Abstract
In this review, we summarize work over the past 15 years on mucin gene expression and regulation in the lung, as well as how mucin gene expression is altered in chronic lung diseases. This field owes a great debt to Carol Basbaum for her pioneering work in dissecting signaling pathways regulating mucin gene expression and for her tremendous energy in promoting the importance of understanding the basic pathogenic mechanisms that drive mucus overproduction in cystic fibrosis, chronic obstructive pulmonary disease, and asthma.
The luminal surface of the airway is coated by mucus, a protective barrier against toxins and pathogens, which clears particles and infectious agents from the airways via mucociliary clearance, an important component of the innate immune system of the lungs (1). Mucus is composed of water, ions, proteins, lipids, and glycoproteins. Mucin glycoproteins are a major macromolecular constituent of normal mucus, comprising 40–50% by weight of dialyzed, lyophilized mucus from healthy individuals (2). In cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease (COPD), the biochemical constituents of mucus are more complex (3).
With chronic infection and/or inflammation, cells and cellular components are included in mucus, creating sputum that contains leukocytes, DNA, proteoglycans, and filamentous actin. Further complicating the sputum mixture are fragments of mucins and other glycoproteins that are digested by leukocyte and bacterial proteases. Historically, biochemical and histochemical analyses revealed that mucins are present at high levels in sputum of patients with CF (4–7), chronic bronchitis (8), and asthma (9), and are a major component of sputum. A few reports indicate that the relative concentration of mucins in sputum is decreased compared with normal mucus due to degradation of mucins or increased amounts of other proteins and proteoglycans (2, 10). Overall, studies to evaluate changes in mucins in lung disease are very limited because purifying mucin glycoproteins is technically difficult due to their viscoelastic properties and heterogeneous structures. Ascertaining primary changes in mucin levels in lung disease has been complicated by secondary effects of proteases and glycosidases in infected sputum. A significant breakthrough in the field was the cloning of mucin genes, which encode the protein backbone of mucins. These studies, starting in the late 1980s and early 1990s, permitted evaluation of mucin gene expression in disease states, unperturbed by proteolytic degradation or by the heterogeneity of sputum constituents.
MUC1 was the first mucin gene cloned and revealed significant information concerning mucin expression and molecular structure. MUC1 was cloned from breast cancer tissue (11) and from pancreatic tumor cells (12). MUC1 cDNA encodes a large domain of tandemly repeating amino acids (tandem repeat domain) rich in serine and threonine residues, which are the putative sites of O-linked glycosylation. Further determination of mucin gene sequences has established that the tandem repeat domains constitute the largest domain of the molecule and that there is no significant homology in tandem repeat sequence between different mucin genes or indeed between homologs of different species. However, there may be variability in size of a single mucin between different individuals because each allele may contain different numbers of repeats. In addition to the tandem repeat domain, MUC1 cDNA encodes a transmembrane domain. These studies demonstrated that there exists a previously unrecognized family of mucin glycoproteins that are tethered to the epithelial apical membrane. Other membrane-tethered glycoproteins that have serine/threonine-rich domains and O-glycosylation are known, but their lack of tandem repeat domains prevent them from being considered as mucin glycoproteins.
The first secreted mucin gene identified, MUC2, was originally cloned from intestinal epithelium (13). Secreted mucins likewise contain the signature feature of mucins, the large tandem repeat domain of amino acids rich in serine and threonine where O-linked glycans are anchored. However, instead of a transmembrane domain, they contain several characteristic modules at their amino and carboxyl termini, including domains with homology to the D domains of the Von Willebrand factor. The D domains are believed to be functionally important for oligomerization of mucin monomers likely affecting mucin biophysical properties. Detailed descriptions of the modular motifs of mucins can be found in two recent reviews (14, 15).
Although the first mucin genes were not cloned from respiratory tract tissues, Craig Gerard and coworkers (16) and Carol Basbaum and colleagues (17) were the first to demonstrate that epithelial mucins are also expressed in the lung by demonstrating that MUC2 mRNA is expressed in the respiratory tract. This was followed by detection of MUC1 mRNA in the respiratory tract (18). Further advancing the field of airway mucin biology were the isolation and characterization of mucin cDNA clones from nasal and airway tissues. These genes, MUC5AC (19) and MUC5B (20), are localized adjacent to each other on chromosome 11p15.5 and are recognized to be two major secreted mucin genes expressed in the respiratory tract.
To date, 18 different human mucin genes have been cloned; at least 12 are expressed as mRNA in the lower respiratory tract (reviewed in Ref. 21). In the healthy lung, MUC1 and MUC4 are expressed at the apical surface of the respiratory epithelium. MUC5AC and MUC2 tend to be expressed in goblet cells of the superficial airway epithelium, while MUC5B, MUC8, and MUC19 are expressed in mucosal cells of submucosal glands. MUC7, a small, secreted, non–cysteine-rich mucin and a subclass of mucin unto itself, is not well-expressed normally and when expressed is localized to serosal cells of submucosal glands. The other mucins shown to be expressed as mRNA in lung tissue include MUC11, MUC13, MUC15, and MUC20; the localization of these genes within different compartments or cells in the lung is not yet known. The availability of mucin gene cDNA sequences has revolutionized the field of mucin biology permitting the development of cDNA probes and antibodies specific to cognate sequences in mucin protein backbones. These tools have been critical in the evaluation of cell-specific expression of mucin genes and proteins, and have enabled the evaluation of mucin expression in healthy lung and in airway diseases.
A major insight/contribution, perhaps initially promulgated by Professor Lynne Reid subsequent to her extensive histopathologic studies of lung tissues (22) and subsequently championed by Carol Basbaum, is that inflammation activated mucin gene expression (23). This concept has had wide-reaching ramifications, as it focused attention on the importance of mucin gene regulation in airway diseases. Importantly, Basbaum and colleagues first brought attention to the role of bacterial products, which are prevalent in CF airways, in regulating mucin expression in epithelial cells. This work further promoted the emerging concept that mucin glycoproteins are an important part of the innate immune defense system in the lung.
A key development that propelled this field has been the cloning of promoter sequences for several respiratory tract mucin genes including MUC1 (24), MUC2 (25, 26), MUC5AC (27), MUC5B (28, 29), and MUC4 (30, 31). Promoter sequences used in reporter chimera constructs have been an extremely useful tool to evaluate gene regulation and intracellular signaling in epithelial cells in vitro. This approach has resulted in a large body of literature defining the regulation of mucin genes in epithelial cells in vitro (reviewed in Ref. 21). Seminal studies, which established the current paradigm of mucin gene regulatory pathways, were initially performed using the MUC2 promoter (32), and more recently confirmed for MUC5AC. MUC2 and MUC5AC have several common active cis-elements in their promoters, including NF-κB, Sp-1, and CREB, suggesting that they share common regulatory pathways.
Mucus obstruction of airways and chronic inflammation are both fundamental features of CF, COPD, and asthma. Disease exacerbations with increased mucus production are also associated with bacterial or viral infections. These clinical observations suggest a link between infection, inflammation, and mucus production. Over the past 15 years, investigators have provided evidence establishing that infectious and inflammatory mediators interact with host epithelial cells and activate intracellular signaling pathways resulting in selective regulation of specific mucin genes. The studies on human mucins have been performed in vitro in lung cancer cell lines or in cultured primary airway epithelial cells to evaluate the effect of single agents and to permit dissection of the activated signaling pathways. Primarily, MUC gene expression has been evaluated and very few reports have correlated the increased mRNA findings with increased specific MUC protein expression. As discussed above, mucin glycoproteins are notoriously difficult to isolate, identify, and quantitate. Even with the development of antibodies for specific mucin proteins, it has been difficult to correlate gene regulatory studies with demonstration of increased protein production. A major conundrum that persists today is that despite evidence that MUC2 gene expression is upregulated in CF airways (33), it has been difficult to demonstrate the presence of MUC2 glycoprotein in normal mucus (34) or CF sputum (35). This contrasts with several reports that MUC5AC and MUC5B are major mucin glycoproteins in sputum from patients with asthma (36, 37), CF (35), and COPD (8). Bacteria relevant to different chronic and acute airway diseases have been evaluated for their effect on mucin regulation. Pseudomonas aeruginosa LPS (33), P. aeruginosa flagellin (38), Staphylococcus aureus lipoteichoic acid (39), Bordetella pertussis (40), and Haemophilus influenza (41) all transcriptionally upregulate MUC2 gene expression. P. aeruginosa LPS (27), H. influenza (42), and Mycobacterium pneumoniae (43) have also been reported to upregulate MUC5AC expression. In addition to bacteria, viral dsRNA also upregulates MUC2 expression (44).
Several cytokines and chemokines relevant to asthma, CF, and COPD also regulate mucin gene expression. TNF-α (45–48), IL-1β (47, 48), and IL-9 (49, 50) upregulate both MUC2 and MUC5AC mRNA expression, whereas IL-6 and IL-17 increase expression of both MUC5AC and MUC5B (51). Interestingly, this is one of the few reports of mediators regulating MUC5B expression, which is particularly surprising considering the large amount of MUC5B mucin protein detected in mucus and sputum.
Other major airway inflammatory mediators also increase MUC5AC expression in vitro, including prostaglandins (46); proteases such as neutrophil elastase (52, 53), human airway trypsin (54), tissue kallikrein (55), metalloproteinases (56); and reactive oxygen species (55, 57, 58). Toxic agents that induce airway inflammation and oxidant stress, including tobacco smoke and pollutants (46, 59–61), also increase MUC5AC expression.
The diverse array of infectious and inflammatory factors described above mediate mucin gene transcription by activating cell surface receptors and signaling via a limited repertoire of intracellular pathways. The initial step in the process requires the activation of cell surface receptors specific for each stimulus—for example, Toll-like receptor 2 for H. influenzae (41), (42), platelet activating factor receptor for S. aureus lipoteichoic acid (39), asialoGM1 for P. aeruginosa flagellin (62), and P2Y2 receptor for dsRNA (44). Activation of the retinoic acid receptor α (47) and the epidermal growth factor receptor (53, 63–65) have been implicated in mucin gene regulation following several different stimuli. The cell surface receptors signal via downstream pathways, usually resulting in direct or indirect activation of the mitogen-activating protein kinase system. The ultimate step leading to MUC2 or MUC5AC upregulation is the activation of several transcription factors including NF-κB, AP-1, Sp-1, or CREB (reviewed in Ref. 21).
Although most published reports on MUC gene regulation have examined transcriptional mechanisms of regulation, there is a growing body of literature providing evidence that post-transcriptional mechanisms of regulation are important in mediating MUC mRNA abundance. TNF-α (46), neutrophil elastase (52, 66) and IL-8 (67) increase MUC5AC mRNA stability. MUC2 mRNA is also increased by post-transcriptional mechanisms after epithelial exposure to 12-O-tetradecanoylphorbol-13-acetate or forskolin (68). MUC4 is post-translationally regulated by TGF-β in rat mammary epithelial cells (69) and post-transcriptionally regulated by neutrophil elastase in primary normal human bronchial epithelial cells (70). Mechanisms whereby mucin mRNA is stabilized are not yet known; future studies are required to determine the cis stability domains in the mRNA sequence, intracellular signals required for RNA-binding protein–mRNA interactions, and the identity of the RNA binding proteins required for mRNA stabilization.
Few studies on the downregulation of mucin gene expression have been reported, although clearly repression of mucin production in chronic airway diseases by pharmacologic agents or anti-inflammatory mediators would be clinically useful. The glucocorticoid, dexamethasone (Dex), decreases the abundance of MUC5AC mRNA in airway epithelial cell lines (71–73), primary differentiated NHBE cells (73), and rat primary differentiated and undifferentiated airway epithelial cells (72). Recent data have demonstrated that the Dex-induced repression of MUC5AC mRNA abundance in airway epithelial cell lines is regulated at the transcriptional, not the post-transcriptional level, and that the ligand-activated glucocorticoid receptor binds to two GRE cis-sites in the MUC5AC promoter (73).
However, it is not yet established whether repression of mucin genes results in concomitant decrease of mucin production in vivo or in vitro. An inhaled glucorticoid reduced CA19–9, a mucin marker in the bronchoalveolar lavage of patients with CF (74). Dex represses mucin synthesis in airway epithelial cell lines (71), but repression of Muc5ac mRNA does not result in decreased production of Muc5ac mucin in differentiated rat airway epithelial cells (72). This has not yet been evaluated in primary differentiated NHBE cells. Identifying mechanisms that effect downregulation of mucin gene expression and mucin production will be important to better understand processes whereby mucus overproduction can be reversed and mucus obstruction averted or overcome.
Mouse models have been extremely useful to test hypotheses of asthma or COPD pathogenesis. Although mouse models are also used to assess mechanisms of mucin gene regulation, the resulting phenotype is far more complex because the stimuli tested typically induce goblet cell metaplasia, thereby resulting in increased MUC gene and protein expression (reviewed in Ref. 20). This is an important point, because the regulatory factors required for goblet cell metaplasia may be different than those required to upregulate MUC expression.
There are several features of murine airways that are different from human airways, which must be considered when interpreting in vivo data on mucin gene regulation. The major secretory cell in murine airways is the Clara cell (75); there are very few resident goblet cells in normal mice. Mice phenotypes vary dramatically by their background strain (76), therefore characteristics ascribed to transgenes or loss of gene function must be evaluated in light of their background. Finally, it has been very challenging to establish murine models of CF lung disease using CFTR-null or CFTR-mutant mice, as the mice do not develop airway disease spontaneously (77). The failure of “CF” mice to develop chronic bronchitis and increased mucus production probably is due to the presence of alternative ion channels in murine airways that compensate for loss of CFTR function (78). Despite these caveats, the use of murine models to study airway diseases gained tremendous momentum with the recognition that Th2 cytokine exposure or allergen sensitization recapitulated many features in mouse airways seen in asthmatic airways, including goblet cell metaplasia. Both cDNA probes and MUC-specific antibodies are available for murine Muc5ac, and many reports have established that Muc5ac expression, not normally present in mouse airways, correlates directly with Alcian blue–periodic acid Schiff staining, the histochemical marker for goblet cells (79, 80).
To date, many mediators that regulate human MUC5AC mRNA expression in vitro may also increase airway goblet cell number and Muc5ac expression in vivo. The observed increase in Muc5ac mRNA or protein expression likely reflects the increased number of goblet cells in mouse airways. Mediators that induce goblet cell metaplasia include cigarette smoke (81, 82), acrolein (83), neutrophil elastase (84), IL-1β (85), IL-6/ IL-17 (86), IL-9 (87), TNF-α (88), LPS (89), M. pneumoniae infection (43), and UTP (90). To confirm that these mediators regulate murine Muc5ac mRNA expression, mRNA steady-state equilibrium and promoter-reporter functional experiments need to be done in vitro with mouse airway epithelial cells. An exception to these reports is the contrasting effect of the Th2 cytokines, IL-13 and IL-4, on mouse lung in vivo versus exposure to human airway epithelial cells in vitro. IL-13 and IL-4 do not increase human MUC5AC mRNA expression (86), although IL-13 (91), and IL-4 (92) induce goblet cell metaplasia in vivo, thereby resulting in an increase in Muc5ac expression. This exception illustrates the principle that signaling pathways that regulate goblet cell metaplasia may be different than the signaling pathways required for MUC gene regulation, and may vary between mice and humans.
The concept that normal mucus has important innate immune properties is becoming an accepted paradigm. However, there is still limited information on the functions attributable directly to the mucin glycoproteins, particularly the secreted mucins. The tremendous progress in understanding transcriptional mechanisms of mucin gene regulation still needs to be extended to the areas of in vivo regulation of Muc expression and of post-transcriptional mechanisms of MUC/Muc gene regulation. Finally, the promise of mucin research is the opportunity to discover new therapies to reduce mucin production and goblet cell hyperplasia. This hope has not yet been actualized and is the driving objective of mucin research for many investigators following the vision of Carol Basbaum.
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