Cell Reports

Article

Crosstalk between the Rb Pathway and AKTSignaling Forms a Quiescence-Senescence SwitchYoshinori 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, Japan2Graduate School of Biomedical Science, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan3Cancer Research Institute, Kanazawa University, Kanazawa 920-1192, Japan4PRESTO, Japan Science Technology Agency, Saitama 332-0012, Japan5CREST, Japan Science Technology Agency, Saitama 332-0012, Japan

*Correspondence: eiji.hara@jfcr.or.jp

http://dx.doi.org/10.1016/j.celrep.2014.03.006This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

SUMMARY

Cell-cycle arrest in quiescence and senescenceis largely orchestrated by the retinoblastoma (Rb)tumor-suppressor pathway, but the mechanismsunderlying the quiescence-senescence switchremain unclear. Here, we show that the crosstalkbetween the Rb-AKT-signaling pathways forms thisswitch by controlling the overlapping functions ofFoxO3a and FoxM1 transcription factors in culturedfibroblasts. In the absence of mitogenic signals,although FoxM1 expression is repressed by the Rbpathway, FoxO3a prevents reactive oxygen species(ROS) production by maintaining SOD2 expression,leading to quiescence. However, if the Rb pathwayis activated in the presence of mitogenic signals,FoxO3a is also inactivated by AKT, thus reducingSOD2 expression and consequently allowing ROSproduction. This situation elicits senescence throughirreparable DNA damage. We demonstrate that thispathway operates in mouse liver, indicating thatthismachinerymay contributemore broadly to tissuehomeostasis in vivo.

INTRODUCTION

Over the last few decades, it has become apparent that onco-

genic proliferative signals are coupled to a variety of growth-

inhibitory responses, such as the induction of apoptotic cell

death or irreversible cell-cycle arrest known as ‘‘cellular senes-

cence’’ (Sharpless and DePinho, 2005). Thus, both apoptosis

and cellular senescence are thought to act as important tumor-

suppression mechanisms (Campisi and d’Adda di Fagagna,

2007; Collado and Serrano, 2010; Kuilman et al., 2010). How-

ever, unlike apoptotic cells, senescent cells remain viable for

long periods of time and are hypothesized to persist and accu-

mulate with age in vivo (Krishnamurthy et al., 2004; Jeyapalan

et al., 2007; Yamakoshi et al., 2009). Thus, the irreversibility of

194 Cell Reports 7, 194–207, April 10, 2014 ª2014 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 acti-

vation 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 irre-

versible 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 subse-

quent 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 deter-

mines whether a cell undergoes quiescence or senescence. We

previously reported that the activation of the Rb pathway pro-

vokes 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 inte-

grate 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 collabo-

rate 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 triplicatemeasurements. 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 quiescence-

senescence switch by regulating the functions of FoxO3a and

FoxM1 Forkhead transcription factors (Eijkelenboom and Bur-

gering, 2013; Lam et al., 2013).

RESULTS

Mitogenic Signals Cooperate with the Rb Pathway toInduce Cellular SenescenceTo 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 d’Adda di

Fagagna, 2007; Dimri et al., 1995) (Figures 1A and 1B, 1

Cell Reports 7, 194–207, April 10, 2014 ª2014 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 mMof LY294002 (PI3K inhibitor) (4, 5, and 6) or 10 mMof 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 7more 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, 194–207, April 10, 2014 ª2014 The Authors

and 2). Note that once the senescent phenotypes were fully

induced, subsequent inactivation of the ER-p16INK4a fusion pro-

tein by the removal of 4-OHT could no longer revoke the senes-

cent cell-cycle arrest (Figures 1A–1C, 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 (Fig-

ures 1A–1C, 6) or serum starvation (Figures 1A–1C, 9) throughout

the 4-OHT treatment, then the vast majority of ER-p16 cells rein-

itiated 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 sig-

nals 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 (Fig-

ures 1A and 1B, 4 and 5). However, because these cells immedi-

ately resumed cell proliferation as soon as the normal culture

conditions were restored (Figures 1A–1C, 6 and 9; Figure S1F),

these seemingly senescent cells were actually quiescent, rather

than senescent. These results are somewhat consistent with pre-

vious 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

thesemarkers 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

the antibodies shown on the left (P, phosphospecific antibody) or to analysis of n

experiments are shown. b-actin was used as a loading control. Error bars indica

(B) ER-p16 cells cultured under the indicated conditions in (A) (3, 6, and 9) were su

mean ± SD.

(C) Enrichment of gH2AX at subtelomeric regions was assayed on chromosome 1

at indicated distances from the chromosome end. The graph shows log2 ratios of

in triplicate, and the average positions are plotted (n = 2).

(D) ER-p16 cells were subjected to transfection with siRNA oligos against either co

three times (at 2-day intervals) throughout 4-OHT treatment (2, 3, 5, 6, 8, and 9)

then subsequently cultured under normal conditions without 4-OHT and siRNA oli

the antibodies shown on the left (P, phosphospecific antibody) or to analysis o

p16INK4a-positive cells are also shown at the bottom. Representative data from

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 su

means ± SD.

(F) ER-p16 cells were cultured under the normal conditions with (4, 5, and 6) or wit

presence (2, 3, 5, and 6) or absence (1 and 4) of 4-OHT for 7 more days. In lane

without 4-OHT and NAC for 7 days further. These cells were then subjected either

antibody) or to analysis of nuclear Flag-tagged p16 staining and ROS intracellula

b-actin was used as a loading control. Error bars indicate mean ± SD of triplicat

(G) ER-p16 cells cultured under the conditions indicated in (F) (3 and 6) were sub

mean ± SD.

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 SenescenceTo 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 consis-

tent with previous reports that the ectopic expression of acti-

vated 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 intracel-

lular 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 (d’Adda di Fagagna, 2008), we

examined DNA damage. Notably, the accumulation of gH2AX

foci and 53BP1 foci and increased level of phosphorylated (acti-

vated) form of Chk2, the signs of DNA damage, were observed

in p16INK4a-induced senescent cells (Figures S2A–S2C, 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 chro-

matin 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

uclear Flag-tagged p16 staining. Representative data from three independent

te mean ± SD of triplicate measurements.

bjected to cell proliferation analysis performed in triplicate. Error bars indicate

2p by ChIP assay followed by real-time PCR using previously validated primers

ER-p16 cells treated with or without 4-OHT. Each PCR reaction was performed

ntrol (1, 2, and 3), ATM sequence 1 (4, 5, and 6), or ATM sequence 2 (7, 8, and 9)

or mock treatment (1, 4, and 7) for 7 days. In lanes 3, 6, and 9, the cells were

gos for 7more days. These cells were subjected either to western blotting using

f nuclear Flag-tagged p16 staining. The percentages of nuclear Flag-tagged

three independent experiments are shown. Error bars indicate mean ± SD of

bjected to cell proliferation analysis performed in triplicate. Error bars indicate

hout (1, 2, and 3) 1 mM of NAC for 7 days. These cells were then cultured in the

s 3 and 6, the cells were then subsequently cultured under normal conditions

to western blotting using the antibodies shown on the left (P, phosphospecific

r levels. Representative data from three independent experiments are shown.

e measurements.

jected to cell proliferation analyses performed in triplicate. Error bars indicate

Cell Reports 7, 194–207, April 10, 2014 ª2014 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

(legend continued on next page)

198 Cell Reports 7, 194–207, April 10, 2014 ª2014 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 produc-

tion 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 acti-

vation induced by mitogenic signaling cooperates with the

p16INK4a/Rb pathway to increase ROS levels, thereby provoking

persistent DDR and a consequent senescent (irreversible) cell-

cycle arrest.

AKT Promotes ROS Production by Blocking the FoxO3a-SOD2 AxisTo explore this idea, we next focused on the downstream medi-

ators 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 FoxO-

family proteins by AKT is known to cause the elevation of intra-

cellular 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 accumula-

tion 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 S2A–S2C, 2, 5 and 8). Moreover,

the depletion of FoxO3a using previously validated siRNA oligos

caused a significant reduction of SOD2 expression and a conse-

quent 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 intra-

cellular 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 over-

expressed in ER-p16 cells (Figures 3A–3C, 9). Furthermore, the

depletion of SOD2 using previously validated siRNA oligos led

to a significant increase in intracellular ROS levels and phosphor-

ylation 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 un-

changed or undetectable in ER-p16 cells, respectively (Fig-

in (A) and ROS levels are also shown at the bottom. Representative data from

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 s

mean ± SD.

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-depen-

dent inactivation of FoxO3a, leading to an irreparable DDR and

consequent senescent (irreversible) cell-cycle arrest.

FoxO3a Binds to the FBEs in the SOD2Gene Promoter inQuiescent Cells, but Not in Senescent CellsWe therefore next examined the regulation of SOD2 gene

expression by FoxO3a. There are five putative Forkhead tran-

scription factor-binding elements (FBEs) within the human

SOD2 gene promoter. A promoter reporter analysis and electro-

phoretic mobility shift analysis (EMSA) revealed that the FBEs

at �1,292 (FBE-2), �1,268 (FBE-3), and �1,181 (FBE-4) up-

stream 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, endoge-

nous FoxO3a only bound to these FBEs if themitogenic 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 se-

nescent cells. However, given that FoxO3a is inactivated by AKT

in proliferating cells, it was puzzling that SOD2 expression was

maintained in proliferating cells (Figures S2A–S2C, 1, 6 and 9).

Because SOD2 expression was repressed when the p16INK4a/

Rb pathway was activated in the presence of AKT signaling (Fig-

ure 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 indi-

rectly regulate SOD2 gene expression in proliferating cells.

FoxM1 Can Substitute for FoxO3a in Proliferating CellsTo substantiate this idea, we searched for evidence that the

E2F target gene product acts as a substitute for FoxO3a in prolif-

erating 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 candi-

date. Its mRNA expression is commonly reduced when senes-

cent cell-cycle arrest is induced by serial passage (replicative

senescence), oncogenic Ras expression (Ras-induced senes-

cence), depletion of DP1 (an essential activator of E2F transcrip-

tion 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 pro-

moter in proliferating HDFs (data not shown). These results, in

three independent experiments are shown. Error bars indicate mean ± SD of

ubjected to cell proliferation analysis performed in triplicate. Error bars indicate

Cell Reports 7, 194–207, April 10, 2014 ª2014 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 rect-

angles on the promoter scheme indicate the deletion of the FBE sites. U2OS cells were transiently cotransfected with the indicated luciferase reporter constructs

(legend continued on next page)

200 Cell Reports 7, 194–207, April 10, 2014 ª2014 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 con-

sequent 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, overexpres-

sion 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 over-

expressed instead of FoxM1 or FoxO3a-TM (Figures S4F and

S4G, lanes 8 and 9), indicating that SOD2 is a critical down-

stream 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 expres-

sion 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 PreventHepatocyte SenescenceFinally, 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 prolifera-

tion 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-

and expression vectors (right panel). Firefly luciferase activity was normalized to th

in triplicate, and representative results from three independent experiments are

ments.

(B–D) The positions of the PCR primers for the ChIP analysis are shown at the top

(1, 4, and 5) or under contact-inhibition (2) or serum-starvation (3) conditions for 7

of 4-OHT for 7 more days. In lane 5, the cells were then subsequently cultured

subjected to ChIP analysis using antibodies against control immunoglobulin G (bla

(red). A schematic representation of the FBEs (black rectangles) in the humanSOD

performed in triplicate, and representative results from three independent exper

cytes are nonproliferating cells, they retain their ability to prolifer-

ate 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 accumu-

lation 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), suggest-

ing 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/FoxO3a-

ROS-DDR pathway plays a role in forming a quiescence-senes-

cence switch in hepatocytes in vivo.

In the livers of juvenile mice, a significant proportion of hepa-

tocytes 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 whichmost 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) accumu-

lation (Figures 6A–6C, 5 and 6). Thus, although other mecha-

nisms 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

e activity of cotransfected Renilla luciferase. The experiments were performed

shown in the right panel. Error bars indicate mean ± SD of triplicate measure-

of the panel (red arrows). ER-p16 cells were cultured under normal conditions

days. The cells were then cultured in the presence (2, 3, 4, and 5) or absence (1)

under normal conditions without 4-OHT for 7 days further. These cells were

ck), FoxO3a (red), or FoxM1 (green), and the PCR primers are shown at the top

2 gene promoter region is shown at the top of the panels. The experimentswere

iments are shown. Error bars indicate mean ± SD of triplicate measurements.

Cell Reports 7, 194–207, April 10, 2014 ª2014 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, 194–207, April 10, 2014 ª2014 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, 194–207, April 10, 2014 ª2014 The Authors 203

were increased in the livers of agedmice as comparedwith those

in the livers of young adult mice (Figure 5B, 3–6). Because SIRT1

has been shown to activate PTEN and thus inhibits phosphoryla-

tion of AKT (Ming et al., 2010), it is tempting to speculate that

the reduction of SIRT1 expressionmay promote phosphorylation

of AKT, at least partly, in aged mice liver. Together, these results

indicated that FoxM1 and FoxO3a are likely to play com-

plementary roles in preventing the onset of senescent (irrevers-

ible) 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 estab-

lished 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 in-

duces 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 sys-

tem, 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 con-

trolling 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 (An-

ders et al., 2011; Miyauchi et al., 2004; Nogueira et al., 2008;

Park et al., 2009), it remained largely unclear how these mole-

cules integrate with the Rb pathway to form a quiescence-

senescence 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 amodel in which although FoxO3a is themain trans-

activator 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 FoxM1mRNA expression is repressed by the acti-

vation 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, 194–207, April 10, 2014 ª2014 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-associ-

ated 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 repli-

cative senescence and Ras-induced senescence in several

different cell types (Bartkova et al., 2006; d’Adda 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 ROS-

generating 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 irrevers-

ibility of senescent cell-cycle arrest (Narita et al., 2003; Satyanar-

ayana et al., 2004; Litovchick et al., 2011; Ivanov et al., 2013), it is

likely that the FoxO3a/FoxM1-ROS-DDR pathway plays impor-

tant 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 S1C–S1E). How-

ever, 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 quies-

cent, but not senescent. Similar results were also reported inmu-

rine 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 se-

nescent cells in culture and in tissues, this clearly needs to be in-

terpreted 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 un-

physiologically 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-Senes-

cence 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 senes-

cence 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 molecu-

lar mechanisms that distinguish between quiescence and senes-

cence 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).

Immunoblotting and Immunoprecipitation Analysis

Immunoprecipitation and immunoblotting were performed as previously

described (Takahashi et al., 2012). The antibodies used are listed in Supple-

mental Experimental Procedures.

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

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, 194–207, April 10, 2014 ª2014 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 theMinistry

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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