INTRACELLULAR ACTIVATION OF GELATINASE-A (72-KDA TYPE-IV COLLAGENASE)BY NORMAL FIBROBLASTS

Normal fibroblasts cultured as monolayers secrete matrix metalloprotei nases (MMP), including gelatinase 4 (72-kDa type IV collagenase) as in active zymogens. Previously we found that normal fibroblasts cultured in a type I collagen lattice (dermal equivalent) secrete active gelati nase A. Here me show that the activation of progelatinase A occurs wit hin the cell and that the activator copurifies with Golgi membranes. C ell extracts of fibroblasts cultured in collagen lattices contain acti ve 62-kDa gelatinase A at least 4-6 h before active enzyme is detected in the culture medium, Pulse-chase experiments confirm these results, The activator is membrane-bound and localizes to the Golgi-enriched f raction, Highly purified plasma membranes from lattice cultures are un able to convert gelatinase A from the zymogen to its active form. The activator may be a metalloproteinase because EDTA prevents activation of exogenous proenzyme by membrane fractions. Membrane-type MMP1, the enzyme thought to be responsible for activation of gelatinase A on the plasma membrane of tumor cells, shows no significant change in either mRNA or protein levels during lattice culture, Intracellular levels o f gelatinase A mRNA and protein increase during the culture period, an d tissue inhibitor of teinases concentration does not change, Because of the greater availability of tissue inhibitor of metalloproteinases- free ee proenzyme as a substrate for the activator, it is possible tha t membrame-type MMP1 is the activating enzyme. In that case, malignant transformation may involve a change in the localization of the activa tor to the plasma membrane.