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）：该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。该复合物在紧密连接 (TJ) 的组装中发挥作用，尽管它在功能结构中不存在。要测试的假设是 JAM-1 是招募 PAR3 的膜锚，PAR3 是 Cdc42-GTP/PAR6 全长/aPKC 亚复合物对接的支架，从而定位“原始”粘附连接的复杂顶端。激活的 aPKC 然后磷酸化有助于形成最终 TJ 的蛋白质。我们新鉴定的 PAR6A-short 不能结合 aPKC 或 Cdc42，但保留半 PDZ 结构域，调节 TJ 形成和/或稳定成熟 TJ。体外模型是WlF-B细胞和肠Caco-2细胞，WlF-B细胞产生极化肝表型，它们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 的形成对于器官功能的恢复至关重要。方法包括分子生物学、形态学（例如活细胞成像）、定量生物化学（例如分级分离、体外结合、蛋白质鉴定）以及使用重组腺病毒来改变/敲低复合物1蛋白质。目标 #1 测试 JAM 的作用。在#1A中，JAM的细胞质或胞质（粘附）结构域中突变的构建体在W1F-B细胞中表达，以确定这些结构域是否/何时在进展为肝表型的过程中发挥作用。 GFP 报告基因的活细胞成像提供了对该过程动态的深入了解。在#1B 中，在肝 Fao 细胞中表达的突变体被用于一种新型微珠测定中，以鉴定 JAM 招募的蛋白质。在#1C 中，突变型 JAM 或 RNAi 在 2/3 肝切除大鼠的肝脏中表达，以确定对 TJ 形成的影响。目标 #2 测试 PAR6 和 PAR6A-short 的作用。在 #2A 中，PAR6A-short 在不同成熟阶段的 tet-off Caco-2 细胞中被诱导，以区分 TJ 在形成与维持中的作用。第二种方法是使用 RNAi 沉默 WIF-B 细胞中的所有 PAR6 或仅沉默 PAR6A-short。在#2B 中，体外测定测试候选物报告结合全长 PAR6 以结合短 PAR6A。在#2C 中，PAR6A-short 或 RNAi 在体内表达，以确定对肝再生的影响。目标#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。