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1.
Robert Terkeltaub Bing Yang Martin Lotz Ru Liu‐Bryan 《Arthritis \u0026amp; Rheumatology》2011,63(7):1928-1937
Objective
Interleukin‐1β (IL‐1β) and tumor necrosis factor α (TNFα) stimulate chondrocyte matrix catabolic responses, thereby compromising cartilage homeostasis in osteoarthritis (OA). AMP‐activated protein kinase (AMPK), which regulates energy homeostasis and cellular metabolism, also exerts antiinflammatory effects in multiple tissues. This study was undertaken to test the hypothesis that AMPK activity limits chondrocyte matrix catabolic responses to IL‐1β and TNFα.Methods
Expression of AMPK subunits was examined, and AMPKα activity was ascertained by the phosphorylation status of AMPKα Thr172 in human knee articular chondrocytes and cartilage by Western blotting and immunohistochemistry, respectively. Procatabolic responses to IL‐1β and TNFα, such as release of glycosaminoglycan, nitric oxide, and matrix metalloproteinases 3 and 13 were determined by dimethylmethylene blue assay, Griess reaction, and Western blotting, respectively, in cartilage explants and chondrocytes with and without knockdown of AMPKα by small interfering RNA.Results
Normal human knee articular chondrocytes expressed AMPKα1, α2, β1, β2, and γ1 subunits. AMPK activity was constitutively present in normal articular chondrocytes and cartilage, but decreased in OA articular chondrocytes and cartilage and in normal chondrocytes treated with IL‐1β and TNFα. Knockdown of AMPKα resulted in enhanced catabolic responses to IL‐1β and TNFα in chondrocytes. Moreover, AMPK activators suppressed cartilage/chondrocyte procatabolic responses to IL‐1β and TNFα and the capacity of TNFα and CXCL8 (IL‐8) to induce type X collagen expression.Conclusion
Our findings indicate that AMPK activity is reduced in OA cartilage and in chondrocytes following treatment with IL‐1β or TNFα. AMPK activators attenuate dephosphorylation of AMPKα and procatabolic responses in chondrocytes induced by these cytokines. These observations suggest that maintenance of AMPK activity supports cartilage homeostasis by protecting cartilage matrix from inflammation‐induced degradation.2.
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Edith Bonnelye Pascal Reboul Nicolas Duval Marco Cardelli Jane E. Aubin 《Arthritis \u0026amp; Rheumatology》2011,63(8):2374-2384
Objective
We reported previously that the orphan nuclear receptor, estrogen receptor–related receptor α (ERRα), is expressed in articular chondrocytes and is dysregulated in a mouse model of inflammatory arthritis. The aim of this study, therefore, was to determine whether ERRα is also dysregulated in patients with osteoarthritis (OA).Methods
ERRα messenger RNA (mRNA) and protein were quantified in normal and OA cartilage samples and in OA chondrocytes in vitro, with and without short‐term treatment with a variety of OA‐associated factors and signaling pathway agonists and inhibitors.Results
ERRα expression was lower in OA than in normal articular cartilage. Interleukin‐1β (IL‐1β) markedly up‐regulated ERRα expression in OA chondrocytes in vitro, and agonist or inhibitor treatment indicated that the up‐regulation was dependent on cyclooxygenase 2 (COX‐2; NS398), prostaglandin E2, cAMP (8‐bromo‐cAMP), and protein kinase A (PKA; KT5720). Treatment with the ERRα inverse agonist XCT790 decreased the expression of SOX9 and the up‐regulation of ERRα by IL‐1β, suggesting autoregulation of ERRα in the IL‐1β pathway. Matrix metalloproteinase 13 (MMP‐13) expression was also decreased by treatment with XCT790 plus IL‐1β versus IL‐1β alone, and the down‐regulation of MMP‐13 mRNA and protein observed with XCT790 alone suggests that the up‐regulation of MMP‐13 by IL‐1β is ERRα‐dependent.Conclusion
We report the first evidence that ERRα expression is regulated by IL‐1β in COX‐2–, cAMP‐, and PKA‐dependent pathways in OA chondrocytes. We confirmed that SOX9 is an ERRα target gene in human, as in rodent, chondrocytes and identified MMP‐13 as a potential new target gene, which suggests that ERRα may both respond to the healing signal and contribute to extracellular degradation in OA cartilage.4.
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Sohel M. Julovi Tadashi Yasuda Makoto Shimizu Teruko Hiramitsu Takashi Nakamura 《Arthritis \u0026amp; Rheumatology》2004,50(2):516-525
Objective
To investigate the mechanism of the inhibitory action of hyaluronan (HA) on interleukin‐1β (IL‐1β)‐stimulated production of matrix metalloproteinases (MMPs) in human articular cartilage.Methods
IL‐1β was added to normal and osteoarthritic (OA) human articular cartilage in explant culture to stimulate MMP production. Articular cartilage was incubated or preincubated with a clinically used form of 800‐kd HA to assess its effect on IL‐1β‐induced MMPs. Levels of secreted MMPs 1, 3, and 13 in conditioned media were detected by immunoblotting; intracellular MMP synthesis in chondrocytes was evaluated by immunofluorescence microscopy. Penetration of HA into cartilage tissue and its binding to CD44 were analyzed by fluorescence microscopy using fluoresceinated HA. Blocking experiments with anti‐CD44 antibody were performed to investigate the mechanism of action of HA.Results
Treatment and pretreatment with 800‐kd HA at 1 mg/ml resulted in significant suppression of IL‐1β‐stimulated production of MMPs 1, 3, and 13 in normal and OA cartilage explant culture. Fluorescence histocytochemistry revealed that HA penetrated cartilage tissue and localized in the pericellular matrix around chondrocytes. HA‐binding blocking experiments using anti‐CD44 antibody demonstrated that the association of HA with chondrocytes was mediated by CD44. Preincubation with anti‐CD44 antibody, which suppressed IL‐1β‐stimulated MMPs, reversed the inhibitory effect of HA on MMP production that was induced by IL‐1β in normal and OA cartilage.Conclusion
This study demonstrates that HA effectively inhibits IL‐1β‐stimulated production of MMP‐1, MMP‐3, and MMP‐13, which supports the clinical use of HA in the treatment of OA. The action of HA on IL‐1β may involve direct interaction between HA and CD44 on chondrocytes.6.
Alwin Scharstuhl Elly L. Vitters Peter M. van der Kraan Wim B. van den Berg 《Arthritis \u0026amp; Rheumatology》2003,48(12):3442-3451
Objective
Osteoarthritis (OA) is a joint disease characterized by osteophyte development, fibrosis, and articular cartilage damage. Effects of exogenous transforming growth factor β (TGFβ) isoforms and bone morphogenetic proteins (BMPs) suggest a role for these growth factors in the pathogenesis of OA. The aim of this study was to elucidate the role of endogenous TGFβ and BMP during papain‐induced OA‐like changes in mice.Methods
We used adenoviral overexpression of TGFβ and BMP antagonists to block growth factor signaling. An adenovirus expressing a secreted, pan–specific TGFβ antagonist called murine latency‐associated peptide 1 (mLAP‐1) was used. In addition, we used intracellular inhibitory Smad6 as a BMP antagonist and Smad7 as a TGFβ/BMP inhibitor. Papain was injected into the knee joints of C57BL/6 mice to induce osteophyte development, synovial thickening, and articular cartilage proteoglycan (PG) loss.Results
Intraarticular injection of papain caused increased protein expression of several TGFβ and BMP isoforms in synovium and cartilage. Adenovirus transfection into the joint resulted in a strong expression of the transgenes in the synovial lining. Overexpression of mLAP‐1, Smad6, and Smad7 led to a significant reduction in osteophyte formation compared with that in controls. Smad6 and Smad7 overexpression also significantly decreased synovial thickening. Furthermore, the secreted TGFβ inhibitor mLAP‐1 increased articular cartilage PG loss.Conclusion
Our results indicate a pivotal role of endogenous TGFβ in the development of osteophytes and synovial thickening, implicating endogenous TGFβ in the pathogenesis of OA. In contrast, the prevention of cartilage damage by endogenous TGFβ signifies the protective role of TGFβ in articular cartilage. This is the first study to demonstrate that endogenous BMPs are involved in osteophyte formation and synovial thickening in experimental OA.7.
Zhiyong Fan Brigitte Bau Huiqing Yang Stephan Soeder Thomas Aigner 《Arthritis \u0026amp; Rheumatology》2005,52(1):136-143
Objective
Interleukin‐1β (IL‐1β) is one potentially important cytokine during cartilage destruction. The aim of this study was to investigate whether there are different effects of low and high concentrations of IL‐1β on the expression level of anabolic genes (type II collagen, aggrecan), catabolic genes (matrix metalloproteinase 1 [MMP‐1], MMP‐2, MMP‐3, MMP‐13, and ADAMTS‐4), and cytokines (IL‐1β, IL‐6, and leukemia inhibitory factor [LIF]) by articular chondrocytes (normal and osteoarthritic). Determination of whether there was a difference in reactivity between normal and osteoarthritic chondrocytes was also a goal of this study.Methods
Gene expression levels were detected by real‐time polymerase chain reaction from isolated (nonpassaged) chondrocytes (normal [n = 6]; osteoarthritic [n = 7]) after stimulation with 0.01 ng, 0.1 ng, 1 ng, and 10 ng/ml IL‐1β.Results
In normal adult articular chondrocytes the expression of both aggrecan and type II collagen genes was significantly down‐regulated, whereas matrix‐degrading proteases (except MMP‐2), as well as the investigated cytokines, were induced by IL‐1β in a dose‐dependent manner. The strongest regulation was found for IL‐6 and LIF. Osteoarthritic chondrocytes showed strongly increased levels of catabolic enzymes and mediators, but were less responsive to further stimulation with IL‐1β.Conclusion
Our study confirms that IL‐1β activity is critically dependent on both the applied concentration and the reactivity of the cells stimulated. The responsiveness appears to be significantly reduced in late‐stage osteoarthritic chondrocytes. However, these cells show high basic expression levels of catabolic enzymes and mediators. Thus, it remains open whether our data indicate that osteoarthritic chondrocytes are per se not responsive to IL‐1β or are already so strongly stimulated (e.g., by IL‐1) during the disease process that they are refractory to further stimulation.8.
Tetsuya Kobayashi Kohei Notoya Takako Naito Satoko Unno Akihiro Nakamura Johanne Martel‐Pelletier Jean‐Pierre Pelletier 《Arthritis \u0026amp; Rheumatology》2005,52(2):479-487
Objective
To evaluate the in vivo therapeutic effect of pioglitazone, a peroxisome proliferator–activated receptor γ (PPARγ) agonist, on the development of lesions in a guinea pig model of osteoarthritis (OA), and to determine the influence of pioglitazone on the synthesis of matrix metalloproteinase 13 (MMP‐13) and interleukin‐1β (IL‐1β) in articular cartilage.Methods
The OA model was created by partial medial meniscectomy of the right knee joint. The guinea pigs were divided into 4 treatment groups: unoperated animals that received no treatment (normal), operated animals (OA guinea pigs) that received placebo, OA guinea pigs that received oral pioglitazone at 2 mg/kg/day, and OA guinea pigs that received oral pioglitazone at 20 mg/kg/day. The animals began receiving medication 1 day after surgery and were killed 4 weeks later. Macroscopic and histologic analyses were performed on the cartilage. The levels of MMP‐13 and IL‐1β in OA cartilage chondrocytes were evaluated by immunohistochemistry.Results
OA guinea pigs treated with the highest dosages of pioglitazone showed a significant decrease, compared with the OA placebo group, in the surface area (size) and grade (depth) of cartilage macroscopic lesions on the tibial plateaus. The histologic severity of cartilage lesions was also reduced. A significantly higher percentage of chondrocytes in the middle and deep layers stained positive for MMP‐13 and IL‐1β in cartilage from placebo‐treated OA guinea pigs compared with normal controls. Guinea pigs treated with the highest dosage of pioglitazone demonstrated a significant reduction in the levels of both MMP‐13 and IL‐1β in OA cartilage.Conclusion
This is the first in vivo study demonstrating that a PPARγ agonist, pioglitazone, could reduce the severity of experimental OA. This effect was associated with a reduction in the levels of MMP‐13 and IL‐1β, which are known to play an important role in the pathophysiology of OA lesions.9.
Richard F. Loeser Cathy S. Carlson Marcello Del Carlo Ada Cole 《Arthritis \u0026amp; Rheumatology》2002,46(9):2349-2357
Objective
To determine whether oxidative damage to cartilage proteins can be detected in aging and osteoarthritic (OA) cartilage, and to correlate the results with the local production of interleukin‐1β (IL‐1β) and the responsiveness of isolated chondrocytes to stimulation with insulin‐like growth factor 1 (IGF‐1).Methods
The presence of nitrotyrosine was used as a measure of oxidative damage. Histologic sections of knee articular cartilage, obtained from young adult and old adult cynomolgus monkeys, which develop age‐related, naturally occurring OA, were evaluated. Each cartilage section was graded histologically on a scale of 0–7 for the presence of OA‐like changes, and serial sections were immunostained using antibodies to nitrotyrosine and IL‐1β. Chondrocytes isolated and cultured from cartilage adjacent to the sections used for immunostaining were tested for their response to IGF‐1 stimulation by measuring sulfate incorporation in alginate cultures. For comparison with the monkey tissues, cartilage sections from human tissue donors and from tissue removed at the time of OA‐related joint replacement surgery were also immunostained for nitrotyrosine and IL‐1β.Results
The presence of nitrotyrosine was associated with aging and with the development of OA in cartilage samples from both monkeys and humans. All sections that were highly positive for IL‐1β also showed staining for nitrotyrosine. However, in a few sections from older adult monkeys and humans, nitrotyrosine was present but IL‐1β was absent, suggesting that some age‐related oxidative damage is independent of IL‐1β. In chondrocytes that were isolated from monkey cartilage positive for nitrotyrosine or IL‐1β, the response to stimulation with IGF‐1 was significantly reduced. In some samples from older adult monkeys, IGF‐1 resistance was seen in cells isolated from tissue that did not stain for nitrotyrosine or IL‐1β.Conclusion
Oxidative damage due to the concomitant overproduction of nitric oxide and other reactive oxygen species is present in both aging and OA cartilage. This damage can contribute to the resistance of chondrocytes to IGF‐1 stimulation, but it is unlikely to be the sole cause of IGF‐1 resistance in these chondrocytes.10.
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Shigeru Miyaki Tomoyuki Nakasa Shuhei Otsuki Shawn P. Grogan Reiji Higashiyama Atsushi Inoue Yoshio Kato Tempei Sato Martin K. Lotz Hiroshi Asahara 《Arthritis \u0026amp; Rheumatology》2009,60(9):2723-2730
Objective
MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue‐specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA‐140 (miR‐140).Methods
To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR‐140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin‐1β (IL‐1β) on miR‐140 expression. Double‐stranded miR‐140 (ds–miR‐140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA.Results
Microarray analysis showed that miR‐140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR‐140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR‐140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL‐1β suppressed miR‐140 expression. Transfection of chondrocytes with ds–miR‐140 down‐regulated IL‐1β–induced ADAMTS5 expression and rescued the IL‐1β–dependent repression of AGGRECAN gene expression.Conclusion
This study shows that miR‐140 has a chondrocyte differentiation–related expression pattern. The reduction in miR‐140 expression in OA cartilage and in response to IL‐1β may contribute to the abnormal gene expression pattern characteristic of OA.19.
Medora M. Hardy Karen Seibert Pamela T. Manning Mark G. Currie B. Mark Woerner Dorothy Edwards Alane Koki Catherine S. Tripp 《Arthritis \u0026amp; Rheumatology》2002,46(7):1789-1803
Objective
To examine cyclooxygenase‐2 (COX‐2) enzyme expression, its regulation by interleukin‐1β (IL‐1β), and the role of prostaglandin E2 (PGE2) in proteoglycan degradation in human osteoarthritic (OA) cartilage.Methods
Samples of human OA articular cartilage, meniscus, synovial membrane, and osteophytic fibrocartilage were obtained at knee arthroplasty and cultured ex vivo with or without IL‐1β and COX inhibitors. COX expression was evaluated by immunohistochemistry and Western blot analysis. The enzymatic activity of COX was measured by conversion of arachidonic acid to PGE2. Cartilage degradation was evaluated by measuring the accumulation of sulfated glycosaminoglycans in the medium.Results
IL‐1β induced robust expression of COX‐2 and PGE2 in OA meniscus, synovial membrane, and osteophytic fibrocartilage explants, whereas low levels were produced in OA articular cartilage. IL‐1β also induced cartilage proteoglycan degradation in OA synovial membrane‐cartilage cocultures. Increased proteoglycan degradation corresponded to the induction of COX‐2 protein expression in, and PGE2 production from, the synovial membrane. Dexamethasone, neutralizing IL‐1β antibody, or the selective COX‐2 inhibitor, SC‐236, attenuated both the IL‐1β‐induced PGE2 production and cartilage proteoglycan degradation in these cocultures. The addition of PGE2 reversed the inhibition of proteoglycan degradation caused by SC‐236.Conclusion
IL‐1β‐induced production of COX‐2 protein and PGE2 was low in OA articular cartilage compared with that in the other OA tissues examined. IL‐1β‐mediated degradation of cartilage proteoglycans in OA synovial membrane‐cartilage cocultures was blocked by the selective COX‐2 inhibitor, SC‐236, and the effect of SC‐236 was reversed by the addition of exogenous PGE2. Our data suggest that induction of synovial COX‐2‐produced PGE2 is one mechanism by which IL‐1β modulates cartilage proteoglycan degradation in OA.20.
Carol G. Chen Daniel Thuillier Emily N. Chin Tamara Alliston 《Arthritis \u0026amp; Rheumatology》2012,64(10):3278-3289