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. The bile salt export pump (BSEP) appears to be responsible for the secretion of bile acids. Impairment of BSEP function or expression by endobiotics and xenobiotics lead to cholestatic liver injury. Elucidation of molecular mechanisms for regulated expression of BSEP will have therapeutic, toxicological, and pharmaceutical significance. The long-term goal of the proposed project is to test the central hypothesis that expression of BSEP is coordinately regulated by endobiotics and xenobiotics through distinct but functionally related mechanisms. The specific aims of this pilot project include: (1) To determine the transactivative role of nuclear receptor FXR in the regulated expression of BSEP by oxysterol 22(R)-OHC; and (2) to determine the interactive modes of oxysterol 22(R)-OHC with the other two FXR activators CDCA and guggulsterone. The first specific aim is designed to test the hypothesis that oxysterol-induced up-regulation of BSEP expression is achieved through activation of nuclear receptor FXR. Two approaches are proposed to test this hypothesis: (a) dominant negative FXR mutants will be constructed and their ability to suppress the up-regulated expression of BSEP by 22(R)-OHC will be tested; and (b) the FXR binding site IR-1 in the BSEP promoter will be mutated and its ability to regulate reporter expression will be tested in response to oxysterol 22(R)-OHC. The second specific aim is designed to test the hypothesis that BSEP expression is coordinately regulated by FXR activators oxysterol 22(R)-OHC, bile acid CDCA, and xenobiotic guggulsterone. Two approaches are proposed to tested the hypothesis: (1) human primary hepatocytes will be treated with 22(R)-OHC, CDCA, and guggulsterone individually or in a combination of the two, followed by measurement of BSEP mRNA levels; (2) Huh 7 cells transfected with BSEP promoter reporters will be treated with 22(R)-OHC, CDCA, and guggulsterone individually or in a combination, followed by measurement of reporter activity.