Abnormal growth patterns are commonly observed in children suffering from chronic inflammatory diseases. These disorders are associated with the increased production of pro-inflammatory cytokines, which inhibit growth plate chondrocyte dynamics. Ceramide, a sphingosine-based lipid second messenger, mediates many of the actions of pro-inflammatory cytokines. Ceramide inhibits IGF-1 signalling and induces apoptosis in numerous cell types. This study determined the effects of C2-ceramide (C2-c) (40 μM, 25 μM, 10 μM), a cell permeable ceramide analogue, on murine growth plate chondrocytes, using the ATDC5 chondrogenic cell line and cultured fetal metatarsals. In ATDC5 cells, C2-c at 40 μM and 25 μM significantly induced apoptosis (63%; P<0.05 and x%) and significantly reduced proliferation (62%; P<0.05 and x%). C2-cat 40 μM significantly reduced fetal metatarsal growth (62%; P<0.05). The effect of C2-c on IGF-1 induced proliferation was examined. In ATDC5 cells, in the presence of IGF-1 (24 h, 100 ng/ml), ceramide (25 μM) induced a 68% reduction in proliferation (P<0.001). However, in the absence of IGF-1, ceramide induced a comparable 61% decrease (P<0.001). C2-c (40 μM) induced the same 31% reduction in metatarsal growth both in the presence and absence of IGF-1 (8 d, 100 ng/ml) (both P<0.001). Therefore, C2-c does not appear to specifically inhibit the pro-proliferative effects of exogenous IGF-1.The effect of C2-c on endogenous IGF-1 induced proliferation was also examined, using AG1024, an IGF-1R and IR blocker (10 μM). In the absence of exogenous IGF-1, AG1024 significantly reduced proliferation (28%, P<0.001). C2-c (25 μM) significantly reduced proliferation compared to AG1024 treatment (55%, P<0.001). C2-c and AG1024 in combination further reduced proliferation compared to C2-c alone (46% P<0.01). Further studies are required to confirm whether the inhibitory effects of pro-inflammatory cytokines in growth plate chondrocytes are mediated through ceramide, influencing the growth of children with inflammatory diseases through a local effect at the growth plate.
01 - 05 Apr 2006
European Society of Endocrinology