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PubMed1999

GCF levels of MMP-3 and MMP-8 following placement of bioresorbable membranes.

Matrix metalloproteinases (MMPs) are responsible for remodeling and degrading extracellular matrix and basement membrane components. MMP-3 and -8 levels were assessed in this study during the early healing phase following a guided tissue regeneration (GTR) procedure. 32 patients, having 2 or 3 walled intrabony defects of PD > or =6 mm, were stratified into 2 groups on the basis of age, sex, smoking status and disease severity. All intrabony defects were treated using the resorbable Guidor membrane but only 1 group of patients was given a pre-operative dosage of antibiotic (3 g amoxycillin). GCF samples for the quantification of MMP-3 and -8 levels were obtained from the intrabony site where a membrane was placed (membrane site), from the non-adjacent site on the adjacent tooth which was involved in the surgical flap (surgical control site), and from a healthy site (healthy) on the contralateral side. The GCF samples taken at baseline, 1 week, 4 weeks and 3 months after surgery were analyzed using an enzyme linked immunoabsorbent essay (ELISA) for MMP-3 and using a time-resolved immunofluorescence assay (IFMA) for MMP-8. MMP-3 was detected in a very low % of sites at baseline while relatively high levels of MMP-8 were detected at all 3 types of sites at baseline. MMP-8 levels increased for all sites at week 1, and this was statistically significant for the membrane site, but at week 4, the levels decreased for both the membrane and the surgical sites. There was no statistically significant difference between the levels of MMP-3 and -8 in the antibiotic and non-antibiotic group. Collagen remodelling occurs during the early wound healing period following surgical and regenerative procedures. The levels of MMP-3 and -8 in GCF appear to reflect these processes. Interestingly, the presence of the membranes appeared to increase the levels of MMP-3 and -8 and may relate to the resorption of the resorbable membrane by host systems.

Absorbable Implants↗

PubMed1995

Gingival crevicular stromelysin, collagenase and tissue inhibitor of metalloproteinases levels in healthy and diseased sites.

The ability of stromelysin (SL), fibroblast-type collagenase (FIB-CL) and tissue inhibitor of metalloproteinases (TIMP), to differentiate between healthy, gingivitis and periodontitis sites was investigated. SL and FIB-CL are members of a family of enzymes which are capable of degrading most of the extracellular matrix macromolecules. Extracellular control of these enzymes is performed by TIMP. 40 patients each provided 3 GCF samples from healthy, gingivitis and periodontitis sites. GCF samples were collected by means of sterile paper strips. GCF samples were eluted into 500 microliters of assay buffer and assays for SL, FIB-CL and TIMP were performed by a sandwich ELISA. The mean amounts of SL and TIMP in diseased sites (gingivitis and periodontitis) were significantly higher than the mean amount of these GCF components in healthy sites (MANOVA p values were: 0.006 for SL and 0.001 for TIMP). GCF SL and TIMP differentiated healthy from diseased sites. Both SL and TIMP showed moderate correlation with clinical indices. FIB-CL was detectable in only 20.8% of all sites and did not correlate with disease status.

Adult↗

PubMed2004

Focal expression and final activity of matrix metalloproteinases may explain irregular dysfunctional endometrial bleeding.

Irregular dysfunctional bleeding of the endometrium (ie, metrorrhagia without organic lesion) is common in women, whether treated or not with ovarian hormones. Several matrix metalloproteinases (MMPs) become normally expressed and/or activated at menstruation and cause extracellular matrix breakdown. We therefore explored whether episodes of irregular dysfunctional bleeding could be associated with untimely MMP activity. By histology, foci of stromal breakdown were exclusively found in the endometrium of metrorrhagic women at bleeding. In these foci, 1) expression of estrogen receptor-alpha and progesterone receptor was altered; 2) collagenase-1 (MMP-1), stromelysin-1 (MMP-3), and gelatinase B (MMP-9) became detected in stromal cells, together with MMP-9 in neutrophils; and 3) gelatinase A (MMP-2) was more expressed and immunolocalized at the membrane of stromal cells. By biochemistry, endometrial lysates from nonbleeding metrorrhagic patients contained more latent and active MMP-2 and -9 than age-matched controls; at bleeding, collagenase activity, MMP-9, and active MMP-2 were strikingly increased whereas tissue inhibitor of metalloproteinases-1 (TIMP-1) was considerably decreased. As a functional assay, in situ gelatin zymography revealed large areas of gelatinolytic activity only in endometrium of bleeding patients. Altogether, these results strongly suggest that inappropriate focal expression and activation of several MMPs, combined with decreased inhibition, trigger irregular dysfunctional endometrial bleeding.

Adult↗

PubMed1994

Regional and temporal changes in the synthesis of matrix metalloproteinases and TIMP-1 during development of the rabbit mandibular condyle.

Connective tissues synthesise and secrete a family of matrix metalloproteinases (MMPs; collagenases, gelatinases and stromelysins) capable of degrading all the components of connective tissue matrices at physiological pH. We document the patterns of synthesis and distribution of MMPs and the tissue inhibitor of metalloproteinases-1 (TIMP-1) within the developing rabbit mandibular condyle using immunofluorescence microscopy. MMPs and TIMP-1 were detected both as bright intracellular accumulations within Golgi vesicles and also as diffuse matrix-bound extracellular deposits. Cells in the articular zone, proliferative zone, condylar cartilage and bone of the mandibular ramus were shown to produce all 3 classes of MMPs and TIMP-1 with the exception of stromelysin, which was not synthesised by cells of the bone of spongiosum. Temporal synthesis of MMPs and TIMP-1 within these regions varied during the period 18 d postcoitum to 14 d postnatum. Our results document unique patterns of MMP and TIMP-1 synthesis during embryonic and early postnatal development of condylar cartilage and support the concept that cells synthesise and secrete MMPs and TIMP-1 before undergoing proliferation and hypertrophy. A comparison of these results with data in the rabbit growth plate show many similarities, but some differences exist that probably reflect differences in the modes of growth of the 2 cartilages.

Animals↗

PubMed2007

Induction of matrix metalloproteinases (MMP3, MMP12 and MMP13) expression in the microglia by amyloid-beta stimulation via the PI3K/Akt pathway.

Alzheimer's disease is characterized by the presence of senile plaques in the brain composed primarily of amyloid-beta peptide. Microglia have been reported to surround these Abeta plaques, which have opposite roles, provoking a microglia-mediated inflammatory response that contributes to neuronal cell loss or the removal of Abeta and damaged neurons. We herein analyzed the process of expression of Matrix metalloproteinases induced by Abeta stimulation. We found that Abeta1-42 induces a high level of MMP3, MMP12 and MMP13 in the microglia. The signal transduction pathway for the expression of these MMPs mRNA induced by Abeta1-42 depends on PI3K/Akt.

Alzheimer Disease↗

PubMed2005

Developmental regulation of Wnt/beta-catenin signals is required for growth plate assembly, cartilage integrity, and endochondral ossification.

Studies have suggested that continuous Wnt/beta-catenin signaling in nascent cartilaginous skeletal elements blocks chondrocyte hypertrophy and endochondral ossification, whereas signaling starting at later stages stimulates hypertrophy and ossification, indicating that Wnt/beta-catenin roles are developmentally regulated. To test this conclusion further, we created transgenic mice expressing a fusion mutant protein of beta-catenin and LEF (CA-LEF) in nascent chondrocytes. Transgenic mice had severe skeletal defects, particularly in limbs. Growth plates were totally disorganized, lacked maturing chondrocytes expressing Indian hedgehog and collagen X, and failed to undergo endochondral ossification. Interestingly, the transgenic cartilaginous elements were ill defined, intermingled with surrounding connective and vascular tissues, and even displayed abnormal joints. However, when activated beta-catenin mutant (delta-beta-catenin) was expressed in chondrocytes already engaged in maturation such as those present in chick limbs, chondrocyte maturation and bone formation were greatly enhanced. Differential responses to Wnt/beta-catenin signaling were confirmed in cultured chondrocytes. Activation in immature cells blocked maturation and actually de-stabilized their phenotype, as revealed by reduced expression of chondrocyte markers, abnormal cytoarchitecture, and loss of proteoglycan matrix. Activation in mature cells instead stimulated hypertrophy, matrix mineralization, and expression of terminal markers such as metalloprotease (MMP)-13 and vascular endothelial growth factor. Because proteoglycans are crucial for cartilage function, we tested possible mechanisms for matrix loss. Delta-beta-catenin expression markedly increased expression of MMP-2, MMP-3, MMP-7, MMP-9, MT3-MMP, and ADAMTS5. In conclusion, Wnt/beta-catenin signaling regulates chondrocyte phenotype, maturation, and function in a developmentally regulated manner, and regulated action by this pathway is critical for growth plate organization, cartilage boundary definition, and endochondral ossification.

ADAM Proteins↗

PubMed2003

A potent, selective inhibitor of matrix metalloproteinase-3 for the topical treatment of chronic dermal ulcers.

The pathology of chronic dermal ulcers is characterized by excessive proteolytic activity which degrades extracellular matrix (required for cell migration) and growth factors and their receptors. The overexpression of MMP-3 (stromelysin-1) and MMP-13 (collagenase-3) is associated with nonhealing wounds, whereas active MMPs-1, -2, -9, and -14 are required for normal wound healing to occur. We describe the synthesis and enzyme inhibition profile of (3R)-3-[([(1S)-2,2-dimethyl-1-(([(1S)-2-methoxy-1-phenylethyl]amino)carbonyl)propyl]amino)carbonyl]-6-(3-methyl-4-phenylphenyl)hexanoic acid (UK-370,106, 7), which is a potent inhibitor of MMP-3 (IC(50) = 23 nM) with >1200-fold weaker potency vs MMP-1, -2, -9, and -14. MMP-13, which may also contribute to the pathology of chronic wounds, was inhibited about 100-fold less potently by compound 7. Compound 7 potently inhibited cleavage of [(3)H]-fibronectin by MMP-3 (IC(50) = 320 nM) but not cleavage of [(3)H]-gelatin by either MMP-2 or -9 (up to 100 microM). Compound 7 had little effect, at MMP-3 selective concentrations, on keratinocyte migration over a collagen matrix in vitro, which is a model of the re-epithelialization process. Following iv (rat) or topical administration to dermal wounds (rabbit), compound 7 was cleared rapidly (t(1/2) = 23 min) from plasma, but slowly (t(1/2) approximately 3 days) from dermal tissue. In a model of chronic dermal ulcers, topical administration of compound 7 for 6 days substantially inhibited MMP-3 ex vivo. These data suggest compound 7 is sufficiently potent to inhibit MMP-3-mediated matrix degradation while leaving unaffected cellular migration mediated by MMPs 1, 2, and 9. These properties make compound 7 a suitable candidate for progression to clinical trials in human chronic dermal wounds, such as venous ulcers.

Administration, Cutaneous↗

PubMed2001

Gene expression by human articular chondrocytes cultured in alginate beads.

Culture of articular chondrocytes in alginate beads offers several advantages over culture in monolayer; cells retain their phenotype for 8 months or longer. Earlier studies of chondrocytes cultured in alginate concentrated on collagen and proteoglycan synthesis. However, gene expression by in situ hybridization (ISH) has not been investigated. The purposes of the present study on human chondrocytes were (a) to modify the ISH procedure for the alginate beads to examine the mRNA expression of alpha1 (II) procollagen, aggrecan, and two matrix metalloproteinases (MMP-3 and MMP-8) thought to be involved in cartilage matrix degradation, and (b) to compare expression in cultured chondrocytes with that in chondrocytes of intact human cartilage. The modifications made for ISH include the presence of CaCl2 and BaCl2 in the fixation and washing steps and exclusion of cetyl pyridinium chloride. By ISH we show that aggrecan, MMP-3, and MMP-8 are continuously expressed during 8 months of culture. The alpha1 (II) procollagen gene is expressed only during the first 2 months of culture and after 3 months its expression is undetectable, which is consistent with its absence in adult articular cartilage. By Western blotting, Type II collagen protein had been synthesized and deposited in both the cell-associated and further-removed matrix compartments at 7 and 14 days of culture. These data indicate that chondrocytes cultured in alginate beads could be preserved for immunohistochemistry and ISH and that culture of human chondrocytes in alginate beads may serve as a good model for studying cartilage-specific phenotype as well as factors that influence cartilage matrix turnover.

Adult↗

PubMed2000

Localisation of matrix metalloproteinases and TIMP-2 in resorbing mouse bone.

There is strong evidence that matrix metalloproteinases (MMPs) play a crucial role during osteogenesis and bone remodelling. Their synthesis by osteoblasts has been demonstrated during osteoid degradation prior to resorption of mineralised matrix by osteoclasts and their activities are regulated by tissue inhibitors of metalloproteinases (TIMPs). For this study we developed and utilised specific polyclonal antibodies to assess the presence of collagenase (MMP13), stromelysin 1 (MMP3), gelatinase A (MMP2), gelatinase B (MMP9) and TIMP-2 in both freshly isolated neonatal mouse calvariae and tissues cultured with and without bone-resorbing agents. Monensin was added towards the end of the culture period in order to promote intracellular accumulation of proteins and facilitate antigen detection. In addition, bone sections were stained for the osteoclast marker, tartrate-resistant acid phosphatase (TRAP). In uncultured tissues the bone surfaces had isolated foci of collagenase staining, and cartilage matrix stained for gelatinase B (MMP9) and TIMP-2. Calvariae cultured for as little as 3 h with monensin revealed intracellular staining for MMPs and TIMP-2 in mesenchymal tissues, as well as in cells lining the bone plates. The addition of cytokines to stimulate bone resorption resulted in pronounced TRAP activity along bone surfaces, indicating active resorption. There was a marked upregulation of enzyme synthesis, with matrix staining for collagenase and gelatinase B observed in regions of eroded bone. Increased staining for TIMP-2 was also observed in association with increased synthesis of MMPs. The new antibodies to murine MMPs should prove valuable in future studies of matrix degradation.

Acid Phosphatase↗

PubMed1996

Expression of immunoreactive matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in human normal livers and primary liver tumors.

Matrix metalloproteinases (MMPs) play an important role in cancer cell invasion by degrading extracellular matrix proteins. However, little is known about the in situ expression of MMP in human normal livers and primary liver tumors. In this study, we therefore examined the in situ expression of immunoreactive MMP and tissue inhibitors of MMP (TIMP) in 10 normal livers, 11 surgically resected intrahepatic cholangiocarcinomas (CCs), and 6 surgically resected hepatocellular carcinomas (HCCs). In normal livers, MMP and TIMP were infrequently and faintly expressed in bile ducts, but were not expressed in hepatocytes. In the 11 CCs, MMP-1, MMP-2, MMP3, MMP-9, TIMP-1, and TIMP-2 were expressed in tumor cells and/or tumor stroma in 11 (100%), 5 (45%), 8 (73%), 3 (27%), 9 (82%), and 9 (82%), respectively. The expression of MMP and TIMP in tumor cells was located in the cytoplasm with a diffuse or granular pattern; that in the tumor stroma was situated in fibroblasts, leukocytes, and extracellular matrix. Their expression was stronger in CC cases with severe invasion than in CC cases with mild invasion. In contrast, MMP and TIMP were not expressed in any cases of HCC. These results show that intrahepatic bile duct cells may neoexpress or overexpress MMP and TIMP after malignant transformation but that hepatocytes do not, and suggest that MMP and TIMP play an important role in CC cell invasion by degrading extracellular matrix proteins.

Adult↗

PubMed1999

Transforming growth factor-beta induces collagenase-3 expression by human gingival fibroblasts via p38 mitogen-activated protein kinase.

Human collagenase-3 (matrix metalloproteinase 13 (MMP-13)) is characterized by exceptionally wide substrate specificity and restricted tissue specific expression. Human skin fibroblasts in culture express MMP-13 only when they are in three-dimensional collagen (Ravanti, L., Heino, J., López-Otín, C., and Kähäri. V.-M. (1999) J. Biol. Chem. 274, 2446-2455). Here we show that MMP-13 is expressed by fibroblasts during normal human gingival wound repair. Expression of MMP-13 by human gingival fibroblasts cultured in monolayer or in collagen gel was induced by transforming growth factor-beta1 (TGF-beta1). Treatment of gingival fibroblasts with TGF-beta1 activated two distinct mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase 1/2 (ERK1/2) in 15 min and p38 MAPK in 1 and 2 h. Induction of MMP-13 expression by TGF-beta1 was blocked by SB203580, a specific inhibitor of p38 MAPK, but not by PD98059, a selective inhibitor of ERK1/2 activation. Adenovirus-mediated expression of dominant negative p38alpha and c-Jun potently inhibited induction of MMP-13 expression in gingival fibroblasts by TGF-beta1. Infection of gingival fibroblasts with adenovirus for constitutively active MEK1 resulted in activation of ERK1/2 and JNK1 and up-regulation of collagenase-1 (MMP-1) and stromelysin-1 (MMP-3) production but did not induce MMP-13 expression. In addition, activation of p38 MAPK by constitutively active MKK6b or MKK3b was not sufficient to induce MMP-13 expression. These results show that TGF-beta-elicited induction of MMP-13 expression by gingival fibroblasts is dependent on the activity of p38 MAPK and the presence of functional AP-1 dimers. These observations demonstrate a fundamental difference in the regulation of collagenolytic capacity between gingival and dermal fibroblasts and suggest a role for MMP-13 in rapid turnover of collagenous matrix during repair of gingival wounds, which heal with minimal scarring.

Cells, Cultured↗

PubMed2001

Activation of latent transforming growth factor beta1 by stromelysin 1 in extracts of growth plate chondrocyte-derived matrix vesicles.

Previous studies have shown that matrix vesicles isolated from cultures of costochondral growth zone chondrocytes and treated with 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] can activate recombinant human latent transforming growth factor beta1 (rhTGF-beta1). It is unknown what enzyme or other factor in the extracellular organelles is responsible for the activation. This study tested the hypothesis that enzymes present in matrix vesicles can activate latent TGF-beta1 and that this is regulated by 1alpha,25(OH)2D3. To do this, we examined the ability of matrix vesicle extracts to activate small latent rhTGF-beta1. In addition, enzymes previously determined to be present in matrix vesicles were screened for their ability to activate small latent rhTGF-beta1. Recombinant human matrix metalloproteinase 2 (rhMMP-2; 72 kDa gelatinase), rhMMP-3 (stromelysin 1), purified human plasminogen, and purified urokinase (plasminogen activator) were each tested at varying concentrations. To assess the role of cell maturation, we used a cell culture model in which chondrocytes are derived from two distinct zones of rat costochondral cartilage, the resting zone and the growth zone. Matrix vesicles were isolated from these cultures and then tested. The results showed that extracts of matrix vesicles produced by both growth zone and resting zone chondrocytes were able to activate small latent rhTGF-beta1. The effects were dose and time dependent, with greater activity being found in extracts of matrix vesicles from the growth zone chondrocyte cultures. Only rhMMP-3 was able to activate small latent rhTGF-beta1, indicating that stromelysin-1, but not MMP-2, plasminogen, or urokinase, was involved. As observed in the extracts, the effect of rhMMP-3 was time and dose dependent. When anti-MMP-3 antibody was added to matrix vesicle extracts from both cell types, activation of small latent rhTGF-beta1 was dose-dependently blocked. Neither 1alpha,25(OH)2D3 nor 24R,25(OH)2D3 had a direct effect on activation of small latent rhTGF-beta1 by the extracts. However, when intact matrix vesicles were treated with 1alpha,25(OH)2D3, their ability to activate small latent rhTGF-beta1 was increased. Inhibition of phospholipase A2 with quinacrine blocked the 1alpha,25(OH)2D3-dependent effect. These results suggest that the ability of 1alpha,25(OH)2D3-treated matrix vesicles to activate small latent TGF-beta1 is via action of the secosteroid on the matrix vesicle membrane, not on the enzymes responsible for activating latent TGF-beta1. Because matrix vesicles isolated from growth zone chondrocytes have been shown to contain increased phospholipase A2 activity after treatment with 1alpha,25(OH)2D3, it is likely that this secosteroid promotes loss of membrane integrity through phospholipase A2-dependent formation of lysophospholipids, resulting in the release of MMP-3 into the matrix, where latent TGF-beta1 is stored. Taken together, the results of the current study show that matrix vesicles produced by growth plate chondrocytes contain MMP-3, that this enzyme is at least partially responsible for activation of small latent TGF-beta1 in the matrix, and that 1alpha,25(OH)2D3 regulates MMP release from matrix vesicles.

Animals↗

PubMed2005

Role of interleukin 6 (IL-6)/IL-6R-induced signal tranducers and activators of transcription and mitogen-activated protein kinase/extracellular.

OBJECTIVE: Studies have described elevated levels of interleukin 6 (IL-6) and its soluble receptor (sIL-6R) in osteoarthritic and rheumatoid joints, as well as the inhibitory effect of this combination on cartilage matrix production. We investigated the ability of IL-6/sIL-6R to modulate gene expression of matrix metalloproteinase (MMP) and ADAMTS (ADAMS with thrombospondin motifs) family members in bovine chondrocytes, and the potential role of signal transducers and activators of transcription (STAT) and mitogen-activated protein kinases (MAPK) in this regulation. METHODS: Primary cultures of bovine chondrocytes were stimulated with IL-6/sIL-6R for 30 min (Western blot and EMSA) or 24 h (RNA measurements) in the presence or absence of the STAT inhibitor parthenolide or the MAPK inhibitor PD 098059. mRNA was assessed by RT-PCR for the expression of MMP (MMP-1, -3, and -13) and 2 ADAMT family members (ADAMTS-4 and -5/11). RESULTS: IL-6/sIL-6R markedly induced activation of STAT and extracellular signal-related kinase (ERK1/2) and the subsequent expression of the collagenases MMP-1 and MMP-13 as well as MMP-3, an aggrecan-degrading enzyme and activator of pro-MMP. Expression of the 2 specific aggrecanases ADAMTS-4 and -5/11 was also elevated by this combination. Both STAT and MAPK signaling pathways were found to contribute to the IL-6/sIL-6R induction mechanisms, the overall effect being dependent on the respective magnitude of response and the crosstalk between the 2 pathways. CONCLUSION: These data indicate that the cartilage-degrading properties of IL-6/sIL-6R are mediated by induction of the aggrecan-degrading enzymes ADAMTS-4, -5/11, and MMP-3, and the collagen-degrading enzymes MMP-1 and -13. STAT and MAPK pathways play a crucial role in IL-6/sIL-6R modulation of these enzymes, suggesting that new strategies in the treatment of osteoarticular diseases might target these transduction cascades.

ADAM Proteins↗

PubMed2003

Induction of stromelysin-1 (MMP-3) by fibroblast growth factor-2 (FGF-2) in FGF-2-/- microvascular endothelial cells requires prolonged activation of extracellular signal-regulated kinases-1 and -2 (ERK-1/2).

Basic fibroblast growth factor (FGF-2) and matrix metalloproteinases (MMPs) play key roles in vascular remodeling. Because FGF-2 controls a number of proteolytic activities in various cell types, we tested its effect on vascular endothelial cell expression of MMP-3 (stromelysin-1), a broad-spectrum proteinase implicated in coronary atherosclerosis. Endothelial cells (EC) from FGF-2-/- mice are highly responsive to exogenous FGF-2 and were therefore used for this study. The results showed that treatment of microvascular EC with human recombinant FGF-2 results in strong induction of MMP-3 mRNA and protein expression. Upregulation of MMP-3 mRNA by FGF-2 requires de novo protein synthesis and activation of the ERK-1/2 pathway. FGF-2 concentrations (5-10 ng/ml) that induce rapid and prolonged (24 h) activation of ERK-1/2 upregulate MMP-3 expression. In contrast, lower concentrations (1-2 ng/ml) that induce robust but transient (<8 h) ERK-1/2 activation are ineffective. Inhibition of ERK-1/2 activation at different times (-0.5 h to +8 h) of EC treatment with effective FGF-2 concentrations blocks MMP-3 upregulation. Thus, FGF-2 induces EC expression of MMP-3 with a threshold dose effect that requires sustained activation of the ERK-1/2 pathway. Because FGF-2 controls other EC functions with a linear dose effect, these features indicate a unique role of MMP-3 in vascular remodeling.

Animals↗

PubMed2002

Substrate binding of gelatinase B induces its enzymatic activity in the presence of intact propeptide.

Expression of gelatinase B (matrix metalloprotease 9) in human placenta is developmentally regulated, presumably to fulfill a proteolytic function. Here we demonstrate that gelatinolytic activity in situ, in tissue sections of term placenta, is co-localized with gelatinase B. Judging by molecular mass, however, all the enzyme extracted from this tissue was found in a proform. To address this apparent incongruity, we examined the activity of gelatinase B bound to either gelatin- or type IV collagen-coated surfaces. Surprisingly, we found that upon binding, the purified proenzyme acquired activity against both the fluorogenic peptide (7-methoxycoumarin-4-yl)-acetic acid (MCA)-Pro-Leu-Gly-Leu-3-(2,4-dinitrophenyl)-l-2,3-diaminopropionyl-Ala-Arg-NH(2) and gelatin substrates, whereas its propeptide remained intact. These results suggest that although activation of all known matrix metalloproteases in vitro is accomplished by proteolytic processing of the propeptide, other mechanisms, such as binding to a ligand or to a substrate, may lead to a disengagement of the propeptide from the active center of the enzyme, causing its activation.

Binding Sites↗

PubMed1996

Changes in the expression of matrix proteases and of the transcription factor c-Ets-1 during progression of precancerous bronchial lesions.

Matrix proteases and the transcription factor c-Ets-1, which regulates in vitro stromelysin 1, collagenase 1, and urokinase type plasminogen activator gene promoters, are frequently expressed in invasive carcinomas. Using in situ hybridization and immunohistochemistry, we analyzed collagenase 1, stromelysins 1 and 3, matrilysin, urokinase type plasminogen activator, and c-Ets-1 gene expression on serial frozen sections of 39 intraepithelial bronchial lesions, including areas of hyperplasia, metaplasia, dysplasia, carcinoma in situ, and corresponding lung carcinomas in 13 patients. In intraepithelial lesions, expression of all matrix proteases was detected in epithelial cells. Conversely, in microinvasive or invasive lesions, a fibroblastic expression was observed. Collagenase 1 and matrilysin were expressed seldomly in intraepithelial lesions and frequently in carcinomas (p = 0.0016 and p < 0.0001, respectively). Stromelysin 1 was expressed inconsistently in 31% of intraepithelial lesions of all grades and in 50% of carcinomas. Stromelysin 3 and urokinase type plasminogen activator were expressed only, but frequently, in preinvasive lesions (dysplasia, carcinoma in situ) and in carcinomas. The expression of stromelysin 3 in fibroblasts started with dysplasia and carcinoma in situ, but was more frequent in invasive than preinvasive lesions (p = 0.0012). c-Ets-1 was more often expressed in carcinomas than in intraepithelial lesions (p < 0.0001) and was always expressed in fibroblasts. Comparing preinvasive lesions adjacent to or at a distance from squamous lung carcinoma, stromelysin 3 epithelial expression was more frequent in preinvasive lesions adjacent to invasive foci than in others (p = 0.036). We conclude that (a) both epithelial expression of matrix proteases in intraepithelial bronchial lesions and their stromal expression in microinvasive and invasive lesions suggest their role in lung tumor development; (b) c-Ets-1 does not act as a transcriptional activator for matrix proteases genes in preinvasion, although it might regulate collagenase 1 gene during lung tumor progression; and (c) matrix proteases might offer new therapeutic targets for chemoprevention of lung cancer.

Bronchial Neoplasms↗

PubMedDate not supplied

Correlation between the appearance of gelatinases in the synovial fluid of patients with rheumatoid arthritis and polymorphonuclear elastase, stromelysin-1, and the tissue inhibitor of metalloproteinase-1.

OBJECTIVE: To clarify whether gelatinases in the synovial fluid are related to rheumatoid arthritis (RA). METHODS: Gelatinases in the synovial fluid of patients with RA were analyzed by gelatin zymography. RESULTS: We classified patients into 3 distinct groups based on the gelatinases present. Although inactive gelatinase A was found in the synovial fluid of all patients with RA, the presence of gelatinase B varied: group I contained none, group II contained inactive gelatinase B, and group III contained active gelatinase B. The presence of gelatinase B was positively correlated with the polymorphonuclear (PMN) elastase content of the synovial fluid. Furthermore, the presence of activated gelatinase B correlated well with the stromelysin-1 content. Conversely, large amounts of the tissue inhibitor of metalloproteinase-1 (TIMP-1) were found in the synovial fluid of patients in group II. CONCLUSION: The appearance and form of gelatinase B may reflect the inflammatory condition of patients with RA.

Arthritis, Rheumatoid↗

PubMed1997

Expression of urokinase-type plasminogen activator, stromelysin 1, stromelysin 3, and matrilysin genes in lung carcinomas.

We have previously shown that the extracellular-matrix-degrading enzymes, urokinase-type plasminogen activator (u-PA), stromelysin 1, stromelysin 3, and matrilysin, may play an important role in the transition from lung preneoplasia to invasive carcinoma. Using in situ hybridization and immunohistochemistry, we analyzed serial frozen sections for the expression of these enzymes in 89 lung carcinomas including 25 neuroendocrine (NE) carcinomas (10 small-cell lung carcinomas, 7 large-cell NE carcinomas, 1 atypical, and 7 typical carcinoids) and 64 non-small-cell, non-NE carcinomas (29 squamous and 7 basaloid carcinomas, 23 adenocarcinomas, and 5 large-cell carcinomas). Proteases, except matrilysin, were more often expressed in stromal than cancer cells. In non-small-cell, non-NE carcinomas, stromal co-expression of u-PA and stromelysin 3 was seen in 80 to 90% of the tumors and was highly correlated (P < 0.0001). Stromal u-PA and stromelysin 3 expression was linked to tumor size (P = 0.01 and 0.03, respectively) and lymph node involvement (P = 0.001 and 0.02, respectively). Epithelial expression of u-PA was correlated to tumor size (P = 0.04). Epithelial expression of stromelysin 3 predominated in squamous and basaloid carcinomas (P = 0.0005) and was inversely correlated to squamous differentiation (P = 0.018). Epithelial expression of matrilysin predominated in adenocarcinomas and large-cell carcinomas (P = 0.07). In NE carcinomas including small-cell lung carcinomas, stromal expression of u-PA was correlated to lymph node metastasis (P = 0.017). Epithelial expression of all enzymes were significantly less frequent in NE than in non-NE tumors. We conclude that 1) epithelial expression of matrix proteases in lung cancer is linked to cell phenotype (NE versus non-NE, squamous versus glandular) and 2) their stromal, rather than epithelial, expression influences local metastasis.

Carcinoma↗