Sphingomyelinase decreases type II collagen expression in bovine articular cartilage chondrocytes via the ERK signaling pathway.
OBJECTIVE
Ceramide, a mediator of proinflammatory cytokine signaling, induces cartilage degradation and reduces type II collagen synthesis in articular cartilage. The accumulation of ceramide is associated with arthritis in Farber's disease. The aim of this study was to investigate the mechanism of ceramide-induced down-regulation of type II collagen.


METHODS
Bovine articular chondrocytes were stimulated with sphingomyelinase (SMase) to increase levels of endogenous ceramide. Components of the ERK pathway were inhibited by Raf-1 kinase inhibitor and the MEK inhibitor, PD98059. Cell extracts were analyzed by Western blotting for ERK-1/2, SOX9, c-Fos, and type II collagen, and the level of c-fos messenger RNA (mRNA) was analyzed by quantitative polymerase chain reaction. Localization of ERK-1/2, SOX9, and c-Fos was assessed by immunocytochemistry and confocal microscopy.


RESULTS
SMase treatment of chondrocytes caused sustained phosphorylation of ERK-1/2 throughout the cytoplasm and nucleus that was reduced by inhibitors of Raf-1 kinase and MEK-1/2. SMase treatment of chondrocytes also induced translocation of c-Fos to the nucleus and phospho-SOX9 to the cytoplasm and increased expression of c-fos mRNA. Type II collagen expression, which was down-regulated by SMase treatment, was restored by the MEK-1/2 inhibitor, PD98059.


CONCLUSIONS
SMase down-regulates type II collagen in articular chondrocytes via activation of the ERK signaling cascade, redistribution of SOX9, and recruitment of c-Fos. This new mechanism for cartilage degradation provides potential targets for future treatment of arthritic disease.
Gilbert
S J
SJ
Cardiff University, Cardiff, Wales, UK. GilbertSJ1@Cardiff.ac.uk
Blain
E J
EJ
Duance
V C
VC
Mason
D J
DJ
D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D024043:Collagen Type II / Q000378:metabolism*; D003470:Culture Media; D015536:Down-Regulation / Q000502:physiology; D017781:Genes, Immediate-Early / Q000502:physiology; D006609:High Mobility Group Proteins / Q000378:metabolism; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D055758:SOX9 Transcription Factor; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism* / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism
D024043:Collagen Type II; D003470:Culture Media; D006609:High Mobility Group Proteins; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D055758:SOX9 Transcription Factor; D014157:Transcription Factors; D019908:Proto-Oncogene Proteins c-raf; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D013108:Sphingomyelin Phosphodiesterase