Abstract
Neutral sphingomyelinases (SMases) are involved in the induction of ceramide-mediated proapoptotic signaling under heat stress conditions. Although ceramide is an important mediator of apoptosis, the neutral SMase that is activated under heat stress has not been identified. In this study, we cloned an Mg(2+)-dependent neutral SMase from a zebrafish embryonic cell cDNA library using an Escherichia coli expression-cloning vector. Screening of the clones using an SMase activity assay with C(6)-7-nitro-2-1,3-benzoxadiazol-4-yl-sphingomyelin as the substrate resulted in the isolation of one neutral SMase cDNA clone. This cDNA encoded a polypeptide of 420 amino acids (putative molecular weight: 46,900) containing two predicted transmembrane domains in its C-terminal region. The cloned neutral SMase 1 acted as a mediator of stress-induced apoptosis. Bacterially expressed recombinant neutral SMase 1 hydrolyzed [choline-methyl-(14)C]sphingomyelin optimally at pH 7.5 in the presence of an Mg(2+) ion. In zebrafish embryonic cells, the endogenous SMase enzyme was localized in the microsomal fraction. In FLAG-tagged SMase-overexpressing cells, neutral SMase 1 colocalized with a Golgi marker in a cytochemical analysis. Inactivation of the enzyme by an antisense phosphorothioate oligonucleotide repressed the induction of ceramide generation, caspase-3 activation, and apoptotic cell death by heat stress. Thus, neutral SMase 1 participates in an inducible ceramide-mediating, proapoptotic signaling pathway that operates in heat-induced apoptosis in zebrafish embryonic cells.
Highlights
Sphingolipids have been implicated as bioactive molecules in animal cells
zebrafish embryonic (ZE) cells and embryos exhibit typical apoptotic features such as membrane blebbing, nuclear and cytoplasmic shrinkage, and nuclear DNA fragmentation [30]. We studied whether such apoptotic processes are mediated by ceramide generation and caspase-3 activation under heat stress
Knockdown Experiment with an Antisense Oligonucleotides—To examine whether ceramide generation by Mg2ϩ-dependent neutral SMase was involved in heat-induced apoptosis, the phosphorothioate oligonucleotide for neutral SMase 1 was used to repress the levels of this protein
Summary
C2-ceramide, N-acetylsphingosine (a ceramide analog); MCA, 4-methylcoumaryl-7-amide; C6-NBD-sphingomyelin, C6-7-nitro-2-1,3-benzoxadiazol-4-yl-sphingomyelin; [14C]phosphatidylcholine, L-3-phosphatidyl[N-methyl-14C]choline,1,2-dipalmitoyl; [14C]sphingomyelin, [N-methyl-14C]sphingomyelin; DAPI, 4Ј,6-diamidino-2-phenylidole; ER, endoplasmic reticulum; FCS, fetal calf serum; HEK, human embryonic kidney; PAF, [1-O-octadecyl-3H]lyso-plateletactivating factor; PBS, phosphate-buffered saline; SMase, sphingomyelinase; LB, Luria-Bertani broth; PARP, poly(ADP-ribose) polymerase; PIPES, 1,4-piperazinediethanesulfonic acid; ZE, zebrafish embryonic; Z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. We found a zebrafish Mg2ϩ-dependent neutral SMase 1 that produces ceramide and causes thalidomide-induced vascular defects in zebrafish embryos [32]. This enzyme may be the mediator of stressinduced ceramide generation and apoptosis. Zebrafish cells showed high Mg2ϩ-dependent neutral SMase activity. We report the expression cloning and biochemical features of this zebrafish neutral SMase 1 as a key enzyme in stress-induced apoptosis and ceramide generation in zebrafish embryonic (ZE) cells. Biochemical characterization showed that his enzyme is a mediator of stressinducible apoptosis in ZE cells
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