Abstract
This study aimed to investigate the role of ceramide in inducing autophagy in osteoblasts, then further investigate the involved downstream signaling cascades. The effect of exogenous ceramide on inducing autophagy in MC3T3-E1 and Primary murine osteoblasts (POB) were assessed by (a) Transmission Electron Microscopy (TEM), Western Blot (WB) analysis, (c) Immunofluorescence microscopy, and (d) Propidium Iodide (PI)-uptake/flow cytometry analysis. C2-ceramide induced the formation of cytoplasmic vacuoles and elevated the expression of the autophagy proteins Atg5, beclin1, and LC3 II. Silencing Atg5 or beclin1 with siRNAs partially rescued the C2-ceramide-induced cell death in osteoblasts yet this silencing did not rescue the C2-ceramide-induced autophagy as assessed by TEM. Hence, these results suggest that C2-ceramide induces Atg5 and beclin1-independent autophagic cell death in osteoblasts. This work highlighted a novel role for ceramide in inducing autophagy in osteoblasts. The use of ceramide or inhibitors of its synthesis could be utilized as a therapeutic tool for modulating osteoblast survival in different metabolic bone diseases.