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Keywords:

  • hydrogen peroxide;
  • osteoblast;
  • mammalian target of rapamycin;
  • cyclin B1;
  • proliferation;
  • cell cycle;
  • G2 cell cycle arrest

Abstract

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

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 μM 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 μM H2O2 treatment for 24–72 hr. A total of 0.5–5 mM of H2O2 but not lower concentrations (5–200 μM) 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 process. Anat Rec, 292:1107–1113, 2009. © 2009 Wiley-Liss, Inc.

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 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 loss-related 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 5′ 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

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

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, β-actin, 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 modified Eagle's medium-high glucose (DMEM) and α-MEM, respectively, supplemented with 10% fetal bovine serum (FBS), 50 units/mL penicillin and 50 μg 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 μM) for various times (24, 48, 72 hr). Six wells were included in each concentration. At the end of treatment, 20 μL MTT (AMRESCO, OH) was added for 4 hr. Then the medium was discarded carefully and 150 μL 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 = (A570 of control − A570 of treated cells)/A570 of control cells × 100%.

Cell Viability 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.

Cell Cycle Analysis

Cell cycle assays were performed to assess the cell cycle progression. Briefly, exponentially growing MC3T3-E1 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 μM 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 Calibur™, 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).

Statistical Analysis

Statistical analyses were performed by ANOVA, and P < 0.05 was considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

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 μM), as expected, the cell viability was not affected compared with the controls (Fig. 1A,B). Cell proliferation, however, was inhibited dose- and time- dependently by 5–200 μM H2O2. Here we show the data for proliferation of MC3T3-E1 cells after treatment with 5–200 μM H2O2 for 48 hr (Fig. 2A) and 200 μM H2O2 for 24, 48, and 72 hr (Fig. 2C), and that of MG63 cells after treatment with 5–200 μM H2O2 for 72 hr (Fig. 2B) and 200 μM H2O2 for 24, 48, and 72 hr (Fig. 2D).

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Figure 1. Effect of H2O2 on cell viability of osteoblasts. (A) MC3T3-E1 and (B) MG63 cells were treated with 0–1,000 μM H2O2 for 72 hr and cell viability was detected by trypan blue dye-exclusion method.

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Figure 2. H2O2 inhibits proliferation of osteoblasts dose- and time-dependently. (A) MC3T3-E1 and (B) MG63 cells were treated with 5, 20, 50, 100, or 200 μM 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 μM 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.

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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 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 μM) 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 μM) 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 μM 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).

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Figure 3. H2O2 inhibits mTOR signaling in osteoblasts. (A) MC3T3-E1 and (B) MG63 cells were incubated with 0–200 μM 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 μM H2O2 for 30 min or with 1,000 μM H2O2 for indicated times, cell lysates were subjected to Western blot analysis with antibodies against S6K, phospho-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 μM H2O2 for 30 min, cell lysates were analyzed as in (C). (E) MG63 were incubated with 200–5,000 μM H2O2 for 30 min, cell lysates were analyzed by Western blot with antibodies as indicated.

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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 μM 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.

Table 1. H2O2 induced G2 cell cycle arrest in osteoblast
 Cell proportion (%)
G0/G1 phaseS phaseG2/M phase
  1. MC-3T3-E1 cells treated with 200 μM 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.

Control77.81 ± 1.3910.85 ± 0.1611.34 ± 1.54
200 μM H2O2 treatment for 24 hr63.06 ± 3.3411.49 ± 2.0125.46 ± 2.42
200 μM H2O2 treatment for 48 hr52.77 ± 2.3714.87 ± 1.3532.36 ± 2.61
200 μM H2O2 treatment for 72 hr40.22 ± 3.5413.19 ± 4.1546.59 ± 5.88
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Figure 4. H2O2 induces G2 cell cycle arrest in osteoblasts. MC3T3-E1 cells treated with 200 μM 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.

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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 μM H2O2 for 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.

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Figure 5. H2O2 but not rapamycin down-regulates cyclin B1 in osteoblasts. MC3T3-E1 cells treated with 200 μM H2O2 for 0, 24, or 48 hr were subjected to Western blot analysis with antibodies against cyclin B1 and β-actin.

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Figure 6. Rapamycin inhibits cell proliferation in osteoblast. MC3T3-E1 cells were treated with 50 nM rapamycin for 24, 48, or 72 hr and cell proliferation was detected by MTT assay.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

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-signal-regulated kinase 1/2 (ERK1/2) and NF-κB (Bai et al.,2004), and that ROS enhances osteoclastgenesis by stimulating receptor activator of NF-κB 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 μM) 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 μM 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 μM H2O2 produced significant lethality involving both apoptosis and necrosis. The concentrations of H2O2 (5–200 μM) 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 μM 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-κB 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 MC3T3-E1 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 μM) 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 μM) 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.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED