REGULATION OF HISTIDINE DECARBOXYLASE GENE EXPRESSION

胃泌素刺激肠嗜铬细胞(ECL)是关键步骤 在导致胃酸分泌的生理途径中。 对ECL细胞或其细胞内机制知之甚少 在胃泌素的作用下被激活。组氨酸基因 脱羧酶(HDC)编码产生组胺的酶(来自 组氨酸)，并在含氧酶的ECL细胞中高表达 粘膜。高胃泌素状态不仅导致ECL的增殖 细胞，但也增加了HDC的mRNA水平，在我们的 实验室进行Northern杂交和原位杂交分析。这个 拟议研究的总体目标是描述独联体的特征-- 针对ECL细胞HDC基因表达的调节机制， 它们控制着它对胃泌素的反应。这个实验室已经克隆了 人和大鼠HDC基因的5‘侧翼调节区， 并将它们连接到一个荧光素酶报告基因。胃泌素治疗多发性硬化症 HDC-荧光素酶双基因转染人胃癌细胞系 构建和克隆的人CCK-B/胃泌素受体导致4- 荧光素酶活性成倍增加。剂量反应曲线、时间进程 CCK-B受体拮抗剂的研究及拮抗作用 L365,260(ICso=5 nm)表示高度特异的反应。删除 分析表明，胃泌素反应元件位于 起始点的100个核苷酸。AGS细胞的转染性研究 还发现了一种可能的胃特异性顺式调节元件 含有同源结构域样的Attta基序。这项提议旨在进一步 描述胃泌素反应元素，以及胃- 通过额外的缺失和突变研究，特定的增强子 在AGS-B细胞和原代ECL细胞制剂中。核蛋白 还将研究与这些顺式作用的DNA元件相互作用的物质。 这些体外研究将通过转基因研究得到扩展，转基因研究 将决定hdc基因中的哪些序列是正确的 ECL细胞在体内的组织特异性和调控表达。这个 人生长激素基因编码杂交体的表达 HDC基因5‘侧翼序列控制的区域将是 检查过了。总体而言，拟议的研究将在分子水平上进行研究 HDC基因表达水平的调节，HDC基因表达的主要靶点之一 ECL细胞中的胃泌素，以及ACID中的一个关键信号机制 分泌物。

Gastrin stimulation of the enterochromaffin-like (ECL) cell is a key step in the physiological pathway that leads to gastric acid secretion. Little is known about the ECL cell, or about the intracellular mechanisms that are activated in response to gastrin. The gene histidine decarboxylase (HDC) encodes the enzyme which produces histamine (from histidine), and which is highly expressed in the ECL cell of the oxyntic mucosa. Hypergastrinemic states lead not only to proliferation of ECL cells, but also to increased HDC mRNA levels, demonstrated in our laboratory by Northern blot and in situ hybridization analysis. The overall objective of the proposed studies is to characterize the cis- regulatory mechanisms which target HDC gene expression to the ECL cell, and which control Its response to gastrin. This laboratory has cloned the 5' flanking regulatory regions of both the human and rat HDC genes, and joined them to a luciferase reporter gene. Gastrin treatment of a gastric cancer (AGS) cell line transfected with both HDC-luciferase constructs and the cloned human CCK-B/gastrin receptor leads to a four- fold increase in luciferase activity. A dose response curve, time course studies, and antagonism with the specific CCK-B receptor antagonist L365,260 (ICso=5nM) indicate a highly specific response. Deletion analysis indicated that the gastrin response element was located within 100 nucleotides of the start site. Transfection studies in AGS cells have also identified a putative stomach specific cis-regulatory element containing a homeodomain-like ATTTA motif. This proposal aims to further characterize the gastrin response element, as well as the stomach- specific enhancer, through additional deletion and mutagenesis studies in AGS-B cells and in primary ECL cell preparations. Nuclear proteins which interact with these cis-acting DNA elements will also be studied. These in vitro studies will be extended through transgenic studies, which will determine which sequences in the HDC gene are necessary for correct tissue-specific and regulated expression in ECL cells in vivo. The expression of a hybrid consisting of the human growth hormone gene coding region under the control of the HDC gene 5' flanking sequences will be examined. Overall, the proposed studies will examine at a molecular level the regulation of HDC gene expression, one of the main targets of gastrin in the ECL cell, as well as a key signaling mechanism in acid secretion.