Activation of Ceramidase and Ceramide Kinase by Vanadate via a Tyrosine Kinase–Mediated Pathway

Abstract

Abstract. Ceramide, a key molecule in the metabolism of sphingolipids, is converted by ceramidase to sphingosine, and phosphorylated by ceramide kinase to form ceramide-1-phosphate (C1P). In this study, we improved on a method of thin-layer chromatography using a fluorescent ceramide, 4-nitrobenzo-2-oxa-1,3-diazole-labeled C6-ceramide (NBD-ceramide) by adding another step for separation of extracted ceramide metabolites by lipophilicity, and determined levels of C1P, caproic acid, sphingomyelin, and glucosylceramide simultaneously. Also we found that 1) treatment of NBD-ceramide-labeled cells (human lung adenocarcinoma A549 cells and Chinese hamster ovary cells) with Na 3 VO 4 increased the amount of NBD-C1P formed within 30 min, 2) the treatment increased production of NBD-caproic acid, a counterpart of sphingosine, by ceramidase within 2 h, 3) expression of ceramide kinase enhanced the Na 3 VO 4 -induced formation of NBD-C1P, and tyrosine kinase inhibitors (herbimycin and genistein) decreased the response, 4) the production of NBD-caproic acid in A549 cells was inhibited by genistein, and 5) the responses for 2 h after Na 3 VO 4 treatment were accompanied by a decrease in the production of NBD-sphingomyelin, not a loss of NBD-ceramide. The improved thin-layer chromatography method was useful for the simultaneous determination of enzymatic activities for ceramide metabolism in cells.

Effects of Glycerophospholipids on Ceramide Kinase Activity: Cardiolipin-Affected Cellular Formation of Ceramide-1-phosphate

“…NBD-labeled ceramide metabolites including NBD-C1P were separated using TLC, and the levels of metabolites were determined as described previously. 14,26,27) Absolute values (pmol/well) of ceramide metabolites in HepG2 cells were similar to those in cells previously reported, 14,26,27) and the relative levels of NBD-sphingomyelin, NBD-glucosylceramide, and NBD-C1P at 30 min after labeling with NBD-ceramide were 5-10 (% of NBD-ceramide), 3-6, and 0.05-0.1%, respectively. Since the ceramide metabolites showed a wide range of values and the absolute values varied depending on experiments, the data in Figs.…”

“…13) Previously, we reported that treatment with orthovanadate, a general inhibitor of tyrosine phosphatase, increased C1P formation via CerK in A549 cells and CHO cells. 14) However, precise mechanisms for the posttranslational regulation of CerK activity have not yet been elucidated.…”

Effects of Hsp90 inhibitors, geldanamycin and its analog, on ceramide metabolism and cytotoxicity in PC12 cells

“…GA and 17-AAG were used at concentrations reported previously (Sano, 2001;Salehi et al, 2006;Clark et al, 2009). The concentrations of other reagents were also the same as those in previous reports (Taniguchi et al, 2007;Kurosawa et al, 2009;Tada et al, 2010). GA, 17-AAG, PMA, and A23187 were dissolved in dimethyl sulfoxide.…”

“…The labeled cells were washed twice with NBD-ceramide-free medium and further incubated for 1 hr with Na 3 VO 4 . Lipid extraction was performed using a chloroform/methanol solution followed by vigorous vortex mixing, and the lipids in the organic and aqueous phases were analyzed on a TLC silica gel 60 plate (#105724, Merck, German) using 1-butanol: acetic acid: water (3:1:1) as the mobile phase, as described (Tada et al, 2010) with minor modifications. The fluorescence was detected with LAS1000-Plus (Fuji-Film, Tokyo, Japan; 470 nm excitation and 515 nm emission).…”

“…The NBD-ceramide used in this study is converted into NBD-sphingomyelin (NBD-SM), NBD-glucosylceramide (NBD-GlcCer), NBD-ceramide-1-phosphate (NBD-C1P), NBD-caproic acid (C6-fatty acid), and non-fluorescent sphingosine in cells by respective enzymes (Tada et al, 2010). First, we analyzed levels of NBD-ceramide metabolites in PC12 cells that were cultured with 5 μM NBD-ceramide for 30 min at 37°C and washed with the ceramide-free medium (Fig.…”