Role of Cytokine Signaling in Intestinal Restitution

细胞因子信号传导在肠道恢复中的作用

• 批准号: 8077964
• 负责人: Narendra Kumar
• 金额: $ 12.79万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8077964
• 关键词: Actins; Biological Assay; Biological Process; Cell Culture Techniques; Cell Cycle Progression; Cell Line; Cell Proliferation; Cell Survival; Cell model; Cell-Free System; Cells; Colitis; Communication; Complex; Cytokine Signaling; Dose; Epithelial; Epithelial Cells; Figs - dietary; Gastrointestinal tract structure; Gene Delivery; Genetic Transcription; Goals; HT29 Cells; Health; Immune; In Vitro; Inflammation; Inflammatory Bowel Diseases; Inflammatory disease of the intestine; Interleukin-2; Intestinal Diseases; Intestines; JAK3 gene; Janus kinase 3; Knockout Mice; MDCK cell; Mediating; Modeling; Molecular; Mucous Membrane; Mus; Phosphorylation; Phosphotransferases; Physiological; Physiology; Play; Protein Tyrosine Kinase; Recruitment Activity; Regulation; Role; Signal Transduction Pathway; Specificity; System; Testing; Tetracyclines; Time; Tyrosine; Tyrosine Phosphorylation; Up-Regulation; Wound Healing; adapter protein; cell motility; cytokine; in vivo; mutant; receptor; src Homology Region 2 Domain; villin

DESCRIPTION (provided by applicant): The intestinal epithelial wound repair functions (Restitution) are compromised during various intestinal disorders including inflammatory bowel disease (IBD). Recently we have shown for the first time that intestinal epithelial mucosa express functional Janus kinase 3 (Jak3) a non-receptor tyrosine kinase the biological functions of which were known to be limited to immune cells ( Kumar et al. JBC 2007). Our preliminary studies show that in an intestinal epithelial cell culture model: (a) 1L-2 (a cytokine produced during intestinal inflammation) promotes mucosal wound repair in a dose dependent manner, (b) Jak3 is activated (tyrosine phosphorylated) by 1L-2, (c) Jak3 forms complex with villin and ShcA in an IL-2 dependent manner, (d) activation by IL-2 leads to tyrosine phosphorylation of villin, [sic] and ShcA in a time dependent manner, (e) IL-2 induces dose dependent up-regulation of transcription of villin and ShcA, (f) Inhibition of Jak3 activation results in loss of tyrosine phosphorylation of villin and a significant decrease in mucosal wound repair. In a cell-free system (a) the SH2 domain of Jak3 directly interacts with tyrosine phosphorylated villin, and (b) Jak3 directly phosphorylates villin in an in-vitro kinase assay. Our hypothesis is that JAK3 plays an essential role in the signal transduction pathways involved in intestinal epithelial restitution during normal physiological condition and during intestinal inflammation. To test the hypothesis the specific objectives of the present proposal are: (a) to determine in a cell free system the specificity of Jak3 interactions with villin and ShcA and its significance on the regulation of Jak3-mediated signal transduction pathways and actin remodeling, (b) to further characterize these in an intestinal cell model, using villin- and Jak3-expressing intestinal cell lines HT-29 CI-19A and Caco-2 (which duplicates the intestinal model) and villin-null and Jak3-expressing MDCK cells to over-express functional mutants of Jak3 in a tetracyclin regulated system, and (c) to determine the in-vivo significance of Jak3 activation and its interaction with villin using gene delivery for Jak3 and its functional mutants in an experiemtnal [sic] colitis model of using wild type, and Jak3 knockout mice. PUBLIC HEALTH RELEVANCE: The long-term goal of the proposed project is to understand how the communication between intestinal epithelial cells and immune cells of the gastrointestinal tract regulate intestinal epithelial functions under normal physiology and why some of these functions are compromised during various intestinal disorder such as inflammatory bowel disease.

描述（由申请人提供）： 肠上皮伤口修复功能（Restitution）在包括炎症性肠病（IBD）在内的各种肠道疾病中受到损害。最近，我们首次证明肠上皮粘膜表达功能性 Janus 激酶 3 (Jak3)，这是一种非受体酪氨酸激酶，已知其生物学功能仅限于免疫细胞 (Kumar 等人，JBC 2007)。我们的初步研究表明，在肠上皮细胞培养模型中：(a) 1L-2（肠道炎症期间产生的细胞因子）以剂量依赖性方式促进粘膜伤口修复，(b) Jak3 被 1L-2 激活（酪氨酸磷酸化），(c) Jak3 以 IL-2 依赖性方式与绒毛蛋白和 ShcA 形成复合物，(d) IL-2 激活导致 绒毛蛋白、[原文如此]和ShcA的酪氨酸磷酸化以时间依赖性方式进行，(e) IL-2诱导绒毛蛋白和ShcA转录的剂量依赖性上调，(f)抑制Jak3激活导致绒毛蛋白酪氨酸磷酸化的丧失和粘膜伤口修复的显着降低。在无细胞系统中，(a) Jak3 的 SH2 结构域直接与酪氨酸磷酸化绒毛蛋白相互作用，(b) 在体外激酶测定中，Jak3 直接磷酸化绒毛蛋白。我们的假设是，JAK3 在正常生理条件和肠道炎症期间肠上皮恢复所涉及的信号转导途径中发挥重要作用。为了检验这一假设，本提案的具体目标是：(a) 在无细胞系统中确定 Jak3 与绒毛蛋白和 ShcA 相互作用的特异性及其对 Jak3 介导的信号转导途径和肌动蛋白重塑的调节的意义，(b) 使用表达绒毛蛋白和 Jak3 的肠细胞系 HT-29 CI-19A 和 Caco-2 在肠细胞模型中进一步表征这些特征 （复制肠道模型）和绒毛无效且表达 Jak3 的 MDCK 细胞在四环素调节系统中过表达 Jak3 的功能突变体，以及（c）在使用野生型和 Jak3 的实验结肠炎模型中使用 Jak3 及其功能突变体的基因递送来确定 Jak3 激活及其与绒毛蛋白相互作用的体内意义 基因敲除小鼠。 公共健康相关性：该项目的长期目标是了解胃肠道肠上皮细胞和免疫细胞之间的通讯如何在正常生理条件下调节肠上皮功能，以及为什么其中一些功能在炎症性肠病等各种肠道疾病期间受到损害。