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.
Lee
A Y
AY
Division of Dermatology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Akers
K T
KT
Collier
M
M
Li
L
L
Eisen
A Z
AZ
Seltzer
J L
JL
D015415:Biomarkers; D002478:Cells, Cultured; D003094:Collagen / Q000494:pharmacology; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018093:Gelatinases / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism*; D011233:Precipitin Tests; D038981:Receptors, Collagen; D013347:Subcellular Fractions / Q000378:metabolism
D015415:Biomarkers; D004792:Enzyme Precursors; D016023:Integrins; D038981:Receptors, Collagen; D003094:Collagen; D018093:Gelatinases; D008666:Metalloendopeptidases; C069860:progelatinase