Ceramide-induced cell death/survival in murine osteoblasts

Abstract

Programmed cell death (PCD) or apoptosis is a naturally occurring cell suicide pathway induced in a variety of cell types. We determined whether ceramide treatment contributes to reduced cell viability and increased PCD in primary osteoblasts and the signalling pathways that are involved. Cell viability was determined by the 3-(4,5-dimethyl-thiozol-2-yl)-2,5-diphenyl tetrazolium bromide assay. We found that C(2)-ceramide (<or=10(-7) M) promoted osteoblast viability, whilst concentrations >or=2 x 10(-6) M significantly reduced osteoblast viability in a dose- and time-dependent manner. The effect of ceramide on cell viability was specific since C(2)-dihydroceramide had no effect. Increasing intracellular ceramide levels with either sphingomyelinase (SMase) or an inhibitor of ceramide metabolism also increased osteoblast apoptosis. Ceramide-induced PCD in osteoblasts was determined by nuclear appearance and DNA fragmentation. PCD was induced by both C(2)-ceramide and SMase. The ability of ceramide (5 x 10(-8) M) to promote osteoblast survival was prevented by a general protein kinase C (PKC) inhibitor and by a PKC zeta inhibitor, whilst osteoblast survival was enhanced in the presence of a protein phosphatase 1 (PP1) inhibitor. Phosphatidylinositol-3 kinase (PI3K) inhibitors had no effect on osteoblast survival. The ability of ceramide (5 x 10(-5) M) to induce apoptosis was prevented by the inhibitors of PP1 and PKC delta, whilst the general PKC and PI3K inhibitors had no effect on it. Our findings suggest that ceramide signals osteoblast survival and apoptosis through different intracellular pathways, and that alteration in the intracellular levels of ceramide may play an important role in bone remodelling.