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培育的肾素表达细胞系，正在与体内转基因分析相结合，以阐明控制小鼠肾素基因转录调控的机制。这一建议完善和测试了一个来自当前数据的机制模型，该模型认为肾素的表达是通过结合在关键的近端启动子元件和远端增强子元件(S)上的细胞特异性和一般性因子的合作来消除或覆盖普遍存在的负调控元件来实现的。利用绿色荧光蛋白进行的转基因报告分析表明，正确地在空间上、时间上和响应生理扰动时确定肾素的已知模式所需的所有调控信息都位于小鼠REN-1c基因5‘侧翼序列的4.1kb以内。在特定的目标下，将使用各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”元件作用的第一类HOX基因的直接下游靶基因。转基因和细胞转染法将被用来评估单个复合体或多个离散增强子(S)是否在体内观察到的关键表达部位(如肾血管和胎盘组织)差异驱动表达。在细胞因子、激素和机械膨胀的反应中，被观察到的介导肾素表达下调的顺式作用元件将通过体外瞬时转染试验被定位在5‘侧翼序列内，然后通过转基因分析检测体内参与。目前的数据表明，被确认为在As4.1细胞中调节肾素高水平表达的顺式作用元件实际上在体内得到了利用。此外，主要成分似乎在进化过程中在包括人类在内的物种中是保守的。识别特定的识别序列及其同源蛋白伙伴和阐明相互作用的机制，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.