My long term objective is to characterize novel molecular mechanisms responsible for the regulation of renin-angiotensin system (RAS) and their implications in the pathogenesis of hypertension and other cardiovascular diseases. Preliminary studies conducted by the applicant and other researchers in this laboratory identified mouse LXRalpha, an orphan transcriptional factor of the nuclear receptor superfamily, as a transcriptional activator of renin expression in response to cAMP. Subsequently, the applicant cloned N-CoR, the nuclear receptor corepressor, in a yeast two-hybrid screen using mLXRalpha as bait and demonstrated that N-CoR binds with mLXRalpha specifically in yeast. My immediate goal is to characterize the cellular and molecular mechanisms of N-CoR regulating tissue-specific renin expression.
The specific aims are: 1) To document mLXRalpha as a cAMP responsive transcriptional factor. Detailed dose response and time course studies will be performed using Northern blot analysis. Transfection/reporter assay will be conducted to further confirm the preliminary result that mLXRalpha confers cAMP responsiveness to the renin promoter through the unique renin CRE element. 2) To analyze N-CoR function in the regulation of renin gene expression in cell culture models. This study will be conducted by cotransfection of N-CoR, and its dominant negative mutant N-CoRI or antisense oligonucleotide targeting N-CoR mRNA, followed by Northern and Western analyses to measure renin expression. Moreover, the tk-luciferase reporter drove by the renin cAMP responsive element (CRE) will be used in a cotransfection and reporter assay to further define N-CoR function. 3) To characterize the interaction between N-CoR and mLXRalpha using in vitro assays. The interaction of N-CoR with mLXRalpha in solution or on DNA (CRE) will be examined by GST pull-down assays and gel mobility shift assays, respectively. mLXRalpha's binding affinity on CRE in the presence and absence of N-CoR will be evaluated by gel shift competition assays. 4) To demonstrate NCoR's inhibitory activity on renin gene expression in vivo. The HVJ-liposome technology will be used to transfer antisense oligonucleotides into mouse liver to block N-CoR expression. Induced renin gene expression in liver is expected. This project will provide a superb training experience in using state-of-the art molecular biology approaches to address an important yet poorly understood problem in cardiovascular biology.
