Objective: To profile the expression of all known members of the matrix metalloproteinase (MMP), ADAMTS, and tissue inhibitor of metalloproteinases (TIMP) gene families in normal cartilage and cartilage from patients with osteoarthritis (OA).Methods: Human cartilage was obtained from femoral heads at joint replacement for OA or following fracture to the femoral neck. Total RNA was purified, and gene expression was assayed using quantitative real‐time polymerase chain reaction.Results: Several members of the above gene families were regulated in OA. Genes that showed increased expression in OA were MMP13, MMP28, and ADAMTS16 (all at P < 0.001), MMP9, MMP16, ADAMTS2, and ADAMTS14 (all at P < 0.01), and MMP2, TIMP3, and ADAMTS12 (all at P < 0.05). Genes with decreased expression in OA were MMP1, MMP3, and ADAMTS1 (all at P < 0.001), MMP10, TIMP1, and ADAMTS9 (all at P < 0.01), and TIMP4, ADAMTS5, and ADAMTS15 (all at P < 0.05). Correlation analysis revealed that groups of genes across the gene families were coexpressed in cartilage.Conclusion: This is the first comprehensive expression profile of all known MMP, ADAMTS, and TIMP genes in cartilage. Elucidation of patterns of expression provides a foundation with which to understand mechanisms of gene regulation in OA and potentially to refine the specificity of antiproteolytic therapies.

Background Oxidative stress is proposed as an important factor in osteoarthritis (OA). Objective To investigate the expression of the three superoxide dismutase (SOD) antioxidant enzymes in OA. Methods SOD expression was determined by real-time PCR and immunohistochemistry using human femoral head cartilage. SOD2 expression in Dunkin–Hartley guinea pig knee articular cartilage was determined by immunohistochemistry. The DNA methylation status of the SOD2 promoter was determined using bisulphite sequencing. RNA interference was used to determine the consequence of SOD2 depletion on the levels of reactive oxygen species (ROS) using MitoSOX and collagenases, matrix metalloproteinase 1 (MMP-1) and MMP-13, gene expression. Results All three SOD were abundantly expressed in human cartilage but were markedly downregulated in end-stage OA cartilage, especially SOD2. In the Dunkin–Hartley guinea pig spontaneous OA model, SOD2 expression was decreased in the medial tibial condyle cartilage before, and after, the development of OA-like lesions. The SOD2 promoter had significant DNA methylation alterations in OA cartilage. Depletion of SOD2 in chondrocytes increased ROS but decreased collagenase expression. Conclusion This is the first comprehensive expression profile of all SOD genes in cartilage and, importantly, using an animal model, it has been shown that a reduction in SOD2 is associated with the earliest stages of OA. A decrease in SOD2 was found to be associated with an increase in ROS but a reduction of collagenase gene expression, demonstrating the complexities of ROS function.

The balance between matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), is pivotal in the remodeling of extracellular matrix. TGF-β has profound effects on extracellular matrix homeostasis, in part via its ability to alter this balance at the level of gene expression. The intracellular signaling pathways by which TGF-β mediates its actions include the Smad pathway, specific to the TGF-β superfamily, but also, for example, mitogen-activated protein kinase pathways; furthermore, cross-talk between the Smads and other signaling pathways modifies the TGF-β response. The reciprocal effect of TGF-β on the expression ofTimp-1 and MMP-1 supports its role in matrix anabolism, yet the mechanisms by which TGF-β inducesTimp-1 and represses induced MMP-1 have remained opaque. Here, we (i) investigate the mechanism(s) by which TGF-β1 induces expression of the Timp-1 gene and (ii) compare this with TGF-β1 repression of phorbol ester-inducedMMP-1 expression. We report that the promoter-proximal activator protein 1 (AP1) site is essential for the response of bothTimp-1 and MMP-1 to TGF-β (induction and repression, respectively). c-Fos, JunD, and c-Jun are essential for the induction of Timp-1 gene expression by TGF-β1, but these AP1 factors transactivate equally well from both Timp-1 andMMP-1 AP1 sites. Smad-containing complexes do not interact with the Timp-1 AP1 site, and overexpression of Smads does not substitute or potentiate the induction of the gene by TGF-β1; furthermore, Timp-1 is still induced by TGF-β1 in Smad knockout cell lines, although to varying extents. In contrast, Smads do interact with the MMP-1 AP1 site and mediate repression of induced MMP-1 gene expression by TGF-β1.

Objectives To investigate the effect of leptin on cartilage destruction. Methods Collagen release was assessed in bovine cartilage explant cultures, while collagenolytic and gelatinolytic activities in culture supernatants were determined by bioassay and gelatin zymography. The expression of matrix metalloproteinases (MMP) was analysed by real-time RT–PCR. Signalling pathway activation was studied by immunoblotting. Leptin levels in cultured osteoarthritic joint infrapatellar fat pad or peri-enthesal deposit supernatants were measured by immunoassay. Results Leptin, either alone or in synergy with IL-1, significantly induced collagen release from bovine cartilage by upregulating collagenolytic and gelatinolytic activity. In chondrocytes, leptin induced MMP1 and MMP13 expression with a concomitant activation of STAT1, STAT3, STAT5, MAPK (JNK, Erk, p38), Akt and NF-κB signalling pathways. Selective inhibitor blockade of PI3K, p38, Erk and Akt pathways significantly reduced MMP1 and MMP13 expression in chondrocytes, and reduced cartilage collagen release induced by leptin or leptin plus IL-1. JNK inhibition had no effect on leptin-induced MMP13 expression or leptin plus IL-1-induced cartilage collagen release. Conditioned media from cultured white adipose tissue (WAT) from osteoarthritis knee joint fat pads contained leptin, induced cartilage collagen release and increased MMP1 and MMP13 expression in chondrocytes; the latter being partly blocked with an anti-leptin antibody. Conclusions Leptin acts as a pro-inflammatory adipokine with a catabolic role on cartilage metabolism via the upregulation of proteolytic enzymes and acts synergistically with other pro-inflammatory stimuli. This suggests that the infrapatellar fat pad and other WAT in arthritic joints are local producers of leptin, which may contribute to the inflammatory and degenerative processes in cartilage catabolism, providing a mechanistic link between obesity and osteoarthritis.

Objective: To use an in vitro model of chondrogenesis to identify microRNAs (miRNAs) with a functional role in cartilage homeostasis.Methods: The expression of miRNAs was measured in the ATDC5 cell model of chondrogenesis using microarray and was verified using quantitative reverse transcription–polymerase chain reaction. MicroRNA expression was localized by in situ hybridization. Predicted miRNA target genes were validated using 3′‐untranslated region‐Luc reporter plasmids containing either wild‐type sequences or mutants of the miRNA target sequence. Signaling through the Smad pathway was measured using a (CAGA)₁₂‐Luc reporter.Results: The expression of several miRNAs was regulated during chondrogenesis. These included 39 miRNAs that are coexpressed with miRNA‐140 (miR‐140), which is known to be involved in cartilage homeostasis and osteoarthritis (OA). Of these miRNAs, miR‐455 resides within an intron of COL27A1 that encodes a cartilage collagen. When human OA cartilage was compared with cartilage obtained from patients with femoral neck fractures, the expression of both miR‐140‐5p and miR‐455‐3p was increased in OA cartilage. In situ hybridization showed miR‐455‐3p expression in the developing limbs of chicks and mice and in human OA cartilage. The expression of miR‐455‐3p was regulated by transforming growth factor β (TGFβ) ligands, and miRNA regulated TGFβ signaling. ACVR2B, SMAD2, and CHRDL1 were direct targets of miR‐455‐3p and may mediate its functional impact on TGFβ signaling.Conclusion: MicroRNA‐455 is expressed during chondrogenesis and in adult articular cartilage, where it can regulate TGFβ signaling, suppressing the Smad2/3 pathway. Diminished signaling through this pathway during the aging process and in OA chondrocytes is known to contribute to cartilage destruction. We propose that the increased expression of miR‐455 in OA exacerbates this process and contributes to disease pathology.

Objective To use a computational approach to investigate the cellular and extracellular matrix changes that occur with age in the knee joints of mice. Methods Knee joints from an inbred C57/BL1/6 (ICRFa) mouse colony were harvested at 3–30 months of age. Sections were stained with H&E, Safranin-O, Picro-sirius red and antibodies to matrix metalloproteinase-13 (MMP-13), nitrotyrosine, LC-3B, Bcl-2, and cleaved type II collagen used for immunohistochemistry. Based on this and other data from the literature, a computer simulation model was built using the Systems Biology Markup Language using an iterative approach of data analysis and modelling. Individual parameters were subsequently altered to assess their effect on the model. Results A progressive loss of cartilage matrix occurred with age. Nitrotyrosine, MMP-13 and activin receptor-like kinase-1 (ALK1) staining in cartilage increased with age with a concomitant decrease in LC-3B and Bcl-2. Stochastic simulations from the computational model showed a good agreement with these data, once transforming growth factor-β signalling via ALK1/ALK5 receptors was included. Oxidative stress and the interleukin 1 pathway were identified as key factors in driving the cartilage breakdown associated with ageing. Conclusions A progressive loss of cartilage matrix and cellularity occurs with age. This is accompanied with increased levels of oxidative stress, apoptosis and MMP-13 and a decrease in chondrocyte autophagy. These changes explain the marked predisposition of joints to develop osteoarthritis with age. Computational modelling provides useful insights into the underlying mechanisms involved in age-related changes in musculoskeletal tissues.