The neutral type 2
sphingomyelinase (nSMase2) hydrolyzes
sphingomyelin and generates
ceramide, a major bioactive
sphingolipid mediator, involved in growth arrest and apoptosis. The role of nSMase2 in apoptosis is debated, and apparently contradictory results have been observed on fibroblasts isolated from nSMase2-deficient
fragilitas ossium (homozygous fro/fro) mice. These mice exhibit a severe neonatal dysplasia, a lack of long bone mineralization and delayed apoptosis patterns of hypertrophic chondrocytes in the growth plate. We hypothesized that apoptosis induced by nutrient deprivation, which mimics the environmental modifications of the growth plate, requires nSMase2 activation. In this study, we have compared the resistance of fro/fro fibroblasts to different death inducers (
oxidized LDL,
hydrogen peroxide and nutrient
starvation). The data show that nSMase2-deficient fro/fro cells resist to apoptosis evoked by nutrient
starvation (
fetal calf serum/
glucose/
pyruvate-free DMEM), whereas wt fibroblasts die after 48h incubation in this medium. In contrast,
oxidized LDL and
hydrogen peroxide are similarly toxic to fro/fro and wt fibroblasts, indicating that nSMase2 is not involved in the mechanism of toxicity evoked by these agents. Interestingly, wt fibroblasts treated with the SMase inhibitor
GW4869 were more resistant to
starvation-induced apoptosis. The resistance of fro/fro cells to
starvation-induced apoptosis is associated with an increased expression of
hyaluronan synthase 2 (HAS2) mRNAs and
protein, which is inhibited by
ceramide. In wt fibroblasts, this HAS2 rise and its protective effect did not occur, but exogenously added HA exhibited a protective effect against
starvation-induced apoptosis. The protective mechanism of HAS2 involves an increased expression of the
heat-shock protein Hsp72, a chaperone with antiapoptotic activity. Taken together, these results highlight the role of nSMase2 in apoptosis evoked by nutrient
starvation that could contribute to the delayed apoptosis of hypertrophic chondrocytes in the growth plate, and emphasize the antiapoptotic properties of HAS2.