Mast cell tryptase activates extracellular-regulated kinases (p44/p42) in airway smooth-muscle cells - Importance of proteolytic events, time course,and role in mediating mitogenesis

We previously reported that mast cell tryptase is a potent mitogen for cult ured airway smooth-muscle cells, but the early intracellular signals mediat ing this response are not known. In many cells, proliferative effects are m ediated by a mitogen-activated protein kinase signaling pathway involving R af-1, MAP kinase kinases (MEKs), and extracellular signal-regulated protein kinases (ERKs) 1 and 2. Therefore, we tested for tryptase-induced activati on of ERK1 and 2 in cultured dog tracheal smooth-muscle cells. Tryptase, in nanomolar concentrations which potently stimulated DNA synthesis, increase d dual phosphorylation of ERKs in cellular lysates as well as ERK2 kinase a ctivity in immunoprecipitates. Pretreatment of cells with the MEK inhibitor PD098059 abolished tryptase-induced increases in DNA synthesis and attenua ted increases in ERK2 activity. irreversible inhibition of tryptase's prote olytic activity, using p-amidino phenylmethanesulfonyl fluoride, attenuated tryptase-induced increases in DNA synthesis and dual phosphorylation of ER Ks by 76% and 40 to 60%, respectively. Tryptase also increased c-fos transc ription as quantified in polymerase chain reactions. In concentrations that caused similar increases in DNA synthesis, tryptase and platelet-derived g rowth factor (PDGF-BB) increased ERK activity (and c-fos transcription) wit h markedly different kinetics, the tryptase-induced responses being slower in onset and more sustained. We conclude that tryptase-induced mitogenesis in airway smooth-muscle cells requires activation of ERK1 and 2; that these responses depend partially, but not completely, upon tryptase's properties as a protease; and that they are slower in onset and more sustained than t hose induced by PDGF-BB.