AP-1 , and a B, HIF-1 k Cells via Activation of NF-Endothelial Cells and Human Endothelial Expression in Rat Liver Sinusoidal Ethanol Augments RANTES/CCL5

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 (cid:2) (HIF-1 (cid:2) ) 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 (cid:2) , and JNK-2, but not JNK-1. EMSA experiments showed increased HIF-1 (cid:2) binding to wild-type hypoxia response elements (HREs; (cid:3) 31 to (cid:3) 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 (cid:3) 22 to (cid:3) 19), HRE-2 (nt (cid:3) 32 to (cid:3) 29), and AP-1 (nt (cid:3) 250 to (cid:3) 244) were required for ethanol-mediated RANTES expression. These results were corroborated by chromatin immunoprecipitation assays showing augmented HIF-1 (cid:2) 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- (cid:1) B led to HIF-1 (cid:2) 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-1 (cid:2) activation independently of hypoxia. The identiﬁcation of HIF-1 (cid:2) and AP-1 in ethanol-induced RANTES expression provides new strategies to ameliorate ethanol-induced inﬂammatory responses. The Journal of Immunology, 2009, 183: 5964–5976. 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. These studies, to our knowledge, show for the ﬁrst time the involvement of HIF-1 (cid:1) , independent of hypoxia, in the regulation of RANTES/CCL5 expression. are expressed change three independent metabolism to activation of two independent signaling pathways. one pathway, activation of AP-1 via PKC, Src kinase, and JNK-2 leads to up-regulation of RANTES. This involves an AP-1 complex consisting of c-Jun, c-Fos, and JunD, but not JunB. involves activation of HIF-1 (cid:1) via crosstalk with NF- (cid:3) the latter involving activation of MEK. The activation of HIF-1 (cid:1) to HRE-binding in the RANTES promoter followed by increased gene transcription. We suggest that ethanol-mediated RANTES generated from LSECs augments the inﬁltration of monocytes/polymorphonuclear neutrophils and T and B cells from circulation into the liver, contributing to B-ATF) fami-lies can form homodimers or heterodimers that constitute the active AP-1 complex. Numerous studies have identiﬁed JNK-medi-ated phosphorylation of c-Jun as inﬂuencing the transcriptional activation of the AP-1 complex (43). We observed that overexpression of c-Jun, c-Jun with c-Fos, and JunD greatly increased RANTES promoter activity, whereas overexpression of either c-Fos or JunB had no effect. These data suggest that c-Jun forms a homodimer or heterodimer with c-Jun, c-Fos, or JunD to augment RANTES promoter activity. Moreover JunD, but not JunB, bound to the RANTES promoter to increase its activity. Recent studies (44) have examined the roles of JunD and JunB in the regulation of hemeoxygenase (HO)-1 promoter activity. These studies show that JunB activates whereas JunD represses HO-1 expression, demonstrating the opposing roles of JunB and JunD in regulating HO-1 (44). JunD has been shown to protect the liver from isch-emia/reperfusion injury by dampening AP-1 transcriptional activation (45). In contrast, our studies showed that JunD activated while JunB had no effect on RANTES expression, suggesting differential roles of JunD in gene expression. These results are in consonance with previous studies (46) wherein JunD is suggested to exert its role in cellular growth control through both transcriptional activation and repression of target genes. We HIF-1