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 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.

Introduction: Recent evidence suggests that tissue accumulation of senescent p16^INK4a-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 p16^INK4a 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 p16^INK4a in the OA-induced SASP and its regulation by microRNAs (miRs). Methods: We used IL-1-beta-treated primary OA chondrocytes cultured in three-dimensional setting or mesenchymal stem cells differentiated into chondrocyte to follow p16^INK4a expression. By transient transfection experiments and the use of knockout mice, we validate p16^INK4a function in chondrocytes and its regulation by one miR identified by means of a genome-wide miR-array analysis. Results: p16^INK4a 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, p16^INK4a 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 p16^INK4a. Accordingly, p16^INK4a expression increased while miR-24 level was repressed upon IL-1-beta addition, in OA cartilage and during in vitro terminal chondrogenesis. Conclusions: We disclosed herein a new role of the senescence marker p16^INK4a and its regulation by miR-24 during OA and terminal chondrogenesis.

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.

Osteoarthritis (OA) is one of the most common joint disorders. Evidence suggests that the infrapatellar fat pad (IFP) is directly involved in OA pathology. However, a comparison between OA versus non-OA IFP is still missing. Thus, the aim of this study was to compare IFP molecular, adipocytes and extracellular matrix characteristics of patients affected by OA, and patients undergoing anterior cruciate ligament (ACL) reconstruction. We hypothesized that not only inflammation but also changes in adipocytes and extracellular matrix (ECM) composition might be involved in OA pathogenesis. Fifty-three patients were enrolled. IFP biopsies were obtained, evaluating: (a) lymphocytic infiltration and vascularization; (b) adipocytes area and number; (c) adipo-cytokines and extracellular matrix gene expression levels; (d) IL-6 and VEGF protein production; (e) collagen fibers distribution. OA IFP was more inflamed and vascularized compared to ACL IFP. OA IFP adipocytes were larger and numerically lower (1.3-fold) than ACL IFP adipocytes. An increase of gene expression of typical white adipose tissue genes was observed in OA compared to ACL IFP. Collagen-types distribution was different in the OA IFP group compared to controls, possibly explaining the change of the biomechanical characteristics found in OA IFP. Statistical linear models revealed that the adipocyte area correlated with BMI in the OA group. In conclusion, inflammation and fibrotic changes of OA IFP could represent novel therapeutic targets to counteract OA.

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.