Interferon-gamma modulates articular chondrocyte and osteoblast metabolism through protein kinase R-independent and dependent mechanisms.

Osteoarthritis (OA) affects multiple tissues of the synovial joint and is characterised by articular cartilage degeneration and bone remodelling. Interferon-γ (IFN-γ) is implicated in osteoarthritis pathology exerting its biological effects via various mechanisms including activation of protein kinase R (PKR), which has been implicated in inflammation and arthritis. This study investigated whether treatment of articular cartilage chondrocytes and osteoblasts with IFN-γ could induce a degradative phenotype that was mediated through the PKR signalling pathway. IFN-γ treatment of chondrocytes increased transcription of key inflammatory mediators (TNF-α, IL-6), matrix degrading enzymes (MMP-13), the transcription factor STAT1, and PKR. Activation of PKR was involved in the regulation of TNF-α, IL-6, and STAT1. In osteoblasts, IFN-γ increased human and mouse STAT1, and human IL-6 through a mechanism involving PKR. ALP, COL1A1 (human and mouse), RUNX2 (mouse), and PHOSPHO1 (mouse) were decreased by IFN-γ. The number of PKR positive cells were increased in post-traumatic OA (PTOA). This study has revealed that IFN-γ propagates inflammatory and degenerative events in articular chondrocytes and osteoblasts via PKR activation. Since IFN-γ and PKR signalling are both activated in early PTOA, these mechanisms are likely to contribute to joint degeneration after injury and might offer attractive targets for therapeutic intervention.

Importance of reference gene selection for articular cartilage mechanobiology studies.

OBJECTIVE: Identification of genes differentially expressed in mechano-biological pathways in articular cartilage provides insight into the molecular mechanisms behind initiation and/or progression of osteoarthritis (OA). Quantitative PCR (qPCR) is commonly used to measure gene expression, and is reliant on the use of reference genes for normalisation. Appropriate validation of reference gene stability is imperative for accurate data analysis and interpretation. This study determined in vitro reference gene stability in articular cartilage explants and primary chondrocytes subjected to different compressive loads and tensile strain, respectively. DESIGN: The expression of eight commonly used reference genes (18s, ACTB, GAPDH, HPRT1, PPIA, RPL4, SDHA and YWHAZ) was determined by qPCR and data compared using four software packages (comparative delta-Ct method, geNorm, NormFinder and BestKeeper). Calculation of geometric means of the ranked weightings was carried out using RefFinder. RESULTS: Appropriate reference gene(s) for normalisation of mechanically-regulated transcript levels in articular cartilage tissue or isolated chondrocytes were dependent on experimental set-up. SDHA, YWHAZ and RPL4 were the most stable genes whilst glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and to a lesser extent Hypoxanthine-guanine phosphoribosyltransferase (HPRT), showed variable expression in response to load, demonstrating their unsuitability in such in vitro studies. The effect of using unstable reference genes to normalise the expression of aggrecan (ACAN) and matrix metalloproteinase 3 (MMP3) resulted in inaccurate quantification of these mechano-sensitive genes and erroneous interpretation/conclusions. CONCLUSION: This study demonstrates that commonly used 'reference genes' may be unsuitable for in vitro cartilage chondrocyte mechanobiology studies, reinforcing the principle that careful validation of reference genes is essential prior to each experiment to obtain robust and reproducible qPCR data for analysis/interpretation.

Protein kinase R plays a pivotal role in oncostatin M and interleukin-1 signalling in bovine articular cartilage chondrocytes.

This study investigated whether treatment of articular cartilage chondrocytes with a combination of oncostatin M (OSM) and interleukin-1 (IL-1) could induce a degradative phenotype that was mediated through the protein kinase R (PKR) signalling pathway. High-density monolayer cultures of full depth, bovine chondrocytes were treated with a combination of OSM and IL-1 (OSM+IL-1) for 7 days. To inhibit the activation of PKR, a pharmacological inhibitor of PKR was added to duplicate cultures. Pro- and active matrix metalloproteinase-9 (MMP9) and MMP9 mRNA were significantly upregulated by OSM+IL-1 through a PKR dependent mechanism. ADAMTS4 and ADAMTS5 mRNA were also upregulated by OSM+IL-1. The upregulation of ADAMTS4 and ADAMTS5 were, in part, mediated through PKR. OSM+IL-1 resulted in a loss of type II collagen, which could not be rescued by PKR inhibition. OSM+IL-1 reduced the expression of COL2A1 (type II collagen), COL9A1 (type IX collagen), COL11A1 (type XI collagen), and ACAN (aggrecan) mRNAs. Expression of type II and XI collagen and aggrecan was reduced further when PKR was inhibited. OSM+IL-1 resulted in an 11-fold increase in TNFa mRNA which was, in part, mediated through the PKR pathway. This study demonstrates, for the first time, that a number of catabolic and pro-inflammatory effects known to be important in human arthritis and induced by OSM and IL-1, are mediated by the PKR signalling pathway.

The effects of cyclic tensile strain on the organisation and expression of cytoskeletal elements in bovine intervertebral disc cells: an in vitro study.

It is still relatively unclear how intervertebral disc (IVD) cells sense a mechanical stimulus and convert this signal into a biochemical response. Previous studies demonstrated that the cytoskeletal elements are mechano-responsive in many cell types and may contribute to mechano-signalling pathways. The objective of this study was to determine the response of cells from the outer annulus fibrosus (OAF) to physiological levels of cyclic tensile strain; further, cells from the nucleus pulposus (NP) were also subjected to an identical loading regime to compare biological responses across the IVD populations. We determined whether the organisation and expression of the major cytoskeletal elements and their associated accessory proteins are responsive to mechanical stimulation in these cells, and whether these changes correlated with either a catabolic or anabolic phenotype. OAF and NP cells from immature bovine IVD were seeded onto Flexcell® type I collagen coated plates. Cells were subjected to cyclic tensile strain (10 %, 1 Hz) for 60 minutes. Post-loading, cells were processed for immunofluorescence microscopy, RNA extracted for quantitative PCR and protein extracted for Western blotting analysis. F-actin reorganisation was evident in OAF and NP cells subjected to tensile strain; strain induced ß-actin at the transcriptional and translational level in OAF cells. ß-tubulin mRNA and protein synthesis increased in strained OAF cells, but vimentin expression was significantly inhibited. Cytoskeletal element organisation and expression were less responsive to strain in NP cells. Tensile strain increased type I collagen and differentially regulated extracellular matrix (ECM)-degrading enzymes' mRNA levels in OAF cells. Strain induced type II collagen transcription in NP cells, but had no effect on the transcription of any other genes analysed. Tensile strain induces different mechano-responses in the organisation and/or expression of cytoskeletal elements and on markers of IVD metabolism. Differential mechano-regulation of anabolic and catabolic ECM components in the OAF and NP populations reflects their respective mechanical environments in situ.

Sphingomyelinase decreases type II collagen expression in bovine articular cartilage chondrocytes via the ERK signaling pathway.

OBJECTIVE: Ceramide, a mediator of proinflammatory cytokine signaling, induces cartilage degradation and reduces type II collagen synthesis in articular cartilage. The accumulation of ceramide is associated with arthritis in Farber's disease. The aim of this study was to investigate the mechanism of ceramide-induced down-regulation of type II collagen. METHODS: Bovine articular chondrocytes were stimulated with sphingomyelinase (SMase) to increase levels of endogenous ceramide. Components of the ERK pathway were inhibited by Raf-1 kinase inhibitor and the MEK inhibitor, PD98059. Cell extracts were analyzed by Western blotting for ERK-1/2, SOX9, c-Fos, and type II collagen, and the level of c-fos messenger RNA (mRNA) was analyzed by quantitative polymerase chain reaction. Localization of ERK-1/2, SOX9, and c-Fos was assessed by immunocytochemistry and confocal microscopy. RESULTS: SMase treatment of chondrocytes caused sustained phosphorylation of ERK-1/2 throughout the cytoplasm and nucleus that was reduced by inhibitors of Raf-1 kinase and MEK-1/2. SMase treatment of chondrocytes also induced translocation of c-Fos to the nucleus and phospho-SOX9 to the cytoplasm and increased expression of c-fos mRNA. Type II collagen expression, which was down-regulated by SMase treatment, was restored by the MEK-1/2 inhibitor, PD98059. CONCLUSION: SMase down-regulates type II collagen in articular chondrocytes via activation of the ERK signaling cascade, redistribution of SOX9, and recruitment of c-Fos. This new mechanism for cartilage degradation provides potential targets for future treatment of arthritic disease.

Chondroitin sulphate impedes the migration of a sub-population of articular cartilage chondrocytes.

OBJECTIVE: To determine whether chondroitin sulphate (CS) impedes the migration of primary articular chondrocytes. DESIGN: Articular chondrocytes were isolated from young and skeletally mature bovine animals. Boyden chambers were used to quantify chondrocyte migration on aggrecan in the presence and absence of CS chains. A novel in vitro model of cell migration into articular cartilage explants was designed to visualise and quantify the migration of labelled chondrocytes into cartilage matrix which had been treated with chondroitinase ABC to remove CS chains present. RESULTS: A consistent trend of increased migration with both age groups of a sub-population of chondrocytes was demonstrated on aggrecan in the absence of CS. These data were supported by results from the in vitro model of chondrocyte migration which demonstrated increasing numbers of a chondrocyte sub-population from both age groups of cartilage migrating into the chondroitinase ABC digested cartilage explants with time in culture. Minimal migration of these chondrocytes was demonstrated into phosphate buffered saline (PBS) treated control explants. CONCLUSIONS: We confirm that a sub-population of chondrocytes isolated from both young and skeletally mature articular cartilages have the ability to migrate. We also demonstrate that CS chains inhibit the migration of these articular chondrocytes and that their removal by chondroitinase ABC digestion enhances the migration of these chondrocytes. Such findings may provide a clinical application for improving cell-based cartilage repair strategies by enhancing integration between endogenous and repair tissue.

Loading alters actin dynamics and up-regulates cofilin gene expression in chondrocytes.

Chondrocyte mechanotransduction in response to mechanical loading is essential for the health and homeostasis of articular cartilage. The actin cytoskeleton has been implicated in cell mechanics and mechanotransduction. This study tests the hypothesis that loading modulates actin dynamics and organisation with subsequent changes in gene expression for actin associated proteins. Chondrocytes were transfected with eGFP-actin, seeded in agarose and subjected to cyclic compression (10 cycles, 1 Hz, 0-15% strain) on the stage of a confocal microscope. Compression resulted in a subsequent reduction in cortical eGFP-actin intensity and a reduction in fluorescence recovery after photobleaching (FRAP), suggesting net cortical actin de-polymerisation, compared to unloaded controls. Cyclic compression for 10 min up-regulated gene expression for the actin depolymerising proteins, cofilin and destrin. Thus mechanical loading alters cortical actin dynamics, providing a potential mechanism through which chondrocytes can adapt their mechanical properties and mechanosensitivity to the local mechanical environment.

F-actin cytoskeletal organization in intervertebral disc health and disease.

The cytoskeleton, which in most cell types, including the intervertebral disc described here, comprises microfilaments, microtubules and intermediate filaments, plays important functions in many fundamental cellular events, including cell division, motility, protein trafficking and secretion. The cytoskeleton is also critical for communication; for example, alterations to the architecture of the F-actin (filamentous actin) cytoskeletal networks can affect communication between the cells and the extracellular matrix, potentially compromising tissue homoeostasis. Although there are limited studies to date, this paper aims to review current knowledge on F-actin cytoskeletal element organization in intervertebral disc cells, how F-actin differs with pathology and its implications for mechanotransduction.

The effect of thymosin beta4 on articular cartilage chondrocyte matrix metalloproteinase expression.

Mechanical loading is paramount in regulating both the anabolic and catabolic activities of articular cartilage chondrocytes, essential for the matrix to retain its functional integrity. We have identified thymosin beta(4) as a putative mechanically regulated gene that may mediate load-enhanced synthesis and activation of matrix metalloproteinases (MMPs) 2 and 9 in articular cartilage. The objective of this study was to confirm the mechanical regulation of thymosin beta(4) and determine its effect on cartilage chondrocyte MMP production. Thymosin beta(4) mRNA expression, analysed by quantitative PCR, revealed a significant 20-fold increase in cartilage loaded for 10 min which was still evident after 30 min of loading. Treatment of primary chondrocytes with 2 and 4 micro x ml(-1) thymosin beta(4) peptide for 4 h significantly increased pro-MMP 9 expression and activation. We postulate a functional role for load-induced thymosin beta(4) in modulating the cytoskeletal organization of articular cartilage chondrocytes to affect MMP expression.

Up-regulation of matrix metalloproteinase expression and activation following cyclical compressive loading of articular cartilage in vitro.

Osteoarthritis (OA) results in articular cartilage degeneration and subchondral bone remodeling. Excessive or abnormal loading of the joint may contribute to matrix destruction by creating an imbalance between proteinases and their inhibitors. This study investigates whether cyclical loading regulates expression and/or activation of metalloproteinases 2 and 9 (MMPs) in articular cartilage explants. Gelatin zymography, reverse zymography, and MMP activity assays of mechanically loaded bovine cartilage explants (0.5 MPa, 1 Hz, 3 h) showed increased expression and activation of MMPs 2 and 9, whereas expression of the tissue inhibitors of MMPs was unaffected. This shows, for the first time that mechanical loading can influence tissue homeostasis generating an imbalance of proteinases and their inhibitors inducing turnover and/or catabolic events in cartilage.