Endoplasmic Reticulum Stress in Hepatic Stellate Cells Promotes Liver Fibrosis via PERK-Mediated Degradation of HNRNPA1 and Up-regulation of SMAD2
Endoplasmic Reticulum Stress in Hepatic Stellate Cells Promotes Liver Fibrosis via PERK-Mediated Degradation of HNRNPA1 and Up-regulation of SMAD2
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DOI：
10.1053/j.gastro.2015.09.039
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发表时间：
2016-01-01
期刊：
影响因子：
29.4
通讯作者：
Kim, Sang Geon
中科院分区：
文献类型：
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作者：
Koo, Ja Hyun;Lee, Hyo Ju;Kim, Sang Geon
背景与目的：内质网（ER）应激与多种疾病有关。肝星状细胞（HSC）有助于肝纤维化的发展。关于 ER 应激和 HSC 激活之间联系的信息很少。我们研究了 HSC 中 ER 应激对肝纤维化进展的影响以及细胞和小鼠中这一过程的调节。方法：测量从慢性丙型肝炎病毒感染和/或纤维化患者收集的 2 组肝脏样本（n = 25 和 n = 44）中的蛋白质和信使 RNA。使用化学试剂在细胞和小鼠中诱导内质网应激。构建慢病毒载体以从 α-平滑肌肌动蛋白启动子表达葡萄糖调节蛋白 78（GRP78；也称为 HSPA5）或异质核核糖核蛋白 A1（HNRNPA1），并将其注射到 C57BL/6 小鼠中进行 HSC 特异性基因表达。从小鼠或大鼠中收集肝组织和 HSC，并使用免疫印迹和定量逆转录聚合酶链反应进行分析。 LX-2 细胞用小干扰 RNA、microRNA 模拟物或过表达载体转染。结果：与轻度纤维化患者相比，重度纤维化患者的肝组织中的肝脏 ER 应激要高得多。 ER 应激诱导 HSC 中的纤维化基因。将葡萄糖调节蛋白 78 靶向慢病毒递送至小鼠 HSC 可以减少肝脏中的纤维积累。在暴露于 ER 应激的患者或小鼠的纤维化肝组织中，SMAD2 水平升高，但 SMAD3 水平没有升高；小干扰 RNA 介导的 SMAD2 敲除减少了 ER 应激介导的 HSC 激活。在大鼠 HSC 中，ER 应激通过降低 SMAD2 表达抑制剂 microRNA 18a (MIR18A) 的水平来增加 SMAD2 信使 RNA 的水平，而不是反式激活 SMAD2 基因。 ER 应激激活的 PKR 样内质网激酶（也称为 EIF2AK3 (PERK)）在 Thr51 磷酸化 HNRNPA1（初级 MIR18A 成熟加工所需的蛋白质），加速其降解。小鼠 HSC 中 HNRNPA1（或其 T51A 突变体）的过度表达可抑制肝纤维化。与轻度纤维化肝组织相比，严重纤维化患者的 HSC 中磷酸化 PERK 水平升高，HNRNPA1 水平降低。结论：HSC 中的 ER 应激通过 MIR18A 失调诱导 SMAD2 过度表达，从而促进肝纤维化。这种失调是由 PERK 磷酸化和 HNRNPA1 不稳定介导的。
BACKGROUND & AIMS: Endoplasmic reticulum (ER) stress has been implicated in a variety of diseases. Hepatic stellate cells (HSCs) contribute to the development of liver fibrosis. Information on the link between ER stress and HSC activation is scarce. We investigated the effects of ER stress in HSCs on the progression of liver fibrosis and the regulation of this process in cells and mice. METHODS: Proteins and messenger RNAs were measured in 2 sets of liver samples (n = 25 and n = 44) collected from patients with chronic hepatitis C virus infection and/or fibrosis. ER stress was induced in cells and mice using chemical agents. Lentiviral vectors were constructed to express glucose-regulated protein 78 (GRP78; also known as HSPA5) or heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) from the alpha-smooth muscle actin promoter and injected into C57BL/6 mice for HSC-specific gene expression. Liver tissues and HSCs were collected from mice or rats and analyzed using immunoblottings and quantitative reverse-transcription polymerase chain reaction. LX-2 cells were transfected with small interfering RNAs, microRNA mimics, or overexpression vectors. RESULTS: Hepatic ER stress was much higher in liver tissues from patients with severe vs mild fibrosis. ER stress induced fibrogenic genes in HSCs. Targeted lentiviral delivery of glucose-regulated protein 78 to HSCs in mice reduced fiber accumulation in liver. Levels of SMAD2, but not SMAD3, were increased in fibrotic liver tissues from patients or mice exposed to ER stress; small interfering RNA-mediated knockdown of SMAD2 reduced ER stress-mediated activation of HSCs. In rat HSCs, ER stress increased levels of SMAD2 messenger RNA by decreasing levels of microRNA 18a (MIR18A), an inhibitor of SMAD2 expression, rather than transactivating the SMAD2 gene. ER stress-activated PKR-like endoplasmic reticulum kinase, also known as EIF2AK3 (PERK) phosphorylated HNRNPA1, a protein required for the maturational processing of primary MIR18A, at Thr51, accelerating its degradation. Overexpression of HNRNPA1 (or its T51A mutant) in HSCs of mice inhibited liver fibrosis. Severe fibrotic liver tissues from patients had increased levels of phosphorylated PERK and reduced levels of HNRNPA1 in HSCs, compared with mild fibrotic liver tissues. CONCLUSIONS: ER stress in HSCs promotes liver fibrosis by inducing overexpression of SMAD2, via dysregulation of MIR18A; this dysregulation is mediated by PERK phosphorylation and destabilization of HNRNPA1.