Pyk2 and Rho kinase in intestinal epithelial migration

Pyk2 和 Rho 激酶在肠上皮迁移中的作用

• 批准号: 7477739
• 负责人: STEVEN S WU
• 金额: $ 18.87万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7477739
• 关键词: Actins; Address; Affect; Agonist; Animals; Area; Cancer Cell Growth; Cell Line; Cells; Closure; Colitis; Communicable Diseases; Cultured Cells; Cytoskeleton; DNA Sequence Rearrangement; Data; Defect; Development; Disease; Environment; Epithelial; Epithelial Cells; Epithelium; Event; Experimental Models; Focal Adhesion Kinase 1; Focal Adhesions; G-Protein-Coupled Receptors; Gastrointestinal tract structure; Growth; HA-1077; Healed; Immigration; In Vitro; Individual; Inflammatory; Injury; Interleukin-1; Intestines; Life; Lung; Measures; Membrane; Modeling; Modification; Morphology; Mucous Membrane; Mus; Pathway interactions; Phospho-Specific Antibodies; Phosphorylation; Phosphotransferases; Physiological; Play; Process; Protein Tyrosine Kinase; Proteins; Public Health; Range; Rate; Receptor Protein-Tyrosine Kinases; Relative (related person); Reporting; Research; Research Personnel; Rho-associated kinase; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Site; Skin; Staining method; Stains; TNF gene; Testing; Therapeutic Intervention; Tyrosine Phosphorylation; Wound Healing; cell motility; cell type; cytokine; fasudil; healing; in vitro Model; in vivo; in vivo Model; injury and repair; intestinal epithelium; intracellular protein transport; kinase inhibitor; migration; prevent; protein localization location; protein tyrosine kinase PYK2; repaired; response; rho; rho GTP-Binding Proteins; wound

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.

描述（由申请人提供）：肠上皮的完整性对于肠道功能和防御疾病至关重要。恢复原状或以填补上皮缺陷的迁移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很重要。该建议的重点是细胞质酪氨酸激酶Pyk2的磷酸化和激活，该酶pyk2与膜局灶性粘附相关，并与生长和细胞骨架重排有关。在肠上皮细胞（IEC）培养物中，pyk2磷酸化是可诱导的，并且部分依赖于Rho及其效应子Rho激酶（ROK）。 ROK的抑制会改变LEC的形态，并增加迁移到人工伤口的迁移区域，从而假设PYK2起负面作用。因此，该应用的长期目标是了解PYK2信号传导和IEC迁移中RHO/ROK途径的作用，以及开发用于将细胞培养（体外）中信号事件与活动物（体内）相关的实验模型。第一个具体目的是表征体外迁移期间PYK2与Rho/Rok途径之间的关系。我们将通过免疫荧光染色检查PYK2的磷酸化和定位，并测量细胞迁移速率，同时抑制ROK或改变PYK2表达。第二个具体目的是测试ROK抑制对小鼠肠损伤模型中PYK2磷酸化，损伤和修复的影响。我们将通过化学诱导结肠炎（通过DSS）对小鼠进行ROK抑制剂，然后测量肠上皮损伤，并使用磷酸化特异性抗体评估PYK2激活。通过阐明上皮恢复涉及的信号传导途径，我们将更好地了解GL上皮中伤口愈合的机制。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措施。该项目对公共卫生的潜在影响在于有望通过防止上皮伤口的发展或促进其闭合来开发肠道损伤修饰的疗法。此外，该研究介绍了可能在不同类型的上皮（例如粘膜，肺和皮肤）中保守的基本机制。因此，这可能与一系列创伤，有毒或传染病过程中的伤口愈合有关。