Tryptase Activates Phosphatidylinositol 3-Kinases Proteolytically Independent from Proteinase Activated Receptor-2 in Cultured Dog Airway Smooth Muscle Cells

Mast cell tryptase is a potent mitogen for many cells in the airways and lung, but the cellular mechanisms for its growth stimulatory effects are poorly understood. Our major goal was to determine whether tryptase activates phosphatidylinositol 3-kinases (PI 3-kinases) in cultured dog tracheal smooth muscle cells to induce its mitogenic effects. After exposure to tryptase, cells were lysed. Immunocomplexes prepared from the lysates using an antibody to the p85 subunit of PI 3-kinase, but not using antiphosphotyrosine antibodies, possessed increased capacity to phosphorylate inositol on its D3 hydroxyl group. Tryptase also increased phosphorylation of Akt, a downstream target of PI-3 kinases. This effect was abolished by one PI 3-kinase inhibitor, wortmannin, and attenuated by another, LY294004, which also blocked tryptase’s mitogenic effects. Treatment of tryptase with p-amidino phenylmethanesulfonyl fluoride, to abolish its proteolytic activity irreversibly, inhibited its stimulatory effects on Akt phosphorylation. Proteinase-activated receptor-2 (PAR2) activating peptides failed to increase Akt phosphorylation in the cultured dog tracheal smooth muscle cells, but the PAR2 activating peptides did induce brisk increases in Akt phosphorylation in MadinDarby Canine Kidney cells. We concluded that tryptase activates PI 3-kinases in cultured dog tracheal smooth muscle cells to induce its potent mitogenic effects. These effects of tryptase on PI 3-kinases appear to occur via novel proteolytic mechanisms independent from PAR2. Also, tryptase, though comparable in mitogenic potency to platelet-derived growth factor (PDGF), induces considerably less tyrosine phosphorylation on proteins than occur in response to PDGF.