Vectorial TGFbeta signaling in polarized intestinal epithelial cells.
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.
Yakovich
Adam J
AJ
Center for Cell and Developmental Biology, Research Institute at Nationwide Children's Hospital, Columbus, Ohio 43205, USA.
Huang
Qin
Q
Du
Jianguo
J
Jiang
Bo
B
Barnard
John A
JA
D000818:Animals; D015153:Blotting, Western; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity; D004285:Dogs; D020895:Enterocytes / Q000166:cytology* / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D062793:Occludin; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D015398:Signal Transduction; D051899:Smad2 Protein / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*; D053781:Transforming Growth Factor beta2 / Q000378:metabolism
D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D020033:Protein Isoforms; D018125:Receptors, Transforming Growth Factor beta; D051899:Smad2 Protein; D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2; D017346:Protein Serine-Threonine Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II