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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.

We recently reported that the marked decrease in cellular ceramide in primary astrocytes is an early event associated with the mitogenic activity of basic fibroblast growth factor (bFGF) (Riboni, L., Viani, P., Bassi, R., Stabieini, A., and Tettamanti, G. (2000) GLIA 32, 137–145). Here we show that a rapid activation of sphingomyelin biosynthesis appears to be the major mechanism responsible for the fall in ceramide levels induced by bFGF. When quiescent astrocytes were treated with bFGF, an increased amount of newly synthesized ceramide (from eitherl-[3H]serine or [3H]sphingosine) was directed toward the biosynthesis of sphingomyelin. Conversely, bFGF did not appear to affect ceramide levels by other metabolic pathways involved in ceramide turnover such as sphingomyelin degradation and ceramide biosynthesis, degradation, and glucosylation. Enzymatic studies demonstrating a relevant and rapid increase in sphingomyelin synthase activity after bFGF treatment have provided a convincing explanation for the activation of sphingomyelin biosynthesis. The bFGF-induced increase in sphingomyelin synthase appears to depend on a post-translational activation mechanism. Moreover, in the presence of brefeldin A, the activation of sphingomyelin biosynthesis was abolished, suggesting that the enzyme is located in a compartment other than the Golgi apparatus. Also the phosphatidylcholine-specific phospholipase C inhibitor D609 exerted a potent inhibitory effect on sphingomyelin biosynthesis. Finally, we demonstrate that inhibition of sphingomyelin biosynthesis by brefeldin A or D609 led to a significant inhibition of bFGF-stimulated mitogenesis. All this supports that, in primary astrocytes, the early activation of sphingomyelin synthase is involved in the bFGF signaling pathway leading to proliferation.

Current studies indicate that ceramide is involved in the regulation of important cell functions, namely cell growth, differentiation, and apoptosis. In the present study, the possible role of ceramide in the differentiation of neuroblastoma Neuro2a cells was investigated. The following results were obtained. (a) Ceramide content of Neuro2a cells, induced to differentiate by retinoic acid (RA) treatment rapidly increased after addition of RA, was maintained at high levels in RA-differentiated cells and returned to the starting levels with removal of RA and reversal of differentiation; under the same conditions, the sphingosine content remained unchanged. (b) After a short pulse with [3H]sphingomyelin or [3H]sphingosine or L-[3H]serine, the metabolic formation of ceramide was markedly higher and more rapid in RA-differentiated than undifferentiated cells. (c) Inhibitors of ceramide biosynthesis (Fumonisin B1, beta-chloroalanine and L-cycloserine) diminished the extent of the differentiating effect of RA and concomitantly Cer content decreased. (d) The activity of neutral sphingomyelinase increased after addition of RA, maintained high levels in RA-differentiated cells, and returned to the initial levels with removal of RA. (e) Experimental conditions that cause an elevation of ceramide content (treatment with sphingosine or ceramide or C2-ceramide or bacterial sphingomyelinase) inhibited cell proliferation and stimulated neurite outgrowth; dihydro-analogues of sphingosine, ceramide, and C2-ceramide had no effect on differentiation. (f) treatment with Fumonisin B1 completely inhibited sphingosine-induced differentiation. These data suggest a specific bioregulatory function of ceramide in the control of Neuro2a cell growth and differentiation and pose the general hypothesis of a mediator role of ceramide in the differentiation of cells of neural origin.