RESUMEN
OBJECTIVE: Endothelial function depends on the equilibrium in the synthesis of vasoactive endothelial factors. It is well known that endothelin and nitric oxide (NO) exhibit reciprocal regulation. We assessed the ability of NO to regulate endothelin-converting enzyme-1 (ECE-1) expression in vascular endothelial cells. METHODS AND RESULTS: Bovine aortic endothelial cells were incubated with 2 different NO donors as well as with a cyclic-GMP analog, dibutyryl-cGMP (dB-cGMP). ECE-1 protein content and mRNA expression were evaluated by Western blot and Northern blot, respectively, promoter activity by transfection experiments, ECE-1 activity by ELISA, and cGMP production by radioimmunoassay. Both NO donors decreased ECE-1 protein content, mRNA expression, and ECE-1 activity. ODQ, an inhibitor of soluble guanylyl cyclase, blocked those effects. NO donors raised cGMP levels, and dB-cGMP mimicked their effects on ECE-1 expression, which were blocked by KT5823, a nonspecific PKG inhibitor. The changes on ECE-1 expression were due to a destabilization on 3'-untranslated region (3'-UTR) of this mRNA, because the activity of a luciferase reporter construct containing the 3'-UTR of the ECE-1 gene was reduced by dB-cGMP in a PKG-dependent manner. The biological relevance of this regulation was confirmed in bovine aortic endothelial cells coincubated with macrophages in the presence of lipopolysaccharide, in eNOS-deficient mice, and in Wistar rats treated with NO donors. In every case, an inverse relationship was observed between NO and ECE-1 protein content. CONCLUSION: Our results support that NO regulates ECE-1 expression through a cGMP/PKG-dependent regulatory mechanism at the post-transcriptional level via the 3'-UTR of the ECE-1 gene.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Metaloendopeptidasas/metabolismo , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico/metabolismo , ARN Mensajero/metabolismo , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/metabolismo , Ácido Aspártico Endopeptidasas/genética , Bovinos , Línea Celular , Técnicas de Cocultivo , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Regulación hacia Abajo/fisiología , Enzimas Convertidoras de Endotelina , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Guanilato Ciclasa/metabolismo , Células HEK293 , Humanos , Riñón/citología , Riñón/efectos de los fármacos , Riñón/embriología , Riñón/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Metaloendopeptidasas/genética , Ratones , Ratones Noqueados , Modelos Animales , Óxido Nítrico Sintasa de Tipo III/deficiencia , Óxido Nítrico Sintasa de Tipo III/genética , ARN Mensajero/efectos de los fármacos , Ratas WistarRESUMEN
The circulating levels of heat shock proteins (HSP) are increased in cardiovascular diseases; however, the implication of this for the fibrotic process typical of such diseases remains unclear. HSP70 can interact with the vascular smooth muscle cells (SMC), the major producer of extracellular matrix (ECM) proteins, through the Toll-like receptors 4 (TLR4). The transforming growth factor type-ß1 (TGF-ß1) is a well known vascular pro-fibrotic cytokine that is regulated in part by AP-1-dependent transcriptional mechanisms. We hypothesized that extracellular HSP70 could interact with SMCs, inducing TGF-ß1 synthesis and subsequent changes in the vascular ECM. We demonstrate that extracellular HSP70 binds to human aorta SMC TLR4, which up-regulates the AP-1-dependent transcriptional activity of the TGF-ß1 promoter. This is achieved through the mitogen activated protein kinases JNK and ERK, as demonstrated by the use of specific blockers and the knockdown of TLR4 with specific small interfering RNAs. The TGF-ß1 upregulation increase the expression of the ECM proteins type I collagen and fibronectin. This novel observation may elucidate the mechanisms by which HSP70 contributes in the inflammation and fibrosis present in atherosclerosis and other fibrosis-related diseases.
Asunto(s)
Matriz Extracelular/metabolismo , Proteínas HSP70 de Choque Térmico/fisiología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Células Cultivadas , Colágeno Tipo I/metabolismo , Fibronectinas/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Receptor Toll-Like 4/metabolismo , Factor de Transcripción AP-1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Regulación hacia ArribaRESUMEN
The aim of our study was to analyze the relationships between atherosclerosis and endothelin-converting enzyme-1 (ECE-1). Four-week-old C57BL/6J [wild-type (WT)] and apolipoprotein E-deficient (apoE) mice were fed with a standard or Western-type fat diet for 8 wks. ApoE showed atherosclerotic lesions in the aorta, higher blood pressure and vascular lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) protein content than WT. ApoE showed a significant increase in ECE-1 protein content and mRNA expression in aorta, lung, and kidney, without changes in heart. When an ECE-1 inhibitor, FR-901533, was administered to them, blood pressure decreased in apoE on fat diet versus apoE on normal diet and WT. ECE-1 and LOX-1 protein content were elevated in peripheral blood mononuclear cells (PBMC) from hypercholesterolemic patients. In order to study the mechanism involved in this ECE-1 up-regulation, bovine aortic endothelial cells (BAEC) were treated with oxidized-low density lipoproteins (oxLDL). OxLDL, but not LDL, increased ECE-1 protein content, mRNA expression and promoter activity. Our results demonstrate that ECE-1 increases in different atherosclerosis situations. Up-regulation of ECE-1 could contribute, at least partially, to the development of hypertension seen in apoE mice, because FR-901533 avoided it. Probably, atherosclerotic situations course with an increase of oxLDL, which is able to induce ECE-1 expression with the subsequent potential pathological effects.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Aterosclerosis/metabolismo , Lipoproteínas LDL/metabolismo , Metaloendopeptidasas/metabolismo , Anciano , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Ácido Aspártico Endopeptidasas/genética , Aterosclerosis/etiología , Aterosclerosis/genética , Aterosclerosis/fisiopatología , Secuencia de Bases , Bovinos , Células Cultivadas , ADN/genética , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Enzimas Convertidoras de Endotelina , Humanos , Hipercolesterolemia/sangre , Hipercolesterolemia/enzimología , Lipoproteínas LDL/farmacología , Masculino , Metaloendopeptidasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Depuradores de Clase E/sangre , Receptores Depuradores de Clase E/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
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