Cytoskeletal rescue of polarized atypical PKC in intestinal junctions under infla

描述（由申请人提供）：紧密连接的开放参与炎症性肠病（IBD）的发病机制。许多研究都集中在酪氨酸激酶在去除紧密连接成分中的作用。然而，还没有人研究非典型蛋白激酶C （aPKC）在炎症中的作用，aPKC是进化上保守的紧密连接组织者。我们的初步数据显示，暴露于促炎信号的肠上皮中aPKC急剧下降。我们还表明，这种作用是转录后的，与Hsp-70-中间丝依赖的aPKC拯救机制有关。我们假设，炎症信号破坏了在根尖结构域募集和维持非典型PKC的翻译后机制，从而导致肠上皮紧密连接通透性增加。我们建议：(1)确定Hsp70伴侣蛋白拯救错误折叠的aPKC的分子机制。结合人肠细胞的组织培养、亚细胞分离和我们实验室开发的体外重建试验，将用于分析aPKC拯救机制的组成部分。(2)表征抗aPKC活性构象丢失或降解的分子机制。Thr/Ser磷酸酶催化亚基的特异性敲除和角蛋白中间丝在通常缺乏它们的人类细胞中的表达，将用于评估保护aPKC免于失去其活性构象或增强拯救机制的可能机制。这些观察结果将在过表达角蛋白中间丝的转基因小鼠中得到验证。(3)确定下调aPKC的促炎信号及其在维持aPKC生理水平的降解和救援机制中的靶点。除了TNF-外，我们将筛选降低aPKC水平的促炎细胞因子，并分析受促炎信号影响的aPKC的激活和降解途径的步骤。同样，这些结果将在动物模型中得到验证

DESCRIPTION (provided by applicant): Opening of tight junctions is involved in the pathogenesis of inflammatory bowel disease (IBD). Many studies have focused on the role of Tyr-kinases in the removal of tight junction components. However, no one has yet studied the role of atypical Protein Kinase C (aPKC), the evolutionarily conserved organizer of tight junctions, in inflammation. Our preliminary data shows a steep decrease of aPKC in intestinal epithelia exposed to pro- inflammatory signals. We have also shown that this effect is post-trancriptional and related to an Hsp-70- intermediate filament- dependent mechansim of rescue of aPKC. We hypothesize that post-translational mechanisms that recruit and maintain atypical PKC at the apical domain are impaired by inflammatory signals, resulting in increased tight junction permeability in the intestinal epithelium. We propose to: (1) Determine the molecular mechanisms by which Hsp70 chaperones rescue misfolded aPKC otherwise targeted for degradation. A combination of tissue culture of human intestinal cells, subcellular fractionation, and an in vitro reconstitution assay developed in our laboratory will be used to analyze the components of the aPKC rescue mechanisms. (2) Characterize molecular mechanisms that can antagonize loss of aPKC active conformation or degradation. Specific knock-down of Thr/Ser phosphatase catalytic subunits and expression of keratin intermediate filaments in human cells that normally lack them will be used to assess possible mechanisms to protect aPKC from losing its active conformation or to enhance the rescue mechanism. These observations will be validated in transgenic mice overexpressing keratin intermediate filaments. (3) Identify pro-inflammatory signals that downregulate aPKC and their targets in the degradation and rescue machinery that maintains aPKC physiologic levels. We will screen for pro-inflammatory cytokines that decrease aPKC levels, aside of TNF-, and analyze the step of the activation and degradation pathways of aPKC that are affected by pro-inflammatory signaling. Again, the results will be validated in an animal model PUBLIC HEALTH RELEVANCE: IBD is a chronic invalidating disease that affects around 3 million mericans. Increased permeability across the epithelial intestinal barrier is not the cause, but it is widely accepted as an important factor to perpetuate the inflammation. The studies in this project are intended to understand the molecular basis of a novel, as yet non studied mechanism to organize and maintain the "tightness" (tight junction competence) of the epithelial barrier.