TRANSCRIPTIONAL REGULATION OF BILE SALT EXPORT PUMP BY ENDOBIOTICS AND XENOBIOTI

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Conversion of cholesterol to bile acids and subsequent secretion into bile represents a major pathway in the elimination of excessive cholesterol. Genetic and biochemical studies have established that the bile salt export pump (BSEP) is responsible for the secretion. Impairment of BSEP function or expression by endobiotics and xenobiotics lead to cholestatic liver injury. On the other hand, enhancement of BSEP expression by endobiotics and xenobiotics has therapeutic effect on cholestasis and hypercholesterolemia. Elucidation of the molecular mechanism for the regulation of BSEP expression will have physiological, toxicological and therapeutic significance. The long-term objective of the proposed project is to elucidate the molecular mechanism by which the expression of BSEP is coordinately regulated by endobiotics and xenobiotics through distinct but functionally related pathways. The specific aims of this pilot project are: specific aim #1 to determine whether hypolipidemic guggulsterone-mediated induction of BSEP expression is achieved through the c-Jun/c-fos activation pathway and specific aim #2 to determine whether nuclear receptor liver homology receptor 1 (LRH-1) transcriptionally regulates BSEP expression. Two experimental approaches are proposed for specific aim #1: (a) super electrophoretic mobility shift assay will be performed to determine whether c-Jun/c-fos binds to the guggulsterone responsive element (GuRE) in the BSEP promoter in vitro; (b) chromatin immunoprecipitation will be performed to determine whether c-Jun/c-fos is recruited to the GuRE in vivo. Two experimental approaches are proposed for specific aim #2: (a) LRH-1 expression will be specifically silenced by RNAi and the effect of LRH-1 knockdown on BSEP expression will be determined; (b) mutational analysis of the potential LRH-1 binding sites in the BSEP promoter will be performed to identify the functional LRH-1 site(s).