REACTION OF MAST-CELL PROTEASES TRYPTASE AND CHYMASE WITH PROTEASE ACTIVATED RECEPTORS (PARS) ON KERATINOCYTES AND FIBROBLASTS

Protease activated receptors (PARs) com pose a family of G protein sig nal transduction receptors activated by proteolysis. In this study, th e susceptibility of PARs expressed on human keratinocytes and dermal f ibroblasts to the human mast cell proteases tryptase and chymase was e valuated. PAR activation was measured by monitoring cytosolic [Ca2+] i n cells loaded with the fluorescent Ca2+ probe Fura-2. Tryptase produc ed transient cytosolic Ca2+ mobilization in keratinocytes, but not in fibroblasts. Ca2+ mobilization in keratinocytes required enzymatically active tryptase, demonstrated desensitization, and was blocked by pre treatment of cells with the PAR-2 peptide agonist SLIGKV, trypsin, or the phospholipase inhibitor U73122. Heparin, a GAG that binds to trypt ase, stabilizing its functional form, also inhibited tryptase-induced Ca2+ mobilization. The maximal response elicited by tryptase was small er than that observed upon treatment of keratinocytes with trypsin, a known activator of PAR-2, and keratinocytes made refractory to tryptas e by pretreatment with the protease remained responsive to trypsin. Pr etreatment of keratinocytes with thrombin, an activator of PAR-1 and - 3 (thrombin receptors), had no detectable effect on the tryptase or tr ypsin responses. These data suggest that in keratinocytes tryplase may be activating a subpopulation of PAR-2 receptors. Treatment of kerati nocytes or fibroblasts with human chymase did not produce Ca2+ mobiliz ation, nor did it affect Ca2+ mobilization produced by trypsin. Howeve r, chymase pretreatment of fibroblasts rapidly inhibited the ability o f these cells to respond to thrombin. Inhibition was dependent on chym ase enzymatic activity and was not significantly affected by the prese nce of heparin. This finding is consistent with studies indicating tha t PAR-1 may be susceptible to proteases with chymotrypsin-like specifi city. These results suggest that the proteases tryptase and chymase se creted from mast cells in skin may affect the behavior of surrounding cells by the hydrolysis of PARs expressed by these cells. (C) 1998 Wil ey-Liss, Inc.