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 mM (53%; P!0 . 05). At 40 mM, C2-ceramide significantly reduced proliferation ([ 3 H]-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 mM) induced an equivalent reduction in proliferation (60%; P!0 . 001). Similarly, C2-ceramide (40 mM) 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 mM) 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.

SOCS2 is the critical regulator of GH action in murine growth plate chondrogenesis

“…(35,65,66) These observations indicate that SOCS1 and a lack of GH effect on metatarsal growth. (43,44,(54)(55)(56)68) However, Scheven and Hamilton did report GH stimulatory effects on metatarsal length, which is in contrast to the data of this current study. (68) Our studies did show that SOCS2 À/À metatarsals increased in growth in response to GH, suggesting that modulation of SOCS2 expression may be a critical method of altering GH insensitivity and raising local IGF-1 concentration.…”

“…GH, IGF-1, or LY-294002 were added at the final concentrations of 100 ng/mL, 100 ng/mL, and 10 mM, respectively, as used previously in similar studies on murine metatarsals. (43,44) Bone lengths were measured using a Nikon Eclipse TE300 microscope with a digital camera attached, using Image Tool (Image Tool Version 3.00, University of Texas Health Life Sciences Center, San Antonio, TX, USA). Bones were incubated in a humidified atmosphere (378C, 5% CO 2 ) for up to 14 days.…”

“…Immunoblotting was performed as described previously. (44) Briefly, lysates were run on 3% to 8% Tris Acetate gels (for STAT protein analysis) or 10% Bis-Tris gels (for SOCS protein analysis) (Invitrogen). Nitrocellulose films were blocked in 5% BSA (Fraction V; Sigma) and probed overnight with primary antibodies (Supplemental Table S1) diluted in 5% BSA.…”

“…(43,44,(54)(55)(56) To determine if this lack of response to GH was because of the inhibitory actions of SOCS2, we measured growth of embryonic metatarsals from WT and SOCS2 À/À mice cultured with GH or IGF-1 over a 13-day period (Fig. 3A,B).…”

Endocrine role of bone: recent and emerging perspectives beyond osteocalcin

“…Recent in vitro results have indicated that sphingolipids are implicated in osteoblast and chondrocyte apoptosis and in the regulation of osteoclastogenesis (Takeda et al 1998, MacRae et al 2006; reviewed by ). In vivo, sphingolipid metabolism plays a critical role in skeletogenesis; mouse models lacking the ceramide-generating neutral sphingomyelinase 2 enzyme (nSMase2/SMPD3 -gene-targeted Smpd3 K/K and fro/fro mice) display gross skeletal abnormalities, including deformed long bones, short-limb dwarfism, hypomineralisation, delayed dentin mineralisation and enamel formation (Aubin et al 2005, Stoffel et al 2005, Alebrahim et al 2014.…”