Abstract. We found that striptease-positive mast cells were abundant in the invasive front of human colon adenocarcinoma by examining 30 cases. Because tryptase has been suggested to be the agonist proteinase for protease-activated receptor-2 (PAR-2), we investigated the effects of stimulation of PAR-2 by tryptase on the cell signaling and proliferation of DLD-1, a human colon carcinoma cell line. PAR-2 stimulation by tryptase induced the increase in [Ca 2+ ] i , which was desensitized by the prior application of PAR-2 activating peptide (AP). The proliferative responses of DLD-1 to tryptase and PAR-2 AP were associated with the phosphorylation of MEK and MAP kinase. Inhibition of MEK by PD98059 completely inhibited the proliferationenhancing effects of tryptase and PAR-2 AP as well as phosphorylation of MAP kinase. Moreover, tryptase and PAR-2 AP stimulated the production of prostaglandin E 2 and the inhibition of prostaglandin synthesis by indomethacin or NS398 resulted in the complete inhibition of the proliferative responses to tryptase and PAR-2 AP. Furthermore, the tryptase-stimulated proliferation of DLD-1 was concentration-dependently inhibited by nafamostat mesilate, a specific inhibitor of tryptase. These results as a whole indicated that tryptase has proliferative effects on DLD-1 through cyclooxygenase-and MAP kinase-dependent manners acting on PAR-2 by its proteolytic activity.

Protease-activated receptor-2 (PAR-2) has been demonstrated to be highly expressed in the gastrointestinal tract. In the present study, we investigated the effects of PAR-2 stimulation on the cell signaling and proliferation of DLD-1, a human colon carcinoma cell line, in comparison with the PAR-1 stimulation. PAR-2 stimulation by agonist peptide SLIGKV concentration-dependently induced the increase in [Ca2+]i and the proliferation of DLD-1 whereas the inverse peptide LSIGKV did not. Trypin (10(-9) M), an agonist protease for PAR-2, also enhanced the proliferation of DLD-1. The proliferative response of DLD-1 to PAR-2 stimulation was associated with the transient phosphorylation of MEK and MAP kinase, but not p38 MAP kinase and JNK. Inhibition of MEK by PD98059 (50 microM) completely inhibited the proliferation-stimulating effects as well as the phosphorylation of MAP kinase induced by PAR-2 agonist peptide (100 microM) and trypsin (10(-9) M). The prolonged treatment with PAR-2 agonist peptide for more than one hour was required for the enhanced proliferative response, suggesting the existence of unknown long-lasting cooperative signaling with MAP kinase cascade. PAR-1 stimulation by the agonist peptide SFLLRN (100 microM) or thrombin (10(-8) M) produced Ca2+ signaling, however, the stimulation neither produced the cell proliferative response nor the activation of MEK-MAP kinase cascade. These results indicated that Ca2+ signaling induced by PARs activation was not enough for inducing the cell proliferation in DLD-1 cells and that stimulation of PAR-2 can induce the activation of MEK-MAP kinase cascade, leading to the growth promoting response.