PERSPECTIVE
Airway Mucus Obstruction: Mucin Glycoproteins, MUC Gene Regulation and Goblet Cell Hyperplasia

Mary Callaghan Rose, Tracey J. Nickola, and Judith A. Voynow

Center for Genetic Medicine Research, Children's Research Institute and Department of Pediatrics and of Biochemistry and Molecular Biology, George Washington University Medical Center, Washington, District of Columbia; and Department of Pediatrics, Duke University Medical Center, Durham, North Carolina

The mucus layer that coats the airway epithelium provides a protective barrier against pathogenic and noxious agents and participates in the mucosal response to inflammation and infection. Airway mucus is composed of water, ions, lung secretions, serum protein transudates, and mucin glycoproteins (mucins). Mucins are the major components of mucus and the macromolecules that impart rheologic properties to airway mucus (1, 2). Airway mucus is overproduced in the upper and/or lower respiratory tracts during acute challenges and in chronic conditions (asthma, cystic fibrosis, bronchitis, and sinusitis), thereby contributing to mucus obstruction of the airways (3). Mucus obstruction is the culmination of several complex processes including mucin (MUC) gene regulation, mucin secretion and goblet cell hyperplasia (GCH) (Figure 1). Insight into each of these processes is limited; more detailed information about fundamental cellular mechanisms will be required to better understand their interrelationships. For example, pathogenic agents and inflammatory mediators initiate secretion of mucins (4) and sustain mucin production by increasing expression of mucin genes (8, 9). Nevertheless, each process has markedly different kinetics and their response to mediators likely involves different cellular signaling and pathways.

View larger version (34K): [in this window] [in a new window]   Figure 1.   Processes that impact on mucus obstruction of the airways. A normal human airway epithelium (left) exposed to pathogens or environmental agents that activate inflammatory and/or immune mediators may initially respond by mucin hypersecretion from goblet cells and submucosal glandular cells (not depicted). Mucin overproduction is maintained by increased expression of MUC genes and of glycosyltransferase genes. In chronic conditions, a hyperplastic airway epithelium (right) results, reflecting division of goblet cells, differentiation of progenitor cells and/or transdifferentiation of airway epithelial cells.

The increasing availability of molecular probes for specific mucin genes and gene products is beginning to provide some insights into the pathogenesis of mucus overproduction. However, detailed information about the expression and regulation of respiratory tract mucins in specific disease entities is still incomplete. The report by Chen and colleagues (10) provides new information on altered expression of a specific mucin gene, MUC5B.

	Mucin Glycoproteins and MUC genes

Structurally, mucins are complex glycoconjugates (Figure 2); their protein backbones and O-glycosides are products of MUC genes and of glycosyltransferase genes, respectively (see Ref. 11). Mucins share a common structural motif of tandem repeats, which are enriched in serine and/or threonine residues and are unique in sequence and size for each mucin. The 1992 Update on mucins by James Gum in AJRCMB highlighted structural information on the first five mucin genes and gene products identified (12). Information on complete coding sequences and on partial sequences later available, as well as tissue and cell-specific localization, has been summarized and reviewed for nine mucin genes (13, 14). MUC11 (15), MUC12 (15), and MUC13 (16) were more recently identified.

View larger version (21K): [in this window] [in a new window]   Figure 2.   Schematic of a secreted mucin glycoprotein. Secreted mucins typically have a central tandem repeat domain (MUC2) or tandem repeat domains interspersed with cysteine-rich domains (MUC5AC and MUC5B). They have cysteine-rich regions, homologous to von Willebrand factor D-domains, at their amino and carboxyl termini. Hundreds of O-glycosides are covalently attached to serine or threonine residues in the MUC protein backbone. Tandem repeats; threonine, serine, and proline-rich domains; unique domain;  cysteine-rich domains; vertical lines, O-glycosides.

There are two major classes of mucins, secreted and membrane-tethered. Secreted mucins are large (> 1,000 kD) and viscoelastic; they aggregate through thiol, ionic, and hydrophobic interactions with proteins as well as other mucins (see Ref. 17). Several secreted mucins (MUC2, MUC5AC, MUC5B, and MUC6), whose genes are clustered on chromosome 11p15 (18), contain domains with significant homology to von Willebrand factor D domains (Figure 2) that are sites for oligomerization. Membrane-tethered mucins (MUC1, MUC3, MUC4, MUC12, and MUC13) contain a transmembrane and a short cytoplasmic domain. Several tethered mucins (MUC3, MUC4, and MUC12) also have epidermal growth factor-like domains near their carboxyl end. Some MUC4 mucins have alternatively-spliced transcripts that lack a transmembrane domain (19) and are present in airway secretions (20). Thus, both secreted and membrane-tethered mucins may potentially contribute to mucus obstruction.

Eight MUC genes (MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC7, and MUC8 [reviewed in Ref. 14] and MUC13 [reviewed in Ref. 16]) are expressed in normal respiratory tract tissues. Other mucin genes (MUC3 and MUC6) are expressed in some lung adenocarcinomas (21). Attention has focused on MUC5AC and MUC5B, as their mRNA is well expressed in normal airway tissues and their gene products have been identified in lung mucus from patients with asthma. MUC5AC mucin, initially isolated as tracheobronchial mucin from the lung mucus of a patient with bronchial asthma (22, 23), was subsequently shown to be present in airway secretions pooled from healthy individuals (24, 25) and in lung mucus from another patient with asthma (24). MUC5AC mRNA levels are moderately increased in bronchial tissue of patients with asthma (26). Further, Muc5ac (murine MUC5AC homolog) mRNA and protein expression, as well as goblet cell metaplasia, is induced in murine models of asthma (27). Thus, MUC5AC and Muc5ac are signature genes in human asthma and in mouse models of allergic asthma. MUC5B mucins are also a major component of lung mucus from a patient with status asthmaticus (28, 29) and with chronic bronchitis (30), thus suggesting that MUC5B is also a major component of lung mucus from patients with obstructive lung diseases.

The report in this issue by Chen and coworkers (10) further elucidates the expression pattern and regulation of MUC5B and indicates that altered MUC5B expression may also occur in other airway disorders. It is well demonstrated that MUC5B mRNA is mainly expressed in the mucosal cells of submucosal glands in healthy airways of adults. In contrast, MUC5AC expression is generally restricted to surface goblet cells in the upper and lower respiratory tracts (31). In the report by Chen and colleagues (10) MUC5B mRNA is localized to the submucosal glands in four subjects with healthy airways; whereas four patients with emphysema and three patients with usual interstitial pneumonitis (UIP) express MUC5B mRNA both in their submucosal gland cells and in surface goblet cells. The UIP and emphysema patients also exhibit GCH. These findings are striking for two reasons. First, it is one of the first reports to demonstrate that a change in MUC expression by goblet cells is a sequelae of pulmonary disease, and therefore likely to affect the composition and biophysical properties of airway mucus. Studies to determine the levels of MUC5B mucin(s) in airway mucus from these patients could be quite informative. Second, it indicates that mucin gene expression may be altered in pulmonary diseases that have classically been considered disorders of the interstitium. These patients may have secondary infections (such as acute bronchitis) or inflammatory conditions (such as gastroesophageal reflux or allergen/air pollutant exposure), resulting in airway inflammation and subsequent overproduction of mucins and airway mucus obstruction.

The observed pattern of MUC5B expression in patients with emphysema and UIP mimics that of MUC5B in early fetal development. At 13 wk of gestation, MUC5B is expressed in the surface epithelium of the trachea and in subepithelial invaginations. At 23 wk, MUC5B expression is largely restricted to submucosal glands of the trachea which are morphologically well differentiated by that timeand bronchioles. Limited MUC5B expression is evident in some cells at the neck of tracheal submucosal gland ducts and in the surface epithelium of trachea and bronchioles (33). Taken together, these observations suggest that expression of MUC5B in emphysema and UIP airways reverts to an early developmental phenotype and reflects either transdifferentiation of ciliated cells to a secretory phenotype or differentiation of progenitor cells.

Interestingly, the atypical localization of MUC5B expression in patients with emphysema and UIP also occurs in normal human tracheobronchial epithelial (NHTBE) cells, which differentiate into a model system of human airway epithelium. NHTBE cells are isolated by protease digestion from dissected airways and thus likely contain mainly surface airway epithelial cells. When grown on an extracellular matrix in serum-free media and maintained at an air- liquid interface, NHTBE cultured cells manifest a mucociliary phenotype that histologically mimics the surface airway epithelium (reviewed in Ref. 34). Expression of MUC5B (35, 36), as well as MUC5AC (35), has been demonstrated by Northern and/or reverse transcriptase/polymerase chain reaction analysis in this model system. Expression of MUC5B mRNA by NHTBE cells isolated from normal and from pathologic tissues has now been demonstrated in this model system, although localization of MUC5B expression to goblet cells was not demonstrated (10). Taken together, these observations suggest that NHTBE cells in culture revert to an airway epithelium typically expressed at 13 wk of fetal development and support the concept that progenitor cells are present in adult airway epithelium (38).

	MUC Gene Regulation

The copious amounts of mucus produced during inflammatory airway diseases likely reflect increased expression of MUC genes (8). Differential regulation of mucin genes by various mediators has been reported by several laboratories, and increased mucin gene expression has been shown to occur though upregulation of MUC transcription and/or mRNA stability (reviewed in Refs. 8 and 9). These in vitro studies have, for the most part, been performed on lung cancer epithelial cell lines, which provide useful model systems for mechanistic studies when the same phenotype is demonstrated both in lung cancer cell lines and in NHTBE cultured cells. For example, neutrophil elastase increases MUC5AC expression in A549 and NHTBE cells (39); Interleukin (IL)-9 increases MUC5AC expression in NCI-H292 and NHTBE cells (40).

To date, most in vitro studies have focused on the regulation of MUC2 and MUC5AC genes by various mediators (reviewed in Refs. 8 and 9). Regulation of MUC5B expression by mediators has not been as well investigated, but several mediators (such as tumor necrosis factor-, 15-HETE, PGE₂, and PMA) that increase MUC5AC expression have no effect on MUC5B expression (41). In this study, a functional role for retinoic acid regulation of the MUC5B 5'-upstream flanking sequence is demonstrated for the first time (10). Retinoic acid is well recognized as a requirement for the differentiation of tracheobronchial epithelial cells into an airway epithelium that histologically mimics hamster (42), human (43), or guinea pig (44) airway epithelium in vivo. However, its role on mucin gene expression is less clear. Somewhat conflicting results have been reported as to which mucin genes exhibit altered expression in NHTBE cells in the presence of retinoic acid (35, 36, 45), perhaps indicating that mucin gene expression reflects subtle differences in the differentiation status of NHTBE cells. Although retinoic acid appears to increase mucin gene expression, the mechanism(s) whereby it regulates MUC5B promoter activity is not yet evident, as no RAR or RXR cis-sequences are present in the 4,169 bases of the MUC5B 5'-upstream sequence (10). Thus, it is not clear whether the increase in MUC5B reporter gene activity after exposure to retinoic acid reflects increased regulation of MUC5B and/or an increased number of MUC5B-expressing cells as the mucociliary phenotype begins to develop.

This study also provides new information on several kilobases of the 5' upstream region of MUC5B (10). The authors clarify the discrepancies in the literature about the transcriptional start site of MUC5B (46, 47), essentially confirming that proposed by Offner and associates (47) and later by Van-Seuningen and colleagues (48). They further demonstrate that the MUC5B genome has 30 exons at the 5' end of the large central exon, not 29 exons as originally suggested (46).

	Goblet Cell Hyperplasia/Metaplasia

Mucus overproduction during inflammatory and obstructive airway diseases is now recognized, at least in the case of asthma (26), as a partial consequence of airway GCH. Goblet cells are normally present in the human airway epithelium, but their differentiation pathways are not yet known. An increase in goblet cell number reflects hyperplasia (cell division) and/or metaplasia (differentiation of progenitor cells or transdifferentiation of airway epithelial cells). MUC5AC is a genetic marker for goblet cells in human airways. This report (10) adds MUC5B to the armamentarium of genetic markers for goblet cells, especially when GCH manifests. Using markers for both mucin genes will be useful to further delineate and distinguish the effect of specific mediators involved in GCH and MUC gene regulation.

The presence of goblet cells in murine airways is now recognized as a phenotypic marker in models of allergic asthma (reviewed in Ref. 49); their appearance reflects goblet cell metaplasia (GCM) as murine airways normally express few, if any, goblet cells (50). Th2 cytokines mediate GCM in murine models of asthma (see Ref. 51). Both IL13 (52, 53) and IL-9 (54) induce airway hyperresponsiveness and GCM in murine models of asthma. IL-13 (27) and IL-9 (54) also induce murine Muc5ac mRNA expression. However, it is not yet known whether these Th2 cytokines initiate airway cell differentiation to goblet cells, which then express Muc5ac as a signature gene, or whether they directly upregulate Muc5ac expression, which then contributes to or influences cell differentiation.

Th2 cytokines that result in GCM in murine airways do not necessarily increase mucin gene expression in human airway epithelial cells. Neither IL-4 nor IL-13 increases MUC5AC expression in NHTBE (55), NCI-H292, A549, or Calu-3 cells (9), although IL-9 transcriptionally upregulates MUC5AC gene expression in NCI-H292 and NHTBE cells (40). Delineating and distinguishing the effects of mediators that are typically present in inflamed or infected airways on GCM/GCH and mucin gene regulation are a prerequisite to elucidating the role of mucins in airway obstruction. Further, elucidating the mechanisms that activate GCM in murine airways should prove applicable to understanding the development and reversal of GCH in human airways.

	Future Directions

The ultimate goal for investigators studying airway mucins and mucus is to circumvent the processes that result in mucin overproduction and thus prevent mucus obstruction of the airways. Two of the three major areas that impact on the pathogenesis of airway mucus obstruction (Figure 1) have been briefly overviewed. Further elucidation of the regulation of specific airway mucin genes by relevant mediators, and identification of the mechanisms that result in GCH, are clearly warranted. However, several topics not overviewed in this article are also important to better understand the pathogenesis of mucus obstruction in human airways. These include: (i) mechanisms that regulate mucin exocytosis from secretory granules; (ii) characterization of specific mucin protein backbones and specific glycosyltransferases in airway mucus from healthy individuals and patients with airway diseases; and (iii) comparative analyses of glycosylation patterns of specific mucins with regard to their core structures or terminal glycans (sialic acid, fucose, or sulfate residues). This would address the long held belief in this field that mucins secreted by patients with airway diseases have altered O-glycosides (reviewed in Ref. 56). The next challenge in this field will be to determine the effect of specific MUC proteins and post-translational modifications on the biophysical properties of airway mucus. This relationship is the link that will tie the molecular properties of mucus to the physiologic manifestations of airway diseases.

	Footnotes

Address correspondence to: M. C. Rose, Ph.D., Rm. 5726, Center for Genetic Research, Children's National Medical Center, 111 Michigan Ave, N.W., Washington, DC 20010. E-mail: Mrose{at}cnmc.org

(Received in original form September 6, 2001).

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