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

To find a transcriptional inhibi-tor for Gb3S, we developed a convenient cell-based chemical screening assay system by constructing a fusion gene construct of the human Gb3S promoter and

2-deoxy-D-glucose as an inhibitor of globotriaosylceramide biosynthesis

Tetsuya Okuda1, Koichi Furukawa2 and Ken-ichi Nakayama1

1 Glycolipids Function Analysis Team, Health Technology Research Center, National Institute of Advanced Industrial Science and Technology, Kagawa, Japan

2 Department of Biochemistry II, Graduate School of Medicine, Nagoya University, Aichi, Japan

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 classi-fied in terms of a difference in their carbohydrate

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 inhibi-tor 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 identi-fied 2-deoxy-d-glucose as a potent inhibitor for the Gb3S promoter In cul-tured 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, cytokine-induced 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

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.

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

abnor-mal expression and⁄ or accumulation can lead to

sev-eral disease states For example, patients with Fabry

disease, an X-linked lysosomal storage disease caused

by deficiency of the Gb3 catabolic enzyme

a-galacto-sidase A, have been found to accumulate Gb3 in

vari-ous 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

recep-tor 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

endo-thelial cells (ECs) Inflammatory mediators, such as

tumor necrosis factor-a (TNF-a) and

lipopolysaccha-ride (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

pre-cursor 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

devel-oped 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

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 pro-moter The pML reporter vector includes the Metridia longa secreted luciferase gene [12] as a reporter gene The human Gb3S promoter has previously been identi-fied in the 5¢-flanking region ()1893 bp to +84 bp

Fig 1 Establishment of Gb3S promoter-driven luciferase secretion cells (A) The scheme of the constructed vector plasmid (pML-Gb3Sp) Open box, Gb3S promoter; bold black arrow, reporter gene (secreted Metridia luciferase); TB, transcriptional blocker; Kan R ⁄ Neo R , kanamycin and neomycin resistance gene (B) Luciferase activity of vector transfectants HeLa cells were transiently

transfect-ed with pML-Gb3Sp (Gb3Sp) or empty vector (pML) The luciferase activity of the culture medium was measured as described in Experi-mental 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 estab-lished, and relative luciferase activities were calculated (filled bar).

from the transcriptional initiation site; GenBank

acces-sion 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

con-trol to monitor background expression levels As

shown in Fig 1B, more than seven-fold overexpression

of luciferase reporter activity was observed in

HML-Gb3 Furthermore, the reporter activity was

moder-ately stronger than that of the transient transfectants

This result is presumably because of the difference in

transfection efficiency between stable and transient

transfectants

Characterization of the reporter activity

of HML-Gb3

For the purposes of chemical screening, we

deter-mined 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 2 (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 4· 105 cells (Fig 2,

right) In this case, the background ratio reached a

plateau at around 2· 105 cells (Fig 2, left) From

these results, we conclude that the optimal culture

time and seeding cell number of HML-Gb3 in the

24-well plate format were 16 h and 2· 105 cells, respectively, for the rapid and highly sensitive moni-toring 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 sub-stances on the reporter activity of HML-Gb3 cells (Fig 3) First, we examined potential inhibitors for transcriptional factor Sp1 (mithramycin A, strepto-zotocin, 2DG, high glucose treatment), which has been

Fig 2 Characterization of the reporter

activ-ity of HML-Gb3 Cells (2 · 10 5 ) 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

exam-ined (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.

Fig 3 Effect of candidate inhibitors on Gb3S promoter activity HML-Gb3 cells were treated with the following substances or con-ditions for 16 h; 1, untreated control; 2, 10 m M 2DG; 3, 30 m M glu-cose (high gluglu-cose treatment); 4, 10 m M streptozotocin; 5, 200 n M

mithramycin A; 6, 100 l M citrate; 7, 10 m M pyruvate; 8, 50 l M spli-tomicin; 9, 100 l M nicotinamide; 10, 20 ngÆmL)1TNF-a; 11, glucose starvation; 12, 10 m M GlcNAc; 13, 10 m M galactose; 14, 10 m M

2-deoxy- D -galactose; 15, 10 m M 2DG with 10 m M pyruvate; 16,

10 m M 2DG with 50 l M splitomicin; 17, 10 m M 2DG with 100 l M

nicotinamide After the treatment with each inhibitor, luciferase activity in the culture medium was measured as described previ-ously 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 = 4.

identified previously as an essential factor for Gb3S

promoter regulation [11] Mithramycin A is a

well-characterized 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

dis-played 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

HML-Gb3, 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

con-cluded that there was a very poor correlation between

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 How-ever, these compounds showed no inhibitory effect on the reporter activity of HML-Gb3 cells (Fig 3, bars 6 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 8 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 2DG-treated HML-Gb3 cells

TNF-a is an inducer of Gb3 expression in ECs [8–10] It has been reported that TNF-a induces Gb3

Fig 4 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 = 2 (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 m M ) for the indicated times (individually for 0, 4, 8 or 16 h) (C) Luciferase activity

of cell lysates (Cell) and culture medium (Medium) from HML-Gb3 cells treated with (+) or without ( )) 2DG (10 m M ) for 16 h Error bars, mean ± SD, n = 4 (D) The num-ber of viable (squares with full lines) and dead (circles with dotted lines) cells after treatment or not with 2DG (10 m M ) 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 = 4.

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

inhi-bition 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-galactose-treated HML-Gb3 cells This result indicates that the

glucose backbone and deoxygenation of the hydroxy

group in carbon position 2 are important in decreasing

the reporter activity of HML-Gb3 cells

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,

pro-longed 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

secre-tion 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

impor-tant Therefore, we used a Gb3 highly expressed

teratocarcinoma (NCCIT cells) in the following experi-ments because the cell viability and growth were unaf-fected by 2DG treatment (Fig 5A) As expected, 2DG treatment for 1 week strongly suppressed Gb3S expres-sion (Fig 5B) The mRNA expresexpres-sion of other major glycolipid synthase genes, such as GM3 (Neu-Aca2,3LacCer) synthase [25] and b1,4-galactosyltrans-ferase 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 accu-mulation 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

Fig 5 The effects of 2DG on NCCIT cells (A) The number of via-ble (squares with full lines) and dead (circles with dotted lines) cells after treatment or not with 2DG (10 m M ) for the indicated days (1, 2, 4 and 7 days) Filled squares and circles, nontreated cells; open squares and circles, 2DG-treated cells Error bars, mean ± SD, n = 4 (B) RT-PCR analysis of glycolipid synthase gene mRNA expression in NCCIT cells treated with (+) or without ( )) 2DG (10 m M ) for 1 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.

generated GSL is the amino-CTH

[lactotriaosylcera-mide (Lc3, GlcNAcb1,3LacCer) or asialo-GM2

(Gal-NAcb1,4LacCer)] 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 1 The cellular Gb3 levels were reduced by

approximately 40% after 2DG treatment, and then its

derivative globotetraosylceramide (Gb4) became

unde-tectable By contrast, the level of the Gb3 precursor

LacCer increased 3.5-fold after 2DG treatment

More-over, 2DG resulted in increased levels of amino-CTH,

which is synthesized from LacCer by enzymes other

than Gb3S Although gangliosides were a minor

com-ponent 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

Fig 6 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 endoglycocerami-dase 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 m M , 7 days) NCCIT cells (bottom panel) The elution positions of stan-dard 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.

Table 1 The composition of neutral GSL 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 Each value is shown as a mean of two

indepen-dent experiments.

GSL

Relative expression level Nontreated 2DG-treated

Amino-CTH 0.21 1.49

Fig 7 HPLC analysis of GSL-derived oligosaccharides of NCCIT cells Oligosaccharides released from gangliosides by endoglyco-ceramidase 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 m M , 7 days) NCCIT cells (bottom panel) The elution pattern of standard 2-AA-labeled oligosaccharides, which were generated from commercially available GSLs (arrow-heads: 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 pat-tern 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.

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 2 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 inflammation-induced 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 O157-infected 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

We have established a simple and convenient method for screening inhibitors of Gb3 biosynthesis by employing a Gb3S promoter assay Using this proce-dure, we successfully identified a glucose analogue 2DG as a candidate inhibitor Furthermore, we found that 2DG treatment strongly repressed Gb3S transcrip-tion and decreased the Gb3 content in the cells Con-versely, 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 tran-scription The expression of gangliosides, although a

Table 2 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.

GSL

Relative expression level Nontreated 2DG-treated

Fig 8 Gb3 expression on the surface of HUVEC (A) Flow

cytomet-ric 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 m M 2DG (2DG), 20 ngÆmL)1TNF-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.

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

sup-pressive 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

star-vation 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

dis-played by other glycolytic inhibitors (Fig 3) Thus,

2DG presumably regulates the Gb3S promoter in a

dif-ferent 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

transcrip-tional factor Sp1, thereby reducing its transcriptranscrip-tional

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

mithra-mycin 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

and Gb3S promoter Previously, it has been reported that Gb3S transcription is up-regulated in mega-karyoblastic leukemia during phorbol 12-myristate 13-acetate (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-reg-ulation 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 verotoxin-induced 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 ther-apy 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

Previously, a therapeutic strategy using small

mole-cular chaperones (1-deoxgalactonojirimycin) has been

proposed [28] This chemical is able to increase

resid-ual enzyme activity by rescuing misfolded mutant

a-galactosidase protein from endoplasmic

reticulum-associated 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 1

(MDR1) has also been proposed as a potential

treat-ment 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

reduc-tion in Gb3 accumulareduc-tion 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

genetic-based 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

asso-ciated 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

More-over, the assay described in this report makes it

possi-ble 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

(Tsu-kuba, 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

2 mm l-glutamine HUVEC, purchased from KURABO

(Osaka, Japan), were maintained in HuMedia-EG2 (KU-RABO) Passages 4–9 were used in these experiments When stimulated by TNF-a (PeproTech, Rocky Hill, NJ, USA), 5· 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 ngÆmL)1 of TNF-a, and incubated for another 24 h All cells were cultured at 37C in a humidi-fied 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 insert-ing 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 poly-adenylation 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 follow-ing 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 per-formed 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

2· 105 HeLa cells with lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions To establish stable mutants, these cells were incubated for 2 weeks in the presence of 400 lgÆmL)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-Glow Secreted Luciferase Reporter Assay kit (Clontech) and Lumi-nescencer JNRII (ATTO, Tokyo, Japan) 2DG, streptozoto-cin and mithramystreptozoto-cin A (Sigma-Aldrich, St Louis, MO, USA) were diluted with culture medium, and added to the

cells 4 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-free; 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

RT-PCR

RT-PCR analysis of the Gb3S gene was carried out

accord-ing to the previously reported method [13] with some

modi-fications 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

SuperScript One-Step RT-PCR System with Platinum

Taq DNA polymerase (Invitrogen), according to the

manu-facturer’s instructions Briefly, total mRNA (0.5 lg) and

forward and reverse primers (5 pmol each) were mixed with

SuperScript 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

ther-mal cycler The reactions were performed using the

follow-ing conditions: 55C for 30 min, 94 C for 2 min, and then

40 cycles [for Gb3S, b1,4-galactosyltransferase 6 (B4GalT6)

and GM3 synthase (GM3S)] or 25 cycles [for

glyceralde-hyde-3-phosphate dehydrogenase (GAPDH)] of 94C for

15 s, 59C for 30 s and 72 C for 30 s, with a final

exten-sion of 72C for 5 min 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

cor-responded 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

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 per-formed as reported previously [11] Briefly, total lipids from

1· 107

cells were sequentially extracted with chloroform– methanol–water 2 : 1 : 0 and 1 : 2 : 0.8 (v⁄ v ⁄ v), respec-tively 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 glycol-ipids 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

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 1· 106

cells or ganglio-side from 2· 106cells or 10 lg of GSL standards in chloro-form–methanol (2 : 1, v⁄ v) were evaporated to dryness in a glass vial The carbohydrate moieties were then digested by the addition of 4 mU of recombinant endoglycocera-midase II (Takara Bio) and incubation at 37C for 16 h in

10 lL of 50 mm sodium acetate buffer (pH 5.0) containing

1 mgÆmL)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 mgÆmL)12-AA, 40 mgÆmL)1sodium acetate trihydrate, 20 mgÆmL)1 boric acid and 45 mgÆmL)1 sodium cyanoborohydride in methanol) and incubated at

80C for 1 h Labeled oligosaccharides were purified using a discovery DPA-6S column (Supelco, Poole, UK) and ana-lyzed 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 fluo-rescence detection⁄ gradient conditions were identical to those described in the published methodology [36]

Flow cytometric analysis

Expression of Gb3 on the cell surface was analyzed by flow cytometry After treatment with 20 ngÆmL)1 TNF-a or

10 mm 2DG, cells were harvested in 0.05% trypsin–EDTA solution Approximately 1· 106

cells were then suspended

in 100 lL of cold NaCl⁄ Pi The suspensions were incubated with 1 lg mouse monoclonal anti-Gb3 IgG3 52 (mAb 52) (Y Kondo et al., unpublished results) on ice, and were