Regulation of sphingomyelinases in cells of the oligodendrocyte lineage.
Controversy exists regarding the nature of the "executioner" sphingomyelinase (SMase) in cells and its subcellular localization. A new fluorescence-based assay with the substrate 6-hexadecanoylamino-4-methylumbelliferyl-phosphorylcholine allowed rapid and reliable microassays of neutral (N) and acid (A) SMase activity in cell extracts from primary cultures of neonatal rat oligodendrocytes (OPC) and a human oligodendroglioma cell line (HOG). Total SMase activity was much higher in OPC than in HOG cells. Both staurosporine and tumor necrosis factor-alpha (TNF-alpha) induced apoptosis and activated NSMase in a multiphasic manner in both OPC and HOG cells. The increase in caspase 8 activity preceded the 1 hr peak of NSMase activation, which was followed by caspase 3 activation. In contrast, ASMase activity, which constituted >90% of the total SMase activity, was unresponsive to proapoptotic drugs. Neither reducing ASMase levels by 50% by pretreatment with desipramine nor inhibiting sphingolipid synthesis by 50% with fumonisin B1 had any effect on cell death. Isolation of sphingolipid-rich plasma membrane microdomains (rafts) from the cells by sucrose density gradient ultracentrifugation revealed an enrichment of sphingomyelin, ceramide, and caspase 8. Proapoptotic drugs such as staurosporine promoted the translocation of NSMase to the raft fraction. In contrast, ASMase, other lysosomal hydrolases, and caspase 3 remained absent from rafts even after staurosporine treatment. The staurosporine-induced concomitant increase of ceramide in the raft fraction and caspase 3 in the cytosol could be mimicked by the addition of exogenous bacterial SMase. We conclude that caspase 8 activates NSMase in rafts in oligodendrocytes and that the downstream apoptotic signal is via caspase 3.
Testai
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FD
Departments of Pediatrics, Biochemistry, and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA.
Landek
M A
MA
Dawson
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G
D000818:Animals; D000831:Animals, Newborn; D001562:Benzimidazoles / Q000378:metabolism; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000502:physiology; D002470:Cell Survival; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D009836:Oligodendroglia / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D009837:Oligodendroglioma / Q000473:pathology; D051381:Rats; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D001562:Benzimidazoles; D004791:Enzyme Inhibitors; D014409:Tumor Necrosis Factor-alpha; C447680:hoechst 32258; D013108:Sphingomyelin Phosphodiesterase; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C505410:Casp3 protein, rat; C505528:Casp8 protein, rat; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases; D019311:Staurosporine