Induction of terminal differentiation in epithelial cells requires polymerization of hensin by galectin 3

During terminal differentiation, epithelia become columnar and develop spec ialized apical membrane structures (microvilli) and functions (regulated en docytosis and exocytosis). Using a clonal intercalated epithelial cell line , we found that high seeding density induced these characteristics, whereas low density seeding maintained a protoepithelial state. When cells were pl ated at low density, but on the extracellular matrix of high density cells, they converted to the more differentiated phenotype. The extracellular mat rix (ECM) protein responsible for this activity was purified and found to b e a large 230-kD protein, which we termed hensin. High density seeding caus ed hensin to be polymerized and deposited in the extracellular matrix, and only this form of hensin was able to induce terminal differentiation. Antib odies to hensin blocked the change in phenotype. However, its purification to homogeneity re-suited in loss of activity, suggesting that an additional protein might be necessary for induction of terminal differentiation. Here , we found that a 29-kD protein specifically associates with hensin in the ECM. Addition of purified p29 restored the activity of homogenously purifie d hensin. Mass fingerprinting identified p29 as galectin 3. Purified recomb inant galectin 3 was able to bind to hensin and to polymerize it in vitro. Seeding cells at high density induced secretion of galectin 3 into the ECM where it bundled hensin. Hence, the high density state causes a secretion o f a protein that acts on another ECM protein to allow the new complex to si gnal the cell to change its phenotype. This is a new mechanism of inside-ou t signaling.