The effect of the selective human MC3 receptor agonist PG992 on high density human chondrocyte micromass cultures activated by IL-1beta.

Introduction Osteoarthritis (OA) is a degenerative joint disease partially mediated by the catabolic cytokine IL-1β, which causes progressive and permanent degeneration of cartilage (1). A potential anti-inflammatory and chondroprotective role for melanocortin peptides has been shown via the human melanocortin-3 (hMC3) receptor subtype. This study aims to assess the chondroprotective and anti-inflammatory effects of the hMC3 receptor agonist PG992 and the partially selective agonist [DTRP8]-γ-MSH on IL-1β induced cell death, pro-inflammatory cytokine and matrix metalloproteinase (MMP) release in human 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 24-well plates. After 48h micromasses were treated with PG992 (Ac-Nle-c[Asp-Trp-Pro-DNal(2)-Arg-Trp-Lys]-NH2) (10.0µg/ml) or [DTRP8]-Y-MSH (3.0µg/ml) for 30mins prior to IL-1β (100pg/ml) stimulation for 6h. Micromasses were harvested for RT-PCR gene expression of MC1, MC3, cell viability studies, alcian blue staining for sulphated glycosaminoglycan (GAG) content and western blot detection for hemeoxygenase-1 (HO-1) expression. Cell free supernatants were analysed for 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. Both cell viability assays MTT and Neutral Red (NR) confirmed that there was a significant increase in micromass cultures treated with PG992 when compared to IL-1β. IL-1 stimulation caused a maximal cell death of 17% and 19% respectively (#p≤0.05), with [DTRP8]-γ-MSH inhibiting cell death by 126% and 133% (*p≤0.05) respectively, whilst PG992 inhibited cell death by 135% and 159% respectively (*p≤0.05). ELISA analysis confirmed that IL-1β stimulation caused a significant increase in IL-6, IL-8 and MMP-1 release. PG992 significantly reduced IL-6 and IL-8 release by 77% and 81% respectively and completely abrogated MMP-1 release. Alcian blue staining showed an increased GAG accumulation in micromasses treated with PG992 (132.1±1.4 µg/µg protein) when compared to IL-1β (81.9±1.2 µg/µg protein), a similar effect was observed for [DTRP8]-Y-MSH (113±1.7 µg/µg protein). IL-1β caused a 33% (0.33 fold) reduction in HO-1 expression compared to control, whilst pre-treatment of micromasses with [DTRP8]-Y-MSH and PG992 caused a significant increase in HO-1 expression with a 2.4 and 2.5 fold increase respectively when compared to stimulus (*p≤0.05). Conclusion: The selective hMC3 receptor agonist PG992 exhibited both chondroprotection and modulation of inflammatory and tissue destructive pathways following IL-1β chondrocyte activation highlighting a role for the hMC3 receptor for treatment of OA