A Novel RhoA/ROCK-CPI-17-MEF2C Signaling Pathway Regulates Vascular Smooth Muscle Cell Gene Expression

Differentiation of vascular smooth muscle cells (VSMC) is a fundamental aspect of normal development and vascular disease. During contraction, VSMCs modulate calcium sensitivity through RhoA/ROCK-mediated inhibition of the myosin light chain phosphatase complex (MLCP). Previous studies have demonstrated that this signaling pathway functions in parallel to increase the expression of smooth muscle genes through the myocardin-family of co-activators. MEF2C fulfills a critical role in VSMC differentiation and regulates myocardin expression, leading us to investigate whether the RhoA/ROCK signaling cascade might regulate MEF2 activity. Depolarization-induced calcium signaling increased the expression of myocardin, which was sensitive to ROCK and p38 MAPK inhibition. We previously identified protein phosphatase 1α (PP1α), a known catalytic subunit of the MLCP in VSMCs, as a potent repressor of MEF2 activity. PP1α inhibition resulted in increased expression of myocardin, while ectopic expression of PP1α inhibited the induction of myocardin by MEF2C. Consistent with these data, shRNA-mediated suppression of a PP1α inhibitor, CPI-17, reduced myocardin expression and inhibited VSMC differentiation, suggesting a pivotal role for CPI-17 in regulating MEF2 activity. These data constitute evidence of a novel signaling cascade that links RhoA-mediated calcium sensitivity to MEF2-dependent myocardin expression in VSMCs through a mechanism involving p38 MAPK, PP1α, and CPI-17. Differentiation of vascular smooth muscle cells (VSMC) is a fundamental aspect of normal development and vascular disease. During contraction, VSMCs modulate calcium sensitivity through RhoA/ROCK-mediated inhibition of the myosin light chain phosphatase complex (MLCP). Previous studies have demonstrated that this signaling pathway functions in parallel to increase the expression of smooth muscle genes through the myocardin-family of co-activators. MEF2C fulfills a critical role in VSMC differentiation and regulates myocardin expression, leading us to investigate whether the RhoA/ROCK signaling cascade might regulate MEF2 activity. Depolarization-induced calcium signaling increased the expression of myocardin, which was sensitive to ROCK and p38 MAPK inhibition. We previously identified protein phosphatase 1α (PP1α), a known catalytic subunit of the MLCP in VSMCs, as a potent repressor of MEF2 activity. PP1α inhibition resulted in increased expression of myocardin, while ectopic expression of PP1α inhibited the induction of myocardin by MEF2C. Consistent with these data, shRNA-mediated suppression of a PP1α inhibitor, CPI-17, reduced myocardin expression and inhibited VSMC differentiation, suggesting a pivotal role for CPI-17 in regulating MEF2 activity. These data constitute evidence of a novel signaling cascade that links RhoA-mediated calcium sensitivity to MEF2-dependent myocardin expression in VSMCs through a mechanism involving p38 MAPK, PP1α, and CPI-17. IntroductionDuring development, vascular smooth muscle cells (VSMCs) 3The abbreviations used are: VSMCvascular smooth muscle cellsMLCPmyosin light chain phosphatasePP1αprotein phosphatase 1αSRFserum response factorMEFmyocyte enhancer factorROCKRhoA-associated kinaseCaMKcalcium/calmodulin-dependent kinaseCPIPKC-potentiated protein phosphatase inhibitorHandheart and neural crest-derived family. migrate to primitive endothelial tubes while simultaneously executing a program of differentiation to contribute to the vascular architecture (1.Drake C.J. Hungerford J.E. Little C.D. Morphogenesis of the first blood vessels.Ann. NY Acad. Sci. 1998; 857: 155-179Crossref PubMed Scopus (120) Google Scholar). Upon incorporation into the vasculature, VSMCs become quiescent and primarily regulate vascular tone (2.Owens G.K. Regulation of differentiation of vascular smooth muscle cells.Physiol. Rev. 1995; 75: 487-517Crossref PubMed Scopus (1380) Google Scholar). However, unlike terminally differentiated striated muscle cell types, VSMCs retain a capacity, referred to as the activated or synthetic phenotype, to proliferate postnatally in response to vascular injury. This activated phenotype is of particular clinical interest, since it plays an important role in most stenotic vascular diseases described to date (3.Owens G.K. Kumar M.S. Wamhoff B.R. Molecular regulation of vascular smooth muscle cell differentiation in development and disease.Physiol. Rev. 2004; 84: 767-801Crossref PubMed Scopus (2536) Google Scholar). The MADS-box (MCM-1, Agamous, Deficiens, Serum Response Factor) transcriptional regulators, serum response factor (SRF) and myocyte enhancer factor 2 (MEF2) play critical roles in the phenotypic modulation of VSMCs, as these transcription factors are known to regulate both immediate early genes involved in proliferation and migration, and, somewhat paradoxically, smooth muscle marker genes involved in the contractile phenotype (4.Miano J.M. Serum response factor: toggling between disparate programs of gene expression.J. Mol. Cell. Cardiol. 2003; 35: 577-593Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar, 5.Creemers E.E. Sutherland L.B. McAnally J. Richardson J.A. Olson E.N. Myocardin is a direct transcriptional target of Mef2, Tead, and Foxo proteins during cardiovascular development.Development. 2006; 133: 4245-4256Crossref PubMed Scopus (108) Google Scholar, 6.Gordon J.W. Pagiatakis C. Salma J. Du M. Andreucci J.J. Zhao J. Hou G. Perry R.L. Dan Q. Courtman D. Bendeck M.P. McDermott J.C. Protein kinase A-regulated assembly of a MEF2·HDAC4 repressor complex controls c-Jun expression in vascular smooth muscle cells.J. Biol. Chem. 2009; 284: 19027-19042Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The cellular signals that direct SRF to these distinct sets of genes have been intensively studied, where SRF physically interacts with the myocardin family of co-activators in contractile VSMCs to induce smooth muscle marker gene expression (7.Wang D.Z. Olson E.N. Control of smooth muscle development by the myocardin family of transcriptional coactivators.Curr. Opin. Genet. Dev. 2004; 14: 558-566Crossref PubMed Scopus (179) Google Scholar). However, in response to proliferative growth factor stimulation, myocardin is displaced from SRF, in favor of an Elk-1 interaction, to target immediate early gene expression, such as c-fos (8.Wang Z. Wang D.Z. Hockemeyer D. McAnally J. Nordheim A. Olson E.N. Myocardin and ternary complex factors compete for SRF to control smooth muscle gene expression.Nature. 2004; 428: 185-189Crossref PubMed Scopus (456) Google Scholar). Recently, calcium signaling induced by depolarization has been shown to increase the expression of both SRF-dependent immediate early genes and smooth muscle marker genes (9.Wamhoff B.R. Bowles D.K. McDonald O.G. Sinha S. Somlyo A.P. Somlyo A.V. Owens G.K. L-type voltage-gated Ca2+ channels modulate expression of smooth muscle differentiation marker genes via a rho kinase/myocardin/SRF-dependent mechanism.Circulat. Res. 2004; 95: 406-414Crossref PubMed Scopus (151) Google Scholar). Interestingly, the induction of c-fos in this model was prevented by calcium/calmodulin-dependent kinase (CaMK) inhibition, and the induction of VSMC marker genes was attenuated by RhoA-associated kinase (ROCK) inhibition (9.Wamhoff B.R. Bowles D.K. McDonald O.G. Sinha S. Somlyo A.P. Somlyo A.V. Owens G.K. L-type voltage-gated Ca2+ channels modulate expression of smooth muscle differentiation marker genes via a rho kinase/myocardin/SRF-dependent mechanism.Circulat. Res. 2004; 95: 406-414Crossref PubMed Scopus (151) Google Scholar). These results suggest that distinct calcium-mediated signaling pathways regulate these seemingly opposing SRF-dependent genes.Much less is known regarding the regulation of MEF2-dependent gene expression in VSMCs. Like SRF, MEF2 regulates the expression of immediate early genes, such as c-jun, and recent studies have suggested that c-jun expression in VSMCs is CaMK-dependent (6.Gordon J.W. Pagiatakis C. Salma J. Du M. Andreucci J.J. Zhao J. Hou G. Perry R.L. Dan Q. Courtman D. Bendeck M.P. McDermott J.C. Protein kinase A-regulated assembly of a MEF2·HDAC4 repressor complex controls c-Jun expression in vascular smooth muscle cells.J. Biol. Chem. 2009; 284: 19027-19042Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). However, MEF2C has also been shown to be genetically upstream of myocardin and of critical importance to VSMC differentiation (5.Creemers E.E. Sutherland L.B. McAnally J. Richardson J.A. Olson E.N. Myocardin is a direct transcriptional target of Mef2, Tead, and Foxo proteins during cardiovascular development.Development. 2006; 133: 4245-4256Crossref PubMed Scopus (108) Google Scholar, 10.Lin Q. Lu J. Yanagisawa H. Webb R. Lyons G.E. Richardson J.A. Olson E.N. Requirement of the MADS-box transcription factor MEF2C for vascular development.Development. 1998; 125: 4565-4574Crossref PubMed Google Scholar). Yet, the signaling pathways that regulate MEF2-dependent myocardin expression in VSMCs remain unknown; however, recent studies suggest that RhoA signaling may be involved (11.Ren J. Albinsson S. Hellstrand P. Distinct effects of voltage- and store-dependent calcium influx on stretch-induced differentiation and growth in vascular smooth muscle.J. Biol. Chem. 2010; 285: 31829-31839Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 12.Martin K. Weiss S. Metharom P. Schmeckpeper J. Hynes B. O'Sullivan J. Caplice N. Thrombin stimulates smooth muscle cell differentiation from peripheral blood mononuclear cells via protease-activated receptor-1, RhoA, and myocardin.Circ. Res. 2009; 105: 214-218Crossref PubMed Scopus (53) Google Scholar). We recently identified protein phosphatase 1α (PP1α) as a potent trans-dominant repressor of MEF2 activity (13.Perry R.L. Yang C. Soora N. Salma J. Marback M. Naghibi L. Ilyas H. Chan J. Gordon J.W. McDermott J.C. Direct interaction between myocyte enhancer factor 2 (MEF2) and protein phosphatase 1α represses MEF2-dependent gene expression.Mol. Cell. Biol. 2009; 29: 3355-3366Crossref PubMed Scopus (32) Google Scholar). Interestingly, in VSMCs PP1α serves as the catalytic subunit of the myosin light chain phosphatase complex (MLCP) and is regulated by RhoA signaling to control calcium sensitivity during contraction (14.Somlyo A.P. Somlyo A.V. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase.Physiol. Rev. 2003; 83: 1325-1358Crossref PubMed Scopus (1667) Google Scholar). In addition, signals emanating from RhoA in VSMCs have been previously shown to activate p38 MAP kinase (MAPK) signaling (15.Deaton R.A. Su C. Valencia T.G. Grant S.R. Transforming growth factor-β1-induced expression of smooth muscle marker genes involves activation of PKN and p38 MAPK.J. Biol. Chem. 2005; 280: 31172-31181Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), a known activator of MEF2 transcriptional activity in multiple cell types (16.Han J. Jiang Y. Li Z. Kravchenko V.V. Ulevitch R.J. Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation.Nature. 1997; 386: 296-299Crossref PubMed Scopus (678) Google Scholar, 17.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. Post-translational control of the MEF2A transcriptional regulatory protein.Nucleic Acids Res. 1999; 27: 2646-2654Crossref PubMed Scopus (93) Google Scholar, 18.Zhao M. New L. Kravchenko V.V. Kato Y. Gram H. di Padova F. Olson E.N. Ulevitch R.J. Han J. Regulation of the MEF2 family of transcription factors by p38.Mol. Cell. Biol. 1999; 19: 21-30Crossref PubMed Scopus (376) Google Scholar). In this report we document for the first time, a novel signaling pathway in VSMCs that links RhoA-mediated regulation of calcium sensitivity to MEF2-dependent expression of myocardin. This pathway involves the de-repression of MEF2 from PP1α inhibition by a two-step mechanism involving p38 MAPK and ROCK-mediated activation of the PP1α inhibitor, CPI-17 (PKC-potentiated protein phosphatase inhibitor of 17 kDa). Thus, this is the first report to identify a dominant signaling cascade that regulates myocardin expression in VSMCs, which may prove critical to our understanding of vascular development and stenotic vascular disease.DISCUSSIONMEF2C plays an essential role in VSMC differentiation and is genetically upstream of the SRF-coactivator, myocardin (5.Creemers E.E. Sutherland L.B. McAnally J. Richardson J.A. Olson E.N. Myocardin is a direct transcriptional target of Mef2, Tead, and Foxo proteins during cardiovascular development.Development. 2006; 133: 4245-4256Crossref PubMed Scopus (108) Google Scholar, 10.Lin Q. Lu J. Yanagisawa H. Webb R. Lyons G.E. Richardson J.A. Olson E.N. Requirement of the MADS-box transcription factor MEF2C for vascular development.Development. 1998; 125: 4565-4574Crossref PubMed Google Scholar). MEF2 proteins are integrators of a number of cellular signaling pathways, and are also regulated by several interacting co-factors that either enhance or repress transcriptional activity. We document in this report that cellular signals emanating from RhoA serve to relieve MEF2C from the repressive effects of PP1α to increase myocardin expression in VSMCs (Fig. 6). Furthermore, we demonstrate, for the first time, that this genetic pathway connecting CPI-17, MEF2C, and myocardin is critical for VSMC differentiation (FIGURE 1, FIGURE 2, FIGURE 3, FIGURE 4, FIGURE 5). In addition, PP1α serves to modulate c-Jun expression through an entirely different mechanism involving recruitment of HDAC4 to MEF2 proteins and phosphatase-dependent regulation of JNK signaling.Signal-dependent Control of PP1αThe cellular distribution and substrate specificity of PP1α is regulated by physical interaction with regulatory subunits, that typically contain a conserved RVXF domain (21.Cohen P.T. Protein phosphatase 1-targeted in many directions.J. Cell Science. 2002; 115: 241-256Crossref PubMed Google Scholar). In VSMCs, PP1α is targeted to the myosin light chains by physical interaction with MYPT1; however, this RVXF domain is also conserved among MADS-box proteins, such as MEF2A-D and may serve to target PP1α to nuclear MEF2 proteins (13.Perry R.L. Yang C. Soora N. Salma J. Marback M. Naghibi L. Ilyas H. Chan J. Gordon J.W. McDermott J.C. Direct interaction between myocyte enhancer factor 2 (MEF2) and protein phosphatase 1α represses MEF2-dependent gene expression.Mol. Cell. Biol. 2009; 29: 3355-3366Crossref PubMed Scopus (32) Google Scholar, 14.Somlyo A.P. Somlyo A.V. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase.Physiol. Rev. 2003; 83: 1325-1358Crossref PubMed Scopus (1667) Google Scholar). Interestingly, SRF also contains a conserved RVXF domain within its MADS-box, yet our data suggest that PP1α cannot overcome myocardin or TGF-β induction of SRF-target genes (Fig. 3F). In addition, the phosphatase activity of PP1α is regulated through interaction with specific inhibitor proteins like Inhibitor 1 and 2 (I1 and I2), and CPI-17. The potency of these inhibitor proteins is regulated by phosphorylation and dephosphorylation by cellular kinases and phosphatases, such as PKA, calcineurin, ROCK, and PKN (14.Somlyo A.P. Somlyo A.V. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase.Physiol. Rev. 2003; 83: 1325-1358Crossref PubMed Scopus (1667) Google Scholar, 21.Cohen P.T. Protein phosphatase 1-targeted in many directions.J. Cell Science. 2002; 115: 241-256Crossref PubMed Google Scholar, 33.Eto M. Regulation of cellular protein phosphatase-1 (PP1) by phosphorylation of the CPI-17 family, C-kinase-activated PP1 inhibitors.J. Biol. Chem. 2009; 284: 35273-35277Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). data that phosphorylation of CPI-17 by ROCK PKN regulates PP1α to modulate gene and have on MEF2 transcriptional activity (13.Perry R.L. Yang C. Soora N. Salma J. Marback M. Naghibi L. Ilyas H. Chan J. Gordon J.W. McDermott J.C. Direct interaction between myocyte enhancer factor 2 (MEF2) and protein phosphatase 1α represses MEF2-dependent gene expression.Mol. Cell. Biol. 2009; 29: 3355-3366Crossref PubMed Scopus (32) Google Scholar). The for this specificity is however, it may be to the distribution of and with the nuclear distribution of CPI-17, the of CPI-17 to compete with MEF2C for PP1α (21.Cohen P.T. Protein phosphatase 1-targeted in many directions.J. Cell Science. 2002; 115: 241-256Crossref PubMed Google have previously shown that PP1α regulates the transcriptional activity of MEF2 proteins through a number of PP1α physically interacts with both the and of and to transcriptional activity PP1α of and PP1α serves to HDAC4 to MEF2 (13.Perry R.L. Yang C. Soora N. Salma J. Marback M. Naghibi L. Ilyas H. Chan J. Gordon J.W. McDermott J.C. Direct interaction between myocyte enhancer factor 2 (MEF2) and protein phosphatase 1α represses MEF2-dependent gene expression.Mol. Cell. Biol. 2009; 29: 3355-3366Crossref PubMed Scopus (32) Google Scholar). We within the cellular of VSMCs, that these previously identified in a where PP1α regulates myocardin expression through direct interaction with MEF2C, and regulates c-jun expression by HDAC4 to MEF2 proteins and dephosphorylation of Furthermore, we identify a nuclear role for CPI-17 in regulating VSMC gene of CPI-17 expression in has that in to its expression in smooth muscle CPI-17 is also in the and J. G. L. Somlyo A. M. of CPI-17 in smooth muscle during development and in Cell Biol. 2009; PubMed Scopus Google Scholar). Interestingly, myocardin and several smooth muscle marker genes, such as expression in striated and smooth muscle types during development Li J. M. F. Yu Lu Owens G.K. M.S. Myocardin is a critical serum response factor in the transcriptional program regulating smooth muscle cell Cell. Biol. 2003; PubMed Scopus Google Scholar, D. Wang Z. Sutherland L. Richardson J.A. Olson E.N. Activation of gene expression by myocardin, a transcriptional for serum response 105: Full Text Full Text PDF PubMed Scopus Google Scholar, E.E. Wang D.Z. Olson E.N. J.M. Myocardin is a for smooth muscle Acad. Sci. A. PubMed Scopus Google Scholar). In light of our evidence the critical role of CPI-17 in the regulation of myocardin expression, these data suggest a role for CPI-17 regulating the development of muscle we novel evidence that PP1α serves as critical of MEF2-dependent gene expression in VSMCs, and for the first that RhoA-mediated signaling plays a fundamental role in myocardin expression through MEF2 These have important to the of vascular smooth muscle development and in the of vascular stenotic and for of VSMC differentiation in cell IntroductionDuring development, vascular smooth muscle cells (VSMCs) 3The abbreviations used are: VSMCvascular smooth muscle cellsMLCPmyosin light chain phosphatasePP1αprotein phosphatase 1αSRFserum response factorMEFmyocyte enhancer factorROCKRhoA-associated kinaseCaMKcalcium/calmodulin-dependent kinaseCPIPKC-potentiated protein phosphatase inhibitorHandheart and neural crest-derived family. migrate to primitive endothelial tubes while simultaneously executing a program of differentiation to contribute to the vascular architecture (1.Drake C.J. Hungerford J.E. Little C.D. Morphogenesis of the first blood vessels.Ann. NY Acad. Sci. 1998; 857: 155-179Crossref PubMed Scopus (120) Google Scholar). Upon incorporation into the vasculature, VSMCs become quiescent and primarily regulate vascular tone (2.Owens G.K. Regulation of differentiation of vascular smooth muscle cells.Physiol. Rev. 1995; 75: 487-517Crossref PubMed Scopus (1380) Google Scholar). However, unlike terminally differentiated striated muscle cell types, VSMCs retain a capacity, referred to as the activated or synthetic phenotype, to proliferate postnatally in response to vascular injury. This activated phenotype is of particular clinical interest, since it plays an important role in most stenotic vascular diseases described to date (3.Owens G.K. Kumar M.S. Wamhoff B.R. Molecular regulation of vascular smooth muscle cell differentiation in development and disease.Physiol. Rev. 2004; 84: 767-801Crossref PubMed Scopus (2536) Google Scholar). The MADS-box (MCM-1, Agamous, Deficiens, Serum Response Factor) transcriptional regulators, serum response factor (SRF) and myocyte enhancer factor 2 (MEF2) play critical roles in the phenotypic modulation of VSMCs, as these transcription factors are known to regulate both immediate early genes involved in proliferation and migration, and, somewhat paradoxically, smooth muscle marker genes involved in the contractile phenotype (4.Miano J.M. Serum response factor: toggling between disparate programs of gene expression.J. Mol. Cell. Cardiol. 2003; 35: 577-593Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar, 5.Creemers E.E. Sutherland L.B. McAnally J. Richardson J.A. Olson E.N. Myocardin is a direct transcriptional target of Mef2, Tead, and Foxo proteins during cardiovascular development.Development. 2006; 133: 4245-4256Crossref PubMed Scopus (108) Google Scholar, 6.Gordon J.W. Pagiatakis C. Salma J. Du M. Andreucci J.J. Zhao J. Hou G. Perry R.L. Dan Q. Courtman D. Bendeck M.P. McDermott J.C. Protein kinase A-regulated assembly of a MEF2·HDAC4 repressor complex controls c-Jun expression in vascular smooth muscle cells.J. Biol. Chem. 2009; 284: 19027-19042Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The cellular signals that direct SRF to these distinct sets of genes have been intensively studied, where SRF physically interacts with the myocardin family of co-activators in contractile VSMCs to induce smooth muscle marker gene expression (7.Wang D.Z. Olson E.N. Control of smooth muscle development by the myocardin family of transcriptional coactivators.Curr. Opin. Genet. Dev. 2004; 14: 558-566Crossref PubMed Scopus (179) Google Scholar). However, in response to proliferative growth factor stimulation, myocardin is displaced from SRF, in favor of an Elk-1 interaction, to target immediate early gene expression, such as c-fos (8.Wang Z. Wang D.Z. Hockemeyer D. McAnally J. Nordheim A. Olson E.N. Myocardin and ternary complex factors compete for SRF to control smooth muscle gene expression.Nature. 2004; 428: 185-189Crossref PubMed Scopus (456) Google Scholar). Recently, calcium signaling induced by depolarization has been shown to increase the expression of both SRF-dependent immediate early genes and smooth muscle marker genes (9.Wamhoff B.R. Bowles D.K. McDonald O.G. Sinha S. Somlyo A.P. Somlyo A.V. Owens G.K. L-type voltage-gated Ca2+ channels modulate expression of smooth muscle differentiation marker genes via a rho kinase/myocardin/SRF-dependent mechanism.Circulat. Res. 2004; 95: 406-414Crossref PubMed Scopus (151) Google Scholar). Interestingly, the induction of c-fos in this model was prevented by calcium/calmodulin-dependent kinase (CaMK) inhibition, and the induction of VSMC marker genes was attenuated by RhoA-associated kinase (ROCK) inhibition (9.Wamhoff B.R. Bowles D.K. McDonald O.G. Sinha S. Somlyo A.P. Somlyo A.V. Owens G.K. L-type voltage-gated Ca2+ channels modulate expression of smooth muscle differentiation marker genes via a rho kinase/myocardin/SRF-dependent mechanism.Circulat. Res. 2004; 95: 406-414Crossref PubMed Scopus (151) Google Scholar). These results suggest that distinct calcium-mediated signaling pathways regulate these seemingly opposing SRF-dependent genes.Much less is known regarding the regulation of MEF2-dependent gene expression in VSMCs. Like SRF, MEF2 regulates the expression of immediate early genes, such as c-jun, and recent studies have suggested that c-jun expression in VSMCs is CaMK-dependent (6.Gordon J.W. Pagiatakis C. Salma J. Du M. Andreucci J.J. Zhao J. Hou G. Perry R.L. Dan Q. Courtman D. Bendeck M.P. McDermott J.C. Protein kinase A-regulated assembly of a MEF2·HDAC4 repressor complex controls c-Jun expression in vascular smooth muscle cells.J. Biol. Chem. 2009; 284: 19027-19042Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). However, MEF2C has also been shown to be genetically upstream of myocardin and of critical importance to VSMC differentiation (5.Creemers E.E. Sutherland L.B. McAnally J. Richardson J.A. Olson E.N. Myocardin is a direct transcriptional target of Mef2, Tead, and Foxo proteins during cardiovascular development.Development. 2006; 133: 4245-4256Crossref PubMed Scopus (108) Google Scholar, 10.Lin Q. Lu J. Yanagisawa H. Webb R. Lyons G.E. Richardson J.A. Olson E.N. Requirement of the MADS-box transcription factor MEF2C for vascular development.Development. 1998; 125: 4565-4574Crossref PubMed Google Scholar). Yet, the signaling pathways that regulate MEF2-dependent myocardin expression in VSMCs remain unknown; however, recent studies suggest that RhoA signaling may be involved (11.Ren J. Albinsson S. Hellstrand P. Distinct effects of voltage- and store-dependent calcium influx on stretch-induced differentiation and growth in vascular smooth muscle.J. Biol. Chem. 2010; 285: 31829-31839Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 12.Martin K. Weiss S. Metharom P. Schmeckpeper J. Hynes B. O'Sullivan J. Caplice N. Thrombin stimulates smooth muscle cell differentiation from peripheral blood mononuclear cells via protease-activated receptor-1, RhoA, and myocardin.Circ. Res. 2009; 105: 214-218Crossref PubMed Scopus (53) Google Scholar). We recently identified protein phosphatase 1α (PP1α) as a potent trans-dominant repressor of MEF2 activity (13.Perry R.L. Yang C. Soora N. Salma J. Marback M. Naghibi L. Ilyas H. Chan J. Gordon J.W. McDermott J.C. Direct interaction between myocyte enhancer factor 2 (MEF2) and protein phosphatase 1α represses MEF2-dependent gene expression.Mol. Cell. Biol. 2009; 29: 3355-3366Crossref PubMed Scopus (32) Google Scholar). Interestingly, in VSMCs PP1α serves as the catalytic subunit of the myosin light chain phosphatase complex (MLCP) and is regulated by RhoA signaling to control calcium sensitivity during contraction (14.Somlyo A.P. Somlyo A.V. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase.Physiol. Rev. 2003; 83: 1325-1358Crossref PubMed Scopus (1667) Google Scholar). In addition, signals emanating from RhoA in VSMCs have been previously shown to activate p38 MAP kinase (MAPK) signaling (15.Deaton R.A. Su C. Valencia T.G. Grant S.R. Transforming growth factor-β1-induced expression of smooth muscle marker genes involves activation of PKN and p38 MAPK.J. Biol. Chem. 2005; 280: 31172-31181Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), a known activator of MEF2 transcriptional activity in multiple cell types (16.Han J. Jiang Y. Li Z. Kravchenko V.V. Ulevitch R.J. Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation.Nature. 1997; 386: 296-299Crossref PubMed Scopus (678) Google Scholar, 17.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. Post-translational control of the MEF2A transcriptional regulatory protein.Nucleic Acids Res. 1999; 27: 2646-2654Crossref PubMed Scopus (93) Google Scholar, 18.Zhao M. New L. Kravchenko V.V. Kato Y. Gram H. di Padova F. Olson E.N. Ulevitch R.J. Han J. Regulation of the MEF2 family of transcription factors by p38.Mol. Cell. Biol. 1999; 19: 21-30Crossref PubMed Scopus (376) Google Scholar). In this report we document for the first time, a novel signaling pathway in VSMCs that links RhoA-mediated regulation of calcium sensitivity to MEF2-dependent expression of myocardin. This pathway involves the de-repression of MEF2 from PP1α inhibition by a two-step mechanism involving p38 MAPK and ROCK-mediated activation of the PP1α inhibitor, CPI-17 (PKC-potentiated protein phosphatase inhibitor of 17 kDa). Thus, this is the first report to identify a dominant signaling cascade that regulates myocardin expression in VSMCs, which may prove critical to our understanding of vascular development and stenotic vascular disease.

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