RESUMO
Platelet-derived growth factor (PDGF)-BB, a potent mitogen for mesenchymal cells, also downregulates expression of multiple smooth muscle (SM) cell (SMC)-specific markers. However, there is conflicting evidence whether PDGF-BB represses SMC marker expression at a transcriptional or posttranscriptional level, and little is known regarding the mechanisms responsible for these effects. Results of the present studies provide clear evidence that PDGF-BB treatment strongly repressed SM alpha-actin, SM myosin heavy chain (MHC), and SM22alpha promoters in SMCs. Of major significance for resolving previous controversies in the field, we found PDGF-BB-induced repression of SMC marker gene promoters in subconfluent, but not postconfluent, cultures. Treatment of postconfluent SMCs with a tyrosine phosphatase inhibitor restored PDGF-BB-induced repression, whereas treatment of subconfluent SMCs with a tyrosine kinase blocker abolished PDGF-BB-induced repression, suggesting that a tyrosine phosphorylation event mediates cell density-dependent effects. On the basis of previous observations that Ets-1 transcription factor is upregulated within phenotypically modulated neointimal SMCs, we tested whether Ets-1 would repress SMC marker expression. Consistent with this hypothesis, results of cotransfection experiments indicated that Ets-1 overexpression reduced transcriptional activity of SMC marker promoter constructs in SMCs, whereas it increased activity of SM alpha-actin promoter in endothelial cells. PDGF-BB treatment increased expression of Ets-1 in cultured SMCs, and SM alpha-actin mRNA expression was reduced in multiple independent clones of SMCs stably transfected with an Ets-1-overexpressing construct. Taken together, results of these experiments provide novel insights regarding possible mechanisms whereby PDGF-BB and Ets-1 may contribute to SMC phenotypic switching associated with vascular injury.
Assuntos
Anticoagulantes/farmacologia , Músculo Liso Vascular/fisiologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , Actinas/genética , Animais , Aorta Torácica/citologia , Becaplermina , Biomarcadores , Contagem de Células , Diferenciação Celular/fisiologia , Células Cultivadas , Luciferases/genética , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Cadeias Pesadas de Miosina/genética , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-ets-1 , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ets , Proteínas Proto-Oncogênicas c-sis , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The interactions between serum response factor (SRF) and CArG elements are critical for smooth muscle cell (SMC) marker gene transcription. However, the mechanisms whereby SRF, which is expressed ubiquitously, contributes to SMC-specific transcription are unknown. Myocardin was recently cloned as a coactivator of SRF in the heart, but its role in regulating CArG-dependent expression of SMC differentiation marker genes has not been clearly elucidated. In this study, we examined the expression and the function of myocardin in SMCs. In adult mice, myocardin mRNA was expressed in multiple smooth muscle (SM) tissues including the aorta, bladder, stomach, intestine, and colon, as well as the heart. Myocardin was also expressed in cultured rat aortic SMCs and A404 SMC precursor cells. Of particular interest, expression of myocardin was induced during differentiation of A404 cells, although it was not expressed in parental P19 cells from which A404 cells were derived. Cotransfection studies in SMCs revealed that myocardin induced the activity of multiple SMC marker gene promoters including SM alpha-actin, SM-myosin heavy chain, and SM22alpha by 9- to 60-fold in a CArG-dependent manner, whereas myocardin short interfering RNA markedly decreased activity of these promoters. Moreover, adenovirus-mediated overexpression of a dominant-negative form of myocardin significantly suppressed expression of endogenous SMC marker genes, whereas adenovirus-mediated overexpression of wild-type myocardin increased expression. Taken together, results provide compelling evidence that myocardin plays a key role as a transcriptional coactivator of SMC marker genes through CArG-dependent mechanisms.
Assuntos
Músculo Liso Vascular/metabolismo , Proteínas Nucleares/genética , Elementos de Resposta/genética , Transativadores/genética , Transcrição Gênica/genética , Células 3T3 , Animais , Biomarcadores , Linhagem Celular , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Expressão Gênica , Regulação da Expressão Gênica , Vetores Genéticos/genética , Camundongos , Músculo Liso Vascular/citologia , Mutação , Miocárdio/citologia , Miocárdio/metabolismo , Proteínas Nucleares/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologia , Ratos , Elementos de Resposta/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Resposta Sérica/metabolismo , Transativadores/fisiologia , TransfecçãoRESUMO
The SAP family transcription factor myocardin functionally synergizes with serum response factor (SRF) and plays an important role in cardiac development. To determine the function of myocardin in the smooth muscle cell (SMC) lineage, we mapped the pattern of myocardin gene expression and examined the molecular mechanisms underlying transcriptional activity of myocardin in SMCs and embryonic stem (ES) cells. The human and murine myocardin genes were expressed in vascular and visceral SMCs at levels equivalent to or exceeding those observed in the heart. During embryonic development, the myocardin gene was expressed abundantly in a precise, developmentally regulated pattern in SMCs. Forced expression of myocardin transactivated multiple SMC-specific transcriptional regulatory elements in non-SMCs. By contrast, myocardin-induced transactivation was not observed in SRF(-/-) ES cells but could be rescued by forced expression of SRF or the SRF DNA-binding domain. Furthermore, expression of a dominant-negative myocardin mutant protein or small-interfering-RNA-induced myocardin knockdown significantly reduced SM22 alpha promoter activity in SMCs. Most importantly, forced expression of myocardin activated expression of the SM22 alpha, smooth muscle alpha-actin, and calponin-h1 genes in undifferentiated mouse ES cells. Taken together, these data demonstrate that myocardin plays an important role in the SRF-dependent transcriptional program that regulates SMC development and differentiation.
