Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase promoter contains a CREB binding site that regulates cAMP action in Caco-2 cells
Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase promoter contains a CREB binding site that regulates cAMP action in Caco-2 cells
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DOI：
10.1042/0264-6021:3450201
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发表时间：
2000-01-15
影响因子：
4.1
通讯作者：
Serra, D
中科院分区：
文献类型：
--
作者：
Eggers, A;Caudevilla, C;Serra, D
cAMP 增加线粒体 (mit.) 3-羟基-3-甲基戊二酰 (HMG)-CoA 合酶基因的转录，该基因编码一种被提议作为生酮控制位点的酶。 Caco-2 细胞与 cAMP 一起孵育，24 小时内 mit.HMG-CoA 合酶 mRNA 水平增加了 4 倍。我们已经鉴定出位于 mit.HMG-CoA 合酶启动子上游 546 bp 处的活性 cAMP 响应元件 (CRE)，它对于诱导二丁酰 cAMP 的表达是必需的。含有与氯霉素乙酰转移酶基因融合的 mit.HMG-CoA 合酶启动子的 CRE 元件的构建体与蛋白激酶 A 和 cAMP 反应元件结合蛋白 (CREB) 的显性失活突变体的共转染表明，对 cAMP 的反应是由转录因子 CREB 介导的。在 Caco-2 细胞的转染测定中，CRE 元件赋予蛋白激酶 A 对异源启动子的反应性。凝胶延迟测定显示 mit.HMG-CoA 合酶 CRE 与重组 CREB 结合。用假定的 mit.HMG-CoA 合酶 CRE 序列和来自 Caco-2 细胞的核蛋白获得的移动带与其他基因（例如生长抑素和磷酸烯醇丙酮酸羧激酶）的 CRE 序列竞争。我们得出结论，cAMP 对 Caco-2 细胞中 mit.HMG-CoA 合酶基因表达的调节是由启动子中的 CRE 序列介导的。
cAMP increases transcription of the mitochondrial (mit.) gene for 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase, which encodes an enzyme that has been proposed as a control site of ketogenesis. The incubation of Caco-2 cells with cAMP increased mit.HMG-CoA synthase mRNA levels 4-fold within 24 h. We have identified an active cAMP-response element (CRE) located 546 bp upstream of the mit.HMG-CoA synthase promoter that is necessary for the induction of expression by dibutyryl cAMP. Co-transfections of constructs, containing the CRE element of the mit.HMG-CoA synthase promoter fused to the gene for chloramphenicol acetyltransferase, with protein kinase A and a dominant-negative mutant of cAMP-response-element-binding protein (CREB) show that the response to cAMP is mediated by the transcription factor CREB. The CRE element confers responsiveness of protein kinase A to a heterologous promoter in transfection assays in Caco-2 cells. Gel-retardation assays revealed that the mit.HMG-CoA synthase CRE binds to recombinant CREB. The shifted band obtained with the putative mit.HMG-CoA synthase CRE sequence and nuclear proteins from Caco-2 cells competed with CRE sequences of other genes such as somatostatin and phosphoenolpyruvate carboxykinase. We conclude that the regulation of the expression of the gene for mit.HMG-CoA synthase in Caco-2 cells by cAMP is mediated by a CRE sequence in the promoter.