Tonic activation of hypoxia‐inducible factor 1α in avascular articular cartilage and implications for metabolic homeostasis

Objective

To determine whether oxygen‐dependent activation patterns of hypoxia‐inducible factor 1α (HIF‐1α) observed in vascularized tissues are conserved within avascular and hypoxic articular cartilage and whether HIF‐1α affects cartilage matrix synthesis.

Methods

Explants of bovine articular cartilage and primary chondrocytes were exposed to normoxia (21% O₂), hypoxia (2% O₂), and simulated hypoxia (21% O₂ plus CoCl₂). Western blot and immunofluorescence analyses of HIF‐1α were performed to determine HIF‐1α activation patterns. To simulate cartilage loss from disease or injury, the top layers of cartilage were removed from osteochondral explants, and the residual cartilage was assessed for HIF‐1α immunolocalization and proteoglycan synthesis.

Results

We demonstrated continuous nuclear translocation of HIF‐1α in deeper layers of intact articular cartilage. HIF‐1α was not completely degraded in chondrocytes exposed to normoxia, but rather, colocalized to the Golgi complex, a finding not previously reported for any cell type. Following alteration of the oxygen gradient by removal of the top layers of cartilage, predominantly perinuclear HIF‐1α was found in the deeper layers. Restoration of intranuclear HIF‐1α to these areas was achieved by hypoxia and simulated hypoxia. Under conditions in which HIF‐1α was inactivated, matrix synthetic activity was altered (P < 0.0001) compared with control cartilage.

Conclusion

These findings demonstrate that hypoxia‐dependent activation of HIF‐1α is highly conserved and that changes in oxygen tensions following cartilage loss from injury or disease alter cartilage metabolism in part by changing HIF‐1α activity. The discovery of tonic activation of HIF‐1α within intact articular cartilage underscores its potential importance to cartilage homeostasis.

     

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.

     

Interleukin‐6 plays an essential role in hypoxia‐inducible factor 2α–induced experimental osteoarthritic cartilage destruction in mice

Objective

Hypoxia‐inducible factor 2α (HIF‐2α) (encoded by Epas1) causes osteoarthritic (OA) cartilage destruction by regulating the expression of catabolic factor genes. We undertook this study to explore the role of interleukin‐6 (IL‐6) in HIF‐2α–mediated OA cartilage destruction in mice.

Methods

The expression of HIF‐2α, IL‐6, and catabolic factors was determined at the messenger RNA and protein levels in primary culture mouse chondrocytes, human OA cartilage, and mouse experimental OA cartilage. Experimental OA in wild‐type, HIF‐2α–knockdown (Epas1^+/−), and Il6^–/– mice was caused by intraarticular injection of Epas1 adenovirus or destabilization of the medial meniscus. The role of IL‐6 was determined by treating with recombinant IL‐6 protein or by injecting HIF‐2α adenovirus (AdEpas1) intraarticularly in mice with or without IL‐6–neutralizing antibody.

Results

We found that Il6 is a direct target gene of HIF‐2α in articular chondrocytes. Both Epas1 and Il6 were up‐regulated in human and mouse OA cartilage, whereas HIF‐2α knockdown in mice led to inhibition of both Il6 expression and cartilage destruction. Treatment with IL‐6 enhanced Mmp3 and Mmp13 expression; conversely, Il6 knockdown inhibited HIF‐2α–induced up‐regulation of Mmp3 and Mmp13. Injection of IL‐6 protein into mouse knee joints triggered OA cartilage destruction, whereas IL‐6 neutralization led to blocking of HIF‐2α–induced cartilage destruction with concomitant modulation of Mmp3 and Mmp13 expression. Moreover, Il6 knockout resulted in inhibition of AdEpas1‐induced and destabilization of the medial meniscus–induced cartilage destruction as well as inhibition of Mmp3 and Mmp13 expression.

Conclusion

Our findings indicate that IL‐6 acts as a crucial mediator of HIF‐2α–induced experimental OA cartilage destruction in mice via regulation of Mmp3 and Mmp13 levels.

     

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.

     

Expression of hypoxia‐inducible factor 1α by macrophages in the rheumatoid synovium: Implications for targeting of therapeutic genes to the inflamed joint

Objective

To determine if the rheumatoid synovium is a suitable target for hypoxia‐regulated gene therapy.

Methods

Sequential sections of wax‐embedded synovial membrane samples were obtained from 10 patients with rheumatoid arthritis (RA), 10 with primary osteoarthritis (OA), and from 6 healthy controls. Membrane sections from each patient were immunostained for hypoxia‐inducible factor 1α (HIF‐1α) and CD68 (a pan–macrophage marker).

Results

HIF‐1α was expressed abundantly by macrophages in most rheumatoid synovia, predominantly close to the intimal layer but also in the subintimal zone. There was markedly lower expression of HIF‐1α in OA synovia, and it was absent from all of the healthy synovia.

Conclusion

These observations indicate that macrophages transduced with a therapeutic gene under the control of a hypoxia‐inducible promoter could be administered to RA patients systemically. Migration of these cells to synovial tissue would result in the transgene being switched on in diseased joints but not in healthy tissues.

     

Growth‐related oncogene α induction of apoptosis in osteoarthritis chondrocytes

Objective

To evaluate the apoptotic effect of the chemokine growth‐related oncogene α (GROα), which we recently reported to be up‐regulated in osteoarthritis (OA) chondrocytes. Chondrocyte apoptosis is considered to be a major determinant of cartilage damage in OA, a disease resulting from the aberrant production of inflammatory mediators (cytokines and chemokines) and effectors (matrix metalloproteinases and reactive oxygen and nitrogen species) by chondrocytes.

Methods

We investigated the apoptotic effect of GROα on isolated human cells and on in vitro–cultured cartilage explants by conventional methods (morphology, detection of DNA fragmentation in situ and in solution, exposure of phosphatidylserine) and by analysis of “early” biochemical events (plasma membrane depolarization, activation of caspase 3, and phosphorylation of c‐Jun N‐terminal kinase/stress‐activated protein kinase).

Results

We clearly demonstrated that GROα was able to initiate a series of morphologic, biochemical, and molecular changes that led to chondrocyte apoptosis. Moreover, we found that additional signals delivered from the extracellular matrix (ECM) were essential in the control of chondrocyte susceptibility to GROα‐induced apoptosis, since cell death was detected only when cells were stimulated after reestablishment of their proper interactions with the ECM, or in cartilage explant samples with reduced ECM, as indicated by decreased Safranin O staining.

Conclusion

GROα can induce apoptosis in articular chondrocytes, and the induction is dependent upon additional signals from the ECM. These findings are relevant to understanding the pathogenesis of OA, in view of the availability of the GROα chemokine in the joint space in the course of this rheumatic disease.

     

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.

     

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.

     

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.

     

Mitochondrial dysfunction in osteoarthritis is associated with down‐regulation of superoxide dismutase 2

Objective

Superoxide dismutase 2 (SOD2) is down‐ regulated in osteoarthritis (OA). This study was undertaken to investigate the functional effects of this down‐regulation in the context of oxidative damage and mitochondrial dysfunction.

Methods

Lipid peroxidation in articular cartilage from OA patients and from lesion‐free control subjects with femoral neck fracture was assessed by measuring malondialdehyde levels using the thiobarbituric acid reactive substances assay. Long‐range polymerase chain reaction amplification and a mitochondrial DNA (mtDNA) strand break assay were used to investigate the presence of somatic large‐scale mtDNA rearrangements in cartilage. Microscale oxygraphy was used to explore possible changes in mitochondrial respiratory activity between OA and control chondrocytes. RNA interference was used to determine the effects of SOD2 depletion on lipid peroxidation, mtDNA damage, and mitochondrial respiration.

Results

OA cartilage had higher levels of lipid peroxidation compared to control cartilage, and lipid peroxidation was similarly elevated in SOD2‐depleted chondrocytes. SOD2 depletion led to a significant increase in mtDNA strand breaks in chondrocytes, but there was no notable difference in the level of strand breaks between OA and control chondrocytes. Furthermore, only very low levels of somatic, large‐scale mtDNA rearrangements were identified in OA cartilage. OA chondrocytes showed less spare respiratory capacity (SRC) and higher proton leak compared to control chondrocytes. SOD2‐depleted chondrocytes also showed less SRC and higher proton leak.

Conclusion

This is the first study to analyze the effects of SOD2 depletion in human articular chondrocytes in terms of changes to oxidation and mitochondrial function. The findings indicate that SOD2 depletion in chondrocytes leads to oxidative damage and mitochondrial dysfunction, suggesting that SOD2 down‐regulation is a potential contributor to the pathogenesis of OA.