Vol. 304, Issue 3, 1048-1054, March 2003

Mechanisms of Down-Regulation of CYP2E1 Expression by Inflammatory Cytokines in Rat Hepatoma Cells

Jukka Hakkola, Yin Hu and Magnus Ingelman-Sundberg

Department of Pharmacology and Toxicology, University of Oulu, Oulu, Finland (J.H.); Astrazeneca R&D Södertälje, Research DMPK, Södertälje, Sweden (Y.H.); and Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden (M.I.-S.)

CYP2E1 is one of the major cytochrome P450 forms whose expression is strongly inhibited by inflammatory cytokines in humans and rodents. In the present study, we have used the Fao rat hepatoma cell line that constitutively expresses CYP2E1 enzyme to investigate mechanisms of cytokine action. The cells were treated with interleukin (IL)-1, tumor necrosis factor- (TNF), or IL-6 for 24 or 72 h, and the expression of CYP2E1 was monitored at the transcriptional, mRNA, and protein levels. All three cytokines decreased the CYP2E1 mRNA levels after 24 h, and the effect was even stronger after 72 h. In contrast, significant inhibition of CYP2E1 protein was seen only after 72 h. In transfection assays using a CYP2E1 5' 3685 to +29-luciferase construct, it was found that IL-6 inhibited gene transcription after 24 h, but a similar effect by IL-1 and TNF was registered only after 72 h. Using 5' deletions of the CYP2E1 5'-reporter construct a responsive region for the IL-6 effect was located to 669 to 507 base pairs in the CYP2E1 5'-flanking region. Interestingly, IL-1, but not TNF, was found to reduce hepatocyte nuclear factor (HNF)-1 binding to the CYP2E1 promotor. However, the transactivation function of HNF-1 was found to be impaired in Fao cells. In mouse primary hepatocytes, IL-1 decreased HNF-1-mediated transactivation. In conclusion, our data indicate that inflammatory cytokines inhibit CYP2E1 expression by multiple mechanisms, including control of HNF-1 function and regulation of other transcriptional factors acting on the CYP2E1 5'-upstream regulatory region. In addition, regulation of factors of importance for the CYP2E1 mRNA stability may be involved.