Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/11/19/M705621200.DC1.html
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 6, pp. 3594 –3606, February 8, 2008
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Heparan Sulfate Regulates Self-renewal and Pluripotency ofEmbryonic Stem Cells*□
S

Received for publication, July 9, 2007, and in revised form, October 10, 2007 Published, JBC Papers in Press, November 16, 2007, DOI 10.1074/jbc.M705621200

Embryonic stem (ES) cell self-renewal and pluripotency are
maintained by several signaling cascades and by expression of
intrinsic factors, such as Oct3/4 and Nanog. The signaling cascades are activated by extrinsic factors, such as leukemia inhibitory factor, bone morphogenic protein, and Wnt. However, themechanism that regulates extrinsic signaling in ES cells is
unknown. Heparan sulfate (HS) chains are ubiquitously present
as the cell surface proteoglycans and are known to play crucial
roles in regulating several signaling pathways. Here we investigated whether HS chains on ES cells are involved in regulating
signaling pathways that are important for the maintenance of ES
cells. RNA interference-mediated knockdown of HS chain elongation inhibited mouse ES cell self-renewal and induced spontaneous differentiation of the cells into extraembryonicendoderm. Furthermore, autocrine/paracrine Wnt/␤-cateninsignaling through HS chains was found to be required for the
regulation of Nanog expression. We propose that HS chains areimportant for the extrinsic signaling required for mouse ES cell
self-renewal and pluripotency.

Embryonic stem (ES)2 cells from the inner cell mass of preimplantation mouse embryos can be used to establish pluripo-* This work was supported by Core Research for Evolutional Science andTechnology of Japan Science and Technology Agency and The Ministry of
Education, Culture, Sports, Science, and Technology Haiteku (2004 –2008).
The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked “advertisement” inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental data 1– 6.
1
To whom correspondence should be addressed: Laboratory of Cell Biology,
Dept. of Bioinformatics, Faculty of Engineering, Soka University, 1-236
Tangi-cho, Hachioji, Tokyo 192-8577, Japan. Tel.: 81-426-91-8140; Fax:
81-426-91-9315; E-mail: shoko@t.soka.ac.jp.
2
The abbreviations used are: ES, embryonic stem; HS, heparan sulfate; mES,
mouse ES; LIF, leukemia inhibitory factor; BMP, bone morphogenic protein;
Id, inhibitor of differentiation; FGF, fibroblast growth factor; siRNA, smallinterfering RNA; MEF, mouse embryonic fibroblast; EB, embryoid body;
BIO, 6-bromoindirubin-3⬘-oxine; MeBIO, 1-methyl-6-bromoindirubin-3⬘-

3594 JOURNAL OF BIOLOGICAL CHEMISTRY

tent cell lines (1, 2). ES cells retain the ability to differentiate
into the representative cell types of all three germ layers of thedeveloping mouse embryo. Human ES cell lines have been
derived (3), and the pluripotency of these cells is a feature providing considerable potential for exploitation in the development of cell replacement therapies to treat human disease.
However, the molecular mechanisms that control pluripotency
and differentiation of ES cells are largely unknown. It will beessential to gain a better understanding of these mechanisms to
exploit the potential of ES cells for therapeutic purposes.
A number of studies have investigated the factors controlling
pluripotency of mouse ES (mES) cells (4). Self-renewal of mES cells
is sustained by signals mediated by the cytokine, leukemia inhibitory factor (LIF) (5, 6). LIF signals through the heteromeric receptor gp130 and the low affinity LIF receptor to induce activation of
STAT3 (7–10). However, exposure of cells to serum is also
required for LIF-mediated maintenance of self-renewal (11).
Treatment of mES cells with the bone morphogenic proteins(BMPs) BMP2 and BMP4 or with growth differentiation factor
6 can substitute for serum. The ability of BMP/growth differentiation factor 6 to promote self-renewal requires co-stimulation with LIF (11). BMP induces the expression of inhibitor ofdifferentiation (Id) genes through activation of Smad signaling,
and the Id gene products suppress expression of genes involvedin the induction of neural differentiation (11). Thus, BMP suppresses neural differentiation and, in combination with LIF, issufficient to sustain self-renewal of mES cells without the needfor feeder cells or serum factors.
Wnt signaling was shown to play a role in the regulation ofself-renewal of both mouse and human ES cells independently
oxine; CS, chondroitin sulfate; BSA, bovine serum albumin; HPLC, high performance liquid chromatography; AP, alkaline phosphatase; Tcf, T-cellspecific factor; SPR, surface plasmon resonance; vWF, von Willebrandfactor; RNAi, RNA interference; PI3K, phosphoinositide 3-kinase; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; FACS, fluorescence-activated cell sorter; IL, interleukin; RT, reverse transcription; EGFP,
enhanced green fluorescent protein; ERK, extracellular signal-regulated
kinase.

VOLUME 283 • NUMBER 6 • FEBRUARY 8, 2008

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

Norihiko Sasaki‡§, Kazuhiko Okishio‡, Kumiko Ui-Tei¶, Kaoru Saigo¶, Akiko Kinoshita-Toyoda§储, Hidenao Toyoda§储,Tomoaki Nishimura**‡‡, Yasuo Suda§**‡‡, Michiko Hayasaka§§, Kazunari Hanaoka§§, Seiji Hitoshi¶¶,Kazuhiro Ikenaka¶¶, and Shoko Nishihara‡§1
From the ‡Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-cho,
Hachioji, Tokyo 192-8577, the ¶Department of Biophysics and Biochemistry, Graduate School of Science, the University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, the 储Department of Bioanalytical Chemistry, Graduate School of Pharmaceutical
Sciences, Chiba University, 1-33 Yayoi, Inage, Chiba 263-8522, the **Department of Nanostructure and Advanced Materials,
Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Kohrimoto, Kagoshima 890-0065, ‡‡Sudx-Biotec
Corporation, KIBC 461, 5-5-2, Minatojima-minami, Chuo-ku, Kobe 650-0047, §§Laboratory of Molecular Embryology, Departmentof Bioscience, Kitasato University School of Science, 1-15-1 Kitasato, Sagamihara, Kanagawa 228, ¶¶Division of Neurobiology andBioinformatics, National Institute for Physiological Sciences, 38 Nishigonaka Myodaiji, Okazaki, Aichi 444-8585, and §Core
Research for Evolutional Science and Technology of Japan Science and Technology Agency, Kawaguchi Center Building,
4-1-8, Hon-cho, Kawaguchi, Saitama 332-0012, Japan

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/11/19/M705621200.DC1.html

HS Chains Are Required for the Maintenance of ES Cells

EXPERIMENTAL PROCEDURES
Cell Culture and Transfection—R1 (27) and E14TG2a (28)mES cell lines were maintained on mouse embryonic fibroblasts (MEFs) inactivated with 10 ␮g/ml mitomycin C (Sigma)
FEBRUARY 8, 2008 • VOLUME 283 • NUMBER 6

in ES medium (Dulbecco’s modified Eagle’s medium supplemented with 15% FBS {Hyclone}, 1% penicillin/streptomycin{Invitrogen}, 0.1 mM mercaptoethanol {Invitrogen}, and 0.1 mM
nonessential amino acids {Invitrogen}) with 1000 units/ml LIF(Chemicon). We generated siRNA expression plasmids targeting EXT1 and EGFP as negative control by inserting of the corresponding double strand DNAs into the BamHI-HindIII site ofpSilencer 3.1-H1 (Ambion). The sequences used for RNAi were
designed as described previously (29): EGFP, 5⬘-GATCCCGCCACAACGTCTATATCATGGGGAAAATCCATGATATAGACGTTGTGGCTTTTTTGGAAA-3⬘; EXT1-1, 5⬘-GATCCCGTCCTACCGCAGTATTCATCTGCTTCCTGTCACAGATGAATACTGCGGTAGGACTTTTTTGGA-3⬘; EXT1-2,
5⬘-GATCCCGGTCTATTCATCAGGATAAAAGCTTCCTGTCACTTTTATCCTGATGAATAGACCTTTTTTA-3⬘. For
transient knockdown of EXT1 mRNA by RNAi, siRNA expression plasmids for EXT1 were transfected into mES cells as follows. Prior to transfection, the mES cells were harvested,
replated at 1 ⫻ 106 cells on gelatin-coated feeder-free 60 mm
tissue culture dishes (Iwaki) in ES medium with LIF, and incubated for 16 h. On day 1, the cells were transfected with an
siRNA expression plasmid (2 ␮g per culture dish) using Lipofectamine 2000 (Invitrogen). On day 2, the cells were harvested
and replated at 3 ⫻ 106 cells on gelatin-coated feeder-free 60
mm tissue culture dishes in ES medium with LIF and 2 ␮g/ml
puromycin (Sigma). In general, puromycin selection of transfected cells was carried out for 24 h. On day 3 (2 days after
transfection), the cells were harvested and analyzed as
described below.
For differentiation into embryoid bodies (EBs), the cells weretransferred on day 3 to low-cell-binding 60 mm dishes (Nunc)
and cultured in ES medium without LIF. The numbers of small
noncystic EBs and large EBs filled with cystic cavity werecounted by microscopic examination. EBs were fixed overnightin 4% paraformaldehyde at 4 °C, dehydrated, embedded in paraffin and sectioned at 10 ␮m. Sections were stained with hematoxylin and eosin (Merck).
For morphological observation and real time PCR analysis ofdifferentiation markers, the cells were replated on day 2 andincubated with puromycin for 3 days.
For exogenous activation of Wnt/␤-catenin signaling, thecells were treated with 2 ␮M 6-bromoindirubin-3⬘-oxine (BIO;
R&D Systems), a specific pharmacological inhibitor of glycogensynthase kinase-3 or 1-methyl-6-bromoindirubin-3⬘-oxine(MeBIO; R & D Systems), an inactive analog of BIO duringtransfection and culture.
FACS Analysis—Two days after transfection, mES cells were
harvested and the cell suspension was incubated with mouse
IgM negative isotype control (Chemicon), anti-HS antibody
10E4 (Seikagaku Corp.) or anti-chondroitin sulfate (CS) antibody 2H6 (Seikagaku Corp.) diluted in FACS buffer (0.5%bovine serum albumin (BSA) and 0.1% sodium azide in PBS).
After washing, the cell suspension was incubated with fluorescein isothiocyanate-conjugated secondary antibody (Sigma)
diluted in FACS buffer. Cell sorting and analysis were performed using a FACSAria Cell Sorter (BD Biosciences).
JOURNAL OF BIOLOGICAL CHEMISTRY

3595

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010of LIF/STAT3 signaling (12). Wnt proteins play roles in theregulation of gene expression, cell proliferation, and differentiation and in the maintenance of cell polarity (13). The bindingof Wnt protein to its cognate receptor, Frizzled, results in theinhibition of glycogen synthase kinase-3. This in turn leads tothe stabilization and nuclear accumulation of ␤-catenin and tochanges in gene transcription. Signaling by this canonical Wnt
pathway has been suggested to result in downstream activationof expression of the homeoprotein Nanog, a transcription factor that is essential for maintenance of the inner cell mass and of
ES cell pluripotency (14, 15). The activation of Nanog sustains
ES cell self-renewal without the use of feeder cells or treatment
with LIF (12).
Heparan sulfate (HS) proteoglycans are ubiquitously present
in the extracellular matrix and on the cell surface. The HS
polysaccharide chains of the proteoglycans are covalently
attached to several core proteins (16). HS chains are synthesized in the Golgi apparatus by several enzymes, including
members of the EXT protein family. The chains consist of
repeating disaccharide units of D-glucuronic acid-N-acetyl-Dglucosamine that are modified differentially by epimerizationand sulfation (16). A large number of physiologically important
molecules can bind to specific sulfated regions of HS chains(17). Genetic studies have shown that HS chains regulate biological functions by interacting with various extracellular signaling molecules, such as members of the fibroblast growth
factor (FGF) family, Wnt/Wingless (Wg), Hedgehog (Hh), and
BMP (18). In Drosophila, for example, analyses of mutations of
the EXT family genes tout-velu (ttv), sister of ttv (sotv), andbrother of ttv (botv) have indicated that HS chains are required
for signaling and distribution of Hh, Wg, and Decapentaplegic(the functional ortholog of mammalian BMP2 and BMP4) during embryogenesis and wing development (19 –22). In mammals, the importance of HS chains in development has been
demonstrated by analyses of mutations of enzymes required for
HS chain modification, and FGF and Indian hedgehog signaling
through HS chains has been suggested to be required during
development (18, 23–26). Thus, there is evidence that HS
chains have essential functions in development, however, it is
not yet clear what role HS chains play in the regulation of early
embryogenesis and in ES cells.
Our current understanding suggests that HS chains may contribute to the maintenance of ES cell self-renewal by regulating
the activities of several signaling pathways, such as LIF/STAT3,
BMP/Smad, and Wnt/␤-catenin. In the present study, weinvestigated the contribution of HS chains to the regulation of
ES cell self-renewal and pluripotency. We used small interfering RNA (siRNA) to knockdown EXT1, which is required for
HS chain elongation. Transfected mES cells grew more slowly
than untreated control cells and differentiated into extraembryonic endoderm even in the presence of LIF and serum. Thisis the first demonstration of the importance of HS chains for the
maintenance of self-renewal and pluripotency of mES cells.

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/11/19/M705621200.DC1.html

HS Chains Are Required for the Maintenance of ES Cells

3596 JOURNAL OF BIOLOGICAL CHEMISTRY

pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM Na3VO4, 10 mM
NaF, protease inhibitors).
To isolate nuclear extracts, cells were first suspended in 100
␮l of buffer (10 mM Hepes, pH 7.4, 2 mM MgCl2,1 mM EDTA, 10
mM KCl, 1 mM dithiothreitol, protease inhibitors). After incubation for 15 min on ice, 12.5 ␮l of 5% Nonidet P-40 was added,
the suspension was vortexed for 10 s, and incubated for a further 5 min on ice. The suspension was centrifuged at 13,000rpm for 30 s. The supernatant was removed and the pellets,
comprising the nuclear extracts, were washed with PBS andlysed with lysis buffer (25 mM Hepes, pH 7.4, 500 mM NaCl, 1
mM dithiothreitol, 10% glycerol, 0.2% Nonidet P-40, 5 mM
MgCl2). The purity of the cell fractionation (cytosol andnucleus) was confirmed in Western blot analysis using an antiYes monoclonal antibody (BD Biosciences) and an anti-Lamin
B1 antibody (Zymed Laboratories Inc.); these antibodies are
specific for the cytosol and nucleus, respectively. Only low levels of cross-contamination were observed (⬍1%).
Ten micrograms of cell lysates or nuclear extracts were separated by 10% SDS-PAGE and transferred onto polyvinylidene
difluoride membranes (Millipore). After blocking, the membranes were incubated with antibodies to STAT3 (BD Biosciences), phosphorylated STAT3 (Tyr-705 BD Biosciences),
ERK-1/2 (Cell Signaling Technology), phosphorylated ERK-1/2(Thr-183 and Thr-185; Sigma), Akt (BD Biosciences), phosphorylated Akt (Ser-472 and Ser-473; BD Biosciences), phosphorylated Smad1 (Ser-463 and Ser-465; Cell Signaling Technology), Yes (Santa Cruz Biotechnology), phosphorylated Src
family (Tyr-416; Cell Signaling Technology), ␤-actin (Sigma),
␤-catenin (Cell Signaling Technology), phosphorylated ␤-catenin (Ser-33/37/Thr41; Cell Signaling Technology), Lamin B1,
Oct3/4 (Santa Cruz Biotechnology), or Nanog (ReproCELL).
The membranes were then incubated with the appropriate peroxidase-conjugated secondary antibody (Cell Signaling Technology). After washing, the membranes were developed with
ECL Plus reagents (GE Healthcare). For detection of phosphorylated Yes, cells were lysed with lysis buffer (30 mM Tris-HCl,
pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM EDTA, 1 mM Na3VO4, 10 mM NaF,
protease inhibitors) and immunoprecipitated with 1 ␮g of antiYes monoclonal antibody and protein G Magnetic Beads (NewEngland Biolabs); this approach was adopted as this is the only
commercially available anti-phosphorylated Src family antibody that cross-reacts with phosphorylated Yes.
Luciferase Reporter Assay and Immunostaining—Transactivation of ␤-catenin on T-cell-specific factor (Tcf) was determined with a luciferase reporter assay. siRNA expression plasmid (2 ␮g) was cotransfected with reporter plasmid such as,
TOPFLASH (2 ␮g, containing three Tcf binding sites, Upstate
Biotechnology) or FOPFLASH (2 ␮g, containing inactive Tcf
binding sites, Upstate Biotechnology) and pCH110 (0.2 ␮g,containing ␤-galactosidase, GE Healthcare) as control of transfection efficiency using Lipofectamine 2000 as described above.
Cell lysates were prepared 3 days after transfection and luciferase activity was measured with Dual-Light威 System (Applied
Biosystems). Luminescence was measured with a Lumat
LB9501 luminometer (Berthold). Luciferase activity was normalized for transfection efficiency by ␤-galactosidase activity.
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High Performance Liquid Chromatography (HPLC) Analysis
of Unsaturated Disaccharides—Two days after transfection,
mES cells were harvested and fluorometric post-column HPLC
analysis of unsaturated disaccharides from HS chains was performed as reported previously (30).
Molecular Size Analysis of HS Chains—One day after transfection, mES cells were harvested, replated at 1.5 ⫻ 106 cells perwell in 6-well 0.2% gelatin-coated plates and incubated in sulfate-free ES medium with LIF, puromycin and 100 ␮Ci/ml
Na235SO4 (ARC). After labeling for 24 h, the cells were washedtwice with PBS and then treated with 1 mg/ml trypsin (WAKO)
for 10 min at 37 °C. The trypsin was neutralized with 2 mg/ml
trypsin inhibitor (Roche Applied Science). After centrifugation,
the supernatants were treated with 0.5 M NaOH at 4 °C overnight and neutralized with 1 M acetic acid. The labeled galactosaminoglycans were eliminated by chondroitinase ABC(Seikagaku Corp.) digestion, desalted in a PD-10 column (GEHealthcare) and resistant HS chains were isolated by anion
exchange chromatography on HiTrap DEAE FF (GE Healthcare) using sodium phosphate buffer (pH 6.0) containing 1.0 M
NaCl as the eluent. The sizes of the purified HS chains wereanalyzed by gel chromatography on a Sephacryl S-300 column(GE Healthcare) (1 ⫻ 44 cm) and eluted with 50 mM Tris-HCl,
pH 7.4, containing 0.2 M NaCl. Fractions (1 ml/fraction) werecollected and analyzed for radioactivity using a scintillation
counter. The purity of the labeled HS was determined by sensitivity to enzyme digestion with 5 milliunits of heparitinase I
and II (Seikagaku Corp.) and heparinase (Seikagaku Corp.).
Estimations of molecular mass values were derived from fractionation of several Dextran molecular size markers (Sigma) by
gel chromatography and staining with orsinol solution.
Proliferation Assay—Two days after transfection, mES cells
were harvested and replated in triplicate at 0.8 ⫻ 104 cells perwell in 96-well 0.2% gelatin-coated plates in ES medium with
LIF. Cell counting kit-8 (Dojindo) was added after 0 h, 24 h or
48 h and incubated further for 2 h. The soluble formazan product was measured at 450 nm.
Self-renewal Assay—Two days after transfection, mES cells
were harvested and replated at 1 ⫻ 104 cells per gelatin coated
60 mm tissue culture dish in ES medium with LIF. For detectionof undifferentiated cells, cells were fixed and stained with
5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium(Nacalai Tesque) 5 days after replating. Alkaline phosphatase(AP) positive colonies were counted by microscopic examination. Colonies of tightly packed and flattened AP-positive cells
were counted as undifferentiated, and colonies of mixtures of
unstained and stained cells and entirely unstained cells withflattened irregular morphology were considered differentiated.
Immunoblotting—Two days after transfection, the culture
solution for the mES cells was replaced with serum-free ES
medium without LIF for 4 h and the cells were stimulated for 20
min with one of the following: 15% FBS, 1000 units/ml LIF, 10
ng/ml BMP4 (R&D Systems), 40 ng/ml basic FGF (Upstate Biotechnology) or 15% FBS plus 1000 units/ml LIF. For depletionof HS chains, mES cells were incubated with 5 milliunits ofheparitinase I and II and heparinase for 2 h before extrinsic
stimulation. Cells were lysed with lysis buffer (50 mM Tris-HCl,Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/11/19/M705621200.DC1.html

HS Chains Are Required for the Maintenance of ES Cells

FEBRUARY 8, 2008 • VOLUME 283 • NUMBER 6

tively; apparent equilibrium constant KD (nM) for binding ofvWF A1 to heparin, 1248.1).
RT-PCR and Real Time PCR—Total RNA was isolated frommES cells by TRIzol Reagent (Invitrogen) and subsequently
reverse transcribed using an oligo-dT primer (Invitrogen) and aSuperscriptII first strand synthesis kit (Invitrogen). Primer setsfor PCR amplification are listed in supplemental data 2. All
cDNAs were amplified in quantitative ranges, which were confirmed by examining various cycles for the samples giving maximum levels of signals in each primer sets. Primers sets andprobes for real time PCR are listed in supplemental data 3. Real
time PCR was performed using an ABI PRISM威 7700 sequence
detection system (Applied Biosystems). The relative amountsof each mRNA were normalized by ␤-actin mRNA in the same
cDNA.

RESULTSHS Chains Are Reduced by Knockdown of EXT1 mRNA—Hereditary multiple exostosis is an autosomal dominant disordercharacterized by the development of benign cartilage-capped
tumors at the juxta-epiphyseal regions of long bones (32). It is
associated with mutation of either EXT1 or EXT2, which
encode glycosyltransferases possessing both D-glucuronic acidand GlcNAc-transferase activities that are necessary for HS
chain elongation (33, 34). In the Golgi apparatus, EXT1 andEXT2 form a hetero-oligomeric complex that exhibits higher
glycosyltransferase activity than either enzyme alone (35, 36).
To examine the effect of reduced HS chain levels in ES cells, weknocked down expression of EXT1 mRNA using RNAi. We
designed two constructs expressing different siRNAs targeting
EXT1 (EXT1-1 and EXT1-2), as described previously (29), andsiRNAs targeting EGFP as a negative control. We represent
mES cells transfected with EGFP siRNA expression plasmids as“control cells” throughout this paper. Real time PCR analysis
performed 2 days after transfection of the cells with EXT1
siRNA expression plasmids showed that the level of EXT1mRNA was reduced to about 20% that of control cells. In
contrast, the expression of other nontargeted genes was
unaffected by this treatment in the two mES lines tested (R1and E14TG2a) (Fig. 1A). The knockdown effect on EXT1 wasmaintained for at least 3 days following puromycin selection.
We used E14TG2a to confirm the same knockdown effect on
mES cells using one siRNA expression plasmid (EXT1-1)
throughout all experiments.
Two days after transfection, we performed FACS analysis
using an anti-HS antibody to determine whether HS chain
expression was reduced by knockdown of EXT1 mRNA. As
shown in Fig. 1B, HS chains were abundant in mES cells,
whereas CS chains were not. We observed a significant reduction in HS chain expression on the cell surface of cells transfected with an EXT1 siRNA expression plasmid compared with
control cells (Fig. 1B). The knockdown effect was higher using
EXT1-1 siRNA compared with EXT1-2 siRNA. Reduction of
HS chain expression was accompanied by a subtle increase in
CS chain synthesis that correlated with the level of reduction of
HS chains. Such an increase in CS chain expression was previously observed in EXT1-deficient ES cells derived from EXT1
knock-out mice (25). The reduction of HS chain expression in
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Relative luciferase activity is defined as the ratio of luciferase
activity of TOPFLASH to that of FOPFLASH.
For immunostaining of ␤-catenin, mES cells were fixed with
4% paraformaldehyde and permeabilized with 0.1% saponin 3days after transfection. After washing and subsequent blocking,cells were stained with an anti-␤-catenin antibody. After washing, cells were stained with fluorescein isothiocyanate-conjugated secondary antibody (Chemicon) and counterstained withpropidium iodide (BD Biosciences). Immunofluorescence
images were taken using an LSM5Pascal confocal laser scanning microscope (Carl Zeiss) with ⫻40/1.3 objectives at room
temperature.
125
I-Labeled Wnt3a Binding Assay—Two micrograms ofrecombinant mouse Wnt3a (R&D Systems) were iodinatedwith 100 ␮Ci of 125I-sodium (ARC) using iodogen-precoatedreaction tubes (Pierce) according to manufacturer’s instructions. Radiolabeled Wnt3a was separated from unincorporated
125
I-sodium on a PD-10 column. The specific activity of theradiolabeled Wnt3a was 1.85 ⫻ 104 cpm/ng protein. For thebinding assay, mES cells were harvested 2 days after transfection and replated in triplicate at 5 ⫻ 105 cells per well in 24-well
0.2% gelatin-coated plates. The cells were allowed to attach for
3 h and then washed three times with ice-cold binding buffer(serum-free ES medium containing 1 mg/ml BSA and 0.1%
sodium azide). After incubating with 80 ng/ml 125I-labeled
Wnt3a either alone or in the presence of 100 ␮g/ml heparin(Sigma) in binding buffer for 3 h at 4 °C, the cells were gently
washed three times with ice-cold binding buffer and lysed with
0.2 N NaOH. The amount of radiolabeling in each extract wascounted with a gamma counter (Aloka).
Surface Plasmon Resonance (SPR) Analysis—Heparin(Nacalai Tesque) was dialyzed against distilled water using an
MWCO3500 membrane (SpectroPore) and lyophilized. Thenthe heparin was conjugated with a monovalent linker molecule
to prepare the ligand conjugate for the immobilization of heparin on the gold-coated chip as previously described (31). The
surface of the gold-coated chip (SUDx-Biotec) was oxidatively
washed with UV ozone cleaner (Structure Probe Inc.) for 20
min. The chips were then immersed in 1 ␮M of the ligandconjugate in 50% (v/v) methanol solution overnight at room
temperature with gentle agitation to prepare Sugar Chips withimmobilized heparin. The Sugar Chips were washed sequentially with water, PBS containing 0.05% Tween 20 and water,and dried at room temperature.
The Sugar Chip with immobilized heparin was set on a prismwith refraction oil (nD ⫽ 1.518, Cargill Laboratories Inc.) in an
SPR apparatus (SPR670M, Moritex). The SPR measurements
were performed at room temperature in accordance with the
manufacturer’s instructions and using Tris-buffered saline (20mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 0.05% Tween
20 and 0.1% BSA as the running buffer at a flow rate of 15
␮l/min. The kinetic binding parameters were calculated using
the software of the manufacturer. We performed binding of
BSA or von Willebrand factor (vWF) A1 to heparin, as negativeand positive controls, respectively (supplemental data 1)(apparent association and dissociation rate constants ka (M⫺1s⫺1) and kd (s⫺1) of vWF A1, 3.51 ⫻ 103, 4.38 ⫻ 10⫺3, respec-

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/11/19/M705621200.DC1.html

HS Chains Are Required for the Maintenance of ES Cells

EXT1
EXT2
GlcAT -I
ChGn-2

140
120
100

Lfringe80

EXT1-1(RNAi)

0
0
100

101
102
103
104
Fluorescence intensity

0
0
100

EXT1-2 (RNAi)

E14TG2aControl80
60

Lfringe40200EXT1-1 (RNAi)

0
100

104
101
102
103
Fluorescence intensity

0100

anti-CS

0
100

128

anti-HS
Control
EXT1-1(RNAi)

104
101
102
103
Fluorescence intensity

0
0
100

Control
EXT1-1(RNAi)

80
60
40

V0

0

Control

6

EXT1-2(RNAi)

8

10 12 14 16
Effluent volume (ml)18

20

E14TG2a140

200 10035

8.5 kDa

120
101
102
103
104
Fluorescence intensity

E14-TG2a128

8.5 kDaanti-CS
Control
EXT1-1(RNAi)

100

Control
EXT1-1(RNAi)

80
V0

60
40
20
0
6

8

10 12 14 16
Effluent volume (ml)

18

20

101
102
103
104
Fluorescence intensity

FIGURE 1. EXT1 siRNA induces efficient knockdown of EXT1 mRNA and reduction of HS chains in mES cells. A, real time PCR analysis of cells 2 days after
transfection. The results are shown as the proportion (%) of expression of each glycosyltransferase mRNA relative to that observed in control cells: GlcAT,
glucuronosyltransferase; ChGn, chondroitin ␤1,4-N-acetylgalactosaminyltransferase; Lfringe, lunatic fringe. The values shown are the means ⫾ S.D. of three
independent experiments. We used two constructs expressing different siRNAs targeting EXT1 (EXT1-1 and EXT1-2). B, FACS analysis of cells 2 days after
transfection using an anti-HS antibody or anti-CS antibody (black and blue lines represent IgM isotype control of control and EXT1-deficient cells, respectively).
Three independent experiments were performed and representative results are shown. C, molecular size analysis of HS chains from mES cell surface by gel
chromatography on a Sephacryl S-300 column. The open squares and closed squares indicate the elution profiles of HS chains from control and EXT1-deficient
cells, respectively. Arrows indicate the elution positions of dextran molecular mass standards.

EXT1-deficient cells was also confirmed by HPLC analysis of
unsaturated disaccharides from HS chains (data not shown).
We also examined the molecular sizes of HS chains derived
from EXT1-deficient cell surface by gel chromatography. As isshown in Fig. 1C, the lengths of HS chains in EXT1-deficient
cells (⬃35kDa) appeared to be reduced compared with control
cells (50 –150 kDa).
HS Chains Are Important for Self-renewal and Proliferationof mES Cells—We performed colony assays on EXT1-deficient
cells to determine whether the resulting reduction in HS chain
expression affected the frequency with which the cells remain
in an undifferentiated state. The number of colonies derived
from EXT1-deficient cells that remained in an undifferentiatedstate fell to about 50% that of control cells, even in the presenceof LIF and serum (Fig. 2A). We next examined proliferation oftransfected mES cells. As shown in Fig. 2B, the rate of proliferation of EXT1-deficient cells decreased significantly comparedwith control cells. These results suggest that the reduction in
HS chain expression inhibited the capacity for both self-renewal and proliferation of mES cells, presumably because of
reduced levels of HS chain-dependent signaling.
The Reduction of HS Chain Expression Increases Spontaneous
Differentiation of mES Cells into Extraembryonic Endoderm—We
compared the morphologies of control and EXT1-deficient
cells. Four days after transfection in the presence of LIF, control
cells appeared to be undifferentiated cells with modest packed
morphology in which the cells preferentially adhered to one

3598 JOURNAL OF BIOLOGICAL CHEMISTRY

another, but differentiated cells with a flattened morphology
also existed at low levels because of the feeder-free culture conditions (Fig. 3A). In contrast, almost all of the EXT1-deficient
cells exhibited a flattened, differentiated morphology that, insome cells, was reminiscent of the stellate morphology of theparietal endoderm (37) (Fig. 3A). Furthermore, expression of
Oct3/4 and Nanog, which are markers of the undifferentiatedstate, was significantly decreased in EXT1-deficient cells compared with control cells (Fig. 3B), indicating that spontaneous
differentiation of EXT1-deficient cells occurred more frequently than of control cells. Upon withdrawal of LIF, control
cells exhibited a variety of flattened and differentiated morphologies (data not shown), suggesting that pluripotency had
been maintained. However, most of the EXT1-deficient cells
exhibited a parietal endoderm-like morphology (data notshown), as was the case in the presence of LIF.
To further characterize the transfected mES cells, we evaluated expression of several germ layer markers by real time PCR
analysis of cells 4 days after transfection (Fig. 3, B–D). In thepresence of LIF, we detected higher expression in EXT1-deficient cells of markers of the extraembryonic endoderm lineage(primitive endoderm, Gata4 and Gata6; parietal endoderm,
Dab2 and LamininB1; and visceral endoderm, Bmp2 and Ihh)than control cells (Fig. 3C), whereas other lineage markers(such as the trophoblast marker, Cdx2; the primitive ectoderm
marker, Fgf-5; the neuroectoderm marker, Isl1; and the mesoderm marker, Brachyury) were weakly expressed (Fig. 3D).
VOLUME 283 • NUMBER 6 • FEBRUARY 8, 2008

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

EXT1
EXT2
GlcAT -I
ChGn-2

100

0

35

20

Radioactivity (cpm)120

EXT1-1 (RNAi)

128
Control
EXT1-2(RNAi)

200 100

100

101
102
103
104
Fluorescence intensity

Relative cell numbers

0

anti-HS

Relative cell numbers

20

Relative cell numbers

128

40

R1

120
Control
EXT1-1(RNAi)

60

Relative cell numbers

Ratio ofglycosyltransferase mRNA (%)

Control

160

ControlC)

anti-CS

Relative cell numbers

R1

180

128

anti-HS

Relative cell numbers

A)

Ratio ofglycosyltransferase mRNA (%)

R1

128

Radioactivity (cpm)B)

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/11/19/M705621200.DC1.html

HS Chains Are Required for the Maintenance of ES Cells
1.2
Ratio of alkaline phosphatase
positive colonies

A)

1
0.8
0.6

0.2

Control EXT1-1 EXT1-2

Control EXT1-1(RNAi)(RNAi)
R1(RNAi)
E14TG2aR1B) 1.6

Control

1.6

EXT1-2(RNAi)

1
0.8
0.6
0.4
0.2

EXT1-1(RNAi)

1.4
1.2
1
0.8
0.6
0.4
0.2

0

24
Time (h)

48

0

0

24
Time (h)48

FIGURE 2. EXT1-deficient cells show decreased potential for self-renewal
and proliferation. A, self-renewal assay. The ratio of AP-positive colonies isshown after normalization against control cells (value ⫽ 1). Approximately
70% of the colonies derived from the control cells remained in an undifferentiated state in feeder-free culture. The values shown are the means ⫾ S.D. fromthree independent experiments, and significant values are indicated; *, p ⬍ 0.01,
in comparison with control. B, proliferation assay. The values shown are themeans ⫾ S.D. from three independent experiments.

These results reflect the morphologies of EXT1-deficient cells
as shown in Fig. 3A. Following withdrawal of LIF, EXT1-deficient cells underwent further differentiation. We observed only
induction of extraembryonic endoderm lineage markers inthese cultures compared with control cells (Fig. 3, C and D).
Upon induction of differentiation of control cells, various
markers of differentiation, including Cdx2, Fgf-5, andBrachyury, and extraembryonic endoderm lineage markers
exhibited a further increase in expression associated with adecrease of expression of Oct3/4 and Nanog, indicating that
pluripotency had been maintained. Our analyses indicate that
HS chains are important for the maintenance of the undifferentiated state and of pluripotency of mES cells and that signaling pathways mediated by HS chains may be involved in signaling pathways that control the differentiation of mES cells into
the extraembryonic endoderm lineage.
We next examined the formation of EBs in suspension culture in control and EXT1-deficient cells. EB formation occurs
during the in vitro differentiation of ES cells into the three germlayers, mesoderm, ectoderm, and endoderm. This differentiation process resembles the formation of the developing conceptus at the egg-cylinder stage. As shown in Fig. 3, E and F, almost
all control cells formed large fluid-filled cystic EBs 6 days after
EB formation, in which differentiation into the three germ layers had begun to proceed. In contrast, 53.02 ⫾ 5.83% of EXT1deficient cells formed noncystic and significantly small EBs.
This suggests that the reduction in HS chain expression inhibFEBRUARY 8, 2008 • VOLUME 283 • NUMBER 6

JOURNAL OF BIOLOGICAL CHEMISTRY

3599

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

Absorbance (450 nm)

EXT1-1(RNAi)

1.2

E14TG2a

1.8

Control

1.4

0

*

*

0.4

0

Absorbance (450 nm)*

ited differentiation of EXT1-deficient cells into the three germlayers, supporting the results that differentiation of EXT1-deficient cells into nonextraembryonic endoderm lineage wasinhibited in normal culture (Fig. 3, D and E). Similar results
were obtained in cells transfected with the second siRNA
expression plasmid targeting EXT1 (EXT1-2) and also in a second mES cell line (E14TG2a; data not shown).
The Reduction of HS Chains Down-regulates SpecificSignaling—Several signaling molecules have been shown to beimportant for the maintenance of mES cell self-renewal, forexample, LIF/STAT3, BMP/Smad, Wnt/␤-catenin, phosphoinositide 3-kinase (PI3K)/Akt, and members of the Src family(7–12, 38, 39). Therefore, we performed Western blot analysis
of cell lysates prepared 2 days after transfection of control and
EXT1-deficient cells to determine whether the activity of signaling molecules was affected by the reduction of HS chain
expression. We observed a similar increase in the level of phosphorylation of Akt and Smad1 in control and EXT1-deficient
cells following exposure to 15% FBS (supplemental data 4), suggesting that the serum-responsive signaling by PI3K and Smadwas not affected by the reduced expression of HS chains. Moreover, we found that treatment of control or EXT1-deficient cellswith LIF or BMP4 gave rise to similar increases in the phosphorylation of STAT3 and Smad1, respectively (Fig. 4, A and B).
Heparitinase treatment was performed to examine the effect of
HS chain depletion on BMP/Smad and LIF/STAT3 signaling.
This treatment led to a reduction in BMP/Smad signaling but
not of LIF/STAT3 signaling (Fig. 4, C and D), demonstratingthat HS chains contribute to BMP signaling but not LIF signaling in mES cells. The level of phosphorylation of ERK in
response to basic FGF treatment was reduced in EXT1-deficient cells compared with the level observed in control cells,suggesting that FGF signaling was reduced in EXT1-deficient
cells (Fig. 4B) and more reduction was detected after heparitinase treatment (Fig. 4, C and D). Phosphorylation of the Src
family member, cYes, which has been implicated in the maintenance of self-renewal of ES cells (38), was activated to a similar extent in both control and EXT1-deficient cells following
treatment with LIF and FBS (Fig. 4, A and B). We observed
comparable results in similar studies with E14TG2a cells (datanot shown).
HS Chains Regulate Autocrine/Paracrine Wnt/␤-Catenin
Signaling in mES Cells—In the absence of feeder cells, weobserved a significantly higher level of phosphorylation of
␤-catenin in EXT1-deficient cells than control cells (Fig. 5A).
This suggests that there was a decrease in autocrine/paracrine
Wnt/␤-catenin signaling in EXT1-deficient cells. Next, we
examined Wnt/␤-catenin signaling using a luciferase reporter
system. We found a significant decrease in luciferase activity in
EXT1-deficient cells compared with control cells under feederfree culture conditions (Fig. 5B). This is consistent with areduction in autocrine/paracrine Wnt/␤-catenin signaling in
EXT1-deficient cells. We subsequently examined the nuclear
localization of ␤-catenin, an indicator of activation of thecanonical Wnt pathway. Accumulation of ␤-catenin was significantly decreased in the nuclei of EXT1-deficient cells compared with control cells (supplemental data 5). We carried out aWestern blot analysis and confirmed that nuclear accumula-Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/11/19/M705621200.DC1.html

HS Chains Are Required for the Maintenance of ES Cells
C)1.6

D)

Gata4

1.2

2

0.8

1.6
1.2
0.8
0.4
0
8

0.4
0
Gata6

2

Relative amounts of mRNA

1*
Control

EXT1-1 (RNAi)

B)

1
0

Oct3/4

Bmp2

0

LIFLIF+
E14TG2a

LIF-

LIF+
R1

LIFLIF+
E14TG2a

LIF-

LIF+
R1

Control
EXT1-1(RNAi)

EXT1-1(RNAi)

Control

Control
EXT1-1(RNAi)
EXT1-2(RNAi)

EXT1-1(RNAi)

Control

Control
EXT1-1(RNAi)

Control
EXT1-1(RNAi)
EXT1-2(RNAi)

Control
EXT1-1(RNAi)
EXT1-2(RNAi)

0

Control
EXT1-1(RNAi)
EXT1-2(RNAi)

1

0.4
0

R1
Ihh

2

Nanog

LIF-

LIF+

0

0.8

Brachyury

4
3
2
1

1

0
1.2

0.4
0
5

1

2

0.4

Isl1

0.8

LamininB1

2

0
3

0.8

2
0
1.2

LIFLIF+
E14TG2aE)

R1

Control

EXT1-1 (RNAi)

Control

EXT1-1 (RNAi)F)

R1

FIGURE 3. EXT1-deficient cells differentiate spontaneously into the extraembryonic endoderm lineage. A, photomicrographs of cells at 4 days after
transfection in the presence of LIF. Representative photographs of control and EXT1-deficient cells from two independent experiments are shown. Scale bars,
100 ␮m. (*, high magnification image of the boxed area in EXT1-deficient R1 cells, stellate morphology of parietal endoderm. Scale bars, 50 ␮m). B–D, real time
PCR analysis of cells 4 days after transfection in the presence or absence of LIF. The results are shown after normalization against control cells in the presenceof LIF (value ⫽ 1). The values shown are the means ⫾ S.D. from two independent experiments. E, representative photomicrographs at 6 days after EB formation.
Scale bars, 200 ␮m. F, representative hematoxylin/eosin sections of EBs at 6 days after EB formation. Scale bars, 200 ␮m.

tion of ␤-catenin was reduced in EXT1-deficient cells compared with control cells (Fig. 5C). Thus, activation of Wnt/␤catenin signaling appeared to be reduced in EXT1-deficient
cells. Similar results were observed in analogous studies using
E14TG2a cells (data not shown).
RT-PCR analysis showed that both R1 and E14TG2a celllines expressed several Wnts, as did MEFs. Wnt2, Wnt4, and
Wnt5a were expressed in all cells, but Wnt3a, Wnt7a, and
Wnt10b were expressed only in the mES cells (Fig. 5D). We
examined the effect of reduction of HS chains on the ability of
Wnt3a to bind to the mES cell surface. EXT1-deficient cells
exhibited a significantly lower specific 125I-Wnt3a binding thancontrol cells (Fig. 5E), indicating that HS chains contribute to
the binding of Wnt3a to the mES cell surface. Furthermore, SPR
analysis (supplemental data 6) confirmed binding of Wnt3a to3600 JOURNAL OF BIOLOGICAL CHEMISTRY

heparin, the structural analogue of HS chains, indicating that
Wnt3a does indeed bind to HS chains on mES cells (apparent
association and dissociation rate constants ka (M⫺1 s⫺1) and kd(s⫺1), 2.22 ⫻ 105, 5.77 ⫻ 10⫺3, respectively, apparent equilibrium constant KD (nM) for binding of Wnt3a to heparin, 26.0).
These results demonstrate that HS chains regulate autocrine/
paracrine Wnt/␤-catenin signaling in mES cells.
HS Chain Regulation of Wnt/␤-Catenin Signaling Contributes to Self-renewal of mES Cells—To determine whether the
regulation of Wnt/␤-catenin signaling by HS chains is necessary for the self-renewal and pluripotency of mES cells, we
examined the effect of exogenous activation of Wnt/␤-cateninsignaling in EXT1-deficient cells using BIO, a specific pharmacological inhibitor of glycogen synthase kinase-3. The level ofluciferase activity of Wnt/␤-catenin signaling in BIO-treated
VOLUME 283 • NUMBER 6 • FEBRUARY 8, 2008

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

Relative amounts of mRNA

1.2

Dab2

2

Control
EXT1-1(RNAi)

E14
TG2a

0
3

EXT1-1(RNAi)

EXT1-2 (RNAi)

4

Control

EXT1-1 (RNAi)

Relative amounts of mRNA

Control

Fgf-5

6Control
EXT1-1(RNAi)
EXT1-2(RNAi)R1

Cdx2

Control
EXT1-1(RNAi)
EXT1-2(RNAi)A)

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/11/19/M705621200.DC1.html

HS Chains Are Required for the Maintenance of ES Cells

LIF+

LIF-

ControlEXT1-1EXT1-2ControlEXT1-1EXT1-2(RNAi) (RNAi)(RNAi) (RNAi)

Phospho-STAT3
STAT3
BMP4-

BMP4+

Relative amounts of
STAT3 phosphorylation(p-STAT3/STAT3)

B)

A)

C)
0.6

LIF-

0

bFGF+

bFGF-

ControlEXT1-1EXT1-2 ControlEXT1-1EXT1-2(RNAi) (RNAi)(RNAi) (RNAi)

Phospho-ERK
ERK

STAT3
Control EXT1-1 EXT1-2 Control EXT1-1 EXT1-2(RNAi) (RNAi)(RNAi) (RNAi)

LIFRelative amounts of
Smad1 phosphorylation(p-Smad1/β-actin)

β-actin

BMP4-

Phospho-Yes
Yes

0.8
β-actin
0.4
0

0.3
0.2
0.1
0
LIF-

Relative amounts of
Smad1 phosphorylation(p-Smad1/β-actin)

Heparitinase

+

-

bFGF-

BMP4+

1.2

bFGF+

Phospho-ERK

*

0.8

*

ERK

0.4
0

Control EXT1-1 EXT1-2 Control EXT1-1 EXT1-2(RNAi) (RNAi)(RNAi) (RNAi)

bFGF+

0.5
0.4
0.3
0.2
0.1
0 Control

EXT1-1 EXT1-2 Control EXT1-1 EXT1-2(RNAi) (RNAi)(RNAi) (RNAi)

LIF-,FBS-

LIF+,FBS+

1
0.8
0.6

*

0.4
0.2
0

BMP4Heparitinase

Relative amounts of
ERK phosphorylation(p-ERK/ERK)

LIF+

Heparitinase+-

Control EXT1-1 EXT1-2 Control EXT1-1 EXT1-2(RNAi) (RNAi)(RNAi) (RNAi)

bFGF-

0.4
Relative amounts ofYes phosphorylation(p-Yes/Yes)

Relative amounts of
STAT3 phosphorylation(p-STAT3/STAT3)

D)

Relative amounts of
ERK phosphorylation(p-ERK/ERK)

LIF+,FBS+

BMP4+

Phospho-Smad1

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

LIF-,FBS-

Heparitinase
+
-

LIF+

1.2

BMP4-

Control EXT1-1EXT1-2 ControlEXT1-1EXT1-2(RNAi) (RNAi)(RNAi) (RNAi)

LIF+

Phospho-STAT3

0.2

ControlEXT1-1EXT1-2 ControlEXT1-1 EXT1-2(RNAi) (RNAi)(RNAi) (RNAi)

Phospho-Smad1

Heparitinase
+
-

0.4

BMP4+
+

-

1.2
0.8

*

0.4
0

bFGFHeparitinase

bFGF+
-

+

FIGURE 4. Specific signaling is decreased in EXT1-deficient cells. A, Western blot analysis of mES cells stimulated with each extrinsic factor. Two independent
experiments were performed, and representative results are shown. B, quantification of Western blots shown in A. The histograms show mean densitometricreadings ⫾ S.D. of the phosphorylated protein/loading controls. Values were obtained from duplicate measurements of two independent experiments andsignificant values are indicated; *, p ⬍ 0.01, in comparison with stimulated control. C, Western blot analysis of mES cells stimulated with each extrinsic factor
after treatment with or without heparitinase. Two independent experiments were performed, and representative results are shown. D, quantification ofWestern blots shown in C. The histograms show mean densitometric readings ⫾ S.D. of the phosphoprotein/loading controls. Values were obtained from
duplicate measurements of two independent experiments, and significant values are indicated; *, p ⬍ 0.01, in comparison with heparitinase untreated andextrinsically stimulated samples.

cells was 2–3-fold higher than in cells treated with MeBIO, an
inactive analogue of BIO (data not shown). Two days after
transfection, the level of Nanog mRNA was significantly upregulated in BIO-treated cells (Fig. 6A), indicating that NanogFEBRUARY 8, 2008 • VOLUME 283 • NUMBER 6

expression is regulated by Wnt/␤-catenin signaling. The levelsof Nanog and Oct3/4 mRNAs in MeBIO-treated EXT1-deficient cells were 30 –50% of those in MeBIO-treated control
cells (Fig. 6A). Untreated cells showed similar results (data not
JOURNAL OF BIOLOGICAL CHEMISTRY

3601

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/11/19/M705621200.DC1.html

HS Chains Are Required for the Maintenance of ES Cells
Nearly all of the colonies derived
from culture of EXT1-deficient cells
A)
E14TG2aβ-catenin(nuclear)R1
in the presence of BIO exhibited aControlEXT1-1EXT1-2 ControlEXT1-1
LaminB1(RNAi)(RNAi)(RNAi)
1.2
tightly packed and AP-positive
Phospho-β-catenin1morphology reminiscent of undifβ-catenin(total)
0.8
*
0.6
ferentiated ES cells cultured on a
*
*
3
0.4
feeder layer. BIO treatment there0.2
2.5*
0fore rescued defective self-renewal
Control EXT1-1
ControlEXT1-1EXT1-2*(RNAi) (RNAi)(RNAi)
2*
R1
E14TG2ain EXT1-deficient cells (Fig. 6C).
1.5
This
rescue effect demonstrates
Wnt1 Wnt2 Wnt3a Wnt4 Wnt5a Wnt7a Wnt10b GAPDH
D)
1
R1
that HS chain-dependent signaling
0.5
by Wnt/␤-catenin contributes to
E14TG2a0
Control EXT1-1
Control EXT1-1EXT1-2(RNAi) (RNAi)(RNAi)
self-renewal of mES cells. However,
MEF
R1
E14TG2athe total numbers of colonies
4
B)
scarcely differed between BIOE)
9
3.5
treated cells and MeBIO-treated83
7
cells (Fig. 6C), indicating that BIO
6
2.5**
treatment had no effect on cell pro5*
2**
4
liferation. In a proliferation assay,
*
*
1.5
*
3
we observed no effect of BIO on
1
2
mES cell proliferation compared1
0.5
0with untreated and MeBIO-treatedControl EXT1-1 EXT1-2
Control EXT1-1
0
Control EXT1-1(RNAi) (RNAi)(RNAi)
ControlEXT1-1 EXT1-2(RNAi) (RNAi)(RNAi)
cells (data not shown). These results
R1
E14TG2aR1
E14TG2ademonstrate that HS chain-deFIGURE 5. Wnt/␤-catenin signaling is decreased in EXT1-deficient cells. A, Western blot analysis of mES cells
after LIF and serum starvation for 4 h at 2 days after transfection. Representative immunoblots are shown. The pendent signaling by autocrine/
histograms show mean densitometric readings ⫾ S.D. of the phospho-␤-catenin/total ␤-catenin after normal- paracrine Wnt/␤-catenin is reization against control cells (value ⫽ 1). Values were obtained from duplicate measurements of two independ- quired for the maintenance ofent experiments, and significant values are indicated; *, p ⬍ 0.01, in comparison with control. B, luciferase
reporter assay. Relative luciferase activities (TOPFLASH/FOPFLASH) are shown as means ⫾ S.D. from three self-renewal but not for the proliferindependent experiments, and significant values are indicated; *, p ⬍ 0.01; **, p ⬍ 0.05, in comparison with ation of mES cells.
control. C, Western blot analysis of mES cells at 3 days after transfection. Representative immunoblots are shown.
Wnt/␤-Catenin Signaling ReguThe histograms show mean densitometric readings ⫾ S.D. of the ␤-catenin/lamin B1 after normalization against
control cells (value ⫽ 1). Values were obtained from duplicate measurements of two independent experiments and lated by HS Chains Is Important forsignificant values are indicated; *, p ⬍ 0.01, in comparison with control. D, RT-PCR analysis of the expression of Pluripotency of mES Cells—Our
several Wnts in mES cells and MEFs. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. E, binding assay of 125Ilabeled Wnt3a. The value of specific 125I-Wnt3a binding (total counts/min bound minus counts/min bound in the next step was to examine the effectpresence of 100 ␮g/ml free heparin) is the mean ⫾ S.D. of three independent experiments, and significant values are of BIO on EXT1-deficient cell pluriindicated; *, p ⬍ 0.01, in comparison with control.
potency in the absence of LIF, inwhich ES cells spontaneously differshown). This suggests that decreased signaling in EXT1-defi- entiated into several lineages (Fig. 3, B–D). The expression ofcient cells affects the expression of Nanog and Oct3/4. BIO differentiation markers was analyzed 4 days after transfection
treatment rescued the level of Nanog mRNA in EXT1-deficient using real time PCR analysis (Fig. 6, D–F). The expression ofcells, but not of Oct3/4 (Fig. 6A). The signaling pathways that various differentiation markers was examined in EXT1-defiregulate Oct3/4 expression are therefore dependent upon HS cient cells treated with either BIO or MeBIO. BIO induced
chain expression but not upon Wnt. Furthermore, the expres- expression of the markers, including Cdx2, Fgf-5, andsion patterns of Nanog and Oct3/4 proteins were correlated Brachyury; expression of the differentiation markers was at awith mRNA levels (Fig. 6B). Recently, it has been demonstrated low level in the MeBIO-treated cells. In the latter treatmentthat the orphan nuclear receptor LRH-1 is required for main- group, only markers of the extraembryonic endoderm lineage
tenance of Oct3/4 expression in mES cells (40). The level of (Gata6, LamininB1, and BMP2) were detected at appreciable
LRH-1 mRNA in EXT1-deficient cells treated with MeBIO was levels (similar to those described in Fig. 3, C and D). Therefore,
also reduced to 30 –50% that in control cells (Fig. 6A), similarly BIO rescued the defective pluripotency of EXT1-deficient cells.
Nanog-deficient mES cells spontaneously differentiate into
to Oct3/4 expression, but was unaffected by BIO treatment (Fig.
6A). This suggests that the reduction in Oct3/4 expression in the extraembryonic endoderm lineage, implicating Nanog in
EXT1-deficient cells is mediated by LRH-1. Overall, our analy- the control of mES cell pluripotency (14, 15). Nanog and Oct3/4
ses demonstrate that Wnt/␤-catenin signaling through HS expression was reduced in EXT1-deficient cells treated withchains regulates Nanog expression but not that of Oct3/4 in MeBIO, whereas treatment with BIO rescued the level of Nanog
expression although only to the same level as in MeBIO-treatedmES cells.
Next, we examined the ability of BIO to rescue self-renewal control cells (Fig. 6D). Thus Wnt/␤-catenin signaling sustains
in EXT1-deficient cells. The number of AP-positive colonies Nanog expression. In control cells, BIO treatment resulted inderived from EXT1-deficient cells treated with MeBIO was sig- reduced expression of various differentiation markers associnificantly reduced compared with that of control cells (Fig. 6C). ated with maintenance of Nanog and Oct3/4 expression com-

3602 JOURNAL OF BIOLOGICAL CHEMISTRY

R1

E14TG2aSpecific 125I-Wnt3a bound(cpm x 103)

Relative amounts of
β-catenin (arbitarydensitometric units)

ControlEXT1-1EXT1-2 Control EXT1-1(RNAi) (RNAi)(RNAi)VOLUME 283 • NUMBER 6 • FEBRUARY 8, 2008

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

Relative luciferase activity

Relative amounts of
β-catenin phosphorylation(arbitary densitometric units)C)Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/11/19/M705621200.DC1.html

HS Chains Are Required for the Maintenance of ES Cells
B)
250
200

Nanog

150

β-actin50
Control

EXT1-1

Control(RNAi)

R1

EXT1-1(RNAi)

E14TG2a160

E14TG2aControl
EXT1-1+ MeBIO BIO MeBIO BIO+ MeBIO BIO MeBIO BIO(RNAi)C)+MeBIO+BIO

Control

Control

1276 ±1371556 ±133

EXT1-1

EXT1-1205 ±13213±67

EXT1-1

EXT1-1301 ±165

220 ±79(RNAi)

2
R1

1.6

0.8
0.4
0

Control

EXT1-1

Control(RNAi)

R1

EXT1-1(RNAi)

E14TG2a80
EXT1-1

E14TG2aControl
EXT1-1+ MeBIO BIO MeBIO BIO+ MeBIO BIO MeBIO BIO

R1Control

40
0Control

EXT1-1(RNAi)

Control(RNAi)(RNAi)(RNAi)(RNAi)E14
TG2a

EXT1-1(RNAi)

Oct3/4

E14TG2a30

Control

EXT1-1(RNAi)

Control

R1

EXT1-1(RNAi)

E14TG2a

1.2
0.8
0.4
0

Control

EXT1-1

Control(RNAi)

R1

EXT1-1(RNAi)

E14TG2a

1.6
1.2
0.8
0.4
0

**

*
ControlEXT1-1ControlEXT1-1(RNAi)(RNAi)+MeBIO

+BIO
R1

Nanog

2

0.8

1

0.4

0
1.4

0.8
0.4

E) 1.6

ControlEXT1-1ControlEXT1-1(RNAi)(RNAi)+MeBIO

ControlEXT1-1ControlEXT1-1(RNAi)(RNAi)+BIO+MeBIO+BIO
E14TG2a

R1
Gata6

1.4
1.2
0.8
0.4
0
1.6
1.4
1.2
0.8
0.4
0
4

+MeBIO+BIO
E14TG2a

Cdx2

0
1.2

Oct3/4

1.2

0Relative amounts of mRNA

F) 1.2

Relative amounts of mRNA

Relative amounts of mRNAD) 3

ControlEXT1-1ControlEXT1-1(RNAi)(RNAi)

Fgf-5

0.8
0.4
0
Isl1

1.2
0.8
0.4
0
1.2

Brachyury

0.8
LamininB1

0.4
0ControlEXT1-1ControlEXT1-1(RNAi)(RNAi)+MeBIO+BIO
R1ControlEXT1-1ControlEXT1-1(RNAi)(RNAi)+MeBIO+BIO
E14TG2a

Bmp2

3
2
1
0

ControlEXT1-1ControlEXT1-1(RNAi)(RNAi)+MeBIO+BIO
R1ControlEXT1-1ControlEXT1-1(RNAi)(RNAi)+MeBIO+BIO
E14TG2aFIGURE 6. Activation of Wnt/␤-catenin signaling by treatment with BIO rescues self-renewal and pluripotency of EXT1-deficient cells. A, real time PCR
analysis of mES cells 2 days after transfection. The value shown is the mean ⫾ S.D. of three independent experiments (white bar, MeBIO-treated; black bar,
BIO-treated). B, Western blot analysis of mES cells at 2 days after transfection. Representative immunoblots are shown. The histograms show mean densitometric readings ⫾ S.D. of the protein/␤-actin. Values were obtained from duplicate measurements of two independent experiments (white bar, MeBIO-treated;black bar, BIO-treated). C, self-renewal assay. Upper panels, photographs of representative colonies. The total number of colonies is indicated at the bottom ofeach image. To the right of each photograph is a high magnification image. Scale bars, 200 ␮m. The ratio of AP-positive colonies is shown in lower panels after
normalization against MeBIO-treated control cells (value ⫽ 1). The value shown is the mean ⫾ S.D. of three independent experiments. Significant values areindicated; *, p ⬍ 0.01; **, p ⬍ 0.03, in comparison with control. D–F, real time PCR analysis of several germ layer markers at 4 days after transfection in theabsence of LIF. The results are shown after normalization against MeBIO-treated control cells (value ⫽ 1). The values shown are the means ⫾ S.D. of two
independent experiments.

pared with MeBIO treatment. This indicates that BIO inhibited
differentiation of control cells. In turn, maintenance of Nanog
expression by autocrine/paracrine Wnt/␤-catenin signaling
through HS chains is important for the maintenance of pluripotency of mES cells.

DISCUSSION
In this study, we have demonstrated that EXT1-deficientmES cells, which express greatly reduced levels of HS chains,
FEBRUARY 8, 2008 • VOLUME 283 • NUMBER 6

proliferate slowly and differentiate spontaneously into theextraembryonic endoderm lineage. Signaling by Wnt and
FGF, but not BMP and LIF, was disrupted in EXT1-deficient
cells. Furthermore, we demonstrated that autocrine/paracrine Wnt/␤-catenin signaling through HS chains was necessary for maintenance of Nanog expression, self-renewal,
and pluripotency in mES cells even in the presence of LIF/
STAT3 and BMP/Smad signaling. Our results indicate that
HS chains are important regulators of the extrinsic signaling
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10

1.6

Ratio of alkaline phosphatase
positive colonies

β-actin20

0(RNAi)(RNAi)

1.2

120

R1
Relative amounts of
LRH-1 mRNA (x 1000/β-actin)

Relative amounts of
Nanog (Nanog /β-actin)

1000EXT1-1

R1
Control

Relative amounts of
Oct3/4 (Oct3/4 /β-actin)

Relative amounts of
Oct3/4 mRNA (x 1000/β-actin)

Relative amounts of
Nanog mRNA (x 1000/β-actin)A)Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/11/19/M705621200.DC1.html

HS Chains Are Required for the Maintenance of ES Cells

3604 JOURNAL OF BIOLOGICAL CHEMISTRY

chains have been shown to play crucial roles in FGF signaling
during development (18).
We did not observe a reduction in BMP/Smad signaling in
response to reduced short HS chains in mES cells (Fig. 4B),
although previous reports have indicated that HS chains contribute to signaling by BMP in Drosophila and Xenopus (18, 43).
The specific decrease in signaling by Wnt and FGF, but not by
BMP, mediated by short HS chains in EXT1-deficient cells may
be because of the effects of reduced HS chain length on theability to accumulate sufficient ligands for their cognate receptors. This is supported by the result that HS chain depletion by
heparitinase treatment led to not only further reduction in FGF
signaling but also a reduction in BMP/Smad signaling (Fig. 4, C
and D).
IL-6 is a heparin/HS-binding cytokine, and HS chains may
regulate paracrine IL-6 signaling (44). Although LIF is an IL-6
family member and might also bind to HS chains, we demonstrated that HS chains are not required for LIF/STAT3 signaling. Neither reduction of HS chains (Fig. 4, A and B) nor HS
chain depletion by heparitinase treatment (Fig. 4, C and D) had
an effect on LIF/STAT3 signaling. Taken together, HS chainscontribute to the regulation of several signaling pathways mediated by Wnt, BMP, and FGF but not LIF in mES cells (Fig. 7).
As shown in Fig. 6C, the total numbers of colonies scarcely
differed between BIO-treated cells and MeBIO-treated cells,
and we observed no effect of BIO on mES cell proliferation(data not shown), indicating that BIO treatment had no effecton cell proliferation. As shown in Fig. 2B, HS chains contributeto mES cell proliferation. Thus, we suggest that HS chain-mediated signaling pathways that are not influenced by Wnt/␤catenin control mES cell proliferation.
Nanog expression was increased in response to BIO treatment in both control and EXT1-deficient cells (Fig. 6A). However, the difference in the extent of increase in the expression of
Nanog mRNA following BIO treatment of control and EXT1deficient cells (Fig. 6A) indicates that other signaling pathways
contribute to the control of its expression through HS chains.
Recently, it has been demonstrated that Nanog transcription isregulated by an interaction between Oct3/4 and Sox2 or a novel
pluripotential cell-specific Sox element-binding protein (45,46). As such, the difference in the extent of the increase in theexpression of Nanog mRNA following BIO treatment of control
and EXT1-deficient cells may be due to a possible reduction in
Oct3/4 and Sox2 or pluripotential cell-specific Sox elementbinding protein mediated by unknown signaling through HS
chains.
Expression of Oct3/4 and LRH-1 mRNA was decreased in
EXT1-deficient cells treated with MeBIO (Fig. 6A), and theirexpression was not affected by BIO treatment in either control or EXT1-deficient cells (Fig. 6A). Thus regulation of
Oct3/4 and LRH-1 expression does not require Wnt/␤-catenin signaling through HS chains. LRH-1 has been shown toregulate the expression of Oct3/4 by binding to its proximalenhancer and promoter (40). LRH-1 has also been shown toplay an important role in the regulation of cell proliferation(47). The signaling pathways controlling the expression of
LRH-1 remain to be determined. The decrease in Oct3/4
expression observed in EXT1-deficient cells may be dependVOLUME 283 • NUMBER 6 • FEBRUARY 8, 2008

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pathways contributing to self-renewal and pluripotency ofmES cells.
We designed constructs expressing two different siRNAs targeting EXT1. Knockdown of EXT1 in response to EXT1-1
siRNA was maintained at ⬃20% of the level observed in control
cells for at least 3 days following selection, even in the absenceof puromycin. However, when EXT1-2 siRNA was used, thelevel of EXT1 mRNA recovered to 50 – 60% of that of control
cells 3 days after selection (data not shown). As a result, theeffects of EXT1 knockdown were somewhat weaker in cells
transfected with the EXT1-2 plasmid (Figs. 2–5), reflecting a
response that correlated with EXT1 mRNA levels. These observations strongly support the conclusion that our results reflect
the function of HS chains.
In this study, we used a transient knockdown system andfound that the efficiency of knockdown decreased daily following the ending of selection. We therefore used puromycin to
select for efficient knockdown in cells throughout the experiments described in Fig. 3 and Fig. 6, D–F. However, in the selfrenewal and proliferation assays (Fig. 2 and Fig. 6C), the cells
had to be replated at low densities, which rendered them susceptible to puromycin-induced cell death. Thus, puromycin
could not be used after replating. These different conditions
underlie the apparent experimental disparities such as thatbetween Fig. 2A, in which undifferentiated colonies derived
from EXT1-deficient cells were present at half of the level ofcontrol cells, and Fig. 3A, in which almost all EXT1-deficient
cells differentiated.
Several signaling molecules, such as LIF/STAT3, BMP/
Smad, Wnt/␤-catenin, PI3K/Akt, and Src family members, arerequired for self-renewal of mES cells (7–12, 38, 39). Until now,
it has been believed that extrinsic stimulation by both LIF andserum, including BMP, was sufficient to maintain mES cell selfrenewal (4). However, in this study, we showed that self-renewal of mES cells was significantly decreased in response to
the reduction in HS chain expression, even when LIF- andserum-mediated signaling were not reduced (Fig. 2 and Fig.
4). As shown in Fig. 5, several Wnts were expressed in mES
cells, indicating that autocrine/paracrine Wnts function inmES cells. Indeed, autocrine/paracrine Wnt/␤-catenin signaling was actually observed in cells cultured under feederfree conditions. Furthermore, we showed that Wnt3a binds
to the mES cell surface mediated by HS chains, and we demonstrated that autocrine/paracrine Wnt/␤-catenin signaling
through HS chains is important for maintenance of self-renewal of mES cells (Fig. 5 and Fig. 6). Thus, we propose thatmaintenance of mES cell self-renewal requires not only LIF
plus serum factors but also autocrine/paracrine Wnts.
Reduction of HS chain expression also led to decreased signaling by FGF (Fig. 4). It has been demonstrated that HS chains
regulate mouse fibroblast cell proliferation through FGF signaling (41). FGF has been also reported to maintain self-renewal of
human ES cells (42), although this effect has yet to be observed
in mES cells. We speculate that FGF signaling through HS
chains may contribute to the maintenance of self-renewal andproliferation of mES cells, if FGF is present in serum or theconditioned medium. This idea is supported by the fact that HS

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/11/19/M705621200.DC1.html

HS Chains Are Required for the Maintenance of ES Cells
Acknowledgment—We thank Aya Juni for guiding ES cell culture.

Wnt

REFERENCES

FGF

Wnt

LIF
?

β-catenin

STAT3
Non-neurallineage

Myc

Primitive
endoderm

Nanog
Oct3/4

Smad

Id
LRH-1

Unknown
factors
Trophectoderm
Neural lineage: HS chains: HS proteoglycan: Signaling mediatedby HS chains: Signaling not
mediated by HS chains

FIGURE 7. HS chains regulate signaling by extrinsic factors required forthe maintenance of self-renewal and pluripotency of mES cells. HS chainscontribute to the regulation of several signaling pathways mediated by
extrinsic factors, such as BMP, Wnt, FGF, and unknown factors, but not to LIF
and unknown serum factors in mES cells. We demonstrated that autocrine/
paracrine Wnt/␤-catenin signaling mediated by HS chains regulates Nanog
expression and sustains self-renewal by suppression of primitive endodermal
differentiation. BMP/Smad signaling is regulated by HS chains (Fig. 4, C and D)
and blocks neural differentiation by induction of Id (11). FGF signaling is also
regulated by HS chains (Fig. 4), but the signaling pathway required for maintenance of self-renewal is unknown in mES cells. From the observations madein this study, we suggest that HS chain-dependent signaling by unknown
factors regulates mES cell proliferation and Oct3/4 expression. LIF/STAT3 signaling is not regulated by HS chains (Fig. 4) and blocks non-neuronal differentiation by induction of Myc (51). *, BMP/Smad signaling is not decreased byshort HS chains (⬃35 kDa) but is decreased by depletion of HS chains.

ent upon the decrease in LRH-1 expression because of
reduced signaling in these unidentified pathways in an HS
chain-dependent manner. Although the signaling pathways
activated downstream of HS chain expression remain to be
determined, those required for LRH-1 expression might regulate the proliferation of mES cells.
In conclusion, we have demonstrated the importance ofHS chains for the regulation of self-renewal and pluripotency of mES cells. These results suggest that it is possible tomodify self-renewal and differentiation of ES cells by the useof RNAi targeted to genes encoding enzymes required for HS
chain synthesis. Signaling by Wnt, FGF, and Notch havebeen shown to be involved in the regulation of self-renewalof hematopoietic and neural stem cells (48 –50). However,the contribution of glycochains to such signaling in thesecells has not yet been determined. Modulation of the glycosylation pathways in stem cells will provide the opportunity
to engineer stem cells to follow specific differentiation programs that may be of use for the development of cell replacement therapies.
FEBRUARY 8, 2008 • VOLUME 283 • NUMBER 6

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BMP

1. Evans, M. J., and Kaufman, M. H. (1981) Nature 292, 154 –156
2. Martin, G. R. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 7634 –7638
3. Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., and Jones, J. M. (1998) Science 282, 1145–1147
4. Chambers, I., and Smith, A. (2004) Oncogene 23, 7150 –7160
5. Smith, A. G., Heath, J. K., Donaldson, D. D., Wong, G. G., Moreau, J., Stahl,M., and Rogers, D. (1988) Nature 336, 688 – 690
6. Williams, R. L., Hilton, D. J., Pease, S., Willson, T. A., Stewart, C. L., Gearing, D. P., Wagner, E. F., Metcalf, D., Nicola, N. A., and Gough, N. M.
(1988) Nature 336, 684 – 687
7. Boeuf, H., Hauss, C., Graeve, F. D., Baran, N., and Kedinger, C. (1997)
J. Cell Biol. 138, 1207–1217
8. Matsuda, T., Nakamura, T., Nakao, K., Arai, T., Katsuki, M., Heike, T., andYokota, T. (1999) EMBO J. 18, 4261– 4269
9. Niwa, H., Burdon, T., Chambers, I., and Smith, A. (1998) Genes Dev. 12,2048 –2060
10. Raz, R., Lee, C. K., Cannizzaro, L. A., d’Eustachio, P., and Levy, D. E. (1999)
Proc. Natl. Acad. Sci. U. S. A. 96, 2846 –2851
11. Ying, Q. L., Nichols, J., Chambers, I., and Smith, A. (2003) Cell 115, 281–292
12. Sato, N., Meijer, L., Skaltsounis, L., Greengard, P., and Brivanlou, A. H.
(2004) Nat. Med. 10, 55– 63
13. Wodarz, A., and Nusse, R. (1998) Annu. Rev. Cell Dev. Biol. 14, 59 – 88
14. Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S., andSmith, A. (2003) Cell 113, 643– 655
15. Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K.,
Maruyama, M., Maeda, M., and Yamanaka, S. (2003) Cell 113, 631– 642
16. Esko, J. D., and Selleck, S. B. (2002) Annu. Rev. Biochem. 71, 435– 471
17. Bernfield, M., Gotte, M., Park, P. W., Reizes, O., Fitzgerald, M. L., Lincecum, J., and Zako, M. (1999) Annu. Rev. Biochem. 68, 729 –777
18. Lin, X. (2004) Development (Camb.) 131, 6009 – 6021
19. Bornemann, D. J., Duncan, J. E., Staatz, W., Selleck, S., and Warrior, R.
(2004) Development (Camb.) 131, 1927–1938
20. Han, C., Belenkaya, T. Y., Khodoun, M., Tauchi, M., and Lin, X. (2004)
Development (Camb.) 131, 1563–1575
21. Takei, Y., Ozawa, Y., Sato, M., Watanabe, A., and Tabata, T. (2004) Development (Camb.) 131, 73– 82
22. The, I., Bellaiche, Y., and Perrimon, N. (1999) Mol. Cell 4, 633– 639
23. Garcia-Garcia, M. J., and Anderson, K. V. (2003) Cell 114, 727–737
24. Li, J. P., Gong, F., Hagner-McWhirter, A., Forsberg, E., Abrink, M., Kisilevsky, R., Zhang, X., and Lindahl, U. (2003) J. Biol. Chem. 278,
28363–28366
25. Lin, X., Wei, G., Shi, Z., Dryer, L., Esko, J. D., Wells, D. E., and Matzuk,
M. M. (2000) Dev. Biol. 224, 299 –311
26. Merry, C. L., Bullock, S. L., Swan, D. C., Backen, A. C., Lyon, M., Beddington, R. S., Wilson, V. A., and Gallagher, J. T. (2001) J. Biol. Chem. 276,
35429 –35434
27. Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., and Roder, J. C.
(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8424 – 8428
28. Smith, A. G., and Hooper, M. L. (1987) Dev. Biol. 121, 1–9
29. Ui-Tei, K., Naito, Y., Takahashi, F., Haraguchi, T., Ohki-Hamazaki, H.,
Juni, A., Ueda, R., and Saigo, K. (2004) Nucleic Acids Res. 32, 936 –948
30. Toyoda, H., Kinoshita-Toyoda, A., and Selleck, S. B. (2000) J. Biol. Chem.
275, 2269 –2275
31. Suda, Y., Arano, A., Fukui, Y., Koshida, S., Wakao, M., Nishimura, T.,
Kusumoto, S., and Sobel, M. (2006) Bioconjug. Chem. 17, 1125–1135
32. Wicklund, C. L., Pauli, R. M., Johnston, D., and Hecht, J. T. (1995) Am. J.
Med. Genet. 55, 43– 46
33. Lind, T., Tufaro, F., McCormick, C., Lindahl, U., and Lidholt, K. (1998)
J. Biol. Chem. 273, 26265–26268
34. McCormick, C., Leduc, Y., Martindale, D., Mattison, K., Esford, L. E.,
Dyer, A. P., and Tufaro, F. (1998) Nat. Genet. 19, 158 –161
35. McCormick, C., Duncan, G., Goutsos, K. T., and Tufaro, F. (2000) Proc.
Natl. Acad. Sci. U. S. A. 97, 668 – 673

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/11/19/M705621200.DC1.html

HS Chains Are Required for the Maintenance of ES Cells
36. Senay, C., Lind, T., Muguruma, K., Tone, Y., Kitagawa, H., Sugahara, K., Lidholt, K., Lindahl, U., and Kusche-Gullberg, M. (2000) EMBO Rep. 1, 282–286
37. Fujikura, J., Yamamoto, E., Yonemura, S., Hosoda, K., Masui, S., Nakao, K.,
Miyazaki Ji, J., and Niwa, H. (2002) Genes Dev. 16, 784 –789
38. Anneren, C., Cowan, C. A., and Melton, D. A. (2004) J. Biol. Chem. 279,
31590 –31598
39. Paling, N. R., Wheadon, H., Bone, H. K., and Welham, M. J. (2004) J. Biol.
Chem. 279, 48063– 48070
40. Gu, P., Goodwin, B., Chung, A. C., Xu, X., Wheeler, D. A., Price, R. R.,
Galardi, C., Peng, L., Latour, A. M., Koller, B. H., Gossen, J., Kliewer, S. A.,
and Cooney, A. J. (2005) Mol. Cell. Biol. 25, 3492–3505
41. Gallagher, J. T. (2001) J. Clin. Investig. 108, 357–361
42. Xu, R. H., Peck, R. M., Li, D. S., Feng, X., Ludwig, T., and Thomson, J. A.
(2005) Nat. Methods 2, 185–190
43. Ohkawara, B., Iemura, S., ten Dijke, P., and Ueno, N. (2002) Curr. Biol. 12,
205–209

44. Mummery, R. S., and Rider, C. C. (2000) J. Immunol. 165, 5671–5679
45. Kuroda, T., Tada, M., Kubota, H., Kimura, H., Hatano, S. Y., Suemori, H.,
Nakatsuji, N., and Tada, T. (2005) Mol. Cell. Biol. 25, 2475–2485
46. Rodda, D. J., Chew, J. L., Lim, L. H., Loh, Y. H., Wang, B., Ng, H. H., andRobson, P. (2005) J. Biol. Chem. 280, 24731–24737
47. Botrugno, O. A., Fayard, E., Annicotte, J. S., Haby, C., Brennan, T., Wendling, O., Tanaka, T., Kodama, T., Thomas, W., Auwerx, J., andSchoonjans, K. (2004) Mol. Cell 15, 499 –509
48. Hitoshi, S., Alexson, T., Tropepe, V., Donoviel, D., Elia, A. J., Nye, J. S.,
Conlon, R. A., Mak, T. W., Bernstein, A., and van der Kooy, D. (2002)
Genes Dev. 16, 846 – 858
49. Panchision, D. M., and McKay, R. D. (2002) Curr. Opin. Genet. Dev. 12,
478 – 487
50. Reya, T., and Clevers, H. (2005) Nature 434, 843– 850
51. Cartwright, P., McLean, C., Sheppard, A., Rivett, D., Jones, K., and Dalton,
S. (2005) Development (Camb.) 132, 885– 896

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

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