The intestinal epithelium is sustained by a highly-dynamic balance of cell proliferation, differentiation, and senescence. Coordination of these processes is essential to anatomic and functional integrity and reestablishing continuity of the barrier after injury by a wide variety of insults. Although a complex variety of regulatory receptors stimulated by their cognate ligands modulate the intestinal epithelium, preliminary studies indicate that members of the fibroblast growth factor family (FGF) acting through an epithelial-associated isoform of the fibroblasts growth factor receptor 3 (FGFR3 IIIb) may play a central role in regulating the intestinal epithelial stem cell compartment. The overall goal of the studies described in this proposal is the delineation of the role of FGFR3 in the regulation of intestinal epithelial proliferation. Preliminary studies have demonstrated the presence of a """"""""new"""""""" FGF, tentatively designated FGF-12, in intestinal epithelial cells with serves as the ligand for FGFR3 IIIb, and characterization of the signaling pathways has demonstrated the participation of a 66 kDa tyrosine phosphorylated protein (pp66 kDa) that distinguishes the FGFR3 signaling complex from that of other receptors. The central role of FGFR3 in the regulation of the intestinal epithelium is supported mucosal abnormalities in mice rendered FGFR3-deficient by targeted gene deletion. Therefore, efforts will focus on the role of the FGFR3 and its ligand in sustaining homeostasis of the intestinal epithelium. The goals of this proposal will be accomplished through three specific aims: 1). Isolation and characterization of FGF-12. This will be accomplished through purification from a colonic carcinoma-derived cell line (HT-29) and colonic mucosa. This will facilitate molecular cloning and expression to define its functional effects using stable transfectants expressing FGFR3 isoforms. 2). Identification of pp66 kDa and determination of its role in FGFR3 specific signal transduction. The pp66 kDa will be purified from human colonic epithelium and then molecularly cloned in order to define the nature of the pp66 kDa interaction with other components of the FGFR3 receptor complex linking it to the MAPK cascade. The functional importance will be validated by blockade of pp66 kDa in situ and assessment of effects on cell responses. 3). Determination of the functional effects of the FGFR3 in vitro and in vivo. Functional effects of stimulation of FGFR3 by the ligand FGF-12, alone and in context of other regulatory peptides on proliferation and differentiation in vitro, will be complemented by analysis of intestinal epithelial kinetics and mucosal function in FGFR3-deficient mice. Reparative responses to injury in the FGFR3-deficient mice will be compared to wild-type mice as well as lines selectively reconstituted with FGFR3 expression in the intestinal epithelium by transgenic approaches.
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| Beck, Paul L; Rosenberg, Ian M; Xavier, Ramnik J et al. (2003) Transforming growth factor-beta mediates intestinal healing and susceptibility to injury in vitro and in vivo through epithelial cells. Am J Pathol 162:597-608 |
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