Effect of ethanol on innate antiviral pathways and HCV replication in human liver cells.
Effect of ethanol on innate antiviral pathways and HCV replication in human liver cells.
复制标题
DOI：
10.1186/1743-422x-2-89
复制
发表时间：
2005-12-02
期刊：
影响因子：
4.8
通讯作者：
Polyak SJ
中科院分区：
文献类型：
--
作者：
Plumlee CR;Lazaro CA;Fausto N;Polyak SJ
酒精滥用降低慢性丙型肝炎患者对干扰素治疗的应答率为了模拟这种表型背后的分子机制，我们表征了乙醇对Huh 7人肝癌细胞、HCV复制子细胞系和原代人肝细胞中Jak-Stat和MAPK通路的影响。高生理浓度的急性乙醇激活Jak-Stat和p38 MAPK途径，并抑制HCV复制在几个独立的复制子细胞系。此外，急性乙醇诱导Stat 1丝氨酸磷酸化，这是部分介导的p38 MAPK通路。相反，当与外源性IFN-α结合时，乙醇抑制IFN对HCV复制的抗病毒作用，包括抑制IFN诱导的Stat 1酪氨酸磷酸化。乙醇的这些作用独立于i）乙醇通过ADH和CYP 2 E1代谢，和ii）乙醇的细胞毒性或细胞抑制作用。在该模型系统中，乙醇直接干扰Jak-Stat途径和HCV复制。丙型肝炎病毒感染是全世界发病率和死亡率的重要原因。由于有进展为慢性感染的倾向，约70%的慢性病毒血症患者出现慢性肝病的组织学证据，包括慢性肝炎、肝硬化和肝细胞癌。对于滥用乙醇的患者来说，情况更加可怕，与不饮酒的HCV患者相比，发展终末期肝病的风险显着更高。重组干扰素α（IFN-α）治疗在8-12%的慢性丙型肝炎患者中产生持续应答（即清除病毒血症）。IFN加利巴韦林联合治疗和聚乙二醇化IFN加利巴韦林治疗可显著改善应答率。然而，超过50%的慢性感染患者仍然没有清除病毒血症。此外，滥用酒精的HCV感染患者对IFN治疗的应答率极低，但所涉及的机制尚未阐明。MAPK在调节分化、细胞生长以及对细胞因子、趋化因子和应激的应答中起重要作用。MAPK信号转导的核心元件由3种激酶组成的模块组成，称为MKKK、MKK和MAPK，它们依次相互磷酸化。目前，在哺乳动物细胞中已经表征了四种MAPK模块：细胞外调节激酶（ERK 1和2）、应激激活/c-Jun N末端激酶（SAPK/JNK）、p38 MAP激酶和ERK 5。有趣的是，乙醇调节MAPK。然而，关于乙醇如何在先天抗病毒途径（例如人类细胞中的Jak-Stat途径）的背景下影响MAPKs的信息极其有限。当IFN-α与其受体结合时，两种受体相关酪氨酸激酶Tyk 2和Jak 1通过磷酸化被激活，并磷酸化保守酪氨酸残基上的Stat 1和Stat 2。Stat 1和Stat 2联合收割机与IRF-9蛋白结合形成转录因子干扰素刺激基因因子3（ISGF-3），其与干扰素刺激反应元件（ISRE）结合，并诱导IFN-α诱导基因（ISG）的转录。ISG介导IFN的抗病毒作用。Stats 1、3、4、5a和5 b的转录活性也受到丝氨酸磷酸化的调节。在位置727（S727）的保守丝氨酸氨基酸上的Stat 1的磷酸化导致ISGF-3转录因子复合物的最大转录活性。虽然p38 MAPK和Jak-Stat通路之间的串扰对于IFN诱导的ISRE转录是必不可少的，但p38不参与IFN诱导的Stat 1丝氨酸磷酸化。然而，细胞应激反应诱导的刺激，如紫外线诱导p38 MAPK介导的Stat 1 S727磷酸化。在目前的报告中，我们假设酒精和HCV蛋白调节MAPK和Jak-Stat途径在人类肝细胞。为了开始解决这些问题，我们表征了急性乙醇对Huh 7细胞、HCV复制子细胞系和原代人肝细胞中Jak-Stat和MAPK通路的相互作用。
Alcohol abuse reduces response rates to IFN therapy in patients with chronic hepatitis C. To model the molecular mechanisms behind this phenotype, we characterized the effects of ethanol on Jak-Stat and MAPK pathways in Huh7 human hepatoma cells, in HCV replicon cell lines, and in primary human hepatocytes. High physiological concentrations of acute ethanol activated the Jak-Stat and p38 MAPK pathways and inhibited HCV replication in several independent replicon cell lines. Moreover, acute ethanol induced Stat1 serine phosphorylation, which was partially mediated by the p38 MAPK pathway. In contrast, when combined with exogenously applied IFN-α, ethanol inhibited the antiviral actions of IFN against HCV replication, involving inhibition of IFN-induced Stat1 tyrosine phosphorylation. These effects of alcohol occurred independently of i) alcohol metabolism via ADH and CYP2E1, and ii) cytotoxic or cytostatic effects of ethanol. In this model system, ethanol directly perturbs the Jak-Stat pathway, and HCV replication. Infection with Hepatitis C virus is a significant cause of morbidity and mortality throughout the world. With a propensity to progress to chronic infection, approximately 70% of patients with chronic viremia develop histological evidence of chronic liver diseases including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The situation is even more dire for patients who abuse ethanol, where the risk of developing end stage liver disease is significantly higher as compared to HCV patients who do not drink. Recombinant interferon alpha (IFN-α) therapy produces sustained responses (ie clearance of viremia) in 8–12% of patients with chronic hepatitis C. Significant improvements in response rates can be achieved with IFN plus ribavirin combination and pegylated IFN plus ribavirin therapies. However, over 50% of chronically infected patients still do not clear viremia. Moreover, HCV-infected patients who abuse alcohol have extremely low response rates to IFN therapy, but the mechanisms involved have not been clarified. MAPKs play essential roles in regulation of differentiation, cell growth, and responses to cytokines, chemokines and stress. The core element in MAPK signaling consists of a module of 3 kinases, named MKKK, MKK, and MAPK, which sequentially phosphorylate each other. Currently, four MAPK modules have been characterized in mammalian cells: Extracellular Regulated Kinases (ERK1 and 2), Stress activated/c-Jun N terminal kinase (SAPK/JNK), p38 MAP kinases, and ERK5. Interestingly, ethanol modulates MAPKs. However, information on how ethanol affects MAPKs in the context of innate antiviral pathways such as the Jak-Stat pathway in human cells is extremely limited. When IFN-α binds its receptor, two receptor associated tyrosine kinases, Tyk2 and Jak1 become activated by phosphorylation, and phosphorylate Stat1 and Stat2 on conserved tyrosine residues. Stat1 and Stat2 combine with the IRF-9 protein to form the transcription factor interferon stimulated gene factor 3 (ISGF-3), which binds to the interferon stimulated response element (ISRE), and induces transcription of IFN-α-induced genes (ISG). The ISGs mediate the antiviral effects of IFN. The transcriptional activities of Stats 1, 3, 4, 5a, and 5b are also regulated by serine phosphorylation. Phosphorylation of Stat1 on a conserved serine amino acid at position 727 (S727), results in maximal transcriptional activity of the ISGF-3 transcription factor complex. Although cross-talk between p38 MAPK and the Jak-Stat pathway is essential for IFN-induced ISRE transcription, p38 does not participate in IFN induction of Stat1 serine phosphorylation. However, cellular stress responses induced by stimuli such as ultraviolet light do induce p38 MAPK mediated Stat1 S727 phosphorylation. In the current report, we postulated that alcohol and HCV proteins modulate MAPK and Jak-Stat pathways in human liver cells. To begin to address these issues, we characterized the interaction of acute ethanol on Jak-Stat and MAPK pathways in Huh7 cells, HCV replicon cells lines, and primary human hepatocytes.