Mechanisms of allergy
Proteinase-activated receptor-2–mediated matrix metalloproteinase-9 release from airway epithelial cells,☆☆,

https://doi.org/10.1067/mai.2000.109058Get rights and content

Abstract

Background: Matrix metalloproteinases (MMPs) digest extracellular matrix components and might be important mediators of tissue remodeling. Proteinase activated receptor-2 (PAR-2) is expressed in a variety of cell types including epithelial cells. PAR-2 receptors are activated by serine proteases such as trypsin and mast cell tryptase and have been implicated in inflammation. Objective: To study the effects of PAR-2–mediated airway epithelial cell activation on the production of MMP-9. Methods: A specific PAR-2–activating peptide and trypsin were used to activate the human airway epithelial cell line A549 as well as primary cultures of small airway epithelial cells (SAEC). MMP-2 and MMP-9 messenger RNA and enzymatic activity were evaluated by RT-PCR and gelatin zymography, respectively. Results: PAR-2–activating peptides upregulated MMP-9 mRNA expression and release of MMP-9 enzymatic activity from airway epithelial cells but had no effect on MMP-2 production. Dexamethasone and budesonide (10–6 to 10–10 mmol) inhibited PAR-2–mediated MMP-9 release. Pretreatment with indomethacin indicated that MMP-9 release was not prostaglandin dependent. Inhibitors of the MAP kinase MEK- 1, and NFκB showed that both pathways are important for PAR-2–mediated MMP-9 release. Trypsin, a physiologic PAR-2 activator, upregulated MMP-9 but also MMP-2 release from airway epithelial cells. Conclusion: PAR-2 receptors appear to play an important role in the regulation of MMP-9 release from airway epithelial cells. As such, these receptors may be critical elements in tissue remodeling in asthma and other inflammatory conditions in the airways. (J Allergy Clin Immunol 2000;106:537-45.)

Section snippets

Material

Pancreatic trypsin, dexamethasone, and MG-132 (Cbz-Leu-Leu-leucinal; Sigma-Aldrich Canada LTD, Oakville, Ontario, Canada); recombinant human TNF (Cedarlane Laboratories Ltd, Ontario, Canada); Trizol, murine Moloney leukemia virus reverse transcriptase, Taq polymerase, oligo-dT primers, (Gibco BRL, Ontario, Canada); Fura-2 (Molecular Probes Inc, Eugene, Ore); Dulbecco’s modified Eagle’s medium (DMEM), FCS, penicillin, streptomycin, and L -glutamine (Biowhittaker, Walkersville, Md) were purchased

PAR-2 activation induces MMP-9 mRNA and enzymatic activity in A549 cells

Trypsin (10 U/mL) induced a sharp increase in the intracellular Ca++ concentration in A549 cells (Fig l), as did the PAR-2–activating peptide tc-LIGRLO-NH2 (70 μmmol/L; Fig 1).

. Free [Ca++] levels in A549 cells after activation with 10 U/mL of trypsin (top panel ) or 70 μmmol/L tc-LIGRLO-NH2 (bottom panel ).

The inactive peptide LRGILS-NH2 had no effect. Although treatment of the cells with trypsin desensitized the PAR-2 response to tc-LIGRLO-NH2 (Fig 1), pretreatment of the cells with

Discussion

In this study, we demonstrated that PAR-2–mediated activation of airway epithelial cells upregulated the synthesis and release of pro–MMP-9. MMP-9 was secreted in the proenzyme form from epithelial cells after PAR-2 activation. Recently, PAR-2 activation has been shown to cause relaxation of airways that was mediated by the release of cyclooxygenase products from airway epithelium,24 which indicates a protective role for airway epithelial cell PAR-2. Our results indicate that the effect of

Acknowledgements

The authors thank Ms Jolanda Sawicka and Dr Greg Sawicki for providing positive controls for MMP-2 and MMP-9 gelatin zymography and TIMP-1 and TIMP-2 Western blot analysis.

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      To our knowledge, the expression of MMP-1 and MMP-13 in chondrocytes following PAR2 activation is a novel observation, although the addition of SLIGKV-NH2 to OA cartilage has previously been shown to increase MMP-1 and MMP-13 immunostaining (22). Aside from the collagenases, other MMPs, including MMP-2 (42), MMP-3 (43), and MMP-9 (44), have been shown to be induced by PAR2 activation in various cell types. Biased agonism following activator peptide stimulation of PAR2 has previously been explored.

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    Supported by an MRC/PMAC Fellowship, MRC Canada, Alberta Heritage Foundation of Medical Research (AHFMR) and Glaxo-Heritage.

    ☆☆

    H.V. is an MRC Scholar and an Alberta Heritage Clinical Investigator. M.D. and R.M. are Alberta Heritage Senior Medical Scholars. A.D.B. holds the Astra Chair in Asthma Research. J.L.W. is an Alberta Heritage Scientist MRC and a Senior Scientist.

    Reprint requests: Redwan Moqbel, PhD, FRCPath, Pulmonary Research Group, Department of Medicine 574 HMRC, University of Alberta, Edmonton, AB, T6G 2S2, Canada.

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