Molecular Mechanisms of Cholestatic Fibrogenesis

项目摘要/摘要 胆汁淤积性纤维化是胆管的一种病理生物学过程，以胆管狭窄为特征， 胆汁淤积和进行性门脉周围纤维化。在胆道纤维化期间，病变的胆管细胞 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)。进展到终末期疾病的特征是夸大的纤维化 对慢性损伤的反应，最终导致门脉周围基质分子沉积，进展为 胆汁性肝硬变。释放旁分泌、胆管细胞衍生的因子，从而激活肝脏 星状细胞(HSC)是胆道疾病发展过程中早期的、重要的和潜在可逆的步骤 纤维化症。ZAST同源增强子2(EZH2)是一种关键的表观遗传调节因子，通过酶介导 组蛋白3(H3K27me3)上赖氨酸27的三甲基化导致转录沉默。我们的新奇系列 研究表明，在正常的胆管细胞中，HSC激活基因的转录是 被EZH2以稳态方式沉默。我们还认为p300乙酰转移酶可以导致H3K27 乙酰化(H3K27ac)启动转录。我们生成了以下新颖的初步数据：1) RNA-SEQ和CHIP-SEQ已经确定了HSC的胆管细胞衍生旁分泌激活剂的网络， 包括纤维连接蛋白(FN)，可被EZH2沉默；2)转化生长因子-β通过 蛋白酶体降解EZH2；3)胆汁性肝纤维化患者肝组织中p300蛋白积聚 和H3K27ac与Fn在门脉周围沉积；4)p300形成转录因子复合体 在转化生长因子-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的过度纤维化。在此基础上 初步数据，我们提出的中心假设是H3K27me3和H3K27ac的变化， 表观遗传学启动胆管细胞和肝星状细胞之间的纤维化串扰。在AIM I中，我们将测试 假设EZH2通过H3K27导致Fn和其他基因靶点的动态平衡沉默 甲基化，这一过程是由转化生长因子-β通过蛋白酶体降解EZH2来调节的。在AIM II中， 我们将评估关于转化生长因子-β下游的p300复合体激活Fn转录的假设。 通过H3K27乙酰化，促进旁分泌HSC活化。在AIM III中，我们将调查 EZH2和p300在H3K27上的相互调节控制胆管细胞Fn的假设 体内沉积、HSC激活和胆汁纤维化。这些研究的意义在于小说 表观遗传调节剂允许胆管细胞中特定的膜信号重写 组蛋白编码并产生致纤维化的旁分泌分子，随后促进HSC的激活。在……里面 反过来，针对这些新发现的通路的表观遗传药理学干预可能会有 预防或逆转导致胆道纤维化的细胞外基质事件的能力。

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.