Abstract
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 β1 to β166 bp, and 32% of it was dependent on the region from β1,033 to β1,600 bp. The mutation of SSRE element (β999βΌβ994 bp) and wildβtype SSRE decoy oligodeoxynucleotides (ODN) did not alter the basal activity and the stimulating activity by lysophosphatidycholine (LPC). The mutation of upstream AP1 element (β1,530βΌβ1,524 bp) did not affect the basal activity, but resulted in near 30% reduction in the stimulating activity by LPC. Moreover, wildβtype 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 (β662βΌβ656 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 β1 bp to β166 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. J. Cell. Biochem. 98: 872β884, 2006. Β© 2006 WileyβLiss, Inc.