title: Ceramide inhibition of chondrocyte proliferation and bone growth is IGF-I independent.
abstract: 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 microM (53%; P < 0.05). At 40 microM, 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 microM) induced an equivalent reduction in proliferation (60%; P < 0.001). Similarly, C2-ceramide (40 microM) 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 microM) 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.
authors.affiliation: Bone Biology Group, Division of Gene Function and Development, Roslin Institute, Roslin, Midlothian EH25 9PS, UK. vicky.macrae@bbsrc.ac.uk
authors.forename: V E
authors.initials: VE
authors.lastname: MacRae
authors.forename: T
authors.initials: T
authors.lastname: Burdon
authors.forename: S F
authors.initials: SF
authors.lastname: Ahmed
authors.forename: C
authors.initials: C
authors.lastname: Farquharson
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western / Q000379:methods; D001846:Bone Development / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D024043:Collagen Type II / Q000032:analysis; D024121:Collagen Type X / Q000032:analysis; D016207:Cytokines / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006132:Growth Plate / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000032:analysis; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I / Q000037:antagonists & inhibitors / Q000494:pharmacology / Q000502:physiology*; D008682:Metatarsal Bones / Q000196:embryology; D051379:Mice; D008815:Mice, Inbred Strains; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D051058:Oncogene Protein v-akt / Q000378:metabolism; D009924:Organ Culture Techniques; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D055758:SOX9 Transcription Factor; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014157:Transcription Factors / Q000032:analysis; D020032:Tyrphostins / Q000494:pharmacology
chemical_list: D015415:Biomarkers; D024043:Collagen Type II; D024121:Collagen Type X; D016207:Cytokines; D006609:High Mobility Group Proteins; C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; C064769:N-acetylsphingosine; D010750:Phosphoproteins; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; C527933:Sox9 protein, mouse; D014157:Transcription Factors; D020032:Tyrphostins; C447031:tyrphostin AG 1024; D007334:Insulin-Like Growth Factor I; D051058:Oncogene Protein v-akt; D048052:Mitogen-Activated Protein Kinase 3; D013110:Sphingosine