Tryptase activates phosphatidylinositol 3-kinases proteolytically independently 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 anti-phosphotyrosine 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, LY-294004, 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 (PAR-2)-activating peptides failed to increase Akt phosphorylation in cultured dog tracheal smooth muscle cells, but the PAR-2-activating peptides did induce brisk increases in Akt phosphorylation in Madin-Darby 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 PAR-2. Also, tryptase, although comparable in mitogenic potency to platelet-derived growth factor (PDGF), induces considerably less tyrosine phosphorylation on proteins than occur in response to PDGF.
Brown
James K
JK
Pulmonary and Critical Care Medicine Section, Dept. of Veterans Affairs Medical Center, and Department of Medicine, University of California San Francisco, CA 94121, USA. jkbrown@itsa.ucsf.edu
Hollenberg
Morley D
MD
Jones
Cary A
CA
D000818:Animals; D000936:Antigen-Antibody Complex; D002478:Cells, Cultured; D004285:Dogs; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D009130:Muscle, Smooth / Q000166:cytology / Q000201:enzymology*; D019869:Phosphatidylinositol 3-Kinases / Q000276:immunology / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D019000:Phosphotyrosine / Q000276:immunology; D021122:Protein Subunits / Q000276:immunology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D044464:Receptor, PAR-2 / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D014132:Trachea; D053802:Tryptases
D000936:Antigen-Antibody Complex; D048049:Extracellular Signal-Regulated MAP Kinases; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D019000:Phosphotyrosine; D021122:Protein Subunits; D051057:Proto-Oncogene Proteins c-akt; D044464:Receptor, PAR-2; D012697:Serine Endopeptidases; D053802:Tryptases