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At least 19 recordsLinked to original sources
PubMed1990

The collagenase activities, interstitial collagenase and type IV collagenase, in human stomach cancer: with special reference to local spreading and lymph node metastasis.

In order to investigate the role of collagenase in cancer invasion and metastasis, two collagenase activities of interstitial collagenase and type IV collagen degrading enzyme (type IV collagenase) were determined in 40 cases of human stomach cancer tissue. Elevated cancers which are known to have a propensity to cause blood-borne metastases showed higher activities of both interstitial collagenase and type IV collagenase than flat or ulcerous type of cancer. Using the parameters of lymph node metastasis vs tumor size or vs depth of cancerous invasion into the stomach wall, classification of the cases was attempted according to the degree of malignancy. In the cases with marked lymph node metastases in spite of small tumor size and/or shallow cancerous invasion into the stomach wall, type IV collagenase activity was higher than that in the cases with lower malignancy (p less than 0.025, p less than 0.05, respectively). These results suggest that collagenase in stomach cancer tissue play an important role in the invasion and metastasis of cancer cells. Type IV collagenase activity in stomach cancer tissue could be one of the useful biological markers for the degree of malignancy.

Adult↗
PubMed1992

Cloning and sequencing of mouse collagenase cDNA. Divergence of mouse and rat collagenases from the other mammalian collagenases.

Mouse collagenase cDNA was cloned and sequenced. The deduced amino acid sequence was compared to those of the other mammalian collagenases and related matrix metalloproteinases. These comparisons, as well as those of some enzymatic properties, show that the rodent (mouse and rat) interstitial collagenases are very similar but differ more from the other interstitial collagenases than does human neutrophil collagenase. This supports the hypothesis that the order Rodentia is an outgroup to the other eutherian (placental) mammalian orders.

Amino Acid Sequence↗
PubMed2000

Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-Jun N-terminal kinase, and nuclear factor kappaB: differential regulation of collagenase 1 and collagenase 3.

OBJECTIVE: To examine the mechanism of interleukin-1 (IL-1)-induced collagenase 3 (matrix metalloproteinase 13 [MMP-13]) gene expression in cultured chondrocytes for the purpose of better understanding how the gene is induced in these cells, and how it contributes to cartilage degradation in osteoarthritis. METHODS: The transcriptional and posttranscriptional responses of the MMP-13 gene to IL-1 were assessed first. Then, direct inhibitors of mitogen-activated protein kinase (MAPK) signaling pathways and a constitutive repressor of nuclear factor kappaB (NF-kappaB) were used to assess the role of each pathway in IL-1-mediated induction of MMP-13. RESULTS: We found that IL-1 induction of MMP-13 requires p38 activity, c-Jun N-terminal kinase (JNK) activity and NF-kappaB translocation. These results suggest that both NF-kappaB and activator protein 1 transcription factors are necessary for IL-1 induction of MMP-13. We also compared the signaling pathways necessary for IL-1 to stimulate collagenase 1 (MMP-1) in articular chondrocytes and chondrosarcoma cells and found that IL-1 induction of MMP-1 requires different pathways from those required by MMP-13. In chondrosarcoma cells, MMP-1 induction depends on p38 and MEK (an MAPK kinase of the extracellular signal-regulated kinase pathway) and does not require JNK or NF-kappaB. In articular chondrocytes, inhibition of MEK had no effect, while inhibition of p38 gave variable results. CONCLUSION: These studies demonstrate, for the first time, that p38, JNK, and NF-kappaB are required for IL-1 induction of MMP-13. The results also highlight the differential requirements for signaling pathways in the induction of MMP-1 and MMP-13. Additionally, they demonstrate that induction of MMP-1 by IL-1 in chondrocytic cells depends on unique combinations of signaling pathways that are cell type-specific.

Chondrocytes↗
PubMed2000

Selective enhancement of collagenase-mediated cleavage of resident type II collagen in cultured osteoarthritic cartilage and arrest with a synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1).

OBJECTIVE: To examine whether type II collagen cleavage by collagenase and loss of proteoglycan are excessive in human osteoarthritic (OA) articular cartilage compared with nonarthritic articular cartilage, and whether this can be inhibited by a selective synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1 [MMP-1]). METHODS: Articular cartilage samples were obtained during surgery from 11 patients with OA and at autopsy from 5 adults without arthritis. The articular cartilage samples were cultured in serum-free medium. A collagenase-generated neoepitope, which reflects cleavage of type II collagen, and proteoglycan glycosaminoglycan (GAG), which predominantly reflects aggrecan release, were assayed in culture media. In addition, cultures were performed using either of 2 synthetic MMP inhibitors, both of which inhibited collagenase 2 (MMP-8) and collagenase 3 (MMP-13), but one of which spared collagenase 1. Cultures were also biolabeled with 3H-proline in the presence and absence of these inhibitors to measure collagen synthesis (as tritiated hydroxyproline) and incorporation in articular cartilage. RESULTS: As a group, cleavage of type II collagen by collagenase was significantly increased in OA cartilage samples. In contrast, proteoglycan (GAG) release was not increased. This release of a collagenase-generated epitope was inhibited by both MMP inhibitors in 2 of 5 nonarthritic samples and in 9 of 11 OA cartilage samples. The inhibitor that spared collagenase 1 was generally more effective and inhibited release from 4 of 5 nonarthritic cartilage samples and the same OA cartilage samples. Group analyses revealed that the inhibition of collagenase neoepitope release by both inhibitors was significant in the OA patient cartilage, but not in the nonarthritic cartilage. Proteoglycan loss was unaffected by either inhibitor. Newly synthesized collagen (predominantly, type II) exhibited increased incorporation in OA cartilage, but only in the presence of the inhibitor that arrested collagenase 1 activity. CONCLUSION: These results further indicate that the digestion of type II collagen by collagenase is selectively increased in OA cartilage, and that this can be inhibited in the majority of cases by a synthetic inhibitor that can inhibit collagenases 2 and 3, but not collagenase 1. The results also suggest that in OA, newly synthesized collagen is digested, but in a different manner than that of resident molecules. Proteoglycan release was not increased in OA cartilage and was unaffected by these inhibitors. Inhibitors of this kind may be of value in preventing damage to type II collagen in human arthritic articular cartilage.

Adult↗
PubMed2001

Induction of collagenase mRNA expression in dermal fibroblasts by IFN-alpha 2b and determination of the IFN-alpha 2b responsive element on 5'-flanking regions of collagenase promoter.

We have previously demonstrated that interferon-alpha2b (IFN-alpha2b) markedly depresses the expression of mRNA for type I procollagen in dermal fibroblasts. In the present study, the effect of various concentrations of IFN-alpha2b on the expression of collagenase mRNA and activity of 5'-flanking regions of collagenase promoter in dermal fibroblasts are presented. The results showed at least a 2-fold increase in the expression of collagenase mRNA in fibroblasts grown at either 70% confluency (40.9 +/- 4.6 vs. 18.5 +/- 1.6, n=4, p<0.05) or 95% confluency (24.7 +/- 6.7 vs. 4.5 +/- 1.6, n=4, p<0.05). The effects of IFN-alpha2b on collagenase mRNA stability and promoter activity were evaluated to determine the mechanism by which IFN-alpha2b increases the expression of collagenase mRNA. IFN-alpha2b-treated and untreated fibroblasts were treated with alpha-amanitin to arrest collagenase mRNA transcription, and total RNA was then harvested at 0, 3, 6, 12, and 24 h. The decay curves of collagenase mRNA as a function of time showed a greater rate of degradation for collagenase mRNA in IFN-alpha2b-treated cells relative to untreated control cells. This difference was more pronounced in cells treated with alpha-amanitin at either 12 or 24 h. To determine the regions of the collagenase promoter that might function as IFN-alpha2b responsive elements, eight different fragments of the collagenase promoter, -518, -300, -171, -161, -127, -91, -74, and -66 to +63 nucleotide (nt), were constructed in a chloramphenicol acetyltransferase (CAT) expression vector. The results of CAT activity of cells transfected with these construct identified three constructs, 171/+63, -161/+63, and -127/+63, as being responsive to IFN-alpha2b treatment in dermal fibroblasts. The CAT activity was increased 279%, 163%, and 261% in -171/+63, -161/+63, and -127/+63-transfected fibroblasts, respectively, in response to IFN-alpha2b treatment relative to untreated control. No significant increase in CAT activity was found in cells transfected with the other constructs of the collagenase promoter. A time response experiment showed a marked increase in CAT activity of cells transfected with either 127/+63 or -171/63 constructs within 6-12 hr of IFN-alpha2b treatment. In conclusion, IFN-alpha2b significantly increases the expression of collagenase mRNA in dermal fibroblasts probably through stimulation of the -127/-91 region of the collagenase promoter. Thus, this region may function as an IFN-alpha2b responsive element on collagenase promoter.

5' Untranslated Regions↗
PubMed1988

New Achromobacter collagenase and its immunological relationship with a vertebrate collagenase.

Evidence is presented that Achromobacter iophagus produces two distinct collagenases. Achromobacter collagenases A and B were separated by high-performance liquid chromatography from partially purified enzyme. The main collagenase, A (EC 3.4.24.8), which has been already described, was eluted in the region of molecular mass 110-90 kDa. A minor collagenase B eluted in the region of 320 kDa, although in SDS-gel electrophoresis the apparent molecular masses of its main active forms were estimated as 55 and 110 kDa. The specificities of collagenases A and B are different. Collagenase A splits in its synthetic substrate Pz-Pro-Leu-Gly-Pro-DArg the bond Leu-Gly, collagenase B does not split this substrate. Both collagenases split bonds Gln-Gly and Leu-Gly in synthetic peptides DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-DArg-OH and DNP-Pro-Leu-Gly-Ile-Ala-Gly-DArg-NH2, respectively. Collagenase B is twice as active as A on the native collagen type I. Both enzymes are inhibited by EDTA. The antibodies raised against the human tooth collagenase specifically inhibited the collagenase B, but did not influence the activity of collagenase A. These results indicate, to our knowledge for the first time, an immunological relationship between a bacterial and a vertebrate collagenase.

Amino Acid Sequence↗
PubMed1998

Collagenase-1 and collagenase-3 synthesis in normal and early experimental osteoarthritic canine cartilage: an immunohistochemical study.

OBJECTIVE: The coexistence of different collagenases in cartilage suggests the possibility of specific roles for these enzymes in the degradation of the collagen network in osteoarthritis (OA). We investigated the in situ synthesis and distribution of collagenase- and collegenase-3 in normal and early experimental OA cartilage. METHODS: The OA model was created on 12 mongrel dogs by sectioning the anterior cruciate ligament of the right stifle joint with a stab wound. Dogs were divided into 3 groups of 4 animals each, and sacrificed at 4, 8, and 12 weeks, respectively. A 4th group (n = 4) of unoperated dogs was used as control. Articular cartilage from femoral condyles and tibial plateaus was examined histologically to grade severity of lesions, and immunohistochemical and morphometric analyses were performed to detect the presence of chondrocytes producing collagenase-1 and -3. RESULTS: In OA dogs, the histologic severity of lesions increased with time, being most severe at 8 and 12 weeks after surgery. In cartilage from OA compared to unoperated dogs, the immunoreactivity was 5-9 times higher (p < 0.0002) for collagenase-1, and 3-6 times higher (p < 0.0002) for collagenase-3, in both femoral condyles and tibial plateaus. Although the cell score increased throughout the cartilage, comparison of the superficial and upper intermediate layers (superficial) with the lower intermediate and deep layers (deep) revealed a significantly higher level for collagenase-1 (p < 0.007) in the superficial layers, contrary to the collagenase-3 data, which indicated a higher level (p < 0.007) in the deep layers. For collagenase- , the cell score increased steadily up to the 12th week, and for collagenase-3, the elevation peaked at 8 weeks. Correlation between the histologic severity and cell score in cartilage specimens from unoperated and OA dogs revealed the highest coefficient for collagenase- at the superficial layers (r = 0.69, p < 0.0001), while for collagenase-3, this was noted at the deep layers (r = 0.65, p < 0.0004). CONCLUSION: The number of chondrocytes involved in the synthesis of collagenase-1 and collegenase-3 increases dramatically in the early phase of OA. However, the difference in the topographic distribution of these enzymes, as well as the variation in their correlation pattern, may reflect a different function allocated for each collagenase in the OA cartilage degradation process.

Animals↗
PubMed1984

Heterogeneity among human collagenases demonstrated by monoclonal antibody that selectively recognizes and inhibits human neutrophil collagenase.

The heterogeneity of human collagenases has been examined using a monoclonal antibody to neutrophil collagenase. This antibody inhibited collagenase activity and, when covalently coupled to Sepharose, bound both latent and active enzyme. Although human neutrophil collagenase was inhibited by the antibody, the activity of human skin and rheumatoid synovial collagenase was not significantly diminished in the presence of the antibody. Competitive inhibition studies also differentiated between these collagenases. Only human neutrophil collagenase effectively blocked the antibody in a competitive enzyme-linked immunosorbent assay while skin and rheumatoid synovial collagenase again failed to interact with the antibody. The unequivocal recognition of neutrophil collagenase as an immunologically distinct entity from other collagenases supports the hypothesis that neutrophil collagenase is a separate gene product from fibroblast or synovial collagenase.

Animals↗
PubMed1998

Comparative immunolocalization studies of collagenase 1 and collagenase 3 production in the rheumatoid lesion, and by human chondrocytes and synoviocytes in vitro.

The degradation of fibrillar type II collagen is a major feature of cartilage destruction in rheumatoid arthritis (RA). Since collagenase 3 is produced by chondrocytes and preferentially degrades type II cartilage collagen, it seemed likely that this enzyme would have a prominent role in the destruction of rheumatoid joints. Using immunolocalization techniques, we have examined and compared the production and distributions of collagenase 1 and collagenase 3 in cells and tissues derived from rheumatoid knee arthroplasties. Primary cultures of chondrocytes stimulated with interleukin-1 beta showed that most of the cells produced collagenase 1, whereas only a minority (approximately 5-10%) produced collagenase 3; a few chondrocytes demonstrated the co-ordinate production of both enzymes. Primary cultures of rheumatoid synoviocytes produced collagenase 1, but not collagenase 3. Both enzymes were demonstrated in the rheumatoid lesion. Collagenase 1 was more commonly observed in both synovium and cartilage (22 of the 28 specimens), was especially prominent at cartilage erosion sites, and most of the positive specimens demonstrated extracellular enzyme. By contrast, collagenase 3 was observed less frequently (7/28 specimens) and was produced by relatively few chondrocytes and synovial cells, this usually being much less than that observed for chondrocytes of osteoarthritic cartilage. These observations suggest different regulatory mechanisms for the production of collagenases 1 and 3 in the rheumatoid lesion, and demonstrate that the distribution and production of collagenase 1 are far more prevalent than those for collagenase 3.

Arthritis, Rheumatoid↗
PubMed1985

Human alveolar macrophages produce a fibroblast-like collagenase and collagenase inhibitor.

Human macrophages have been implicated in connective tissue remodeling; however, little is known about their direct effects upon collagen degradation. We now report that human alveolar macrophages in culture produced both a collagenase and a collagenase inhibitor. The collagenase was secreted in latent form and could be activated by exposure to trypsin. Collagenase production could be increased three- to fourfold by incubating the cells with lipopolysaccharide, but synthesis was largely unaffected by exposure to phorbol myristate acetate. By several criteria, macrophage collagenase was the same as the collagenase secreted by human skin fibroblasts: (a) they were antigenically indistinguishable in double immunodiffusion; (b) both degraded type III collagen preferentially to type I, had little activity against type II collagen, and none against types IV and V, and (c) their affinity for susceptible collagens was equivalent, Michaelis constant = 1-2 microM. Collagenase inhibitory activity was also present in the macrophage-conditioned medium, and was accounted for by an antigen that showed immunologic and functional identity with the collagenase inhibitor secreted by human skin fibroblasts. The amount of inhibitor released by unstimulated cells, approximately 100 ng/10(6) cells per 24 h, was substantially augmented by both phorbol and lipopolysaccharide, although considerable variability in response to these agents was observed between macrophage populations derived from different subjects. As negligible quantities of collagenase or collagenase inhibitor were detectable intracellularly, it appeared that both proteins were secreted rapidly after synthesis. Thus, human macrophages have the capacity to modulate collagen degradation directly by production of collagenase and collagenase inhibitor.

Cells, Cultured↗
PubMed1991

Collagenase production at the border of granulation tissue in a healing wound: macrophage and mesenchymal collagenase production in vivo.

We demonstrated the cells producing collagenase and the time course of collagenase-production at early stages of wound healing, using histology and two immunohistochemical procedures on cross sections of rat skin harvested 0, 3, 5, 7 and 12 days after full-thickness incisions. A monospecific rabbit polyclonal antibody to neutral collagenase purified from rat myometrial cells was used to demonstrate collagenase production. Specificity of this reaction was confirmed by blocking the reaction with excess homogeneously purified antigen. Macrophages were simultaneously labelled using a mouse anti-rat monoclonal antibody recognizing exclusively mature macrophages. Intracellular collagenase was not reliably detectable at day 0, but was prominent at days 3 and 5 and thereafter declined. Double labeling technique showed occasional macrophages producing collagenase in the developing granulation tissue, but most cells labeled as macrophages were negative for collagenase. Most activity was found in fibroblasts adjacent to granulation tissue elements. Since the granulation tissue parallels revascularization in a dendritic pattern, a cross section at three days typically shows an annulus of collagenase-positive cells surrounding a branch of the active granulation tissue. At days 5, 7 and 12 after wounding the pattern of collagenase expression became indistinct as more tissue was involved in the granulation process. However, double-labelling for macrophages and collagenase showed the dichotomy between collagenase expression and presence of macrophages to persist. The finding that collagenase is produced in connective tissue adjacent to granulation tissue suggests an inductive process, possibly due to diffusion of cytokines produced by granulation tissue elements.

Animals↗
PubMed1990

[New direct assay method of type IV collagenase in tissue homogenate and biochemical role of collagenase against type I, and IV collagens to the invasion of the stomach and lung cancer].

In order to study the role of both collagenases against type I and type IV collagen (type I and type IV collagenase) with regard to tumor invasion and metastasis, the activities of both collagenases in tissue homogenate in each 40 cases of stomach and lung cancers were investigated. The direct assay method of type IV collagenase in tissue was established through modification of Liotta's method. In stomach cancer, the part of advancing front of cancer showed the highest activity of type I collagenase. The adjacent mucosa to cancer also showed high activity of type IV collagenase. The cancer tissues that had the remarkable finding of vascular invasion of cancer cells showed high activity of type IV collagenase. In lung cancer, the correlation between the size of cancer mass and activity of type I collagenase was shown. Squamous cell carcinoma in comparison to adenocarcinoma had higher activity of type I collagenase and poor activity of type IV collagenase. These results suggested that the activity of type I collagenase might participate in local invasion and the activity of type IV collagenase might be associated with vascular invasion of cancer through disruption of basement membrane and they could be one of the useful biochemical tumor marker to represent the growth and metastatic pattern.

Adenocarcinoma↗
PubMed1990

Independent expression and cellular processing of Mr 72,000 type IV collagenase and interstitial collagenase in human tumorigenic cell lines.

The regulation of Mr 72,000 type IV collagenase and interstitial collagenase expression was studied in vitro. Three tumorigenic human cell lines were used, together with human fetal lung fibroblasts as a nontumorigenic control. Mr 72,000 type IV collagenase was expressed constitutively by all four cell lines, whereas only A2058 melanoma cells exhibited constitutive expression of interstitial collagenase. Treatment of cells with transforming growth factor beta 1 (TGF-beta 1) and 12-O-tetradecanoylphorbol-13-acetate (TPA) revealed an opposite pattern of regulation of these two metalloproteinases. Specifically, TPA increased interstitial collagenase mRNA levels in each cell line and decreased type IV collagenase mRNA levels in control fibroblasts and the tumorigenic cell lines, HT-1080 and A2058. TGF-beta 1 treatment increased type IV collagenase mRNA levels in each cell line and decreased interstitial collagenase mRNA levels in A2058 melanoma cells. Interstitial collagenase mRNA induction was accompanied in all cell lines by elevated interstitial procollagenase in the conditioned medium, as detected by zymography. Changes in Mr 72,000 type IV collagenase expression revealed a more complex pattern of regulation. TPA and TGF-beta 1 treatment of HT-1080 cells resulted in the appearance of two bands of gelatinolytic activity with a molecular weight of approximately 62,000 and 59,000. The Mr 62,000 species was also induced by TGF-beta 1 treatment of A2058 cells. Addition of affinity-purified radiolabeled Mr 72,000 type IV procollagenase to TPA-treated HT-1080 cells demonstrated that both species were products of the Mr 72,000 proenzyme and that exogenous proenzyme could be processed by these cells. Western blot analysis with specific antipeptide antibodies revealed that both the Mr 62,000 and 59,000 species were derived from the Mr 72,000 proenzyme by amino-terminal cleavage. There was no evidence for cellular processing of either interstitial procollagenase or the Mr 92,000 type IV procollagenase. These results demonstrate that the Mr 72,000 type IV collagenase is under the control of different regulatory elements from interstitial collagenase, at the level of both mRNA expression and cellular processing, and that this processing appears to be the result of a phorbol ester and TGF-beta 1-inducible cellular activation mechanism. The ratio of active enzyme species to latent Mr 72,000 proenzyme may provide a better correlation with invasive potential than overall levels of this widely expressed metalloproteinase.

Blotting, Western↗
PubMedDate not supplied

The levels of collagenase, tissue inhibitor of metalloproteinases-1 (TIMP-1), collagenase approximately TIMP-1 complexes and glycosaminoglycan (GAG) in sequential samples of synovial fluid aspirated from patients with osteoarthritis.

OBJECTIVE: Collagen turnover in connective tissues is thought to be controlled by the balance between the levels of interstitial collagenase and tissue inhibitor of metalloproteinases (TIMP-1). The aim of this study was to measure the level of total collagenase (MMP-1), TIMP-1, collagenase approximately TIMP-1 complex and glycosaminoglycan (GAG) in sequential samples of osteoarthritic knee synovial fluid from well documented patients to determine if these parameters changed with time and correlated with clinical indices. METHODS: Twenty-one patients were recruited and randomly allocated to receive tiaprofenic acid, indomethacin or naproxen. Total collagenase, TIMP-1, collagenase approximately TIMP-1 complex and GAG were measured in 80 osteoarthritic synovial fluids taken over a period of six months. RESULTS: The majority of fluids contained a molar excess of TIMP-1 over collagenase, although in seven fluids collagenase was present in excess; six of these samples were from a single patient. GAG levels were relatively unchanged over the six months studied. CONCLUSION: The levels of collagenase and TIMP-1 varied between patients and over time in individual patients. No collagenase approximately TIMP-1 complex was found in any fluid. There was no significant difference in the median levels of collagenase, TIMP-1 or GAG in the different treatment groups. High levels of collagenase were found in one patient with a crystal related disease. These immunoassays give valuable information on the levels of collagenase and TIMP-1 in individual patients with time and may help to determine the mechanisms controlling the turnover of cartilage collagen in different arthritides.

Adult↗
PubMed1984

Effect of dexamethasone, indomethacin, and lipopolysaccharide on the secretion of interstitial collagenase and type V collagenase by cultured macrophages.

The rabbit alveolar macrophage secretes at least two collagenolytic metalloproteinases in vitro including an interstitial collagenase and a type V collagenase. Using assays previously shown to discriminate between these two activities, the secretion of these two enzyme activities was investigated. Both enzyme activities accumulated in culture over 11 days and the release of both were similarly inhibited by cycloheximide. Collagenolytic activity was negligible in cell lysates. The interstitial collagenase was found in a latent form but the type V collagenase activity was active in the culture medium. When cultured in the presence of dexamethasone, the secretion of both the enzymes were identically inhibited in a dose-dependent manner. Indomethacin was an effective inhibitor of secretion of both collagenases at a concentration of 10(-5) M but not at lower concentrations. Finally, bacterial lipopolysaccharide stimulated the secretion of both type V and interstitial collagenase by these cells. These studies indicate that, like the interstitial collagenase, the type V collagenase is released from the cell as synthesized and is not stored intracellularly. Protein synthesis is necessary for the release of both these collagenases. Furthermore, the release of type V collagenase responded to dexamethasone, indomethacin, and lipopolysaccharide in a manner identical to the secretion of the interstitial collagenase suggesting that synthesis and secretion of these two enzymes are regulated in a similar manner.

Animals↗
PubMed1996

The new collagenase, collagenase-3, is expressed and synthesized by human chondrocytes but not by synoviocytes. A role in osteoarthritis.

Recently, a new human collagenase, collagenase-3 has been identified. Since collagen changes are of particular importance in cartilage degeneration, we investigated if collagenase-3 plays a role in osteoarthritis (OA). Reverse transcriptase-PCR analysis revealed that in articular tissues collagenase-3 was expressed by the chondrocytes but not by the synoviocytes. Northern blot analysis of the chondrocyte mRNA revealed the presence of two major gene transcripts of 3.0 and 2.5 kb, and a third one of 2.2 kb was occasionally present. Compared to normal, OA showed a significantly higher (3.0 kb, P < or = 0.05; 2.5 kb, P < or = 0.03) level of collagenase-3 mRNA expression. Collagenase-3 had a higher catalytic velocity tate (about fivefold) than collagenase-1 on type II collagen. With the use of two specific antibodies, we showed that human chondrocytes had the ability to produce collagenase-3 as a proenzyme and as a glycosylated doublet. The chondrocyte collagenase-3 protein is produced in a significantly higher (P < or = 0.04) level in OA (approximately 9.5-fold) than in normal. The synthesis and expression of this new collagenase could also be modulated by two proinflammatory cytokines, IL-1 beta and TNF-alpha, in a time- and dose-dependent manner. This study provides novel and interesting data on collagenase-3 expression and synthesis in human cartilage cells and suggest its involvement in human OA cartilage patho-physiology.

Aged↗
PubMed1993

The measurement of collagenase, tissue inhibitor of metalloproteinases (TIMP), and collagenase-TIMP complex in synovial fluids from patients with osteoarthritis and rheumatoid arthritis.

OBJECTIVE: To compare the levels of collagenase, tissue inhibitor of metalloproteinases (TIMP), and collagenase-TIMP complex in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA). This study aims to clarify existing data from previously used enzyme or inhibitor activity assays performed following separation by gel filtration, by using a 1-step enzyme-linked immunosorbent assay (ELISA) for each component. METHODS: Total collagenase, free TIMP, and collagenase-TIMP complex were measured using a newly developed, specific double-antibody sandwich ELISA: RESULTS: Levels of both collagenase and TIMP were significantly higher in RA patients (collagenase 1,560 +/- 150 ng/ml [mean +/- SEM], TIMP 1,610 +/- 130 ng/ml; n = 80) than OA patients (collagenase 420 +/- 90 ng/ml, TIMP 1,050 +/- 60 ng/ml; n = 80), with the difference being especially striking for collagenase. Sixteen RA fluids had detectable levels of collagenase-TIMP complex, compared with only 3 OA fluids. CONCLUSION: The level of total collagenase in SF is greater in RA than OA, while levels of free TIMP show more overlap between the 2 diseases; this may simply reflect the increased inflammation seen in RA, or it may reflect a different disease mechanism.

Arthritis, Rheumatoid↗
PubMed1994

Adsorption of collagenase to particulate titanium: a possible mechanism for collagenase localization in periprosthetic tissue.

Osteolysis is a central feature of aseptic loosening of orthopaedic joint prostheses. This destructive process is believed to result from phagocytosis of implant wear debris by periprosthetic and synovial macrophages and the subsequent release of proinflammatory mediators, including collagenase. Isolated murine macrophages were cultured in vitro with particulate titanium in order to explore the mechanism of macrophage activation by particulate wear debris. The results, in which the amount of secreted, soluble collagenase in culture supernatants was inversely proportional to titanium concentration, suggested that titanium strongly adsorbed secreted collagenase. This inference was confirmed by direct binding assays in which particulate titanium coated with adsorbed collagenase bound an alkaline phosphatase conjugated anti-collagenase antibody, but not a conjugated anti-IgG antibody. Adsorption of collagenase was not influenced by preincubation of titanium particles with albumin. The adsorbed collagenase remained enzymatically active as indicated by its ability to hydrolyze a synthetic peptide substrate. These results demonstrate that particulate titanium stimulates collagenase production by macrophages and then strongly adsorbs the secreted proinflammatory enzyme. The process of macrophage stimulation, collagenase secretion, and adsorption may represent an important mechanism for localization and concentration of collagenase in periprosthetic and synovial tissue, a mechanism that ultimately triggers bone resorption through osteoclast activation.

Adsorption↗