Role of Cytokine Signaling in Intestinal Restitution

点击翻译按钮获取中文摘要

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.