Of Myd88 Associates with TLR5 to Inhibit Recruitment Receptor In

Published January 16, 2012, doi:10.4049/jimmunol.1102405 The Journal of Immunology

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

Kosuke Kato,* Erik P. Lillehoj,† Yong Sung Park,* Tsuyoshi Umehara,* Nicholas E. Hoffman,‡ Muniswamy Madesh,‡ and K. Chul Kim*

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 counterregulates airway inflammation by suppressing TLR signaling. In this article, we elucidate the mechanism by which MUC1 inhibits TLR5 signaling. Overexpression of MUC1 in HEK293 cells dramatically reduced Pseudomonas aeruginosa-stimulated IL-8 expression and decreased the activation of NF-kB and MAPK compared with cells not expressing MUC1. However, overexpression of MUC1 in HEK293 cells did not affect NF-kB or MAPK activation in response to TNF-a. Overexpression of MyD88 abrogated the ability of MUC1 to inhibit NF-kB activation, and MUC1 overexpression inhibited flagellin-induced association of TLR5/MyD88 compared with controls. The MUC1 cytoplasmic tail associated with TLR5 in all cells tested, including HEK293T cells, human lung adenocarcinoma cell line A549 cells, and human and mouse primary airway epithelial cells. Activation of epidermal growth factor receptor tyrosine kinase with TGF-a–induced phosphorylation of the MUC1 cytoplasmic tail at the Y46EKV sequence and increased association of MUC1/TLR5. Finally, in vivo experiments demonstrated increased immunofluorescence colocalization of Muc1/TLR5 and Muc1/phosphotyrosine staining patterns in mouse airway epithelium and increased Muc1 tyrosine phosphorylation in mouse lung homogenates following P. aeruginosa infection. In conclusion, epidermal growth factor receptor 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 Journal of Immunology, 2012, 188: 000–000.

UC1 (MUC1 in humans, Muc1 in animals) is a trans- tandem repeats, whereas the C-terminal subunit consists of a membrane glycoprotein expressed on the apical sur- 58-aa EC juxtamembranous region, a transmembrane domain, M face of mucosal epithelial cells, as well as the surface and a highly conserved cytoplasmic tail (CT) containing multiple of hematopoietic cells (1). MUC1 was originally cloned in phosphorylation sites (1). The 72-aa MUC1 CT contains seven epithelial-derived human breast and pancreatic cancer cells (2, tyrosine residues whose phosphorylation status has been associ- 3), where it is uniformly expressed on the cell surface, as well as ated with intracellular signal transduction in cancer cells (5). in the cytosol and nucleus (4). The MUC1 protein consists of two In the airways, MUC1 expressed on exposed epithelia con- noncovalently associated polypeptide subunits: an N-terminal tributes to the first line of defense against inhaled pathogens; extracellular (EC) subunit and a C-terminal subunit. The MUC1 however, the physiological role of MUC1 during airway inflam- ectodomain is composed almost entirely of variable numbers of mation, particularly its CTwith multiple signaling motifs, is largely unknown. Previously, we reported that Muc1 knockout mice intranasally infected with P. aeruginosa displayed greater levels *Department of Physiology, Center for Inflammation, Translational and Clinical of airway inflammation compared with wild-type littermates. The Lung Research, Temple University School of Medicine, Philadelphia, PA 19140; †Department of Pediatrics, University of Maryland School of Medicine, Baltimore, anti-inflammatory effect of MUC1 was mediated through inhibi- MD 21201; and ‡Department of Biochemistry, Temple University School of Medi- tion of the proinflammatory TLR5-signaling pathway (6, 7), and cine, Philadelphia, PA 19140 P. aeruginosa upregulated MUC1 expression in airway epithelial Received for publication August 18, 2011. Accepted for publication December 14, cells (8). Therefore, it was concluded that increased MUC1/Muc1 2011. expression subsequent to production of proinflammatory media- This work was supported by U.S. Public Health Service Grants HL-47125 and HL- 81825 (to K.C.K.), AI-83463 (to E.P.L.), and HL-86699 (to M.M.). tors led to attenuation of TLR signaling via a negative-feedback mechanism (7). Address correspondence and reprint requests to Prof. K. Chul Kim, Department of Physiology, Temple University School of Medicine, 3420 N. Broad Street, MRB-410, TLRs comprise 10-member (human) or 12-member (mouse) Philadelphia, PA 19140. E-mail address: [email protected] families of innate immune receptors that initiate intracellular Abbreviations used in this article: CT, cytoplasmic tail; EGFR, epidermal growth signaling following ligation of pathogen-associated molecular factor receptor; IP, immunoprecipitation; MEF, mouse embryonic fibroblast; MTSE, primary mouse tracheal surface epithelial; MUC1 DCT, deletion of the MUC1 cyto- patterns (9). With the exception of TLR3, all TLRs initiate sig- plasmic tail domain; NHBE, normal human bronchial/tracheal epithelial; PAK, Pseu- naling by recruitment of the MyD88 adaptor protein that bridges domonas aeruginosa strain K; TAK1, TGF-b–activated kinase 1; TIR, Toll/IL-1R; the intracellular region of TLRs with downstream effector mole- TRAF6, TNFR-associated factor 6. cules, such as IRAK1, a serine/threonine kinase, and TNFR-asso- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 ciated factor 6 (TRAF6), an E3 ubiquitin ligase, to activate the

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102405 2 MECHANISM OF CROSS-TALK BETWEEN MUC1 AND TLR5

NF-kB and MAPK pathways, resulting in the release of proin- bacteria at 60˚C for 30 min and stored at 280˚C. Pervanadate, a general flammatory cytokines and chemokines. protein tyrosine phosphatase inhibitor, was freshly prepared immediately 3 In addition to NF-kB and MAPKs, TLR5 activates the PI3K before its use as a 100 stock solution by mixing 600 mMH2O2 and 0.2 mMNaVO (11). pathway, a known mechanism for negative regulation of TLR5 3 4 signaling (10). The MUC1 CT contains a consensus binding site Cells for PI3K (5, 11), leading to the suggestion that PI3K may be re- Mouse embryonic fibroblast (MEF) cells were obtained from embryonic sponsible for MUC1-induced suppression of TLR5 signaling. day 13 embryos and maintained in DMEM supplemented with 10% FBS However, our previous study demonstrated that although MUC1 (13). Primary mouse tracheal surface epithelial (MTSE) cells were pre- activated the PI3K pathway in response to flagellin, a TLR5 ago- pared from 8–12-wk-old C57BL/6 mice and cultured as described (6). nist, PI3K was not responsible for MUC1-induced suppression Normal human bronchial/tracheal epithelial (NHBE) cells were obtained of TLR5 signaling (12). In this study, evidence is presented that from Lonza Walkersville (Walkersville, MD) and cultured at an air/liquid interface, according to the manufacturer’s protocol. A549 cells (CCL-185), MUC1 expression disrupts TLR5–MyD88 interaction and down- a human lung epithelial cell line, as well as HEK293 cells (CRL-1573) stream proinflammatory signaling and that epidermal growth factor and HEK293T cells (CRL-11268), human embryonic kidney cell lines, receptor (EGFR) activation promotes this anti-inflammatory effect were purchased from American Tissue Culture Collection (Manassas, VA). of MUC1 through tyrosine phosphorylation of the MUC1 CT. A549 cells were cultured in Opti-MEM medium supplemented with 5% FBS and antibiotics. HEK293 cells were stably transfected with the pcDNA3.1 empty vector (HEK293-pcDNA3.1 cells) or with a MUC1 Materials and Methods cDNA cloned in pcDNA3.1 using Lipofectamine 2000 (Invitrogen, Reagents Carlsbad, CA). HEK293T cells were used for transient transfection. Both types of cells were subsequently transfected with FLAG-tagged human All reagents were purchased from Sigma (St. Louis, MO), unless otherwise TLR5 cloned into the HindIII and AgeI sites of the pDsRed-Monomer- stated. The sources of the Abs and reagents were phospho-p38 (Thr180/ Hyg-N1 vector (Clontech Laboratories, Palo Alto, CA). Stable clones were Tyr182) (3D7) rabbit monoclonal, phospho-p44/42 (Thr202/Tyr204) rab- isolated in the presence of hygromycin (100 mg/ml) and referred to as bit monoclonal, phosphotyrosine (P-Tyr-100) mouse monoclonal, EGFR HEK293-TLR5 and HEK293-TLR5/MUC1 cells, respectively; they were rabbit monoclonal, AG1478 (Cell Signaling Technology, Beverly, MA), cultured in DMEM containing penicillin (100 U/ml), streptomycin (100 MyD88 (HFL296) rabbit polyclonal, MUC1 ectodomain (GP1.4) mouse mg/ml), hygromycin (100 mg/ml), and 10% FBS. monoclonal, normal rabbit IgG, normal mouse IgG (Santa Cruz Biotech- nology, Santa Cruz, CA), TLR5 (IMG-664A) mouse monoclonal (Imgenex, Western blot and immunoprecipitation analyses San Diego, CA), HRP-conjugated goat anti-mouse IgG and anti-rabbit IgG secondary Abs (KPL, Gaithersburg, MD), P. aeruginosa strain K (PAK) Western blot analysis was performed as described (12). For direct immu- flagellin rabbit antiserum (Dr. Dan Wozniak, Wake Forest University, noprecipitation (IP) analysis, the cells were extracted with lysis buffer (0.5 Winston-Salem, NC), MUC1-pcDNA3.1 expression plasmid and MUC1 ml/100-mm dish), and equal amounts of protein were incubated with anti- CT (CT2) hamster Ab (Dr. Sandra J. Gendler, Mayo Clinic College of FLAG Ab (10 mg), anti-MUC1 CT (CT2) Ab (40 ml), anti-MUC1 ecto- Medicine, Scottsdale, AZ), TRAF6 expression plasmid (Dr. Yong Lin, domain (GP1.4) Ab (30 ml), or isotype-matched normal IgG (Santa Cruz) Lovelace Respiratory Research Institute, Albuquerque, NM), MyD88 and at 4˚C for 16 h with continuous agitation. Protein G-agarose beads (Pierce, IRAK1 expression plasmids (Dr. Katherine Fitzgerald, University of Rockford, IL) were added to precipitate immune complexes and the beads Massachusetts Medical School, Worcester, MA), and human rTNF-a were washed five times at 4˚C with lysis buffer and eluted by boiling in (Prospec, Rehovot, Israel). PAK flagellin was purified, as described (6). SDS-PAGE loading buffer. Immune complexes were separated by SDS- Heat-inactivated PAK (1.0 3 109 CFU/ml) was prepared by incubating PAGE and subjected to Western blot analysis.

FIGURE 1. Effect of MUC1 expression on TLR5-dependent IL-8 promoter activity and activation of NF-kB, p38, and ERK1/2. HEK293-pcDNA3.1, HEK293-TLR5, and HEK293-TLR5/ MUC1 cells (A–D)orMuc12/2 MEF cells transfected with pcDNA3.1 empty vector or MUC1 expression plasmid (E, F) were untreated (CON) or treated for 6 h with heat-inactivated PAK (1.0 3 107 CFU/ml) or ﬂagellin (1.0 mg/ml). Equal protein amounts of cell lysates were subjected to Western blotting with the indicated Abs (A, D, F) or luciferase assays for IL-8 promoter activity (B)or NF-kB reporter activity (C, E). Each bar represents the mean 6 SEM (n =3). The results are representative of three independent experiments. *p , 0.05 compared with untreated controls, †p . 0.05 compared with untreated controls. The Journal of Immunology 3

Luciferase reporter assays for ELAM-1 and IL-8 vector increased NF-kB activation, as well as enhanced p38 and An NF-kB–dependent ELAM-1–luciferase reporter plasmid (pELAM1- ERK1/2 phosphorylation, compared with untreated controls (Fig. luc) was generated, as described elsewhere (14), by cloning a fragment 1E,1F). However, all three of these effects were substantially 2 2 (2241 to 254 bp) of human E-selectin promoter into the pGL3 reporter reduced in Muc1 / MEF cells overexpressing human MUC1 plasmid (Promega, Madison, WI). Briefly, cells were transiently trans- compared with Muc12/2 MEF-pcDNA3.1 cells. fected with the pIL-8–luciferase (15) or pELAM-1–luciferase reporter genes with a Renilla luciferase reporter construct, using Lipofectamine MUC1 expression does not affect TNF-a/TNFR signaling 2000, according to the manufacturer’s protocol. In addition, the cells were transfected with expression plasmids for TRAF6, IRAK1, or MyD88. The To determine whether inhibition of NF-kB and MAPK by MUC1 total amount of plasmid DNA was kept constant by addition of pcDNA3.1 could be replicated in another innate immune ligand/receptor for each transfection. At 24–36 h posttransfection, the cells were treated system, the effects of MUC1 expression on TNF-a/TNFR- with appropriate stimuli for 6 h, and cell lysates were assayed for firefly mediated activation of NF-kB and MAPK were assessed. As be- and Renilla luciferase activities using the Dual-luciferase Reporter System (Promega) and an L-Max II luminometer (Molecular Devices, Sunnyvale, fore, flagellin-induced NF-kB activation was suppressed in CA). Relative luciferase activity was determined by normalizing with HEK293-TLR5/MUC1 cells compared with HEK293-TLR5 cells Renilla luciferase activity. (Fig. 2A). By contrast, TNF-a–induced NF-kB activities were equal in both cell types. (Note the different numerical scales on Experimental P. aeruginosa lung infection the two ordinate axes.) Combining the flagellin and TNF-a stimuli C57BL/6 Muc12/2 mice and Muc1+/+ littermates have been described in HEK293-TLR5 cells produced an NF-kB activation response 7 (16). Mice were intranasally infected with 1.0 3 10 CFU PAK in a 40-ml that was ∼2-fold greater than that achieved by the individual suspension, as described (6). Left lung lobes were homogenized in 1.0 ml agonists. However, NF-kB activation in HEK293-TLR5/MUC1 lysis buffer and subjected to IP, as described above. The remaining lobes were fixed with 10% paraformaldehyde and embedded in paraffin, and cells in response to flagellin plus TNF-a was equal to that pro- 5-mm sections were made. All procedures were approved by the Institu- duced by TNF-a alone. Additionally, TNF-a–stimulated phos- tional Animal Care and Use Committee of Temple University. phorylations of p38 and ERK1/2 were not suppressed in HEK293- TLR5/MUC1 cells compared with HEK293-TLR5 cells (Fig. 2B). Immunofluorescence These combined data suggested that MUC1 attenuates activation Paraffin sections on glass slides were deparaffinized in xylene, rehydrated, of NF-kB and MAPK mediated by TLR5 but not by TNFR. blocked in normal goat serum, and incubated overnight at 4˚C with primary Abs (1:100 dilution) and for 1 h at room temperature with fluorescent- MUC1 interferes with flagellin-induced association of conjugated secondary Abs (1:500). Nuclei were counterstained with DAPI. TLR5/MyD88 Coverslips were applied in antifade solution (Invitrogen); samples were visualized by laser scanning confocal microscopy (LSM 510; Zeiss, Because activation of NF-kB and MAPK by TLR5 and TNFR Stuttgart, Germany), and images were analyzed using LSM 510 software. requires prior activation of TGF-b–activated kinase 1 (TAK1), and the signaling pathways leading to activation of TAK1 by these Statistical analysis Differences between groups were assessed by comparing mean 6 SEM values using the Student t test and were considered significant at p , 0.05.

Results MUC1 expression attenuates TLR5-dependent IL-8 promoter activity and activation of NF-kB, p38, and ERK1/2 Airway epithelial cells sense P. aeruginosa infection through recognition of flagellin by TLR5 (17). Because MUC1 expression inhibits TLR5-driven NF-kB activation and IL-8 production by airway epithelia (6, 18), we sought to determine whether MUC1 suppresses TLR5 signaling and, if so, to elucidate the mechanism by building upon previous studies using the well-established HEK293 cell culture system (6, 11, 12, 18–20). Stable clones of HEK293-pcDNA3.1, HEK293-TLR5, and HEK293-TLR5/MUC1 cells were established in HEK293 cells not expressing MUC1 (Fig. 1A). P. aeruginosa-induced IL-8 promoter activity was sig- nificantly enhanced in HEK293-TLR5 cells compared with HEK293-pcDNA3.1 cells (Fig. 1B). TLR5-dependent IL-8 promoter activation was completely suppressed in HEK293-TLR5/ MUC1 cells. TLR5 regulates IL-8 gene expression through NF-kB and MAPK pathways (21). Therefore, we asked whether MUC1 attenuates TLR5-dependent activation of NF-kB and/or MAPK. Treatment of HEK293-TLR5 cells with flagellin increased NF-kB–dependent reporter (ELAM-1) activity compared with untreated controls (Fig. 1C). However, NF-kB activity was FIGURE 2. Effect of MUC1 expression on TNF-a–induced activation completely suppressed in HEK293-TLR5/MUC1 cells. Similarly, of NF-kB, p38, and ERK1/2. HEK293-TLR5 and HEK293-TLR5/MUC1 cells were untreated or treated for 6 h with flagellin (1.0 mg/ml), TNF-a treatment of HEK293-TLR5 cells with flagellin augmented the (100 ng/ml), or flagellin plus TNF-a. Equal protein amounts of cell lysates phosphorylation of p38 and ERK1/2 compared with controls, and were subjected to luciferase assay to measure NF-kB reporter activity (A) both effects were drastically reduced in HEK293-TLR5/MUC1 and Western blotting with the indicated Abs (B). Each bar represents the cells (Fig. 1D). Identical results were observed in an indepen- mean 6 SEM (n = 3). Note the different ordinate scales in A. The results 2/2 dent cell culture system. P. aeruginosa treatment of Muc1 are representative of three independent experiments. *p , 0.05 compared MEF cells previously transfected with the pcDNA3.1 empty with untreated controls, †p . 0.05 compared with untreated controls. 4 MECHANISM OF CROSS-TALK BETWEEN MUC1 AND TLR5

FIGURE 3. Effect of MyD88, IRAK1, or TRAF6 overexpression on MUC1-mediated suppression of TLR5 signaling. A and C, HEK293-TLR5 and HEK293-TLR5/MUC1 cells were untreated or treated with flagellin (1.0 mg/ml) or heat-inactivated PAK (1.0 3 107 CFU/ml) for 30 min. Equal protein amounts of cell lysates were used for IP with anti-FLAG Ab (TLR5) or isotype-matched normal mouse IgG, and immunoprecipitated proteins were subjected to Western blotting with the indicated Abs. B, HEK293-TLR5 and HEK293-TLR5/MUC1 cells were transfected with TRAF6 (left panel), IRAK1 (middle panel), or MyD88 (right panel) expression plasmids. The cells were untreated or treated for 6 h with flagellin (1.0 mg/ml). Equal protein amounts of cell lysates were subjected to luciferase assay to measure NF-kB reporter activity. Each bar represents the mean 6 SEM value (n = 3). The results are representative of three independent experiments. *p , 0.05. receptors are distinct, the data presented above suggested that treated HEK293-TLR5 and HEK293-TLR5/MUC1 cells exhibited cross-talk between MUC1 and TLR5 signaling occurs upstream of equal flagellin/TLR5 binding (Fig. 3A), suggesting that MUC1 did TAK1. Therefore, experiments were performed to identify the not block binding of flagellin to its cognate receptor. Next, we possible site(s) between TLR5 and TAK1 that is/are suppressed by determined whether MUC1-mediated suppression of TLR5 sig- MUC1. Engagement of TLR5 by flagellin triggers the recruitment naling was blocked following overexpression of TRAF6, IRAK1, of MyD88 to the intracellular Toll/IL-1R (TIR) domain of TLR5. or MyD88. Although overexpression of TRAF6 or IRAK1 did not MyD88 subsequently binds to and activates IRAK1 (22), and reverse the inhibitory effect of MUC1 (Fig. 3B), MyD88 over- activated IRAK1 phosphorylates TRAF6, thereby inducing Lys63- expression completely abrogated MUC1-dependent suppression of linked autoubiquitination and activation of TAK1 (23). Flagellin- flagellin-induced NF-kB activation (Fig. 3B). Overexpression of

FIGURE 4. Association of MUC1 and TLR5 in HEK293T cells. HEK293T cells were untransfected or transiently transfected with expression plasmids for TLR5-FLAG and/or full-length MUC1 (A) or MUC1 DCT (B). At 24 h posttransfection, equal protein amounts of cell lysates were used for IP with isotype-matched normal mouse IgG, anti-FLAG (TLR5-FLAG), anti-CT2 (full-length MUC1), or anti-MUC1 ectodomain (GP1.4) (MUC1 DCT). Immunoprecipitated proteins were subjected to Western blotting with the indicated Abs. Protein expression levels of TLR5 and MUC1 were veriﬁed in the same lysates used for IP. The results are representative of two or three independent experiments. The Journal of Immunology 5

TRAF6, IRAK1, and MyD88 following transient transfection was MUC1/TLR5 association. Treatment of A549 cells with perva- confirmed by Western blotting (data not shown). Finally, treatment nadate, a broad-spectrum tyrosine phosphatase inhibitor, increased of HEK293-TLR5 cells with flagellin or heat-inactivated P. aer- constitutive MUC1 CT tyrosine phosphorylation and augmented uginosa increased association of TLR5/MyD88 compared with MUC1/TLR5 association compared with untreated cells (Fig. 6A). untreated cells (Fig. 3C). In contrast, such association was not To investigate the possible role of the Y46 residue in MUC1 CT observed in HEK293-TLR5/MUC1 cells. Taken together, these phosphorylation and association of MUC1/TLR5, HEK293T cells results suggested that MUC1 overexpression abrogates flagellin- were cotransfected with TLR5 and a CD8/MUC1 chimeric protein stimulated recruitment of MyD88 to TLR5, thus inhibiting NF-kB (26): either one with the CD8 EC and transmembrane domains and activation; however, this inhibitory effect is fully reversible upon the MUC1 CT (CD8/MUC1-WT) or CD8/MUC1-Y46F, in which MyD88 overexpression. Tyr-46 residue was mutated to phenylalanine (11, 26) (Fig. 6B). Pervanadate treatment increased both constitutive tyrosine phos- MUC1 associates with TLR5 phorylation of CD8/MUC1-WT (Fig. 6C, lower panel) and asso- Two possible mechanisms were considered for MUC1’s ability to ciation of CD8/MUC1-WT and TLR5 (Fig. 6C, upper panel) reversibly block the interaction between MyD88 and TLR5: compared with untreated cells. However, CD8/MUC1-Y46F binding of MUC1 to MyD88 and/or binding of MUC1 to TLR5. exhibited significant decreases in both constitutive tyrosine Reciprocal coimmunoprecipitation studies revealed that MUC1 phosphorylation and association with TLR5 compared with CD8/ constitutively forms a protein complex with TLR5, but not with MUC1-WT (Fig. 6C). Next, we determined whether Y46 is im- MyD88 (data not shown), in lysates of HEK293T cells transiently portant for the suppressive effect of MUC1 on PAK-induced transfected with MUC1 and TLR5 (Fig. 4A). Deletion of the MUC1 CT domain (MUC1 DCT) almost completely abated its association with TLR5 (Fig. 4B), suggesting that the MUC1 CT domain is necessary to interact with TLR5. Constitutive interaction between endogenously expressed MUC1 and TLR5 was also demonstrated in lysates of A549 cells, as well as in primary MTSE cells and NHBE cells (Fig. 5A). In addition, association of MUC1/ TLR5 in NHBE cells was increased 5-fold over basal levels following treatment with P. aeruginosa (Fig. 5B). In summary, these results indicated that MUC1 binds to TLR5 through its CT domain. Activation of EGFR induces MUC1 CT tyrosine phosphorylation and increases association of MUC1/TLR5 Tyrosine residues in the MUC1 CT are targets of cytosolic and receptor protein kinases, and these phosphotyrosines serve as docking sites for oncoproteins (24). For example, EGFR phosphorylates the MUC1 CT at a Y46EKV sequence that, upon phosphorylation, serves as a binding site for the kinase (25). Therefore, we investigated constitutive and EGFR-induced CT tyrosine phosphorylation and the role of phosphorylation in

FIGURE 6. Phosphorylation of the MUC1 CT at Y46 by EGFR increases MUC1/TLR5 association. A and C–H, A549 cells or HEK293T cells, transiently transfected with expression plasmids for TLR5 plus CD8/MUC1-WT or CD8/MUC1-Y46F, were untreated or treated for 30 min with pervanadate (0.2 mM), AG1478 (100 nM), or TGF-a (100 ng/ FIGURE 5. Association of MUC1 and TLR5 in human airway epithelial ml). Equal amounts of cell lysates were used for IP with anti-CT2, anti- cells. A, Equal protein amounts of lysates from A549, MTSE, and NHBE CD8 (CD8/MUC1), or anti-FLAG (TLR5-FLAG), and immunoprecipi- cells were used for IP with isotype-matched normal IgG or anti-CT2 to tated proteins were subjected to Western blotting with the indicated Abs. precipitate MUC1. Immunoprecipitated proteins were subjected to Western B, Schematic illustration of the CD8/MUC1 chimeric protein. D, For blotting with the indicated Abs. B, NHBE cells were untreated (CON) or ELAM-1–luciferase reporter assay, cells were untreated or treated with treated for 48 h with heat-inactivated PAK (1.0 3 107 CFU/ml). Equal PAK and assayed as in Fig. 1. Luciferase activity of pcDNA3.1 transfec- protein amounts of cell lysates were immunoprecipitated as in A (left tants treated with PAK was set as 100%. Each bar represents the mean 6 panel). The density of each TLR5 band that was normalized to the density SEM (n = 4). Transfection efﬁciency of CD8/MUC1-WT and CD8/MUC1- of the corresponding MUC1 CT band is presented. The results are repre- Y46F was veriﬁed by Western blotting. The results are representative of sentative of two or three independent experiments. three independent experiments. *p , 0.01. 6 MECHANISM OF CROSS-TALK BETWEEN MUC1 AND TLR5

NF-kB activation, as shown in Fig. 1. Overexpression of CD8/ Discussion MUC1-WT and CD8/MUC1-Y46F decreased PAK-induced MUC1/Muc1 expression counterregulates airway inflammation ELAM-1 activity by 65% and 25%, respectively, compared with during P. aeruginosa infection (6, 8), and this anti-inflammatory pcDNA3.1 control, suggesting that Y46 of MUC1 CT plays an activity is attributed to its ability to suppress TLR5 signaling (6, important role in suppressing PAK-induced TLR5 signaling (Fig. 12, 18). However, the mechanism of cross-talk between TLR5 6D). Even in the absence of pervanadate, treatment of A549 cells and MUC1/Muc1 is unknown. In this article, we demonstrated the with an EGFR agonist, TGF-a, increased tyrosine phosphoryla- suppressive effect of MUC1 expression on TLR5-dependent IL-8 tion of the MUC1 CT and enhanced association of MUC1/TLR5, promoter activity (Fig. 1B), as well as NF-kB and MAPK acti- as well as MUC1/EGFR (Fig. 6E). Identical results were observed vation (Figs. 1C–F), which correlated with reduced TLR5/MyD88 using epidermal growth factor as the stimulus (data not shown). association (Fig. 3C) and increased MUC1/TLR5 association Similarly, TGF-a stimulated tyrosine phosphorylation of CD8/ (Figs. 4–6). NF-kB inhibition was completely reversed upon MUC1-WT (Fig. 6F, lower panel) and increased association of MyD88 overexpression (Fig. 3B). Increased MUC1 CT phos- CD8/MUC1-WT with TLR5 (Fig. 6F, upper panel) compared with phorylation, likely at the Y46EKV site, and greater MUC1/TLR5 untreated cells. However, TGF-a treatment failed to increase as- association were associated with TGF-a–dependent EGFR acti- sociation of CD8/MUC1-Y46F with TLR5 (Fig. 6G). In addition, vation (Fig. 6). In vivo experiments established increased Muc1 treatment of CD8/MUC1-WT–transfected cells with a selective CT tyrosine phosphorylation in mouse lung homogenates fol- EGFR inhibitor, AG1478, abrogated TGF-a–induced association lowing P. aeruginosa infection (Fig. 7) and greater Muc1/TLR5 of CD8/MUC1-WT/TLR5 (Fig. 6H). In conclusion, these data and Muc1/phosphotyrosine immunofluorescence colocalization in suggested that EGFR-dependent Y46 phosphorylation of the infected mouse airway epithelium (Fig. 8). Together, these results MUC1 CT increases association of MUC1 CT with TLR5, hence suggested a mechanism whereby EGFR tyrosine-phosphorylates presumably enhances MUC1-mediated TLR5 suppression. the MUC1 CT, thus increasing its association with TLR5 and competitively and reversibly inhibiting recruitment of MyD88 to Immunofluorescence colocalization of Muc1 and TLR5 and TLR5 and subsequent proinflammatory signal transduction. tyrosine phosphorylation of the Muc1 CT in vivo Flagellin binding to the TLR5 ectodomain induces receptor P. aeruginosa infection of mice was shown to activate EGFR in homodimerization, resulting in a protein conformational change airway epithelium (27). Therefore, we asked whether P. aerugi- in its CT domain and allowing recruitment of the MyD88 adapter nosa airway infection of mice stimulates tyrosine phosphorylation protein (22). The data presented in this study indicated that of the Muc1 CT in vivo and increases Muc1/TLR5 interaction. MUC1 mediates its anti-inflammatory effects at the level of Mice were infected intranasally with P. aeruginosa, and lung the TLR5 intracellular domain and are entirely consistent with homogenates were subjected to Muc1 CT IP and phosphotyrosine the previous publication demonstrating that transfection of immunoblot analysis at 0, 4, and 24 h postinfection. Airway in- RAW264.7 cells with MUC1 DCT, but not MUC1 DEC, abolished fection was confirmed by increased inflammatory cell infiltrate the ability of MUC1 to inhibit TLR-driven TNF-a production into the lungs at 4 and 24 h (data not shown). Muc1 CT tyrosine (18). This effect was achieved not only with the MyD88- phosphorylation was increased at 24 h postinfection compared dependent TLR2, TLR4, TLR7, and TLR9, but also with TLR3, with 0 and 4 h (Fig. 7A). Identical results were observed in the which signals through TRIF rather than MyD88. Given that all reciprocal approach when lung homogenates were subjected to TLRs and many adaptor proteins share the conserved TIR domain phosphotyrosine IP, followed by Muc1 CT immunoblotting (Fig. mediating homo- and heterotypic interactions, it seems very likely 7B). As a negative control, a 25-kDa tyrosine-phosphorylated CT that MUC1 suppresses TLR signaling by associating with receptor protein band was not detected in the lungs of P. aeruginosa- TIR domains, thus acting as a steric hindrance/decoy receptor and infected Muc12/2 mice (Fig. 7C). Finally, laser-scanning confocal excluding recruitment of MyD88 and TRIF to their respective immunofluorescence microscopy was used to assess the Muc1, receptors. Interestingly, the MUC1 CT contains an amino acid TLR5, and phosphotyrosine staining patterns in lungs of unin- sequence (R17DTYHP) that is homologous to the consensus fected and P. aeruginosa-infected mice. In uninfected animals, no RDXF1F2G motif (where F represents a hydrophobic residue, evidence of Muc1/TLR5 or Muc1/phosphotyrosine colocalization and X represents any residue) of the TIR domain and that was was observed (Fig. 8). However, epithelial cell apical colocali- shown to be responsible for heteromeric interaction between zation of the Muc1/TLR5 and Muc1/phosphotyrosine immuno- TLR2/4 and MyD88 (28). However, the possible role of the staining patterns was seen in P. aeruginosa-infected mice at 24 h MUC1 CT “TIR domain-like” sequence in TLR signal transduc- postinfection (Fig. 8). tion remains speculative.

FIGURE 7. P. aeruginosa infection increases tyrosine phosphorylation of the Muc1 CT in vivo. Muc1+/+ and Muc12/2 mice were intranasally infected with PAK (1.0 3 107 CFU/mouse). At 0, 4, and 24 h postinfection, lung homogenates were prepared, and equal amounts of protein were used for IP with anti-CT2 Ab (A, C) or anti-phosphotyrosine Ab (B). Immunoprecipitated proteins were subjected to Western blotting with the indicated Abs. Each lane represents a lung homogenate from an individual mouse. The results are representative of two or three independent experiments. The Journal of Immunology 7

FIGURE 8. Colocalization of Muc1 and TLR5 in mouse airway epithelium in vivo. Parafﬁn-embedded mouse lung sections from uninfected (Control) and PAK intranasally infected Muc1+/+ mice were processed for immunostaining with anti-CT2 and anti-TLR5 (A) or anti-phosphotyrosine (B) Abs, followed by incubation with ﬂuorescein-conjugated secondary Abs and DAPI to counterstain nuclei. Scale bars, 20 mm. Arrowheads indicate areas of colocalization.

EGFR regulates innate immune responses in the airways, in- crete mechanism not involving its ectodomain. These multidi- cluding mucin secretion by goblet cells, and chemokine production mensional effects may relate to the functional difference between and proliferation by epithelial cells (29). As with all EGFR ligands, the two organs, with the former constituting an impenetrable TGF-a is synthesized in a latent form as a membrane-tethered barrier against commensal bacteria and the latter dealing with the precursor protein on the surface of airway epithelial cells. Pro- timely resolution of inflammation to maintain its vital function of teolytic cleavage of pro-TGF-a by TNF-a converting enzyme gas exchange. precedes EGFR activation and proinflammatory signaling (30). It is also apparent that the MUC1 CT exhibits functional ac- Like TGF-a, TNF-a converting enzyme is initially synthesized in tivities apart from its ability to directly block TLR signaling. an inactive form that is activated by a variety of diverse stimuli, Ahmad et al. (44, 45) demonstrated that the MUC1 C-terminal including airway bacterial pathogens (31), cigarette smoke (32), subunit promotes TNF-a–induced activation of NF-kB in human and reactive oxygen species (33), the last of which are upregulated breast cancer cells. In contrast, we reported that the MUC1 CT by TLRs and dual oxidase (33). Based on the current results, binds to the IKKg subunit to inhibit H. pylori-dependent NF-kB a second function can now be ascribed to activated EGFR apart activation in a human gastric cancer cell line (46). It is unknown from proinflammatory signaling: tyrosine phosphorylation of the whether IKK/NF-kB are recruited to the inner leaflet 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-a (8, 29, 34, 35). It is tempting to speculate that simultaneous upregulation 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 coun- terregulation of airway inflammation. In addition to its anti-inflammatory properties mediated through its CT, a growing body of evidence suggests that the MUC1 EC regions contribute to the pathogenesis of microorganisms that colonize and infect mucosal surfaces (36). MUC1/Muc1 is an EC adhesion site for P. aeruginosa on airway epithelia (19, 37, 38), as well as for Escherichia coli and Salmonella enterica on intestinal epithelia (39, 40). 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, 41). McAuley et al. (42) demonstrated that Muc12/2 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 FIGURE 9. Schematic illustration of the proposed mechanism through damage and exaggerated penetration through the intestinal barrier, which MUC1 negatively regulates TLR5 signaling. Step 1, TLR5 on ep- eventually resulting in systemic infection. As originally proposed ithelial cells senses bacteria-derived flagellin. Step 2, Activated TLR5 triggers MyD88-dependent signaling to induce the release of inflammatory by Gendler (43), the heterodimeric nature of the MUC1 protein mediators, which results in recruitment of leukocytes into the site of in- may provide a mechanism for rapid ectodomain shedding that fection to clear the bacteria. Step 3, Inflammatory products, such as neu- concurrently signals to the cell interior, through its CT, the pres- trophil elastase and TNF-a, upregulate MUC1 expression. Step 4, EGFR ence of an invading pathogen. Although Muc1 acts as a decoy activated by TLRs or other means phosphorylates the MUC1 CT at Y46. receptor for invading bacteria in the intestinal tract, it concurrently Step 5, MUC1 binds to TLR5, which interferes with the recruitment of plays an anti-inflammatory role in the respiratory tract by a dis- MyD88 to TLR5. 8 MECHANISM OF CROSS-TALK BETWEEN MUC1 AND TLR5 plasma membrane by the MUC1 CT or whether the membrane- 11. Wang, H., E. P. Lillehoj, and K. C. Kim. 2003. Identification of four sites of stimulated tyrosine phosphorylation in the MUC1 cytoplasmic tail. Biochem. bound CT is released into the cytoplasm to interact with IKK/NF- Biophys. Res. Commun. 310: 341–346. kB. Nonetheless, apically polarized MUC1 presumably favors its 12. Kato, K., W. Lu, H. Kai, and K. C. Kim. 2007. Phosphoinositide 3-kinase is ability to interact with TLR5 and, hence, selectively suppresses activated by MUC1 but not responsible for MUC1-induced suppression of Toll- like receptor 5 signaling. Am. J. Physiol. Lung Cell. Mol. Physiol. 293: L686– TLR5-induced NF-kB activity per se. Interestingly, exposure of L692. polarized airway epithelial cells to cigarette smoke redistributed 13. Vasir, B., Z. Wu, K. Crawford, J. Rosenblatt, C. Zarwan, A. Bissonnette, apical MUC1 into the cytosol, suggesting that exogenous insults D. Kufe, and D. Avigan. 2008. 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