OPEN
ORIGINAL ARTICLE
Activation of PDK-1 maintains mouse embryonic stem cell
self-renewal in a PKB-dependent manner
LS Ling1,2
, D Voskas2
and JR Woodgett1,2
The phosphatidylinositol 30
kinase (PI3K) pathway is involved in many cellular processes including cell proliferation, survival and
glucose transport, and is implicated in various disease states, such as cancer and diabetes. Although there have been numerous
studies dissecting the role of PI3K signaling in different cell types and disease models, the mechanism by which PI3K signaling
regulates embryonic stem (ES) cell fate remains unclear. It is believed that in addition to proliferation and tumorigenesis, PI3K
activity may also be important for ES cell self-renewal. Paling et al. reported that the inhibition of PI3K led to a reduction in the
ability of leukemia inhibitory factor to maintain self-renewal, causing cells to differentiate. Studies in our lab have revealed that ES
cells completely lacking glycogen synthase kinase-3 (GSK-3) remain undifferentiated compared with wild-type ES cells. GSK-3 is
negatively regulated by PI3K, suggesting that PI3K may have a vital role in maintaining pluripotency in ES cells through GSK-3. By
using a modified Flp recombinase system, we expressed activated alleles of 3-phosphoinositide-dependent protein kinase-1 and
protein kinase B to create stable, isogenic ES cell lines to further study the role of the PI3K signaling pathway in stem cell fate
determination. In vitro characterization of the transgenic cell lines revealed a strong tendency toward the maintenance of
pluripotency, and this phenotype was found to be independent of canonical Wnt signal transduction. In summary, PI3K signaling is
sufficient to maintain the self-renewal and survival of stem cells. As this pathway is frequently mutationally activated in cancers, its
effect on suppressing differentiation may contribute to its oncogenicity.
Oncogene advance online publication, 4 March 2013; doi:10.1038/onc.2013.44
Keywords: PDK-1; GSK-3; embryonic stem cell; pluripotency
INTRODUCTION
The phosphatidylinositol 3-kinases (PI3Ks) are a family of
conserved intracellular lipid kinases that phosphorylate the
30
-hydroxyl group of phosphatidylinositol and phosphoinositides,
producing PtdIns(3)P, PtdIns(3,4)P2 (PIP2) and PtdIns(3,4,5)P3
(PIP3).1
The various isoforms of PI3Ks are divided into three main
classes. Class I PI3Ks are involved in many important physiological
processes and are further subdivided into class IA: those with a
p110 catalytic subunit (a, b or d) and a p85 regulatory subunit (a, b
or g) that are activated by growth factor receptor tyrosine kinases
and class IB: those with a p110g catalytic subunit and a p101 or
p84 regulatory subunit that are activated by G-protein-coupled
receptors.2
Class II and III PI3Ks are further divided based on
differences in substrate preference, mechanism of activation and
structural homology.3
The PI3K signaling pathway is a complex pathway involved in
the regulation of proliferation, cell growth, survival, glucose
transport, vesicle trafficking, cell adhesion, cell motility and
cytoskeletal organization. Insulin and other growth factors
stimulate PI3K; the resulting lipid products act as second
messengers and bind to N-terminal pleckstrin homology domains
of proteins, such as 3-phosphoinositide-dependent protein kinase-1
(PDK-1) and protein kinase B (PKB), to allosterically modify their
activity or translocate them to the plasma membrane where they
can bind to receptors and activate signal transduction.4
The PI3K
pathway is one of the pathways most frequently activated in
human cancers, and two point mutations of PI3K p110a in
particular, E545K and H1047R, lead to constitutive activation of the
pathway.5
Previous studies have suggested that PI3K signaling
may have a vital role in maintaining stem cell pluripotency and
giving embryonic stem (ES) cells their tumor-like proliferative
properties. A large-scale transcriptional study implicated
components of the PI3K pathway in maintaining human ES cell
viability and pluripotency.6
Selective inhibition of class-IA PI3K
catalytic isoforms increased differentiation and reduced
proliferation.7
Phosphatase and tensin homolog deleted on
chromosome ten (PTEN) inhibits PI3K signaling and negatively
regulates ES cell proliferation and teratoma formation.8
PTEN-null
ES cells have enhanced tumorigenicity and PI3K signaling with
increased PKB activation and Cyclin D1 levels.9
It has been
suggested that the activation of PKB via myristoylation is sufficient
to maintain the undifferentiated phenotype of ES cells without the
addition of leukemia inhibitory factor (LIF).10
Glycogen synthase
kinase-3 (GSK-3) is negatively regulated by PI3K, and studies
performed by Doble et al.11
revealed that GSK-3 double knockout
(DKO) mouse ES cells remained undifferentiated, further
implicating the importance of the PI3K and Wnt pathways in
regulating stem cell pluripotency.
Several studies have also employed the use of PI3K inhibitors.
Paling et al.12
reported that the inhibition of PI3K with the
inhibitor LY294002 mitigated the ability of LIF to maintain mouse
embryonic stem (mES) cell self-renewal and caused cells to
differentiate. Transcriptome profiling revealed several markers of
pluripotency to be downregulated, such as Nanog, Esrrb, Tbx-3
and Tcl-1.13
Cell proliferation was also inhibited with the addition
of LY294002, leading to the accumulation of mES cells in G1 phase
1
Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada and 2
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Correspondence: Dr JR Woodgett, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Room 982, Toronto, Ontario, Canada M5G 1x5.
E-mail: woodgett@lunenfeld.ca
Received 10 April 2012; revised 4 December 2012; accepted 11 January 2013
Oncogene (2013), 1–12
& 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13
www.nature.com/onc
followed by apoptotic cell death.14
Of note, LY294002 is a
nonspecific PI3K inhibitor and also inhibits mammalian target of
rapamycin (mTOR), which is crucial for ES cell proliferation.15
Here,
we analyzed the effects of expressing activated forms of
components of the PI3K signaling pathway in ES cells on
pluripotency. In order to further dissect the molecular
mechanism of self-renewal under the control of PI3K signaling,
we generated constitutively active isogenic mouse ES cell lines of
PDK-1 and PKB, and characterized the pluripotentiality of the cell
lines. In accordance with previous literature, activation of the PI3K
signaling pathway appears to maintain stem cell self-renewal. We
analyzed roles for PKB-mediated effects on Wnt signaling in
mediating this pluripotent phenotype.
RESULTS
Role of PI3K pathway in maintenance of pluripotency of ES cells
To generate isogenic ES cell lines expressing activated alleles of
genes of interest, we employed a modified Flp-In system
(Figure 1a). The activated mutants of genes were inserted into
the GSK-3b locus that had been targeted by an FRT-puromycin
cassette. In the generation of the host control cell line, almost 700
individual colonies were picked and screened following puromycin
selection, and 4 positive colonies were identified. As expected,
the host control ES cells (GSK-3bþ / À
ES cells) exhibited a decrease
in GSK-3b protein expression compared with R1 ES cells
(Supplementary Figure 1a). PDK-1 was made constitutively active
by the addition of a Src myristoylation signal on the N-terminus,
which localizes the protein to the cell membrane.16
Three
independent, activated PDK-1 ES cell lines were generated with
a myc-his tag on the N-terminus and a V5 tag at the C-terminus
resulting in the increased protein size in the transgenic cell lines
(Figure 1b). All three transgenic cell lines showed increased
phosphorylation of PKB on threonine 308 and p70S6K on
threonine 389, demonstrating activation of the pathway
(Figure 1b). In addition, proper localization of myr-PDK-1 at the
cell membrane was visualized by confocal microscopy following
immunofluorescent staining for the V5 epitope (Figure 1c).
To assess the pluripotency of the myr-PDK-1 cells, we withdrew
LIF from the media and observed the morphological changes of
Figure 1. Modified Flp-In system. (a) A targeting vector is introduced to knock out a single allele of GSK-3b and create a host cell line with a
FRT-puro cassette. Co-transfection of Flp recombinase and a gene of interest previously cloned into a vector containing a V5 epitope and FRThygro
cassette results in homologous recombination at the FRT sites. Positive colonies are resistant to hygromycin and sensitive to puromycin.
(b) Western blot analysis of transgenic myr-PDK-1 cell lines. (c) Membrane localization of myr-PDK-1 by immunofluorescent staining of V5;
confocal, Â 40 magnification.
PDK-1 in PKB-dependent embryonic stem cell self-renewal
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the cells grown in culture. Interestingly, the myr-PDK-1 cells
tended to remain as distinct, round colonies in monolayer culture,
whereas the host control ES cells, similar to R1 wild-type ES cells,
showed evidence of morphological differentiation and spread
throughout the dish (Figure 2a). The myr-PDK-1 colonies maintained
positive staining for alkaline phosphatase (AP) activity even
in the absence of LIF for 5 days, a further indication that these cells
remained pluripotent (Figure 2a). However, all cells comprising
each colony did not always exhibit positive staining. For example,
a number of colonies consisted entirely of AP-positive cells,
whereas others consisted of both positive and negative cells
(Figure 2b).
Figure 2. Analysis of pluripotent markers on myr-PDK-1 cells. (a, b) Alkaline phosphatase activity. Cells were grown in monolayer and stained
for alkaline phosphatase activity on day 5; phase contrast, Â 10 magnification. (c) Gross morphology of embryoid bodies; phase contrast, Â 5
magnification. (d) Oct-4 immunofluorescent expression of myr-PDK-1 transgenic embryoid bodies at day 10; confocal, Â 40 magnification.
Ctrl, control.
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We proceeded to induce differentiation of the cells by
generating embyroid bodies using the hanging drop method.
After 3 days as hanging drops, the embryoid bodies (EBs) were
then each placed into the well of a 96-well tissue culture plate and
maintained for weeks. After a few days in the plate, contractile
beating was observed in both the myr-PDK-1 and control EBs.
However, the myr-PDK-1 cells retained a tightly compact
morphology even at 33 days, whereas the host control and R1
embyroid bodies grew in what appeared to be an uncontrolled
manner and differentiated (Figure 2c). Immunofluorescent staining
of the embyroid bodies for Oct-4, a marker of pluripotency,
revealed that the V5-epitope-tagged myr-PDK-1 cells were
positive for Oct-4 expression, whereas the control cells extinguished
Oct-4 expression (Figure 2d).
Previously, Watanabe et al.10
showed that myr-PKB expressing
ES cells remain undifferentiated. We therefore generated
transgenic ES cell lines expressing a constitutively activated PKB
(PKB-DD) in which the two activatory phosphorylation sites are
mutated to aspartic acid to partially mimic phosphorylation,17
and
detected a similar phenotype to the myr-PDK-1 cells in terms of
AP activity, overall EB morphology and immunofluorescent
staining for Oct-4 (Supplementary Figure 2). These data are
consistent with the notion that PDK-1 acts through PKB/Akt to
promote maintenance of pluripotentiality.
To gain insight into the mechanism underlying the maintenance
of de-differentiation of the myr-PDK-1 cell lines, we
employed a downstream block and used a PKB inhibitor that
specifically inhibits its activity by targeting the pleckstrin
homology domain.18
Cells grown for several weeks in the
presence of the PKB inhibitor (long-term treatment) showed a
marked decrease in PKB serine 473 phosphorylation and in PDK-1
expression (Figure 3a and Supplementary Figure 3). Interestingly,
addition of the PKB inhibitor eliminated AP activity observed in
untreated myr-PDK-1 cells (Figure 3b), and similarly ablated Oct-4
expression from the myr-PDK-1 and PKB-DD cells within 8 h of
inhibitor treatment (Figure 3c), suggesting that the inhibition of
PKB led to the loss of pluripotency. In initial experiments, longterm
treatment with the PKB inhibitor appeared to significantly
suppress the translation of transgenic and endogenous PDK-1,
complicating the interpretation of the data. However, experiments
Figure 3. Treatment of myr-PDK-1 ES cells with PKB inhibitor. (a) Western blot analysis of whole-cell lysates after growing cells long term
in inhibitor. (b) Cells grown in monolayer stained for alkaline phosphatase activity on day 5; phase contrast, Â 10 magnification.
(c) Immunofluorescent staining of cells grown in monolayer after 8 h treatment with PKBi; confocal, Â 40 magnification. Ctrl, control; GAPDH,
glyceraldehyde 3-phosphate dehydrogenase.
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conducted with a different, specific anti-PDK-1 antibody showed
no change in PDK-1 protein levels (Supplementary Figure 1b). In
an experiment where cells were grown in monolayer in the
presence of PKB inhibitor for 2–3 weeks before forming EBs and
then maintained in the presence of PKB inhibitor, control EBs
appeared smaller overall and myr-PDK-1 EBs consistently lost
transgene expression (as judged by loss of V5 epitope staining;
Supplementary Figure 3). Thus, we investigated the effects of
rapamycin treatment on myr-PDK-1 and PKB-DD cells. As the cells
were unable to survive long-term exposure to rapamycin, we
examined Oct-4 staining in the myr-PDK-1 cells in the presence of
rapamycin for 8 h. Rapamycin treatment did not affect Oct-4
expression levels as assessed by immunofluorescent staining
(Figure 4a). We further examined the effects of the rapamycin
treatment by western blot analysis, and saw that 8 h of treatment
resulted in decreased mTOR activity, evident by decreased
phosphorylation of p70S6K on threonine 371, and otherwise no
effect on p70S6K phospho-T389, endogenous PKB or transgenic
PDK-1 expression levels (Figure 4b). Our results indicate that
activation of PDK-1 maintains mouse ES cell self-renewal in a PKBdependent
manner in the absence of LIF.
It has previously been shown that PI3K and JAK/STAT pathways
act in parallel to maintain pluripotency.19
To assess roles for the
JAK/STAT and other pathways associated with maintaining
Figure 4. Treatment of myr-PDK-1ES cells with rapamycin (rapa). (a) Immunofluorescent staining of cells grown in monolayer after 8 h
stimulation with rapa; confocal, Â 40 magnification. (b) Western blot analysis of whole-cell lysates. Ctrl, control; GAPDH, glyceraldehyde
3-phosphate dehydrogenase.
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pluripotent phenotypes, we tested myr-PDK-1 and PKB-DD ES cells
for the activation of STAT-3 and ERK in the presence and absence
of LIF (Supplementary Figure 1c). No significant differences in
either expression or phosphorylation of STAT-3 and ERK were
observed, suggesting that the persistence of undifferentiated myrPDK-1
and PKB-DD cells in the absence of LIF is independent of
these pathways.
Does PI3K-dependent maintenance of pluripotency require
stabilization of b-catenin?
GSK-3 has a key role in promoting the phosphorylation and destabilization
of b-catenin in the Wnt pathway. Wnt leads to escape
of b-catenin from GSK-3 phosphorylation and allows the protein
to accumulate and induce transcription through interaction with
TCF/LEF DNA-binding proteins (reviewed in Huang and He;20
Wu
et al.;21
and MacDonald et al.22
). Several studies have suggested
that PI3K signaling may regulate b-catenin through inactivation of
GSK-3.23,24
We therefore investigated the phosphorylation state of
GSK-3 and expression levels of b-catenin in myr-PDK-1 and PKBDD
cells. Even though GSK-3a and -3b phosphorylation at Ser 21
and 9, respectively, were increased as expected in these
transgenic ES cell lines, we observed no apparent difference in
localization (Figure 5a) or in cytosolic protein accumulation of bcatenin
(Figure 5b). To further test for PI3K pathway regulation of
b-catenin, we used a GSK-3b mutant S9A cell line, which expresses
only a mutant of GSK-3b that is unresponsive to PI3K signaling
due to mutation of the PKB/Akt phosphorylation site, on a GSK-3
knockout background. These cells were transiently transfected
with PKB-DD-V5 and assessed for b-catenin accumulation. We
observed no difference in b-catenin localization (Figure 6a)
or cytosolic protein levels (Figure 6b) between GSK-3b S9A and
GSK-3b WT cells or upon expression of PKB-DD-V5. Axin-2
transcript levels were similarly unaffected (Figure 6c). Based on
these results, we conclude that in our myr-PDK-1 and PKB-DD ES
cells, activation of the PI3K pathway does not affect canonical Wnt
signaling. We further examined Oct-4 protein expression levels in
whole-cell lysates of GSK-3 S9A and GSK-3b-WT cells transiently
transfected with myr-PDK-1-V5. Again, GSK-3 does not appear to
affect the effects of myr-PDK-1 on Oct-4 expression levels
(Figure 6d). Taken together, we conclude that the maintenance
of pluripotency by PI3K signaling is independent of b-catenin in
our system.
Differentiation and proliferation capacity of transgenic ES cells in
teratomas
To further assess the differentiation capacity and proliferation
potential of myr-PDK-1 and PKB-DD ES cells, we performed
teratoma assays. Equal numbers of host control, myr-PDK1 or PKBDD
ES cells were injected subcutaneously into the hindlimbs of
SCID-beige mice and allowed to develop for 3 weeks. Mice
Figure 5. Analysis of b-catenin expression in myr-PDK-1 and PKB-DD cells. (a) Immuno-fluorescent staining of myr-PDK-1 cells with b-catenin.
(b) Western blot analysis of myr-PDK-1 and PKB-DD cytosolic cell lysates for b-catenin protein expression and whole-cell lysates for phosphoGSK-3
and pan-GSK-3 levels. Ctrl, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
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Figure 6. Examination of cross talk between PI3K and Wnt signaling via GSK-3. (a) Analysis of PKB-mediated regulation of b-catenin expression
via GSK-3 by immunofluorescent staining following transient transfection with PKB-DD-V5. (b) Western blot analysis of cytosolic cell lysates
after transient transfection with PKB-DD-V5. (c) Semi-quantitative reverse transcription–PCR for axin-2 (1 Â and 10 Â template dilutions) after
transient transfection with PKB-DD. (d) Western blot analysis of whole-cell lysates of GSK-3b S9A and GSK-3b WT cells transiently transfected
with myr-PDK-1-V5. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
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Figure 7. Immunohistology of 3-week-old teratomas. (a) Teratomas are stained with hematoxylin and eosin stain. Teratomas appear purple
and the surrounding muscle fibers pink. Host control (Ctrl) teratomas are small and remain encapsulated, whereas myr-PDK1 teratomas grow
into the surrounding muscle tissue. Whole slide scanning, scale bar ¼ 500 mm. (b) Teratomas were stained for V5 (brownish red) to illustrate
areas of transgene expression. myr-PDK1 and PKB-DD expression is patchy throughout the teratoma. Scale bar ¼ 100 mm. (c) An equal number
of cells were plated and allowed to grow in standard tissue culture conditions. Cells were counted on days 3 and 5. (d) Immunohistological
staining of Ki67 and cleaved Caspase-3 (brown) in teratomas. Scale bar ¼ 100 mm.
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bearing myr-PDK1 and PKB-DD teratomas exhibited very large
masses, in some cases encompassing almost the entire hindlimb
and impeding mobility. In contrast, mice injected with host control
cells appeared unaffected and moved around normally. Upon
dissection, a substantial size difference between control and
transgenic teratomas was noticeable (Figure 7a). In addition,
transgenic teratomas appeared to grow into surrounding muscle
tissue, whereas host control teratomas remained encapsulated
(Figure 7a). Teratomas were stained for V5 to detect transgene
expression. V5 staining was patchy in both myr-PDK1 and PKB-DD
teratomas as the transgene did not appear to be expressed in
every cell (Figure 7b). Unlike in cell culture where ES cells were
maintained in the presence of hygromycin, the loss of transgene
expression in the animal was likely due to teratoma growth in the
absence of selection.
In tissue culture, myr-PDK1 cells required more frequent
passaging than host control and R1 cell lines; PKB-DD cells
required even more frequent passaging. When the same number
of cells were initially plated and allowed to grow over a period of 5
days, myr-PDK1 cells grew at a faster rate than controls and PKBDD
cells grew most rapidy (Figure 7c). Furthermore, staining for
proliferation and apoptosis markers, Ki67 and cleaved Caspase-3,
respectively, revealed an increase in Ki67 and a reduction in
Caspase-3 intensity in myr-PDK-1 and PKB-DD teratomas compared
with host cell controls (Figure 7d). Collectively, these results
support roles for the PI3K signaling in enhancing the proliferation
and survival of ES cells.
DISCUSSION
In this study, we generated stable isogenic myr-PDK-1 ES cell lines
to examine the role that constitutively active mutants of the PI3K
pathway (PDK-1 and PKB/Akt) have in ES cell self-renewal. Several
groups have previously demonstrated roles for components of the
PI3K signaling cascade in maintaining pluripotency, mostly based
on loss-of-function studies.6,10,12,25
Inhibition of PI3K signaling has
additionally been shown to reduce the self-renewal capacity of
induced pluripotent stem cells.26
Consistent with stabilization of
pluripotency, we demonstrate here that activated myr-PDK-1 ES
cells retain AP activity in the absence of LIF, and EBs appear
morphologically undifferentiated and display sustained Oct-4
staining compared with controls.
Although PDK-1 targets multiple downstream substrates,
including PKB, S6K, RSK, SGK and atypical PKC isoforms,27
the
effect of myr-PDK-1 on pluripotency appears to occur via its
activation of PKB, as treatment of myr-PDK-1 ES cells and EBs with
PKB inhibitors reversed the LIF-independent pluripotent phenotype.
Similar to studies demonstrating a role for an activated
myristoylated form of PKB in promoting LIF-independent self-
renewal,10,19
we also showed that activated PKB-DD ES cells and
EBs exhibit LIF-independent pluripotency. To probe the molecular
mechanism(s) by which myr-PDK-1 and PKB-DD may mediate LIFindependent
pluripotency, roles for downstream targets of these
protein kinases were examined, including mTOR complex-1
(mTORC-1) and GSK-3. Both myr-PDK-1 and PKB-DD ES cells
were treated with rapamycin, an inhibitor of mTOR. PDK-1 and
PKB activate mTORC-1 signaling to promote protein synthesis and
cell growth.28
Upon treatment with an inhibitor of mTOR, no
change was observed in Oct-4 staining or pluripotency, indicating
that another pathway downstream of PDK-1 and PKB is
responsible for the maintenance of Oct-4 expression and
pluripotency in the absence of LIF.
It is well established that PKB/Akt phosphorylates and inhibits
GSK-3.29
Furthermore, inhibition of GSK-3 has been linked to the
maintenance of an undifferentiated pluripotent stem cell
state.30,31
Through its association with the destruction complex,
GSK-3 targets b-catenin for degradation. The ability for PKB
to stabilize b-catenin in the cytoplasm through its inhibition of
GSK-3 has been tested by a number of groups.24,32–35
Here, we
investigated whether this mechanism has a role in the
maintenance of a PKB-mediated LIF-independent pluripotent
stem cell state. Consistent with several other studies,36–38
neither myr-PDK-1 nor PKB-DD ES cells enhanced b-catenin
levels, either in the presence of a wild-type or mutant version of
GSK-3 that is unresponsive to PKB. Furthermore, no changes were
observed in Oct-4 levels, suggesting that the maintenance of
PKB-mediated LIF-independent pluripotency is also GSK-3
independent. This result supports the hypothesis that PI3K and
Wnt signaling pathways are insulated and do not cross talk—at
least in ES cells (Figure 8).
In mediating ES cell pluripotency, PI3K signaling has previously
been implicated downstream of both cytokines and growth
factors.12,19
For example, both LIF and insulin-like growth factor
have been shown to activate PKB in ES cells.13,19
More specifically,
LIF-mediated PKB activation positively regulates Tbx-3 and
subsequently, Nanog and Oct-4 expression.13,19
It has been
suggested that Jak-STAT and PI3K-PKB signal in parallel pathways
downstream of LIF to maintain pluripotency.19
Recently, Oct-4
has been shown to be phosphorylated by PKB in embryonal
carcinoma cells, an event that was associated with increased survival
and maintenance of expression of nanog.39
The ability to generate
stable isogenic ES cell lines targeting additional components within
the PI3K and other pathways should allow further dissection of the
functional hierarchy of signaling proteins that control ES cell
pluripotency and provide further insight into appropriate targets
for drug-mediated modulation of ES self-renewal.
MATERIALS AND METHODS
Cell culture
ES cells were maintained on a mouse embryonic fibroblast (MEF) feeder
layer in ES cell culture medium (ES-Dulbecco’s modified Eagle’s medium)
consisting of high glucose Dulbecco’s modified Eagle’s medium (Invitrogen,
Burlington, ON, Canada) supplemented with 15% ES cell-qualified
fetal bovine serum (Hyclone, Thermo Fisher Scientific, Ottawa, ON,
Canada), 50 U penicillin/streptomycin (Invitrogen), 2 mM GlutaMAX (Invitrogen),
0.1 mM MEM non-essential amino acids (Invitrogen), 1 mM sodium
pyruvate (Invitrogen), 0.1 mM 2-mercaptoethanol (Sigma, Oakville, ON,
Canada) and 1000 U/ml LIF (Millipore, Billerica, MA, USA) at 37 1C, 5% CO2.
Before experiments, feeders were removed and ES cells were grown on
plates coated with 0.1% gelatin (Sigma). For treatments, ES cells were
grown in 1 mM PKBi (Calbiochem, Billerica, MA, USA) or 100 ng/ml
rapamycin (Calbiochem).
Figure 8. Separate PI3K and Wnt signaling pathways. GSK-3 is a key
regulator in both the PI3K and Wnt signal transduction pathways. In
the context of the myr-PDK-1 and PKB-DD transgenic ES cell lines,
the PI3K signaling pathway does not cross talk with the canonical
Wnt signaling pathway through GSK-3. APC, adenomatous polyposis
coli; LRP, lipoprotein-related protein; GF, growth factor.
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Generation of GSK-3bþ / À
mouse ES cells as host cell line
R1 ES cells, first established by Nagy et al.,40
were provided by the stem
cell core facility and used to generate the GSK-3bþ / À
host cell line. The
targeting vector, pTVB_deltaX2_NR_FRT/Pur_AgeIBclI (a gift of B. Doble)
was linearized with the NdeI restriction enzyme (NEB, Whitby, ON, Canada),
purified using QiaxII beads (Qiagen, Mississauga, ON, Canada), and
electroporated into approximately 1 Â 106
R1 ES cells using a BIO-RAD
Gene Pulser (Bio-Rad, Mississauga, ON, Canada) with a single pulse of 250 V
at 500 mF. After 48 h, cells were treated with 1.2 mg/ml puromycin
(Invitrogen). Colonies were picked and split into two replica flat-bottom
96-well plates. One was used for screening by PCR and the other was
allowed to propagate. Approximately 500 colonies were picked and
screened for homologous recombination by pooled-PCR using primers
that flank the short arm of homology. The forward primer (50
-CACAAT
GCGACTGACCCACTTCCCTTTC-30
) is found in pTVB_deltaX2_NR_FRT/
Pur_AgeIBclI upstream of the short arm and the reverse primer
(50
-CTTAACTACCGGTGGATGTGGAATGTG-30
) is in the endogenous GSK-3b
locus. Four colonies were found to be positive for homologous
recombination by pooled-PCR. These were subsequently confirmed by
single PCRs for the short arm, the puromycin gene and the FRT-puro
cassette, which replaces exon 2. The primer sequences used for puromycin
were forward: 50
-ATCGAGCGGGTCACCGAGCT-30
and reverse: 50
-TTGC
GGGTCATGCACCAGGT-30
and those used to flank GSK-3b exon 2 were
forward: 50
-GGGGCAACCTTAATTTCATT-30
and reverse: 50
-TCTGGGCTA
TAGCTATCTAGTAACG-30
.
Cloning of Flp-In constructs
In order to clone the genes of interest into the Flp-In vector, pEF5/FRT/V5D-TOPO
(Invitrogen), the forward cloning primer consisted of the TOPO
recognition sequence (CACC) followed by a full Kozak sequence and the
first 20 to 29 nucleotides of the gene of interest. The design of the reverse
primer involved removing the stop codon of the gene so that a fusion
could be formed in frame with the V5 epitope.
Generation of stable Flp-In ES cell lines
Lipofectamine 2000 (Invitrogen) was used to co-transfect 0.2 mg of
pEF_myr-myc-his-PDK-1_FRT_V5 or pEF_HA-PKB-DD_FRT_V5 and 1.8 mg
of the pOG44 construct (Invitrogen), which expresses Flp recombinase, into
GSK-3b host cells. Cells were placed in a six-well plate (Corning, Tewksbury,
MA, USA) and selected with 250 mg/ml hygromycin (InvivoGen, San Diego,
CA, USA) after 48 h. Colonies were picked and expanded from 96-well
plates to replica 24-well plates, where 1.2 mg/ml puromycin selection
medium was used in place of hygromycin for one of the replica plates.
Proper integration of the transgene into the knockout GSK-3b locus will
result in colonies that are hygromycin resistant and puromycin sensitive.
Colonies were further expanded into 6 cm dishes and lysed with RIPA
buffer for western blot analysis of V5 expression.
GSK-3 knockout and knock-in ES cell lines
GSK-3 DKO, GSK-3b S9A and GSK-3b WT ES cell lines were previously
generated and described by Doble et al.11
Briefly, GSK-3 DKO ES cells lack
both GSK-3a and -3b alleles. GSK-3b S9A and GSK-3b WT ES cells were
generated by knocking the S9A or WT form of GSK-3b back into GSK-3 DKO
ES cells.
Cell counting
A total of 200 000 cells were plated in triplicate for each time point and
grown under standard ES cell tissue culture conditions. On days 3 and 5,
adherent and floating cells from each plate were collected and counted
using a Z2 Coulter Particle Count and Size Analyzer (Beckman Coulter,
Mississauga, ON, Canada).
Cytosolic lysate preparation and western blot analysis
Cells were rinsed twice with phosphate-buffered saline (PBS) and scraped
into 500 ml of cold hypotonic lysis buffer: 50 mM Tris pH 7.4, 1 mM EDTA,
complete protease inhibitor tablet (Roche, Mississauga, ON, Canada),
phosphatase inhibitor cocktail (Sigma), 1 mM sodium orthovanadate, 10 mM
NaF and 10 mM b-glycerophosphate. Cells were incubated on ice for
15–20 min, and then pelleted by centrifuging in an ultra-centrifuge at
80 000 r.p.m. for 30 min at 4 1C. The supernatant containing the cytosolic
fraction was transferred into a new Eppendorf tube, and protein levels
were quantified using the BCA Protein Assay (Pierce, Thermo Fisher
Scientific, Ottawa, ON, Canada).
Protein lysates containing sample buffer and TECEP (Pierce) were boiled
for 10 min and loaded onto an 8% SDS–PAGE gel, then transferred onto
polyvinylidene difluoride membrane (Millipore) after electrophoresis via
semi-dry transfer. After primary antibody incubation, the membrane was
washed with TBST and incubated with either goat-anti-mouse or goat-antirabbit
(Bio-Rad) secondary antibodies. The membrane was washed again
and then exposed to film (Kodak) using ECL reagent (Molecular Probes,
Burlington, ON, Canada).
AP activity
A single-cell suspension of 1000cells/cm2
were seeded onto a 0.1% gelatincoated
well of a six-well plate (Corning) and grown in monolayer in
differentiation medium. On day 5, cells were washed in PBS, fixed in 2%
paraformaldehyde/PBS for 30min at room temperature, washed again in
PBS, and stained in the dark using the Vector Blue Phosphatase Substrate Kit
III (Vector Laboratories, Burlington, ON, Canada, SK-5300) according to
manufacturer’s instructions.
EB assay
Differentiation medium used to maintain EBs were the same as ESDulbecco’s
modified Eagle’s medium but with 5% fetal bovine serum and
no LIF supplementation. EBs were generated using the hanging drop
method. Thirty-microliter drops containing 800 cells/drop were plated onto
the lid of a 10-cm2
tissue culture dish (Corning) containing 5 ml of PBS to
prevent the drops from drying out. EBs were maintained as hanging drops
in a tissue culture incubator at 37 1C and 5% CO2 for 3 days and then
transferred to ultra-low-binding 96-well plates (Costar, Tewksbury, MA,
USA); differentiation medium was changed every other day.
Immunofluorescent staining and microscopy
Whole EBs were washed in PBS, fixed with ice-cold 4% paraformaldehyde/
PBS overnight at 4 1C, permeabilized twice with 0.5% Triton X-100/PBS
(PBT) for 15 min at room temperature, blocked in 2% bovine serum
albumin/PBT for 1 h, and incubated with primary antibody overnight at
4 1C. EBs were then washed three times in PBS before adding secondary
antibody and TOPRO-3 iodide (Invitrogen) and incubating overnight at
4 1C. Primary and secondary antibodies were diluted to final working
concentrations in 2% bovine serum albumin/PBT. EBs were mounted with
ProLong gold anti-fade reagent (Invitrogen) after being gently washed five
times in PBS. Images were obtained with a Zeiss LSM510 confocal
microscope and associated software (Toronto, ON, Canada).
Teratoma assay
Cells were grown for two passages on 0.1% gelatin-coated dishes to
eliminate feeder fibroblasts, trypsinized into single-cell suspensions, and
resuspended in PBS to a density of 107
cells/ml. Using a 25-G needle, these
cells (200 ml) were injected subcutaneously into the hindlimbs of SCIDbeige
mice (C.B-Igh-1b
GbmsTac-Prkdcscid
-Lystbg
N7; Jackson Laboratory,
Bar Harbor, ME, USA). Teratomas were collected after 3 weeks. The tissues
were fixed with 10% neutral-buffered formalin overnight at room
temperature then stored in 70% ethanol for subsequent paraffin
embedding, sectioning and immunohistological staining.
Hematoxylin and eosin stain and immunohistological staining and
microscopy
Sections from paraffin-embedded teratomas were dewaxed and rehydrated
using standard procedures. For hematoxylin and eosin stain
staining, sections were treated with iodine for 5 min, washed, treated with
3% sodium thiosulphate for 1 min, and washed under running tap water.
Subsequently, slides were stained in Harris’ Hematoxylin for 5 min, washed
and rinsed in 95% ethanol before staining with eosin for 1 min. Slides were
then thoroughly washed in running tap water, dehydrated in a series of
alcohol rinses, and cleared in three changes of xylene. For immunohistological
staining, antigen retrieval was performed using 10 mM sodium
citrate buffer for 20 min at 98 1C. Antigen detection and 3,30
-diaminobenzidine
(DAB) color development were performed using kits according to
the manufacturers’ recommendations (Vector Laboratories and DAKO
(Burlington, ON, Canada) respectively). Images were obtained with an
Olympus microscope.
PDK-1 in PKB-dependent embryonic stem cell self-renewal
LS Ling et al
10
Oncogene (2013), 1 – 12 & 2013 Macmillan Publishers Limited
Antibodies
The following primary antibodies were used for western blot analysis,
immunofluorescent and/or immunohistological staining: mouse anti-V5
(Invitrogen, R960-25), mouse b-actin (Abcam, ab6276), mouse anti-GAPDH
(Abcam, ab8245), mouse anti-b-catenin (BD Biosciences, 610154), rabbit
anti-b-catenin (Cell Signaling Technology, Whitby, ON, Canada, (CST) 9562),
rabbit anti-Oct-4 (Santa Cruz, sc-9081), rabbit anti-PDK-1 (CST 3062, 5662),
rabbit anti-PKB (CST 9272), rabbit anti-PKB-S473 (CST 9271, 4060), rabbit
anti-PKB-T308 (CST 9275), rabbit anti-p70S6K-T389 (CST 9206), rabbit antip70S6K-T371
(CST 9208), rabbit anti-p44/42MAPK (CST 4695), rabbit
anti-p44/42MAPK-T202/Y204 (CST 9101), mouse anti-STAT-3 (CST 9139),
rabbit anti-STAT-3-Y705 (CST 9145), mouse anti-GSK-3a/b (Millipore 4G1E),
rabbit anti-Ki67 (Thermo Scientific, SP6), and rabbit anti-cleaved Caspase-3
(Cell Signaling Technology, 9661).
For western blot analysis, horseradish peroxidase-conjugated secondary
antibodies were purchased from Bio-Rad: goat anti-mouse (170–6516) and
goat anti-rabbit (170–6515). For immunofluorescent staining, fluorochrome-conjugated
secondary antibodies were purchased from Invitrogen:
goat anti-mouse Alexa Fluor 488 (A11029) and goat anti-rabbit Alexa Fluor
468 (A11011).
Reverse transcription–PCR
RNA was extracted from cells using TRIzol Reagent (Invitrogen)
followed by cDNA synthesis using SuperScript II Reverse Transcriptase
(Invitrogen) according to manufacturer’s instructions. The template for
each PCR reaction was the cDNA obtained from 50 ng total RNA, and the
reactions were incubated in a BIO-RAD DNA Engine Dyad Peltier Thermal
Cycler at 94 1C for 2 min, 30 cycles of 94 1C for 30 s, 56 1C for 30 s and 68 1C
for 1 min, followed by a final extension at 68 1C for 7 min. Primer sequences
were forward: 50
-AAGCCTGGCTCCAGAAGATCACAA-30
and reverse:
50
-TTTGAGCCTTCA GCATCCTCCTGT-30
for Axin211
and forward: 50
-TAGG
CACCAGGGTGTGATGG-30
and reverse: 50
-CATGGCTGGGGTGTTGAAGG-30
for b-actin.13
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
We thank Brad Doble for generously sharing reagents and protocols, Malgosia
Kownacka for providing feeder cells, Mike Parsons and Elizabeth Rubie for technical
expertise and advice, and Eric Ho and Satish Patel for troubleshooting and helpful
discussions. LSL was supported by a NSERC Canada Graduate Scholarship and DV was
supported by a Heart and Stroke Foundation of Canada Fellowship. These studies
were funded by a CIHR operating grant (FRN 12858).
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