Implications of Fibroblast Growth Factor Receptor-3 Signaling in Intestinal Epithelial Cells

Brodrick, Brooks Barrett, Department of Pharmacology, University of Virginia
Cohn, Steven, Department of Pharmacology, University of Virginia

Fibroblast growth factor receptors (FGFRs) are receptor tyrosine kinases that mediate cellular growth and differentiation during normal gut development and injuryrepair. During normal intestinal development robust FGFR-3 expression is restricted to the lower portion of the crypt where stem cells reside. Mice lacking FGFR-3 develop ~50 0.000000ewer intestinal crypts and have a paucity of clonogenic stem cells. Activation of the -catenin/Tcf-4 complex is crucial for maintaining intestinal stem cells and for Paneth cell lineage allocation/differentiation. Crypt epithelial cells in FGFR-3 -/- mice show decreased levels of -catenin protein and reduced transcription of some -catenin/Tcf-4 target genes. All differentiated intestinal epithelial cell lineages are present in these mice, but there is a significant reduction in the number of Paneth cells and in the expression of Paneth cell lineage markers. Caco2 cells, a colon carcinoma derived epithelial cell line that expresses FGFR-3, sustained -catenin/Tcf-4 transcriptional activity when treated with FGF9, a FGFR-3 ligand, or co-transfected with a constituitively active mutant (K650E) of FGFR-3. Furthermore, Caco2 cells expressing K650E possessed high levels of nuclear localization of -catenin, necessary for -catenin/Tcf-4 transcriptional activation. Caco2 cells treated with FGF9 or co-transfected with K650E showed high-level expression of mRNA for human defensin 5 (HD5) and defensin 6 (HD6), two Paneth cell markers. Expression of a dominant negative Tcf4 construct or shRNA for -catenin dramatically reduced HD5 expression. Inhibitor studies indicated that activation of MEK1/2 and p38 MAPK by FGFR-3 is also required for the induction of Paneth cell markers in addition to and independent of the effect of FGFR-3 on Tcf4/catenin activity. These findings suggest that FGFR-3 signaling can directly modulate - catenin/Tcf-4 transcriptional activity and may play a role in Paneth cell specification possibly through the -catenin/Tcf-4 signaling complex. Paneth cells are ideally ii positioned adjacent to epithelial crypt stem cells where they provide essential niche signals that maintain the integrity and viability of stem cells. Our present studies strongly suggest a novel role for FGFR-3 signaling in regulating intestinal epithelial cell differentiation. Defining the mechanism(s) by which FGFR-3 signaling regulates Paneth cell differentiation will enhance our understanding of the role of Paneth cells in the stem cell niche. iii DEDICATION I would like to dedicate this to my parents, who always believed in me and encouraged me to never give up, and to my sister, who taught me you can achieve anything you work hard at. I love you.

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PHD (Doctor of Philosophy)
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