ABSTRACT
Mucus lining the airway luminal surface serves as a primary physicochemical barrier for the lung against airborne particles and chemicals. The major constituent of mucus responsible for this property is a mixture of glycoproteins collectively referred to as mucins. Mucins are produced by two types of cells in the airway-goblet cells in the surface epithelium and mucous cells in the submucosal gland. At present, 13 genes have been identified as mucin-producing genes, five of which have been shown to be expressed in the airway (1)—MUC1, MUC2, MUC4, MUC5/5AC, and MUC5B. Mucins derived from MUC genes are secreted into the airway lumen, except for MUC1 mucin, which is present as a transmembrane glycoprotein on the surface of secretory cells (2). Although it seems likely that MUC1 mucin is also released into the lumen, as previously shown for other cell types including various tumor cells (3) and primary uterine epithelial cells (4), its contribution to the total amount of the secreted mucins in the airway appears to be negligible (5). Excellent reviews are available elsewhere 217
(2,6). In this chapter, we will focus on the potential role of MUC1 mucins in the mucociliary clearance of the airway with some background information on the structure and function of MUC1 mucins. By convention, human mucin genes will be referred to as MUC and nonhuman mucin genes as Muc throughout this chapter. STRUCTURE AND FUNCTION OF MUC1 MUCIN
MUC1 mucins are highly glycosylated transmembrane proteins with high molecular mass and are widely expressed on the apical surface of most secretory epithelial cells. The human MUC1 gene is localized on chromosome lq21-24 (7), and its deduced amino acid sequence indicates four characteristic domains-the N-terminal signal sequence, the extracellular (EC) domain containing tandem repeats, the transmembrane (TM) domain, and the C-terminal cytoplasmic (CT) domain (8-12). A schematic structure of MUC1 mucin is seen in Fig. 1. The EC domain contains 20-amino-acid tandem repeats that seem to occur 21-125 times as result of genetic polymorphism (13). In contrast, the EC domain of the hamster Mucl gene contains only 12 tandem repeats of 20 amino acids (14). This repetitive region and the regions adjacent to it comprise most of the EC portion of the molecule, extending 200-500 nm above the plasma membrane (15). The C-terminal CT domain contains 69 (human) or 68 (hamster) amino acids with a high percentage of tyrosine, serine, and threonine residues, which are potential phosphorylation sites. While the EC domain shows low sequence identity among species, the amino acid sequence of the CT domain is very similar (88%) (14). Based on their anatomical location and numerous phosphorylation sites, MUC1 mucins have been suggested to function as a receptor. Cell surface
mucins are abundantly expressed in most carcinoma cells and have been shown to reduce intercellular (15,16) as well as matrix adhesion (17) through their extended and rigid structure (15). They are also expressed by normal epithelial cells in various glandular epithelial tissues, including the respiratory, gastrointestinal, and female reproductive tracts (18-20), and have been shown to be associated with cell differentiation in mammary (21), uterine epithelial (20), and airway epithelial cells (14). Similar to cancer cells, Mucl mucins in uterine epithelial cells have been shown to exhibit an antiadhesive property, suggesting that they play an important role in maintaining the prereceptive phase in the uterus (22). The role of MUC1 mucins in the airway, however, is unknown. MUC1 MUCINS AND INTRACELLULAR SIGNALING
The deduced amino acid sequence of MUC1 mucin reveals that some of the tyrosine residues present in the CT domain of MUC1 mucins, once phos-phorylated, constitute docking sites for proteins known to be involved in signal transduction, such as PI-3'-kinase, PLCyl, Src and Grb2. Despite the presence of potential adaptor protein docking sites, MUC1 mucins are different from the typical receptor tyrosine kinase (RTK) in that they do not contain autophosphorylation sites. In this aspect, MUC1 mucins rather resemble the structure of a prototypical cytokine receptor known to have no autophosphorylation activity. Unlike the cytokine receptors, however, MUC1 mucins do not contain consensus sequence motifs known for JAKs (Janus-kinases). Furthermore, judging from their structure, it seems quite unlikely that MUC1 mucins can form a dimer as do RTK molecules following ligand-receptor binding. Collectively, it seems unclear at this point to which of the known receptor types MUC1 mucins belong.Recently, Zrihan-Licht et al. (23) demonstrated, for the first time, the presence of phosphorylation on the CT domain of MUC1 mucins in a breast cancer cell line. Using the same cell line, Pandey et al. (24) showed that MUC1 mucins can directly interact with the SH2 domain of an adaptor protein, Grb2. They also demonstrated that the MUCl/Grb2 complex associates with a guanine nucleotide exchange protein, Sos, supporting a role for MUC1 mucin in intracellular signaling. A ligand responsible for tyrosine phosphorylation of the CT domain of MUC1 mucins has been identified in the breast cancer cell as the EC domain of MUC1 mucins containing a tandem repeat array (25). The signaling mechanism of MUC1 mucins, however, remains to be uncovered. MUC1 MUCINS PRODUCED BY AIRWAY EPITHELIAL CELLS
Primary hamster tracheal surface epithelial (HTSE) cells grown on a thick collagen gel synthesize and secrete mucins at confluence (26,27). This HTSE cell
culture system has been used extensively for studying the regulation of goblet cell mucin secretion (28). Many years ago we made an interesting observation that human neutrophil elastase can release mucins from HTSE cells (29). Some of these released mucins were derived from the apical surface of goblet cells, indicating for the first time the presence of mucins on the surface of airway goblet cells. Further characterization of the plasma membrane of HTSE cells revealed the presence of two different types of membrane mucins-one almost indistinguishable from secreted mucins and the other immunoprecipitable with anti-MUC1 antibody (30). We then cloned a full-length Mucl cDNA from our HTSE cell cDNA library and found that expression of Mucl mRNA correlated with goblet cell differentiation (14). The degree of its expression in HTSE cells was very high and almost comparable to that of MCF-7 cells (a breast cancer cell line). Based on both the localization and molecular structure of Mucl mucins in the airway, it seemed highly likely that they serve a receptor function in airway epithelial cells. PSEUDOMONAS ADHESION TO EPITHELIAL CELLS
Pseudomonas aeruginosa (PA) is an opportunistic pathogen responsible for a wide range of infections, one of the most debilitating being chronic pulmonary infection in cystic fibrosis (CF) patients. In CF, nonmucoid strains of PA initially colonize the upper respiratory tract of patients before converting into mucoid alginate-producing variants (31). The latter are almost exclusively associated with hyperviscous bronchial secretions of CF patients. A plethora of information has amassed with respect to the interactions between PA and airway mucus (32). Although the exact pathophysiology of PA infection in CF is still unclear, it is currently thought that the initial stage of infection involves adhesion to airway epithelial cells (33) through asialoglycolipids present on the cell surface (34). It was recently shown that adhesion of PA to these glycolipids on airway epithelial cells results in translocation of NF-kB and initiation of IL-8 expression (35), indicating an important role in initiating an epithelial proinflammatory response to PA adhesion. MUC1 MUCINS ARE ADHESION SITES FOR PSEUDOMONAS AERUGINOSA
Based on the structure of MUC1 mucins and their location in the airway, we hypothesized that Mucl mucins may serve as an adhesion site for PA. In testing the hypothesis, we first established a cell line that expresses both Mucl mRNA and protein by stable transfection of a full-length cDNA of hamster Mucl (14) into CHO cells. In contrast to CHO-Mucl cells, the parent CHO cells do not express Mucl. Our binding experiments revealed that PA adhesion to CHO-Mucl
cells was significantly greater than to CHO cells and the increase in PA adhesion to CHO-Mucl cells was completely abolished following treatment with neutrophil elastase (Fig. 2). Since neutrophil elastase cleaves the EC domain of Mucl mucins (data not shown), as predicted from our previous publication (29), these results indicate that Mucl mucins serve as an adhesion site for PA. A POTENTIAL ROLE OF MUC1 MUCINS IN THE AIRWAY
The physiological significance of PA adhesion to MUC1 mucins remains unknown. Transiently inspired PA are normally trapped by airway secretions and removed by mucociliary clearance mechanisms. It might be possible that MUC1 mucins serve as a secondary defense barrier against airborne bacteria that manage to escape the gel phase of airway mucus, the primary physical barrier. Bacteria “ trapped” by MUC1 mucins might be cleared from the airway by some as yet unidentified proteolytic cleavage. In other membrane receptor-ligand systems, it has been demonstrated that ligand binding induces intracellular signaling events, ultimately leading to release of the receptor from the cell surface. For example,
Fan and Derynck (37) recently showed that growth factor activation of cell surface RTKs induces protease-mediated release of the EC domain of TGF-a through activation of the ERK MAP kinase signaling pathway. As discussed above, MUC1 mucin interaction with Grb2 and Sos proteins similarly implicates its association with a MAP kinase pathway. It is tempting to speculate that PA adhesion to cell-associated MUC1 mucin activates one or more intracellular MAP kinase cascades and that one of the resulting consequences is release of the MUC1 mucin-PA complex from epithelial cells and clearance from the airway. Our preliminary data showed that PA adhesion to CHO-Mucl cells results in an increase in tyrosine phosphorylation of Mucl mucins (data not shown), strongly suggesting a role of Mucl mucins in airway infection and inflammation. REFERENCES
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