ANGIOTENSIN REGULATION OF GENE EXPRESSION

血管紧张素II受体(AIIR)潜在机制的鉴定 基因表达的中介调控是我们理解的核心 血管紧张素II(AII)如何通过 对生物体的生理和病理后果。而当 AIIR的结构和短期膜或细胞质效应 由配体占据的受体启动的已被广泛应用 在调查中，很少有研究直接涉及到 AIIR控制基因转录。在这项建议中，委托人 目的是确定AIIR激活的分子机制。 编码酪氨酸羟基酶基因的表达 儿茶酚胺生物合成中的限速酶。 儿茶酚胺能细胞是心血管和神经系统的效应细胞 AII的其他动态平衡功能。我们已经证明，从长远来看， 大鼠肾上腺髓质细胞对儿茶酚胺的诱导性释放 AIIR与TH基因的转录激活有关。 我们已经确定了第二信使系统和蛋白质的作用 蛋白酪氨酸酶参与了这种激活。参与AIIR激活的通路 与尼古丁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域 调节所有II的激活和调节它的其他区域 激活。介导AII刺激的启动子区域不同 从参与突触激活的TH基因的区域。这个 参与AIIR调控的特定启动子序列仍然存在 以确定身份。一种检测DNA-蛋白质结合的体外实验 与调节区形成的几种不同的蛋白质复合体 TH的发起人。刺激AIIR诱导形成特定的 具有TH基因调控区的核蛋白复合体 推动者。这种效果是AIIR独有的，没有被复制。 其他激活基因表达的处理(即刺激 尼古丁受体或蛋白激酶C)。核能的本质 形成AIIR诱导的复合体的蛋白质和与之结合的其他复合体 启动子的调控区域是未知的，将在 建议进行的研究。它们在基因调控中的作用 AIIR的表达将被确定。 具体而言，目标是：(I)确定个别监管机构 基因启动子中参与转录的元件 AIIR的调节；(Ii)进一步鉴定核蛋白 与TH启动子AIIR调控元件相互作用；和(Iii) 精选核蛋白(S)的纯化和克隆 由AIIR转录激活。为了实现这些目标，我们有 开发了一种体外模型，允许研究 TH基因的调节元件和反式作用因子 参与由本地AIR发起的监管级联 激活。含有启动子和启动子的重组基因 荧光素酶报告基因在原代培养中瞬时表达 未转化肾上腺髓质细胞的表达及AIIR的调节作用 被检查过了。DNA亲和蛋白的选择将按顺序进行 鉴定、纯化和克隆介导血管紧张素转换酶的核蛋白 AIIR的转录效应。未来的研究将确定 AIIR控制特定功能的机制 转录因子。对这些机制的阐明将 有助于更好地理解如何长期 产生AII的动态平衡和病理效应。

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.