Division of Gastroenterology and Hepatology, Mayo Clinic and Foundation, Rochester, MN.
Gastroenterology Research Unit, Mayo Clinic and Foundation, Rochester, MN.
Hepatology. 2019 Nov;70(5):1674-1689. doi: 10.1002/hep.30706. Epub 2019 Jun 22.
During biliary disease, cholangiocytes become activated by various pathological stimuli, including transforming growth factor β (TGF-β). The result is an epigenetically regulated transcriptional program leading to a pro-fibrogenic microenvironment, activation of hepatic stellate cells (HSCs), and progression of biliary fibrosis. This study evaluated how TGF-β signaling intersects with epigenetic machinery in cholangiocytes to support fibrogenic gene transcription. We performed RNA sequencing in cholangiocytes with or without TGF-β. Ingenuity pathway analysis identified "HSC Activation" as the highly up-regulated pathway, including overexpression of fibronectin 1 (FN), connective tissue growth factor, and other genes. Bioinformatics identified enhancer of zeste homologue 2 (EZH2) as an epigenetic regulator of the cholangiocyte TGF-β response. EZH2 overexpression suppressed TGF-β-induced FN protein in vitro, suggesting FN as a direct target of EZH2-based repression. Chromatin immunoprecipitation assays identified an FN promoter element in which EZH2-mediated tri-methylation of lysine 27 on histone 3 is diminished by TGF-β. TGF-β also caused a 50% reduction in EZH2 protein levels. Proteasome inhibition rescued EZH2 protein and led to reduced FN production. Immunoprecipitation followed by mass spectrometry identified ubiquitin protein ligase E3 component N-recognin 4 in complex with EZH2, which was validated by western blotting in vitro. Ubiquitin mutation studies suggested K63-based ubiquitin linkage and chain elongation on EZH2 in response to TGF-β. A deletion mutant of EZH2, lacking its N-terminal domain, abrogates both TGF-β-stimulated EZH2 degradation and FN release. In vivo, cholangiocyte-selective knockout of EZH2 exacerbates bile duct ligation-induced fibrosis whereas MDR2 mice are protected from fibrosis by the proteasome inhibitor bortezomib. Conclusion: TGF-β regulates proteasomal degradation of EZH2 through N-terminal, K63-linked ubiquitination in cholangiocytes and activates transcription of a fibrogenic gene program that supports biliary fibrosis.
在胆道疾病中，胆管细胞受到各种病理刺激的激活，包括转化生长因子-β (TGF-β)。其结果是一个受表观遗传调控的转录程序，导致纤维生成的微环境、肝星状细胞 (HSCs) 的激活和胆道纤维化的进展。本研究评估了 TGF-β信号在胆管细胞中与表观遗传机制的相互作用如何支持纤维生成基因的转录。我们对有或没有 TGF-β的胆管细胞进行了 RNA 测序。通路分析鉴定出“HSC 激活”是高度上调的通路，包括纤维连接蛋白 1 (FN)、结缔组织生长因子和其他基因的过表达。生物信息学鉴定出增强子的锌指同源物 2 (EZH2) 是胆管细胞 TGF-β反应的表观遗传调节剂。EZH2 过表达在体外抑制 TGF-β诱导的 FN 蛋白，表明 FN 是 EZH2 抑制的直接靶点。染色质免疫沉淀试验鉴定出 FN 启动子元件，其中 TGF-β导致 EZH2 介导的组蛋白 3 赖氨酸 27 的三甲基化减少。TGF-β还导致 EZH2 蛋白水平降低 50%。蛋白酶体抑制可挽救 EZH2 蛋白并减少 FN 产生。免疫沉淀后进行质谱分析鉴定出 E3 泛素连接酶 E3 成分 N-识别蛋白 4 与 EZH2 复合物，在体外通过 Western 印迹验证。泛素突变研究表明，TGF-β反应中 EZH2 上存在 K63 基泛素连接和链延伸。缺乏其 N 端结构域的 EZH2 缺失突变体，可消除 TGF-β刺激的 EZH2 降解和 FN 释放。在体内，胆管细胞选择性敲除 EZH2 可加重胆管结扎诱导的纤维化，而 MDR2 小鼠通过蛋白酶体抑制剂硼替佐米免受纤维化的影响。结论：TGF-β通过胆管细胞中的 N 端、K63 连接泛素化调节 EZH2 的蛋白酶体降解，并激活支持胆道纤维化的纤维生成基因程序的转录。
