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.

     

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.

     

SirT1 enhances survival of human osteoarthritic chondrocytes by repressing protein tyrosine phosphatase 1B and activating the insulin‐like growth factor receptor pathway

Objective

The protein deacetylase SirT1 inhibits apoptosis in a variety of cell systems by distinct mechanisms, yet its role in chondrocyte death has not been explored. We undertook the present study to assess the role of SirT1 in the survival of osteoarthritic (OA) chondrocytes in humans.

Methods

SirT1, protein tyrosine phosphatase 1B (PTP1B), and PTP1B mutant expression plasmids as well as SirT1 small interfering RNA (siRNA) and PTP1B siRNA were transfected into primary human chondrocytes. Levels of apoptosis were determined using flow cytometry, and activation of components of the insulin‐like growth factor receptor (IGFR)/Akt pathway was assessed using immunoblotting. OA and normal knee cartilage samples were subjected to immunohistochemical analysis.

Results

Expression of SirT1 in chondrocytes led to increased chondrocyte survival in either the presence or the absence of tumor necrosis factor α/actinomycin D, while a reduction of SirT1 by siRNA led to increased chondrocyte apoptosis. Expression of SirT1 in chondrocytes led to activation of IGFR and the downstream kinases phosphatidylinositol 3‐kinase, phosphoinosite‐dependent protein kinase 1, mTOR, and Akt, which in turn phosphorylated MDM2, inhibited p53, and blocked apoptosis. Activation of IGFR occurs at least in part via SirT1‐mediated repression of PTP1B. Expression of PTP1B in chondrocytes increased apoptosis and reduced IGFR phosphorylation, while down‐regulation of PTP1B by siRNA significantly decreased apoptosis. Examination of cartilage from normal donors and OA patients revealed that PTP1B levels are elevated in OA cartilage in which SirT1 levels are decreased.

Conclusion

For the first time, it has been demonstrated that SirT1 is a mediator of human chondrocyte survival via down‐regulation of PTP1B, a potent proapoptotic protein that is elevated in OA cartilage.

     

Semaphorin 3A is expressed in human osteoarthritic cartilage and antagonizes vascular endothelial growth factor 165–promoted chondrocyte migration: An implication for chondrocyte cloning

Objective

Vascular endothelial growth factor 165 (VEGF₁₆₅) and its receptors, including neuropilin 1 (NRP‐1), are overexpressed in human osteoarthritic (OA) articular cartilage, although their functional roles in the cartilage are not fully understood. An axon‐guidance molecule, semaphorin 3A (Sema3A), which binds to NRP‐1, acts as an antagonist of VEGF signaling in endothelial cells. The aim of this study was to examine the expression of Sema3A and the functions of the VEGF₁₆₅/Sema3A/NRP‐1 axis in OA cartilage.

Methods

The expression of Sema3A in OA and normal cartilage samples was examined by real‐time polymerase chain reaction and immunohistochemical analyses. Functional analyses of VEGF₁₆₅ and Sema3A were carried out using OA chondrocytes in culture. The migration activity of chondrocytes was examined in a monolayer wound assay. The effects of Sema3A on VEGF₁₆₅‐induced up‐regulation of matrix metalloproteinases (MMPs) and intracellular signaling were also studied in cultured chondrocytes.

Results

Sema3A expression was significantly elevated in OA cartilage as compared to normal cartilage. Sema3A immunoreactivity directly correlated with the Mankin score and with chondrocyte cloning. VEGF₁₆₅ promoted the migration of chondrocytes, and this activity was suppressed by VEGF receptor 2 tyrosine kinase inhibitors. Sema3A antagonized the chondrocyte migration promoted by VEGF₁₆₅, and the activity was blocked by a selective inhibitor of, or small interfering RNA for, Sema3A. VEGF₁₆₅‐induced overexpression of MMPs and phosphorylation of ERK and focal adhesion kinase in chondrocytes were inhibited by Sema3A.

Conclusion

Our findings provide the first evidence that Sema3A is overexpressed, with a direct correlation with cloning, in OA cartilage and that it suppresses the VEGF₁₆₅‐promoted migration of chondrocytes. Our findings also suggest that Sema3A plays a role in chondrocyte cloning through inhibition of cell migration in OA cartilage.

     

Pioglitazone,a peroxisome proliferator–activated receptor γ agonist,reduces the progression of experimental osteoarthritis in guinea pigs

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.

     

Accelerated,aging‐dependent development of osteoarthritis in α1 integrin–deficient mice

Objective

Cell–matrix interactions regulate chondrocyte differentiation and survival. The α1β1 integrin is a major collagen receptor that is expressed on chondrocytes. Mice with targeted inactivation of the integrin α1 gene (α1‐KO mice) provide a model that can be used to address the role of cell–matrix interactions in cartilage homeostasis and osteoarthritis (OA) pathogenesis.

Methods

Knee joints from α1‐KO and wild‐type (WT) BALB/c mice were harvested at ages 4–15 months. Knee joint sections were examined for inflammation, cartilage degradation, and loss of glycosaminoglycans (by Safranin O staining). Immunohistochemistry was performed to detect the distribution of α1 integrin, matrix metalloproteinases (MMPs), and chondrocyte apoptosis.

Results

In WT mice, the α1 integrin subunit was detected in hypertrophic chondrocytes in the growth plate and in a subpopulation of cells in the deep zone of articular cartilage. There was a marked increase in α1‐positive chondrocytes in the superficial and upper mid‐zones in OA‐affected areas in joints from old WT mice. The α1‐KO mice showed more severe cartilage degradation, glycosaminoglycan depletion, and synovial hyperplasia as compared with the WT mice. MMP‐2 and MMP‐3 expression was increased in the OA‐affected areas. In cartilage from α1‐KO mice, the cellularity was reduced and the frequency of apoptotic cells was increased. These results suggest that the α1 integrin subunit is involved in the early remodeling process in OA cartilage.

Conclusion

Deficiency in the α1 integrin subunit is associated with an earlier deregulation of cartilage homeostasis and an accelerated, aging‐dependent development of OA.

     

Cartilage repair in a rat model of osteoarthritis through intraarticular transplantation of muscle‐derived stem cells expressing bone morphogenetic protein 4 and soluble flt‐1

Objective

The control of angiogenesis during chondrogenic differentiation is an important issue affecting the use of stem cells in cartilage repair, especially with regard to the persistence of regenerated cartilage. This study was undertaken to investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the blocking of VEGF with its antagonist, soluble Flt‐1 (sFlt‐1), on the chondrogenesis of skeletal muscle‐derived stem cells (MDSCs) in a rat model of osteoarthritis (OA).

Methods

We investigated the effect of VEGF on cartilage repair in an immunodeficiency rat model of OA after intraarticular injection of murine MDSCs expressing bone morphogenetic protein 4 (BMP‐4) in combination with MDSCs expressing VEGF or sFlt‐1.

Results

In vivo, a combination of sFlt‐1– and BMP‐4–transduced MDSCs demonstrated better repair without osteophyte formation macroscopically and histologically following OA induction, when compared with the other groups. Higher differentiation/proliferation and lower levels of chondrocyte apoptosis were also observed in sFlt‐1– and BMP‐4–transduced MDSCs compared with a combination of VEGF‐ and BMP‐4–transduced MDSCs or with BMP‐4–transduced MDSCs alone. In vitro experiments with mixed pellet coculture of MDSCs and OA chondrocytes revealed that BMP‐4–transduced MDSCs produced the largest pellets, which had the highest gene expression of not only type II collagen and SOX9 but also type X collagen, suggesting formation of hypertrophic chondrocytes.

Conclusion

Our results demonstrate that MDSC‐based therapy involving sFlt‐1 and BMP‐4 repairs articular cartilage in OA mainly by having a beneficial effect on chondrogenesis by the donor and host cells as well as by preventing angiogenesis, which eventually prevents cartilage resorption, resulting in persistent cartilage regeneration and repair.

     

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.

     

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.

     

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.

     

Anti‐Fas–induced apoptosis in chondrocytes reduced by hyaluronan: Evidence for CD44 and CD54 (intercellular adhesion molecule 1) involvement

Objective

To investigate the in vitro effect of therapeutic hyaluronan (HA) of 500–730 kd on anti‐Fas–induced apoptosis of chondrocytes from osteoarthritis (OA) patients, and to assess its mechanism of action by analyzing the role of the 2 HA receptors, CD44 and CD54 (intercellular adhesion molecule 1 [ICAM‐1]).

Methods

Chondrocytes isolated from human OA knee cartilage were cultured and the effect of HA on both spontaneous and anti‐Fas–induced apoptosis was evaluated. Apoptosis was analyzed by JAM test (for quantitative analysis of fragmented DNA), cell death detection immunoassay (for quantitative analysis of oligonucleosome), TUNEL assay, and electron microscopy. Blocking experiments with anti‐CD44 and anti‐CD54 alone or in combination were performed to investigate the HA mechanism of action.

Results

Both quantitative tests demonstrated that anti‐Fas significantly induced apoptosis of isolated OA chondrocytes. HA at 1,000 μg/ml significantly reduced the anti‐Fas–induced apoptosis of chondrocytes but did not affect spontaneous chondrocyte apoptosis. These data were also confirmed by TUNEL staining and by electron microscopy morphologic evaluation. The antiapoptotic effects of HA on anti‐FAS–induced chondrocyte apoptosis were significantly decreased by both anti‐CD44 (mean ± SD 57 ± 12% inhibition) and anti–ICAM‐1 (31 ± 22% inhibition). The mixture of the 2 antibodies had an additive effect, since the rate of inhibition increased to 87 ± 13%.

Conclusion

These data demonstrate that 500–730‐kd HA exerts an antiapoptotic effect on anti‐FAS–induced chondrocyte apoptosis by binding its specific receptors (CD44 and ICAM‐1). Furthermore, this HA fraction may be able to slow down chondrocyte apoptosis in OA by regulating the processes of cartilage matrix degradation.