076P London, UK
Pharmacology 2017



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

V. C. Can1, I. C. Locke2, P. Grieco3, S. J. Getting11Life Sciences, University of Westminster, London, United Kingdom, 2Biomedical Sciences, University of Westminster, London, United Kingdom, 3Pharmacy and CIRPEB, University of Naples Federico II, Naples, Italy.

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]-γ-MSH (3.0μg/ml) for 30mins prior to IL-1β (100pg/ml) stimulation for 6h. Micromasses were harvested for RT-PCR gene expression of hMCand hMCreceptors, 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≤0.05vs.control or *p≤0.05vs.stimulus.

Results: RT-PCR showed hMC1 and hMC3 receptor expression on micromass C-20/A4 cells. Cell viability (MTT and Neutral Red) showed that IL-1β 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% respectively, whilst PG992 inhibited cell death by 135% and 159% respectively (*p≤0.05). 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 treated with PG992 (132.1±1.4μg/ml) compared to IL-1β (81.9±1.2μg/ml), a similar effect was observed for [DTRP8]-γ-MSH (113±1.7μg/ml). IL-1β caused a 33% (0.33 fold) (#p≤0.05) reduction in the anti-inflammatory protein HO-1 compared to control, whilst pre-treatment with PG992 and [DTRP8]-γ-MSH caused significant increases in HO-1 expression with a 2.5 and 2.4 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.


(1) Kaneva MK et al. (2014). Biochem Pharmacol 92: 336-47.

(2) Getting SJ et al. (2006). Mol Pharmacol 70: 1850-1855.

(3) Greco KV et al. (2011). Biochem Pharmacol 82: 1919-29.