Although osteoarthritis (OA) is not an inflammatory arthritis, a characteristic feature of OA is increased production of pro-inflammatory cytokines, such as interleukin 1beta (IL-1b), by articular chondrocytes. In fact, the degree of articular inflammation is often associated with disease progression; indicating that this process probably contributes to articular damage. Suppressor of cytokine signalling (SOCS) proteins are, as the name suggests, inhibitors of cytokine signalling that function via the JAK/STAT pathway (Janus kinase/signal transducers and activators of transcription). Eight SOCS proteins, SOCS1-SOCS7 and CIS-1 (cytokine-inducible SH2-domain-1 with similar structure to the other SOCS proteins) have been identified, of which, SOCS1-3 and CIS-1 are the best characterised. Reduced expression of SOCS proteins would be predicted to result in increased cytokine responsiveness and thereby could contribute to OA pathology. 1) To compare the expression of SOCS1-3 and CIS-1 in normal and OA human articular chondrocytes and 2) to analyze the effects of IL-1b on SOCS1-3 and CIS-1 mRNA expression.BACKGROUND
OBJECTIVES
Many patients suffering from osteoarthritis (OA) take daily glucosamine (GlcN) in the hope of slowing down disease progression and ameliorating pain. However, the physiological basis of this effect is not known. We previously presented preliminary data suggesting that GlcN prevented the increase in interleukin-1beta (IL-1b) expression caused by addition of inflammatory cytokines to cultures of healthy human articular chondrocytes. Previous studies had also shown that, in OA, epigenetic DNA methylation loss at specific CpG sites in relevant promoters ‘unsilences’ the genes and that this DNA de-methylation underlies the aberrant gene expression of proteases (Arthritis Rheum 52;3110-24). Furthermore, exogenous inflammatory cytokines have the capacity to cause DNA de-methylation in the IL-1b promoter (Arthritis Rheum. 2009, 60, 3303-3313). The aims of the present study were to investigate whether GlcN not only prevents the increased IL-1b expression, but also inhibits epigenetic unsilencing by preventing the cytokine-induced loss of DNA methylation. Healthy chondrocytes were isolated from the articular cartilage of four femoral heads, after operations following femoral neck fracture (ethic permission was obtained). The chondrocytes were cultured for 5 weeks in four treatment groups: no treatment (control); with IL-1b and oncostatin M (IL1b+OSM); with 2.0mM GlcN; and with IL1b+OSM+GlcN. Total RNA and genomic DNA were extracted. The % DNA methylation at the CpG site at -299bp (previously identifies as the crucial CpG site) was determined after bisulphite modification with a pyrosequencer. Gene expression of IL-1B was quantified by SybrGreen-based qRT-PCR.INTRODUCTION
METHODS
