Molecular Mechanisms of Cholestatic Fibrogenesis

胆汁淤积纤维形成的分子机制

• 批准号: 10553630
• 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/10553630
• 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; Epithelium; Event; Extracellular Matrix; Fibronectins; Fibrosis; Foundations; Gene Expression; Gene Silencing; Gene Targeting; Genes; Genetic; Genetic Models; Genetic Transcription; Goals; Grant; 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; Progressive Disease; Regulation; Role; Signal Transduction; Signaling Molecule; Surgical Models; System; Techniques; Testing; Therapeutic; Transcription Initiation; Transforming Growth Factor beta; Writing; bile duct; cholangiocyte; clinically relevant; congenital hepatic fibrosis; 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; pharmacologic; 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.

项目总结/摘要 胆汁淤积性纤维形成是胆管的病理生物学过程，其特征在于胆管狭窄， 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， 高度分泌，释放各种旁分泌信号分子，随后激活肝脏 星状细胞（HSC）。向终末期疾病进展的特征是过度的纤维化 对慢性损伤的反应，最终导致基质分子在门静脉周围沉积， 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）是胆道疾病进展中的早期、重要且潜在可逆的步骤。 纤维化zeste同源物增强子2（EZH 2）是一种关键的表观遗传调节因子， 组蛋白3上赖氨酸27的三甲基化（H3 K27 me 3）以沉默转录。我们的新产品线 研究表明，在正常胆管细胞中，HSC激活基因的转录是 被EZH 2稳态沉默。我们还提出，p300乙酰转移酶可以导致H3 K27 乙酰化（H3 K27 ac）以启动转录。我们得到了以下新的初步数据：1） RNA-seq和ChIP-seq已经鉴定了HSC的胆管细胞衍生的旁分泌激活剂的网络， 包括纤连蛋白（FN），其被EZH 2沉默; 2）TGF-β通过以下途径诱导沉默丧失： EZH 2的蛋白酶体降解; 3）患有胆汁性纤维化的人肝脏显示p300的积累 沿着H3 K27 ac与FN在门静脉周围沉积; 4）p300与FN形成转录因子复合物 TGF-β下游，并导致FN启动子上的H3 K27 ac;和 体内抑制EZH 2导致胆道疾病小鼠模型中过度的纤维化。基于此 根据初步数据，我们提出了中心假设，即H3 K27 me 3和H3 K27 ac的变化， 表观遗传学引发胆管细胞和HSC之间的纤维化串扰。在目标I中，我们将测试 EZH 2通过H3 K27导致FN和其他基因靶点的稳态沉默的亚假设 甲基化，并且该过程由TGF-β通过EZH 2的蛋白酶体降解来调节。在Aim II中， 我们将评估TGF-β下游的p300复合物激活FN转录的亚假设 通过H3 K27乙酰化并促进旁分泌HSC活化。在目标III中，我们将研究 亚假设EZH 2和p300在H3 K27相互调节控制胆管细胞FN 沉积、HSC活化和胆纤维化。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异性膜信号重写 组蛋白编码并产生纤维化旁分泌分子，其随后促进HSC活化。在 反过来，针对这些新发现的具有表观遗传药理学的途径的干预措施可能具有 预防或逆转驱动胆道纤维化的细胞外基质事件的能力。