Redox sensitive mechanism regulating iNOS expression in hepatocytes are unknown. The PI's preliminary data suggest that oxidative stress transcriptionally regulates interleukin 1-beta mediated iNOS expression via direct effects upon the iNOS promoter.
The specific aim of this proposal is to functionally map the iNOS promoter to identify regions conferring the redox sensitivity. The PI has already established the transient transfection/expression assays and proposes to use deletion and site- directed mutagenesis to map the relevant areas. He describes the use of EMSA to identify the presence of relevant transcription factors and, if observed, the use of functional cloning methods to identify any novel transcription factors. Proof of relevance using antisense strategies and in an in vivo model is proposed.
