Regulation of Intracellular Ceramide Content in B16 Melanoma Cells

Abstract

We previously reported that ceramide released from glycosphingolipids (GSLs) by endoglycoceramidase was directly metabolized to GSLs, and thus the content of GSLs was constantly maintained in B16 melanoma cells (Ito, M., and Komori, H. (1996) J. Biol. Chem. 271, 12655-12660). In this study, the metabolism of ceramide released from sphingomyelin (SM) by bacterial sphingomyelinase (SMase) was examined using B16 cells and their GSL-deficient mutant counterpart GM95 cells. Treatment of B16 melanoma cells with bacterial SMase effectively hydrolyzed SM on the plasma membrane. Under these conditions, NeuAc␣2,3Gal␤1,4Glc␤1,1ceramide was significantly increased. Interestingly, UDP-glucose: ceramide glucosyltransferase-1 (GlcT-1) activity and GSL synthesis, but not SM synthesis or sphingosine generation, were found to be up-regulated by SMase treatment. The up-regulation of GSL synthesis seemed to occur at both the transcriptional and post-translational steps of GlcT-1 synthesis. Accumulation of ceramide by bacterial SMase was much higher in GM95 cells than in the parental cells. When the enzyme was removed from the culture medium, the intracellular ceramide level in B16 cells, but not that in the mutant cells, normalized. No rapid restoration of SM in either of the cell lines was observed after removal of the enzyme. SMase treatment strongly inhibited DNA synthesis in GM95 cells but not that in B16 cells. In the presence of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of GlcT-1, SMase treatment markedly increased the ceramide content and thus inhibited DNA synthesis in B16 cells. Our study provides the first evidence that GlcT-1 functions to regulate the level of intracellular ceramide by glycosylation of the ceramide when it is present in excess.

Perturbation of membrane microdomains reduces mitogenic signaling and increases susceptibility to apoptosis after T cell receptor stimulation

“…Ceramide glycosylation in the ER contributes to the maintenance of ceramide homeostasis in the intracellular organelles. 38 Although the fluidity of the intracellular membranes of asmase 7/7 cells is reduced, cholesterol and ceramide levels are not significantly altered in membrane preparations of wild-type compared to aSMasedeficient cells.…”

Ceramidase Enhances Phospholipase C-induced Hemolysis by Pseudomonas aeruginosa

“…Because Cer is toxic to host cells, it should be excluded by the defense system of the host. For example, Komori et al (45,46) reported that Cer generated in the outer leaflet of the plasma membrane by bacterial SMase was metabolized to glycosphingolipids in B16 melanoma cells through activation of a glucosylceramide synthase. Recently, Huitema et al (47) reported the cloning of human SM syntheses (SMS1 and SMS2) and demonstrated that SMS1 is located in the endoplasmic reticulum, whereas SMS2 is present in the plasma membrane.…”

Modulation of Hepatitis C Virus Genome Replication by Glycosphingolipids and Four-Phosphate Adaptor Protein 2

“…Here, we took advantage of pharmacological inhibitors of glucosylceramide synthase (UGCG), N-butyldeoxynojirimycin (NB-DNJ) (54-57) and D, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) (58,59), to determine the role of glucosylceramide in HCV RNA synthesis. Since UGCG activity is required to generate the glucosylceramide (Fig.…”