Hydrogen Peroxide Induces G2 Cell Cycle Arrest and Inhibits Cell

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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*
                                Department of Cell Biology, School of Basic Medical Sciences,
                                      Southern Medical University, Guangzhou, China
                         Institute of Orthopedics, Southern Medical University, Guangzhou, China
                       Department of Urology, Guangzhou General Hospital of Guangzhou Command,
                                                     Guangzhou, China
                    Institute of Genetic Engineering, Southern Medical University, Guangzhou, China

                               Reactive oxygen species (ROSs) are involved in osteoporosis by inhibi-
                          ting 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 mani-
                          fested by dephosphorylation of S6K (T389), 4E-BP1 (T37/46), and
                          S6(S235/236) in MC3T3-E1 and MG63 cells. Rapamycin, which could in-
                          hibit 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
                          process. Anat Rec, 292:1107–1113, 2009. V 2009 Wiley-Liss, Inc.

                          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 activ-
ities of osteoblasts, the bone-forming cells, and osteo-
clasts, the bone-resorbing cells. Any loss of osteoblastic         Grant sponsor: National Natural Sciences Foundation of
activity or an increase in osteoclastic activity would           China and Program for New Century Excellent Talents in
ultimately lead to osteoporosis. So proliferation and dif-       University; Grant numbers: 30771027 and 30870955.
ferentiation of osteoblasts and osteoclasts are very im-           *Correspondence to: Xiao-Chun Bai or Shen-Qiu Luo, Depart-
portant for the pathogenesis of osteoporosis (Erlebacher         ment of Cell Biology, School of Basic Medical Sciences, Southern
et al., 1995; Manolagas and Jilka, 1995; Manolagas,              Medical University, Guangzhou 510515, China. Fax: 86-20-
2000).                                                           61648208. E-mail: or
   Oxidative stress, resulting from excessive levels of            Received 7 February 2009; Accepted 15 April 2009
reactive oxygen species (ROSs) such as superoxides               DOI 10.1002/ar.20925
anions and hydrogen peroxide (H2O2), represents a                Published online in Wiley InterScience (www.interscience.wiley.
major cause of cellular damage and death in a plethora           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 osteoclast-
genesis whereas antioxidants suppress osteoclast differ-
entiation and activity; (iii) ROS prevents osteoblastic
differentiation whereas antioxidants enhance differentia-
tion 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 loss-
related diseases.
   The mammalian target of rapamycin, mTOR, serves
as a signal integrator of many upstream signals, includ-
ing 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 (Saba-
tini, 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 dis-
tinct mechanisms: (i) mTOR phosphorylates and inacti-
vates 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 pro-
tein synthesis of 50 capped mRNAs; (ii) mTOR phospho-
rylates 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 conse-
quently, ceases cell growth (Wullschleger et al., 2006;          Fig. 1. Effect of H2O2 on cell viability of osteoblasts. (A) MC3T3-E1
Soulard and Hall, 2007).                                       and (B) MG63 cells were treated with 0–1,000 lM H2O2 for 72 hr and
   Although the roles and mechanisms of ROS in regula-         cell viability was detected by trypan blue dye-exclusion method.
tion of cell differentiation in osteoblasts have been stud-
ied (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      modified Eagle’s medium-high glucose (DMEM) and a-
and its progenitor in bone marrow makes an important           MEM, respectively, supplemented with 10% fetal bovine
contribution to the amount of differentiated and func-         serum (FBS), 50 units/mL penicillin and 50 lg per strep-
tional osteoblasts, and that mTOR plays a central role in      tomycin in a humidified atmosphere of 5% CO2. Cultures
cell growth and proliferation, this study examined the         were trypsinized upon confluence and subcultured into
effect of hydrogen peroxide on mTOR signaling, cell            12-, 6-, or 96-well plates for further experiments.
cycle progression, and cell proliferation in mouse osteo-
blast cell line, MC3T3-E1, and in human osteoblast-like
                                                               Cell Proliferation Assay
cell line, MG63.
                                                                 Methyl thiazolyl tetrazolium (MTT) assays were per-
            MATERIAL AND METHODS                               formed to assess the rate of cell proliferation. Briefly,
                                                               MC3T3-E1 and MG63 cells were planted into 96-well
Reagents                                                       plates. After incubation overnight, the medium was
  Catalase, dimethyl sulphoxide (DMSO), were pur-              replaced with fresh medium with or without H2O2 at
chased form Sigma-Aldrich (St. Louis, MO). Antibodies          indicated concentrations(5, 20, 50, 100, 200 lM) for vari-
against phospho-S6K(T389), 4E-BP1, phospho-4E-BP1              ous times (24, 48, 72 hr). Six wells were included in
(T37/46), and phospho-S6(S235/236) were purchased              each concentration. At the end of treatment, 20 lL MTT
from Cell Signaling Inc. (Beverly, MA), anti-S6, S6K, b-       (AMRESCO, OH) was added for 4 hr. Then the medium
actin, and cyclin B1 antibodies were from Santa Cruz           was discarded carefully and 150 lL DMSO was added.
Biotech (Santa Cruz, CA).                                      Absorbance was recorded at 570 nm with The Universal
                                                               Microplate Reader (Bio-Tek instruments) using wells
                                                               without cells as blanks. All experiments were performed
Cell Culture
                                                               in triplicate. The inhibition rate of cell proliferation was
  Human osteoblast-like cell line MG63 and mouse               calculated by formula: % inhibition ¼ (A570 of control
osteoblast cell line MC3T3-E1 were grown in Dulbecco’s         À 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 time-      MG63 cells were treated with 200 lM H2O2 for 24, 48, or 72 hr and
dependently. (A) MC3T3-E1 and (B) MG63 cells were treated with 5,         cell proliferation was detected by MTT assay. Inhibition rates were cal-
20, 50, 100, or 200 lM H2O2 for 48 hr and 72 hr, respectively, and cell   culated as described in Materials and Methods.
proliferation was detected by MTT assay. (C) MC3T3-E1 and (D)

Cell Viability Analysis                                                   Statistical Analysis
  Cells were treated with different concentrations (0–                      Statistical analyses were performed by ANOVA, and
1 mM) of H2O2. After 72 hr, cell viability was deter-                     P < 0.05 was considered statistically significant.
mined by counting the viable cell number with a hemo-
cytometer after staining with trypan blue.
                                                                          Dose- and Time-Dependent Inhibition of
Cell Cycle Analysis
                                                                          Proliferation by H2O2 in Osteoblasts
  Cell cycle assays were performed to assess the cell
cycle progression. Briefly, exponentially growing MC3T3-                      Cellular responses elicited by H2O2 depend upon the
E1 cells were synchronized at the G1/S boundary after                     severity of the damage, which is further influenced by
starvation with basal medium for 24 hr, followed by                       the cell type and the magnitude of the dose of the expo-
incubation in the presence or absence of 200 lM H2O2                      sure (Finkel and Holbrook, 2000; Temple et al., 2005). In
for 24, 48, and 72 hr. At the indicated intervals, cells                  our experiments, MC3T3-E1 and MG63 cells underwent
were harvested and measured by cell cycle detection kit                   severe cell death after high doses of H2O2 (0.5 or 1 mM)
(KEY GEN, Nanjing, China) following manufacturer’s                        treatment for 72 hr as determined by the Trypan Blue
instructions. The cell cycle distribution was analyzed by                 dye-exclusion method. After an exposure to low doses of
flow cytometry (FACS CaliburTM, BD) immediately. The                       H2O2 (5–200 lM), as expected, the cell viability was not
percentage of cells in G0/G1, S, and G2/M phases were                     affected compared with the controls (Fig. 1A,B). Cell pro-
calculated using FCS Express software.                                    liferation, however, was inhibited dose- and time-
                                                                          dependently by 5–200 lM H2O2. Here we show the data
                                                                          for proliferation of MC3T3-E1 cells after treatment with
Western Blot Analysis                                                     5–200 lM H2O2 for 48 hr (Fig. 2A) and 200 lM H2O2 for
   After treatment, cells were lysed immediately in                       24, 48, and 72 hr (Fig. 2C), and that of MG63 cells after
Laemmli buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS,                         treatment with 5–200 lM H2O2 for 72 hr (Fig. 2B) and
10% glycerol, 50 mM dithiothreitol, 0.01% bromophenol                     200 lM H2O2 for 24, 48, and 72 hr (Fig. 2D).
blue) for 5 min at 95 C. Cell lysates were analyzed by
SDS/PAGE and transferred electrophoretically to Nitro
                                                                          H2O2 Inhibits mTOR Signaling in Osteoblasts
cellulose membrane (Bio-Rad Corp, Hercules, CA). Blots
were probed with specific antibodies and immunoreac-                          It has been shown recently that rapamycin inhibits pro-
tive proteins were revealed by the enhanced chemilumi-                    liferation and differentiation of MC3T3-E1 cells and
nescence (ECL) kit (Santa Cruz Biotechnology Inc., CA).                   mouse bone marrow stromal cells (BMSCs). It suggests
1110                                                               LI ET AL.

  Fig. 3. H2O2 inhibits mTOR signaling in osteoblasts. (A) MC3T3-E1           S6K(T389), 4E-BP1, phospho-4E-BP1 (T37/46), S6, and phospho-S6
and (B) MG63 cells were incubated with 0–200 lM H2O2 for 30 min, cell         (S235/236). (D) MC-3T3-E1 cells were pretreated with 500 U/mL cata-
lysates were subjected to Western blot analysis with antibodies against       lase or not, then incubated with 1,000 lM H2O2 for 30 min, cell lysates
S6, phospho-S6 (S235/236), 4E-BP1, and phospho-4E-BP1 (T37/46).               were analyzed as in (C). (E) MG63 were incubated with 200–5,000 lM
(C) MC3T3-E1 cells were incubated with 200–5,000 lM H2O2 for 30 min           H2O2 for 30 min, cell lysates were analyzed by Western blot with anti-
or with 1,000 lM H2O2 for indicated times, cell lysates were subjected        bodies as indicated.
to Western blot analysis with antibodies against S6K, phospho-

                                   TABLE 1. H2O2 induced G2 cell cycle arrest in osteoblast
                                                                                         Cell proportion (%)
                                                                 G0/G1 phase                    S phase               G2/M phase
               Control                                           77.81    Æ   1.39           10.85   Æ   0.16         11.34   Æ   1.54
               200 lM H2O2 treatment for 24 hr                   63.06    Æ   3.34           11.49   Æ   2.01         25.46   Æ   2.42
               200 lM H2O2 treatment for 48 hr                   52.77    Æ   2.37           14.87   Æ   1.35         32.36   Æ   2.61
               200 lM H2O2 treatment for 72 hr                   40.22    Æ   3.54           13.19   Æ   4.15         46.59   Æ   5.88
               MC-3T3-E1 cells treated with 200 lM H2O2 for 24, 48, or 72 hr were subjected to cell cycle anal-
               ysis as described in Materials and Methods. All values are presented as mean Æ SD of 3
                                              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 Æ SD of 3 experiments.

  Fig. 5. H2O2 but not rapamycin down-regulates cyclin B1 in osteo-
blasts. MC3T3-E1 cells treated with 200 lM H2O2 for 0, 24, or 48 hr
were subjected to Western blot analysis with antibodies against cyclin     Fig. 6. Rapamycin inhibits cell proliferation in osteoblast. MC3T3-
B1 and b-actin.                                                          E1 cells were treated with 50 nM rapamycin for 24, 48, or 72 hr and
                                                                         cell proliferation was detected by MTT assay.
that mTOR may play an important role in regulation of
cell growth and proliferation in osteoblasts (Singha et al.,
                                                                         H2O2 Induces G2 Cell Cycle Arrest
2008). The phosphorylation of S6K on T389, 4E-BP1 on
T37/46, and S6 on S235/236 may represent the activity of                 in Osteoblasts
mTOR, as these sites are specifically phosphorylated by                     It has been demonstrated that inhibition of mTOR by
mTOR both in vitro and in vivo and are inhibited by rapa-                nutrient starvation or rapamycin inhibits cell growth
mycin treatment. To determine whether mTOR is                            and induces G1 cell cycle arrest in some cell types (Shi
involved in H2O2-induced suppression of osteoblast prolif-               et al., 2005; Law et al., 2006). In MC3T3-E1 cells, how-
eration, MC3T3-E1 and MG63 cells were incubated with                     ever, the percentage of cells in G2/M phase increased
different doses (0–5,000 lM) of H2O2 for different times (5              about two, three, or fourfold compared with control after
min to 4 hr). However, we did not see any significant                     incubation with 200 lM H2O2 for 24, 48, or 72 hr,
changes in phosphorylation of S6 (S235/236) and 4E-BP1                   respectively (Table 1; Fig. 4). Accordingly, the percentage
(T37/46) after incubation with low concentrations (5–200                 of cells in G0/G1 phase decreased time-dependently (Ta-
lM) of H2O2 for 30 min (Fig. 3A,B) which could prevent                   ble 1; Fig. 4). It is suggested that H2O2 induces a G2 cell
proliferation of osteoblast (Fig. 1). But higher concentra-              cycle arrest in osteoblasts.
tions (0.5–5 mM) of H2O2 suppressed mTOR signaling
dose- and time-dependently, manifested by a dephospho-                   H2O2 but not Rapamycin Down-Regulates
rylation of S6K (T389), 4E-BP1 (T37/46), and S6(S235/
                                                                         Levels of Cyclin B1 in Osteoblasts
236) in MC3T3-E1 and MG63 cells (Fig. 3C,E). The only
exception was that, in MC3T3-E1 cells, 200 lM H2O2 or                      Cyclin B complexes with p34(cdc2) to form the matu-
short-time exposure (5 or 15 min) of 1 mM H2O2 somehow                   ration-promoting factor (MPF), which plays an impor-
enhanced phosphorylation of S6K (T389; Fig. 3C). This                    tant role in cell cycle progression in G2/M phase. To
action of H2O2 on mTOR inhibition could be reversed by                   elucidate whether cyclin B is regulated by H2O2,
H2O2 scavenger, catalase (Fig. 3D).                                      MC3T3-E1 cells were incubated with 200 lM H2O2 for
1112                                                    LI ET AL.

24 and 48 hr and protein level of cyclin B1 was detected      reported recently to inhibit cell proliferation in MC3T3-
by Western blot. As shown in Fig. 5, H2O2 down-regu-          E1 and mouse BMSCs by decreasing the levels of cyclin
lated cyclin B1 time-dependently. Rapamycin, which            A and cyclin D1 (Singha et al., 2008). It has been sug-
could inhibit cell proliferation (Fig.6), however, did not    gested that ROS could affect the mTOR pathway both
reduce the protein level of cyclin B1 in MC3T3-E1 cells.      positively and negatively in various cell types (Corra-
It demonstrated that H2O2 induced G2 arrest via a             detti and Guan, 2006; Reiling and Sabatini, 2006). To
mTOR-independent mechanism. Down-regulation of                determine if mTOR signaling is involved in ROS-induced
cyclin B1 might be responsible for the induction of G2        inhibition of cell proliferation in osteoblasts, the effects
cell cycle arrest and inhibition of cell proliferation by     of ROS on mTOR signaling in osteoblasts were examined
H2O2 in osteoblasts.                                          for the first time. We demonstrated that higher concen-
                                                              trations of H2O2 (500–5000 lM) inhibited mTOR signal-
                                                              ing dose- and time-dependently, manifested by a
                     DISCUSSION                               dephosphorylation of S6K (T389), 4E-BP1 (T37/46), and
   Recently, it has been suggested that ROS may play an       S6(S235/236) in MC3T3-E1, MG63 (Fig. 3). However, low
important role in postmenopausal bone loss by generat-        concentrations (5–200 lM) of H2O2 which could prevent
ing a more oxidized bone microenvironment (Lean et al.,       proliferation of osteoblast did not affect mTOR activity.
2003, 2005). But the mechanisms of the actions of ROS         Moreover, inhibition of mTOR signaling often induces a
and the cellular targets that regulate bone mass are          G1 cell cycle arrest that correlates with down-regulation
poorly understood. It has been shown that ROS stimu-          of cyclin D1 levels in some cell types. We found that
lates osteoclastogenesis whereas antioxidants suppress        H2O2 induced G2 cell cycle arrest instead of G1 arrest
osteoclast differentiation and activity (Aitken et al.,       and decreased levels of cyclin B1. On the contrary, rapa-
2004; Ha et al., 2004). Most reports about the effects of     mycin did not down-regulate protein level of cyclin B1.
ROS on osteoblasts focus on ROS prevention of cell dif-       It is suggested that suppression of mTOR signaling may
ferentiation and induction of cell death (apoptosis and       not be involved in prevention of cell proliferation by
necrosis) (Mody et al., 2001; Bai et al., 2004; Liu et al.,   H2O2 in osteoblasts. The significance of H2O2-induced
2004; Fatokun et al., 2006, 2008). We have previously         inhibition of mTOR signaling in osteoblasts and whether
shown that ROS inhibits osteoblastic differentiation of       it is related to cellular apoptosis or necrosis should be
BMSCs and calvarial osteoblasts by extracellular-signal-      further investigated.
regulated kinase 1/2 (ERK1/2) and NF-jB (Bai et al.,
2004), and that ROS enhances osteoclastgenesis by stim-
ulating receptor activator of NF-jB ligand (RANKL)                               LITERATURE CITED
expression in osteoblast (Bai et al., 2005). We found no
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