Cell Reports

Article
Crosstalk between the Rb Pathway and AKT
Signaling Forms a Quiescence-Senescence Switch
Yoshinori Imai,1,2 Akiko Takahashi,1 Aki Hanyu,1 Satoshi Hori,1 Seidai Sato,1 Kazuhito Naka,3 Atsushi Hirao,3
Naoko Ohtani,1,4 and Eiji Hara1,5,*
1Division

of Cancer Biology, The Cancer Institute, Japanese Foundation for Cancer Research, Koto-ku, Tokyo 135-8550, Japan
School of Biomedical Science, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan
3Cancer Research Institute, Kanazawa University, Kanazawa 920-1192, Japan
4PRESTO, Japan Science Technology Agency, Saitama 332-0012, Japan
5CREST, Japan Science Technology Agency, Saitama 332-0012, Japan
*Correspondence: eiji.hara@jfcr.or.jp
http://dx.doi.org/10.1016/j.celrep.2014.03.006
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
2Graduate

SUMMARY

Cell-cycle arrest in quiescence and senescence

is largely orchestrated by the retinoblastoma (Rb)
tumor-suppressor pathway, but the mechanisms
underlying the quiescence-senescence switch
remain unclear. Here, we show that the crosstalk
between the Rb-AKT-signaling pathways forms this
switch by controlling the overlapping functions of
FoxO3a and FoxM1 transcription factors in cultured
fibroblasts. In the absence of mitogenic signals,
although FoxM1 expression is repressed by the Rb
pathway, FoxO3a prevents reactive oxygen species
(ROS) production by maintaining SOD2 expression,
leading to quiescence. However, if the Rb pathway
is activated in the presence of mitogenic signals,
FoxO3a is also inactivated by AKT, thus reducing
SOD2 expression and consequently allowing ROS
production. This situation elicits senescence through
irreparable DNA damage. We demonstrate that this
pathway operates in mouse liver, indicating that
this machinery may contribute more broadly to tissue
homeostasis in vivo.

INTRODUCTION
Over the last few decades, it has become apparent that oncogenic proliferative signals are coupled to a variety of growthinhibitory responses, such as the induction of apoptotic cell
death or irreversible cell-cycle arrest known as cellular senescence (Sharpless and DePinho, 2005). Thus, both apoptosis
and cellular senescence are thought to act as important tumorsuppression mechanisms (Campisi and dAdda di Fagagna,
2007; Collado and Serrano, 2010; Kuilman et al., 2010). However, unlike apoptotic cells, senescent cells remain viable for
long periods of time and are hypothesized to persist and accumulate with age in vivo (Krishnamurthy et al., 2004; Jeyapalan
et al., 2007; Yamakoshi et al., 2009). Thus, the irreversibility of
194 Cell Reports 7, 194207, April 10, 2014 2014 The Authors

cell-cycle arrest is crucial for the tumor-suppression properties

of cellular senescence (Sharpless and DePinho, 2005).
Numerous biochemical and genetic studies have revealed that
although several different signaling pathways can induce cellular
senescence, these pathways eventually merge toward the activation of the retinoblastoma tumor suppressor gene product
(Rb) and its family members (p107 and p130) (Chandler and
Peters, 2013). For example, activation or overexpression of
Rb-family proteins is sufficient to induce senescence-like irreversible cell-cycle arrest (Gil and Peters, 2006), and ablation of
all three Rb-family genes renders mouse embryonic fibroblasts
(MEFs) completely insensitive to senescence-inducing signals
(Dannenberg et al., 2000; Sage et al., 2000). Importantly, once
the Rb-family proteins are fully activated, particularly by the
p16INK4a cyclin-dependent kinase (CDK) inhibitor, the cell-cycle
arrest becomes irreversible and is no longer revoked by subsequent inactivation of the Rb pathway (Dai and Enders, 2000;
Beausejour et al., 2003; Takahashi et al., 2006). These findings
placed the Rb pathway at the heart of the cellular machinery
that establishes the irreversibility of senescent cell-cycle arrest.
In addition to cellular senescence, Rb-family proteins also
induce cellular quiescence (van den Heuvel and Dyson, 2008),
the state of reversible cell-cycle arrest required for tissue
homeostasis in multicellular organisms (Cheung and Rando,
2013). For instance, the Rb pathway is activated in quiescent
cells, and MEFs lacking all three Rb-family genes are insensitive
to quiescence-inducing signals following contact inhibition or
serum starvation (Dannenberg et al., 2000; Sage et al., 2000).
These results raised the question of how the Rb pathway determines whether a cell undergoes quiescence or senescence. We
previously reported that the activation of the Rb pathway provokes quiescence or senescence, depending on the absence
or presence of mitogenic signals, respectively, in a conditionally
immortalized human fibroblast (Takahashi et al., 2006). However,
to date, it remains largely unclear how mitogenic signals integrate with the Rb pathway to establish senescent cell-cycle
arrest, especially in nonimmortalized normal human cells.
In the present study, we set out to fill this gap by identifying the
downstream mediators of the mitogenic signaling that collaborate with the Rb pathway in normal human cells. Here, we
show that the crosstalk between the Rb pathway and AKT

Figure 1. Cooperation between Mitogenic Signaling and the Rb Pathway Is Required to Establish Senescent Cell-Cycle Arrest
(A) ER-p16 cells were cultured under normal conditions (1, 2, and 3) or under contact-inhibition (4, 5, and 6) or serum-starvation (7, 8, and 9) conditions for 7 days.
These cells were then cultured in the presence (2, 3, 5, 6, 8, and 9) or absence (1, 4, and 7) of 4-OHT for 7 more days. In lanes 3, 6, and 9, the cells were then
subsequently cultured under normal conditions without 4-OHT for 7 days further. The experimental conditions and representative micrographs of SA b-gal
staining and immunofluorescence staining for Flag-tagged p16 (red) are shown. DNA was stained with DAPI (blue).
(B) Total cell lysates were prepared at indicated culture conditions in (A) (lane number corresponds to culture condition in A) and were subjected to western blot
analysis using the antibodies shown left (P, phosphospecific antibody). The percentages of nuclear Flag-tagged p16 staining and SA b-gal staining in (A) are also
shown at the bottom. The representative data from three independent experiments are shown. Error bars indicate mean SD of triplicate measurements. Vinculin
was used as a loading control.
(C) ER-p16 cells cultured under the indicated conditions in (A) (3, 6, and 9) were subjected to cell proliferation analyses performed in triplicate. Error bars indicate
mean SD.

signaling plays important roles in forming a quiescencesenescence switch by regulating the functions of FoxO3a and
FoxM1 Forkhead transcription factors (Eijkelenboom and Burgering, 2013; Lam et al., 2013).
RESULTS
Mitogenic Signals Cooperate with the Rb Pathway to
Induce Cellular Senescence
To explore the molecular mechanisms underlying the cell-fate
switch between quiescence and senescence in normal human
cells, we first generated the TIG-3 strain of primary normal

human diploid fibroblasts (HDFs) expressing a fusion protein

of estrogen receptor (ER) and Flag-tagged human p16INK4a
(ER-p16 cells). Upon the addition of a modified ligand for the
ER, 4-hydroxy-tamoxifen (4-OHT), nuclear accumulation of
the ER-p16INK4a fusion protein (a sign of p16INK4a activation)
and consequent dephosphorylation of the Rb protein (a sign of
Rb activation) were observed (Figures 1A and 1B, 1 and 2).
This was accompanied by various senescent phenotypes,
including senescence-associated (SA) b-galactosidase (b-gal)
activity and enlarged cell morphology, the two most widely
used markers for cellular senescence (Campisi and dAdda di
Fagagna, 2007; Dimri et al., 1995) (Figures 1A and 1B, 1
Cell Reports 7, 194207, April 10, 2014 2014 The Authors 195

Figure 2. AKT Signaling Is Required for Senescent Cell-Cycle Arrest

(A) ER-p16 cells were cultured under normal conditions (1, 2, and 3) or with 20 mM of LY294002 (PI3K inhibitor) (4, 5, and 6) or 10 mM of AKT inhibitor (7, 8, and 9) for
7 days. These cells were then cultured in the presence (2, 3, 5, 6, 8, and 9) or absence (1, 4, and 7) of 4-OHT for 7 more days. In lanes 3, 6, and 9, the cells were then
subsequently cultured under normal conditions without 4-OHT and chemical inhibitors for 7 days further. Cells were then subjected to western blot analysis using

(legend continued on next page)

196 Cell Reports 7, 194207, April 10, 2014 2014 The Authors

and 2). Note that once the senescent phenotypes were fully
induced, subsequent inactivation of the ER-p16INK4a fusion protein by the removal of 4-OHT could no longer revoke the senescent cell-cycle arrest (Figures 1A1C, 3). This was not the case
when p16INK4a was depleted by a previously validated small
interfering RNA (siRNA) prior to the 4-OHT treatment in ER-p16
cells (Figures S1A and S1B), thus confirming that these effects
were due to the activation of the p16INK4a/Rb pathway and not
to any unforeseen side effects of the 4-OHT treatment. Notably,
if the mitogenic signals were blocked by contact inhibition (Figures 1A1C, 6) or serum starvation (Figures 1A1C, 9) throughout
the 4-OHT treatment, then the vast majority of ER-p16 cells reinitiated cell proliferation as soon as normal culture conditions
(at low confluency in 10% serum medium without 4-OHT) were
restored, suggesting that cooperation between mitogenic signals and the Rb pathway is indeed required for the establishment
of senescent (irreversible) cell-cycle arrest. These results also
indicate that ER-p16 cells are an ideal model system to study
the cooperation between mitogenic signals and the Rb pathway
toward senescent cell-cycle arrest in normal human cells.
In the course of this study, we unexpectedly noted that
prolonged (more than 14 days) serum starvation induced SA
b-gal activity and enlarged cell morphology in multiple strains
of primary HDFs (Figures 1A and 1B, 7 and 8; Figures S1C
S1E). Moreover, SA b-gal activity was also detected when
ER-p16 cells were rendered quiescent by contact inhibition (Figures 1A and 1B, 4 and 5). However, because these cells immediately resumed cell proliferation as soon as the normal culture
conditions were restored (Figures 1A1C, 6 and 9; Figure S1F),
these seemingly senescent cells were actually quiescent, rather
than senescent. These results are somewhat consistent with previous observations that the SA b-gal activity and the enlarged cell
morphology are not always associated with cellular senescence
(Dimri et al., 1995; Lin et al., 1998). Thus, we decided not to use
these markers for the detection of senescent cells in the following
experiments in this study. Nevertheless, these results further
support the idea that cooperation between mitogenic signals

and the Rb pathway is indeed required for the establishment of

senescent (irreversible) cell-cycle arrest in normal human cells.
AKT Signaling Is Required for Cellular Senescence
To gain mechanistic insights into the cooperation between
mitogenic signals and the Rb pathway, we next sought to identify
the mitogenic signaling pathway required for the establishment
of irreversible cell-cycle arrest. Using a collection of 282 small
chemical inhibitors (SCADS inhibitor kit) against key downstream
molecules activated by mitogenic signaling (Kawada et al.,
2006), we found that the blockage of the phosphatidylinositol
3-kinase (PI3K)-AKT pathway throughout the 4-OHT treatment
rendered p16INK4a incapable of inducing irreversible cell-cycle
arrest in ER-p16 cells (Figures 2A and 2B, 6 and 9). This is consistent with previous reports that the ectopic expression of activated AKT provoked senescence-like cell-cycle arrest by
elevating the intracellular levels of reactive oxygen species
(ROS) (Miyauchi et al., 2004; Nogueira et al., 2008). Indeed, the
phosphorylated (activated) form of AKT and elevated intracellular ROS levels were observed in p16INK4a-induced senescent
cells (Figures S2A and S2B, 2 and 3), but not in quiescent cells
(Figures S2A and S2B, 4, 5, 7 and 8). Because excess levels of
ROS are known to provoke irreparable DNA damage, a major
cause of cellular senescence (dAdda di Fagagna, 2008), we
examined DNA damage. Notably, the accumulation of gH2AX
foci and 53BP1 foci and increased level of phosphorylated (activated) form of Chk2, the signs of DNA damage, were observed
in p16INK4a-induced senescent cells (Figures S2AS2C, 2 and
3), although to a lesser extent than in replicative senescent cells
or Ras-induced senescent cells (Figures S2D and S2E, 2 and 4).
Moreover, the gH2AX foci were enriched at the subtelomeric
regions in p16INK4a-induced senescent cells (Figure 2C), similar
to previous observations in senescent cells, as judged by chromatin immunoprecipitation (ChIP) analysis (Fumagalli et al.,
2012; Hewitt et al., 2012).
It is also worth mentioning that the siRNA-mediated depletion
of ataxia telangiectasia mutated (ATM), a critical downstream

the antibodies shown on the left (P, phosphospecific antibody) or to analysis of nuclear Flag-tagged p16 staining. Representative data from three independent
experiments are shown. b-actin was used as a loading control. Error bars indicate mean SD of triplicate measurements.
(B) ER-p16 cells cultured under the indicated conditions in (A) (3, 6, and 9) were subjected to cell proliferation analysis performed in triplicate. Error bars indicate
mean SD.
(C) Enrichment of gH2AX at subtelomeric regions was assayed on chromosome 12p by ChIP assay followed by real-time PCR using previously validated primers
at indicated distances from the chromosome end. The graph shows log2 ratios of ER-p16 cells treated with or without 4-OHT. Each PCR reaction was performed
in triplicate, and the average positions are plotted (n = 2).
(D) ER-p16 cells were subjected to transfection with siRNA oligos against either control (1, 2, and 3), ATM sequence 1 (4, 5, and 6), or ATM sequence 2 (7, 8, and 9)
three times (at 2-day intervals) throughout 4-OHT treatment (2, 3, 5, 6, 8, and 9) or mock treatment (1, 4, and 7) for 7 days. In lanes 3, 6, and 9, the cells were
then subsequently cultured under normal conditions without 4-OHT and siRNA oligos for 7 more days. These cells were subjected either to western blotting using
the antibodies shown on the left (P, phosphospecific antibody) or to analysis of nuclear Flag-tagged p16 staining. The percentages of nuclear Flag-tagged
p16INK4a-positive cells are also shown at the bottom. Representative data from three independent experiments are shown. Error bars indicate mean SD of
triplicate measurements. b-actin was used as a loading control.
(E) ER-p16 cells cultured under the conditions indicated in (D) (3, 6, and 9) were subjected to cell proliferation analysis performed in triplicate. Error bars indicate
means SD.
(F) ER-p16 cells were cultured under the normal conditions with (4, 5, and 6) or without (1, 2, and 3) 1 mM of NAC for 7 days. These cells were then cultured in the
presence (2, 3, 5, and 6) or absence (1 and 4) of 4-OHT for 7 more days. In lanes 3 and 6, the cells were then subsequently cultured under normal conditions
without 4-OHT and NAC for 7 days further. These cells were then subjected either to western blotting using the antibodies shown on the left (P, phosphospecific
antibody) or to analysis of nuclear Flag-tagged p16 staining and ROS intracellular levels. Representative data from three independent experiments are shown.
b-actin was used as a loading control. Error bars indicate mean SD of triplicate measurements.
(G) ER-p16 cells cultured under the conditions indicated in (F) (3 and 6) were subjected to cell proliferation analyses performed in triplicate. Error bars indicate
mean SD.

Cell Reports 7, 194207, April 10, 2014 2014 The Authors 197

Figure 3. AKT Promotes ROS Production by Blocking the FoxO3a-SOD2 Axis

(A) ER-p16 cells were retrovirally transduced with either empty vector (1, 2, and 3), Myc-tagged FoxO3a-TM (4, 5, and 6), or Myc-tagged SOD2 (7, 8, and 9) under
normal culture conditions. These cells were then cultured in the presence (2, 3, 5, 6, 8, and 9) or absence (1, 4, and 7) of 4-OHT for 7 days. In lanes 3, 6, and 9, the
cells were subsequently cultured under normal conditions without 4-OHT for 7 more days. The experimental conditions and representative micrographs of
immunofluorescence staining for Flag-tagged p16 (red), FoxO3a (red), and DNA damage foci (g-H2AX foci [red] and 53BP1 foci [green]) are shown. DNA was
stained with DAPI (blue).
(B) Total cell lysates were prepared in culture conditions indicated in (A) (lane number corresponds to culture condition in A) and subjected either to western blot
analysis using the antibodies shown on the left (P, phosphospecific antibody) or to analysis of ROS intracellular levels. The percentages of positively stained cells

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198 Cell Reports 7, 194207, April 10, 2014 2014 The Authors

mediator of the DNA damage response (DDR) pathway,

rendered p16INK4a incapable of inducing irreversible cell-cycle
arrest in ER-p16 cells (Figures 2D and 2E, 6 and 9). Identical
results were obtained using two different sets of previously
validated siRNAs against ATM, precluding the possibility of
off-target effects (Figures 2D and 2E, 6 and 9). These results,
together with the observation that the inhibition of ROS production by N-acetylcysteine (NAC) treatment throughout the 4-OHT
treatment abolished the onset of p16INK4a-induced senescence
(Figures 2F and 2G, 6), led us to consider that the AKT activation induced by mitogenic signaling cooperates with the
p16INK4a/Rb pathway to increase ROS levels, thereby provoking
persistent DDR and a consequent senescent (irreversible) cellcycle arrest.
AKT Promotes ROS Production by Blocking the FoxO3aSOD2 Axis
To explore this idea, we next focused on the downstream mediators of the AKT signaling pathway. The FoxO-family proteins,
members of the Forkhead transcription factor family, are critical
downstream mediators of AKT signaling (Eijkelenboom and
Burgering, 2013). The phosphorylation (inactivation) of FoxOfamily proteins by AKT is known to cause the elevation of intracellular ROS levels through the repression of detoxifying
enzymes, such as manganese superoxide dismutase (SOD2)
(Brunet et al., 1999; Kops et al., 2002; Miyamoto et al., 2007).
Therefore, we examined the contribution of FoxO3a, the most
abundant FoxO family transcription factor in ER-p16 cells, to
the regulation of the ROS level. Notably, the nuclear accumulation of FoxO3a (a sign of FoxO3a activation) and the expression
levels of SOD2 were both inversely correlated with levels of
phosphorylated (activated) AKT and intracellular ROS in
ER-p16 cells treated with 4-OHT in the presence or absence of
mitogenic signaling (Figures S2AS2C, 2, 5 and 8). Moreover,
the depletion of FoxO3a using previously validated siRNA oligos
caused a significant reduction of SOD2 expression and a consequent increase of intracellular ROS levels in HDFs cultured in low
(0.2%) serum medium, but not in normal (10%) serum medium
(Figures S3A and S3B). Conversely, ectopic expression of the
constitutively active form of FoxO3a (FoxO3a-TM), which is
resistant to phosphorylation by AKT (Brunet et al., 1999),
throughout the 4-OHT treatment resulted in the upregulation
of SOD2 expression and the consequent reduction of intracellular ROS levels, thereby rendering ER-p16 cells resistant
to p16INK4a-induced senescent cell-cycle arrest (Figures 3A
3C, 6). Similar results were also observed when SOD2 was overexpressed in ER-p16 cells (Figures 3A3C, 9). Furthermore, the
depletion of SOD2 using previously validated siRNA oligos led
to a significant increase in intracellular ROS levels and phosphorylation of Chk2 (a DDR), regardless of the serum concentration in
the culture medium (Figures S3C and S3D).
Because the expression levels of SOD1and SOD3 were unchanged or undetectable in ER-p16 cells, respectively (Fig-

ure S2A), it seems possible that SOD2, but not SOD1 or SOD3,
plays key role in detoxifying ROS in ER-p16 cells. Collectively,
these results indicated that mitogenic signaling cooperates
with the p16INK4a/Rb pathway to increase the intracellular ROS
levels through the reduction of SOD2 expression via AKT-dependent inactivation of FoxO3a, leading to an irreparable DDR and
consequent senescent (irreversible) cell-cycle arrest.
FoxO3a Binds to the FBEs in the SOD2 Gene Promoter in
Quiescent Cells, but Not in Senescent Cells
We therefore next examined the regulation of SOD2 gene
expression by FoxO3a. There are five putative Forkhead transcription factor-binding elements (FBEs) within the human
SOD2 gene promoter. A promoter reporter analysis and electrophoretic mobility shift analysis (EMSA) revealed that the FBEs
at 1,292 (FBE-2), 1,268 (FBE-3), and 1,181 (FBE-4) upstream of the transcription start site respond to the ectopic
expression of FoxO3a-TM (Figure 4A, 1, 2, 9, 10, 13, 15, 16,
and 17 and data not shown). Concordantly, moreover, endogenous FoxO3a only bound to these FBEs if the mitogenic signaling
was blocked by culturing under high-density or serum-starvation
conditions (Figures 4B and 4C, 2 and 3). These observations
support our idea that mitogenic signaling works cooperatively
with the p16INK4a/Rb pathway to increase the intracellular ROS
levels through the AKT-dependent inactivation of FoxO3a in senescent cells. However, given that FoxO3a is inactivated by AKT
in proliferating cells, it was puzzling that SOD2 expression was
maintained in proliferating cells (Figures S2AS2C, 1, 6 and 9).
Because SOD2 expression was repressed when the p16INK4a/
Rb pathway was activated in the presence of AKT signaling (Figure S2A, 2) and Rb is known to play key roles in repressing the
E2F target genes in senescent cells (Chicas et al., 2010), we
reasoned that the SOD2 expression may be maintained by the
E2F transcription factor in proliferating cells. Unexpectedly,
however, we were unable to find a consensus E2F recognition
sequence within 10 kb upstream of the transcription start site
of the human SOD2 gene promoter, implying that E2F may indirectly regulate SOD2 gene expression in proliferating cells.
FoxM1 Can Substitute for FoxO3a in Proliferating Cells
To substantiate this idea, we searched for evidence that the
E2F target gene product acts as a substitute for FoxO3a in proliferating cells. Combining multiple data sets of DNA microarray
analyses, we found FoxM1, another member of the Forkhead
transcription factor family (Lam et al., 2013), as a strong candidate. Its mRNA expression is commonly reduced when senescent cell-cycle arrest is induced by serial passage (replicative
senescence), oncogenic Ras expression (Ras-induced senescence), depletion of DP1 (an essential activator of E2F transcription factors), or p16INK4a expression (ER-p16 cells treated with
4OHT) in HDFs (Figures S4A and S4B). We also confirmed that
both E2F1 and E2F3 bound to and activated FoxM1 gene promoter in proliferating HDFs (data not shown). These results, in

in (A) and ROS levels are also shown at the bottom. Representative data from three independent experiments are shown. Error bars indicate mean SD of
triplicate measurements. b-actin was used as a loading control.
(C) ER-p16 cells cultured under the conditions indicated in (A) (3, 6, and 9) were subjected to cell proliferation analysis performed in triplicate. Error bars indicate
mean SD.

Cell Reports 7, 194207, April 10, 2014 2014 The Authors 199

Figure 4. FoxO3a and FoxM1 Regulate Human SOD2 Gene Promoter Activity
(A) Schematic representation of the reporter constructs of human SOD2 gene promoter used in the promoter-reporter analysis (left panel). The Forkhead
transcription factor-binding elements (FBEs) were at the following locations from the transcription start site are shown as black rectangles: FBE-1 (from 4,987
to 4,980), FBE-2 (from 1,292 to 1,285), FBE-3 (from 1,268 to 1,261), FBE-4 (from 1,181 to 1,174), and FBE-5 (from 928 to 921). The white rectangles on the promoter scheme indicate the deletion of the FBE sites. U2OS cells were transiently cotransfected with the indicated luciferase reporter constructs

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200 Cell Reports 7, 194207, April 10, 2014 2014 The Authors

conjunction with a previous observation that CDK4 and CDK6,

targets of p16INK4a, stabilize FoxM1 protein (Anders et al.,
2011), suggest that p16INK4a blocks FoxM1 expression at both
the mRNA and protein levels in senescent cells. Note that the
depletion of FoxM1 using previously validated siRNA oligos
caused a significant reduction of SOD2 expression and a consequent elevation of intracellular ROS levels in HDFs cultured
in normal, but not low, serum medium (Figure S4C). Conversely,
the ectopic expression of FoxM1 resulted in the elevation of
SOD2 expression and the reduction of ROS levels, rendering
p16INK4a incapable of inducing senescent (irreversible) cell-cycle
arrest, despite the presence of mitogenic signaling in ER-p16
cells (Figures S4D and S4E, 6). Importantly, moreover, the
promoter-reporter analysis data, in conjunction with the ChIP
analysis and EMSA, revealed that FoxM1 activates the SOD2
gene promoter through binding to FBE2, FBE3, and FBE5 in
proliferating cells, but not in nonproliferating cells (Figures 4A,
1, 2, 6, 9, 13, 15, 16, and 17; Figures 4B and 4D, 1; and data
not shown).
It should also be noted that FoxM1 overexpression blunted
the effects of FoxO3a depletion on SOD2 expression, ROS
levels, and DDR in quiescent HDFs cultured in low serum
medium (Figure S4F lanes 2, 3, 5, and 6). In contrast, overexpression of FoxO3a-TM blunted the effects of FoxM1 depletion on
SOD2 expression, ROS levels, and DDR in proliferating HDFs
cultured in normal serum medium (,Figure S4G, lanes 2, 3, 5,
and 6). Similar results were also observed when SOD2 was overexpressed instead of FoxM1 or FoxO3a-TM (Figures S4F and
S4G, lanes 8 and 9), indicating that SOD2 is a critical downstream mediator of the FoxO3a/FoxM1-ROS pathway, at least
in the HDFs used in this study. Together, these results clearly
indicate that although FoxO3a is the main transactivator of
SOD2 gene expression in quiescent HDFs, FoxM1 assumes
this role when FoxO3a is inactivated by mitogenic signaling
and is aided by the concomitant upregulation of FoxM1 expression via E2F in proliferating HDFs. Thus, it appears that FoxO3a
and FoxM1 complement each other in preventing the onset of
cellular senescence in cultured HDFs.
FoxO3a and FoxM1 Complementally Prevent
Hepatocyte Senescence
Finally, we asked whether our observations applied in vivo using
the mouse as a model organism. The mammalian liver has the
unique capacity to regenerate its growth and mass and is a
well-characterized biological system for studying cell proliferation and quiescence in vivo. In rodents, the liver mass increases
several-fold in the first 4 postnatal weeks, until the liver/body
weight ratio approaches adult levels. Although adult hepato-

cytes are nonproliferating cells, they retain their ability to proliferate and regenerate damaged hepatic tissue after toxic injury
and/or infections, indicating that adult hepatocytes are in the
quiescent state (Fausto, 2004). In contrast, age-related defects
in hepatocyte proliferation are known to be associated with the
appearance of multiples senescence markers, such as accumulation of DNA damage foci, increased expression of p16INK4a,
and diminished expression of FoxM1 (Krishnamurthy et al.,
2004; Wang et al., 2001, 2009; Yamakoshi et al., 2009), suggesting that aged hepatocytes are likely to be in the senescent state.
These notions, together with the observation that the ectopic
expression of FoxM1 in mouse hepatocytes prevented the
progression of age-related proliferation defects (Wang et al.,
2001), prompted us to examine whether the FoxM1/FoxO3aROS-DDR pathway plays a role in forming a quiescence-senescence switch in hepatocytes in vivo.
In the livers of juvenile mice, a significant proportion of hepatocytes are positive for Ki67 expression (a proliferation marker)
(Figures 5A and 5B, 1 and 2), and we detected substantial levels
of FoxM1, but not FoxO3a, bound to the regions of functional
FBEs (FEB-4 and FEB-5) of the mouse SOD2 gene promoter
(Figure 5C, 1 and 2; Figure S5, 1 and 6). In the livers of young
adult mice, in which most of the hepatocytes are in the quiescent
state (Figures 5A and 5B, 3 and 4), FoxO3a, but not FoxM1, was
bound to the same region of the mouse SOD2 gene promoter
(Figure 5C, 3 and 4). This was accompanied by reduced
FoxM1 expression, diminished AKT phosphorylation, and
consequent nuclear accumulation (activation) of FoxO3a in the
livers of young adult mice (Figures 5A and 5B, 3 and 4). Note
that lower SOD2 expression was observed in the livers of young
adult mice lacking the FoxO3a gene, accompanied by increased
ROS levels and DNA damage foci (gH2AX and 53BP1) accumulation (Figures 6A6C, 5 and 6). Thus, although other mechanisms may also be involved, the simplest explanation for this
result is that FoxO3a acts as a substitute for FoxM1 in preventing
the elevation of ROS levels and the consequent onset of cellular
senescence by maintaining SOD2 expression in the quiescent
hepatocytes of young adult mice livers. Importantly, neither
FoxM1 nor FoxO3a bound to the SOD2 gene promoter in the
livers of older mice (Figure 5C, 5 and 6), coinciding with the
reduction of both FoxM1 expression level and the nuclear
FoxO3a level (Figures 5A and 5B, 5 and 6). Concordantly, the
signs of cellular senescence, such as increase of p16INK4a
expression, reduction of SIRT1 expression, decreased SOD2
levels, elevated ROS levels, and accumulation of DNA damage
foci (Abdelmohsen et al., 2007; Kuilman et al., 2010), were all
observed in the livers of older mice (Figures 5A and 5B, 5
and 6). Notably, moreover, the levels of AKT phosphorylation

and expression vectors (right panel). Firefly luciferase activity was normalized to the activity of cotransfected Renilla luciferase. The experiments were performed
in triplicate, and representative results from three independent experiments are shown in the right panel. Error bars indicate mean SD of triplicate measurements.
(BD) The positions of the PCR primers for the ChIP analysis are shown at the top of the panel (red arrows). ER-p16 cells were cultured under normal conditions
(1, 4, and 5) or under contact-inhibition (2) or serum-starvation (3) conditions for 7 days. The cells were then cultured in the presence (2, 3, 4, and 5) or absence (1)
of 4-OHT for 7 more days. In lane 5, the cells were then subsequently cultured under normal conditions without 4-OHT for 7 days further. These cells were
subjected to ChIP analysis using antibodies against control immunoglobulin G (black), FoxO3a (red), or FoxM1 (green), and the PCR primers are shown at the top
(red). A schematic representation of the FBEs (black rectangles) in the human SOD2 gene promoter region is shown at the top of the panels. The experiments were
performed in triplicate, and representative results from three independent experiments are shown. Error bars indicate mean SD of triplicate measurements.

Cell Reports 7, 194207, April 10, 2014 2014 The Authors 201

Figure 5. Complementary Roles of FoxO3a and FoxM1 in Preventing Hepatocyte Senescence In Vivo
(A) Hematoxylin and eosin (H&E) staining and immunohistochemistry for endogenous p16INK4aexpression, Ki67 expression, FoxO3a expression, gH2AX foci,
and 53BP1 foci were performed using biopsy samples of livers from 3-week-old (1 and 2), 24-week-old (3 and 4), and 109-week-old (5 and 6) wild-type female
CD-1 mice.
(B) Biopsy samples of livers from 3-week-old, 24-week-old, and 109-week-old wild-type female CD-1 mice were subjected to western blot analysis using the
antibodies shown on the left or to the analysis of ROS intracellular levels (lane number corresponds to that in A) (P, phosphospecific antibody). Vinculin was used
as a loading control. The percentages of positively stained cells in (A) are also shown at the bottom. Error bars indicate mean SD of triplicate measurements.
(C) Indicated mouse livers were subjected to the ChIP analysis using antibodies against control immunoglobulin G (black), FoxO3a (red), or FoxM1 (green), and
PCR primers are shown at the top (red arrows). A schematic representation of the FBEs (black rectangles) in the mouse SOD2 gene promoter region is shown at
the top of the panels (see also Figure S5). Experiments were performed in triplicate, and representative results from three independent experiments are shown.
Error bars indicate mean SD of triplicate measurements.

202 Cell Reports 7, 194207, April 10, 2014 2014 The Authors

Figure 6. Roles of FoxO3a in Preventing Hepatocyte Senescence In Vivo

(A) Hematoxylin and eosin (H&E) staining and immunofluorescence analysis of Ki67 expression, FoxO3a expression, gH2AX foci, and 53BP1 foci were performed
using biopsy samples of livers from 3-week-old (WT) or 24-week-old (WT or FoxO3a null) female C57BL/6 mice.
(B) Biopsy samples of livers from 3-week-old and 24-week-old female C57BL/6 mice of the indicated genotype were subjected either to western blot analysis
using the antibodies shown on the left or to analysis of ROS intracellular levels (lane number corresponds to that in A) (P, phosphospecific antibody). Vinculin was
used as a loading control. The percentages of positively stained cells in (A) are also shown at the bottom. Error bars indicate mean SD of triplicate
measurements.
(C) Indicated mouse livers were subjected to ChIP analysis using the antibodies against control immunoglobulin G (black), FoxO3a (red), or FoxM1 (green), and
PCR primers are shown at the top (red arrows). A schematic representation of the FBEs (black rectangles) in mouse SOD2 gene promoter region is shown at the
top of the panels (see also Figure S5). The experiments were performed in triplicate, and representative results from three independent experiments are shown.
Error bars indicate mean SD of triplicate measurements.

Cell Reports 7, 194207, April 10, 2014 2014 The Authors 203

were increased in the livers of aged mice as compared with those

in the livers of young adult mice (Figure 5B, 36). Because SIRT1
has been shown to activate PTEN and thus inhibits phosphorylation of AKT (Ming et al., 2010), it is tempting to speculate that
the reduction of SIRT1 expression may promote phosphorylation
of AKT, at least partly, in aged mice liver. Together, these results
indicated that FoxM1 and FoxO3a are likely to play complementary roles in preventing the onset of senescent (irreversible) cell-cycle arrest in hepatocytes, and the disruption of this
complementary network may provoke hepatocyte senescence
during the aging process in vivo.
DISCUSSION
One of the important questions remaining in the field of cellular
senescence is how the irreversibility of cell-cycle arrest is established in senescent cells (Sharpless and DePinho, 2005; Baker
and Sedivy, 2013). Although the Rb pathway is known to play
key roles in the implementation of cellular senescence, it also induces cellular quiescence, a reversible form of cell-cycle arrest
(Cheung and Rando, 2013). Therefore, we wondered how the
Rb pathway determines whether a cell undergoes reversible
(quiescent) or irreversible (senescent) cell-cycle arrest in normal
human cells. To address this issue, we generated primary HDFs
expressing an ER-p16INK4a fusion protein. Using this cell system, in conjunction with a collection of chemical inhibitors
against various mitogenic signaling molecules, we found that
AKT signaling and the Rb pathway cooperatively regulate the
cell-fate choice between quiescence and senescence by controlling the complementary roles of FoxO3a and FoxM1. It
should be noted that although AKT, FoxO3a, and FoxM1 were
previously reported to be involved in cellular senescence (Anders et al., 2011; Miyauchi et al., 2004; Nogueira et al., 2008;
Park et al., 2009), it remained largely unclear how these molecules integrate with the Rb pathway to form a quiescencesenescence switch.
In the present study, we revealed that the levels of intracellular
ROS, critical inducers of cellular senescence (Finkel, 2011), are
fine-tuned by the complementary roles of FoxM1 and FoxO3a
and their different response to mitogenic signals in HDFs. Our
results draw a model in which although FoxO3a is the main transactivator of SOD2 gene expression in quiescent HDFs, FoxM1
assumes this role when FoxO3a is inactivated by mitogenic
signaling through AKT activation, aided by the concomitant
upregulation of FoxM1 mRNA expression via E2F activation in
proliferating HDFs (Figures 7A and 7B). In senescent HDFs,
however, FoxO3a is inactivated by mitogenic signaling via AKT
activation and FoxM1 mRNA expression is repressed by the activation of Rb pathway, thus causing the reduction of SOD2 levels
and the consequent elevation of ROS levels, which could
provoke accumulation of irreparable DNA damage, resulting in
the establishment of senescent (irreversible) cell-cycle arrest
(Figure 7C). Note that although it was previously speculated
that FoxM1 and FoxO3a bind to different FBEs (Park et al.,
2009), we showed here that FoxM1 and FoxO3a bind to the
same FBEs in the SOD2 gene promoter, at least in cultured
HDFs and murine livers (Figures 4 and 5). These results provide
additional support for the model described above.
204 Cell Reports 7, 194207, April 10, 2014 2014 The Authors

There are, however, some caveats to our model. For example,

it is unclear whether the pathway described here also plays key
roles in other cell types. Moreover, as it has recently become
apparent that cellular senescence can be induced by a variety
of different stresses (Chandler and Peters, 2013; Naylor et al.,
2013), the pathways leading to the establishment of senescent
cell-cycle arrest are likely to be more complex than previously
envisaged. For instance, the induction of senescence-associated secretory phenotype (SASP) and the downregulation of
the transcriptional repressor HES1 (Rodier and Campisi, 2011;
Sang et al., 2008) were reportedly required for the establishment
of senescent cell-cycle arrest in certain settings. Although these
phenomena were not observed in p16INK4a-induced senescence
in HDFs (data not shown) as reported previously (Coppe et al.,
2011; Takahashi et al., 2012), it is possible that SASP and other
mechanisms are contributing to the establishment of senescent
cell-cycle arrest, depending on the cellular context and/or type
of initiating insult. However, it should be noted that the elevation
of ROS levels and the accumulation of DNA damage reportedly
play key roles in the induction of senescent phenotypes in replicative senescence and Ras-induced senescence in several
different cell types (Bartkova et al., 2006; dAdda di Fagagna
et al., 2003; Di Micco et al., 2006; Lee et al., 1999; Takai et al.,
2003). Moreover, recent reports suggest the existence of ROSgenerating positive-feedback loops that could sustain DDR in
senescent cells (Takahashi et al., 2006; Moiseeva et al., 2009;
Passos et al., 2010). Taken together, although we cannot rule
out the existence of other pathways contributing to the irreversibility of senescent cell-cycle arrest (Narita et al., 2003; Satyanarayana et al., 2004; Litovchick et al., 2011; Ivanov et al., 2013), it is
likely that the FoxO3a/FoxM1-ROS-DDR pathway plays important roles in forming a quiescence-senescence switch, at least
in certain settings.
It is also worth mentioning that both SA b-gal activity and
enlarged cell morphology were induced by prolonged serum
starvation in HDFs (Figures 1A and 1B; Figures S1CS1E). However, because these cells immediately resumed cell proliferation
as soon as the normal culture conditions were restored (Figures
1A and 1C, 9; Figure S1F), as judged by time-lapse video analysis
(data not shown), these seemingly senescent cells were quiescent, but not senescent. Similar results were also reported in murine cells (Yegorov et al., 1998), and SA b-gal activity is known to
be induced by contact inhibition (Dimri et al., 1995; Severino
et al., 2000). These data, in conjunction with the observation
that SA b-gal activity is dispensable for the implementation of
cellular senescence (Lee et al., 2006), suggest that although SA
b-gal activity is regarded as the gold standard for identifying senescent cells in culture and in tissues, this clearly needs to be interpreted with caution. In this study, we therefore examined ROS
levels, DDR, and the irreversibility of cell-cycle arrest.
It should be noted that inadequate culture environments
can elicit phenotypes closely resembling cellular senescence
(Ramirez et al., 2001). For example, the oxygen levels in standard
cell culture conditions (20%) were suggested to be unphysiologically high (Parrinello et al., 2003). However, similar
results were obtained in ER-p16 cells regardless of high (20%)
or low (3%) oxygen conditions (data not shown). Moreover, signs
of the FoxO3a/FoxM1-ROS-DDR pathway were observed in

Figure 7. A Model of Quiescence-Senescence Switch

In quiescent cells, FoxO3a plays key roles in
preventing ROS production by inducing SOD2
expression (A), whereas FoxM1 assumes this role
when FoxO3a is inactivated by mitogenic signaling
in proliferating cells (B). However, activation of
the Rb pathway by the induction of p16INK4a, for
example, in the presence of mitogenic signals
provokes ROS production by inactivating both
FoxO3a and FoxM1. High levels of ROS cause
accumulation of irreparable DNA damage, thereby
causing senescent (irreversible) cell-cycle arrest (C).

mouse liver parenchymal cells during the aging process in vivo

(Figures 5 and 6). Thus, it is likely that our findings do not simply
reflect tissue culture stress but instead represent at least certain
aspects of the cell-fate choice between quiescence and senescence in vivo. Although further work is required to understand
exactly where and when the pathway described here plays a
role in vivo, our work expands our understanding of the molecular mechanisms that distinguish between quiescence and senescence and opens up new possibilities for its control toward the
fight against cancer and aging.
EXPERIMENTAL PROCEDURES
Cell Culture, RNAi, and ChIP Analysis
Cell culture, RNAi, and ChIP analysis were performed as previously described
(Takahashi et al., 2012).

Analysis of Intracellular ROS Levels

Relative ROS levels were measured as described previously (Takahashi et al.,
2012).
Mice
CD-1 mice and C57BL/6 mice (both wild-type and FoxO3a / ) (Miyamoto
et al., 2007) were cared for using protocols approved by the Committee for
the Use and Care of Experimental Animals of the Japanese Foundation for
Cancer Research.

ACCESSION NUMBERS
Microarray data have been deposited to the NCBI Gene Expression Omnibus
(http://www.ncbi.nlm.nih.gov/gds) under the accession numbers GSE49860,
GSE49861, GSE49862, and GSE49863.

SUPPLEMENTAL INFORMATION
Immunoblotting and Immunoprecipitation Analysis
Immunoprecipitation and immunoblotting were performed as previously
described (Takahashi et al., 2012). The antibodies used are listed in Supplemental Experimental Procedures.

Supplemental Information includes Supplemental Experimental Procedures

and five figures and can be found with this article online at http://dx.doi.org/
10.1016/j.celrep.2014.03.006.

Cell Reports 7, 194207, April 10, 2014 2014 The Authors 205

ACKNOWLEDGMENTS
We thank the SCADS Inhibitor Kit Screening Committee supported by the
Ministry of Education, Culture, Sports, Science and Technology (MEXT) for
providing us with SCADS inhibitor kits. This work was supported by grants
from the Japan Science and Technology Agency (JST), MEXT, and the Ministry
of Health, Labour, and Welfare of Japan (MHLW). Y.I. was supported by a
fellowship from the Japan Society for Promotion of Science (JSPS).
Received: October 12, 2013
Revised: January 13, 2014
Accepted: March 3, 2014
Published: April 3, 2014

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