Cockroach allergen extract stimulates protease-activated receptor-2 (PAR-2) expressed in mouse lung fibroblast.
OBJECTIVE
To investigate whether cockroach allergen extract can stimulate Protease-activated receptor 2 (PAR-2) expressed in mouse lung fibroblast.


METHODS
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.


METHODS
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 microM), and trans-cinnamoyl-LIGRO agonist peptide (up to 400 microM).


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.
Kondo
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Bayer Research Center Kyoto, Bayer Yakuhin, Ltd., 6-5-1-3 Kunimidai, 619-0216 Kizu-cho, Soraku-gun, Kyoto, Japan. shin1kondo@ybb.ne.jp
Helin
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Shichijo
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Bacon
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KB
D000485:Allergens / Q000276:immunology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003058:Cockroaches / Q000276:immunology*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D008168:Lung / Q000166:cytology; D051379:Mice; D008810:Mice, Inbred C57BL; D044464:Receptor, PAR-2 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D000485:Allergens; D044464:Receptor, PAR-2; D002118:Calcium