Regulation of ceramide metabolism in glioma cells : evidence on the role of phosphatidylinosotol-4-kinases

A great number of evidence supports the role of ceramide (Cer), a key intermediate of sphingolipid metabolism as signalling molecule in mechanisms governing growth, differentiation and death in many cell types including glial cells. Different studies demonstrate that in glial cells Cer exerts antiproliferative and proapoptotic effects, and strongly support that Cer-signaling is altered in glial tumors. The control of Cer levels is a very complex process which involves both specific enzymes localized in different subcellular compartments and the regulation of Cer intracellular movements. Recent evidence suggests that the biological effects exerted by Cer depends on the variation of Cer levels in specific subcellular compartments, therefore correlated to the regulation of its intracellular traffic. Notwithstanding the mechanisms involved in Cer transport play a crucial role in Cer metabolism and signalling, the information on this process is very limited. Recently, a protein (CERT) that acts as ceramide carrier protein between endoplasmic reticulum (ER) and Golgi apparatus and is involved in sphingomyelin (SM) biosynthesis has been identified. The amino-terminal region of CERT contains a pleckstrin homology (PH) domain with selectivity towards phosphatidylinositol-4-phosphate (PI4P), to allow its targeting to the Golgi apparatus. In addition, recent evidence demonstrates that the interaction of oxysterol-binding protein (OSBP) with ER and Golgi apparatus is regulated by its PH domain and is involved in the vesicle -mediated Cer transport from ER to the the Golgi for SM biosynthesis. This suggests the possibility that phosphoinositides, in particular PI4P a lipid known to be enriched in Golgi membranes, contributes to the regulation of ceramide metabolism and signalling. A number of different isoforms of PI4-kinase (PI4-K) have been characterized. The isoenzymes differ from subcellular distribution and sensivity to inhibitors. Most of the isoenzymes are inhibited by phenylarsine oxide (PAO), whereas only the class III PI4-Ks, mainly associated to Golgi membranes, are sensitive to wortmannin (WT). In the present study we investigate the effect of PI4-Ks on ceramide metabolism and traffic in C6 glioma cells. Treatment of C6 glioma cells with both PAO and WT results in a dose-dependent decrease of phosphatidylinositol phosphorylation. In pulse/chase experiments with 3H sphingosine both inhibitors promote a dose-dependent increase of 3H-Cer with a concomitant decrease of 3H-SM and in a lesser extent of 3H-glucosylceramide. Metabolic experiments performed with N-hexanoyl-[3H]sphingosine and 3H-SM allow to exclude an effect of PI4-K inhibitors and/or polyphosphoinositides directly on SM-synthase, SMase or glucosylceramide-synthase activity. Moreover, PI4-K inhibitors strongly affect the intracellular distribution of BODIPY-Cer. In fact in cells treated with WT and PAO the accumulation of the fluorescence in the perinuclear region representative of the Golgi apparatus was strongly reduced. Staining with NBD Cer a specific marker of the Golgi demonstrate the integrity of Golgi apparatus in WT and PAO treated cells.Taken together these results strongly suggest that the inhibition of a PI4-K localized in the Golgi apparatus impairs the biosynthesis of sphingomyelin and in a lesser extent of glucosylceramide, possibly reducing the dockng sites, namely PI4P, for ceramide targeting to the Golgi apparatus. This could represent a further element in the cross-talk between phosphoinositides and sphingolipid signaling involved in the control of glial cell fate.