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 H(2)O(2) 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 H(2)O(2) 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 H(2)O(2) or 2 muM 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 H(2)O(2) or CsA. Our results suggest that the chondrogenesis-inhibiting effect of H(2)O(2) 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.
Zákány
Róza
R
Department of Anatomy, Histology and Embryology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Hungary.
Szíjgyártó
Zsolt
Z
Matta
Csaba
C
Juhász
Tamás
T
Csortos
Csilla
C
Szucs
Kornélia
K
Czifra
Gabriella
G
Bíró
Tamás
T
Módis
László
L
Gergely
Pál
P
D000818:Animals; D019703:Calcineurin / Q000235:genetics / Q000502:physiology*; D065095:Calcineurin Inhibitors; D002356:Cartilage / Q000196:embryology*; D002642:Chick Embryo; D019902:Chondrocytes / Q000187:drug effects / Q000502:physiology; D016572:Cyclosporine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D018878:Limb Buds / Q000502:physiology; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009924:Organ Culture Techniques; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055758:SOX9 Transcription Factor; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D065095:Calcineurin Inhibitors; D004791:Enzyme Inhibitors; D005419:Flavonoids; D006609:High Mobility Group Proteins; D055758:SOX9 Transcription Factor; D014157:Transcription Factors; D016572:Cyclosporine; D006861:Hydrogen Peroxide; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019703:Calcineurin; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one