MUC1/Muc1 expression counter-regulates airway inflammation during Pa infection (6
), and this anti-inflammatory activity is attributed to its ability to suppress TLR5 signaling (6
). However, the mechanism of crosstalk between TLR5 and MUC1/Muc1 is unknown. In this report, we now demonstrate the suppressive effect of MUC1 expression on TLR5-dependent IL-8 promoter activity (), as well as NF-κB and MAPK activation (), that correlated with reduced TLR5/MyD88 association () and increased MUC1/TLR5 association (, ). NF-κB inhibition was completely reversed upon MyD88 overexpression (). Increased MUC1 CT phosphorylation, likely at the Y46
EKV site, and greater MUC1/TLR5 association were associated with TGF-α-dependent EGFR activation (). In vivo
experiments established increased Muc1 CT tyrosine phosphorylation in mouse lung homogenates following Pa infection () and greater Muc1/TLR5 and Muc1/phosphotyrosine immunofluorescence co-localization in infected mouse airway epithelium (). Together, these results suggest a mechanism whereby EGFR tyrosine-phosphorylates the MUC1 CT, thus increasing its association with TLR5 and competitively and reversibly inhibiting recruitment of MyD88 to TLR5 and subsequent proinflammatory signal transduction.
Flagellin binding to the TLR5 ectodomain induces receptor homodimerization resulting in a protein conformational change in its CT domain and allowing recruitment of the MyD88 adapter protein (22
). The data presented in this study indicate that MUC1 mediates its anti-inflammatory effects at the level of the TLR5 intracellular domain, and are entirely consistent with the previous publication demonstrating that transfection of RAW264.7 cells with MUC1ΔCT, but not MUC1ΔEC, blocked its ability to inhibit TLR-driven TNF-α production (18
). This effect was achieved not only with the MyD88-dependent TLR2, TLR4, TLR7, and TLR9, but also with TLR3 which signals through TRIF rather than MyD88. Given that all TLRs and many adaptor proteins share the conserved TIR domain mediating homo- and heterotypic interactions, it seems very likely that MUC1 suppresses TLR signaling by associating with receptor TIR domains, thus acting as a steric hindrance/decoy receptor and excluding recruitment of MyD88 and TRIF to their respective receptors. Interestingly, the MUC1 CT contains an amino acid sequence (R17
DTYHP) that is homologous to the consensus RDXΦ1
G motif (where Φ represents a hydrophobic residue, and X any residue) of the TIR domain and that has been shown to be responsible for heteromeric interaction between TLR2/4 and MyD88 (28
). However, the possible role of the MUC1 CT “TIR domain-like” sequence in TLR signal transduction remains speculative.
EGFR regulates innate immune responses in the airways, including mucin secretion by goblet cells, and chemokine production and proliferation by epithelial cells (29
). As with all EGFR ligands, TGF-α is synthesized in a latent form as a membrane-tethered precursor protein on the surface of airway epithelial cells. Proteolytic cleavage of pro-TGF-α by TNF-α converting enzyme (TACE) precedes EGFR activation and proinflammatory signaling (30
). Like TGF-α, TACE is initially synthesized in an inactive form that is activated by a variety of diverse stimuli, including airway bacterial pathogens (31
), cigarette smoke (32
), and reactive oxygen species (ROS) (33
), the latter of which are up-regulated by TLRs and dual oxidase (33
). On the basis of the current results, a second function can now be ascribed to activated EGFR apart from proinflammatory signaling, namely tyrosine phosphorylation of the MUC1 CT and MUC1/TLR5 protein interaction. Of note, airway epithelial cell expression levels of both MUC1 and EGFR are increased by a common proinflammatory cytokine, TNF-α (8
). It is tempting to speculate that simultaneous up-regulation of MUC1 and EGFR in the vicinity of an ongoing inflammatory response facilitates sequential steps of EGFR-mediated tyrosine phosphorylation of MUC1, MUC1/TLR5 interaction, and counter-regulation of airway inflammation.
In addition to its anti-inflammatory properties mediated through its CT, a growing body of evidence suggests that the MUC1 extracellular regions contribute to the pathogenesis of microorganisms that colonize and infect mucosal surfaces (36
). MUC1/Muc1is an extracellular adhesion site for Pa
on airway epithelia (19
), and for Escherichia coli
and Salmonella enteric
on intestinal epithelia (39
). Helicobacter pylori
binds to the MUC1 ectodomain on gastric epithelial cells and MUC1 ectodomain shedding acts as a releasable decoy to block infection by this pathogen (36
). McAuley et al
) demonstrated that Muc1−/−
mice are more susceptible to infection by gastrointestinal Campylobacter jejuni
compared with Muc1+/+
littermates. Increased bacterial colonization by C. jejuni
was accompanied by severe epithelial damage and exaggerated penetration through the intestinal barrier, eventually resulting in systemic infection. As originally proposed by Gendler (43
), the heterodimeric nature of the MUC1 protein may provide a mechanism for rapid ectodomain shedding that concurrently signals to the cell interior, through its CT, the presence of an invading pathogen. While Muc1 acts as a decoy receptor for invading bacteria in the intestinal tract, it concurrently plays an anti-inflammatory role in the respiratory tract by a discrete mechanism not involving its ectodomain. These multidimensional effects may relate to the functional difference between the two organs - the former constituting an impenetrable barrier against commensal bacteria, whereas the latter dealing with the timely resolution of inflammation in order to maintain its vital function of gas exchange.
It is also apparent that the MUC1 CT exhibits functional activities apart from its ability to directly block TLR signaling. Ahmad et al
) demonstrated that the MUC1 C-terminal subunit promotes TNF-α-induced activation of NF-κB in human breast cancer cells. In contrast, we reported that the MUC1 CT binds to the IKKγ subunit to inhibit H. pylori
-dependent NF-κB activation in a human gastric cancer cell line (46
). It is currently unknown whether IKK/NF-κB are recruited to the inner leaflet of the plasma membrane by the MUC1 CT, or whether the membrane-bound CT is released into the cytoplasm to interact with IKK/NF-κB. Nonetheless, apically-polarized MUC1 presumably favors its ability to interact with TLR5 and, hence, selectively suppress TLR5-induced NF-κB activity per se
. Interestingly, exposure of polarized airway epithelial cells to cigarette smoke re-distributed apical MUC1 into the cytosol, suggesting that exogenous insults can affect the subcellular localization of MUC1 and, by implication, its functional properties in the lung (47
). Elucidation of the factors controlling MUC1 cellular and functional heterogeneity deserves further investigation.
In conclusion, we propose the following sequence of events during airway Pa infection in the context of the anti-inflammatory role of MUC1/Muc1 (). Host defense against the pathogen is mediated primarily by mucociliary clearance and phagocytosis. During the early stage of infection, TLR5 on respiratory epithelial cells (and, perhaps, resident macrophages) sense Pa through its interaction with flagellin (step 1) and triggers MyD88-dependent signaling (step 2) to induce inflammatory mediators which result in recruitment of leukocytes into the site of infection to clear the bacteria. The inflammatory products generated during this process, such as neutrophil elastase and TNF-α (8
) (step 3), up-regulate MUC1/Muc1 expression during the late stage of infection following clearance of the pathogen from the airways. Activation of EGFR by TLRs and/or alternative mechanisms, stimulates phosphorylation of the MUC1 CT at Y46
(step 4), leading to MUC1/TLR5 interaction (step 5), thus interfering with the recruitment of MyD88 to TLR5 and down-regulating the inflammatory response. Ongoing studies in our laboratory are directed at testing this hypothetical model.
Figure 9 Schematic illustration of the proposed mechanism through which MUC1 negatively regulates TLR5 signaling. Step 1, TLR5 on epithelial cells sense bacteria-derived flagellin. Step 2, activated TLR5 triggers MyD88-dependent signaling to induce the release (more ...)