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Originally published In Press as doi:10.1074/jbc.M011265200 on June 6, 2001

J. Biol. Chem., Vol. 276, Issue 31, 29028-29036, August 3, 2001
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Bone Morphogenetic Protein-2 Promotes Osteoblast Apoptosis through a Smad-independent, Protein Kinase C-dependent Signaling Pathway*

Eric HayDagger, Jérome Lemonnier, Olivia Fromigué, and Pierre J. Marie§

From the Laboratory of Osteoblast Biology and Pathology, INSERM U 349, Affiliated CNRS, Lariboisière Hospital, 75475 Cedex 10 Paris, France

Received for publication, December 14, 2000, and in revised form, May 22, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-beta (TGF-beta ) 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-beta 2 inhibits apoptosis in human osteoblasts. Studies of the mechanisms of action showed that BMP-2 increases the Bax/Bcl-2 ratio, whereas TGbeta -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 cytochrome c-caspase-9-caspase-3, -6, -7 cascade in human osteoblasts.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bone formation is a complex process that involves the recruitment and proliferation of osteoprogenitor cells and their differentiation into osteoblasts (1-3). 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 (4). 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 (5-9). Moreover, the frequency of osteoblast apoptosis controls osteoblast lifespan and bone formation during the postnatal life (10). Osteotropic hormones that regulate osteoblastic cell proliferation and differentiation were found to control osteoblast apoptosis (10, 11). Some local regulatory cytokines are also known to modulate apoptosis in osteoblasts. Among them, tumor necrosis factor-alpha , interleukin-1 and -6, insulin-like growth factor-1, and fibroblast growth factor signaling induce pro- or antiapoptotic effects on osteoblasts (12-16), 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-beta (TGF-beta ) family that play essential roles in osteogenesis (17-19). BMPs play a pivotal role in the commitment and differentiation of cells of osteoblastic lineage (20, 21). BMP-2, a prototype of BMPs, promotes osteoblast maturation by increasing the expression of the transcription factor Runx2, previously referred to as Cbfa1/Pebpalpha A/AML3, and the expression of osteoblast marker genes (22-25). In nonskeletal cell types, BMPs were found to regulate apoptosis, although both pro- and antiapoptotic effects have been reported (26-33). Members of the Msx family or the cyclin-dependent kinase inhibitor p21 have been implicated in the proapoptotic activity of BMPs (26, 29, 31). However, the signaling pathways by which BMPs induce the death program remain largely unknown (34). 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 (35). TGF-beta 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 (36-38). The Smad signaling pathway has been shown to play a role in BMP-2-induced osteoblast differentiation (39, 40). However, other signaling pathways, such as extracellular signal-regulated kinase (ERK1/2), protein kinase C, and cAMP-dependent protein kinase A (PKA) (41-43) may also be involved in the BMP-induced effects on bone cells. Another cascade is activated by TGF-beta and BMP-4 and involves TGF-beta -activated kinase-1 (TAK1), a member of the mitogen-activated protein kinase (MAPK) kinase family, p38, and c-Jun N-terminal kinase (44, 45). The TAK1-p38 kinase pathway was recently found to be involved in BMP-2-induced apoptosis (44, 46). 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 (25, 47). 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-beta 2 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Treatments-- Establisment and characterization of primary human calvaria cells and IHNC cells have been previously described in detail (25, 47). 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) (25, 47). 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 (25, 47). 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-beta 2 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.

Plasmids and Transfection-- 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-beta -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 beta -galactosidase activity in transfected cells and by the expression of Smad1 in transfected cells by Western blot and immunocytochemical analyses. The number of beta -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.

Detection of Apoptotic Cells-- 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-beta 2 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 (48). 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.

Western Blot Analysis-- 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 mM Na3VO4) 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 (49), 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-beta -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.

Cytochrome c Measurements-- 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 (50). 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).

Determination of Caspase Activity-- 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 mM phenylmethylsulfonyl 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.

Protein Kinase Assay-- 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 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 0.5 mM EGTA, 0.05% Triton X-100, 10 mM beta -mercaptoethanol, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 10 mM phenylmethylsulfonyl fluoride). PKC activity was determined by measuring the transfer of 32P-labeled phosphate to a biotinylated peptide substrate (AAKIQASFRGHMARKK) that is specific for PKC activity (51) using the Signa TECTTM PKC Assay System (Promega).

Selective Inhibition of Signaling Pathways-- 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 (52); 2'-amino-3-methoxyflavone (PD-98059; Biomol), a specific inhibitor of MEK activation (53); and 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazol (SB 203580; Calbiochem), a highly specific inhibitor of p38 MAPK (54). 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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

BMP-2 Promotes Apoptosis in Osteoblasts-- 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 (25, 47). 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. 1A). In contrast to the effect of rhBMP-2, treatment with rhTGF-beta 2 (10 ng/ml) decreased by 2-fold the number of TUNEL-positive primary human calvaria osteoblasts (Fig. 1, A and 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-beta 2 reduced the number of apoptotic cells, confirming the effect of these factors documented in primary human calvaria osteoblasts (Fig. 1, B and 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-beta 2 (10 ng/ml, 24 h) decreased the number of trypan blue-stained IHNC cells (+rhTGF-beta 2: 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).


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  		Fig. 1.   BMP-2 promotes apoptosis in human osteoblasts. Primary human calvaria cells (A) and IHNC cells (B) were treated with rhTGF-beta 2 (10 ng/ml), rhBMP-2 (50 ng/ml), or the DNA-damaging agent etoposide (50 µM) for 24 h and stained with TUNEL (arrows), and the percentage of cells that were TUNEL-positive was counted (C). All cells treated with etoposide were TUNEL-positive. Data are the mean ± S.E. of 3-5 cultures. a, a significant difference with controls; b, a significant difference with rhBMP-2-treated cells (p < 0.05). D and E show that BMP-2 induces apoptosis independently of the presence of serum or cell growth. IHNC cells were cultured in the presence (10% FCS) or absence (0% FCS) of serum from 48 to 72 h of culture and then treated with rhBMP-2 (50 ng/ml) for 24 h, and the total cell number (D) or TUNEL-positive cells (E) were counted. Data are the mean ± S.E. of four cultures. a, values significantly different from rhBMP-2-untreated cells (p < 0.05).

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 for 24 h and treated with rhBMP-2 for 24 h. As shown in Fig. 1D, IHNC cells continued to proliferate from 24 to 48 h after serum withdrawal, whereas the presence of 10% serum enhanced cell growth. As shown in Fig. 1E, serum withdrawal for 24 h increased the number of TUNEL-positive cells. The addition of rhBMP-2 for 24 h further enhanced apoptosis either in the presence or in the absence of serum (Fig. 1E). These observations indicate that BMP-2 induces apoptosis in human osteoblasts independently of the presence of serum growth factors or of cell growth.

BMP-2 Promotes Osteoblast Apoptosis through Caspase-9-- To investigate the downstream events involved in BMP-2-induced apoptosis, we first analyzed the changes in the proapoptotic protein Bax and anti-apoptotic protein Bcl-2 levels. Western blot analysis showed that treatment of IHNC cells with rhBMP-2 increased Bax protein levels by 66%. In contrast, rhTGF-beta 2 had no effect (Fig. 2A). Additionally, rhBMP-2 markedly decreased Bcl-2 levels, which led to an increase in the Bax/Bcl-2 ratio by 600% (Fig. 2B). Another signaling pathway leading to apoptosis involves the release of cytochrome c into the cytoplasm (50). The released cytochrome c cooperates with molecules such as Apaf-1 to activate caspase-9 and thereby caspase-3 (55, 56). To determine if BMP-2-induced apoptosis in osteoblasts involves cytochrome c, the amounts of cytochrome c in mitochondria and cytosol were determined by Western blot analysis. As shown in Fig. 2C, the BMP-2-induced apoptosis was associated with decreased mitochondrial cytochrome c content and increased cytochrome c release in the cytosol. Cox-4, a mitochondrial marker used as a control of purity of the biological samples, was not found in the cytoplasm but was present in the mitochondrial extracts, validating the purity of the preparation. This indicates that the induction of apoptosis by BMP-2 in human osteoblasts is associated with mitochondrial release of cytochrome c.


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  		Fig. 2.   Effects of BMP-2 on Bax and Bcl-2 protein levels and cytochrome c release. A, IHNC cells were treated with rhBMP-2 (50 ng/ml) or rhTGF-beta 2 (10 ng/ml) for 24 h, and the levels of the proapoptotic protein Bax and the antiapoptotic protein Bcl-2 were determined by Western blot analysis, scanned by densitometric analysis, and corrected for beta -actin. B, distinct effects of BMP-2 and TGF-beta 2 on Bax/Bcl-2 ratio. C, Western blot analysis showing that rhBMP-2 induced release of cytochrome c from the mitochondria to the cytoplasm. The mitochondrial component Cox-4 was used as control of purity of the biological samples. Results shown are representative of at least two independent experiments.

Changes in Bax and Bcl-2 are known to balance apoptotic signals through activation of caspases (57, 58). We thus investigated the implication of initiator caspases and effector caspases in BMP-2-induced apoptosis. Biochemical analyses showed that treatment with rhBMP-2 increased caspase-9 activity in IHNC cells, consistent with the release of cytochrome c in the cytosol (Fig. 3A). Furthermore, rhBMP-2 increased effector caspase-3, -6, and -7 activity. This effect was dose-dependent in a range of 10-100 ng/ml (not shown) with a maximal effect at 50 ng/ml (Fig. 3B). In contrast, rhTGF-beta 2 decreased effector caspase-3, -6, and -7 activity (Fig. 3B), consistent with its inhibitory effect on apoptosis (Fig. 1, A-C). To confirm the implication of caspases in the proapoptotic effect of BMP-2, IHNC cells were treated with rhBMP-2 in the presence of specific caspase inhibitors. As shown in Fig. 3C, the stimulatory effect of rhBMP-2 on the number of apoptotic cells was suppressed by the anti-caspase-9 Z-LEHD-FMK, confirming the implication of caspase-9 activation in BMP-2-induced apoptosis. In contrast, the anti-caspase-8 agent (Z-IETD-FMK) had no effect on rhBMP-2-induced apoptosis (Fig. 3D). Caspase-9 is known to promote effector caspases leading to apoptosis (59). Accordingly, treatment with the broad anti-effector caspase-3, -6, and -7 Z-DEVD-FMK compound suppressed the stimulatory effect of rhBMP-2 on apoptosis (not shown). These data indicate that osteoblast apoptosis induced by BMP-2 involves activation of caspase-9, but not caspase-8, and subsequent activation of effector caspase-3, -6, and -7 in human osteoblasts.


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  		Fig. 3.   BMP-2-induced apoptosis involves caspase-9 activation. IHNC cells were treated with rhBMP-2 (50 ng/ml) or rhTGF-beta 2 (10 ng/ml) for 24 h, and caspase-9 (A) and caspase-3, -6, and -7 (B) activities were determined. C, the percentage of TUNEL-positive apoptotic cells was determined in IHNC cells treated with rhBMP-2 (+rhBMP-2) or the vehicle (-rhBMP-2), in the absence (Control) or presence of specific caspase-9 inhibitor (Z-LEDH-FMK; 10 µg/ml) or caspase-8 inhibitor (Z-IETD-FMK; 10 µg/ml). The data are the mean ± S.E. of four values. *, p < 0.05 versus corresponding nonrhBMP-2-treated cells. Results shown are representative of at least two independent experiments. Note that caspase-9 inhibitor abolished the proapoptotic effect of BMP-2.

Forced Expression of Dominant Negative Smad1 Suppresses BMP-2-induced Runx2 but Not BMP-2-induced Apoptosis in IHNC Cells-- We then determined the signaling pathway that mediates the BMP-2-induced apoptosis in human osteoblasts. One known signaling pathway implicated in the BMP-2 promoting effect on murine osteoblast differentiation involves Smad proteins (39, 40). Smad1 is required for the BMP-2 inducing effect on osteoblast differentiation (39, 60). To investigate whether the Smad pathway is involved in BMP-2-induced apoptosis, we established transfectants with a dominant negative Smad1 expression vector and determined Smad1 expression. The efficiency of transfection was first validated by determination of beta -galactosidase activity. As shown in Fig. 4A, beta -galactosidase was expressed in about one-third of transfected cells (arrows). Quantification of labeled cells revealed that the number of beta -galactosidase-positive cells was 29.7 ± 2.0% (S.E.) after 72 h of transfection, indicating persistent transfection efficiency at this late time point. To confirm the efficiency of the transient transfection, we examined nuclear Smad1 staining revealed by immunocytochemistry. As shown in Fig. 4A, cells transfected with the pcDNA vector alone present nuclear Smad1 staining, whereas DN Smad1-transfected cells had reduced expression of Smad1 in the nucleus (arrows). Quantification of labeled cells showed that the fraction of DN Smad1-transfected cells without nuclear Smad1 staining was 31.2 ± 2.5% compared with 8.9 ± 0.8% in pcDNA-transfected cells (p < 0.001). This different nuclear Smad1 immunostaining was found in different experiments at 72 h post-transfection, showing efficiency of the DN Smad1 transfection at this late time point. Moreover, Western blot analysis using an antibody recognizing the Smad1 protein domain that has not been truncated (T) showed increased Smad1 levels in IHNC cells forced to express the DN Smad1 vector compared with pcDNA-transfected cells at 48 h post-transfection (Fig. 4B). These results confirm the data shown in Fig. 4A, indicating that transfected cells overexpressed the DN Smad1 protein at 72 h post-transfection.


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  		Fig. 4.   Forced expression of DN Smad1 suppresses Cbfa1/Runx2 expression. A, IHNC cells were transfected with truncated Smad1 (T-Smad1) or pcDNA control vector. Efficiency of transfection was assessed by the expression of beta -galactosidase (arrows) and by the absence of nuclear Smad1 staining in DN Smad1-transfected cells compared with pcDNA-transfected cells, revealed by immunocytochemistry at 72 h post-transfection. (arrows). B, Western blot analysis showing Smad1 protein levels in transfected cells at 48 h post-transfection. C, IHNC cells transfected with pcDNA or T-Smad1 were treated with rhBMP-2 (50 ng/ml) or the vehicle for 24 h and Cbfa1/Osf2 protein levels were determined by Western blot analysis and corrected for beta -actin. Results shown are representative of at least two independent experiments. Note that the promoting effect of rhBMP-2 on Cbfa1 protein levels was abolished in cells overexpressing truncated Smad1.

To establish that the forced expression of the DN Smad1 was functional in transfected cells, we examined the expression of the osteoblast transcription factor Runx2 in response to BMP-2. The expression of Runx2 is promoted by BMP-2 (22, 47), and this factor is essential for the expression of several osteoblast differentiation marker genes (61). As shown in Fig. 4C, treatment with rhBMP-2 for 24 h increased Runx2 protein levels by 2-fold in IHNC cells but not in cells overexpressing the DN Smad1 vector. The BMP-2-induced-Runx2 expression was decreased by about 30% in DN Smad1-transfected cells, which is consistent with the 30% transfection efficiency and with the decreased Smad1 nuclear localization in the DN Smad1-transfected cells (Fig. 4C). Thus, overexpression of the DN Smad1 effectively blocked the BMP-2-induced expression of the osteoblast transcription factor Runx2 in IHNC cells, indicating that Smad1 is required for expression of this transcription factor in human osteoblasts.

We then examined whether overexpression of DN Smad1 in transfected IHNC cells suppressed BMP-2-induced apoptosis. As shown in Fig. 5A, rhBMP-2 increased apoptosis similarly in IHNC cells overexpressing the DN Smad1 and in cells transfected with the vector alone, as revealed by TUNEL analysis. This suggests that Smad1 is not required for apoptosis induced by BMP-2. To confirm this finding, we examined the changes in caspase-9 and downstream effector caspases in IHNC cells overexpressing DN Smad1. As shown in Fig. 5B, treatment with rhBMP-2 increased caspase-9 activity to the same levels in DN Smad1-transfected cells and vector-transfected cells. Similarly, rhBMP-2 increased caspase-3, -6, and -7 activity to the same extent in DN Smad1 and pcDNA-transfected cells (Fig. 5C). These data indicate that, although the transient transfection with DN Smad1 reduced Runx2 expression, neither caspase activity nor apoptosis induced by BMP-2 was abolished, which supports the assertion that BMP-2-induced apoptosis is independent of Smad1 signal transduction pathway.


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  		Fig. 5.   Overexpression of dominant negative Smad1 does not inhibit BMP-2-induced apoptosis. A, IHNC cells transfected with truncated Smad1 (T-Smad1) or pcDNA vector were treated with rhBMP-2 (50 ng/ml) or the vehicle (Control) for 24 h, and the number of apoptotic cells was determined (A). Caspase-9 activity (B) and caspase-3, -6, and -7 (caspase-like) activity (C) were also measured. The data are the mean ± S.E. of four values. *, p < 0.05 versus control cells. Results shown are representative of at least two independent experiments. Note that the proapoptotic effect of rhBMP-2 was similar in cells overexpressing truncated Smad1 and in pcDNA-transfected cells.

Effects of MAPK Inhibitors on BMP-2-induced Apoptosis-- The results described above suggest the existence of a signaling pathway other than the Smad pathway for the proapoptotic effect of BMP-2 in IHNC cells. We therefore examined which pathway might be involved in the BMP-2-induced apoptosis in IHNC cells. Signaling pathways including PKA, PKC, and p38 MAPK have been suggested to be involved in apoptosis induced by various stimulations (46, 62-67). Moreover, PKA, PKC, and p38 MAPK have been reported to mediate BMP effects on osteoblast differentiation (68, 69). We thus examined whether the BMP-2-induced apoptosis may be mediated by activation of these pathways using specific kinase inhibitors. As shown in Fig. 6A, pretreatment of IHNC cells with calphostin C, a selective inhibitor of PKC, completely blocked the promoting effect of rhBMP-2 on the number of TUNEL-positive cells. In contrast, pretreatment with the p38 MAPK inhibitor SB203580 did not block the BMP-2-induced apoptosis in IHNC cells (Fig. 6B). Likewise, PD-98059, which inhibits MEK, a kinase that lies upstream of ERK, did not suppress apoptosis induced by BMP-2. The PKA inhibitor H89 also had no effect on BMP-2-induced apoptosis (Fig. 6B). These data suggest that PKC but not PKA, p38 MAPK, or MEK is involved in the apoptosis induced by BMP-2 in osteoblastic cells.


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  		Fig. 6.   Distinct effects of signaling inhibitors on BMP-2-induced apoptosis. IHNC cells were pretreated for 2 h with calphostin C (2 µM), a specific inhibitor of PKC (A); SB203580 (25 µM), a specific p38 inhibitor; PD-98059 (25 µM), a specific inhibitor of ERK pathway; or H89 (25 µM), a specific PKA inhibitor (B) or the vehicle and then treated with rhBMP-2 (50 ng/ml) or the vehicle (Control) in the presence of the inhibitor or the vehicle for 24 h, and the percentage of TUNEL-positive apoptotic cells was recorded. The data are the mean ± S.E. of four values. *, p < 0.05 versus control cells. Results shown are representative of at least two independent experiments. Note that calphostin C but not other inhibitors abolished rhBMP-2-induced apoptosis.

Essential Role of PKC in BMP-2-induced Apoptotic Mechanisms in IHNC Cells-- We then examined whether treatment of IHNC cells with BMP-2 leads to direct activation of PKC. Fig. 7 shows that treatment of IHNC cells with rhBMP-2 significantly enhanced the protein kinase activity of PKC at 5 and 10 min. In contrast, rhTGF-beta 2 had no effect on PKC activity. These results indicate that BMP-2 transiently activates the PKC pathway in IHNC cells. In order to further confirm the role of PKC in BMP-2-induced apoptosis in osteoblasts, we examined the effect of selective PKC inhibitors on the mechanisms involved in the proapoptotic effect of BMP-2. Western blot analysis confirmed that rhBMP-2 increased the Bax/Bcl-2 ratio in IHNC cells. The amplitude of increase differed from our previous results (Fig. 2) due to the distinct solvents (Me2SO versus Dulbecco's modified Eagle's medium) used in the two types of experiments. We found that, in the presence of the PKC inibitor calphostin C, the promoting effect of rhBMP-2 on Bax protein level was suppressed (Fig. 8A). Furthermore, the rhBMP-2-induced inhibitory effect on Bcl-2 level was abolished by calphostin C (Fig. 8A). As a result, the promoting effect of rhBMP-2 on the Bax/Bcl-2 ratio was blocked by calphostin C (Fig. 8A). To further document the implication of PKC in the BMP-2-induced apoptosis, we determined the effect of PKC inhibitors on the activity of caspase-9 and downstream effector caspase-3, -6, and -7. As shown in Fig. 8B, pretreatment with calphostin C completely blocked the induction of caspase-9 activity by rhBMP-2. Moreover, calphostin C reduced the basal caspase-3, -6, and -7 activity and abolished the effector caspase activity promoted by rhBMP-2 (Fig. 8C). These results indicate that PKC plays an essential role in the proapoptotic effect of BMP-2 in IHNC cells.


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  		Fig. 7.   BMP-2 transiently promotes PKC activity. IHNC cells were treated with rhBMP-2 (50 ng/ml), rhTGF-beta 2 (10 ng/ml), or the vehicle (Control), and PKC activity was determined by the transfer of 32P-labeled phosphate to a biotinylated peptide substrate that is specific for PKC activity. The data are the mean ± S.E. of four values. *, p < 0.05 versus control cells.


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  		Fig. 8.   Inhibition of PKC suppresses downstream events involved in BMP-2-induced apoptosis. IHNC cells were pretreated for 2 h with calphostin C (2 µM) or the vehicle and then treated with rhBMP-2 (50 ng/ml) or the vehicle (control) for 24 h in the presence or absence of the PKC inhibitor calphostin C. A, the levels of the proapoptotic protein Bax and antiapoptotic protein Bcl-2 were determined by Western blot, and the levels were corrected for beta -actin. B, caspase-9 and caspase-3, -6, and -7 activities were determined. The data are the mean ± S.E. of four values. *, p < 0.05 versus control cells. Note that calphostin C suppressed the effect of BMP-2 on Bax/Bcl-2 and caspase activities.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

BMPs are multifunctional proteins that play an essential role in the control of osteoblast differentiation. We show in this report a novel function of BMP-2 in osteoblasts; BMP-2 induces molecular events leading to apoptosis in human osteoblasts, an effect that is mediated by activation of PKC. We first demonstrated that BMP-2 increases the number of apoptotic cells in primary human calvaria osteoblasts as well as in immortalized human calvaria osteoblasts and that this effect is independent of serum growth factors and cell growth. The proapoptotic effect of BMP-2 clearly differs from that of TGF-beta 2, which has an antiapoptotic effect, suggesting a distinct regulatory role of these members of the TGF-beta superfamily on human osteoblast survival. This is consistent with our previous finding showing that BMP-2 and TGF-beta have antagonistic effects on cell proliferation and osteoblast marker gene expression in human osteoblasts (70). Although BMP-2 was previously found to exert pro- and antiapoptotic effects in other cell types (28, 32), our data are the first to show that BMP-2 is involved in the control of apoptosis in human osteoblasts.

Apoptosis is a multiple step process implicating upstream induction phases and downstream execution stages (71-74). We studied the nature of the pathways involved in the BMP-2-induced-apoptosis in IHNC cells. One upstream pathway that plays a central role in controlling cell death involves the apoptotic promoter Bax and the inhibitory protein Bcl-2. The heterodimerization of these molecules leads to a balance of apoptotic signals through activation of caspases (57, 58). Our finding that BMP-2 promotes Bax and decreases Bcl-2 protein levels suggests that the proapoptotic effect of BMP-2 involves these regulatory proteins. This is supported by our finding that BMP-2 increases caspase-9 activity, which is a downstream enzyme activated by Bax and inhibited by Bcl-2 family proteins (59). BMP-2 also increases the release of cytochrome c into cytoplasm, indicating that this pathway may be involved, in cooperation with Apaf-1 (55, 56), in caspase-9 activation. In contrast, caspase-8, which is associated with the Fas/FADD apoptotic pathway (71), does not appear to be involved in rhBMP-2-induced apoptosis in IHNC cells. Downstream events involve effector caspases, which cleave intracellular substrates during the execution phase of apoptosis and contribute to protease and nuclease activation (71). Our finding that BMP-2 activates effector caspases-3, -6, and -7 in IHNC cells and that specific inhibitors of caspase-9 and effector caspases abolish the BMP-2-induced apoptosis demonstrate the implication of these enzymes in the proapoptotic effect of BMP-2 in IHNC cells. Therefore, BMP-2-induces apoptosis in human osteoblasts by mechanisms implicating Bax/Bcl-2 and mitochondrial cytochrome c release, leading to activation of caspase-9 and effector caspases, DNA degradation, and ultimately to cell death.

The signaling cascade by which BMP-2 induces apoptosis is not fully understood and may vary with the cell type (34). Different signaling pathways have been suggested to transduce BMP-2 signals from receptor serine/threonine kinases to the nucleus (35-38). Smad proteins have been shown to transduce signals induced by BMP and TGF-beta binding to their receptors. BMP-2 signaling involves phosphorylation of Smad1, which cooperates with Smad4 to transactivate genes (35-38). The Smad1 signaling pathway has been previously found to be involved in the promoting effect of BMP-2 on osteoblast differentiation marker genes (39, 40, 60). Therefore, it can be postulated that activation of Smad1 may be involved in apoptosis in osteoblasts. Here, we demonstrate that transfection with a vector that induces DN Smad1 overexpression did not block the BMP-2-induced apoptosis in IHNC cells and did not suppress the activation of apoptotic mechanisms, caspase-9, and effector caspases that are involved in the proapoptotic effect of BMP-2. These conclusions were drawn from transient transfection assays allowing persistent expression of DN Smad1 for 72 h, thus allowing studies on apoptosis up to this time point. The validity of the assay was confirmed by the finding that forced expression of DN Smad1 blocked the BMP-2-induced expression of Runx2, a transcription factor that serves an important role in regulating genes associated with osteoblast differentiation (61). This indicates that Smad1 is essential for induction of Runx2 expression by BMP-2 but not for BMP-2-induced apoptosis in human osteoblasts. Recent studies in other cell types have shown that overexpression of Smad6, which blocks the Smad pathway by inhibiting Smad1 (46, 75), can block BMP-2-induced apoptosis, suggesting an implication of Smad proteins in apoptosis in these cells. In contrast, our data indicate that BMP-2-induced apoptosis in IHNC cells is independent of Smad1 activation. Thus, the BMP-2-induced apoptosis in human osteoblasts cells is most likely mediated by a Smad-independent pathway.

It has been reported that BMP-2 may transduce signals by pathways distinct from Smads. BMP-2 was reported to activate protein kinase A, protein kinase C, ERKs, and Tak1-p38 in distinct cell types (41-44). Other reports showed the possible implication of p38 and ERK in apoptosis induced by BMPs (45, 46). We examined the possible involvement of these kinases in the BMP-2 signaling pathway that induces apoptosis in human osteoblasts. Our data show that BMP-2 transiently activates PKC in IHNC cells, suggesting that the PKC signaling pathway may be involved in the BMP-2-induced apoptosis in these cells. To clearly demonstrate a causal relationship between PKC activation and BMP-2-induced apoptosis, we employed specific pharmacological inhibitors of signaling pathways. The PKC inhibitor calphostin C inhibited the BMP-2-activation of apoptosis, whereas other inhibitors such as H89 and PD-98059, which inhibit PKA and ERK signaling pathways, respectively, do not inhibit BMP-2-induced apoptosis in IHNC cells. This suggests that PKC plays a key role in the apoptotic effect of BMP-2 in human osteoblasts. Presumably, suppression of PKC activity blocks the activation of downstream targets required for cell death. To further examine the role of PKC in BMP-2-induced apoptosis, we investigated the effects of the selective PKC signaling inhibitor calphostin C on the mechanisms involved in apoptosis. Calphostin C suppressed Bax/Bcl-2 and the activation of caspase-9 and effector caspases induced by BMP-2, further indicating that BMP-2 exerts its proapoptotic effect in large part through PKC activation. This is the first demonstration that PKC is involved in BMP-2-induced apoptosis in bone cells. Activation of PKC has been previously observed to contribute to apoptotic signaling (76). However, the effect is dependent on the PKC isoforms involved. Novel PKC isoforms appear to be proapoptotic, whereas classical and atypical PKC isoenzymes are associated with cell survival in a number of cell types (76). Several PKC isoforms have been identified in osteoblasts (77). We recently showed that PKCalpha /beta 1 isoforms are expressed in IHNC cells (78). Although the target PKC isoenzyme for the proapoptotic action of BMP-2 remains to be determined, PKC is clearly important for activation of mechanisms leading to apoptosis in human osteoblasts.

Another pathway involving TAK1-p38 MAPK cascade has been shown to be involved in apoptosis induced by BMP-2 in other cells (45, 46). Because we have not examined whether BMP-2 could activate TAK1 in IHNC cells, we cannot completely exclude the possibility that the TAK1-p38 signaling pathway may be involved in the BMP-2-induced apoptosis in IHNC cells. However, we found that SB203580, a specific inhibitor of p38 MAPK, had no effect on BMP-2-induced apoptosis. Moreover, the BMP-2-induced apoptosis was completely blocked by the PKC inhibitor, suggesting that the TAK1-p38 signal cascade is not essential for BMP-2-induced apoptotic signal in IHNC cells. Taken together, our findings indicate that p38, PKA, and Smad pathways do not appear to be involved in BMP-2-induced apoptosis in IHNC cells and suggest that this regulation appears to be mediated by a single pathway.

In summary, we demonstrate that BMP-2 is a molecular mediator of apoptosis in osteoblasts. We also show that the BMP-2-induced apoptosis is Smad-independent and PKC-dependent. In addition, we report that the induction of apoptosis by BMP-2 is mediated by changes in Bax/Bcl-2 expression, cytochrome c release, activation of caspase-9, and effector caspases. These results suggest a novel role for BMP-2 as a regulatory factor of osteoblast apoptosis and provide insight into the mechanisms used by BMP-2 to regulate apoptosis in human osteoblasts.

    ACKNOWLEDGEMENTS

We thank S. Roman-Roman (Avantis, Romainville, France) for the gift of vectors, Dr. G. Karsenty (Baylor College, Houston, TX) for the gift of the mouse Cbfa1/Osf2 antibody, and Genetics Institute (V. Rosen, Cambridge, MA) for the generous gift of rhBMP-2.

    FOOTNOTES

* This work was supported in part by a GIP Fonds de Recherche Hoechst Marion Roussel grant (GIP/HMR/INSERM).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a grant from Genset France.

§ To whom correspondence should be addressed. Tel.: 33-1-49-95-63-58; Fax: 33-1-49-95-84-52; E-mail: pierre.marie@inserm.lrb.ap-hop-paris.fr.

Published, JBC Papers in Press, June 6, 2001, DOI 10.1074/jbc.M011265200

    ABBREVIATIONS

The abbreviations used are: BMP, bone morphogenetic protein-2; TGF-beta , transforming growth factor-beta ; ERK, extracellular signal-regulated kinase; PKA, cAMP-dependent protein kinase A; MAPK, mitogen-activated protein kinase; TAK1, TGF-beta -activated kinase-1; IHNC, immortalized human neonatal calvaria; DN, dominant negative; rhBMP-2, recombinant human BMP-2; rhTGF-beta 2, recombinant human TGF-beta 2; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; FCS, fetal calf serum; PKC, protein kinase C; Z-, benzyloxycarbonyl-; FMK, phenylmethylketone; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase.

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DISCUSSION
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