Darren A. Plumb

In vivo cyclic compression causes cartilage degeneration and subchondral bone changes in mouse tibiae

OBJECTIVEnAlterations in the mechanical loading environment in joints may have both beneficial and detrimental effects on articular cartilage and subchondral bone, and may subsequently influence the development of osteoarthritis (OA). Using an in vivo tibial loading model, the aim of this study was to investigate the adaptive responses of cartilage and bone to mechanical loading and to assess the influence of load level and duration.nnnMETHODSnCyclic compression at peak loads of 4.5N and 9.0N was applied to the left tibial 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. Changes in articular cartilage and subchondral bone were analyzed by histology and micro-computed tomography.nnnRESULTSnMechanical loading promoted cartilage damage in both age groups of mice, and the severity of joint damage increased with longer duration of loading. Metaphyseal bone mass increased with loading in young mice, but not in adult mice, whereas epiphyseal cancellous bone mass decreased with loading in both young and adult mice. In both age groups, articular cartilage thickness decreased, and subchondral cortical bone thickness increased in the posterior tibial plateau. Mice in 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.nnnCONCLUSIONnThis noninvasive loading model permits dissection of temporal and topographic changes in cartilage and bone and will enable investigation of the efficacy of treatment interventions targeting joint biomechanics or biologic events that promote OA onset and progression.

E74-like Factor 3 (ELF3) Impacts on Matrix Metalloproteinase 13 (MMP13) Transcriptional Control in Articular Chondrocytes under Proinflammatory Stress

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-1β stimulation in chondrocytes, and the IL-1β-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-1β-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.

Collagen XXVII is developmentally regulated and forms thin fibrillar structures distinct from those of classical vertebrate fibrillar collagens.

We have generated an antiserum to the variable domain of mouse collagen XXVII, a recently discovered novel member of the fibrillar collagen family. Collagen XXVII protein was first detectable in the mouse at embryonic day 12.5 (E12.5). By E14.5, the protein localized to cartilage, developing dermis, cornea, the inner limiting membrane of the retina, and major arteries of the heart. However, at E18.5, collagen XXVII protein was no longer apparent in most tissues and appeared restricted mainly to cartilage where expression continued into adulthood. Type XXVII collagen immunolocalized to 10-nm-thick nonstriated fibrils that were distinct from fibrils formed by the classical fibrillar collagens. The transient nature of its expression and unusual fibrillar structure suggest that collagen XXVII plays a developmental role distinct from those of the classical fibrillar collagens.

GADD45β Enhances Col10a1 Transcription via the MTK1/MKK3/6/p38 Axis and Activation of C/EBPβ-TAD4 in Terminally Differentiating Chondrocytes

GADD45β (growth arrest- and DNA damage-inducible) interacts with upstream regulators of the JNK and p38 stress response kinases. Previously, we reported that the hypertrophic zone of the Gadd45β−/− mouse embryonic growth plate is compressed, and expression of type X collagen (Col10a1) and matrix metalloproteinase 13 (Mmp13) genes is decreased. Herein, we report that GADD45β enhances activity of the proximal Col10a1 promoter, which contains evolutionarily conserved AP-1, cAMP-response element, and C/EBP half-sites, in synergism with C/EBP family members, whereas the MMP13 promoter responds to GADD45β together with AP-1, ATF, or C/EBP family members. C/EBPβ expression also predominantly co-localizes with GADD45β in the embryonic growth plate. Moreover, GADD45β enhances C/EBPβ activation via MTK1, MKK3, and MKK6, and dominant-negative p38αapf, but not JNKapf, disrupts the combined trans-activating effect of GADD45β and C/EBPβ on the Col10a1 promoter. Importantly, GADD45β knockdown prevents p38 phosphorylation while decreasing Col10a1 mRNA levels but does not affect C/EBPβ binding to the Col10a1 promoter in vivo, indicating that GADD45β influences the transactivation function of DNA-bound C/EBPβ. In support of this conclusion, we show that the evolutionarily conserved TAD4 domain of C/EBPβ is the target of the GADD45β-dependent signaling. Collectively, we have uncovered a novel molecular mechanism linking GADD45β via the MTK1/MKK3/6/p38 axis to C/EBPβ-TAD4 activation of Col10a1 transcription in terminally differentiating chondrocytes.

ADAM17 Controls Endochondral Ossification by Regulating Terminal Differentiation of Chondrocytes

ABSTRACT Endochondral ossification is a highly regulated process that relies on properly orchestrated cell-cell interactions in the developing growth plate. This study is focused on understanding the role of a crucial regulator of cell-cell interactions, the membrane-anchored metalloproteinase ADAM17, in endochondral ossification. ADAM17 releases growth factors, cytokines, and other membrane proteins from cells and is essential for epidermal growth factor receptor (EGFR) signaling and for processing tumor necrosis factor alpha. Here, we report that mice lacking ADAM17 in chondrocytes (A17ΔCh) have a significantly expanded zone of hypertrophic chondrocytes in the growth plate and retarded growth of long bones. This abnormality is caused by an accumulation of the most terminally differentiated type of chondrocytes that produces a calcified matrix. Inactivation of ADAM17 in osteoclasts or endothelial cells does not affect the zone of hypertrophic chondrocytes, suggesting that the main role of ADAM17 in the growth plate is in chondrocytes. This notion is further supported by in vitro experiments showing enhanced hypertrophic differentiation of primary chondrocytes lacking Adam17. The enlarged zone of hypertrophic chondrocytes in A17ΔCh mice resembles that described in mice with mutant EGFR signaling or lack of its ligand transforming growth factor α (TGFα), suggesting that ADAM17 regulates terminal differentiation of chondrocytes during endochondral ossification by activating the TGFα/EGFR signaling axis.

Collagen XXVII organises the pericellular matrix in the growth plate.

In order to characterise the function of the novel fibrillar type XXVII collagen, a series of mice expressing mutant forms of the collagen were investigated. Mice harboring a glycine to cysteine substitution in the collagenous domain were phenotypically normal when heterozygote and displayed a mild disruption of growth plate architecture in the homozygous state. Mice expressing an 87 amino acid deletion in the collagenous domain of collagen XXVII were phenotypically normal as heterozygotes whereas homozygotes exhibited a severe chondrodysplasia and died perinatally from a lung defect. Animals expressing the 87 amino acid deletion targeted specifically to cartilage were viable but severely dwarfed. The pericellular matrix of proliferative chondrocytes was disrupted and the proliferative cells exhibited a decreased tendency to flatten and form vertical columns. Collagen XXVII plays an important structural role in the pericellular extracellular matrix of the growth plate and is required for the organisation of the proliferative zone.

Human chondrocyte cultures as models of cartilage-specific gene regulation.

The human adult articular chondrocyte is a unique cell type that has reached a fully differentiated state as an end point of development. Within the cartilage matrix, chondrocytes are normally quiescent and maintain the matrix constituents in a low-turnover state of equilibrium. Isolated chondrocytes in culture have provided useful models to study cellular responses to alterations in the environment such as those occurring in different forms of arthritis. However, expansion of primary chondrocytes in monolayer culture results in the loss of phenotype, particularly if high cell density is not maintained. This chapter describes strategies for maintaining or restoring differentiated phenotype by culture in suspension, gels, or scaffolds. Techniques for assessing phenotype involving primarily the analysis of synthesis of cartilage-specific matrix proteins as well as the corresponding mRNAs are also described. Approaches for studying gene regulation, including transfection of promoter-driven reporter genes with expression vectors for transcriptional and signaling regulators, chromatin immunoprecipitation, and DNA methylation are also described.

Mouse Models of Osteoarthritis: Surgical Model of Posttraumatic Osteoarthritis Induced by Destabilization of the Medial Meniscus

The surgical model of destabilization of the medial meniscus (DMM) has become a gold standard for studying the onset and progression of posttraumatic osteoarthritis (OA). The DMM model mimics clinical meniscal injury, a known predisposing factor for the development of human OA, and permits the study of structural and biological changes over the course of the disease. In addition, when applied to genetically modified or engineered mouse models, this surgical procedure permits dissection of the relative contribution of a given gene to OA initiation and/or progression. This chapter describes the requirements for the surgical induction of OA in mouse models, and provides guidelines and tools for the subsequent histological, immunohistochemical, and molecular analyses. Methods for the assessment of the contributions of selected genes in genetically modified strains are also provided.

Progressive cell-mediated changes in articular cartilage and bone in mice are initiated by a single session of controlled cyclic compressive loading.

We previously showed that repetitive cyclic loading of the mouse knee joint causes changes that recapitulate the features of osteoarthritis (OA) in humans. By applying a single loading session, we characterized the temporal progression of the structural and compositional changes in subchondral bone and articular cartilage. We applied loading during a single 5‐minute session to the left tibia of adult (26‐week‐old) C57Bl/6 male mice at a peak load of 9.0N for 1,200 cycles. Knee joints were collected at times 0, 1, and 2 weeks after loading. The changes in articular cartilage and subchondral bone were analyzed by histology, immunohistochemistry (caspase‐3 and cathepsin K), and microcomputed tomography. At time 0, no change was evident in chondrocyte viability or cartilage or subchondral bone integrity. However, cartilage pathology demonstrated by localized thinning and proteoglycan loss occurred at 1 and 2 weeks after the single session of loading. Transient cancellous bone loss was evident at 1 week, associated with increased osteoclast number. Bone loss was reversed to control levels at 2 weeks. We observed formation of fibrous and cartilaginous tissues at the joint margins at 1 and 2 weeks. Our findings demonstrate that a single session of noninvasive loading leads to the development of OA—like morphological and cellular alterations in articular cartilage and subchondral bone. The loss in subchondral trabecular bone mass and thickness returns to control levels at 2 weeks, whereas the cartilage thinning and proteoglycan loss persist.

Overexpression of TIMP-3 in chondrocytes produces transient reduction in growth plate length but permanently reduces adult bone quality and quantity

Bone development and length relies on the growth plate formation, which is dependent on degradative enzymes such as MMPs. Indeed, deletion of specific members of this enzyme family in mice results in important joint and bone abnormalities, suggesting a role in skeletal development. As such, the control of MMP activity is vital in the complex process of bone formation and growth. We generated a transgenic mouse line to overexpress TIMP3 in mouse chondrocytes using the Col2a1-chondrocyte promoter. This overexpression in cartilage resulted in a transient shortening of growth plate in homozygote mice but bone length was restored at eight weeks of age. However, tibial bone structure and mechanical properties remained compromised. Despite no transgene expression in adult osteoblasts from transgenic mice in vitro, their differentiation capacity was decreased. Neonates, however, did show transgene expression in a subset of bone cells. Our data demonstrate for the first time that transgene function persists in the chondro-osseous lineage continuum and exert influence upon bone quantity and quality.