MacRae V E, Burdon T, Ahmed S F, Farquharson C
Bone Biology Group, Division of Gene Function and Development, Roslin Institute, Roslin, Midlothian EH25 9PS, UK.
J Endocrinol. 2006 Nov;191(2):369-77. doi: 10.1677/joe.1.06958.
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.
促炎细胞因子会抑制生长板发育。然而，其潜在的作用机制尚不清楚。这些效应可能由神经酰胺介导，神经酰胺是一种基于鞘氨醇的脂质第二信使，在多种慢性炎症疾病中水平会升高。为了验证这一假设，我们测定了细胞可渗透的神经酰胺类似物C2-神经酰胺对ATDC5软骨生成细胞系生长以及对培养的胎鼠跖骨的影响。在ATDC5细胞中，40微摩尔（82%；P < 0.05）和25微摩尔（53%；P < 0.05）的C2-神经酰胺均显著诱导细胞凋亡。在40微摩尔时，C2-神经酰胺显著降低细胞增殖（[3H]-胸腺嘧啶核苷摄取/毫克蛋白）（62%；P < 0.05）。根据软骨生成和分化标志物（sox 9、胶原蛋白II和胶原蛋白X）的表达判断，C2-神经酰胺并未显著改变细胞的分化状态。胰岛素样生长因子-I（IGF-I）信号通路是骨骼生长的主要自分泌/旁分泌调节因子。无论有无IGF-I，25微摩尔的C2-神经酰胺均诱导细胞增殖出现同等程度的降低（60%；P < 0.001）。同样，无论有无IGF-I，40微摩尔的C2-神经酰胺均使胎鼠跖骨生长降低31%（均为P < 0.001）。此外，在存在IGF-I和胰岛素受体阻滞剂AG1024的情况下，C2-神经酰胺降低了ADCT5的增殖。因此，C2-神经酰胺依赖性抑制似乎独立于IGF介导的骨骼生长刺激。事实上，生化研究表明，25微摩尔的C2-神经酰胺预处理并未改变IGF-I刺激的胰岛素受体底物-1、Akt或P44/42丝裂原活化蛋白激酶的磷酸化。总之，C2-神经酰胺通过一种不依赖IGF-I的机制抑制生长板软骨细胞的增殖并诱导其凋亡。
