Chondrocyte AMP‐activated protein kinase activity suppresses matrix degradation responses to proinflammatory cytokines interleukin‐1β and tumor necrosis factor α

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.

     

Bone morphogenetic protein and transforming growth factor β inhibitory Smads 6 and 7 are expressed in human adult normal and osteoarthritic cartilage in vivo and are differentially regulated in vitro by interleukin‐1β

Objective

Bone morphogenetic protein (BMP) and transforming growth factor β (TGFβ) are potent anabolic factors in adult articular chondrocytes. In this study, we investigated whether intracellular inhibitors of BMP and TGFβ signaling, inhibitory Smad6 (I‐Smad6) and I‐Smad7, are expressed in articular chondrocytes in normal and osteoarthritic (OA) cartilage, and whether their expression shows a correlation with the anabolic activity of OA chondrocytes in vivo and after interleukin‐1β (IL‐1β) stimulation in vitro.

Methods

RNA isolated directly from normal and OA human knee cartilage as well as from cultured articular chondrocytes was analyzed by (quantitative) polymerase chain reaction technology. Immunolocalization of the I‐Smads was performed on tissue sections and compared with the anabolic cellular activity as documented by in situ hybridization experiments for aggrecan and type II collagen.

Results

Both Smad6 and Smad7 were expressed in all samples of normal and OA cartilage. Immunostaining (including confocal microscopy) confirmed the presence of Smad6 and Smad7 in the majority of normal and degenerated articular chondrocytes; localization was mostly cytoplasmic. No correlation between expression of the main anabolic genes and expression of the I‐Smads was found. In cultured articular chondrocytes, stimulation with IL‐1β showed up‐regulation of Smad7, whereas Smad6 was down‐regulated.

Conclusion

Both Smad6 and Smad7 are expressed in adult human articular chondrocytes. The primarily cytoplasmic localization suggests permanent activation of the I‐Smads in articular cartilage in vivo. No evidence was found that up‐regulation or down‐regulation of I‐Smads in OA cartilage correlates directly with the anabolic (or catabolic) activity of articular chondrocytes. The regulation in chondrocytes of Smad6 and Smad7 expression by IL‐1β suggests a potentially important role of IL‐1β signaling in chondrocytes, via indirect influencing of the BMP/TGFβ signaling cascade.

     

Regulation of plasminogen activator inhibitor 1 expression in human osteoarthritic chondrocytes by fluid shear stress: Role of protein kinase Cα

Objective

To test a fluid flow system for the investigation of the influence of shear stress on expression of plasminogen activator inhibitor 1 (PAI‐1) in human osteoarthritic (OA) articular chondrocytes (from lesional and nonlesional sites) and human SW‐1353 chondrocytes.

Methods

Human SW‐1353 chondrocytes and OA and normal human articular chondrocytes were cultured on type II collagen–coated glass plates under static conditions or placed in a flow chamber to form a closed fluid‐circulation system for exposure to different levels of shear stress (2–20 dyn/cm²). Real‐time polymerase chain reaction was used to analyze PAI‐1 gene expression, and protein kinase C (PKC) inhibitors and small interfering RNA were used to investigate the mechanism of shear stress–induced signal transduction in SW‐1353 and OA (lesional and nonlesional) articular chondrocytes.

Results

There was a significant reduction in PAI‐1 expression in OA chondrocytes obtained from lesional sites compared with those obtained from nonlesional sites. In SW‐1353 chondrocytes subjected to 2 hours of shear flow, moderate shear stresses (5 and 10 dyn/cm²) generated significant PAI‐1 expression, which was regulated through PKCα phosphorylation and Sp‐1 activation. These levels of shear stress also increased PAI‐1 expression in articular chondrocytes from nonlesional sites and from normal healthy cartilage through the activation of PKCα and Sp‐1 signal transduction, but no effect of these levels of fluid shear stress was observed on OA chondrocytes from lesional sites.

Conclusion

OA chondrocytes from lesional sites and those from nonlesional sites of human cartilage have differential responses to shear stress with regard to PAI‐1 gene expression, and therefore diverse functional consequences can be observed.

     

Estrogen receptor–related receptor α regulation by interleukin‐1β in prostaglandin E2– and cAMP‐dependent pathways in osteoarthritic chondrocytes

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.

     

Molecular phenotyping of human chondrocyte cell lines T/C‐28a2, T/C‐28a4, and C‐28/I2

Objective

Because the immortalized chondrocyte cell lines C‐28/I2, T/C‐28a2, and T/C‐28a4 have become a common tool in cartilage research, permitting investigations in a largely unlimited and standardized manner, we investigated the molecular phenotype of these cell lines by gene expression profiling.

Methods

Complementary DNA–array analysis as well as online quantitative polymerase chain reaction were used to identify the gene expression profiles of the 3 cell lines cultured in monolayer and alginate beads, as compared with the expression profiles of cultured human adult primary chondrocytes.

Results

A similar, but not identical, gene expression profile was established for all 3 cell lines. SOX9 was expressed at a significant level in all 3 cell lines. Extracellular matrix proteins and matrix‐degrading proteases were rarely expressed. In contrast, genes involved in the cell cycle were strongly up‐regulated, as compared with the expression levels in physiologic chondrocytes.

Conclusion

The expression of SOX9, the master gene of chondrocytic cell differentiation, reflects the basically chondrocytic phenotype of these cells. However, the major issue appears to be that these cell lines mainly proliferate and show less expression of genes involved in matrix synthesis and turnover. In this respect, C‐28/I2 cells display the highest levels of matrix‐anabolic and matrix‐catabolic genes and thus are presumably preferable for use in investigating chondrocyte anabolic and catabolic activity and its regulation. None of the 3 cell lines appears to be a direct substitute for primary chondrocytes. A successful approach will have to validate the findings obtained with chondrocyte cell lines by using primary chondrocytes or cartilage‐tissue cultures. This would permit the establishment of reproducible in vitro models and subsequently allow investigators to relate the findings to the physiologic situation.

     

Freshly isolated osteoarthritic chondrocytes are catabolically more active than normal chondrocytes,but less responsive to catabolic stimulation with interleukin‐1β

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.

     

Lectin‐like oxidized low‐density lipoprotein receptor 1 mediates matrix metalloproteinase 3 synthesis enhanced by oxidized low‐density lipoprotein in rheumatoid arthritis cartilage

Objective

To investigate for the presence of oxidized low‐density lipoprotein (ox‐LDL) and lectin‐like oxidized LDL receptor 1 (LOX‐1) in cartilage specimens from rheumatoid arthritis (RA) joints and to determine whether the interaction of ox‐LDL with LOX‐1 can induce matrix metalloproteinase 3 (MMP‐3) in articular cartilage explant culture.

Methods

Human articular cartilage specimens obtained from patients with RA, osteoarthritis (OA), and femoral neck fractures were examined for LOX‐1 and ox‐LDL by confocal fluorescence microscopy. The association between ox‐LDL and LOX‐1 was evaluated by immunofluorescence analysis. Articular cartilage specimens from patients with femoral neck fractures were incubated with ox‐LDL, with or without preincubation with neutralizing anti–LOX‐1 antibody. MMP‐3 synthesis by chondrocytes in explant cartilage was evaluated by immunofluorescence, and protein secretion into conditioned medium was monitored by immunoblotting and enzyme‐linked immunosorbent assay.

Results

The majority of the RA chondrocytes stained positively with both anti–LOX‐1 and anti–ox‐LDL antibodies; however, no positive cells were found in OA and normal cartilage specimens. Anti–LOX‐1 antibody suppressed the binding of DiI‐labeled ox‐LDL to chondrocytes in explant culture, suggesting that the interaction was mediated by LOX‐1. In contrast to native LDL, ox‐LDL induced MMP‐3 synthesis by articular chondrocytes in association with the induction of LOX‐1, which resulted in enhanced secretion of MMP‐3 into the culture medium. Anti–LOX‐1 antibody reversed ox‐LDL–stimulated MMP‐3 synthesis to control levels.

Conclusion

Ox‐LDL, principally mediated by LOX‐1, enhanced MMP‐3 production in articular chondrocytes. Increased accumulation of ox‐LDL with elevated expression of LOX‐1 in RA cartilage indicates a specific role of the receptor–ligand interaction in cartilage pathology in RA.

     

Genetic inhibition of fibroblast growth factor receptor 1 in knee cartilage attenuates the degeneration of articular cartilage in adult mice

Objective

Fibroblast growth factor (FGF) family members are involved in the regulation of articular cartilage homeostasis. The aim of this study was to investigate the function of FGF receptor 1 (FGFR‐1) in the development of osteoarthritis (OA) and its underlying mechanisms.

Methods

FGFR‐1 was deleted from the articular chondrocytes of adult mice in a cartilage‐specific and tamoxifen‐inducible manner. Two OA models (aging‐associated spontaneous OA, and destabilization‐induced OA), as well as an antigen‐induced arthritis (AIA) model, were established and tested in Fgfr1‐deficient and wild‐type (WT) mice. Alterations in cartilage structure and the loss of proteoglycan were assessed in the knee joints of mice of either genotype, using these 3 arthritis models. Primary chondrocytes were isolated and the expression of key regulatory molecules was assessed quantitatively. In addition, the effect of an FGFR‐1 inhibitor on human articular chondrocytes was examined.

Results

The gross morphologic features of Fgfr1‐deficient mice were comparable with those of WT mice at both the postnatal and adult stages. The articular cartilage of 12‐month‐old Fgfr1‐deficient mice displayed greater aggrecan staining compared to 12‐month‐old WT mice. Fgfr1 deficiency conferred resistance to the proteoglycan loss induced by AIA and attenuated the development of cartilage destruction after surgically induced destabilization of the knee joint. The chondroprotective effect of FGFR‐1 inhibition was largely associated with decreased expression of matrix metalloproteinase 13 (MMP‐13) and up‐regulation of FGFR‐3 in mouse and human articular chondrocytes.

Conclusion

Disruption of FGFR‐1 in adult mouse articular chondrocytes inhibits the progression of cartilage degeneration. Down‐regulation of MMP‐13 expression and up‐regulation of FGFR‐3 levels may contribute to the phenotypic changes observed in Fgfr1‐deficient mice.

     

MicroRNA‐140 is expressed in differentiated human articular chondrocytes and modulates interleukin‐1 responses

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.

     

Inhibition of interleukin‐1β‐stimulated production of matrix metalloproteinases by hyaluronan via CD44 in human articular cartilage

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.

     

Catabolic stress induces features of chondrocyte senescence through overexpression of caveolin 1: Possible involvement of caveolin 1–induced down‐regulation of articular chondrocytes in the pathogenesis of osteoarthritis

Objective

Articular chondrocyte senescence is responsible, at least in part, for the increased incidence of osteoarthritis (OA) with increased age. Recently, it was suggested that caveolin 1, a 21–24‐kd membrane protein, participates in premature cellular senescence. Caveolin 1 is the principal structural component of caveolae, vesicular invaginations of the plasma membrane. This study was undertaken to investigate whether the catabolic factors oxidative stress and interleukin‐1β (IL‐1β) induce features of premature senescence of articular chondrocytes through up‐regulation of caveolin 1 expression.

Methods

Caveolin 1 expression was investigated in human OA cartilage by real‐time polymerase chain reaction and in rat OA cartilage by immunohistologic analysis. We studied whether IL‐1β and H₂O₂ induce caveolin 1 expression in OA chondrocytes and analyzed the relationship between cellular senescent phenotypes and caveolin 1 expression in human chondrocytes.

Results

In human and rat OA articular cartilage, caveolin 1 positivity was associated with cartilage degeneration. Both IL‐1β and H₂O₂ up‐regulated caveolin 1 messenger RNA and protein levels, and both treatments induced marked expression of senescent phenotypes: altered cellular morphology, cell growth arrest, telomere erosion, and specific senescence‐associated β‐galactosidase activity. Caveolin 1 overexpression induced p38 MAPK activation and impaired the ability of chondrocytes to produce type II collagen and aggrecan. In contrast, down‐regulation of caveolin 1 with antisense oligonucleotide significantly inhibited the features of chondrocyte senescence induced by catabolic factors. Caveolin 1 induction and stresses with both IL‐1β and H₂O₂ up‐regulated p53 and p21 and down‐regulated phosphorylated retinoblastoma (Rb), suggesting that the p53/p21/Rb phosphorylation pathway, as well as prolonged p38 MAPK activation, may mediate the features of chondrocyte senescence induced by stress.

Conclusion

Our findings suggest that IL‐1β and oxidative stress induce features of premature senescence in OA chondrocytes, mediated, at least in part, by stress‐induced caveolin 1 expression. This indicates that caveolin 1 plays a role in the pathogenesis of OA via promotion of chondrocyte down‐regulation.