RESUMO
Protease-activated receptor 2 (PAR2) has been implicated in the pathogenesis of airway inflammation. We report that epithelial PAR2 stimulation with trypsin (0.05-1 U/ml) or an agonist peptide (SLIGKV-NH2, 1-100 microM) for 0.5-3 h dose- and time-dependently enhanced neutrophil adhesion to alveolar type II epithelial cells (A549 cells) and that this stimulation also induced the formation of epithelial actin filaments. Both responses in neutrophil adhesion and epithelial actin reorganization were reduced by a Rho inhibitor, mevastatin and by a Rho-associated kinase (ROCK) inhibitor, Y-27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide). Neutrophil adherence was also inhibited by an inhibitor of actin polymerization, cytochalasin D and a tyrosine kinase inhibitor, genistein. Further, the PAR2-mediated tyrosine phosphorylation of focal adhesion kinase (FAK), a major cytoskeleton protein, was detected, and this response was inhibited by mevastatin or Y-27632. These results suggest that PAR2 stimulation of alveolar epithelial cells enhances neutrophil adhesion presumably at least in part through Rho/ROCK signal-mediated actin cytoskeleton reorganization associated with the tyrosine phosphorylation of FAK.
Assuntos
Neutrófilos/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptor PAR-2/fisiologia , Transdução de Sinais , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Amidas/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citocalasina D/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Genisteína/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Pulmão/citologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Receptor PAR-2/agonistas , Fatores de Tempo , Tripsina/farmacologia , Quinases Associadas a rhoRESUMO
The protease-activated receptor-2 (PAR-2) has been implicated in airway inflammation. Here, we examined the interaction between PAR-2 and lipopolysaccharide (LPS), a major proinflammatory factor, using cultured guinea pig tracheal epithelial cells. In fura2-loaded cells, LPS (1 microg/ml) transiently increased intracellular Ca(2+) concentrations ([Ca(2+)]i), this effect being abolished by a Ca(2+) channel blocker, verapamil, and Ca(2+) removal. Prestimulation of PAR-2 with trypsin (0.1-1 U/ml) or an agonist peptide (SLIGRL-NH(2), 1 microM) for 60 min inhibited the LPS-induced [Ca(2+)]i increase. Such an inhibitory effect of trypsin was abolished by inhibitors of protein kinase C (PKC), chelerythrine and staurosporine. A PKC activator, phorbol 12,13-dibutylate, also reduced the LPS response. Trypsin also inhibited a transient increase in [Ca(2+)]i caused by a Ca(2+) channel opener, Bay K 8644. When the trypsin-pretreated cells were incubated in normal buffer for 10-60 min before LPS exposure, the effect of trypsin on the Ca(2+) response to LPS diminished in a time-dependent manner. Such a recovery was slowed by incubation with a protein phosphatase inhibitor, okadaic acid. Further, trypsin induced sustained activations of PKCalpha and -epsilon. Thus, PAR-2 stimulation reduced the epithelial cell response to LPS, probably through the inactivation of Ca(2+) channels via PKC-mediated phosphorylation.
Assuntos
Cálcio/metabolismo , Células Epiteliais/metabolismo , Lipopolissacarídeos/metabolismo , Receptor PAR-2/metabolismo , Mucosa Respiratória/citologia , Traqueia/anatomia & histologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Células Cultivadas , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Cobaias , Isoenzimas/metabolismo , Lipopolissacarídeos/farmacologia , Ácido Okadáico/farmacologia , Dibutirato de 12,13-Forbol/farmacologia , Proteína Quinase C/metabolismo , Receptor PAR-2/agonistas , Receptor PAR-2/imunologia , Mucosa Respiratória/metabolismo , Traqueia/metabolismo , Tripsina/metabolismoRESUMO
The protease-activated receptor-2 (PAR-2), a G protein-coupled receptor activated by trypsin, contributes to the pathogenesis of inflammatory disease including asthma. Here, we examined the mechanisms by which stimulation of PAR-2 induces an increase in intracellular Ca2+ concentration ([Ca2+]i) in guinea pig tracheal epithelial cells. Trypsin (0.01-3 units/ml) dose-dependently induced a transient increase in [Ca2+]i, the increase being blocked by soybean trypsin inhibitor (SBTI 1 microM). An increase in [Ca2+]i was also induced by an agonist peptide for PAR-2 (SLIGRL-NH2, 0.001-10 microM) but not by thrombin (3 units/ml, an activator for PAR-1, PAR-3 or PAR-4). Repeated or cross stimulation of trypsin or SLIGRL-NH2 caused marked desensitization of the [Ca2+]i response. These responses of [Ca2+]i to trypsin and SLIGRL-NH2 were attenuated by a phospholipase C inhibitor, U-73122, and a Ca2+-ATPase inhibitor, thapsigargin (100 nM), while removal of Ca2+ and a L-type Ca2+-channel blocker, verapamil, were without significant effects. Further, trypsin was without effect on the rate of fura 2 quenching by Mn2+ entry as an indicator of Ca2+ influx. Thus, stimulation of PAR-2 appears to increase [Ca2+]i through the mobilization of Ca2+ from intracellular stores probably via phospholipase Cbeta-linked generation of a second messenger.