title: Pseudomonas aeruginosa stimulates tyrosine phosphorylation of and TLR5 association with the MUC1 cytoplasmic tail through EGFR activation.
abstract: BACKGROUND
  MUC1 is a membrane-tethered mucin expressed on the surface of epithelial and hematopoietic cells. Previous studies have established that MUC1 attenuates airway inflammation in response to Pseudomonas aeruginosa (Pa) through suppression of Toll-like receptor (TLR) signaling. Here, we elucidate the mechanism through which the MUC1 cytoplasmic tail (CT) inhibits TLR5 signaling in response to Pa and its flagellin in primary normal human bronchial epithelial (NHBE) cells.
  
  
  METHODS
  NHBE and human and mouse macrophages were stimulated with Pa or flagellin and transforming growth factor-α (TGF-α) and tumor necrosis factor-α (TNF-α) levels in cell culture supernatants were measured by ELISA. NHBE cells were stimulated with Pa, flagellin, or TNF-α and MUC1-CT, and epidermal growth factor receptor (EGFR) levels were measured by immunoblotting. NHBE cells were stimulated with Pa and MUC1-CT/TLR5 and MUC1-CT/EGFR association were detected by co-immunoprecipitation.
  
  
  RESULTS
  Stimulation of NHBE cells with Pa and flagellin each increased release of the EGFR ligand, TGF-α, from NHBE cells. Both stimuli also activated EGFR tyrosine phosphorylation in these same cells. By contrast, stimulation of NHBE cells with Pa failed to induce TNF-α release, whereas stimulation of human or mouse macrophages with Pa promoted TNF-α release. Stimulation of NHBE cells with recombinant TNF-α increased both MUC1 and EGFR protein levels, and stimulation of these cells with Pa enhanced MUC1-CT tyrosine phosphorylation and increased MUC1-CT/TLR5 and MUC1-CT/EGFR protein association, in an EGFR-dependent manner.
  
  
  CONCLUSIONS
  These results indicate that in response to Pa or flagellin, EGFR associates with and tyrosine phosphorylates MUC1-CT in primary NHBE cells, leading to increased MUC1-CT association with TLR5. Based on prior studies in tumor cells, increased MUC1-CT/TLR5 association in NHBE cells is predicted to competitively inhibit Pa/flagellin-stimulated TLR5 activation, reduce TLR5-dependent cell signaling, and down-regulate airway inflammation. Given that MUC1 is a universal suppressor of TLR signaling, the results from this study suggest that abnormal interactions between MUC1 and EGFR or TLRs may lead to the development of chronic inflammatory diseases. Thus, this is an important finding from the clinical point of view.
authors.affiliation: Department of Otolaryngology, University of Arizona College of Medicine, 1656 E Mabel St, MRB-419, Tucson, AZ, 85724, USA. kosukekato@oto.arizona.edu.|Department of Physiology and Center for Inflammation, Translational and Clinical Lung Research, Temple University School of Medicine, Philadelphia, PA, 19140, USA. kosukekato@oto.arizona.edu.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Kato
authors.affiliation: Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD, 21201, USA. elillehoj@peds.umaryland.edu.
authors.forename: Erik P
authors.initials: EP
authors.lastname: Lillehoj
authors.affiliation: Department of Otolaryngology, University of Arizona College of Medicine, 1656 E Mabel St, MRB-419, Tucson, AZ, 85724, USA. kckim@oto.arizona.edu.|Department of Physiology and Center for Inflammation, Translational and Clinical Lung Research, Temple University School of Medicine, Philadelphia, PA, 19140, USA. kckim@oto.arizona.edu.
authors.forename: Kwang Chul
authors.initials: KC
authors.lastname: Kim
mesh_terms: D000328:Adult; D000818:Animals; D002460:Cell Line; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D005408:Flagellin / Q000494:pharmacology; D006801:Humans; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D008810:Mice, Inbred C57BL; D018396:Mucin-1 / Q000378:metabolism*; D010766:Phosphorylation; D011550:Pseudomonas aeruginosa; D051198:Toll-Like Receptor 5 / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D018396:Mucin-1; D051198:Toll-Like Receptor 5; D014409:Tumor Necrosis Factor-alpha; D005408:Flagellin; D014443:Tyrosine; D066246:ErbB Receptors
keywords: EGFR; Flagellin; Inflammation; MUC1; Normal human bronchial epithelial cells; Pseudomonas aeruginosa; TLR5