DOG MASTOCYTOMA-CELLS SECRETE A 92-KD GELATINASE ACTIVATED EXTRACELLULARLY BY MAST-CELL CHYMASE

Gelatinolytic metalloproteinases implicated in connective tissue remod eling and tumor invasion are secreted from several types of cells in t he form of inactive zymogens, In this report, characterization of gela tinase activity secreted by the BR line of dog mastocytoma cells revea ls a phorbol-inducible, similar to 92-kD, Ca2+- and Zn2+-dependent pro enzyme cleaved over time to smaller, active forms, Incubation of cells with the general serine protease inhibitor, PMSF, prevented proenzyme cleavage and permitted its purification free of activation products, The NH2-terminal 13 amino acids of the purified mastocytoma progelatin ase are 50-67% identical to those of human, mouse, and rabbit 92-kD pr ogelatinase (gelatinase B; matrix metalloproteinase-9). Degranulation of mastocytoma cells using ionophore A23187 greatly accelerated proenz yme cleavage, suggesting that a serine protease present in secretory g ranules hydrolyzed the progelatinase to active fragments, To identify the activating protease, cells were coincubated with ionophore and a p anel of selective serine protease inhibitors, Soybean trypsin inhibito r and succinyl-L-Ala-Ala-Pro-Phe-chloromethylketone, which inhibit mas t cell chymase, prevented progelatinase activation. Inhibitors of tryp tase and dog mast cell protease (dMCP)-3, i.e., aprotinin or bis(5-ami dino-2-benzimidazolyl) methane (BABIM), did not. In further experiment s using highly purified enzymes, mastocytoma cell chymase activated 92 -kD progelatinase in the absence of other enzymes or cofactors; trypta se and dMCP-3, however, had no effect. These data demonstrate that dog mastocytoma cells secrete a metalloproteinase related to progelatinas e B that is directly activated outside of the cell by exocytosed chyma se, and provide the first demonstration of a cell that activates a mat rix metalloproteinase it secretes by cosecreting an activating enzyme, In mastocytomas, this pathway may facilitate tumor invasion of surrou nding tissues, and in normal mast cells, it could play a role in tissu e remodeling and repair.