Bone Morphogenetic Protein-2 Inhibits Serum Deprivation-induced Apoptosis of Neonatal Cardiac Myocytes through Activation of the Smad1 Pathway
2001; Elsevier BV; Volume: 276; Issue: 33 Linguagem: Inglês
10.1074/jbc.m101463200
ISSN1083-351X
AutoresMasahiro Izumi, Yasushi Fujio, Keita Kunisada, Shinji Negoro, Eiro Tone, Masanobu Funamoto, Tomoaki Osugi, Yuichi Oshima, Yoshikazu Nakaoka, Tadamitsu Kishimoto, Keiko Yamauchi‐Takihara, Hisao Hirota,
Tópico(s)Cardiac Fibrosis and Remodeling
ResumoBone morphogenetic protein (BMP)-2 has been shown to induce ectopic expression of cardiac transcription factors and beating cardiomyocytes in non-precardiac mesodermal cells, suggesting that BMP-2 is an inductive signaling molecule that participates in cardiac development. However, direct evidence of the effects of BMP-2 on cardiac myocytes has not been reported. To examine the role of BMP-2 and its receptors, we studied the ability of BMP-2 to promote survival of isolated neonatal rat cardiac myocytes. BMP receptors IA, IB, and II and activin receptor I were found to be expressed in myocytes, and BMP-2 phosphorylated Smad1 and p38 MAPK. Interestingly, BMP-2 promoted survival and inhibited apoptosis of serum-deprived myocytes, although it did not strongly induce hypertrophic growth. To explore the mechanisms for this protective effect, an adenovirus-based vector system was used. Similar to BMP-2, Smad1 promoted survival that was repressed by Smad6. Moreover, BMP-2 and Smad1 enhanced the expression of the anti-apoptotic molecule Bcl-xL. Antisense oligonucleotides to bcl-xLattenuated the survival effected by BMP-2. Overall, our findings suggest that BMP-2 prevents apoptosis of myocytes by induction of Bcl-xLvia a Smad1 pathway and might be a novel survival factor without any hypertrophic effect on myocytes. Bone morphogenetic protein (BMP)-2 has been shown to induce ectopic expression of cardiac transcription factors and beating cardiomyocytes in non-precardiac mesodermal cells, suggesting that BMP-2 is an inductive signaling molecule that participates in cardiac development. However, direct evidence of the effects of BMP-2 on cardiac myocytes has not been reported. To examine the role of BMP-2 and its receptors, we studied the ability of BMP-2 to promote survival of isolated neonatal rat cardiac myocytes. BMP receptors IA, IB, and II and activin receptor I were found to be expressed in myocytes, and BMP-2 phosphorylated Smad1 and p38 MAPK. Interestingly, BMP-2 promoted survival and inhibited apoptosis of serum-deprived myocytes, although it did not strongly induce hypertrophic growth. To explore the mechanisms for this protective effect, an adenovirus-based vector system was used. Similar to BMP-2, Smad1 promoted survival that was repressed by Smad6. Moreover, BMP-2 and Smad1 enhanced the expression of the anti-apoptotic molecule Bcl-xL. Antisense oligonucleotides to bcl-xLattenuated the survival effected by BMP-2. Overall, our findings suggest that BMP-2 prevents apoptosis of myocytes by induction of Bcl-xLvia a Smad1 pathway and might be a novel survival factor without any hypertrophic effect on myocytes. bone morphogenetic protein BMP receptor mitogen-activated protein kinase transforming growth factor-β-activated kinase-1 MAP kinase kinase c-Jun N-terminal kinase leukemia inhibitory factor extracellular signal-regulated kinase polymerase chain reaction fetal calf serum brain natriuretic peptide phosphate-buffered saline 3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling enzyme-linked immunosorbent assay activin receptor I signal transducer and activator of transcription Bone morphogenetic protein (BMP)1-2, a member of the transforming growth factor-β superfamily, signals through the heterotetrameric complex of type I and II serine/threonine kinase receptors (1Massague J. Weis-Garcia F. Cancer Surv. 1996; 27: 41-64PubMed Google Scholar, 2ten Dijke P. Miyazono K. Heldin C.H. Curr. Opin. Cell Biol. 1996; 8: 139-145Crossref PubMed Scopus (236) Google Scholar). Downstream of this receptor complex, at least two distinct intracellular pathways have been suggested to mediate inductive signals from the cell membrane to the nucleus. One pathway involves a family of transcription factors collectively known as Smad proteins. Thus far, eight mammalian Smad proteins, specified as Smad1–8, have been isolated. They can be classified into three subgroups by their structures and functions: pathway-restricted, common mediator, and inhibitory Smad proteins. Smad1, Smad5, and probably Smad8/MADH6 are pathway-restricted Smad proteins, which are activated by BMP receptors. Smad4 is a common mediator Smad, whereas Smad6 and Smad7 are classified in the inhibitory Smad subgroup. Following binding of BMPs to their receptors, Smad1 and Smad5 are phosphorylated by BMP receptors, form hetero-oligomeric complexes with Smad4, translocate into the nucleus, and modulate transcription of a variety of target genes (3Heldin C.H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3327) Google Scholar, 4Derynck R. Zhang Y. Feng X.H. Cell. 1998; 95: 737-740Abstract Full Text Full Text PDF PubMed Scopus (948) Google Scholar, 5Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3975) Google Scholar). Another pathway is the MAPK cascade initiated by TAK-1 (6Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1172) Google Scholar). TAK-1 was originally identified as a member of the MAPK kinase kinase family activated in response to transforming growth factor-β and BMP-4 (6Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. 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BMP-2 was originally identified as a molecule that induces bone and cartilage formation and is now considered a multifunctional cytokine (10Wozney J.M. Rosen V. Celeste A.J. Mitsock L.M. Whitters M.J. Kriz R.W. Hewick R.M. Wang E.A. Science. 1988; 242: 1528-1534Crossref PubMed Scopus (3334) Google Scholar, 11Hogan B.L. Genes Dev. 1996; 10: 1580-1594Crossref PubMed Scopus (1718) Google Scholar). Interestingly, BMP-2 induces ectopic expression of cardiac transcription factors and beating cardiomyocytes in non-precardiac mesodermal chick cells, and BMP-2-deficient mouse embryos exhibit a defect in cardiac development manifested by abnormal development of the heart (12Rosen V. Thies R.S. Trends Genet. 1992; 8: 97-102Abstract Full Text PDF PubMed Scopus (229) Google Scholar, 13Reddi A.H. Curr. Opin. Genet. Dev. 1994; 4: 737-744Crossref PubMed Scopus (328) Google Scholar). These data suggest that BMPs are inductive signaling molecules that participate in development of the heart. In addition to cardiac development, BMP-2 has been reported to exert both pro-apoptotic and anti-apoptotic effects, depending on cell types and circumstances (14Yokouchi Y. Sakiyama J. Kameda T. Iba H. Suzuki A. Ueno N. Kuroiwa A. Development. 1996; 122: 3725-3734Crossref PubMed Google Scholar, 15Iwasaki S. Iguchi M. Watanabe K. Hoshino R. Tsujimoto M. Kohno M. J. Biol. Chem. 1999; 274: 26503-26510Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 16Bhatia M. Bonnet D. Wu D. Murdoch B. Wrana J. Gallacher L. Dick J.E. J. Exp. Med. 1999; 189: 1139-1148Crossref PubMed Scopus (332) Google Scholar). Therefore, it is of interest to examine whether BMP-2 might also regulate survival of cardiac myocytes. Recent evidence that apoptosis of cardiac myocytes is a feature in several myocardial disease states, including ischemic heart disease and congestive heart failure, has raised hopes that inhibition of myocyte apoptosis can prevent the loss of contractile cells and thus provide a new target in a multimodal therapeutic approach to cardiac disease (17Haunstetter A. Izumo S. Circ. Res. 1998; 82: 1111-1129Crossref PubMed Scopus (722) Google Scholar). Several groups have reported that insulin-like growth factor-1, cardiotrophin-1, leukemia inhibitory factor (LIF), and neuregulin reduce myocyte apoptosis after ischemia, serum withdrawal, myocyte stretch, and treatment with the cardiotoxic hemotherapeutic drug doxorubicin (18Buerke M. Murohara T. Skurk C. Nuss C. Tomaselli K. Lefer A.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8031-8035Crossref PubMed Scopus (361) Google Scholar, 19Li Q. Li B. Wang X. Leri A. Jana K.P. Liu Y. Kajstura J. Baserga R. Anversa P. J. Clin. Invest. 1997; 100: 1991-1999Crossref PubMed Scopus (435) Google Scholar, 20Fujio Y. Kunisada K. Hirota H. Yamauchi-Takihara K. Kishimoto T. J. Clin. Invest. 1997; 99: 2898-2905Crossref PubMed Scopus (178) Google Scholar, 21Zhao Y.Y. Sawyer D.R. Ragavendra R. Baliga R. Opel D.J. Han X. Marchionni M.A. Kelly R.A. J. Biol. Chem. 1998; 273: 10261-10269Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 22Sheng Z. Knowlton K. Chen J. Hoshijima M. Brown J.H. Chien K.R. J. Biol. Chem. 1997; 272: 5783-5791Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). However, these factors simultaneously induce cardiac hypertrophy. Although cardioprotective factors can thus be considered clinically suitable for treating heart failure, the hypertrophic effect accompanying their cardioprotective function may promote structural and geometric changes in the left ventricle that are commonly referred to as remodeling. Progressive, adverse remodeling of the myocardium may lead to ventricular dilatation and congestive heart failure (23Zannad F. Alla F. Dousset B. Peres A. Pitt B. Circulation. 2000; 102: 2700-2706Crossref PubMed Scopus (845) Google Scholar). Our study demonstrates that human BMP-2 promotes survival of neonatal rat cardiac myocytes in vitro without any significant change in cell size. This survival effect is accompanied by a marked reduction in the proportion of apoptotic cells in BMP-2-treated cultures. We also describe the survival effect of BMP-2 involving an increase in Bcl-xL via a Smad1 signaling pathway. These data support the concept that the BMP-2/Smad1 signaling system plays an important role in regulation of the myocardium and suggest that BMP-2 as an attenuator of apoptosis in cardiac myocytes has therapeutic and prognostic potential. Recombinant human BMP-2 (80 mg/ml; Yamanouchi Co., Ltd., Tokyo), recombinant human LIF (106 units/ml; PeproTech EC, Ltd., London), and norepinephrine (1 mg/ml; Sankyo Co., Ltd., Tokyo) were used this study. Anti-Smad1, anti-Smad6, anti-p38 MAPK, anti-ERK1/2, anti-JNK, anti-Bcl-xL, and anti-Bcl-2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-α-tubulin monoclonal antibody was from Calbiochem, and phospho-Smad1, p38 MAPK, and ERK1/2 were from New England Biolabs Inc. (Beverly, MA). All other chemicals were reagents of molecular biology grade obtained from standard commercial sources. Primary cultures of neonatal cardiac myocytes were prepared from the ventricles of 1–2-day-old Wistar rats obtained from Kiwa Dobutsu (Wakayama, Japan) as described previously (24Kunisada K. Hirota H. Fujio Y. Matsui H. Tani Y. Yamauchi-Takihara K. Kishimoto T. Circulation. 1996; 94: 2626-2632Crossref PubMed Scopus (168) Google Scholar). 10 µg of total RNA was prepared by the acid guanidinium thiocyanate/phenol/chloroform method (25Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63088) Google Scholar). Total RNA (0.1 µg) was subjected to first-strand synthesis using oligo(dT) (Amersham Pharmacia Biotech, Uppsala) and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) at 37 °C for 2 h, and the reaction was stopped by incubation at 70 °C for 10 min. The primers used for gene amplification of BMP-2, BMP receptors (BMPRs), and β-tubulin by PCR were synthesized according to the sequence previously reported (26Roelen B.A. Goumans M.J. van Rooijen M.A. Mummery C.L. Int. J. Dev. Biol. 1997; 41: 541-549PubMed Google Scholar, 27Iantosca M.R. McPherson C.E. Ho S.Y. Maxwell G.D. J. Neurosci. Res. 1999; 56: 248-258Crossref PubMed Scopus (32) Google Scholar, 28Kunisada K. Negoro S. Tone E. Funamoto M. Osugi T. Yamada S. Okabe M. Kishimoto T. Yamauchi-Takihara K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 315-319Crossref PubMed Scopus (222) Google Scholar). Neonatal rat cardiac myocytes were cultured at a cell density of 3 × 105/ml in six-well dishes in duplicate. After being cultured with Medium 199 with 10% fetal calf serum (FCS) for 24 h, the medium was changed to Medium 199 with 1% FCS and incubated for 24 h. Thereafter, cardiac myocytes were cultured without serum for another 12 h. The cells were then stimulated with BMP-2. Western blotting was performed as previously described (24Kunisada K. Hirota H. Fujio Y. Matsui H. Tani Y. Yamauchi-Takihara K. Kishimoto T. Circulation. 1996; 94: 2626-2632Crossref PubMed Scopus (168) Google Scholar). Northern blotting was performed as previously described (29Boise L.H. Gonzalez-Garcia M. Postema C.E. Ding L. Lindsten T. Turka L.A. Mao X. Nunez G. Thompson C.B. Cell. 1993; 74: 597-608Abstract Full Text PDF PubMed Scopus (2917) Google Scholar). RNAs from cardiac myocytes were prepared as described above. Mouse bcl-x cDNA, kindly donated by Dr. Y. Tsujimoto (Osaka University Medical School), was used as a probe. Mouse brain natriuretic peptide (BNP) cDNA, kindly donated by Dr. K. R. Chien (University of California, San Diego, CA), was used as a probe. Mouse junB cDNA was purchased from American Type Culture Collection. For examination of changes in myocyte phenotypes produced by BMP-2, LIF, and norepinephrine, cardiac myocytes were grown on slides and fixed in 2% (w/v) formaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100/phosphate-buffered saline (PBS). Following three PBS washes, the chamber slides were incubated in 1% bovine serum albumin/PBS for 1 h to block nonspecific sites. Subsequently, the cells were stained for 1 h in 0.05% Tween 20 and 1% bovine serum albumin/PBS with a mouse anti-rabbit α-actinin monoclonal antibody. Following another three PBS washes, the slides were incubated for 30 min in 0.05% Tween 20 and 1% bovine serum albumin/PBS with rhodamine B isothiocyanate-conjugated anti-mouse IgG as a secondary antibody and finally mounted on glass coverslips. To measure the cell-surface areas, light photomicrographs of beating cells (100 cells from six visual fields) were obtained with an Olympus IMT-2 microscope, and cell surfaces were traced with the Macscope program (Mitani Co., Tokyo). To examine the effect of BMP-2 on protein synthesis, the incorporation of [3H]leucine was measured. Cultured myocytes were treated with BMP-2 (80 ng/ml), LIF (1 × 103 units/ml), or norepinephrine (2 µg/ml) and co-incubated with [3H]leucine (1 µCi/ml) for 24 h. The cells were washed three times with PBS and then treated with 5% trichloroacetic at 4 °C for 10 min to precipitate the protein. The precipitates were dissolved in 0.1 n NaOH, and aliquots were counted with a scintillation counter. Cell viability was quantitated with the MTS (inner salt) cell respiratory assay (Promega, Madison, WI) (30Berridge M.V. Tan A.S. Arch. Biochem. Biophys. 1993; 303: 474-482Crossref PubMed Scopus (1098) Google Scholar,31Cory A.H. Owen T.C. Barltrop J.A. Cory J.G. Cancer Commun. 1991; 3: 207-212Crossref PubMed Scopus (1298) Google Scholar). Neonatal rat cardiac myocytes were cultured at a density of 2 × 104/100 µl in 96-well dishes. After culturing in Medium 199 with 10% FCS for 24 h, the medium was changed to Medium 199 with 1% FCS, and the cells were incubated for 24 h. Next, the cardiac myocytes were cultured without serum for another 12 h and stimulated with BMP-2 or LIF. 48 h later, MTS was incubated with the cells for 1 h at 37 °C in a humidified 5% CO2 atmosphere. The absorbance was then recorded at 490 nm with a 96-well plate reader. Each experiment was carried out in triplicate and repeated in three independent experiments. Apoptosis was evaluated with the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay and cell death detection ELISA. The TUNEL assay was performed as previously described (32Negoro S. Oh H. Tone E. Kunisada K. Fujio Y. Kenneth W. Kishimoto T. Yamauchi-Takihara K. Circulation. 2001; 103: 555-561Crossref PubMed Scopus (179) Google Scholar), and the cells were analyzed by fluorescence microscopy. For quantitative analysis, 600 α-actinin-positive cardiac myocytes were counted, and the number of TUNEL-positive cells among them was expressed as a percentage. The cell death detection assay (Roche Molecular Biochemicals) measures the presence of the soluble histone·DNA complex as a result of DNA fragmentation (33Thome M. Schneider P. Hofmann K. Fickenscher H. Meinl E. Neipel F. Mattmann C. Burns K. Bodmer J.L. Schroter M. Scaffidi C. Krammer P.H. Peter M.E. Tschopp J. Nature. 1997; 386: 517-521Crossref PubMed Scopus (1140) Google Scholar). For this assay, neonatal rat cardiac myocytes were cultured at a density of 5 × 105/1 ml in 6-cm dishes. After culturing in Medium 199 with 10% FCS for 24 h, the medium was changed to Medium 199 with 1% FCS, and the cells were incubated for 24 h. Next, the cardiac myocytes were cultured without serum for another 12 h and stimulated with BMP-2. 48 h later, all cells were collected in lysis buffer and subjected to capture ELISA according to the manufacturer's protocol. Each experiment was carried out in triplicate and repeated in three independent experiments. The antisense and sense phosphorothioate oligonucleotides were designed as described by Wang et al.(34Wang Z. Karras J.G. Howard R.G. Rothstein T.L. J. Immunol. 1995; 155: 3722-3725PubMed Google Scholar): antisense, CTGAGACATTTTTAT and sense, ATAAAAATGTCTCAG. This region of murine bcl-x L is completely identical to that of rat bcl-x L (35Shiraiwa N. Inohara N. Okada S. Yuzaki M. Shoji S. Ohta S. J. Biol. Chem. 1996; 271: 13258-13265Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Antisense oligonucleotide assays were performed according to previously described methods (36Ackermann E.J. Taylor J.K. Narayana R. Bennett C.F. J. Biol. Chem. 1999; 274: 11245-11252Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The recombinant adenoviruses expressing Smad proteins were kindly donated by Dr. K. Miyazono (Tokyo University Medical School) (37Fujii M. Takeda K. Imamura T. Aoki H. Sampath T.K. Enomoto S. Kawabata M. Kato M. Ichijo H. Miyazono K. Mol. Biol. Cell. 1999; 10: 3801-3813Crossref PubMed Scopus (368) Google Scholar). The recombinant viruses were purified and concentrated as described previously (38Becker T.C. Noel R.J. Coats W.S. Gomez-Foix A.M. Alam T. Gerard R.D. Newgard C.B. Methods Cell Biol. 1994; 43: 161-189Crossref PubMed Scopus (562) Google Scholar). They were prepared for experiments at high multiplicity of infections. 36 h after plating, cardiac myocytes were transfected with adenoviral vectors in Medium 199 with 10% FCS at a multiplicity of infection of 20 and incubated for 12 h. After removal of the viral suspension, cardiac myocytes were serum-starved for 6 h and stimulated with reagents. The adenoviral vector expressing β-galactosidase (referred to as Ad-β-gal) was used as a control. Data are presented as means ± S.E. Statistical analysis was carried out using unpaired Student'st tests. A p value of <0.05 was considered significant. mRNA expression of BMP receptors was analyzed in cardiac myocytes grown in the absence or presence of BMP-2 (80 ng/ml). Cultures were assayed for the expression of BMP-2, BMPR-IA, BMPR-IB, BMPR-II, and activin receptor I (ActR-I) mRNAs by reverse transcription-PCR after 3 h in culture. As shown in Fig.1, mRNAs for BMPR-IB, ActR-I, and BMPR-II were readily apparent in control cultures, whereas mRNAs for BMP-2 and BMPR-IA were less abundant. Cultures grown in the presence of BMP-2 expressed BMP-2, BMPR-IA, BMPR-II, and ActR-I mRNAs. Although this PCR method is not quantitative, the induction of BMP-2 and BMPR-IA mRNAs following exposure to BMP-2 was striking. These observations indicate that treatment with BMP-2 can activate heart cells through its receptors. To determine whether BMP-2 transduces signals in neonatal cardiac myocytes, we examined the phosphorylation of Smad1, p38 MAPK, ERK1/2, and JNK after BMP-2 stimulation by Western blot analysis. After 8 h of culture in serum-deprived medium, cardiac myocytes were stimulated with BMP-2 (80 ng/ml) for the times indicated (Fig. 2 A). Smad1 was rapidly phosphorylated within 15 min in cardiac myocytes, followed by a decrease within 2 h. As shown in Fig. 2 B, BMP-2 also activated Smad1 in a dose-dependent manner. The amounts of Smad1 protein were the same for each period (Fig. 2, A andB, lower panels). In the MAPK family, p38 MAPK was rapidly phosphorylated within 5 min, whereas ERK1/2 and JNK were not activated (Fig. 2 C). The induction of immediate-early genes precedes the late phenotypic changes in cardiomyocytes following stimulation of G-protein-coupled receptorsin vitro (39Shubeita H.E. McDonough P.M. Harris A.N. Knowlton K.U. Glembotski C.C. Brown J.H. Chien K.R. J. Biol. Chem. 1990; 265: 20555-20562Abstract Full Text PDF PubMed Google Scholar, 40Ito H. Hirata Y. Hiroe M. Tsujino M. Adachi S. Takamoto T. Nitta M. Taniguchi K. Marumo F. Circ. Res. 1991; 69: 209-215Crossref PubMed Scopus (462) Google Scholar, 41Iwaki K. Sukhatme V.P. Shubeita H.E. Chien K.R. J. Biol. Chem. 1990; 265: 13809-13817Abstract Full Text PDF PubMed Google Scholar) and cardiac hypertrophy in vivo(42Izumo S. Nadal-Ginard B. Mahdavi V. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 339-343Crossref PubMed Scopus (745) Google Scholar). We therefore determined whether stimulation of cardiomyocytes with BMP-2 would induce junB gene expression. Stimulation with BMP-2 resulted in the rapid and transient induction ofjunB mRNA (Fig.3 A). BMP-2 also inducedjunB mRNA expression in a dose-dependent manner (Fig. 3 B). Since the reactivation of an embryonic pattern of gene expression is a hallmark of cardiomyocyte hypertrophy, we therefore determined whether stimulation of cardiomyocytes with BMP-2 would induce BNP gene expression. As shown in Fig. 3 C, enhancement of BNP mRNA expression was detected within 6 h, with a maximum level at 12 h, followed by a decrease within 24 h. Neonatal rat ventricular cardiomyocytes respond to various hypertrophic stimuli by an increase in individual cell size, assembly of myofibrils, and induction of the BNP gene. To assess the effects of BMP-2 on these morphological features of a hypertrophic response, cardiomyocytes were treated with BMP-2 (80 ng/ml) and stained with an anti-α-actinin antibody. Unstimulated cells and LIF/norepinephrine-treated cells served as negative and positive controls, respectively. Interestingly, BMP-2 did not induce a strong hypertrophic response in cardiac myocytes in comparison with LIF and norepinephrine, but did induce BNP gene expression (Fig.4 A). To quantify hypertrophic effects, we measured the cell-surface area and the incorporation of [3H]leucine as described under “Experimental Procedures.” As shown in Fig. 4 (B and C), the increase in cell size and [3H]leucine incorporation induced by BMP-2 was significantly less than those induced by LIF or norepinephrine. These findings suggest that the hypertrophic effects of BMP-2 are significantly less than those of LIF and norepinephrine. Primary cultures of neonatal rat ventricular myocytes maintained in serum-free medium exhibited a gradual reduction in cell numbers. We performed an MTS cell respiration assay to measure cell viability in primary cultures of neonatal rat cardiac myocytes. The myocytes were cultured in the presence or absence of BMP-2 for 48 h. As shown in Fig.5 A, BMP-2 promoted myocardial cell survival in a dose-dependent manner. LIF-treated cells served as positive controls. On the other hand, BMP-2 could not promote survival of cardiac non-myocytes (data not shown). We examined next whether the survival effect of BMP-2 was mediated by inhibition of programmed cell death (apoptosis). We performed the TUNEL assay and cell death detection ELISA on primary cultures of neonatal rat cardiac myocytes. About 18% of the cardiac myocytes maintained in serum-deprived medium for 48 h showed positive TUNEL staining (Fig. 5 B). As shown in Fig. 5 C, BMP-2 (80 ng/ml) significantly reduced TUNEL-positive cells. Cell death detection ELISA also demonstrated that BMP-2 significantly suppressed serum deprivation-induced nuclear fragmentation (Fig. 5 D). These results indicate that BMP-2 acts as a survival factor at least in part by preventing programmed cell death in neonatal rat cardiac myocytes. To investigate the molecular mechanism that relates to the anti-apoptotic effect of BMP-2, we examined the expression ofbcl-x L and bcl-2, which are anti-apoptotic members of bcl-2 families. Neonatal rat cardiac myocytes were treated with BMP-2 (80 ng/ml) for the indicated periods of time. Upon examination by Northern blot analysis, expression of bcl-x L and bcl-2 mRNAs showed enhancement of bcl-x L gene expression in cardiac myocytes, with the maximum level attained at 24 h, whereas no enhancement of bcl-2 gene expression was detected (Fig.6, A and B). We also examined the expression of Bcl-xL and Bcl-2 proteins by Western blot analysis in cardiac myocytes treated with BMP-2 (80 ng/ml) for 24 h. A high level of Bcl-xL was also detected in BMP-2-treated cells, but no up-regulation of Bcl-2 (Fig. 6,C and D). These results indicate that BMP-2 may promote cardiac myocyte survival through the induction of Bcl-xL. To demonstrate the specificity of bcl-x L antisense oligonucleotides, cardiac myocytes were treated with bcl-x L antisense or sense oligonucleotides in the presence or absence of BMP-2 for 48 h. The myocytes were subsequently examined with the MTS cell respiratory assay. In the presence of antisense oligonucleotides, the increase in Bcl-xL induced by BMP-2 was inhibited, but not in the presence of sense oligonucleotides (Fig.7 A). Antisense oligonucleotides against bcl-x L mRNA canceled the BMP-2-mediated protective effect on cell survival (Fig.7 B). Although the sense nucleotides also inhibited BMP-2-mediated cell protection, their inhibitory effect was significantly less than that of antisense oligonucleotides (Fig.7 B). We investigated the molecular mechanisms that regulate bcl-x L gene expression upon BMP-2 stimulation in cardiac myocytes using an adenovirus-based vector system that allows for highly efficient transfection of DNAs into many cell types. In our study, we demonstrated that activation of BMP receptors resulted in the activation of downstream signaling pathways, including Smad1 and p38 MAPK. To identify the downstream effector for BMP-2-mediated protection of cell survival through the induction ofbcl-x L, the effects of Smad1 on cell survival andbcl-x L induction were examined. We performed an MTS respiratory cell assay, a TUNEL assay, and cell death detection ELISA in cultured cardiac myocytes transfected with adenoviral vectors expressing Smad1 (referred to as Ad-Smad1) in the presence or absence of BMP-2 for 48 h. Interestingly, the phosphorylation of Smad1 was detected in cultured cardiac myocytes transfected with Ad-Smad1 (Fig.8 A). As shown in Fig.8 B, Smad1 promoted cell survival, which was enhanced by the presence of BMP-2. TUNEL-positive cells were significantly reduced in cardiac myocytes transfected with Ad-Smad1 compared with those in untransfected cardiac myocytes (Fig. 8 C). Cell death detection ELISA also showed that fragmented DNA was significantly reduced in cardiac myocytes transfected with Ad-Smad1 (Fig.8 D). Altogether, these results indicate that the survival effect of Smad1 is mediated by inhibition of apoptosis. Moreover, the expression of the bcl-x L gene and protein was increased in cultured cardiac myocytes transfected with Ad-Smad1 (Fig.9, A–D). These findings suggest that BMP-2 promotes cardiac muscle survival through the induction of bcl-x L via the Smad signaling pathway.Figure 9Smad1 induces bcl-x Lin neonatal rat cardiac myocyte primary cultures. A, neonatal rat cardiac myocytes were transfected with Ad-Smad1 (Smad1(adeno)) or Ad-β-gal (β-gal(adeno)). Next, the myocytes were cultured without serum for another 24 h. bcl-x L mRNA expression was examined by Northern blot analysis. B, densitometry was used to assess the relative intensity of the band forbcl-x L mRNA as a ratio in comparison with the intensity of the band for 28 S rRNA. The data are expressed as relative intensity. Values represent the means ± S.D. (n = three experiments). *, p < 0.01versus control. C, cardiac myocytes were transfected with Ad-Smad1 or Ad-β-gal with BMP-2 (80 ng/ml). Next, the myocytes were cultured without serum for another 24 h. The cell lysates were Western-blotted with anti-Bcl-xLantibody. D, densitometry was used to assess the relative intensity of the band for Bcl-xL as a ratio in comparison with the intensity of the band for α-tubulin. The data are expressed as relative intensity. Values are the means ± S.D. (n = three experiments). *, p < 0.01versus control.View Large Image Figure ViewerDownload (PPT) To examine whether Smad6 inhibits the Smad1-mediated protect
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