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Ceramide is an important lipid messenger involved in mediating a variety of cell functions including apoptosis. In this study, we show that antisense bax inhibits cytochrome c release, poly(ADP-ribose)polymerase cleavage and cell death induced by ceramide in HL-60 cells. In addition, ceramide induces translocation of Bax to mitochondria. The addition of the broad spectrum caspase inhibitor zVAD-fmk prevented ceramideinduced apoptotic cell death but did not inhibit translocation of Bax and mitochondrial cytochrome c release. Furthermore, ceramide inhibits the expression of the antiapoptotic protein BclxL with an increase in the ratio of Bax to Bcl-xL. These data provide direct evidence that Bax plays an important role in regulating ceramide-induced apoptosis. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Ceramide induces cell cycle arrest and apoptotic cell death associated with increased levels of p27(kip1). The aim of this study was to examine the effects of ceramide on p27(kip1) protein levels as a measure of cell cycle arrest and apoptosis. Results showed that ceramide increased p27(kip1) protein levels through activation of protein phosphatase 2A (PP2A) in PC-3 prostate cancer cells. Treatment of cells with the PP2A inhibitor okadaic acid or with PP2A-Cα siRNA inhibited ceramide-induced enhanced p27(kip1) protein expression and Akt dephosphorylation, and prevented Skp2 downregulation. Overexpression of constitutively active Akt attenuated ceramide-induced Skp2 downregulation and p27(kip1) upregulation. In addition, ceramide stimulated binding of the PP2A catalytic subunit PP2A-Cαβ to Akt as assessed by immunoprecipitation experiments, indicating that PP2A is involved in the induction of p27(kip1) via inhibition of Akt pathway. Finally, whether PP2A can regulate p27(kip1) expression independently of Akt pathway was determined. Knockdown of PP2A-Cα with siRNA reduced p27(kip1) levels in the presence of Akt inhibitor. These data reveal that PP2A is a regulator of ceramide-induced p27(kip1) expression via Akt-dependent and Akt-independent pathways.

Abstract:Many different stimuli such as bioactive agents and environmental stresses are known to cause the activation of sphingomyelinase (SMase), which hydrolyzes sphingomyelin to generate ceramide as a second messenger playing a key role in differentiation and apoptosis in various cell types. Here we identified multiple forms of the membrane-associated neutral SMase (N-mSMase) activity in bovine brain. They could be classified into two groups according to extracting agents: group T-mSMase, extracted with 0.2% Triton X-100, and group S-mSMase, extracted with 0.5 M (NH 4 ) 2 SO 4 . Group TmSMase was further separated into four forms of TmSMase: ␣, ␤, ␥, and ␦, which were extensively purified from 40,000-g pellets of bovine brain homogenates by 3,150-, 5,275-, 1,665-, and 2,556-fold over the membrane extracts, respectively, by sequential use of several column chromatographies. On the other hand, S-mSMase was eluted as two active peaks of S-mSMase ⑀ and in a phenyl-5PW hydrophobic HPLC column and further purified by 1,119-and 976-fold over 40,000-g pellets of the homogenates, respectively. These highly purified NmSMase enzyme preparations migrated as several bands on sodium dodecyl sulfate -polyacrylamide gel electrophoresis and showed many different features in biochemical properties such as pH dependence, Mg 2ϩ requirements, and effects of detergents. Taken together, our data strongly suggest that mammalian brain N-mSMase may exist as multiple forms different in both its chromatographic profiles and biochemical properties. Key Words: Neutral sphingomyelinase -Ceramide -PurificationMammalian brain.

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