Báo cáo khoa học: A novel, promoter-based, target-specific assay identifies 2-deoxy-D-glucose as an inhibitor of globotriaosylceramide biosynthesis docx

A novel, promoter-based, target-specific assay identifies 2-deoxy-D-glucose as an inhibitor of globotriaosylceramide biosynthesis Tetsuya Okuda1, Koichi Furukawa2 and Ken-ichi Nakayama1 Glycolipids Function Analysis Team, Health Technology Research Center, National Institute of Advanced Industrial Science and Technology, Kagawa, Japan Department of Biochemistry II, Graduate School of Medicine, Nagoya University, Aichi, Japan Keywords Fabry disease; glycosphingolipid; glycosyltransferase; hemolytic uremic syndrome; promoter Correspondence T Okuda, Glycolipids Function Analysis Team, Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi, Takamatsu, Kagawa 761-0395, Japan Fax: +81 87 869 3593 Tel: +81 87 869 3563 E-mail: t-okuda@aist.go.jp (Received 27 April 2009, revised 22 June 2009, accepted 15 July 2009) doi:10.1111/j.1742-4658.2009.07215.x Abnormal biosynthesis of globotriaosylceramide (Gb3) is known to be associated with Gb3-related diseases, such as Fabry disease The Gb3 synthase gene (Gb3S) codes for a1,4-galactosyltransferase, which is a key enzyme involved in Gb3 biosynthesis in vivo Transcriptional repression of Gb3S is a way to control Gb3 biosynthesis and may be a suitable target for the treatment of Gb3-related diseases To find a transcriptional inhibitor for Gb3S, we developed a convenient cell-based chemical screening assay system by constructing a fusion gene construct of the human Gb3S promoter and a secreted luciferase as reporter Using this assay, we identified 2-deoxy-d-glucose as a potent inhibitor for the Gb3S promoter In cultured cells, 2-deoxy-d-glucose markedly reduced endogenous Gb3S mRNA levels, resulting in a reduction in cellular Gb3 content and a corresponding accumulation of the precursor lactosylceramide Moreover, cytokineinduced expression of Gb3 on the cell surface of endothelial cells, which is closely related to the onset of hemolytic uremic syndrome in O157-infected patients, was also suppressed by 2-deoxy-d-glucose treatment These results indicate that 2-deoxy-d-glucose can control Gb3 biosynthesis through the inhibition of Gb3S transcription Furthermore, we demonstrated the general utility of our novel screening assay for the identification of new inhibitors of glycosphingolipid biosynthesis Introduction Glycosphingolipid (GSL) is commonly found as a component of the cell membrane in eukaryotic cells GSL is composed of ceramide and various ceramide- linked carbohydrate chains A large number of GSLs are found in mammalian tissues, which can be classified in terms of a difference in their carbohydrate Abbreviations 2-AA, anthranilic acid; 2DG, 2-deoxy-D-glucose; asialo-GM2, GalNAcb1,4LacCer; B4GalT6, b1,4-galactosyltransferase 6; BDNF, brain-derived neurotrophic factor; EC, endothelial cell; FITC, fluorescein isothiocyanate; GlcNAc, N-acetylglucosamine; HUVEC, human umbilical vein endothelial cell; GA1, asialo-GM1 (Galb1,3GalNAcb1,4LacCer); Gal, galactose; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Gb3, globotriaosylceramide (Gala1,4LacCer); Gb3S, Gb3 synthase gene; Gb4, globotetraosylceramide (GalNAcb1,3Gala1,4LacCer); GD1a, NeuAca2,3Galb1,3GalNAcb1,4(NeuAca2,3)LacCer; GD1b, Galb1,3GalNAcb1,4(NeuAca2,8NeuAca2,3)LacCer; GM1, Galb1,3GalNAcb1,4 (NeuAca2,3)LacCer; GM2, GalNAcb1,4(NeuAca2,3)LacCer; GM3, NeuAca2,3LacCer; GM3S, GM3 synthase; GSL, glycosphingolipid; GT1b, NeuAca2,3Galb1,3GalNAcb1,4(NeuAca2,8NeuAca2,3)LacCer; HUS, hemolytic uremic syndrome; LacCer, lactosylceramide (Galb1,4Glcb1Cer); Lc3, lactotriaosylceramide (GlcNAcb1,3LacCer); LPS, lipopolysaccharide; mAb, monoclonal antibody; MDR1, multiple drug resistance protein 1; TNF-a, tumor necrosis factor-a; TPA, phorbol 12-myristate 13-acetate; TRE, TPA response element FEBS Journal 276 (2009) 5191–5202 ª 2009 The Authors Journal compilation ª 2009 FEBS 5191 A novel strategy for the inhibition of glycosphingolipid biosynthesis structure [1] Globotriaosylceramide (Gb3) is the initial structure of the globo-series GSL [2] Gb3 is known as the blood group Pk antigen [3], or the CD77 antigen, which is associated with a subset of immature B-cell or Burkitt’s lymphomas [4] Although the molecular function of Gb3 is poorly understood, recent reports have indicated that abnormal expression and ⁄ or accumulation can lead to several disease states For example, patients with Fabry disease, an X-linked lysosomal storage disease caused by deficiency of the Gb3 catabolic enzyme a-galactosidase A, have been found to accumulate Gb3 in various tissues [5] As a result of a defect in this enzyme, depositions of Gb3 are found in numerous tissues, notably the vascular endothelium, causing a systemic disorder in patients, which functionally affects the skin, eyes, kidney, heart and autonomic nervous system Gb3 also plays a role in the well-characterized receptor for verotoxin, a product of Escherichia coli O157 strain [6,7] Infection of E coli O157 is frequently associated with hemolytic uremic syndrome (HUS), resulting from vetoroxin-induced damage to endothelial cells (ECs) Inflammatory mediators, such as tumor necrosis factor-a (TNF-a) and lipopolysaccharide (LPS), enhance the Gb3 expression level in ECs through the up-regulation of Gb3S transcription These events are considered to be a progression towards the onset of HUS [8–10] Gb3 is synthesized from its precursor lactosylceramide (LacCer, Galb1,4Glcb1Cer) and UDP-galactose by a1,4-galactosyltransferase (EC 2.4.1.228) in the mammalian cell A single gene, the Gb3 ⁄ CD77 synthase gene (Gb3S), codes for a1,4-galactosyltransferase Indeed, targeted disruption of Gb3S results in the complete absence of Gb3 and its derivatives in vivo [10] From these observations, we reasoned that the transcriptional inhibition of Gb3S may be an effective means of treatment of Gb3-related diseases Previously, we have identified and characterized the human Gb3S promoter [11] The Gb3S promoter is specifically activated in cells that express Gb3, which indicates the importance of this promoter activity for Gb3 expression In this study, we developed a simple and convenient assay for monitoring Gb3S promoter activity by using a secreted luciferase reporter gene Because this assay is able to measure Gb3S promoter activity in a one-step reaction, the method can be used to readily identify potential transcriptional inhibitors of Gb3S Using this assay, we identified 2-deoxy-d-glucose (2DG) as a candidate inhibitor for Gb3S transcription, and confirmed that 5192 T Okuda et al treatment with 2DG suppresses Gb3 biosynthesis in vivo Results Development of Gb3S promoter-driven luciferase secretion cells To identify a novel inhibitor for Gb3 biosynthesis, we have recently developed a cell-based screening assay using the pML reporter vector and the Gb3S promoter The pML reporter vector includes the Metridia longa secreted luciferase gene [12] as a reporter gene The human Gb3S promoter has previously been identified in the 5¢-flanking region ()1893 bp to +84 bp Fig Establishment of Gb3S promoter-driven luciferase secretion cells (A) The scheme of the constructed vector plasmid (pMLGb3Sp) Open box, Gb3S promoter; bold black arrow, reporter gene (secreted Metridia luciferase); TB, transcriptional blocker; KanR ⁄ NeoR, kanamycin and neomycin resistance gene (B) Luciferase activity of vector transfectants HeLa cells were transiently transfected with pML-Gb3Sp (Gb3Sp) or empty vector (pML) The luciferase activity of the culture medium was measured as described in Experimental procedures The relative luciferase activity was determined from the ratio of the activity in the transfectant with pML (open bar) After G418 selection, stable pML-Gb3p or pML mutants were established, and relative luciferase activities were calculated (filled bar) FEBS Journal 276 (2009) 5191–5202 ª 2009 The Authors Journal compilation ª 2009 FEBS T Okuda et al from the transcriptional initiation site; GenBank accession number AB473818) of the human Gb3S gene locus [11,13] We amplified this region by PCR and inserted it into the pML reporter vector as shown in Fig 1A The resultant plasmid (pML-Gb3Sp) was transfected into Gb3-positive HeLa cells The stable mutant HML-Gb3 (HeLa cells stably transfected with pML-Gb3Sp) was subsequently established by G418 selection A stable transfectant of pML empty vector, named HML, was also established as a negative control to monitor background expression levels As shown in Fig 1B, more than seven-fold overexpression of luciferase reporter activity was observed in HMLGb3 Furthermore, the reporter activity was moderately stronger than that of the transient transfectants This result is presumably because of the difference in transfection efficiency between stable and transient transfectants A novel strategy for the inhibition of glycosphingolipid biosynthesis 24-well plate format were 16 h and · 105 cells, respectively, for the rapid and highly sensitive monitoring of reporter activity We used these conditions in all subsequent experiments Identification of 2DG as a transcriptional inhibitor of Gb3S We examined the effects of a number of bioactive substances on the reporter activity of HML-Gb3 cells (Fig 3) First, we examined potential inhibitors for transcriptional factor Sp1 (mithramycin A, streptozotocin, 2DG, high glucose treatment), which has been Characterization of the reporter activity of HML-Gb3 For the purposes of chemical screening, we determined the optimal culture time and seeding cell number of HML-Gb3 in a 24-well plate format The time course of the reporter activity of HML-Gb3 is shown in Fig (left) The reporter activity of HML-Gb3 increased in proportion to the culture time and reached a plateau at around 48 h, whereas the background ratio (HML-Gb3 ⁄ HML) reached a plateau after around 16 h The reporter activity of HML-Gb3 also increased with cell number and reached a plateau at around · 105 cells (Fig 2, right) In this case, the background ratio reached a plateau at around · 105 cells (Fig 2, left) From these results, we conclude that the optimal culture time and seeding cell number of HML-Gb3 in the Fig Effect of candidate inhibitors on Gb3S promoter activity HML-Gb3 cells were treated with the following substances or conditions for 16 h; 1, untreated control; 2, 10 mM 2DG; 3, 30 mM glucose (high glucose treatment); 4, 10 mM streptozotocin; 5, 200 nM mithramycin A; 6, 100 lM citrate; 7, 10 mM pyruvate; 8, 50 lM splitomicin; 9, 100 lM nicotinamide; 10, 20 ngỈmL)1 TNF-a; 11, glucose starvation; 12, 10 mM GlcNAc; 13, 10 mM galactose; 14, 10 mM 2-deoxy-D-galactose; 15, 10 mM 2DG with 10 mM pyruvate; 16, 10 mM 2DG with 50 lM splitomicin; 17, 10 mM 2DG with 100 lM nicotinamide After the treatment with each inhibitor, luciferase activity in the culture medium was measured as described previously The luciferase activity of the HML cell (HML) is indicated as a negative control The results represent relative luciferase activity as a percentage of the untreated control Error bars, mean ± SD, n = Fig Characterization of the reporter activity of HML-Gb3 Cells (2 · 105) of HML-Gb3 (filled squares) or HML (open circles) were seeded into a 24-well (15.49 mm diameter) culture plate Luciferase activity in the culture medium was then measured at the relevant incubation time (time course, top left) A variety of cell numbers were examined (cell number, top right) The luciferase activity was then measured after 16 h of incubation The ratios (HML-Gb3 ⁄ HML) of these experiments are shown in the bottom panels FEBS Journal 276 (2009) 5191–5202 ª 2009 The Authors Journal compilation ª 2009 FEBS 5193 A novel strategy for the inhibition of glycosphingolipid biosynthesis T Okuda et al Fig The effect of 2DG on the HML-Gb3 cells (A) Dose-dependent inhibitory effect of 2DG on luciferase activity of HML-Gb3 cells HML-Gb3 cells were treated with the indicated concentration of 2DG for 16 h The results represent the relative luciferase activity as a percentage of the luciferase activity of untreated cells Error bars, mean ± SD, n = (B) Time-dependent alteration of luciferase activity of HML-Gb3 cells after 2DG treatment The HML-Gb3 cells were incubated in the presence (open squares) or absence (filled squares) of 2DG (10 mM) for the indicated times (individually for 0, 4, or 16 h) (C) Luciferase activity of cell lysates (Cell) and culture medium (Medium) from HML-Gb3 cells treated with (+) or without ()) 2DG (10 mM) for 16 h Error bars, mean ± SD, n = (D) The number of viable (squares with full lines) and dead (circles with dotted lines) cells after treatment or not with 2DG (10 mM) for the indicated times (0, 24, 48 and 72 h) Filled squares and circles, non-treated cells; open squares and circles, 2DG-treated cells Error bars, mean ± SD, n = identified previously as an essential factor for Gb3S promoter regulation [11] Mithramycin A is a wellcharacterized inhibitor of Sp1 transcriptional activity This compound inhibits the interaction of Sp1 protein with its consensus DNA sequence [14,15] Treatment with 2DG, streptozotocin or high levels of glucose has been reported to down-regulate the transcriptional activity of Sp1 protein via O-GlcNAc modification [16–19] Surprisingly, only 2DG displayed a strong inhibitory effect in the reporter activity assays using HML-Gb3 cells 2DG had a strong dose-dependent suppressive effect on the Gb3S promoter (Fig 4A), which was not displayed by any of the other chemicals examined Because 2DG is a well-known glycolytic inhibitor, as a result of its inhibitory effect on glucose hexokinase [16,20,21], it was suspected that the observed decrease in reporter activity was caused by a depletion of ATP in these cells As expected, glucose starvation of HMLGb3, which diminishes ATP production, decreased the reporter activity of HML-Gb3 cells (Fig 3, bar 11) However, the decreased reporter activity by 2DG could not be salvaged by treatment with pyruvate, which directly activates the tricarboxylic acid cycle to generate ATP (Fig 3, bar 15) Therefore, we concluded that there was a very poor correlation between 5194 the suppression of ATP generation and the effect of 2DG on the Gb3S promoter It is known that glycolytic inhibition stress induces gene silencing [21] Indeed, 2DG, citrate and pyruvate are known to act as glycolytic inhibitors, which could induce gene silencing via the same mechanism However, these compounds showed no inhibitory effect on the reporter activity of HML-Gb3 cells (Fig 3, bars and 7) Previously, 2DG has been identified as an activator for a class III histone deacetylase SIRT1, which induced gene silencing through chromatin remodeling [22] To confirm the relationship between 2DG and SIRT1 in HML-Gb3 cells, we examined the effects of the SIRT1 inhibitors (splitomicin and nicotinamide) [22,23] on 2DG treated HML-Gb3 cells Treatment with these SIRT1 inhibitors slightly enhanced reporter activity in HML-Gb3 control cells (Fig 3, bars and 9) This result indicates that the reporter activity is partly affected by SIRT1 activity in the cells However, reporter activity in 2DG-treated HML-Gb3 cells did not recover These results indicate that SIRT1 is unrelated to the decrease in reporter activity in 2DGtreated HML-Gb3 cells TNF-a is an inducer of Gb3 expression in ECs [8–10] It has been reported that TNF-a induces Gb3 FEBS Journal 276 (2009) 5191–5202 ª 2009 The Authors Journal compilation ª 2009 FEBS T Okuda et al expression via transcriptional up-regulation of Gb3S [10,11] The parent cell line of HML-Gb3 is also known to be sensitive to TNF-a [24] Thus, we fully anticipated that TNF-a treatment would enhance the reporter activity of HML-Gb3 cells However, no such change was observed after TNF-a treatment (Fig 3, bar 10) To determine the key structure in 2DG for the inhibition of the reporter activity of HML-Gb3 cells, we examined the effects of structural analogues of 2DG, such as N-acetylglucosamine (GlcNAc), galactose (Gal) and 2-deoxy-d-galactose on HML-Gb3 cells (Fig 3, bars 12 and13) After treatment with these compounds, we found a slight, but significant, decrease in reporter activity only in the 2-deoxy-d-galactosetreated HML-Gb3 cells This result indicates that the glucose backbone and deoxygenation of the hydroxy group in carbon position are important in decreasing the reporter activity of HML-Gb3 cells A novel strategy for the inhibition of glycosphingolipid biosynthesis teratocarcinoma (NCCIT cells) in the following experiments because the cell viability and growth were unaffected by 2DG treatment (Fig 5A) As expected, 2DG treatment for week strongly suppressed Gb3S expression (Fig 5B) The mRNA expression of other major glycolipid synthase genes, such as GM3 (NeuAca2,3LacCer) synthase [25] and b1,4-galactosyltransferase 6, which is a LacCer synthase [26], were unaffected by 2DG treatment Next, we examined the GSL components in NCCIT cells before and after treatment with 2DG TLC analysis showed that the major neutral GSL of the NCCIT cell was Gb3 (Fig 6B) After 2DG treatment, the level of Gb3 markedly decreased, which was followed by an accumulation of LacCer and the appearance of another GSL (Fig 6A, asterisk) Based on the HPLC elution time (Fig 6B, asterisk), it seems that the newly 2DG inhibits Gb3 biosynthesis via the transcriptional repression of Gb3S in cells To confirm whether 2DG inhibits Gb3S promoter activity, we characterized several other effects of 2DG on HML-Gb3 cells Because 2DG treatment represses human papillomavirus early in gene transcription [16,20], which is essential for HeLa cell viability, prolonged exposure to 2DG causes cell growth inhibition and death (Fig 4D) These effects were observed in cells 24 h after 2DG treatment Indeed, almost all cells were dead within 72 h However, decreased reporter activity of HML-Gb3 cells was detected immediately after 2DG treatment (Fig 4B) This result supports the observation that 2DG toxicity barely affects the reporter activity of HML-Gb3 16 h after treatment Our assay system used a secreted form of luciferase as reporter Hence, there is the possibility that the apparent transcriptional repression of Gb3S could be the result of 2DG-induced inhibition of protein secretion Thus, we measured the reporter activity in the cell lysate from HML-Gb3 cells (Fig 4C) Decreased reporter activity was observed in the culture medium of HML-Gb3 cells after 2DG treatment However, no reporter activity could be detected in the cell lysate prepared from the same cells From these results, we conclude that 2DG treatment almost certainly represses Gb3S promoter activity in HML-Gb3 cells To verify this conclusion, we analyzed the expression levels of Gb3S mRNA and GSLs after 2DG treatment In order to decrease endogenous Gb3 or Gb3S mRNA levels, long-term exposure of 2DG seems to be important Therefore, we used a Gb3 highly expressed Fig The effects of 2DG on NCCIT cells (A) The number of viable (squares with full lines) and dead (circles with dotted lines) cells after treatment or not with 2DG (10 mM) for the indicated days (1, 2, and days) Filled squares and circles, nontreated cells; open squares and circles, 2DG-treated cells Error bars, mean ± SD, n = (B) RT-PCR analysis of glycolipid synthase gene mRNA expression in NCCIT cells treated with (+) or without ()) 2DG (10 mM) for week Expression of GAPDH mRNA in the cells was monitored as an internal control Gb3S, Gb3 synthase; B4GalT6, b1,4-galactosyltransferase 6; GM3S, GM3 synthase FEBS Journal 276 (2009) 5191–5202 ª 2009 The Authors Journal compilation ª 2009 FEBS 5195 A novel strategy for the inhibition of glycosphingolipid biosynthesis T Okuda et al Fig Neutral GSL analysis of 2DG-treated NCCIT cells (A) TLC analysis for neutral glycolipids from NCCIT cells, visualized by orcinol– H2SO4 Lane 1, standard neutral glycolipids, lane 2, glycolipids from NCCIT cells; lane 3, glycolipids from 2DG-treated NCCIT cells The experiment was performed with a solvent system consisting of chloroform–methanol–water (60 : 35 : 8, v ⁄ v ⁄ v) Asterisk indicates a newly appeared GSL (B) HPLC analysis of neutral GSL-derived oligosaccharides Oligosaccharides released from neutral GSLs by endoglycoceramidase were labeled with the fluorescent compound 2-AA and analyzed using an HPLC system, as described in Experimental procedures GSLs were purified from NCCIT cells (top panel) or 2DG-treated (10 mM, days) NCCIT cells (bottom panel) The elution positions of standard 2-AA-labeled oligosaccharides, which were generated from commercially available GSLs (1, LacCer; 2, Gb3; 3, Gb4; 4, GA1), are shown as arrowheads Asterisk indicates estimated elution areas of amino-CTH generated GSL is the amino-CTH [lactotriaosylceramide (Lc3, GlcNAcb1,3LacCer) or asialo-GM2 (GalNAcb1,4LacCer)] To determine the expression levels of each of the GSLs, we carried out semi-quantitative HPLC analyses (Fig 6B) The results are shown in Table The cellular Gb3 levels were reduced by approximately 40% after 2DG treatment, and then its derivative globotetraosylceramide (Gb4) became undetectable By contrast, the level of the Gb3 precursor LacCer increased 3.5-fold after 2DG treatment Moreover, 2DG resulted in increased levels of amino-CTH, which is synthesized from LacCer by enzymes other than Gb3S Although gangliosides were a minor component compared with neutral GSLs in NCCIT cells, HPLC analysis also detected these compounds (Fig 7) GM3 was found to be a major ganglioside in NCCIT Table The composition of neutral GSL in NCCIT cells before and after 2DG treatment The relative expression level of each GSLderived oligosaccharide is represented as a ratio of the LacCer level in nontreated cells Each value is shown as a mean of two independent experiments Relative expression level GSL Nontreated 2DG-treated LacCer Amino-CTH Gb3 Gb4 Others 1.00 0.21 3.24 0.19 0.33 3.47 1.49 1.94 0.03 0.49 5196 Fig HPLC analysis of GSL-derived oligosaccharides of NCCIT cells Oligosaccharides released from gangliosides by endoglycoceramidase were labeled with the fluorescent compound 2-AA and analyzed using an HPLC system as described in Experimental procedures Gangliosides were purified from NCCIT cells (middle panel) or 2DG-treated (10 mM, days) NCCIT cells (bottom panel) The elution pattern of standard 2-AA-labeled oligosaccharides, which were generated from commercially available GSLs (arrowheads: 1, LacCer; 2, GM3; 3, GM2; 4, GA1; 5, GM1; 6, GD1a; 7, GD1b; 8, GT1b), are shown in the top panel Only the elution pattern of GM3-derived oligosaccharide was detected as a double peak This result is presumably derived from the difference in the molecular species of sialic acid in the GM3 oligosaccharide FEBS Journal 276 (2009) 5191–5202 ª 2009 The Authors Journal compilation ª 2009 FEBS T Okuda et al A novel strategy for the inhibition of glycosphingolipid biosynthesis Table The composition of gangliosides in NCCIT cells before and after 2DG treatment The relative expression level of each GSL-derived oligosaccharide is represented as a ratio of the LacCer level in nontreated cells ND, not detected Relative expression level GSL Nontreated 2DG-treated GM3 GM2 GM1 GD1a GD1b Others 0.12 0.03 ND 0.04 0.08 0.15 0.15 0.08 0.02 0.02 0.01 0.05 cells, and some complex-type gangliosides were also detected After 2DG treatment, increased expression of GM3, GM2 [GalNAcb1,4(NeuAca2,3)LacCer] and GM1 [Galb1,3GalNAcb1,4(NeuAca2,3)LacCer] was detected We also detected a slight decrease in the level of some complex gangliosides following treatment with 2DG These changes are summarized in Table From these observations, we concluded that 2DG treatment inhibits cellular Gb3 synthesis through the transcriptional inhibition of Gb3S, but the effect is considerably restricted to Gb3 in GSL synthesis of NCCIT cells The effects of 2DG on the cell surface expression of Gb3 in ECs Recent studies have demonstrated that inflammationinduced Gb3 expression on the cell surface of ECs is closely related to the onset of HUS in O157-infected patients [8,9] In particular, enhancement of Gb3 expression levels by cytokine stimulation in ECs is considered as a progression step to HUS in O157infected patients We found that Gb3 expression on the cell surface could be detected in primary cultured human umbilical vein endothelial cells (HUVECs) by flow cytometric analysis using a Gb3-specific monoclonal antibody (mAb) 52, which is enhanced by TNF-a stimulation (Fig 8) Using this model, we examined whether 2DG could control Gb3 expression We treated HUVEC with 10 mm of 2DG for 24 h with or without TNF-a stimulation In both cases, 2DG treatment significantly suppressed the cell surface Gb3 expression at constantly low levels Discussion Fig Gb3 expression on the surface of HUVEC (A) Flow cytometric analysis of HUVEC stained by mAb 52 The cell surface Gb3 was stained with primary mouse monoclonal anti-Gb3 IgG3 52 (mAb 52) and FITC-labeled secondary antibody (thin line) before (untreated) or after treatment for 24 h with 10 mM 2DG (2DG), 20 ngỈmL)1 TNF-a (TNF) or both (TNF + 2DG) These negative controls were prepared by primary treatment with control mouse IgG and secondary FITC-labeled antibody (thin line with dark shading) The numbers of mAb 52-stained HUVEC in the marked areas (M) are shown in (B) as the percentage of total cells (Gb3-positive cells) Error bars, mean ± SE, n = 4, from two independent experiments *P < 0.05 We have established a simple and convenient method for screening inhibitors of Gb3 biosynthesis by employing a Gb3S promoter assay Using this procedure, we successfully identified a glucose analogue 2DG as a candidate inhibitor Furthermore, we found that 2DG treatment strongly repressed Gb3S transcription and decreased the Gb3 content in the cells Conversely, 2DG caused an increase in the level of the Gb3 precursor lactosylceramide and other neutral glyocolipids in the NCCIT teratocarcinoma cell line A similar result was found by analyzing the glycolipid composition of genetically engineered Gb3S null mice tissues [10] These results are entirely consistent with the concept that a reduction in cellular Gb3 content is a result of the inhibitory effect of 2DG on Gb3S transcription The expression of gangliosides, although a FEBS Journal 276 (2009) 5191–5202 ª 2009 The Authors Journal compilation ª 2009 FEBS 5197 A novel strategy for the inhibition of glycosphingolipid biosynthesis minor glycolipid component of NCCIT cells, was detectable by HPLC analysis (Fig 7) Indeed, our results showed that 2DG treatment also affected the expression levels of gangliosides in the NCCIT cells Moreover, an increase in the expression of GM3 and its derivatives results in a slight decrease in the ratio of some complex-type gangliosides These alterations are mainly caused by the increased expression of GM3 as a result of the accumulation of its precursor LacCer From these observations, we concluded that the suppressive effect of 2DG is substantially targeted towards the synthesis of Gb3 or its derivatives in NCCIT cells 2DG, a nonmetabolizable glucose analogue, acts as a glycolytic inhibitor by inhibiting glucose hexokinase Thus, 2DG has frequently been used as a glucose starvation mimetic Recently, its repressive effect on gene transcription has been reported [16,20,21], and some of the mechanisms have already been investigated [16,21] For example, 2DG treatment reduces the expression of brain-derived neurotrophic factor (BDNF) and its receptor TrkB by generating a repressive chromatin environment around the BDNF promoter [21] This event is caused by glycolytic inhibition stress, because other glycolytic inhibitors, such as citrate or pyruvate, also reduce BDNF promoter activity The repressive effect of 2DG on the Gb3S promoter was not displayed by other glycolytic inhibitors (Fig 3) Thus, 2DG presumably regulates the Gb3S promoter in a different way to that of other inhibitors of the BDNF promoter In addition, it has been reported that one of the mechanisms for generating a repressive chromatin environment by 2DG is the activation of the class III histone deacetylase SIRT1 [22] We examined the effects of SIRT1 inhibitors on the 2DG-decreased reporter activity of HML-Gb3 cells However, we were unable to detect any changes in the reporter activity of these cells (Fig 3) It has also been reported that 2DG [16], as well as streptozotocin [17] and high glucose treatment [18,19], enhances O-GlcNAc modification of the transcriptional factor Sp1, thereby reducing its transcriptional capability [17] Previously, we have identified the Sp1 protein as a key regulator for the Gb3S promoter [11] Thus, we expected that the inhibitory effect of 2DG on the Gb3S promoter would be controlled in this manner However, we could not detect any changes in the Gb3S promoter activity after treatment with high glucose or streptozotocin (Fig 3) Even mithramycin A, which directly inhibits the binding between Sp1 and its consensus DNA sequence [14,15], did not show an inhibitory effect (Fig 3) Our results raise the possibility that 2DG indirectly down-regulates Gb3S transcription through the interaction between the Sp1 5198 T Okuda et al and Gb3S promoter Previously, it has been reported that Gb3S transcription is up-regulated in megakaryoblastic leukemia during phorbol 12-myristate 13acetate (TPA)-induced differentiation [27] TPA is a strong inducer for the transcription of several genes, which are controlled by TPA response elements (TREs) As TRE is absent from the transcriptional regulatory domain of Gb3S [11], TPA-induced up-regulation of Gb3S should be regulated by TRE-induced gene products 2DG treatment also induces various changes in the expression levels of several genes, which suggests that 2DG suppresses Gb3S transcription through a very complicated process In this study, we could not elucidate the precise silencing mechanism of the Gb3S gene by 2DG We are currently investigating the means by which 2DG regulates the Gb3S promoter Several reports have indicated that inflammatory mediators, such as LPS and cytokines, enhance the expression level of Gb3 on the EC surface [8,9] It is known that Gb3 is the sole receptor for verotoxin in vivo [10] and that HUS is caused by verotoxininduced damage to ECs Thus, the enhancement of Gb3 expression on ECs by cytokines induced by E coli infection is considered as a progression step for the onset of HUS in O157-infected patients In this study, we demonstrated that 2DG could suppress Gb3 expression on the surface of ECs even after TNF-a stimulation (Fig 8) It has been considered that this event is based on the up-regulation of Gb3S gene mRNA expression Although we could not detect any enhancement of Gb3S promoter activity by TNF-a treatment (Fig 3), increased Gb3 expression in ECs was able to suppress the effect of 2DG treatment This result indicates that basal Gb3S promoter activity is needed for TNF-a to induce the expression of Gb3 in ECs Thus, the suppression of promoter activity may be a method of preventing the progression of HUS in O157-infected patients Hence, the development of a nontoxic inhibitor for the suppression of Gb3 promoter activity could be a useful treatment for HUS in O157-infected patients Fabry disease is also a major target for this study In this disease, the accumulation of Gb3 is observed in a number of tissues, which causes a systemic disorder [5] Because a genetic deficit of a-galactosidase A, a Gb3 catabolic enzyme, is the cause of this disease, enzyme replacement therapy is performed with a recombinant a-galactosidase Unfortunately, this therapy is very costly because it uses a large amount of recombinant enzyme Thus, an alternative more economical approach needs to be developed to treat this condition FEBS Journal 276 (2009) 5191–5202 ª 2009 The Authors Journal compilation ª 2009 FEBS T Okuda et al Previously, a therapeutic strategy using small molecular chaperones (1-deoxgalactonojirimycin) has been proposed [28] This chemical is able to increase residual enzyme activity by rescuing misfolded mutant a-galactosidase protein from endoplasmic reticulumassociated degradation Such a therapeutic strategy is anticipated to be effective for some Fabry disease patients carrying missense mutations in the coding region of the a-galactosidase A gene that lead to misfolding of the mutant protein Direct blockade of neutral GSL synthesis by the inhibition of multiple drug resistance protein (MDR1) has also been proposed as a potential treatment for Fabry disease [29] MDR1 can translocate glucosylceramide into the Golgi apparatus for neutral GSL synthesis, including Gb3 [30] A recent report has shown that the inhibition of MDR1 by cyclosporin A results in a reduction in Gb3 accumulation in several tissues of a Fabry model mouse Reduction of Gb3 by substrate deprivation using a synthetic inhibitor for glucosylceramide synthase has also been proposed as a potential means of treating Fabry disease [31] This study reported a drastic reduction in Gb3 accumulation in Fabry model mice and cell lines from Fabry disease patients [32] by treatment with glucosylceramide synthase inhibitors There is, however, no abnormality in the geneticbased deficient form of Gb3S in mice [10] or humans [33,34] We believe that specific inhibition of Gb3S transcription by a chemical agent will be a primary target for the treatment of Fabry disease with no associated adverse effects Although 2DG cannot be used directly as a drug for the treatment of Gb3-related diseases because of its intrinsic toxicity, modification of the molecule promises to be a way forward Moreover, the assay described in this report makes it possible to efficiently screen for further drug candidates to combat this disease We believe that the development of the principles outlined in this study will bring about the identification of molecules of therapeutic utility Experimental procedures Cell culture HeLa cells, provided by the RIKEN CELL BANK (Tsukuba, Japan), were maintained in Dulbecco’s modified Eagle’s minimal essential medium supplemented with 10% fetal bovine serum Human teratocarcinoma cells, NCCIT, were obtained from the American Type Culture Collection (Manassas, VA, USA), and were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and mm l-glutamine HUVEC, purchased from KURABO A novel strategy for the inhibition of glycosphingolipid biosynthesis (Osaka, Japan), were maintained in HuMedia-EG2 (KURABO) Passages 4–9 were used in these experiments When stimulated by TNF-a (PeproTech, Rocky Hill, NJ, USA), · 105 cells were seeded onto a culture dish (100 mm in diameter) and then incubated for 24 h After incubation, medium was replaced with fresh HuMedia-EG2 containing 20 ngỈmL)1 of TNF-a, and incubated for another 24 h All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2 Construction of plasmids The pMet-Luciferase (pML) reporter vector (Clontech, Mountain View, CA, USA) was used for the reporter assay In order to improve the sensitivity of the reporter assay, the background transcription was reduced by inserting a synthetic transcriptional blocker into the 5¢-upstream region of the multiple cloning site of this vector (Fig 1A) Specifically, the blocker was composed of adjacent polyadenylation and transcription pause sites [35] The promoter region of the Gb3S gene was amplified by PCR using HeLa cell genomic DNA as a template The following PCR primers were used: 5¢-TGAGTCGACTCAG CTCTTGGAGGGGCAACA-3¢ and 5¢-GCGCGCACAAA TGTCGCCTCCAGAACA-3¢ The amplified product was then digested with SalI and BamHI and subcloned into the corresponding recognition sites of the pML vector This DNA insert comprised the )1893 bp to +84 bp region of the 5¢-flanking region of the Gb3S gene, as reported previously [11] All PCR experiments were performed using PrimeSTAR HS DNA polymerase (Takara Bio, Shiga, Japan) The DNA insert in each plasmid construct was verified by sequencing Establishment of stable transfectants and the luciferase assay An aliquot of 0.4 lg of each plasmid was transfected into · 105 HeLa cells with lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions To establish stable mutants, these cells were incubated for weeks in the presence of 400 lgỈmL)1 of G418 The G418-resistant clones were subsequently isolated For the luciferase assay, transient or stable transfectants were incubated with 500 lL of culture medium for 16 h in a 24-well (15.49 mm diameter) cell culture plate In this assay, the reporter protein (luciferase) was secreted into the culture medium A 50 lL aliquot of culture medium from each transfectant was used to measure luciferase activity The luciferase activity was measured using the Ready-To-GlowÔ Secreted Luciferase Reporter Assay kit (Clontech) and Luminescencer JNRII (ATTO, Tokyo, Japan) 2DG, streptozotocin and mithramycin A (Sigma-Aldrich, St Louis, MO, USA) were diluted with culture medium, and added to the FEBS Journal 276 (2009) 5191–5202 ª 2009 The Authors Journal compilation ª 2009 FEBS 5199 A novel strategy for the inhibition of glycosphingolipid biosynthesis cells h after seeding After a further 16 h, the culture medium was collected and analyzed for luciferase activity Cell lysates for measuring the intracellular luciferase activity were prepared as follows The cells in each well were lysed with 100 lL of lysis buffer containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mm phenylmethylsulfonyl fluoride and a proteinase inhibitor cocktail (Complete mini EDTA-freeÔ; Roche, Penzberg, Germany) in NaCl ⁄ Pi Cell viability assays HML-Gb3 (2 · 105) or NCCIT (1 · 105) cells were seeded into a 24-well plate in culture medium with or without 10 mm 2DG After the indicated culture time (shown in Figs 4D, 5A), the cells were harvested and 0.1 vol of 0.4% (w ⁄ v) trypan blue was added to the cell suspension The number of stained and unstained cells was then determined using a hemocytometer T Okuda et al CAGGTCCACC-3¢ The PCR products were analyzed by agarose gel electrophoresis (1.5% gel) and the DNA was visualized using ethidium bromide under UV illumination Glycolipid extraction and TLC analysis Glycolipid extraction and TLC immunostaining were performed as reported previously [11] Briefly, total lipids from · 107 cells were sequentially extracted with chloroform– methanol–water : : and : : 0.8 (v ⁄ v ⁄ v), respectively Gangliosides and neutral glycolipids were separated by column chromatography using DEAE Sephadex A-25 (Sigma-Aldrich) and Iatrobeads 6RS-8060 (Mitsubishi Kagaku Iatron, Tokyo, Japan), respectively Purified glycolipids were analyzed on HPTLC plates (Merck, Darmstadt, Germany) with a solvent system consisting of chloroform– methanol–water (60 : 35 : 8, v ⁄ v ⁄ v) Glycolipids were visualized by orcinol–H2SO4 HPLC analysis RT-PCR RT-PCR analysis of the Gb3S gene was carried out according to the previously reported method [13] with some modifications Total RNA was isolated using Trizol reagent (Invitrogen) from NCCIT cells before and after treatment with 10 mm 2DG The amplification of the target gene cDNA was carried out using gene-specific primers and a SuperScriptÔ One-Step RT-PCR System with PlatinumÔ Taq DNA polymerase (Invitrogen), according to the manufacturer’s instructions Briefly, total mRNA (0.5 lg) and forward and reverse primers (5 pmol each) were mixed with SuperScriptÔ RT ⁄ Platinum Taq Mix (0.5 lL) in reaction buffer (25.0 lL) containing dNTP (0.2 mm) and MgSO4 (1.2 mm) in distilled water, and these were reacted in a thermal cycler The reactions were performed using the following conditions: 55 °C for 30 min, 94 °C for min, and then 40 cycles [for Gb3S, b1,4-galactosyltransferase (B4GalT6) and GM3 synthase (GM3S)] or 25 cycles [for glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] of 94 °C for 15 s, 59 °C for 30 s and 72 °C for 30 s, with a final extension of 72 °C for For the amplification of Gb3S cDNA, the sense primer 5¢-TGGAAGTTCGGCGGCATC TA-3¢ and the antisense primer 5¢-CAGGGGGC AGGGTGGTGACG-3¢ were used The PCR products corresponded to nucleotides +550 to +844 of the ORF region of the Gb3S gene For amplification of B4GalT6, GM3S and GAPDH cDNA, the following primers were used: for B4GalT6, the forward primer 5¢-TGAACAGACTGGCA CACAACC-3¢ and the reverse primer 5¢-TGTCAGCCC ACTTACACCAC-3¢; for GM3S, the forward primer 5¢CGTCCCCACAATCGGTGTCA-3¢ and the reverse primer 5¢-ACCACTCCCTCTTTGACCAG-3¢; for GAPDH, the forward primer 5¢-CCACCCATGGCAAATTCCATGGCA -3¢ and the reverse primer 5¢-TCTAGACGGCAGGT 5200 Semi-quantitative analysis of GSLs was carried out by HPLC using a published carbohydrate fluorescent labeling method for GSLs described by Neville et al [36] with slight modifications Neutral GSLs from · 106 cells or ganglioside from · 106 cells or 10 lg of GSL standards in chloroform–methanol (2 : 1, v ⁄ v) were evaporated to dryness in a glass vial The carbohydrate moieties were then digested by the addition of mU of recombinant endoglycoceramidase II (Takara Bio) and incubation at 37 °C for 16 h in 10 lL of 50 mm sodium acetate buffer (pH 5.0) containing mgỈmL)1 sodium cholate The liberated oligosaccharide was fluorescently labeled using anthranilic acid (2-AA; Sigma-Aldrich) Samples were sequentially mixed with 80 lL of labeling mixture (30 mgỈmL)1 2-AA, 40 mgỈmL)1 sodium acetate trihydrate, 20 mgỈmL)1 boric acid and 45 mgỈmL)1 sodium cyanoborohydride in methanol) and incubated at 80 °C for h Labeled oligosaccharides were purified using a discovery DPA-6S column (Supelco, Poole, UK) and analyzed using a TSK gel-amide 80 column (Tosoh, Tokyo, Japan) and the HPLC system LC Module I plus (Waters, Milford, MA, USA) The chromatography system and fluorescence detection ⁄ gradient conditions were identical to those 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Establishment of stable transfectants and the luciferase assay An aliquot of 0.4 lg of each plasmid was transfected into · 105 HeLa cells with lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) according... 14646–14652 Ishii A, Ohta M, Watanabe Y, Matsuda K, Ishiyama K, Sakoe K, Nakamura M, Inokuchi J, Sanai Y & Saito M (1998) Expression cloning and functional characterization of human cDNA for ganglioside... of Gb3 markedly decreased, which was followed by an accumulation of LacCer and the appearance of another GSL (Fig 6A, asterisk) Based on the HPLC elution time (Fig 6B, asterisk), it seems that