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Cellular and Molecular Biology of Airway Mucins
Abstract
Airway mucus constitutes a thin layer of airway surface liquid with component macromolecules that covers the luminal surface of the respiratory tract. The major function of mucus is to protect the lungs through mucociliary clearance of inhaled foreign particles and noxious chemicals. Mucus is comprised of water, ions, mucin glycoproteins, and a variety of other macromolecules, some of which possess anti-microbial, anti-protease, and anti-oxidant activities. Mucins comprise the major protein component of mucus and exist as secreted and cell-associated glycoproteins. Secreted, gel-forming mucins are mainly responsible for the viscoelastic property of mucus, which is crucial for effective mucociliary clearance. Cell-associated mucins shield the epithelial surface from pathogens through their extracellular domains and regulate intracellular signaling through their cytoplasmic regions. However, neither the exact structures of mucin glycoproteins, nor the manner through which their expression is regulated, are completely understood. This chapter reviews what is currently known about the cellular and molecular properties of airway mucins.
1. INTRODUCTION
1.1. Airway Mucus Research—Historical View
The importance of mucus in the clearance of inhaled particles from the airways has been recognized for nearly 50 years (Adler and Li, 2001). Beginning in the 1960s, several important descriptive studies utilized the alcian blue/PAS stain to identify mucosubstances in different regions of healthy and diseased airways (Lamb and Reid, 1969). By the 1970s, more mechanistic studies of mucus production were performed using bronchial or tracheal organ culture techniques (Ellis and Stahl, 1973; Boat et al., 1974). At this time, classical particle clearance physiologic techniques were used to investigate the flow of mucus over airway surfaces. While it was clear that what we now refer to a mucociliary clearance through the combined effects of mucus secretion and ciliary beating were of paramount importance for the self-cleansing property of the airways, it was also recognized that the factors, which normally control this mechanism, were largely unknown (Randell and Boucher, 2006). During the first half of the 1980s, improved in vitro airway cell culture techniques, and advancements in detection and quantification of glycoproteins in mucus, were developed and applied to study the regulation of mucus secretion (Adler et al., 1981; Cheng et al., 1981; Wu and Smith, 1982; Adler et al., 1987; Whitcutt et al., 1988). Beginning in 1985, our laboratory began a systematic investigation of the cellular and molecular properties of the major glycoprotein component of airway mucus, the mucins. Among others, our overarching goals were (1) to elucidate the mechanisms of mucin production in the airway, (2) to investigate the structure–function relationship of lung mucin glycoproteins, and (3) to identify the role of mucins in the airway response to harmful agents, particularly the role of MUC1 mucin during exposure to infectious pathogens.
1.2. Role of Mucus in Airway Health and Disease
Mucus is a viscous, gel-like material consisting of various macromolecules, inorganic salts, and water. Mucus is produced by mucous cells found in the surface epithelium (e.g. goblet cells), mucous glands, and mixed glands containing both serous and mucous cells, of the respiratory, gastrointestinal, urogenital, and visual and auditory systems in mammals. Airways mucus, sometimes referred to as the airway surface liquid, serves as the first line of defense against harmful inhaled particles (Adler and Li, 2001; Lillehoj and Kim, 2002).The characteristic gel-like property of mucus is believed to be attributable mainly to the presence of high molecular weight, polydisperse glycoproteins, or mucins. Airway mucins are produced by goblet cells of surface epithelia and mucous cells of submucosal glands. Both the quality and quantity of mucin production determines the viscoelastic property of mucus, which is critical for efficient mucociliary clearance. Viscoelasticity refers to the combined viscous (resistance to flow) and elastic (returning to original shape) characteristics of mucus.
1.2.1. Two-layer Model of Mucociliary Clearance
The current bipartite model of the airway surface liquid proposes that mucociliary clearance is mediated through two distinct, yet interacting layers, a high-viscosity gel (mucus) layer that overlies a low-viscosity sol, or periciliary liquid layer (PCL) (Knowles and Boucher, 2002; Livraghi and Randell, 2007; Fahy and Dickey, 2010). Mucin glycoproteins containing terminal cysteine-rich domains form intermolecular disulfide bonds resulting in polymers that impart a mesh-like property on the gel layer. The gel layer with its trapped foreign particles is propelled out of the airways by the rapid and coordinated action of cilia beating within the PCL. The PCL extends to the height of the cilia and provides a relatively low resistance solution conducive to cilia beating. A recently postulated gel-on-brush model further refines this mechanism by proposing that membrane-tethered mucins and other high molecular weight glycoconjugates within the PCL prevent mucins in the gel layer from penetrating the interciliary space, thereby maintaining its low-viscosity state and stabilizing mucociliary transport (Button et al., 2012). All components of the ciliary clearance system, including mucus, the PCL, and cilia, are critically important for its normal operation, and defects in any one element may lead to severe airway dysfunction and disease.
1.2.2. Regulation of Mucus Production
Regulation of mucus production is essential for normal lung function. Mucus overproduction contributes to the morbidity and mortality of airways diseases, among the most important being chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), asthma, and chronic bronchitis (Rose and Voynow, 2006; Rogers, 2007). Infectious agents and host inflammatory mediators activate mucin gene expression in many of these chronic lung diseases. Microbial pathogens and host response molecules also drive airway remodeling through goblet cell hyperplasia (GCH), which refers to increased goblet cell numbers and goblet cell metaplasia (GCM), the reversible differentiation of non-goblet airway epithelial cells into goblet cells. Both pathological processes sustain airway mucin overproduction and contribute to airway obstruction by mucus. However, it is important to note that although they may be pathologically linked, GCH and GCM arise from distinct cellular and molecular pathways that may or may not be related to mucin overproduction. The precise mechanisms that regulate GCH and GCM are unknown. Effective treatment of airway diseases resulting from mucus overproduction will only be achieved once the complexity of these pathways and processes are completely understood.
1.2.3. Non-mucin Proteins of Mucus
Because the conventional view of the role of airway mucus relates to mucociliary clearance, the initial focus of airway mucus research was focused on the regulation of expression of mucin glycoproteins. Based on their anatomical location in the airways, as well as their relatively complex glycoprotein structure, it was suggested early in the history of mucus research that mucins possess multifaceted properties that are necessary for host defense against inhaled harmful substances, including antimicrobial, antiprotease, and anti-oxidant activities (Jacquot et al., 1992).These properties are related, in part, to the ability of mucin glycoproteins to non-covalently associate with other macromolecules present in mucus. For example, Kim et al. (1989, 1996) demonstrated that not only were airway mucins extremely hydrophobic in character, but also were tightly associated with other molecules present in airway secretion, and that these associations were resistant to the chaotropic effects of 4–6 M guanidine hydrochloride. Given that the biophysical dimensions of mucin glycoproteins exceed the average diameter of mucin secretory granules, it has been questioned how these highly hydrophilic macromolecules are organized within these organelles (Perez-Vilar, 2007). It was postulated that the hydrophobic property of mucins is necessary for efficient packaging of the large mucin molecules (>106 Da) within secretory granules (Kim, 1991a). Recent proteomics analysis of airway mucus secretions has revealed that mucins are intimately associated with other proteins that possess antimicrobial, antiprotease, antioxidant, and anti-inflammatory properties (Kesimer et al., 2009; Ali et al., 2011). Thus, mucins resemble a large “aircraft carrier” bearing a variety of “weapons” to be used against invading pathogens (Kim, 2012). It has been suggested that the association of mucins with these bioactive molecules occurs within secretory granules prior to exocytosis, such that the latter can interact with invading pathogens more effectively upon exocytosis (Kim and Singh, 1990a, 1990b). How and when such associations take place inside the goblet cell, and the manner through which the associated molecules are packaged into mucous granules, remain to be discovered.
1.2.4. Major Mucins of the Airways—MUC1, MUC4, MUC5AC, MUC5B, MUC16
The mucin family of glycoproteins is classified into those that are secreted by epithelial cells and form the mucus gel and those that are embedded in the epithelial cell membrane. By convention, all mucin glycoproteins are designated MUC in humans and Muc in animals, followed by an Arabic numeral indicating their order of discovery. Mucin genes are correspondingly designated as MUC and Muc, respectively. Five major mucins are expressed in the airways: MUC1, MUC4, MUC5AC, MUC5B, and MUC16 (Table 4.1). A review by Sheehan et al.(2006) described the roles of airway mucins in protecting and stabilizing the ciliated surface, and in assembling the mucous gel ovelaying the airway epithelium. Other reviews that may be consulted for additional information on airway mucins genes and their encoded proteins include Gendler (2001), Gendler and Spicer (1995), Hattrup and Gendler (2008), Kim and Lillehoj (2008), Lillehoj and Kim (2002), Rose (1992), Rose and Voynow (2006),Thornton et al. (2008), Turner and Jones (2009),Voynow et al. (2006), and Voynow and Rubin (2009). Although focused exclusively on intestinal mucins, a recent review by McGuckin et al.(2011) on the interaction of mucins with intestinal pathogens facilitates a better understanding of the role of mucins during respiratory tract infection. In this review, we will discuss extensively on the role of MUC1 mucin during airway infection in Section 5. More immediately, we will briefly summarize the roles of mucus and mucins in major human airway diseases characterized by mucus overproduction.
Table 4.1
Comparision of the major airway mucins
| Mucin | Secreted | Membrane | Chromosome* | AA per repeat** |
|---|---|---|---|---|
| MUC1 | − | + | 1q21 | 20 |
| MUC4 | − | + | 3q29 | 16 |
| MUC5AC | + | − | 11p15.5 | 5/8 |
| MUC5B | + | − | 11p15.5 | 29 |
| MUC16 | − | + | 19p13.3 | 156 |
2. MUCUS, MUCINS, AND COPD
2.1. Role of Mucins in the Airways
2.1.1. General Functions of Mucins
Normally, mucus acts like a raft floating above the ciliated epithelia, capturing potentially harmful microbes, inhaled particles, inflammatory cells, and cell debris as a result of its gel-like structure and adhesive property, and transporting the trapped substances out of the airways via the aid of ciliary beating (Knowles and Boucher, 2002).The mucous layer may also provide a physical barrier over the epithelium protecting it against microorganisms and insoluble material. In this regard, mucus functions to maintain the local molecular environment with respect to proper hydration, ionic composition, and the concentration and accessibility of other macromolecules. In essence, mucus constitutes the first line of innate defense of the respiratory tract against potentially injurious substances. These essential functions of mucus (mucociliary clearance and barrier function) are primarily conferred by the mucin glycoproteins, particularly MUC5AC and MUC5B, the major gel-forming mucins in the airways.
The other important functions of gel-forming mucins are the capture, retention, and release of biologically active molecules (Cebo et al., 2001). Among the molecules that mucins have been shown to be reversibly associated with are cytokines, growth factors, and trefoil factors (TFFs). These association/dissociation properties with accessory proteins may allow mucins to regulate inflammation and immune responses, and to influence postinjury epithelial repair. For example, mucins directly interact with interleukins, such as IL-1, IL-4, IL-6, and IL-7, as well as indirectly through interaction with specific lectins that are associated with these molecules. Because mucins also bind to pathogens, these intermolecular interactions may allow mucins to serve as bridges between inflammatory mediators and microorganisms, thereby facilitating the resolution of inflammation.
TFFs are relatively small polypeptides that are expressed by most mucin-producing epithelia, including those of the respiratory tract. TFFs bind to mucins (Kindon et al., 1995; Tomasetto et al., 2000), and regulate mucous viscosity (Thim et al., 2002). By way of these actions, TFFs may enhance the protective capabilities of the airway mucosal defensive barrier. Studies of trefoil peptides in gastrointestinal epithelial cells found that TFFs enhance cell migration in vitro, and promote epithelial restitution and mucosal repair in vivo (Podolsky, 1997; Wong et al., 1999). In a murine model of asthma, trans-differentiating airway Clara cells specifically expressed TFF1, which was stored in a distinct subset of secretory granules (Kouznetsova et al., 2007). Royce et al. (2011) reported that TFF2 regulates airway remodeling in animals models of asthma, and that TFF2-deficient mice with symptoms of asthma had increased GCH and subepithelial collagen thickness. Lung transcript profiling in mice identified TFF2 as a candidate gene whose gene product regulated the lung function (Ganguly et al., 2007). Oertel et al. (2001) demonstrated that human recombinant TFF2 and TFF3 stimulated the migration of human airway epithelial cells in chemotactic and two-dimensional wound repair assays, either alone or in concert with the epidermal growth factor (EGF). TFF3 facilitated airway epithelial ciliated cell differentiation, and its expression was associated with differentiation of humanized tracheal xenografts in vivo and air–liquid interface cell culture models in vitro. Further, exogenous TFF3 promoted the differentiation of respiratory ciliated cells in an EGF receptor-dependent manner (LeSimple et al., 2007). Wiede et al. (1999) demonstrated the presence of TFF3, but not TFF1 or TFF2, in airway mucosa and in the sputum of subjects with chronic bronchitis. Reduction in lung TFF3 mRNA expression was found in a rat model of COPD induced by passive smoking plus intratracheal administration of LPS (Li et al., 2013). Notwithstanding these previous studies, the role of TFFs in normal airway and in the repair process of injured COPD epithelium is essentially unknown and requires further investigation.
With respect to the membrane-associated mucins, MUC1, MUC4, and MUC16, their role involves the activation of intracellular signal transduction pathways, control of inflammation and immune responses to infectious agents, and regulation of cell differentiation and proliferation (Hollingsworth and Swanson, 2004). The role of MUC1 in intracellular signaling is discussed below in greater detail (Section 5). MUC4 is proposed to play a protective role in airway and other epithelia (Carraway et al., 2009). Through its extracellular EGF-like domain, MUC4 interacts with the receptor tyrosine kinase, ErbB2, and controls ErbB2 and ErbB3 tyrosine phosphorylation. MUC4 can also modulate cell apoptosis, regulate cell–cell adhesion, and serve as tumor marker or target for cancer therapy. MUC16, or cancer antigen-125 (CA-125), has also found application as a tumor marker that may be elevated in some patients with specific types of cancers (Bast et al., 1998). Its intracellular region contains a polybasic amino acid sequence (RRRKK) that interacts with the ezrin/radixin/moesin (ERM) family of proteins (Blalock et al., 2007), including the Janus kinase 2 ( JAK2) (Lakshmanan et al., 2012).
The functional description of mucins in mediating signal transduction and regulating cell differentiation and proliferation are primarily derived from studies in cancer cells, with limited descriptions for individual mucins from airway epithelial cells. The following sections summarize the known functions of the best characterized mucins in the airway, with emphasis on COPD where appropriate.
2.1.2. Secreted Mucins
There are four secreted mucins in the lung: MUC2, MUC5AC, MUC5B, and MUC19. Among these, MUC5AC and MUC5B are the major secreted airway mucins. MUC2 is the major intestinal mucin, expressed by goblet cells of the small intestine and colon, although its expression has been described in diseased lungs of humans and rats (Jany et al., 1991; Ohmori et al., 1994). However, MUC2 does not appear to be a major component of lung mucus, according to immunoassay and proteomics analyses of normal and disease-derived sputum (Hovenberg et al., 1996b;Thornton et al., 1996; Kesimer et al., 2009; Ali et al., 2011). Muc2 knockout mice exhibited defects in goblet cell development in the colon concomitant with an absence of mucus, and leading to colonic inflammation and spontaneous development of colitis and colorectal cancer (Velcich et al., 2002;Van der Sluis et al., 2006). The anti-inflammatory and tumor suppressive roles of MUC2 in the colon are not fully understood. MUC19, the major salivary glandular mucin, has also been identified in the tracheolarynx (Das et al., 2010), and in the mouse lung (Young et al., 2007).The functions of MUC2 and MUC19 in the lung are unknown.
MUC5AC and MUC5B are the major components of the mucus gel in normal airways, and are believed to contribute to the barrier function and the rheology of airway mucus (Thornton et al., 2008). The MUC5AC and MUC5B genes exist as overlapping sequences on chromosome 11 in the region p15.5, which also contains the genes for MUC2 and MUC6 (Fig. 4.1). The MUC5AC gene product is produced by goblet cells (Hovenberg et al., 1996b), whereas MUC5B is a submucosal gland mucin (Wickstrom et al., 1998). Both MUC5AC and MUC5B protein backbones consist of multidomain structures with central, O-glycosylated tandem repeats flanked on either side by D, B, C, and CK domains that exhibit sequence homologies with the corresponding domains of the prepro-von Willebrand factor (Fig. 4.2) (Moniaux et al., 2001). MUC5AC has been widely used as a marker for GCM (Zuhdi Alimam et al., 2000). MUC5B was reported as the major mucin present in the apical secretions of in vitro air–liquid interface cultures of primary normal human bronchial epithelial (NHBE) cells and in induced sputum (Kesimer et al., 2009; Ali et al., 2011). MUC5AC may function by facilitating general mucociliary clearance, whereas MUC5B may be more relevant to the clearance of specific pathogens or airway irritants (Thornton and Sheehan, 2004).
The MUC5AC and MUC5B genes exist as overlapping sequences on chromosome 11p15.5, in the vicinity of the genes for MUC2 and MUC6. (For color version of this figure, the reader is referred to the online version of this book.)
Relative sizes of the different domains are not drawn to scale. PTS, proline/threonine/serine. (For color version of this figure, the reader is referred to the online version of this book.)
MUC5AC is upregulated in the airways during a variety of airway diseases, including COPD, CF, and asthma. MUC5AC expression was increased in bronchial submucosal glands of stable COPD patients (Caramori et al., 2009). During allergic airway inflammation, MUC5AC expression was highly induced, whereas MUC5B expression remained constant (Young et al., 2007; Evans et al., 2009; Roy et al., 2011). Utilizing a quantitative Western blotting assay, Thornton and Sheehan (2004) measured the levels of MUC2, MUC5AC, and the different glycoforms of MUC5B in sputum. Greater MUC5B levels, particularly its low-charge glycoform, were found in the sputa of CF and COPD patients compared with secretions from normal subjects and subjects with asthma. Collectively, MUC5AC production is thought to be more relevant to the pathogenesis of asthma (Ordonez et al., 2001; Hallstrand et al., 2007; Evans et al., 2009), whereas MUC5B is believed to be more related to COPD and CF (Thornton and Sheehan, 2004; Kirkham et al., 2008). Further, the study by Thornton and Sheehan (2004) revealed that airways mucus, even in normal individuals, was comprised of variable amounts of MUC5AC and MUC5B. This variability may be related to differences in genetic polymorphisms in the respective mucin genes. For example, a direct correlation was found between the length of the MUC5AC variable numbers of tandem repeats (VNTR) region, and in particular, with a 6.4 kb Hinf I VNTR fragment and the severity of CF lung disease (Guo et al., 2011). A MUC5B gene promoter polymorphism has been associated with pulmonary fibrosis (Seibold et al., 2011). A comprehensive analysis of MUC5AC and MUC5B in COPD and other airway diseases associated with mucus overproduction remains to be performed. These studies are now closer to realization with the recent availability of Muc5ac and Muc5b knockout mice (Roy et al., 2010; Hasnain et al., 2011).
2.1.3. Membrane-Tethered Mucins
The membrane-tethered mucins MUC1, MUC4, MUC11, MUC15, MUC16 and MUC20 have been identified in the lung, with MUC1, MUC4 and MUC16 being the predominant ones (Fig. 4.3). Cell surface mucins are single-pass, transmembrane glycoproteins that are involved in assorted functions, including protection of the epithelial surface from infectious pathogens, regulating intracellular signaling cascades, and controlling cell differentiation and proliferation. Among the three major airway membrane mucins, MUC1 is unique in that its intracellular region contains multiple serine, threonine, and tyrosine residues as potential sites of phosphorylation (Fig. 4.4). Many of these sites are located within the consensus amino acid sequence motifs for binding of signaling proteins. The role of MUC1 in the airways is discussed below in greater detail. MUC4 is broadly expressed in the small intestine, colon, stomach, cervix, and lung (Gendler and Spicer, 1995). MUC4 is an intermembranous ligand for the receptor tyrosine kinase, ErbB2 (Carraway et al., 1999, 2009). Binding of MUC4 to ErbB2 may competitively inhibit the interaction of ErbB2 with its soluble ligands, thereby regulating cell proliferation and growth. In COPD, the airway epithelium is chronically exposed to neutrophil elastase (NE), a major inflammatory protease released by infiltrating neutrophils. NE upregulated MUC4 mRNA and protein expression in normal human bronchial epithelial cells in vitro, suggesting MUC4 may also play a role in lung inflammation (Fischer et al., 2003). MUC16 has been mostly studied in ovarian cancer, but is known to be expressed in normal airway epithelia and submucosal glands (Davies et al., 2007).The blood level of MUC16 in COPD patients was significantly higher than that in control subjects, and directly correlated with systolic pulmonary arterial pressure (Yilmaz et al., 2011). Other than these limited reports, the function of MUC16 in the airways is unknown.
Relative sizes of the different domains are not drawn to scale. Although four tandem repeats are shown for each molecule, the actual numbers can vary between 25 and 125 (MUC1), 145 and 395 (MUC4), and >60 (MUC16). TR, tamdem repeat; SEA, sea urchin sperm protein, enterokinase, agrin domain; TM, transmembrane domain; CT, cytoplasmic tail; IR, imperfect repeats; US, unique sequence; Cys, cysteine-rich domain; NIDO, nodogen-like domain; AMOP, adhesion-associated domain in MUC4 and other proteins; VWD, von Willebrand factor type d-like domain; EGF, epidermal growth factor-like domain; NT, NH2-terminal domain; →, proteolytic cleavage sites. (For color version of this figure, the reader is referred to the online version of this book.)
The relative locations of tyrosine, serine and threonine residues as potential phosphorylation site, and the consensus binding motifs for phosphatidylinositol 3-kinase (PI3K), phospholipase Cγ1 (PLCγ1), c-Src, β-catenin (β-cat), Grb2, ezrin/radixin/moesin (ERM) family proteins are indicated. aa, amino acid. (For color version of this figure, the reader is referred to the online version of this book.)
MUC1 was the first mucin to be genetically cloned and remains the best characterized. MUC1 is expressed on the surface of most secretory epithelia and on some hematopoietic cells (Chang et al., 2000; Gendler, 2001). MUC1 is a type-I transmembrane protein consisting of a highly glycosylated, large extracellular (EC) domain made up of varying numbers (25–125) of 20-amino acid tandem repeats (VNTRs), a hydrophibic transmembrane domain, and a cytoplasmic tail (CT). The glycosylated VNTRs of MUC1 confer an extended, rod-like structure that can project 200–500 nm above the cell surface. Both anti-adhesive and adhesive properties of MUC1 have been proposed based on its glycosylated ectodomain. Because of its large, extended EC domain conformation, MUC1 may sterically inhibit intercellular adhesion between adjacent cells (Hilkens et al., 1992; Wesseling et al., 1995, 1996). Conversely, the presence of sialyl Lewisa and sialyl Lewisx carbohydrates on the MUC1 ectodomain was associated with leukocyte adhesion to E-selectin-expressing endothelial cells (Zhang et al., 1996). MUC1 may also bind to intercellular adhesion molecule-1 (ICAM-1) on endothelial cells and on antigen-presenting cells (APCs), thus facilitating tumor cell metastasis, and aiding T cell-APC interactions (Regimbald et al., 1996). At the COOH-terminus of the molecule, the MUC1 CT domain contains binding sites for numerous intracellular signaling molecules, including c-Src, ErbB family members, glycogen synthase kinase3β (GSK3β), protein kinase Cδ (PKCδ), β-catenin, p120 catenin, Grb-2, p53, heat shock protein 70 (HSP70), and HSP90 (Kim and Lillehoj, 2008). Many of these binding sites are phosphorylated in response to extracellular stimulation, implicating a role for the MUC1 CT in signal transduction.
MUC1 has been suggested to be an oncoprotein based on the results from a variety of independent studies. These include its ability to (1) stimulate cell proliferation via β-catenin-, ErbB-, and estrogen receptor α (ERα)-dependent mechanisms (Schroeder et al., 2002, 2003;Wei et al., 2006), (2) facilitate cell survival through regulation of the FOXO3a and p53 transcription factors (Yin et al., 2004; Wei et al., 2007), (3) promote PyV MT- and Wnt-1 mediated oncogenesis (Al Masri and Gendler, 2005), (4) confer resistance to apoptosis induced by genotoxic agents, reactive oxygen species, and hypoxia (Ahmad et al., 2012), and (5) support tumor metastasis by interacting with ICAM-1 (Rahn et al., 2005). Interaction with β-cetenin and nuclear translocation of the MUC1 CT initiates epithelial to mesenchymal transition (EMT) of pancreatic cancer cells, resulting in increased invasiveness and metastasis (Roy et al., 2011). The secreted form of MUC1 (shed MUC1) seems also to be required for EMT (Horn et al., 2009).
The MUC1 EC domain can be untethered from the cell surface by proteolytic cleavage, both spontaneously and upon stimulation by tumor necrosis factor-α (TNF-α) or the tumor promoter, phorbol 12-myristate 13-acetate (PMA). Stimulated MUC1 shedding is mediated by cellular proteases, which include NE (Kim et al., 1987; Blalock et al., 2008), TNF-α converting enzyme (TACE) (Thathiah et al., 2003), membrane-type 1-matrix metalloprotease (MT1-MMP) (Thathiah and Carson, 2004), MMP-14 (Lindén et al., 2009), and γ-secretase (Julian et al., 2009). The functional significance of MUC1 shedding is not fully understood. It was suggested that shed MUC1, and possibly other membrane-tethered mucins, may form a mucous gel in the immediate vicinity of the apical cell surface, likely serving as a protective barrier against invading pathogens and chemicals (Sheehan et al., 2006). How and when the membrane glycoproteins are cleaved remains largely unknown and will be important questions to address in the context of airway infection and inflammation. Given the ability of the MUC1 ectodomain to bind to invading bacteria (Lillehoj et al., 2001; Lindén et al., 2009; Kato et al., 2010), it is possible that shed MUC1 may serve as a decoy receptor to prevent the direct interaction of bacteria with the epithelial cell surface and to serve as a vehicle facilitating bacterial clearance during infection (Lindén et al., 2009). Shed Muc1 may also contribute to mucociliary clearance under normal conditions, and to mucus obstruction of the airways during disease states. Interestingly, mucus accumulation in the small intestine was observed in a CFTR knockout mouse model of CF, which could not be attributed to overproduction of Muc2, Muc3, or Muc5ac (Parmley and Gendler, 1998). Rather, backcrossing of the CFTR-deficient animals to Muc1 knockout mice prevented gastrointestinal mucus accumulation and improved survival, implicating shed Muc1 in the mucus accumulation. In addition, evidence exists to indicate that shed MUC1 may also suppress immune responses by (1) regulating leukocyte motility, (2) providing an impenetrable barrier around target cells, thereby preventing access by immune effector cells, (3) direct inactivation of immune cells through receptor–ligand interactions, and/or (4) sequestering cytokines, such as transforming growth factor-α (TGF-α) and TGF-β (Hattrup and Gendler, 2008).
In addition to its predominant expression by epithelial cells, MUC1 also is expressed by various subsets of naïve and activated T cells, including CD4+, CD8+, and Th17 cells (Agrawal et al., 1998; Chang et al., 2000; Konowalchuk and Agrawal, 2012; Nishida et al., 2012), dendritic cells (Wykes et al., 2002; Cloosen et al., 2004), monocytes (Leong et al., 2003), and macrophages (Lu et al., unpublished data). Muc1 null mice had defects in the T cell development, and dysfunctional natural killer and dentritic cells (Gendler, 2001). Muc1-deficient mice exhibited aberrant differentiation of bone marrow progenitor cells into myeloid-derived suppressor cells as a consequence of downregulation of β-catenin levels that occured in the absence of Muc1 expression (Poh et al., 2009). Finally, murine dendritic cells lacking Muc1 had constitutively activated Toll-like receptor (TLR) signaling (Williams et al., 2010).
In conclusion, MUC1 expression in the airway may play important roles in mucociliary clearance, regulation of immune responses, and the resolution of lung inflammation. However, the specific role of MUC1 in COPD airways is unknown. Given that MUC1 expression increases in the COPD lung (Ishikawa et al., 2011a, 2011b), it is not unreasonable to predict that overexpression of MUC1 may contribute to lung pathology in COPD patients through (1) immunosuppression of the systemic and local lung immune systems via the MUC1 EC and CT domains, (2) promotion of airway remodeling and mucous cell differentiation through stimulation of EMT, (3) binding to and protection of bacteria against phagocytosis or immune cell killing, (4) contributing to airway mucus gel obstruction, and/or (5) disruption of intraepithelial cell adhesion through interaction with β-catenin in c-Src- and GSK3β-dependent manners. On the other hand, it might also be possible that the loss of the anti-inflammatory function of MUC1 due to mutation (s) in its CT domain (Kim and Lillehoj, 2008), for example as a result of long-term exposure to cigarette smoke (Pleasance et al., 2010), may have resulted in a failure to control inflammation leading to chronic inflammatory lung diseases such as COPD. Future studies will be required to formally test these, or other, hypotheses.
2.2. Mucus Hypersecretion in COPD Airways
Chronic obstructive pulmonary disease (COPD) is one of the most common lung diseases in the world. COPD is comprised of two commonly coexisting clinical entities, chronic bronchitis and emphysema. The primary risk factor for COPD is chronic tobacco smoking. Mucus hypersecretion is a prominent feature of COPD, manifested by an increased amount of sputum. Excess mucus has been associated with several of the pathological features of COPD, most notably an increased frequency and duration of microbial infection, decreased lung function, and increased morbidity and mortality (Vestbo, 2002). Normally, in the small airways of healthy individuals, goblet cells are absent or sparse (Jeffery, 1998; Williams et al., 2006). However, in COPD subjects, elevated numbers of goblet cells are evident with excessive mucus production (Saetta et al., 2000). The components of sputum derive mainly from the central airways with some contribution from the peripheral airways. Overproduction of mucus in peripheral airways mainly contributes to airflow obstruction in COPD patients (Alexis et al., 2001).
Mucus hypersecretion is often associated with changes in both the location and profile of mucin gene expression in COPD airways. These changes are reflected in both the membrane-tethered mucins as well as the secreted, gel-forming mucins. For example, the membrane-associated MUC1 mucin is capable of untetheing its extracellular region from the airway epithelial cell surface into the airway lumen in vivo. Ectodomain shedding may be responsible for the appearance of increased levels of murine Muc1 glycoprotein in the bronchoalveolar lavage fluid (BALF) of mice with cigarette smoke-induced COPD (Lu et al., unpublished data). Ishikawa et al. (2011a, 2011b) recently reported that the sputum level of KL-6 (MUC1) was significantly higher in COPD patients compared with nonsmokers, smokers, and prolonged coughers with normal lung function. The increased sputum MUC1 levels were positively correlated with smoking history, age, and levels of sputum macrophages and eosinophils. MUC1 was more prominently expressed in the bronchiolar/alveolar epithelium in COPD than in the control lungs.
MUC5AC and MUC5B, which are the most predominant gel-forming mucins in the airways, are present at higher levels in mucus from diseased airways (Williams et al., 2006). More specifically, MUC5AC and MUC5B expression levels in COPD patients are remarkably increased, and their relative expression patterns are altered compared with non-COPD smokers and normal subjects. Studies analyzing the mucin components of airway sputum from COPD patients have revealed that MUC5AC and MUC5B are also the major mucins in this airway component, with MUC5B being the predominant component (Kirkham et al., 2002, 2008). In peripheral airways, Caramori et al. (2004) reported that COPD was associated with increased expression of MUC5B in the bronchiolar lumen and increased MUC5AC in the bronchiolar epithelium. In COPD patients, the expression of MUC5AC was increased not only in the surface epithelium, but also in submucosal glands, and the elevated MUC5AC directly correlated with smoking history, and inversely correlated with the Forced Expiratory Volume in One Second (FEV1), a measure of lung function (Caramori et al., 2009). MUC5B, which is normally expressed in the submucosal gland in the bronchioles, was also found to be present in the surface epithelium (Kirkham et al., 2008).The expression and localization of MUC2, MUC4, and MUC6 in the peripheral airways were found not to be changed by the smoking history and the presence of COPD (Hovenberg et al., 1996a; Kirkham et al., 2002; Caramori et al., 2004). Other mucins, such as MUC7 and MUC8, have not been evaluated, but are postulated to change during COPD according to the following evidence. First, MUC7 was induced by cigarette smoke extract and bacterial lipopolysaccharide (LPS) exposure in human airway epithelial cells in vitro and in mice in vivo (Fan and Bobek, 2010). Second, MUC8 was induced in airway epithelial cells by the high-mobility group box-1 protein (HMGB1), a recently identified proinflammatory mediator that is active in various inflammatory diseases (Kim et al., 2012, in press).
2.3. Mucin Regulation Related to COPD
A growing body of evidence indicates that mucus hypersecretion in COPD is induced by microbial products, airborne pollutants, and mediators of inflammation. Both viral and bacterial components directly upregulated mucin gene expression (Shimizu et al., 1996; Mata et al., 2011). Inhalation of sulfur dioxide, ozone, cigarette smoke, or acorlein upregulated Muc5ac expression, stimulated neutrophilic inflammation, and induced GCM in rat and mouse airway epithelia (Wagner et al., 2003; Sueyoshi et al., 2004; Bein and Leikauf, 2011; Nie et al., 2012). Chemical irritant-induced airway inflammation appeared to mediate the development of GCM. Airway instillation of NE, an endogenous mediator of Muc5ac expression, stimulated bronchial GCM in hamsters and in mice (Breuer et al., 1993; Voynow et al., 2004). Th1 and Th2 cytokines (predominantly IL-9 and IL-13), IL-1β, TNF-α, TGF-α, IL-6, and IL-17 also regulated mucin gene expression and GCM in vivo (Turner and Jones, 2009). The downstream signaling cascades involved in mucin upregulation and GCM involve the epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 6 (STAT6), forkhead box protein A2 (FoxA2), SAM domain-containing prostate-derived Ets factor (SPDEF), and nuclear factor-κB (NF-κB) (Turner and Jones, 2009). Activation of hypoxia inducible factor-1 (HIF-1) signaling was recently found to contribute to GCM in COPD patients (Polosukhin et al., 2011). Finally, notch signaling was critical for negative regulation of Muc5ac expression and GCM during postnatal mouse lung development (Tsao et al., 2011). Of these known signaling pathways involved in regulating mucin production and influencing GCM, the EGFR and Th2 cytokine pathways seem to have the greatest potential for therapeutic intervention to inhibit of excessive mucus production during lung diseases (Lai and Rogers, 2010).
3. STRUCTURE OF AIRWAY MUCINS
Currently, 22 human mucin genes have been cloned, of which 16 have been identified in the lung (MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC7, MUC8, MUC11, MUC13, MUC15, MUC16, MUC18, MUC19, MUC20, MUC21, and MUC22) (Lillehoj and Kim, 2002; Rose and Voynow, 2006; Davies et al., 2007; Itoh et al., 2008; Hijikata, 2011). All mucins are high molecular-weight glycoproteins characterized by the presence of VNTRs. Mucin VNTRs consist of sequential replicates of amino acid sequences unique to each mucin that are enriched in serine, threonine, and proline amino acid residues (Fig. 4.5). Serine and threonine residues are the sites of covalent attachment of glycan side chains of the peptide backbone through O-glysosidic linkages with N-acetylgalactosamine of the oligosaccharides. Examination of the predicted amino acid sequences of the cloned mucin genes revealed that there are two types of mucins, membrane-tethered and secreted mucins. Secreted mucins are further subdivided into gel-forming and nongel-forming mucins. In the airways, MUC2, MUC5AC, MUC5B, MUC7 and MUC19 are secreted and gel-forming mucins, while MUC1, MUC4, MUC8, MUC11, MUC13, MUC15, MUC16, MUC18, MUC20 and MUC21 are membrane-bound mucins (Thornton et al., 2008; Hijikata et al., 2011).
The total number of amino acids (aa) in each repeat is indicated at the end of each sequence. Points (….) designate intervening sequences not listed to minimize space. MUC14, MUC15, and MUC18 do not contain tandem repeats. MUC19 has at least seven different repeats ranging from 5 to 16 amino acids (Rose and Voynow, 2006). *, degenerate repeats; a,Gendler et al., 1990 (GenBank accession no. J05581); b,Gum et al., 1997 (GenBank accession no. AAC02272); c,Porchet et al., 1991 (GenBank accession no. Q99102); d,Williams et al., 1999 (GenBank accession no. AAD55679); e,Williams et al., 1999 (GenBank accession no. AAD55678); f,Williams et al., 2001 (GenBank accession no. AAK56861); g,Yin et al., 2002 (GenBank accession no. AAK74120); h,Gum et al., 2002 (GenBank accession no. AK026404); i,Higuchi et al., 2004 (GenBank accession no. AB098731); j,Gum et al., 1989 (GenBank accession no. J04638); k,Aubert et al., 1991 (GenBank accession no. CCA84031); l,Dufosse et al., 1993 (GenBank accession no. S35049); m,Toribara et al., 1993 (GenBank accession no. B46629); n,Bobek et al., 1996 (GenBank accession no. NP689504); oSachdev, direct submission (GenBank accession no. AAA58346); p,Lapensee et al., 1997 (GenBank accession no. NP_002548). *The first repeat of MUC9 is shown. Subsequent repeats are degenerate (Rose and Voynow, 2006).
3.1. Gel-forming Airway Mucins—MUC5AC, MUC5B
Initial cloning experiments identified three genes that were thought to encode distinct mucins and were originally designated as MUC5A, MUC5B, and MUC5C. Subsequent studies demonstrated that MUC5A and MUC5C were identical and its designation was changed to MUC5AC (Guyonnet-Duperat et al., 1995).To date, there is a discrepancy regarding the total number of exons present in the 150 kb MUC5AC gene. The full size 5′ UTR of MUC5AC has not yet been determined, but it is estimated that the mRNA length is approximately 17.5 kb. Within the NH2-terminal region of the MUC5AC protein are located 4 cysteine-rich D domains, similar to the von Willebrand factor and responsible for intermolecular disulfide bond formation between individual MUC5AC glycoproteins (Gendler and Spicer, 1995). The MUC5AC NH2-terminus also contains a putative leucine zipper motif not found in any other mucin identified so far, but its function is unknown (van de Bovenkamp et al., 1998). Within the central region of the MUC5AC molecule, coded by a single large exon, are nine cysteine domains (Cys1-Cys9). Cys1–Cys5 are interspersed by domains rich in serine, threonine, and proline (STP), but with no repetitive sequences, whereas the Cys5– Cys9 domains are interspersed by four VNTR domains, each being eight amino acids in length. The COOH-terminal region of MUC5AC has a single D domain, as well as the B, C, and CK domains. Like the D domains, the CK domain also participates in the formation of disulfide-linked polymers.
MUC5B mucin is the second major respiratory tract mucin (Wickstrom et al., 1998). MUC5B is unique in the mucin superfamily because its repeat region is degenerate and non-tandem (Dufosse et al., 1993). Due to numerous amino acid insertions and deletions, only 22 of a possible 55 complete repeats are present. Nevertheless, the sequence remains mucin-like with a high percentage of serine, threonine, and proline residues and is heavily O-glycosylated. The central region of MUC5B contains a single, contiguous exon of 10,713 base pairs (3570 amino acids) that may be the biggest exon described for a vertebrate gene.
3.2. Membrane-tethered Airway Mucins—MUC1, MUC4, MUC16
MUC1, MUC4, and MUC16 are the three major membrane mucins expressed in the airways. The presence of mucin-like glycoproteins on the airway surface epithelium was first documented by Kim et al. (1987). Subsequently, the full-length membrane mucin gene, MUC1, was cloned (Gendler et al., 1990; Lan et al., 1990) and expression of MUC1 protein in lung tissue explants as well as cultured airway epithelial cells was independently reported by Pemberton et al. (1992) and Hollingsworth et al. (1992), respectively. MUC1 is expressed by mucosal epithelial cells as well as hematopoietic cells, including lymphocytes and dendritic cells (Gendler, 2001). Its expression in other hematopoietic cells is less clear, although MUC1-expressing corneal endothelial cells have been described (Jung et al., 2002). While the full-length MUC1 molecule is embedded in the plasma membrane through its hydrophobic transmembrane region, its ectodomain is releasable into the airway lumen (Kesimer et al., 2009; Ali et al., 2011), as previously shown for other cell types including tumor cells and primary uterine epithelial cells (Gendler and Spicer, 1995; Pimental et al., 1996). MUC4 and MUC16 are additional membrane-bound mucins expressed in the respiratory tract. Their respective roles in the airways have been described above (Section 2.1.1).
3.3. Airway Mucin Glycosylation
Mucins are predominantly O-glycosylated molecules with a relatively lesser amount of N-linked oligosaccharides. O-glycosylation is initiated in the Golgi when an N-acetylgalactosaminyl peptidyltransferase adds N-acetylgalactosamine (GalNAc) to a serine or threonine residue on the mucin polypeptide chain (Rose and Voynow, 2006). Stepwise addition of additional glycan moieties (galactose [Gal], N-acetylglucosamine [GlcNAc], fucose [Fuc] and sialic acid [SA]) by specific glycosyltransferases generates the completed glycan side chain. More than 10 glycosyltransferases are involved in synthesis of mucin O-linked glycans (Brockhausen and Schachter, 1997). While the pattern and composition of mucin O-glycans is relatively complex, their structures can be divided into four major core structures (Fig. 4.6). Core types 1 and 2 are formed by the transfer of Gal to the O-linked GalNAc residue. Core types 3 and 4 are formed by the addition of GlcNAc to the GalNAc moiety. Subsequent incorporation of another GlcNAc to form a second branch off of the GalNAc residue generates a core 2 structure from core 1, and a core 4 structure form core 3, respectively. All core structures and both branches can be elongated by further additions of Gal, GalNAc, Fuc, and SA residues. Finally, addition of sulfate to Gal or GlcNAc residues adds additional structural heterogeneity and complexity.
The MUC1 tandem repeat is shown. GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; Gal, galactose; SA, sialic acid; Fuc, fucose. (For color version of this figure, the reader is referred to the online version of this book.)
Airway mucin glycosylation may be altered in disease states (Rose, 1992). All four mucin O-glycan core types have been identified in patients with chronic bronchitis (Van Halbeek et al., 1994). In the case of CF, Thomsson et al. (1998) isolated and identified more than 60 different oligosaccharides from airway mucins, with sizes of up to 15 monosaccharide units, some being unique to the respiratory mucins. Comparison of the mucin glycans from patients with chronic bronchitis and CF indicated differences in the glycosylation process and suggested that bacterial infection in the latter group influenced the expression of specific glycosyltransferases in the human bronchial mucosa (Davril et al., 1999). Similarly, in a comprehensive analysis of oligosaccharides from CF and non-CF individuals, 260 compositional types of O-glycans were identified with CF mucins containing a higher proportion of sialylated and sulfated O-glycans compared with non-CF mucins (Xia et al., 2005). Changes in respiratory mucin glycosylation in CF may be responsible for differential bacterial binding to airway epithelial cells and thus increased tendency for lung infection (Roussel and Lamblin, 2003). However, O-linked glycans released from purified mucins from CF and non-CF patients were also reported to have no observable differences (Holmén et al., 2004; Schulz et al., 2005), and the glycosylation patterns of MUC5AC in a cell line containing wild type or mutant CFTR were identical (Leir et al., 2005). Further analysis of mucin oligosaccharides in airway diseases may shed light on these apparent discrepant results.
4. MUCIN SECRETION
The lack of treatment for airway obstruction that was frequently experienced in the clinical setting prompted research on the regulation of airway mucin secretion (Fahy, 2002; Fahy and Dickey, 2010; Voynow and Rubin, 2009). However, the unavailability of good experimental models, combined with the limited knowledge of mucin biochemistry, kept the field from moving forward. A major breakthrough came with the availability of an in vitro cell culture technique to grow tracheal epithelial (TE) cells from various animals (Wu and Smith, 1982; Lee et al., 1984), and a gel filtration chromatography method to measure mucins secreted from these cultures (Cheng et al., 1981). Briefly, cells were metabolically radiolabeled with sugars, most notably with 3H-GlcNAc, and the spent culture media were subjected to multiple gel filtration chromatographic steps to separate the high molecular weight glycoconjugates. Kim (1985) first demonstrated the requirement of a thick collagen matrix for the production of mucin-like glycoproteins from cultured TE cells, and also was the first to biochemically characterize the mucins produced from these cells by chromatographically separating mucins from other secreted glycoconjugates, such as proteoglycans (Kim et al., 1985, 1989; Kim, 1991a). Thus, the establishment of both the TE cell culture system and the gel filtration mucin assay has made it possible to move the field forward, yielding most of our current understanding of the pharmacology of airway mucin secretion (Kim and Brody, 1989; Kim, 1991b).
Later, the TE culture system was further improved by incorporating an air–liquid interface (ALI) culture modification whereby the cells are grown on a porous membrane support, and the apical (upper) chamber was exposed to air while the basolateral (lower) chamber was bathed in media (Adler et al., 1987, 1990; Whitcutt et al., 1988). The ALI culture system has been extensively used to study the functional aspects of airway epithelial cells in the context of various pulmonary diseases. A number of reviews on the regulation of airway mucin production are available elsewhere, including those summarizing mucin secretion (Kim, 1991b; Rogers, 2002; Kim et al., 2003; Fahy and Dickey, 2010), mucin gene expression (Rose and Voynow, 2006; Thai et al., 2008;Turner and Jones, 2009), and GCM (Tesfaigzi, 2008; Evans and Koo, 2009; Curran and Cohn, 2010). For the remainder of this section, we will focus on the regulation of airway mucin secretion at the cellular level.
4.1. Cells Expressing Mucins in the Lung
MUC1 was the first mucin gene cloned from breast cancer and pancreatic tumor cells, and based on the amino acid sequence predicted from its nucleotide sequence, it was suggested to be embedded in the plasma membrane through a single-pass transmembrane domain (Gendler et al., 1990; Lan et al., 1990). The first secreted mucin gene identified, MUC2, was originally cloned from intestinal epithelium (Gum et al., 1989) and was the first mucin to be identified at the mRNA level in the airways (Gerard et al., 1990; Jany et al., 1991). This was followed by the detection of MUC1 mRNA in bronchial epithelial cells (Hollingsworth et al., 1992), and subsequently by identification of MUC5AC and MUC5B expression in the lungs (Meerzaman et al., 1994; Desseyn et al., 1997). MUC1 and MUC4 proteins are expressed on the apical surface of airway epithelial cells, while MUC2 and MUC5AC are expressed in goblet cells of the superficial airway epithelium (Voynow et al., 2006). In the submucosal glands, MUC5B, MUC8, and MUC19 are expressed in mucosal cells, whereas MUC7 is localized to serosal cells. Cellular localization of other mucin glycoproteins known to be expressed at the mRNA in the airways (MUC11, MUC13, MUC15, and MUC20) remains to be clarified.
4.2. Regulation of Mucin Secretion
Airway mucus constitutes the first line of defense against inhaled pathogens, and its proper quantity and quality are crucial to maintain its function. Much research has focused on understanding the regulation of mucin secretion with the goal of discovering therapies to control mucus hypersecretion that is frequently manifested in patients suffering from COPD, CF, asthma, chronic bronchitis, bronchiectasis, and other relevant lung diseases. In particular, studies addressing the pharmacology of mucin secretion using primary TE cell culture systems and the gel filtration column assay were a major focus in the 1980s. In general, there do not appear to be significant interspecies’ differences, either as intact animals or in primary TE cell culture models, in mucin release in response to various pharmacological agents. It is worth mentioning, however, that most of our current knowledge on mucin secretion is based on the measurement of a mixture of high molecular weight mucins using the gel filtration assay. In the future, it will be necessary to investigate the secretion and function of individually purified mucin glycoproteins in the lung.
4.3. Mucin Gene Transcription, mRNA Stability, Translation, and Protein Degradation
A variety of infectious and inflammatory mediators provoke mucin gene transcription. In general, these agents act by binding to cell surface receptors that, in turn, stimulate intracellular signaling cascades leading to activation of transcription factors, including NF-κB, AP-1, Sp1, and CREB, that bind to mucin gene promoters and regulate gene transcription (Rose and Voynow, 2006). For example, the TGF-β-Smad signaling pathway cooperates with NF-κB to mediate nontypeable Haemophilus influenzae (NTHi)-induced MUC2 gene transcription (Jono et al., 2002). Similarly, NTHi regulates MUC5AC gene transcription through a p38 mitogen-activated protein kinase (MAPK) signaling response (Wang et al., 2002). Perrais et al. (2002) identified an EGF → EGFR → Ras → Raf → extracellular signal-regulated kinase (ERK) → Sp1 pathway that regulated MUC2 and MUC5AC gene transcription by human airway epithelial cells. Other cell surface receptors that have been shown to activate mucin gene expression include the P2Y2 receptor (Londhe et al., 2003), retinoic acid receptor α (Koo et al., 2002), and platelet-activating factor receptor (Lemjabbar and Basbaum, 2002).
While its congate surface receptor is currently unknown, NE efficiently upregulates both secreted (Voynow et al., 1999) and membrane-bound (Fischer et al., 2003; Kuwahara et al., 2005) mucin gene expression, and is recognized as one of the most potent mucin secretagogues. Kuwahara et al. (2005) demonstrated that A549 cells, a human lung alveolar carcinoma cell line, treated with NE exhibited significantly higher MUC1 protein levels in cell lysates compared with cells treated with vehicle alone. MUC1 protein shed into cell-conditioned medium was rapidly and completely degraded by NE. Actinomycin D blocked NE-stimulated MUC1 expression, suggesting a mechanism of increased gene transcription. By real time RT-PCR, quantitatively greater MUC1 mRNA levels were measured in NE-treated A549 cells compared with controls. However, NE did not affect MUC1 mRNA stability, implying increased de novo transcription induced by the protease. Transfection of cells with a MUC1 gene promoter-luciferase reporter demonstrated that NE stimulated MUC1 promoter activity, which was completely blocked by the Sp1 inhibitor, mithramycin A. NE-driven MUC1 promoter activity also was inhibited by mutation of a putative Sp1 binding site at −99/-90 bp relative to the MUC1-transcription start site. An electrophoretic mobility shift assay (EMSA) revealed that treatment of A549 cells with NE increased the binding of Sp1 to the −99/-90 bp site. These results indicated that NE-dependent MUC1 gene transcription was mediated through increased binding of Sp1 to the −99/-90 segment of the MUC1 promoter. In support of this conclusion, Morris and Taylor-Papadimitriou (2001) and Kovarik et al. (1993, 1996) reported that the Sp1 site at −99/-90 was crucial for cell- and tissue-specific regulation of MUC1 gene expression.
Posttranscriptional mechanisms of mucin gene regulation have also been reported. TNF-α (Borchers et al., 1999), NE (Voynow et al., 1999), and IL-8 (Bautista et al., 2001) increased MUC5AC mRNA stability in vitro. Voynow et al. (1999) reported that treatment of A549 cells with NE increased MUC5AC mRNA and protein levels through a mechanism involving increased transcript stability. MUC2 mRNA levels in intestinal epithelial cells were increased by posttranscriptional mechanisms after epithelial exposure to PMA or forskolin (Velcich and Augenlicht, 1993). MUC4 was posttranslationally regulated by TGF-β in rat mammary epithelial cells (Price-Schiavi et al., 1998), and Fischer et al. (2003) showed that treatment of primary NHBE cells with NE increased MUC4 mRNA levels by prolonging its half-life from 5 to 21 h. In the primary rat tracheal surface epithelial (RTSE) cells, the glucocorticoid, dexamethasone (DEX), dose-dependently suppressed Muc5ac mRNA levels, while the levels of cellular Muc5ac protein were unchanged (Lu et al., 2005). DEX-enhanced translation of the rat Muc5ac gene transcript and increased the stability of intracellular Muc5ac protein by a mechanism not involving proteasomal degradation. Thus, whereas DEX inhibited the levels of rat Muc5ac mRNA in primary RTSE cells, the levels of Muc5ac protein remained unchanged, as a consequence of increases in both translation and protein stability. By contrast, DEX suppressed MUC5AC mRNA levels and MUC5AC protein secretion in dose-dependent manners in human A549 cells, indicating that some of the effects of DEX differed when comparing primary cells with the transformed cell line. Further studies are required to elucidate the mechanisms whereby MUC2, MUC4, and MUC5AC mRNA and/or protein stabilities are regulated.
4.4. Mucin Secretagogues
4.4.1. Neutrophil Elastase
NE, the major protease produced by neutrophils during airway inflammation, was the first mucin secretagogue identified using the in vitro primary TE cell culture system and the gel filtration mucin assay. Kim et al. (1987) demonstrated that NE stimulated mucin release from the primary hamster TE cells. The source of these secreted mucins was mainly from the apical surface of the cultured cells, and not from an intracellular pool. This finding was confirmed by others, including Breuer et al. (1989), and constituted the first report, suggesting the presence of mucins on the surface of airway epithelial cells that were releasable by NE through a proteolytic mechanism. These cell surface mucins were later identified as MUC1/Muc1. In addition to the release of cell surface mucins, NE was also shown to stimulate mucin secretion from secretory granules (Breuer et al., 1987, 1993). Thus, NE can release mucins from goblet cells via exocytosis of mucin granules, as well as from the surface of epithelial cells via proteolytic cleavage of MUC1/Muc1. However, the detailed mechanistic pathways of both processes and their regulation in health and disease are largely unknown.
4.4.2. Nucleotides
While studying the role of guanine nucleotide-binding proteins, or G proteins, in the context of mucin granule exocytosis, Kim and Lee (1991) first discovered that activation of the P2u receptor on the airway goblet cells by ATP or UTP resulted in a massive secretion of mucin glycoproteins. The stimulatory effect of mucin secretion by the nucleotides was confirmed by Davis et al. (1992), who demonstrated nucleotide-stimulated mucin granule exocytosis by cultured goblet cells. In addition to the pharmacology of ATP on mucin release, these nucleotides have also been shown to play an important role for constitutive mucin secretion in normal physiology. Briefly, mechanical strain caused the release of cellular ATP into the extracellular space (Homolya et al., 2000) which, in turn, stimulated mucin release from airway goblet cells by activation of specific nucleotide receptors, P2u or P2Y2, that respond to both ATP and UTP with equal potency (Kim et al., 1996). In the light of the presence of smooth muscle tonicity in the airways, this mechanism of mucin release might be responsible for basal or constitutive secretion as initially demonstrated by Kim et al. (1993). Later, Chen et al. (2001) reported that although ATP and UTP increased mucin secretion by airway epithelial cells through activation of the same cell surface receptor, UTP, but not ATP, increased expression of the MUC5AC and MUC5B genes, suggesting that secretion and gene expression by these nucleotides involved different signaling pathways. Given the identical signaling pathway utilizing by these two nucleotides (i.e. the P2Y2 receptor), the presence of two separate endpoints (MUC5AC and MUC5B) remains to be explained.
4.4.3. Nitric Oxide
Nitric oxide (NO), an endogenous product of l-arginine that is important for vascular tone, has been shown to release mucins indirectly in response to various inflammatory agents (Adler et al., 1995), oxidative stress (Wright et al., 1996), and directly through PKC- and ERK-dependent pathways (Song et al., 2007). However, the stimulatory effect of NO on the regulation of mucin secretion has been challenged by Kim et al. (2006).
4.5. Agents that Inhibit Mucin Secretion
While goblet cell mucin pharmacology was focused mainly on stimulation, there are a few agents that have been shown to suppress mucin secretion.
4.5.1. Glucocorticoids
Glucocorticoids regulate the expression of vaious types of genes, including mucins, through the glucocorticoid receptors (GR). Normally, activation of GR results in its nuclear translocation and binding to glucocorticoid response elements (GRE) in the promoters of target genes. Chen et al. (2012) demonstrated that the DEX suppressed MUC5AC gene expression in primary NHBE cells through two GREs in the MUC5AC gene promoter.
4.5.2. Poly-cationic Peptides
Cationic peptides such as eosinophil major basic protein (Kim et al., 1999), poly-l-lysine, and poly-l-arginine (Ko et al., 1999) suppressed mucin release from the primary hamster TE cells without cytotoxicity. The mechanism of mucin suppression by these polycationic peptides remains to be elucidated, but appears to involve decreased mRNA stability and protein translation (Kim et al., unpublished data).
4.5.3. MARCKS-related Peptide
Li et al. (2001a) showed that myristoylated arginine-rich C kinase substrate (MARCKS), a protein involved in granule/vesicle exocytosis, in general, is also involved in mucin secretion from airway goblet cells. Recently, they have been able to demonstrate that a MARCKS-related peptide can suppress mucin hypersecretion in animal models (Singer et al., 2004; Green et al., 2011).
4.5.4. Macrolide Antibiotic Analogs
Macrolide antibiotic analogs have also been shown to inhibit mucus production, most likely through their anti-inflammatory effects (Tamaoki et al., 1995;Tamaoki, 2004).
4.5.5. PDE5 Inhibitor
Sidenafil, a PDE5 inhibitor, has been shown to suppress acrolein-induced inflammation as well as GCM and Muc5ac production in vivo by the NO/cGMP pathway (Wang et al., 2009).
4.6. Goblet Cell Metaplasia (GCM) and Goblet Cell Hyperplasia
GCM and GCH are induced in response to exposure of the airways to a multitude of endogenous and exogenous mediators (Fig. 4.7).These mediators bind to surface receptors on the airway epithelial cells that activate intracellular signaling pathways, ultimately leading to the expression of a variety of transcription factors that bind to the promoters of genes to regulate cell phenotype. For example, the Th2 cytokine, IL-13, drives ciliated cells to convert into goblet cells through the thyroid transcription factor 1 (TTF-1), FoxA2, and SPDEF transcription factors (Curran and Cohn, 2010). Many exogenous mediators that drive GCM and GCH during prolonged stimulation also induce airway mucin gene expression under normal conditions. Expression levels of mucin gene transcripts and proteins are regulated by their respective degradative processes. Mucin proteins that escape degradation are glycosylated and packaged into mucin granules. Under appropriate extracellular stimulation, these granules traffic to and fuse with the apical cell membrane through the docking actions of MARCKS, soluble N-ethyl-maleimide-sensitive factor-attachment protein receptor (SNARE), and mammalian uncoordinated (Munc) proteins (Rogers, 2007).The cell biology of mucin exocytosis has been reviewed by Davis and Dickey (2008). Finally, the membrane-associated protein, calcium-dependent chloride channel 1 (CLCA1), or the mouse homolog, Gob5, is expressed in goblet cell mucus secretory granules where it has been suggested to play a role in secreting chloride anions into the granule lumen and contributing to the salt and water composition of secreted mucus (Thai et al., 2008). Further, overexpression of CLCA1 increased MUC5AC levels, whereas knockdown of Gob5 decreased Muc5ac levels. Thus, CLCA1 may also play a functional role in regulating MUC5AC gene expression in airway epithelial cells prior to secretion. While all of these mechanisms and pathways are operative under normal conditions, prolonged stimulation by many of these mediators leads to abnormally increased mucin gene expression, mucus hypersecretion, and GCM/GCH.
Exogenous and endogenous mediators bind to surface receptors (R) on the airway epithelial cells that activate intracellular signaling pathways, ultimately leading to the expression of a variety of transcription factors that bind to the promoters of mucin genes. Mucin gene transcription leads to mRNAs that are translated, glycosylated, and packaged into secretory granules with CLCA1 on their membranes. MARCKS, SNARE, and Munc proteins mediate granule exocytosis. Other mechanisms to control mucin secretion involve degradation of their mRNAs and proteins prior to granule packaging. (For color version of this figure, the reader is referred to the online version of this book.)
5. PHYSIOLOGIC ROLE OF MUC1 MUCIN IN THE AIRWAYS
5.1. Structure and Expression of MUC1 in the Airways
5.1.1. MUC1 Ectodomain—Carbohydrates and VNTRs
MUC1 is a highly glycosylated transmembrane protein of a large molecular mass (>300 kDa) that is widely expressed on the apical surface of most secretory epithelial cells. The human MUC1 gene is localized on chromosome 1q21-24 (Swallow et al., 1987), and its deduced amino acid sequence indicated three distinct regions: (1) the NH2-terminus consisting of a putative signal peptide and degenerate repeats, (2) the major portion of the protein, which is the tandem repeat region, and (3) the COOH-terminus consisting of degenerate tandem repeats and a unique sequence containing a transmembrane sequence and a cytoplasmic tail (Gendler et al., 1990; Lan et al., 1990). Similar protein domain structures have been reported for all nonhuman Muc1 mucins that have been analyzed (Spicer et al., 1995). The extracellular domain of all MUC1/Muc1 glycoproteins contains 20-amino acid VNTRs that are repeated in humans between 25 and 125 times (Aplin et al., 1994). Interspecies comparisons between mammalian MUC1/Muc1 VNTRs reveal 15%–100% amino acid sequence identities (Fig. 4.8). This repetitive region, and the regions adjacent to it, comprises most of the extracellular portion of the molecule, extending 200–500 nm above the plasma membrane (Hilkens et al., 1992). This long ectodomain projection has been proposed to be responsible for the observation that aberrant overexpression of MUC1 by cancer cells reduces cell–cell and cell-matrix adhesion (van de Wiel-van Kemenade et al., 1993; Wesseling et al., 1995; Kondo et al., 1998). Similarly, Muc1 expression in baboon uterine epithelial cells has been shown to confer an anti-adhesive property that was suggested to play an important role in maintaining the pre-receptive phase in the uterus (Hild-Petito et al., 1996).
Dashes (−) indicate identical residues. aHomo sapiens (Gendler et al., 1990; Genbank accession no. J05581); bHylobates lar (Spicer et al., 1995; GenBank accession no. L41624); cPapio anubis (GenBank accession no. XP_003892817.1); dMus musculus domesticus (Spicer et al., 1991; GenBank accession no. M64928); eRattus norvegicus (DeSouza et al., 1998; GenBank accession no. AAB62948); fOryctolagus cuniculus (Spicer et al., 1995; GenBank accession no. AAB64380); gMesocricetus auratus (Park et al., 1996; GenBank accession no. U36918); hBos taurus (Pallesen et al., 2001; GenBank accession no. CAC81810); iCapra hircus (Sacchi et al., 2004; GenBank accession no. AY388993); jOvis aries (Rasero et al., 2007; GenBank accession no. ABD96017.1); kBos grunniens(Zhao et al., direct submission; GenBank accession no. ABG49529.1); lCanis lupus familiaris (Ishiguro et al., 2007; GenBank accession no. NM_001194977.1).
5.1.2. MUC1 Ectodomain Shedding—the SEA Domain and Autoproteolysis
The MUC1 ectodomain undergoes proteolytic cleavage during protein translation in the endoplasmic reticulum (Hilkens and Buijs, 1988; Ligtenberg et al., 1992). However, the two subunits remain noncovalently associated during posttranslational glycosylation and trafficking to the cell surface. In fact, the MUC1 heterodimer remains intact in the presence of urea, β-mercaptoethanol, high temperatures, or acidic conditions, but dissociates in the presence of sodium dodecyl sulfate (Ligtenberg et al., 1992; Julian and Carson, 2002).The MUC1 cleavage site was mapped to a Gly316-Ser317 peptide bond located 59 amino acids proximal to the transmembrane region (numbered as in Gendler et al., 1990) (Parry et al., 2001). Proteolysis occurs within the SEA (sea urchin sperm protein, enterokinase, agrin) domain, a 120-amino acid domain that is highly conserved in several abundantly glycosylated, mucin-like proteins (Levitin et al., 2005; Macao et al., 2006). In vitro incubation of purified uncleaved MUC1 protein, in the absence of any additional cellular components, resulted in molecular self-cleavage, which was enhanced by the nucleophile, hydroxylamine. Based on these and other experiments, it was concluded that MUC1 undergoes autoproteolysis mediated by conformational strain-dependent protonation of the amide nitrogen of the critical serine residue, followed by N → O-acyl shift and peptide bond hydrolysis (Johansson et al., 2009).
Soluble forms of the MUC1 ectodomain are present in the conditioned media of in vitro cultured epithelial cells as well as in sera of cancer patients (Boshell et al., 1992; McGuckin et al., 1994; Pimental et al., 1996; Zhang et al., 1996;Treon et al., 2000; Julian and Carson, 2002). Some studies have implicated cellular proteases in MUC1 ectodomain shedding, independent of the known autoproteolysis site (see Section 2.2.) (Thathiah et al., 2003; Thathiah and Carson, 2004). Because the two MUC1 subunits dissociate in vitro only under nonphysiological conditions, it is unclear whether intracellular cleavage at the Gly316-Ser317 peptide bond is sufficient to permit shedding or if additional proteolysis is required. In this regard, evidence has been published indicating that Ser317-to-Ala site-directed mutagenesis at the cleavage site blocked MUC1 proteolysis and inhibited ectodomain shedding (Lillehoj et al., 2003).
5.1.3. Alternative Splicing of MUC1 Gene Transcripts
Another possible explanation for soluble forms of MUC1 is alternative mRNA splicing. At least, 12 splice variants of the MUC1 gene transcript have been described (Imbert-Fernandez et al., 2011). Some of the more well-characterized of these are schematically illustrated in Fig. 4.9. Smorodinsky et al. (1996) reported that alternative splicing caused a translation stop codon normally present in an intron to be introduced into the MUC1 coding sequence prior to the hydrophobic transmembrane domain. The resulting gene product, MUC1/SEC, was secreted into cell culture medium. MUC1/SEC later was identified as a binding partner of another MUC1 isoform, MUC1/Y, a membrane-tethered protein arising as a consequence of deletion of the VNTR region by alternative splicing (Baruch et al., 1999). Another differential splicing transcript, MUC1/Z, is similar to MUC1/Y in its lack of the VNTR domain (Oosterkamp et al., 1997). Two additional MUC1 splice variants that differ in the NH2-terminus are MUC1/A and MUC1/B (Imbert-Fernandez et al., 2011). MUC1/A differs from MUC1/B by having a 9-amino acid insertion prior to the VNTR region. As a result of this insertion, MUC1/A is predicted to undergo altered cleavage of its signal peptide, thereby potentially modifying its intracellular trafficking and/or subsequent posttranslational processing. The MUC1 splice variants, CT80 and CT58, encode transmembrane proteins with intracellular regions that differ from the normal MUC1 CT sequence (Hinojosa-Kurtzberg et al., 2003). As a consequence, CT80 and CT58 lack some of the known signaling sites in the MUC1 intracellular region, suggesting that they may exhibit different signaling functions compared with the normally spliced MUC1 gene product. Additional studies are needed to assess the functions of theses MUC1 CT splice variants.
The top line illustrates the intron-exon organization of the MUC1 gene with seven exons (e1–e7) and six introns (i1–i6). The relative contributions of each exon to the MUC1 protein structure are indicated by green (leader peptide), blue (tandem repeats), purple (juxtamembrane region of ectodomain), brown (transmembrane [TM] region), and red (cytoplasmic region). The MUC1/SEC splice variant arises as a result of absence of splicing at the e2-i2 boundary, with the incorporation of an 11 amino acid segment (yellow) prior to a stop codon before the TM region. MUC1/Y and MUC1/Z splice variants are missing all or most of the tandem repeats. The CT80 and CT58 splice variants are missing e7 with unique 32- and 10-amino acid extensions (yellow) at the end of their cytoplasmic regions. (For interpretation of the references to color in this figure legend, the reader is referred to the online version of this book.)
5.1.4. The MUC1 Cytoplasmic Tail (CT)
The MUC1 COOH-terminal CT region contains multiple potential phosphorylation sites (Fig. 4.10). Specifically, the 72-amino acid human MUC1 CT contains nine serine (S24, S39, S40, S44, S50, S56, S57, S59, and S69), six threonine (T19, T28, T31, T41, T61, and T68), and seven tyrosine residues (Y8,Y20,Y26,Y29,Y35,Y46, and Y60) (numbered beginning with the first residue of the intracellular sequence). Interspecies amino-acid sequence comparisons between 19 mammalian MUC1/Muc1 proteins reveal 15%–100% identities in their intracellular regions, with the majority of potential phosphorylation sites being conserved. Many of the tyrosine residues are located within consensus sequence binding motifs and are constitutively phosphorylated in cancer cells, which may be necessary for interaction of the MUC1 CT with signaling kinases and adapter proteins and progression to the cancer phenotype (Zrihan-Licht et al., 1994; Pandy et al., 1995; Li et al., 1998a, 2001b, 2001c, 2003, 2004; Singh et al., 2007). These include phosphatidylinositol 3-kinase (PI3K) (Y20HPM), Shc (Y26PTY), phospholipase Cγ (PLCγ) (Y35VPP), c-Src and EGFR (Y46EKV), and Grb-2 (Y60TNP). Zrihan-Licht et al. (1994) first suggested that the pattern of MUC1 CT tyrosine phosphorylation was similar to that of some cytokine and growth factor receptors. But unlike cytokine/ growth factor receptors, the MUC1 CT is not capable of autophosphorylation. Other signaling proteins bind to non-tyrosine sites, including GSK3β (D42RSP), PKCδ (T41DRS), and β-catenin (S50AGNGGSSL) (Li et al., 2001b; Ren et al., 2002). Still other binding partners of the MUC1 CT are estrogen receptor α, p53, p120ctn, all ErbB members, adenomatous polyposis coli (APC), HSP70, HSP90 and calcium-modulating cyclophilin ligand (CAML) (Li and Kufe, 2001; Schroeder et al., 2001; Hattrup et al., 2004; Ren et al., 2006; Wei et al., 2006, 2007; Guang et al., 2009). Consensus sequences resembling an ITAM (immunoreceptor tyrosine-based activation motif) and ITIM (immunoreceptor tyrosine-based inhibitory motif) are present in the CT (Gendler, 2001).These latter motifs may be relevant to MUC1 expression by leukocytes. Given the receptor-like structure of the MUC1 protein, there has been interest in identifying possible ligands that may stimulate intracellular signaling upon binding to the MUC1 ectodomain. Among those that have been identified is Pseudomonas aeruginosa. The remainder of this review briefly summarizes the relationship between MUC1 and P. aeruginosa, followed by a detailed discussion of our studies on the role of MUC1 in the airways during the host inflammatory response to lung pathogens.
Dashes (−) indicate identical residues. The red boxes indicate the amino acid sequences for binding of PI3K, PLCγ1, GSK3β, c-Src, β-catenin, and Grb2.The asterisks (*) indicate spaces to maintain sequence alignment. aHomo sapiens (Gendler et al., 1990; GenBank accession no. J05581); bPan troglodytes (NCBI reference sequence XP_003308490.1); cPan paniscus (NCBI reference sequence XP_003817115.1); dPongo abelii (NCBI reference sequence XP_002810115.2); eHylobates lar (Spicer et al., 1995; GenBank accession no. L41624); fPapio anubis (NCBI reference sequence XP_003892818.1); gSaimiri boliviensis (NCBI reference sequence XP_003937852.1); hCallithrix jacchus (NCBI reference sequence XP_002760128.1); iOtolemur garnettii (NCBI reference sequence XP_003804060.1); jMus musculus (Spicer et al., 1991; GenBank accession no. M64928); kRattus norvegicus (DeSouza et al., 1998; GenBank accession no. AAB62948); lOryctolagus cuniculus (Spicer et al., 1995; GenBank accession no. AAB64380); mMesocricetus auratus (Park et al., 1996; GenBank accession no. U36918); nCavia cutleri (Spicer et al., 1995; GenBank accession no. L41546); oSus scrofa (Liu et al., direct submission; GenBank accession no. AAO63589); pBos taurus (Pallesen et al., 2001; GenBank accession no. CAC81810); qOvis aries (NCBI reference sequence XP_004003716.1); rFelis catus (NCBI reference sequence XP_003999784.1); sCanis lupus familiaris (Ishiguro et al., 2007; GenBank accession no. NM_001194977.1).
5.2. MUC1 and P. aeruginosa
5.2.1. P. aeruginosa Lung Infection and Inflammation in Cystic Fibrosis (CF)
P. aeruginosa is an opportunistic pathogen responsible for a wide range of infections, one of the most debilitating being chronic pulmonary infection in CF. CF is a recessive genetic disease with mutations in the CF transmembrane conductance regulator (CFTR) protein, and characterized by abnormal transport of chloride and sodium across respiratory epithelia with concomitant neutrophil-dominated airway inflammation. In CF, non-mucoid strains of the bacteria initially colonize the respiratory tract of patients before converting into mucoid, alginate-producing variants (Hoiby, 1974; Govan and Harris, 1988).The latter are almost exclusively associated with hyperviscous mucous secretions of CF patients. P. aeruginosa bacteria isolated from the airway secretions of CF patients are tightly bound to mucins (Ramphal et al., 1987; Reddy, 1992; Sajjan et al., 1992). P. aeruginosa LPS transcriptionally activates MUC5AC gene expression, providing evidence directly linking bacterial infection to mucus overproduction in CF patients (Li et al., 1998b). Although the exact pathophysiology of P. aeruginosa infection in CF remains to be completely clarified, it is currently thought that the initial stage of colonization involves bacterial adhesion to airway epithelial cells (Woods et al., 1980; Ramphal and Pier, 1985; Saiman et al., 1990, 1992; Saiman and Prince, 1993; Imundo et al., 1995; Zar et al., 1995). DiMango et al. (1995) demonstrated that asialo-gangliosides on the surface of airway epithelial cells are responsible for adhesion of P. aeruginosa. Bacteria binding to the glycolipids stimulated the expression and release of IL-8, a potent neutrophil chemoattractant. Airway epithelial cells in CF patients showed increased levels of surface asialo-gangliosides (Saiman and Prince, 1993). DiMango and coauthors (1998) subsequently reported that the increased expression of IL-8 in CF epithelial cells was caused by activation of a proinflammatory NF-κB signaling pathway.
5.2.2. Adhesion of P. aeruginosa to Muc1-Transfected CHO Cells
Based on the receptor-like structure of MUC1, and the ability of P. aeruginosa to bind to secreted mucins, we hypothesized that MUC1 mucins on the cell surface are sites for bacterial adhesion. Chinese hamster ovary (CHO) cells, which do not express endogenous Muc1 mucin, were stably transfected with the hamster Muc1 cDNA, and binding of P. aeruginosa was examined (Kim et al., 2001; Lillehoj et al., 2001). CHO-Muc1 cells expressed both Muc1 mRNA and protein based on Northern and Western blot analyses, and Muc1 protein on the cell surface was degraded by NE. CHO-Muc1 cells exhibited significantly increased bacterial adhesion compared with cells transfected with the empty vector (CHO-X) using both the mucoid CF3 and the non-mucoid P. aeruginosa strain K (PAK) bacterial strains. Additionally, adhesion of P. aeruginosa was completely abolished by proteolytic cleavage of Muc1 from the cell surface using NE, or by deletion of the Muc1 extracellular domain. These results provided a model system for studying epithelial cell responses to bacterial adhesion that leads to airway inflammation in general and CF in particular.
Adhesins are microbial components that facilitate bacterial adhesion to host cells and are required for colonization of mucosal surfaces (Coutte et al., 2003). Because previous studies had identified P. aeruginosa flagellin and pilin, the structural proteins of flagella and pili respectively, as bacterial adhesins for binding to airway epithelial cells, we studied the proteins that were responsible for P. aeruginosa binding to CHO-Muc1 cells using genetic and biochemical approaches. CHO-Muc1 and CHO-X cells were compared for adhesion of PAK (Lillehoj et al., 2002). Wild-type PAK and isogenic mutant strains lacking pili (PAK/NP) or flagella cap protein (PAK/fliD−) had significantly increased adherence to CHO-Muc1 cells, whereas flagellin-deficient (PAK/fliC−) bacteria were equally adherent to both cell types. Further, P. aeruginosa binding to CHO-Muc1 cells was blocked by pretreatment of the bacteria with an antibody to P. aeruginosa flagellin, or by pretreatment of CHO-Muc1 cells with purified flagellin. Thus, flagellin is an adhesin of P. aeruginosa that was responsible for its binding to Muc1 mucin on the epithelial cell surface.
5.2.3. Adhesion of P. aeruginosa to Airway Epithelial Cells
Further studies were necessary to extend these results to human airway epithelial cells. More specifically, Muc1 overexpressed in CHO cells exhibits an uncommon pattern of O-glycosylation (Bäckström et al., 2003), and the tandem repeats of human MUC1 (GSTAPPAHGVTSAPDTRPAP) and hamster Muc1 (GSSAPVTSSATNAPTTPVHS) share only eight common amino acids (underlined). The knockdown of MUC1 expression in bronchial (NuLi-1) or alveolar (A549) epithelial cells by small interfering (si)RNA significantly reduced PAK binding to the cells compared with a negative control siRNA using two independent bacterial adhesion assays (Kato et al., 2010). Conversely, overexpression of MUC1 in human embryonic kidney (HEK)293T cells, which do not express MUC1 endogenously, increased bacterial adherence compared with MUC1-non-expressing cells. By confocal microscopy, P. aeruginosa and MUC1 were colocalized on the surface of NuLi-1 cells. Combined with our prior studies, these results suggest that MUC1 serves as a binding site for P. aeruginosa bacteria on the surface of airway epithelial cells. This conclusion is consistent with prior studies that have documented MUC1/Muc1 as an epithelial adhesion site for Helicobacter pylori (Lindén et al., 2004; McGuckin et al., 2007; Costa et al., 2008; Lindén et al., 2009), Campylobacter jejuni (McAuley et al., 2007), Escherichia coli (Sando et al., 2009; Parker et al., 2010), and Salmonella enterica (Parker et al., 2010).
5.3. Modulation of Innate Immune Response by MUC1
5.3.1. Anti-inflammatory Role of MUC1 in the Airways
Bacterial adhesion to the airway epithelial cell surface receptors often activates intracellular signaling pathways that culminate in the expression of a variety of host mediators that drive innate and acquired immune responses to neutralize bacterial colonization (Rastogi et al., 2001; Gómez and Prince, 2008). Because our prior studies identified MUC1/Muc1 as an adhesion site for P. aeruginosa (Kim et al., 2001; Lillehoj et al., 2001; Kato et al., 2010), we initially predicted that airway proinflammatory responses would be attenuated in the setting of reduced or deficient MUC1/Muc1 expression. To test this hypothesis, Muc1-knockout (Muc1−/−) mice and their wild-type littermates (Muc1+/+) were experimentally infected in the airways with P. aeruginosa and the degree of lung inflammation was compared in the two mouse strains at 4 h postinfection (Lu et al., 2006). Surprisingly, Muc1−/− mice showed increased proinflammatory cytokine (TNF-α) and chemokine (KC, mouse ortholog of human IL-8) levels in BALF compared with Muc1+/+ mice. Correspondingly, increased numbers of neutrophils were seen in BALF of Muc1−/− vs. Muc1+/+ mice. Muc1−/− mice also had higher levels of TNF-α and KC in BALF following in vivo treatment with purified P. aeruginosa flagellin, greater TNF-α levels in spent cell culture media of alveolar macrophages treated in vitro with flagellin, and higher levels of KC in media of primary TE cells treated with flagellin compared with Muc1+/+ mice and cells. Finally, a MUC1-targeting RNA interference approach was utilized to rule out the possibility that these results were due to genetic compensation by other molecules in Muc1−/− mice. The knockdown of MUC1 by siRNA in primary NHBE cells enhanced flagellin-induced IL-8 production compared with the negative control siRNA. In conclusion, these results suggested that MUC1 plays an anti-inflammatory role during airway P. aeruginosa infection.
5.3.2. MUC1 Inhibition of TLR5 Signaling
P. aeruginosa flagellin engages TLR5 to stimulate downstream signaling and innate immune responses (Zhang et al., 2005). Because TLR5 and MUC1/Muc1 are flagellin receptors and both are expressed by airway epithelial cells, we speculated that these two signaling molecules may crosstalk through their common ligand. To explore the relationship between MUC1 and TLR5 in response to flagellin, HEK293T cells were stably transfected with a MUC1-expressing plasmid, and flagellin-driven IL-8 production was determined following the transient transfection with a TLR5-expressing plasmid, or with an empty vector negative control (Lu et al., 2006). Flagellin-treated HEK293T cells transfected with the empty vector control had a 55% greater IL-8 production compared with PBS-treated cells, due to low levels of endogenous TLR5. Flagellin-treated cells transfected with TLR5 had a 164% greater IL-8 production compared with control-treated cells. However, cotransfection of the cells with TLR5 plus MUC1 completely abolished the increased IL-8 production in response to flagellin treatment (Kato et al., 2012). Cotransfection of the cells with TLR5 plus a MUC1 CT deletion mutant restored flagellin-driven IL-8 production that was equal to that of cells expressing TLR5 alone. In summary, these data indicated not only that overexpression of MUC1 in HEK293T cells inhibited flagellin-dependent TLR5 signaling, but also that this effect was mediated by the MUC1 CT.
A number of reports indicated that PI3K suppresses inflammation during the early stage of bacterial infection (Fukao and Koyasu, 2003). For example, Yu et al. (2006) demonstrated that TLR5-mediated PI3K activation negatively regulated flagellin-induced proinflammatory gene expression in the human colon epithelial cell line, T84. Phosphoinositides in the inner leaflet of the plasma membrane are produced by activated PI3K, leading to membrane recruitment of Akt. Phosphorylation of Akt by PDK1 and TORC2 stimulates a diverse array of downstream cellular activities, including increased cellular proliferation and survival, and decreased TLR signaling. Because the MUC1 CT contains a consensus PI3K binding site at Y20HPM, we next focused on the possible role of PI3K and Akt in mediating the suppressive effect of MUC1 on flagellin-induced proinflammatory response in the airway epithelial cells (Kato et al., 2007). HEK293T cells overexpressing a CD8/MUC1 chimeric protein were utilized for these experiments. CD8/MUC1 contains the extracellular and transmembrane domains of CD8 and the MUC1 CT (Meerzaman et al., 2000).Treatment of CD8/MUC1-HEK293T cells with anti-CD8 antibody stimulates MUC1 CT tyrosine phosphorylation, including Y20 (Wang et al., 2003).Treatment of CD8/MUC1-HEK293T cells with anti-CD8 antibody stimulated recruitment of the PI3K regulatory subunit p85 to the MUC1 CT and increased Akt phosphorylation (Kato et al., 2007). MUC1-PI3K interaction and Akt phosphorylation did not occur in cells expressing a tyrosine-to-phenylalanine substitution at the critical Y20 residue. However, mutation of Y20, or pharmacological inhibition of PI3K by wortmannin, failed to block MUC1-induced suppression of flagellin-induced TLR5 signaling. It was concluded that whereas PI3K was downstream of MUC1 activation and negatively regulated TLR5 signaling, PI3K was not responsible for MUC1-induced counter-regulation of TLR5 signaling.
5.3.3. MUC1/Muc1 Inhibition of General TLR Signaling
Two experimental systems were used to evaluate the ability of MUC1/Muc1 to inhibit general TLR signaling, Muc1 knockout mice and HEK293T cells overexpressing human MUC1. Peritoneal and alveolar macrophages from Muc1+/+ and Muc1−/− mice were treated with agonists for TLR2 (Pam3Cys), TLR3 (polyI:C), TLR4 (LPS), TLR7 (loxoribine), or TLR9 (CpG DNA), and TNF-α levels in spent culture media were measured by ELISA (Ueno et al., 2008). Macrophages from Muc1−/− mice produced significantly higher levels of TNF-α in response to all ligands tested compared with cells from Muc1+/+ mice. Identical results were seen using mouse primary TE cells treated with the TLR2 agonist. Next, we examined the effect of MUC1 expression on TLR-driven activation of NF-κB, a proinflammatory transcription factor that undergoes nuclear translocation following ligand engagement of TLRs (Kawai and Akira, 2007). HEK293T cells were transiently transfected with plasmids encoding an NF-κB-luciferase reporter gene and TLR2, TLR3, TLR4, TLR5, TLR7, or TLR9 in the presence or absence of MUC1. The cells were treated with the respective agonists and relative luciferase activity was measured. NF-κB activation was drastically induced by all TLR ligands tested, and the increased NF-κB activation was suppressed by MUC1 in a dose-dependent fashion in all cases. Finally, RAW264.7 mouse macrophages endogenously expressing TLRs were transiently transfected with plasmids encoding full-length MUC1, deletion mutants containing only the MUC1 EC or CT regions, or an empty vector control prior to stimulation with various TLR ligands and measurement of TNF-α levels in culture supernatants. While empty vector-transfected RAW264.7 cells responded to all the TLR ligands tested with a significant increase in TNF-α levels, the cells transfected with full-length MUC1 showed a dose-dependent suppression of TLR-driven TNF-α production. Identical results were observed in cells transfected with the MUC1 EC deletion mutant that still retained the MUC1 CT region. However, the inhibitory effect was lost in cells transfected with the MUC1 CT deletion mutant. In summary, these results indicated that MUC1 is a universal negative regulator of TLR signaling and that the CT domain of MUC1 is required for its anti-inflammatory effect.
5.3.4. Inhibition of Lung Inflammation in Response to Respiratory Syncytial Virus (RSV ) and Nontypeable H. Influenzae (NTHi)
Prior studies by ourselves (Lu et al., 2006; Guang et al., 2010) and others (DeSouza et al., 1999; Kardon et al., 1999; McGuckin et al., 2007) have demonstrated that Muc1−/− mice display enhanced inflammatory responses to a variety of bacterial pathogens compared with Muc1+/+ littermates. To assess the role of MUC1/Muc1 in the pulmonary response to microbial pathogens other than P. aeruginosa, we conducted additional studies using respiratory syncytial virus RSV and NTHi infection, two major human airway pathogens. In the first investigation, A549 cells were treated with RSV and the levels of TNF-α and MUC1 proteins were monitored temporally during the course of infection by ELISA and Western blot analysis, respectively (Li et al., 2010). Following RSV infection, an early increase in TNF-α levels in culture supernatants was followed by a later increase in MUC1 levels in cell lysates, suggesting that the increased TNF-α may stimulate increased production of MUC1. This supposition was supported by the observation that pretreatment of the cells with soluble TNF-α receptor (TNFR) inhibited RSV-induced MUC1 protein expression. The knockdown of MUC1 expression by siRNA, but not by a negative control siRNA, increased RSV-stimulated TNF-α levels. Conversely, MUC1 overexpression decreased TNF-α levels production compared with normal MUC1 expressing cells.
In the second study, treatment of A549 cells with a lysate of NTHi increased early IL-8 levels and later MUC1 protein levels in dose- and time-dependent manners, compared with cells treated with the vehicle control (Kyo et al., 2011). Both effects were attenuated following transfection of the cells with a TLR2-targeting siRNA, compared with a non-targeting control siRNA. NTHi-induced IL-8 release in A549 cells was suppressed by MUC1 overexpression and enhanced by MUC1 knockdown. NHTi-induced TNF-α release upregulated MUC1 protein levels, which was completely inhibited by pretreatment with a soluble TNFR. Finally, primary TE cells from Muc1−/− mice exhibited increased in vitro NTHi-stimulated KC production compared with TE cells from Muc1+/+ mice. We concluded that NTHi-induced TNF-α production upregulated MUC1 protein expression through its interaction with TNFR, which in turn suppressed further increases in TNF-α levels. Combined with the results for the RSV infection model system, these results suggested a hypothetical feedback loop model whereby airway pathogens activate TLR on airway epithelial cells, leading to an early increase in TNF-α and IL-8 production, which subsequently upregulate MUC1 expression, leading to later suppression of TLR signaling and decreased cytokine and chemokine production. Further details of this theoretical feedback loop mechanism can be found in our prior review articles (Kim and Lillehoj, 2008; Kim, 2012; Kim et al., in press).
5.4. Molecular Mechanism of Crosstalk between MUC1 and TLRs—Current Working Model
5.4.1. TNF-α is a Key Regulator of MUC1 Expression during Airway P. aeruginosa Infection
TNF-α, a major proinflammatory mediator during airway infection, upregulated MUC1 expression (Koga et al., 2007) in airway epithelia and was required for NE-induced MUC1 upregulation (Kuwahara et al., 2007). To assess the contribution of TNF-α for increased MUC1 levels during airway infection, Muc1−/− and TNFR−/− mice, and their wild type littermates, were infected with P. aeruginosa and TNF-α and MUC1 levels were monitored at various times postinfection (Choi et al., 2011). Muc1 levels in uninfected Muc1+/+ mice lungs were relatively low and increased steadily postinfection, reaching maximum levels at 2–4 days before returning to baseline levels at day 7. However, TNFR−/− mice failed to upregulate Muc1 expression following P. aeruginosa infection. Additionally, greater numbers of inflammatory cells were present in the BALF of Muc1−/− or TNFR−/− mice compared with their wild-type controls. Both Muc1−/− and TNFR−/− mice were unable to resolve bacteria-induced lung inflammation. These results not only supported the previous observations that TNF-α upregulated MUC1 expression (Koga et al., 2007), but also confirmed that TNF-α production was required for P. aeruginosa-induced Muc1 upregulation. Further, these data allowed us to answer a critical question on the role of MUC1 in the airways, namely whether the anti-inflammatory activity of MUC1 is beneficial or harmful during bacterial lung infection. It is likely that the anti-inflammatory role of MUC1 comes into play at a late stage of infection, mainly as a result of the increased levels of TNF-α produced at the early stage. In summary, the existence of a negative feedback loop during TLR-driven airway inflammation involving TNF-α (pro-inflammatory) and MUC1 (anti-inflammatory) provides a novel mechanism of control to prevent hyperinflammatory airway diseases. This hypothetical mechanism is schematically illustrated in Fig. 4.11.
(A) During acute P. aeruginosa (PA) lung infection, TLR5 is the key receptor for bacterial flagellin and triggers MyD88-dependent signaling to induce inflammatory mediators including TNF-α that result in recruitment of leukocytes into the site of infection to clear the bacteria. (B) During the inflammation phase, TNF-α up-regulates MUC1 and EGFR expression through the TNFR (Burgel and Nadel, 2008; Choi et al., 2011). Activation of EGFR by TLRs and/or alternative mechanisms stimulates phosphorylation of the MUC1 CT at the Y46 residue. (C) During the resolution phase, Y46-phosphorylated MUC1 CT interacts with the intracellular region of TLR5, thereby blocking recruitment of MyD88 and inhibiting inflammatory signaling (Kato et al., 2012). N, neutrophil; M, macrophage. (For color version of this figure, the reader is referred to the online version of this book.)
5.4.2. MUC1 Associates with TLR5 to Inhibit Recruitment of MyD88
An important question that is only beginning to be answered is how the increased levels of MUC1 can suppress TLR signaling during airway infection and inflammation. To specifically address the molecular mechanism through which MUC1 counter-regulates TLR-dependent inflammation, we conducted several experiments examining protein–protein interaction between MUC1, TLR5, and MyD88, a major adapter protein involved in TLR signaling (Kato et al., 2012). Overexpression of MUC1 in HEK293T cells dramatically reduced P. aeruginosa- and flagellin-stimulated IL-8 expression, and decreased NF-κB, ERK1/2, and p38 activation, compared with MUC1 nonexpressing cells. Overexpression of MUC1 in HEK293T cells, however, did not affect NF-κB or MAPK activation in response to TNF-α. Since the TLR5 and the TNF-α signaling pathways are initiated with two different receptors, but converge at the point of activation of TAK to share a common downstream signaling pathway, namely, NF-κB and MAPK activation, these results suggested that the site of interaction between TLR5 and MUC1 should be between TLR5 and TAK activation, i.e. either MyD88, IRAK1, or TRAF6. Overexpression of MyD88, but neither IRAK1 nor TRAF6, abrogated the ability of MUC1 to inhibit NF-κB activation, and MUC1 overexpression inhibited flagellin-induced association of TLR5 with MyD88, compared with the respective controls. Finally, association between the MUC1 CT and TLR5 was demonstrated in HEK293T and A549 cell lines, as well as in human and mouse primary airway epithelial cells.
5.4.3. Activation of EGFR Increases Association of MUC1 with TLR5
The EGFR receptor tyrosine kinase regulates innate immune responses in the airways, including mucin secretion by goblet cells, and chemokine production and proliferation by epithelial cells (Burgel and Nadel, 2008). The EGFR ligand, TGF-α, is synthesized in a latent form as a membrane-tethered precursor protein on the surface of airway epithelial cells where it undergoes proteolytic cleavage by TACE in response to P. aeruginosa LPS and activation of a TLR4 → dual oxidase → ROS signaling cascade. Based on this information, we performed several experiments to investigate the relationship between TGF-α, MUC1/Muc1, and TLR5 in P. aeruginosa-treated airway epithelial cells (Kato et al., 2012). Activation of EGFR by soluble TGF-α stimulated phosphorylation of the MUC1 CT at the Y46EKV sequence and increased the association between MUC1 CT and TLR5 in A549 cells. Finally, in vivo experiments demonstrated increased immunofluorescence colocalization of Muc1 with TLR5 and greater Muc1 phosphotyrosine immunostaining patterns in mouse airway epithelium, as well as augmented Muc1 tyrosine phosphorylation in the mouse lung homogenates, following P. aeruginosa infection. Taken together with the data presented in Section 5.4.2, these combined results suggested that EGFR phosphorylates the MUC1 CT, leading to increased MUC1-CT/TLR5 association, thereby competitively inhibiting recruitment of MyD88 to TLR5. As discussed above, both TNF-α and TGF-α are produced during the airway P. aeruginosa infection. Therefore, it is tempting to speculate that simultaneous upregulation of MUC1 and activation of EGFR in the vicinity of an ongoing inflammatory response facilitates the sequential steps of EGFR-mediated tyrosine phosphorylation of the MUC1 CT and MUC1/TLR5 interaction that precede resolution of airway inflammation.
5.5. Perspective
Although the role of MUC1 mucin in the airway remains to be completely elucidated, the fact that MUC1 is present on the surface of the airway epithelial cells is extremely interesting in view of its receptor-like characteristics. Our recent findings that MUC1/Muc1 served as an adhesion site for P. aeruginosa in human and animal model systems, and that P. aeruginosa induced phosphorylation of the MUC1 CT domain, strongly suggest a possible role for this mucin in the regulation of bacterial-driven inflammation. Further, a growing body of evidence suggests that MUC1 acts during the resolution phase of inflammation through its ability to inhibit TLR signaling in the lung. It remains to be determined whether the MUC1 ectodomain-dependent airway epithelial binding properties of P. aeruginosa, and the counter-regulatory effects of the MUC CT on acute P. aeruginosa inflammation, can be extended to other airway pathogens. Additionally, in the context of the pathophysiological role of MUC1 in the lung, it is intriguing to ask whether the level MUC1 expression, or the intactness of its CT domain, is associated with the genesis of chronic inflammatory lung disease. In this regard, our recent report, using an in vivo model of murine repetitive airway P. aeruginosa infection, demonstrated that Muc1 deficiency exacerbated airspace enlargement compared with Muc1-expressing mice (Umehara et al., 2012). Given that MUC1 promotes a diverse array of signal transduction pathways that are associated with cell proliferation and the anti-apoptotic response (Kufe, in press), it is not unreasonable to propose that MUC1 may also regulate the postinjury repair response of airway epithelial cells.
6. CONCLUDING REMARKS
Although the role of MUC1 mucin in the airways remains to be fully determined, the fact that it is present on the surface of the airway epithelial cells is extremely interesting in view of its receptor-like characteristics. Our recent finding that extracellular P. aeruginosa can stimulate the phosphorylation of the MUC1 intracellular domain suggests a possible role for this mucin in the host response to microbial infection. With the rapidly and constantly accumulating information on the cellular signaling pathways involved in various cell functions, the information obtained from future studies hopefully will enable us to predict other possible functions of MUC1 in epithelial cell biology in general, and in airway epithelia in particular. Pharmacological strategies that lead to better treatments and prevention of airway infection and inflammation are expected benefits of these future studies.
Acknowledgments
The work cited in this manuscript has been funded by grants from the National Institutes of Health (AI073988, HL047125, HL049362, HL063742, and HL081825), the Cystic Fibrosis Foundation, the American Lung Association, and the Maryland Industrial Partnerships. The authors wish to thank all of our previous colleagues and coworkers who have contributed to this work.
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