The cleavage of pro-urokinase type plasminogen activator by stromelysin-1.

Membrane binding of urokinase type plasminogen activator (u-PA) is thought to play a pivotal role in connective tissue remodeling and invasive processes. We compare the ability of different matrix-metalloproteinases involved in connective tissue turnover to cleave pro-urokinase type plasminogen activator between the catalytic domain and the receptor binding part to investigate a potential role for matrix-metalloproteinases in the regulation of membrane-associated proteolytic activity. We employed several forms of human stromelysin-1 (full length, C-truncated, and recombinant catalytic domain), rabbit C-truncated stromelysin-1, the human gelatinases A and B and the human catalytic domain of neutrophil collagenase. The gelatinases and the collagenase did not separate the receptor binding domain of pro-urokinase type plasminogen activator from the catalytic domain, whereas all stromelysin-1 forms cleaved the glutamic acid 143-leucine 144 bond of pro-urokinase type plasminogen activator. This reaction could be inhibited by specific inhibitors of matrix metalloproteinases and was not affected by inhibitors of serine proteinases. The M(r) 31000 cleavage product with leucine 144 as N-terminus displayed no proteolytic activity towards the pro-urokinase type plasminogen activator substrate pyroGlu-Gly-Arg-pNA-HCI (S2444), but it could be activated by an additional treatment with plasmin. Comparison between full length stromelysin-1 and its C-truncated forms, showed that both exhibited the same cleavage properties towards pro-urokinase type plasminogen activator. Thus, the cleavage of pro-urokinase type plasminogen activator by stromelysin-1 is not influenced by the presence or absence of the C-terminal domain. The recombinant catalytic domain of MMP-3 generated pro-urokinase type plasminogen activator, whereas incubation of pro-urokinase type plasminogen activator with the native forms of human or rabbit stromelysin-1 led to a moderate activation of pro-uPA due to an additional cleavage that is catalyzed by a serine proteinase.

Substance P induces the secretion of gelatinase A from human synovial fibroblasts.

We investigated the secretion of the matrix metalloproteinases, interstitial collagenase (matrix metalloproteinase-1), gelatinase A (matrix metalloproteinase-2) and stromelysin-1 (matrix metalloproteinase-3) in human synovial fibroblasts after stimulation with the neuropeptide substance P. Human synovial fibroblasts were stimulated with substance P or interleukin-1 beta (IL-1 beta). In the cell culture media gelatinase A, interstitial collagenase and stromelysin-1 were identified and their activities towards different substrates were determined. Substance P in synovial fibroblasts induced an increase in the overall matrix metalloproteinase activity towards the dinitrophenyl-labelled peptide by 85%, against an increase of 124% after stimulation with IL-1 beta. In case of substance P stimulation, the increase in activity reflects a significantly enhanced secretion of gelatinase A, whereas no significant increase of stromelysin-1 and collagenase secretion could be observed. The matrix metalloproteinase pattern showing the highest gelatinase A secretion was obtained after stimulation with substance P. This pattern was very pronounced and differed very clearly from the pattern seen after IL-1 beta stimulation which caused a significant rise in collagenase and stromelysin-1 activity. We assume that distinct stimulation pathways are involved and that the neuropeptide (substance P), which is always present in the inflamed joint, plays its own and separate role in proliferative processes leading to the cartilage destruction.

Activation of progelatinase A and progelatinase A/TIMP-2 complex by membrane type 2-matrix metalloproteinase.

C-terminal truncated membrane-type 2 matrix metalloproteinase (MT2-MMP1-269), comprising prodomain and catalytic domain, was expressed as a soluble protein in Escherichia coli. Unlike the corresponding form of MT1-MMP, which can be isolated as a 31 kDa protein, MT2-MMP1-269 proved to be comparatively instable, and already the freshly isolated preparation displayed several proteins in SDS-PAGE representing MT2-MMP1-269 (33 kDa) and four N-truncated forms with N-termini methionine32 (30 kDa), isoleucine37 (30 kDa), leucine84 (24 kDa), and leucine93 (22 kDa), the catalytic domain. After thawing of frozen preparations the 33 and the 30 kDa proforms were no longer detectable in SDS-PAGE, and only the 24 and 22 kDa forms remained. The catalytic domain of MT2-MMP activated progelatinase A as well as the progelatinase A/TMP-2 complex by cleaving the 72 kDa progelatinase A to yield 67 kDa gelatinase A, which is then transformed into 62 kDa gelatinase A. The 62 kDa form is about twice as active as the 67 kDa form towards the synthetic substrate N-(2,4)-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg. No significant difference in activity was found between free and complexed gelatinase A forms. the activation of the progelatinase A/TIMP-2 complex proceeds in two steps: At first MT2-MMP is inhibited by the progelatinase A/TIMP-2/MT2-MMP, complex, whereby a ternary complex, progelatinase A/TIMP-2/ MT-2MMP is generated. This ternary complex is then activated by excess MT2-MMP. Our results suggest a mechanism for spatially regulated extracellular gelatinase A activity mediated by activation with membrane-type MMPs; Free gelatinase A is released into the extracellular space, while gelatinase A/TIMP-2 bound to MT-MMP remains anchored on the cell surface.

Progelatinase B forms from human neutrophils. complex formation of monomer/lipocalin with TIMP-1.

The three forms of neutrophil gelatinase B-monomer, homodimer and monomer/lipocalin complex-, were isolated from phorbolester stimulated neutrophil granulocytes by chromatography on gelatin-Sepharose and heparin-Ultrogel. On average, about 50% of the monomer/lipocalin complex was found to be complexed with TIMP-1. After activation with trypsin monomer, homodimer and monomer/lipocalin complex displayed a specific activity of about 2000 mU/mg towards the substrate N-(2,4)-dinitrophenyl-Pro-Gln-Gly-lle-Ala-Gly-Gln-D-Arg, whereas the monomer/lipocalin/TIMP-1 complex could be activated to a specific activity of only 200 mU/mg. The ternary monomer/lipocalin/TIMP-1 complex behaves like the progelatinase A-TIMP-2 complex and the progelatinase B-TIMP-1 complex in that it is an inhibitor for active metalloproteinases (MMPs) and, after activation, a gelatinase with a pronouncedly reduced activity. When the monomer/lipocalin/TIMP-1 complex inhibits an MMP, a quaternary complex monomer/lipocalin/TIMP-1/MMP is generated which after activation shows a sixfold higher proteolytic activity than the active ternary complex.

Further development of flow-through electrophoresis.

Recent improvements in the field of flow-through-electrophoresis are presented. These improvements make it possible to run flow through electrophoresis with the same precision, but more economically and more easily than the formerly described apparatus. This was achieved by limiting the original system to slab gels only. This leads to a considerable reduction in the size of the apparatus and to simplification of its handling. Although the gel width is reduced to 10 mm, the apparatus is suitable for the analysis of mg as well as microgram quantities. The usefulness of this method is demonstrated by the analysis of samples obtained during the isolation and purification of collagen type II, and by the analysis of insulin samples and other substances. The procedure is also suitable for SDS-electrophoresis under flow-through conditions, enabling a simple and fast separation of proteins and determination of their molecular masses.

Generation and activity of the ternary gelatinase B/TIMP-1/LMW-stromelysin-1 complex.

Incubation of progelatinase B, isolated from human polymorphonuclear leukocytes, with TIMP-1 leads to the formation of the progelatinase B/TIMP-1 complex. This complex behaves like a Janus in a similar manner as we previously described for the progelatinase A/TIMP-2 complex. It shows the properties of TIMP-1 and is a better inhibitor for gelatinase A than for gelatinase B. Treatment with trypsin leads to activation of the binary complex. The activity, however, amounts only to slightly more than 10% of the activity of free gelatinase B, not complexed with TIMP-1. When the progelatinase B/TIMP-1 complex inhibits an active matrix metalloproteinase, a ternary complex is generated that after activation displays a distinct higher proteolytic activity than the active binary complex. The active binary complex cannot be transformed into the active ternary complex.

Synovial and peritoneal macrophages in organoid culture.

Cultivation of macrophages and their progenitors has been very useful for elucidation of function, behaviour and morphology of these cells. The purpose of this contribution is to describe a new in vitro system (organoid, high density or micromass culture) which proved to be convenient for cultivation of macrophages derived from human synovial fluid and tissue and mouse peritoneal fluid. Using this method, highly differentiated and functionally active macrophages of marked purity and long maintenance (up to 2 weeks) could be obtained even after previous cultivation and subcultivation in monolayer culture. The macrophages were identified by electron microscopy and immunomorphology using HLA-DR-DP, CD-68 (markers for human macrophages), anti-human-polymorphonuclear leukocyte-gelatinase and F4/80 (a mouse macrophage surface marker). The significance of this method as a research tool in the study of cartilage degradation by macrophages in co-cultures is stressed.

Activity of ternary gelatinase A-TIMP-2-matrix metallo-proteinase complexes.

The progelatinase A-TIMP-2 complex behaves like a Janus. Like TIMP (tissue inhibitor of metalloproteinases) it inhibits active matrix metalloproteinases, and activation with 4-aminophenylmercury acetate leads to a gelatinolytic activity. This activity, however, amounts only to less than 10% of that of free gelatinase A not complexed with TIMP-2. When the progelatinase A-TIMP-2 complex inhibits an active matrix metalloproteinase, a ternary complex is generated. After activation with 4-aminophenylmercury acetate this ternary complex displays a more than tenfold proteolytic activity compared to activated gelatinase A-TIMP-2 complex, thus reaching the activity of free gelatinase A. The activity of the ternary complex is nearly independent from the bound matrix metalloproteinase. When the progelatinase A-TIMP-2 complex is activated at first with 4-aminophenylmercury acetate the generation of the ternary complex is made impossible and not such a significant enhancement of activity is observed. These results suggest that gelatinase A-TIMP-2 complex may be a matrix metalloproteinase of the 'second step': It starts its proteolytic attack after it has switched off the activity of other matrix metalloproteinases.

Isolation of latent 31-kDa C-truncated stromelysin and 21-kDa stromelysin from rabbit synovial fibroblasts: an alternative activation pathway for stromelysin.

The processing of culture medium of rabbit synovial fibroblasts led to the isolation of three stromelysin-1 (MMP-3) cleavage products: A 31-kDa protein, which represents a C-truncated latent stromelysin-1, an active stromelysin-1 of 21 kDa, that originates from the 31-kDa proform by activation. A third protein had a molecular mass of 25 kDa representing the C-terminal part of prostromelysin-1 and is missing in the C-truncated latent stromelysin-1. The activation process of human prostromelysin-1 in vitro is known to lead to an active stromelysin-1 with a relative molecular mass of 45 kDa by removing the N-terminal prodomain. This active stromelysin-1 is further processed to a lower molecular mass active form of 28 kDa. Our results obtained for the highly homologous rabbit stromelysin-1 indicate that another activation pathway is possible. In a first step prostromelysin-1 is hydrolysed between Met261-Glu generating a C-truncated latent stromelysin-1, which is activated by cleavage of the Thr83-Phe bond to the 21-kDa stromelysin-1. The latent C-truncated stromelysin-1 is slowly converted even at 4 degrees C into the active form. In the presence of 50 microM ZnCl2 this activation was prevented for at least three weeks. The activation rate is largely enhanced by aminophenylmercury acetate and especially by trypsin. The differences of the 21-kDa stromelysin-1 to a 28-kDa stromelysin-1 isolated from human rheumatoid synovial fluids described earlier are discussed.

A trypsin sensitive stromelysin isolated from rheumatoid synovial fluid is an activator for matrix metalloproteinases.

The processing of synovial fluids of patients suffering from rheumatoid arthritis led to the characterization of a neutral metalloproteinase with polymorphonuclear leukocyte progelatinase and polymorphonuclear leukocyte procollagenase activating properties. The activator exhibits a relative molecular mass of M(r) 27,000 and is an active form of stromelysin. Thus, it reacts specifically with antibodies raised against human stromelysin, splits polymorphonuclear leukocyte progelatinase in a manner characteristic of stromelysin, and is inhibited by EDTA as well as by a tissue inhibitor of metalloproteinases (TIMP-2). The activator shows a high specificity for the matrix metalloproteinases, polymorphonuclear leukocyte progelatinase and polymorphonuclear leukocyte procollagenase. It shows only weak hydrolysis of casein and gelatin, and it does not activate fibroblast M(r) 72,000 progelatinase. Brief treatment with trypsin does not lead to a significant change in the activator's relative molecular mass, but induces a rapid loss of its activating activity for polymorphonuclear leukocyte progelatinase, while its proteolytic activity against the synthetic substrate, N-(2,4)-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg, is increased about 3-fold. The same tryptic treatment does not affect the activator's proteolytic activity towards casein and gelatin.

Characterization of a gelatinase from human rheumatoid synovial fluid cells.

A metalloproteinase with a specificity for gelatin was isolated from serum-free medium of cultures of rheumatoid synovial fluid. The enzyme showed all the properties of a leukocyte gelatinase. In addition to gelatin this proteinase cleaved the synthetic substrate dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (Dnp-peptide) rapidly, while casein was a much poorer substrate. This proteinase showed no enzymatic activity against collagen type I, was secreted in a latent form and could be activated by trypsin or organomercurial compounds, such as mersalylic acid or 4-aminophenyl-mercury acetate. The latent enzyme had an apparent molecular mass of 130,000-150,000 estimated by gel filtration or 97,000 by electrophoresis on polyacrylamide gel containing sodium dodecyl sulphate. When analysed by immunoblotting the enzyme was recognized by antibodies raised against human polymorphonuclear leukocyte gelatinase. Although we found synovial fibroblasts to be largely present in the cell cultures we could not detect any fibroblast gelatinase activity.

The complex between a tissue inhibitor of metalloproteinases (TIMP-2) and 72-kDa progelatinase is a metalloproteinase inhibitor.

Human rheumatoid synovial cells in culture secrete both 72-kDa progelatinase and a complex consisting of 72-kDa progelatinase and a 24-kDa inhibitor of metalloproteinases, TIMP-2. In addition, the culture medium contains TIMP-1, the classical inhibitor of metalloproteinases, with a molecular mass of 30 kDa. TIMP-1 does not form a complex with free 72-kDa progelatinase. Free progelatinase and progelatinase complexed with TIMP-2 can be activated with the organomercury compound p-aminophenylmercury acetate. The activated complex shows less than 10% the enzyme activity of activated free gelatinase. The progelatinase-TIMP-2 complex could be shown to be an inhibitor for other metalloproteinases, such as gelatinase and collagenase secreted by human rheumatoid synovia fibroblasts, as well as for the corresponding enzymes from human neutrophils.

Ultrastructural changes induced by alpha-sarcin in a human pulmonary tumor grown in naked mice.

alpha-Sarcin is a cytotoxic polypeptide produced by Aspergillus giganteus. It suppresses protein synthesis in yeast and wheat germ extracts and has a purine-specific RNase activity. The substance has been tested for its antitumor properties in a series of induced tumor systems in mice such as sarcoma and carcinoma among others. Although some of the in vitro effects of alpha-Sarcin on certain cellular components have been elucidated, the biological effects leading to cellular damage are still obscure. In this work we analysed the morphological changes in tumor cells derived from human pulmonary adenocarcinoma heterotransplanted and grown in naked mice, induced shortly (24 hours) after a single intratumoral injection of alpha-Sarcin (0.4 mg/tumor). The results obtained were: 1) swelling of mitochondria; 2) cell necrosis with partial removal of necrotic cells by phagocytosis; 3) thickening of interlobular connective tissue; 4) hyperplasia of goblet-cell-like clear cells. The mode of action concerning these cellular changes is presently uncertain. In view of the severity of these structural alterations it seems conceivable that alpha-Sarcin may enter the cell undergoing interactions with different intracellular structures. This would require a selective membrane permeabilization, perhaps induced upon formation of complexes with negatively-charged membrane phospholipids.