College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea.
Department of Internal Medicine, Seoul Metropolitan Government Seoul National University Boramae Medical Center, Seoul, Korea.
Gastroenterology. 2016 Jan;150(1):181-193.e8. doi: 10.1053/j.gastro.2015.09.039. Epub 2015 Oct 3.
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.
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 α-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.
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.
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.
内质网（ER）应激与多种疾病有关。肝星状细胞（HSCs）参与肝纤维化的发展。关于 ER 应激与 HSC 激活之间联系的信息很少。我们研究了 ER 应激在 HSCs 中对肝纤维化进展的影响，并在细胞和小鼠中研究了这一过程的调节。
使用化学试剂诱导细胞和小鼠产生 ER 应激。使用慢病毒载体构建从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）的表达载体，并将其注射到 C57BL/6 小鼠中以进行 HSC 特异性基因表达。从小鼠或大鼠中收集肝组织和 HSCs，并用免疫印迹和定量逆转录聚合酶链反应进行分析。用小干扰 RNA、microRNA 模拟物或过表达载体转染 LX-2 细胞。
严重纤维化患者肝组织中肝 ER 应激明显高于轻度纤维化患者。ER 应激诱导 HSCs 产生纤维生成基因。在小鼠中靶向性地将葡萄糖调节蛋白 78 递送至 HSCs，可减少肝脏中的纤维堆积。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；用小干扰 RNA 敲低 SMAD2 可减少 ER 应激介导的 HSCs 激活。在大鼠 HSCs 中，ER 应激通过降低 microRNA 18a（MIR18A）的水平来增加 SMAD2 信使 RNA 的水平，MIR18A 是 SMAD2 表达的抑制剂，而不是反式激活 SMAD2 基因。激活的蛋白激酶 R 样内质网激酶，也称为 EIF2AK3（PERK），在 Thr51 处磷酸化 HNRNPA1，这是初级 MIR18A 成熟加工所必需的蛋白，加速其降解。在小鼠的 HSCs 中超表达 HNRNPA1（或其 T51A 突变体）可抑制肝纤维化。与轻度纤维化肝组织相比，严重纤维化患者的肝组织中 HSCs 的磷酸化 PERK 水平升高，HNRNPA1 水平降低。
HSCs 中的 ER 应激通过诱导 SMAD2 的过表达来促进肝纤维化，这是通过 MIR18A 的失调来实现的；这种失调是由 PERK 磷酸化和 HNRNPA1 的不稳定介导的。
