Effects of the selective MC3 agonists PG990 and PG992 on Interleukin-1 beta induced chondrocyte cell death and pro-inflammatory cytokine release

V. C. Can1, I. C. Locke1, P. Grieco2, S. J. Getting1. 1Life Sciences, University of Westminster, London, UNITED KINGDOM, 2Department of Pharmacy and CIRPEB, University of Naples Federico II, Naples, ITALY.

Introduction: The catabolic cytokine IL-1β plays a critical role in the development of osteoarthritis, activating chondrocytes to secrete pro-inflammatory cytokines and matrix metalloproteinases (MMPs). These degrade the extracellular matrix promoting chondrocyte apoptosis, leading to progressive and permanent degeneration of cartilage (1). A role for melanocortin peptides exerting anti-inflammatory and chondroprotection effects have been shown, although the receptor subtype involved is unclear. This study aims to assess the chondroprotection and anti-inflammatory effects of the selective human melanocortin-3 (hMC3) receptor agonists PG990, PG992 and D[Trp8]-γ-MSH on IL-1β induced cell death, pro-inflammatory cytokine and matrix metalloproteinase release.

Methods: Human C-20/A4 chondrocytic cells were treated with D[Trp8]-γ-MSH (3.0μg/ml), PG990 (Ac-Nle-c[D-P-P-DNal(2′)-R-W-K]-NH2) or PG992 (Ac-Nle-c[D-W-P-DNal(2′)-R-W-K]-NH2) (1.0-30.0μg/ml) for 30mins prior to cAMP analysis, Total Collagen expression, or IL-1β (5000pg/ml) stimulation for 24h. Cell viability was determined by MTT, whilst, IL-8 and MMP-1 release were detected by ELISA in cell free supernatants. Data are expressed as Mean±SD of n=4-8 determinations in triplicate. *p<0.05 vs. control or #p<0.05 vs. stimulus.

Results: PG990 and PG992 caused cAMP accumulation with maximal accumulation at 1.0μg/ml and 3.0μg/ml respectively indicating hMC3 receptor-functionality, whilst increasing total collagen expression by PG990 (1.0μg/ml) of 113.9±1.1μg/ml (*p<0.05) and PG992 (3.0μg/ml) of 143.5±0.2μg/ml (*p<0.05) compared to control (104.5±0.1μg/ml). IL-1β stimulation caused a maximal cell death of 25% (*p<0.05) which was completely abrogated in the presence of D[Trp8]-γ-MSH, PG990 (1.0μg/ml) and PG992 (3.0μg/ml). IL-1β caused a significant increase in IL-8 and MMP-1 release, which was not inhibited by D[Trp8]-γ-MSH. However, PG990 (1.0μg/ml) and PG992 (3.0μg/ml) reduced IL-8 by 80% (#p<0.05) and 47% (#p<0.05) respectively, whilst MMP-1 was reduced only by PG992 (3.0μg/ml) by 16% (#p<0.05).

Conclusion: These data highlight that the selective hMC3 receptor agonists (PG990 and PG992) exhibited chondroprotection and modulation of IL-8 following IL-1β activation of chondrocytes at 24h, whilst D[Trp8]-γ-MSH only exerted chondroprotective effects. This data highlights a role for the hMC3receptor in exerting anti-inflammatory and chondroprotective properties.

References:

(1) Aida Y et al. (2005), Life Sci 77: 3210-3221.

(2) Wang L et al. (2015). Int J Clinical Exp Pathol, 8: 298-308.