Phosphatidylinositol 3-Kinase/AKT Pathway Regulates the Endoplasmic Reticulum to Golgi Traffic of Ceramide in Glioma Cells
A LINK BETWEEN LIPID SIGNALING PATHWAYS INVOLVED IN THE CONTROL OF CELL SURVIVAL*
- Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, Laboratorio Interdisciplinare di Tecnologie Avanzate, via Fratelli Cervi 93, 20090 Segrate (Milan), Italy
- 2 To whom correspondence should be addressed: Dept. of Medical Chemistry, Biochemistry and Biotechnology, L.I.T.A. via F.lli Cervi, 93 20090 Segrate (Milan) Italy. Tel.: 390250330370; Fax: 390250330365; E-mail: paola.viani{at}unimi.it.
Abstract
Different lines of evidence indicate that both aberrant activation of the phosphatidylinositol 3-OH kinase (PI3K)/Akt survival pathway and down-regulation of the death mediator ceramide play a critical role in the aggressive behavior, apoptosis resistance, and adverse clinical outcome of glioblastoma multiforme. Furthermore, the inhibition of the PI3K/Akt pathway and the up-regulation of ceramide have been found functional to the activity of many cytotoxic treatments against glioma cell lines and glioblastomas as well. A reciprocal control between PI3K/Akt and ceramide signaling in glioma cell survival/death is suggested by data demonstrating a protective role of PI3K/Akt on ceramide-induced cell death in glial cells. In this study we investigated the role of the PI3K/Akt pathway in the regulation of the ceramide metabolism in C6 glioma cells, a cell line in which the PI3K/Akt pathway is constitutively activated. Metabolic experiments performed with different radioactive metabolic precursors of sphingolipids and microscopy studies with fluorescent ceramides demonstrated that the chemical inhibition of PI3K and the transfection with a dominant negative Akt strongly inhibited ceramide utilization for the biosynthesis of complex sphingolipids by controlling the endoplasmic reticulum (ER) to Golgi vesicular transport of ceramide. These findings constitute the first evidence for a PI3K/Akt-dependent regulation of vesicle-mediated movements of ceramide in the ER-Golgi district. Moreover, the findings also suggest the activation of the PI3K/Akt pathway as crucial to coordinate the biosynthesis of membrane complex sphingolipids with cell proliferation and growth and/or to maintain low ceramide levels, especially as concerns those treatments that promote ceramide biosynthesis in the ER.
Footnotes
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↵3 The abbreviations used are: GBM, glioblastoma multiforme; ER, endoplasmic reticulum; PI3K, phosphatidylinositol-3-OH kinase; PIP2, phosphatidylinositol(4,5)P2; PIP3, phosphatidylinositol(3,4,5)P3; PH, pleckstrin homology; PTEN, phosphatase and tensin homologue; Cer, ceramide; Wm, wortmannin; BFA, brefeldin A; BSA, bovine serum albumin; CERT, ceramide transfer protein; [3H]Sph, d-erythro-[3-3H]sphingosine; [14C]SM, [choline-methyl-14C]sphingomyelin; [3H]C6-Cer, N-[hexanoyl-6-3H]d-erythro-hexanoyl-sphingosine; HPTLC, high performance thin layer chromatography; NBD-C6-Cer, 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl) sphingosine; FAPP, four phosphate adaptor protein; EGF-FAPP1-PH, PH domain of FAPP1 protein fused with enhanced green fluorescent protein; DN-AKT, dominant negative mutant of Akt; PBS, phosphate-buffered saline; siRNA, small interfering RNA; SMS, sphingomyelin synthase; GCS, glucosylceramide synthase; SMase, sphingomyelinase; PI4K, phosphatidylinositol 4-OH kinase; PI4P, phosphatidylinositol 4-phosphate; GlcCer, glucosylceramide; SM, sphingomyelin; SREBP, sterol regulatory element-binding protein; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; N-SMase, neutral SMase; A-SMase, acidic SMase.
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↵* This work was supported by grants from the University of Milan (to P. G., L. R., and P. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 Both authors contributed equally to this work.
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- Received November 25, 2008.
- Revision received December 17, 2008.
- The American Society for Biochemistry and Molecular Biology, Inc.
