Abstract.
Objective: To investigate whether cockroach allergen extract can stimulate Protease-activated receptor 2 (PAR-2) expressed in mouse lung fibroblast.
Materials: We established an immortalized lung fibroblast cell line, DM5, from PAR-2 deficient mice. By stable transfection with either an empty vector (DM5/EV) or an expression vector encoding mouse PAR-2 cDNA (DM5/Par2), a pair of lung fibroblast cell lines with or without functional PAR-2 expression were prepared.
Treatment: The cells were exposed to cockroach allergen extract {up to 800 protein nitrogen unit (PNU)/ml}, trypsin (up to 100 nM), SLIGRL agonist peptide (up to 500 μM), and trans-cinnamoyl-LIGRO agonist peptide (up to 400 μM).
Methods: The cells were loaded with Fluo-3 calcium indicator and mobilization of intracellular calcium with the stimuli was monitored by a fluorometric plate reader. Extracellular signal-regulated kinase (ERK) phosphorylation was examined by Western blot analysis using an anti-phospho ERK antibody.
Results: The cockroach extract induced intracellular calcium transients in a concentration dependent manner in DM5/Par2 but not in DM5/EV. The activity was abolished when the cockroach extract was heat denatured or pre-incubated with PMSF (phenylmethanesulfonyl fluoride) prior to the assay. Concomitantly, ERK phosphorylation was seen in DM5/Par2 with the cockroach extract but not with a heat-denatured extract. The responses were sensitive to an inositol-1,4,5 triphosphate antagonist (2-APB) indicating that calcium was mobilized from intracellular store.
Conclusions: Cockroach allergen extract can activate PAR-2 and thereby stimulate mouse lung fibroblasts likely through protease(s). The present study proposes a potential mechanism for cockroach antigens, similar to house dust mite antigens, in the etiology of respiratory diseases.
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Received 29 February 2004; returned for revision 12 April 2004; accepted by M. Katori 22 April 2004
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Kondo, S., Helin, H., Shichijo, M. et al. Cockroach allergen extract stimulates protease-activated receptor-2 (PAR-2) expressed in mouse lung fibroblast. Inflamm. res. 53, 489–496 (2004). https://doi.org/10.1007/s00011-004-1287-8
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DOI: https://doi.org/10.1007/s00011-004-1287-8