ARTHRITIS & RHEUMATISM Vol. 58, No. 1, January 2008, pp 209–220 DOI 10.1002/art.23172 © 2008, American College of Rheumatology Sphingomyelinase Decreases Type II Collagen Expression in Bovine Articular Cartilage Chondrocytes via the ERK Signaling Pathway S. J. Gilbert, E. J. Blain, V. C. Duance, and D. J. Mason Objective. Ceramide, a mediator of proinflamma- tory 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 ana- lyzed 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 confo- cal 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 chon- drocytes also induced translocation of c-Fos to the nucleus and phospho-SOX9 to the cytoplasm and in- creased expression of c-fos mRNA. Type II collagen expression, which was down-regulated by SMase treat- ment, was restored by the MEK-1/2 inhibitor, PD98059. Conclusion. SMase down-regulates type II colla- gen in articular chondrocytes via activation of the ERK signaling cascade, redistribution of SOX9, and recruit- ment of c-Fos. This new mechanism for cartilage degra- dation provides potential targets for future treatment of arthritic disease. Ceramide is an important lipid second messenger for tumor necrosis factor  (TNF), a proinflammatory cytokine that is widely implicated in the pathogenesis of arthritic diseases (1). Ceramide activates intracellular signaling cascades that are involved in a variety of cellular functions such as proliferation, differentiation, necrosis, and apoptosis (2). Studies of articular cartilage suggest that ceramide plays a role in cartilage degener- ation (3–5), and our group recently showed that activa- tion of the ceramide signaling pathway disrupts cartilage matrix homeostasis, resulting in down-regulation of type II collagen (6). In addition, several metabolites of cer- amide have been implicated in cartilage degradation and arthritic disease (7,8). Farber’s disease, in which a lack of ceramidase causes excess ceramide accumulation within the cartilage and bone, is associated with joint pain and arthritis-like joint degeneration (9,10). This finding is important, because it provides direct evidence for a role of ceramide in human arthritic disease. TNF can increase the level of endogenous cer- amide through 2 main pathways: the catabolic pathway involving hydrolysis of the membrane lipid sphingomye- lin by endosomal acidic and membrane-bound neutral sphingomyelinases (SMases) (11–13), and de novo syn- thesis (14). Hydrolysis of sphingomyelin at the plasma membrane is also achieved by the application of exoge- nous SMase, an enzyme with properties similar to those of neutral SMase, and leads to a transient increase in intracellular ceramide formation (15), the magnitude of which increases with increasing doses of SMase (16). Exogenously applied short-chain ceramides mimic sev- eral actions of TNF (12,17), emphasizing the role of lipid second messengers in TNF signaling (18). In several cell types, ceramide regulates a variety of protein Supported by the Arthritis Research Campaign, UK (grants 16436 and 14874) and the School of Biosciences, Cardiff University. S. J. Gilbert, PhD, E. J. Blain, PhD, V. C. Duance, PhD, D. J. Mason, PhD: Cardiff University, Cardiff, Wales, UK. Address correspondence and reprint requests to S. J. Gilbert, PhD, School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3US, Wales, UK. E-mail: GilbertSJ1@Cardiff.ac.uk. Submitted for publication May 4, 2007; accepted in revised form September 14, 2007. 209