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B, HIF-1
, and AP-11
*Department of Biochemistry and Molecular Biology,
Department of Molecular Microbiology and Immunology, and
Department of Pathology, Southern California Research Center for Alcoholic Liver and Pancreatic Diseases and Cirrhosis, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033; and
Department of Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, CA 90073
Chronic alcohol consumption leads to liver inflammation and cirrhosis. Alcoholic liver disease patients have increased levels of hepatic RANTES/CCL5. However, less is known about the molecular mechanisms for ethanol-induced RANTES up-regulation. In this study, we observed that liver sinusoidal endothelial cells derived from ethanol-fed rats (E-rLSECs) showed severalfold increases in RANTES and hypoxia-inducible factor 1
(HIF-1) mRNAs compared with control rLSECs (C-rLSECs). Similar effects were seen in acute ethanol treatment of isolated rLSECs and human dermal microvascular endothelial cells. Ethanol-induced RANTES mRNA expression required ethanol metabolism, p38 MAPK, HIF-1, and JNK-2, but not JNK-1. EMSA experiments showed increased HIF-1 binding to wild-type hypoxia response elements (HREs; –31 to –9 bp) within the RANTES promoter in response to ethanol. RANTES promoter analysis showed that cis elements proximal to the transcription start site, HRE-1 (nt –22 to –19), HRE-2 (nt –32 to –29), and AP-1 (nt –250 to –244) were required for ethanol-mediated RANTES expression. These results were corroborated by chromatin immunoprecipitation assays showing augmented HIF-1 binding to HRE-1. Additionally, promoter analysis revealed c-Jun, c-Jun/c-Fos, and JunD, but not JunB, bound to the AP-1 site of the RANTES promoter. Ethanol-mediated activation of NF-
B led to HIF-1 activation and concomitant RANTES expression. Plasma of ethanol-fed c-Junflox/flox-Mx-1-Cre mice showed attenuated levels of RANTES compared with ethanol-fed control mice, supporting the role of c-Jun in ethanol-induced RANTES expression. Our studies showed that ethanol-mediated RANTES/CCL5 expression occurs via HIF-1 activation independently of hypoxia. The identification of HIF-1 and AP-1 in ethanol-induced RANTES expression provides new strategies to ameliorate ethanol-induced inflammatory responses.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by pilot project funding from National Institutes of Health Grants P50-AA011999 (to V.K.K.), P50-AA011999 (to H.T.), R24-AA012885 (to H.T. for support of animal and morphological core facilities, including the Non-Parenchymal Liver Cell Core), T32-AA07578 (predoctoral fellowship to S.Y.), CA108302 (to K.M.), and P30-DK048522 (for support of the Analytical-Metabolic-Instrumentation Core, University of Southern California Research Center for Liver Diseases).
2 Address correspondence and reprint requests to Dr. Vijay K. Kalra, Department of Biochemistry and Molecular Biology, University of Southern California Keck School of Medicine, Los Angeles, CA 90033. E-mail address: vkalra{at}usc.edu
3 Abbreviations used in this paper: LSEC, liver sinusoidal endothelial cell; ChIP, chromatin immunoprecipitation; C-rLSEC, control rat LSEC; DPI, diphenyleneiodonium chloride; E-rLSEC, ethanol-fed rat LSEC; HIF, hypoxia-inducible factor; HMEC, human microvascular endothelial cell; HO, hemeoxygenase; HRE, hypoxia response element; MKK, MAPK kinase; PHD, prolylhydroxylase; poly(I:C), polyinosinic:polycytidylic acid; PKC, protein kinase C; PP1, protein phosphatase 1; qRT-PCR, quantitative RT-PCR; rLSEC, rat LSEC; scRNA, scrambled RNA; siRNA, small interfering RNA; wt, wild type.
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