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 ... -  《-》

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 ... -  《-》

The role of ANK interactions with MYBBP1a and SPHK1 in catabolic events of articular chondrocytes.

To determine the role of progressive ankylosis protein (ANK)/Myb-binding protein 1a (MYBBP1a) and sphingosine kinase 1 (SPHK1) interactions in catabolic events of articular chondrocytes. ANK/MYBBP1a and SPHK1 interactions were identified using yeast two-hybrid screening and co-immunoprecipitation. To determine the role of these interactions in catabolic events of articular chondrocytes, ank/ank and wild type (WT) mouse chondrocytes transfected with full-length or mutant ank expression vectors (EVs) or femoral heads were treated with interleukin-1beta (IL-1β) in the absence or presence of SPHK inhibitor. Catabolic marker mRNA levels were analyzed by real time PCR; proteoglycan loss using safranin O staining and MMP-13 immunostaining were determined in femoral head explants; NF-κB activity was determined by transfecting chondrocytes with an NF-κB-specific luciferase reporter and analyzing nuclear translocation of p65 by immunoblotting; MYBBP1a nuclear or cytoplasmic amounts were determined by immunohistochemistry and immunoblotting. The ANK N-terminal region interacted with SPHK1, whereas a cytoplasmic C-terminal loop interacted with MYBBP1a. Lack of ANK/MYBBP1a and SPHK1 interactions in ank/ank chondrocytes resulted in increased MYBBP1a nuclear amounts and decreased SPHK1 activity, and consequently decreased NF-κB activity, catabolic marker mRNA levels, proteoglycan loss, and MMP-13 immunostaining in IL-1β-treated articular chondrocytes or femoral heads. Transfection with full-length ank EV reduced nuclear MYBBP1a amounts and fully restored SPHK and NF-κB activities in IL-1β-treated ank/ank chondrocytes, whereas transfection with P5L or F376del mutant ank reduced nuclear MYBBP1a or increased SPHK activity, respectively, and consequently either transfection only partially restored NF-κB activity. ANK/MYBBP1a and SPHK1 interactions stimulate catabolic events in IL-1β-mediated cartilage degradation.

Minashima T ，Campbell KA ，Hadley SR ，Zhang Y ，Kirsch T ... -  《-》

Modulation of TGF-beta signaling by proinflammatory cytokines in articular chondrocytes.

Roman-Blas JA ，Stokes DG ，Jimenez SA 《OSTEOARTHRITIS AND CARTILAGE》

MicroRNA-558 regulates the expression of cyclooxygenase-2 and IL-1β-induced catabolic effects in human articular chondrocytes.

Cyclooxygenase-2 (COX-2) is a major prostaglandin E2 (PGE2) synthetic enzyme and is involved in the pathogenesis of chronic inflammation and pain in osteoarthritis (OA). The objective of this study was to directly address whether microRNA (miR)-558 can control the interleukin (IL)-1β-mediated induction of COX-2 and catabolic effects in human articular chondrocytes. Total RNA was extracted from the cartilage tissues of normal and OA donors or cultured human articular chondrocytes. The expression of miR-558 was quantified by TaqMan assay. To investigate the repressive effect of miR-558 on COX-2 expression, human chondrocytes and chondrogenic SW1353 cells were transfected with mature miR-558 or an antisense inhibitor (anti-miR-558). The expression of COX-2 protein was determined by Western blot analysis and the involvement of miR-558 in IL-1β-induced catabolic effects was examined by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Direct interaction between miR-558 and the putative site in the 3'-untranslated region (UTR) of COX-2 messenger RNA (mRNA) was validated by luciferase reporter assay. Normal human articular cartilage expressed miR-558, and its expression was significantly lower in OA cartilage. Stimulation with IL-1β led to a significant reduction in miR-558 expression in normal and OA chondrocytes. IL-1β-induced activation of MAP kinase (MAPK) and nuclear factor-κB (NF-κB) decreased miR-558 expression and induced COX-2 expression in chondrocytes. The overexpression of miR-558 directly suppressed the luciferase activity of a reporter construct containing the 3'-UTR of human COX-2 mRNA and significantly inhibited IL-1β-induced upregulation of COX-2, while treatment with anti-miR-558 enhanced IL-1β-induced COX-2 expression and reporter activity in chondrocytes. Interestingly, IL-1β-induced activation of NF-κB and expression of matrix metalloproteinase (MMP)-1 and MMP-13 was significantly inhibited by miR-558 overexpression. These findings demonstrated that cartilage homeostasis is influenced by miR-558, which directly targets COX-2 and regulates IL-1β-stimulated catabolic effects in human chondrocytes.

Park SJ ，Cheon EJ ，Kim HA 《-》