REGULATION OF HUMAN ALDOSTERONE SYNTHASE

描述：(改编自《研究人员摘要》)该项目旨在利用人类遗传学、细胞培养和转基因小鼠的方法，进一步研究人类醛固酮合成酶(CYP11B2)的调控。拟议的研究将扩展申请者正在进行的合作项目，该项目定义了CYP11B2近端5‘侧翼区的转录调控元件。将确定CYP11B2或其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。此外，还将鉴定CYP11B2-CYP11B1区域的其他多态，确定与已鉴定等位基因的连锁不平衡，并确定它们对醛固酮生产和CYP11B2表达的功能影响。我们将研究细胞色素P11B2在肾上腺外组织中的表达，包括确定细胞色素P11B2是否在人体心脏内表达。在啮齿动物心肌细胞和/或人脐静脉内皮细胞中，调控CYP11B2报告结构表达的顺式作用元件将被确定。通过确定在CYP11B2周围的染色质中是否存在组织特异性DNase I超敏位点，将确定影响CYP11B2表达的可能的基因座控制区，并将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。调控细胞色素P11B2表达的钙信号通路将通过定义调控细胞色素P11B2转录的特异性钙调素蛋白激酶(S)来实现，方法是将含有活性突变体(I、II和IV型)的重组逆转录病毒导入H295R肾上腺细胞。采用原位杂交和免疫组织化学方法检测正常人体肾上腺组织中钙调素蛋白激酶(S)的表达。这些研究应该为心血管疾病的遗传风险因素提供潜在的机制。

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.