Metallothionein protects bone marrow stromal cells against hydrogen peroxide-induced inhibition of osteoblastic differentiation

Cell Biology International 28 (2004) 905e911 www.elsevier.com/locate/cellbi

Metallothionein protects bone marrow stromal cells against hydrogen peroxide-induced inhibition of osteoblastic differentiation An-Ling Liua,*, Zhong-Ming Zhangb, Bi-Feng Zhua, Zhao-Hui Liaoa, Zhu Liua a

Yingdong College of Biotechnology, Shaoguan University, Shaoguan, Guangdong Province 512005, PR China Department of Orthopaedics and Spinal Surgery, NanFang Hospital, The First Military Medical University, Guangzhou 510515, PR China

b

Received 6 April 2004; revised 2 September 2004; accepted 13 September 2004

Abstract Metallothionein (MT), a cysteine-rich, metal-binding protein, is involved in homeostatic regulation of essential metals and protection of cells against oxidative injury. It has been shown that oxidative stress is associated with pathogenesis of osteoporosis and is capable of inhibiting osteoblastic differentiation of bone cells by nuclear factor-kB (NF-kB). In this study, the effect of MT on oxidative stress-induced inhibition of osteoblast differentiation was examined. 50e200 mM hydrogen peroxide-induced oxidative stress suppressed the osteoblastic differentiation process of primary mouse bone marrow stromal cells (BMSCs), manifested by a reduction in the differentiation marker alkaline phosphatase (ALP). The presence of exogenous MT (20e500 mM) or induction of endogenous MT by ZnCl2 (50e200 mM) could protect BMSCs against H2O2-induced inhibition of osteoblastic differentiation, manifested by a resumption of H2O2-inhibited ALP activity and ALP positive cells. Furthermore, adding exogenous MT or inducing endogenous MT expression impaired H2O2-stimulated NF-kB signaling. These data indicate the ability of MT to protect BMSCs against oxidative stress-induced inhibition of osteoblastic differentiation. Ó 2004 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Metallothionein; Oxidative stress; Osteoblast; Differentiation; Hydrogen peroxide; Osteoporosis

1. Introduction Bone is continuously destroyed and reformed in vertebrates in order to maintain bone volume and calcium homeostasis throughout their lives. Osteoblasts and osteoclasts are specialized cells responsible for bone formation and resorption. Any loss of osteoblastic activity or increase in osteoclastic activity would

* Corresponding author. Tel.: C86 20 61360535; fax: C86 751 8120069. E-mail address: [email protected] (A.-L. Liu).

ultimately lead to osteoporosis, characteristics of lower bone mineral densities (BMD), a decrease in bone mass, and weaker bones that are more likely to fracture (Manolagas and Jilka, 1995). The differentiation of osteoblasts and osteoclasts are believed to be very important in the pathogenesis of osteoporosis (Boyle, et al., 2003; Manolagas, 2000). Osteoblasts are derived from osteoprogenitors that reside in the bone marrow. Although evidence suggests that many cytokines, hormones, signaling pathways and transcription factors may be involved in osteoblast differentiation, the signaling mechanisms that contribute to decreased osteoblastic differentiation in osteoporosis

1065-6995/$ - see front matter Ó 2004 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2004.09.004

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Nomenclature ALP alkaline phosphatase BMD bone mineral densities BMSCs bone marrow stromal cells ERKs extracellular signal-regulated kinases ECL enhanced chemiluminescence HSF heat shock factor IkB NF-kB inhibitory proteins MAPK mitogen-activated protein kinase MT Metallothionein NF-kB nuclear factor-kB ROS reactive oxygen species XXO xantine/xanthine oxidase

are not well known (Ducy et al., 1997; Harada and Rodan, 2003). Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, can cause severe damage to DNA, protein and lipids. High levels of oxidant produced during normal cellular metabolism (e.g. mitochondrial electron transport) or from environmental stimuli (e.g. cytokines, UV radiation) perturb the normal redox balance and shift cells into a state of oxidative stress (Finkel and Holbrook, 2000). Oxidative stress has been implicated in a wide variety of disease processes, including atherosclerosis, diabetes, neurodegenerative disorders and the process of aging (Finkel and Holbrook, 2000). At the cellular level, oxidant injury elicits a wide spectrum of responses, ranging from proliferation to growth or differentiation arrest, senescence and cell death, by activating numerous major signaling pathways, such as nuclear factor-kB (NF-kB), mitogen-activated protein kinases (MAPKs), p53 and heat shock factor (HSF) (Martindale and Holbrook, 2002). Recently, several lines of evidence have found a close association between oxidative stress and pathogenesis of osteoporosis. A marked decrease in plasma antioxidants are found in aged osteoporotic women (Maggio et al., 2003); estrogen deficiency causes bone loss by lowering thiol antioxidants in osteoclasts (Lean et al., 2003); there is a biochemical link between increased oxidative stress and reduced BMD in aged men and women (Basu et al., 2001); dietary antioxidant vitamin intake has a beneficial effect on BMD in postmenopausal women (Morton et al., 2001); oxidative stress is able to inhibit osteoblastic differentiation of bone cells by extracellularsignal-regulated kinases (ERKs) and NF-kB (Mody et al., 2001; Bai et al., 2004). Metallothioneins (MTs) are intracellular, ubiquitous, low molecular, cysteine-rich, metal-binding proteins which have four major isoforms (MT-I, MT-II, MTIII and MT-IV) (Vasak and Hasler, 2000). MT synthesis is induced by various stimuli, such as cadmium, zinc,

mercury, oxidative stress and anticancer agents. Although MT-null mice enjoy apparently good health and the critical biological roles of MTs have been questioned, a number of roles associated with cellular and tissue stress have been attributed to MTs. These roles include the detoxification of heavy metals, homeostatic regulation of essential metals and protection of tissues against various forms of oxidative injury (Tapiero and Tew, 2003). MTs can protect against alcoholic liver injury through inhibition of oxidative stress (Zhou et al., 2002). MT-III has been found to be markedly reduced in the brain of patients with Alzheimer’s disease and several other neurodegenerative diseases (Chung and West, 2004). These observations prompted us to explore the role of MT in osteoblastic differentiation of bone cells during oxidative stress. Here we demonstrate that MT is capable of protecting mouse bone marrow stromal cells (BMSCs) against hydrogen peroxide-induced inhibition of osteoblastic differentiation.

2. Materials and methods 2.1. Cell culture and treatment BMSCs were isolated from 1 month-old mice and cultivated in aMEM supplemented with 10% fetal bovine serum (Life Technologies Inc., Gaithersburg, MD, USA). Cultures were trypsinized upon confluence and propagated to passage 2 before being subcultured onto 24-well plates, 100-mm petri dishes or glass slides for further experiment. BMSCs were pretreated with 50e200 mM ZnCl2 for 16 h (then the media was changed), or pretreated with 20e500 mM MT (from rabbit liver, containing about 7% Cd C Zn) (SigmaeAldrich, St. Louis, MO, USA) for 30 min, then incubated with or without different concentrations of H2O2 for various times in osteoblastic differentiation medium (aMEM containing 10 mM bglycerophosphate, 50 mg/ml ascorbic acid and 10ÿ8 M dexamethasone, SigmaeAldrich, St. Louis, MO, USA). 2.2. Cell viability analysis BMSCs subcultured into 24-well plates were treated with different concentrations of H2O2 once confluent. After 24 h, cell viability was determined using Trypan Blue. 2.3. Measurement of MT expression MT concentration was determined by 109Cdeheme affinity assay. High affinity of MT for cadmium coupled with a good stability at high temperature is the basis for

A.-L. Liu et al. / Cell Biology International 28 (2004) 905e911

the determination. Cells treated with or without ZnCl2 were harvested and resuspended in 10 mM TriseHCl, pH 7.5, and sonicated briefly. The supernatant was used for determination of total MT protein. Total protein levels were determined by BCA assay and used to normalize MT levels. Free 109Cd was added to the heatdenatured supernatant. The mixture was incubated at room temperature for 10 min, then hemoglobin solution was added to remove the excess cadmium. After heating, cooling and centrifuging, the supernatant was transferred to a g-counting tube. The amount of radioactivity in the supernatant fraction was determined using a scintillation counter. Results were expressed as 109 Cd-bound protein (nmol) per gram of total protein. 2.4. Alkaline phosphatase (ALP) staining Cells subcultured on glass slides in differentiation medium were treated as above once confluent. After 4 days, the cell cultures were fixed with neutral formaldehyde and subjected to modified Gomori’s ALP staining. The ratio of ALP-positive cells to total cells (500 cells were counted per group) was quantified microscopically. 2.5. Determination of ALP activity BMSCs subcultured into 24 well plates were treated as above, once confluent, for 4 days. To quantify ALP activity, a semiquantitative method was used, using a-naphthyl phosphate as the substrate and Fast Blue salt (SigmaeAldrich Corp. St. Louis, MO, USA) as the diazonium salt. Briefly, cells were washed three times with ice-cold Tris-buffered saline, pH 7.4, and scraped immediately upon addition of ice-cold 50 mM Trisbuffered saline, and the collected lysates were sonicated for 20 s at 4  C. Protein levels were determined by BCA assay and were used to normalize ALP activity. The kinase assay was performed in assay buffer (10 mM MgCl2 and 0.1 M alkaline buffer, pH 10.3) containing 10 mM p-nitrophenylphosphate in alkaline buffer (3.71 mg/ml assay buffer) as the substrate. Tubes were incubated in a 37  C water bath and timed. The reaction was stopped by the addition of 0.3 N NaOH. Reaction mixtures were transferred into cuvettes, and absorbance was read at OD405. The relative ALP activity is defined as mM of p-nitrophenol phosphate hydrolyzed per minute per microgram of total protein (units). 2.6. Western blot Cells subcultured into 100 mm dishes were treated, then treated cells (1 ! 107) were washed with cold PBS and lysed in Laemmli buffer (62.5 mM TriseHCl pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.01% bromophenol blue) for 5 min at 95  C. Cell lysates were analysed by SDS/PAGE and transferred

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electrophoretically to polyvinylidene difluoride membrane (Bio-Rad Corp Hercules, CA, USA). Blots were probed with antibodies specific to MT (rabbit polyclonal antibody reacts with all metallothionein isoforms from a broad range of mammalian species, from Santa Cruz Biotechnology, CA, USA, Cat. sc11377), b-actin and phosphorylated inhibitor of NF-kB a (IkBa) (Santa Cruz Biotechnology, CA, USA). Immunoreactive proteins were revealed using an enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology, CA, USA). 2.7. Statistical analysis Each experiment was repeated a minimum of three times. Statistical analyses were performed by Student’s t test. Data were reported as the mean G SD.

3. Results 3.1. Hydrogen peroxide inhibits osteoblastic differentiation of BMSCs 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 exposure (Martindale and Holbrook, 2002). In our experiments, BMSCs underwent severe cell death after treatment with high doses of H2O2 (500 mM) for 1 or 4 days, as determined by the Trypan Blue dye-exclusion method. Using low doses (50, 100 or 200 mM) of H2O2, however, the cell viability of BMSCs was not significantly affected, compared to the control (Fig. 1A). The osteoprecursor cells in bone marrow stromal cultures can spontaneously differentiate into osteoblasts, with the expression of ALP (Ducy et al., 1997; Harada and Rodan, 2003). To determine whether H2O2-induced oxidative stress inhibits osteoblastic differentiation of bone cells, we first tested the effects of H2O2 on ALP activity during the differentiation of BMSCs. Low doses (50, 100 or 200 mM) of H2O2 were added to confluent BMSCs cultured in differentiation medium for 4 days. The expression of ALP was measured by a quantitative ALP assay. Protein levels were measured by BCA assay and were used to normalize ALP activity. We found that H2O2 inhibited ALP activity in BMSCs. In the presence of 50, 100 or 200 mM H2O2, ALP activity was significantly (P ! 0.01) less than in controls, suggesting that H2O2 reduced the expression of early markers of osteoblastic differentiation (Fig. 1B). Furthermore, we used histochemical ALP staining to confirm the results obtained from the ALP assay. Consistent with ALP assay data, 50, 100 or 200 mM H2O2 significantly (P ! 0.01) reduced ALP positive cells in differentiated BMSCs (Fig. 1C).

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effect (Fig. 2A, B). These results demonstrate that exogenous MT can protect BMSCs against H2O2induced inhibition of osteoblastic differentiation.

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3.3. Pre-induction of MT with zinc attenuated the inhibitory effect of H2O2 on osteoblastic differentiation in BMSCs

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MT genes in higher species are rapidly induced in vitro and in vivo by a variety of stimuli, including metals, hormones, cytokines, oxidants, stress and irradiation (Haq et al., 2003). We further investigated whether an increase in MT levels reduces the inhibitory effect of H2O2 on osteoblast differentiation by upregulation of endogenous MT expression, using ZnCl2. Treatment of mouse BMSCs with ZnCl2 for 16 h was found to increase the level of MT protein in a dosedependent manner, as detected by Western blotting (Fig. 3A) and 109Cdeheme affinity assay (Fig. 3B). H2O2 was added when the media was changed after incubating with ZnCl2 for 16 h. ZnCl2 then dose-dependently

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Fig. 1. Low doses of H2O2 inhibit osteoblastic differentiation of BMSCs. (A) BMSCs isolated from 1 month old mice were treated with 50e500 mM H2O2 for 1 or 4 days and cell viability was detected by the Trypan Blue dye-exclusion method. (B) Low doses (50e200 mM) of H2O2 were added to confluent BMSCs cultured in differentiation medium (aMEM containing 10 mM b-glycerophosphate and 10ÿ8 M dexamethasone) for 4 days, then cells were subjected to an ALP activity assay. (C) BMSCs treated as described in (B) and ALP expression measured by ALP staining and the ratio of ALP positive cells to total cells counted microscopically. Con, control; *P ! 0.01 versus controls. Data are the mean G SD for three independent experiments.

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It is known that MT is capable of scavenging oxygen free radicals and is involved in the protection of tissues against various forms of oxidative injury (Tapiero and Tew, 2003). Changes in different MT isoforms’ expression and localization occur during differentiation (Quaife et al., 1994). To elucidate the effects of MT on oxidative stress-induced inhibition of osteoblastic differentiation, BMSCs were incubated with 20e500 mM MT for 30 min and then treated with 100 mM H2O2 in differentiation medium for 4 days. It was found that 100 or 500 mM MT pretreatment significantly (P ! 0.01) rescued the decrease in ALP activity and ALP positive cells induced by H2O2, while 20 mM MT had no notable

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These results suggest that high doses of H2O2 induce cell death, while low doses suppress ALP expression during the osteoblastic differentiation of mouse BMSCs.

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MT(µM) Fig. 2. MT protects BMSCs against H2O2-induced inhibition of osteoblastic differentiation. Confluent BMSCs were incubated with or without 20e500 mM MT for 30 min and then treated with or without 100 mM H2O2 in differentiation medium (aMEM containing 10 mM bglycerophosphate and 10ÿ8 M dexamethasone) for 4 days. (A) Treated cells were subjected to ALP activity assay. (B) Treated cells were subjected to ALP staining and the ratio of ALP positive cells to total cells were counted microscopically. Con, control; *P ! 0.01 versus controls; **P ! 0.01 versus column H2O2 (no MT). Data are the mean G SD for three independent experiments.

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inflammation, apoptosis, cell proliferation and differentiation (Pahl, 1999) and oxidative stress is known to be an activator of NF-kB (Li and Karin, 1999). Activation of NF-kB occurs via phosphorylation of inhibitory IkB proteins, followed by proteasome-mediated degradation of IkB, resulting in the release and nuclear translocation of active NF-kB. It has been shown that activation of NF-kB is required for oxidative stress-inhibited osteoblastic differentiation of bone cells (Bai et al., 2004). To determine whether MT has an effect on H2O2-induced NF-kB activation during osteoblastic differentiation of BMSCs, cells were pre-incubated with MT for 30 min or with various concentrations of ZnCl2 for 16 h to induce MT and then treated with 100 mM H2O2 in differentiation medium for 12 h. Cell lysates were subjected to Western blot analysis with anti-IkBa, phospho-IkBa and b-actin antibodies. We found that an increase in IkBa phosphorylation was induced by H2O2 treatment, accompanied by a reduction in levels of IkBa protein; ZnCl2 or MT-pretreatment decreased H2O2-stimulated IkBa phosphorylation and degradation (Fig. 4). Our results demonstrate that MT inhibits H2O2-activated NF-kB signaling during osteoblastic differentiation of BMSCs.

ZnCl2 (µM)

Fig. 3. Induction of MT attenuated the inhibitory effect of H2O2 on osteoblastic differentiation in BMSCs. (A) BMSCs were treated with 50, 100 or 200 mM ZnCl2 for 16 h and the cell lysates were subjected to Western blot analysis with anti-MT and b-actin antibodies. (B) BMSCs were pretreated with 50, 100 or 200 mM ZnCl2 for 16 h and total MT expression was determined by 109Cdeheme affinity assay. (C) Confluent BMSCs were pre-incubated with or without 50, 100 or 200 mM ZnCl2 for 16 h, then the media was changed and cells were incubated with or without 100 mM H2O2 in differentiation medium for 4 days. Cells were then subjected to ALP activity assay. (D) Confluent BMSCs were treated as (C) and cells were subjected to ALP staining and the ratio of ALP positive cells to total cells was counted microscopically. Con, control; *P ! 0.01 versus controls; **P ! 0.01 versus column H2O2 (no ZnCl2). Data are the mean G SD for three independent experiments.

reversed the decrease of ALP activity (Fig. 3C) and ALP positive cells (Fig. 3D) induced by H2O2. Moreover, 100 mM ZnCl2 alone neither enhanced activity of ALP (Fig. 3C), nor increased ALP positive cells (Fig. 3D) in BMSCs. These results suggest that enhanced expression of MT counteracts the inhibitory action of H2O2 in osteoblastic differentiation of BMSCs. 3.4. MT inhibits H2O2-activated NF-kB signaling during osteoblastic differentiation of BMSCs NF-kB transcription factors are involved in regulating large numbers of genes related to immune function,

4. Discussion The decrease in osteoblastic differentiation is an important factor in the pathogenesis of osteoporosis. It has previously been shown that H2O2 (1 mM) or xantine/xanthine oxidase (XXO)-induced oxidative stress is able to inhibit bone cell differentiation of a mouse preosteoblastic cell line (MC3T3-E1) and of a marrow stromal cell line (M2-10B4) that undergoes osteoblastic differentiation (Mody et al., 2001). More Actin

p-IκBα

IκBα H2O2 50

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Fig. 4. MT inhibits H2O2-activated NF-kB signaling during osteoblastic differentiation of BMSCs. Confluent BMSCs were preincubated with or without 50 or 100 mM ZnCl2 for 16 h, or pre-incubated with 500 mM MT for 30 min and then treated with or without 100 mM H2O2 in differentiation medium for 12 h. Cell lysates were subjected to Western blot analysis with anti-IkBa, phospho-IkBa or b-actin antibodies.

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recent data from rabbit BMSCs and calvarial osteoblasts demonstrated that a much lower dose of H2O2 (0.1 mM) inhibited expression of osteoblastic differentiation markers, while a higher dose of H2O2 (1 mM) induced cell death (Bai et al., 2004). Our results from primary mouse BMSCs agree with these studies, but the dose of H2O2 in our experiments (0.1 mM) is much lower than that in Mody’s and consistent with that in Bai’s. The discrepancy between these results may be caused by the different cell type and different source, dose and duration of oxidative stimulus. Metal ions, such as zinc and cadmium, are robust inducers of MT and increase its transcription by activating metal-regulatory transcription factor-1 (Saydam et al., 2001). Due to its abundant cysteine residues (25e30%), MT can bind metal ions with high affinity and has a role in metal detoxification and in the protection of cells against free radical injury. Here we showed that adding exogenous MT or inducing endogenous MT expression could protect BMSCs against H2O2-induced inhibition of osteoblastic differentiation, manifested by the rescue of an H2O2-inhibited osteoblastic differentiation marker (ALP) (Fig. 2 and Fig. 3). MT has free radical scavenging properties and is known to function like glutathione. The ability of MT to scavenge hydroxyl and superoxide radicals and function like superoxide dismutase in microorganisms has been demonstrated (Thornalley and Vasak, 1985). We deduce that the protective role of MT against H2O2-induced inhibition of osteoblastic differentiation may be due to its action as a scavenger of H2O2. On the other hand, although studies have demonstrated that decreased antioxidants (especially thiol antioxidants) and increased oxidative stress are involved in osteoporosis, caused by aging and estrogen deficiency (Basu et al., 2001; Lean et al., 2003; Maggio et al., 2003), and MT-III is markedly reduced in the brain of patients with age-related neurodegenerative diseases (Chung and West, 2004), the significance of MT protection of BMSCs against H2O2-induced inhibition of osteoblastic differentiation in the pathogenesis of osteoporosis remains unclear. Ongoing studies in our laboratory are exploring the changes in expression of different MT isoforms in aged osteoporotic men and postmenopausal women. NF-kB is a transcription factor that is involved in gene activation and plays a pivotal role in a diverse array of cellular activities associated with the regulation of cell growth, differentiation, death, and development. In bone, its major role was emphasized by the phenotype of NF-kB knockout mice exhibiting osteoporosis, mainly due to an impairment in osteoclastogenesis and osteoclastic function (Iotsova et al., 1997). At the same time, several reports point to the negative regulation of osteoblast differentiation by NF-kB in MC3T3 cells (Deyama et al., 2001) and the human osteosarcoma cell line Saos-2 (Andela et al., 2002). A

recent study also showed that oxidative stress-induced phosphorylation of IkBa and activation of NF-kB are essential for inhibition of rabbit BMSC and calvarial osteoblast differentiation elicited by H2O2 (Bai et al., 2004). Here we show that MT inhibits H2O2-activated NF-kB signaling during osteoblastic differentiation of BMSCs (Fig. 4). Our results are consistent with several studies on the relationship between MT and NF-kB (Kanekiyo et al., 2002; Kim et al., 2003; Papouli et al., 2002; Sakurai et al., 1999), and further implicates NF-kB in MT protection against H2O2-induced inhibition of osteoblastic differentiation. In summary, we have shown in this report that addition of exogenous MT or induction of endogenous MT can protect BMSCs against H2O2-induced inhibition of osteoblastic differentiation, manifested by the rescue of an H2O2-inhibited osteoblastic differentiation marker. Exogenous and endogenous MT inhibit H2O2activated NF-kB signaling during osteoblastic differentiation of BMSCs. These data suggest the ability of MT to protect against oxidative stress-induced inhibition of osteoblastic differentiation.

Acknowledgements This work was supported by a grant from the National Natural Sciences Foundation of China (No. 30300397).

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