Neutral sphingomyelinase-induced ceramide triggers germinal vesicle breakdown and oxidant-dependent apoptosis in Xenopus laevis oocytes.
ABSTRACT Ceramide regulates many cellular processes, including cell growth, differentiation, and apoptosis. Although the effects of exogenous bacterial neutral sphingomyelinase (SMase) in Xenopus laevis oocytes have been investigated, its microinjection into oocytes has not been reported previously. Thus, we compared the incubation versus microinjection of the neutral Bacillus cereus sphingomyelinase (bSMase) to examine whether the topology of ceramide generation determines its effects on the fate of oocytes. In agreement with previous findings, incubation of mature stage VI oocytes with bSMase increased ceramide levels in oocyte extracts over time, causing the germinal vesicle breakdown indicative of maturation, without evidence of cytotoxicity. In contrast, bSMase microinjection, which increased ceramide levels in a time- and dose-dependent manner, resulted in oocyte apoptosis characterized by reactive oxygen species (ROS) generation, reduced glutathione (GSH) depletion in cytosol and mitochondria, release of cytochrome c and Smac/Diablo from mitochondria, and caspase-3 activation. Microinjection of acidic SMase from human placenta recapitulated the apoptotic effects of bSMase microinjection. Preincubation of oocytes with GSH-ethyl ester before bSMase microinjection prevented ROS generation and mitochondrial downstream events, thus protecting oocytes from bSMase-induced death. These findings show a divergent action of bSMase-induced ceramide on oocyte maturation or apoptosis depending on the intracellular site where ceramide is generated.
Article: Probiotic sonicates selectively induce mucosal immune cells apoptosis through ceramide generation via neutral sphingomyelinase.[show abstract] [hide abstract]
ABSTRACT: Probiotics appear to be beneficial in inflammatory bowel disease, but their mechanism of action is incompletely understood. We investigated whether probiotic-derived sphingomyelinase mediates this beneficial effect. Neutral sphingomyelinase (NSMase) activity was measured in sonicates of the probiotic L. brevis (LB) and S. thermophilus (ST) and the non-probiotic E. coli (EC) and E. faecalis (EF). Lamina propria mononuclear cells (LPMC) were obtained from patients with Crohn's disease (CD) and Ulcerative Colitis (UC), and peripheral blood mononuclear cells (PBMC) from healthy volunteers, analysing LPMC and PBMC apoptosis susceptibility, reactive oxygen species (ROS) generation and JNK activation. In some experiments, sonicates were preincubated with GSH or GW4869, a specific NSMase inhibitor. NSMase activity of LB and ST was 10-fold that of EC and EF sonicates. LB and ST sonicates induced significantly more apoptosis of CD and UC than control LPMC, whereas EC and EF sonicates failed to induce apoptosis. Pre-stimulation with anti-CD3/CD28 induced a significant and time-dependent increase in LB-induced apoptosis of LPMC and PBMC. Exposure to LB sonicates resulted in JNK activation and ROS production by LPMC. NSMase activity of LB sonicates was completely abrogated by GW4869, causing a dose-dependent reduction of LB-induced apoptosis. LB and ST selectively induced immune cell apoptosis, an effect dependent on the degree of cell activation and mediated by bacterial NSMase. These results suggest that induction of immune cell apoptosis is a mechanism of action of some probiotics, and that NSMase-mediated ceramide generation contributes to the therapeutic effects of probiotics.PLoS ONE 01/2011; 6(3):e16953. · 4.09 Impact Factor
Article: Nongenomic steroid- and ceramide-induced maturation in amphibian oocytes involves functional caveolae-like microdomains associated with a cytoskeletal environment.[show abstract] [hide abstract]
ABSTRACT: Stimulation of full-grown amphibian oocytes with progesterone initiates a nontranscriptional signaling pathway that converges in the activation of Cdc2/cyclin B and reentry into meiosis. We observed that cholesterol depletion mediated by methyl-beta-cyclodextrin (MbetaCD) inhibited meiotic maturation, suggesting involvement of membrane rafts. In the present study, we further characterized caveolae-like membranes from Rhinella arenarum oocytes biochemically and functionally. The identification by mass spectrometry of a nonmuscle myosin heavy-chain associated with caveolar membranes showed evidence of direct involvement of the underlying cytoskeletal environment in the structure of oocyte rafts. Biophysical analysis using the fluorescent probe Laurdan revealed that MbetaCD-mediated cholesterol depletion affected membrane lipid order. In line with this finding, cholesterol removal also affected the localization of the raft marker lipid GM1. Results demonstrated that ceramide is an effective inducer of maturation that alters the distribution of the raft markers caveolin-1, SRC, and GM1, while progesterone seems not to affect membrane microdomain integrity. Cholesterol depletion had a greater effect on ceramide-induced maturation, thus suggesting that ceramide is an inducer more vulnerable to changes in the plasma membrane. MbetaCD treatment delayed tyrosine phosphorylation and MAPK activation in progesterone-induced maturation. Functional studies regarding tyrosine phosphorylation raise the possibility that the hormone receptor is located in the nonraft membrane in the absence of ligand and that it translocates to the caveola when it binds to progesterone. The presence of raft markers and the finding of signaling molecules from MAPK cascade functionally associated to oocyte light membranes suggest that this caveolae-rich fraction efficiently recreates, in part, maturation signaling.Biology of Reproduction 06/2011; 85(4):808-22. · 4.01 Impact Factor