The long-term goal of my laboratory is to elucidate the role of transforming growth factor-alpha (TGFalpha) and related peptides in epithelial cell biology with a particular focus on the neoplastic consequences of their dysregulation. TGFalpha is a member of a family of peptides that bind the EGF receptor (EGFR); these include EGFR); these include EGF, amphiregulin (AR), heparin-binding EGF-like growth factor (HB- EGF) and betacellulin (BTC). During the previous granting period, we have established valuable in vitro (polarized epithelial cells) and in vivo (transgenic and null mice) models to examine the actions of these growth factors. We have demonstrated that TGFalpha is delivered preferentially to the basolateral compartment of polarized epithelial cells; EGFR is restricted to this compartment and thus TGFalpha acts in an autocrine manner. In contrast, EGF has been immunolocalized to the apical compartment by laser scanning confocal microscopy (LSCM). Thus EGF could not act in an autocrine manner in this system since tight junctions prevent access of this apically sorted EGF to basolateral EGFRs in intact monolayers. We submit that the study of the action of growth factors in polarized epithelial cells offers a significant in vitro advance in that it stimulates the in vivo context in which there is spatial compartmentalization of ligands and receptors. The study of mouse models that we have developed (MMTV-TGFalpha transgenic mice and EGFR null mice) has provided important insights into the mechanisms of action of TGFalpha and the EGFR in vivo. Experiments are proposed to define the mechanism underlying basolateral sorting of TGFalpha and to examine the trafficking of EGF, AR, HB-EGF and BTC in polarized epithelial cells. Mechanistic studies of EGFR-regulated release of prostaglandins into the basolateral compartment of polarized colorectal cancer cell lines represent a novel line of investigation with important clinical implications. The biological consequences (mitogenicity, apoptosis) of cross-induction of TGFalpha family members will be examined in nontransformed rat intestinal epithelial cells. These in vitro studies will be complemented by generating mice with a targeted disruption of AR and crossing homozygous AR-deficient progeny to TGFalpha deficient mice, as well as by pursuing further studies of EGFR null mice.
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