Patel N A, Chalfant C E, Yamamoto M, Watson J E, Eichler D C, Cooper D R
Departments of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa, USA.
FASEB J. 1999 Jan;13(1):103-13. doi: 10.1096/fasebj.13.1.103.
Acute hyperglycemia may contribute to the progression of atherosclerosis by regulating protein kinase C (PKC) isozymes and by accelerating vascular smooth muscle cell (VSMC) proliferation. We investigated acute glucose regulation of PKCbeta gene expression in A10 cells, a rat aortic smooth muscle cell line. Western blot analysis showed that PKCbetaII protein levels decreased with high glucose (25 mM) compared to normal glucose (5.5 mM), whereas PKCbetaI levels were unaltered. PKCbeta mRNA levels were depleted by 60-75% in hyperglycemic conditions. To elucidate whether high glucose regulated PKCbeta expression via the common promoter for PKCbetaI and PKCbetaII, deletion constructs of the PKCbeta promoter ligated to CAT as reporter gene were transfected into A10 cells. Construct D (-411 to +179CAT) showed quenching in high glucose (25 mM) and suggested the involvement of a carbohydrate response element in the 5' promoter region of the PKCbeta gene. In actinomycin D-treated A10 cells, a 60% decrease in PKCbeta mRNA with high glucose treatment indicated that posttranscriptional destabilization by glucose was also occurring. We have demonstrated that glucose-induced posttranscriptional destabilization of PKCbetaII message is mediated via a nuclease activity present in the cytosol. The specificity of glucose to posttranscriptionally destabilize PKCbetaII mRNA, but not the PKCbetaI mRNA, was confirmed in both A10 cells and primary cultures from human aorta.
急性高血糖可能通过调节蛋白激酶C（PKC）同工酶和加速血管平滑肌细胞（VSMC）增殖来促进动脉粥样硬化的发展。我们研究了大鼠主动脉平滑肌细胞系A10细胞中PKCβ基因表达的急性葡萄糖调节作用。蛋白质印迹分析表明，与正常葡萄糖（5.5 mM）相比，高葡萄糖（25 mM）条件下PKCβII蛋白水平降低，而PKCβI水平未改变。在高血糖条件下，PKCβ mRNA水平降低了60 - 75%。为了阐明高葡萄糖是否通过PKCβI和PKCβII的共同启动子调节PKCβ表达，将与CAT作为报告基因连接的PKCβ启动子缺失构建体转染到A10细胞中。构建体D（-411至+179CAT）在高葡萄糖（25 mM）条件下显示出淬灭，提示PKCβ基因5'启动子区域存在碳水化合物反应元件。在用放线菌素D处理的A10细胞中，高葡萄糖处理使PKCβ mRNA减少60%，表明葡萄糖也在发生转录后去稳定作用。我们已经证明，葡萄糖诱导的PKCβII信使转录后去稳定作用是通过存在于细胞质中的核酸酶活性介导的。在A10细胞和人主动脉原代培养物中均证实了葡萄糖对PKCβII mRNA转录后去稳定的特异性，而对PKCβI mRNA则无此作用。
