REGULATION OF HISTIDINE DECARBOXYLASE

组氨酸脱羧酶在肠嗜铬细胞中高表达，负责L-组氨酸向组胺的转化。在初步的研究中，我们已经证明了HDC基因在转录和转录后都受到胃泌素的调控。HDC基因中介导胃泌素反应性的启动子元件被鉴定为两个新的顺式作用元件(GAS-RE1和GAS-RE2)，它们位于转录起始点下游，分别与52kD和35kD蛋白结合。胃泌素通过MAP依赖的途径激活HDC转录，导致GAS-REBP1和GAS-REBP2增加启动子结合。GAS-REBP2的候选基因已经从人胃的cDNA文库中克隆出来，它似乎代表了一种新的35kD的DNA结合蛋白。胃泌素刺激也导致HDC过渡后的酶活性，很可能是通过增加翻译来实现的。HDC的激活也是通过翻译后将74 kDa的HDC前体蛋白切割成更活跃的54 kD的酶形式来实现的。在初步研究中，我们已经将这个切割位点定位在一个8个氨基酸的区域，并证明了这个区域的缺失抑制了切割和酶的激活。此外，我们还发现，HDC蛋白的表达导致了HDC启动子活性的反馈抑制，并且这种抑制不依赖于酶的活性，并且是由N-末端的肽序列介导的。最后，我们已经证明了4.8kb的小鼠嗜铬粒蛋白A启动子足以靶向ECL细胞和介导转基因小鼠的胃泌素反应。拟议的研究旨在通过体外和体内两种方法进一步研究HDC基因的调控。(1)GAS-REBP2候选蛋白的进一步鉴定及其在HDC基因调控中的作用。(2)准确确定HDC的酶切位点，分析胃泌素对HDC的翻译调控。(3)绘制介导转录抑制的N-末端HDC序列图谱，并探讨其抑制机制。(4)利用HDC-GFP报告基因构建，对ECL细胞特异性表达所必需的顺式作用DNA序列进行HDC基因分析。总体而言，这些研究将提供对胃泌素调节HDC基因表达和酶活性的机制的更多了解。

Histidine decarboxylase (HDC) is highly expressed in enterochromaffin-like (ECL) cells, and carries out the conversion of L-histidine to histamine. In preliminary studies, we have demonstrated that the HDC gene is regulated both transcriptionally and post-transcriptionally by gastrin in transfected gastric cell lines. The promoter elements mediating gastrin responsiveness in the HDC gene have been identified as two novel cis-acting elements (GAS-RE1 and GAS-RE2) which are located in tandem downstream of the transcriptional start site, and bind to 52 kD and 35 kD proteins, respectively. The activation of HDC transcription by gastrin occurs through a MAP kinase-dependent pathway leading to increased promoter binding by GAS-REBP1 and GAS-REBP2. A candidate for GAS-REBP2 has been cloned from a human stomach cDNA library, and appears to represent a novel 35 kD DNA binding protein. Gastrin stimulation also leads to post-transitional HDC enzymatic activity, most likely through increases in translation. HDC activation also occurs through post-translational cleavage of the 74 kDa HDC precursor protein to a more active 54 kD enzymatic form. In preliminary studies, we have localized this cleavage site to an 8 amino acid region, and demonstrated that deletion of this region inhibits cleavage and enzymatic activation. Further, we have shown that expression of HDC protein leads to feedback inhibition of HDC promoter activity, and that this inhibition is independent of enzyme activity and mediated by N-terminal peptide sequences. Finally, we have shown that 4.8 kb of the mouse chromogranin A promoter are sufficient for targeting ECL cells and mediating gastrin responsiveness in transgenic mice. The proposed studies are aimed at investigating further the regulation of the HDC gene, using both in vitro and in vivo approaches. (1) The candidate GAS-REBP2 protein will be further characterized, and its role in HDC gene regulation investigated. (2) The HDC enzymatic cleavage site will be precisely defined, and translational regulation of HDC by gastrin will be analyzed. (3) The N-terminal HDC sequences mediating transcriptional inhibition will be mapped, and mechanisms of inhibition explored. (4) Finally, the cis-acting DNA sequences which are necessary for ECL cell-specific expression will be analyzed for the HDC gene using HDC-GFP reporter gene constructs. Overall, these studies will provide greater understanding of the mechanisms involved in the regulation of HDC gene expression and enzymatic activity by gastrin.