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: 7196770
• 负责人: STEVEN S WU
• 金额: $ 23.1万
• 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 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7196770
• 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身体抵御疾病至关重要。恢复或迁移以填充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非常重要。该提案重点关注细胞质酪氨酸激酶 Pyk2 的磷酸化和激活，该激酶与膜粘着斑相关，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 磷酸化具有强烈诱导性，部分依赖于 Rho 及其效应器 Rho 激酶 (ROK)。抑制 ROK 会改变 lEC 的形态并增加迁移到人工伤口的面积，从而得出 Pyk2 发挥负面作用的假设。因此，本申请的长期目标是了解 Pyk2 信号传导和 Rho/ROK 通路在 IEC 迁移中的作用，以及开发将细胞培养物（体外）中的信号传导事件与活体中的信号传导事件相关联的实验模型。动物（体内）。第一个具体目标是表征体外迁移过程中 Pyk2 与 Rho/ROK 通路之间的关系。我们将通过免疫荧光染色检查 Pyk2 的磷酸化和定位，并测量细胞迁移率，同时抑制 ROK 或改变 Pyk2 表达。第二个具体目标是在小鼠肠道损伤模型中测试 ROK 抑制对 Pyk2 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 Pyk2 激活。通过阐明参与上皮恢复的信号通路，我们将更好地理解胃肠道上皮伤口愈合的机制；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。该项目对公众健康的潜在影响在于有望开发出改善肠道损伤的疗法，通过阻止上皮伤口的发展或促进其闭合。此外，这项研究还探讨了不同类型上皮（例如粘膜、肺和皮肤）中可能保守的基本机制；因此，它可能与一系列创伤性、毒性或传染病过程中的伤口愈合相关。