Ceramide inhibition of chondrocyte proliferation and bone growth is IGF-I independent

    1. C Farquharson1
    1. 1Bone Biology Group, Division of Gene Function and Development, Roslin Institute, Roslin, Midlothian EH25 9PS, UK
    2. 2Bone and Endocrine Research Group, Royal Hospital for Sick Children, Glasgow, UK
    1. (Requests for offprints should be addressed to V E MacRae; Email: vicky.macrae{at}bbsrc.ac.uk)


    Proinflammatory cytokines inhibit growth plate development. However, their underlying mechanisms of action are unclear. These effects may be mediated by ceramide, a sphingosine-based lipid second messenger, which is elevated in a number of chronic inflammatory diseases. To test this hypothesis, we determined the effects of C2-ceramide, a cell permeable ceramide analogue, on the growth of the ATDC5 chondrogenic cell line and on cultured fetal mice metatarsals. In ATDC5 cells, C2-ceramide significantly induced apoptosis at both 40 (82%; P < 0.05) and 25 μM (53%; P < 0.05). At 40 μM, C2-ceramide significantly reduced proliferation ([3H]-thymidine uptake/mg protein) (62%; P < 0.05). C2-ceramide did not markedly alter the differentiation state of the cells as judged by the expression of markers of chondrogenesis and differentiation (sox 9, collagen II and collagen X). The IGF-I signalling pathway is the major autocrine/paracrine regulator of bone growth. Both in the presence and absence of IGF-I, C2-ceramide (25 μM) induced an equivalent reduction in proliferation (60%; P < 0.001). Similarly, C2-ceramide (40 μM) induced a 31% reduction in fetal metatarsal growth both in the presence and absence of IGF-I (both P < 0.001). Furthermore, C2-ceramide reduced ADCT5 proliferation in the presence of AG1024, an IGF-I and insulin receptor blocker. Therefore, C2-ceramide-dependent inhibition appears to be independent of IGF-mediated stimulation of bone growth. Indeed, biochemical studies demonstrated that C2-ceramide (25 μM) pretreatment did not alter IGF-I-stimulated phosphorylation of insulin receptor substrate-1, Akt or P44/42 MAP kinase. In conclusion, C2-ceramide inhibits proliferation and induces apoptosis in growth plate chondrocytes through an IGF-I independent mechanism.

    • Received in final form 11 July 2006
    • Accepted 28 July 2006
    • Made available online as an Accepted Preprint 6 November 2006
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