Cecilia L. Dragomir

Differential expression of GADD45β in normal and osteoarthritic cartilage: Potential role in homeostasis of articular chondrocytes

OBJECTIVE Our previous study suggested that growth arrest and DNA damage-inducible protein 45beta (GADD45beta) prolonged the survival of hypertrophic chondrocytes in the developing mouse embryo. This study was undertaken, therefore, to investigate whether GADD45beta plays a role in adult articular cartilage. METHODS Gene expression profiles of cartilage from patients with late-stage osteoarthritis (OA) were compared with those from patients with early OA and normal controls in 2 separate microarray analyses. Histologic features of cartilage were graded using the Mankin scale, and GADD45beta was localized by immunohistochemistry. Human chondrocytes were transduced with small interfering RNA (siRNA)-GADD45beta or GADD45beta-FLAG. GADD45beta and COL2A1 messenger RNA (mRNA) levels were analyzed by real-time reverse transcriptase-polymerase chain reaction, and promoter activities were analyzed by transient transfection. Cell death was detected by Hoechst 33342 staining of condensed chromatin. RESULTS GADD45beta was expressed at higher levels in cartilage from normal donors and patients with early OA than in cartilage from patients with late-stage OA. All chondrocyte nuclei in normal cartilage immunostained for GADD45beta. In early OA cartilage, GADD45beta was distributed variably in chondrocyte clusters, in middle and deep zone cells, and in osteophytes. In contrast, COL2A1, other collagen genes, and factors associated with skeletal development were up-regulated in late OA, compared with early OA or normal cartilage. In overexpression and knockdown experiments, GADD45beta down-regulated COL2A1 mRNA and promoter activity. NF-kappaB overexpression increased GADD45beta promoter activity, and siRNA-GADD45beta decreased cell survival per se and enhanced tumor necrosis factor alpha-induced cell death in human articular chondrocytes. CONCLUSION These observations suggest that GADD45beta might play an important role in regulating chondrocyte homeostasis by modulating collagen gene expression and promoting cell survival in normal adult cartilage and in early OA.

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

OBJECTIVE Alterations 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. METHODS Cyclic 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. RESULTS Mechanical 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. CONCLUSION This 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.

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.

Identification of MoKA, a Novel F-Box Protein That Modulates Krüppel-Like Transcription Factor 7 Activity

ABSTRACT KLF7, a member of the Krüppel-like transcription factor family, is believed to regulate neurogenesis and cell cycle progression. Here, a yeast two-hybrid screen for KLF7 cofactors in the developing nervous system identified a novel 140-kDa protein named MoKA, for modulator of KLF7 activity. Interaction between MoKA and KLF7 was confirmed by the in vitro glutathione S-transferase pull-down assay and by coimmunoprecipitation of the proteins overexpressed in mammalian cells. Functional assays documented that MoKA is a KLF7 coactivator, and in situ hybridizations identified the developing nervous system and the adult testes as two sites of MoKA and Klf7 coexpression. Chromatin immunoprecipitation experiments demonstrated KLF7 binding to the p21WAF1/Cip1 gene while transient transfection assays documented KLF7 stimulation of the p21WAF1/Cip1 proximal promoter. Additional tests revealed that distinct structural motifs of MoKA direct interaction with KLF7 and shuttling between the nucleus and cytoplasm of asynchronously cycling cells. Altogether, our results strongly suggest that MoKA and KLF7 interact functionally to regulate gene expression during cell differentiation and identify the cell cycle regulator p21WAF1/Cip1 as one of the targeted genes.

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.

Acute inflammation with induction of anaphylatoxin C5a and terminal complement complex C5b-9 associated with multiple intra-articular injections of hylan G-F 20: a case report

OBJECTIVE The purpose of this case report was to investigate local immune mechanisms present during an acute inflammatory flare initiated by viscosupplementation with hylan G-F 20 in a patient with osteoarthritis (OA) and past meniscectomy. EXPERIMENTAL DESIGN A patient with a history of bilateral OA and partial left knee meniscectomy, who had received three injections of hylan G-F 20, was diagnosed with an acute flare reaction in the left knee. Her chart was evaluated for clinical, radiological, and laboratory findings and for clinical follow-up. Histopathological synovial examination and real-time polymerase chain reaction (RT-PCR) for genes with major roles in local inflammation and enzyme-linked immunosorbent assays (ELISAs) for markers of complement activation and cytokines were performed. To study the impact of the inflammatory and immune features we compared the case patient with groups of three representative OA and three rheumatoid arthritis (RA) patients. RESULTS The patient exhibited evidence of highly increased acute phase reactant C-reactive protein (CRP) in the blood. The pathological examination of the synovial membrane identified abundant fibrinous exudate with numerous particles of hyaluronan surrounded by a dense infiltrate of neutrophils and eosinophils. The synovium had moderate hypertrophy and sclerosis as well as an inflammatory infiltrate predominantly composed of T lymphocytes and macrophages with scattered perivascular eosinophils and neutrophils. Immunoperoxidase staining identified numerous deposits of C5b-9 in the fibrinous exudates and the synovial membrane of the patient. Similar findings were observed in the RA patients, whereas deposits were rare in OA synovial samples. In addition, both anaphylatoxin C5a and the terminal complement complex C5b-9 were present at high levels, comparable to those in RA patients. The levels of mRNA for interleukin-1 beta (IL-1β), IL-6, and the neutrophil marker myeloperoxidase (MPO) were markedly increased compared to those in the RA and OA patients. CONCLUSIONS This present study is indicative of a pseudo-septic acute inflammatory reaction in response to local accumulation of hylan G-F 20 with the activation of complement and local invasion of pro-inflammatory cells.