Background. The molecular mechanisms controlling intestinal epithelial cell differentiation are poorly defined because of the difficulty of growing normal intestinal cells. We have taken advantage of the ability of the Caco-2 cell line to acquire a glandular phenotype in 3-dimensional (3-D) culture systems to investigate the role of α2 and α3 integrins in morphologic differentiation. Methods. Caco-2 cells transfected with sense or antisense DNA constructs of α2 or α3 integrins were grown in 3-D Matrigel or collagen I in the presence or absence of integrin function-blocking antibodies. We used light and confocal microscopy, BrDU incorporation, TUNEL assay, a fluorometric adhesion assay, FACS analysis, and Western blot analysis to study the effect of extracellular matrix (ECM) and integrins on morphology, polarization, proliferation, apoptosis, cell adhesion, and integrin expression. Results. Compared to collagen I, Caco-2 cells cultured in 3-D Matrigel display cytoskeletal and adherens junction rearrangements and decreased proliferation consistent with cellular differentiation. These changes, which are inhibited by α2 and α3 blocking monoclonal antibodies and α2 and α3 antisense DNA transfection, were associated with an increase in α3 integrin expression. Conclusions. We demonstrated that signaling through both constitutively expressed α2 integrin and Matrigel-induced α3 integrin expression is required to acquire a differentiated phenotype in Caco-2 cells.
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