Coordinated interactions between signaling networks govern the balance of cell fate decisions in human embryonic stem cells. In this issue, Singh et al. (2012) report that PI3K/Akt signaling switches Activin/Smad activity between pro-self-renewal and prodifferentiation by regulating ERK and GSK3β/β-catenin signaling

Chen, Ye-Guang

Li, Zhongwei

Wang, Xiao-Fan

PubMed

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Where PI3K/Akt Meets Smads: The Crosstalk Determines Human Embryonic Stem Cell Fate 

Cell Stem Cell
PreviewsWhere PI3K/Akt Meets Smads: The Crosstalk
Determines Human Embryonic Stem Cell FateYe-Guang Chen,1,* Zhongwei Li,1 and Xiao-Fan Wang2,*
1State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084, China
2Department of Pharmacology and Cancer Biology, Duke University Medical Center, P.O. Box 3813, Durham, NC 27710, USA
*Correspondence: ygchen@tsinghua.edu.cn (Y.-G.C.), wang0011@mc.duke.edu (X.-F.W.)
DOI 10.1016/j.stem.2012.02.008
Coordinated interactions between signaling networks govern the balance of cell fate decisions in human
embryonic stem cells. In this issue, Singh et al. (2012) report that PI3K/Akt signaling switches Activin/
Smad activity between pro-self-renewal and prodifferentiation by regulating ERK and GSK3b/b-catenin
signaling.Human embryonic stem cells (hESCs)
hold great promise in regenerative medi-
cine. Understanding the detailed sig-
naling network that dictates hESC self-
renewal and directed differentiation will
undoubtedly contribute to future applica-
tions. Several extrinsic signaling path-
ways have been implicated in maintaining
hESC pluripotency, including Activin/
Nodal, fibroblast growth factor (FGF),
insulin-like growth factor (IGF), and Wnt
signaling (Pera and Tam, 2010). How-
ever, these signaling pathways are often
studied in isolation and in undefined
medium conditions. Thus, how these sig-
naling pathways, as a network, coordi-
nate to influence hESC fate in a defined
context is unclear. In the current issue
of Cell Stem Cell, Singh et al. (2012) em-
ployed a chemically defined medium to
examine the combinatorial input of sig-
naling pathways that govern hESC fate
determination.
In this definedmedium, only three types
of recombinant growth factors were in-
cluded to maintain hESC self-renewal:
Heregulin (H), Activin A (A), and IGF-1 (I).
Heregulin and IGF-1 are known potent
activators of PI3K/Akt signaling, whereas
Activin A activates Smad2/3, which func-
tion to regulate transcription in the nu-
cleus. The authors found that omission
of Heregulin and IGF-1 from the medium
(HI medium) or blockage of PI3K/Akt
activity induced the expression of mes-
endoderm genes such as Brachyury,
Eomes, Goosecoid, and MixL1, whereas
self-renewal could be maintained by ex-
pressing constitutively active Akt (caAkt)
in hESCs cultured in HI medium. Inter-
estingly, simultaneous blockage of Acti-
vin/Smad signaling also abolished induc-tion of these mesendoderm genes in
the HI condition. As Activin/Smad2/3
signaling is required for expression of
the pluripotency gene Nanog, these re-
sults suggest that robust PI3K/Akt activity
collaborates with Activin/Smad signal-
ing to maintain hESC self-renewal, while
weak PI3K/Akt activity switches the
function of Activin/Smad to promote
differentiation.
The authors further investigated the
possible downstream effectors of PI3K/
Akt. They observed that inhibition of
PI3K/Akt signaling enhanced phosphory-
lation of ERK at T202 and Y204 and
GSK3b at S9, indicating ERK activation
and GSK3b inactivation, respectively.
Likewise, addition of Heregulin and IGF-1
or ectopic expression of caAkt in the HI
medium blocked ERK and GSK3b phos-
phorylation. Thus, PI3K/Akt suppresses
ERK activity but activates GSK3b. In
agreement, inhibition of ERK activity
attenuated mesendoderm marker gene
expression and retained Nanog expres-
sion in the HI medium, suggesting a
negative role for ERK in maintenance of
hESC self-renewal. Consistent with a
previous report (Ding et al., 2005), the
authors showed that Raf/MEK/ERK sig-
naling negatively controls GSK3b activity.
As Akt can directly bind to and negatively
regulate Raf activity via phosphorylation
(Rommel et al., 1999), these data reveal
that PI3K/Akt promotes hESC self-
renewal by interfering with Raf/MEK/ERK
signaling, leading to enhanced GSK3b
activity.
GSK3b is a critical component of the
canonical Wnt pathway, and the inactiva-
tion of GSK3b leads to accumulation of
b-catenin in the nucleus and drives theCell Stem Celltranscriptional regulation of target genes.
Thus, the observed inactivation of GSK3b
by PI3K/Akt inhibition and ERK activation
may lead to activation of b-catenin. In-
deed, PI3K/Akt inhibition enhanced the
transcriptional activity of b-catenin, and
this effect could be blocked by inhibition
of ERK activity. These results suggest
that PI3K/Akt and ERK regulate hESC
fate determination by influencing the
activities of the Wnt signaling medi-
ators GSK3b and b-catenin. Activation of
b-catenin was previously reported to sus-
tain self-renewal in hESCs (Sato et al.,
2004). However, Singh et al. (2012) found
that blockage of GSK3b by the pharma-
cological inhibitor BIO induced mesen-
doderm gene expression under the +HAI
condition. This result could be explained
by the dose effect of BIO: low BIO dose
stabilizes Myc, which contributes to self-
renewal, whereas high BIO dose pro-
motes b-catenin-mediated expression of
differentiation genes.
Finally, the authors tested whether
b-catenin affects the outcome of Activin/
Smad2/3 signaling. They found that Acti-
vin/Smad2/3 signaling is required for the
BIO-inducedmesendoderm gene expres-
sion, and ectopic expression of an active
form of Smad3 can enhance MixL1 pro-
moter reporter activity, indicating that
Smad2/3 may induce mesendoderm
gene expression in collaboration with
b-catenin. Based on their findings, the
authors proposed a model for mainte-
nance of hESC self-renewal (Figure 1). In
this model, Heregulin and IGF-1 synergis-
tically activate PI3K/Akt signaling and
thus suppress Raf/MEK/ERK activity,
leading to high GSK3b activity and
low b-catenin activity; the low b-catenin10, March 2, 2012 ª2012 Elsevier Inc. 231
Figure 1. Heregulin and IGF-1 via PI3K/Akt and Activin
via Smad2/3 Cooperate to Maintain Self-Renewal of
hESCs
Red indicates the activated state of signaling molecules,
whereas blue indicates the repressive state. The dashed line
indicates the regulation with unclear mechanisms.
Cell Stem Cell
Previewsactivity does not allow the moderate
level of Activin/Smad2/3 activity to
initiate mesendoderm gene expres-
sion and thereby maintains hESCs
in their self-renewal state.
This study has convincingly linked
the PI3K/Akt pathway to GSK3b/
b-catenin signaling and revealed
the collaboration between b-catenin
and Smad activities in controlling
hESC fate determination, but it also
raised some interesting questions.
Functions of ERK were previously
regarded to be quite different in
mouse and human ESCs. A number
of laboratories have demonstrated
a prodifferentiation role for ERK in
mESCs (Li et al., 2012; Ying et al.,
2008). However, in hESCs, ERK
was thought to act as a pro-self-
renewal signal (Armstrong et al.,
2006; Li et al., 2007). One possible
explanation for this discrepancy
could be that different culture con-
ditions dictate different signal re-
quirements to maintain hESC self-
renewal. In the defined medium of
the present study, the two robust
PI3K/Akt stimulators Heregulin and
IGF-1 keep the ERK activity lowand maintain the capacity for self-renewal
in cooperation with Activin/Smad2/3.
However, in other culture media contain-
ing multiple undefined growth factors,
high ERK activity may promote self-
renewal in collaboration with signals other
than Activin. This idea is supported by
a recent study showing that inhibition of
ERK activity renders hESCs more sensi-
tive to BMP-induced differentiation (Na
et al., 2010). Although the present study
showed that PI3K/Akt switches Activin/
Smad2/3 activity from prodifferentiation232 Cell Stem Cell 10, March 2, 2012 ª2012to pro-self-renewal mainly via inhibition
of b-catenin activity, the contribution of
direct regulation of Smad2/3 activity by
PI3K/Akt remains to be determined. It is
also unclear how PI3K/Akt inhibition leads
to enhanced Smad2/3 activity. Whether
loss of function of b-catenin substitutes
the requirement of PI3K/Akt activation in
maintenance of hESC self-renewal also
awaits investigation. As Smad2/3 can
interact with the b-catenin/TCF complex
to drive gene transcription, it would be
interesting to investigate how Smad2/3Elsevier Inc.collaborates with b-catenin to stim-
ulate expression of differentiation
genes, but not pluripotency genes,
in hESCs. Moreover, systematic in-
vestigation of the expression pat-
tern of downstream target genes in
response to different combinatorial
signal inputs will also shed light
on the underlying mechanisms of
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Where PI3K/Akt Meets Smads: The Crosstalk Determines Human Embryonic Stem Cell Fate

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