Acetaldehyde and Epithelial Junctions: Role of PP2A and Occludin Phosphorylation

性状（由申请方提供）：乙醛是乙醇消耗的毒性代谢产物。它可以破坏肠上皮细胞之间的紧密连接，使毒素进入体内。这会增加酒精性肝病和某些癌症的风险。这种破坏涉及闭合蛋白和其他紧密连接蛋白的去磷酸化。PP 2A是一种丝氨酸/苏氨酸磷酸酶，已知在调节紧密连接完整性中起作用。我的初步数据表明，乙醛破坏紧密连接和粘附连接的稳定性，并通过PP 2A依赖性机制促进上皮细胞向间充质细胞的转化。该项目的长期目标是阐明乙醇诱导结肠癌风险增加的机制。我的第一个具体目标是确定PP 2A和Thr的occludin，一种紧密连接蛋白的去磷酸化作用，在乙酰丙酮酸诱导的紧密连接破坏。这将通过检查乙酰丙酮酸诱导的破坏与跨上皮电阻，菊粉流量，和免疫荧光共聚焦显微镜的紧密连接。通过Fostriecin（一种PP 2A抑制剂）和通过siRNA敲低PP 2A表达的保护作用，将用于检查PP 2A在破坏紧密连接和通过乙醛促进细胞迁移中的作用。我的第二个具体目标是确定乙醛诱导上皮间充质转化（EMT）的PP 2A依赖性机制。我们将通过相差显微镜和免疫荧光共聚焦显微镜以及RT-PCR和Western印迹来检测乙酰丙酮酸诱导的EMT，以揭示基因转录和蛋白表达的变化。我们将使用fostriecin和2D和3D培养条件下的occludin苏氨酸突变体的产生和生长，以研究PP 2A和苏氨酸去磷酸化在乙酰丙酮诱导的EMT中的作用。 公共卫生相关性：乙醛是一种毒素，当一个人饮用酒精饮料时，结肠会产生。这种毒素打开了紧密连接，这是肠道细胞之间的屏障，可以保护身体免受结肠中物质的侵害。屏障的破坏会增加肝脏疾病，胰腺炎和癌症的风险，因此我正在研究乙醛如何破坏紧密连接蛋白之间的连接以及如何保持这些连接的完整性。

DESCRIPTION (provided by applicant): Acetaldehyde is a toxic metabolite of ethanol consumption. It can disrupt tight junctions between intestinal epithelial cells, allowing toxins into the body. This raises the risk of alcoholic liver disease and certain cancers. This disruption involves dephosphorylation of occludin and other tight junction proteins. PP2A, a serine/threonine phosphatase, is known to play a role in regulating tight junctional integrity. My preliminary data suggests that acetaldehyde destabilizes tight junctions and adherens junctions and promotes epithelial- to-mesenchymal transition by a PP2A-dependent mechanism. The long-range goal of this project is to elucidate the mechanisms involved in the ethanol-induced increase in risk for colon cancer. My first specific aim is to determine the role of PP2A and Thr-dephosphorylation of occludin, a tight junction protein, in acetaldehyde-induced disruption of tight junctions. This will be achieved by examining acetaldehyde-induced disruption of tight junctions with transepithelial resistance, inulin flux, and immunofluorescence confocal microscopy. Protection by fostriecin, a PP2A inhibitor, and by siRNA to knock down PP2A expression, will be used to examine the role of PP2A in disruption of tight junctions and in promotion of cell migration by acetaldehyde. My second specific aim is to determine that acetaldehyde induces epithelial-to-mesenchymal transition(EMT) by a PP2A-dependent mechanism. We will examine acetaldehyde-induced EMT by phase- contrast and immunofluorescence confocal microscopy as well as RT-PCR and Western blot to reveal changes in gene transcription and protein expression. We will use fostriecin and the generation and growth of occludin threonine mutants under 2D and 3D culture conditions to examine the roles of PP2A and threonine dephosphorylation in acetaldehyde-induced EMT. PUBLIC HEALTH RELEVANCE: Acetaldehyde is a toxin produced in the colon when a person drinks alcoholic beverages. This toxin opens the tight junction, a barrier between intestinal cells that protects the body from substances in the colon. Disruption of the barrier increases the risk of liver disease, pancreatitis, and cancer, so I am studying how acetaldehyde breaks connections between tight junction proteins and how to keep these connections intact.