期刊
BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION
卷 1676, 期 3, 页码 251-260出版社
ELSEVIER
DOI: 10.1016/j.bbaexp.2004.01.002
关键词
m-epoxide hydrolase; gene expression; GATA-4
资金
- NIDDK NIH HHS [R01 DK 25836] Funding Source: Medline
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 EPHXI promoter activity up to 7.5-fold in a dose-dependent manner. Endogenous EPHXI 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, EPHXI 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. (C) 2004 Elsevier B.V. All rights reserved.
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