Restoration of the mucous phenotype by retinoic acid in retinoid-deficienthuman bronchial cell cultures: Changes in mucin gene expression

Retinoid-deficient cultures of airway epithelial cells undergo squamous dif ferentiation. Treatment of such cultures with retinoic acid (RA) leads to r estoration of the mucous phenotype. The purpose of our study was to charact erize the cellular and molecular changes following RA treatment of retinoid -deficient human tracheobronchial epithelial cell cultures. Of particular i nterest was to determine when during the conversion of the squamous to the mucous phenotype the mucin genes MUC2, MUC5AC, and MUC5B were expressed. We used cornifin alpha and secreted mucin as markers to monitor the squamous and mucous phenotypes, respectively. Our studies showed that the RA respons iveness of the cultures progressively decreased with protracted retinoid de ficiency, requiring higher RA concentrations to restore the mucous phenotyp e. Within 12 h after the start of RA treatment, cornifin ct expression decr eased, signaling the beginning of a change in cellular phenotype. At 24 h a fter addition of RA to the cultures, a significant number of mucous cells a ppeared, and at 72 h mucin was secreted in measurable amounts. Induction of mucin gene expression occurred sequentially: MUC2, MUC5AC, and MUC5B mRNAs were upregulated at 24, 48, and 72 h, respectively. When cultures maintain ed in 10(-8) M RA were treated with 10(-6) M RA, MUC2 but not MUC5AC and MU C5B mRNA levels were upregulated within 6 h. Our study indicates that MUC2 mRNA is an early marker of mucous differentiation, whereas MUC5AC and MUC5B mRNAs are expressed during more advanced stages Of mucous differentiation. Our studies further suggest that each of the mucin genes is regulated by d istinct mechanisms.