Human endothelial nitric oxide synthase (eNOS) plays a crucial role in maintaining blood pressure homeostasis and vascular integrity. It, therefore, is very essential to elucidate the regulation of it. In the current study, a red fluorescent protein (RFP) reporter system containing human eNOS promoter was first constructed, being characteristics of real time morphologic and quantitative analysis for the same sample. It was observed by DNA sequence deletion that 68% of the basal activity of the promoter was controlled by the region from -1 to -166 bp, and 32% of it was dependent on the region from -1,033 to -1,600 bp. The mutation of SSRE element (-999 to -994 bp) and wild-type SSRE decoy oligodeoxynucleotides (ODN) did not alter the basal activity and the stimulating activity by lysophosphatidylcholine (LPC). The mutation of upstream AP1 element (-1,530 to -1,524 bp) did not affect the basal activity, but resulted in near 30% reduction in the stimulating activity by LPC. Moreover, wild-type AP1 decoy ODN also remarkably attenuated it. It was proved by EMSA analysis that LPC indeed enhanced the activity of AP1 transcriptional factor binding to AP1 element. However, the role of AP1 was dependent on the presence of SP1, which was proved by the combining mutation of AP1 with SP1. The mutation of downstream AP1 element (-662 to -656 bp) had no influence on the basal and stimulating activities by LPC. These results strongly suggest that the main functional region of the promoter is from -1 bp to -166 bp, that the upstream AP1 participates in the activation of the promoter by LPC on the premise of the presence of SP1, and that the downstream AP1 and SSRE do not involve the basal and stimulating activity by LPC.

Human endothelial nitric oxide synthase (eNOS) plays a pivotal role in maintaining blood pressure homeostasis and vascular integrity. It has recently been reported that mitogen-activated protein kinases (MAPKs) are intimately implicated in expression of eNOS. However detailed mechanism mediated by them remains to be clarified. In this study, eNOS gene transactivity in human umbilical vein endothelial cells was up-regulated by stimulation of lysophosphatidylcholine (LPC). The stimulation of LPC highly activated both extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK), with differences in the dynamic processes of activation between them. Unexpectedly, p38 MAPK could not be activated by the stimulation of LPC. The activation of JNK signalling pathway by overexpression of JNK or its upstream kinase active mutant up-regulated the transactivity of eNOS significantly, but the activation of p38 signalling pathway down-regulated it largely. The inhibition of either ERK1/2 or JNK signalling pathway by kinase-selective inhibitors could markedly block the induction of the transactivity by LPC. It was observed by electrophoretic mobility shift assay that LPC stimulated both SP1 and AP1 DNA binding activity to go up. Additionally using decoy oligonucleotides proved that SP1 was necessary for maintaining the basal or stimulated transactivity, whereas AP1 contributed mainly to the increase of the stimulated transactivity. These findings indicate that the up-regulation of the eNOS gene transactivity by LPC involves the enhancement of SP1 transcription factor by the activation of JNK and ERK1/2 signalling pathways and AP1 transcription factor by the activation of JNK signalling pathway.

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.

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