Tang K, Wu H, Mahata S K, Taupenot L, Rozansky D J, Parmer R J, O'Connor D T
Department of Medicine and Center for Molecular Genetics, University of California, and Department of Veterans Affairs Medical Center, San Diego, California 92161, USA.
J Biol Chem. 1996 Nov 8;271(45):28382-90. doi: 10.1074/jbc.271.45.28382.
To explore stimulus-transcription coupling in pheochromocytoma cells, we studied the biosynthetic response of chromogranin A, the major soluble protein co-stored and co-released with catecholamines, to chromaffin cells' physiologic nicotinic cholinergic secretory stimulation. Chromogranin A mRNA showed a time-dependent 3.87-fold response to nicotinic stimulation, and a nuclear run-off experiment indicated that the response occurred at a transcriptional level. Transfected chromogranin A promoter/luciferase reporter constructs were activated by nicotinic stimulation, in time- and dose-dependent fashions, in both rat PC12 pheochromocytoma cells and bovine chromaffin cells. Cholinergic subtype agents indicated that nicotinic stimulation was required. Promoter deletions established both positive and negative nicotinic response domains. Transfer of candidate promoter domains to a heterologous (thymidine kinase) promoter conferred region-specific nicotinic responses onto that promoter. A proximal promoter domain (from -93 to -62 base pairs) was activated in copy number- and distance-dependent fashion, and thus displayed features of a promoter element. Its activation was sufficient to account for the overall positive response to nicotine. Within this proximal region, a cAMP response element (CRE) was implicated as a major nicotinic response element, since a CRE point-gap mutation decreased nicotinic induction, transfer of CRE to a thymidine kinase promoter augmented the promoter's response to nicotine, and nicotine activated the CRE-binding protein CREB through phosphorylation at serine 133. We conclude that secretory stimulation of pheochromocytoma cells also activates the biosynthesis of the major secreted protein (chromogranin A), that the activation is transcriptional, and that a small proximal domain, including the CRE box, is, at least in part, both necessary and sufficient to account for the positive response to nicotine.
为了探究嗜铬细胞瘤细胞中的刺激-转录偶联，我们研究了嗜铬粒蛋白A（一种与儿茶酚胺共同储存和释放的主要可溶性蛋白）对嗜铬细胞生理性烟碱型胆碱能分泌刺激的生物合成反应。嗜铬粒蛋白A mRNA对烟碱型刺激呈现出时间依赖性的3.87倍反应，核转录实验表明该反应发生在转录水平。在大鼠PC12嗜铬细胞瘤细胞和牛嗜铬细胞中，转染的嗜铬粒蛋白A启动子/荧光素酶报告基因构建体被烟碱型刺激以时间和剂量依赖性方式激活。胆碱能亚型试剂表明需要烟碱型刺激。启动子缺失确定了正性和负性烟碱型反应结构域。将候选启动子结构域转移到异源（胸苷激酶）启动子上，使该启动子具有区域特异性烟碱型反应。一个近端启动子结构域（从-93到-62碱基对）以拷贝数和距离依赖性方式被激活，因此表现出启动子元件的特征。其激活足以解释对尼古丁的总体阳性反应。在这个近端区域内，一个环磷酸腺苷反应元件（CRE）被认为是主要的烟碱型反应元件，因为CRE点间隙突变降低了烟碱型诱导，将CRE转移到胸苷激酶启动子上增强了该启动子对尼古丁的反应，并且尼古丁通过丝氨酸133的磷酸化激活了CRE结合蛋白CREB。我们得出结论，嗜铬细胞瘤细胞的分泌刺激也激活了主要分泌蛋白（嗜铬粒蛋白A）的生物合成，这种激活是转录性的，并且一个小的近端结构域，包括CRE框，至少部分地既是对尼古丁阳性反应的必要条件也是充分条件。
