Downregulation of human endothelial nitric oxide synthase promoter activity by p38 mitogen-activated protein kinase activation

Abstract

Human endothelial nitric oxide synthase (eNOS) plays a crucial role in maintaining blood pressure homeostasis and vascular integrity. eNOS gene expression may be upregulated by a signaling pathway, including PI-3Kgamma--> Jak2--> MEK1 --> ERK1/2--> PP2A. It remains unclear whether other mitogen-activated protein kinase (MAPK) family members, such as JNK, p38 kinase, and ERK5/BMK1, also modulate eNOS gene expression. Our purpose, therefore, is to shed light on the effect of the p38 MAPK signaling pathway on the regulation of eNOS promoter activity. The results showed that a red fluorescent protein reporter gene vector containing the full length of the human eNOS promoter was first successfully constructed, expressing efficiently in ECV304 cells with the characteristics of real time observation. The wild-types of p38alpha, p38beta, p38gamma, and p38delta signal molecules all markedly downregulated promoter activity, which could be reversed by their negative mutants, including p38alpha (AF), p38beta (AF), p38gamma (AF), and p38delta (AF). Promoter activity was also significantly downregulated by MKK6b (E), an active mutant of an upstream kinase of p38 MAPK. The reduction in promoter activity by p38 MAPK could be blocked by treatment with a p38 MAPK specific inhibitor, SB203580. Moreover, the activation of endogenous p38 MAPK induced by lipopolysaccharide resulted in a prominent reduction in promoter activity. These findings strongly suggest that the activation of the p38 MAPK signaling pathway may be implicated in the downregulation of human eNOS promoter activity.