Hepatocyte Nuclear Factor (HNF) 1 and HNF4 Mediate Hepatic Multidrug Resistance Protein 2 Up-Regulation during Hepatitis C Virus Gene Expression Ishtiaq Qadri, Mieko Iwahashi, Gerd A. Kullak-Ublick, and Francis R. Simon Department of Pediatrics and Graduate Program in Toxicology (I.Q.) and Division of Gastroenterology/Hepatology (M.I., F.R.S.), University of Colorado Health Sciences Center, Aurora, Colorado; and Laboratory of Molecular Gastroenterology and Hepatology, University Hospital, Zurich, Switzerland (G.A.K.-U.) Received February 13, 2006; accepted April 28, 2006 ABSTRACT Hepatitis C virus (HCV) is known to induce hepatic oxidative stress that is implicated in the up-regulation of multidrug resis- tance proteins (MRPs). The relationship between increased prooxidant production, MRPs, and HCV has not been investi- gated. Here, we report that a homeodomain-containing tran- scription factor, hepatocyte nuclear factor (HNF) 1, plays a central role in liver gene regulation during HCV gene expression and/or subgenome replication. MRP2 protein and mRNA ex- pression were increased and MRP2 promoter activity was in- creased 7-fold. Mutations within the putative HNF1 binding site of the human MRP2 promoter abrogated HCV-induced activa- tion, implicating HNF1 in the induction of MRP2 by HCV. The mechanism by which HNF1-mediated activation occurs seems to be transcriptional, because the regulated expression of HNF4, which is known to control HNF1 expression, was also increased. Consistent with this finding, HNF1 mRNA was in- creased 10-fold. A promoter-luciferase construct of the human HNF1 gene was activated in an HNF4-dependent manner, and a mutant construct lacking the HNF4 binding site was not activated in HCV-positive cells. Consistent with this hypothesis, HNF4 protein and mRNA levels as well as HNF4 promoter activity and DNA binding activity were increased. The expres- sion of HNF1 seems to play a critical role in the induction of hepatic MRP2 secondary to HCV subgenomic replication. The ability of HCV to induce HNF1 and HNF4 is attributed to 1) increased oxidative stress and 2) direct protein-protein interac- tions between HCV nonstructural component (NS) 5A and HNF1, leading to enhanced HNF1 DNA binding. In conclusion, we describe a novel mechanism by which HCV gene expres- sion may induce adaptive responses involving MRP2 via HNF1 activation. This may constitute, in part, the cellular detoxifica- tion task force during HCV infection. Hepatitis C virus (HCV) infection poses a serious health problem, because the virus is the causative agent of chronic hepatitis worldwide. HCV infection typically produces a pro- longed, insidious course that may progress to liver fibrosis, cirrhosis, insulin resistance, and eventually hepatocellular carcinoma (World Health Organization, 1999). HCV is a pos- itive-strand RNA virus that is co- and post-translationally cleaved to produce at least 10 polypeptides, including three structural components (Core, E1, and E2) and seven non- structural components (NS2 to NS5B) (Reed and Rice, 2000). HCV nonstructural proteins direct viral replication from a ribonucleoprotein replication complex that is associated with the endoplasmic reticulum (Hijikata et al., 1993). Although the potential mechanism(s) of HCV-induced cell injury and disease progression is unclear, emerging evidence includes the relationship between liver damage and production of oxygen-derived free radicals, glutathione (GSH) depletion, tumor suppressor p53 protein inactivation, and sustained release of inflammatory cytokines (Multu-Turkoglu et al., 1997; Qadri et al., 2002, 2004; Liu et al., 2003; Abdalla et al., 2005). Transcriptional regulators such as hepatocyte nuclear fac- tor (HNF) 1 and HNF4, nuclear factor-B (NF-B), signal This work was supported from an American Liver Foundation, Herman Lopetta Memorial Award (to I.Q.) and National Institutes of Health Grant DK015851 (to F.R.S.). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.023499. ABBREVIATIONS: HCV, hepatitis C virus; NS, nonstructural; GSH, glutathione; HNF, hepatocyte nuclear factor; NF-B, nuclear factor-B; STAT, signal transducer and activator of transcription; MRP, multidrug resistant protein; ROS, reactive oxygen species; ABC, ATP-binding cassette; NAC, N-acetylcysteine; DMEM, Dulbecco’s modified Eagle’s medium; bp, base pair(s); PCR, polymerase chain reaction; Rn, normalized reporter signal; hMRP, human multidrug resistant protein; PAGE, polyacrylamide gel electrophoresis; LUC, luciferase; GST, glutathione S-transferase; EMSA, electrophoresis mobility shift assay; ALU, arbitrary light unit; DCF, dichlorofluorescein; tH 2 O 2 , tert-hydrogen peroxide; tBHQ, tert 8-bromo-7-hydroxyquinoline; IFN, interferon. 0026-895X/06/7002-627–636$20.00 MOLECULAR PHARMACOLOGY Vol. 70, No. 2 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 23499/3125267 Mol Pharmacol 70:627–636, 2006 Printed in U.S.A. 627 by guest on November 14, 2013 molpharm.aspetjournals.org Downloaded from