Bisphosphonates and Estrogens Inhibit Osteocyte Apoptosis via Distinct Molecular Mechanisms Downstream of Extracellular Signal-regulated Kinase Activation

Both estrogens and bisphosphonates attenuate osteocyte apoptosis by activating the extracellular signal-regulated kinases (ERKs). However, whereas estrogens activate ERKs via an extranuclear function of the estrogen receptor, bisphosphonates do so by opening connexin 43 hemichannels. Here, we demonstrated that the signaling events downstream of ERKs induced by these two stimuli are also distinct. Inhibition of osteocyte apoptosis by estrogens requires nuclear accumulation of ERKs and activation of downstream transcription factors. On the other hand, anti-apoptosis induced by bisphosphonates requires neither transcription nor ERK-dependent transcription factors. Instead, the effect of bisphosphonates is abolished when ERKs are restricted to the nucleus by blocking CRM1/exportin1-mediated nuclear protein export or by expressing nuclear-anchored ERKs, but it is unaffected in cells expressing cytoplasmic-anchored ERKs. Connexin 43/ERK-mediated anti-apoptosis induced by bisphosphonates requires the kinase activity of the cytoplasmic target of ERKs, p90RSK, which in turn phosphorylates the pro-apoptotic protein BAD and C/EBPβ. Phosphorylation of BAD renders it inactive, whereas phosphorylation of C/EBPβ leads to binding of pro-caspases, thus inhibiting apoptosis independently of the transcriptional activity of this transcription factor. Consistent with the evidence that estrogens and bisphosphonates phosphorylate diverse targets of ERKs, probably resulting from activation of spatially distinct pools of these kinases, the two agents had additive effects on osteocyte survival. Both estrogens and bisphosphonates attenuate osteocyte apoptosis by activating the extracellular signal-regulated kinases (ERKs). However, whereas estrogens activate ERKs via an extranuclear function of the estrogen receptor, bisphosphonates do so by opening connexin 43 hemichannels. Here, we demonstrated that the signaling events downstream of ERKs induced by these two stimuli are also distinct. Inhibition of osteocyte apoptosis by estrogens requires nuclear accumulation of ERKs and activation of downstream transcription factors. On the other hand, anti-apoptosis induced by bisphosphonates requires neither transcription nor ERK-dependent transcription factors. Instead, the effect of bisphosphonates is abolished when ERKs are restricted to the nucleus by blocking CRM1/exportin1-mediated nuclear protein export or by expressing nuclear-anchored ERKs, but it is unaffected in cells expressing cytoplasmic-anchored ERKs. Connexin 43/ERK-mediated anti-apoptosis induced by bisphosphonates requires the kinase activity of the cytoplasmic target of ERKs, p90RSK, which in turn phosphorylates the pro-apoptotic protein BAD and C/EBPβ. Phosphorylation of BAD renders it inactive, whereas phosphorylation of C/EBPβ leads to binding of pro-caspases, thus inhibiting apoptosis independently of the transcriptional activity of this transcription factor. Consistent with the evidence that estrogens and bisphosphonates phosphorylate diverse targets of ERKs, probably resulting from activation of spatially distinct pools of these kinases, the two agents had additive effects on osteocyte survival. Previous work from our group (1Plotkin L.I. Weinstein R.S. Parfitt A.M. Roberson P.K. Manolagas S.C. Bellido T. J. Clin. Investig. 1999; 104: 1363-1374Crossref PubMed Google Scholar, 2Plotkin L.I. Manolagas S.C. Bellido T. J. Biol. Chem. 2002; 277: 8648-8657Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar) demonstrates that bisphosphonates prevent apoptosis of osteocytes and other cells of the osteoblastic lineage via the opening of hemichannels formed by connexin (Cx) 1The abbreviations used are: Cx, connexin; ERK, extracellular signal-regulated kinase; wt, wild type; dn, dominant negative; GFP, green fluorescent protein; nGFP, nuclear green fluorescent protein; nRFP, nuclear red fluorescent protein; CREB, cAMP-responsive element-binding protein; α-MEM, α-minimal essential medium; HA, hemagglutinin; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. 43 leading to activation of Src and the extracellular signal-regulated kinases (ERKs). Remarkably, Cx43 (but not other connexins) confers de novo responsiveness to these agents to connexin-naïve cells. The ability of Cx43 to transduce pro-survival signals requires both the pore-forming domain of Cx43 and the C-terminal portion of the protein, which is physically associated with the kinase Src (3Kanemitsu M.Y. Loo L.W. Simon S. Lau A.F. Eckhart W. J. Biol. Chem. 1997; 272: 22824-22831Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 4Loo L.W. Kanemitsu M.Y. Lau A.F. Mol. Carcinog. 1999; 25: 187-195Crossref PubMed Scopus (45) Google Scholar). Src activation and its interaction with Cx43 are indispensable for the anti-apoptotic effects of bisphosphonates (2Plotkin L.I. Manolagas S.C. Bellido T. J. Biol. Chem. 2002; 277: 8648-8657Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Similar to bisphosphonates, ligand-induced activation of sex steroid receptors also attenuates osteocyte and osteoblast apoptosis by a mechanism that requires activation of ERKs and Src. However, the molecular events that lead to activation of the Src/ERK pathway by estrogens or androgens are different from the ones involved in bisphosphonate action. Thus, either class of sex steroids activate ERKs via an extranuclear function of their receptors that can be mediated by the ligand binding domain of the proteins (5Kousteni S. Bellido T. Plotkin L.I. O'Brien C.A. Bodenner D.L. Han L. Han K. DiGregorio G.B. Katzenellenbogen J.A. Katzenellenbogen B.S. Roberson P.K. Weinstein R.S. Jilka R.L. Manolagas S.C. Cell. 2001; 104: 719-730Abstract Full Text Full Text PDF PubMed Google Scholar). In addition, kinase-dependent activation of transcription factors and gene transcription is required for the anti-apoptotic effect of sex steroids on osteoblastic cells (6Kousteni S. Han L. Chen J.R. Almeida M. Plotkin L.I. Bellido T. Manolagas S.C. J. Clin. Investig. 2003; 111: 1651-1664Crossref PubMed Google Scholar). Heretofore, the mechanism by which bisphosphonates prevent apoptosis downstream of ERKs, however, has remained unknown. We report that, unlike the anti-apoptotic effect of ERK activation induced by sex steroids, anti-apoptotic effects triggered by the Cx43/Src/ERK pathway do not require gene transcription or ERK-dependent transcription factors. Instead, they depend on the cytoplasmic localization of ERKs and the phosphorylation of the ERK-activated kinase p90RSK and its cytoplasmic targets BAD and C/EBPβ. Moreover, and consistent with the evidence that estrogens and bisphosphonates phosphorylate diverse targets of ERKs, the two agents had additive effects on osteocyte survival, supporting the notion that they activate spatially distinct pools of these kinases. DNA Constructs and Transient Transfections—Wild type (wt) HA-BAD and the HA-BAD mutants in which Ser have been replaced by Ala (triple mutant, S112A/S136A/S155A, and single mutants, S112A, S136A, and S155A) were provided by X-M. Zhou (Apoptosis Technology, Inc. Cambridge, MA) (7Zhou X.M. Liu Y. Payne G. Lutz R.J. Chittenden T. J. Biol. Chem. 2000; 275: 25046-25051Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Dominant negative (dn) A-CREB and A-C/EBPβ, dnRunx2, dnSTAT3 (Tyr705), wt and kinase-deficient (K-) MEK, wt and K- p90RSK2, wtC/EBPβ, C/EBPβ T217A, ElkC, and dnElk-1 and GAL4-luciferase were provided by C. Vinson (NCI, National Institutes of Health, Bethesda, MD) (8Ahn S. Olive M. Aggarwal S. Krylov D. Ginty D.D. Vinson C. Mol. Cell. Biol. 1998; 18: 967-977Crossref PubMed Google Scholar), P. Ducy (Baylor College of Medicine, Houston, TX) (9Ducy P. Starbuck M. Priemel M. Shen J. Pinero G. Geoffroy V. Amling M. Karsenty G. Genes Dev. 1999; 13: 1025-1036Crossref PubMed Google Scholar), M. Saunders (GlaxoSmithKline) (10Kaptein A. Paillard V. Saunders M. J. Biol. Chem. 1996; 271: 5961-5964Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), N. G. 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Krutovskikh (Unit of Multistage Carcinogenesis, International Agency for Research, Lyon, France) (19Krutovskikh V.A. Yamasaki H. Tsuda H. Asamoto M. Mol. Carcinog. 1998; 23: 254-261Crossref PubMed Scopus (52) Google Scholar), respectively. The constructs encoding the nuclear green fluorescent protein (nGFP) or the nuclear red fluorescent protein (nRFP) were described (1Plotkin L.I. Weinstein R.S. Parfitt A.M. Roberson P.K. Manolagas S.C. Bellido T. J. Clin. Investig. 1999; 104: 1363-1374Crossref PubMed Google Scholar, S. Bellido T. Plotkin L.I. O'Brien C.A. Bodenner D.L. Han L. Han K. DiGregorio G.B. Katzenellenbogen J.A. Katzenellenbogen B.S. Roberson P.K. Weinstein R.S. Jilka R.L. Manolagas S.C. Cell. 2001; 104: 719-730Abstract Full Text Full Text PDF PubMed Google Scholar). the constructs used in this have been to were described (2Plotkin L.I. Manolagas S.C. Bellido T. J. Biol. Chem. 2002; 277: 8648-8657Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, S. Bellido T. Plotkin L.I. O'Brien C.A. Bodenner D.L. Han L. Han K. DiGregorio G.B. Katzenellenbogen J.A. Katzenellenbogen B.S. Roberson P.K. Weinstein R.S. Jilka R.L. Manolagas S.C. Cell. 2001; 104: 719-730Abstract Full Text Full Text PDF PubMed Google Scholar). Cell cells from were described (1Plotkin L.I. Weinstein R.S. Parfitt A.M. Roberson P.K. Manolagas S.C. Bellido T. J. Clin. Investig. 1999; 104: 1363-1374Crossref PubMed Google Scholar, Y. J. 1997; PubMed Google Scholar). from wild type or from were provided by A. F. Lau (University of at and described B.J. J.M. E. Lau A.F. Cell Growth 1997; 8: Google Scholar). of cells were with to the of ERK and to ERK2 in the with to the localization of the cells were in for by with for or were with or for the cells were in with the stimuli for to The of cells nuclear accumulation of was by cells in the nucleus with the a cells from by were for of cells were either by or by the nuclear of cells with or nRFP, described (1Plotkin L.I. 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We that the of both and induced in nuclear accumulation or transcription with a and Moreover, the of both agents at in of ERK phosphorylation In addition, or a of both agents apoptosis whereas with of either neither induced ERK phosphorylation nor of both agents and these not that and do not with the anti-apoptotic of but that, in these agents additive effects on ERK activation and The that, unlike stimuli that in gene the anti-apoptotic by the opening of Cx43 hemichannels by bisphosphonates in cells Instead, in this activation of the pro-apoptotic protein BAD and the of a domain in C/EBPβ of these events from of ERKs in the and in they are abolished when ERKs are restricted to the In addition, we that activation C/EBPβ leads to a and the of a domain which C/EBPβ to to and the activation of (14Buck M. Poli V. Hunter T. Chojkier M. Mol. Cell. 2001; 8: 807-816Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). 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