Molecular Mechanisms of Stellate Cell Activation in Liver Fibrosis

描述（由申请人提供）： 肝星状细胞（hepatic stellate cells，HSC）是肝脏的主要间充质细胞类型，在创伤反应中起关键作用。在损伤时，HSC经历成肌纤维细胞转分化（MTD）以参与瘢痕形成，并且如果损伤持续则引起肝纤维化和肝硬化。我们迄今为止的研究表明，这种MTD是由形态发生素如Necdin、Wnt和δ样同源物（DLK 1）的再活化引起的。Necdin通过其与典型Wnt 10 b启动子的推定Necdin位点（GN盒）的结合直接上调Wnt 10 b。Wnt 10 b和DLK 1相互正向交叉调节，并在表观遗传学上抑制HSC静止基因Ppar β以驱动HSC MTD。这种表观遗传调控是由甲基-CpG结合蛋白MeCP 2的水平增加引起的，MeCP 2结合Ppar启动子以招募HDAC和辅助阻遏物HP-1 β，同时刺激EZH 2的表达，从而在3'外显子处进行H3 K27二甲基化或三甲基化以沉默基因。我们的研究结果还表明， Wnt途径通过抑制通常阻断MeCP 2翻译的miR-212和miR-132来增加MeCP 2蛋白。我们还证明了部分肝切除术后肝再生中短暂激活的HSC表达DLK 1，DLK 1通过旁分泌方式刺激肝细胞DLK 1表达以支持早期肝脏生长。HSC-肝细胞与DLK 1串扰的这一概念也得到了我们在急性CCl 4损伤模型中的初步结果的支持，表明活化的HSC的初始DLK 1诱导之后是再生肝细胞中的DLK 1诱导。 为了扩展我们的研究，我们将测试以下假设：1）经典Wnt途径通过下调miR-212和miR-132以及随后的MeCP 2介导的Ppark表观遗传抑制来激活HSC; 2）HSC衍生的DLK 1是一种新的有丝分裂原，其通过HSC-肝细胞串扰启动肝再生。我们将通过在HSC中典型的Wnt拮抗或激活下表达miR-212/132模拟物或抗miR-212/132来检测Wnt介导的MeCP 2诱导中miR-212/132减少的因果关系（Aim 1a和B）。一旦因果关系建立，我们将确定经典Wnt途径是否或如何表观遗传或转录抑制miR-212/132（目标1-c和d）。为了测试DLK 1的新的促有丝分裂作用，将使用急性和慢性CCl 4小鼠模型通过免疫染色和分析分离的HSC与肝细胞来确定DLK 1的时间和细胞类型特异性表达，并测试抗DLK 1抗体施用对肝纤维化和再生的影响（目的2-a和B）。为了确定活化的HSC与肝细胞表达的DLK各自的作用，我们将使急性和慢性CCl 4肝毒性作用于由Dlk 1+（m）/flox（p）小鼠与β 1（I）胶原启动子-Cre或白蛋白-Cre小鼠杂交产生的复合小鼠，以确定各自的条件性敲除对肝纤维化和再生的影响。总的来说，这些努力将确定负责HSC MTD中Ppar β抑制的MeCP 2上调的上游机制以及HSC衍生的DLK 1在肝纤维化和再生中的作用，并为肝纤维化的潜在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.