Human microsomal epoxide hydrolase (mEH) is a bifunctional protein that plays an important role in the sodium-dependent hepatic uptake of bile acids and in the metabolism of numerous xenobiotic carcinogens. Aberrant expression of mEH caused by polymorphisms in regulatory elements of the mEH gene (EPHX1) has been shown to result in defects in bile acid uptake resulting in hypercholanemia, and along with poly- morphisms in the coding region, in the altered metabolism of numerous carcinogens resulting in alterations in the susceptibility to various forms of cancer. Despite the critical role that mEH plays in bile acid transport and xenobiotic metabolism, the mechanisms that serve to regulate EPHX1 expression by endogenous and exogenous factors are now just beginning to be elucidated by studies from our laboratory. The long-term objectives of this research program are therefore to characterize the regulation of EPHX1 expression, further elucidate the role of mEH in mediating bile acid transport and to identify and functionally characterize human EPHX1 polymorphisms that affect transport function in hypercholanemic subjects.
The specific aims of this proposal are A) to elucidate the transcriptional mechanisms involved in the bile acid induction of EPHX1 expression mediated by HNF-3beta, PPARalpha and PXR/CAR;B) to characterize mEH mediated transport of bile acids into hepatocytes and into the lumen of the endoplasmic reticulum where bile acid detoxification may take place via glucuronidation and C) to characterize the effects of human EPHX1 polymorphisms at regulatory sites or in the coding region on the transcription of EPHX1 and/or functional expression of mEH as related to the etiology of hypercholanemia. EPHX1 expression studies will use promoter activity assays, Northern and Western blot analyses, transcription factor expression vectors, footprinting, site-directed mutagenesis, electrophoretic mobility shift assays and chromatin immunoprecipitation. Transports studies will be done on wild type and mEH knockout mice. Polymorphism studies will utilize sequencing, genotyping and functional expression procedures. These studies should substantially increase our knowledge of this important transport/metabolic protein that plays a critical role in numerous physiological processes and in the etiology of several diseases including cholestatic liver disease.
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