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• W2017293256 abstract "Bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-β (TGF-β) family, regulates osteoblast differentiation and bone formation. Here we show a novel function of BMP-2 in human osteoblasts and identify a signaling pathway involved in this function. BMP-2 promotes apoptosis in primary human calvaria osteoblasts and in immortalized human neonatal calvaria osteoblasts, as shown by terminal deoxynucleotidyl transferase-mediated nick end labeling analysis. In contrast, TGF-β2 inhibits apoptosis in human osteoblasts. Studies of the mechanisms of action showed that BMP-2 increases the Bax/Bcl-2 ratio, whereas TGβ-2 has a negative effect. Moreover, BMP-2 increases the release of mitochondrial cytochrome c to the cytosol. Consistent with these results, BMP-2 increases caspase-9 and caspase-3, -6, and -7 activity, and an anti-caspase-9 agent suppresses BMP-2-induced apoptosis. Overexpression of dominant-negative Smad1 effectively blocks BMP-2-induced expression of the osteoblast transcription factor Runx2 but not the activation of caspases or apoptosis induced by BMP-2, indicating that the Smad1 signaling pathway is not involved in the BMP-2-induced apoptosis. The proapoptotic effect of BMP-2 is PKC-dependent, because BMP-2 increases PKC activity, and the selective PKC inhibitor calphostin C blocks the BMP-2-induced increased Bax/Bcl-2, caspase activity, and apoptosis. In contrast, the cAMP-dependent protein kinase A inhibitor H89, the p38 MAPK inhibitor SB203580, and the MEK inhibitor PD-98059 have no effect. The results show that BMP-2 uses a Smad-independent, PKC-dependent pathway to promote apoptosis via a Bax/Bcl-2 and cytochromec-caspase-9-caspase-3, -6, -7 cascade in human osteoblasts. Bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-β (TGF-β) family, regulates osteoblast differentiation and bone formation. Here we show a novel function of BMP-2 in human osteoblasts and identify a signaling pathway involved in this function. BMP-2 promotes apoptosis in primary human calvaria osteoblasts and in immortalized human neonatal calvaria osteoblasts, as shown by terminal deoxynucleotidyl transferase-mediated nick end labeling analysis. In contrast, TGF-β2 inhibits apoptosis in human osteoblasts. Studies of the mechanisms of action showed that BMP-2 increases the Bax/Bcl-2 ratio, whereas TGβ-2 has a negative effect. Moreover, BMP-2 increases the release of mitochondrial cytochrome c to the cytosol. Consistent with these results, BMP-2 increases caspase-9 and caspase-3, -6, and -7 activity, and an anti-caspase-9 agent suppresses BMP-2-induced apoptosis. Overexpression of dominant-negative Smad1 effectively blocks BMP-2-induced expression of the osteoblast transcription factor Runx2 but not the activation of caspases or apoptosis induced by BMP-2, indicating that the Smad1 signaling pathway is not involved in the BMP-2-induced apoptosis. The proapoptotic effect of BMP-2 is PKC-dependent, because BMP-2 increases PKC activity, and the selective PKC inhibitor calphostin C blocks the BMP-2-induced increased Bax/Bcl-2, caspase activity, and apoptosis. In contrast, the cAMP-dependent protein kinase A inhibitor H89, the p38 MAPK inhibitor SB203580, and the MEK inhibitor PD-98059 have no effect. The results show that BMP-2 uses a Smad-independent, PKC-dependent pathway to promote apoptosis via a Bax/Bcl-2 and cytochromec-caspase-9-caspase-3, -6, -7 cascade in human osteoblasts. bone morphogenetic protein-2 transforming growth factor-β extracellular signal-regulated kinase cAMP-dependent protein kinase A mitogen-activated protein kinase TGF-β-activated kinase-1 immortalized human neonatal calvaria dominant negative recombinant human BMP-2 recombinant human TGF-β2 terminal deoxynucleotidyl transferase-mediated nick end labeling fetal calf serum protein kinase C benzyloxycarbonyl- phenylmethylketone mitogen-activated protein kinase/extracellular signal-regulated kinase kinase Bone formation is a complex process that involves the recruitment and proliferation of osteoprogenitor cells and their differentiation into osteoblasts (1Aubin J.E. Liu F. Principles of Bone Biology. Academic Press, Inc., San Diego, CA1996: 51-67Google Scholar, 2Stein G.S. Lian J.B. Stein J.L. Van Wijnen A.J. Montecino M. Physiol. Rev. 1996; 76: 593-629Crossref PubMed Scopus (400) Google Scholar, 3Marie P.J. Advances in Organ Biology. JAI Press, Stamford, CT1998: 445-473Google Scholar). At the end of the formation period, osteoblasts die by apoptosis, or programmed cell death, recognized by chromatin condensation, nuclear fragmentation, DNA degradation, and formation of membrane blebbing (4Manolagas S.C. Endocr. Rev. 2000; 21: 115-137Crossref PubMed Scopus (2028) Google Scholar). Recent observations indicate that apoptosis has a major impact on skeletal development and remodeling. Apoptosis is essential for the elimination of osteoblasts during skeletal development (5Garcia-Martinez V. Macias D. Ganan Y. Garcia-Lobo J.M. Francia M.V. Fernandez-Teran M.A. Hurle J.M. J. Cell Sci. 1993; 106: 201-208PubMed Google Scholar, 6Buckland R.A. Collinson J.M. Graham E. Davidson D.R. Hill R.E. Mech. Dev. 1998; 71: 143-150Crossref PubMed Scopus (79) Google Scholar, 7Furtwangler J.A. 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Bone Miner Res. 1998; 13: 793-802Crossref PubMed Scopus (472) Google Scholar). Some local regulatory cytokines are also known to modulate apoptosis in osteoblasts. Among them, tumor necrosis factor-α, interleukin-1 and -6, insulin-like growth factor-1, and fibroblast growth factor signaling induce pro- or antiapoptotic effects on osteoblasts (12Tsuboi M. Kawakami A. Nakashima T. Matsuoka N. Urayama S. Kawabe Y. Fujiyama K. Kiriyama T. Aoyagi T. Maeda K. Eguchi K. J. Lab. Clin. Med. 1999; 134: 222-231Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 13Hill P.A. Tumber A. Meikle M.C. Endocrinology. 1997; 138: 3849-3858Crossref PubMed Scopus (186) Google Scholar, 14Bellido T. O'Brien C.A. Roberson P.K. Manolagas S.C. J. Biol. Chem. 1998; 273: 21137-21144Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 15Mansukhani A. Bellosta P. Sahni M. Basilico C. J. Cell Biol. 2000; 149: 1297-1308Crossref PubMed Scopus (267) Google Scholar, 16Lemonnier J. Haÿ E. Delannoy Ph. Fromigué O. Lomri A. Modrowski M. Marie P.J. Am. J. Pathol. 2001; 158: 1833-1842Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), indicating an important role for local regulatory factors in the control of osteoblast apoptosis. Bone morphogenetic proteins (BMPs)1 are members of the transforming growth factor-β (TGF-β) family that play essential roles in osteogenesis (17Urist M.R. Science. 1965; 150: 893-899Crossref PubMed Scopus (4520) Google Scholar, 18Reddi A.H. Huggins C.B. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 1601-1605Crossref PubMed Scopus (685) Google Scholar, 19Wozney J.M. Mol. Reprod. Dev. 1992; 32: 160-167Crossref PubMed Scopus (616) Google Scholar). BMPs play a pivotal role in the commitment and differentiation of cells of osteoblastic lineage (20Yamaguchi A. Semin. Cell Biol. 1995; 6: 165-173Crossref PubMed Scopus (140) Google Scholar,21Marie P.J. J. Cell Eng. 1997; 2: 92-99Google Scholar). BMP-2, a prototype of BMPs, promotes osteoblast maturation by increasing the expression of the transcription factor Runx2, previously referred to as Cbfa1/PebpαA/AML3, and the expression of osteoblast marker genes (22Lee M.H. Javed A. Kim H.J. Shin H.I. Gutierrez S. Choi J.Y. Rosen V. Stein J.L. van Wijnen A.J. Stein G.S. Lian J.B. Ryoo H.M. J. Cell. Biochem. 1999; 73: 114-125Crossref PubMed Scopus (241) Google Scholar, 23Yamaguchi A. Katagiri T. Ikeda T. Wozney J.M. Rosen V. Wang E.A. Kahn A.J. Suda T. Yoshiki S. J. Cell Biol. 1991; 113: 681-687Crossref PubMed Scopus (657) Google Scholar, 24Harris S.E. Bonewald L.F. Harris M.A. Sabatini M. Dallas S. Feng J.Q. Ghosh-Choudhury N. Wozney J. Mundy G.R. J. Bone Miner Res. 1994; 9: 855-863Crossref PubMed Scopus (304) Google Scholar, 25Hay E. Hott M. Graulet A.M. Lomri A. Marie P.J. J. Cell. Biochem. 1999; 72: 81-93Crossref PubMed Scopus (83) Google Scholar). In nonskeletal cell types, BMPs were found to regulate apoptosis, although both pro- and antiapoptotic effects have been reported (26Marazzi G. Wang Y. Sassoon D. Dev. Biol. 1997; 186: 127-138Crossref PubMed Scopus (133) Google Scholar, 27Schmidt C. Christ B. Patel K. Brand-Saberi B. Dev. Biol. 1998; 202: 253-263Crossref PubMed Scopus (37) Google Scholar, 28Song Q. Mehler M.F. Kessler J.A. Dev. Biol. 1998; 196: 119-127Crossref PubMed Scopus (62) Google Scholar, 29Jernvall J. Aberg T. Kettunen P. Keranen S. Thesleff I. Development. 1998; 125: 161-169PubMed Google Scholar, 30Mohan R.R. Kim W.J. Mohan R.R. Chen L. Wilson S.E. Invest. Ophthalmol. Vis. Sci. 1998; 39: 2626-2636PubMed Google Scholar, 31Ferrari D. Lichtler A.C. Pan Z.Z. Dealy C.N. Upholt W.B. Kosher R.A. Dev. Biol. 1998; 197: 12-24Crossref PubMed Scopus (100) Google Scholar, 32Iantosca M.R. McPherson C.E. Ho S.Y. Maxwell G.D. J. Neurosci. Res. 1999; 56: 248-258Crossref PubMed Scopus (32) Google Scholar, 33Rodriguez-Leon J. Merino R. Macias D. Ganan Y. Santesteban E. Hurle J.M. Nat. Cell Biol. 1999; 1: 125-126Crossref PubMed Scopus (84) Google Scholar). Members of the Msx family or the cyclin-dependent kinase inhibitor p21 have been implicated in the proapoptotic activity of BMPs (26Marazzi G. Wang Y. Sassoon D. Dev. Biol. 1997; 186: 127-138Crossref PubMed Scopus (133) Google Scholar, 29Jernvall J. Aberg T. Kettunen P. Keranen S. Thesleff I. Development. 1998; 125: 161-169PubMed Google Scholar, 31Ferrari D. Lichtler A.C. Pan Z.Z. Dealy C.N. Upholt W.B. Kosher R.A. Dev. Biol. 1998; 197: 12-24Crossref PubMed Scopus (100) Google Scholar). However, the signaling pathways by which BMPs induce the death program remain largely unknown (34Merino R. Ganan Y. Macias D. Rodriguez-Leon J. Hurle J.M. Ann. N. Y. Acad. Sci. 1999; 887: 120-132Crossref PubMed Scopus (56) Google Scholar). Moreover, no evidence exists to suggest that BMPs have an induction role of apoptosis in osteoblasts. BMPs signal through type I and II serine/threonine kinase receptors that phosphorylate the downstream target proteins Smads. Activation of type I BMP receptor phosphorylates Smad1, Smad5, and presumably Smad8 and associates with Smad4 in a heteromeric complex that is translocated to the nucleus, where it activates transcription (35Heldin C.H Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3358) Google Scholar). TGF-β binding to receptors leads to phosphorylated Smad2 and Smad3, which associate with Smad4, and the complex can translocate to the nucleus to regulate transcriptional activity (36Derynck R. Zhang Y. Feng X.H. Cell. 1998; 95: 737-740Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar, 37Nakao A. Imamura T. Souchelnytskyi S. Kawabata M. Ishisaki A. Oeda E. Tamaki K. Hanai J. Heldin C.H. Miyazono K. ten Dijke P. EMBO J. 1997; 16: 5353-5362Crossref PubMed Scopus (916) Google Scholar, 38Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3999) Google Scholar). The Smad signaling pathway has been shown to play a role in BMP-2-induced osteoblast differentiation (39Yamamoto N. Akiyama S. Katagiri T. Namiki M. Kurokawa T. Suda T. Biochem. Biophys. Res. Commun. 1997; 238: 574-580Crossref PubMed Scopus (202) Google Scholar, 40Nishimura R. Kato Y. Chen D. Harris S.E. Mundy G.R. Yoneda T. J. Biol. Chem. 1998; 273: 1872-1879Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). However, other signaling pathways, such as extracellular signal-regulated kinase (ERK1/2), protein kinase C, and cAMP-dependent protein kinase A (PKA) (41Lee Y.S. Chuong C.M. J. Cell. Physiol. 1997; 170: 153-165Crossref PubMed Scopus (80) Google Scholar, 42Palcy S. Goltzman D. Biochem. J. 1999; 343: 21-27Crossref PubMed Scopus (54) Google Scholar, 43Lou J. Tu Y. Li S. Manske P.R. Biochem. Biophys. Res. Commun. 2000; 268: 757-762Crossref PubMed Scopus (133) Google Scholar) may also be involved in the BMP-induced effects on bone cells. Another cascade is activated by TGF-β and BMP-4 and involves TGF-β-activated kinase-1 (TAK1), a member of the mitogen-activated protein kinase (MAPK) kinase family, p38, and c-Jun N-terminal kinase (44Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1178) Google Scholar, 45Yamaguchi K. Nagai S. Ninomiya-Tsuji J. Nishita M. Tamai K. Irie K. Ueno N. Nishida E. Shibuya H. Matsumoto K. EMBO J. 1999; 18: 179-187Crossref PubMed Scopus (326) Google Scholar). The TAK1-p38 kinase pathway was recently found to be involved in BMP-2-induced apoptosis (44Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1178) Google Scholar, 46Kimura N. Matsuo R. Shibuya H. Nakashima K. Taga T. J. Biol. Chem. 2000; 275: 17647-17652Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). However, the molecular events underlying the effect of BMP-2 on apoptotic pathways in bone cells are not known. We have recently shown that BMP-2 has the capacity to induce osteoblast differentiation marker genes in primary human calvaria osteoblasts as well as in immortalized human neonatal calvaria (IHNC) osteoblastic cells (25Hay E. Hott M. Graulet A.M. Lomri A. Marie P.J. J. Cell. Biochem. 1999; 72: 81-93Crossref PubMed Scopus (83) Google Scholar, 47Hay E. Lemonnier J. Modrowski D. Lomri A. Lasmoles F. Marie P.J. J. Cell. Physiol. 2000; 183: 117-128Crossref PubMed Scopus (58) Google Scholar). In the present study, we have determined the effect of BMP-2 on apoptosis and investigated the signaling pathway that mediates the control of apoptosis by BMP-2 in human osteoblasts. We show here that BMP-2 promotes the cell death signaling pathway, whereas TGF-β2 inhibits apoptosis in human osteoblasts. Further experiments utilizing a dominant negative (DN) Smad1 vector showed that the apoptotic signal induced by BMP-2 in IHNC cells is independent of the Smad1 pathway. We also provide evidence that the BMP-2-induced apoptosis in human osteoblasts is mediated by activation of PKC, leading to activation of caspase-9, effector caspases, and DNA fragmentation. Establisment and characterization of primary human calvaria cells and IHNC cells have been previously described in detail (25Hay E. Hott M. Graulet A.M. Lomri A. Marie P.J. J. Cell. Biochem. 1999; 72: 81-93Crossref PubMed Scopus (83) Google Scholar, 47Hay E. Lemonnier J. Modrowski D. Lomri A. Lasmoles F. Marie P.J. J. Cell. Physiol. 2000; 183: 117-128Crossref PubMed Scopus (58) Google Scholar). Both primary human calvaria cells and IHNC cells express similar osteoblast phenotypic characteristics (alkaline phosphatase, type 1 collagen, the osteoblast transcription factor Runx2, and osteocalcin) (25Hay E. Hott M. Graulet A.M. Lomri A. Marie P.J. J. Cell. Biochem. 1999; 72: 81-93Crossref PubMed Scopus (83) Google Scholar, 47Hay E. Lemonnier J. Modrowski D. Lomri A. Lasmoles F. Marie P.J. J. Cell. Physiol. 2000; 183: 117-128Crossref PubMed Scopus (58) Google Scholar). Primary human calvaria cells and IHNC cells were cultured in Dulbecco's modified Eagle's medium supplemented with glutamine (292 mg/liter), 10% heat-inactivated fetal calf serum (FCS), and antibiotics (100 IU/ml penicillin and 100 µg/ml streptomycin). Previous studies showed that recombinant human BMP-2 (rhBMP-2) (kindly provided by Genetics Institute, Cambridge, MA) induces a dose-dependent stimulatory effect on alkaline phosphatase activity, an early osteoblast marker, with a maximal stimulatory effect at 50 ng/ml in these cells (25Hay E. Hott M. Graulet A.M. Lomri A. Marie P.J. J. Cell. Biochem. 1999; 72: 81-93Crossref PubMed Scopus (83) Google Scholar, 47Hay E. Lemonnier J. Modrowski D. Lomri A. Lasmoles F. Marie P.J. J. Cell. Physiol. 2000; 183: 117-128Crossref PubMed Scopus (58) Google Scholar). Subsequent studies were therefore performed at this optimal dose. To analyze the effect of serum withdrawal on cell proliferation and survival, IHNC cells were cultured for 24 h in the presence of 10% FCS or in serum-free conditions and then treated with rhBMP-2 or rhTGF-β2 for 24 h before detection of apoptosis. The cells cannot be cultured longer than 72 h in serum-deprived conditions, because they detach from the substrate. Truncated Rmad-1, the mouse homologue of human Smad1, was achieved by substitution of an AGC codon by the stop codon TAA at position 1623. This missmatch mutation causes a deletion of 118 base pairs in the NH2 domain, which is responsible for the nuclear assignment of the molecule. Truncated DNA was cloned in Neo pcDNA3.1 (+) (InVitrogen). The vector alone was used as control. Stable transfection cannot not be conducted in IHNC cells, because these cells do not escape senescence crisis. We thus performed transient transfections allowing effective expression of DN Smad1 for 72 h, which was sufficient to determine the changes in apoptosis induced by BMP-2 occurring at 24–48 h of treatment. Cells were plated at 5000 cells/cm2 the day before transfection. IHNC cells were cotransfected with the plasmid (15 µg/100-mm dish) and pSV-β-galactosidase control vector (Promega) at a 10:1 ratio, by calcium phosphate precipitation according to standard procedures described by the manufacturer (Profection mammalian transfection systems; Promega). After 16 h, the transfection medium was replaced with fresh medium (1% bovine serum albumin, serum-free) overnight. Efficiency of transfection was controlled by determination of β-galactosidase activity in transfected cells and by the expression of Smad1 in transfected cells by Western blot and immunocytochemical analyses. The number of β-galactosidase-positive cells and the number of cells showing absence of a nuclear Smad1 immunostaining were counted 72 h post-transfection. Apoptosis and the activity of caspases in transfected cells were determined as described below. To detect apoptotic nuclei, DNA cleavage was assessed by the TUNEL assay as described by the manufacturer (Roche Molecular Biochemicals). Primary human calvaria cells or IHNC cells (5000/cm2) cultured on Labtek chambers in serum-deprived conditions (1% BSA, serum-free) or in the presence of 10% FCS for 24 h were treated with rhBMP-2 or rhTGF-β2 for 24 h and then fixed with paraformaldehyde at room temperature for 5 min. Endogenous peroxidase was quenched with 3% H2O2, and the cells were permeabilized with 0.1% Triton X-100, at 4 °C for 2 min and incubated for 1 h at 37 °C with the TUNEL reaction mixture containing the terminal deoxynucleotidyl transferase. Incorporated fluorescein was detected by sheep anti-fluorescein antibody conjugated with horseradish peroxidase. The TUNEL signal was detected with peroxidase-labeled antidigoxigenin antibody, revealed with diaminobenzidine, and mounted. TUNEL-positive cells were detected by brown nuclei and nuclear fragmentation. Positive controls consisted of cells treated for 24 h with 50 µm etoposide, a topoisomerase II inhibitor that induces DNA damage and nuclear fragmentation associated with apoptosis (48Ritke M.K. Rusnak J.M. Lazo J.S. Allan W.P. Dive C. Heer S. Yalowich J.C. Mol. Pharmacol. 1994; 46: 605-611PubMed Google Scholar). Additional positive controls consisted of cells treated with DNase I for 10 min. Negative controls were obtained by omitting the transferase from the reaction. In each experiment, the number of total and TUNEL-positive cells was counted. To further determine cell viability in vitro, trypan blue staining was used for determination of dead cells by dye exclusion. After the addition of trypan blue (0.4%), the percentage of primary human calvaria cells or IHNC cells exhibiting both nuclear and cytoplasmic trypan blue staining (dead cells) was determined. A total of 1500 cells were counted for each cell type, and the results were expressed as a percentage of total cells. IHNC cells (10,000/cm2) cultured in the presence or absence of rhBMP-2 were washed twice with cold phosphate-buffered saline and scrapped in 1 ml of ice-cold lysis buffer (10 mm Tris-HCl, 5 mm EDTA, 150 mm NaCl, 30 mm sodium pyrophosphate, 50 mm NaF, and 1 mmNa3VO4) containing 10% glycerol and protease inhibitors (Roche Molecular Biochemicals). Protein samples were solubilized in 4× Laemmli SDS loading buffer and boiled at 95 °C for 5 min. Fifty micrograms of proteins, determined using the DC Protein assay (Bio-Rad), were resolved on 12% acrylamide gel and then transferred onto polyvinylidene difluoride-Hybond-P membranes (Amersham Pharmacia Biotech). Blots were saturated overnight with 1% blocking solution (Roche Molecular Biochemicals) in TBS buffer (50 mm Tris-HCl, 150 mm NaCl) and 0.1% Tween 20. Membranes were then incubated with mouse monoclonal anti-Cbfa1/Osf2 (49Xiao G. Wang D. Benson M.D. Karsenty G. Franceschi R.T. J. Biol. Chem. 1998; 273: 32988-32994Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar), polyclonal anti-human Bax (0.5 µg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), monoclonal anti-Bcl-2 (0.5 µg/ml; Santa Cruz Biotechnology), monoclonal anti-Smad1 (0.5 µg/ml; Santa Cruz Biotechnology), or polyclonal anti-β-actin (1.5 µg/ml; Sigma) in 0.5% blocking buffer. After 1 h at room temperature, the membranes were washed twice with TBS plus 0.1% Tween 20 and 0.5% blocking buffer and incubated for 1 h with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Following incubation with appropriate secondary antibodies, the membranes were washed, and the signals were visualized with BM chemiluminescence blotting substrate (Roche Molecular Biochemicals). The specific bands on the autoradiograms were quantitated by densitometry. IHNC cells were treated with rhBMP-2 for 24 h, and mitochondrial and cytosolic fractions were prepared by differential centrifugation in buffer containing sucrose as described (50Yang J. Liu X. Kim C.N. Ibrado A.M. Cai J. Peng T.I. Jones D.P. Wang X. Science. 1997; 275: 1081-1082Crossref PubMed Scopus (4422) Google Scholar). Protein samples (400 µg) were loaded on SDS-15% polyacrylamide gels, subjected to electrophoresis, and then transferred to nitrocellulose membranes. Western blots were probed with primary rabbit polyclonal anti-cytochrome c antibody or a monoclonal mouse antibody recognizing Cox-4, a component of the mitochondrial membrane (CLONTECH) and then probed with appropriate secondary horseradish peroxidase-conjugated antibodies and developed with BM chemiluminescence blotting substrate (Roche Molecular Biochemicals). To identify the caspases involved in rhBMP-2-induced apoptosis, IHNC cells (10,000/cm2) were cultured in 1% bovine serum albumin/serum-free medium in the presence or absence of rhBMP-2 (50 ng/ml). After 24 h, the cells were lysed in 400 µl of lysis buffer (10 mm Tris, pH 7.4, 200 mm NaCl, 5 mm EDTA, 10% glycerol, 1% Nonidet P-40) for 30 min on ice and stored at −20 °C. The activity of effector caspases (caspase-3, -6, and -7) and initiator caspases (caspase-8 and -9) was determined by the cleavage of synthetic fluorogenic substrates containing the amino acid sequence recognized by specific caspases. The substrates were as follows: DEVD (Asp-Glu-Val-Asp) for caspase-3-like; IETD (Ile-Glu-Thr-Asp) for caspase-8; LEHD (Leu-Glu-His-Asp) for caspase-9, combined with a fluorophore (7-amino-4-methylcoumarin). Upon cleavage of the substrate by caspases, free 7-amino-4-methylcoumarin fluorescence emission was detected using a spectrofluorimeter. For the assay, aliquots of 100 µl were incubated for 2 h at 37 °C with 200 µl of reaction buffer (0.1 mmphenylmethylsulfonyl fluoride, 10 mm dithiothreitol, 10 mm Hepes/NaOH, pH 7.4) containing 5 µl of specific substrate (20 µm final concentration). The fluorescence released in samples was measured by excitation at 367 nm, and analysis was made at 440 nm. The negative control was buffer mix, and the positive control was free 7-amino-4-methylcoumarin (10 µm in phosphate-buffered saline). Results were expressed as arbitrary units and corrected for protein content. To further determine the role of caspases in the BMP-2-induced apoptosis, cells were treated with rhBMP-2 (50 ng/ml) for 24 h in the presence of specific caspase-3, -6, -7, -8, or -9 inhibitors (10 µg/ml), and the number of TUNEL-positive apoptotic cells was determined as described above. For direct analysis of PKC activation, IHNC cells (10,000/cm2) were cultured in Dulbecco's modified Eagle's medium with 0% FCS plus 1% bovine serum albumin for 24 h and then treated with rhBMP-2 (50 ng/ml) or the vehicle for 10–60 min. The cells were lysed in lysis buffer (25 mmTris-HCl (pH 7.4), 0.5 mm EDTA, 0.5 mm EGTA, 0.05% Triton X-100, 10 mm β-mercaptoethanol, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 10 mm phenylmethylsulfonyl fluoride). PKC activity was determined by measuring the transfer of32P-labeled phosphate to a biotinylated peptide substrate (AAKIQASFRGHMARKK) that is specific for PKC activity (51Chen S.J. Klann E. Gower M.C. Powell C.M. Sessoms J.S. Sweatt J.D. Biochemistry. 1993; 32: 1032-1039Crossref PubMed Scopus (70) Google Scholar) using the Signa TECTTM PKC Assay System (Promega). To determine the signal transduction pathways involved in rhBMP-2-induced apoptosis, we used selective inhibitors of signaling pathways. We used calphostin C (Biomol Research Laboratories, Plymouth, PA), a potent and selective inhibitor of PKC (52Tamaoki T. Nakano H. Bio/Technology. 1990; 8: 732-735Crossref PubMed Scopus (248) Google Scholar); 2′-amino-3-methoxyflavone (PD-98059; Biomol), a specific inhibitor of MEK activation (53Alessi D.R. Cuenda P. Cohen D.T. Dudley D.T. Satiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3259) Google Scholar); and 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazol (SB 203580; Calbiochem), a highly specific inhibitor of p38 MAPK (54Kramer R.M. Roberts E.F. Um S.L. Borsh-Haubold A.G. Watson S.P. Fisher M.J. Jakubowski J.A. J. Biol. Chem. 1996; 271: 27723-27729Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). IHNC cells were pretreated for 2 h with the indicated signaling inhibitor or the vehicle and then treated with rhBMP-2 (50 ng/ml) or the vehicle in the presence of the inhibitor or the vehicle for 24 h. Apoptotic cells were then detected by TUNEL analysis as described above. We previously showed that rhBMP-2 promotes osteoblast marker genes and differentiation in primary human calvaria osteoblasts as well as in the corresponding IHNC cell line (25Hay E. Hott M. Graulet A.M. Lomri A. Marie P.J. J. Cell. Biochem. 1999; 72: 81-93Crossref PubMed Scopus (83) Google Scholar, 47Hay E. Lemonnier J. Modrowski D. Lomri A. Lasmoles F. Marie P.J. J. Cell. Physiol. 2000; 183: 117-128Crossref PubMed Scopus (58) Google Scholar). To determine whether BMP-2 induces apoptosis in osteoblasts, we tested the effect of BMP-2 on DNA fragmentation revealed by TUNEL analysis in normal primary human calvaria osteoblasts as well as in IHNC cells. As shown in Fig.1, A and C, treatment with rhBMP-2 (50 ng/ml) for 24 h increased by 2-fold the number of TUNEL-positive apoptotic cells in primary human calvaria osteoblasts. TUNEL positivity in these cells reflected true apoptosis because all cells treated with etoposide were TUNEL-positively stained, confirming the validity of the TUNEL assay for detection of true apoptosis (Fig. 1 A). In contrast to the effect of rhBMP-2, treatment with rhTGF-β2 (10 ng/ml) decreased by 2-fold the number of TUNEL-positive primary human calvaria osteoblasts (Fig. 1, Aand C). In the IHNC cell line, the basal number of apoptotic cells was higher than in primary human calvaria cells, as expected from this immortalized cell line (Fig. 1, B and C). In these cells, BMP-2 also had a proapoptotic effect. rhBMP-2 increased the number of TUNEL-positive IHNC cells, and rhTGF-β2 reduced the number of apoptotic cells, confirming the effect of these factors documented in primary human calvaria osteoblasts (Fig. 1, Band C). To know if BMP-2-induced apoptosis was associated with decreased cell viability, we examined the effect of rhBMP-2 using trypan blue staining. We found that rhBMP-2 (50 ng/ml, 24 h) increased by 42% the number of unviable trypan blue-stained IHNC cells (controls: 15 ± 0.6% (S.E.) versus +rhBMP-2: 21.3 ± 1.6%, p < 0.05). In contrast, rhTGF-β2 (10 ng/ml, 24 h) decreased the number of trypan blue-stained IHNC cells (+rhTGF-β2: 12.5 ± 0.8% versus controls: 15 ± 0.6%, p < 0.05). Additional experiments using ethidium bromide/acridine orange staining indicated that BMP-2 induced apoptosis but not necrosis (not shown). To determine the influence of serum withdrawal on the effect of BMP-2 on cell survival, IHNC cells were grown in the presence or absence of serum fo" @default.
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• W2017293256 title "Bone Morphogenetic Protein-2 Promotes Osteoblast Apoptosis through a Smad-independent, Protein Kinase C-dependent Signaling Pathway" @default.
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• W2017293256 doi "https://doi.org/10.1074/jbc.m011265200" @default.
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