Human cartilage is a complex tissue of matrix proteins that vary in amount and orientation from superficial to deep layers and from loaded to unloaded zones. A major challenge to efforts to repair cartilage by stem cell-based and other tissue engineering strategies is the inability of the resident chondrocytes to lay down new matrix with the same structural and resilient properties that it had upon its original formation. This is particularly true for the collagen network, which is susceptible to cleavage once proteoglycans are depleted. Thus, a thorough understanding of the similarities and particularly the marked differences in mechanisms of cartilage remodeling during development, osteoarthritis, and aging may lead to more effective strategies for preventing cartilage damage and promoting repair. To identify and characterize effectors or regulators of cartilage remodeling in these processes, we are using culture models of primary human and mouse chondrocytes and cell lines and mouse genetic models to manipulate gene expression programs leading to matrix remodeling and subsequent chondrocyte hypertrophic differentiation, pivotal processes which both go astray in OA disease. Matrix metalloproteinase (MMP)-13, the major type II collagen-degrading collagenase, is regulated by stress-, inflammation-, and differentiation-induced signals that not only contribute to irreversible joint damage (progression) in OA, but importantly, also to the initiation/onset phase, wherein chondrocytes in articular cartilage leave their natural growth-and differentiation-arrested state. Our work points to common mediators of these processes in human OA cartilage and in early through late stages of OA in surgical and genetic mouse models.

Objectives Alterations in the mechanical loading environment in joints may have both beneficial and detrimental effects on articular cartilage and subchondral bone and subsequently influence the development of osteoarthritis (OA). We used an in vivo tibial loading model to investigate the adaptive responses of cartilage and bone to mechanical loading and to assess the influence of load level and duration. Methods We applied cyclic compression of 4.5 and 9.0N peak loads to the left tibia via the knee joint of adult (26-week-old) C57Bl/6 male mice for 1, 2, and 6 weeks. Only 9.0N loading was utilized in young (10-week-old) mice. The changes in articular cartilage and subchondral bone were analyzed by histology and microcomputed tomography. Results Loading promoted cartilage damage in both age groups, with increased damage severity dependent upon the duration of loading. Metaphyseal bone mass increased in the young mice, but not in the adult mice, whereas epiphyseal cancellous bone mass decreased with loading in both young and adult mice. Articular cartilage thickness decreased, and subchondral cortical bone thickness increased in the posterior tibial plateau in both age groups. Both age groups developed periarticular osteophytes at the tibial plateau in response to the 9.0N load, but no osteophyte formation occurred in adult mice subjected to 4.5N peak loading. Conclusion This non-invasive loading model permits dissection of temporal and topographical changes in cartilage and bone and will enable investigation of the efficacy of treatment interventions targeting joint biomechanics or biological events that promote OA onset and progression.

The type XXVII collagen gene codes for a novel vertebrate fibrillar collagen that is highly conserved in man, mouse, and fish (Fugu rubripes). The pro␣1(XXVII) chain has a domain structure similar to that of the type B clade chains (␣1(V), ␣3(V), ␣1(XI), and ␣2(XI)). However, compared with other vertebrate fibrillar collagens (types I, II, III, V, and XI), type XXVII collagen has unusual molecular features such as no minor helical domain, a major helical domain that is short and interrupted, and a short chain selection sequence within the NC1 domain. Pro␣1(XXVII) mRNA is 9 kb and expressed by chondrocytes but also by a variety of epithelial cell layers in developing tissues including stomach, lung, gonad, skin, cochlear, and tooth. By Western blotting, type XXVII antisera recognized multiple bands of 240 -110 kDa in tissue extracts and collagenous bands of 150 -140 kDa in the conditioned medium of the differentiating chondrogenic ATDC5 cell line. Phylogenetic analyses revealed that type XXVII, together with the closely related type XXIV collagen gene, form a new, third clade (type C) within the vertebrate fibrillar collagen family. Furthermore, the exon structure of the type XXVII collagen gene is similar to, but distinct from, those of the genes coding for the type A or B clade pro␣ chains.Fibril-forming or fibrillar collagens are one of the most ancient families of extracellular matrix molecules being found throughout the metazoan kingdom from the simplest (porifera (sponges)) to the most complex animals (vertebrates). Fibrillar collagens form major structural elements in extracellular matrices as diverse as the evolutionarily "primitive" mesoglea of cnidarians (1) to the highly specialized connective tissues of vertebrates (e.g. bone, cartilage, skin, and tendon) (2). Molecular features shared by all members of this family include a highly conserved C-terminal noncollagenous (NC1) domain and a long collagenous domain of ϳ1000 amino acid residues.Phylogenetic analyses have revealed that the previously known vertebrate fibrillar collagens fall into two related but distinct groups or clades (3, 4). The type A clade consists of the pro␣1(I), pro␣2(I), pro␣1(II), pro␣1(III), and pro␣2(V), whereas the type B clade contains the remaining chains encoding types V and XI collagens (with the exception of pro␣3(XI), which is derived from the COL2A1 gene). The division of the vertebrate fibrillar collagens into two clades is supported by two further observations. Firstly, the exon structures of the genes are virtually identical within a clade yet distinct between clades (5). Secondly, the members of each clade share homologous Nterminal noncollagenous domains (von Willebrand factor type C domain for type A and TSPN 1 for type B clade members) with the exception of the pro␣2(I) chain, where the N-terminal noncollagenous domain appears to have been deleted (6).We have recently described how the members of the two clades of vertebrate fibrillar collagens have apparently arisen early during vertebrate evolution from a single ...

Matrix metalloproteinase (MMP)-13 has a pivotal, rate-limiting function in cartilage remodeling and degradation due to its specificity for cleaving type II collagen. The proximal MMP13 promoter contains evolutionarily conserved E26 transformation-specific sequence binding sites that are closely flanked by AP-1 and Runx2 binding motifs, and interplay among these and other factors has been implicated in regulation by stress and inflammatory signals. Here we report that ELF3 directly controls MMP13 promoter activity by targeting an E26 transformation-specific sequence binding site at position ؊78 bp and by cooperating with AP-1. In addition, ELF3 binding to the proximal MMP13 promoter is enhanced by IL-1␤ stimulation in chondrocytes, and the IL-1␤-induced MMP13 expression is inhibited in primary human chondrocytes by siRNA-ELF3 knockdown and in chondrocytes from Elf3 ؊/؊ mice. Further, we found that MEK/ERK signaling enhances ELF3-driven MMP13 transactivation and is required for IL-1␤-induced ELF3 binding to the MMP13 promoter, as assessed by chromatin immunoprecipitation. Finally, we show that enhanced levels of ELF3 co-localize with MMP13 protein and activity in human osteoarthritic cartilage. These studies define a novel role for ELF3 as a procatabolic factor that may contribute to cartilage remodeling and degradation by regulating MMP13 gene transcription.The matrix metalloproteinases (MMPs) 3 are a family of enzymes that coordinately degrade components of the extracellular matrix in physiological/normal matrix remodeling processes (1) and in disease states wherein their aberrant and enhanced expression contributes to exacerbated matrix degradation (2, 3). Type II collagen is a major constituent of articular cartilage that contributes to its structural and functional properties by conferring tensile strength, and its degradation is the pivotal event that determines the irreversible progression of osteoarthritis (OA), in which articular cartilage is slowly and progressively destroyed (2). OA occurs in conjunction with changes in the synovium and subchondral bone that are associated with dysregulated chondrocyte physiology exemplified in part by the abnormal expression of catabolic and anabolic gene products (2). In this context, proinflammatory cytokines have been shown to trigger a diverse array of intracellular signaling pathways leading to the overexpression of a variety of matrix-degrading enzymes, including MMPs (2).Because collagen degradation is mediated almost exclusively by MMPs, those with higher collagenolytic activity (collagenases) are the rate-limiting, major players in irreversible cartilage destruction (4), and MMP13 (collagenase 3) plays a very prominent role here. MMP13 preferentially and more potently cleaves type II collagen compared with other collagenases (5-7). Moreover, MMP13 levels and activity are enhanced in OA cartilage, associated with degenerative changes and co-localizing with MMP13-specific type II collagen cleavage products, inflammatory cytokines, and their receptors (4,8). Furt...