ANGIOTENSIN REGULATION OF GENE EXPRESSION

血管紧张素II受体（AIIR）的机制鉴定- 介导的基因表达调控是我们理解的核心 血管紧张素II（AII）如何对细胞产生持久的影响， 对机体的生理和病理后果。而 AIIR的结构和短期膜或细胞质效应 由配体占据的受体引发的免疫反应已经被广泛地 调查，很少有研究直接解决的机制， AIIR控制基因转录。在这一建议中，主要 目的是确定AIIR激活的分子机制， 编码酪氨酸羟化酶（THE）的基因的表达， 儿茶酚胺生物合成的限速酶。 儿茶酚胺能细胞是心血管和心血管疾病的既定效应器， AII的其他稳态功能。我们已经证明， 诱导肾上腺髓质细胞释放儿茶酚胺 AIIR与TH基因的转录激活有关。 我们已经描述了第二信使系统和蛋白质的作用 介导这种激活的激酶。参与AIIR激活的途径 与烟碱受体诱导过程中使用的不同 TH基因。我们已经确定了基因启动子区， 介导AII激活和调节此激活的其他区域 activation.介导AII刺激的启动子区域不同 从参与TH基因的突触激活的区域。的 参与AIIR调控的特异性启动子序列仍然存在， 以供识别。检测到体外DNA-蛋白结合试验 与调控区形成的几种不同的蛋白质复合物 的TH启动子。刺激AIIR诱导形成特异性 TH基因调控区的核蛋白复合物 启动子这种效应是AIIR所独有的， 激活THE基因表达（即刺激 烟碱受体或蛋白激酶C）。核的性质 形成AIR-induced complex和其他complex的蛋白质 启动子调控区是未知的，将在 建议的研究。它们在调节THE基因中的作用 将确定AIIR的表达。 具体目标是：（一）确定个别监管机构 基因启动子中的元件，参与转录 调节AIIR;（ii）进一步表征核蛋白， 与TH启动子AIIR调控元件相互作用;和（iii） 纯化和克隆选择的核蛋白， 通过AIIR的转录激活。为了实现这些目标，我们 开发了一种体外模型，可以研究 TH基因的调节元件和反式作用因子， 参与从本地AIIR发起的监管级联 activation.由THE启动子和 荧光素酶报告基因在原代培养物中瞬时表达 非转化肾上腺髓质细胞的增殖及其受AIIR的调节 进行了检查。将采用DNA亲和蛋白选择， 鉴定、纯化和克隆介导细胞凋亡的核蛋白， AIIR的转录效应。未来的研究将决定 AIIR控制特定功能的机制 转录因子阐明这些机制将 有助于更好地了解长期 产生AII的稳态和病理作用。

Identification of mechanisms underlying angiotensinII receptor (AIIR)- mediated regulation of gene expression is central to our understanding of how angiotensin II (AII) exerts long-lasting effects on cells with physiological and pathological consequences to the organism. While the structure of AIIR and the short-term membrane or cytoplasmic effects initiated by the ligand-occupied receptor have been extensively investigated, few studies have directly addressed mechanisms by which AIIR controls gene transcription. In this proposal the principal objective is to determine molecular mechanisms by which AIIR activates expression of the gene which encodes tyrosine hydroxylase (THE), the rate limiting enzyme in catecholamine biosynthesis. Catecholaminergic cells are established effectors of cardiovascular and other homeostatic functions of AII. We have shown that the long-term induction of catecholamine release from the adrenal medullary cells by AIIR is associated with the transcriptional activation of the TH gene. We have characterized second messenger systems and the roles of protein kinases mediating this activation. Pathways involved in AIIR activation were different from those utilized during nicotinic receptor induction of the TH gene. We have identified THE gene promoter region that mediates AII activation and additional regions that modulate this activation. The promoter region mediating AII stimulation was different from the region involved in synaptic activation of the TH gene. The specific promoter sequences involved in the regulation by AIIR remain to be identified. An in vitro DNA-protein binding assay detected several different protein complexes forming with the regulatory region of the TH promoter. Stimulation of AIIR induced formation of a specific nuclear protein complex with the regulatory region of the TH gene promoter. This effect was unique to AIIR, and was not reproduced by other treatments which activate THE gene expression (i.e. stimulation of nicotinic receptors or protein kinase C). The nature of nuclear proteins forming AIIR-induced complex and other complexes binding to THE promoter regulatory regions are unknown, and will be elucidated in the proposed studies. Their role in the regulation of THE gene expression by AIIR will be determined. Specifically the aims are: (i) to identify individual regulatory elements in THE gene - promoter that participate in transcriptional regulation by AIIR; (ii) to further characterize nuclear proteins which interact with the TH promoter AIIR regulatory elements; and (iii) to purify and clone selected nuclear protein(s) that mediate transcriptional activation by AIIR. To achieve these goals we have developed an in vitro model which allows the investigation of regulatory elements of the TH gene and trans-acting factors that participate in the regulatory cascades initiated from native AIIR activation. The recombinant genes consisting of THE promoter and luciferase reporter gene are transiently expressed in primary cultures of nontransformed adrenal medullary cells and their regulation by AIIR is examined. DNA-affinity protein selection will be employed in order to identify, purify and clone nuclear proteins that mediate the transcriptional effects of AIIR. Future studies will determine mechanisms by which the AIIR controls the function of specific transcriptional factors. Elucidation of these mechanisms will contribute towards a better understanding of how the long-term homeostatic and pathologic effects of AII are produced.