Abstract
Ceramide is an important lipid messenger involved in mediating a variety of cell functions including apoptosis. However, mechanisms responsible for ceramide-induced apoptosis remain unclear. We investigated the possibility that ceramide may decrease antiapoptotic signaling in cells by inhibiting Akt kinase activity. Our data show that C2-ceramide induces apoptosis in HMN1 motor neuron cells and decreases both basal and insulin- or serum-stimulated Akt kinase activity 65-70%. These results are consistent with decreased Akt kinase activity being involved in the apoptotic effects of ceramide. This possibility is further supported by studies showing that constitutively active Akt kinase decreases C2-ceramide-induced death of HMN1 cells as well as COS-7 cells. Decreased Akt activity is not due to ceramide activating the ceramide-activated protein phosphatase or to a direct inhibition of Akt kinase by ceramide, suggesting that ceramide acts upstream of Akt kinase to decrease its activity. Treating cells with C2-ceramide does not affect phosphorylation of insulin receptor substrate-1, interactions between insulin receptor substrate-1 and p85, or insulin-stimulated phosphatidylinositol 3-kinase activity, suggesting that the effects of C2-ceramide on Akt kinase are not mediated through modulating phosphatidylinositol 3-kinase. In sum, our results suggest that inhibition of the key antiapoptotic kinase, Akt, may play an important role in ceramide-induced apoptosis.
Highlights
Apoptosis is an evolutionarily conserved form of cell death critical for tissue homeostasis
C2-ceramide Induces Apoptosis in HMN1 Cells—The cellpermeable ceramide analogue, C2-ceramide, is toxic to HMN1 cells, with 90% of the cells dying by 5 h following exposure to 50 M C2-ceramide (Fig. 1A)
Active Akt Kinase Inhibits C2 Ceramide-induced Death of HMN1 and COS-7 Cells—To further address whether the inhibition of Akt kinase activity following C2ceramide treatment may represent an important aspect of ceramide-induced apoptosis, we examined the effect of increasing Akt kinase activity on ceramide-induced death of HMN1 cells
Summary
Materials—C2-ceramide, C2-dihydroceramide, C8-ceramide, and MOWIOL were obtained from Calbiochem. In Vitro Immunocomplex Kinase Assay for Akt Kinase—Log phase HMN1 cells were subcultured and grown for 24 h in DMEM containing 10% fetal bovine serum, followed by the removal of serum for 24 h prior to performing the assays. Cells were washed in ice-cold phosphatebuffered saline and lysed at the indicated times in ice-cold lysis buffer (20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 10 mM NaCl, 1 mM EGTA, 1 g/ml leupeptin, 10 g/ml aprotinin, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 mM Na3VO4), and supernatants were collected after centrifugation at 13,000 ϫ g for 15 min at 4 °C. Proteins were resolved in 10% SDSpolyacrylamide gels, electrophoretically transferred onto polyvinylidene difluoride transfer membrane, incubated with sheep anti-Akt antibody, and visualized by ECL. Thin layer chromatography plates were developed in CHCl3/CH3OH/H2O/NH4OH (60:47:11.3:2), dried, visualized, and quantified by autoradiography
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