Regulation of mucous differentiation and mucin gene expression in the tracheobronchial epithelium
Introduction
The mucociliary epithelium of the conducting airways fulfills many functions that are essential for maintaining the health of the respiratory tract. One of the critical functions is to produce secretions such as mucins, which protect the airways against microbial, particulate and chemical toxins that contaminate the breathing air. Virtually all forms of airway inflammation are associated with mucus hypersecretion, which can lead to airway obstruction. An important goal of respiratory medicine is to elucidate the mechanisms involved in the regulation of mucin synthesis and secretion.
To date, 12 mucin genes, designated MUCs1–4, 5AC, 5B, 7–8 (Rose and Gendler, 1997), MUC9 (Lagow et al., 1999) and MUCs11–12, (Williams et al., 1999) have been identified. Three of these mucin genes, MUC2, MUC5AC and MUC5B, encode polymeric mucins that are expressed by airway epithelium; however, so far, only two major components of airway mucus have been identified, namely MUC5AC and MUC5B (Hovenberg et al., 1996a, Hovenberg et al., 1996b, Thornton et al., 1997, Wickstrom et al., 1998).
Vitamin A and its derivatives, so-called retinoids, have been shown to regulate growth and differentiation of tracheobronchial epithelial cells (Gray et al., 1996, Guzman et al., 1996, Koo et al., 1999, Koo et al., 2000, Yoon et al., 1997, Yoon et al., 1999). Our efforts have been focused on the elucidation of mechanisms by which retinoic acid (RA), a prototypical retinoid, regulates differentiation of airway epithelium and controls mucin gene expression.
In the studies reported here, we show that the effects of RA are modified by thyroid hormone (T3). Both RA and T3 are ligands of closely related nuclear receptors that act as transcriptional factors (Beato, 1991). We also examined the role of epidermal growth factor (EGF) in the regulation of mucin gene expression, because EGF has been shown in vivo, as well as in vitro, to effect mucous differentiation as well as mucin production (Stahlman et al., 1988, VanScott et al., 1988, St. George et al., 1991, Guzman et al., 1995, Takeyama et al., 1999).
Section snippets
Air–liquid interface cultures
Passage-2, NHTBE cells (Clonetics Corp., La Jolla, CA) were seeded onto uncoated, semipermeable Transwell clear membranes (Corning Costar, Cambridge, MA) in serum-free, hormone and growth factor-supplemented medium (all media supplements were purchased from Sigma, St. Louis, MO), containing all-trans retinoic acid (RA, Sigma). For the complete medium formulation, see Gray et al. (1996). Cultures were grown submerged for the first 7 days, at which time, the air–liquid interface was created.
Development of the mucociliary phenotype and dependence of the expression of eicosanoid and nitric oxide enzymes on the state of differentiation
To investigate factors and mechanisms involved in the regulation of epithelial differentiation, we developed the NHTBE air–liquid interface culture system using early passage, airway epithelial cells. Following cell seeding, there is a rapid growth phase in which the epithelium is composed entirely of poorly differentiated, non-polarized cells. As the cell density increases and the cultures reach confluency (∼day 10–day 14), the epithelium becomes visibly polarized with many cells containing
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