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Phosphatidylinositol 3-Kinase Regulates Bone Morphogenetic Protein-2 (BMP-2)-induced Myocyte Enhancer Factor 2A-dependent Transcription of BMP-2 Gene in Cardiomyocyte Precursor Cells*

  • Nandini Ghosh-Choudhury
    Correspondence
    To whom correspondence may be addressed.
    Affiliations
    Departments of Pathology, San Antonio, Texas 78229-3900

    Departments of South Texas Veterans Health Care System, San Antonio, Texas 78229-3900
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  • Sherry L. Abboud
    Footnotes
    Affiliations
    Departments of Pathology, San Antonio, Texas 78229-3900

    Departments of South Texas Veterans Health Care System, San Antonio, Texas 78229-3900
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  • Lenin Mahimainathan
    Affiliations
    Departments of Medicine, University of Texas Health Science Center, San Antonio, Texas 78229-3900
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  • Bysani Chandrasekar
    Footnotes
    Affiliations
    Departments of Medicine, University of Texas Health Science Center, San Antonio, Texas 78229-3900
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  • Goutam Ghosh Choudhury
    Correspondence
    To whom correspondence may be addressed
    Affiliations
    Departments of Medicine, University of Texas Health Science Center, San Antonio, Texas 78229-3900

    Departments of Geriatric Research, Education, and Clinical Center, San Antonio, Texas 78229-3900
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  • Author Footnotes
    * This study was supported in part by a Dept. of Veterans Affairs Medical Research Service Merit Review Award and Research Excellence Area Program Award, by NIDDK, National Institutes of Health (NIH) Grant RO1 DK55815 (to G. G. C.), by a Department of Defense Breast Cancer Award (DAMD17-99-1-9400), and by a Veterans Affairs VISN 17 Grant (to N. G. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ”advertisement“ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    The on-line version of this article (available at http://www.jbc.org) contains Supplemental Fig. S1.
    ∥ Supported by NIH Grant AR-42306.
    ‡‡ Supported by American Heart Association Grant-in-Aid 0150105N and NHLBI, NIH Grant HL68020.
Open AccessPublished:March 27, 2003DOI:https://doi.org/10.1074/jbc.M302277200
      The growth and differentiation factor bone morphogenetic protein-2 (BMP-2) regulates cardiac development during vertebrate embryogenesis. In cardiac precursor cells, BMP-2 has recently been shown to induce expression of cardiac transcription factors, including myocyte enhancer factor 2A (MEF-2A). The specific signal transduction mechanism by which BMP-2 regulates these actions is not known. We investigated the role of phosphatidylinositol (PI) 3-kinase in regulating these processes in cardiomyocyte precursor CL6 cells. BMP-2 increased PI 3-kinase activity in these cells in a time-dependent manner, resulting in increased expression of sarcomeric myosin heavy chain (MHC) and MEF-2A. Inhibition of PI 3-kinase abolished these actions of BMP-2, indicating the involvement of PI 3-kinase in these processes. Furthermore, BMP-2 stimulated specific protein·DNA complex formation when an MEF-2 DNA recognition element was used as probe. Antibody supershift assay confirmed the presence of MEF-2A in this protein·DNA complex. Inhibition of PI 3-kinase activity completely prevented the MEF-2A·DNA complex formation. BMP-2 also increased transcription of a reporter gene driven by an MEF-2-specific DNA element in a PI 3-kinase-dependent manner. Ectopic expression of MEF-2A increased BMP-2 transcription to the same extent induced by BMP-2, indicating that MEF-2A may participate in BMP-2 autoregulation in CL6 cells. Expression of dominant negative PI 3-kinase completely abolished BMP-2-induced as well as MEF-2A-mediated BMP-2 transcription. Furthermore expression of MEF-2A increased MHC expression in a PI 3-kinase-dependent manner. Together these data provide the first evidence that BMP-2-induced PI 3-kinase signaling regulates MEF-2A expression and define a mechanism of MEF-2A-dependent BMP-2 transcription.
      Bone morphogenetic proteins were originally identified as growth and differentiation factors for osteogenic cells and are now considered as multifunctional polypeptides (
      • Sakou T.
      ). BMPs
      The abbreviations used are: BMPs, bone morphogenetic proteins; MEF, myocyte enhancer factor; PI, phosphatidylinositol; MHC, myosin heavy chain; EMSA, electrophoretic mobility shift assay; PKC, protein kinase C.
      1The abbreviations used are: BMPs, bone morphogenetic proteins; MEF, myocyte enhancer factor; PI, phosphatidylinositol; MHC, myosin heavy chain; EMSA, electrophoretic mobility shift assay; PKC, protein kinase C.
      play important roles in the development of many organs, including lung, kidney, gut, skin, teeth, and heart (
      • Hogan B.L.
      ). BMP-2, a member of this family of proteins, is expressed during mouse embryonic heart development (
      • Abdelwahid E.
      • Rice D.
      • Pelliniemi L.J.
      • Jokinen E.
      ). A similar expression pattern of BMP-2 is conserved in Xenopus, where this growth and differentiation factor is expressed in the heart progenitor cells and in the mature heart (
      • Knochel S.
      • Dillinger K.
      • Koster M.
      • Knochel W.
      ). During chick embryogenesis, the tissues that express BMP-2 are in contact with the precursor cells committed to cardiac muscle lineage. Furthermore, introduction of BMP-2 in vivo induces expression of cardiac-specific transcription factors (
      • Schultheiss T.M.
      • Burch J.B.
      • Lassar A.B.
      ). Finally, abnormal heart development leads to embryonic lethality in BMP-2 null mice (
      • Zhang H.
      • Bradley A.
      ).
      The MEF-2 family of transcription factors has been shown to play a pivotal role in myogenesis of smooth muscle, skeletal, and cardiac cells (
      • Olson E.N.
      • Perry M.
      • Schulz R.A.
      ). MEF-2 DNA binding elements are present in many cardiac muscle genes. MEF-2A, MEF-2C, and MEF-2D bind the consensus DNA element with the same specificity, whereas MEF-2B binds with relatively reduced affinity (
      • Black B.L.
      • Olson E.N.
      ). Addition of noggin, a BMP-2 antagonist, to chick embryo explants induced loss of cardiac-specific transcription factors, including MEF-2A (
      • Schlange T.
      • Andree B.
      • Arnold H.H.
      • Brand T.
      ). One of the major kinases that regulate MEF-2 transcriptional activity is the p38 mitogen-activated protein kinase (
      • McKinsey T.A.
      • Zhang C.L.
      • Olson E.N.
      ). More recently, PI 3-kinase, which is activated by the receptor and nonreceptor tyrosine kinases, has been shown to regulate MEF-2 transcriptional activity and myogenesis (
      • Jiang B.H.
      • Zheng J.Z.
      • Vogt P.K.
      ,
      • Tamir Y.
      • Bengal E.
      ). Thus PI 3-kinase is required for insulin-like growth factor-1 receptor tyrosine kinase-induced myogenesis in culture and during mouse embryogenesis (
      • Florini J.R.
      • Ewton D.Z.
      • Coolican S.A.
      ,
      • Powell-Braxton L.
      • Hollingshead P.
      • Warburton C.
      • Dowd M.
      • Pitts-Meek S.
      • Dalton D.
      • Gillett N.
      • Stewart T.A.
      ).
      BMP-2 acts as a survival factor for neonatal cardiac myocytes via Smad 1, the downstream target of BMP receptor (
      • Izumi M.
      • Fujio Y.
      • Kunisada K.
      • Negoro S.
      • Tone E.
      • Funamoto M.
      • Osugi T.
      • Oshima Y.
      • Nakaoka Y.
      • Kishimoto T.
      • Yamauchi-Takihara K.
      • Hirota H.
      ). BMP-2 also induces expression of cardiac transcription factors in embryonic teratocarcinoma cells (
      • Monzen K.
      • Shiojima I.
      • Hiroi Y.
      • Kudoh S.
      • Oka T.
      • Takimoto E.
      • Hayashi D.
      • Hosoda T.
      • Habara-Ohkubo A.
      • Nakaoka T.
      • Fujita T.
      • Yazaki Y.
      • Komuro I.
      ). The mechanism by which BMP-2 regulates cardiac development and cardiac gene expression is largely unknown. Here we show that BMP-2 activates PI 3-kinase in P19CL6 embryonic carcinoma cells, which differentiate along the cardiomyocyte lineage. BMP-2-induced expression and DNA binding of MEF-2 transcription factor are dependent upon PI 3-kinase activity. In addition we show that BMP-2-induced PI 3-kinase activity regulates MEF-2-dependent reporter gene transcription. Finally, we demonstrate that BMP-2-induced BMP-2 gene transcription is regulated by MEF-2 in a PI 3-kinase-dependent manner.

      EXPERIMENTAL PROCEDURES

      Materials—Recombinant BMP-2 was obtained from Genetics Institute, Cambridge, MA. Na3VO4, Nonidet P-40, and phenylmethylsulfonyl fluoride were purchased from Sigma. Aprotinin was obtained from Bayer. Antibody against the p85 and p110 subunits of PI 3-kinase, MEF-2A, and noggin were obtained from Santa Cruz Biotechnology. Anti-phosphotyrosine antibody was purchased from Upstate Biotechnology Inc. MF-20 antibody recognizing sarcomeric MHC was obtained from the Developmental Studies Hybridoma Bank, University of Iowa. Tissue culture reagents and LipofectAMINE were obtained from Invitrogen. A dual luciferase assay kit was purchased from Promega Inc. Nuclear fraction extraction reagents were obtained from Pierce. The pSRαΔp85 plasmid encoding for a dominant negative regulatory subunit for PI 3-kinase and Ad Myr-p110 adenovirus vector expressing the constitutively active p110 catalytic subunit of PI 3-kinase were gifts from Dr. Wataru Ogawa, Kobe University, Japan. The MEF-2A expression plasmid was obtained from Dr. Richard Prywes (Columbia University, New York).
      Cell Culture—P19CL6 cells, described as CL6 in this report, and CL6-Noggin cells expressing noggin were kind gifts of Dr. I. Komuro, Department of Cardiovascular Medicine, University of Tokyo, Japan. CL6 cells undergo differentiation in the presence of 1% Me2SO into beating cardiomyocyte and express cardiac-specific genes (
      • Monzen K.
      • Shiojima I.
      • Hiroi Y.
      • Kudoh S.
      • Oka T.
      • Takimoto E.
      • Hayashi D.
      • Hosoda T.
      • Habara-Ohkubo A.
      • Nakaoka T.
      • Fujita T.
      • Yazaki Y.
      • Komuro I.
      ). The cells were grown in α-minimal essential medium with 10% fetal bovine serum. The cells were serum-deprived for 24 h to make them quiescent. To induce MEF-2A expression, the cells were grown in 1% Me2SO or in the indicated concentration of BMP-2.
      Adenovirus Infection—CL6 cells were infected with a multiplicity of infection of 50 for Ad Myr-p110, essentially as described before (
      • Ghosh-Choudhury N.
      • Abboud S.L.
      • Nishimura R.
      • Celeste A.
      • Mahimainathan L.
      • Choudhury G.G.
      ).
      Immunofluorescence—Immunofluorescence detection of sarcomeric MHC was performed essentially as described previously (
      • Ghosh-Choudhury N.
      • Abboud S.L.
      • Nishimura R.
      • Celeste A.
      • Mahimainathan L.
      • Choudhury G.G.
      ). Briefly, treated cells in eight-well chamber slides were fixed in cold methanol for 15 min followed by blocking with donkey IgG for 15 min. The cells were then incubated with MF-20 for 30 min. Stained cells were treated with Cy-tagged secondary anti-mouse antibody for 30 min. The cells were visualized using a confocal microscope (Olympus FluoView 500) and photographed using FluoView software.
      Immunoprecipitation, Immunoblotting, and PI 3-kinase Assay— Cells were lysed in radioimmune precipitation assay buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 0.05% aprotinin, and 1% Nonidet P-40). Cleared cell lysates were prepared by centrifugation at 10,000 × g for 30 min at 4 °C. Protein concentration was determined in the lysate, and an equal amount of protein was immunoprecipitated with the p85 regulatory subunit of PI 3-kinase or anti-phosphotyrosine antibody. The immunoprecipitates were used in the PI 3-kinase assay using PI as substrate in the presence of [γ-32P]ATP, and the products were separated by thin layer chromatography followed by autoradiography (
      • Choudhury G.G.
      • Karamitsos C.
      • Hernandez J.
      • Gentilini A.
      • Bardgette J.
      • Abboud H.E.
      ,
      • Choudhury G.G.
      • Grandaliano G.
      • Jin D.C.
      • Katz M.S.
      • Abboud H.E.
      ). Immunoblotting of the lysates was performed using appropriate antibodies as described previously (
      • Choudhury G.G.
      ,
      • Ghosh Choudhury G.
      • Kim Y.S.
      • Simon M.
      • Wozney J.
      • Harris S.
      • Ghosh-Choudhury N.
      • Abboud H.E.
      ,
      • Ghosh-Choudhury N.
      • Ghosh-Choudhury G.
      • Celeste A.
      • Ghosh P.M.
      • Moyer M.
      • Abboud S.L.
      • Kreisberg J.
      ,
      • Ghosh-Choudhury N.
      • Woodruff K.
      • Qi W.
      • Celeste A.
      • Abboud S.L.
      • Ghosh Choudhury G.
      ).
      Electrophoretic Mobility Shift Assay—CL6 cells were incubated with BMP-2 in the presence and absence of Ly294002. Nuclear extracts were prepared using a kit according to the method provided by the vendor. An MEF-2 DNA probe was prepared by annealing the oligonucleotide 5′-GATCGCTCTAAAAATAACCCTGTCG-3′ with its complementary strand and labeling the double-stranded oligonucleotide consensus sequence using [γ-32P]ATP and T4 polynucleotide kinase (
      • Martin J.F.
      • Schwarz J.J.
      • Olson E.N.
      ). The EMSA was performed using 10 μg of the nuclear extract as described previously (
      • Choudhury G.G.
      • Ghosh-Choudhury N.
      • Abboud H.E.
      ,
      • Bardgette J.
      • Abboud H.E.
      • Choudhury G.G.
      ). To determine the specificity of protein·DNA interaction, the nuclear extract was incubated with a 100-fold excess of cold double-stranded oligonucleotide and incubated farther with the labeled probe. For supershift analysis, the nuclear extracts were incubated with the MEF-2A or cyclin D1 antibody on ice for 30 min before binding reaction was performed as described before (
      • Choudhury G.G.
      • Ghosh-Choudhury N.
      • Abboud H.E.
      ,
      • Ghosh Choudhury G.
      • Ricono J.M.
      ,
      • Bardgette J.
      • Abboud H.E.
      • Choudhury G.G.
      ).
      Transfection and Luciferase Assay—The BMP-2-LUC reporter plasmid, in which the firefly luciferase gene is driven by a 2.7-kb 5′-flanking sequence of BMP-2 gene, has been described before (
      • Ghosh-Choudhury N.
      • Abboud S.L.
      • Nishimura R.
      • Celeste A.
      • Mahimainathan L.
      • Choudhury G.G.
      ,
      • Ghosh-Choudhury N.
      • Harris M.A.
      • Feng J.Q.
      • Mundy G.R.
      • Harris S.E.
      ,
      • Ghosh-Choudhury N.
      • Windle J.J.
      • Koop B.A.
      • Harris M.A.
      • Guerrero D.L.
      • Wozney J.M.
      • Mundy G.R.
      • Harris S.E.
      ). BMP-2-LUC reporter plasmid was cotransfected with different expression plasmids using LipofectAMINE Plus reagent as described (
      • Choudhury G.G.
      ,
      • Ghosh Choudhury G.
      • Kim Y.S.
      • Simon M.
      • Wozney J.
      • Harris S.
      • Ghosh-Choudhury N.
      • Abboud H.E.
      ,
      • Ghosh Choudhury G.
      • Ricono J.M.
      ,
      • Ghosh-Choudhury N.
      • Choudhury G.G.
      • Harris M.A.
      • Wozney J.
      • Mundy G.R.
      • Abboud S.L.
      • Harris S.E.
      ). A cytomegalovirus promoter-driven Renilla luciferase plasmid was included in the transfection mix to correct for transfection efficiency. Luciferase activity was determined using a dual luciferase assay kit.

      RESULTS

      BMP-2 Stimulates PI 3-kinase Activity—CL6 cells, a clonal derivative of embryonic P19 teratocarcinoma cells, undergo differentiation into beating cardiomyocytes when treated with Me2SO in a BMP-2-dependent manner (
      • Monzen K.
      • Shiojima I.
      • Hiroi Y.
      • Kudoh S.
      • Oka T.
      • Takimoto E.
      • Hayashi D.
      • Hosoda T.
      • Habara-Ohkubo A.
      • Nakaoka T.
      • Fujita T.
      • Yazaki Y.
      • Komuro I.
      ). To investigate the role of PI 3-kinase in response to BMP-2, CL6 cells were incubated with 100 ng/ml BMP-2 for different periods of time. The lysates were then immunoprecipitated with an antibody recognizing the p85 regulatory subunit of PI 3-kinase. The immunoprecipitates were used in immunocomplex PI 3-kinase assays. BMP-2 increased PI 3-kinase activity in a time-dependent manner (Fig. 1A). The mechanism by which PI 3-kinase is activated by receptor and nonreceptor tyrosine kinases is by direct association of the SH-2 domain of the regulatory subunit with the phosphotyrosine of the proteins (
      • Shepherd P.R.
      • Withers D.J.
      • Siddle K.
      ). Therefore, we assayed PI 3-kinase activity in the anti-phosphotyrosine immunoprecipitates from BMP-2-treated CL6 cells. BMP-2 significantly increased PI 3-kinase activity in the anti-phosphotyrosine immunoprecipitates (Fig. 1B). These data indicate that activation of BMP receptor serine threonine kinase in CL6 cells activated PI 3-kinase in the tyrosine-phosphorylated protein fraction.
      Figure thumbnail gr1
      Fig. 1Effect of BMP-2 on PI 3-kinase activity in CL6 cardiomyocyte precursor cells.A, serum-deprived CL6 cells were incubated with 100 ng/ml BMP-2 for the indicated periods of time. Equal amounts of cleared cell lysates were immunoprecipitated with anti-p85 regulatory subunit antibody of PI 3-kinase, followed by immune complex kinase assay as described under ”Experimental Procedures.“ The 3-phosphoinositide was separated by TLC. B, the cells were stimulated with BMP-2 for 15 min. PI 3-kinase assay was performed in anti-phosphotyrosine immunoprecipitates and analyzed by TLC. Arrows indicate the position of PI 3-phosphate.
      BMP-2-induced PI 3-kinase Is Required for Cardiomyocyte Differentiation—To examine the role of PI 3-kinase in BMP-2-induced cardiomyocyte differentiation, we used Ly294002, a pharmacological inhibitor of PI 3-kinase. Incubation of CL6 cell with this inhibitor completely blocked BMP-2-induced PI 3-kinase activity (Fig. 2A). Mature cardiomyocytes express sarcomeric MHC. During differentiation of CL6 cells to cardiomyocytes, MHC is highly expressed and serves as a marker for mature cardiomyocytes (
      • Monzen K.
      • Shiojima I.
      • Hiroi Y.
      • Kudoh S.
      • Oka T.
      • Takimoto E.
      • Hayashi D.
      • Hosoda T.
      • Habara-Ohkubo A.
      • Nakaoka T.
      • Fujita T.
      • Yazaki Y.
      • Komuro I.
      ). To test the effect of PI 3-kinase on BMP-2-induced cardiomyocyte differentiation, CL6 cells were incubated with Ly294002 in the presence and absence of BMP-2. The cells were stained with anti-MHC antibody MF-20. As expected, BMP-2 efficiently stimulated expression of MHC, suggesting induction of mature cardiomyocytes (Fig. 2B, compare panel b with panel a). Inhibition of PI 3-kinase activity by Ly294002 abolished BMP-2-induced MHC expression (Fig. 2B, compare panel d with panel b). To further confirm the involvement of PI 3-kinase, we used an adenovirus vector (Ad Myrp110) containing the constitutively active myristoylated p110 catalytic subunit of PI 3-kinase. Infection of CL6 cells with this adenovirus vector showed expression of p110 within 24 h (Fig. 2C). To test the effect of PI 3-kinase on MHC expression, CL6 cells were infected with Ad Myr-p110. The cells were stained with MF-20. Expression of the constitutively active PI 3-kinase increased expression of sarcomeric MHC (Fig. 2D). These data indicate that PI 3-kinase is required for MHC-expressing mature cardiomyocyte formation in response to BMP-2.
      Figure thumbnail gr2
      Fig. 2PI 3-kinase regulates expression of MHC in CL6 cardiomyocyte precursor cells.A, PI 3-kinase inhibitor blocks BMP-2-induced PI 3-kinase activity. CL6 cells were incubated with 12.5 μm Ly294002 for 1 h before incubation with 100 ng/ml BMP-2 for 15 min. PI 3-kinase activity was determined in the anti-phosphotyrosine immunoprecipitates as described in . The arrow indicates the position of PI 3-phosphate. B, PI 3-kinase inhibitor prevents MHC expression. CL6 cells in eight-chamber slides were treated with Ly294002 followed by BMP-2. The cells were stained with MF-20 antibody as described under ”Experimental Procedures.“ C, expression of constitutively active p110 subunit of PI 3-kinase. CL6 cells were infected with a multiplicity of infection of 50 for Ad Myr-p110 for the indicated periods of time (
      • Ghosh-Choudhury N.
      • Abboud S.L.
      • Nishimura R.
      • Celeste A.
      • Mahimainathan L.
      • Choudhury G.G.
      ,
      • Choudhury G.G.
      ). 25 μg of cleared cell lysates was immunoblotted with anti-p110 antibody to detect the expression of the protein. The bottom panel shows immunoblot analysis of the same sample with actin antibody. D, expression of constitutively active PI 3-kinase increases MHC expression. CL6 cells were infected with Ad Myr-p110 or with a control virus Ad GFP as described (
      • Ghosh-Choudhury N.
      • Abboud S.L.
      • Nishimura R.
      • Celeste A.
      • Mahimainathan L.
      • Choudhury G.G.
      ,
      • Choudhury G.G.
      ) After infection, the cells were grown in serum-deprived medium for 48 h. The cells were stained with MF-20 antibody as described under ”Experimental Procedures.“
      PI 3-kinase Regulates BMP-2-induced MEF-2A Expression— Me2SO-mediated cardiomyocyte differentiation of CL6 cells has previously been shown to induce MEF-2C expression (
      • Monzen K.
      • Shiojima I.
      • Hiroi Y.
      • Kudoh S.
      • Oka T.
      • Takimoto E.
      • Hayashi D.
      • Hosoda T.
      • Habara-Ohkubo A.
      • Nakaoka T.
      • Fujita T.
      • Yazaki Y.
      • Komuro I.
      ). However, it is not known whether MEF-2A, which is also a marker for heart development, is expressed in CL6 cells (
      • Schlange T.
      • Andree B.
      • Arnold H.H.
      • Brand T.
      ). To address this and to examine the effect of BMP-2, CL6 cells were incubated with different concentrations of BMP-2. The lysates were immunoblotted with an antibody that recognizes MEF-2A. BMP-2 at a concentration of 40–100 ng/ml increased MEF-2A protein abundance (Fig. 3A). Me2SO also induced MEF-2A expression in these cells (Fig. 3B).
      Figure thumbnail gr3
      Fig. 3Effect of BMP-2 on MEF-2A protein expression in CL6 cells.A, BMP-2 increases MEF-2A protein expression. Quiescent cells were incubated with different concentrations of BMP-2 for 24 h. Equal amounts of proteins were immunoblotted with the MEF-2A antibody. The lower panel shows immunoblot analysis of the same samples with anti-actin antibody to demonstrate equal loading. B, Me2SO increases MEF-2A expression. Quiescent CL6 cells were incubated with 1% Me2SO. Equal amounts of protein were immunoblotted with MEF-2A antibody. The bottom panels show the immunoblot analysis of the same samples with anti-actin antibody. C, inhibition of PI 3-kinase blocks MEF-2A expression. Quiescent CL6 cells were treated with Ly294002 for 1 h. Cells were then incubated either with BMP-2 or Me2SO or BMP-2 plus Me2SO. Equal amounts of protein were immunoblotted with anti-MEF-2A antibody. The bottom panel shows the immunoblot of the same samples with actin antibody to show equal loading.
      To test the effect of PI 3-kinase on BMP-2-induced MEF-2A expression, we incubated CL6 cells with Ly294002 in the presence or absence of BMP-2. Cells were also treated with Me2SO or Me2SO plus Ly294002. As expected, BMP-2 and Me2SO alone increased MEF-2A expression (Fig. 3C, compare lanes 3 and 2 with lane 1). Addition of both BMP-2 and Me2SO did not further increase the abundance of MEF-2A (Fig. 3C, lane 4). Inhibition of PI 3-kinase activity abolished both BMP-2-induced and Me2SO-induced MEF-2A expression (Fig. 3C, compare lane 7 with 3 and lane 6 with 2, respectively). These data indicate that PI 3-kinase regulates a common target, which modulates MEF-2A expression in CL6 cells in response to BMP-2 as well as Me2SO.
      PI 3-kinase Regulates BMP-2-induced MEF-2A DNA Binding—Because BMP-2 increased MEF-2A protein expression, we tested whether the growth and differentiation factor stimulates the DNA binding activity of the transcription factor. Nuclear extracts isolated from BMP-2-treated CL6 cells were incubated with a 32P-labeled MEF-2 consensus oligonucleotide, and the products were analyzed by electrophoretic mobility shift assay. BMP-2 increased formation of protein·DNA complex (Fig. 4A, compare lane 2 with 1). Inclusion of a 100-fold excess of cold oligonucleotide completely prevented the protein·DNA complex formation, indicating the specificity of DNA-protein interaction (Fig. 4A, compare lane 3 with 2). To test the involvement of MEF-2A in the formation of protein·DNA complex, an antibody specific for MEF-2A was used in the EMSA. MEF-2A antibody inhibited the protein·DNA complex formation (Fig. 4B, compare lane 3 with 2). Note that the antibody partially supershifted the complex (Fig. 4B, lane 3, indicated by the bracket). A heterologous antibody against cyclin D1 did not have any effect on protein·DNA complex formation (Fig. 4B, lane 4). These data indicate that BMP-2 not only regulates the expression of MEF-2A in CL6 cells but also modulates its DNA binding activity to its cognate DNA element.
      Figure thumbnail gr4
      Fig. 4PI 3-kinase regulates BMP-2-induced MEF-2A DNA binding in CL6 cells.A, BMP-2 increases MEF-2A DNA binding. Quiescent CL6 cells were incubated with BMP-2. Nuclear extracts (10 μg) were used in EMSA as described under ”Experimental Procedures“ (
      • Choudhury G.G.
      • Ghosh-Choudhury N.
      • Abboud H.E.
      ,
      • Bardgette J.
      • Abboud H.E.
      • Choudhury G.G.
      ). For competition, 100-fold excess cold oligonucleotide was used with the nuclear extracts before the probe was added. The protein·DNA complex was separated by 5% polyacrylamide gel electrophoresis. The arrow indicates the specific protein·DNA complex. B, supershift analysis of BMP-2-induced protein·DNA complex. Nuclear extracts were incubated with MEF-2A antibody or nonspecific cyclin D1 antibody for 30 min on ice before incubation with the MEF-2A probe. Protein·DNA complexes were separated as described in panel A. The arrow indicates the protein·DNA complex. The bracket indicates the supershifted MEF-2A·DNA complex. C, inhibition of PI 3-kinase blocks BMP-2-induced MEF-2A DNA binding. CL6 cells were incubated with Ly294002 for 1 h before stimulation with BMP-2. Nuclear extracts were used in EMSA as described in panel A. The arrow indicates the protein·DNA complex.
      PI 3-kinase regulates receptor tyrosine kinase-induced transcription of target genes, which modulate cell survival (
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Hu L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ). However, nothing is known about regulation of BMP-receptor serine threonine kinase-induced PI 3-kinase-dependent MEF-2 transcriptional activation. Therefore, to assess the functional consequences of BMP-2-induced PI 3-kinase activation in MEF-2A DNA binding, an electrophoretic mobility shift assay was carried out with nuclear extracts isolated from CL6 cells incubated with BMP-2 alone and BMP-2 plus Ly294002. Inhibition of PI 3-kinase activity by this pharmacological agent abolished BMP-2-induced MEF-2A·DNA complex formation (Fig. 4C, compare lane 4 with 2). These data indicate that activation of PI 3-kinase regulates BMP receptor serine threonine kinase-induced MEF-2-DNA interaction.
      PI 3-kinase Regulates BMP-2-induced MEF-2-dependent Transcription—Transcriptional activation of MEF-2 family transcription factors is mediated by tyrosine kinase-dependent PI 3-kinase. For example, activation of insulin-like growth factor-1 receptor tyrosine kinase induces myogenic differentiation via PI 3-kinase-dependent increase in MEF-2 transcriptional activity (
      • Tamir Y.
      • Bengal E.
      ). To directly examine the effect of PI 3-kinase on MEF-2-dependent transcription in BMP-2 receptor serine threonine kinase activation, we constructed a reporter plasmid in which four copies of the MEF-2 DNA binding element were cloned upstream of the SV-40 basal promoter driving luciferase cDNA (Fig. 5A). The reporter plasmid was transfected into CL6 cells. Incubation of these transiently transfected cells with BMP-2 increased transcription of the reporter gene, indicating that BMP-2 stimulates transcription from the MEF-2 DNA element (Fig. 5B). Furthermore, cotransfection of MEF-2A cDNA with the reporter plasmid resulted in a significant increase in the transcriptional activity demonstrating that the reporter construct is responsive to the MEF-2A transcription factor (Fig. 5B). Addition of BMP-2 to these cotransfected cells showed no further significant additive effect. To test the involvement of PI 3-kinase in the BMP-2-induced transcription of this reporter gene, transiently transfected cells were incubated with the PI 3-kinase inhibitor, Ly294002, before addition of BMP-2. Inhibition of PI 3-kinase significantly blocked BMP-2-induced transcription of the reporter gene driven by the MEF-2 DNA element (Fig. 5C). To confirm this observation, reporter construct was cotransfected with a dominant negative 85-kDa subunit of PI 3-kinase. Transiently transfected cells were incubated with BMP-2. Dominant negative PI 3-kinase significantly prevented BMP-2-induced transcription of the reporter gene (Fig. 5D). These data indicate that BMP-2-induced PI 3-kinase activity is necessary for transcriptional activation of MEF-2.
      Figure thumbnail gr5
      Fig. 5PI 3-kinase regulates MEF-2A transcriptional activity.A, schematic showing the structure of the reporter plasmid in which luciferase gene is driven by four copies of MEF-2 DNA consensus element (
      • Martin J.F.
      • Schwarz J.J.
      • Olson E.N.
      ) from SV-40 basal promoter. B, BMP-2 and MEF-2A regulate reporter transcription. The reporter construct in panel A was cotransfected with vector or MEF-2A expression plasmid into CL6 cells. A Renilla luciferase plasmid was included with each transfection mix. Transiently transfected cells were incubated with BMP-2. Cell lysates were used for luciferase activity as described under ”Experimental Procedures.“ C, PI 3-kinase regulates BMP-2-induced reporter gene transcription. The same reporter plasmid in panel A was cotransfected with the Renilla plasmid into CL6 cells. The transiently transfected cells were treated with Ly294002 for 1 h before addition of BMP-2. Cell lysates were assayed for luciferase activity as described under ”Experimental Procedures.“ D, dominant negative PI 3-kinase inhibits BMP-2-induced reporter gene transcription. The reporter plasmid in panel A was cotransfected with vector or pSRαΔp85 coding for dominant negative p85 subunit of PI 3-kinase. The cells were treated with BMP-2, and the lysates were assayed for luciferase activity as described under ”Experimental Procedures.“
      MEF-2ARegulatesBMP-2GeneTranscriptioninaPI3-kinase-dependent Manner—We have shown previously that BMP-2 autoregulates its own transcription in osteogenic cells (
      • Ghosh-Choudhury N.
      • Windle J.J.
      • Koop B.A.
      • Harris M.A.
      • Guerrero D.L.
      • Wozney J.M.
      • Mundy G.R.
      • Harris S.E.
      ,
      • Ghosh-Choudhury N.
      • Choudhury G.G.
      • Harris M.A.
      • Wozney J.
      • Mundy G.R.
      • Abboud S.L.
      • Harris S.E.
      ). To investigate the involvement of PI 3-kinase in BMP-2 transcription, we used a reporter construct in which the firefly luciferase gene is driven by the promoter of the BMP-2 gene (BMP-2-LUC) (
      • Ghosh-Choudhury N.
      • Windle J.J.
      • Koop B.A.
      • Harris M.A.
      • Guerrero D.L.
      • Wozney J.M.
      • Mundy G.R.
      • Harris S.E.
      ). This plasmid was transfected either with vector alone or dominant negative p85 subunit of PI 3-kinase into CL6 cells. Incubation of transiently transfected cells with BMP-2 increased the reporter gene expression, indicating that BMP-2 autoregulates its own transcription in these cells similar to autoregulation in osteoblasts (Fig. 6A) (
      • Ghosh-Choudhury N.
      • Harris M.A.
      • Feng J.Q.
      • Mundy G.R.
      • Harris S.E.
      ,
      • Ghosh-Choudhury N.
      • Windle J.J.
      • Koop B.A.
      • Harris M.A.
      • Guerrero D.L.
      • Wozney J.M.
      • Mundy G.R.
      • Harris S.E.
      ). However, transfection of dominant negative PI 3-kinase completely blocked BMP-2-induced BMP-2 transcription (Fig. 6A). These data indicate that, in CL6 cardiomyocyte precursor cells, PI 3-kinase regulates BMP-2 gene transcription induced by BMP-2.
      Figure thumbnail gr6
      Fig. 6PI 3-kinase regulates MEF-2A-dependent BMP-2 transcription.A, dominant negative PI 3-kinase inhibits BMP-2-induced BMP-2 transcription in CL6 cells. BMP-2-LUC reporter plasmid was cotransfected into CL6 cells with either vector or pSrαΔp85, which encodes dominant negative p85 subunit of PI 3-kinase. The cells were incubated with BMP-2. Lysates were assayed for luciferase activity as described under ”Experimental Procedures.“ B, expression of MEF-2A stimulates BMP-2 transcription. CL6 cells were cotransfected with BMP-2-LUC reporter plasmid and MEF-2A expression vector. Also vector-transfected cells were incubated with BMP-2. Cell lysates were used for luciferase assay as described under ”Experimental Procedures.“ C, dominant negative PI 3-kinase blocks MEF-2A-mediated BMP-2 transcription. BMP-2-LUC reporter plasmid was cotransfected with MEF-2A expression vector and pSRαΔp85. Cell lysates were used for luciferase activity as described under ”Experimental Procedures.“
      To investigate the role of MEF-2 family of transcription factors in regulating the transcription of BMP-2, we analyzed the 5′-flanking sequence of BMP-2 gene. This analysis revealed the presence of three MEF-2 elements (
      • Ghosh-Choudhury N.
      • Choudhury G.G.
      • Harris M.A.
      • Wozney J.
      • Mundy G.R.
      • Abboud S.L.
      • Harris S.E.
      ). To examine involvement of MEF-2A in BMP-2 transcription, we transfected the BMP-2-LUC reporter plasmid along with the MEF-2A expression vector into CL6 cells. Ectopic expression of MEF-2A increased BMP-2 promoter activity to an extent similar to that induced by BMP-2 alone (Fig. 6B), suggesting that MEF-2A regulates BMP-2 gene transcription in CL6 cells. Next, we examined the involvement of PI 3-kinase in the regulation of the MEF-2A-dependent transcription of BMP-2 gene. BMP-2-LUC was cotransfected with the MEF-2A expression vector alone or along with dominant negative PI 3-kinase. As expected, MEF-2A increased transcription of the BMP-2 gene (Fig. 6C). Expression of dominant negative PI 3-kinase completely blocked the MEF-2A-dependent BMP-2 transcription (Fig. 6C). Together these data indicate that BMP-2-induced PI 3-kinase may regulate MEF-2A transcriptional activity, which in turn regulates BMP-2 gene transcription.
      MEF-2A Induces Formation of Mature Cardiomyocyte-expressing MHC—Because MEF-2A regulates BMP-2 expression and BMP-2 is necessary for differentiation of CL6 cells to form mature cardiomyocytes (
      • Monzen K.
      • Shiojima I.
      • Hiroi Y.
      • Kudoh S.
      • Oka T.
      • Takimoto E.
      • Hayashi D.
      • Hosoda T.
      • Habara-Ohkubo A.
      • Nakaoka T.
      • Fujita T.
      • Yazaki Y.
      • Komuro I.
      ), we examined the effect of MEF-2A expression on this process. CL6 cells were transfected with MEF-2A expression vector. The transfected cells were stained with the MF-20 antibody for sarcomeric MHC, which is expressed in mature cardiomyocyte. Expression of MEF-2A significantly increased expression of MHC in cardiomyocytes (Fig. 7, compare panel B with A). To address the involvement of PI 3-kinase, we treated the MEF-2A-transfected cells with Ly294002. The cells were then stained with MF-20 antibody. Inhibition of PI 3-kinase by the pharmacological inhibitor significantly blocked MEF-2A-induced MHC expression (Fig. 7, compare panel D with B). These data demonstrate that PI 3-kinase regulates MEF-2A-induced cardiomyocyte differentiation of CL6 cells.
      Figure thumbnail gr7
      Fig. 7MEF-2A regulates MHC expression. CL6 cells were transfected with the vector or MEF-2A expression plasmid (A and B). At 4 h post-transfection, the cells were incubated with Ly294002 (C and D). After 48 h, the cells were stained with MF-20 antibody as described under ”Experimental Procedures.“

      DISCUSSION

      The present study shows that, in cardiomyocyte precursor cells, BMP-2 activates PI 3-kinase. We demonstrate that PI 3-kinase regulates differentiation of precardiac cells to MHC-expressing mature cardiomyocytes. We show that BMP-2-induced expression of MEF-2A transcription factor, its DNA binding, and transcriptional activity requires activation of PI 3-kinase. Also we demonstrate that PI 3-kinase modulates autoregulation of BMP-2 gene transcription in these cells. Furthermore, we provide the first evidence that MEF-2A regulates transcription of BMP-2 gene and that this MEF-2A-dependent transcriptional regulation is PI 3-kinase-sensitive.
      The role of PI 3-kinase in growth factor and cytokine-induced signal transduction where receptor and nonreceptor tyrosine kinases are involved is well established (
      • Shepherd P.R.
      • Withers D.J.
      • Siddle K.
      ). PI 3-kinase regulates many biological activities, including proliferation, migration, endocytosis, protein transport, and secretion (
      • Toker A.
      • Cantley L.C.
      ). A role for PI 3-kinase has recently been shown in adipocyte, osteogenic, and myogenic differentiation (
      • Jiang B.H.
      • Zheng J.Z.
      • Vogt P.K.
      ,
      • Ghosh-Choudhury N.
      • Abboud S.L.
      • Nishimura R.
      • Celeste A.
      • Mahimainathan L.
      • Choudhury G.G.
      ,
      • Kaliman P.
      • Vinals F.
      • Testar X.
      • Palacin M.
      • Zorzano A.
      ,
      • Kulik G.
      • Klippel A.
      • Weber M.J.
      ,
      • Pinset C.
      • Garcia A.
      • Rousse S.
      • Dubois C.
      • Montarras D.
      ). BMP-2 is known to play important roles in cardiac development (
      • Zhang H.
      • Bradley A.
      ). However, the molecular basis for its effect on the differentiation process is poorly understood. We have shown here that BMP-2, which induces differentiation of precursor cells to mature cardiomyocytes, increases PI 3-kinase activity in these cells (Fig. 1A).
      PI 3-kinase consists of a catalytic p110 subunit and a regulatory SH-2 domain-containing p85 subunit (
      • Shepherd P.R.
      • Withers D.J.
      • Siddle K.
      ). One mechanism of tyrosine kinase-induced activation of this lipid kinase depends upon its association via its SH-2 domain to the phosphotyrosine residue of the tyrosine-phosphorylated proteins, which include the tyrosine kinase itself and its substrates (
      • Shepherd P.R.
      • Withers D.J.
      • Siddle K.
      ). This mechanism demonstrates direct physical association of PI 3-kinase activity with the tyrosine-phosphorylated protein fraction. Indeed, BMP-2-stimulated PI 3-kinase activity in anti-phosphotyrosine immunoprecipitates suggests the presence of PI 3-kinase in a tyrosine-phosphorylated signaling complex (Fig. 1B). Because BMP receptors possess serine threonine kinase activity and do not undergo tyrosine phosphorylation themselves, it remains to be identified with which tyrosine-phosphorylated protein PI 3-kinase is associated upon BMP-2 activation of CL6 cells.
      BMP-2 is necessary for cardiomyocyte development in vivo and in vitro (
      • Schultheiss T.M.
      • Burch J.B.
      • Lassar A.B.
      ,
      • Zhang H.
      • Bradley A.
      ,
      • Monzen K.
      • Shiojima I.
      • Hiroi Y.
      • Kudoh S.
      • Oka T.
      • Takimoto E.
      • Hayashi D.
      • Hosoda T.
      • Habara-Ohkubo A.
      • Nakaoka T.
      • Fujita T.
      • Yazaki Y.
      • Komuro I.
      ). Sarcomeric MHC is expressed in mature cardiomyocyte. It is established that BMP-2 increases MHC expression during cardiomyocyte differentiation (
      • Monzen K.
      • Shiojima I.
      • Hiroi Y.
      • Kudoh S.
      • Oka T.
      • Takimoto E.
      • Hayashi D.
      • Hosoda T.
      • Habara-Ohkubo A.
      • Nakaoka T.
      • Fujita T.
      • Yazaki Y.
      • Komuro I.
      , Fig. 2B). However, the signal transduction mechanism by which MHC is expressed in mature cardiomyocytes is not precisely known. We demonstrate that PI 3-kinase regulates BMP-2-induced MHC expression during cardiomyocyte differentiation (Fig. 2, B and D).
      BMP-2 exerts its biological effect by receptor-mediated serine phosphorylation of BMP-specific Smad 1, Smad 5, and Smad 8 (
      • Sakou T.
      ). After phosphorylation, the Smad proteins heterodimerize with Smad 4 to translocate to the nucleus and directly stimulates transcription of target genes necessary for biological activity. Thus ectopic expression of Smad 1 has been shown to promote survival of cardiac myocytes (
      • Izumi M.
      • Fujio Y.
      • Kunisada K.
      • Negoro S.
      • Tone E.
      • Funamoto M.
      • Osugi T.
      • Oshima Y.
      • Nakaoka Y.
      • Kishimoto T.
      • Yamauchi-Takihara K.
      • Hirota H.
      ). BMP-2 also stimulates expression of cardiac-specific genes such as MEF-2A during cardiomyocyte differentiation (
      • Schlange T.
      • Andree B.
      • Arnold H.H.
      • Brand T.
      ). Homozygous mutation in MEF-2C, a member of the MEF-2 family of transcription factor, is embryonically lethal due to defect in cardiac development (
      • Lin Q.
      • Schwarz J.
      • Bucana C.
      • Olson E.N.
      ). The phenotype is very similar to that seen in BMP-2 null mice (
      • Zhang H.
      • Bradley A.
      ). During mouse embryogenesis, MEF-2C is expressed in the precardiogenic mesoderm at embryonic day E7.75, whereas MEF2A is expressed beginning at E8.25 (
      • Edmondson D.G.
      • Lyons G.E.
      • Martin J.F.
      • Olson E.N.
      ,
      • Molkentin J.D.
      • Firulli A.B.
      • Black B.L.
      • Martin J.F.
      • Hustad C.M.
      • Copeland N.
      • Jenkins N.
      • Lyons G.
      • Olson E.N.
      ). BMP-2 is detectable as early as E7.5 (
      • Zhang H.
      • Bradley A.
      ), indicating that this growth and differentiation factor could play a role in expression of MEF-2A. Here we show that, in CL6 cardiomyocyte precursor cells, BMP-2 stimulated expression of MEF-2A (Fig. 3A). These data correlate with the expression pattern of BMP-2 and MEF-2A and a role of BMP-2 in MEF-2A expression during mouse cardiac development (
      • Zhang H.
      • Bradley A.
      ,
      • Edmondson D.G.
      • Lyons G.E.
      • Martin J.F.
      • Olson E.N.
      ,
      • Molkentin J.D.
      • Firulli A.B.
      • Black B.L.
      • Martin J.F.
      • Hustad C.M.
      • Copeland N.
      • Jenkins N.
      • Lyons G.
      • Olson E.N.
      ).
      The signaling mechanism regulating the expression of MEF-2A transcription factor is largely unknown. Recently, PI 3-kinase has been shown to regulate the transcriptional activity of MEF-2 in myogenic cells (
      • Xu Q.
      • Wu Z.
      ). However, PI 3-kinase does not have any effect on expression of MEF-2A (
      • Tamir Y.
      • Bengal E.
      ). In contrast to this observation, we demonstrate that BMP-2-induced expression of MEF-2A is regulated by PI 3-kinase in CL6 cardiomyocyte precursor cells (Fig. 3C).
      PI 3-kinase has been shown to regulate insulin-like growth factor-1-induced myogenic differentiation (
      • Kaliman P.
      • Vinals F.
      • Testar X.
      • Palacin M.
      • Zorzano A.
      ,
      • Pinset C.
      • Garcia A.
      • Rousse S.
      • Dubois C.
      • Montarras D.
      ,
      • Xu Q.
      • Wu Z.
      ). Expression of constitutively active PI 3-kinase increased transcription of myogenin (
      • Xu Q.
      • Wu Z.
      ). This increase in myogenin transcription was ascribed to the increase in MEF-2-mediated transcriptional activity (
      • Xu Q.
      • Wu Z.
      ). In another study, Tamir and Bengal (
      • Tamir Y.
      • Bengal E.
      ) showed that PI 3-kinase regulates the transcriptional activity of MEF-2 without having any effect on its DNA binding property. In contrast, in CL6 cardiomyocyte precursor cells, we show that BMP-2 increases MEF-2A·DNA interaction, and inhibition of PI 3-kinase activity blocks this DNA binding, resulting in significant reduction in MEF-2-dependent transcription of a reporter gene (Figs. 4 and 5). The reduction in protein·DNA complex formation and transcription from the MEF-2 DNA element may be in part due to the decreased expression of MEF-2A we observed in the presence of PI 3-kinase inhibition (Fig. 3). On the other hand, PI 3-kinase has been shown to increase the phosphorylation of MEF-2A, which regulates the transcriptional activity of this protein (
      • Tamir Y.
      • Bengal E.
      ). Therefore, we cannot completely rule out the possibility of post-translational modification of MEF-2A in regulating its DNA binding, resulting in its transcriptional activation in response to BMP-2 in CL6 cells.
      We have shown previously that BMP-2 autoregulates its expression during osteogenesis (
      • Ghosh-Choudhury N.
      • Windle J.J.
      • Koop B.A.
      • Harris M.A.
      • Guerrero D.L.
      • Wozney J.M.
      • Mundy G.R.
      • Harris S.E.
      ). This is one of the mechanisms by which BMP-2 maintains its sustained effect during osteoblast differentiation. Expression of the BMP-2 ortholog dpp is also regulated by dpp signaling during retinal differentiation in Drosophila (
      • Chanut F.
      • Heberlein U.
      ). Here we have shown BMP-2 autoregulates also its own transcription in cardiomyocyte precursor cells (Fig. 6A). Others have shown that, during cardiac development in mouse, BMP-2 and MEF-2A are expressed in precardial mesoderm, thereby suggesting a possible role of each protein in the other's expression (
      • Zhang H.
      • Bradley A.
      ,
      • Edmondson D.G.
      • Lyons G.E.
      • Martin J.F.
      • Olson E.N.
      ,
      • Molkentin J.D.
      • Firulli A.B.
      • Black B.L.
      • Martin J.F.
      • Hustad C.M.
      • Copeland N.
      • Jenkins N.
      • Lyons G.
      • Olson E.N.
      ). In fact, we have shown that BMP-2 stimulates MEF-2A expression in CL6 cells (Fig. 3). Analysis of the BMP-2 5′-flanking sequence revealed the presence of an MEF-2 consensus DNA element in the BMP-2 promoter (
      • Ghosh-Choudhury N.
      • Choudhury G.G.
      • Harris M.A.
      • Wozney J.
      • Mundy G.R.
      • Abboud S.L.
      • Harris S.E.
      ). The observation that expression of exogenous MEF-2A increased the transcription from BMP-2 promoter indicates that the DNA elements present in the BMP-2 5′-flanking sequence are responsive to this transcription factor (Fig. 6B). How this transcriptional activation is mediated is not known at this point. However, it is known that activation of the BMP receptor increases the transcriptional activity of the BMP-specific Smad proteins. Interaction between transforming growth factor β-specific Smad 2 and MEF-2A has been described in C2C12 myoblasts (
      • Quinn Z.A.
      • Yang C.C.
      • Wrana J.L.
      • McDermott J.C.
      ). In fact this interaction significantly increased the transcription of the target reporter gene, indicating the relevance of the physical association of MEF-2A and Smad 2. Therefore, our observation that MEF-2A significantly increased BMP-2 transcription may represent the presence of cross-talk between BMP-specific Smad(s) and MEF-2A or direct interaction of these transcription factors.
      The cystine knot protein noggin binds BMPs in the two hydrophobic patches, which directly interact with the BMP receptor and block BMP signal transduction (
      • Groppe J.
      • Greenwald J.
      • Wlater E.
      • Rodriguez-Leon J.
      • Economldes A.N.
      • Kwiatkowskl W.
      • Affolter M.
      • Vale W.W.
      • Belmonte J.C.I.
      • Choe S.
      ,
      • Zimmerman I.B.
      • De Jesus-Excober J.M.
      • Harland R.M.
      ,
      • Brunet L.J.
      • McMahon J.A.
      • McMahon A.P.
      • Harland R.M.
      ). Thus noggin prevents cardiac gene expression, including MEF-2A in chick embryo explant (
      • Schlange T.
      • Andree B.
      • Arnold H.H.
      • Brand T.
      ). Similar to the precardiac parental cells, precardiac cells expressing noggin do not undergo cardiac differentiation in response to Me2SO, indicating that sustained action of BMP-2 is necessary for this process (
      • Monzen K.
      • Shiojima I.
      • Hiroi Y.
      • Kudoh S.
      • Oka T.
      • Takimoto E.
      • Hayashi D.
      • Hosoda T.
      • Habara-Ohkubo A.
      • Nakaoka T.
      • Fujita T.
      • Yazaki Y.
      • Komuro I.
      ,
      • Jamali M.
      • Karamboulas C.
      • Rogerson P.J.
      • Skerjanc I.S.
      ). We also show that CL6 cells expressing noggin are incapable of activating MEF-2A in response to BMP-2 (Supplemental Fig. S1).
      Because BMP-2 stimulates MEFF-2A expression (Fig. 3A) and MEF-2A increases transcription of BMP-2 gene (Fig. 6), one possible explanation of this observation may be that BMP-2-mediated expression of MEF-2A may maintain the level of BMP-2 via its increased transcription. This pathway may represent one of the mechanisms of BMP-2 autoregulation, which maintains the level of BMP-2 required for cardiomyocyte differentiation (Fig. 8). Furthermore our data show that ectopic expression of MEF-2A increased expression of MHC (Fig. 7). Whether this effect of MEF-2A is due to its effect on BMP-2 expression, which in turn induces cardiomyocyte differentiation, is yet to be established. However, we demonstrate that MEF-2A-dependent MHC expression is PI 3-kinase-sensitive (Fig. 7).
      Figure thumbnail gr8
      Fig. 8Scheme of BMP-2-induced MEF-2A-dependent BMP-2 expression and cardiac differentiation. It is established (
      • Schultheiss T.M.
      • Burch J.B.
      • Lassar A.B.
      ,
      • Zhang H.
      • Bradley A.
      ) that BMP-2 stimulates BMP-specific Smads to regulate cardiac differentiation (indicated by black arrows). We demonstrate that BMP-2 increases PI 3-kinase activity, which regulates MEF-2A-dependent transcription of BMP-2 suggesting a plausible mechanism of autoregulation of BMP-2 expression. Also, increased MEF-2A expression regulates MHC expression, a marker for cardiac differentiation (green arrows). The dotted red arrow indicates a plausible interaction between BMP-specific Smad and MEF-2A (yet to be identified). This notion is based on the observation that Smad 2, a transforming growth factorβ-specific Smad, has been shown to interact directly with MEF-2A (
      • Quinn Z.A.
      • Yang C.C.
      • Wrana J.L.
      • McDermott J.C.
      ).
      In the present study, we provide the evidence that PI 3-kinase modulates BMP-2 gene transcription in CL6 cells (Fig. 6A). Several downstream targets of PI 3-kinase have been identified (
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Ahmadi K.
      • Timms J.
      • Katso R.
      • Driscoll P.C.
      • Woscholski R.
      • Parker P.J.
      • Waterfield M.D.
      ,
      • Wishart M.J.
      • Taylor G.S.
      • Dixon J.E.
      ). One is the Akt serine threonine kinase, which has been shown to play important roles in myogenic, osteogenic, and adipose differentiation (
      • Jiang B.H.
      • Zheng J.Z.
      • Vogt P.K.
      ,
      • Ghosh-Choudhury N.
      • Abboud S.L.
      • Nishimura R.
      • Celeste A.
      • Mahimainathan L.
      • Choudhury G.G.
      ,
      • Kaliman P.
      • Vinals F.
      • Testar X.
      • Palacin M.
      • Zorzano A.
      ,
      • Kulik G.
      • Klippel A.
      • Weber M.J.
      ,
      • Pinset C.
      • Garcia A.
      • Rousse S.
      • Dubois C.
      • Montarras D.
      ). Other targets of PI 3-kinase are the novel isoforms of PKC (
      • Parekh D.B.
      • Ziegler W.
      • Parker P.J.
      ). PKC[epsis ] and PKCδ have recently been shown to induce phosphorylation of MEF-2A, resulting in increased MEF-2A transcriptional activity (
      • Ornatsky O.I.
      • Cox D.M.
      • Tangirala P.
      • Andreucci J.J.
      • Quinn Z.A.
      • Wrana J.L.
      • Prywes R.
      • Yu Y.T.
      • McDermott J.C.
      ). In cardiomyocytes, the target of PI 3-kinase that modulates MEF-2A function has not yet been identified.
      In summary, we have shown that BMP-2 regulates expression of MHC in cardiomyocytes in a PI 3-kinase-sensitive manner. We have also shown that BMP-2 stimulates expression of MEF-2A transcription factor. Our data demonstrate that PI 3-kinase regulates its expression, DNA recognition, and transcriptional activity. Finally, we have provided the first evidence that BMP-2-induced BMP-2 transcription is mediated in a PI 3-kinase-sensitive and MEF-2-dependent manner. Taken together our data indicate that BMP-2 and MEF-2A orchestrate cardiac development in a concerted way using the PI 3-kinase signal transduction pathway.

      Acknowledgments

      We thank Dr. Dan Riley for critically reading the manuscript. We also thank Drs. Richard Prywes and Wataru Ogawa for providing the adenovirus vector and plasmid constructs and Dr. A. Celeste for providing us recombinant BMP-2 used in this study.

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