Metalloproteinase and tissue inhibitor of metalloproteinase expression in the murine STR/ort model of osteoarthritis.

OBJECTIVE: To study the temporal expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the STR/ort mouse model of osteoarthritis, using in situ hybridization with oligonucleotide probes and specific antisera for each protein. METHODS: In situ hybridization and immunolocalization experiments were performed on serial cryosections of knee joints from STR/ort and control CBA mice. The mRNA was localized using digoxygenin-labeled probes. RESULTS: MMP2, MMP3, MMP7, MMP9, MMP13, MT1-MMP and TIMP2 mRNA was detected in the tibial articular chondrocytes of STR/ort mice at all ages (12, 18, 24, 30 and 35 weeks). Levels were always higher than in age-matched CBA mice. Neither MMP8 nor TIMP1 mRNA was detected in murine cartilage. The location and distribution of each of the MMP mRNA transcripts varied within the tibial plateau. Immunolocalization consistently detected MMP3 and MT1-MMP in articular cartilage and MMP13 in calcified cartilage. Other proteases and their inhibitors were not detected in either of these cartilages but MMP2 and MMP9 were immunolocalized in bone marrow cells and growth cartilage respectively. CONCLUSION: Expression of all the detected MMPs and TIMP-2 is up-regulated in STR/ort mice at the mRNA level. However, failure to detect protein expression for MMPs 2, 7, 9, 13 and TIMPs 1 and 2 in murine chondrocytes by immunohistochemistry indicates that the changes in mRNA levels in STR/ort mice must be interpreted with caution.

Regulation of matrix metalloproteinases in a model of colonic wound healing in a rabbit.

PURPOSE: Matrix metalloproteinases occur in the colon at an anastomosis but not in the normal colon. Matrix metalloproteinase synthesis can be regulated by cytokines, for example interleukin-1 beta and growth factors, such as transforming growth factor beta and basic fibroblast growth factor. The aim of this study was to investigate the regulation of matrix metalloproteinases at an anastomosis by identifying the cell types that synthesize matrix metalloproteinases, examining factors that might regulate their synthesis, determining whether they occur in an active form, and assessing the effect of suture type on these parameters. METHODS: An anastomosis was formed in the distal colon of rabbits using either polyglactin or polydioxanone and the animals were killed six hours or seven days later. The distribution of matrix metalloproteinases and cytokines and the cell types were assessed by immunohistochemistry. Matrix metalloproteinase-2, matrix metalloproteinase-3, and matrix metalloproteinase-9 were detected also by zymography. RESULTS: Immunohistochemistry showed that matrix metalloproteinases were restricted to the suture line. Although zymography demonstrated that matrix metalloproteinase-2 was present mainly in an active form, matrix metalloproteinase-9 and matrix metalloproteinase-3 were present in the pro-form. The active form of matrix metalloproteinase-3 occurred more often in the polydioxanone-sutured rabbits. With the exception of matrix metalloproteinase-9, the matrix metalloproteinases were synthesized by fibroblasts. Interleukin-1 beta and transforming growth factor beta were more widespread than in the normal colon and were localized adjacent to the matrix metalloproteinases. Basic fibroblast growth factor was also more widespread postoperatively but occurred deeper in the anastomosis than the matrix metalloproteinases. CONCLUSIONS: This study has shown that interleukin-1 beta and transforming growth factor beta may regulate the synthesis of the matrix metalloproteinases by fibroblasts and that minor differences that occur in the matrix metalloproteinase profile are dependent on the suture type.

Immunolocalization of matrix metalloproteinases in partial-thickness defects in pig articular cartilage. A preliminary report.

BACKGROUND: Partial-thickness defects in mature articular cartilage do not heal spontaneously. Attempts at repair often result in limited integration between the repair tissue and the surrounding cartilage, with formation of chondrocyte clusters adjacent to a zone of cartilage necrosis. In wound repair, spatially and temporally controlled expression of matrix metalloproteinases and their inhibitors have been implicated in proteolytic degradation of damaged extracellular matrix components, but the sequence of events following damage to cartilage is unknown. To determine this sequence, we studied the distribution of matrix metalloproteinases and their inhibitors during early in vivo repair of partial-thickness defects in pig articular cartilage. METHODS: With use of a model that elicits the ingrowth of mesenchymal cells into partial-thickness defects, partial-thickness defects were created in knee joint cartilage. The distributions of matrix metalloproteinase-1, 2, 3, 9, 13, and 14; tissue inhibitors of metalloproteinase-1 and 2; and the neoepitope DIPEN341 specifically generated following matrix metalloproteinase cleavage of aggrecan were determined by immunolocalization of repair tissue and surrounding cartilage excised from immature pigs during the first eight weeks of repair and from adult minipigs at eight days and three weeks. RESULTS: Synthesis of matrix metalloproteinase-13 was usually confined to hypertrophic chondrocytes in immature cartilage and to the radial zone in adult cartilage. Following injury, strong induction of matrix metalloproteinase-13 synthesis was observed in chondrocyte clusters surrounding lesions in all of the animals. The migration of macrophages into defects was prominent at two and eight days, with synthesis and deposition of matrix metalloproteinase-9 onto damaged cartilage matrix and newly synthesized matrix in the defect. The DIPEN341 neoepitope was localized to damaged cartilage matrix at eight days and six weeks, indicating partial degradation of aggrecan. Focal synthesis of matrix metalloproteinase-1, 3, and 14 and of tissue inhibitor of metalloproteinase-1 occurred at later times, suggesting continuous remodeling of the increasingly compact repair tissue. CONCLUSIONS: The expression of matrix metalloproteinase-13 by normal hypertrophic chondrocytes and the induction of synthesis in chondrocyte clusters adjacent to the zone of cartilage necrosis suggest that this enzyme participates in the pericellular proteolysis required for lacunar expansion. The localization of matrix metalloproteinase-9 to damaged cartilage matrix suggested matrix proteolysis, which was confirmed with DIPEN341 localization. Reduced matrix metachromasia persisted and was colocalized with DIPEN341 at six weeks. However, under the conditions investigated, there was only limited proteolytic degradation in the zone of cartilage necrosis. This may render the zone mechanically weakened, thereby contributing to subsequent instability of the region, and may form a barrier to integration of repair tissue with viable cartilage. CLINICAL RELEVANCE: Osteoarthritis initially involves the superficial layers of cartilage. The development of procedures to promote the healing or repair of early defects will have major advantages in terms of disease alleviation as well as economic importance. Identification of the enzymes involved in the early repair of partial-thickness defects in articular cartilage is clinically relevant because proteolysis of damaged matrix has to take place in order for repair tissue to integrate with surrounding healthy cartilage.

Stromelysin-2 is upregulated during normal wound repair and is induced by cytokines.

Stromelysin-2 is a matrix metalloproteinase that degrades in vitro several protein components relevant to wound repair such as collagens III and IV, gelatin, nidogen, laminin-1, proteoglycans, and elastin. Furthermore, it can activate other matrix metalloproteinases, such as collagenase-1 (matrix metalloproteinase-1) and collagenase-2 (matrix metalloproteinase-8), as well as 92 kDa gelatinase. The aim of this study was to determine in a large variety of wounds (normally healing dermal and mucosal wounds, suction blisters, ex vivo cultures, diabetic, decubitus, rheumatic, and venous ulcers) and keratinocyte cultures, which factors contribute to stromelysin-2 expression and how it is induced in relation to other matrix metalloproteinases. Our results show that stromelysin-2 mRNA and protein are upregulated later (at 3 d) than matrix metalloproteinase-1 in normally healing wounds and ex vivo explants, in which stromelysin-2 is invariably expressed by keratinocytes migrating over dermal matrix. The number of keratinocytes expressing stromelysin-2 was greatest in chronic inflamed diabetic and venous ulcers compared with rheumatoid and decubitus ulcers, six of which had no signal. In keratinocyte cultures, tumor necrosis factor-alpha, epidermal growth factor, and transforming growth factor-beta1 induced stromelysin-2 expression as measured by quantitative reverse transcriptase-polymerase chain reaction, whereas different matrices did not upregulate the mRNA. Immunostaining demonstrated stromal transforming growth factor-beta1 in contact with the stromelysin-2-positive keratinocytes. Our results suggest that stromelysin-2 expression is important for the normal repair process and is upregulated by cytokines rather than cell-matrix interactions. Stromelysin-2 is most likely to participate in the remodeling of the newly formed basement membrane, and is not overexpressed in retarded wound healing.

Generation and novel distribution of matrix metalloproteinase-derived aggrecan fragments in porcine cartilage explants.

We have studied aggrecan catabolism mediated by matrix metalloproteinases (MMPs) in a porcine cartilage culture system. Using antibodies specific for DIPEN(341) and (342)FFGVG neoepitopes, we have detected MMP-derived fragments in conditioned medium and cultured cartilage, by radioimmunoassay, Western blotting, and immunolocalization. Radioimmunoassay revealed that the amount (pmol of epitope/mg of total glycosaminoglycan) of (342)FFGVG epitope released from cartilage remained constant over a 5-day culture period and was not increased by IL-1alpha or retinoate. However, the proportion (pmol of epitope/mg of released glycosaminoglycan) of (342)FFGVG epitope released was decreased upon stimulation, consistent with the involvement of a non-MMP proteinase, such as aggrecanase. The data suggest that in vitro MMPs may be involved in the base-line catabolism of aggrecan. Immunolocalization experiments showed that DIPEN(341) and ITEGE(373) epitopes were increased by treatment with IL-1alpha and retinoate. Confocal microscopy revealed that ITEGE(373) epitope was largely intracellular but with matrix staining in the superficial zone, whereas DIPEN(341) epitope was cell-associated and widely distributed in the matrix. Surprisingly, the majority of (342)FFGVG epitope, determined by radioimmunoassay and Western blotting, was retained in the tissue despite the absence of a G1 domain anchor. Interleukin-1alpha stimulation caused a marked increase in tissue DIPEN(341) and (342)FFGVG epitope, and the (342)FFGVG fragments retained in the tissue were larger than those released into the medium. Active porcine aggrecanase was unable to cleave (342)FFGVG fragments at the downward arrowGlu(373) downward arrowAla(374) bond but cleaved intact aggrecan at this site, suggesting that (342)FFGVG fragments are not substrates for aggrecanase. The apparent retention of large (342)FFGVG fragments within cartilage, and their resistance to N-terminal cleavage by aggrecanase suggests that (342)FF6V6 fragments may have a role in cartilage homeostasis.

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.

Stromelysin (MMP-3) synthesis is up-regulated in estrogen-deficient mouse osteoblasts in vivo and in vitro.

Sex steroids are important regulators of bone cell function and osteoblast-derived matrix metalloproteinases (MMPs) are key mediators of bone resorption during the initial stage of osteoid removal prior to osteoclast attachment. To investigate the mechanism of bone loss following estrogen deficiency, we examined the effects of estrogen on osteoblast synthesis of MMPs and tissue inhibitor of metalloproteinases (TIMPs). Immunolocalization in mouse bone samples ex vivo and primary mouse osteoblast (MOB) cultures was used to document the synthesis of mouse interstitial collagenase (MMP-13), stromelysin-1 (MMP-3), gelatinase-A (MMP-2), and gelatinase-B (MMP-9). Endosteal bone lining cells from distal femoral head and lumbar vertebral body showed an increase in the pattern of synthesis of stromelysin-1 following ovariectomy, compared with sham-operated controls; the synthesis of other MMPs was unaffected. The expression of all classes of MMPs and TIMP-1 and TIMP-2 by MOB in culture was demonstrated by reverse transcriptase-polymerase chain reaction. Following the withdrawal of 17beta-estradiol, MOB cultures showed a significant increase in the number of cells synthesizing stromelysin-1; this effect was enhanced by stimulation with either interleukin-1 or interleukin-6. Northern blot analysis showed only a slight increase in stromelysin-1 mRNA message following the withdrawal of 17beta-estradiol. Our data show an unexpected up-regulation of stromelysin-1 synthesis by osteoblasts both in vivo and in vitro following estrogen withdrawal. Although this effect was not reflected in a significant change in stromelysin-1 mRNA expression in vitro, there is evidence to suggest a role for this enzyme in the early stages of bone loss during the pathogenesis of osteoporosis.

Matrix metalloproteinase-2 activation in human hepatic fibrosis regulation by cell-matrix interactions.

Matrix metalloproteinase-2 (MMP-2) is involved in extracellular matrix remodeling. It is secreted as a proenzyme and activated by membrane type-MMPs (MT-MMP), such as MT1-MMP. In liver fibrosis, MMP-2 is highly expressed in myofibroblasts and may have a profibrogenic role. The mechanisms of its activation in the liver are still unclear. The aim of this work was to show that pro-MMP-2 is efficiently activated in human fibrotic liver and to investigate the role of cell-matrix interactions in this process. Liver specimens obtained from patients with active cirrhosis were compared to normal liver specimens. Human hepatic myofibroblasts were cultured either on plastic, fibronectin, laminin, or on collagen I gels. MMP-2 activity was visualized by gelatin zymography. MMP-2 active form (59 kd) was detected in active cirrhosis but not in normal liver. Myofibroblasts cultured on plastic, fibronectin, or laminin predominantly expressed inactive pro-MMP-2 (66 kd). In contrast, myofibroblasts cultured on collagen I markedly activated the enzyme. Similar results were obtained using membrane fractions from cells previously cultured on collagen or plastic. Activation was inhibited by the tissue inhibitor of metalloproteinases-2 but not by tissue inhibitor of metalloproteinases-1, implicating a MT-MMP-mediated process. Culture on collagen I up-regulated MT1-MMP protein detected by Western blotting, but decreased MT1-MMP mRNA. This study shows that MMP-2 is activated in fibrotic liver. It suggests that interactions between collagen I and myofibroblasts promote this process through a post-translational increase of MT1-MMP expression in these cells.

Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) distribution in normal and pathological human bone.

Degradation of skeletal connective tissue is regulated, at least in part, by the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinase (TIMPs), their natural inhibitors. The balance between MMPs and TIMPs may therefore be a determinant of normal bone turnover, and imbalance could thus lead to reduced organization of bone structure. To test this hypothesis, the cellular expression of MMPs and TIMP-1 was investigated by immunohistochemistry in human neonatal rib and osteophytic and heterotopic bone; these differ in their structure, with heterotopic bone showing the least and normal rib the most organized development. In all samples, high levels of MMPs were expressed. Collagenase and stromelysin-2 were detected in chondrocytes, osteoblasts, and osteoclasts, whereas gelatinase-B was confined to osteoclasts and mononuclear cells. Matrix-associated stromelysin-1 was present in fibrous tissue and osteoid. In contrast, the expression of TIMP-1 varied markedly between the three types of bone. In heterotopic bone only occasional low level TIMP-1 expression was detected in chondrocytes and osteoblasts. Osteophytic bone showed varying levels of TIMP-1, which was matrix-bound in fibrous tissue and cell-associated in osteoblasts, chondrocytes, and occasional mononuclear cells. In both types of bone, expression of TIMP-1 by osteoclasts was absent despite large numbers of these cells. Neonatal rib bone showed consistent expression of TIMP-1, particularly in chondrocytes, osteoblasts, and lining cells. In contrast to pathological bone, many osteoclasts were TIMP-1 positive. These results suggest that, in heterotopic and osteophytic bone, the low levels of TIMP-1, and in particular its absence in osteoclasts, may partly explain the more poorly organized bone formation in these pathological bone samples. Furthermore, TIMP-1 may play a role in the regulation of bone modeling and remodeling in normal developing human bone.

Stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10) expression in developing human bone: potential roles in skeletal development.

Stromelysin, a member of the matrix metalloproteinase family, demonstrates wide substrate specificity with the ability to degrade proteoglycan, fibronectin, laminin, casein, and the nonhelical region of collagen. The two forms of stromelysin (SL), types 1 (MMP-3) and 2 (MMP-10), share 82% sequence homology, but exhibit differences in cellular synthesis and inducibility by cytokines and growth factors in vitro. However, the distribution of the two isoforms in bone has not been reported. We investigated the presence of SL-1 and SL-2 in human osteophytic and neonatal rib bone using immunohistochemistry and, combined with a new method of in situ zymography, determined the activity of the immunolocalized stromelysins. Latent SL-1 was strongly expressed in the extracellular matrix in fibrous tissue surrounding areas of endochondral ossification in osteophytes, and adjacent to the periosteum of fetal rib bone. Active SL-1 expression was detected in osteocytes and the matrix surrounding osteocytic lacunae. SL-2 showed intense cell-associated staining at sites of resorption in areas of endochondral ossification and in resorptive cells at the chondro-osseous junction, which correlated with enzyme activity detected by zymography. Within the rib, active SL-2 expression was localized in chondrocytes of the growth plate, whereas only occasional SL-1 signal was evident. Vascular areas showed strong SL-2 staining with some proteolytic activity. SL-2, but not SL-1, was strongly expressed in osteoclasts and most mononuclear cells within the marrow. At sites of bone formation both isoforms were expressed by osteoblasts with SL-1 also present in osteoid. These results demonstrate, for the first time, the differential expression of SL-1 and SL-2 in developing human bone, indicating specific roles for the two isoforms. In situ zymography demonstrates that SL-2 is produced in an active form with associated degradation, whereas SL-1, in a matrix-bound proenzyme form, may act as a reservoir for later activation.

Induction of matrix metalloproteinase activation cascades based on membrane-type 1 matrix metalloproteinase: associated activation of gelatinase A, gelatinase B and collagenase 3.

SW1353 chondrosarcoma cells cultured in the presence of interleukin-1, concanavalin A or PMA secreted procollagenase 3 (matrix metalloproteinase-13). The enzyme was detected in the culture medium by Western blotting using a specific polyclonal antibody raised against recombinant human procollagenase 3. Oncostatin M enhanced the interleukin-1-induced production of procollagenase 3, whereas interleukin-4 decreased procollagenase 3 synthesis. The enzyme was latent except when the cells had been treated with concanavalin A, when a processed form of 48 kDa, which corresponds to the active form, was found in the culture medium and collagenolytic activity was detected by degradation of 14C-labelled type I collagen. The concanavalin A-induced activation of procollagenase 3 coincided with the processing of progelatinase A (matrix metalloproteinase-2) by the cells, as measured by gelatin zymography. In addition, progelatinase B (matrix metalloproteinase-9) was activated when gelatinase A and collagenase 3 were in their active forms. Concanavalin A treatment of SW1353 cells increased the amount of membrane-type-1 matrix metalloproteinase protein in the cell membranes, suggesting that this membrane-bound enzyme participates in an activation cascade involving collagenase 3 and the gelatinases. This cascade was effectively inhibited by tissue inhibitors of metalloproteinases-2 and -3. Tissue inhibitor of metalloproteinases-1, which is a much weaker inhibitor of membrane-type 1 matrix metalloproteinase than tissue inhibitors of metalloproteinases-2 and -3 [Will, Atkinson, Butler, Smith and Murphy (1996) J. Biol. Chem. 271, 17119-17123], was a weaker inhibitor of the activation cascade.

Effect of colonic obstruction on the distribution of matrix metalloproteinases during anastomotic healing.

BACKGROUND: Anastomotic dehiscence is common after surgery for colonic obstruction. The strength of an anastomosis is dependent on collagen, which is degraded by matrix metalloproteinases (MMPs). The aim of this study was to determine the distribution of the MMPs and their inhibitor, tissue inhibitor of metalloproteinases (TIMP) 1 in an experimental model of colonic obstruction, with and without resection and anastomosis. METHODS: The distal colon of rabbits was obstructed with a Silastic ring for 24 h and then either the ring was removed or the obstructed segment was resected and an anastomosis formed. Rabbits were killed immediately or at intervals for up to 7 days after operation. The distribution of the MMPs and TIMP-1 was examined by indirect immunofluorescence. RESULTS: MMPs and TIMP-1 were present throughout the descending colon for 24 h in both groups. They persisted to the third day in rabbits with an anastomosis but by day 7 were restricted to the suture line. Their presence correlated with microscopic damage. CONCLUSION: The extensive distribution of the MMPs suggests that these enzymes contribute to anastomotic dehiscence, but only in the immediate postoperative period.

MT1-MMP on the cell surface causes focal degradation of gelatin films.

The membrane-type matrix metalloproteinases (MT-MMPs) are a subclass of the matrix metalloproteinase (MMP) family which uniquely possess a C-terminal transmembrane domain and are initiators of an activation cascade for progelatinase A (MMP-2). Recent studies have shown that they can also efficiently directly degrade a number of matrix macromolecules. We now show that cells expressing MT1-MMP on their cell surfaces cause subjacent proteolysis of a gelatin film and that this proteolysis is inhibited by TIMP-2 but not by TIMP-1. These data indicate that expression of MT1-MMP on the cell surface may lead to both progelatinase A activation and extracellular matrix degradation.

Role of matrix metalloproteinases in healing of colonic anastomosis.

PURPOSE: The aim of this study was to compare the distribution of the matrix metalloproteinases (MMPs) during anastomotic healing in a normal colon with that in an ischemic colon in a rabbit. This family of enzymes degrades all components of connective tissue and has been implicated as a cause of anastomotic dehiscence. METHODS: A left-sided anastomosis was formed in the distal colon of one group of rabbits, and in the other group, 9 cm of distal colon was made ischemic before resection and anastomosis 12 hours later. Tissues from the anastomosis and sites around the colon were removed at 12 hours, 1 day, and 3 days after anastomosis and, also, at 7 days in the normal group. Distribution of the MMPs and their inhibitor, tissue inhibitor of metalloproteinases (TIMP), was localized by indirect immunofluorescence. RESULTS: In rabbits having only an anastomosis, the MMPs and TIMP-1 were, at all times, seen solely in the anastomotic segment and were strictly confined to the immediate vicinity of the suture line. While in rabbits with an ischemic colon before anastomosis, the MMPs initially extended several centimeters proximally and distally from the suture line. By the third day, however, there were only minor differences between the two models. CONCLUSION: Distribution of the MMPs and TIMP-1 in normal healing is consistent with a role in the remodeling of colonic anastomosis, but when healing of the colon is compromised, these enzymes are more widespread and may contribute to anastomotic dehiscence.

Distribution of matrix metalloproteinases and their inhibitor, TIMP-1, in developing human osteophytic bone.

Connective tissues synthesise and secrete a family of matrix metalloproteinases (MMPs) which are capable of degrading most components of the extracellular matrix. Animal studies suggest that the MMPs play a role in bone turnover. Using specific polyclonal antisera, immunohistochemistry was used to determine the patterns of synthesis and distribution of collagenase (MMP-1), stromelysin (MMP-3), gelatinase A (MMP-2) and gelatinase B (MMP-9) and of the tissue inhibitor of metalloproteinases-1 (TIMP-1) within developing human osteophytic bone. The different MMPs and TIMP showed distinct patterns of localisation. Collagenase expression was seen at sites of vascular invasion, in osteoblasts synthesising new matrix and in some osteoclasts at sites of resorption. Chondrocytes demonstrated variable levels of collagenase and stromelysin expression throughout the proliferative and hypertrophic regions, stromelysin showing both cell-associated and strong matrix staining. Intense gelatinase B expression was observed at sites of bone resorption in osteoclasts and mononuclear cells. Gelatinase A was only weakly expressed in the fibrocartilage adjacent to areas of endochondral ossification. There was widespread but variable expression of TIMP-1 throughout the fibrous tissue, cartilage and bone. These results indicate that MMPs play a role in the development of human bone from cartilage and fibrous tissue and are likely to have multiple functions.

Development of recombinant adenoviruses that drive high level expression of the human metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 and -2 genes: characterization of their infection into rabbit smooth muscle cells and human MCF-7 adenocarcinoma cells.

Remodelling of the extracellular matrix resulting from increased secretion of metalloproteinase enzymes (MMPs) is implicated in many pathological conditions, including rheumatoid arthritis, restenosis following balloon angioplasty, atherosclerosis and cancer cell invasion and metastasis. Clear definition of the normal and pathological function of individual MMPs will benefit from approaches that use gene transfer to produce increases in MMP levels that mimic those observed in pathological conditions. Similarly, gene transfer methods leading to controlled increases in levels of the tissue inhibitor of metalloproteinases (TIMPs) will help to define the function of MMPs both in vitro and in vivo. Gene transfer of TIMPs may also have therapeutic potential in pathological conditions where inhibition of MMP activity may be beneficial. We have used the adenovirus serotype 5 vector system to generate replication-deficient recombinant adenoviruses capable of expressing the MMP-9, TIMP-1 or -2 genes. High level expression is driven by the cytomegalovirus major immediate early promoter (CMV IEP). Efficient and selective over-production of each recombinant protein was shown by immunofluorescence in either rabbit smooth muscle cells (SMC) or human MCF-7 adenocarcinoma cells. High level secretion directly dependent on the multiplicity of infection (MOI) was observed for each functional transgene by gelatin zymography. Using a quantitative ELISA assay, levels of recombinant TIMP-1 were detected when SMC were infected with as low as three plaque forming units (pfu) of virus per cell in vitro. A linear increase in TIMP-1 secretion was observed up to 1000 pfu/cell of virus (0.75 ng/10(4) cells/24 h at 3 pfu/cell to 1243 ng/10(4) cells/24h at 1000 pfu/cell). Similar levels of secretion of MMP-9 and TIMP-2 were observed by Western blot analysis using the same MOI of adenovirus. Thus, recombinant adenoviruses are an efficient and flexible system for high level expression of MMPs and TIMPs and will be useful tools in the study of matrix remodelling in vivo and in vitro.

Production of collagenase by human osteoblasts and osteoclasts in vivo.

Studies in some animal species have demonstrated the production of metalloproteinases by bone cells, suggesting that they may play a role in bone modeling and remodeling. The aim of the present study was to investigate the expression of collagenase in human bone in situ, using heterotopic and osteophytic bone. Immunohistochemistry was performed on chilled sections of bone, using well characterized polyclonal antibodies to human collagenase. The heterotopic and osteophytic bone exhibited high turnover and both bone modeling and remodeling were evident. Collagenase expression by osteoblasts was demonstrated in cells synthesising matrix and in lining cells; the strongest signal was seen in areas of de novo matrix formation, where bridges of woven bone were being formed between areas of mineralized bone. Collagenase was also present in some osteoclasts associated with eroded bone surfaces and in some mononuclear cells that were present in resorption cavities and in the bone marrow. Our results provide the first demonstration, in situ, of collagenase in human bone and suggest that it may play a role in human bone modeling and remodeling. Production of collagenase by active osteoblasts and lining cells suggest that it may be involved both in matrix formation and activation of bone remodeling. The presence of collagenase in osteoclasts provides further evidence that metalloproteinases may play a role in bone resorption.

Cellular mechanisms for human procollagenase-3 (MMP-13) activation. Evidence that MT1-MMP (MMP-14) and gelatinase a (MMP-2) are able to generate active enzyme.

Gelatinase A and membrane-type metalloproteinase (MT1-MMP) were able to process human procollagenase-3 (Mr 60,000) to the fully active enzyme (Tyr85 N terminus; Mr 48,000). MT1-MMP activated procollagenase-3 via a Mr 56,000 intermediate (Ile36 N terminus) to 48,000 which was the result of the cleavage of the Glu84-Tyr85 peptide bond. We have established that the activation rate of procollagenase-3 by MT1-MMP was enhanced in the presence of progelatinase A, thereby demonstrating a unique new activation cascade consisting of three members of the matrix metalloproteinase family. In addition, procollagenase-3 can be activated by plasmin, which cleaved the Lys38-Glu39 and Arg76-Cys77 peptide bonds in the propeptide domain. Autoproteolysis then resulted in the release of the rest of the propeptide domain generating Tyr85 N-terminal active collagenase-3. However, plasmin cleaved the C-terminal domain of collagenase-3 which results in the loss of its collagenolytic activity. Concanavalin A-stimulated fibroblasts expressing MT1-MMP and fibroblast-derived plasma membranes were able to process human procollagenase-3 via a Mr 56,000 intermediate form to the final Mr 48,000 active enzyme which, by analogy with progelatinase A activation, may represent a model system for in vivo activation. Inhibition experiments using tissue inhibitor of metalloproteinases, plasminogen activator inhibitor-2, or aprotinin demonstrated that activation in the cellular model system was due to MT1-MMP/gelatinase A and excluded the participation of serine proteinases such as plasmin during procollagenase-3 activation. We have established that progelatinase A can considerably potentiate the activation rate of procollagenase-3 by crude plasma membrane preparations from concanavalin A-stimulated fibroblasts, thus confirming our results using purified progelatinase A and MT1-MMP. This new activation cascade may be significant in human breast cancer pathology, where all three enzymes have been implicated as playing important roles.

Matrix metalloproteinases in the formation of human synovial joint cavities.

Matrix metalloproteinases (MMPs) have been implicated in tissue remodelling in growth and development. A histochemical study of human fetal limbs was undertaken to assess the presence, and consequently the possible role, of MMPs and their inhibitor TIMP-1 (tissue inhibitor of metalloproteinases-1) in synovial joint cavity formation. Cryostat sections of fetal limbs from 7 to 14 wk gestation were stained with specific antibodies to collagenase (MMP-1), gelantinases A (MMP-2) and B (MMP-9), stromelysin (MMP-3) and TIMP-1. Immunoreactive (IR) MMP-1, MMP-2 and MMP-3 were seen chiefly in chondrocytes, but in all cases in zones distant from the joint line before cavity formation. IR-MMP-1 and MMP-2 were also localised both in synovium and on the articular surfaces of joints after cavity formation. In addition IR-MMP-2 was seen in a "collar' of perichondrium alongside the hypertrophic zone of chondrocytes and weakly in bone marrow spaces. IR-MMP-9 was seen in neutrophil leucocytes and in bone marrow spaces. IR-TIMP-1 was generally distributed in connective tissue cells. No IR-MMP (1, 2,3 or 9) was seen along potential joint lines before or at the time of cavity formation, nor was there aspecific decrease in IR-TIMP-1 at this site. These findings confirm a role for metalloproteinases in developmental processes such as cartilage remodelling and bone marrow space formation. MMP-1 and MMP-2 may be involved in the remodelling of developing synovial tissue and the articular surfaces subsequent to cavity formation. However, we have failed to find evidence to indicate that the loss of tissue strength at the joint line which allows synovial joint cavity formation relates to high local levels of MMPS.