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

GADD45beta enhances Col10a1 transcription via the MTK1/MKK3/6/p38 axis and activation of C/EBPbeta-TAD4 in terminally differentiating chondrocytes.

GADD45beta (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 Gadd45beta(-/-) 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 GADD45beta 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 GADD45beta together with AP-1, ATF, or C/EBP family members. C/EBPbeta expression also predominantly co-localizes with GADD45beta in the embryonic growth plate. Moreover, GADD45beta enhances C/EBPbeta activation via MTK1, MKK3, and MKK6, and dominant-negative p38alphaapf, but not JNKapf, disrupts the combined trans-activating effect of GADD45beta and C/EBPbeta on the Col10a1 promoter. Importantly, GADD45beta knockdown prevents p38 phosphorylation while decreasing Col10a1 mRNA levels but does not affect C/EBPbeta binding to the Col10a1 promoter in vivo, indicating that GADD45beta influences the transactivation function of DNA-bound C/EBPbeta. In support of this conclusion, we show that the evolutionarily conserved TAD4 domain of C/EBPbeta is the target of the GADD45beta-dependent signaling. Collectively, we have uncovered a novel molecular mechanism linking GADD45beta via the MTK1/MKK3/6/p38 axis to C/EBPbeta-TAD4 activation of Col10a1 transcription in terminally differentiating chondrocytes.

Roles of inflammatory and anabolic cytokines in cartilage metabolism: signals and multiple effectors converge upon MMP-13 regulation in osteoarthritis.

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 of 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 metalloproteinases (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.

Inducible knockout of CHUK/IKKα in adult chondrocytes reduces progression of cartilage degradation in a surgical model of osteoarthritis.

CHUK/IKKα contributes to collagenase-driven extracellular matrix remodeling and chondrocyte hypertrophic differentiation in vitro, in a kinase-independent manner. These processes contribute to osteoarthritis (OA), where chondrocytes experience a phenotypic shift towards hypertrophy concomitant with abnormal matrix remodeling. Here we investigated the contribution of IKKα to OA in vivo. To this end, we induced specific IKKα knockout in adult chondrocytes in AcanCreERT2/+; IKKαf/f mice treated with tamoxifen (cKO). Vehicle-treated littermates were used as wild type controls (WT). At 12 weeks of age, WT and cKO mice were subjected to the destabilization of medial meniscus (DMM) model of post-traumatic OA. The cKO mice showed reduced cartilage degradation and collagenase activity and fewer hypertrophy-like features at 12 weeks after DMM. Interestingly, in spite of the protection from structural articular cartilage damage, the postnatal growth plates of IKKα cKO mice after DMM displayed abnormal architecture and composition associated with increased chondrocyte apoptosis, which were not as evident in the articular chondrocytes of the same animals. Together, our results provide evidence of a novel in vivo functional role for IKKα in cartilage degradation in post-traumatic OA, and also suggest intrinsic, cell-autonomous effects of IKKα in chondrocytes that control chondrocyte phenotype and impact on cell survival, matrix homeostasis, and remodeling.

Transcription factor KLF7 is important for neuronal morphogenesis in selected regions of the nervous system.

The Krüppel-like transcription factors (KLFs) are important regulators of cell proliferation and differentiation in several different organ systems. The mouse Klf7 gene is strongly active in postmitotic neuroblasts of the developing nervous system, and the corresponding protein stimulates transcription of the cyclin-dependent kinase inhibitor p21waf/cip gene. Here we report that loss of KLF7 activity in mice leads to neonatal lethality and a complex phenotype which is associated with deficits in neurite outgrowth and axonal misprojection at selected anatomical locations of the nervous system. Affected axon pathways include those of the olfactory and visual systems, the cerebral cortex, and the hippocampus. In situ hybridizations and immunoblots correlated loss of KLF7 activity in the olfactory epithelium with significant downregulation of the p21waf/cip and p27kip1 genes. Cotransfection experiments extended the last finding by documenting KLF7's ability to transactivate a reporter gene construct driven by the proximal promoter of p27kip1. Consistent with emerging evidence for a role of Cip/Kip proteins in cytoskeletal dynamics, we also documented p21waf/cip and p27kip1 accumulation in the cytoplasm of differentiating olfactory sensory neurons. KLF7 activity might therefore control neuronal morphogenesis in part by optimizing the levels of molecules that promote axon outgrowth.

Elf3 Contributes to Cartilage Degradation in vivo in a Surgical Model of Post-Traumatic Osteoarthritis.

The E-74 like factor 3 (ELF3) is a transcription factor induced by inflammatory factors in various cell types, including chondrocytes. ELF3 levels are elevated in human cartilage from patients with osteoarthritis (OA), and ELF3 contributes to the IL-1ß-induced expression of genes encoding Mmp13, Nos2, and Ptgs2/Cox2 in chondrocytes in vitro. Here, we investigated the contribution of ELF3 to cartilage degradation in vivo, using a mouse model of OA. To this end, we generated mouse strains with cartilage-specific Elf3 knockout (Col2Cre:Elf3f/f) and Comp-driven Tet-off-inducible Elf3 overexpression (TRE-Elf3:Comp-tTA). To evaluate the contribution of ELF3 to OA, we induced OA in 12-week-old Col2Cre:Elf3f/f and 6-month-old TRE-Elf3:Comp-tTA male mice using the destabilization of the medial meniscus (DMM) model. The chondrocyte-specific deletion of Elf3 led to decreased levels of IL-1ß- and DMM-induced Mmp13 and Nos2 mRNA in vitro and in vivo, respectively. Histological grading showed attenuation of cartilage loss in Elf3 knockout mice compared to wild type (WT) littermates at 8 and 12 weeks following DMM surgery that correlated with reduced collagenase activity. Accordingly, Elf3 overexpression led to increased cartilage degradation post-surgery compared to WT counterparts. Our results provide evidence that ELF3 is a central contributing factor for cartilage degradation in post-traumatic OA in vivo.

Identification of genes regulated by transcription factor KLF7 in differentiating olfactory sensory neurons.

Gene targeting in mice has recently demonstrated that transcription factor KLF7 plays a critical role in neurite outgrowth and neuronal survival. Here we extended this genetic evidence by establishing the transcriptional profile of differentiating olfactory sensory neurons (OSNs) in Klf7(-/-) mice, and by identifying relevant genes that are directly regulated by KLF7. Functional clustering of DNA microarray data revealed that loss of KLF7 affects primarily the activity of genes involved in OSN differentiation, axonal growth, cytoskeletal dynamics, cell adhesion and synaptogenesis. Cell transfection experiments, on the other hand, demonstrated that the promoters of the genes encoding the OSN-specific OMP and the adhesion molecule L1 are both activated by KLF7 binding to CACCC motifs. Collectively, these results advance knowledge of transcriptional regulation of olfactory neurogenesis and KLF7 action.

Identification of MoKA, a novel F-box protein that modulates Krüppel-like transcription factor 7 activity.

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 p21(WAF1/Cip1) gene while transient transfection assays documented KLF7 stimulation of the p21(WAF1/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 p21(WAF1/Cip1) as one of the targeted genes.

Mice without transcription factor KLF7 provide new insight into olfactory bulb development.

Recent genetic studies have excluded that peripheral innervation plays a substantial role in the initial outgrowth of the olfactory bulb. Mice without Kruppel-like factor 7 activity die at birth and display hypoplastic olfactory bulbs which lack peripheral innervation. Here, we report that incomplete penetrance of the mutation is responsible for partial bulb innervation in a small fraction of Klf7 null mice. Analysis of the partially innervated bulbs of mutant embryos, newborns and adult mice revealed an obligatory correlation with local restoration of laminar architecture, neuronal cell differentiation and neuronal activity. The degree of normal OB maturation in Klf7-/- OBs was proportional to the degree of peripheral innervation. These findings therefore indicate that peripheral innervation contributes to bulb maturation late in development by promoting cell morphogenesis and differentiation.

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. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1941-1949, 2016.

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.

ADAM17 controls endochondral ossification by regulating terminal differentiation of chondrocytes.

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.

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.

Differential expression of GADD45beta 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.