Hippo Signaling Mediates the Development of Liver Fibrosis

 描述(由申请人提供)：本提案的主要目标是研究肝脏中的河马信号通路及其在肝纤维化发病机制中的作用。这些研究的结果将影响我们对肝脏发育和损伤后恢复的理解。河马信号通路是肝脏大小、细胞增殖和细胞命运的重要调节因子。河马途径负向调节YAP。肝细胞中高水平的YAP可将这些细胞转化为肝祖细胞(HPC)样表型。快速分裂的HPC与纤维化的发展有关，已知YAP直接激活与这一过程相关的基因。肝硬变的肝脏样本经常显示核YAP染色(河马失活的迹象)，肝星状细胞(HSC)的激活与YAP的增加有时间上的联系。这些观察表明，灭活上皮细胞中的河马信号可以激活促纤维化基因程序。这项提议将验证和剖析这一观察的机制。在AIM 1中，将检查几种纤维化肝损伤模型，以确定Hippo信号是否被失活。这将与上皮区功能丧失研究相辅相成，以确定是否取消河马信号将最大限度地减少纤维化的发展。在AIM 2中，表达YAP的肝细胞将通过细胞分离和单细胞测序技术进行检测，以确定特定的肝细胞类型是否对YAP表达最敏感，以及这种细胞类型是否可能协调肝纤维化的发展。目的3，重点确定YAP表达的肝细胞中哪些分泌因子可能激活HSCs。这将使用RNA干扰和Cas9-Crispr技术，从YAP过度表达的肝细胞中敲除由微阵列表达和芯片序列数据定义的候选分子的表达。这一筛选将在体外进行，随后将在体内进行验证。了解Hippo信号是否是肝纤维化发生的共同机制，可能协调这一过程的上皮细胞的特征，以及潜在的激活HSCs的分泌因子，将是开发治疗肝纤维化的治疗方法的重要信息。

 DESCRIPTION (provided by applicant): The overarching goal of this proposal is to investigate the Hippo signaling pathway in the liver and how it contributes to the pathogenesis of hepatic fibrosis. The outcome of these studies will impact on our understanding of hepatic development and recovery after injury. The Hippo signaling pathway is an important regulator of liver size, cellular proliferation and cell fate. The Hippo pathway negatively regulates Yap. High levels of Yap in hepatocytes convert these cells into a hepatic progenitor cell (HPC)-like phenotype. Rapidly dividing HPCs are associated with the development of fibrosis, and it is known that Yap directly activates genes associated with this process. Cirrhotic liver samples often display nuclear Yap staining (a sign of Hippo inactivation) and hepatic stellate cell (HSC) activation is temporally associated with increasing Yap. These observations suggest that inactivating Hippo signaling in epithelial cells can activate a profibrotic gene program. This proposal will validate and dissect the mechanism of this observation. In AIM 1, several fibrogenic liver injury models will be examined to see if Hippo signaling is inactivated. This will be complemented with loss of function studies in the epithelial compartment to determine if abrogating Hippo signaling will minimize the development of fibrosis. In AIM 2, Yap-expressing hepatocytes will be examined by cellular fractionation and using single-cell sequencing techniques to determine if a priori, a particular hepatocyte "type" is most sensitive to Yap expression and if this cell-type likely orchestrates the development of liver fibrosis. AIM 3, focuses on determining which secreted factors from Yap-expressing hepatocytes likely activate HSCs. This will be performed using RNA interference and Cas9-Crispr technologies to knockdown expression of candidate molecules defined by microarray expression and ChIP-Seq data from Yap overexpressing hepatocytes. This screen will be performed in vitro followed by in vivo validation. Understanding if Hippo signaling is a common mechanism for the development of hepatic fibrosis, the characteristics of the epithelial cells that may orchestrate this process, and potential secreted factors to activate HSCs will be important information in developing therapeutics to treat liver fibrosis.