Vectorial TGFβ signaling in polarized intestinal epithelial cells

Abstract

Polarized gastrointestinal epithelial cells form tight junctions that spatially separate apical and basolateral cell membrane domains. These domains harbor functionally distinct proteins that contribute to cellular homeostasis and morphogenesis. Transforming growth factor beta (TGFbeta) is a critical regulator of gastrointestinal epithelial cell growth and differentiation. Functional assays of vectorial TGFbeta signaling and immunofluorescence techniques were used to determine the localization of TGFbeta receptors and ligand secretion in polarizing Caco-2 cells, a colon cancer cell line. Results were compared to the nontransformed MDCK cell line. In both Caco-2 and MDCK cells, addition of TGFbeta1 to the basolateral medium resulted in phosphorylation of Smad2. No phosphorylation was observed when TGFbeta1 was added to the apical chamber, indicating that receptor signaling is localized at the basolateral membrane. In support of this, immunofluorescence and biotinylation assays show receptor localization along the basolateral membrane. Secretion of TGFbeta1 from MDCK and Caco-2 cells into the apical or basolateral medium was measured by ELISA. Interestingly, secretion was exclusively apical in the nontransformed MDCK line and basolateral in transformed Caco-2 cells. Collectively, these results show basolateral domain specificity in localization of the TGFbeta receptor signaling apparatus. These observations have important implications for understanding the biology of TGFbeta in polarized epithelia, including elements of communication between epithelial and mesenchymal layers, and will prove useful in the design of therapeutics that target TGFbeta function.