LOCALIZATION AND ACTIVATION OF TYPE-IV COLLAGENASE GELATINASE AT ENDOTHELIAL FOCAL CONTACTS/

The cell-surface localization and site of activation of type IV collag enases/gelatinases (matrix metalloproteinases, MMP) in bovine pulmonar y microvascular endothelial (BPMVE) cells was examined. Sucrose densit y centrifugation of plasma membranes and immunofluorescent staining of whole cells indicated association of 72 kDa (MMP-2) and 96 kDa (MMP-9 ) type IV collagenase/gelatinases with the plasma membrane. Incubation of the BPMVE cells with rhodaminated MMP-9 demonstrated colocalizatio n with beta(1)-integrin, indicating incorporation into the focal conta cts. The focal contacts were extracted with saponin, and associated pr oteolytic activity was examined by zymography. The focal contacts cont ained latent MMP-2, and stimulation of the cells with cytochalasin D o r with 8-bromoadenosine 3',5'-cyclic monophosphate with 3-isobutyl-1-m ethylxanthine increased both latent and activated MMP-9 in the focal c ontacts. Addition of these stimuli in unconditioned culture medium did not produce this effect, indicating that the MMP-9 in focal contact e xtracts was derived from previously secreted enzyme. The activated met alloproteinase degraded extracellular matrix collagens and was inhibit ed by 1,10-phenanthroline. These findings indicate that endothelial ce lls release MMP into the extracellular milieu and then concentrate and activate MMP-9 from medium at the focal contacts.