Roles Of AP-2α In Atherosclerosis And The HeLa Cell Apoptosis

PartⅠInvolvement of transcription factor AP-2αin atherosclerosis by its direct regulation to CD36Aims:The transcription factor activator protein-2α(AP-2α) regulates cell-type specific gene expression during development, differentiation and cancer,but its role in atherosclerosis has so far not been explored.This study addresses the potential role of AP-2αin the foam cell formation of atherosclerotic lesions through its direct regulation to CD36 gene.Methods:Immunohistochemistry and double immunofluorescent staining were used to detect the expression of AP-2αin apoE^-/-mice and human atherosclerotic lesions.Relative quantitative real-time RT-PCR,western blot and immunofluorescent staining were used to detect the effect of AP-2αon CD36 expression in rat vascular smooth muscle cells(VSMCs).Oil Red O staining and high performance liquid chromatography analysis were performed to assess the lipid accumulation in VSMCs.To demonstrate the direct regulation of AP-2αto the promoter of CD36 gene,Luciferase assay,electrophoretic mobility shift assay,and chromatin immunoprecipitation assay were performed in VSMCs.Results:AP-2αwas highly expressed mostly in foam cells of atherosclerotic aortas whether in apoE^-/-mice or in human.AP-2α up-regulated the mRNA and protein expression of CD36 gene and promoted the lipid accumulation in VSMCs.The promoter activity of CD36 gene was enhanced by AP-2αover-expression and there were two AP-2 binding sites in human CD36 promoter region.Mutation of the two AP-2 binding sites abolished the enhanced effect by AP-2α.Conclusions: AP-2αmay function in atherogenesis by directly enhancing the expression of CD36 and consequently increasing the lipid uptake.PartⅡInvolvement of Transcription Factor AP-2αin Doxazosin-Induced HeLa Cell ApoptosisAims:To investigate the pro-apoptotic effects ofα1-adrenergic inhibitor doxazosin in HeLa cells and the potential involvement of transcription factor activator protein-2α(AP-2α)in doxazosin-induced apoptosis.Methods:The HeLa cells were exposed to various concentrations of doxazosin for 16h.Apoptosis was detected using a DNA fragmentation assay,Hoechst 33258 staining,and flow cytometric analysis.The expression of AP-2αand caspase-3 was detected by relative quantitative RT-PCR and Western blot assays,respectively.After the respective transfections of the HeLa cells with AP-2αoverexpressing constructs and an antisense oligonucleotide against AP-2α,apoptosis was assessed by flow cytometric analysis,and the expression of AP-2αand caspase-3 was detected by relative quantitative RT-PCR and Western blot assays.The colorimetric assay was performed to detect the caspase-3 activity.Results:Treatment with various concentrations of doxazosin for 16h increased the apoptotic rate and total cell death rate of the HeLa cells in a dose-dependent manner and upregulated the expression of AP-2αand caspase-3 in a dose-dependent manner.A dose-dependent increase was observed in the caspase-3 activity.Overexpressing AP-2αled to the increased rate of doxazosin-induced apoptosis and the total cell death, whereas doxazosin-induced apoptosis and the total cell death in HeLa cells decreased by antisense AP-2α.Furthermore,overexpressing AP-2αincreased the expression and activity of caspase-3,whereas antisense AP-2αin part abolished the increased effects of doxazosin on caspase-3 expression and activity.Conclusions:Doxazosin induces apoptosis in HeLa cells in a dose-dependent manner,and transcription factor AP-2αis functionally involved in doxazosin-induced HeLa cell apoptosis.