Mst1-FoxO Signaling Protects Naı ¨ve T Lymphocytes from
Cellular Oxidative Stress in Mice
Juhyun Choi1., Sangphil Oh1., Dongjun Lee1, Hyun Jung Oh1, Jik Young Park2, Sean Bong Lee2, Dae-Sik
1National Research Laboratory of Molecular Genetics, Department of Biological Sciences, Biomedical Research Center, Korea Advanced Institute of Science and
Technology, Daejeon, South Korea, 2Genetics of Development and Disease Branch, National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of
Health, Bethesda, Maryland, United States of America
Background: The Ste-20 family kinase Hippo restricts cell proliferation and promotes apoptosis for proper organ
development in Drosophila. In C. elegans, Hippo homolog also regulates longevity. The mammalian Ste20-like protein
kinase, Mst1, plays a role in apoptosis induced by various types of apoptotic stress. Mst1 also regulates peripheral naı ¨ve T
cell trafficking and proliferation in mice. However, its functions in mammals are not fully understood.
Methodology/Principal Findings: Here, we report that the Mst1-FoxO signaling pathway plays a crucial role in survival, but
not apoptosis, of naı ¨ve T cells. In Mst12/2mice, peripheral T cells showed impaired FoxO1/3 activation and decreased FoxO
protein levels. Consistently, the FoxO targets, Sod2 and catalase, were significantly down-regulated in Mst12/2T cells,
thereby resulting in elevated levels of intracellular reactive oxygen species (ROS) and induction of apoptosis. Expression of
constitutively active FoxO3a restored Mst12/2T cell survival. Crossing Mst1 transgenic mice (Mst1 Tg) with Mst12/2mice
reduced ROS levels and restored normal numbers of peripheral naı ¨ve T cells in Mst1 Tg;Mst12/2progeny. Interestingly,
peripheral T cells from Mst12/2mice were hypersensitive to c-irradiation and paraquat-induced oxidative stresses, whereas
those from Mst1 Tg mice were resistant.
Conclusions/Significance: These data support the hypothesis that tolerance to increased levels of intracellular ROS
provided by the Mst1-FoxOs signaling pathway is crucial for the maintenance of naı ¨ve T cell homeostasis in the periphery.
Citation: Choi J, Oh S, Lee D, Oh HJ, Park JY, et al. (2009) Mst1-FoxO Signaling Protects Naı ¨ve T Lymphocytes from Cellular Oxidative Stress in Mice. PLoS
ONE 4(11): e8011. doi:10.1371/journal.pone.0008011
Editor: Matt Kaeberlein, University of Washington, United States of America
Received September 3, 2009; Accepted November 4, 2009; Published November 24, 2009
Copyright: ? 2009 Choi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by grants from the NRL (National Research Laboratory) Program, a Nuclear Research Grant, and the 21stCentury Frontier
Functional Human Genome Project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
Maintenance of T cell homeostasis is critical for normal
functioning of the immune system. Fas, TNF, and ROS effectively
promote the elimination of antigen-specific activated T cells and
limit autoimmunity [1,2]. Intracellular redox status is a physio-
logical regulator of T cell activation and apoptosis during normal
T cell development in the thymus, and also regulates the immune
response in peripheral lymphoid organs . Recent studies have
shown that the transcription factor, FoxO1, is critical for
maintaining naı ¨ve T cells in peripheral lymphoid organs by virtue
of its regulation of L-selectin (CD62L), CCR7 and interleukin 7
receptor a (IL-7Ra/CD127) expression [4,5]. However, naı ¨ve T
cell homeostatic mechanisms in protective immunity are not fully
In mammals, Mst1 (mammalian sterile 20-like 1) kinase, which is
a key component of the ‘‘Hippo’’ signaling pathway, has been
implicated in regulating the cell cycle, apoptosis and cellular
responses to oxidative stress . Recently, Mst1-deficient (Mst12/2)
mice were shown to possess decreased numbers of peripheral T
cells, mostly naı ¨ve T cells; those cells that remained showed
increased proliferation after T cell receptor (TCR) ligation in vitro
and impaired lymphocyte homing to the spleen and lymph nodes
[7,8]. Additionally, defective egress of mature thymocytes from
Mst12/2thymus were also reported [7,9]. The Rassf adapter
protein, Rapl (also named Nore1b), is known to activate Mst1 by
regulating its localization and kinase activity during lymphocyte
migration to lymphoid organs . These observations suggest that
the Rapl-Mst1 pathway can modulate cell proliferation and T-cell
migration [7,8]. Consistent with these recent findings, we observed
a similar phenotype in Mst12/2mice. In addition, we identified a
new and important role for the Mst1-FoxO signaling pathway in
regulating the homeostasis of peripheral naı ¨ve T cells.
Results and Discussion
Mst1 Deficiency Causes Systemic Lymphopenia
To define the physiological functions of Mst1 in higher
organisms, we generated mice lacking Mst1. Lymphoid tissues
from Mst12/2mice displayed a paucity of T and B cells in the
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spleen and reduced numbers of T cells in lymph nodes; in
particular, a large number of cells in white pulp and T cell zones
appeared to be absent (Figure S1A and B). We confirmed that the
numbers of T cells in spleen, lymph nodes, and peripheral blood of
Mst1+/+and Mst1+/2mice were not different, indicating the
haplosufficiency of Mst1 with respect to leukocyte generation
(Figure S2). Mst1+/+or Mst1+/2mice were thus used as controls.
Consistent with recent findings from Mst1-deficient mice [7-9], we
also observed the reduced numbers of both CD4+and CD8+
peripheral T cells in Mst12/2mice (Figure S2A). Most of the
absent T cells in peripheral blood, spleen, and lymph nodes were
naı ¨ve T cells, not effector/memory T cells (Figure S2B). Although
the ratio of single-positive CD4 and CD8 cells appeared to be
slightly increased in the thymus from Mst12/2mice (Figure S1E),
the thymic architecture and the actual numbers of these cells were
similar in Mst1+/+, Mst1+/2, and Mst12/2mice (Figure S1C and
D). The thymic development of Mst12/2T cells bearing TCRs
was also normal, as was their environmental epithelium (unpub-
lished data). Thus, we concluded that the reduced number of
peripheral naı ¨ve T cells from Mst12/2mice does not result from
impaired T cell development or thymic development.
Mst1 Deficiency Causes Naı ¨ve T Cell Death
Mst1 promotes cellular oxidative stress and/or growth factor
deprivation-induced apoptosis in neurons [6,11]. Therefore, we
first tested whether Mst1 deficiency affected the survival of T cells.
Interestingly, many of the peripheral CD4+and CD8+T cells in
Mst12/2mice rapidly underwent apoptosis (Figure 1A, top).
There was also a loss of mitochondrial transmembrane potential
(ym) in these Mst12/2peripheral CD4+and CD8+T cells
(Figure 1A, bottom), confirming apoptosis. Thus, spontaneous cell
death of peripheral Mst12/2T cells might be responsible for the
reduced numbers of peripheral T cells in Mst12/2mice.
To explore the basis for this reduced cellularity and/or
apoptosis of peripheral T cells in Mst12/2mice, we first crossed
Fas- and FasL-defective mice (lpr and gld, respectively) with
Mst12/2mice. Neither Mst12/2;Faslpr/lprnor Mst12/2;FasLgld/gld
mice exhibited a restoration of peripheral T cells to normal levels
(Figure S3), indicating that the apoptosis of Mst12/2T cells was
independent of Fas and FasL. An examination of serum cytokines
and chemokines revealed no significant differences between
Mst1+/2and Mst12/2mice. Notably, the levels of IL-7, which is
critical for the maintenance of both naı ¨ve and memory T cell
homeostasis , and tumor necrosis factor (TNF), which is
associated with apoptosis of activated T cells , were similar in
the sera of Mst1+/2and Mst12/2mice (Figure S4).
FoxO Pproteins Are Inactivated in Mst12/2Peripheral T
Mst1 phosphorylates and activates FoxO3a on serine 207,
promoting cellular oxidative stress-induced apoptosis in neurons
and longevity in Caenorhabditis elegans . Phosphorylation of
FoxO1 on serine 212 by Mst1 also regulates neuronal cell death
under conditions of growth-factor deprivation . Akt and Mst1
exhibit mutually antagonistic functions, and FoxO proteins can be
inactivated by Akt [14–16]. On the basis of these reports, we
examined the expression levels of FoxO1/3 proteins in peripheral
CD4+, CD8+, naı ¨ve (CD62LhiCD44lo), and effector/memory
(CD62LloCD44hi) T cells. Interestingly, Mst12/2T cells displayed
reduced levels of FoxO3a and FoxO1 proteins, and the
phosphorylated form of FoxO1/3 (Figures 1B and S5). Thus, we
hypothesized that Mst1 is required for maintaining normal levels
of FoxO1/3 proteins, and further postulated that inactivation of
these proteins might disrupt peripheral naı ¨ve T cell homeostasis.
In primary neurons, FoxO3a subcellular localization is
reciprocally regulated by MST1, which promotes nuclear
localization by phosphorylating FoxO3a on serine 207 in response
to hydrogen peroxide (H2O2), and by Akt, which phosphorylates
FoxO proteins and induces their translocation to the cytoplasm
where they are subsequently degraded [6,16]. Using immuno-
staining to examine the subcellular localization of FoxO3a in
peripheral T cells, we found that FoxO3a was primarily localized
to the nucleus in Mst1+/2T cells (,85%). By contrast, the
majority of FoxO3a was localized in the cytoplasm of peripheral
Mst12/2T cells (,65%), and its staining intensity was significantly
weaker than in Mst1+/2T cells (Figure 1C). Consistent with this
observation, FoxO1/3 was detected in an activated, phosphory-
lated state in a significant proportion (,37%) of Mst1+/2
peripheral T cell nuclei, whereas negligible numbers of similarly
stained T cells were detected in Mst12/2mice (Figure 1D). We
were unable to specifically detect FoxO1 protein because FoxO1
antibodies suitable for immunostaining are unavailable. These
data show that Mst1 modulates FoxO1/3 nuclear localization and
activation in peripheral T cells.
Expression of Sod2 and Catalase Are Significantly Down-
Regulated in Mst12/2Peripheral T Cells
Recent reports have shown that FoxO1 deletion in T cells
induces a reduction in the number of peripheral naı ¨ve T cells due
to a defect in the expression of lymphocyte-trafficking molecules
(L-selectin and CCR7) and IL-7Ra [4,5], indicating a critical role
for FoxO1 in homeostasis of naı ¨ve T cells. On the basis of these
reports and our observation that FoxO1/3 expression was reduced
in Mst12/2T cells, we examined the expression of L-selectin,
CCR7 and IL-7Ra in Mst12/2naı ¨ve T cells. Although IL-7Ra
expression was slightly reduced in peripheral blood, the cell
surface levels of these proteins did not show statistically significant
difference between Mst1+/+and Mst12/2naı ¨ve T cells (Figure 2).
Thus, it is likely that a reduction in FoxO1 protein levels, unlike
ablation of the FoxO1 gene, does not significantly affect the
expression of these molecules in Mst12/2mice.
FoxO family factors stimulate transcription of Sod2 and catalase
(Cat), which are important in reducing cellular oxidative stress
[17,18]. Thus, we then examined the expression levels of these
FoxO targets in T cells. Consistent with a role for the Mst1-FoxO
pathway in regulating Sod2 and Cat expression, quantitative PCR
analyses showed that Sod2 and catalase mRNA levels were
significantly decreased in T cells derived from Mst12/2spleens
and lymph node compared to those from Mst1+/2mice (Figure 3).
Consistent with reduced mRNA levels, their protein levels were
also reduced in Mst12/2T cells (Figure S5) Thus, in the absence
of Mst1, reducing and/or inactivating FoxO1/3 could result in
decreases in Sod2 and catalase expression in CD4+and CD8+T
Increased ROS in Mst12/2Peripheral T Cells Induces
Because Sod2 and catalase were down-regulated in Mst12/2T
cells, we tested whether the levels of intracellular ROS were
increased in T cells from Mst12/2mice, and thus might account
for the increase in apoptosis. Although the overall levels of
intracellular ROS in thymic T cells were similar in Mst1+/2and
Mst12/2mice, intracellular ROS levels increased gradually during
the progression from double-positive to single-positive T cells in
the thymus (unpublished data). Interestingly, the peripheral CD4+
and CD8+T cells in Mst12/2mice showed elevated ROS levels
compared to Mst1+/2mice (Figure 4A). Increased ROS can
Role of Mst1 in Naive T Cells
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Figure 1. Increased apoptosis and dysregulation of FoxO1/3 proteins in peripheral T cells from Mst12 2/ /2 2mice. (A) Top: Apoptotic cell
death of peripheral blood T cells from Mst1+/2and Mst12/2mice (n=13) was detected. The percentage of Annexin V-positive cells was determined
by normalization to the percentage of Mst1+/2Annexin V-positive cells (defined as 100%). Error bars indicate SEM. **, p,0.01; ***, p,0.001. Bottom:
Mitochondrial transmembrane potential (ym) of CD4+and CD8+T cells in peripheral blood from Mst1+/2and Mst12/2mice was determined by
staining with Rhodamine 123. CCCP-treated cells were used as a control (grey). FACS profiles shown are representative of three independent
experiments. (B) FoxO1/3 protein levels and phosphorylation were decreased in Mst12/2T cells. CD4+/CD62Lhior CD4+/CD62LloT cells from spleens
were purified by MACS. Cell lysates were then analyzed by immunoblotting. (C) Immunostaining of FoxO3a in peripheral blood T cells from Mst1+/2
and Mst12/2mice. Lymphocytes were stained for CD4 (green) and FoxO3a (red), or CD8 (red) and FoxO3a (green); nuclei were stained with DAPI
(blue). (D) Immunostaining of p-FoxO1/3 in peripheral blood T cells from Mst1+/2and Mst12/2mice. Lymphocytes were stained for CD4 (green) and
p-FoxO1/3 (red), or CD8 (red) and p-FoxO1/3 (green); nuclei were stained with DAPI (blue).
Role of Mst1 in Naive T Cells
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produce DNA double-strand breaks (DSBs) in mammalian cells
. We therefore examined cH2AX, an established measure of
DSBs, in T cells. As expected, CD4+and CD8+T cells from
Mst12/2mice showed an increase in cH2AX signals compared
with Mst1+/2T cells (Figure S6), indicating the presence of DSBs.
Intracellular ROS can regulate Bcl-2 levels in activated T cells ,
and the Bim/Bcl-2 balance is critical for maintaining naı ¨ve and
memory T cell homeostasis . Therefore, we also investigated
Figure 2. Surface expression of L-selectin, CCR7, and IL-7Ra in Mst12 2/ /2 2peripheral T cells. L-selectin (n$6), CCR7 (n$3), and CD127 (n$3)
expression on naive T cells from peripheral blood (PB), spleen (Sp), lymph node (LN) were quantified by FACS. MFI, mean fluorescent intensity.
*, p,0.05; **, p,0.01; ***, p,0.001.
Figure 3. Dysregulation of FoxO1/3 results in down-regulation of Sod2 and catalase. Quantitative RT-PCR analysis of Sod2 and catalase
mRNA expression in lymphocytes from thymus, spleen, and inguinal lymph nodes of Mst1+/2and Mst12/2mice. Lymphocytes from spleen and lymph
nodes were further purified into CD4+and CD8+T cells by MACS. Target mRNA expression levels were normalized to endogenous b-actin. Fold
changes were calculated by measuring Mst12/2/Mst1+/2ratios. Data are pooled from three independent experiments of triplicate. Error bars indicate
SEM. *, p,0.05; **, p,0.01; ***, p,0.001.
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Bcl-2 involvement in the T cell death associated with increased
ROS in Mst12/2mice. Bcl-2 mRNA expression was decreased
about 2- to 3-fold in Mst12/2compared with Mst1+/2T cells;
however, the expression level of BimEL, an antagonist of Bcl-2, in
Mst12/2T cells was increased compared with controls (Figure 4B).
Thus, elevated intracellular ROS in Mst12/2T cells ultimately
decreased Bcl-2 levels, creating a disruption in the balance
between Bcl-2 and BimELthat might contribute to the apoptosis of
peripheral Mst12/2T cells.
To determine whether the increased apoptosis of Mst12/2T
cells was primarily due to ROS, we incubated Mst12/2T cells
from peripheral blood with the antioxidant, N-acetyl-L-cysteine
(NAC). At an NAC concentration of 10 ng/ml, intracellular ROS
was reduced (Figure 4C, left) and apoptosis was decreased in
Mst12/2CD4+and CD8+T cells (Figure 4C, right), suggesting
that the elevated levels of ROS in peripheral Mst12/2T cells
resulted in cell death. Although NAC treated Mst1+/2T cells had
lower ROS levels than untreated cells, apoptosis in these cells was
not affected by NAC treatment. Although NAC treated Mst12/2
CD8+T cells have modestly lower levels of ROS compared with
untreated Mst12/2CD8+T cells, it is also noted that ROS levels
in NAC treated Mst12/2CD8+T cells were not reduced as much
as in CD4+T cells. These differences in response to NAC
treatment between CD4+and CD8+T cells may account for
smaller change in Mst12/2CD8+T cell death compared to CD4+
T cell death. Taken together, these results suggest that Mst1-
deficient naı ¨ve T cells fail to activate FoxO proteins, resulting in a
failure to induce Sod2 and catalase expression, the accumulation
of intracellular ROS, and increased apoptosis in single-positive T
We next generated Mst1 wild-type transgenic (Mst1 Tg) and
kinase-dead transgenic (Mst1KD Tg) mice, then tested whether the
observed defects were corrected in the Mst1 Tg;Mst12/2or
Mst1KD Tg;Mst12/2progeny. In Mst1 Tg;Mst12/2mice,
intracellular ROS levels in CD4+and CD8+T cells were
decreased to levels comparable to those found in Mst1+/2
peripheral T cells (Figure 5A), and the numbers of peripheral
naı ¨ve T cells were recovered to an even greater extent in Mst1
Figure 4. Mst1 is important in ROS regulation and peripheral T cell survival. (A) Intracellular ROS levels in peripheral blood CD4+and CD8+
T cells from Mst1+/2and Mst12/2mice were detected by staining with DCF-DA. Data are representative of three independent experiments. (B)
Quantitative RT-PCR analysis of Bcl-2 and BimELmRNA expression in splenic CD4+and CD8+T cells (n=5 for Bcl-2 and n=3 for BimEL). **, p,0.01. (C)
ROS levels (left) and apoptotic cell death (right) in peripheral blood T cells from Mst1+/2and Mst12/2mice (n$5 for each) with or without NAC
(10 ng/ml) treatment were determined. Relative FITC-median values of DCF-DA fluorescence were analyzed for CD4+and CD8+populations. The
percentage of Annexin V-positive cells was determined by normalization to the percentage of Mst1+/2Annexin V-positive cells (defined as 100%).
Error bars indicate SEM.
Role of Mst1 in Naive T Cells
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Tg;Mst12/2mice (Figure 5B). In Mst1 Tg;Mst12/2mice,
FoxO3a, Sod2, and catalase protein levels in T cells were also
recovered to levels comparable to those in Mst1+/2T cells (Figure
S5). Notably, expression of kinase-dead Mst1 in Mst1KD
Tg;Mst12/2mice failed to restore normal numbers of peripheral
blood T cells (Figure 5B), indicating that Mst1 kinase activity
toward FoxO1/3 is critical for peripheral T cell homeostasis.
Expression of FoxO3a-S207D Rescues the Mst1-Null
To determine whether Mst1-mediated phosphorylation of
FoxO is sufficient to promote the neutralization of elevated
intracellular ROS in peripheral T cells, we infected Mst12/2bone
marrow cells with a retrovirus expressing a constitutively active
FoxO3a-S207D mutant (FoxO3a-SD), and then transplanted
infected bone marrow into lethally irradiated recipient mice. This
construct conferred a 3- to 4-fold survival advantage on peripheral
Mst12/2CD8+T cells, examined 4 weeks after transplantation,
compared to those transplanted with cells transduced with a
control pMSCV-IRES-GFP (Mig) virus (Figure 5C; p=0.014). In
contrast, transplanted bone marrow cells transduced with a non-
phosphorylatable S207A FoxO3a mutant (FoxO3a-SA) were
much less effective in rescuing the population of Mst12/2CD8+
T cells than were FoxO3a-SD-transduced cells (Figure 5C;
p=0.067). Interestingly, the Mst12/2CD4+T cell population
showed maximum survivability in peripheral blood after trans-
Figure 5. Expression of Mst1 and FoxO3a-SD rescues the peripheral T cell phenotypes of Mst12 2/ /2 2mice. (A) Flow cytometric analysis of
ROS levels in peripheral blood T lymphocytes from Mst1+/2, Mst12/2, and Mst1 Tg;Mst12/2mice. FITC-median values (FITC-MV) of DCF-DA
fluorescence are indicated. Data are representative of three independent experiments. (B) Naı ¨ve and effector/memory T cell subsets of peripheral
blood from Mst1+/2, Mst12/2, Mst1 Tg;Mst12/2, and Mst1KD Tg;Mst12/2mice (n$3) were quantified by FACS. *, p,0.05; **, p,0.01; ***, p,0.001,
compared with Mst1+/2lymphocytes. (C) Bone marrow cells from Mst12/2mice were transduced with Mig, Mig-FoxO3a, Mig-FoxO3a-SD, or Mig-
FoxO3a-SA retroviral constructs, and 3,66105GFP-positive cells were intravenously injected into irradiated normal C57BL/6J mice. After 4 weeks,
peripheral blood GFP+CD4+and GFP+CD8+T cells were quantified by FACS (n=5), and the values were expressed as a percentage of GFP-positive
lymphocytes in each group. (D) Relative numbers of surviving cells in peripheral blood from Mst1+/2, Mst1 Tg;Mst1+/2, and Mst12/2mice after
irradiation or paraquat injection. Left: Mice were whole-body irradiated at a dose of 5 Gy, and tail blood was collected before and 72 h after
irradiation (n=3). Number of surviving T cells after irradiation was divided by the pre-irradiation number for each genotype and expressed relative to
the Mst1+/2T-cell survival fraction. *, p,0.05; **, p,0.01. Right: Paraquat (5 mg/kg) was intraperitoneally injected into mice (n=3) and tail blood was
collected before, 48 h, 96 h, and 144 h after injection. The number of surviving T cells was expressed as a fraction of cell numbers before paraquat
injection for each genotype. Error bars indicate SEM.
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plantation with bone marrow cells transduced with wild-type
FoxO3a. The responsiveness of FoxO3a-SD could be different in
peripheral CD4+and CD8+T cells in the context of oxidative
Mst1 Confers ROS Resistance in Lymphocytes
Lymphocytopenia can be induced by a variety of cytotoxic
stresses, including chemotherapy and radiation. Radiation gener-
ates oxygen-derived free radicals in peripheral lymphocytes .
To address the capacity of Mst1 in T cells to protect against
irradiation-induced lymphopenia, we examined peripheral T cell
viability after exposure to c-irradiation (5 Gy). Seventy-two hours
after c-irradiation, we found dramatic increases in the viability of
peripheral CD4+(,2.5-fold) and CD8+(,4-fold) T cells from
Mst1 Tg;Mst+/2mice compared to Mst1+/2T cells (Figure 5D,
left). Using sublethal doses (5 mg/kg) of N,N’-dimethyl-4,4’-
bipyridinium dichloride (paraquat), a herbicide that induces the
formation of ROS , we found that intracellular ROS levels
were dramatically increased in peripheral CD4+and CD8+T cells
of Mst1+/2and Mst12/2mice within 24 hours (unpublished data).
However, overexpression of Mst1 (in Mst1 Tg;Mst+/2mice)
effectively neutralized intracellular ROS in peripheral T cells in
association with approximately a 3.5-fold increase in T cell
numbers 6 days after injection (Figure 5D, right).
These results suggest that Mst1 plays a role in promoting
survival of naı ¨ve T cells by detoxifying ROS through the Mst1-
FoxO-Sod2/catalase pathway (Figure 6). How does regulating
intracellular ROS maintain peripheral naı ¨ve T cell homeostasis?
The peripheral T cell population needs to be maintained at a near-
constant number. Naı ¨ve T cell maintenance in the periphery
depends on the appropriate stimulation of TCR, whereas effector/
memory cells can survive in the absence of TCR ligation [23–25].
Consistent with recent reports [7,8], we confirmed that Mst1 is
activated by TCR ligation in T cells (Figure S7). Importantly, we
observed that FoxO protein levels and phosphorylation of FoxO
proteins were reduced in Mst12/2T cells, and found that T cells
from Mst12/2mice exhibited higher levels of apoptosis (Figures 1
and 4C, right). Our findings thus suggest that the Mst1-FoxO-
Sod2/catalase pathway might tightly modulate intracellular ROS
in CD4+and CD8+T cells to maintain levels optimal for cell
Importantly, two recent studies showed that FoxO1 is critical
for the maintenance of naı ¨ve T cell homeostasis by controlling the
expression of IL-7Ra, L-selectin, and CCR7 [4,5]. FoxO1-
deficient T cells have defects in both survival and homing to
secondary lymphoid organs. Mst12/2T cells also have defects in
homing capacity [7,9], and the Rap1-Rapl-Mst1 pathway has
been previously suggested to play a role in controlling T-cell
trafficking [10,26]. However, unlike FoxO1-deficient T cells,
Mst1-deficient T cells appeared to show insignificant changes in
the expression of IL-7Ra, L-selectin, CCR7, but Sod2 and
catalase were significantly reduced (Figures 2, 3 and S5). It is
possible that although the residual levels of FoxO proteins in
Mst12/2T cells are too low to promote sufficient expression of
Sod2 and catalase to protect cells from oxidative damage, they are
high enough to maintain expression of other FoxO transcriptional
targets involved in T cell trafficking and survival. Thus, increased
ROS in Mst12/2naı ¨ve T cells might also result in a homing defect
to secondary lymphoid organs. In addition to the Rap1-Rapl-Mst1
pathway [7,10,26], the Mst1-FoxO pathway might also be
important for T cell trafficking. Unlike Mst12/2mice, which
show a deprivation of peripheral naı ¨ve T cells, Rapl-deficient mice
exhibit concentrations of peripheral blood lymphocytes compara-
ble to those of wild-type mice . We also found that Nore1/
Rassf5-deficient mice, which are deficient for both Nore1A and
Nore1B/Rapl splice variants, exhibited normal numbers of
peripheral blood lymphocytes, even though the numbers of T
cells in lymph node and spleen were reduced (Figure S8A). It is
also reported that Mst12/2thymocytes have defects in egress from
thymus, which result in lymphopenia in peripheral lymphoid
organs [7,9]. However, we did not observe the accumulation of
CD4 or CD8 single positive thymocytes in thymus from Mst12/2
mice (Figure S1D). It is interesting that Rapl-deficient mice also
have defects in thymocyte egress and ratio of CD4 or CD8 single
positive thymocytes were increased, but actual cell numbers of
these cells and numbers of T cells from peripheral blood were
apparently normal . Thus, the Rap1-Rapl-Mst1 pathway
could be responsible for controlling the T cell trafficking to
secondary lymphoid organs. By controlling intracellular ROS
levels, the Mst1-FoxO pathway is likely only important for the
survival of peripheral blood naı ¨ve T cells, a function that is
independent of the Rap1-Rapl-Mst1 pathway. We also found that
apoptosis was increased in splenic CD4+and CD8+T cells (Figure
S9A), but their ROS levels were not different from those of Mst1+/+
(Figure S9B). However, these cells showed decreased protein levels
of FoxO1/3 (Figures 1B and S5), Catalase and Sod2 (Figure S5).
Therefore, environmental differences between spleen and periph-
eral blood might have differentially affected ROS levels in splenic
and peripheral blood T cells of Mst12/2. In addition, Mst12/2
CD4+T cells were rescued by FoxO3a wild-type and FoxO3a-
S207A mutant but not by FoxO3a-S207D mutant (Figure 4C).
Based on these results, we can not exclude the possibility that there
might be still other functional mechanisms for regulating T cell
survival by FoxO1/3 and/or Mst1 can regulate T cell homeostasis
through other unknown factors.
Previous studies in yeast and mammalian cells have shown that
Mst1 promotes cell death in response to oxidative stress by
phosphorylating histone H2B [27,28]. In primary neurons, Mst1
enhances cell death by directly activating FoxO1/3 [6,11]. In
sharp contrast, our in vivo mouse study demonstrates for the first
time that Mst1 is crucial for cell survival rather than cell death in
response to intracellular ROS, at least in naı ¨ve T cells, and it
accomplishes this function by regulating FoxO proteins. Thus,
Mst1 appears to act in a cell context-dependent manner to
differentially regulate cell death and survival by modulating
Figure 6. Proposed model of Mst1-FoxO pathway in naı ¨ve T
cells. In peripheral T cells, Mst1 is activated after TCR ligation, followed
by FoxO1/3 activation, which is important for ROS regulation and
survival of naı ¨ve T cells. Mst1 might also be activated by Rapl through
TCR ligation during migration to lymphoid organs, resulting in LFA-1
clustering, as recently reported by others [7,8]. Arrow size indicates
intensity of activation of its target.
Role of Mst1 in Naive T Cells
PLoS ONE | www.plosone.org7 November 2009 | Volume 4 | Issue 11 | e8011
FoxO1/3 function. It will be interesting to determine whether the
loss of Mst1 promotes cell survival in response to oxidative stresses
in primary neurons.
The Hippo signaling pathway restricts cell proliferation and
promotes apoptosis in Drosophila . Consistent with this,
epithelial tissues of WW452/2mice also displayed hyperprolifera-
tion and defects in apoptosis . Hippo signaling has also been
proposed to regulate mammalian organ size , but neither
Mst12/2nor Mst22/2mice showed any epithelial hyperplasia or
increase in the size of any organs (unpublished data). Notably,
Mst22/2T cells showed no reduction in the numbers of T cells in
peripheral lymphoid organs (Figure S8B), suggesting that Mst2 is
dispensable for maintaining naı ¨ve T cells homeostasis. In addition,
Mst12/2;Mst22/2mice died at about E8.5 with severe growth
retardation, failed placental development, impaired yolks sac and
embryo vascular patterning, and increased apoptosis in placentas
and embryos . Thus, although Mst1 and Mst2 kinases are
functionally similar, both play essential roles in early mouse
development. Taken together, these observations suggest that
Mst1, but not Mst2, plays a distinct role in maintaining naı ¨ve T
Finally, we showed that overexpression of Mst1 rescues T cells
from apoptosis caused by abnormally elevated intracellular ROS
during exposure to anticancer treatments. Thus, Mst1 may
represent a potential target of therapeutic strategies to ameliorate
lymphopenic side effects in patients undergoing radiation
treatment or chemotherapy.
Materials and Methods
Animal care and experimentation were performed in accor-
dance with procedures approved by the KAIST (Korea Advanced
Institute of Science and Technology) Animal Care and Use
Generation of Mst12/2and Mst22/2mice was described in a
separate publication . Nore12/2mice were obtained from Dr.
Generation of Antibodies
An antibody to Mst1 was generated as previously described
. An antibody to p-FoxO1 (S212)/p-FoxO3 (S207) was
generated by injecting rabbits with the phosphopeptide antigen,
C-SAGWKNpSIRHNLS. Antibodies were affinity purified from
immune sera using the appropriate antigens.
Flow Cytometric Analysis
Thymus, spleen, and inguinal lymph nodes were filtered
through a 40-mm Cell Strainer (BD Falcon), and the resulting
lymphocytes were isolated by centrifugation over Lymphocyte
Separation Media (Mediatech Cellgro). Peripheral blood was lysed
with Ack lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM
Na2EDTA, pH 7.2) to obtain leukocytes. Isolated cells were
stained with antibodies against CD4 (GK1.5), CD8a (53-6.7;
Southern Biotech), CD44 (IM7), CCR7 (EBI-1), CD127 (SB/199;
eBioscience), and CD62L (MEL-14; BD Pharmingen). Intracellu-
lar ROS levels were assessed by prestaining 106cells with
antibodies against CD4 and CD8, followed by staining with
DCF-DA (Invitrogen) at a concentration of 20 mM for 30 min at
37uC. Apoptosis was determined in freshly isolated lymphocytes by
first prestaining for CD4 and CD8, after which lymphocytes were
stained with Annexin V staining kit (BD Pharmingen). The
mitochondrial transmembrane potential (ym) of lymphocytes was
determined by staining 106lymphocytes (prestained for CD4 and
CD8) with Rhodamine 123 at a final concentration of 4 mg/ml for
20 min at 37uC. Cells treated with 20 mM CCCP for 20 min
before Rhodamine 123 staining were used as a control. Analyses
were performed on a triple-laser LSRII flow cytometer with DiVA
software (Beckton Dickinson).
Magnetic-Activated Cell Sorting
Lymphocytes were isolated from spleen and lymph nodes. CD4+
and CD8+T cells were purified using CD4 and CD8a microbeads,
respectively, and CD4+/CD62LhiT cells were purified using
CD4+CD62L+T Cell Isolation Kit II with a VarioMacs magnetic
cell sorter (Miltenyi Biotec).
Magnetic-sorted lymphocytes were lysed in 0.5% NP-40 lysis
buffer containing protease and phosphatase inhibitors. Whole-cell
lysates were separated by SDS-PAGE on 7.5%–12% gradient gels,
transferred to nitrocellulose membranes, and immunoblotted with
antibodies against Mst1, FoxO1, FoxO3a (Cell Signaling), and p-
FoxO1 (S212)/p-FoxO3 (S207) (Biosource).
For intracellular staining, T lymphocytes were prestained for
CD4 and CD8, fixed with 4% paraformaldehyde, permeabilized
withSAPbuffer, andstainedwithantibodies against FoxO3a and p-
FoxO1(S212)/p-FoxO3(S207) (generated by our laboratory). Sam-
ples were analyzed by confocal microscopy (LSM510, Carl Zeiss).
Constructs and Bone Marrow Transplantation
Human FoxO3a-S207D and FoxO3a-S207A cDNAs were
generated and cloned into the pMSCV-IRES-GFP retroviral
backbone, MIG (Clontech). Retroviruses were produced by
transiently transfecting 293T cells with the plasmids together with
pEQPAM3 and pVSV-G (Clontech). Retroviruses were trans-
duced into bone marrow mononuclear (BMMN) cells harvested
from femurs and tibiae of 4–6-week-old Mst12/2mice using
‘‘spinfection’’ . These virally transduced BMMN cells were
injected into the tail vein of irradiated (12 Gy total: 7 Gy followed
by 5 Gy 3 h later) normal C57BL/6J mice. After 4 wk, blood was
collected and GFP-positive cells were analyzed by FACS.
In Vitro NAC Treatment
For in vitro NAC experiments, peripheral blood was collected
and lysed with Ack lysis buffer containing NAC (10 ng/ml,
Sigma). The concentration of NAC was maintained in all reagents
used in subsequent steps of DCF-DA staining and Annexin V
Total RNA from freshly isolated or magnetically sorted
lymphocytes from each organ was isolated using TRIzol Reagent
(Intron) according to the manufacturer’s instructions and treated
with DNaseI. RNA was reverse transcribed using Superscript III
(Invitrogen). Quantitative RT-PCR reactions were performed in
triplicate using iQ SYBR Green Supermix and the iQ5 Multicolor
Real-Time PCR Detection System, and data were analyzed using
iQ5 optical system software (Bio-Rad). Gene-specific primer
sequences were obtained from Primer Bank (http://pga.mgh.
harvard.edu/primerbank). PCR primers used were 59-CAG ACC
TGC CTT ACG ACT ATG G-39 and 59-CTC GGT GGC GTT
GAG ATT GTT-39 for Sod2; 59-AGA GAG CGG ATT CCT
Role of Mst1 in Naive T Cells
PLoS ONE | www.plosone.org8 November 2009 | Volume 4 | Issue 11 | e8011
GAG AGA-39 and 59-ACC TTT CCC TTG GAG TAT CTG G-
39 for catalase; 59-ATG CCT TTG TGG AAC TAT ATG GC-39
and 59-GGT ATG CAC CCA GAG TGA TGC-39 for Bcl-2; 59-
GAC AGA ACC GCA AGG TAA TCC-39 and 59-ACT TGT
CAC AAC TCA TGG GTG-39 for BimEL; and 59-AGG TCA
TCA CTA TTG GCA ACG A-39 and 59-CAC TTC ATG ATG
GAA TTG AAT GTA GTT-39 for b-actin.
Statistical analyses of differences between two groups were
performed using two-tailed Student’s t-tests. A p-value ,0.05 was
considered statistically significant.
control and Mst12/2mice. (A) H&E staining of splenic tissue
sections. Rp: red pulp; Wp: white pulp. (B) H&E staining of lymph
node tissue sections. TZ: T cell zone; BF: B cell follicle. (C) H&E
staining of thymus sections. M: medulla; C: cortex. (D) Thymocyte
subset numbers quantified from Mst1+/+(solid bars), Mst1+/2(grey
bars), and Mst12/2(open bars) mice by FACS (n$5). Error bars
indicate SEM. (E) Representative FACS profiles of T cell subsets
in thymus from Mst1+/+and Mst12/2mice.
Found at: doi:10.1371/journal.pone.0008011.s001 (3.80 MB TIF)
Reduced numbers of peripheral T cells in Mst12/2
mice. (A) Total lymphocytes, CD4+T cells and CD8+T cells in
spleen (n$6), inguinal lymph nodes (n$4), and peripheral blood
(n$8) from Mst1+/+(solid bars), Mst1+/2(grey bars), and Mst12/2
(open bars) mice (age 6–8 weeks) were quantified. **, p,0.01;
***, p,0.001; n.s., not significant, compared with Mst1+/+
lymphocytes. (B) NaA˜¯ve (CD62LhiCD44lo) and effector/memory
(CD62LloCD44hi) T cell subset numbers in spleen (n$4), lymph
nodes (n$4), and peripheral blood (n$6) from Mst1+/+(solid bars),
Mst1+/2(grey bars) and Mst12/2(open bars) mice were quantified
by FACS. Error bars indicate SEM. **, p,0.01; ***, p,0.001,
compared with Mst1+/+lymphocytes.
Found at: doi:10.1371/journal.pone.0008011.s002 (0.29 MB TIF)
Inhibition of Fas-FasL interaction in Mst12/2mice
did not increase the number of peripheral lymphocytes. Faslpr/lpror
FasLgld/gldmice were crossed with Mst12/2mice, and CD4+and
CD8+T cell numbers in peripheral blood collected from progeny
(5-6-weeks old) were quantified. Data show that CD4+or CD8+T
cells were not rescued in Mst12/2;Faslpr/lpror Mst12/2;FasLgld/gld
mice compared to Mst12/2mice (n=3). Error bars indicate SEM.
Found at: doi:10.1371/journal.pone.0008011.s003 (0.15 MB TIF)
Analysis of mouse cytokine levels in serum from Mst1+/2
and Mst12/2mice. Blood from Mst1+/2(solid bars) and Mst12/2
(grey bars) mice collected by tail bleeding was allowed to clot for
2 hours at room temperature before centrifuging for 20 minutes at
approximately 20006g. Relative levels of cytokines and chemokines
were determined by assaying sera according to the manufacturer’s
instruction (Proteome Profiler Array; R&D systems).
Found at: doi:10.1371/journal.pone.0008011.s004 (0.67 MB TIF)
Histology of spleen, lymph nodes, and thymus from
Mst12/2peripheral T cells. Western blot analysis of splenocytes
Downstream targets of FoxO1/3 are reduced in
from Mst1+/2, Mst12/2and Mst1 Tg;Mst12/2mice. CD4+and
CD8+T cells from spleens were purified by MACS. Cell lysates
were then analyzed by immunoblotting. Similar results were
obtained from three independent experiments.
Found at: doi:10.1371/journal.pone.0008011.s005 (0.45 MB TIF)
c-H2AX levels in peripheral blood CD4+and CD8+
T cells from Mst1+/2and Mst12/2mice. For phospho-Histone
H2AX detection, lymphocytes (prestained for CD4 and CD8)
were fixed with 4% paraformaldehyde, permeabilized with SAP
buffer (0.1% saponin, 0.05% NaN3 in Hank’s Balanced Salt
Solution) and stained with anti-phospho-Histone H2AX-FITC
Found at: doi:10.1371/journal.pone.0008011.s006 (0.24 MB TIF)
analysis of wild-type T lymphocytes after TCR stimulation.
Splenocytes were purified by MACS. Purified CD4+(26106cells)
or CD8+(16106cells) T lymphocytes were cultured on 24-well
plates containing pre-bound anti-CD3 or anti-CD3/CD28
(10 ug/ml) antibodies for 24 h. Lysates were separated by SDS-
PAGE and immunoblotted for FoxO3a, p-FoxO1/3, MST1, and
Found at: doi:10.1371/journal.pone.0008011.s007 (0.35 MB TIF)
Mst1 is activated after TCR stimulation. Western blot
mice. (A) Total lymphocytes, CD4+T cells, CD8+T cells, and
naA˜¯ve (CD62LhiCD44lo) and effector/memory (CD62LloCD44hi)
T cell subset numbers in spleen, inguinal lymph nodes, and
peripheral blood were quantified from wild-type (solid bars), and
Nore12/2(open bars) mice (age 6-8 weeks, n=3 for each organ
from one experiment). (B) T cell subset numbers in spleen, lymph
nodes, and peripheral blood from wild-type (solid bars) and
Mst22/2(open bars) mice (age 6–8 weeks, n$3 for each organ
from two independent experiments) were quantified. Error bars
Found at: doi:10.1371/journal.pone.0008011.s008 (0.26 MB TIF)
Peripheral T cell subsets from Nore12/2and Mst22/2
cells from spleen. (A) Apoptotic cell death of peripheral blood T
cells from Mst1+/+and Mst12/2mice (n$3) was detected. Relative
percentage of Annexin V-positive cells was determined. Error bars
indicate SEM. (B) Intracellular ROS levels in splenic CD4+and
CD8+T cells from Mst1+/+and Mst12/2mice were detected by
staining with DCF-DA (n=3). Relative FITC-median values of
DCF-DA fluorescence were analyzed for CD4+and CD8+
populations. Error bars indicate SEM.
Found at: doi:10.1371/journal.pone.0008011.s009 (0.16 MB TIF)
Cell death and intracellular ROS levels in Mst12/2T
We thank Drs. Jaeyul Kwon, Woong-Kyung Suh, and Pamela Schwartz-
berg for critically reading the manuscript.
Conceived and designed the experiments: JC SO DSL. Performed the
experiments: JC SO DL. Analyzed the data: JC SO DL. Contributed
reagents/materials/analysis tools: HJO JYP SBL. Wrote the paper: JC SO
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