In the context of OA disease, NF-κB transcription factors can be triggered by a host of stress-related stimuli including pro-inflammatory cytokines, excessive mechanical stress and ECM degradation products. Activated NF-κB regulates the expression of many cytokines and chemokines, adhesion molecules, inflammatory mediators, and several matrix degrading enzymes. NF-κB also influences the regulated accumulation and remodeling of ECM proteins and has indirect positive effects on downstream regulators of terminal chondrocyte differentiation (including β-catenin and Runx2). Although driven partly by pro-inflammatory and stress-related factors, OA pathogenesis also involves a “loss of maturational arrest” that inappropriately pushes chondrocytes towards a more differentiated, hypertrophic-like state. Growing evidence points to NF-κB signaling as not only playing a central role in the pro-inflammatory stress-related responses of chondrocytes to extra- and intra-cellular insults, but also in the control of their differentiation program. Thus unlike other signaling pathways the NF-κB activating kinases are potential therapeutic OA targets for multiple reasons. Targeted strategies to prevent unwanted NF-κB activation in this context, which do not cause side effects on other proteins or signaling pathways, need to be focused on the use of highly specific drug modalities, siRNAs or other biological inhibitors that are targeted to the activating NF-κB kinases IKKα or IKKβ or specific activating canonical NF-κB subunits. However, work remains in its infancy to evaluate the effects of efficacious, targeted NF-κB inhibitors in animal models of OA disease in vivo and to also target these strategies only to affected cartilage and joints to avoid other undesirable systemic effects.

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.

IntroductionRecent evidence suggests that tissue accumulation of senescent p16INK4a-positive cells during the life span would be deleterious for tissue functions and could be the consequence of inherent age-associated disorders. Osteoarthritis (OA) is characterized by the accumulation of chondrocytes expressing p16INK4a and markers of the senescence-associated secretory phenotype (SASP), including the matrix remodeling metalloproteases MMP1/MMP13 and pro-inflammatory cytokines interleukin-8 (IL-8) and IL-6. Here, we evaluated the role of p16INK4a in the OA-induced SASP and its regulation by microRNAs (miRs).MethodsWe used IL-1-beta-treated primary OA chondrocytes cultured in three-dimensional setting or mesenchymal stem cells differentiated into chondrocyte to follow p16INK4a expression. By transient transfection experiments and the use of knockout mice, we validate p16INK4a function in chondrocytes and its regulation by one miR identified by means of a genome-wide miR-array analysis.Resultsp16INK4a is induced upon IL-1-beta treatment and also during in vitro chondrogenesis. In the mouse model, Ink4a locus favors in vivo the proportion of terminally differentiated chondrocytes. When overexpressed in chondrocytes, p16INK4a is sufficient to induce the production of the two matrix remodeling enzymes, MMP1 and MMP13, thus linking senescence with OA pathogenesis and bone development. We identified miR-24 as a negative regulator of p16INK4a. Accordingly, p16INK4a expression increased while miR-24 level was repressed upon IL-1-beta addition, in OA cartilage and during in vitro terminal chondrogenesis.ConclusionsWe disclosed herein a new role of the senescence marker p16INK4a and its regulation by miR-24 during OA and terminal chondrogenesis.

Objective. Osteoarthritic (OA) chondrocytes behave in an intrinsically deregulated manner, characterized by chronic loss of healthy cartilage and inappropriate differentiation to a hypertrophic-like state. IKK␣ and IKK␤ are essential kinases that activate NF-B transcription factors, which in turn regulate cell differentiation and inflammation. This study was undertaken to investigate the differential roles of each IKK in chondrocyte differentiation and hypertrophy.Methods. Expression of IKK␣ or IKK␤ was ablated in primary human chondrocytes by retrotransduction of specific short-hairpin RNAs. Micromass cultures designed to reproduce chondrogenesis with progression to the terminal hypertrophic stage were established, and anabolism and remodeling of the extracellular matrix (ECM) were investigated in the micromasses using biochemical, immunohistochemical, and ultrastructural techniques. Cellular parameters of hypertrophy (i.e., proliferation, viability, and size) were also analyzed.Results. The processes of ECM remodeling and mineralization, both characteristic of terminally differentiated hypertrophic cells, were defective following the loss of IKK␣ or IKK␤. Silencing of IKK␤ markedly enhanced accumulation of glycosaminoglycan in conjunction with increased SOX9 expression. Ablation of IKK␣ dramatically enhanced type II collagen deposition independent of SOX9 protein levels but in association with suppressed levels of runt-related transcription factor 2. Moreover, IKK␣-deficient cells retained the phenotype of cells in a pre-hypertrophic-like state, as evidenced by the smaller size and faster proliferation of these cells prior to micromass seeding, along with the enhanced viability of their differentiated micromasses.Conclusion. IKK␣ and IKK␤ exert differential roles in ECM remodeling and endochondral ossification, which are events characteristic of hypertrophic chondrocytes and also complicating factors often found in OA. Because the effects of IKK␣ were more profound and pleotrophic in nature, our observations suggest that exacerbated IKK␣ activity may be responsible, at least in part, for the characteristic abnormal phenotypes of OA chondrocytes.

Material

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...

Osteoarthritis (OA), a whole-joint disease driven by abnormal biomechanics and attendant cell-derived and tissue-derived factors, is a rheumatic disease with the highest prevalence, representing a severe health burden with a tremendous economic impact. Members of the nuclear factor κB (NF-κB) family orchestrate mechanical, inflammatory and oxidative stress-activated processes, thus representing a potential therapeutic target in OA disease. The two pivotal kinases, IκB kinase (IKK) α and IKKβ, activate NF-κB dimers that might translocate to the nucleus and regulate the expression of specific target genes involved in extracellular matrix remodelling and terminal differentiation of chondrocytes. IKKα, required for the activation of the so-called non-canonical pathway, has a number of NF-κB-independent and kinase-independent functions in vivo and in vitro, including controlling chondrocyte hypertrophic differentiation and collagenase activity. In this short review, we will discuss the role of NF-κB signalling in OA pathology, with emphasis on the functional effects of IKKα that are independent of its kinase activity and NF-κB activation.

Objective. To link matrix metalloproteinase 13 (MMP-13) activity and extracellular matrix (ECM) remodeling to alterations in regulatory factors leading to a disruption in chondrocyte homeostasis.Methods. MMP-13 expression was ablated in primary human chondrocytes by stable retrotransduction of short hairpin RNA. The effects of MMP-13 knockdown on key regulators of chondrocyte differentiation (SOX9, runt-related transcription factor 2 [RUNX-2], and ␤-catenin) and angiogenesis (vascular endothelial growth factor [VEGF]) were scored at the protein level (by immunohistochemical or Western blot analysis) and RNA level (by real-time polymerase chain reaction) in high-density monolayer and micromass cultures under mineralizing conditions. Effects on cellular viability in conjunction with chondrocyte progression toward a hypertrophic-like state were assessed in micromass cultures. Alterations in SOX9 subcellular distribution were assessed using confocal microscopy in micromass cultures and also in osteoarthritic cartilage.Results. Differentiation of control chondrocyte micromasses progressed up to a terminal phase, with calcium deposition in conjunction with reduced cell viability and scant ECM. MMP-13 knockdown impaired ECM remodeling and suppressed differentiation in conjunction with reduced levels of RUNX-2, ␤-catenin, and VEGF. MMP-13 levels in vitro and ECM remodeling in vitro and in vivo were linked to changes in SOX9 subcellular localization. SOX9 was largely excluded from the nuclei of chondrocytes with MMP-13-remodeled or -degraded ECM, and exhibited an intranuclear staining pattern in chondrocytes with impaired MMP-13 activity in vitro or with more intact ECM in vivo.Conclusion. MMP-13 loss leads to a breakdown in primary human articular chondrocyte differentiation by altering the expression of multiple regulatory factors.The maturation of chondrocytes is arrested in articular cartilage, which prevents their differentiation toward a more terminal hypertrophic-like phenotype (1). The mechanisms maintaining articular chondrocyte homeostasis are perturbed in osteoarthritis (OA), in which chondrocytes recapitulate aspects of endochon-