Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC77

Poster Communications

MC1 and MC3 activation inhibits caspase-3 production and activity, promotes cell-viability, IL-10 and HO-1 release from human C20/A4 chondrocytes

M. K. Kaneva1, M. J. Kerrigan2, I. C. Locke1, S. J. Getting1

1. Cell Communication Group, Human and Health Sciences, University of Westminster, London, United Kingdom. 2. University of Greenwich, London, United Kingdom.

Chondrocyte cell death due to inflammation is an important risk factor predisposing to osteoarthritis and exacerbating the disease progression1, 2. Here, we report protective effect of the melanocortin receptor(MC) pan-agonist α-MSH and the selective MC3 agonist DTRP8-γ-MSH against TNF-α-induced apoptosis2in human C20/A4 chondrocytes as assessed by determination of caspase-3 levels, caspase-3/7 activity and cell viability using MTT assay. We show that melanocortins significantly increase the anti-inflammatory proteins heme oxygenase (HO)-1 and interleukin(IL)-10. C20/A4 chondrocytes were plated at 2.0x104cells/well, treated with PBS, α-MSH or DTRP8-γ-MSH (0.1-30.0µg/ml) ± TNF-α (60.0pg/ml) for 6h with cell viability determined via MTT assay and caspase-3/7 activity via Caspase-3/7 Glo Assay. In separate experiments, cells were plated at 1.5x106cells/cm2 and treated with PBS, TNF-α (60.0pg/ml), or TNF-α + αMSH/DTRP8-γ-MSH (3.0µg/ml), with HO1 and caspase-3 levels evaluated by western blot. Cell-free supernatants were collected and analysed for IL-10 production via commercially available ELISA. TNF-α (60.0pg/ml) caused 25.87% cell death, determined by MTT Assay, which was reduced in dose-dependent manner by treatment with the peptides; maximal inhibition of cell death was achieved at 1.0-3.0µg/ml (αMSH) and 3.0-10.0µg/ml DTRP8-γ-MSH (p<0.01,n=3). Treatment of cells with TNF-α increased caspase-3/7 activity 5.7-fold compared to PBS, but was attenuated in the presence of melanocortins in a concentration-dependent manner (p<0.01). Pre-treatment of cells with the MC3/4 antagonist SHU9119 fully reversed the effect of DTRP8-γ-MSH, but had no effect on αMSH action on these parameters. α-MSH and DTRP8-γ-MSH (3.0µg/ml) inhibited TNF-α activated caspase-3 production assessed by western blot by 49.8% and 42.1% respectively (p<0.01,n=3). The anti-inflammatory proteins HO1 and IL-10 were evaluated following treatment with TNF-α (60.0pg/ml) alone and in the presence of αMSH/DTRP8-γ-MSH (3.0µg/ml). TNF-α led to significant increase in HO1 levels which increased 3-fold in the presence of the peptides compared to PBS (p<0.001,n=3); IL-10 release following peptide treatment resulted in a bell-shaped response with maximal release of 48.83 ± 2.24 pg/ml and 37.13±3.23 pg/ml for αMSH (1.0µg/ml) and DTRP8-γ-MSH (3.0µg/ml) respectively at 6 h. Treatment of C20/A4 chondrocytes with αMSH and DTRP8-γ-MSH prevented TNF-α induced cell death, causing an inhibition of caspase 3/7 activity and an increase in IL-10 and HO-1 production. These results indicate a protective role of these peptides against TNF-α induced chondrocyte apoptosis and induction of anti-inflammatory proteins and thus could represent a promising novel approach for treatment of OA.

Where applicable, experiments conform with Society ethical requirements