REGULATION OF HISTIDINE DECARBOXYLASE GENE EXPRESSION

胃泌素刺激肠嗜铬样（ECL）细胞是一个关键步骤 导致胃酸分泌的生理途径。 对ECL细胞或细胞内机制知之甚少 它们会被胃泌素激活。 组氨酸基因 脱羧酶（HDC）编码产生组胺的酶（从 组氨酸），并且其在泌酸酶的ECL细胞中高度表达。 粘膜高胃泌素状态不仅导致ECL增殖， 细胞，但也增加了HDC mRNA水平，在我们的研究中证实， 实验室通过北方印迹和原位杂交分析。 的 拟议研究的总体目标是表征顺式- 将HDC基因表达靶向ECL细胞的调节机制， 并控制其对胃泌素的反应 这个实验室克隆了 人和大鼠HDC基因的5 ′侧翼调控区， 并将它们连接到荧光素酶报告基因上。 胃泌素治疗a 转染了HDC-荧光素酶的胃癌（AGS）细胞系 构建体和克隆的人CCK-B/胃泌素受体导致四个- 荧光素酶活性的倍数增加。 剂量反应曲线，时间过程 与特异性CCK-B受体拮抗剂的拮抗作用 L365，260（IC 50 = 5 nM）表明高度特异性反应。 删除 分析表明，胃泌素反应元件位于 起始位点的100个核苷酸。 AGS细胞中的转染研究 还鉴定了一种假定的胃特异性顺式调节元件 含有同源结构域样ATTTA基序。 该提案旨在进一步 表征胃泌素反应元件，以及胃- 特异性增强子，通过额外的缺失和诱变研究 在AGS-B细胞和原代ECL细胞制备物中。 核蛋白 与这些顺式作用DNA元件相互作用的分子也将被研究。 这些体外研究将通过转基因研究得到扩展， 将确定HDC基因中的哪些序列是正确的 体内ECL细胞中的组织特异性和调节性表达。 的 由编码人生长激素基因组成的杂合体的表达 在HDC基因5'侧翼序列的控制下的区域将是 考察 总的来说，拟议的研究将在分子水平上进行检查， 水平调节HDC基因表达，这是HDC的主要靶点之一。 胃泌素在ECL细胞，以及一个关键的信号机制，在酸 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.