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.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Clinical Investigator Award (CIA) (K08)
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Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
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University of Iowa
Schools of Medicine
Iowa City
United States
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Murthy, S; Mathur, S; Bishop, W P et al. (1997) Inhibition of apolipoprotein B secretion by IL-6 is mediated by EGF or an EGF-like molecule in CaCo-2 cells. J Lipid Res 38:206-16
Murthy, S; Mathur, S N; Varilek, G et al. (1996) Cytokines regulate apolipoprotein B secretion by Caco-2 cells: differential effects of IL-6 and TGF-beta 1. Am J Physiol 270:G94-102
Bishop, W P; Lin, J; Stein, C A et al. (1995) Interruption of a transforming growth factor alpha autocrine loop in Caco-2 cells by antisense oligodeoxynucleotides. Gastroenterology 109:1882-9
Green, M D; Bishop, W P; Tephly, T R (1995) Expressed human UGT1.4 protein catalyzes the formation of quaternary ammonium-linked glucuronides. Drug Metab Dispos 23:299-302
Varilek, G W; Neil, G A; Bishop, W P et al. (1995) Nerve growth factor synthesis by intestinal epithelial cells. Am J Physiol 269:G445-52
Bishop, W P; Wen, J T (1994) Regulation of Caco-2 cell proliferation by basolateral membrane epidermal growth factor receptors. Am J Physiol 267:G892-900
Varilek, G W; Neil, G A; Bishop, W P (1994) Caco-2 cells express type I interleukin-1 receptors: ligand binding enhances proliferation. Am J Physiol 267:G1101-7
Mathur, S N; Born, E; Bishop, W P et al. (1993) Effect of okadaic acid on apo B and apo A-I secretion by CaCo-2 cells. Biochim Biophys Acta 1168:130-43