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Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_I | Pages 72 - 72
1 Jan 2011
Glaysher M Roach H
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Osteoarthritis (OA) is characterised by the progressive destruction of articular cartilage by matrix-degrading enzymes. Although initially produced by synovial fibroblasts, these enzymes are derived from OA chondrocytes as the disease progresses. Inflammatory cytokines (IL-1beta, TNF-alpha, oncostatin M) are known to induce the aberrant expression of the proteases and IL-1beta in vitro. We wanted to investigate whether the transcription factor NF-kB, which is frequently involved in signal transduction, is a mediator of the effects of inflammatory cytokines. Hence we determined the effects of NF-kB inhibition on the expression of IL-1beta, MMP-13 and MMP-3, which was induced in healthy chondrocytes by culturing with TNF-alpha/OsM.

Chondrocytes were isolated from the healthy cartilage of femoral heads obtained from patients after hemiarthroplasty following a femoral neck fracture (n=4). The chondrocytes from each patient were divided into four experimental groups: untreated control culture; culture with TNF-alpha/OsM; culture with TNF-alpha/OsM in presence of an NF-kB inhibitor; and culture with TNF-alpha/OsM treated + control peptide. Two inhibitors of NF-kB nuclear translocation were employed: an NF-kB p65 (ser276) inhibitory peptide (Imgenex) and (E)-2-Fluoro-4′-methoxystilbene, an analogue of resveratol (Merck). Cells were grown in monolayer culture for two weeks and received two rounds of treatment. Once confluent, cells were harvested and total RNA was extracted, using a Qiagen kit. RNA was reverse transcribed into cDNA and the expression of IL-1beta, MMP-3 and MMP-13 was analysed by conventional RT-PCR.

No expression of IL-1beta was found in control cultures but expression was induced, as expected, following treatment with TNF-alpha/OsM. Presence of the NF-kB inhibitor reduced IL-1beta expression, but did not abolish it completely, as suggested by reduced intensity of the PCR band. This was seen in all four samples. Similarly, NF-kB inhibition attenuated MMP-13 expression in three patients, but in one patient MMP-13 was already expressed in control cultures and no change was observed in the treated groups. MMP-3 was uniformly expressed across all experimental groups and was unaffected by NF-kB inhibition.

NF-kB is the generic name for a family of transcription factors, of which the p65-p50 heterodimer is the most prevalent. NF-kB is normally sequestered within the cytoplasm in an inactive form by binding to inhibitory kB (IkB) proteins. Activation involves degradation of IkB and nuclear translocation of NF-kB. The present results show that the cytokine-induced expression of IL-1beta and MMP-13 in healthy chondrocytes involves nuclear translocation of NF-kB.