Molecular Mechanisms of Cholestatic Fibrogenesis

胆汁淤积纤维形成的分子机制

• 批准号: 10338129
• 负责人: Robert Christian Huebert
• 金额: $ 35.78万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-15 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10338129
• 关键词: Acetylation; Acetyltransferase; Agonist; Area; Bile Duct Diseases; Biliary; Biliary cirrhosis; Biochemical; ChIP-seq; Cholestasis; Chronic; Cicatrix; Coculture Techniques; Collagen; Complex; Coupled; Data; Deposition; Disease; Ductal Epithelial Cell; EP300 gene; Enhancers; Epigenetic Process; Epithelial; Event; Extracellular Matrix; Fibronectins; Fibrosis; Foundations; Gene Expression; Gene Silencing; Gene Targeting; Genes; Genetic; Genetic Models; Genetic Transcription; Goals; Grant; Hepatic; Hepatic Stellate Cell; Histone Code; Histones; Homeostasis; Homologous Gene; Human; Injury; Intervention; Investigation; Knock-out; Lead; Ligation; Liver; Lysine; Mediating; Membrane; Mesenchymal; Methylation; Microfluidics; Molecular; Molecular Target; Organ Donor; Paracrine Communication; Pathogenesis; Pathologic; Pathway interactions; Patients; Pharmacology; Process; Regulation; Role; Signal Transduction; Signaling Molecule; Surgical Models; System; Techniques; Testing; Therapeutic; Transforming Growth Factor beta; Writing; base; bile duct; cholangiocyte; clinically relevant; design; dietary; effective therapy; end stage disease; end stage liver disease; epigenetic regulation; epigenomics; feeding; fibrogenesis; gene network; histone methyltransferase; histone modification; in vivo; liver transplantation; mouse model; mutant; novel; p300/CBP-Associated Factor; paracrine; prevent; primary sclerosing cholangitis; programs; promoter; response; stellate cell; targeted treatment; transcription factor; transcriptome sequencing

PROJECT SUMMARY / ABSTRACT Cholestatic fibrogenesis is a pathobiological process of the bile ducts, characterized by biliary strictures, cholestasis, and progressive peri-portal fibrosis. During biliary fibrosis, diseased cholangiocytes become highly secretory, releasing a variety of paracrine signaling molecules that subsequently activate hepatic stellate cells (HSC). Progression toward end stage disease is characterized by an exaggerated fibrogenic response to chronic injury, culminating in peri-portal deposition of matrix molecules that progresses to biliary cirrhosis. The release of paracrine, cholangiocyte-derived factors that subsequently activate hepatic stellate cells (HSC) is an early, important, and potentially reversible step in the progression of biliary fibrosis. Enhancer of zeste homologue 2 (EZH2) is a key epigenetic regulator that enzymatically mediates the tri-methylation of lysine 27 on histone 3 (H3K27me3) to silence transcription. Our novel line of investigation proposes that, in normal cholangiocytes, transcription of HSC-activating genes is homeostatically silenced by EZH2. We also propose that the p300 acetyltransferase can lead to H3K27 acetylation (H3K27ac) to initiate transcription. We have generated the following novel preliminary data: 1) RNA-seq and ChIP-seq have identified a network of cholangiocyte-derived paracrine activators of HSCs, including fibronectin (FN), that are silenced by EZH2; 2) TGF-β induces loss of silencing through proteasomal degradation of EZH2; 3) Human livers with biliary fibrosis demonstrate accumulation of p300 and H3K27ac along with peri-portal deposition of FN; 4) p300 forms a transcription factor complex downstream of TGF-β and leads to H3K27ac on the FN promoter; and 5) Genetic or pharmacologic inhibition of EZH2 in vivo leads to exaggerated fibrosis in mouse models of biliary disease. Based on this preliminary data, we propose the central hypothesis that changes in H3K27me3 and H3K27ac, epigenetically initiate fibrogenic cross talk between cholangiocytes and HSC. In Aim I, we will test the subhypothesis that EZH2 leads to homeostatic silencing of FN and other gene targets through H3K27 methylation and this process is regulated by TGF-β through proteasomal degradation of EZH2. In Aim II, we will evaluate the subhypothesis that a p300 complex downstream of TGF-β activates FN transcription through H3K27 acetylation and promotes paracrine HSC activation. In Aim III, we will investigate the subhypothesis that reciprocal regulation at H3K27 by EZH2 and p300 controls cholangiocyte FN deposition, HSC activation, and biliary fibrosis in vivo. The significance of these studies lies in the novel concept that epigenetic regulators allow specific membrane signals in cholangiocytes to re-write the histone code and generate fibrogenic paracrine molecules that subsequently promote HSC activation. In turn, interventions targeting these newly discovered pathways with epigenetic pharmacology may have the capability to prevent or reverse the extracellular matrix events that drive biliary fibrosis.

项目概要/摘要 胆汁淤积性纤维化是胆管的病理生物学过程，其特征是胆管狭窄， 胆汁淤积和进行性门静脉周围纤维化。在胆道纤维化过程中，患病的胆管细胞变成 高度分泌，释放多种旁分泌信号分子，随后激活肝脏 星状细胞（HSC）。向终末期疾病进展的特点是过度纤维化 对慢性损伤的反应，最终导致基质分子在门静脉周围沉积，进而发展为 胆汁性肝硬化。旁分泌、胆管细胞衍生因子的释放，随后激活肝细胞 星状细胞（HSC）是胆道疾病进展中早期、重要且可能可逆的一步。 纤维化。 zeste 同源物 2 (EZH2) 的增强子是酶介导的关键表观遗传调节因子 组蛋白 3 (H3K27me3) 上赖氨酸 27 的三甲基化可沉默转录。我们的小说系列 研究表明，在正常胆管细胞中，HSC 激活基因的转录是 被 EZH2 稳态沉默。我们还提出 p300 乙酰转移酶可以导致 H3K27 乙酰化 (H3K27ac) 以启动转录。我们生成了以下新颖的初步数据：1) RNA-seq 和 ChIP-seq 已鉴定出 HSC 胆管细胞衍生的旁分泌激活剂网络， 包括被 EZH2 沉默的纤连蛋白 (FN)； 2) TGF-β通过以下方式诱导沉默的丧失 EZH2 的蛋白酶体降解； 3) 患有胆道纤维化的人类肝脏表现出 p300 的积累 和 H3K27ac 以及 FN 门静脉周围沉积； 4) p300形成转录因子复合物 TGF-β 下游并导致 FN 启动子上的 H3K27ac； 5) 遗传或药理学 体内抑制 EZH2 会导致胆道疾病小鼠模型中纤维化过度。基于此 初步数据，我们提出H3K27me3和H3K27ac变化的中心假设， 表观遗传启动胆管细胞和 HSC 之间的纤维化相互作用。在目标 I 中，我们将测试 EZH2 通过 H3K27 导致 FN 和其他基因靶标的稳态沉默的子假设 甲基化，并且该过程由 TGF-β 通过蛋白酶体降解 EZH2 来调节。在目标二中， 我们将评估 TGF-β 下游的 p300 复合物激活 FN 转录的子假设 通过 H3K27 乙酰化并促进旁分泌 HSC 激活。在目标 III 中，我们将调查 EZH2 和 p300 对 H3K27 的相互调节控制胆管细胞 FN 的子假设 体内沉积、HSC 活化和胆道纤维化。这些研究的意义在于小说 表观遗传调节因子允许胆管细胞中的特定膜信号重写的概念 组蛋白编码并产生纤维化旁分泌分子，随后促进 HSC 激活。在 反过来，针对这些新发现的表观遗传药理学途径的干预措施可能具有 预防或逆转导致胆道纤维化的细胞外基质事件的能力。