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Therapeutic Potential of Melanocortins as Chondroprotective Drugs Current treatments for osteoarthritis (OA) are palliative and there is a pharmaceutical demand for new chondroprotective and restorative drugs. Prophylactic application of endogenous melanocortin peptides activate 7-transmembrane GPCR melanocortin (MC) receptors to mitigate chondrocyte activation and cartilage degradation [1,2]. Several studies indicate that hereditary DNA methylation patterns of normally silenced catabolic and inflammatory genes, such as IL1 β and MMP13, are activated in OA chondrocytes providing a novel link between epigenetic status and arthritic disease. In this study we investigated the properties of melanocortins given therapeutically to chondrocytes, questioning also if they affected the epigenetic markers DNA methyltransferase-1 (DNMT1) and histone deacetylase-1 (HDAC1), crucial for healthy and tissue-specific gene expression. C28/I2 chondrocytes were plated in 3D-micromasses at a density of 0.5x106 cells in 20µl DMEM medium (10%FCS) for 24h prior to further 24 h culture in serum-free DMEM [3]. Micromasses were stimulated with TNF-α (0.1ng/ml) for 24h, following which α-MSH or PG901 (0.1-10.0 µg/ml) were added together with fresh TNF-α for additional 24h. Then, RNA was extracted (RNeasy Plus Mini Kit, Qiagen), reverse transcribed into cDNA (SuperScript III) and used in qPCR (PowerSYBR Green) to detect expression of IL6, matrix metalloprotease (MMP)1, MMP13, aggrecan (AGG), type II collagen (COL2A1), DNMT1 and HDAC1. Results are expressed as arbitrary units based on calculation of 2- ΔΔ Ct method – relative amount of target genes were normalized to GAPDH and to untreated controls, with expression set to 1.0. Data are Mean ± SEM of 3 experiments in triplicate. Statistical analyses were conducted with One-way ANOVA. C28/I2 chondrocytes – grown in 3D-micromasses – display basal IL6 and MMP1 expression – but not MMP13 [1] and produce significant amounts of COL2A1 and AGG products. TNF-α stimulates IL6, MMP1, MMP13 gene expression (˜3-fold increases, P<0.05), whilst COL2A1 and AGG expression is downregulated (˜2.5-3.0 fold, P<0.05). Added 24h after the cytokine, both α-MSH, natural melaconortin [0.3-3µg/ml], and PG901, synthetic MC5-specific ligand [1-3µg/ml], counteracted its effects on the catabolic IL6, MMP1 and MMP13, and significantly upregulated the anabolic genes COL2A1 and AGG (P<0.05). In the context of epigenetic mechanisms, TNFα down-regulated DNMT1 and HDAC1 by ˜2-fold and this effect was abolished by both of the melanocortin peptides (P<0.05). These findings support the current wisdom on the link between epigenetic status and arthritic disease, and simultaneously establish new avenues for the actions of MC receptor agonists on chondrocyte reactivity. These data may lead the way to exploit melanocortin biology for the development of novel chondroprotective therapies. Kaneva MK, Kerrigan MJ, Grieco P, Curley GP, Locke IC, Getting SJ (2012). Chondroprotective and anti-inflammatory role of melanocortin peptides in TNF-alpha activated human C-20/A4 chondrocytes. Br J Pharmacol 167(1): 67-79. Grassel S, Opolka A, Anders S, Straub RH, Grifka J, Luger TA, et al. (2009). The melanocortin system in articular chondrocytes: melanocortin receptors, pro-opiomelanocortin, precursor proteases, and a regulatory effect of alpha-melanocyte-stimulating hormone on proinflammatory cytokines and extracellular matrix components. Arthritis Rheum 60(10): 3017-3027. Greco KV, Iqbal AJ, Rattazzi L, Nalesso G, Moradi-Bidhendi N, Moore AR, et al. (2011). High density micromass cultures of a human chondrocyte cell line: a reliable assay system to reveal the modulatory functions of pharmacological agents. Biochem Pharmacol 82(12): 1919-1929. Funded by The William Harvey Research Foundation.
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