Membrane-Tethered MUC1 Mucin Is Phosphorylated by Epidermal Growth Factor Receptor in Airway Epithelial Cells and Associates with TLR5 To Inhibit Recruitment of MyD88

Abstract

MUC1 is a membrane-tethered mucin glycoprotein expressed on the apical surface of mucosal epithelial cells. Previous in vivo and in vitro studies established that MUC1 counter-regulates airway inflammation by suppressing TLR signaling. In this report, we elucidate the mechanism by which MUC1 inhibits TLR5 signaling. Overexpression of MUC1 in human embryonic kidney HEK293 (293) cells dramatically reduced Pseudomonas aeruginosa (Pa)-stimulated IL-8 expression, and decreased the activation of NF-κB and MAPK compared with MUC1 non-expressing cells. Overexpression of MUC1 in 293 cells, however, did not affect NF-κB or MAKP activation in response to TNF-α. Overexpression of MyD88 abrogated the ability of MUC1 to inhibit NF-κB activation, and MUC1 overexpression inhibited flagellin-induced association of TLR5/MyD88, compared with controls. The MUC1 cytoplasmic tail (MUC1 CT) associated with TLR5 in all cells tested, including 293T cells, human lung adenocarcinoma cell line A549 cells, and human and mouse primary airway epithelial cells. Activation of EGFR tyrosine kinase with TGF-α induced phosphorylation of the MUC1 CT at the Y46 EKV sequence and increased association of MUC1/TLR5. Finally, in vivo experiments demonstrated increased immunofluorescence co-localization of Muc1/TLR5 and Muc1/phosphotyrosine staining patterns in mouse airway epithelium and increased Muc1 tyrosine phosphorylation in mouse lung homogenates following Pa infection. In conclusion, EGFR tyrosine phosphorylates MUC1, leading to an increase in its association with TLR5, thereby competitively and reversibly inhibiting recruitment of MyD88 to TLR5 and downstream signaling events. This unique ability of MUC1 to control TLR5 signaling suggests its potential role in the pathogenesis of chronic inflammatory lung diseases.

The cell surface mucin MUC1 limits the severity of influenza A virus infection

“…This results in inhibition of TLR signaling and a dampening of airway inflammation. This pathway has been elucidated in TLR5-dependent activation of primary epithelial cells in Pseudomonas aeruginosa infection, 14,15 but is also consistent with findings of negative regulation of other TLR molecules 12 and with the antiinflammatory effect of MUC1 during respiratory syncytial virus infection 13 and Haemophilus influenzae infection 44 of lung epithelial cells in vitro.…”

“…In response to airway infection by Pseudomonas aeruginosa 12 and respiratory syncytial virus, 13 TLR activation in airway epithelial cells and macrophages induces the production of inflammatory mediators such as IL-8 (KC in the mouse) and TNFa to recruit effector cells. 14,15 In turn, MUC1 is upregulated and acts to suppress TLR signaling. 16 In addition, production of the antiinflammatory IL-10 and type I interferon (IFN) has been shown to correlate with increased MUC1 expression.…”

Suppression of IL-8 production in gastric epithelial cells by MUC1 mucin and peroxisome proliferator-associated receptor-γ

“…Muc1 Ϫ/Ϫ mice experimentally infected in the stomach with H. pylori or in the lungs with P. aeruginosa or respiratory syncytial virus, had higher levels of TNF-␣ and KC (murine IL-8) and enhanced NF-B activation in the respective epithelia compared with Muc1 ϩ/ϩ littermates. These effects appeared to be mediated through the action of the MUC1 cytoplasmic domain and, more specifically, by one or more of the potential intracellular tyrosine phosphorylation sites, since anti-inflammatory activity was lost using an intracellular tyrosine deficient MUC1 mutant (24). Similar studies by others confirmed that murine MUC1 also counterregulates inflammatory responses against Staphylococcus, Streptococcus, and Corynebacterium, although the responsible mechanism was not described (21).…”

Membrane-Tethered MUC1 Mucin Counter-Regulates the Phagocytic Activity of Macrophages

“…In gastric epithelia, MUC1 contributes to the physicochemical barrier against infectious pathogens (12). On airway epithelial cells, MUC1-EC is an adhesion site for Pseudomonas aeruginosa (Pa) (13,14), whereas MUC1-CT interacts with Toll-like receptors (TLRs) (15,16). Binding of MUC1-CT to TLR5 competitively inhibits recruitment of MyD88 to the TLR5 cytosolic Toll/IL-1 receptor domain, thereby suppressing TLR5 signaling and downstream inflammatory responses (15).…”

“…On airway epithelial cells, MUC1-EC is an adhesion site for Pseudomonas aeruginosa (Pa) (13,14), whereas MUC1-CT interacts with Toll-like receptors (TLRs) (15,16). Binding of MUC1-CT to TLR5 competitively inhibits recruitment of MyD88 to the TLR5 cytosolic Toll/IL-1 receptor domain, thereby suppressing TLR5 signaling and downstream inflammatory responses (15). After experimental Pa lung infection, Muc1 null mice mount hyperinflammatory airway responses and exhibit enhanced bacterial clearance from the lungs compared with wild-type (WT) mice (17).…”