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Hippo Signaling Mediates the Development of Liver Fibrosis

Hippo 信号传导介导肝纤维化的发展

基本信息

批准号：
9588492
负责人：
DEAN YIMLAMAI
金额：
$ 15.59万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-01 至 2020-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9588492
关键词：
Ablation Binding Bioinformatics CRISPR/Cas technology Candidate Disease Gene Cell Line Cell Proliferation Cell Separation Cell division Cell surface Cells ChIP-seq Characteristics Chemicals Chronic Cicatrix Cirrhosis Complement Cues Data Development Disease Epithelial Epithelial Cells Equilibrium Failure Fibrosis Fractionation Functional disorder Gatekeeping Gene Expression Gene Expression Profiling Gene Targeting Genes Genetic Genetic Transcription Genomics Goals Growth Hepatic Hepatic Stellate Cell Hepatocyte High-Throughput RNA Sequencing Human Hypertrophy In Situ In Vitro Inflammatory Infiltrate Injury Liver Liver Fibrosis Liver Regeneration Liver parenchyma Lobe Mediating Mediator of activation protein Mesenchymal Metabolic Modeling Mus Natural regeneration Nuclear Organ Outcome Study Partial Hepatectomy Pathogenesis Pathway interactions Phenotype Phosphotransferases Population Process RNA RNA Interference Recovery Recruitment Activity Reverse Transcriptase Polymerase Chain Reaction Role Sampling Signal Pathway Signal Transduction Small Interfering RNA Staining method Stains Supporting Cell Techniques Therapeutic Trans-Activators Transcription Coactivator Validation cell type improved in vivo inhibitor/antagonist insight interest knock-down liver development liver injury loss of function overexpression programs public health relevance response single cell sequencing stem cells stem-like cell transcriptome sequencing

项目摘要

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.

描述（由申请人提供）：本提案的总体目标是研究肝脏中的Hippo信号通路及其如何促进肝纤维化的发病机制。这些研究的结果将影响我们对损伤后肝脏发育和恢复的理解。Hippo信号通路是肝脏大小、细胞增殖和细胞命运的重要调节剂。Hippo通路负调节雅普。肝细胞中高水平的雅普可将这些细胞转化为肝祖细胞（HPC）样表型。快速分裂的HPC与纤维化的发展相关，并且已知雅普直接激活与该过程相关的基因。肝硬化肝样品通常显示核雅普染色（Hippo失活的标志），并且肝星状细胞（HSC）活化与增加的雅普暂时相关。这些观察结果表明，在上皮细胞中失活Hippo信号传导可以激活促纤维化基因程序。本提案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观察Hippo信号传导是否失活。这将与上皮区室中的功能丧失研究相补充，以确定废除Hippo信号传导是否会最大限度地减少纤维化的发展。在AIM 2中，将通过细胞分级分离和使用单细胞测序技术来检查表达Yap的肝细胞，以确定是否先验的特定肝细胞“类型”对雅普表达最敏感，以及该细胞类型是否可能协调肝纤维化的发展。AIM 3的重点是确定哪些来自表达Yap的肝细胞的分泌因子可能激活HSC。这将使用RNA干扰和Cas9-Crispr技术进行，以敲低由来自过表达雅普的肝细胞的微阵列表达和ChIP-Seq数据定义的候选分子的表达。该筛选将在体外进行，然后进行体内验证。了解Hippo信号传导是否是肝纤维化发展的常见机制，可能协调这一过程的上皮细胞的特征，以及激活HSC的潜在分泌因子，将是开发治疗肝纤维化疗法的重要信息。