Ethanol augments RANTES/CCL5 expression in rat liver sinusoidal endothelial cells and human endothelial cells via activation of NF-kappa B, HIF-1 alpha, and AP-1.
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 1alpha (HIF-1alpha) 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-1alpha, and JNK-2, but not JNK-1. EMSA experiments showed increased HIF-1alpha 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-1alpha 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-kappaB led to HIF-1alpha activation and concomitant RANTES expression. Plasma of ethanol-fed c-Jun(flox/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-1alpha activation independently of hypoxia. The identification of HIF-1alpha and AP-1 in ethanol-induced RANTES expression provides new strategies to ameliorate ethanol-induced inflammatory responses.
Yeligar
Samantha M
SM
Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
Machida
Keigo
K
Tsukamoto
Hidekazu
H
Kalra
Vijay K
VK
D000818:Animals; D002460:Cell Line; D018946:Chemokine CCL5 / Q000096:biosynthesis*; D042783:Endothelial Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D000431:Ethanol / Q000008:administration & dosage / Q000494:pharmacology*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism* / Q000502:physiology; D008099:Liver / Q000098:blood supply / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D016328:NF-kappa B / Q000378:metabolism* / Q000502:physiology; D016755:Proto-Oncogene Proteins c-jun / Q000172:deficiency / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D018808:Transcription Factor AP-1 / Q000378:metabolism* / Q000502:physiology; D015854:Up-Regulation / Q000187:drug effects / Q000276:immunology
D018946:Chemokine CCL5; C497444:Hif1a protein, rat; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016328:NF-kappa B; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D000431:Ethanol