RENIN-EXPRESSING CELL LINES FOR HYPERTENSION RESEARCH

As4.1细胞（ATCC #CRL2193）是我们通过转基因靶向肿瘤发生开发的肾源性肾素表达细胞系，目前正与体内转基因分析一起用于阐明小鼠肾素基因转录调控的机制。这一建议完善并测试了一个基于当前数据的机制模型，该模型表明肾素的表达是通过细胞特异性和一般因子结合在关键的近端启动子元件和远端增强子元件上的合作来实现的，从而废除或覆盖了普遍存在的功能负调控元件。利用EGFP进行的转基因报告基因分析表明，正确指定肾素在空间、时间和生理扰动下的已知模式所需的所有调控信息，都位于小鼠肾素-1c基因5'侧序列4.1 kb内。在具体目标下，将使用多种方法来详细检查我们的发现，即Ren基因是通过近端启动子“开关样”元件作用的1类Hox基因的直接下游靶点。转基因和细胞转染检测将用于评估单个复合物或多个离散增强子是否在体内观察到的关键表达位点（如肾脉管系统和胎盘组织）差异驱动表达。在As4.1细胞对细胞因子、激素和机械膨胀的反应中，顺式作用元件被观察到介导肾素表达的有效下调，在通过转基因分析测试体内参与之前，将通过体外瞬时转染测定在5'侧翼序列中定位。目前的数据表明，在As4.1细胞中被鉴定为介导肾素高水平表达的关键顺式作用元件实际上在体内被利用。此外，主要元素似乎在包括人类在内的所有物种的进化中都是保守的。特异性识别序列及其同源蛋白伴侣的鉴定和相互作用机制的阐明，将为肾素-血管紧张素系统在正常生理和异常病理生理状态中的作用提供基础知识，并有助于深入了解其在脊椎动物进化中的作用。

As4.1 cells (ATCC #CRL2193), a renin-expressing cell line of renal origin that we developed by transgene-targeted tumorigenesis, are being used in concert with transgenic analyses in vivo to elucidate mechanisms governing transcriptional regulation of mouse renin genes. This proposal refines and tests a mechanistic model derived from current data which suggests that renin expression is achieved through cooperation of cell- specific and general factors binding at a crucial, proximal promoter element and distal enhancer element(s) to abrogate or override negative regulatory elements that are ubiquitously functional. Transgenic reporter assays, utilizing EGFP, indicate that all regulatory information required to correctly specify known patterns of renin spatially, temporally, and in response to physiological perturbation, are resident within 4.1 kb of the 5' flanking sequence of the mouse Ren-1c gene. Under the Specific Aims, a variety of approaches will be used to examine in detail our finding that Ren genes are immediate downstream targets of Class 1 Hox genes acting through a proximal promoter "switch-like" element. Transgenic and cellular transfection assays will be used to assess whether a single complex, or multiple discrete, enhancer(s) are differentially driving expression at key sites of expression observed in vivo such as the renal vasculature and placental tissue. Cis-acting elements observed to mediate potent downregulation of renin expression in As4.1 cells in response to cytokine, hormone and mechanical distention will be mapped within the 5' flanking sequence by in vitro transient-transfection assays prior to testing involvement in vivo by transgenic analysis. Current data suggest that cis-acting elements identified as critical to mediating high level expression of renin in As4.1 cells are, in fact, utilized in vivo. Moreover, the major elements appear to be conserved evolutionarily across species, including human. Identification of the specific recognition sequences, their cognate protein partners and elucidation of the mechanism of interaction, should provide fundamental knowledge on the role of the renin-angiotensin system in normal physiology and abnormal pathophysiological states, as well as afford insight into its role in vertebrate evolution.