2-(4-Aminophenyl)acetic acid ethyl ester

A Mr 92,000 metalloprotease, originally observed in neutrophils, has been found to be secreted by various normal and malignant cells of fibroblastic, hematopoietic, and epithelial origin. The responsiveness of the various cell types to the tumor promoter phorbol ester (phorbol myristate acetate) to secrete this enzyme and a corresponding Mr 72,000 gelatinase has been determined using gelatin zymograms. The latent zymogen form of the Mr 92,000 enzyme has been purified from phorbol myristate acetate-stimulated HT1080 human fibrosarcoma cells using sequential gelatin-Sepharose affinity chromatography and gel filtration. Selective elution from gelatin-Sepharose allows for a distinct separation of the Mr 92,000 gelatinase from the Mr 72,000 gelatinase. A fraction of the tumor cell derived latent Mr 92,000 enzyme is isolated as an apparent complex with human tissue inhibitor of metalloproteases, which is partially dissociated in sodium dodecyl sulfate and completely dissociated upon reduction of disulfide bonds and upon p-aminophenylmercuric acetate treatment. Organomercurial treatment rapidly allows for autoactivation of the proenzyme to active Mr 83,000 and Mr 75,000 species. At physiological pH, the enzyme rapidly degrades gelatin into small fragments and slowly cleaves native type V collagen at an apparent single site. Native type IV collagen is degraded to a much lesser extent. The NH2-terminal amino acid sequence of the Mr 92,000 proenzyme has been determined and is distinct from the Mr 72,000 gelatinase/type IV collagenase which is constitutively produced by fibroblasts. The Mr 92,000 enzyme is also immunologically distinct from the Mr 72,000 enzyme but immunologically cross-reactive with the neutrophil, high molecular weight gelatinase. The Mr 92,000 enzyme constitutes a distinct member of the matrix metalloprotease family. Its substrate specificity implies a broad physiological role, acting on basement membrane type V collagen as well as on denatured (gelatinized) collagens and thus may be involved in the invasive and migratory phenotype of human cells.

It has been proposed that proteases are important in endothelial cell behavior. We examined the contribution of the gelatinase/type IV collagenase system in an in vitro model of endothelial differentiation. Human umbilical vein endothelial cells rapidly align and form networks of tubes when cultured on a basement membrane preparation, Matrigel. Zymograms of culture supernates demonstrate a 72-kD and a 92-kD gelatinase activity; the cells produce most of the 72-kD gelatinase, whereas the 92-kD activity is derived entirely from the Matrigel. Addition of antibodies against type IV gelatinase/collagenase decreases the area of the tube network. Both tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, similarly decrease tube formation when added to cultures. Conversely, exogenous recombinant 72-kD gelatinase increases tube-forming activity. The effects of the anti-gelatinase antibodies and the TIMPs are not additive. Inhibition by either antibodies or TIMPs is greatest when they are added at culture initiation, suggesting that the protease activity is important in the early steps of morphogenesis. However, culture of the cells on Matrigel does not increase early expression of mRNA for the 72-kD gelatinase. Expression of message for the enzyme actually decreases during the course of the assay, while transcription of mRNAs for TIMPs increases, further supporting the concept that collagenases facilitate an early event in tube formation. These data demonstrate that gelatinase/type IV collagenase activity is important in endothelial cell morphogenesis on Matrigel, and suggest a role for collagenases in formation of new capillaries in vivo.

The role of matrix metalloproteinase-9 (MMP-9, 92 kDa gelatinase/type IV collagenase) in invasion of mononuclear phagocytes was studied with U937 monoblastoid cells. 12-o-tetradecanoyl 13-phorbol acetate (TPA) differentiated them to macrophage-like cells with induction of MMP-9, and tumor necrosis factor alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) stimulated the production of MMP-9 by TPA-treated cells. TNF alpha also induced the production of MMP-9 by TPA-untreated U937 cells without morphological differentiation. Other agents including dimethyl sulfoxide (DMSO), all-trans-retinoic acid (all-trans-RA), platelet-derived growth factor and 3';5'-cyclic monophosphate had no effects on MMP-9 production by TPA-treated or -untreated cells, but all-trans-RA and DMSO did have a morphological effect on the differentiation of the cells. These data suggest that MMP-9 production by U937 cells is regulated by a mechanism independent of the differentiation to macrophage-like cells. MMP-9 was purified to homogeneity as an inactive zymogen with M(r) 92,000 (proMMP-9) from TPA-differentiated U937 cells treated with TNF alpha. ProMMP-9 was activated by p-aminophenylmercuric acetate (APMA) generating an active species of M(r) 67,000. Trypsin and cathepsin G also attained activation of the zymogen to its full activity obtained by APMA activation, but plasmin, leukocyte elastase, thrombin and plasma kallikrein had no ability to activate it. APMA-activated MMP-9 degraded type I gelatin readily and cleaved native collagen types III, IV and V. Invasion assays using reconstituted basement membrane coupled with a type IV collagenolysis assay showed good correlations between invasiveness, type IV collagenolysis and proMMP-9 production. Invasion was significantly inhibited by EDTA, alpha 2-macroglobulin and tissue inhibitor of metalloproteinases-1, but not by inhibitors of cathepsin G and leukocyte elastase. These data suggest that MMP-9 plays an important role in the invasion of mononuclear phagocytes through basement membranes.