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and MicroRNA-1991
*Department of Biochemistry and Molecular Biology and
Department of Pathology, The Southern California Research Center for ALPD 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 inflammation and cirrhosis of the liver. In this study, we observed that liver sinusoidal endothelial cells (LSEC) derived from ethanol-fed rats showed several fold increases in the mRNA expression of endothelin-1 (ET-1), hypoxia-inducible factor-1
(HIF-1), and inflammatory cytochemokines compared with control rat LSEC. We also observed the same results in acute ethanol-treated LSEC from control rats and human dermal microvascular endothelial cells. Ethanol-mediated ET-1 expression involved NADPH oxidase and HIF-1 activation. Furthermore, ethanol increased the expression of the ET-1 cognate receptor ET-BR in Kupffer cells and THP-1 monocytic cells, which also involved HIF-1 activation. Promoter analysis and chromatin immunoprecipitation showed that hypoxia response element sites in the proximal promoter of ET-1 and ET-BR were required for the binding of HIF-1 to up-regulate their expression. We showed that microRNAs, miR-199 among several microRNAs, attenuated HIF-1 and ET-1 expression, while anti-miR-199 reversed the effects, suggesting that ethanol-induced miR-199 down-regulation may contribute to augmented HIF-1 and ET-1 expression. Our studies, for the first time to our knowledge, show that ethanol-mediated ET-1 and ET-BR expression involve HIF-1, independent of hypoxia. Additionally, ethanol-induced ET-1 expression in rat LSEC is regulated by miR-199, while in human endothelial cells, ET-1 expression is regulated by miR-199 and miR-155, indicating that these microRNAs may function as novel negative regulators to control ET-1 transcription and, thus, homeostatic levels of ET-1 to maintain microcirculatory tone.
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 (V.K.K.) from National Institutes of Health Grant P50-AA011999 (to H.T.) and its animal and morphological core facilities, Grants R24-AA012885 (Non-Parenchymal Liver Cell Core; to H.T.) and T32-AA07578 (Predoctoral Fellowship to S.Y.).
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: HIF-1, hypoxia-inducible factor-1; ChIP, chromatin immunoprecipitation; DPI, diphenylene iodonium; ET-1, endothelin-1; ET-BR, endothelin-B receptor; HMEC-1, human dermal microvascular endothelial cell line; HRE, hypoxia response element; KC, Kupffer cell; LSEC, liver sinusoidal endothelial cell; miRNA, microRNA; PHD, prolylhydroxylase; PlGF, placenta growth factor; scRNA, scrambled RNA; siRNA, small interfering RNA; UTR, untranslated region; PMN, polymorphonuclear leukocyte.
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