Development of Polarity in Hepatic Cells

肝细胞极性的发展

• 批准号: 7110343
• 负责人: Ann L Hubbard
• 金额: $ 41.79万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7110343
• 关键词: RNA interference; biological signal transduction; cell adhesion molecules; cell growth regulation; cell population study; cellular polarity; hepatectomy; histogenesis; immunofluorescence technique; immunoprecipitation; laboratory rat; liver cells; liver regeneration; membrane activity; membrane biogenesis; membrane proteins; phosphoprotein phosphatase; protein kinase C; tight junctions

DESCRIPTION (provided by applicant): This project studies proteins ("complex 1") that participate in specifying polarity. A membrane protein, junctional adhesion molecule (JAM-1), PAR3, PAR6 (two PDZ proteins), and atypical protein kinase C (aPKC) form complex 1. The complex plays a role in assembly of tight junctions (TJ), even though it is absent from the functional structure. The hypothesis to be tested is that JAM-1 is a membrane anchor that recruits PAR3, a scaffold to which a Cdc42-GTP/PAR6 full length/aPKC sub-complex docks, thus locating the complex apical of "primordial" adherens junctions. Activated aPKC then phosphorylates proteins that help form the final TJ. Our newly- identified PAR6A-short, which cannot bind aPKC or Cdc42 but retains a semi-PDZ domain, regulates TJ formation and/or stabilizes mature TJs. In vitro models are WlF-B cells, which develop a polarized hepatic phenotype, and intestinal Caco-2 cells, which express proteins in a tetracycline-inducible manner. In vivo experiments extend in vitro results to the regenerating liver, where TJ formation is crucial to recovery of organ function. Approaches are molecular biology, morphology (eg, live cell imaging), quantitative biochemistry (eg, fractionation, in vitro binding, protein identification) and use of recombinant adenoviruses to alter/knock-down complex 1 proteins. Aim #1 tests JAM's role. In #1A, constructs mutated in the cytoplasmic or ectoplasmic (adhesive) domain of JAM are expressed in WlF-B cells to determine if/when these domains function in progression to an hepatic phenotype. Live-cell imaging of GFP-reporters provides insight into the dynamics of the process. In #1B, mutants expressed in hepatic Fao cells are used in a novel bead assay to identify the proteins JAM recruits. In #1C, mutant JAM or RNAi is expressed in livers of 2/3 hepatectomized rats to determine the effects on TJ formation. Aim #2 tests the roles of PAR6 and PAR6A-short. In #2A, PAR6A-short is induced in tet-off Caco-2 cells at various stages of maturation to distinguish a role in formation versus maintenance of TJs. A second approach is use of RNAi to silence all PAR6 or just PAR6A-short in WIF-B cells. In #2B, in vitro assays test candidates reported to bind full-length PAR6 for binding to PAR6A-short. In #2C, PAR6A-short or RNAi is expressed in vivo to determine the effects on liver regeneration. Aim #3 uses pharmacological approaches to test the roles of aPKC and protein phosphatase 2A in the development of WlF-B polarity.

描述（由申请人提供）：本项目研究参与指定极性的蛋白质（“复合物1”）。膜蛋白、连接粘附分子（JAM-1）、PAR3、PAR6（两种PDZ蛋白）和非典型蛋白激酶C （aPKC）形成复合物1。该复合体在紧密连接（TJ）的组装中起作用，尽管它在功能结构中缺失。要验证的假设是JAM-1是募集PAR3的膜锚，PAR3是Cdc42-GTP/PAR6全长/aPKC亚复合物的支架，从而定位“原始”粘附连接的复杂顶端。然后，激活的aPKC磷酸化帮助形成最终TJ的蛋白质。我们新发现的PAR6A-short不能结合aPKC或Cdc42，但保留半pdz结构域，调节TJ的形成和/或稳定成熟的TJ。体外模型是WlF-B细胞和肠道Caco-2细胞，前者具有极化的肝脏表型，后者以四环素诱导的方式表达蛋白质。体内实验将体外结果扩展到再生肝脏，其中TJ的形成对器官功能的恢复至关重要。方法包括分子生物学、形态学（如活细胞成像）、定量生物化学（如分离、体外结合、蛋白质鉴定）和利用重组腺病毒改变/敲除复合体1蛋白。目标1测试JAM的角色。在#1A中，JAM细胞质或外质（粘附）结构域突变的构建体在WlF-B细胞中表达，以确定这些结构域是否/何时在肝脏表型的进展中起作用。gfp报告的活细胞成像提供了深入了解过程的动态。在#1B中，在肝Fao细胞中表达的突变体被用于一种新的头部试验，以鉴定JAM招募的蛋白质。在#1C中，2/3的肝切除大鼠肝脏中表达突变JAM或RNAi，以确定对TJ形成的影响。Aim #2测试PAR6和PAR6A-short的作用。在#2A中，PAR6A-short在不同成熟阶段的Caco-2细胞中被诱导，以区分在TJs的形成和维持中的作用。第二种方法是使用RNAi沉默wifi - b细胞中的所有PAR6或仅par6a短链。在#2B中，体外实验测试了报道的与PAR6A-short结合的全长PAR6的候选物。在#2C中，体内表达PAR6A-short或RNAi以确定对肝脏再生的影响。Aim #3使用药理学方法来测试aPKC和蛋白磷酸酶2A在WlF-B极性发展中的作用。