Type I collagen fibers account for 90% of the organic matrix of bone. The degradation of this collagen is a major event during bone resorption, but its mechanism is unknown. A series of data obtained in biological models strongly suggests that the recently discovered cysteine proteinase cathepsin K plays a key role in bone resorption. Little is known, however, about the actual action of cathepsin K on type I collagen. Here, we show that the activity of cathepsin K alone is sufficient to dissolve completely insoluble collagen of adult human cortical bone. We found that the collagenolytic activity of cathepsin K is directed both outside the helical region of the molecule, i.e. the typical activity of cysteine proteinases, and at various sites inside the helical region, hitherto believed to resist all mammalian proteinases but the collagenases of the matrix metalloproteinase family and the neutrophil elastase. This property of cathepsin K is unique among mammalian proteinases and is reminiscent of bacterial collagenases. It is likely to be responsible for the key role of cathepsin K in bone resorption.The only mammalian proteinases that have been shown to attack the native triple helical region of type I collagen are the collagenases of the MMP 1 family (1-3) and the neutrophil serine elastase (4) They cleave the type I collagen triple helix across all three chains (i.e. two ␣1 chains and one ␣2 chain) only at a specific point three-quarters of the way to the Nterminal end of the collagen molecule. Proteinases with broad specificity, such as cysteine proteinases, attack only the extrahelical regions that are located at either end of native collagen (telopeptides) and that represent only 4% of the molecule (5). Because the telopeptides are involved in intra-and intermolecular links, this attack may separate individual molecules. The latter proteinases may also attack destabilized triple helices, acting thereby as gelatinases. At 37°C, such a destabilization may transiently affect a small proportion of collagen molecules, because the melting temperature of soluble collagen is only a few degrees higher. It has also been emphasized that when collagen molecules are cross-linked and arranged in insoluble fibers they become more resistant to proteolysis (6). However, the co-operation of proteinases with distinct specificities toward the chemical bonds of collagen fibers has been shown to favor the efficiency of collagenolysis (7).Insoluble type I collagen fibers constitute 90% of the organic matrix of bone, and their degradation is necessary for bone resorption (8). The test tube experiments that have been performed so far showed that it is difficult to achieve complete degradation of adult lamellar bone with a single bone proteinase (9, 10). On the other hand, various biological approaches have shown that both MMPs and cysteine proteinases participate in the bone resorption processes (8,(11)(12)(13). Representatives of these two types of proteinases were identified in osteoclasts, the cells responsible for bone resorptio...

Bone development requires the recruitment of osteoclast precursors from surrounding mesenchyme, thereby allowing the key events of bone growth such as marrow cavity formation, capillary invasion, and matrix remodeling. We demonstrate that mice deficient in gelatinase B/matrix metalloproteinase (MMP)-9 exhibit a delay in osteoclast recruitment. Histological analysis and specialized invasion and bone resorption models show that MMP-9 is specifically required for the invasion of osteoclasts and endothelial cells into the discontinuously mineralized hypertrophic cartilage that fills the core of the diaphysis. However, MMPs other than MMP-9 are required for the passage of the cells through unmineralized type I collagen of the nascent bone collar, and play a role in resorption of mineralized matrix. MMP-9 stimulates the solubilization of unmineralized cartilage by MMP-13, a collagenase highly expressed in hypertrophic cartilage before osteoclast invasion. Hypertrophic cartilage also expresses vascular endothelial growth factor (VEGF), which binds to extracellular matrix and is made bioavailable by MMP-9 (Bergers, G., R. Brekken, G. McMahon, T.H. Vu, T. Itoh, K. Tamaki, K. Tanzawa, P. Thorpe, S. Itohara, Z. Werb, and D. Hanahan. 2000. Nat. Cell Biol. 2:737–744). We show that VEGF is a chemoattractant for osteoclasts. Moreover, invasion of osteoclasts into the hypertrophic cartilage requires VEGF because it is inhibited by blocking VEGF function. These observations identify specific actions of MMP-9 and VEGF that are critical for early bone development.

Upon termination of bone matrix synthesis, osteoblasts either undergo apoptosis or differentiate into osteocytes or bone lining cells. In this study, we investigated the role of matrix metalloproteinases (MMPs) and growth factors in the differentiation of osteoblasts into osteocytes and in osteoblast apoptosis. The mouse osteoblast cell line MC3T3-E1 and primary mouse calvarial osteoblasts were either grown on two-dimensional (2-D) collagen-coated surfaces, where they morphologically resemble flattened, cuboidal bone lining cells, or embedded in three-dimensional (3-D) collagen gels, where they resemble dendritic osteocytes constituting a network of cells. When MC3T3-E1 osteoblasts were grown in a 3-D matrix in the presence of an MMP inhibitor (GM6001), the cell number was dose-dependently reduced by approximately 50%, whereas no effect was observed on a 2-D substratum. In contrast, the murine mature osteocyte cell line, MLO-Y4, was unaffected by GM6001 under all culture conditions. According to TUNEL assay, the osteoblast apoptosis was increased 2.5-fold by 10 M GM6001. To investigate the mechanism by which MMPs mediate the survival of osteoblasts, we examined the effect of GM6001 on MC3T3-E1 osteoblasts in the presence of extracellular matrix components and growth factors, including tenascin, fibronectin, laminin, collagenase-cleaved collagen, gelatin, parathyroid hormone, basic fibroblast growth factor, vascular epidermal growth factor, insulin-like growth factor, interleukin-1, and latent and active transforming growth factor-␤ (TGF-␤). Only active TGF-␤, but not latent TGF-␤ or other agents tested, restored cell number and apoptosis to control levels. Furthermore, we found that the membrane type MMP, MT1-MMP, which is produced by osteoblasts, could activate latent TGF-␤ and that antibodies neutralizing endogenous TGF-␤ led to a similar decrease in cell number as GM6001. Whereas inhibitors of other protease families did not induce osteoblast apoptosis, an inhibitor of the p44/42 mitogen-activated protein kinase showed the same but non-synergetic effect as GM6001. These findings suggest that MMP-activated TGF-␤ maintains osteoblast survival during trans-differentiation into osteocytes by a p44/42-dependent pathway.The skeleton is a dynamic tissue that is continuously remodeling to sustain calcium homeostasis, repair microfractures, and react to strain and stress of the skeleton. The remodeling process is a complex process and relies on the coupling between bone resorption and formation that involves osteoclasts, osteoblasts, and osteocytes.The constant regeneration of bone emphasizes the delicate balance between bone resorption and bone formation, which otherwise may lead to pathological conditions such as osteoporosis or osteopetrosis. The investigation of the cellular actions of the major players of bone remodeling may therefore contribute significantly to the discovery of new and better drugs for the treatment of osteoporosis (1).The major pharmaceutical interventions for treatment and prevention of osteop...

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