Bcl2 Deficiency Activates FoxO through Akt Inactivation and Accelerates Osteoblast Differentiation
et al. (2014) Bcl2 Deficiency Activates FoxO through Akt Inactivation and Accelerates Osteoblast
Differentiation. PLoS ONE 9(1): e86629. doi:10.1371/journal.pone.0086629
Bcl2 Deficiency Activates FoxO through Akt Inactivation and Accelerates Osteoblast Differentiation
Takeshi Moriishi 0
Yosuke Kawai 0
Hisato Komori 0
Satoshi Rokutanda 0
Yutaka Eguchi 0
Yoshihide Tsujimoto 0
Izumi Asahina 0
Toshihisa Komori 0
Irina V. Lebedeva, Columbia University, United States of America
0 1 Department of Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan, 2 Department of Regenerative Oral Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan, 3 Department of Oral and Maxillofacial Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan, 4 Department of Molecular Genetics, Osaka University Medical School , Osaka , Japan
Osteoblast apoptosis plays an important role in bone development and maintenance, and is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging. Although Bcl2 subfamily proteins, including Bcl2 and Bcl-XL, inhibit apoptosis, the physiological significance of Bcl2 in osteoblast differentiation has not been fully elucidated. To investigate this, we examined Bcl2-deficient (Bcl22/2) mice. In Bcl22/2 mice, bromodeoxyuridine (BrdU)positive osteoblasts were reduced in number, while terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive osteoblasts were increased. Unexpectedly, osteoblast differentiation was accelerated in Bcl22/2 mice as shown by the early appearance of osteocalcin-positive osteoblasts. Osteoblast differentiation was also accelerated in vitro when primary osteoblasts were seeded at a high concentration to minimize the reduction of the cell density by apoptosis during culture. FoxO transcription factors, whose activities are negatively regulated through the phosphorylation by Akt, play important roles in multiple cell events, including proliferation, death, differentiation, longevity, and stress response. Expressions of FasL, Gadd45a, and Bim, which are regulated by FoxOs, were upregulated; the expression and activity of FoxOs were enhanced; and the phosphorylation of Akt and that of FoxO1 and FoxO3a by Akt were reduced in Bcl22/2 calvariae. Further, the levels of p53 mRNA and protein were increased, and the expression of p53-target genes, Pten and Igfbp3 whose proteins inhibit Akt activation, was upregulated in Bcl22/2 calvariae. However, Pten but not Igfbp3 was upregulated in Bcl22/2 primary osteoblasts, and p53 induced Pten but not Igfbp3 in vitro. Silencing of either FoxO1 or FoxO3a inhibited and constitutively-active FoxO3a enhanced osteoblast differentiation. These findings suggest that Bcl2 deficiency induces and activates FoxOs through Akt inactivation, at least in part, by upregulating Pten expression through p53 in osteoblasts, and that the enhanced expression and activities of FoxOs may be one of the causes of accelerated osteoblast differentiation in Bcl22/2 mice.
Funding: This study was supported by the Ministry of Education, Science, Sports and Culture and Technology Grant-in-Aid for Scientific Research. 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.
Osteoblast apoptosis plays an important role in bone
development and maintenance. All major regulators of bone metabolism,
including estrogen, androgen, parathyroid hormone (PTH), locally
produced factors like interleukin 6 (IL-6)-type cytokine, bone
morphogenetic proteins (BMPs), insulin-like growth factor-1
(IGF1), Wnts, PTH-related peptide (PTHrP), mechanical forces, and
oxidative stress, modulate osteoblast and osteocyte apoptosis . It
is estimated that 6080% of osteoblasts that originally assembled
at the resorption pit die by apoptosis. Further, bone loss caused by
sex steroid deficiency, glucocorticoid excess, or aging is due in part
to osteoblast apoptosis, and PTH, bisphosphonate, and calcitonin
exert anabolic action on bone by inhibiting osteoblast and
osteocyte apoptosis, , , , , , , , . Bcl2
subfamily proteins, including Bcl2 and Bcl-XL, inhibit apoptosis
through prevention of the release of caspase activators from
mitochondria by inhibiting Bax subfamily proteins . Thus, the
bone loss caused by sex steroid deficiency, glucocorticoid excess, or
aging might be inhibited by Bcl2; however, the physiological
significance of Bcl2 in osteoblast differentiation and bone
development and maintenance has not been fully investigated.
Activation of phosphatidylinositol 3-kinase (PI3K) by a number
of growth factors, including insulin and insulin-like growth factors
(IGF), results in the production of
phosphatidylinositol-(3,4,5)triphosphate (PIP3), and this in turn causes localization of the
kinase Akt to the plasma membrane. At the plasma membrane,
Akt can be phosphorylated by Pdk1 and mTORC2 (consisting of
the kinase mTOR, Rictor, Sin1, and mLST8 complex), leading to
its full activation. Activated Akt phosphorylates a subset of targets,
including the FoxO family of transcription factors, which include
FoxO1, FoxO3a, and FoxO4. Phosphorylated FoxO factors
interact with the adaptor 14-3-3, which promotes relocalization
to the cytoplasm. Oxidative stress opposes nuclear export by
alternative phosphorylation of FoxO factors. Phosphorylation
mediated by JNK and Mst1, which are activated by oxidative
stress, promotes translocation to the nucleus , , . p53,
which senses various intrinsic and extrinsic stress signals, induces
the negative regulators, including Igfbp3 and Pten, in the
PI3KAkt pathway to shut down cell growth and division to avoid the
introduction of infidelity into the process of cell growth and
division , . Igfbp3 binds to free IGF-1 and prevents it from
binding to the IGF-1 receptors, and Pten reverses the effects of
PI3K by dephosphorylating PIP3 .
Recently, FoxO-dependent oxidative defense was shown to be
important for bone formation and bone mass homeostasis ,
. FoxOs inhibit osteoblast apoptosis through the suppression of
oxidative stress . Further, FoxO1 regulates osteoblast
proliferation through the interaction with ATF4, a transcription factor
regulating amino acid import, as well as through the suppression of
p19ARF and p16 and downstream activation of their target
protein p53 . Further, FoxO1 has been shown to regulate
osteoblast differentiation , .
The previous reports showed that osteoblast apoptosis was
unchanged or increased, and osteoblast differentiation was
unchanged or inhibited in Bcl2-deficient (Bcl22/2) primary
osteoblasts compared with wild-type primary osteoblasts in vitro
, . However, we found that osteoblast differentiation is
inhibited in osteoblast-specific Bcl2 transgenic mice . Further,
we found that differentiation of the primary osteoblasts from Bcl2
transgenic mice is also inhibited in vitro, but that it is affected by
apoptosis, because osteoblast apoptosis reduces cell density and
leads to the deceleration of osteoblast differentiation . Thus,
we examined osteoblast proliferation, apoptosis, and
differentiation in the bone tissues of Bcl22/2 mice to evaluate the
physiological roles of Bcl2 in osteoblasts. Contrary to the previous
reports, osteoblast differentiation was accelerated in Bcl22/2 mice.
The differentiation of Bcl22/2 primary osteoblasts was also
accelerated in vitro, when the cells were seeded at a high
concentration to minimize the reduction of the cell density by
apoptosis during culture. Thus, we further pursued the mechanism
of enhanced osteoblast differentiation in vivo using bone tissues.
Here, we show that the deletion of Bcl2 accelerated osteoblast
differentiation, at least in part, through the Akt-FoxO pathway.
Materials and Methods
Prior to the study, all experiments were reviewed and approved
by the Animal Care and Use Committee of Nagasaki University
Graduate School of Biomedical Sciences. (Permit Number:
Bcl22/2 mice were generated as previously described .
Briefly, ES cells derived from 129/Ola were injected into the
blastocysts recovered from the mating of B6C3F1 (C57BL/6 x
C3H F1) with C57BL/6, and the chimeric mice were mated with
ICR. Wild-type, heterozygous, and homozygous mice were
obtained by brother-sister mating of heterozygous mice.
The bone histomorphometric analysis was performed by
measuring the area and perimeter of trabecular bone of femurs
at 2 weeks of age with Image J using the H-E stained
paraffinembedded sections. For histological analyses of the long bones,
mice were sacrificed and fixed in 4% paraformaldehyde/0.01 M
phosphate-buffered saline, and the long bones were decalcified in
10% EDTA (pH7.4) and embedded in paraffin. Sections (37 mm
thick) were stained with hematoxylin and eosin (H-E) or stained for
TUNEL using the ApopTagH system (Intergen, Burlington, MA),
or subjected to in situ hybridization using Col1a1, osteopontin,
and osteocalcin probes . For the BrdU incorporation study,
mice of 2 weeks of age were injected intraperitoneally with 100 mg
BrdU/gram body weight and sacrificed 1 hour later. Sections were
stained with the BrdU staining kit (Zymed, San Francisco, CA). In
the counting of TUNEL-positive or BrdU-positive osteoblastic
cells, only the cells in the distal primary spongiosa of femurs, which
were recognized as osteoblastic cells from the morphology and
attachment to the trabecular bone, were counted.
Real-time RT-PCR and Western Blot Analyses
Total RNA was extracted using ISOGEN (Wako, Osaka,
Japan), and real-time RT-PCR was performed as previously
described . Primer sequences are shown in Table S1. We
normalized the values to that of Gapdh. Western blot analysis was
performed using the following antibodies: anti-Akt,
anti-phosphorylated Akt, anti-FoxO1, anti-FoxO3a, anti-phosphorylated
FoxO1 (Thr24)/FoxO3a (Thr32), anti-JNK, anti-phosphorylated
JNK, anti-Mst1, and anti-phosphorylated Mst1 antibodies (Cell
Signaling, Danvers, MA); anti-phosphorylated FoxO3a (S207)
antibody (Invitrogen, Tokyo, Japan); and anti-actin antibody
(Santa Cruz Biotechnology, Santa Cruz, CA).
In situ Hybridization
For in situ hybridization, we prepared
digoxigenin-11-UTPlabeled single-stranded RNA probes using a DIG RNA labeling kit
(Roche Biochemica) according to the manufacturers instructions.
We used a 0.32 kb fragment of Col1a1 cDNA , a 1.2 kb
fragment of mouse osteopontin cDNA , and a 0.47 kb fragment
of mouse osteocalcin cDNA  to generate antisense and sense
probes. We carried out hybridization as previously described 
and counterstained the sections with methyl green.
Cell Culture Experiments
Primary osteoblasts were isolated from newborn calvaria by
sequential digestion with 0.1% collagenase A and 0.2% dispase.
Osteoblastic cells from the third to fifth fraction were pooled,
plated on 48-well plates at a density of 2.56104/well and 24-well
plates at a density of 56104/well, and used for MTT, osteoblast
differentiation, and TUNEL assays. To examine osteoblast
differentiation, staining for alkaline phosphatase (ALP) activity
and mineralization was performed as previously described .
Quantification of mineralization was performed using VHX-1000
(KEYENCE) and Image J. TUNEL-positive cells were detected
using the ApopTagH system (Intergen, Burlington, MA).
FoxO3aAAA triple mutant (FoxO3a-TM) adenovirus was a gift from K.
Walsh (Boston University Medical School) . In FoxO3a-TM,
the three phosphorylation sites, Thr-32, Ser-253, and Ser-315,
were replaced by alanine residues. MC3T3-E1 cells were infected
with the retrovirus vector (pSIREN-RetroQ, Takara Bio, Inc.
Otsu, Japan) expressing each shRNA for GFP, FoxO1, and FoxO3a,
and cultured for 3 days in the presence of puromycin. BMP2
(100 ng/ml) was added to the medium at confluence. A p532/2
osteoblast cell line, which was established from p532/2 calvarial
cells, was infected with human p53-expressing retrovirus or empty
retrovirus. Retrovirus was constructed by inserting full length
human p53 cDNA into pDON-5 (Takara Bio, Inc.).
Primary osteoblasts from wild-type and Bcl22/2 mice were
transfected with the Gadd45a promoter construct  and
pRLCMV by FuGENE 6 (Roche Diagnostics, Tokyo, Japan).
Luciferase activity was normalized to Renilla luciferase activity
using pRL-CMV (Promega, Madison, WI).
Statistical analyses were performed using Students t-test.
Ekuseru-Toukei 2010 (Social Survey Research Information Co.,
Ltd., Tokyo, Japan). Data are presented as the mean 6 S.D. A
Pvalue of less than 0.05 was considered significant.
Increase in Bone Mass and Osteoblast Apoptosis in
As Bcl22/2 mice died at approximately 23 weeks of age, bone
histomorphometric analysis was performed on the trabecular bone
of femurs at 2 weeks of age (Fig. 1A). The bone volume was
increased in Bcl22/2 mice and the density of osteoblasts in Bcl22/
2 mice was similar to that in wild-type mice. In contrast, the
density of osteoclasts was reduced in Bcl22/2 mice. The
percentage of BrdU-positive osteoblastic cells in Bcl22/2 mice
was less than that in wild-type mice (Fig. 1B, C, F), while the
percentage of TUNEL-positive osteoblastic cells was increased in
Bcl22/2 mice compared with wild-type mice (Fig. 1D, E, G). The
percentage of TUNEL-positive osteocytes in Bcl22/2 mice was
similar to that in wild-type mice (Fig. 1H). The expression of
apoptosis-related genes, including Fas, FasL, p53, Noxa, Bax, Bid,
Bim, Bad, Bnip3l, was increased in calvaria of Bcl22/2 mice
compared with wild-type mice (Fig. 1I).
Osteoblast Differentiation was Accelerated in Bcl22/2
We examined the expression of osteoblast differentiation marker
genes, including Runx2, Osterix, Col1a1, osteopontin, and osteocalcin, in
calvariae of Bcl22/2 mice by real-time RT-PCR analysis. Runx2
and Osterix are upregulated in preosteoblasts, Col1a1 and osteopontin
are upregulated in immature osteoblasts, and osteocalcin is
upregulated in mature osteoblasts , . The expressions of
all of these markers were increased in Bcl22/2 mice compared
with wild-type mice (Fig. 2A). Further, we examined osteoblast
differentiation by in situ hybridization at birth and 2 weeks of age.
Col1a1-expressing cells and osteopontin-expressing cells were
increased at birth and 2 weeks of age in Bcl22/2 mice compared
with wild-type mice, reflecting the increased bone volume and
similar osteoblast density compared with those in wild-type mice
(Fig. 1A, 2DG, NQ). In wild-type mice, there were few
osteocalcin-expressing cells and its expression level was low at birth,
but both the number and expression level were increased in the
bone collar and the trabecular bone near the bone collar but not in
the other trabecular bone at 2 weeks of age (Fig. 2H, J, R, T). In
Bcl22/2 mice, however, osteocalcin-expressing cells were apparently
present in both the bone collar and trabecular bone at birth and
they were observed in the entire trabecular bone at 2 weeks of age
(Fig. 2I, K, S, U). These findings indicate that osteoblast
differentiation was accelerated in Bcl22/2 mice.
Proliferation, Differentiation, and Apoptosis of Bcl22/2
Osteoblasts in vitro
MTT assays showed that proliferation of Bcl22/2 osteoblasts
was reduced compared with that of wild-type osteoblasts (Fig. 3A).
Primary osteoblasts isolated from Bcl22/2 mice were seeded at a
concentration of 2.56104/cm2, ALP activity and the osteoblast
marker gene expression were examined after 6 days, and
mineralization was examined after 17 days (Fig. 3BD). The
ALP activity, mineralization, and the expression of ALP, Col1a1,
osteopontin, and osteocalcin were similar to those from wild-type mice
(Fig. 3BD). However, apoptosis of the proliferating osteoblasts
should affect the results of the MTT assay. Further, apoptosis
during culture should affect osteoblast differentiation, because
osteoblast differentiation in vitro is largely dependent on the cell
density . Thus, we examined apoptosis during osteoblast
proliferation and differentiation in vitro. Osteoblast apoptosis was
significantly increased not only during proliferation but also during
differentiation in Bcl22/2 osteoblasts (Fig. 3E).
To minimize the reduction of cell density by apoptosis, primary
osteoblasts isolated from wild-type, Bcl2+/2, and Bcl22/2 mice
were seeded at a higher concentration (26105/cm2) and ALP
activity and the osteoblast marker gene expression were examined
after 2 days (Fig. 3F, H). ALP activity and the expression of
osteopontin, ALP, and Osterix were increased in Bcl22/2 primary
osteoblasts compared with those in wild-type primary osteoblasts.
After 8 days, the mineralization was similar between wild-type and
Bcl22/2 primary osteoblasts, but osteocalcin mRNA was increased
in Bcl22/2 primary osteoblasts (Fig. 3F, G, I). Although ALP
activity was slightly increased in Bcl2+/2 primary osteoblasts
compared with wild-type primary osteoblasts, the mineralization
and the osteoblast marker gene expression were similar between
Bcl2+/2and wild-type primary osteoblasts (Fig. 3FI).
Upregulation and Activation of FoxOs in Bcl22/2
As Bcl2-deficiency enhanced osteoblast differentiation in vivo,
we examined the mechanism of the accelerated osteoblast
differentiation in vivo by directly analyzing the newborn calvariae.
The expressions of FasL, Gadd45a, and Bim, which are regulated by
FoxOs, were upregulated in Bcl22/2 calvariae (Fig. 1I). As FoxO1
enhances osteoblast differentiation , , FoxOs might be
involved in enhanced osteoblast differentiation in Bcl22/2 mice.
Thus, we first examined the expression and activity of FoxOs. The
expressions of FoxO1, FoxO3a, and FoxO4 mRNAs were increased
in Bcl22/2 calvariae compared with wild-type calvariae, and the
promoter activity of Gadd45a was enhanced in Bcl22/2 primary
osteoblasts compared with wild-type primary osteoblasts (Fig. 4A,
B). FoxO proteins are inactivated through the phosphorylation by
Akt. Akt itself is activated by phosphorylation (Fig. 4H) , ,
. Thus, we examined the activation state of Akt and FoxOs by
examining their phosphorylation. The phosphorylation of Akt was
markedly reduced in Bcl22/2 calvariae compared with wild-type
calvariae, although similar levels of Akt protein were detected
(Fig. 4C). Protein levels of FoxO1 and FoxO3a were increased,
whereas the phosphorylation of Thr24 in FoxO1 and that of
Thr32 in FoxO3, which are phosphorylated by Akt, were reduced
(Fig. 4C). These findings indicate that FoxO proteins were
activated by the inactivation of Akt. FoxO proteins are also
activated through the phosphorylation by JNK and Mst1, and
JNK and Mst1 are activated by phophorylation (Fig. 4H) ,
. Protein levels of JNK and Mst1 and the levels of their
phosphorylated forms were mildly reduced, and the
phosphorylation of S207 in FoxO3a, which is phosphorylated by JNK and
Mst1, was also mildly reduced in Bcl22/2 calvariae compared
with wild-type calvariae. These findings indicate that FoxO
proteins were not activated by JNK and Mst1. These findings
indicate that FoxO proteins were activated in Bcl22/2 primary
osteoblasts through the reduction in Akt phosphorylation but not
through the increase in JNK and Mst1 phosphorylation (Fig. 4H).
We further examined why Akt phosphorylation was inhibited in
Bcl22/2 calvariae. p53 has been shown to induce the expression of
Pten and Igfbp3, whose proteins inhibit Akt phosphorylation
Figure 1. Bone morphometric analysis, BrdU and TUNEL staining, and real-time RT-PCR analysis of apoptosis-related genes in
Bcl22/2 mice. (A) Bone histomorphometric analysis. The trabecular bone volume (bone volume/tissue volume, BV/TV), number of osteoblasts (N.Ob/
B.Pm), and number of osteoclasts (N.Oc/B.Pm) were compared in femurs between 6 wild-type and 4 Bcl22/2 mice at 2 weeks of age. B.Pm, bone
perimeter. (BH) BrdU labeling (B, C) and TUNEL staining (D, E) of sections of femurs from wild-type mice (B, D) and Bcl22/2 mice (C, E). Bars = 50 mm.
BrdU-positive osteoblastic cells (F), TUNEL-positive osteoblastic cells (G), and TUNEL-positive osteocytes (H) were counted and shown as a percentage
of the number of osteoblastic cells or osteocytes. wild-type mice, n = 7; Bcl22/2 mice, n = 5 in F. wild-type mice, n = 8; Bcl22/2 mice, n = 5 in G and H.
(I) Real-time RT-PCR analysis of apoptosis-related genes. RNA was directly extracted from newborn calvariae of wild-type and Bcl22/2 mice. wild-type
mice, n = 6; Bcl22/2 mice, n = 15. *vs. wild-type mice. *P,0.05, **P,0.01.
(Fig. 4H) , , . As p53 mRNA expression was increased
in Bcl22/2 calvariae (Fig. 1I), we confirmed that the protein level
of p53 was also increased in Bcl22/2 calvariae (Fig. 4D). Further,
Pten and Igfbp3 expression was increased in Bcl22/2 calvariae
In the culture of primary osteoblasts, the expression of p53 and
Pten but not Igfbp3 was increased in Bcl22/2 primary osteoblasts
compared with those in wild-type primary osteoblasts (Fig. 4F). To
examine whether p53 induces Pten and Igfbp3, we introduced p53
to p532/2 osteoblast cell line. Pten mRNA but not Igfbp3 mRNA
was induced by p53 (Fig. 4G). Further, FoxO3a mRNA was not
induced by p53 (data not shown). These findings suggest that Akt
phosphorylation was reduced, at least in part, by the induction of
Pten through upregulated p53 (Fig. 4CH).
FoxOs Enhanced Osteoblast Differentiation
To examine whether FoxOs are able to enhance osteoblast
differentiation, a constitutively active form of FoxO3a
(FoxO3aFigure 2. Expression of bone matrix protein genes in Bcl22/2 mice. (A) Real-time RT-PCR analysis of Bcl2, Runx2, Osterix, Col1a1, osteopontin,
and osteocalcin. RNA was directly extracted from newborn calvariae of wild-type (wt) and Bcl22/2 mice. The values of wild-type mice were defined as
1, and relative levels are shown. wild-type mice, n = 6; Bcl22/2 mice, n = 15. *vs. wild-type mice. *P,0.05, **P,0.01, ***P,0.001. (BU) In situ
hybridization analysis of Col1a1, osteopontin, and osteocalcin. The sections of femurs from Bcl2+/2 mice (B, D, F, H, J), Bcl22/2 mice (C, E, G, I, K, M, O,
Q, S, U), and wild-type mice (L, N, P, R, T) at birth (BK) and at 2 weeks of age (LU) were stained with HE (B, C, L, M) or hybridized with Col1a1 (D, E,
N, O), osteopontin (F, G, P, Q), and osteocalcin (HK, RU) probes. Boxed regions in H, I, R, and S are magnified in J, K, T, and U, respectively. Arrows in I
indicate the appearance of osteocalcin-expressing cells in the bone collar. Similar results were obtained in two newborn mice and three 2-week-old
mice in each genotype and representative data are shown. In situ hybridization using the sense probes showed no significant signals (data not
shown). Bars: 100 mm (BI, LS); 50 mm (J, K, T, U).
expression were examined at day 6, and mineralization was examined at day 17. The value of primary osteoblasts from wild-type mice was set as 1
and the relative level is shown. n = 3. Similar results were obtained in three independent experiments and representative data are shown. (E)
Frequencies of TUNEL-positive cells during culture. Primary osteoblasts from calvariae of 6 wild-type and 10 Bcl22/2 mice were stained for TUNEL
before confluence, at confluence, and at 3 and 9 days after confluence. n = 425. Similar results were obtained in two independent experiments and
representative data are shown. (FI) Differentiation of primary osteoblasts from calvariae of Bcl22/2 mice. Primary osteoblasts were seeded at a
concentration of 26105/cm2 (day 0), 50 mg/ml ascorbic acid and 10mM b-glycerophosphate were added at day 1, ALP activity was examined at day 2
(F), mineralization was examined at day 9 (G), and the osteoblast marker gene expression was examined at day 2 (H) and 9 (I) by real-time RT-PCR.
n = 7 in G; n = 5 in H; n = 10212 in I. Similar results were obtained in three independent experiments and representative data are shown. *vs. wild-type
primary osteoblasts. *P,0.05; **, ##P,0.01; ***p,0.001.
TM) was introduced into primary osteoblasts (Fig. 5AC).
FoxO3a-TM enhanced ALP activity, mineralization, and the
expression of Runx2, Osterix, ALP, and osteocalcin. Further, retroviral
introduction of shRNA of either FoxO1 or FoxO3a into MC3T3-E1
cells reduced mineralization (Fig. 5DF). These findings suggest
that FoxOs may be involved in the enhanced osteoblast
differentiation in Bcl22/2 mice.
The proliferation was reduced and apoptosis was increased, but
the differentiation was accelerated in osteoblasts and mature
osteoblasts were increased in Bcl22/2 mice. Therefore, our
findings indicate that bone mass was increased in Bcl22/2 mice
not only due to the decrease in osteoclast number but also due to
the acceleration of osteoblast differentiation.
The finding that osteoblast differentiation was accelerated in
Bcl22/2 mice was unexpected, because previous reports indicated
that osteoblast differentiation was unaffected or inhibited in
Bcl22/2 mice based on the data of in vitro differentiation of
Bcl22/2 osteoblasts , . The culture of primary osteoblasts
seeded at the concentration of 2.56104/cm2 also showed that the
differentiation of Bcl22/2 osteoblasts was unaffected in vitro.
However, the culture of primary osteoblasts seeded at the higher
concentration (26105/cm2) showed that the differentiation of
Bcl22/2 osteoblasts was accelerated. We recently reported that
overexpression of Bcl2 inhibits osteoblast differentiation in vivo
and in vitro . However, the inhibition of osteoblast
differentiation by over-expressed Bcl2 in vitro was dependent on the cell
density seeded, because overexpression of Bcl2 enhanced
osteoblast differentiation by increasing cell density through the
inhibition of apoptosis in vitro . Therefore, the discrepancy
in osteoblast differentiation in Bcl22/2 mice between our data and
previous reports was likely to be explained by the reduction in the
cell density during culture due to the increased apoptosis in Bcl22/
2 osteoblasts. Indeed, we cannot completely exclude the possibility
that the decreased number and dysfunction of osteoclasts in
Bcl22/2 mice indirectly affected the osteoblast differentiation
rather than in a cell autonomous manner.
In Bcl22/2 calvariae, mRNAs for FoxO1, FoxO3a, FoxO4, and
their target genes, including FasL, Gadd45a, and Bim, were
upregulated, and the promoter activity of Gadd45a was
enhanced in Bcl22/2 primary osteoblasts. Further, the
phosphorylation of FoxO1 and FoxO3a by Akt was reduced due to
the suppression of Akt, at least in part, through the upregulation
of Pten and Igfbp3, while the phosphorylation of FoxO3a by
JNK and Mst1 was not enhanced, suggesting that FoxOs were
activated in Bcl22/2 osteoblasts through the PI3K-Akt signaling
pathway. As Pten and Igfbp3 are target genes of p53 , ,
the activation of FoxOs by Akt may be dependent on p53 but
not Bcl2 itself. The expressions of Pten and Igfbp3 were
upregulated in Bcl22/2 calvarial tissues, whereas the expression
of Pten but not Igfbp3 was upregulated in Bcl22/2 primary
osteoblasts. Further, introduction of p53 induced the expression
of Pten but not Igfbp3. These findings indicate that upregulation
of p53 is sufficient for Pten induction in vivo and in vitro, but
that it is not sufficient for Igfbp3 induction in vitro. Therefore,
the molecules, which cooperate with p53 for Igfbp3 induction,
may be insufficient in vitro. Indeed, it is possible that other cell
types including lymphocytes, in which apoptosis is accelerated
, , contributed to the induction of Igfbp3 in Bcl22/2
calvarial tissues. p53 also inhibits FoxO3a activity by inducing
SGK, by directly inhibiting the transcriptional activity, or by
inducing FoxO3a degradation through Mdm2 , , .
Therefore, p53 seems to regulate FoxO activity positively or
negatively depending on the cell type and cell conditions. We
also showed the transcriptional upregulation of FoxOs in Bcl22/
2 calvariae. Recently, it has been shown that FoxO3a is a
target gene of p53 , . Further, FoxO1 and FoxO4 genes
are regulated by FoxO3a . Therefore, the increased p53
may be responsible for the upregulation of FoxO1, FoxO3a, and
FoxO4 mRNA expression in Bcl22/2 calvariae. However, the
introduction of p53 failed to induce FoxO3a mRNA in vitro
(data not shown). Therefore, the mechanism of the increase of
FoxOs mRNA in Bcl22/2 mice still remains to be clarified.
p53 has been shown to inhibit osteoblast differentiation ,
. However, it is evident in vitro but not in vivo, because the
calvarial bone volume is mildly reduced in p532/2 mice
compared with wild-type mice . Since the deletion of p53
enhances proliferation and inhibits apoptosis, p53 deletion
should increase the cell density in culture, leading to the
acceleration of osteoblast differentiation in vitro, because
osteoblast differentiation is dependent on the cell density in vitro
. Similarly, the increase in osteoblast number due to
increased proliferation and reduced apoptosis should also lead
to an increase in bone formation in p532/2 mice as previously
reported . Therefore, the function of p53 in osteoblast
differentiation needs to be further investigated.
Although osteoblast proliferation was not examined in vivo in
previously reported Bcl22/2 mice , , we showed that
the number of proliferating osteoblasts was reduced in Bcl22/2
mice. Further, we observed a reduction in the number of
Bcl22/2 primary osteoblasts in the MTT assay, suggesting that
Bcl2 enhances osteoblast proliferation. However, it could also
have been caused by increased apoptosis during culture.
Previous reports showed that Bcl2 inhibits cell proliferation by
facilitating G0 arrest and delaying G0 to S phase transition in
hematopoietic cells and fibroblasts , and various groups
showed that p27 as well as p130 was elevated in
Bcl2overexpressing cells during arrest , , , , although
overexpression of Bcl2 in myocytes promoted proliferation .
Thus, it is possible that the decrease in proliferating osteoblasts
in Bcl22/2 mice was mostly a reflection of enhanced osteoblast
differentiation, although the activation of FoxOs should have
affected both proliferation and differentiation of osteoblasts in
Bcl22/2 mice .
In summary, osteoblast differentiation was enhanced in
Bcl22/2 mice, at least in part, through FoxOs. FoxOs were
and 10mM b-glycerophosphate were added at day 1, and mRNA was extracted at day 4. The expression of p53, Pten, and Igfbp3 was examined by
real-time RT-PCR. Similar results were obtained in two independent experiments and representative data are shown. n = 12213. *vs. empty retrovirus.
**P,0.01, ***p,0.001. (H) Schematic presentation of the signaling pathway for FoxO activation. p53 induces Pten mRNA and Igfbp3 mRNA. Pten and
Igfbp3 inhibit Akt activation. Akt inhibits the activation of FoxOs. Activation of JNK and Mst1 activate FoxOs. p53 failed to induce Igfbp3 in vitro (G).
Dotted arrows indicate that the activation did not occur in Bcl22/2 mice (C).
activated through the suppression of Akt, at least in part, by
upregulation of Pten through p53. Although osteoblast apoptosis
is in part responsible for osteoporosis in sex steroid deficiency,
glucocorticoid excess, and aging, our findings suggest that the
stresses toward apoptosis
may have a positive effect on
We thank N. Motoyama and T. Sakai for Gadd45a promoter construct, K.
Ito for p53-expressing retrovirus, and C. Fukuda for secretarial assistance.
Conceived and designed the experiments: TK TM. Performed the
experiments: TM YK SR. Analyzed the data: TM. Contributed
reagents/materials/analysis tools: HK YE YT IA. Wrote the paper: TK.
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