Mitogenic responses mediated through the proteinase-activated receptor-2 are induced by expressed forms of mast cell alpha- or beta-tryptases

The proteinase-activated receptor-2 (PAR-2) is the second member of a putative larger class of proteolytically activated receptors that mediate cell activation events by receptor cleavage or synthetic peptidomimetics corresponding to the newly generated N-terminus. To further study the previously identified mitogenic effects of PAR-2, we used the interleukin-l (IL-3)-dependent murine lymphoid cell line, BaF3, for generation of stable cell lines expressing PAR-2 (BaF3/PAR-2) or the noncleavable PAR-2 mutant PAR-2(Arg36-->Ala36). Only BaF3 cells expressing either wild-type or mutated receptor exhibited mitogenic responses when grown in IL-3-deficient media supplemented with PAR-2 activating peptide (SLIGRL, PAR(39-44)). This effect was dose dependent with an EC50 of similar to 80 mu mol/L, sustained at 24, 48, and 72 hours, and was also demonstrable using thrombin receptor peptide TR42-47. Because tryptase shares similar to 70% homology with trypsin (previously shown to activate PAR-2), we studied recombinantly expressed forms of alpha- and beta-tryptases as candidate protease agonists for PAR-2. Hydrolytic activity of the chromogenic substrate tosyl-glycyl-prolyl-argly-4-nitroanilide acetate was present as a sharp peak at M-r similar to 130, confirming the presence of secretable and functionally active homotetrameric cu-and beta-tryptases in transfected COS-1 cells. Dose-dependent proliferative responses were evident using either secreted form of tryptase with maximal responses seen at similar to 3 pmol/L (0.1 U/L). Receptor proteolysis was necessary and sufficient for mitogenesis because active site-blocked tryptase failed to induce this response, and proliferative responses were abrogated in BaF3 cells expressing PAR-2(Arg36-->Ala36). These results specifically identify both forms of mast cell tryptases as serine protease agonists for PAR-2 and have implications for elucidating molecular mechanisms regulating cellular activation events mediated by proteases generated during inflammatory, fibrinolytic, or hemostatic-regulated pathways. (C) 1997 by The American Society of Hematology.