Transcriptional Activation of the p21 Gene by Interleukin-6 Type Cytokines

Abstract

The cyclin-dependent kinase inhibitor p21WAF1,CIP1,SDI1 plays a critical role in cell differentiation, and it has been shown to confer resistance to apoptosis. Based on this, and on evidence that activation of the gp130/signal transducer and activator of transcription (STAT) signal transduction pathway by interleukin (IL)-6 type cytokines promotes differentiation and prevents apoptosis in osteoblastic cells, we have investigated the possibility that p21 is a downstream effector of this signaling pathway in osteoblasts. We report that either oncostatin M (OSM) or IL-6 plus soluble IL-6 receptor increased the levels of p21 mRNA and protein in the osteoblast-like human osteosarcoma cell line MG63 and stimulated the activity of a 2.4-kilobase pair segment of the human p21 gene promoter. Further, nuclear extracts from cytokine-stimulated MG63 cells formed protein-DNA complexes with a 19-base pair nucleotide fragment of the p21 promoter containing a single STAT response element. The identity of the binding proteins as Stat3 and Stat1 was demonstrated with specific antibodies. In addition, and in support of a mediating role of STATs in the activation of the p21 promoter, overexpression of Stat3 potentiated the cytokine effect on the p21 promoter; whereas a dominant negative Stat3, or a mutation of the STAT response element on the promoter, significantly reduced the cytokine effect. Finally, antisense oligonucleotides complementary to p21 mRNA inhibited OSM-induced stimulation of alkaline phosphatase expression and antagonized the protective effect of OSM on anti-Fas-induced apoptosis. These results demonstrate that p21 is a downstream effector of gp130/Stat3 activation and a critical mediator of the pro-differentiating and anti-apoptotic effects of IL-6 type cytokines on human osteoblastic cells. The cyclin-dependent kinase inhibitor p21WAF1,CIP1,SDI1 plays a critical role in cell differentiation, and it has been shown to confer resistance to apoptosis. Based on this, and on evidence that activation of the gp130/signal transducer and activator of transcription (STAT) signal transduction pathway by interleukin (IL)-6 type cytokines promotes differentiation and prevents apoptosis in osteoblastic cells, we have investigated the possibility that p21 is a downstream effector of this signaling pathway in osteoblasts. We report that either oncostatin M (OSM) or IL-6 plus soluble IL-6 receptor increased the levels of p21 mRNA and protein in the osteoblast-like human osteosarcoma cell line MG63 and stimulated the activity of a 2.4-kilobase pair segment of the human p21 gene promoter. Further, nuclear extracts from cytokine-stimulated MG63 cells formed protein-DNA complexes with a 19-base pair nucleotide fragment of the p21 promoter containing a single STAT response element. The identity of the binding proteins as Stat3 and Stat1 was demonstrated with specific antibodies. In addition, and in support of a mediating role of STATs in the activation of the p21 promoter, overexpression of Stat3 potentiated the cytokine effect on the p21 promoter; whereas a dominant negative Stat3, or a mutation of the STAT response element on the promoter, significantly reduced the cytokine effect. Finally, antisense oligonucleotides complementary to p21 mRNA inhibited OSM-induced stimulation of alkaline phosphatase expression and antagonized the protective effect of OSM on anti-Fas-induced apoptosis. These results demonstrate that p21 is a downstream effector of gp130/Stat3 activation and a critical mediator of the pro-differentiating and anti-apoptotic effects of IL-6 type cytokines on human osteoblastic cells. Fine orchestration of the activity of cyclins, Cdk, 1The abbreviations used are: Cdkcyclin-dependent kinaseIL-6interleukin-6IL-11interleukin-11LIFleukemia inhibitory factorOSMoncostatin Mgp130glycoprotein 130STATsignal transducer and activator of transcriptionsIL-6Rsoluble IL-6 receptorFBSfetal bovine serumAPalkaline phosphataseMEMminimum essential mediumMTS3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner saltHRPhorseradish peroxidaselucluciferaseLDHlactate dehydroxylaseγ-IFNγ-interferonANOVAanalysis of varianceSBESTAT binding elementdnStat3dominant negative Stat3kbkilobase pair(s)CMVcytomegalovirusPBSphosphate-buffered saline. and inhibitors of such kinases governs the progression of cells from one phase of their division cycle to the next. Increased expression of Cdk inhibitors is now recognized as a general mechanism for the arrest of cell division (1Hunter T. Cell. 1993; 75: 839-841Abstract Full Text PDF PubMed Scopus (370) Google Scholar, 2Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3215) Google Scholar). One of these Cdk inhibitors, designated as p21WAF1,CIP1,SDI1 because of its original identification by three independent groups as wild type p53 activated fragment 1 (WAF1) (3el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Volgestein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7936) Google Scholar), Cdk-interacting protein 1 (CIP1) (4Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5239) Google Scholar), or senescence cell-derived inhibitor (SDI1) (5Noda A. Ning Y. Venable S.F. Pereira-Smith O.M. Smith J.R. Exp. Cell Res. 1994; 211: 90-98Crossref PubMed Scopus (1315) Google Scholar), binds and inactivates several cyclin/Cdk complexes including the ones involved in the transition between G1 and S phases, and inhibits the replication factor PCNA (proliferating cell nuclear antigen) (6Luo Y. Hurwitz J. Massague J. Nature. 1995; 375: 159-161Crossref PubMed Scopus (516) Google Scholar). To date, p21 has been implicated in three different processes: DNA damage repair, differentiation, and apoptosis. In the former, p21 gene expression is stimulated by the tumor suppressor gene p53, and the protein arrests cells in the G1 phase in order to allow the DNA damage repair process to be performed before cells reenter the S phase (7Sheikh M.S. Li X.S. Chen J.C. Shao Z.M. Ordonez J.V. Fontana J.A. Oncogene. 1994; 9: 3407-3415PubMed Google Scholar, 8Dulic V. Kaufmann W.K. Wilson S.J. Tlsty T.D. Lees E. Harper J.W. Elledge S.J. Reed S.I. Cell. 1994; 76: 1013-1023Abstract Full Text PDF PubMed Scopus (1417) Google Scholar). In the latter two processes, p21 expression increases independently of p53. In this case, the resulting halt of cell division allows cells to progress along their differentiation pathway (9Parker S.B. Eichele G. Zhang P. Rawls A. Sands A.T. Bradley A. Olson E.N. Harper J.W. Elledge S.J. Science. 1995; 267: 1024-1027Crossref PubMed Scopus (1023) Google Scholar, 10Halevy O. Novitch B.G. Spicer D.B. Skapek S.X. Rhee J. Hannon G.J. Beach D. Lassar A.B. Science. 1995; 267: 1018-1021Crossref PubMed Scopus (1092) Google Scholar, 11Liu M. Lee M.-H. Cohen M. Bommakanti M. Freedman L.P. Genes Dev. 1996; 10: 142-153Crossref PubMed Scopus (841) Google Scholar, 12Jiang H. Lin J. Su Z. Collart F.R. Huberman E. Fisher P.B. Oncogene. 1994; 9: 3397-3406PubMed Google Scholar, 13Matsumura I. Ishikawa J. Nakajima K. Oritani K. Tomiyama Y. Miyagawa J. Kato T. Miyazaki H. Matsuzawa Y. Kanakura Y. Mol. Cell. Biol. 1997; 17: 2933-2943Crossref PubMed Scopus (172) Google Scholar, 14Jiang H. Lin J. Su Z.Z. Herlyn M. Kerbel R.S. Weissman B.E. Welch D.R. Fisher P.B. Oncogene. 1995; 10: 1855-1864PubMed Google Scholar). Interestingly, in differentiating muscle cells, neuroblastoma, and melanoma cells, p21 also protects against programmed cell death (15Wang J. Walsh K. Science. 1996; 273: 359-361Crossref PubMed Scopus (463) Google Scholar, 16Poluha W. Poluha D.K. Chang B. Crosbie N.E. Schonhoff M.C. Kilpatrick D.L. Ross A.H. Mol. Cell. Biol. 1996; 16: 1335-1341Crossref PubMed Scopus (225) Google Scholar, 17Gorospe M. Cirielli C. Wang X. Seth P. Capogrossi M.C. Holbrook N.J. Oncogene. 1997; 14: 929-935Crossref PubMed Scopus (292) Google Scholar). cyclin-dependent kinase interleukin-6 interleukin-11 leukemia inhibitory factor oncostatin M glycoprotein 130 signal transducer and activator of transcription soluble IL-6 receptor fetal bovine serum alkaline phosphatase minimum essential medium 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt horseradish peroxidase luciferase lactate dehydroxylase γ-interferon analysis of variance STAT binding element dominant negative Stat3 kilobase pair(s) cytomegalovirus phosphate-buffered saline. It is now established that cytokines such as IL-6 can influence bone resorption and formation by regulating the production of osteoclasts and osteoblasts (reviewed in Ref. 18Manolagas S.C. Jilka T. of Scholar). and OSM by cells of the in response to and and differentiation from In addition, the of and the differentiation of and osteoblastic cells from and human S.C. Jilka T. of Scholar, T. S.C. 1997; PubMed Scopus Google Scholar, T. M. T. Lin H. P. N. B. E. S.C. Google Scholar, K. S.C. Jilka J. Res. 1995; Scholar). IL-6 type cytokines osteoblastic cell apoptosis by serum tumor or the pathway R.S. T. S.C. J. Res. PubMed Scopus Google Scholar, Smith C. S.C. J. Res. 1997; Scholar). The for these cytokines a as T. T. Cell. 1994; 76: Full Text PDF PubMed Scopus Google Scholar). of signaling by of the kinase of kinases N. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). the and nuclear of transcription of 1995; Full Text PDF PubMed Scopus Google Scholar, Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). of evidence that p21WAF1,CIP1,SDI1 is cell differentiation and resistance to apoptosis in cell we have investigated this gene is by IL-6 type cytokines and it plays a role in their on osteoblasts. these we established human osteosarcoma cell of osteoblastic cells and has been used as a of this cell type of Scholar). MG63 cells levels of the OSM receptor and levels of the binding of the IL-6 receptor T. N. V. J. 1996; PubMed Scopus Google the signal pathway can be activated by OSM or by the of IL-6 with We have that such activation to cell cycle arrest and a in the expression of the T. S.C. 1997; PubMed Scopus Google Scholar), and prevents apoptosis R.S. T. S.C. J. Res. PubMed Scopus Google Scholar). we evidence that activation of the signal transduction pathway by IL-6 type cytokines to the activation of the p21WAF1,CIP1,SDI1 and that p21 is a critical mediator of the and anti-apoptotic of these cytokines on osteoblastic cells. IL-6 was from and human OSM from and from and and from was from and from from from p21 was by R. Smith of (5Noda A. Ning Y. Venable S.F. Pereira-Smith O.M. Smith J.R. Exp. Cell Res. 1994; 211: 90-98Crossref PubMed Scopus (1315) Google Scholar). was from human p21 promoter in was by Freedman M. Lee M.-H. Cohen M. Bommakanti M. Freedman L.P. Genes Dev. 1996; 10: 142-153Crossref PubMed Scopus (841) Google Scholar). Finally, the expression containing the for human Stat3 and dominant negative Stat3 by The E. R. J. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google and A. V. M. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), osteosarcoma MG63 cells H. A. J.C. P. PubMed Scopus Google in with and in a of in in 1 1 1 and was in a for in for protein on and to for 1 in containing and and with to either p53, or by for 1 with the with by to the of the of the in the was performed by was from or and was as T. N. V. J. 1996; PubMed Scopus Google Scholar). was by in to and by for with for p21 or for and a performed as T. S.C. 1997; PubMed Scopus Google Scholar). to of MG63 cells in medium for and stimulated with OSM for with and nuclear extracts with and for on in cells of the containing for in cells of 1 and and for and for and to the binding element from the promoter in the p21 promoter (3el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Volgestein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7936) Google Scholar, M. Su W.S. Z. Y. X. Science. 1996; PubMed Scopus Google was and The was for with of nuclear proteins in a containing 1 and 1 in the or of wild type or performed by the nuclear proteins with either or for to the of the of MG63 cells in and the with of a containing a segment of the human p21 gene promoter segment in the STAT binding segment was as the luciferase (3el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Volgestein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7936) Google Scholar, 11Liu M. Lee M.-H. Cohen M. Bommakanti M. Freedman L.P. Genes Dev. 1996; 10: 142-153Crossref PubMed Scopus (841) Google Scholar), with of the containing the luciferase gene and the with of the expression for Stat3 or the E. R. J. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). cells with OSM for the and luciferase activity was in cell to the was with a and luciferase activity was luciferase a was as the of the p21 containing a mutation in the from the promoter was used to and the wild type from a containing a segment of the human p21 gene promoter the luciferase M. Lee M.-H. Cohen M. Bommakanti M. Freedman L.P. Genes Dev. 1996; 10: 142-153Crossref PubMed Scopus (841) Google Scholar). The mutation a that was used for the of and it was by A. PubMed Scopus Google Scholar). and for The was with medium to a of 1 and to the cells. oligonucleotides on the p21WAF1,CIP1,SDI1 is complementary to the the is complementary to the of the is complementary to the and the is complementary to the activity was as T. S.C. 1997; PubMed Scopus Google Scholar). MG63 cells in medium containing and in to was and with medium containing and cells with or OSM in the or of 1 oligonucleotides for and cells in The of cells was on the of the to a with and three with of was to of containing and was to and and and in a cells three with and 1 of and of to and and the as of to of human osteoblast-like MG63 cells was as R.S. T. S.C. J. Res. PubMed Scopus Google Scholar). for in containing in the or of or in with The medium was with containing with or for and cells by cells with cells from the and in was and the of cells nuclear and was a cells We have demonstrated that the of cells with the of cells, that osteoblasts be by either R.S. T. S.C. J. Res. PubMed Scopus Google Scholar). the analysis of the of and the variance for the of two A. K. of Scholar). for the of the groups as the with a 1 to a the the analysis of the the effects of OSM on in the of oligonucleotides OSM by of the by the of the These by was with the the and of In addition, analysis to antisense groups to the with was also of by and was to the of of between in p21 protein for the pro-differentiating and anti-apoptotic effects of OSM on osteoblasts. cells with the oligonucleotides for before the of cells and proteins by was performed with and as in cells with or and in the or of the oligonucleotides for The OSM by and with the or with the with antisense reduced OSM response with that of the or with that of the results in two cells in the or of or in with was by of and the of cells was as the of in cells between by with by MG63 cells in the of OSM in the levels of p21 as by analysis effect was as as of and for p21 levels to increases in p21 protein expression IL-6 in with was used in these of OSM 1 was in the levels of p53 protein in cells with OSM 1 with the results of the p21 protein was by in MG63 cells and OSM increased the was with the with the to the was activation increases p21 protein MG63 cells on in the or (OSM) of OSM for with and with or with plus a of the to the was of evidence that that p21 protein levels by the of the protein M. M. Smith J.R. G.J. Genes Dev. 1996; 10: PubMed Scopus Google Scholar), we investigated OSM p21 protein this cells with OSM for and was to protein and cell from and different The of of p21 was the in cells and cells, that OSM the of the p21 protein We p21 mRNA expression in and MG63 cells. shown in OSM increased the of p21 mRNA as as 1 The effect was and to levels by To was we the of p21 mRNA in the of In this cells with for and with OSM for the effect of In and OSM-induced p21 levels significantly increased in the of that p21 mRNA levels be by The of mRNA levels be either to of to of or to a of To activation the of the p21 transcription was by to or cells. mRNA was different to of and the of p21 mRNA was by analysis The of of p21 mRNA in cells was to that in cells, that the of p21 mRNA is by the OSM-induced in p21 mRNA from To activation the activity of the p21 promoter, MG63 cells with a fragment of the p21 promoter from the luciferase with OSM and luciferase activity was different the of the cytokine OSM increased luciferase with a effect of The effect of OSM was in cells with the with a Stat3 expression The luciferase activity with either in the or of Stat3 in cells with OSM of the and the that the fragment of the p21 promoter used in a designated (3el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Volgestein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7936) Google Scholar, M. Su W.S. Z. Y. X. Science. 1996; PubMed Scopus Google Scholar), we this element by and the of the mutation on the of OSM to the activity of the promoter. with either the wild type or with the of the significantly reduced the response of the promoter to OSM stimulation that this element a role in the OSM-induced activation of the promoter. extracts from cells or with OSM for and performed as a extracts from cells with OSM a that the and its binding was by a of wild type by a to Stat3, a reduced the of the protein-DNA the of to the of the protein-DNA it reduced the of the To the role of Stat3 in the OSM-induced stimulation of the p21 promoter, we a dominant negative of the Stat3 by mutation of to A. V. M. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). of MG63 cells with expression for significantly reduced the OSM-induced in activity of the wild type p21 promoter the of the expression was also with a of the from the promoter to the kinase promoter The inhibited the activity of this to the as the p21 promoter, that binding of activated Stat3 to increases the activity of the p21 promoter. The of the stimulation of p21 gene expression by IL-6 type cytokines to their effects was investigated antisense to the of p21 shown by the results of the analysis in the OSM-induced in p21 protein levels was in cells to antisense oligonucleotides complementary to a the the the OSM-induced stimulation of activity in MG63 cells antisense complementary to the of the and the of the p21 inhibitory effects on OSM-induced in the of the effect of OSM on the expression of the p21 protein with the p21 antisense the protective effect of OSM on apoptosis of MG63 cells used as a negative in these influence on the effects of The results of the in this demonstrate for the that activation of the signaling pathway and Stat3 by IL-6 type cytokines to the activation of the p21WAF1,CIP1,SDI1 gene in human osteoblastic cells. that stimulation of p21 expression is for the and anti-apoptotic effects of IL-6 type cytokines on this cell These in with a role of p21 as a downstream effector of the of cytokines that as by evidence that Stat1 and and p21 and that p21 the cell arrest effect of on cells and the of differentiation by on a leukemia cell line I. Ishikawa J. Nakajima K. Oritani K. Tomiyama Y. Miyagawa J. Kato T. Miyazaki H. Matsuzawa Y. Kanakura Y. Mol. Cell. Biol. 1997; 17: 2933-2943Crossref PubMed Scopus (172) Google Scholar, M. Su W.S. Z. Y. X. Science. 1996; PubMed Scopus Google Scholar). The p21 gene promoter three designated and M. Su W.S. Z. Y. X. Science. 1996; PubMed Scopus Google Scholar). The fragment of the p21 promoter used in the of these three it is that we have the of the effect of IL-6 type cytokines on the activity of this Stat3 binds to binds to and and Stat1 binds to three I. Ishikawa J. Nakajima K. Oritani K. Tomiyama Y. Miyagawa J. Kato T. Miyazaki H. Matsuzawa Y. Kanakura Y. Mol. Cell. Biol. 1997; 17: 2933-2943Crossref PubMed Scopus (172) Google Scholar, M. Su W.S. Z. Y. X. Science. 1996; PubMed Scopus Google Scholar). In of this evidence and the that Stat3 is the STAT activated by IL-6 type cytokines N. Z. Science. 1995; 267: PubMed Scopus Google Scholar), is to be the element of the three for the of the IL-6 type We have that IL-6 type cytokines arrest MG63 cell the G1 phase T. S.C. 1997; PubMed Scopus Google Scholar). These with the that of p21 the stimulation of with the that of a specific is with cell cycle and that p21 a critical role this In support of this, of p21 in a and cell cycle arrest have been the differentiation of a of cell (9Parker S.B. Eichele G. Zhang P. Rawls A. Sands A.T. Bradley A. Olson E.N. Harper J.W. Elledge S.J. Science. 1995; 267: 1024-1027Crossref PubMed Scopus (1023) Google Scholar, 10Halevy O. Novitch B.G. Spicer D.B. Skapek S.X. Rhee J. Hannon G.J. Beach D. Lassar A.B. Science. 1995; 267: 1018-1021Crossref PubMed Scopus (1092) Google Scholar, 11Liu M. Lee M.-H. Cohen M. Bommakanti M. Freedman L.P. Genes Dev. 1996; 10: 142-153Crossref PubMed Scopus (841) Google Scholar, 12Jiang H. Lin J. Su Z. Collart F.R. Huberman E. Fisher P.B. Oncogene. 1994; 9: 3397-3406PubMed Google Scholar, 13Matsumura I. Ishikawa J. Nakajima K. Oritani K. Tomiyama Y. Miyagawa J. Kato T. Miyazaki H. Matsuzawa Y. Kanakura Y. Mol. Cell. Biol. 1997; 17: 2933-2943Crossref PubMed Scopus (172) Google Scholar, 14Jiang H. Lin J. Su Z.Z. Herlyn M. Kerbel R.S. Weissman B.E. Welch D.R. Fisher P.B. Oncogene. 1995; 10: 1855-1864PubMed Google Scholar, B. A. C. Oncogene. 1994; 9: Google Scholar, N. Hannon G. Beach G. Tokino T. Kinzler K.W. Volgestein B. T. Genes Dev. 1997; 9: Scopus Google Scholar). cell cycle arrest in the G1 phase has been shown to the differentiation overexpression of the Cdk inhibitors p21 cells to a M. Lee M.-H. Cohen M. Bommakanti M. Freedman L.P. Genes Dev. 1996; 10: 142-153Crossref PubMed Scopus (841) Google Scholar). overexpression of the Cdk inhibitors p21 and in gene expression S.X. Rhee J. Spicer D.B. Lassar A.B. Science. 1995; 267: PubMed Scopus Google Scholar). In shown we have that IL-6 type two to differentiation in and also p21 levels in human osteoblast-like MG63 cells. we have in the expression of p21 in of bone cells in the of established of with the evidence in this these that p21 be a general mechanism of osteoblastic cell Cdk inhibitors that involved in cell differentiation in with the in differentiation of cells p21 and M. Lee M.-H. Cohen M. Bommakanti M. Freedman L.P. Genes Dev. 1996; 10: 142-153Crossref PubMed Scopus (841) Google Scholar). In line with this we have that the expression of activation also increases in the levels of and in MG63 cells These for the that OSM-induced in was by p21 antisense In to the evidence for a role of p21 in cell differentiation, it has been shown that muscle cells or tumor cells levels of p21 against apoptosis (15Wang J. Walsh K. Science. 1996; 273: 359-361Crossref PubMed Scopus (463) Google Scholar, 16Poluha W. Poluha D.K. Chang B. Crosbie N.E. Schonhoff M.C. Kilpatrick D.L. Ross A.H. Mol. Cell. 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IL-6 is for bone it is for the bone with of The in this report that p21 is a downstream effector of of the IL-6 type cytokines on osteoblastic cells. it is that p21 is also for the in bone cell formation by IL-6 of The of the role of p21 in of bone cell formation in In the evidence that p21 is a response gene for cytokines that the signaling and a critical effector of their and anti-apoptotic in osteoblastic cells. be to the activation of the p21WAF1,CIP1,SDI1 gene is for the established and anti-apoptotic effects of this of cytokines on their and cell We and for A. and for critical of the and the of and for