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PubMed2005

Divergent effects of matrix metalloproteinases 3, 7, 9, and 12 on atherosclerotic plaque stability in mouse brachiocephalic arteries.

Matrix metalloproteinases (MMPs) are thought to be involved in the growth, destabilization, and eventual rupture of atherosclerotic lesions. Using the mouse brachiocephalic artery model of plaque instability, we compared apolipoprotein E (apoE)/MMP-3, apoE/MMP-7, apoE/MMP-9, and apoE/MMP-12 double knockouts with their age-, strain-, and sex-matched apoE single knockout controls. Brachiocephalic artery plaques were significantly larger in apoE/MMP-3 and apoE/MMP-9 double knockouts than in controls. The number of buried fibrous layers was also significantly higher in the double knockouts, and both knockouts exhibited cellular compositional changes indicative of an unstable plaque phenotype. Conversely, lesion size and buried fibrous layers were reduced in apoE/MMP-12 double knockouts compared with controls, and double knockouts had increased smooth muscle cell and reduced macrophage content in the plaque, indicative of a stable plaque phenotype. ApoE/MMP-7 double knockout plaques contained significantly more smooth muscle cells than controls, but neither lesion size nor features of stability were altered in these animals. Hence, MMP-3 and MMP-9 appear normally to play protective roles, limiting plaque growth and promoting a stable plaque phenotype. MMP-12 supports lesion expansion and destabilization. MMP-7 has no effect on plaque growth or stability, although it is associated with reduced smooth muscle cell content in plaques. These data demonstrate that MMPs are directly involved in atherosclerotic plaque destabilization and clearly show that members of the MMP family have widely differing effects on atherogenesis.

Animals↗

PubMed1990

Mechanisms of activation of tissue procollagenase by matrix metalloproteinase 3 (stromelysin).

The mechanism of activation of tissue procollagenase by matrix metalloproteinase 3 (MMP-3)/stromelysin was investigated by kinetic and sequence analyses. MMP-3 slowly activated procollagenase by cleavage of the Gln80-Phe81 bond to generate a fully active collagenase of Mr = 41,000. The specific collagenolytic activity of this species was 27,000 units/mg (1 unit = 1 microgram of collagen digested in 1 min at 37 degrees C). Treatment of procollagenase with plasmin or plasma kallikrein gave intermediates of Mr = 46,000. These intermediates underwent rapid autolytic activation, via cleaving the Thr64-Leu65 bond, to give a collagenase species of Mr = 43,000 that exhibited only about 15% of the maximal specific activity. Similarly, (4-aminophenyl)mercuric acetate (APMA) activated procollagenase by intramolecular cleavage of the Val67-Met68 bond to generate a collagenase species of Mr = 43,000, but with only about 25% of the maximal specific activity. Subsequent incubation of the 43,000-Mr species with MMP-3 resulted in rapid, full activation and generated the 41,000-Mr collagenase by cleaving the Gln80-Phe81 bond. In the case of the proteinase-generated 43,000-Mr species, the action of MMP-3 was approximately 24,000 times faster than that on the native procollagenase. This indicates that the removal of a portion of the propeptide of procollagenase induces conformational changes around the Gln80-Phe81 bond, rendering it readily susceptible to MMP-3 activation. Prolonged treatment of procollagenase with APMA in the absence of MMP-3 also generated a 41,000-Mr collagenase, but this species had only 40% of the full activity and contained Val82 and Leu83 as NH2 termini. Thus, cleavage of the Gln80-Phe81 bond by MMP-3 is crucial for the expression of full collagenase activity. These results suggest that the activation of procollagenase by MMP-3 is regulated by two pathways: one with direct, slow activation by MMP-3 and the other with rapid activation in conjunction with tissue and/or plasma proteinases. The latter event may explain an accelerated degradation of collagens under certain physiological and pathological conditions.

Amino Acid Sequence↗

PubMed1996

Degradation of cross-linked fibrin by matrix metalloproteinase 3 (stromelysin 1): hydrolysis of the gamma Gly 404-Ala 405 peptide bond.

Matrix metalloproteinases (MMPs) can degrade a number of proteins that constitute the extracellular matrix. Previous studies have shown that atherosclerotic plaques contain substantial amounts of fibrin(ogen)-related antigen, and more recently, MMPs have been identified in such lesions. The hypothesis that MMPs play a role in the degradation of fibrinogen (Fg) and cross-linked fibrin (XL-Fb) was investigated. Fibrinogen became thrombin-unclottable when treated with matrix metalloproteinase 3 (MMP-3, stromelysin 1) but not with matrix metalloproteinase 2 (MMP-2, gelatinase A). Incubation of XL-Fb clots (made with 125I-Fg) with MMP-3 resulted in complete lysis after 24 h. A D monomer-like fragment was generated by MMP-3 degradation of fibrinogen, XL-Fb, and fragment DD. Immunoreactivity with monoclonal antibody (MoAb)/4-2 (anti-gamma 392-406) but not with MoAb/4A5 (anti-gamma 397-411) suggested that a major cleavage site was within the sequence participating in the cross-linking of two gamma-chains. NH2-terminal sequence analysis of they gamma-chain of the D monomer-like fragment and of a dipeptide isolated from the MMP-3 digest of XL-fibrin identified the hydrolysis of the gamma Gly 404-Ala 405 peptide bond. These data indicate that the degradation of Fg and XL-Fb by MMP-3 is specific and different from plasmin. This mechanism of fibrinolysis might be of relevance in wound healing, inflammation, atherosclerosis, and other pathophysiological processes.

Amino Acid Sequence↗

PubMed1998

Molecular cloning and cartilage gene expression of equine stromelysin 1 (matrix metalloproteinase 3).

OBJECTIVE: To clone and determine molecular structure of equine stromelysin 1 (matrix metalloproteinase 3) and examine stromelysin expression in articular cartilage. SAMPLES AND PROCEDURE: Total RNA was harvested from equine arthritic cartilage specimens and was used for reverse transcription and polymerase chain reaction amplification to develop overlapping complementary DNA (cDNA) clones. Four cDNA sequences were ligated into plasmid (pGEM3Z) constructs and subcloned into bacterial expression vectors, and sequence was determined by automated dye terminator sequencing. Stromelysin mRNA expression was assessed in normal and arthritic cartilage and synovium by northern blotting. Interleukin 1 (IL-1) regulation of stromelysin transcriptional activity in articular chondrocytes cultured in the presence of 0, 20, and 50 ng of IL-1 alpha/ml was assessed by northern blotting of total RNA isolated from the cell layer and probed with 32P-labeled stromelysin cDNA. RESULTS: 4 overlapping clones provided the full-length cDNA sequence of equine stromelysin, including portions of untranslated 5' and 3' regions, and the entire translated portion coding for the stromelysin prepropeptide. The coding region of 1,431 base pairs was well conserved between species, with 86, 83, and 78% sequence homology to that of human, rabbit, and mouse stromelysin, respectively. Predicted amino-acid (AA) sequence data indicated highly conserved features. Comparison of the equine AA sequence revealed 89, 88, and 84% homology to the AA structure in human, rabbit, and mouse stromelysin, respectively. Minimal stromelysin mRNA expression was evident in normal cartilage and synovium, and increased expression was evident in arthritic cartilage. Marked dose-dependent up-regulation of stromelysin transcriptional activity was evident in chondrocyte cultures exposed to 20 and 50 ng of IL-1/ml. CONCLUSIONS: Stromelysin DNA sequence in horses is similar to that in people and rodents. Constitutive stromelysin message amounts in normal cartilage and synovium are low, but considerably increased in arthritic cartilage and in chondrocytes exposed to IL-1.

Amino Acid Sequence↗

PubMed2007

Crucial role of macrophages in matrix metalloproteinase-mediated cartilage destruction during experimental osteoarthritis: involvement of matrix metalloproteinase 3.

OBJECTIVE: To explore the involvement of synovial macrophages in early cartilage damage in osteoarthritis (OA), and to identify the role of matrix metalloproteinase 3 (MMP-3) in the pathology of early and late OA. METHODS: The role of synovial macrophages in MMP-mediated damage in OA was studied by depleting synovial macrophages prior to elicitation of a collagenase-induced instability model of OA. The expression of MMP in synovium and cartilage was monitored using TaqMan analysis. In spontaneous and induced OA, cartilage pathology was scored in MMP-3-knockout mice and control mice, by histologic assessment and VDIPEN staining. RESULTS: On day 14 following induction of OA, MMP-mediated neoepitopes were detected in cartilage from mice with mild experimental OA (mean +/- SD positively stained surface area 20 +/- 3.2%). Remarkably, by depleting synovial macrophages prior to induction of OA, the generation of MMP-induced neoepitopes was largely prevented (mean +/- SD positively stained surface area 5 +/- 1%; P< 0.001), indicating an important role for synovial macrophages in the occurrence of MMP-mediated cartilage damage. We observed a strong decrease in MMP-3 and MMP-9 expression in synovial but not cartilage tissue in macrophage-depleted joints. Among 2-year-old mice, spontaneous OA-like changes in the lining layer were significantly decreased in MMP-3-knockout mice compared with control mice. Even more striking was the 67% reduction in the occurrence of severe cartilage damage in MMP-3-knockout mice. In addition, MMP-mediated VDIPEN expression was significantly decreased, indicating reduced MMP-mediated cartilage breakdown. CONCLUSION: The results of this study prove that MMP-3 is involved in the generation of severe cartilage damage in murine OA. Synovial macrophages are crucial in early MMP activity and appear to mediate MMP production in synovium rather than cartilage.

Animals↗

PubMed1992

A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 3 (stromelysin-1) using monoclonal antibodies.

A one-step sandwich enzyme immunoassay (EIA) for matrix metalloproteinase 3 (MMP-3; stromelysin-1) was developed. The assay system used two simultaneous immunoreactions using a solid phase monoclonal antibody and a horseradish peroxidase-labeled monoclonal antibody (Fab'). The sensitivity of the assay system was 20 micrograms/l and linearity was obtained between 31 and 500 micrograms/l. The EIA system was capable of measuring both precursor and active forms of MMP-3 as well as the forms of MMP-3 complexed with tissue inhibitors of metalloproteinases. MMP-3 levels as measured by this assay are significantly higher in the sera of patients with rheumatoid arthritis as compared to those of healthy subjects and patients with osteoarthritis. Immunoblot analyses showed that in the sera and synovial fluids of patients with rheumatoid arthritis, MMP-3 is present in the 59- and 57-kDa precursor forms.

Animals↗

PubMed1996

Characterization of the 46-kDa intermediates of matrix metalloproteinase 3 (stromelysin 1) obtained by site-directed mutation of phenylalanine 83.

The precursor of matrix metalloproteinase 3 (MMP-3/ stromelysin 1) is activated in vitro by proteinases or mercurial compounds by stepwise processes which include the initial formation of short-lived intermediates and the subsequent intermolecular cleavage of the His82-Phe83 bond to generate the fully activated mature MMP-3 (Nagase, H., Enghild, J. J., Suzuki, K., and Salvesen, G. (1990) Biochemistry 29, 5783-5789). To study the enzymatic properties of the intermediates we have mutated either His82 or Phe83 to Arg to obtain a stable MMP-3 intermediate. The mutant proteins were expressed in Chinese hamster ovary K-1 cells using a mammalian expression system. The proMMP-3(H82R) mutant was activated by chymotrypsin, elastase, and 4-aminophenylmercuric acetate to the 45-kDa MMP-3 with similar mechanism and kinetics as the wild-type. In contrast, the activation of the proMMP-3(F83R) mutant by proteinases or 4-aminophenylmercuric acetate resulted in 46-kDa forms, which retained 13, 14, or 15 amino acids of the pro-domain depending on the activators. The proteinase-activated MMP-3(F83R) intermediates exhibited little enzymatic activity, but they were partially active after treatment with SH-reacting reagents. These molecules could bind to the tissue inhibitor of metalloproteinases-1 and alpha 2-macroglobulin. However, the SH group of Cys75 of the intermediates was not modified by SH-reagents, indicating that the enzymatic activity generated by SH-reagents resulted from molecular perturbation of the enzyme rather than their interaction with Cys75. When gelatin and transferrin were digested with the 46-kDa intermediates the products were different from those generated by the wild-type MMP-3, suggesting an alteration in substrate specificity. The treatment of proMMP-3 with trypsin resulted in the formation of a 45-kDa MMP-3 with an NH2-terminal Thr85, whose activity and substrate specificity were similar to those of the 46-kDa MMp-3(F83R) obtained from the proMMP-3(F83R) mutant. These observations indicate that the correct processing at the His82-Phe83 bond is critical for expression of the full activity and the specificity of MMP-3.

Amino Acid Sequence↗

PubMed1999

Expression and tissue localization of membrane-type 1, 2, and 3 matrix metalloproteinases in human astrocytic tumors.

Three different membrane-type matrix metalloproteinases (MT1-, MT2-, and MT3-MMPs) are known to activate in vitro the zymogen of MMP-2 (pro-MMP-2, progelatinase A), which is one of the key MMPs in invasion and metastasis of various cancers. In the present study, we have examined production and activation of pro-MMP-2, expression of MT1-, MT2-, and MT3-MMPs and their correlation with pro-MMP-2 activation, and localization of MMP-2, MT1-MMP, and MT2-MMP in human astrocytic tumors. The sandwich enzyme immunoassay demonstrates that the production levels of pro-MMP-2 in the anaplastic astrocytomas and glioblastomas are significantly higher than that in the low-grade astrocytomas (P<0.05 and P<0.01, respectively), metastatic brain tumors (P<0.05), or normal brains (P<0.01). Gelatin zymography indicates that the pro-MMP-2 activation ratio is significantly higher in the glioblastomas than in other astrocytic tumors (P<0.01), metastatic brain tumors (P<0.01), and normal brains (P<0.01). The quantitative reverse transcription polymerase chain reaction analyses demonstrate that MT1-MMP and MT2-MMP are expressed predominantly in glioblastoma tissues (17/17 and 12/17 cases, respectively), and their expression levels increase significantly as tumor grade increases. MT3-MMP is detectable in both astrocytic tumor and normal brain tissues, but the mean expression level is approximately 50-fold lower compared with that of MT1-MMP and MT2-MMP in the glioblastomas. The activation ratio of pro-MMP-2 correlates directly with the expression levels of MT1-MMP and MT2-MMP but not MT3-MMP. In situ hybridization indicates that neoplastic astrocytes express MT1-MMP and MT2-MMP in the glioblastoma tissues (5/5 cases and 5/5 cases, respectively). Immunohistochemically, MT1-MMP and MT2-MMP are localized to the neoplastic astrocytes in glioblastoma samples (17/17 cases and 12/17 cases, respectively), which are also positive for MMP-2. In situ zymography shows gelatinolytic activity in the glioblastoma tissues but not in the normal brain tissues. These results suggest that both MT1-MMP and MT2-MMP play a key role in the activation of pro-MMP-2 in the human malignant astrocytic tumors and that the gelatinolytic activity is involved in the astrocytic tumor invasion.

Adenocarcinoma↗

PubMed2002

Increased expression of membrane type 3-matrix metalloproteinase in human atherosclerotic plaque: role of activated macrophages and inflammatory cytokines.

BACKGROUND: Matrix metalloproteinases (MMPs) are thought to play a prominent role in atherogenesis and destabilization of plaque. Pericellularly localized membrane-type (MT)-MMPs activate secreted MMPs. We investigated the hypothesis that MT3-MMP is expressed in human atherosclerotic plaques and is regulated by locally produced inflammatory cytokines and oxidized low-density lipoprotein (Ox-LDL). METHODS AND RESULTS: Expression and cellular localization of MT3-MMP in normal and atherosclerotic human coronary arteries were examined using specific antibodies. Abundant MT3-MMP expression was noted in medial smooth muscle cells (SMCs) of normal arteries. In atherosclerotic arteries, MT3-MMP expression was observed within complex plaques and colocalized with SMCs and macrophages (Mphi). Cultured human monocyte-derived Mphi constitutively expressed MT3-MMP mRNA and proteolytically active protein, as demonstrated by mRNA analyses, immunoblotting, and gelatin zymography, respectively. Ox-LDL, tumor necrosis factor-alpha, or macrophage colony-stimulating factor caused dose- and time-dependent increases in steady-state levels of MT3-MMP mRNA in cultured Mphi. This correlated with a 2- to 4-fold increase in levels of MT3-MMP immunoreactive protein and enzymatic activity in Mphi membranes. Confocal microscopy and flow cytometry confirmed induction and spatial distribution of MT3-MMP protein from intracellular domains to the Mphi plasma membrane by Ox-LDL, tumor necrosis factor-alpha, or macrophage colony-stimulating factor. CONCLUSIONS: MT3-MMP is expressed by SMCs and Mphi in human atherosclerotic plaques. Proinflammatory molecules cause a progressive increase in the expression of MT3-MMP in cultured Mphi. Our results suggest a mechanism by which inflammatory molecules could promote Mphi-mediated degradation of extracellular matrix and thereby contribute to plaque destabilization.

Arteriosclerosis↗

PubMed1997

Expression and tissue localization of membrane-types 1, 2, and 3 matrix metalloproteinases in human invasive breast carcinomas.

Activation of the zymogen of matrix metalloproteinase 2 (proMMP-2, progelatinase A) possibly is one of the key steps in invasion and metastasis of various human carcinomas. Three different membrane-type MMPs (MT-MMPs), MT1-, MT2-, and MT3-MMPs are thought to be activators of proMMP-2 in the tissues. MT4-MMP is structurally different from the other three enzymes, and its function as proMMP-2 activator is uncertain. In the present study of human invasive breast carcinomas, we examined a correlation between the expression of MT1-, MT2-, and MT3-MMPs, immunolocalization of MT1- and MT2-MMPs, and proMMP-2 activation. Northern blot analysis demonstrated the predominant expression of MT1-MMP mRNA in carcinoma tissues (20 of 20 cases), whereas MT2-MMP was detected in only 25% of the cases (5 of 20 cases), and no detectable expression of MT3-MMP was observed. The expression levels of MT1-MMP but not MT2-MMP correlated well with the presence of lymph node and distant metastases, clinical stages, and size of tumors. Immunohistochemically, MT1-MMP was localized predominantly in the carcinoma cells in all of the samples (32 of 32 cases). Immunostaining of MT2-MMP in the carcinoma cells was observed in only 38% of the cases (12 of 32 cases). Immunoblot analysis of tumor homogenates confirmed the presence of these MT-MMPs. Activation of proMMP-2 was significantly higher in the carcinoma samples with lymph node or distant metastasis compared to carcinoma without metastasis, normal control, or fibrocystic disease (P < 0.05). An increase in the activation ratio of proMMP-2 correlated directly with the expression of MT1-MMP but not MT2-MMP, as measured by either Northern blot analysis or immunostaining. These results suggest that MT1-MMP may play a key role in human breast carcinoma invasion and metastasis by being predominantly responsible for activation of proMMP-2.

Adult↗

PubMed1998

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in loose artificial hip joints.

The role of extracellular matrix metalloproteinase enzymes and the tissue inhibitors of metalloproteinase in the periprostetic connective tissue matrix of loose artificial hip joints is reviewed. In the periprosthetic granulomatous interface connective tissues between bone and implants and inner cellular regenerating pseudocapsular tissues, matrix metalloproteinase 1, matrix metalloproteinase 2, matrix metalloproteinase 3, matrix metalloproteinase-9, and membrane type 1 matrix metalloproteinase enzymes can be shown in the light of immunohistochemistry, enzyme activity analysis, and messenger ribonucleic acid levels. Tissue inhibitors of metalloproteinase 1 and tissue inhibitors of metalloproteinase 2 also are found in the corresponding tissues. Analysis of matrix metalloproteinase and tissue inhibitors of metalloproteinase interaction shows imbalance between the enzymes and the endogenous inhibitors in favor of matrix metalloproteinase. This induces pathologic connective tissue remodeling in the interface and pseudocapsule. The data suggest that matrix metalloproteinase and tissue inhibitors of metalloproteinase system participate in the extracellular matrix degradation and tissue remodeling in artificial hip joints, and may contribute to the periprosthetic weakening, implant loosening, and osteolysis around implants. More evidence for their active involvement is sought by intervention studies with type specific matrix metalloproteinase inhibitors.

Animals↗

PubMed2003

Matrix metalloproteinase 3 in parturition, premature rupture of the membranes, and microbial invasion of the amniotic cavity.

OBJECTIVE: Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that are expressed in many inflammatory conditions and contribute to connective tissue breakdown. Stromelysin 1 [matrix metalloproteinase 3 (MMP-3)], a novel member of this family, is produced in the context of infection and is able to activate the latent forms of other MMPs. The purpose of this study was to determine if parturition (either term or preterm), premature rupture of the membranes (PROM), and microbial invasion of the amniotic cavity are associated with changes in amniotic fluid concentrations of MMP-3. STUDY DESIGN: A cross-sectional study was conducted, which included women who underwent transabdominal amniocentesis (n = 365) in the following categories: (1) mid-trimester with a subsequent normal pregnancy outcome (n = 84) and a subsequent fetal loss (n = 10); (2) preterm labor with intact membranes without microbial invasion of the amniotic cavity who delivered at term (n = 36), or prematurely (n = 50), and preterm labor with microbial invasion of the amniotic cavity (n = 25); (3) preterm PROM with (n = 25) and without (n = 26) microbial invasion of the amniotic cavity; (4) term with intact membranes in the absence of microbial invasion of the amniotic cavity, in labor (n = 52) and not in labor (n = 31); and (5) term with PROM in the absence of microbial invasion of the amniotic cavity and not in labor (n = 26). MMP-3 concentrations in amniotic fluid were measured by a sensitive and specific immunoassay that was validated for amniotic fluid. MMP-3 concentrations were normalized using logarithmic transformation for statistical analysis. Parametric statistics were used and a p value < 0.05 was considered statistically significant. RESULTS: (1) MMP-3 was detected in 99.5% (363/365) of amniotic fluid samples, and its concentration did not change with advancing gestational age. (2) Spontaneous parturition at term and preterm was associated with a significant increase in amniotic fluid MMP-3 concentrations (p = 0.04 and p = 0.002, respectively). (3) Spontaneous rupture of membranes in term and preterm gestations was not associated with significant changes in amniotic fluid MMP-3 concentrations. (4) Intra-amniotic infection was associated with a significant increase in amniotic fluid MMP-3 concentrations in both women with preterm labor and intact membranes (p = 0.03), and women with preterm PROM (p = 0.02). (5) Subsequent fetal loss after genetic amniocentesis was not associated with significant changes in mid-trimester concentrations of amniotic fluid MMP-3. CONCLUSIONS: (1) MMP-3 is a physiologic constituent of amniotic fluid. (2) MMP-3 may play a role in the mechanisms of human parturition and in the regulation of the host response to intrauterine infection.

Amnion↗

PubMed1989

Interaction of human rheumatoid synovial collagenase (matrix metalloproteinase 1) and stromelysin (matrix metalloproteinase 3) with human alpha 2-macroglobulin and chicken ovostatin. Binding kinetics and identification of matrix metalloproteinase cleavage sites.

The homologous proteinase inhibitors, human alpha 2-macroglobulin (alpha 2M) and chicken ovostatin, have been compared with respect to their "bait" region sequences and interactions with two human matrix metalloproteinases, collagenase and stromelysin. A stretch of 34 amino acid residues of the ovostatin bait region sequence was determined and the matrix metalloproteinase cleavage sites identified. Collagenase cleaved a X-Leu bond where X was unidentified, whereas the major cleavage site by stromelysin was at the Gly-Phe bond, 4 residues on the COOH-terminal side of the collagenase cleavage site. Collagenase cleaved the alpha 2M bait region at the Gly679-Leu680 bond, and stromelysin at Gly679-Leu680 and Phe684-Tyr685 bonds. Sequence similarity in the bait region of members of the alpha-macroglobulin family is strikingly low. The kinetic studies indicate that alpha 2M is a 150-fold better substrate for collagenase than type I collagen. Structural predictions based on the bait region sequences suggest that a collagen-like triple helical structure is not a prerequisite for the efficient binding of tissue collagenase to a substrate. The binding of stromelysin to alpha 2M is slower than that of collagenase. Stromelysin reacts with ovostatin even more slowly. Despite the preference of chicken ovostatin for metalloproteinases, human alpha 2M, a far less selective inhibitor, reacts more rapidly with collagenase and stromelysin. These results suggest that alpha 2M may play an important role in regulating the activities of matrix metalloproteinases in the extracellular space.

Amino Acid Sequence↗

PubMed1991

Production of matrix metalloproteinase 3 (stromelysin) by cultured ovine endometrial cells.

Ovine endometrial cells (stromal plus epithelial) in primary culture release matrix metalloproteinase 3 (stromelysin, MMP-3), the enzyme that degrades various extracellular components including the basement membrane components collagen IV and laminin. The enzyme was detected in tissue culture medium by specific enzyme assay. A low level of MMP-3 activity was released from the cells (0.12 +/- 0.02 unit per 10(6) cells per 48 h; 1 unit degrades 1 microgram of reduced, carboxymethylated transferrin min-1 at 37 degrees C), but following stimulation by phorbol myristate acetate (PMA) a mean 4.4-fold increase in MMP-3 production was observed (to 0.53 +/- 0.13 unit per 10(6) cells per 48 h). The enzyme was released primarily in the form of its proenzyme, which could be activated in vitro by (4-aminophenyl)mercuric acetate (APMA). Stimulation of enzyme release from cells by PMA was not seen before 24 h and required PMA at a dose of 10 nM or higher. This finding was substantiated by Western blot analysis. Immunocytochemistry of cells that had been treated with PMA for 48 h, and with the ionophore, monensin, for 4 h, confirmed the potential of the endometrial cells to produce stromelysin. The release of MMP-3 from ovine endometrial cells suggests that this enzyme may have a role in assisting the passage of syncytial processes through the basement membrane during the process of implantation.

Animals↗

PubMed1990

Stepwise activation mechanisms of the precursor of matrix metalloproteinase 3 (stromelysin) by proteinases and (4-aminophenyl)mercuric acetate.

The mechanisms of activation of the precursor of human matrix metalloproteinase 3 (proMMP-3/prostromelysin) by proteinases and (4-aminophenyl)mercuric acetate (APMA) were investigated by kinetic and sequence analyses. Incubation of proMMP-3 with neutrophil elastase, plasma kallikrein, plasmin, or chymotrypsin at 37 degrees C resulted in the formation of MMP-3 of Mr = 45,000 by cleaving of the His82-Phe83 bond. Since this bond is unlikely to be cleaved by these proteinases it was postulated that an initial attack of an activator proteinase on proMMP-3 creates an intermediate form, which is then processed to a more stable form of Mr = 45,000. To test this hypothesis proMMP-3 was incubated with these serine proteinases under conditions that minimize the action of MMP-3. This led to the accumulation of major intermediates of Mr = 53,000 and two minor forms of Mr = 49,000 and 47,000. The 53,000 Mr intermediate generated by human neutrophil elastase resulted from cleavage of the Val35-Arg36 whereas plasma kallikrein cleaved the Arg36-Arg37 and Lys38-Asp39 bonds and chymotrypsin the Phe34-Val35 bond, all of which are located near the middle of the propeptide. Conversion of these intermediates to the fully active 45,000 Mr form of MMP-3 resulted from a bimolecular reaction of the intermediates. A similar short-lived intermediate of Mr = 46,000 generated by APMA was a result of the intramolecular cleavage of the Glu68-Val69 bond, and it was then converted to a stable MMP-3 of Mr = 45,000 by a intermolecular reaction of MMP-3. However, MMP-3 failed to activate proMMP-3.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1989

Synovial procollagenase activation by human mast cell tryptase dependence upon matrix metalloproteinase 3 activation.

Mast cells have been implicated in the pathogenesis of the matrix degradation observed in the cartilaginous and osseous structures of the rheumatoid joint. We previously reported that human mast cell tryptase, a 134-kD granule-associated neutral protease, is present in rheumatoid synovium and can activate collagenase in crude culture medium in vitro. the present study attempts to depict the precise mechanism of this activation. To express full activation of latent collagenase, matrix metalloproteinase 3 (MMP-3) or stromelysin, can be activated by tryptase in a time and dose-dependent manner. Tryptase was not capable of generating active collagenase in the crude media from cultured rheumatoid synoviocytes depleted of proMMP-3 by immunoadsorption. In addition, the function of the tissue inhibitor of metalloproteinases (TIMP) was not altered by tryptase, and SDS-PAGE analysis revealed no degradation of TIMP by tryptase. The tryptase dependent activation of synoviocyte procollagenase thereby appears to be entirely dependent upon its ability to activate proMMP-3.

Blotting, Western↗

PubMed1998

Expression and tissue localization of membrane-types 1, 2, and 3 matrix metalloproteinases in human urothelial carcinomas.

PURPOSE: Three different membrane-type matrix metalloproteinases (MT1, 2, 3-MMP) which can activate proMMP-2 (progelatinase A) are thought to have an important role in various human carcinoma invasions and metastases. We examined the mRNA expression of MT-MMPs and the tissue immunolocalization of MT1-MMP in human urothelial carcinomas. MATERIALS AND METHODS: mRNA was extracted from 27 clinical urothelial carcinomas and 10 normal urothelial mucosa tissues remote from the tumor. RT-PCR using specific primers was performed, and PCR products were hybridized to 32P-labeled internal probes and analyzed by a bioimage analyzer. Immunolocalization was studied using a monoclonal antibody against MT1-MMP (114-6G6). RESULTS: MT1-MMP and MT2-MMP mRNA expressions in urothelial carcinomas were significantly higher than those in the normal mucosa. In contrast, MT3-MMP mRNA was little expressed in both tissues, and the amount of MT3-MMP mRNA appeared to be much lower than MT1-MMP and MT2-MMP in the tissue samples. In terms of the tumor multiplicity, MT1-MMP and MT2-MMP mRNA expressions in the group of multiple tumors were significantly higher than those in the solitary tumor group. The carcinoma cells were immunostained for MT1-MMP predominantly in invasive and superficial carcinoma cells. The immunoreactivity was more intense in the invasive type than in the superficial type. CONCLUSIONS: It is suggested that MT1-MMP and MT2-MMP play an important role in the development of human urothelial carcinomas and reflect some aspects of the pathogenesis of multifocal occurrence. In spite of the possible contribution to the invasive and metastatic phenotype, MT1-MMP mRNA and its product are thought to be expressed already in the clinical superficial stage in some cases of this tumor type.

Carcinoma, Transitional Cell↗

PubMed2004

Association of matrix metalloproteinase 3 promoter genotype with disease outcome in rheumatoid arthritis.

Matrix metalloproteinases (MMPs) are implicated in joint destruction in rheumatoid arthritis (RA). We investigated whether the 5A/6A polymorphism within the MMP-3 (stromelysin-1) gene promoter region is associated with disease outcome in 254 patients with established RA. Patients homozygous for the MMP-3 6A allele had more radiographic damage (measured by Larsen score) than those with other genotypes (109.8 vs 91.1, P=0.04). Patients with the 6A/6A genotype also had more functional impairment and higher serum proMMP-3 levels, although only the latter was significant (P=0.002). A possible association was found between homozygosity for the 6A allele and carriage of the RA-associated HLA-DRB1 shared epitope (SE). Combination of these factors was associated with more severe disease than the SE alone. The data suggest that the MMP-3 6A/6A genotype is associated with worse RA outcome, and that this genotype may have an additive effect with the SE on disease severity.

Adult↗