MacArthur L, Koller K J, Eiden L E
Section on Molecular Neuroscience, National Institute of Mental Health, Bethesda, Maryland 20892.
Mol Pharmacol. 1993 Sep;44(3):545-51.
The bovine enkephalin gene is responsive to multiple signaling pathways in primary chromaffin cell cultures. We examined the effects of activation of the calcium and protein kinase A pathways on accumulation of enkephalin peptide and mRNA, gene transcription, and chromatin structure in the 5' region of the gene. We show here that the increase of enkephalin mRNA and peptide after depolarization of chromaffin cells with KCl or activation of adenylate cyclase with forskolin is preceded by an increase in enkephalin gene transcription. Both enkephalin peptide and mRNA were reduced by co-treatment of KCl- or forskolin-stimulated cultures with phorbol esters. Three enhancer sequences that were previously shown to be responsive to calcium, protein kinase A, and phorbol esters in the human gene in vitro were identified in the bovine enkephalin promoter, identifying a potential locus of control for these pathways in vivo. DNase I hypersensitivity mapping identified two tissue-specific sites that are associated with enkephalin gene expression in adrenal medulla and chromaffin cells; site 1 is in the promoter, which contains the three enhancer elements, and site 2 is in the first intron. These results suggest that regulation of the enkephalin gene in primary chromaffin cells by the calcium, protein kinase A, and protein kinase C signaling pathways occurs by modulation of transcription factor activity at several discrete loci on the enkephalin gene.
牛脑啡肽基因在原代嗜铬细胞培养物中对多种信号通路有反应。我们研究了钙和蛋白激酶A信号通路的激活对脑啡肽肽和mRNA的积累、基因转录以及该基因5'区域染色质结构的影响。我们在此表明，用氯化钾使嗜铬细胞去极化或用福司可林激活腺苷酸环化酶后，脑啡肽mRNA和肽的增加之前是脑啡肽基因转录的增加。用佛波酯共同处理氯化钾或福司可林刺激的培养物后，脑啡肽肽和mRNA均减少。在牛脑啡肽启动子中鉴定出三个增强子序列，这些序列先前在体外人基因中显示对钙、蛋白激酶A和佛波酯有反应，从而确定了这些信号通路在体内的一个潜在控制位点。DNA酶I超敏图谱鉴定出两个与肾上腺髓质和嗜铬细胞中脑啡肽基因表达相关的组织特异性位点；位点1在启动子中，其中包含三个增强子元件，位点2在第一个内含子中。这些结果表明，钙、蛋白激酶A和蛋白激酶C信号通路对原代嗜铬细胞中脑啡肽基因的调控是通过调节脑啡肽基因上几个离散位点的转录因子活性来实现的。
