Molecular Mechanisms of Stellate Cell Activation in Liver Fibrosis

描述(由申请人提供)： 肝星状细胞(HSCs)是肝脏的主要间充质细胞类型，在创伤反应中起关键作用。肝星状细胞损伤后，发生肌成纤维细胞转分化(MTD)，参与瘢痕形成，如果损伤持续，则会导致肝纤维化和肝硬变。到目前为止，我们的研究表明，这种MTD是由Necdin、Wnt和Delta-like同系物(DLK1)等形态致病原的重新激活引起的。Necdin通过与典型的WNT10b启动子的Necdin位点(GN Box)结合，直接上调WNT10b。WNT10b和DLK1相互正向交叉调节，表观上抑制HSC静止基因PPAR，从而驱动HSC MTD。这种表观遗传调控是由于甲基-CpG结合蛋白MeCP2水平的增加，该蛋白与PPAR？启动子结合，招募HDAC和共抑制因子HP-1？，同时刺激EZH2的表达，从而导致H3K27在3‘外显子上的二甲基化或三甲基化，从而使基因沉默。我们的结果还表明，正则的 WNT途径通过抑制通常阻止MeCP2翻译的miR-212和miR-132来增加MeCP2蛋白。我们还证明，肝部分切除后肝再生过程中短暂激活的HSC表达DLK1，DLK1通过旁分泌方式刺激肝细胞DLK1表达，以支持早期肝脏生长。我们在急性CCl4损伤模型中的初步结果也支持了HSC-肝细胞与DLK1的相互作用的概念，该结果表明，在再生肝细胞中，最初激活的HSC诱导DLK1之后是DLK1诱导。为了扩大我们的研究范围，我们将检验假设：1)规范的Wnt途径通过下调miR-212和miR-132激活HSCs，从而导致MeCP2介导的PPAR？表观遗传抑制；2)HSC来源的DLK1是一种新的有丝分裂原，通过HSC-肝细胞串扰启动肝再生。我们将通过在HSC中表达典型的Wnt拮抗或激活(目标1a和b)下的miR-212/132模拟或抗miR-212/132来测试在Wnt介导的MeCP2诱导中miR-212/132减少的因果关系。一旦因果关系确定，我们将确定规范的Wnt途径是否或如何从表观遗传或转录上抑制miR-212/132(目标1-c和d)。为了测试DLK1的新的促有丝分裂作用，将使用急性和慢性CCl4小鼠模型，通过分离的HSC和肝细胞的免疫染色和分析来确定DLK1的时间和细胞类型的特异性表达，并测试抗DLK1抗体对肝纤维化和再生的影响(目标2-a和b)。为了确定活化的HSCs和肝细胞表达的DLK的各自作用，我们将Dlk1+(M)/Flox(P)小鼠与1(I)胶原启动子-Cre或白蛋白-Cre小鼠杂交产生的CCl4急性和慢性肝毒性作用，以确定各自的条件基因敲除对肝纤维化和再生的影响。总之，这些努力将确定MeCP2上调在HSC MTD中抑制PPAR的上游机制以及HSC衍生的DLK1在肝纤维化和再生中的作用，并为潜在的治疗靶点提供新的见解。 慢性肝病。

DESCRIPTION (provided by applicant): Hepatic stellate cells (HSCs) constitute the major mesenchymal cell type of the liver and play pivotal roles in a wound response. Upon injury, HSCs undergo myofibroblastic trans- differentiation (MTD) to participate in scar formation and cause liver fibrosis and cirrhosis if injury is sustained. Our research to date demonstrates this MTD is caused by re-activation of morphogens such as Necdin, Wnt, and delta-like homolog (DLK1). Necdin directly upregulates Wnt10b via its binding to a putative Necdin site (GN box) of the canonical Wnt10b promoter. Wnt10b and DLK1 positively cross-regulate each other and epigenetically repress the HSC quiescence gene Ppar¿ to drive HSC MTD. This epigenetic regulation is caused by increased levels of the methyl-CpG binding protein MeCP2 which binds to the Ppar¿ promoter to recruit HDAC and the co-repressor HP-1¿ while stimulating expression of EZH2 for consequent H3K27 di- or tri-methylation at the 3' exons to silence the gene. Our results also suggest that canonical Wnt pathway increases MeCP2 protein via suppression of miR-212 and miR-132 which normally block MeCP2 translation. We also demonstrate HSCs transiently activated in liver regeneration after partial hepatectomy express DLK1 which stimulates hepatocyte DLK1 expression via a paracrine manner to support early liver growth. This notion of HSC-hepatocyte crosstalk with DLK1 is also supported by our preliminary results in acute CCl4 injury model indicating initial DLK1 induction by activated HSCs is followed by DLK1 induction in regenerating hepatocytes. To extend our research, we will test the hypotheses: 1) canonical Wnt pathway activates HSCs via downregulation of miR-212 and miR-132 and consequent MeCP2-mediated Ppar¿ epigenetic repression and 2) HSC-derived DLK1 is a novel mitogen which initiates liver regeneration via HSC-hepatocyte crosstalk. We will test the causality of reduced miR-212/132 in Wnt-mediated MeCP2 induction by expressing miR-212/132 mimics or anti-miR-212/132 under canonical Wnt antagonism or activation in HSCs (Aim 1a and b). Once the causality is established, we will determine whether or how canonical Wnt pathway epigenetically or transcriptionally repress miR-212/132 (Aim 1-c and d). To test the novel mitogenic role of DLK1, acute and chronic CCl4 mouse models will be used to determine temporal and cell-type specific expression of DLK1 by immunostaining and analysis of isolated HSCs vs. hepatocytes and to test the effects of anti-DLK1 antibody administration on liver fibrosis and regeneration (Aim 2-a and b). To determine respective roles of DLK expressed by activated HSCs vs. hepatocytes, we will render acute and chronic CCl4 hepatotoxicity to compound mice generated from Dlk1+(m)/flox(p) mice crossed with ¿1(I) collagen promoter-Cre or Albumin-Cre mice to determine the effects of respective conditional knockout on liver fibrosis and regeneration. Collectively, these efforts will define upstream mechanisms of MeCP2 upregulation responsible for Ppar¿ repression in HSC MTD and the role of HSC-derived DLK1 in liver fibrosis and regeneration, and provide new insights into potential therapeutic targets for chronic liver disease.