Alleviation of Murine Osteoarthritis by Cartilage-Specific Deletion of IκBζ.

IκBζ, an atypical IκB family member, regulates gene expression in the nucleus as a transcriptional cofactor. Although IκBζ has been extensively studied in the immune system, its specific roles in osteoarthritis (OA) are currently unknown. The objective of this study was to investigate the potential role of IκBζ in chondrocyte catabolism and OA pathogenesis. We also determined the molecular mechanism underlying its relationship to the transcription factor NF-κB. We determined expression levels of IκBζ in mouse chondrocytes treated with interleukin-1β (IL-1β), in human OA cartilage, and in mouse experimental OA cartilage. Adenovirus-mediated overexpression and small interfering RNA knockdown of IκBζ were performed to determine the impact of IκBζ on catabolic gene expression in vitro. Cartilage-specific IκBζ-transgenic and -knockout mice were generated and used for in vivo studies. Experimental and spontaneous OA were induced by surgical destabilization of the medial meniscus and by aging, respectively. Coimmunoprecipitation assay was used to examine the association between IκBζ and NF-κB subunits. IκBζ was highly up-regulated in chondrocytes in response to IL-1β and in OA cartilage of human and mouse knee joints. Overexpression of IκBζ in chondrocytes promoted spontaneous OA development by activating chondrocyte catabolism. Genetic ablation of IκBζ in chondrocytes abolished catabolic gene induction by IL-1β and protected against the development of experimental OA. IκBζ formed complexes with NF-κB members to regulate catabolic factor expression. These findings demonstrate a critical role for IκBζ in OA pathogenesis. Inhibition of IκBζ function might be an effective therapeutic approach for OA treatment.

Choi MC ，MaruYama T ，Chun CH ，Park Y ... -  《-》

Annexin A6 interacts with p65 and stimulates NF-κB activity and catabolic events in articular chondrocytes.

ANXA6, the gene for annexin A6, is highly expressed in osteoarthritic (OA) articular chondrocytes but not in healthy articular chondrocytes. This study was undertaken to determine whether annexin A6 affects catabolic events in these cells. Articular chondrocytes were isolated from Anxa6-knockout mice, wild-type (WT) mice, and human articular cartilage in which ANXA6 was overexpressed. Cells were treated with interleukin-1β (IL-1β) or tumor necrosis factor α (TNFα), and expression of catabolic genes and activation of NF-κB were determined by real-time polymerase chain reaction and luciferase reporter assay. Anxa6(-/-) and WT mouse knee joints were injected with IL-1β or the medial collateral ligament was transected and partial resection of the medial meniscus was performed to determine the role of Anxa6 in IL-1β-mediated cartilage destruction and OA progression. The mechanism by which Anxa6 stimulates NF-κB activity was determined by coimmunoprecipitation and immunoblot analysis of nuclear and cytoplasmic fractions of IL-1β-treated Anxa6(-/-) and WT mouse chondrocytes for p65 and Anxa6. Loss of Anxa6 resulted in decreased NF-κB activation and catabolic marker messenger RNA (mRNA) levels in IL-1β- or TNFα-treated articular chondrocytes, whereas overexpression of ANXA6 resulted in increased NF-κB activity and catabolic marker mRNA levels. Annexin A6 interacted with p65, and loss of Anxa6 caused decreased nuclear translocation and retention of the active p50/p65 NF-κB complex. Cartilage destruction in Anxa6(-/-) mouse knee joints after IL-1β injection or partial medial meniscectomy was reduced as compared to that in WT mouse joints. Our data define a role of annexin A6 in the modulation of NF-κB activity and in the stimulation of catabolic events in articular chondrocytes.

Campbell KA ，Minashima T ，Zhang Y ，Hadley S ，Lee YJ ，Giovinazzo J ，Quirno M ，Kirsch T ... -  《-》

Insulin-Like Growth Factor II (IGF-II) Inhibits IL-1β-Induced Cartilage Matrix Loss and Promotes Cartilage Integrity in Experimental Osteoarthritis.

Osteoarthritis (OA) is a widespread chronic joint disease characterized by articular cartilage destruction and accompanied by pain and disability. In this study, we found that the expression of Insulin-like Growth Factor II (IGF-II) was reduced in articular cartilage in human OA patients as well as in the murine experimental OA model of destabilization of the medial meniscus (DMM). In primary human articular chondrocytes, ectopic expression of lentiviral IGF-II inhibited pro-inflammatory cytokine IL-1β-induced NF-κB activation as well as catabolic gene expression. Interestingly, IGF-II did not significantly alter the phosphorylation states of ERK1/2 or Akt, which are kinases typically activated by IGF-I. Instead, it induced the activity of phospholipase C (PLC) and a PLC inhibitor blocked the inhibitory activity of IGF-II against IL-1β, suggesting that this activity is mediated through PLC. Furthermore, IGF-II increased cartilage matrix levels and decreased MMP13 protein expression in explanted human OA cartilage cultures in vitro. In the in vivo DMM model, intraarticular injection of lentiviral IGF-II led to enhanced cartilage matrix levels and decreased MMP13 protein expression, as well as reduced osteophyte formation and subchondral bone sclerosis. Therefore, our results suggest that IGF-II can promote cartilage integrity and halt knee joint destruction in OA.

Uchimura T ，Foote AT ，Smith EL ，Matzkin EG ，Zeng L ... -  《-》

Inhibition of BATF/JUN transcriptional activity protects against osteoarthritic cartilage destruction.

The basic leucine zipper transcription factor, ATF-like (BATF), a member of the Activator protein-1 family, promotes transcriptional activation or repression, depending on the interacting partners (JUN-B or C-JUN). Here, we investigated whether the BATF/JUN complex exerts regulatory effects on catabolic and anabolic gene expression in chondrocytes and contributes to the pathogenesis of osteoarthritis (OA). Primary cultured mouse chondrocytes were treated with proinflammatory cytokines (interleukin-1β, IL-6 or tumour necrosis factor-α) or infected with adenoviruses carrying the Batf gene (Ad-Batf). Expression of BATF and JUN was examined in human and mouse experimental OA cartilage samples. Experimental OA in mice was induced by destabilisation of the medial meniscus or intra-articular injection of Ad-Batf. The chromatin immunoprecipitation assay was used to examine the binding of BATF and JUN to the promoter regions of candidate genes. Overexpression of BATF, which forms a heterodimeric complex with JUN-B and C-JUN, induced upregulation of matrix-degrading enzymes and downregulation of cartilage matrix molecules in chondrocytes. BATF expression in mouse joint tissues promoted OA cartilage destruction, and conversely, knockout of Batf in mice suppressed experimental OA. Pharmacological inhibition of BATF/JUN transcriptional activity reduced the expression of matrix-degrading enzymes and protected against experimental OA in mice. BATF/JUN-B and BATF/C-JUN complexes play important roles in OA cartilage destruction through regulating anabolic and catabolic gene expression in chondrocytes. Our findings collectively support the utility of BATF as a therapeutic target for OA.

Rhee J ，Park SH ，Kim SK ，Kim JH ，Ha CW ，Chun CH ，Chun JS ... -  《-》

Lithium protects against cartilage degradation in osteoarthritis.

To determine the actions of lithium chloride (LiCl) on catabolic events in human articular chondrocytes, and the effects of LiCl on the progression and severity of cartilage degradation in interleukin-1β (IL-1β)-treated mouse knee joints and after surgical induction of osteoarthritis (OA) in a mouse model. Human articular chondrocytes were treated with LiCl followed by IL-1β, and the expression levels of catabolic genes were determined by real-time polymerase chain reaction. To understand the mechanism by which LiCl affects catabolic events in articular chondrocytes after IL-1β treatment, the activation of NF-κB was determined using luciferase reporter assays, and the activities of MAPKs and the STAT-3 signaling pathway were determined by immunoblot analysis of total cell lysates. Cultures of mouse femoral head explants treated with IL-1β and a mouse model of surgically induced OA were used to determine the effects of LiCl on proteoglycan loss and cartilage degradation. LiCl treatment resulted in decreased catabolic marker messenger RNA levels and activation of NF-κB, p38 MAPK, and STAT-3 signaling in IL-1β-treated articular chondrocytes. Furthermore, LiCl directly inhibited IL-6-stimulated activation of STAT-3 signaling. Consequently, the loss of proteoglycan and severity of cartilage destruction in LiCl-treated mouse knee joints 8 weeks after OA induction surgery or in LiCl-treated mouse femoral head explants after IL-1β treatment were markedly reduced compared to that in vehicle-treated joints or explants. LiCl reduced catabolic events in IL-1β-treated human articular chondrocytes and attenuated the severity of cartilage destruction in IL-1β-treated mouse femoral head explants and in the knee joints of mice with surgically induced OA, acting via inhibition of the activities of the NF-κB, p38, and STAT-3 signaling pathways.

Minashima T ，Zhang Y ，Lee Y ，Kirsch T ... -  《-》