Mitochondrial ceramide increases in UV-irradiated HeLa cells and is mainly derived from hydrolysis of sphingomyelin.
Sphingolipids are important signaling molecules in many biologic processes, but little is known about their organelle-specific roles. Using HeLa cells, we investigated the effects of UV and etoposide-induced apoptosis on the contents of sphingomyelin (SM) and ceramide in subcellular compartments. UV irradiation of HeLa cells increased the levels of SM in all subcellular fractions, but the change was most dramatic in mitochondria. Using diacylglycerol kinase assays to quantify ceramide, we found that the levels of ceramide in mitochondria increased as early as 2 h after UV irradiation and remained elevated at 6 h. The increase in mitochondrial SM and ceramide was inhibited by D609, an inhibitor of sphingomyelinase and SM synthase. The inhibition of sphingolipid production correlated with protection of the mitochondrial transmembrane potential and prevention of cytochrome c release following UV irradiation. In contrast, myriocin, an inhibitor of the de novo ceramide synthesis pathway, only partially suppressed the production of ceramides in mitochondria and cannot suppress UV-induced apoptosis. Fumonicin B1, an inhibitor of ceramide synthase, can only prevent mitochondrial ceramide synthesis and UV-induced apoptosis in a small degree. These results indicate that mitochondrial ceramide production in UV-irradiated HeLa cells is not mediated by the de novo synthesis pathway, but mainly through SM hydrolysis.
Dai
Qiang
Q
Huntsman Cancer Institute and Section of Oncology at the University of Utah, 2000 Circle of Hope, Suite 5244, Salt Lake City, UT 84112, USA.
Liu
Jihua
J
Chen
Jun
J
Durrant
David
D
McIntyre
Thomas M
TM
Lee
Ray M
RM
D017209:Apoptosis / Q000502:physiology / Q000528:radiation effects; D001952:Bridged-Ring Compounds / Q000494:pharmacology; D002518:Ceramides / Q000138:chemical synthesis* / Q000528:radiation effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D009636:Norbornanes; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism* / Q000528:radiation effects; D013859:Thiocarbamates; D013871:Thiones / Q000494:pharmacology; D017855:Transferases (Other Substituted Phosphate Groups) / Q000037:antagonists & inhibitors / Q000378:metabolism; D014466:Ultraviolet Rays
D001952:Bridged-Ring Compounds; D002518:Ceramides; D004791:Enzyme Inhibitors; D009636:Norbornanes; D013109:Sphingomyelins; D013859:Thiocarbamates; D013871:Thiones; C046498:tricyclodecane-9-yl-xanthogenate; D017855:Transferases (Other Substituted Phosphate Groups); C019863:ceramide cholinephosphotransferase; D013108:Sphingomyelin Phosphodiesterase