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Hydrogen Peroxide Induces G2 Cell Cycle Arrest and Inhibits Cell Proliferation in Osteoblasts.

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THE ANATOMICAL RECORD 292:1107–1113 (2009)
Hydrogen Peroxide Induces G2 Cell
Cycle Arrest and Inhibits Cell
Proliferation in Osteoblasts
MING LI,1,2 LI ZHAO,1,2 JUN LIU,3 AN-LING LIU,4 WEI-SEN ZENG,1
SHEN-QIU LUO,1* AND XIAO-CHUN BAI1,2*
1
Department of Cell Biology, School of Basic Medical Sciences,
Southern Medical University, Guangzhou, China
2
Institute of Orthopedics, Southern Medical University, Guangzhou, China
3
Department of Urology, Guangzhou General Hospital of Guangzhou Command,
Guangzhou, China
4
Institute of Genetic Engineering, Southern Medical University, Guangzhou, China
ABSTRACT
Reactive oxygen species (ROSs) are involved in osteoporosis by inhibiting osteoblastic differentiation and stimulating osteoclastgenesis. Little
is known about the role and how ROS controls proliferation of osteoblasts.
Mammalian target of rapamycin, mTOR, is a central regulator of cell
growth and proliferation. Here, we report for the first time that 5–
200 lM hydrogen peroxide (H2O2) dose- and time-dependently suppressed
cell proliferation without affecting cell viability in mouse osteoblast cell
line, MC3T3-E1, and in human osteoblast-like cell line, MG63. Further
study revealed that protein level of cyclin B1 decreased markedly and the
percentage of the cells in G2/M phase increased about 2-4 fold by 200 lM
H2O2 treatment for 24–72 hr. A total of 0.5–5 mM of H2O2 but not lower
concentrations (5–200 lM) of H2O2 inhibited mTOR signaling, as manifested by dephosphorylation of S6K (T389), 4E-BP1 (T37/46), and
S6(S235/236) in MC3T3-E1 and MG63 cells. Rapamycin, which could inhibit mTOR signaling and cell proliferation, however, did not reduce the
protein level of cyclin B1. In a summary, H2O2 prevents cell proliferation
of osteoblasts by down-regulating cyclin B1 and inducing G2 cell cycle
arrest. Inhibition of mTOR signaling by H2O2 may not be involved in this
C 2009 Wiley-Liss, Inc.
process. Anat Rec, 292:1107–1113, 2009. V
Key words: hydrogen peroxide; osteoblast; mammalian target
of rapamycin; cyclin B1; proliferation; cell cycle;
G2 cell cycle arrest
Bone mass is controlled by the numbers and the activities of osteoblasts, the bone-forming cells, and osteoclasts, the bone-resorbing cells. Any loss of osteoblastic
activity or an increase in osteoclastic activity would
ultimately lead to osteoporosis. So proliferation and differentiation of osteoblasts and osteoclasts are very important for the pathogenesis of osteoporosis (Erlebacher
et al., 1995; Manolagas and Jilka, 1995; Manolagas,
2000).
Oxidative stress, resulting from excessive levels of
reactive oxygen species (ROSs) such as superoxides
anions and hydrogen peroxide (H2O2), represents a
major cause of cellular damage and death in a plethora
C 2009 WILEY-LISS, INC.
V
Grant sponsor: National Natural Sciences Foundation of
China and Program for New Century Excellent Talents in
University; Grant numbers: 30771027 and 30870955.
*Correspondence to: Xiao-Chun Bai or Shen-Qiu Luo, Department of Cell Biology, School of Basic Medical Sciences, Southern
Medical University, Guangzhou 510515, China. Fax: 86-2061648208. E-mail: baixc15@fimmu.com or luosq888@163.com
Received 7 February 2009; Accepted 15 April 2009
DOI 10.1002/ar.20925
Published online in Wiley InterScience (www.interscience.wiley.
com).
1108
LI ET AL.
of pathological conditions including osteoporosis (Finkel
and Holbrook, 2000; Finkel, 2003; Weitzmann and
Pacifici, 2006). Recent evidences have demonstrated that
(i) in postmenopausal osteoporosis, estrogen deficiency
induces bone loss through increased ROS production
(Lean et al., 2003, 2005); (ii) ROS stimulates osteoclastgenesis whereas antioxidants suppress osteoclast differentiation and activity; (iii) ROS prevents osteoblastic
differentiation whereas antioxidants enhance differentiation of osteoblast (Mody et al., 2001; Aitken et al., 2004;
Bai et al., 2004; Ha et al., 2004; Liu et al., 2004). These
findings suggest that ROS may represent an important
target for the treatment and/or prevention of bone lossrelated diseases.
The mammalian target of rapamycin, mTOR, serves
as a signal integrator of many upstream signals, including growth factors, nutrients, energy levels, and
stresses. Consequently, one critical function of mTOR is
to integrate these signals into a decision to positively or
negatively influence cell growth and proliferation (Sabatini, 2006; Wullschleger et al., 2006; Tsang et al., 2007;
Rosner et al., 2008). mTOR elicits its pleiotropic function
mainly through controlling protein synthesis by two distinct mechanisms: (i) mTOR phosphorylates and inactivates 4E-BP1, a translation repressor that binds to and
inhibits the translation initiation factor 4E (eIF-4E). On
phosphorylation by mTOR, 4E-BP1 is inactivated and
eIF-4E is released, thus resulting in an increased protein synthesis of 50 capped mRNAs; (ii) mTOR phosphorylates and activates the ribosomal protein S6 kinase
(S6K), which phosphorylates S6 ribosomal protein, a
component of the S40 ribosome subunit, thus facilitating
protein translation. Rapamycin, in complex with
FKBP12, specifically inhibits mTOR function, and consequently, ceases cell growth (Wullschleger et al., 2006;
Soulard and Hall, 2007).
Although the roles and mechanisms of ROS in regulation of cell differentiation in osteoblasts have been studied (Mody et al., 2001; Bai et al., 2004; Jin et al., 2008),
little is known about how ROS controls proliferation of
osteoblasts. Considering that proliferation of osteoblast
and its progenitor in bone marrow makes an important
contribution to the amount of differentiated and functional osteoblasts, and that mTOR plays a central role in
cell growth and proliferation, this study examined the
effect of hydrogen peroxide on mTOR signaling, cell
cycle progression, and cell proliferation in mouse osteoblast cell line, MC3T3-E1, and in human osteoblast-like
cell line, MG63.
MATERIAL AND METHODS
Reagents
Catalase, dimethyl sulphoxide (DMSO), were purchased form Sigma-Aldrich (St. Louis, MO). Antibodies
against phospho-S6K(T389), 4E-BP1, phospho-4E-BP1
(T37/46), and phospho-S6(S235/236) were purchased
from Cell Signaling Inc. (Beverly, MA), anti-S6, S6K, bactin, and cyclin B1 antibodies were from Santa Cruz
Biotech (Santa Cruz, CA).
Cell Culture
Human osteoblast-like cell line MG63 and mouse
osteoblast cell line MC3T3-E1 were grown in Dulbecco’s
Fig. 1. Effect of H2O2 on cell viability of osteoblasts. (A) MC3T3-E1
and (B) MG63 cells were treated with 0–1,000 lM H2O2 for 72 hr and
cell viability was detected by trypan blue dye-exclusion method.
modified Eagle’s medium-high glucose (DMEM) and aMEM, respectively, supplemented with 10% fetal bovine
serum (FBS), 50 units/mL penicillin and 50 lg per streptomycin in a humidified atmosphere of 5% CO2. Cultures
were trypsinized upon confluence and subcultured into
12-, 6-, or 96-well plates for further experiments.
Cell Proliferation Assay
Methyl thiazolyl tetrazolium (MTT) assays were performed to assess the rate of cell proliferation. Briefly,
MC3T3-E1 and MG63 cells were planted into 96-well
plates. After incubation overnight, the medium was
replaced with fresh medium with or without H2O2 at
indicated concentrations(5, 20, 50, 100, 200 lM) for various times (24, 48, 72 hr). Six wells were included in
each concentration. At the end of treatment, 20 lL MTT
(AMRESCO, OH) was added for 4 hr. Then the medium
was discarded carefully and 150 lL DMSO was added.
Absorbance was recorded at 570 nm with The Universal
Microplate Reader (Bio-Tek instruments) using wells
without cells as blanks. All experiments were performed
in triplicate. The inhibition rate of cell proliferation was
calculated by formula: % inhibition ¼ (A570 of control
A570 of treated cells)/A570 of control cells 100%.
H2O2 INDUCES G2 ARREST IN OSTEOBLASTS
1109
Fig. 2. H2O2 inhibits proliferation of osteoblasts dose- and timedependently. (A) MC3T3-E1 and (B) MG63 cells were treated with 5,
20, 50, 100, or 200 lM H2O2 for 48 hr and 72 hr, respectively, and cell
proliferation was detected by MTT assay. (C) MC3T3-E1 and (D)
MG63 cells were treated with 200 lM H2O2 for 24, 48, or 72 hr and
cell proliferation was detected by MTT assay. Inhibition rates were calculated as described in Materials and Methods.
Cell Viability Analysis
Statistical Analysis
Cells were treated with different concentrations (0–
1 mM) of H2O2. After 72 hr, cell viability was determined by counting the viable cell number with a hemocytometer after staining with trypan blue.
Statistical analyses were performed by ANOVA, and
P < 0.05 was considered statistically significant.
Cell Cycle Analysis
Cell cycle assays were performed to assess the cell
cycle progression. Briefly, exponentially growing MC3T3E1 cells were synchronized at the G1/S boundary after
starvation with basal medium for 24 hr, followed by
incubation in the presence or absence of 200 lM H2O2
for 24, 48, and 72 hr. At the indicated intervals, cells
were harvested and measured by cell cycle detection kit
(KEY GEN, Nanjing, China) following manufacturer’s
instructions. The cell cycle distribution was analyzed by
flow cytometry (FACS CaliburTM, BD) immediately. The
percentage of cells in G0/G1, S, and G2/M phases were
calculated using FCS Express software.
Western Blot Analysis
After treatment, cells were lysed immediately in
Laemmli buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS,
10% glycerol, 50 mM dithiothreitol, 0.01% bromophenol
blue) for 5 min at 95 C. Cell lysates were analyzed by
SDS/PAGE and transferred electrophoretically to Nitro
cellulose membrane (Bio-Rad Corp, Hercules, CA). Blots
were probed with specific antibodies and immunoreactive proteins were revealed by the enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology Inc., CA).
RESULTS
Dose- and Time-Dependent Inhibition of
Proliferation by H2O2 in Osteoblasts
Cellular responses elicited by H2O2 depend upon the
severity of the damage, which is further influenced by
the cell type and the magnitude of the dose of the exposure (Finkel and Holbrook, 2000; Temple et al., 2005). In
our experiments, MC3T3-E1 and MG63 cells underwent
severe cell death after high doses of H2O2 (0.5 or 1 mM)
treatment for 72 hr as determined by the Trypan Blue
dye-exclusion method. After an exposure to low doses of
H2O2 (5–200 lM), as expected, the cell viability was not
affected compared with the controls (Fig. 1A,B). Cell proliferation, however, was inhibited dose- and timedependently by 5–200 lM H2O2. Here we show the data
for proliferation of MC3T3-E1 cells after treatment with
5–200 lM H2O2 for 48 hr (Fig. 2A) and 200 lM H2O2 for
24, 48, and 72 hr (Fig. 2C), and that of MG63 cells after
treatment with 5–200 lM H2O2 for 72 hr (Fig. 2B) and
200 lM H2O2 for 24, 48, and 72 hr (Fig. 2D).
H2O2 Inhibits mTOR Signaling in Osteoblasts
It has been shown recently that rapamycin inhibits proliferation and differentiation of MC3T3-E1 cells and
mouse bone marrow stromal cells (BMSCs). It suggests
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LI ET AL.
S6K(T389), 4E-BP1, phospho-4E-BP1 (T37/46), S6, and phospho-S6
(S235/236). (D) MC-3T3-E1 cells were pretreated with 500 U/mL catalase or not, then incubated with 1,000 lM H2O2 for 30 min, cell lysates
were analyzed as in (C). (E) MG63 were incubated with 200–5,000 lM
H2O2 for 30 min, cell lysates were analyzed by Western blot with antibodies as indicated.
Fig. 3. H2O2 inhibits mTOR signaling in osteoblasts. (A) MC3T3-E1
and (B) MG63 cells were incubated with 0–200 lM H2O2 for 30 min, cell
lysates were subjected to Western blot analysis with antibodies against
S6, phospho-S6 (S235/236), 4E-BP1, and phospho-4E-BP1 (T37/46).
(C) MC3T3-E1 cells were incubated with 200–5,000 lM H2O2 for 30 min
or with 1,000 lM H2O2 for indicated times, cell lysates were subjected
to Western blot analysis with antibodies against S6K, phospho-
TABLE 1. H2O2 induced G2 cell cycle arrest in osteoblast
Cell proportion (%)
G0/G1 phase
Control
200 lM H2O2 treatment for 24 hr
200 lM H2O2 treatment for 48 hr
200 lM H2O2 treatment for 72 hr
77.81
63.06
52.77
40.22
1.39
3.34
2.37
3.54
S phase
10.85
11.49
14.87
13.19
0.16
2.01
1.35
4.15
G2/M phase
11.34
25.46
32.36
46.59
1.54
2.42
2.61
5.88
MC-3T3-E1 cells treated with 200 lM H2O2 for 24, 48, or 72 hr were subjected to cell cycle analysis as described in Materials and Methods. All values are presented as mean SD of 3
experiments.
H2O2 INDUCES G2 ARREST IN OSTEOBLASTS
1111
Fig. 4. H2O2 induces G2 cell cycle arrest in osteoblasts. MC3T3-E1 cells treated with 200 lM H2O2 for
24, 48, or 72 hr were subjected to cell cycle analysis as described in Materials and Methods. All values
are presented as means SD of 3 experiments.
Fig. 5. H2O2 but not rapamycin down-regulates cyclin B1 in osteoblasts. MC3T3-E1 cells treated with 200 lM H2O2 for 0, 24, or 48 hr
were subjected to Western blot analysis with antibodies against cyclin
B1 and b-actin.
that mTOR may play an important role in regulation of
cell growth and proliferation in osteoblasts (Singha et al.,
2008). The phosphorylation of S6K on T389, 4E-BP1 on
T37/46, and S6 on S235/236 may represent the activity of
mTOR, as these sites are specifically phosphorylated by
mTOR both in vitro and in vivo and are inhibited by rapamycin treatment. To determine whether mTOR is
involved in H2O2-induced suppression of osteoblast proliferation, MC3T3-E1 and MG63 cells were incubated with
different doses (0–5,000 lM) of H2O2 for different times (5
min to 4 hr). However, we did not see any significant
changes in phosphorylation of S6 (S235/236) and 4E-BP1
(T37/46) after incubation with low concentrations (5–200
lM) of H2O2 for 30 min (Fig. 3A,B) which could prevent
proliferation of osteoblast (Fig. 1). But higher concentrations (0.5–5 mM) of H2O2 suppressed mTOR signaling
dose- and time-dependently, manifested by a dephosphorylation of S6K (T389), 4E-BP1 (T37/46), and S6(S235/
236) in MC3T3-E1 and MG63 cells (Fig. 3C,E). The only
exception was that, in MC3T3-E1 cells, 200 lM H2O2 or
short-time exposure (5 or 15 min) of 1 mM H2O2 somehow
enhanced phosphorylation of S6K (T389; Fig. 3C). This
action of H2O2 on mTOR inhibition could be reversed by
H2O2 scavenger, catalase (Fig. 3D).
Fig. 6. Rapamycin inhibits cell proliferation in osteoblast. MC3T3E1 cells were treated with 50 nM rapamycin for 24, 48, or 72 hr and
cell proliferation was detected by MTT assay.
H2O2 Induces G2 Cell Cycle Arrest
in Osteoblasts
It has been demonstrated that inhibition of mTOR by
nutrient starvation or rapamycin inhibits cell growth
and induces G1 cell cycle arrest in some cell types (Shi
et al., 2005; Law et al., 2006). In MC3T3-E1 cells, however, the percentage of cells in G2/M phase increased
about two, three, or fourfold compared with control after
incubation with 200 lM H2O2 for 24, 48, or 72 hr,
respectively (Table 1; Fig. 4). Accordingly, the percentage
of cells in G0/G1 phase decreased time-dependently (Table 1; Fig. 4). It is suggested that H2O2 induces a G2 cell
cycle arrest in osteoblasts.
H2O2 but not Rapamycin Down-Regulates
Levels of Cyclin B1 in Osteoblasts
Cyclin B complexes with p34(cdc2) to form the maturation-promoting factor (MPF), which plays an important role in cell cycle progression in G2/M phase. To
elucidate whether cyclin B is regulated by H2O2,
MC3T3-E1 cells were incubated with 200 lM H2O2 for
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LI ET AL.
24 and 48 hr and protein level of cyclin B1 was detected
by Western blot. As shown in Fig. 5, H2O2 down-regulated cyclin B1 time-dependently. Rapamycin, which
could inhibit cell proliferation (Fig.6), however, did not
reduce the protein level of cyclin B1 in MC3T3-E1 cells.
It demonstrated that H2O2 induced G2 arrest via a
mTOR-independent mechanism. Down-regulation of
cyclin B1 might be responsible for the induction of G2
cell cycle arrest and inhibition of cell proliferation by
H2O2 in osteoblasts.
DISCUSSION
Recently, it has been suggested that ROS may play an
important role in postmenopausal bone loss by generating a more oxidized bone microenvironment (Lean et al.,
2003, 2005). But the mechanisms of the actions of ROS
and the cellular targets that regulate bone mass are
poorly understood. It has been shown that ROS stimulates osteoclastogenesis whereas antioxidants suppress
osteoclast differentiation and activity (Aitken et al.,
2004; Ha et al., 2004). Most reports about the effects of
ROS on osteoblasts focus on ROS prevention of cell differentiation and induction of cell death (apoptosis and
necrosis) (Mody et al., 2001; Bai et al., 2004; Liu et al.,
2004; Fatokun et al., 2006, 2008). We have previously
shown that ROS inhibits osteoblastic differentiation of
BMSCs and calvarial osteoblasts by extracellular-signalregulated kinase 1/2 (ERK1/2) and NF-jB (Bai et al.,
2004), and that ROS enhances osteoclastgenesis by stimulating receptor activator of NF-jB ligand (RANKL)
expression in osteoblast (Bai et al., 2005). We found no
reports concerning the roles and targets of ROS during
proliferation of osteoblast. In this study, we demonstrated that the cell viability was not affected by low
doses (5–200 lM) of H2O2 in mouse osteoblast cell line
MC3T3-E1 and human osteoblast-like cell line MG63
(Fig. 1A,B). Cell proliferation, however, was suppressed
dose- and time-dependently (Fig. 2A–D) by 5–200 lM of
H2O2. Cell cycle analysis showed that the percentage of
cells in G2/M phase increased about two, three, or fourfold compared with controls (Table 1; Fig. 4). Further
study revealed that protein level of cyclin B1 decreased
markedly by H2O2 exposure (Fig. 5), which might be responsible for the induction of a G2 cell cycle arrest by
H2O2. These data showed for the first time that, H2O2
reduced cyclin B1 expression, induced G2 cell cycle
arrest and prevented cell proliferation in osteoblasts. In
accordance with previous reports (Bai et al., 2004; Fatokun et al., 2006), exposure of MC3T3-E1 cells to concentrations of more than 500 lM H2O2 produced significant
lethality involving both apoptosis and necrosis. The concentrations of H2O2 (5–200 lM) for inhibition of cell proliferation in this study were consistent with the amount
for prevention of cell differentiation in osteoblasts (Bai
et al., 2004). It is interesting that 5–200 lM of H2O2
inhibited cell proliferation in growth medium but prevented cell differentiation in differentiation medium.
Ongoing studies in our laboratory are exploring whether
ERK1/2 and NF-jB signaling are involved in this process, and whether H2O2 down-regulates cyclin B1 and
induces G2/M arrest via proteasome-mediated ubiquitination and degradation.
mTOR plays a central role in cell growth and proliferation. A specific inhibitor of mTOR, rapamycin has been
reported recently to inhibit cell proliferation in MC3T3E1 and mouse BMSCs by decreasing the levels of cyclin
A and cyclin D1 (Singha et al., 2008). It has been suggested that ROS could affect the mTOR pathway both
positively and negatively in various cell types (Corradetti and Guan, 2006; Reiling and Sabatini, 2006). To
determine if mTOR signaling is involved in ROS-induced
inhibition of cell proliferation in osteoblasts, the effects
of ROS on mTOR signaling in osteoblasts were examined
for the first time. We demonstrated that higher concentrations of H2O2 (500–5000 lM) inhibited mTOR signaling dose- and time-dependently, manifested by a
dephosphorylation of S6K (T389), 4E-BP1 (T37/46), and
S6(S235/236) in MC3T3-E1, MG63 (Fig. 3). However, low
concentrations (5–200 lM) of H2O2 which could prevent
proliferation of osteoblast did not affect mTOR activity.
Moreover, inhibition of mTOR signaling often induces a
G1 cell cycle arrest that correlates with down-regulation
of cyclin D1 levels in some cell types. We found that
H2O2 induced G2 cell cycle arrest instead of G1 arrest
and decreased levels of cyclin B1. On the contrary, rapamycin did not down-regulate protein level of cyclin B1.
It is suggested that suppression of mTOR signaling may
not be involved in prevention of cell proliferation by
H2O2 in osteoblasts. The significance of H2O2-induced
inhibition of mTOR signaling in osteoblasts and whether
it is related to cellular apoptosis or necrosis should be
further investigated.
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