MT1-MMP is a membrane-bound matrix metalloproteinase (MT-MMP) capable of mediating pericellular proteolysis of extracellular matrix components. MT1-MMP is therefore thought to be an important molecular tool for cellular remodeling of the surrounding matrix. To establish the biological role of this membrane proteinase we generated MT1-MMP-deficient mice by gene targeting. MT1-MMP deficiency causes craniofacial dysmorphism, arthritis, osteopenia, dwarfism, and fibrosis of soft tissues due to ablation of a collagenolytic activity that is essential for modeling of skeletal and extraskeletal connective tissues. Our findings demonstrate the pivotal function of MT1-MMP in connective tissue metabolism, and illustrate that modeling of the soft connective tissue matrix by resident cells is essential for the development and maintenance of the hard tissues of the skeleton.

We demonstrate the direct involvement of increased collagenase activity in the cleavage of type II collagen in osteoarthritic human femoral condylar cartilage by developing and using antibodies reactive to carboxy-terminal (COL2-3/ 4C short ) and amino-terminal (COL2-1/4N1) neoepitopes generated by cleavage of native human type II collagen by collagenase matrix metalloproteinase (MMP)-1 (collagenase-1), MMP-8 (collagenase-2), and MMP-13 (collagenase-3). A secondary cleavage followed the initial cleavage produced by these recombinant collagenases. This generated neoepitope COL2-1/4N2. There was significantly more COL2-3/ 4C short neoepitope in osteoarthritis (OA) compared to adult nonarthritic cartilages as determined by immunoassay of cartilage extracts. A synthetic preferential inhibitor of MMP-13 significantly reduced the unstimulated release in culture of neoepitope COL2-3/4C short from human osteoarthritic cartilage explants. These data suggest that collagenase(s) produced by chondrocytes is (are) involved in the cleavage and denaturation of type II collagen in articular cartilage, that this is increased in OA, and that MMP-13 may play a significant role in this process. ( J. Clin. Invest. 1997. 99:1534-1545.)

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A new immunoassay was developed to detect denaturation of type II collagen in osteoarthritis (OA). A peptide, al(II)-CBllB, located in the CB11 peptide of type II collagen, was synthesized and used to produce a monoclonal antibody (COL2-3/4m) of the IgG,(K) isotype. This reacts with a defined epitope in denatured but not native type II collagen and the a3 chain of type XI collagen. The latter is present in very small amounts (about 1% wt/wt) in cartilage relative to the al (II) chain. By using an enzyme-linked immunosorbent assay, type II collagen denaturation and total type II collagen content were determined. The epitope recognized by the antibody was resistant to cleavage by a-chymotrypsin and proteinase K which were used to extract al (II)-CB11B from the denatured (a-chymotrypsin soluble) and residual native (proteinase K soluble) collagen a-chains, respectively, present in human femoral articular cartilage. Type II collagen content was significantly reduced from a mean (range) of 14% (9.2-20.8%) of wet weight in 8 normal cartilages to 10.3% (7.4-15.0%) in 16 OA cartilages. This decrease, which may result in part from an increased hydration, was accompanied by an increase in the percent denaturation of type II collagen in OA to 6.0% of total type II collagen compared with 1.1% in normal tissue. The percent denaturation was ordinarily greater in the more superficial zone than in the deep zone of OA cartilage. (J. Clin. Invest.

Many proteins found in mineralized tissues have been proposed to function as regulators of the mineralization process, either as nucleators or inhibitors of hydroxyapatite (HA) formation. We have studied the HA-nucleating and HA-inhibiting properties of proteins from bone [osteocalcin (OC), osteopontin (OPN), osteonectin (ON) and bone sialoprotein (BSP)], dentine [phosphophoryn (DPP)] and calcified cartilage [chondrocalcin (CC)] over a wide range of concentrations. Nucleation of HA was studied with a steady-state agarose gel system at sub-threshold [Ca] x [PO4] product. BSP and DPP exhibited nucleation activity at minimum concentrations of 0.3 microgram/ml (9 nM) and 10 micrograms/ml (67 nM) respectively. OC, OPN, ON and CC all lacked nucleation activity at concentrations up to 100 micrograms/ml. Inhibition of HA formation de novo was studied with calcium phosphate solutions buffered by autotitration. OPN was found to be a potent inhibitor of HA formation [IC50 = 0.32 microgram/ml (0.01 microM)] whereas OC was of lower potency [IC50 = 6.1 micrograms/ml (1.1 microM)]; BSP, ON and CC all lacked inhibitory activity at concentrations up to 10 micrograms/ml. The effect of OPN on HA formation de novo is mainly to inhibit crystal growth, whereas OC delays nucleation. These findings are consistent with the view that BSP and DPP may play roles in the initiation of mineralization in bone and dentine respectively. OPN seems to be the mineralized tissue protein most likely to function in the inhibition of HA formation, possibly by preventing phase separation in tissue fluids of high supersaturation.

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The degeneration of articular cartilage is a key feature of osteoarthritis (OA)'. It is characterized by a loss of the tensile strength (1-3) of this tissue. The incidence of OA increases progressively with age (4). This is in association with a progressive reduction in tensile properties during aging (5). Whereas the large aggregating proteoglycan (now called aggrecan) is responsible for the compressive stiffness of cartilage (6, 7), it is the collagen fibrillar network that determines the tensile properties of this tissue ( 1, 2). These collagen fibrils are primarily composed of type H collagen (-90-95%) but also contain type IX collagen, covalently bound to type II, and type XI collagen which together comprise 2-4% of the total collagen (8).These biomechanical changes are indicative of damage to the collagen fibrils in aging and OA. To investigate these changes at the molecular level first polyclonal (9) and then monoclonal (10) antibodies were developed that only react with epitopes on the collagen a, (II) chains when the triple helix has been denatured, such as occurs following cleavage by interstitial collagenase (MMP1) which results in unwinding of the triple helix.Using a monoclonal antibody in an immunoassay, quantitative evidence was obtained to indicate that there is indeed increased denaturation of type II collagen in osteoarthritic cartilage (10). In the study presented here we describe the use of this antibody to determine where, in both aging and osteoarthritis, this damage to type II collagen occurs. We show that it starts at the articular surface and spreads into the mid and deep zones with increasing degeneration, denaturation being first observed around chondrocytes implicating these cells as primary mediators of collagen degradation in aging and OA. Tissue. Full depth human articular femoral condylar cartilages (0.5-1.0 cm2 surface area) were removed within 15 h of death with a sharp scalpel from the anterior loaded regions of the adult knee joint from a total of 11 individuals, of various ages and sexes, with no observable arthritic joint abnormalities nor recent (2-3 mo) chemotherapy (see Table I). A total of 51 patients underwent total knee arthroplasty for osteoarthritis diagnosed using the criteria of the American College of Rheumatology. Site matched femoral condylar cartilages that remained were immediately removed to the laboratory. By definition, osteophytic cartilages were excluded from this study. Single samples from 30 patients (see Table I) and 12 multiple samples from three other patients 1. Abbreviation used in this paper: OA, osteoarthritis. MethodsType II Collagen Denaturation 2859 J. Clin. Invest.

IntroductionDegenerative joint diseases including osteoarthritis (OA) are common, particularly in the elderly. Early signs of OA include progressive loss from articular cartilage of the proteoglycan aggrecan, reflected by a loss of safranin O staining, excessive damage to type II collagen, and general degeneration and fibrillation of the cartilage surface, resulting ultimately in a loss of articular cartilage (1).One of the primary targets of this disease is type II collagen, the major structural collagen found in articular cartilage in healthy individuals. There is ordinarily a strict balance between the production of type II collagen and degradation of this protein by catabolic enzymes during normal remodeling of cartilage (1). Pathological conditions such as OA are characterized by a loss of this balance with increased proteolysis (1-5) and upregulation of the synthesis of type II procollagen (5) and aggrecan (6).Matrix metalloproteinases (MMPs) comprise a family of zinc-dependent enzymes that degrade extracellular matrix components. MMPs are synthesized in articulating joints by synovial cells and chondrocytes. In mature articular cartilage, chondrocytes maintain the cartilage-specific matrix phenotype. Elevated expression of MMPs is associated with cartilage degradation (1). MMP-13, also known as human collagenase-3, is thought to play an important role in type II collagen degradation in articular cartilage and especially in OA (4, 7-9). Type II collagen is the preferred substrate for MMP-13 (4, 7, 10). Expression and contents of MMP-1 (collagenase-1) and 11,12), expression of MMP-8 (collagenase-2), and collagenase activity (4,8) are upregulated in human OA cartilage.Spontaneous development of focal sites degeneration has been described in aging guinea pigs (13). Sublines of the inbred STR/ORT strain of mice also develop spontaneous OA with aging (14). Mice exhibit upregulated expression of MMP-13 and collagenase activity is upregulated in focal lesions (15). In guinea pigs, MMP-1 and MMP-13 are also upregulated in OA lesions associated with increased collagenase activity (16). It has been suggested that increased collagenase-3 (MMP-13) activity plays a pivotal role in the pathogenesis of osteoarthritis (OA). We have used tetracycline-regulated transcription in conjunction with a cartilage-specific promoter to target a constitutively active human MMP-13 to the hyaline cartilages and joints of transgenic mice. Postnatal expression of this transgene resulted in pathological changes in articular cartilage of the mouse joints similar to those observed in human OA. These included characteristic erosion of the articular cartilage associated with loss of proteoglycan and excessive cleavage of type II collagen by collagenase, as well as synovial hyperplasia. These results demonstrate that excessive MMP-13 activity can result in articular cartilage degradation and joint pathology of the kind observed in OA, suggesting that excessive activity of this proteinase can lead to this disease.