Role of AP1 element in the activation of human eNOS promoter by lysophosphatidylcholine.
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.
Xing
Feiyue
F
Department of Biochemistry, National Specialized Laboratory and the Key Laboratory of Ministry of Education, Jinan University, Guangzhou 510632, People's Republic of China. fyxlj@hotmail.com
Jiang
Yong
Y
Liu
Jing
J
Zhao
Kesen
K
Mo
Yongyan
Y
Qin
Qinghe
Q
Wang
Jingzhen
J
Ouyang
Jianming
J
Zeng
Yaoying
Y
D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D008244:Lysophosphatidylcholines / Q000378:metabolism / Q000494:pharmacology*; D051379:Mice; D016296:Mutagenesis; D009154:Mutation; D041681:NIH 3T3 Cells; D052250:Nitric Oxide Synthase Type III / Q000096:biosynthesis* / Q000235:genetics; D020218:Response Elements / Q000502:physiology*; D018808:Transcription Factor AP-1 / Q000378:metabolism*
D008244:Lysophosphatidylcholines; D018808:Transcription Factor AP-1; D052250:Nitric Oxide Synthase Type III