Overexpression of Bcl2 in osteoblasts inhibits osteoblast differentiation and induces osteocyte apoptosis.
ABSTRACT Bcl2 subfamily proteins, including Bcl2 and Bcl-X(L), inhibit apoptosis. As osteoblast apoptosis is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging, bone loss might be inhibited by the upregulation of Bcl2; however, the effects of Bcl2 overexpression on osteoblast differentiation and bone development and maintenance have not been fully investigated. To investigate these issues, we established two lines of osteoblast-specific BCL2 transgenic mice. In BCL2 transgenic mice, bone volume was increased at 6 weeks of age but not at 10 weeks of age compared with wild-type mice. The numbers of osteoblasts and osteocytes increased, but osteoid thickness and the bone formation rate were reduced in BCL2 transgenic mice with high expression at 10 weeks of age. The number of BrdU-positive cells was increased but that of TUNEL-positive cells was unaltered at 2 and 6 weeks of age. Osteoblast differentiation was inhibited, as shown by reduced Col1a1 and osteocalcin expression. Osteoblast differentiation of calvarial cells from BCL2 transgenic mice also fell in vitro. Overexpression of BCL2 in primary osteoblasts had no effect on osteoclastogenesis in co-culture with bone marrow cells. Unexpectedly, overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteocytes, which had a reduced number of processes, gradually died with apoptotic structural alterations and the expression of apoptosis-related molecules, and dead osteocytes accumulated in cortical bone. These findings indicate that overexpression of BCL2 in osteoblasts inhibits osteoblast differentiation, reduces osteocyte processes, and causes osteocyte apoptosis.
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ABSTRACT: Background Osteocytes form a network through gap junction-coupled cell processes and canaliculi throughout bone; this network extends to the osteoblasts in the bone surface. The osteocyte network is considered to function in mechanosensing and mechanotransduction. However, the lack of suitable animal models makes it difficult to clarify the function of osteocytes. Highlight Any kind of osteocyte death results in necrosis, whereby the intracellular contents, including immunostimulatory molecules, which activate osteoclastogenesis, are released through the canaliculi to the bone surface. This leads to enhanced bone resorption in the damaged region of the bone. Overexpression of Bcl2 in osteoblasts reduces the number of osteoblast processes, resulting in a reduction in the numbers of osteocyte processes and canaliculi. The osteocytes gradually die without enhancement of bone resorption because a severe reduction in the number of canaliculi interrupts the release of intracellular contents to the bone surface. Bcl2 transgenic mice at 4 months of age, in which the osteocyte network is disrupted, are an appropriate mouse model for the evaluation of osteocyte function. These mice show that the osteocyte network enhances bone resorption and inhibits bone formation under physiological conditions, and that these osteocyte functions are augmented under unloaded conditions. Under such conditions, Rankl upregulation in osteoblasts and Sost upregulation in osteocytes are, at least in part, responsible for enhanced bone resorption and suppressed bone formation, respectively. Conclusion Dead osteocytes induce bone resorption, while live osteocytes enhance bone resorption and inhibit bone formation under physiological conditions, while their functions are augmented under unloaded conditions.Journal of Oral Biosciences 08/2014;
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ABSTRACT: Caspase-3 and -7 are generally known for their central role in the execution of apoptosis. However, their function is not limited to apoptosis and under specific conditions activation has been linked to proliferation or differentiation of specialised cell types. In the present study, we followed the localisation of the activated form of caspase-7 during intramembranous (alveolar and mandibular bones) and endochondral (long bones of limbs) ossification in mice. In both bone types, the activated form of caspase-7 was detected from the beginning of ossification during embryonic development and persisted postnatally. The bone status was investigated by microCT in both wild-type and caspase-7-deficient adult mice. Intramembranous bone in mutant mice displayed a statistically significant decrease in volume while the mineral density was not altered. Conversely, endochondral bone showed constant volume but a significant decrease in mineral density in caspase-7 knock-out mice. Cleaved caspase-7 was present in a number of cells that did not show signs of apoptosis. PCR array analysis of the mandibular bone of caspase-7-deficient versus wild-type mice pointed to a significant decrease in mRNA levels for Msx1 and Smad1 in early bone formation. These observations might explain the decrease in the alveolar bone volume of adult knock-out mice. In conclusion, this study is the first to report a non-apoptotic function of caspase-7 in osteogenesis and also demonstrates further specificities in endochondral versus intramembranous ossification.Cell Death & Disease 08/2014; 5:e1366. · 5.18 Impact Factor
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ABSTRACT: 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 (Bcl2(-/-)) mice. In Bcl2(-/-) 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 Bcl2(-/-) 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 Bcl2(-/-) 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 Bcl2(-/-) calvariae. However, Pten but not Igfbp3 was upregulated in Bcl2(-/-) 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 Bcl2(-/-) mice.PLoS ONE 01/2014; 9(1):e86629. · 3.53 Impact Factor
Overexpression of Bcl2 in Osteoblasts Inhibits Osteoblast
Differentiation and Induces Osteocyte Apoptosis
Takeshi Moriishi1, Zenjiro Maruyama1,2, Ryo Fukuyama3, Masako Ito4, Toshihiro Miyazaki1, Hideki
Kitaura5,8, Hidetake Ohnishi1,6, Tatsuya Furuichi1,9, Yosuke Kawai1,7, Ritsuko Masuyama1, Hisato
Komori1, Kenji Takada6, Hiroshi Kawaguchi2, Toshihisa Komori1*
1Department of Cell Biology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan, 2Department of Sensory and Motor System Medicine,
University of Tokyo, Tokyo, Japan, 3Laboratory of Pharmacology, Hiroshima International University, Kure, Japan, 4Department of Radiology and Radiation Biology,
Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan, 5Division of Orthodontic and Biomedical Engineering, Graduate School of Biomedical
Sciences, Nagasaki University, Nagasaki, Japan, 6Department of Orthodontics and Dentofacial Orthopedics, Faculty of Dentistry, Osaka University, Osaka, Japan,
7Department of Regenerative Oral Surgery, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan, 8Division of Orthodontics and Dentofacial
Orthopedics, Graduate School of Dentistry, Tohoku University, Sendai, Japan, 9Laboratory Animal Facility, Research Center for Medical Sciences, School of Medicine, Jikei
University, Tokyo, Japan
Bcl2 subfamily proteins, including Bcl2 and Bcl-XL, inhibit apoptosis. As osteoblast apoptosis is in part responsible for
osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging, bone loss might be inhibited by the upregulation of
Bcl2; however, the effects of Bcl2 overexpression on osteoblast differentiation and bone development and maintenance
have not been fully investigated. To investigate these issues, we established two lines of osteoblast-specific BCL2 transgenic
mice. In BCL2 transgenic mice, bone volume was increased at 6 weeks of age but not at 10 weeks of age compared with
wild-type mice. The numbers of osteoblasts and osteocytes increased, but osteoid thickness and the bone formation rate
were reduced in BCL2 transgenic mice with high expression at 10 weeks of age. The number of BrdU-positive cells was
increased but that of TUNEL-positive cells was unaltered at 2 and 6 weeks of age. Osteoblast differentiation was inhibited, as
shown by reduced Col1a1 and osteocalcin expression. Osteoblast differentiation of calvarial cells from BCL2 transgenic mice
also fell in vitro. Overexpression of BCL2 in primary osteoblasts had no effect on osteoclastogenesis in co-culture with bone
marrow cells. Unexpectedly, overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteocytes,
which had a reduced number of processes, gradually died with apoptotic structural alterations and the expression of
apoptosis-related molecules, and dead osteocytes accumulated in cortical bone. These findings indicate that overexpression
of BCL2 in osteoblasts inhibits osteoblast differentiation, reduces osteocyte processes, and causes osteocyte apoptosis.
Citation: Moriishi T, Maruyama Z, Fukuyama R, Ito M, Miyazaki T, et al. (2011) Overexpression of Bcl2 in Osteoblasts Inhibits Osteoblast Differentiation and
Induces Osteocyte Apoptosis. PLoS ONE 6(11): e27487. doi:10.1371/journal.pone.0027487
Editor: Eliana Saul Furquim Werneck Abdelhay, Instituto Nacional de Ca ˆncer, Brazil
Received September 9, 2011; Accepted October 18, 2011; Published November 17, 2011
Copyright: ? 2011 Moriishi 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 work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology, the ‘‘Ground-based Research
Program for Space Utilization’’ promoted by the Japan Space Forum, the Nakatomi Foundation, and the President’s Discretionary Fund of Nagasaki University,
Japan. 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
Bone is a dynamic tissue that is constantly undergoing
remodeling by osteoblasts and osteoclasts, and bone volume is
determined by the differentiation and function of osteoblasts and
osteoclasts. Osteoblasts, which differentiate from multipotent
mesenchymal cells, express bone matrix protein genes at different
levels depending on the maturity of the cells. Mesenchymal cells
and preosteoblasts weakly express Col1a1, but osteoblasts show
increased levels. Immature osteoblasts express osteopontin, and
mature osteoblasts strongly express osteocalcin. Mature osteoblasts
are finally embedded in the bone matrix to become osteocytes.
Osteocytes located in lacunae establish an extensive intracellular
and extracellular communication system via gap junction-coupled
cell processes and canaliculi, through which cell processes pass
throughout bone, and the communication system is extended to
osteoblasts on the bone surface , . The lacunocanalicular
network formed by osteocytes is thought to be an ideal
mechanosensory system and suitable for mechanotransduction,
by which mechanical energy is converted into electrical and/or
biochemical signals , , , , , .
Osteoblast apoptosis plays an important role in bone develop-
ment and maintenance. It is estimated that 60–80% 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 caused in part by osteoblast apoptosis, and
PTH, bisphosphonate, and calcitonin exert anabolic action on
bone by inhibiting osteoblast 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, bone loss caused by sex steroid deficiency, glucocor-
ticoid excess, or aging might be inhibited by the upregulation of
Bcl2; however, the effects of overexpression of Bcl2 in osteoblast
differentiation and bone development and maintenance have not
been fully investigated.
PLoS ONE | www.plosone.org1 November 2011 | Volume 6 | Issue 11 | e27487
Osteocyte apoptosis is a relatively common event in both
pathological and healthy human bone and osteocyte apoptosis
caused by microdamage precedes intracortical remodeling, raising
the possibility that the apoptotic process might generate a signal
used in targeted osteoclastic bone resorption . Estrogen
withdrawal and glucocorticoids induce osteocyte apoptosis, and
estrogen and bisphosphonates attenuate osteocyte apoptosis ,
, . The message transmitted by osteocyte apoptosis is
considered to travel through the canalicular network to the surface
of bone tissue and to be sent on to progenitor cells, leading to the
initiation signal for remodeling, thereby stimulating the bone
resorption/formation cycle . Distinct from osteoblasts, osteo-
cyte apoptosis represents cumulative death because cellular debris
cannot be removed by phagocytes until the surrounding bone is
resorbed, and TUNEL reactivity is retained in osteocyte lacunae
long after osteocyte death .
To evaluate the effects of the overexpression of BCL2 in
osteoblasts, we examined osteoblast-specific BCL2 transgenic mice.
Here, we show that overexpression of BCL2 in osteoblasts
inhibited osteoblast differentiation, reduced osteocyte processes,
and caused osteocyte apoptosis.
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:
To generate transgenic mice with osteoblasts that express
human BCL2, BCL2 cDNA was inserted into the mammalian
expression vector pNASSb (CLONTECH, Shiga, Japan) by
replacing the b-galactosidase gene at Not I sites, and the 2.3 kb
osteoblast-specific promoter region of mouse Col1a1  was
inserted into pNASSb at EcoRI-XhoI sites. The 2.3 kb Col1a1
promoter was a kind gift from B. de Crombrugghe (University of
Texas M. D. Anderson Cancer Center). Transgenic mice were
generated as previously described , and transgenic lines were
maintained against a B6C3H F1background. Until 2 weeks of age,
both genders were used for analyses, but only male mice were used
for analyses after 4 weeks of age. Tg(H) were mated with p53+/2
mice , and p532/2tg(H) was finally generated. p53+/2mice
were a kind gift from M. Katsuki (National Institute for Basic
Biology). 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.
Bone histomorphometric analyses were performed as previously
described . For assessment of dynamic histomorphometric
indices, mice were injected with calcein 6 d and 2 d before
sacrifice at a dose of 0.16 mg/10 g body weight. 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 (pH 7.4) and
embedded in paraffin. Sections (3–7 mm thick) were stained with
hematoxylin and eosin (H-E) or stained for TUNEL using the
ApopTagH system (Intergen, Burlington, MA), or subjected to
immunohistochemistry using monoclonal anti-human BCL2
antibody (Abcam, Cambridge, UK). For the BrdU incorporation
study, mice of 2 and 6 weeks of age were injected intraperitoneally
with 100 mg BrdU/g 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 cells in the distal primary spongiosa
of femurs, which were recognized as osteoblastic cells from their
morphology and attachment to the trabecular bone, were counted.
Bone canalicular staining (silver impregnation staining) was
performed according to the method previously described .
Ultrastructural analysis was performed using a transmission
electron microscope (H-7100; Hitachi, Tokyo, Japan) as previously
described . To observe the three-dimensional ultrastructure of
osteocytes, the HCl-collagenase method  was applied. The
treated specimens were observed under a scanning electron
microscope (S-3500N; Hitachi).
Real-time RT-PCR and Western blot analyses
Muscle, connective tissue, and periosteum were removed from
femurs and tibiae, and the bones were cut at the metaphyses. After
hematopoietic cells in the diaphyses of femurs and tibiae were
flushed out with PBS, osteoblast-enriched cells were collected
using a micro-intertooth brush (Kobayashi Pharmaceutical Co.
Ltd. Osaka, Japan). The remaining bone was used as a source of
osteocyte-enriched cells. Nearly complete removal of osteoblasts
from the endosteum by the micro-intertooth brush was confirmed
using a scanning electron microscope (Miniscope TM-1000;
Hitachi). Total RNA was extracted using ISOGEN (Wako, Osaka,
Japan), and real-time RT-PCR was performed as previously
described . Primer sequences are shown (Table S1). We
normalized the values to those of Gapdh. Using osteoblast-enriched
or osteocyte-enriched whole-cell lysates, Western blot analysis was
performed using the following antibodies: anti-BCL2, anti-p53,
anti-Cul1, anti-Bim, anti-Cyclin D1, anti-t-Bid, anti-Bad, and anti-
Bcl-XL (BD); anti-Bax, anti-p21, anti-p27, anti-p16, anti-actin
(Santa Cruz Biotechnology, Santa Cruz, CA); anti-p57 (Sigma, St.
Louis, MO); anti-cleaved caspase-3 (Chemicon, Temecula, CA);
anti-Hif-1a (Zymed, San Francisco, CA); and anti-Mcl1 (Abcam).
Quantitative micro-CT analysis was performed with a micro-
CT system (mCT-20; Scanco Medical, Bru ¨ttisellen, Switzerland).
Data from scanned slices were used for three-dimensional analysis
to calculate femoral morphometric parameters. Trabecular bone
parameters were measured on a distal femoral metaphysis.
Approximately 2.4 mm (0.5 mm from the growth plate) were
cranio-caudally scanned and 200 slices in 12 mm increments were
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 and
used for MTT assay and osteoblast differentiation. To examine
osteoblast differentiation, staining for alkaline phosphatase (ALP)
activity and mineralization was performed as previously described
. Mineralization was quantified using VHX-1000 (KEY-
ENCE) and Image J. Primary calvarial cells derived from C57BL/
6 embryos at embryonic day 18.5 were also prepared by culturing
small pieces of calvaria in three-dimensional collagen gel for 10–14
days as described previously , and used for the analyses of
osteoblast differentiation, apoptosis, and osteoclastogenesis. To
examine apoptosis, calvarial cells were infected with EGFP-
expressing retrovirus or human BCL2- and EGFP-expressing
retrovirus, which was generated by a bicistronic expression vector
(human BCL2 internal ribosome entry site (IRES)-EGFP), plated
on 8-well chamber slides (Nalge Nunc, Rochester, NY) at a density
Bcl2 and Osteoblast Differentiation
PLoS ONE | www.plosone.org2November 2011 | Volume 6 | Issue 11 | e27487
Figure 1. Generation of BCL2 transgenic mice and micro-CT and bone histomorphometric analyses. (A) Diagram of the DNA construct
used to generate transgenic mice that express human BCL2 under the control of the Col1a1 promoter. *Intron from SV40 containing splice donor and
acceptor sites, **polyadenylation signal from SV40. (B) Northern blot analysis of the transgene. The expression level of the transgene was examined
by Northern blot analysis using total RNA that had been extracted from the femurs of two BCL2 transgenic mouse lines (tg(L) and tg(H)) and wild-type
mice (wt) at 5 weeks of age. Twenty micrograms of RNA were loaded and Gapdh was used as an internal control. (C, D) Immunohistochemical analysis
using anti-human BCL2 antibody. Sections of trabecular bone (C) and cortical bone (D) from tg(L) at 2 weeks of age were reacted with anti-human
BCL2 antibody, which does not react with mouse Bcl2. Scale bars=50 mm. (E) Real-time RT-PCR analysis of the transgene expression. RNA was
extracted from bone (Bo), brain (Br), heart (He), lung (Lu), stomach (St), liver (Li), kidney (Ki), testis (Te), muscle (Mu), and skin (Sk) of tg(L) at 10 weeks
of age. (F–I) Micro-CT analysis. F and H, Two-dimensional axial image of distal femoral metaphysis of male mice at 6 weeks of age (F) and 10 weeks of
age (H). G and I, Trabecular bone volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) at 6 weeks of age (G) and 10 weeks of
Bcl2 and Osteoblast Differentiation
PLoS ONE | www.plosone.org3November 2011 | Volume 6 | Issue 11 | e27487
of 16104/well, and treated with PBS or 1 mM hydrogen peroxide
for 3 hrs after confluence. TUNEL-positive cells were detected
using the ApopTagH system (Intergen, Burlington, MA). The co-
culture of primary osteoblasts and bone marrow cells was
performed as previously described . In all of the retrovirus
infection experiments, the amount of retrovirus was adjusted to
obtain similar EGFP signals in the EGFP-expressing retrovirus
and human BCLl2- and EGFP-expressing retrovirus infections.
Statistical analyses were performed using Student’s t-test.
P,0.05 was considered significant.
Bone mass increased in Bcl2 transgenic mice at 6 weeks
but not 10 weeks of age
We established two lines of human BCL2 transgenic mice
under the control of mouse 2.3 kb Col1a1 promoter with
different expression levels, a transgenic line with low expression
of BCL2 (tg(L)) and a transgenic line with high expression of
BCL2 (tg(H)) (Fig. 1A, B). Transgene expression was examined
by immunohistochemistry at 2 weeks of age, and was
specifically detected in osteoblasts and young osteocytes
(Fig. 1C, D). We also examined the expression of the transgene
in other tissues by real-time RT-PCR. Although the transgene
was weakly detected in the skin as previously described , it
was barely detectable in the brain, heart, lung, stomach, liver,
kidney, testis, and muscle (Fig. 1E). We examined the bone
phenotypes at a growing stage (6 weeks of age) and an adult
stage (10 weeks of age). On micro-computed tomography
(micro-CT) analysis at 6 weeks of age, bone volume, trabecular
number, and trabecular thickness were increased in tg(L)
compared with wild-type mice (Fig. 1F, G). Bone histomorpho-
metric analysis showed that the osteoblast surface was markedly
increased in tg(L) compared with wild-type mice at 6 weeks of
age (osteoblast surface: wild-type mice; 33.969.2%, tg(L);
*60.465.3%, n=6–8, *p,0.01); however, the parameters of
trabecular bone in tg(L) and tg(H) were comparable to those in
wild-type mice at 10 weeks of age on micro-CT analysis
(Fig. 1H, I). At 10 weeks of age, peripheral quantitative
computed tomography (pQCT) analysis showed that the
mineral density of cortical bone was reduced in tg(H) but not
in tg(L) compared with that in wild-type mice (Figure S1). Bone
histomorphometric analysis at 10 weeks of age showed that the
numbers of osteoblasts and osteocytes were increased in both
tg(L) and tg(H) compared with those in wild-type mice, but that
the bone volume, osteoid thickness, and bone formation rate
were not increased in tg(L) and reduced in tg(H) compared with
those in wild-type mice, irrespective of the similar levels of
osteoclast parameters among wild-type mice, tg(L), and tg(H)
(Fig. 1J). The phenotypic differences observed in BCL2
transgenic mice at 6 and 10 weeks of age were likely to be
due to the change in transgene expression, because the level of
transgene expression at 10 weeks of age was about half of that
at 6 weeks of age in real-time RT-PCR analysis (data not
Osteoblast proliferation was increased without
augmentation of apoptosis and osteoblast differentiation
was impaired in BCL2 transgenic mice
As the osteoblast density was increased in BCL2 transgenic mice,
we examined the effect of BCL2 on osteoblast proliferation by
BrdU labeling. The percentage of BrdU-positive cells was
increased mildly in tg(L) and markedly in tg(H) at 2 weeks of
age and increased similarly in tg(L) and tg(H) at 6 weeks of age
compared with that in wild-type mice at the respective age
(Fig. 2A–C, G). We also examined osteoblast apoptosis by terminal
deoxynucleotidyl transferase-mediated deoxyuridine triphosphate
nick end labeling (TUNEL). Similar percentages of TUNEL-
positive osteoblastic cells were observed among wild-type mice,
tg(L), and tg(H) (Fig. 2D–F, H).
As osteoblast proliferation was increased in BCL2 transgenic
mice, we examined the expression of cell cycle-related molecules
by real-time RT-PCR and Western blot analyses using osteoblast-
enriched samples from femurs and tibiae (Fig. 2I, J). The mRNA
and protein levels of cyclin D1, p21, and Cul1 were increased and
the protein levels of p53, p27, and p16 were increased.
Next, we examined the expression of osteoblast differentiation
markers. Real-time RT-PCR analysis showed similar expression
levels of Col1a1, osteopontin, and osteocalcin in tg(L), but reductions in
Col1a1 and osteocalcin expression and an increase in osteopontin
expression in tg(H) compared with the respective levels in wild-
type mice at 2 weeks of age (Fig. 2K). Similar results were also
obtained by Northern blot analysis at 4 weeks of age and in situ
hybridization at 8 weeks of age (Figure S2). These findings indicate
that osteoblast differentiation was impaired in tg(H). Since the
osteoblast density but neither the bone volume nor bone formation
rate were increased in tg(L) and tg(H) at 10 weeks of age (Fig. 1J),
osteoblast function should have been impaired in both tg(L) and
Overexpression of BCL2 in osteoblasts inhibits osteoblast
differentiation and apoptosis but has no effect on
osteoclastogenesis in vitro
In contrast to enhanced osteoblast proliferation in BCL2
transgenic mice, overexpression of BCL2 had no effect on
osteoblast proliferation in vitro (Figs. 2A–C, G, 3A). To examine
the effect of Bcl2 on osteoblast differentiation, we first introduced
BCL2 into primary osteoblasts from wild-type mice by retrovirus
and examined ALP activity. ALP activity in BCL2-introduced cells
was increased compared with that in EGFP-introduced cells when
the cells were seeded at 1.56104cells/well, whereas it was reduced
compared with that in EGFP-introduced cells when seeded at
56104cells/well (Fig. 3B). To investigate why ALP activity was
dependent on cell density, we examined the effect of BCL2 on
apoptosis in vitro, because cell density is critical for osteoblast
differentiation. Overexpression of BCL2 in primary osteoblasts
significantly reduced osteoblast apoptosis with or without hydro-
gen peroxide treatment (Fig. 3C–G). To minimize the effect of
BCL2 on apoptosis during culture, primary osteoblasts from tg(H)
were seeded at the high concentration (56104cells/well). ALP
activity, mineralization, and the expressions of ALP and osteocalcin
were reduced compared with in wild-type mice (Fig. 3H–J).
age (I) are shown. Trabecular bone parameters were measured on distal femoral metaphysis. Data are presented as the mean 6 S.D. of 4–18 male
mice. (J) Bone histomorphometric analysis. The trabecular bone volume (bone volume/tissue volume, BV/TV), osteoid thickness (O.Th), number of
osteoblasts (N.Ob/B.Pm), number of osteoclasts (N.Oc/B.Pm), eroded surface (ES/BS), number of osteocytes (N.Ot/Ar), mineral apposition rate (MAR),
mineralizing surface (MS/BS), and bone formation rate (BFR/BS) were compared among male wild-type mice (blue), tg(L) (red), and tg(H) (yellow) at 10
weeks of age. Data are presented as the mean 6 S.D. of 5–10 mice. *vs. wild-type mice. *P,0.05, **P,0.01. B.Pm, bone perimeter; BS, bone surface.
Bcl2 and Osteoblast Differentiation
PLoS ONE | www.plosone.org4 November 2011 | Volume 6 | Issue 11 | e27487
Figure 2. Analyses of osteoblast proliferation, apoptosis, and differentiation in BCL2 transgenic mice. (A–H) BrdU labeling (A–C) and
TUNEL staining (D–F) of sections of distal femoral metaphysis from wild-type mice (A, D), tg(L) (B, E), and tg(H) (C, F) at 2 weeks of age. The sections
were counterstained with hematoxylin (A–C) and methyl green (D–F). Scale bars=50 mm. BrdU-positive osteoblastic cells (G) and TUNEL-positive
osteoblastic cells (H) of wild-type mice (blue), tg(L) (red), and tg(H) (yellow) were counted and shown as a percentage of the number of osteoblastic
cells. Data are presented as the mean 6 S.D. of 4–12 mice. *vs. wild-type mice. *P,0.05, **P,0.01. (I) Real-time RT-PCR analysis. RNA was extracted
from the osteoblast-enriched fraction of femurs and tibiae of male wild-type mice (blue) and tg(H) (yellow) at 6 weeks of age and the expression of
cell cycle-related genes was examined. The values of wild-type mice were defined as 1, and relative levels are shown. Data are presented as the mean
6 S.D. of 5–8 mice. *vs. wild-type mice. *P,0.05. (J) Western blot analysis. Proteins were extracted from the osteoblast-enriched fraction of femurs
and tibiae of male wild-type mice (wt) and tg(H) at 6 weeks of age. The ratios of the intensities of the bands are shown against b-actin. Similar results
were obtained in three independent experiments and representative data are shown. (K) Real-time RT-PCR analysis of Col1a1, osteopontin, and
osteocalcin. RNA was extracted from femurs of wild-type mice (wt), tg(L), and tg(H) at 2 weeks of age. The values of wild-type mice were defined as 1,
and relative levels are shown. Data are presented as the mean 6 S.D. of 5 mice. *vs. wild-type mice. *P,0.05.
Bcl2 and Osteoblast Differentiation
PLoS ONE | www.plosone.org5November 2011 | Volume 6 | Issue 11 | e27487
Figure 3. Analyses of osteoblast proliferation, apoptosis, and differentiation and osteoclastogenesis in vitro. (A) MTT assays. MTT
assays were performed using wild-type primary osteoblasts infected with EGFP-expressing or BCL2-and-EGFP-expressing retrovirus. Data are
presented as the mean 6 S.D. of 16 wells. Similar results were obtained in two independent experiments and representative data are shown. (B) ALP
activity. Primary osteoblasts prepared using three-dimensional collagen gel were infected with EGFP-expressing or BCL2- and EGFP-expressing
retrovirus and seeded on 24-well plates at the indicated cell densities, and ALP activity was examined after culture for 6 days (1.56104cells/well) or 4
days (56104cells/well). Similar results were obtained in two independent experiments and representative data are shown. (C–G) Analysis of apoptosis
in vitro. Primary osteoblasts were infected with EGFP-expressing (C, E) or BCL2- and EGFP-expressing (D, F) retrovirus. At confluence, the cells were
treated with PBS (C, D) or 1 mM H2O2(E, F) for 3 h and TUNEL staining was performed. The cells were counterstained with hematoxylin. Scale
bars=50 mm. The percentage of TUNEL-positive cells is shown as the mean 6 S.D. of 5 wells (G). *vs. EGFP. **, ##P,0.01. Similar results were
obtained in two independent experiments and representative data are shown. (H–J) Differentiation of primary osteoblasts from tg(H). Primary
osteoblasts were prepared from newborn calvariae of wild-type mice and tg(H), seeded on 24 well plates at the density of 56104cells/well, and ALP
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To investigate the osteoblast function of supporting osteoclas-
togenesis, we co-cultured calvarial cells and bone marrow cells.
Retroviral introduction of either EGFP or BCL2 into primary
osteoblasts showed similar numbers of tartrate-resistant acid
phosphatase (TRAP)-positive cells and a similar resorption area
in the co-culture (Fig. 3K–R).
Osteocyte apoptosis in BCL2 transgenic mice
Surprisingly, empty lacunae or lacunae containing cell debris
were increased in BCL2 transgenic mice, and the increase was
already evident in the distal femoral metaphysis of tg(H) at 1 week
of age (Figure S3). To examine whether the osteocytes died by
apoptosis, we performed TUNEL staining. About 1% of lacunae
in the cortical bone at the diaphyses of femurs were TUNEL-
positive in wild-type mice from 2 to 10 weeks of age, whereas the
corresponding percentage was about 10% in both tg(L) and tg(H)
at 2 and 4 weeks of age, 20% in tg(L) and 40% in tg(H) at 5–6
weeks of age, and 50% in tg(L) and 60% in tg(H) at 10 weeks of
age (Figs. 4, 5A). After the death of osteocytes, the lacunae
contained only cell debris but TUNEL reactivity was retained in
the lacunae (Fig. 4), because the debris of dead osteocytes cannot
be eliminated until the surrounding bone is resorbed. Thus,
TUNEL-positive lacunae accumulated during bone development.
To further confirm that the osteocytes in BCL2 transgenic mice
died by apoptosis, we observed them by transmission electron
microscopy, which is the most reliable tool for evaluation of the
type of cell death (Fig. 5B, C). Apoptotic structural alterations of
osteocytes, such as cytoplasmic shrinkage, chromatin condensa-
tion, and nuclear disintegration, were observed in the lacunae of
the cortical bone of tibiae of BCL2 transgenic mice (Fig. 5C);
however, we did not observe cellular swelling, disturbance of
plasma membranes and membranes of cytoplasmic organelles, or
swollen, electron-lucent nuclei, all of which are features of
necrosis. Canaliculi and osteocyte processes were abundant and
densely connected in wild-type mice, whereas the numbers of
canaliculi and osteocyte processes were reduced depending on the
expression levels of the transgene and were sparsely connected in
BCL2 transgenic mice (Fig. 5D–I, L, data not shown). Observation
by polarized microscopy showed lamellar collagen deposition in
the cortical bone of both wild-type mice and tg(L), although the
collagen fibers were mildly disorganized in tg(L), probably due to
the death of osteocytes (Fig. 5J, K).
Expression of apoptosis-related genes in osteocytes and
failure to rescue apoptosis by p53 deletion in BCL2
In accordance with the appearance of apoptotic cells, p53, Bax,
Bim, Noxa, and Vegf were upregulated in the osteocyte-enriched
samples from tg(H) compared with the respective levels in those
from wild-type mice (Fig. 6A). On Western blot analyses using
osteoblast-enriched samples, the protein levels of p53, Bax, Bim,
and t-Bid in tg(H) were slightly increased and the protein level of
cleaved caspase-3 in tg(H) was similar to the respective level in
wild-type mice (Fig. 6B). On Western blot analyses using
osteocyte-enriched samples, however, protein levels of p53, HIF-
1a, Bax, Bim, t-Bid, and cleaved caspase-3 were apparently
increased and that of Mcl1 was decreased in tg(H) compared with
the respective level in wild-type mice (Fig. 6C), further supporting
the histological observation that osteocytes died by apoptosis. As
p53 expression was markedly increased in the osteocytes of BCL2
transgenic mice, we mated tg(H) with p53+/2mice and finally
generated p532/2tg(H) mice (Fig. 6D–G). The deletion of p53
failed to inhibit osteocyte apoptosis in tg(H) (Fig. 6H).
We analyzed male BCL2 transgenic mice, and osteoblast density
was increased in tg(L) and tg(H) but the bone formation rate was
similar or reduced in tg(L) and tg(H), respectively, compared with
wild-type mice. Although the differentiation stage of osteoblasts
was not examined in the previously reported BCL2 transgenic
mice, we found that osteoblast differentiation was inhibited in a
manner dependent on transgene expression, as shown by the
staining and von Kossa staining were performed after culture for 4 days and 15 days, respectively (H). I, Quantification of mineralization. RNA was
extracted after culture for 4 days and real-time RT-PCR analysis was performed (J). The value of primary osteoblasts from wild-type mice was set as 1
and the relative levels are shown in I and J. Data are presented as the mean 6 S.D. of 3 mice. *vs. wild-type mice, **p,0.01, ***p,0.001. Similar
results were obtained in two independent experiments and representative data are shown. (K–R) Osteoclastogenesis in vitro. Primary osteoblasts
were infected with retrovirus expressing either EGFP (K, M, O) or BCL2-EGFP (L, N, P), and cultured with bone marrow cells for 6 days. EGFP signal (K,
L), TRAP staining (M, N), dentin slices (O, P), number of multinucleated TRAP-positive cells (Q), and Pit area (R) are shown. Scale bars=20 mm (K, L);
50 mm (M, N); 200 mm (O, P). Similar results were obtained in two independent experiments and representative data are shown.
Figure 4. Osteocyte apoptosis in cortical bone. H-E (A–C, G–I, M–
O) and TUNEL (D–F, J–L, P–R) staining of cortical bone at the diaphyses
of femurs of wild-type mice (A, D, G, J, M, P), tg(L) (B, E, H, K, N, Q), and
tg(H) (C, F, I, L, O, R) at 2 weeks (A–F), 4 weeks (G–L), and 10 weeks of
age (M–R). Scale bars=0.1 mm.
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reduction in Col1a1 and osteocalcin expression and the increase in
osteopontin expression. Further, the inhibition of osteoblast differ-
entiation was also observed in ex vivo experiments; however, the
differentiation of osteoblasts derived from the previous BCL2
transgenic mice was shown to be enhanced in vitro . This
controversial result may have been caused by an increase in the
cell density of primary osteoblasts from BCL2 transgenic mice
compared with wild-type mice during culture, because the
frequency of apoptosis in wild-type primary osteoblasts fell to
one third by the introduction of BCL2 (Fig. 3G) and the increased
cell density accelerated osteoblast differentiation.
Although osteoblast proliferation was not examined in vivo in
previously reported BCL2 transgenic mice , we showed that
osteoblast proliferation was enhanced in BCL2 transgenic mice;
however, introduction of BCL2 failed to enhance the proliferation
of wild-type primary osteoblasts (Fig. 3A), and primary osteoblasts
from previous BCL2 transgenic mice showed similar proliferation
to those from wild-type mice . Further, previous reports
showed that Bcl2 inhibited 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 Bcl2-overexpressing cells during arrest ,
, , , although overexpression of Bcl2 in myocytes
promoted proliferation . Therefore, BCL2 may have no
intrinsic ability to enhance osteoblast proliferation, and it is
possible that the population of proliferating osteoblasts was
increased by the deceleration of osteoblast differentiation in
BCL2 transgenic mice. The upregulation of cyclinD1 and Cul1,
which is a component of an ubiquitin ligase complex targeting p21
and p27 , , in BCL2 transgenic mice may reflect the
accumulation of proliferating osteoblasts. Osteoblast apoptosis was
not reduced in BCL2 transgenic mice; however, osteoblast
apoptosis should have been suppressed in BCL2 transgenic mice
because of an increase of proliferating osteoblasts, in which
apoptosis would be increased by replication stress , and the
suppression of apoptosis should have contributed to the increase in
the number of proliferating osteoblasts.
A increase in osteocyte apoptosis was not observed in the
previous BCL2 transgenic mice . The discrepancy of the
osteocyte phenotype may be due to differences in the genetic
background because the previous BCL2 transgenic mice were CD-
1 outbred mice. We observed an increase in osteocyte apoptosis
not only in BCL2 transgenic mice with a B6C3H F1background
but also in those with a C57BL/6 background, which were
generated by backcrossing with C57BL/6 mice ten times (data not
shown). Outbred mice may have been resistant to osteocyte
apoptosis caused by overexpression of BCL2 in osteoblasts. It is
also possible that the expression level of the transgene in the
previous report was insufficient to induce osteocyte apoptosis.
In our transgenic mice, overexpression of BCL2 in osteoblasts
led to a reduction in the number of osteocyte processes. This
suggests that Bcl2 alters cytoskeletal organization. Recently, it was
reported that Bcl2 is able to form a complex with actin and
gelsolin, which functions to decrease gelsolin-severing activity to
increase actin polymerization, and to suppress cell adhesion,
spreading, and motility . Further, Bcl2 is an independent
Figure 5. Histological analysis of osteocytes. (A) Frequency of
TUNEL-positive lacunae. The number of TUNEL-positive lacunae was
counted at 2 weeks, 4 weeks, 6 weeks, and 10 weeks of age, and
presented as a percentage of the total number of lacunae in the cortical
bone of femurs. (B, C) Images of osteocytes by transmission electron
microscope (TEM) in the cortical bone of tibiae from male wild-type
mouse (B) and tg(H) (C) at 10 weeks of age. Bars: 2 mm. (D–G)
Canalicular staining of femurs from male wild-type mouse (D, F), tg(H)
(E), and tg(L) (G) at 10 weeks of age. F and G show magnified views of
osteocyte lacunae and canaliculi. Bars=50 mm (D, E); 5 mm (F, G). (H, I)
Images of young osteocytes by scanning electron microscope in the
cortical bone of femurs from male wild-type mouse (H) and tg(H) (I) at
10 weeks of age. Bars: 10 mm. (J, K) Polarized microscopy of cortical
bone at diaphyses of femurs in male wild-type mouse (J) and tg(L) (K) at
10 weeks of age. Bars: 50 mm. (L) Number of canaliculi. The number of
canaliculi derived from each lacuna was counted. Four lacunae in the
diaphysis of the femur were examined in each mouse. Data are
presented as the mean 6 S.D. of 7 male wild-type mice and 6 male tg(L)
at 10 weeks of age. *vs. wild-type mice, ***p,0.001.
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Figure 6. Expression of apoptosis-related molecules and TUNEL staining of cortical bone in p532/2tg(H). (A) Real-time RT-PCR analysis
of apoptosis-related genes and Vegf. RNA was extracted from osteocyte-enriched samples of tibiae and femurs of male wild-type mice (blue) and
tg(H) (yellow). The values of wild-type mice were defined as 1, and relative levels are shown. Data are presented as the mean 6 S.D. of 4–10 mice at
5–6 weeks of age. *vs. wild-type mice. *P,0.05, **P,0.01. (B, C) Western blot analysis of apoptosis-related molecules using cell extract from
osteoblast-enriched fraction (B) and osteocyte-enriched fraction (C) in male wild-type mice (wt) and tg(H) at 5–6 weeks of age. b-actin was used as an
internal control. The anti-Bcl2 antibody used here reacts with both mouse and human Bcl2. Similar results were obtained in three independent
experiments and representative data are shown. (D–H) Failure to rescue osteocyte apoptosis by the deletion of p53. H-E (D, E) and TUNEL (F, G)
staining of sections of cortical bone at the diaphyses of femurs of male tg(H) (D, F) and p532/2tg(H) (E, G) and the frequencies of TUNEL-positive
lacunae (H) at 10 weeks of age. Scale bars: 0.1 mm. Data are presented as the mean 6 S.D. of 3 mice.
Bcl2 and Osteoblast Differentiation
PLoS ONE | www.plosone.org9November 2011 | Volume 6 | Issue 11 | e27487
indicator of a favorable prognosis for all types of early-stage breast
cancer , . Thus, the reduction in osteocyte processes in
BCL2 transgenic mice may show the function of Bcl2, which
modulates cytoskeletal reorganization.
The reduction in osteocyte processes in BCL2 transgenic mice
would result in a limited supply of oxygen, nutrients, and survival
factors to osteocytes. In accordance with this hypothesis, Hif1-a
protein, which is stabilized in hypoxia , and p53 protein,
which is stabilized by hypoxia, nutrient deprivation, and
withdrawal of survival factors in addition to DNA damage ,
, were markedly increased, and their target genes, including
Bax and Noxa, were upregulated in osteocytes of BCL2 transgenic
mice. The expression of Mcl-1, which is a key member of the Bcl2
family of pro-survival proteins and undergoes proteasomal-
dependent degradation during anoxia and growth factor with-
drawal, was reduced in osteocytes , . Further, Bim, which
is upregulated by FOXO after the withdrawal of growth factors
, and a truncated form of Bid (t-Bid), which is a product of
caspase-8 mediated cleavage of Bid through death receptor
signaling , increased. The failure to rescue osteocyte apoptosis
by p53 deletion also indicates that multiple signaling pathways
were involved in osteocyte apoptosis. These findings strongly
suggest that osteocyte apoptosis in BCL2 transgenic mice was
caused by an insufficient supply of oxygen, nutrients, and survival
factors, probably due to the reduction of osteocyte processes.
Our BCL2 transgenic mouse with accumulated dead osteocytes
is a useful model to analyze the function of osteocytes because a
repair process, which replaces dead osteocytes with new osteocytes
by bone resorption and formation, was not evident in the mice
irrespective of the massive accumulation of dead osteocytes. The
functions of osteocytes in a physiological condition and an
unloaded condition were examined using these mice (submitted
to another journal). The gradual accumulation of death osteocytes
and the reduction in the number of osteocyte processes, which will
restrict the secretion of inflammation-inducible molecules to bone
marrow, may have limited the repair reaction. We also successfully
identified using BCL2 transgenic mice that pyruvate dehydroge-
nase 4 (Pdk4) is one of the molecules responsible for bone loss in
In conclusion, osteoblast apoptosis is in part responsible for
osteoporosis in sex steroid deficiency, glucocorticoid excess and
aging, however, BCL2 inhibited osteoblast differentiation, im-
paired osteoblast function, and reduced the number of osteocyte
processes. Thus, our results improve the comprehension of cellular
mechanisms involved in osteoporosis.
equal cross-divisions from metaphyses to diaphyses of femurs was
measured in male wild-type mice (blue circles), tg(L) (red triangles),
and tg(H) (yellow circles) of 10 weeks of age. Data are presented as
the mean 6 SD of 6 mice. *vs. wild-type mice, **p,0.01. pQCT
analysis was performed using an XCT Research SA (Stratec
Medizintechnick). The mineral density in cortical bone was
analyzed using the threshold value, 690 mg/cm3.
pQCT analysis. The cortical bone density in 15
analyses. (A) Northern blot analysis of the bone matrix protein
genes including Col1a1, osteopontin, and osteocalcin. RNA was
extracted from the femurs of two male BCL2 transgenic mouse
lines (tg(L) and tg(H)) and wild-type mice (wt) at 4 weeks of age.
Twenty micrograms of RNA was loaded and Gapdh was used as an
internal control. (B–J) The expression of Col1a1 (B–D), osteopontin
(E–G), and osteocalcin (H–J) in male wild-type mice (B, E, H), tg(L)
(C, F, I), and tg(H) (D, G, J) was examined by in situ hybridization
at 8 weeks of age. Serial sections from tibiae were used for in situ
hybridization and counterstained with methyl green. Scale bars:
Northern blot and in situ hybridization
at 1 week of age. Sections from femurs of wild-type mice (A),
tg(L) (B), and tg(H) (C) were stained with H-E, and the cortical
bones are shown. Arrows in C indicate the lacunae containing cell
debris. Scale bars=0.1 mm.
Histological analysis of BCL2 transgenic mice
We thank B. de Crombrugghe for the Col1a1 promoter, M. Katsuki for
p53+/2mice, N. Kanatani for the generation of BCL2 transgenic mice, and
C. Fukuda for secretarial assistance.
Conceived and designed the experiments: TK. Performed the experiments:
T. Moriishi ZM RF MI T. Miyazaki H. Kitaura HO TF YK RM H.
Komori. Analyzed the data: KT H. Kawaguchi. Wrote the paper: TK.
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