Ganesan Murali, Tikhanovich Irina, Vangimalla Shiva Shankar, Dagur Raghubendra Singh, Wang Weimin, Poluektova Larisa I, Sun Yimin, Mercer David F, Tuma Dean, Weinman Steven A, Kharbanda Kusum K, Osna Natalia A
Research Service, Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, Nebraska.
Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska.
Cell Mol Gastroenterol Hepatol. 2017 Oct 16;5(2):101-112. doi: 10.1016/j.jcmgh.2017.10.004. eCollection 2018.
BACKGROUND & AIMS: Alcohol-induced progression of hepatitis C virus (HCV) infection is related to dysfunction of innate immunity in hepatocytes. Endogenously produced interferon (IFN)α induces activation of interferon-stimulated genes (ISGs) via triggering of the Janus kinase-signal transducer and activator of transcription 1 (STAT1) pathway. This activation requires protein methyltransferase 1-regulated arginine methylation of STAT1. Here, we aimed to study whether STAT1 methylation also depended on the levels of demethylase jumonji domain-containing 6 protein (JMJD6) and whether ethanol and HCV affect JMJD6 expression in hepatocytes.
Huh7.5-CYP (RLW) cells and hepatocytes were exposed to acetaldehyde-generating system (AGS) and 50 mmol/L ethanol, respectively. JMJD6 messenger RNA and protein expression were measured by real-time polymerase chain reaction and Western blot. IFNα-activated cells either overexpressing JMJD6 or with knocked-down JMJD6 expression were tested for STAT1 methylation, ISG activation, and HCV RNA. In vivo studies have been performed on C57Bl/6 mice (expressing HCV structural proteins or not) or chimeric mice with humanized livers fed control or ethanol diets.
AGS exposure to cells up-regulated JMJD6 expression in RLW cells. These results were corroborated by ethanol treatment of primary hepatocytes. The promethylating agent betaine reversed the effects of AGS/ethanol. Similar results were obtained in vivo, when mice were fed control/ethanol with and without betaine supplementation. Overexpression of JMJD6 suppressed STAT1 methylation, IFNα-induced ISG activation, and increased HCV-RNA levels. In contrast, JMJD6 silencing enhanced STAT1 methylation, ISG stimulation by IFNα, and attenuated HCV-RNA expression in Huh7.5 cells.
We conclude that arginine methylation of STAT1 is suppressed by JMJD6. Both HCV and acetaldehyde increase JMJD6 levels, thereby impairing STAT1 methylation and innate immunity protection in hepatocytes exposed to the virus and/or alcohol.
酒精诱导的丙型肝炎病毒（HCV）感染进展与肝细胞固有免疫功能障碍有关。内源性产生的干扰素（IFN）α通过触发Janus激酶-信号转导和转录激活因子1（STAT1）途径诱导干扰素刺激基因（ISG）的激活。这种激活需要蛋白甲基转移酶1调节的STAT1精氨酸甲基化。在此，我们旨在研究STAT1甲基化是否也依赖于含jumonji结构域6蛋白（JMJD6）的去甲基酶水平，以及乙醇和HCV是否影响肝细胞中JMJD6的表达。
将Huh7.5-CYP（RLW）细胞和肝细胞分别暴露于乙醛生成系统（AGS）和50 mmol/L乙醇中。通过实时聚合酶链反应和蛋白质免疫印迹法检测JMJD6信使核糖核酸和蛋白质表达。对过表达JMJD6或JMJD6表达被敲低的IFNα激活细胞进行STAT1甲基化、ISG激活和HCV核糖核酸检测。对C57BL/6小鼠（表达或不表达HCV结构蛋白）或喂食对照或乙醇饮食的人源化肝脏嵌合小鼠进行了体内研究。
AGS暴露于细胞中上调了RLW细胞中JMJD6的表达。原代肝细胞乙醇处理证实了这些结果。促甲基化剂甜菜碱逆转了AGS/乙醇的作用。当给小鼠喂食对照/乙醇且补充或不补充甜菜碱时，在体内也获得了类似结果。JMJD6的过表达抑制了STAT1甲基化、IFNα诱导的ISG激活，并增加了HCV核糖核酸水平。相反，JMJD6沉默增强了STAT1甲基化、IFNα对ISG的刺激，并减弱了Huh7.5细胞中HCV核糖核酸的表达。
我们得出结论，JMJD6抑制STAT1的精氨酸甲基化。HCV和乙醛均增加JMJD6水平，从而损害暴露于病毒和/或酒精的肝细胞中的STAT1甲基化和固有免疫保护。
