Characterization of mucins from cultured normal human tracheobronchial epithelial cells

Early-passage normal human tracheobronchial epithelial (NHTBE) cells grown in air-liquid interface cultures in medium containing retinoids differentia te into a mucociliary epithelium over a 2- to 3-wk period and express incre asing mRNA levels of the airway mucin genes MUC5AC and MUC5B as the culture s age; the levels of MUC2 mRNA were very low throughout the study. Using sp ecific antibodies to MUC5AC and MUC5B mucins, we noted a gradual increase i n these two mucins in the intracellular and apically secreted pools as a fu nction of time. A low level of MUC2 mucin was detected, which did not chang e with time. The intracellular and apically secreted mucins isolated from d ay 14 and day 21 cultures by density gradient centrifugation were similar i n density to those previously isolated from human respiratory mucus secreti ons. The sedimentation rate of the apically secreted mucins indicated that they were highly oligomerized, polydisperse macromolecules similar to those previously documented from in vivo secretions. In contrast, the cell-assoc iated mucins from the cultured NHTBE cells were much smaller, possibly only monomers and dimers. Anion-exchange chromatography detected no differences in charge density between the reduced and carboxymethylated cell-associate d and secreted forms of the MUC5AC and MUC5B mucins. The MUC5AC mucin was o f similar charge density to its in vivo counterpart; however, MUC5B was mor e homogeneous than that found in vivo. Finally, evidence is presented for a n intracellular NH2-terminal cleavage of the MUC5B mucins. These studies in dicate that the mucins produced by cultured NHTBE cells are similar to thos e found in human airways, suggesting that this cell culture model is suited for studies of respiratory mucin biosynthesis, processing, and assembly.