Renin, an aspartyl proteinase involved in the regulation of cardiovascular homeostasis, exhibits tissue specific regulation. Our previous studies of the mechanisms of mouse renin gene expression using promoter-reporter gene experiments and gel mobility shift assays suggest that tissue regulated expression is dependent on a complex interaction of transcriptional factors [cAMP responsive element (CRE) binding protein and negative regulatory element (NRE) binding protein]. These trans acting factors interact with a cis acting DNA element, known as the CRE/NRE region which contains a CRE and a NRE that share an overlapping sequence. We have hypothesized that when the NRE binding protein binds to this region, renin expression is inhibited. When the CRE binding protein binds to this region, it blocks NRE binding, thereby permitting constitutive renin expression. In the kidney, CRE binding protein and NRE binding protein compete for binding to this sequence with the CRE binding protein having a greater affinity. In extrarenal sites such as submandibular gland (SMG), the NRE binding protein is able to bind effectively to the sequence, since the CRE binding protein is inactivated by an inhibitory protein that is present in the extrarenal tissue, thereby inhibiting mouse Ren-l expression. The human renin gene also contains an overlapping CRE and NRE, suggesting that its tissue expression may be regulated by similar cis- trans interactions. These data are derived from experiments using in vitro biochemical methods and noncognate cell systems and need to be verified in vivo. In this proposal, we plan to elucidate the molecular mechanism of tissue regulated renin gene expression in mouse and human at the cell and in vivo levels using cognate cell culture systems and an in vivo gene transfer approach. These studies will be performed in renin expressing cells in culture derived from mouse SMG and kidney as well as a human leiomyosarcoma cell line and primary cultures of human choriodecidual cells. In vivo, we will examine tissue renin expression in mouse SMG and kidney. We will test the hypothesis that the relative activity of the CRE binding protein vs. the NRE binding protein in specific tissues regulates renin gene expression via their competition for the overlapping sequence; and that the mutations of the promoter region that affect the function of CRE and/or the NRE will result in alterations in tissue regulated expression. Both the cell culture studies and the in vivo studies will be performed using a combination of gene transfer techniques and """"""""decoy"""""""" approach to test the hypothesis. In summary, the above studies will employ cognate cell culture and in vivo gene transfer to examine tissue regulated expression of renin in mouse and human. The performance of these experiments is a logical extension of the previous in vitro/biochemical studies and will provide an understanding of the mechanism of tissue regulated expression of renin.
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