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. Getting¹. ¹Life 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 hMC₁ and hMC₃ receptors, 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.

References:

(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.