Collagenase-3 (MMP-13) is expressed by tumor cells in invasive vulvar squamous cell carcinomas.
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Collagenase-3 (MMP-13) is a human matrix metalloproteinase specifically expressed by invading tumor cells in squamous cell carcinomas (SCCs) of the head and neck. Here, we have further elucidated the role of MMP-13 in tumor invasion by examining its expression in invasive malignant tumors of the female genital tract. Using in situ hybridization, expression of MMP-13 mRNA was detected in 9 of 12 vulvar SCCs, primarily in tumor cells, but not in intact vulvar epithelium, in cervical SCCs (n = 12), or in endometrial (n = 11) or ovarian adenocarcinomas (n = 8). MMP-13 expression was especially abundant in vulvar carcinomas showing metastasis to lymph nodes and was associated with expression of membrane type 1 MMP by tumor cells and gelatinase-A (MMP-2) by stromal cells, as detected by immunohistochemistry. MMP-13 mRNAs were detected in 9 of 11 cell lines established from vulvar carcinomas and in 4 of 6 cell lines from cervical carcinomas, whereas endometrial (n = 10) and ovarian (n = 9) carcinoma cell lines were negative for MMP-13 mRNA. No correlation was detected between MMP-13 expression and p53 gene mutations in vulvar SCC cell lines. However, MMP-13 expression was detected in 5 of 6 vulvar and cervical SCC cell lines harboring HPV 16 or 68 DNA. These results show that MMP-13 is specifically expressed by malignantly transformed squamous epithelial cells, including vulvar SCC cells, and appears to serve as a marker for their invasive capacity. (+info)
Leukemia inhibitory factor and oncostatin M stimulate collagenase-3 expression in osteoblasts.
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Leukemia inhibitory factor (LIF) and oncostatin M (OSM) have multiple effects on skeletal remodeling. Although these cytokines modestly regulate collagen synthesis in osteoblasts, their effects on collagenase expression and collagen degradation are not known. We tested whether LIF and OSM regulate the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in osteoblast-enriched cells isolated from fetal rat calvariae. LIF and OSM increased collagenase-3 (MMP-13) mRNA and immunoreactive protein levels in a time- and dose-dependent manner. LIF and OSM enhanced the rate of transcription of the collagenase gene and stabilized collagenase mRNA in transcriptionally arrested cells. LIF and OSM failed to regulate the expression of gelatinase A (MMP-2) and B (MMP-9). LIF and OSM modestly stimulated the expression of TIMP-1 but did not alter the expression of TIMP-2 and -3. In conclusion, LIF and OSM stimulate collagenase-3 and TIMP-1 expression in osteoblasts, and these effects may be involved in mediating the bone remodeling actions of these cytokines. (+info)
Differential patterns of response to doxycycline and transforming growth factor beta1 in the down-regulation of collagenases in osteoarthritic and normal human chondrocytes.
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OBJECTIVE: To investigate the ability of doxycycline, transforming growth factor beta1 (TGFbeta1), and phorbol myristate acetate (PMA) to modulate collagenase synthesis in osteoarthritic (OA) chondrocytes. METHODS: Levels of fibroblast collagenase (matrix metalloproteinase 1 [MMP-1]), neutrophil collagenase (MMP-8), and collagenase 3 (MMP-13) proteins and messenger RNA (mRNA) were measured in chondrocytes isolated from involved and uninvolved areas of OA cartilage and from normal human chondrocytes, after treatment with doxycycline, TGFbeta1, and PMA. RESULTS: Chondrocytes isolated from cartilage immediately adjacent to the OA lesion had, on average, 1.8-3.9-fold higher basal levels of MMP mRNA. These cells down-regulated collagenase proteins and mRNA upon incubation with TGFbeta1. In contrast, chondrocytes from areas located more distant from the macroscopic lesion increased MMP-13 mRNA, while MMP-1 and MMP-8 decreased after stimulation with TGFbeta1. Discoordinate regulation was observed after stimulation with PMA, with an increase in MMP-1 and MMP-8 but a decrease in MMP-13. Incubation of OA chondrocytes with doxycycline (1-10 microg/ml), at pharmacologically achievable levels, decreased levels of mRNA of all 3 collagenases, but not G3PDH. In addition, doxycycline inhibited the increase in mRNA for these enzymes in normal chondrocytes stimulated with tumor necrosis factor alpha. CONCLUSION: These findings suggest that regulation of MMP-1, MMP-8, and MMP-13 in OA chondrocytes, although mediated by differing pathways, can be decreased by treatment with doxycycline at low concentrations. Our data provide a rationale for the use of doxycycline in the treatment of OA. (+info)
Maturational disturbance of chondrocytes in Cbfa1-deficient mice.
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Cbfa1, a transcription factor that belongs to the runt-domain gene family, plays an essential role in osteogenesis. Cbfa1-deficient mice completely lacked both intramembranous and endochondral ossification, owing to the maturational arrest of osteoblasts, indicating that Cbfa1 has a fundamental role in osteoblast differentiation. However, Cbfa1 was also expressed in chondrocytes, and its expression was increased according to the maturation of chondrocytes. Terminal hypertrophic chondrocytes expressed Cbfa1 extensively. The significant expression of Cbfa1 in hypertrophic chondrocytes was first detected at embryonic day 13.5 (E13.5), and its expression in hypertrophic chondrocytes was most prominent at E14.5-16.5. In Cbfa1-deficient mice, whose entire skeleton was composed of cartilage, the chondrocyte differentiation was disturbed. Calcification of cartilage occurred in the restricted parts of skeletons, including tibia, fibula, radius, and ulna. Type X collagen, BMP6, and Indian hedgehog were expressed in their hypertrophic chondrocytes. However, osteopontin, bone sialoprotein, and collagenase 3 were not expressed at all, indicating that they are directly regulated by Cbfa1 in the terminal hypertrophic chondrocytes. Chondrocyte differentiation was severely disturbed in the rest of the skeleton. The expression of PTH/PTHrP receptor, Indian hedgehog, type X collagen, and BMP6 was not detected in humerus and femur, indicating that chondrocyte differentiation was blocked before prehypertrophic chondrocytes. These findings demonstrate that Cbfa1 is an important factor for chondrocyte differentiation. (+info)
Activation of neutrophil collagenase in periodontitis.
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Neutrophil collagenase (matrix metalloproteinase 8 [MMP-8]) is an important mediator of tissue destruction in inflammatory diseases. Studies of anaerobic periodontal infections have shown that active MMP-8 in gingival crevicular fluid is associated with the degradation of periodontal tissues in progressive periodontitis whereas the latent enzyme is predominant in gingivitis. Since the activation of MMP-8 appears to be a crucial step in periodontitis, we have examined the activation of MMP-8 in gingival crevicular fluid samples by using a soluble biotinylated collagen substrate. Analysis of gingival crevicular fluid in periodontitis, gingivitis, and controls revealed sixfold (P < 0.001)-higher levels of active collagenase in periodontitis (n = 12) samples compared to gingivitis (n = 17) samples, which exhibited low levels of activity, while controls (n = 25) showed no activity. After gingival crevicular fluid was collected, no further activation of latent collagenase occurred in vitro. Although both MMP-1 and MMP-8, but not MMP-13, could be detected by immunoblots, blocking antibodies to MMP-1 showed that collagenase activity was largely contributed by MMP-8, which was localized to the matrix of diseased tissues. The MMP-8 in gingival crevicular fluid migrated primarily as a 60-kDa form with smaller amounts of a 78-kDa species, whereas MMP-8 isolated from peripheral neutrophils migrated at 70 and 89 kDa, corresponding to active and latent forms of the enzyme, respectively. Most of the MMP-8 in the 60- and 70-kDa bands selectively bound to tissue inhibitor of metalloproteinase 2 and collagen, indicating that most, but not all, of the enzyme in these bands was in an activated form. However, the amounts of the 78- and 60-kDa forms from gingival crevicular fluid in different samples did not correlate (r2 = 0.028) with the latent and active enzyme measured by collagenase assay. Collectively, these studies have identified distinct forms of latent and active MMP-8 in gingival crevicular fluid that appear to result from a unique activation mechanism that occurs in periodontitis. The complexity of MMP-8 activation is further indicated by the presence of latent, activated, and superactivated forms of MMP-8 in the 60- and 70-kDa bands obtained from gingival crevicular fluid and neutrophil samples, respectively. (+info)
Collagenase 3 is a target of Cbfa1, a transcription factor of the runt gene family involved in bone formation.
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Collagenase 3 (MMP-13) is a recently identified member of the matrix metalloproteinase (MMP) gene family that is expressed at high levels in diverse human carcinomas and in articular cartilage from arthritic patients. In addition to its expression in pathological conditions, collagenase 3 has been detected in osteoblasts and hypertrophic chondrocytes during fetal ossification. In this work, we have evaluated the possibility that Cbfa1 (core binding factor 1), a transcription factor playing a major role in the expression of osteoblastic specific genes, is involved in the expression of collagenase 3 during bone formation. We have functionally characterized a Cbfa motif present in the promoter region of collagenase 3 gene and demonstrated, by cotransfection experiments and gel mobility shift assays, that this element is involved in the inducibility of the collagenase 3 promoter by Cbfa1 in osteoblastic and chondrocytic cells. Furthermore, overexpression of Cbfa1 in osteoblastic cells unable to produce collagenase 3 leads to the expression of this gene after stimulation with transforming growth factor beta. Finally, we show that mutant mice deficient in Cbfa1, lacking mature osteoblasts but containing hypertrophic chondrocytes which are also a major source of collagenase 3, do not express this protease during fetal development. These results provide in vivo evidence that collagenase 3 is a target of the transcriptional activator Cbfa1 in these cells. On the basis of these transcriptional regulation studies, together with the potent proteolytic activity of collagenase 3 on diverse collagenous and noncollagenous bone and cartilage components, we proposed that this enzyme may play a key role in the process of bone formation and remodeling. (+info)
Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatous plaques.
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BACKGROUND: Several recent studies attempted to classify plaques as those prone to cause clinical manifestations (vulnerable, atheromatous plaques) or those less frequently associated with acute thrombotic complication (stable, fibrous plaques). Defining the cellular and molecular mechanisms that underlie these morphological features remains a challenge. Because interstitial forms of collagen determine the biomechanical strength of the atherosclerotic lesion, this study investigated expression of the collagen-degrading matrix metalloproteinase (MMP) interstitial collagenase-3 (MMP-13) and the previously studied MMP-1 in human atheroma and used a novel technique to test the hypothesis that collagenolysis in atheromatous lesions exceeds that in fibrous human atherosclerotic lesions. METHODS AND RESULTS: Human carotid atherosclerotic plaques, similar in size, were separated by conventional morphological characteristics into fibrous (n=10) and atheromatous (n=10) lesions. Immunohistochemical and Western blot analysis demonstrated increased levels of MMP-1 and MMP-13 in atheromatous versus fibrous plaques. In addition, collagenase-cleaved type I collagen, demonstrated by a novel cleavage-specific antibody, colocalized with MMP-1- and MMP-13-positive macrophages. Macrophages, rather than endothelial or smooth muscle cells, expressed MMP-13 and MMP-1 on stimulation in vitro. Furthermore, Western blot analysis demonstrated loss of interstitial collagen type I and increased collagenolysis in atheromatous versus fibrous lesions. Finally, atheromatous plaques contained higher levels of proinflammatory cytokines, activators of MMPs. CONCLUSIONS: This report demonstrates that atheromatous rather than fibrous plaques might be prone to rupture due to increased collagenolysis associated with macrophages, probably mediated by the interstitial collagenases MMP-1 and MMP-13. (+info)
In situ zymographic localisation of type II collagen degrading activity in osteoarthritic human articular cartilage.
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OBJECTIVES: Chondrocytic matrix metalloproteinases (MMPs) are believed to be important in osteoarthritic cartilage degradation. The cartilage lesion of osteoarthritis (OA) is focal and often progressive. During its development chondrocytes differentially up and down regulate production of mRNA for individual MMPs. This observation has potential implications for understanding the disease processes that lead to progressive cartilage loss in OA and designing appropriate targeted treatment. The complex regulation of MMP mediated effects means there is a pressing need to establish whether visualisation of MMP mRNA or protein equates to enzyme activity. The technique of in situ zymography (ISZ) offers a way of examining diseased human tissue for in vivo production of an excess of degrading enzyme over inhibitor. The primary objective of this study was to assess, and if positive follow, collagen II degrading activity in cartilage during development of the OA lesion. A secondary objective was to assess whether there was any correlation between sites of collagen II degrading activity and expression of the collagenase (MMP-13), recently implicated in type II collagen degredation in this lesion. METHODS: Biopsied human normal and osteoarthritic cartilage, showing various degrees of damage, was examined by in situ zymography, with and without enzyme inhibitors, to establish sites of type II collagenase activity. Paired samples were probed for MMP-13 mRNA using 35S-labelled oligonucleotide probes. Comparative analyses were performed. RESULTS: In situ zymography showed collagen II degrading activity over chondrocytes only in osteoarthritic cartilage. Distribution and amount varied with the extent of cartilage damage and position of chondrocytes, being greatest in deep cartilage and in cartilage lesions where fissuring was occurring. The enzyme causing the degradation behaved as a matrix metalloproteinase. MMP-13 mRNA expression codistributed with the type II collagenase activity. CONCLUSION: In OA, chondrocytes can degrade type II collagen. The type II collagen degrading activity varies in site and amount as the cartilage lesion progresses and throughout codistributes with MMP-13 mRNA expression. (+info)