Regulation of human microsomal epoxide hydrolase gene (EPHX1) expression by the transcription factor GATA-4.
Microsomal epoxide hydrolase (mEH) is a bifunctional protein that plays a crucial role in the metabolism of numerous xenobiotics as well as in mediating the hepatic sodium-dependent uptake of bile acids that are involved in numerous physiological processes including the regulation of cholesterol metabolism. The transcription factors and nuclear receptors that control the constitutive and inducible expression of the mEH gene (EPHX1), however, have not been described. To characterize these factors, a series of 5'-deletion constructs have been transfected into human liver-derived HepG2 cells as well as non-hepatic HeLa cells. Promoter activity analysis indicated the presence of a positive regulatory element in the -80/-70 bp region. Sequence analysis revealed a putative GATA site at -79/-74 bp as well as an additional site at -31/-26 bp. Electrophoretic mobility shift assays with an anti-GATA-4 antibody confirmed that GATA-4 bound to these two sites with a dissociation constant of 1.56 nM (-79 site) and 0.65 nM (-31 site). Coexpression of GATA-4 stimulated EPHX1 promoter activity up to 7.5-fold in a dose-dependent manner. Endogenous EPHX1 message in HepG2 cells was also significantly increased by overexpression of GATA-4. Mutating the -79 element resulted in a 65% loss of promoter activity, while mutating the -31 element had no effect on basal activity but greatly reduced the response to additional GATA-4. In HeLa cells, which do not express GATA-4, EPHX1 activity was negligible; however, activity could be reconstituted by the addition of exogenous GATA-4. These results demonstrate that GATA-4 plays a critical role in regulating EPHX1 expression.
Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, Los Angeles, CA 90033, USA.
Qin-shi
QS
Zhu
Bin
B
Qian
Daniel
D
Levy
D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004851:Epoxide Hydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D050991:GATA4 Transcription Factor; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D015534:Trans-Activators / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014162:Transfection
D004268:DNA-Binding Proteins; D050991:GATA4 Transcription Factor; D012333:RNA, Messenger; D015534:Trans-Activators; D014157:Transcription Factors; D004851:Epoxide Hydrolases