The primary goal of the proposed research is to better understand mechanisms regulating the proliferation of the intestinal epithelium with particular emphasis on the role of the epidermal growth factor (EGF) receptor and its EGF and transforming growth factor alpha (TGFalpha). EGF has been shown to stimulate mucosal proliferation when administered enterally or parenterally to laboratory animals. However, studies of cellular mechanisms governing this response are extremely difficult in vivo. In this proposal, a tissue culture model of the intestinal epithelium (Caco-2 cells) will be used to examine important aspects of EGF action on enterocytes. Specific topics of study will include 1) the apical/basolateral membrane distribution and function of EGF receptor, 2) the effect of cellular differentiation on cellular responsiveness to EGF, 3) the cellular physiology of EGF-stimulated tyrosine kinase and phosphatase activity, and 4) the possible role of TGFalpha in autoregulation of mucosal growth. Caco-2 cells are human colon carcinoma cells which different spontaneously to resemble normal enterocytes. Preliminary data show that EGF receptor is localized largely to the basolateral membrane of these cells, that basolateral administration of EGF stimulates their proliferation, and that EGF actuates both tyrosine kinase and tyrosine phosphatase activity. Other experiments suggest that autocrine secretion of EGF or TGFalpha may drive the proliferation of these cells. These findings have many potential implications for understanding regulatory processes in living animals and humans. The proposed experiments seek to expand on these observations at the cellular and epithelial level using the Caco-2 cell model. Cells will be grown on porous membranes to allow free access to apical and basolateral surfaces of the monolayer. Phosphotyrosine Western blots will be used to investigate regulation of tyrosine kinase and phosphatase activity in response to apical or basolateral EGF. The relationship of enterocytic differentiation to EGF receptor expression and function will be studied in detail, as will the possible polarized secretion of ligands for the EGF receptor into apical or basolateral media. Cell surface expression of TGFalpha precursor, a membrane-bound molecular capable of activating EGF receptor on neighboring cells, will be looked for the protein of mRNA level. We anticipate that these studies will result in significant new advances in the understanding of processes regulating mucosal proliferation.
