Pyk2 and Rho kinase in intestinal epithelial migration

描述（由申请人提供）：肠上皮的完整性对肠道功能和身体抵御疾病至关重要。恢复或迁移以填补上皮缺陷需要单个细胞的运动性，因此了解调节该过程的细胞内信号传导机制是重要的。该提案的重点是磷酸化和激活的细胞质酪氨酸激酶Pyk 2，这与膜局灶性粘连，并牵连在生长和细胞骨架重排。在肠上皮细胞（IEC）培养物中，Pyk 2磷酸化是强烈诱导的，并且部分依赖于Rho及其效应物Rho激酶（ROK）。ROK的抑制改变了IEC的形态，并增加了迁移到人工伤口的面积，导致Pyk 2起负面作用的假设。因此，本申请的长期目标是了解Pyk 2信号传导和Rho/ROK通路在IEC迁移中的作用，以及开发用于将细胞培养物（体外）中的信号传导事件与活动物（体内）中的信号传导事件相关联的实验模型。第一个具体目标是表征Pyk 2和Rho/ROK途径在体外迁移过程中的关系。我们将通过免疫荧光染色检查Pyk 2的磷酸化和定位，并测量细胞迁移率，同时抑制ROK或改变Pyk 2表达。第二个具体目标是测试ROK抑制对小鼠肠创伤模型中Pyk 2磷酸化、损伤和修复的影响。我们将化学诱导结肠炎（通过DSS），给小鼠施用ROK抑制剂，然后测量肠上皮损伤并使用磷酸特异性抗体评估Pyk 2活化。通过阐明上皮恢复中涉及的信号传导途径，我们将更好地理解GI上皮中伤口愈合的潜在机制;体内模型的开发将支持和验证体外数据，并允许测试潜在的治疗干预。该项目对公共卫生的潜在影响在于有希望通过预防上皮伤口的发展或促进其闭合来开发用于修改肠损伤的疗法。此外，这项研究解决了可能在不同类型的上皮细胞（如粘膜、肺和皮肤）中保存的基本机制;因此，它可能与一系列创伤性、毒性或感染性疾病过程中的伤口愈合有关。

DESCRIPTION (provided by applicant): The integrity of the intestinal epithelium is essential to gut function and defense of the body against disease. Restitution, or migration to fill epithelial defects, requires motility of individual cells and it is thus important to understand the intracellular signaling mechanisms which regulate this process. This proposal focuses on phosphorylation and activation of the cytoplasmic tyrosine kinase Pyk2, which associates with membrane focal adhesions and is implicated in growth and cytoskeletal rearrangement. In intestinal epithelial cell (IEC) cultures, Pyk2 phosphorylation is strongly inducible and partially dependent on Rho and its effector Rho kinase (ROK). Inhibition of ROK alters the morphology of lECs and increases the area of migration into an artificial wound, leading to the hypothesis that Pyk2 plays a negative role. Therefore, the long-term objective of this application is to understand the roles of Pyk2 signaling and the Rho/ROK pathway in IEC migration, as well as to develop experimental models for correlating signaling events in cell culture (in vitro) with those in living animals (in vivo). The first Specific Aim is to characterize the relationship between Pyk2 and the Rho/ROK pathway during in vitro migration. We will examine the phosphorylation and localization of Pyk2 via immunofluorescent staining, and measure the rate of cell migration, while inhibiting ROK or altering Pyk2 expression. The second Specific Aim is to test the effects of ROK inhibition on Pyk2 phosphorylation, injury, and repair in a mouse intestinal wounding model. We will chemically induce colitis (via DSS), administer ROK inhibitor to the mice, then measure intestinal epithelial damage and assess Pyk2 activation using phospho-specific antibodies. By elucidating the signaling pathways involved in epithelial restitution, we will better understand the mechanisms underlying wound healing in the Gl epithelium; the development of in vivo models will support and validate in vitro data, and allow testing of potential therapeutic interventions. The potential impact of this project on public health lies in the promise of developing therapies for the modification of intestinal injury, either by preventing the development of epithelial wounds or promoting their closure. Furthermore, this research addresses basic mechanisms that may be conserved across different types of epithelium, such as mucous membranes, lung, and skin; it may thus prove relevant to wound healing in a range of traumatic, toxic, or infectious disease processes.