S Kondo1, H Helin, M Shichijo, K B Bacon. 1. Bayer Research Center Kyoto, Bayer Yakuhin, Ltd., 6-5-1-3 Kunimidai, 619-0216 Kizu-cho, Soraku-gun, Kyoto, Japan. shin1kondo@ybb.ne.jp
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 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.
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 mousePAR-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 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.