REGULATION OF HUMAN ALDOSTERONE SYNTHASE

产品说明：本项目旨在利用人类遗传学、细胞培养和转基因小鼠方法，进一步研究人醛固酮合酶（CYP 11B 2）的调控。拟定的研究将扩展申请人正在进行的合作项目，该项目已定义了CYP 11B 2近端5'侧翼区的转录调控元件。将确定CYP 11B 2中或附近等位基因的表型效应，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。将确定CYP 11B 2-CYP 11B 1区域的其他多态性，确定与已确定等位基因的连锁不平衡，并确定其对醛固酮产生和CYP 11B 2表达的功能影响。将研究肾上腺外组织中的CYP 11B 2表达，包括确定CYP 11B 2是否在人心脏内表达。将鉴定调节啮齿动物心肌细胞和/或人脐静脉内皮细胞中CYP 11B 2报告基因构建体表达的顺式作用元件。将通过确定CYP 11B 2周围的染色质中是否存在组织特异性DNA酶I超敏位点来鉴定影响CYP 11B 2表达的可能基因座控制区，并通过产生适当的转基因小鼠品系来确认推定基因座控制区的功能。通过确定调节CYP 11B 2转录的特异性CaM激酶来阐明调节CYP 11B 2表达的钙信号通路，所述特异性CaM激酶通过转染质粒并将含有组成型活性CaM激酶突变体（I型、II型和IV型）的重组逆转录病毒转导到H295 R肾上腺皮质细胞中来实现。将使用原位杂交和免疫组织化学检查正常人肾上腺中CaM激酶的表达。这些研究应该为深入了解心血管疾病遗传风险因素的潜在机制提供帮助。

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.