Abstract
Dysregulated osteoblast programmed cell death (PCD) is implicated in metabolic bone diseases such as osteoporosis. Ceramide, a precursor to all complex sphingolipids, is a secondary messenger in PCD. However, the mechanisms of ceramide induced-PCD are poorly characterized. Thus, the aim of this study was to investigate the nature and mechanism of ceramide-induced PCD in osteoblasts. The MTT assay, propidium iodide uptake with flow cytometric analysis, and TUNEL staining were used to assess the effect of exogenous ceramide on cell viability and apoptosis in MC3T3-E1 cells and primary murine osteoblasts. Western blotting and immunofluorescence approaches were used to elucidate the underlying signaling mechanisms. Ceramide reduced MC3T3-E1 and primary osteoblast viability and induced apoptosis in a dose-dependent manner. Ceramide failed to activate the executioner caspases-3 and -7 and poly (ADP-ribose) polymerase (PARP), while a selective inhibitor of caspase-8 abrogated ceramide effects on osteoblast viability. Ceramide induced p38 and ERK activation, but only p38 was involved in ceramide-induced osteoblast PCD. Our study highlights a novel role for ceramide in inducing caspase-8 and p38-dependent osteoblast PCD. Understanding cell death mechanisms in osteoblasts could help in the development of new targeted therapeutics that selectively identify cells with pathogenic programmed cell death pathways.