Role of Cytokine Signaling in Intestinal Restitution

描述(由申请人提供)： 肠上皮损伤修复功能(恢复)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，我们首次发现肠上皮细胞表达功能性Janus kinase3(JAK3)，这是一种非受体酪氨酸激酶，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(一种在肠道炎症过程中产生的细胞因子)以剂量依赖的方式促进粘膜损伤修复，(B)JAK3被1L-2激活(酪氨酸磷酸化)，(C)JAK3以IL-2依赖的方式与Villin和SHCA形成复合体，(D)IL-2激活以时间依赖的方式导致Villin、[SIC]和SHCA的酪氨酸磷酸化，(E)IL-2诱导Villin和SHCA转录依赖上调，(F)抑制JAK3的激活会导致绒毛蛋白酪氨酸磷酸化的丧失，并显著降低粘膜伤口的修复能力。在无细胞体系中，(A)JAK3的SH2结构域直接与酪氨酸磷酸化的绒毛蛋白相互作用，以及(B)JAK3在体外的激酶试验中直接磷酸化绒毛蛋白。我们的假设是，JAK3在正常生理状态和肠道炎症过程中参与肠上皮修复的信号转导通路中发挥重要作用。为了验证这一假说，本提案的具体目标是：(A)在无细胞系统中确定JAK3与Villin和SHCA相互作用的特异性及其在JAK3介导的信号转导通路和肌动蛋白重塑调节中的意义，(B)在肠道细胞模型中进一步表征这些作用，使用表达Villin和JAK3的肠道细胞系HT-29 CI-19A和Caco-2(复制肠道模型)和表达Villin和JAK3的MDCK细胞在四环素调控系统中过表达JAK3的功能突变，(C)在野生型结肠炎模型和JAK3基因敲除小鼠中，通过JAK3及其功能突变体的基因传递，确定JAK3的激活及其与绒毛蛋白的相互作用的体内意义。 与公共卫生相关：拟议项目的长期目标是了解肠道上皮细胞和胃肠道免疫细胞之间的通信在正常生理条件下如何调节肠道上皮功能，以及为什么在各种肠道疾病(如炎症性肠病)期间，这些功能会受到影响。

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.