Hydrogen peroxide inhibits formation of cartilage in chicken micromass cultures and decreases the activity of calcineurin: implication of ERK1/2 and Sox9 pathways

Calcineurin was found as a positive regulator of chondrogenesis in chondrifying chicken micromass cultures (HDCs), as cyclosporine A (CsA) reduced both the amount of cartilage and the expression of mRNAs of aggrecan and the chondrogenic transcription factor Sox9. Cartilage formation was inhibited by H2O2 in a concentration-dependent manner without loss of cellular viability or severe decrease of cell number. Expression of both the mRNA and the unphosphorylated protein Sox9 was decreased, while its phosphorylation was stimulated by either H2O2 or CsA. Oxidative stress decreased the activity of calcineurin but the phosphorylation of the member of MAPK family ERK1/2 was extremely elevated either by 1 mM H2O2 or 2 μM CSA. The ERK inhibitor PD098059 attenuated the depletion of cartilage matrix as well as decreased the expression and phosphorylation of Sox9 in cultures treated with H2O2 or CsA. Our results suggest that the chondrogenesis-inhibiting effect of H2O2 is mediated, at least partly, by inhibition of calcineurin and by activation of ERK1/2. We also propose a regulatory role of calcineurin in the phosphorylation level of either ERK1/2 or Sox9 and a positive role of ERK1/2 in regulating both the expression level and the phosphorylation state of Sox9 in chicken HDCs.