performed by Student's t test to evaluate differences between the two groups. Analysis of variance was performed when the examined experimental groups exceeded three groups. Tukey's multiple comparison test was applied as post hoc test. p values <0.05 were considered to be statistically significant. Cortical bone is the major component of adult bone mass. Shn-2 deficiency reduced cortical bone mass with respect to the levels of cortical thickness and cortical bone area in femora (Fig. 1, A-C) and in calvarial bone (Fig. 1, D and F). Because the size and length of femur of Shn-2-deficient mice were smaller than wild type (21Takagi T. Harada J. Ishii S. Nat. Immunol. 2001; 2: 1048-1053Crossref PubMed Scopus (66) Google Scholar), we also evaluated the values of cortical thickness and cortical area normalized against those of femur length and found that Shn-2 deficiency reduced cortical bone parameters after normalization as well (data not shown). This observation was further supported by reduction in BMD in Shn-2-deficient mice (Fig. 1, G and H). Thus, Shn-2 deficiency reduced total bone mass. With respect to dynamic parameters, Shn-2 deficiency suppressed the levels of BFR as well as mineral apposition rate (MAR) and mineralizing surface (MS/BS) in the periosteal regions of cortical bone (Fig. 1I). These observations indicated that SHN-2 is required for the activities of individual osteoblastic cells (mineral apposition rate) as well as the mineralizing surface (MS/BS) to maintain BFR. We also counted osteoblast number as defined by Parfitt et al. (29Parfitt A.M. Drezner M.K. Glorieux F.H. Kanis J.A. Malluche H. Meunier P.J. Ott S.M. Recker R.R. J. Bone Miner. Res. 1987; 2: 595-610Crossref PubMed Scopus (4914) Google Scholar) because MS/BS represent osteoblast surface but do not represent the number of osteoblast. Although Shn-2 deficiency tended to suppress the number of osteoblast, it was not statistically significant (data not shown). Molecular bases for such Shn-2 deficiency phenotype in bone mass were further examined based on mRNA expression levels in whole bone in vivo. Messenger RNA expression levels of osteoblastic phenotype-related genes such as osterix and osteocalcin were suppressed in the bones of the Shn-2-deficient mice (Fig. 1, J and K). Expression of other maker genes, including Col1a1, Runx2, and Alp, tended to be decreased in Shn-2-deficient mice though they were not statistically significant. Therefore, Shn-2 deficiency suppressed bone formation activity in vivo at the messenger RNA levels. We examined the bones of mice at younger stages, including the time points of 1, 4, and 8 weeks after birth. Although overall contour of the cortical bone in the cross-section of the midshaft was similar (Fig. 2A), cortical bone volume (Fig. 2B) and cortical thickness (Fig. 2C) were less in Shn-2-deficient mice compared with wild type 1 week after birth; this reduction was similarly observed in 4-, 8-, and 12-week-old mice (Fig. 2, A-C). In younger mice such as 3 days old, examination of skeletal preparation did not reveal major defects (Fig. 2D). Thus, SHN-2 is required for the regulation of osteoblasts. Although Shn-2 deficiency resulted in overall reduction in the levels of parameters in bone mass as represented by the data in whole limb bone BMD (Fig. 1, G and H), there was a regionally and temporally limited mild increase in bone volume/tissue volume in the cancellous bone envelope in the metaphysis of Shn-2-deficient mice (Fig. 3A). This phenotype was limited to 8- and 12-week-old mice and was not observed in 4-week-old mice (Fig. 3, A and B). These data indicated that Shn-2 deficiency reduced bone mass (including cortical and cancellous) after mice were 1 week old. However, they were associated with regional (limited to metaphyseal region) cancellous bone increase at the adult stage (8 and 12 weeks). To further elucidate this point, in addition to cortical bone we conducted analyses on the dynamic bone formation parameters in cancellous bone envelope. The data revealed that Shn-2 deficiency suppressed the levels of cancellous MS/BS, mineral apposition rate, and BFR (Fig. 3C), consistent with the observation on these parameters in cortical bone (Fig. 1I). Although Shn-2 deficiency suppressed bone formation activities in cancellous bone, trabecular bone volume was increased in the metaphysis of long bone. To see whether this phenotype was restricted to long bone or not, we also evaluated trabecular bone volume in spine. Trabecular bone volume at the lumbar spine also increased in 12-week-old Shn-2-deficient mice compared with wild type (Fig. 3, A and D). Previously reported animal models, such as mice deficient in SHN-3, TOB, and CIZ, only revealed osteoblastic phenotype, but not osteoclastic phenotype (5Yoshida Y. Tanaka S. Umemori H. Minowa O. Usui M. Ikematsu N. Hosoda E. Imamura T. Kuno J. Yamashita T. Miyazono K. Noda M. Noda T. Yamamoto T. Cell. 2000; 103: 1085-1097Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 23Jones D.C. Wein M.N. Oukka M. Hofstaetter J.G. Glimcher M.J. Glimcher L.H. Science. 2006; 312: 1223-1227Crossref PubMed Scopus (189) Google Scholar, 30Morinobu M. Nakamoto T. Hino K. Tsuji K. Shen Z.J. Nakashima K. Nifuji A. Yamamoto H. Hirai H. Noda M. J. Exp. Med. 2005; 201: 961-970Crossref PubMed Scopus (62) Google Scholar). In contrast to these previous models, Shn-2 mutation “suppressed” bone resorption parameters, including osteoclast number/BS and osteoclast surface/BS levels (Fig. 3, E and G). Thus, Shn-2 deficiency has a unique feature in that it causes reduction in both osteoblastic and osteoclastic activities in vivo to lead to low turnover state in bone. To better understand how SHN-2 acts in cells, we examined Shn-2 expression in osteoblastic cells. We tested whether SHN-2 is functionally involved in the differentiation of osteoblastic cells using bone marrow cells of the wild type and Shn-2-deficient mice. Shn-2 deficiency suppressed the levels of mineralized nodule formation in bone marrow cells cultured in the presence of β-glycero-phosphate and ascorbic acid (Fig. 4, A and B). During the differentiation of osteoblasts in these bone marrow cell cultures, BMP signaling was reported to play a critical role (31Lecanda F. Avioli L.V. Cheng S.L. J. Cell Biochem. 1997; 67: 386-396Crossref PubMed Scopus (248) Google Scholar, 32Takeuchi Y. Watanabe S. Ishii G. Takeda S. Nakayama K. Fukumoto S. Kaneta Y. Inoue D. Matsumoto T. Harigaya K. Fujita T. J. Biol. Chem. 2002; 277: 49011-49018Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Therefore, we examined SHN-2 involvement in BMP signaling. For those experiments, we prepared cells outgrown from the explants of minced calvarial bones. The basal levels of ALP in these wild type and Shn-2-deficient cells derived from the calvaria were similar (Fig. 4C, lane 1 versus 3). BMP-2 enhanced ALP activity ∼3-fold in wild type cells, whereas Shn-2 deficiency suppressed this BMP effect on ALP down to 2-fold (Fig. 4C, lane 2 versus 4; p < 0.05). To further examine Shn-2 expression in osteoblastic cell line, we examined MC3T3E1 cells. Shn-2 mRNA was expressed in osteoblastic MC3T3E1 cells (Fig. 5A). During the cultures of MC3T3E1 cells, Shn-2 mRNA levels increased ∼2.5-fold within 4 days in culture along with the increase in the expression levels of alkaline phosphatase (Fig. 5B, Alp). After day 4 in culture, Shn-2 mRNA levels stayed at steadily high levels, whereas the mRNA levels of osteoblastic differentiation marker genes, such as Alp, Runx2, osterix, and type I collagen (Col1a1) gradually increased up to day 21 (Fig. 5B). Therefore, Shn-2 expression levels were increased along with maturation of osteoblasts during the early differentiation stage.FIGURE 5Shn-2 expression in osteoblast-like cells in vitro. A, Shn-2 mRNA expression was detected in bone in vivo and in MC3T3E1 cells. B, MC3T3E1 cells were cultured for 21 days. RNA was extracted at the indicated time points. Expression levels of Col1a1, Runx2, osterix, Alp, and Shn-2 were estimated based on real-time PCR system. Expression levels of genes related to osteoblastic phenotypes were gradually increased in these cells. Expression level of Shn-2 was enhanced in these cells up to day 4 and kept at similar expression level up to day 21.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Shn-2 overexpression in osteoblastic cell line MC3T3E1 promoted BMP-2 enhancement in the expression levels of Alp, osterix, and Runx2 mRNAs (Fig. 6, A-C). These data revealed that SHN-2 is a positive regulator of osteoblastic differentiation, which acts at least in part by modulating BMP actions in these cells. To examine the mode of SHN-2 modulation of BMP signaling, BMP-induced transcriptional events were studied. Shn-2 overexpression by itself enhanced ∼3-fold the expression of luciferase reporter gene linked to the BMP response elements, 12 × GCCG, in MC3T3E1 cells (Fig. 6D, lane 1 versus 3). This enhancement was similar to that observed in the cells receiving BMP treatment alone (Fig. 6D, lane 2 versus 3). Under this condition, Shn-2 overexpression enhanced luciferase levels ∼2-fold more than those observed in the BMP-induced transcriptional activation (Fig. 6D, lane 2 versus 4). To see whether SHN-2 affects transcription of authentic promoter of gene encoding osteoblastic phenotype, a 1.1-kb osteocalcin promoter construct was transfected into MC3T3E1 cells. Overexpression of Shn-2 enhanced the activity of osteocalcin promoter (Fig. 6E). These observations indicate that SHN-2 enhances bone formation and osteoblastic differentiation at least in part through its action on transcriptional events that are involved in osteoblastic differentiation. Shn-2-deficient mice exhibited in vivo osteoclastic phenotype in bone whereas Shn-3-deficient mice did not (23Jones D.C. Wein M.N. Oukka M. Hofstaetter J.G. Glimcher M.J. Glimcher L.H. Science. 2006; 312: 1223-1227Crossref PubMed Scopus (189) Google Scholar). We therefore pursued the mode of this SHN-2 action in osteoclas-togenesis. Bone marrow cells were cultured in the presence of vitamin D3 and dexamethasone. Shn-2 deficiency suppressed the development of osteoclasts in the bone marrow cell culture (Fig. 7, A and B). As bone marrow cell cultures contain both stromal cells and progenitor cells for osteoclasts, we wished to test whether SHN-2 contributes to osteoclastogenesis in progenitor cells. For this purpose, spleen cells, which contain osteoclast progenitors but less stromal cells, were subjected to osteoclastogenesis assay in cultures in the presence of M-CSF and soluble receptor activator of NF-κB ligand (RANKL). Shn-2 deficiency in spleen cells reduced the development of osteoclasts in culture (Fig. 7C). These data revealed that SHN-2 acts as an endogenous modulator to support osteoclastogenesis. With regard to osteoclast function, Shn-2-deficient cells could form an actin ring and resorb dentin slice (data not shown), suggesting that absence of SHN-2 impaired in RANKL-dependent osteoclast development but not function. To examine Shn-2 expression during osteoclastic cell differentiation, RAW264.7 cells were treated with RANKL. RAW264.7 cells differentiated into tartrate-resistant acid phosphatase-positive multinucleated cells in the presence of RANKL without M-CSF as reported previously (33Hirotani H. Tuohy N.A. Woo J.T. Stern P.H. Clipstone N.A. J. Biol. Chem. 2004; 279: 13984-13992Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Therefore, the effects of RANKL alone could be observed in these cells. Shn-2 mRNA expression levels in RAW264.7 cells were enhanced by RANKL treatment (Fig. 7D). Thus, Shn-2 expression is under the control of RANKL signaling, and at the same time it is supporting osteoclast development. Shn-2 deficiency suppressed the function of osteoblast, which is the main source of RANKL and OPG. Impaired osteoclastogenesis in Shn-2 deficiency would be caused by the suppression of the induction of RANKL by osteoblasts. Thus, we analyzed expression levels of Rankl as well as Opg in bone. We found that Rankl expression levels were enhanced by Shn-2 deficiency (Fig. 8A), whereas expression levels of Opg tended to decrease in Shn-2 deficiency without statistical significance (Fig. 8B). Rankl/Opg ratio significantly increased in Shn-2-deficient mice (Fig. 8C). Therefore, impaired osteoclastogenesis in Shn-2 deficiency occurred despite the elevated Rankl. Because osteoclastogenesis is under the control of RANKL signaling, we analyzed expression levels of the genes downstream of this signaling. Expression level of c-fos was decreased in the bones of Shn-2-deficient mice (Fig. 8D). Because c-fos-deficient mice were reported to exhibit severe osteopetrosis due to impaired osteoclastogenesis (34Grigoriadis A.E. Wang Z.Q. Cecchini M.G. Hofstetter W. Felix R. Fleisch H.A. Wagner E.F. Science. 1994; 266: 443-448Crossref PubMed Scopus (1079) Google Scholar) and c-fos targets NFATc1 (35Boyle W.J. Simonet W.S. Lacey D.L. Nature. 2003; 423: 337-342Crossref PubMed Scopus (4976) Google Scholar, 36Zelzer E. Olsen B.R. Nature. 2003; 423: 343-348Crossref PubMed Scopus (221) Google Scholar), we also examined the expression level of Nfatc1. Expression levels of Nfatc1 were decreased in the bones of Shn-2-deficient mice (Fig. 8E). These results suggest that SHN-2 is involved in the RANKL-induced signals to promote osteoclast development. Although the interactions between RANKL and BMP on osteoclastogenesis remain unclear, BMP has been recently reported to positively regulate osteoclastic activities in vivo (37Okamoto M. Murai J. Yoshikawa H. Tsumaki N. J. Bone Miner. Res. 2006; 21: 1022-1033Crossref PubMed Scopus (172) Google Scholar). Our preliminary data also indicated that BMP treatment enhanced RANKL-induced osteoclastogenesis in wild type spleen cells, whereas such BMP enhancement in osteoclastogenesis was suppressed in Shn-2-deficient cells. We report here that SHN-2 is a novel transcriptional regulator of bone cell function and bone mass. Shn-2 deficiency caused reduction in whole bone BMD in vivo and suppressed bone formation as well as bone resorption parameters. Thus, SHN-2 plays a role in both arms of bone metabolism involved in bone turnover and remodeling. Shn-2 deficiency resulted in reduction in osterix and down-stream osteocalcin gene expression. We also found that overexpression of Shn-2 enhanced the levels of this gene. Thus, SHN-2 would be a modulator of at least one of the two master regulatory molecules of osteoblastic differentiation, osterix. Overexpression experiments indicated that SHN-2 by itself enhanced BMP response element-dependent transcription, possibly due to the enhancement of endogenous BMP. This SHN-2 action was observed to be augmented in the case of the simultaneous presence of exogenously added BMP. Furthermore, even in the absence of BMP, SHN-2 alone enhanced the osteocalcin gene in the 1.1-kb promoter, indicating that SHN-2 is acting on the gene either alone or in combination with the endogenous BMP signaling. We also observed that in the absence of SHN-2, suppression of osterix in bone was observed. However, Runx2 suppression was not obvious, suggesting that SHN-2 may act to modulate the function of osteoblastic master regulatory molecules more at the stages of osterix action that is downstream to the RUNX2 action. As it is obvious that SHN-2 per se is dispensable for osteoblastic differentiation, the importance of SHN-2 activity is to modify the levels of differentiation in osteoblastic cell lineages. Shn-2 deficiency decreased the length of long bone (data not shown) as well as the body weight (20Jin W. Takagi T. Kanesashi S.N. Kurahashi T. Nomura T. Harada J. Ishii S. Dev. Cell. 2006; 10: 461-471Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 21Takagi T. Harada J. Ishii S. Nat. Immunol. 2001; 2: 1048-1053Crossref PubMed Scopus (66) Google Scholar). However, the reason why Shn-2 deficiency causes short stature remains unclear. In growth plate, the columnar structures and primary spongiosa appeared normal. As Shn-2 deficiency slightly increased the length of growth plate, Shn-2 deficiency may result in failure to remodel calcified cartilage. As observed in in vivo analyses, one of the important points in SHN-2 actions is to regulate both osteoclastic and osteoblastic cells. Molecular analyses of osteoclastic activities indicated that deficiency of Shn-2 per se suppressed c-fos and Nfatc1 expression levels in vivo. In the presence of RANKL, Shn-2 enhanced the levels of osteoclastic differentiation in RAW264.7 cells and also the promoter activity of Nfatc1 (data not shown). Therefore, in osteoclastic differentiation SHN-2 would again be a transcriptional modulator of the promoter of Nfatc1, possibly by working together with other transcriptional factors or modulators such as NFATc1 by itself. Bone formation and bone resorption would be often regulated simultaneously in the same direction (2Martin T.J. Sims N.A. Trends Mol. Med. 2005; 11: 76-81Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar, 38Harada S. Rodan G.A. Nature. 2003; 423: 349-355Crossref PubMed Scopus (1135) Google Scholar, 39Winslow M.M. Pan M. Starbuck M. Gallo E.M. Deng L. Karsenty G. Crabtree G.R. Dev. Cell. 2006; 10: 771-782Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar), called coupling. Such a coupling event is important for the body to maintain bone mass. For instance, enhancement of bone formation in the event of loss of bone due to the increase in bone resorption would benefit the maintenance of bone mass (39Winslow M.M. Pan M. Starbuck M. Gallo E.M. Deng L. Karsenty G. Crabtree G.R. Dev. Cell. 2006; 10: 771-782Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 40Felsenberg D. Boonen S. Clin. Ther. 2005; 27: 1-11Abstract Full Text PDF PubMed Scopus (275) Google Scholar). Although understanding the mechanisms of this coupling is important, molecular bases for these coupling phenomena have not yet been fully elucidated (34Grigoriadis A.E. Wang Z.Q. Cecchini M.G. Hofstetter W. Felix R. Fleisch H.A. Wagner E.F. Science. 1994; 266: 443-448Crossref PubMed Scopus (1079) Google Scholar, 36Zelzer E. Olsen B.R. Nature. 2003; 423: 343-348Crossref PubMed Scopus (221) Google Scholar, 41Epstein S. Mayo Clin. Proc. 2005; 80: 379-388Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Compensatory mechanisms would provide for repair of lost bone to resume bone mass level under physiological conditions. However, in the case of postmenopausal osteoporosis, enhancement in bone resorption exceeds that of bone formation to end up with eventual bone loss (1Raisz L.G. J. Clin. Investig. 2005; 115: 3318-3325Crossref PubMed Scopus (1290) Google Scholar, 36Zelzer E. Olsen B.R. Nature. 2003; 423: 343-348Crossref PubMed Scopus (221) Google Scholar). As SHN-2 acts positively in both bone formation and resorption, this molecule plays a role at least in part to support the coupling events during the remodeling cycle to maintain adult bone mass. Our preliminary experiments of co-culture assays using osteoblasts and spleen cells from wild type and Shn-2-deficient mice revealed that Shn-2 deficiency in both osteoblasts and osteoclasts suppressed osteoclasto-genesis (data not shown). This observation supported that SHN-2 plays a role in both osteoblasts and osteoclast to support osteoclastogenesis. Shn-2 deficiency has been reported to suppress adipogenesis by reducing the BMP signaling in fat tissue (20Jin W. Takagi T. Kanesashi S.N. Kurahashi T. Nomura T. Harada J. Ishii S. Dev. Cell. 2006; 10: 461-471Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) and lymphogenesis by suppressing conversion from the double positive thymocytes into single positive cells, respectively (21Takagi T. Harada J. Ishii S. Nat. Immunol. 2001; 2: 1048-1053Crossref PubMed Scopus (66) Google Scholar, 22Kimura M.Y. Hosokawa H. Yamashita M. Hasegawa A. Iwamura C. Watarai H. Taniguchi M. Takagi T. Ishii S. Nakayama T. J. Exp. Med. 2005; 201: 397-408Crossref PubMed Scopus (54) Google Scholar). BMP has been recently reported to positively regulate osteoclastic activities in vivo (37Okamoto M. Murai J. Yoshikawa H. Tsumaki N. J. Bone Miner. Res. 2006; 21: 1022-1033Crossref PubMed Scopus (172) Google Scholar) and also has been implicated in regulation of immune cells (42Bleul C.C. Boehm T. J. Immunol. 2005; 175: 5213-5221Crossref PubMed Scopus (134) Google Scholar). Our observations on SHN-2 action in bone coincides with these notions on the close relationship between bone, fat, and immune system although a direct relationship among those still needs to be elucidated. It is intriguing to note those mice deficient in Shn-2 and those deficient in Shn-3 exhibit opposite phenotypes, i.e. osteopenia versus osteosclerosis, respectively (23Jones D.C. Wein M.N. Oukka M. Hofstaetter J.G. Glimcher M.J. Glimcher L.H. Science. 2006; 312: 1223-1227Crossref PubMed Scopus (189) Google Scholar). In addition, Shn-2-deficient mice revealed osteoclastic phenotype whereas Shn-3-deficient mice did not. Although zinc finger motifs are present in both molecules, overall homology is relatively low (<30%) (17Wu L.C. Gene Expr. 2002; 10: 137-152Crossref PubMed Scopus (60) Google Scholar). These features suggest that the two molecules would not compensate each other but rather they would be required for distinct functions in the regulation of bone metabolism. In conclusion, SHN-2 is a novel regulator of bone cells that plays a role in the maintenance of the bone mass, acting via positive regulation of transcription factors required for the functions of osteoblasts and osteoclasts.

Systemic hormonal control exerts its effect through the regulation of local target tissues, which in turn regulate upstream signals in a feedback loop. The parathyroid hormone (PTH) axis is a well defined hormonal signaling system that regulates calcium levels and bone metabolism. To understand the interplay between systemic and local signaling in bone, we examined the effects of deficiency of the bone matrix protein osteopontin (OPN) on the systemic effects of PTH specifically within osteoblastic cell lineages. Parathyroid hormone receptor (PPR) transgenic mice expressing a constitutively active form of the receptor (caPPR) specifically in cells of the osteoblast lineage have a high bone mass phenotype. In these mice, OPN deficiency further increased bone mass. This increase was associated with conversion of the major intertrabecular cell population from hematopoietic cells to stromal/osteoblastic cells and parallel elevations in histomorphometric and biochemical parameters of bone formation and resorption. Treatment with small interfering RNA (siRNA) for osteopontin enhanced H223R mutant caPPR-induced cAMP-response element (CRE) activity levels by about 10-fold. Thus, in addition to the well known calcemic feedback system for PTH, local feedback regulation by the bone matrix protein OPN also plays a significant role in the regulation of PTH actions.

Cnot7 is a recently identified regulator of spermatogenesis in adult mice. Because Cnot7 binds to Tob, a BMP inhibitor shown to be involved in bone metabolism, we examined whether Cnot7 is involved in bone mass regulation by using adult Cnot7 deficient mice. Cnot7-/- mice exhibited a high bone mass phenotype. This was associated with an increase in bone formation rate but not with any alteration in bone resorption parameters. On BMP treatment, Cnot7-/- cells expressed higher levels of alkaline phosphatase compared with control cells. Direct BMP2 injection induced larger bone mass in Cnot7-/- calvaria than control in vivo. These observations revealed that Cnot7 is an endogenous suppressor of bone mass and inhibits BMP actions in osteoblasts.The molecular mechanisms involved in the determination of bone mass have been gradually understood based on recent analyses. Cnot7 (Ccr4-Not complex 7) is a component of transcriptional Ccr4-Not complex, is conserved from yeast to human, and binds to Tob, but its function in bone is not understood.To elucidate the role of involvement of Cnot7 in bone mass determination, we examined the bone of adult male Cnot7-null and heterozygous mice based on microCT analyses, histomorphometry, cell cultures, and in vivo BMP assays.Cnot7-/- mice showed an increase in bone mass levels by >50% compared with controls. Analyses of the histomorphometric parameters indicated that bone formation activity in Cnot7-/- mice was enhanced, whereas bone resorption activity was not altered. These effects on osteoblasts were cell autonomous because mineralized nodule formation was enhanced in the cultures of bone marrow cells prepared from Cnot7-/- mice. In vitro analyses to elucidate Cnot7 effects revealed that BMP-induced expression of alkaline phosphatase in Cnot7-/- calvaria-derived osteoblastic cells was enhanced compared with controls. Moreover, BMP injection-induced new bone formation in vivo was enhanced in Cnot7-/- mice.These observations indicated that Cnot7 is an endogenous suppressor of bone mass in adult mice and inhibits BMP actions.

Abstract JunD is an activator protein‐1 (AP‐1) component though its function in skeletal system is still not fully understood. To elucidate the role of JunD in the regulation of bone metabolism, we analyzed JunD‐deficient mice. JunD deficiency significantly increased bone mass and trabecular number. This bone mass enhancement was due to JunD deficiency‐induced increase in bone formation activities in vivo. Such augmentation of bone formation was associated with simultaneous increase in bone resorption while the former was dominant over the latter as accumulation of bone mass occurred in JunD‐deficient mice. In a pathological condition relevant to postmenopausal osteoporosis, ovariectomy reduced bone mass in wild type (WT) mice as known before. Interestingly, JunD deficiency suppressed ovariectomy‐induced increase in bone resorption and kept high bone mass. In addition, JunD deficiency also enhanced new bone formation after bone marrow ablation. Examination of molecular bases for these observations revealed that JunD deficiency enhanced expression levels of c‐jun, fra‐1 , and fra‐2 in bone in conjunction with elevated expression levels of runx2 , type I collagen , and osteocalcin . Thus, JunD is involved in estrogen depletion‐induced osteopenia via its action to suppress bone formation and to enhance bone resorption. J. Cell. Biochem. 103: 1037–1045, 2008. © 2008 Wiley‐Liss, Inc.