Regulation Of The Transcriptional Activity Of Human Endothelial Nitric Oxide Synthase Gene Promoter By P38α Mitogen Activated Protein Kinase

AIM:To investigate the effect of the p38a MAPK signaling pathway on the regulation of human endothelial nitric oxide synthase (eNOS) promoter activity. METHODS: Use the plasmid mini kit to extract diverse kinds of plamids, including pDsRedl-Nl, pGL3-BASIC, pGL2-eNOS, pRL-TK, pcDNA3, p38a and p38a(AF), then use agarose gel electrophoresis to identify these plasmids, and use the spectrophotometer to quantitate them. Using different concentration of transfection reagent, the human umbilical vein endothelial cell-12s(HUVEC-12) were transfected with pDsRedl-Nl, and then the fluorescence intensity was observed by fluorescence inverted phase contrast microscope to determine the optimal concentration for transfection. To establish a dual-Luciferase assay model, the cells were cotransfected with a pGL2-eNOS plus pGL3-BASIC, pcDNA3, p38a or negative mutant p38a(AF), as well as a pRL-TK vector as an internal control. The transcription activity of human eNOS promoter was determined through a dual-Luciferase reporter gene system. The effect of p38a MAPK inhibitor SB203580 on eNOS promoter activity was estimated also by the dual-Luciferase reporter gene system, and Western blot was used to analyze phosphorylated and unphosphorylated p38 proteins. To observe the relationship between the change of eNOS promoter activity and p38 protein expression, the siRNA was used to silence p38a gene. RESULTS:The plasmid p38a markedly downregulated the promoter activity which could be reversed by its negative mutant p38a (AF). The expression of phosphorylated p38 protein was remarkably decreased by inhibitor SB203580, but the level of unphosphorylated p38 protein was not siganificantly altered. Following the remarkable decrease of the phosphorylated p38 protein expression, the eNOS promoter activity became enhanced. Differfint concentration of siRNA had differfint silent effect on p38a gene. At the concentration of 100 nM, the silent effect was more significant than other kinds of concentration, followed by a remarkable decrease in p38 protein expression. As the stimulation time of siRNA lasted, the siRNA silent effect changed. At the time of 48h, the silent effect was most significant campared with other time points, followed by the least p38 protein expression. Choosing the optimized concentration and time point of siRNA, the expression of p38 protein was remarkably decreased, and the eNOS promoter activity was enhanced. In a word, these findings demonstrated that the phosphorylation of p38a could decrease the eNOS promoter activity, which might be reversed by the negative mutant p38a (AF). Both inhibitor SB203580 and siRNA could reduce the level of phosphorylation of p38a. On the contray, the eNOS promoter activity was increased. CONCLUSION: The activation of the p38aMAPK signaling pathway downregulates the human eNOS promoter activity.