The effect of the selective MC3 agonist PG990 on high density human chondrocyte micromass cultures activated by TNF-alpha.
V.C. Can , I.C. Locke, P. Grieco and S.J. Getting
Introduction: Osteoarthritis (OA) is a degenerative joint disease partially mediated by the catabolic cytokine TNF-α, leading to progressive and permanent degeneration of cartilage (1). A potential anti-inflammatory and chondroprotective role for melanocortin peptides has
been shown (2), although the receptor subtype involved is unclear. This study aims to assess the chondroprotective and anti-inflammatory effects of the selective melanocortin-3
(hMC3) receptor agonist PG990 and partially selective agonist [DTRP8]- γ-MSH on TNF-α induced cell death, pro-inflammatory inflammatory cytokine and matrix metalloproteinase (MMP) release in chondrocyte micromass cultures.
Methods: Micromass cultures of the human chondrocytic cell line C-20/A4 were obtained by seeding cells at a density of 25.0 x 106 viable cells/mL into individual wells of 24-well plates. After 48h, the micromasses were treated with [DTRP8]- γ-MSH (3.0μg/ml-1) or PG990 (10.0μg/ml-1) for 30mins prior to TNF-α (60pg/ml-1) stimulation for 6h. Micromasses were
harvested for Alcian blue matrix staining to measure sulphated glycosaminoglycan (GAG) content, cell viability and RT-PCR gene expression analysis of MC1, MC3, IL-6, IL-8 and
MMP-1. Cell free supernatants were collected to measure IL-6, IL-8 and MMP-1 release by ELISA. Data are expressed as Mean±SD of n=4 determinations in triplicate. #p≤0.05 vs.
control or *p≤0.05 vs. stimulus.
Results: RT-PCR showed MC1 and MC3 expression on micromass C-20/A4 cells. Alcian Blue staining showed an increased GAG accumulation in micromasses treated with PG990
(121.3±2.4 μg/μg protein) when compared to TNF-α (79.3±1.7 μg/μg protein), a similar effect was observed for [DTRP8]- γ-MSH (109±2.9 μg/μg protein). MTT showed an increase in cell
viability in the presence of PG990 (162.5% increase when compared to TNF-α). Gene expression of IL-6, IL-8 and MMP-1 in micromasses pre-treated with PG990 was significantly reduced when compared to cells exposed only to TNF-α (expression normalised to β-actin). IL-6, IL-8 and MMP-1 expression in TNF-α only treated cells was 5.60±0.04,
5.70±0.05 and 6.90±0.09 respectively and all were significantly reduced in the presence of PG990, which was 0.7±0.05 (87.5% reduction), 0.9±0.01 (84.2% reduction) and 1.1±0.04(84.1% reduction) respectively. A similar effect was observed in [DTRP8]- γ-MSH expression(85.7% reduction, 80.7% reduction and 88.4% reduction respectively). IL-6, IL-8 and MMP-1 release detected by ELISA was significantly reduced by PG990 (IL-6: 89.3±1.2, IL-8:142±2.9 and MMP-1: 297±3.3 pg/mL) when compared to TNF-α. A similar effect was also
observed in [DTRP8]- γ-MSH.
Conclusion: The selective hMC3 agonist PG990 exhibits chondroprotective and modulation of inflammatory and tissue destructive pathways following TNF-α activation of chondrocytes. This data highlights a potential role for MC3 receptor as a novel target for treatment of OA.
 Kaneva MK, et al. (2014), Biochem Pharmacol 92:336-47.
 Getting SJ, et al. (2006), Mol Pharmacol 70:1850–1855.