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Pseudomonas aeruginosa stimulates tyrosine phosphorylation of and TLR5 association with the MUC1 cytoplasmic tail through EGFR activation

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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.

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Abbreviations

AEC:

Airway epithelial cell

CFU:

Colony forming unit

CT:

Cytoplasmic tail

EGFR:

Epidermal growth factor receptor

ELISA:

Enzyme-linked immunosorbent assay

HRP:

Horseradish peroxidase

IL:

Interleukin

Mac:

Macrophage

IB:

Immunoblot

IP:

Immunoprecipitation

NK-κB:

Nuclear factor-κB

NHBE:

Normal human bronchial epithelial

Pa:

Pseudomonas aeruginosa

PAK:

Pseudomonas aeruginosa strain K

PAMPs:

Pathogen-associated molecular patterns

PI3K:

Phosphoinositide 3-kinase

TACE:

Tumor necrosis factor-α converting enzyme

TGF-α:

Transforming growth factor-α

TLR:

Toll-like receptor

TNF-α:

Tumor necrosis factor-α

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Acknowledgments

This work was supported by National institutes of Health Grant RO1 HL-047125. The authors thank Alec Hanss, Audriana Hurbon, and Nicole Morgan for technical assistance through their summer research program at the University of Arizona (Tucson, AZ).

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Authors and Affiliations

  1. Department of Otolaryngology, University of Arizona College of Medicine, 1656 E Mabel St, MRB-419, Tucson, AZ, 85724, USA

    Kosuke Kato & Kwang Chul Kim

  2. Department of Physiology and Center for Inflammation, Translational and Clinical Lung Research, Temple University School of Medicine, Philadelphia, PA, 19140, USA

    Kosuke Kato & Kwang Chul Kim

  3. Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD, 21201, USA

    Erik P. Lillehoj

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  1. Kosuke Kato
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Correspondence to Kwang Chul Kim.

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Responsible Editor: Artur Bauhofer.

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Kato, K., Lillehoj, E.P. & Kim, K.C. Pseudomonas aeruginosa stimulates tyrosine phosphorylation of and TLR5 association with the MUC1 cytoplasmic tail through EGFR activation. Inflamm. Res. 65, 225–233 (2016). https://doi.org/10.1007/s00011-015-0908-8

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  • DOI: https://doi.org/10.1007/s00011-015-0908-8

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