RESUMO
Inducible nitric-oxide synthase (iNOS) plays a central role in the regulation of vascular function and response to injury. A central mediator controlling iNOS expression is transforming growth factor-beta (TGF-beta), which represses its expression through a mechanism that is poorly understood. We have identified a binding site in the iNOS promoter that interacts with the nuclear heterodimer TCF11/MafG using chromatin immunoprecipitation and mutation analyses. We demonstrate that binding at this site acts to repress the induction of iNOS gene expression by cytokines. We show that this repressor is induced by TGF-beta1 and by Smad6-short, which enhances TGF-beta signaling. In contrast, the up-regulation of TCF11/MafG binding could be suppressed by overexpression of the TGF-beta inhibitor Smad7, and a small interfering RNA to TCF11 blocked the suppression of iNOS by TGF-beta. The binding of TCF11/MafG to the iNOS promoter could be enhanced by phorbol 12-myristate 13-acetate and suppressed by the protein kinase C inhibitor staurosporine. Moreover, the induction of TCF11/MafG binding by TGF-beta and Smad6-short could be blocked by staurosporine, and the effect of TGF-beta was blocked by the selective protein kinase C inhibitor calphostin C. Consistent with the in vitro data, we found suppression of TCF11 coincident with iNOS up-regulation in a rat model of endotoxemia, and we observed a highly significant negative correlation between TCF11 and nitric oxide production. Furthermore, treatment with activated protein C, a serine protease effective in septic shock, blocked the down-regulation of TCF11 and suppressed endotoxin-induced iNOS. Overall, our results demonstrate a novel mechanism by which iNOS expression is regulated in the context of inflammatory activation.
Assuntos
Regulação Enzimológica da Expressão Gênica , Fator 1-beta Nuclear de Hepatócito/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Elementos de Resposta , Fator de Crescimento Transformador beta1/metabolismo , Animais , Carcinógenos/farmacologia , Células Cultivadas , Dimerização , Modelos Animais de Doenças , Endotoxemia/enzimologia , Endotoxemia/genética , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Fator 1-beta Nuclear de Hepatócito/antagonistas & inibidores , Fator 1-beta Nuclear de Hepatócito/genética , Humanos , Inflamação/enzimologia , Inflamação/genética , Fator de Transcrição MafG/genética , Fator de Transcrição MafG/metabolismo , Masculino , Mutação , Naftalenos/farmacologia , Óxido Nítrico Sintase Tipo II/genética , Proteína Quinase C/antagonistas & inibidores , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Elementos de Resposta/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína Smad6/genética , Proteína Smad6/metabolismo , Proteína Smad7/genética , Proteína Smad7/metabolismo , Estaurosporina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
OBJECTIVE: Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands reduce lesion formation in animal models of atherosclerosis by mechanisms that have not been defined completely. We hypothesized that PPARgamma ligands stimulate endothelial-derived nitric oxide release (*NO) to protect the vascular wall. METHODS AND RESULTS: The PPARgamma ligands, 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2) or ciglitazone, stimulated a PPAR response element-luciferase reporter construct in transfected porcine pulmonary artery endothelial cells (PAECs), demonstrating that PPARgamma was transcriptionally functional. Treatment with 15d-PGJ2 or ciglitazone significantly increased release of *NO from PAECs or human aortic endothelial cells and augmented calcium ionophore-induced *NO release from human umbilical vein endothelial cells measured by chemiluminescence analysis of culture media. Increases in *NO release caused by treatment with 15d-PGJ2 occurred at 24 hours, but not after 1 to 16 hours, and were abrogated by treatment with the transcriptional inhibitor alpha-amanitin. Overexpression of PPARgamma or treatment with 9-cis retinoic acid also enhanced PAEC *NO release. Neither 15d-PGJ2 nor ciglitazone altered eNOS mRNA, whereas 15d-PGJ2, but not ciglitazone, decreased eNOS protein. CONCLUSIONS: Taken together, these findings demonstrate that PPARgamma ligands stimulate *NO release from endothelial cells derived from multiple vascular sites, through a transcriptional mechanism unrelated to eNOS expression.