REGULATION OF HUMAN ALDOSTERONE SYNTHASE

摘要：本项目旨在利用人类遗传学、细胞培养和转基因小鼠等方法进一步研究人醛固酮合成酶（CYP11B2）的调控。拟议的研究将扩展申请人正在进行的合作项目，该项目已经定义了CYP11B2近5'侧翼区域的转录调控元件。CYP11B2等位基因或其附近等位基因的表型效应将被确定，包括对醛固酮排泄、心脏大小、血压和心肌梗死风险的可能影响。将鉴定CYP11B2- cyp11b1区域的其他多态性，确定与已鉴定的等位基因的连锁不平衡，并确定它们对醛固酮产生和CYP11B2表达的功能影响。CYP11B2在肾上腺外组织中的表达将被研究，包括确定CYP11B2是否在人类心脏中表达。将鉴定在啮齿动物心肌细胞和/或人脐静脉内皮细胞中调节CYP11B2报告基因表达的顺式作用元件。通过确定CYP11B2周围染色质中是否存在组织特异性DNAse I超敏位点，可以确定影响CYP11B2表达的可能的基因座控制区，并通过生产合适的转基因小鼠品系来确认可能的基因座控制区的功能。通过将含有组成型活性CaM激酶突变体（I、II和IV型）的重组逆转录病毒转染到H295R肾上腺皮质细胞，定义调节CYP11B2转录的特定CaM激酶(s)，从而阐明调节CYP11B2表达的钙信号通路。CaM激酶在正常人肾上腺中的表达将通过原位杂交和免疫组织化学进行检测。这些研究应该为心血管疾病遗传风险因素的潜在机制提供见解。

DESCRIPTION: (Adapted from the Investigator's Abstract) This project aims to further study the regulation of human aldosterone synthase (CYP11B2) using human genetics, cell culture and transgenic mouse approaches. The proposed studies will extend the applicants' ongoing collaborative project that has defined transcriptional regulatory elements in the proximal 5' flanking region of CYP11B2. The phenotypic effects of allelism in or near CYP11B2 will be determined, including possible effects on aldosterone excretion, heart size, blood pressure and risk of myocardial infarction. Other polymorphisms in the CYP11B2-CYP11B1 region will be identified, linkage disequilibrium with already identified alleles determined, and their functional effects on aldosterone production and CYP11B2 expression defined. CYP11B2 expression in extra-adrenal tissues will be studied, including determining whether CYP11B2 is expressed within the human heart. Cis-acting elements regulating expression of CYP11B2 reporter constructs in rodent cardiac myocytes and/or human umbilical vein endothelial cells will be identified. Possible locus control regions affecting expression of CYP11B2 will be identified by determining if tissue-specific DNAse I hypersensitivity sites exist in chromatin surrounding CYP11B2, and functioning of putative locus control regions will be confirmed by producing appropriate strains of transgenic mice. Calcium signaling pathways regulating expression of CYP11B2 will be elucidated by defining the specific CaM Kinase(s) that regulate CYP11B2 transcription by transfecting plasmids and transducing recombinant retrovirus containing constitutively active CaM kinase mutants (types I, II, and IV) into H295R adrenocortical cells. Expression of the CaM kinase(s) in normal human adrenals will be examined using in situ hybridization and immunohistochemistry. These studies should provide insight into mechanisms underlying a genetic risk factor for cardiovascular disease.