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.

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.

There is evidence to suggest that the synthesis of type II collagen is increased in osteoarthritis (OA). Using an immunoassay, we show that the content of the C-propeptide of type II procollagen (CPII), released extracellularly from the newly synthesized molecule, is directly related to the synthesis of this molecule in healthy and osteoarthritic articular cartilages. In OA cartilage, CPII content is often markedly elevated (mean 7.6-fold), particularly in the mid and deep zones, reaching 29.6% of the content in newborn. Synthesis is also directly related to total collagen II content in OA, suggesting its importance in maintaining collagen content and cartilage structure. The release of CPII from cartilage is correlated directly with cartilage content. However, the increase in CPII in OA cartilage is not reflected in serum, where a significant reduction is observed. Together these studies provide evidence for alterations in procollagen II synthesis in vivo in patients with OA. ( J. Clin. Invest. 1998. 102:2115-2125 . )

The osteocyte is the terminally differentiated state of the osteogenic mesenchymal progenitor immobilized in the bone matrix. Despite their numerical prominence, little is known about osteocytes and their formation. Osteocytes are physically separated in the bone matrix but seemingly compensate for their seclusion from other cells by maintaining an elaborate network of cell processes through which they interact with other osteocytes and bone-lining cells at the periosteal and endosteal surfaces of the bone. This highly organized architecture suggests that osteocytes make an active contribution to the structure and maintenance of their environment rather than passively submitting to random embedding during bone growth or repair. The most abundant matrix protein in the osteocyte environment is type-I collagen and we demonstrate here that, in the mouse, osteocyte phenotype and the formation of osteocyte processes is highly dependent on continuous cleavage of type-I collagen. This collagenolytic activity and formation of osteocyte processes is dependent on matrix metalloproteinase activity. Specifically, a deficiency of membrane type-1 matrix metalloproteinase leads to disruption of collagen cleavage in osteocytes and ultimately to the loss of formation of osteocyte processes. Osteocytogenesis is thus an active invasive process requiring cleavage of collagen for maintenance of the osteocyte phenotype.

Objective. To determine the sites of cleavage and denaturation of type II collagen (CII) by collagenase(s) in healthy and osteoarthritic (OA) human articular cartilage and their relationship to the distribution of matrix metalloproteinase 1 (MMP-1) and MMP-13.Methods. Single (per subject) full-depth specimens from femoral condylar cartilage were isolated from articulating surfaces at autopsy from 8 subjects without arthritis and during arthroplasty from 10 patients with OA. Fixed frozen sections of cartilage were examined by immunoperoxidase localization, using antibodies to the collagenase-generated cleavage site in CII, to an intrachain epitope recognized only in denatured CII, and to MMP-1 and MMP-13 (proenzyme, activated enzyme, or enzyme/inhibitor complex).Results. Staining for collagen cleavage, denaturation, and both MMPs was weak to moderate and was frequently observed in pericellular sites in cartilage from younger, nonarthritic subjects. In specimens from older subjects, this staining was often more widespread and of greater intensity. Similar staining was usually, but not always, seen for all antibodies. In OA cartilage, staining was often stronger and more intense than that in normal cartilage from older subjects, and the distribution of staining was often similar for the different antibodies. Pericellular staining in the deep zone was frequently more pronounced in arthritic cartilage and extended to territorial and sometimes interterritorial sites. In very degenerate specimens, staining was distributed throughout most of the cartilage matrix.Conclusion. These observations provide evidence for the presence of limited cleavage and denaturation of CII restricted to mainly pericellular and superficial sites in cartilage from younger, healthy subjects, where MMP-1 and MMP-13 are also selectively localized. Collagen degradation is more extensive and often more pronounced in cartilage from older, nonarthritic subjects. Characteristic changes in early OA are similar to those seen with aging in cartilage from older, healthy subjects, with collagen damage and collagenases concentrated closer to the articular surface. There was usually a close correspondence between the cleavage and denaturation of CII and the sites at which these collagenases were detected, suggesting that both MMPs are involved in the physiology and pathology. There was no evidence that the damage to CII is ordinarily initiated in sites other than at and near the articular surface and around chondrocytes.Degradation and loss of articular cartilage are fundamental features of osteoarthritis (OA).