RENIN-EXPRESSING CELL LINES FOR HYPERTENSION RESEARCH

As4.1细胞（ATCC #CRL 2193），一种表达肾素的细胞系的肾起源，我们开发的转基因靶向肿瘤，正在使用的音乐会与转基因分析在体内阐明机制，小鼠肾素基因的转录调控。该提议改进并测试了源自当前数据的机制模型，该模型表明，通过在关键的近端启动子元件和远端增强子元件处结合的细胞特异性和一般因子的合作来实现肾素表达，以消除或覆盖普遍功能的负调节元件。利用EGFP的转基因报告基因分析表明，正确指定已知的肾素模式所需的所有调控信息在空间上，时间上，并响应生理扰动，都驻留在小鼠Ren-1c基因的5'侧翼序列的4.1 kb内。在特定目标下，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”元件起作用的1类Hox基因的直接下游靶标。将使用转基因和细胞转染测定来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（如肾血管系统和胎盘组织）差异驱动表达。在通过转基因分析测试体内参与之前，通过体外瞬时转染测定将观察到的响应于细胞因子、激素和机械扩张而介导As4.1细胞中的肾素表达的有效下调的顺式作用元件定位在5'侧翼序列内。目前的数据表明，顺式作用元件确定为关键介导高水平表达的肾素在As4.1细胞，事实上，在体内使用。此外，主要元素似乎在物种（包括人类）之间进化上是保守的。识别特定的识别序列，其同源蛋白质的合作伙伴和相互作用的机制的阐明，应提供基础知识的作用，在正常生理和异常的病理生理状态下的肾素-血管紧张素系统，以及提供洞察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.