RESUMO
Phenotypic switching of smooth muscle cells (SMCs) plays a central role in the development of vascular diseases such as atherosclerosis and restenosis. However, the factors regulating expression of the human myocardin (Myocd) gene, the master gene regulator of SMC differentiation, have yet to be identified. In this study, we sought to identify the critical factors regulating Myocd expression in human SMCs. Using deletion/genetic reporter analyses, an upstream repressor region (URR) was localised within the Myocd promoter, herein termed PrmM. Bioinformatic analysis revealed three evolutionary conserved Klf4 sites within the URR and disruption of those elements led to substantial increases in PrmM-directed gene expression. Furthermore, ectopic expression established that Klf4 significantly decreased Myocd mRNA levels and PrmM-directed gene expression while electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP) assays confirmed specific binding of endogenous Klf4, and not Klf5 or Klf2, to the URR of PrmM. Platelet-derived growth factor BB (PDGF-BB), a potent inhibitor of SMC differentiation, reduced Myocd mRNA levels and PrmM-directed gene expression in SMCs. A PDGF-BB-responsive region (PRR) was also identified within PrmM, overlapping with the previously identified URR, where either siRNA knockdown of Klf4 or the combined disruption of the Klf4 elements completely abolished PDGF-BB-mediated repression of PrmM-directed gene expression in SMCs. Moreover, ChIP analysis established that PDGF-BB-induced repression of Myocd gene expression is most likely regulated by enhanced binding of Klf4 and Klf5 to a lesser extent, to the PRR of PrmM. Taken together, these data provide critical insights into the transcriptional regulation of the Myocd gene in vascular SMCs, including during SMC differentiation.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Fatores de Transcrição Kruppel-Like/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas Nucleares/genética , Transativadores/genética , Transcrição Gênica , Sequência de Bases , Becaplermina , Células Cultivadas , Primers do DNA , Humanos , Fator 4 Semelhante a Kruppel , Músculo Liso Vascular/citologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-sis/farmacologia , RNA Mensageiro/genéticaRESUMO
Recent genome-wide analyses have implicated alternative polyadenylation - the process of regulated mRNA 3' end formation - as a critical mechanism that influences multiple steps of mRNA metabolism in addition to increasing the protein-coding capacity of the genome. Although the functional consequences of alternative polyadenylation are well known, protein factors that regulate this process are poorly characterized. Previously, we described an evolutionarily conserved family of neuronal splice variants of the CstF-64 mRNA, ßCstF-64, that we hypothesized to function in alternative polyadenylation in the nervous system. In the present study, we show that ßCstF-64 mRNA and protein expression increase in response to nerve growth factor (NGF), concomitant with differentiation of adrenal PC-12 cells into a neuronal phenotype, suggesting a role for ßCstF-64 in neuronal gene expression. Using PC-12 cells as model, we show that ßCstF-64 is a bona fide polyadenylation protein, as evidenced by its association with the CstF complex, and by its ability to stimulate polyadenylation of luciferase reporter mRNA. Using luciferase assays, we show that ßCstF-64 stimulates polyadenylation equivalently at the two weak poly(A) sites of the ß-adducin mRNA. Notably, we demonstrate that the activity of ßCstF-64 is less than CstF-64 on a strong polyadenylation signal, suggesting polyadenylation site-specific differences in the activity of the ßCstF-64 protein. Our data address the polyadenylation functions of ßCstF-64 for the first time, and provide initial insights into the mechanism of alternative poly(A) site selection in the nervous system.