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Department of Cellular Pathology, St. George’s Hospital Medical School, London SW17 0RE, United Kingdom
Address all correspondence and requests for reprints to: Professor T. J. Chambers, Department of Cellular Pathology, St. George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, United Kingdom. E-mail: tchamber{at}sghms.ac.uk.
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Abstract |
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B ligand (RANKL) and in osteoclasts by 17ß-estradiol. We therefore tested the effect of overexpression of Gpx in osteoclasts by stable transfection of RAW 264.7 (RAW) cells, which are capable of osteoclastic differentiation in response to RANKL, with a Gpx-expression construct. Osteoclast formation was abolished. The Gpx expression construct also suppressed RANKL-induced nuclear factor-B activation and increased resistance to oxidation of dihydrodichlorofluorescein by exogenous hydrogen peroxide. We therefore tested the role of hydrogen peroxide in the loss of bone caused by estrogen deficiency by administering pegylated catalase to mice. We found that catalase prevented ovariectomy-induced bone loss. These results suggest that hydrogen peroxide is the reactive oxygen species responsible for signaling the bone loss of estrogen deficiency. |
Introduction |
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The cell responsible for bone resorption is the osteoclast. It is derived from cells of the mononuclear phagocyte system, which are induced to osteoclastic differentiation and function through the actions of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-B ligand (RANKL), cytokines that are expressed physiologically by cells of the osteoblastic lineage (see Refs.1, 2, 3).
Several of the intracellular signals essential for osteoclast formation, including nuclear factor-B (NFB), c-Jun amino-terminal kinase, phosphatidylinositol 3-kinase, and p38 MAPK, are sensitive to reactive oxygen species (ROS) (4), and it has become clear that osteoclastic differentiation and function are stimulated by ROS (5, 6, 7). Moreover, osteoclasts contain a nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase (8), an enzyme that is capable of cytokine-induced generation of ROS. Recently we found evidence that estrogen deficiency causes osteoclastic hyperresorption by lowering antioxidant defenses in osteoclastic cells, thus entraining a ROS-mediated increase in osteoclastic differentiation and function (7). We also found that systemic administration of the antioxidants ascorbate or N-acetyl cysteine prevented the loss of bone that normally occurs in mice rendered estrogen deficient by ovariectomy and that buthionine sulfoximine, which depletes tissues of glutathione, the major cellular antioxidant, caused bone loss. Osteoclast formation has been shown to depend on NFB activation (9, 10). NFB is a known target for activation by ROS (11, 12, 13), and we found that oxidants induced, and estrogen and antioxidants suppressed, activation in osteoclasts of NFB.
The central role of ROS in bone loss caused by estrogen deficiency led us to characterize the antioxidant defense system of osteoclasts. We found that glutathione peroxidase 1 (Gpx) was the predominantly expressed antioxidant enzyme and that its expression in osteoclasts was increased by RANKL and 17ß-estradiol. We therefore tested the effect of Gpx overexpression by transfection of RAW 264.7 (RAW) cells with a plasmid that coded for expression of Gpx. We found that Gpx overexpression abrogated osteoclast formation, suggesting a role for Gpx and hydrogen peroxide in the modulation of osteoclastic differentiation. We then went on to test the role of hydrogen peroxide in vivo by administering mice-pegylated catalase (CAT) and found that CAT prevented bone loss after ovariectomy. These results suggest that estrogen deficiency causes bone loss by lowering the content of Gpx in osteoclasts, so enabling an increase in hydrogen peroxide, which leads to increased osteoclasts.
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Materials and Methods |
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were purchased from Insight Biotechnology Ltd. (Wembley, Middlesex, UK). 17ß-Estradiol and 17-estradiol were from Sigma, and ICI 182,780 was from Tocris Cookson Ltd. (Avonmouth, Bristol, UK).
Isolation and culture of bone marrow cells
Bone marrow cells were isolated from male MF1 mice and cultured as previously described (14). Briefly, 5- to 8-wk-old mice were killed by cervical dislocation. Femora and tibiae were aseptically removed and dissected free of soft tissue. The bone ends were cut, and the marrow was flushed out into a petri dish by slowly injecting PBS at one end of the bone using a sterile 21-gauge needle. The bone marrow suspension was carefully agitated through a 21-gauge needle to obtain a single cell suspension. Bone marrow cells were then washed, resuspended in EMEM/FCS, and incubated at a density of 3 x 105 cells/ml for 24 h in a 75-cm2 flask (Greiner, Stonehouse, Gloucester, UK) with M-CSF (5 ng/ml) to deplete cell preparations of stroma. After 24 h, nonadherent cells were harvested, washed, and resuspended in EMEM/FCS for subsequent incubation.
Real-time PCR analysis of gene expression in bone marrow-derived osteoclasts
Next, 9 x 106 M-CSF-dependent nonadherent bone marrow precursors were added to 75 cm2 tissue culture flasks and cells incubated for 5 d in the presence of M-CSF or M-CSF (50 ng/ml) and RANKL (50 ng/ml). Cultures were fed every 2–3 d and 2 h before harvesting RNA. After incubation, cultures were washed to remove nonadherent cells and total RNA harvested using RNeasy minikits (Qiagen, Crawley, Sussex, UK). Four micrograms total RNA were reverse transcribed for 1 h at 42 C using 200 pmol of random hexamers (Amersham Biosciences, Amersham, Bucks, UK), 50 µmol deoxynucleotide triphosphates, and 600 U Muloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies, Paisley, UK) in a 50-µl reaction. This was then diluted to a final volume of 100 µl. Real-time PCR was carried out using the I-Cycler (Bio-Rad Laboratories, Hemel Hempstead, Herts, UK) using SYBR Green for detection of PCR products. Two microliters of either external plasmid standards or a cDNA was added to a final reaction volume of 25 µl containing 200 µM primers, 200 µM deoxynucleotide triphosphates, 3 mM MgCl2, 0.625 U platinum Taq polymerase, 0.25 U AmpErase UNG, and 2.5 µl 10 x SYBR Green PCR buffer (universal PCR master mix; Applied Biosystems, Warrington, Chesire, UK). Standard curves were generated using plasmid clones containing the corresponding cDNA (see Table 1
for primers, product lengths, and accession numbers). The linear range of the assay was determined by the amplification of log serial dilutions of plasmids from 500 to 5 x 106. Copy number relative to ß-actin (15) copy number in the same sample was calculated for each sample. At the end of the PCR run, a melt-curve analysis was performed to ensure product specificity.
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Similarly, bone marrow-derived osteoclasts (bone marrow precursors grown in M-CSF and RANKL as above for 5 d) were incubated in ICI 182,780 (10–7 M) and 17-estradiol (10–9 M) in phenol red-free EMEM with charcoal-stripped serum for 24 h. For Northern analysis, 10 µg total RNA were size separated in a 1.2% agarose gel, blotted, and hybridized for mouse Gpx1 or ß-actin as previously described (16). Probes were labeled with [-32P]dATP using the Megaprime DNA labeling system (Amersham).
Vector construction
The full-length coding sequences for mouse Gpx 1 (NM_008160) was cloned from mouse bone marrow macrophage RNA after reverse transcription using Muloney murine leukemia virus (Invitrogen Life Technologies), using standard procedures. The sequences with a FLAG tag on the antisense primer was amplified using Gpx1 sense, ATG TGT GCT GCT CGG CTC; Gpx antisense, TTA CTT GTC ATC GTC GTC CTT GTA GTC GGA GTT GCC AGA CTG CTG. The resultant amplicon was subcloned into pGEM-Teasy (Promega, Southampton, Hants, UK) and sequenced. Gpx1-FLAG was excised from pGEM-Teasy with NotI, subcloned into the NotI site of pcDNA3.1(+), and screened for orientation with ApaI.
Transfection of RAW cells
The 2 x 105 RAW cells were transfected with 1 µg pcDNA3.1(+) vector DNA, either empty or containing Gpx1-FLAG using FuGene 6 transfection reagent (Roche Diagnostics Ltd., Lewes, East Sussex, UK), according to their instructions. Cells were incubated in serum-free medium containing the Fugene/DNA mix for 6 h. For stable transfectants, cells were incubated in fresh medium containing 10% FCS for 48 h and then selected with 750 µg/ml G418 (Invitrogen Life Technologies). After 10 d, surviving cells were cloned and expanded.
Osteoclast formation by RAW cells expressing Gpx1
The 104 RAW cells that had either been stably transfected with empty vector or the Gpx1 expression vector were added to a 96-well plate (Greiner) containing a Thermanox coverslips (Invitrogen Life Technologies) and 200 µl culture medium per well, with or without RANKL (100 ng/ml) or TNF (100 ng/ml). Cultures were fed every 2–3 d by replacing 120 µl medium with an equal volume of fresh medium and cytokines. Coverslips were assessed for tartrate-resistant acid phosphatase (TRAP) positivity.
TRAP cytochemistry
Osteoclast formation in cultures of transfected RAW cells was evaluated by quantification of TRAP-positive multinuclear cells. After incubation, cells on coverslips were fixed in 10% formalin for 10 min, washed, permeabilized in acetone for 10 min, washed, and stained for acid phosphatase in the presence of 0.05 M sodium tartrate using the Leucognost-AP cytochemical reagent kit (VWR International Ltd., Poole, Dorset, UK). Cells were counterstained with hematoxylin. TRAP-positive cells with three or more nuclei (multinuclear cells) were counted. However, TRAP-positive cells after treatment with TNF were mononuclear. These were counted.
NFB p50 nuclear binding and competitor assay
RAW cells stably transfected with either empty vector or Gpx1-expression vector were treated with 50 ng/ml RANKL for 1 h before nuclear extracts were harvested as per manufacture’s instruction (BD Biosciences Clontech, Palo Alto, CA). The transfactor colorimetric kit (BD Biosciences Clontech) was used to detect the NFB p50 nuclear binding from the nuclear extracts. An increase in the OD630 correlates to an increase in the NFB p50 nuclear binding. Twenty micrograms of nuclear extract were used. Two hundred nanograms of competitor Oligo decreases the signal because transcription factor binding is competed away and is used to show specificity of binding activity.
Dihydrodichlorofluorescein diacetate (DCFH-DA) assay
DCFH-DA was used to assess intracellular ROS. This probe diffuses readily into cells. Once inside, the ester groups are hydrolyzed by intracellular esterases, releasing the dichloro derivative. This is oxidized to the fluorescent parent dye by intracellular ROS (17). RAW cells stably transfected with empty vector or the Gpx1 expression vector were seeded at 2 x 105 per well in black 96-well plates. After 24 h cells were washed and treated with 10 µM DCFH-DA for 15 min. Increasing concentrations of H2O2 were added for a further 10 min. Cells were washed and read on a fluorescent plate reader in 100 µl PBS (excitation 485 nm/emission 530 nm wavelengths). Cells were then lysed in 0.1% Triton X-100 and the lysate used for protein determination by Coomassie blue method (Coomassie Plus 200, Pierce, Rockford, IL).
Effect of CAT on ovariectomy-induced bone loss
Six- to 8-wk-old female MF1 mice were obtained from Harlan Olac (Oxon, UK). Animals were ovariectomized or sham ovariectomized. Mice were administered pegylated CAT (Sigma) 250 U/animal ip or vehicle (PBS) daily. All animals were pair fed to sham-ovariectomized PBS-control mice and killed after 2 wk. Weights of the mice (grams) before (and after) the experiment (± SEM) were as follows: sham/vehicle, 27.7 ± 0.4 (28.8 ± 0.5); ovariectomized/vehicle, 27.0 ± 1.0 (31.2 ± 0.9); sham/CAT, 27.5 ± 0.6 (28.4 ± 0.5); ovariectomized/CAT, 27.8 ± 0.5 (30.4 ± 0.5). Success of ovariectomy was confirmed by absence of ovaries and atrophy of uteri. Femora were removed, cleaned of soft tissue, fixed for 24 h in 10% phosphate buffered formalin, demineralized in 10% buffered EDTA for 7 d, dehydrated through graded alcohols, and embedded in paraffin wax. Bones were processed for histomorphometric analysis as described (18). Histomorphometry of the distal femur was performed on cancellous bone at least 0.3 mm beyond the growth plate to exclude any primary spongiosa. For each bone, two fields in each of three sections were measured to include at least 6 cm of bone surface.
Statistical analysis.
Statistical analysis was by ANOVA (Fisher’s protected least significant difference test) for multiple comparisons.
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Results |
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). We also found that Gpx expression was augmented in osteoclasts by 17ß-estradiol. This increase was inhibited by the pure antiestrogen ICI 182,780 (Fig. 2). The ability of RANKL and 17ß-estradiol, but not the nonreceptor binding stereoisomer 17-estradiol, to stimulate Gpx expression was confirmed by Northern analysis (Fig. 2). The magnitude of the increase in Gpx expression in osteoclasts by 17ß-estradiol is similar to the magnitude of the changes in osteoclastic parameters seen after ovariectomy (see Fig. 6).
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), compared with the two clones of cells transfected with empty vector. Similar virtual abolition of osteoclastic differentiation by the Gpx expression vector was observed in RAW cells incubated with TNF. Whereas TRAP-positive cells were induced by TNF in RAW cells stably transfected with empty vector, none were observed in cultures of RAW cells transfected with the Gpx expression construct (Fig. 3).
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Stable transfection of RAW cells with Gpx expression vector strongly suppresses NFB activation and DCF-fluorescence
RANKL has been shown to activate the transcription factor NFB, activation of which has been shown to be essential for osteoclast formation and activation. It is also known that NFB activation is augmented by ROS. We therefore compared the ability of RANKL to augment NFB activity in RAW cells stably transfected with the Gpx expression vector vs. RAW cells transfected with empty vector. Stably transfected RAW cells with either empty vector or Gpx were incubated with 50 ng RANKL for 1 h and nuclear extracts harvested. We found that whereas NFB was strongly activated in empty-RAW cells (Fig. 4), activation was essentially abolished in Gpx-RAW cells. Gpx-RAW cells also showed a substantially greater ability to resist the oxidation of DCF caused by exogenous hydrogen peroxide (Fig. 5). These results suggest that endogenous hydrogen peroxide is required for NFB activation and osteoclastic differentiation by RANKL.
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B activation suggests that modulation of hydrogen peroxide by Gpx might play a role in estrogen-deficiency bone loss. To test this possibility, we administered CAT, which has been shown to transit from the circulation (19) to ovariectomized or sham-ovariectomized mice. We found that, whereas ovariectomized mice showed a substantial reduction in bone volume, this was prevented by CAT (Fig. 6
). This was associated with normalization of the ovariectomy-induced increases in bone resorption. CAT did not affect the histomorphometric indices of sham-ovariectomized bone and had no effect on the body weight of the mice. These data strongly suggest that hydrogen peroxide is the cause of estrogen deficiency bone loss. The absence of effect of CAT on the bone of sham-ovariectomized mice suggests that only when Gpx levels fall does hydrogen peroxide escape cells in sufficient quantity to influence bone cell behavior. |
Discussion |
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B-activation by RANKL and TNF and by increased resistance to hydrogen peroxide-induced oxidation of DCF in Gpx-transfected cells. The physiological significance of these observations was supported by experiments in which we found that administration of catalase prevented the loss of bone that normally follows ovariectomy in mice. Both estrogen, which inhibits, and RANKL, which stimulates osteoclastic differentiation, induced Gpx expression in osteoclasts. With the proviso that the expression construct increases Gpx enzyme activity by a factor of 10 in the RAW cells (data not shown), the ability of Gpx overexpression in RAW cells to suppress osteoclastic differentiation suggests that this is a mechanism through which estrogen suppresses osteoclastic differentiation and that the induction of Gpx by RANKL represents negative feedback inhibition, analogous to the induction of the osteoclastogenesis-inhibitory interferon-ß by RANKL (20). It is known that osteoclasts express NADPH oxidase and generate large quantities of ROS during bone resorption (8). Therefore, induction of Gpx by RANKL might reflect ROS-induced activation of signaling cascades that lead to increased expression of the antioxidant defense proteins, which, our experiments suggest, represents a negative feedback inhibition of osteoclastic differentiation.
In addition to augmentation of signaling pathways for the induction of antioxidant defense proteins, ROS will be expected to augment other ROS-sensitive pathways in osteoclasts. In fact, osteoclasts are a prime candidate for regulation by ROS: their activity is dependent on several intracellular signals that are sensitive to ROS, including NFB, c-Jun amino-terminal kinase, phosphatidylinositol 3-kinase, and p38 MAPK (4). Consistent with this, we found that enhanced expression of Gpx suppressed activation of NFB, a transcription factor that is crucial for osteoclast formation and function (9, 10), in osteoclasts. Because ROS potently inhibit tyrosine phosphatases (21, 22), many phosphorylation-dependent signaling cascades are likely to be influenced by ROS in osteoclasts, thereby providing further mechanisms through which modulation of osteoclastic activity might occur.
The ability of Gpx overexpression to abrogate osteoclastic differentiation suggests that among ROS, it is specifically hydrogen peroxide that is crucial for osteoclastic differentiation. Among ROS, hydrogen peroxide has the characteristics most suited to act as both an intra- and intercellular signal because it has a relatively long half-life and is membrane permeant (23). It has moreover been shown to directly stimulate osteoclast formation and function (5, 7), and CAT suppresses osteoclastic differentiation in vitro (24). In fact, many cytokines and growth factors have been shown to activate cells through NADPH oxidase-mediated hydrogen peroxide production, and we showed that suppression of osteoclastic differentiation by Gpx overexpression was associated with suppression of DCF-fluorescence by exogenous hydrogen peroxide. Thus, hydrogen peroxide not only augments osteoclastic differentiation and function but also is essential for osteoclastic differentiation.
The ability of CAT to prevent osteopenia after ovariectomy establishes that hydrogen peroxide is responsible for estrogen-deficiency bone loss. The source of the hydrogen peroxide is uncertain, but the osteoclast has been shown to be a major source in bone (8). CAT did not detectably influence the bones of intact mice, suggesting that hydrogen peroxide appears in the extracellular fluid of bone increase under conditions of estrogen deficiency. We found that Gpx expression in osteoclasts is lower in the absence of estrogen so that we would anticipate a tendency for this to occur after ovariectomy.
Our finding that CAT administration prevents the bone loss caused by ovariectomy represents strong evidence that our in vitro findings have significance for the pathophysiology of bone. The observation that Gpx is decreased in the plasma of osteopenic patients supports the potential significance of our findings for bone pathophysiology in women (25). The effect of CAT on bone loss also has further significance. CAT, unlike hydrogen peroxide, is unable to diffuse across cell membranes. Therefore, suppression of bone loss by CAT is due to degradation of hydrogen peroxide in the extracellular space. This suggests that hydrogen peroxide causes bone loss through a paracrine or autocrine action. Thus, in addition to the direct effect of hydrogen peroxide on signal cascades essential for osteoclast formation, demonstrated by the ability of Gpx overexpression to abrogate osteoclastic differentiation, hydrogen peroxide might also indirectly stimulate osteoclasts. A common consequence of the exposure of cells to hydrogen peroxide is the induction of expression cytokines such as TNF, IL-1, and IL-6, which have been strongly implicated in estrogen-deficiency bone loss (26, 27). Recently it was shown that TNF synergizes strongly with RANKL for osteoclast formation and activation (28, 29) and that mice lacking TNF signaling do not lose bone after ovariectomy (27). This makes it possible that the autocrine-paracrine effects of hydrogen peroxide-induced TNF expression in osteoclasts, osteoblasts, or other cells in the bone microenvironment might contribute to the bone loss caused by hydrogen peroxide. Similarly, hydrogen peroxide might cause bone loss by up-regulation or down-regulation of the expression by bone cells of RANKL or osteoprotegerin, the decoy receptor for RANKL, or through the induction of apoptosis in osteoblastic cells (30).
These results have important implications for bone biology and the treatment of osteoporosis. The results predict that not only the bone loss of estrogen deficiency but also that seen in other situations in which ROS have been implicated, such as in aging and inflammation, might be caused by a prolonged augmentation of ROS signaling in bone cells. Thus, ROS generated by inflammatory tissue in or adjacent to bone might cause the loss of bone that is a feature of diseases such as rheumatoid arthritis or periodontitis. Furthermore, osteoporosis has recently been noted in two mouse models of premature aging associated with oxidative damage (31, 32) in which osteopenia is presumed to be the consequence of oxidative damage. It may be that estrogen deprivation, by lowering osteoclastic thiol antioxidant levels, leads to oxidative damage and that this, rather than signal modulation, causes bone loss. Alternatively, signal modulation by the increased oxidant stress in these models of premature aging might account for the bone loss. Whichever mechanism underlies the bone loss, our results predict that osteoporosis should be prevented by therapies that increase the degradation of hydrogen peroxide in bone.
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Footnotes |
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First Published Online November 4, 2004
Abbreviations: CAT, Catalase; DCFH-DA, dihydrodichlorofluorescein diacetate; EMEM, MEM with Earle’s salts; FCS, fetal calf serum; Gpx, glutathione peroxidase 1; M-CSF, macrophage colony-stimulating factor; NADPH, nicotinamide adenine dinucleotide phosphate reduced; NFB, nuclear factor-B; RANKL, receptor activator of NF-B ligand; RAW, RAW 264.7; ROS, reactive oxygen species; TRAP, tartrate-resistant acid phosphatase.
Received August 4, 2004.
Accepted for publication October 27, 2004.
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References |
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and its receptor CCR1 are the major chemokine ligand/receptor species expressed by osteoclasts. J Cell Biochem 87:386–393[CrossRef][Medline]
potently activates osteoclasts, through a direct action independent of and strongly synergistic with RANKL. Endocrinology 143:1108–1118 induces osteoclastogenesis by direct stimulation of macrophages exposed to permissive levels of RANK ligand. J Clin Invest 106:1481–1488[Medline]
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