Intracellular activation of gelatinase A (72-kDa type IV collagenase) by normal fibroblasts.

Normal fibroblasts cultured as monolayers secrete matrix metalloproteinases (MMP), including gelatinase A (72-kDa type IV collagenase) as inactive zymogens. Previously we found that normal fibroblasts cultured in a type I collagen lattice (dermal equivalent) secrete active gelatinase A. Here we show that the activation of progelatinase A occurs within the cell and that the activator copurifies with Golgi membranes. Cell extracts of fibroblasts cultured in collagen lattices contain active 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 fraction. Highly purified plasma membranes from lattice cultures are unable 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 of gelatinase A mRNA and protein increase during the culture period, and tissue inhibitor of metalloproteinases concentration does not change. Because of the greater availability of tissue inhibitor of metalloproteinases-free proenzyme as a substrate for the activator, it is possible that membrane-type MMP1 is the activating enzyme. In that case, malignant transformation may involve a change in the localization of the activator to the plasma membrane.