The general hypothesis of this grant proposal is that intestinal subepithelial myofibroblasts (ISEMF) are the source of soluble factors that modulate epithelial growth, differentiation, and repair and play a pivotal role in mucosal immunophysiology and cancer. Located at the interface between the epithelium and lamina propria, ISEMF modulate information transfer between these tissue compartments. Through elaboration of basement membrane components, prostaglandins (PGs). and growth factors, ISEMF modulate processes of epithelial cell growth, differentiation, and wound repair. One major pathway by which ISEMF influence epithelial and lamina propria cells is through the production of PGs by cyclooxygenase-2 (COX-2). Data indicate that the bulk of mucosal PG synthesis occurs in the lamina propria, possibly in ISEMF. Because COX-2 regulation appears to be different in the various cell types thus far studied, this proposed research will test the hypothesis that ISEMF are major sites of COX-2 expression and that pro- and antiinflammatory agents act in part by inducing or inhibiting COX-2 expression in ISEMF.
Specific Aims : 1. Determine the mechanisms by which proinflammatory cytokines (IFN-gamma, TNF-alpha, IL-1) and eicosanoids (arachidonic acid, various HETEs) induce COX-2 gene expression in ISEMF, 2. Analyze the mechanisms responsible for inhibition of COX-2 gene expression in ISEMF by antiinflammatory cytokines (IL-4, IL-13) and therapeutic corticosteroids, and 3. Characterize COX-2 expression in human ISEMF in situ and in primary ISEMF cultures from normal small intestine and colon and from various inflammatory diseases such as ulcerative colitis, Crohn's disease, and collagenous colitis and various neoplastic states such as adenomatous and hamartomatous polyps and both sporadic and familial adenomatous polyposis colon cancers. We will use an ISEMF cell line (18Co) isolated from human colon, reporter gene constructs containing full length and mutant COX-2 regulatory elements, gel shift assays, in vitro and in vivo promoter footprinting to determine the role of NF-kappaB/cRel, cEBP/NF-IL6, CREB/ATF, and other transcription factors in the induction and inhibition of COX-2 expression in ISEMF. Key findings from studies with 18Co will be confirmed in primary cultures of ISEMF from normal and various disease states. Using immunohistocytochemistry and in situ hybridization techniques we will identify the cellular sites of COX-2 gene expression in normal and diseased tissue and thus shed light on the role of ISEMF in gastrointestinal disease. These studies should define the processes which mediate signalling between intestinal subepithelial myofibroblasts and other immune and non-immune cells, and how these processes are disrupted in pathological states such as IBD, fibrotic disorders, and cancer.
| Pinchuk, Irina V; Beswick, Ellen J; Saada, Jamal I et al. (2011) Human colonic myofibroblasts promote expansion of CD4+ CD25high Foxp3+ regulatory T cells. Gastroenterology 140:2019-30 |
| Powell, D W; Pinchuk, I V; Saada, J I et al. (2011) Mesenchymal cells of the intestinal lamina propria. Annu Rev Physiol 73:213-37 |
| Mifflin, R C; Pinchuk, I V; Saada, J I et al. (2011) Intestinal myofibroblasts: targets for stem cell therapy. Am J Physiol Gastrointest Liver Physiol 300:G684-96 |
| Pinchuk, I V; Mifflin, R C; Saada, J I et al. (2010) Intestinal mesenchymal cells. Curr Gastroenterol Rep 12:310-8 |
| Kosinski, Cynthia; Stange, Daniel E; Xu, Chuanrui et al. (2010) Indian hedgehog regulates intestinal stem cell fate through epithelial-mesenchymal interactions during development. Gastroenterology 139:893-903 |
| Mittal, Sahil; Mifflin, Randy; Powell, Don W (2009) Cancer stem cells: the other face of Janus. Am J Med Sci 338:107-12 |
| Pinchuk, Irina V; Saada, Jamal I; Beswick, Ellen J et al. (2008) PD-1 ligand expression by human colonic myofibroblasts/fibroblasts regulates CD4+ T-cell activity. Gastroenterology 135:1228-1237, 1237.e1-2 |
| Kosinski, Cynthia; Li, Vivian S W; Chan, Annie S Y et al. (2007) Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors. Proc Natl Acad Sci U S A 104:15418-23 |
| Di Mari, J F; Saada, J I; Mifflin, R C et al. (2007) HETEs enhance IL-1-mediated COX-2 expression via augmentation of message stability in human colonic myofibroblasts. Am J Physiol Gastrointest Liver Physiol 293:G719-28 |
| Pinchuk, Irina V; Beswick, Ellen J; Saada, Jamal I et al. (2007) Monocyte chemoattractant protein-1 production by intestinal myofibroblasts in response to staphylococcal enterotoxin a: relevance to staphylococcal enterotoxigenic disease. J Immunol 178:8097-106 |
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