Restoration of the Mucous Phenotype by Retinoic Acid in Retinoid-Deficient Human Bronchial Cell Cultures: Changes in Mucin Gene Expression

Abstract

Retinoid-deficient cultures of airway epithelial cells undergo squamous differentiation. Treatment of such cultures with retinoic acid (RA) leads to restoration of the mucous phenotype. The purpose of our study was to characterize the cellular and molecular changes following RA treatment of retinoid-deficient human tracheobronchial epithelial cell cultures. Of particular interest 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 responsiveness of the cultures progressively decreased with protracted retinoid deficiency, requiring higher RA concentrations to restore the mucous phenotype. Within 12 h after the start of RA treatment, cornifin alpha expression decreased, signaling the beginning of a change in cellular phenotype. At 24 h after addition of RA to the cultures, a significant number of mucous cells appeared, 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 maintained in 10(-8) M RA were treated with 10(-6) M RA, MUC2 but not MUC5AC and MUC5B 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 distinct mechanisms.

Epigenetic regulation (DNA methylation, histone modifications) of the 11p15 mucin genes (MUC2, MUC5AC, MUC5B, MUC6) in epithelial cancer cells

“…Therefore, from our studies it can be hypothesized that epigenetic silencing of MUC2 and MUC5B would be a specific mechanism used by cancer cells to maintain their undifferentiated state or by normal cells not yet engaged in a differentiation process. These findings have important ramifications for digestive and respiratory cancer diagnosis and prognosis and are in agreement with previous studies that showed expression of the 11p15 mucins when goblet cells reached terminal differentiation (Koo et al, 1999;Blache et al, 2004) or in differentiated tumours (Copin et al, 2001;Sylvester et al, 2001). As such, MUC2 and MUC5B may be considered as markers of differentiated mucus-secreting cells and screening for methylation of key CpG sites identified in this report may serve as a useful diagnostic/ prognostic tool to identify cancer cells undergoing dedifferentiation.…”

Regulation of Airway Mucin Gene Expression

“…Some investigators have postulated that MUC2 is not detected in expectorated mucus because its high insolubility makes it harder to assay (30). Others have also noted that MUC2 expression occurs early during goblet cell hyperplasia/metaplasia and precedes that of MUC5AC expression, suggesting MUC2 to be an early marker for mucous cell differentiation (13). Numerous inflammatory mediators can stimulate MUC2 expression in airway epithelial cells in culture.…”

“…Thus, most of the data on mucins in the respiratory tracts have focused on these two mucins. However, the other mucins are expressed at the mRNA level, both in vitro and in vivo, on airway epithelial cells (13,14). The significance of these other mucins in the contribution to airway mucus rheology and goblet/mucous cell metaplasia is not as clear as that of these two gel-forming mucins.…”

“…There are reports of MUC2 induction by RA in human cultured airway epithelial cells, and RAR-α is apparently the primary mediator of this effect (13,56). Interestingly, RA treatment of primate airway epithelial cells appears to inhibit MUC2 expression, suggesting that some species differences may exist as to RA's effect on mucin (56,57).…”

“…At approximately week 23, its expression on the surface is reduced, and its expression in the glands is more predominant (14). When primary airway epithelial cells are plated in cultures and mucociliary differentiation is initiated in airliquid interface and RA treatment, upregulation and coexpression of both MUC5AC and MUC5B occur, suggesting that cultured cells show some similarity to surface airway cells during fetal development (13). In addition, pathological examination of certain human lung diseases such as emphysema and usual interstitial pneumonia has shown that MUC5B messages can be upregulated in surface airway epithelial cells in association with mucous cell hyperplasia/metaplasia (116).…”