A crucial role for thiol antioxidants in estrogen-deficiency bone loss (original) (raw)

Media and reagents. Bone marrow cells were incubated in MEM with Earle’s salts (Sigma-Aldrich, Poole, Dorset, United Kingdom) supplemented with 10% FBS (Autogen Bioclear UK Ltd., Calne, United Kingdom) and 2 mM glutamine, 100 IU/ml benzylpenicillin, and 100 μg/ml streptomycin (Sigma-Aldrich). Incubations were performed at 37°C in 5% carbon dioxide in humidified air. Recombinant human (rh)M-CSF was provided by Chiron Corp. (Emeryville, California, USA); soluble rhRANKL was from Insight Biotechnology (Wembley, Middlesex, United Kingdom). All other reagents were obtained from Sigma-Aldrich unless otherwise stated.

Animals. The animals used were female 6- to 8-week-old Wistar rats or MF-1 mice from the St. George’s Hospital Medical School colony unless otherwise stated.

Assessment of effect of ovariectomy and estrogen on oxidant defenses. Groups of six rats or mice were subjected to ovariectomy or a sham operation, followed by pair feeding. Three weeks later, a single dose of 17-β estradiol (10 μg/kg), or 17-α estradiol (100 μg/kg), or vehicle was administered subcutaneously in corn oil. All animals were killed 24 hours later. Success of the ovariectomy was confirmed by absence of ovaries and atrophy of uteri. Femora were rapidly cleaned and bone marrow harvested into ice-cold heparinized water and homogenized using a Polytron homogenizer. Liver and spleen were weighed and homogenized in ice-cold water. The homogenates were divided into two equal parts. To one, 0.1 vol of 1% Triton X-100 was added; to the other, an equal volume of 10% sulfosalicylic acid was added. The Triton extract was centrifuged at 10,000 g for 10 minutes at 4°C, and the supernatant was used for enzyme assays. Protein concentration was determined using Coomassie blue (Pierce, Tattenhall, United Kingdom), with BSA as protein standard.

Glutathione was measured in the samples after deproteinization with sulfosalicylic acid. Total glutathione (reduced glutathione [GSH] + oxidized glutathione [GSSG]) was measured using the GSH reductase–dithionitrobenzoic acid (DTNB) recycling procedure according to Tietze (23). GSSG was assayed as above after derivatization of GSH in the sample with 2-vinylpyridine (24). Glutathione reductase was assayed with a kit from Calbiochem (La Jolla, California, USA) according to the manufacturer’s instructions. Thioredoxin and thioredoxin reductase were assayed by the NADPH-dependent reduction of DTNB at 412 nm in the insulin-reducing assay (25).

Effect of NAC and ascorbate on ovariectomy-induced bone loss. Mice (six per group) were subjected to ovariectomy or sham ovariectomy. NAC (100 mg/kg/day intraperitoneally) was administered twice per day. Ascorbate was administered at a dose sufficient to increase tissue glutathione levels (26). For this, 1 mmol/kg or vehicle were administered intraperitoneally at 7:00 am and 6:00 pm each day. Ascorbate was dissolved immediately before use in isosmolar ice-cold saline and adjusted to pH 6.8 with 2 M NaOH. After 14 days, animals were killed, and bone was prepared for assessment of bone volume, analysis of parameters of bone resorption, and bone formation, as described (27). There was no significant change in the weight of mice during the experimental period. Ovariectomy was confirmed by uterine atrophy.

Effect of BSO on mouse bone. Groups of six female mice (Harlan Olac Ltd., Oxon, United Kingdom) were administered BSO (2 mmol/kg intraperitoneally) at 7:00 am and 6:00 pm each day for 3 weeks. BSO was also included in the drinking water (20 mM). Calcein was injected 1 and 6 days before killing the animals. Bones were processed for static and dynamic analysis as described (27).

Effect of 17-β estradiol on osteoblastic cells. Calvarial osteoblasts were obtained from neonatal female rats. Calvariae were dissected free of periosteum and associated soft tissues and incubated in MEM containing bacterial collagenase (1 mg/ml, type II) at 37°C for 100 minutes. The tissue fragments were then agitated, and suspended cells were removed and centrifuged at 250 g for 3 minutes. The cells were resuspended in MEM/FBS (2 × 105 cells/ml) in 75-cm2 flasks and incubated until confluent. Cells were then resuspended in trypsin/EDTA, washed, and incubated at 4 × 105 cells/well in six-well plates (Helena Biosciences Europe, Sunderland, Tyne and Weir, United Kingdom) in MEM/FBS for 24 hours. Medium was then removed and incubation was continued for 18 hours in 17-β estradiol (10–9 M) or vehicle, in phenol red–free MEM, and 10% charcoal-stripped FBS. To prepare charcoal-stripped serum, FBS was incubated with activated charcoal (10 mg/ml) for 2 hours at 37°C. The serum was then centrifuged and the supernatant passed through a 0.22-μm filter (Millipore, Molsheim, France).

Osteoblast-like UMR 106 cells and MC3T3-E1 cells were added to six-well plates (4 × 106 cells/well) and similarly allowed to settle for 24 hours in MEM/FBS before replacement of medium with vehicle/17-β estradiol, phenol red–free MEM, and charcoal-stripped serum.

Cells were then scraped into suspension with a cell scraper and assayed for total glutathione and glutathione reductase, as described above.

Effects on in vitro–generated osteoclasts. Osteoclasts were generated from nonadherent murine bone marrow cells as described previously (28). Briefly, bone marrow cells were obtained from 3- to 4-week-old female MF-1 mice and incubated for 24 hours in M-CSF (5 ng/ml) at 3 × 105 cells/ml. This incubation of bone marrow cells at low density in M-CSF for 24 hours efficiently depletes the cell preparations of stroma: stromal cells were not seen at any stage in the cultures, and no cells survived in cultures subsequently incubated without M-CSF. After 24 hours, nonadherent bone marrow cells were washed, resuspended, and incubated in MEM/FBS with M-CSF (50 ng/ml) and RANKL (30 ng/ml) for 5 days. Cultures were fed every 2–3 days.

To test the effect of estrogen on thiol antioxidant mechanisms, osteoclasts were generated in six-well plates for 5 days. Medium was then removed, and incubation was continued for 18 hours in M-CSF, RANKL, and 17-β estradiol (10–9 M), ICI 182,780 (107 M; Tocris Cookson Ltd., Bristol, United Kingdom), or vehicle, in phenol red–free MEM and 10% charcoal-stripped FBS. Cells were scraped into suspension with a cell scraper and assayed for oxidized and reduced glutathione and glutathione, and thioredoxin reductases, as described above.

For assessment of the effect of agents on osteoclastic differentiation, nonadherent bone marrow cells were incubated for 5 days in the wells of 96-well plates (Helena Biosciences) in RANKL, M-CSF, with or without BSO (10 μM), NAC (30 mM), or hydrogen peroxide (1 μM). Cells were then fixed in 10% formalin for 10 minutes and stained for tartrate-resistant acid phosphatase (TRAP). For this, cells were permeabilized in acetone for 10 minutes, washed, and stained for acid phosphatase in the presence of 0.05 M sodium tartrate. The substrate used was napthol AS-B1 phosphate. The number of TRAP-positive cells containing at least three nuclei was counted microscopically using an eyepiece graticule.

For assessment of NF-κB activation, osteoclast cultures were washed and incubated in M-CSF plus BSO (100 μM) or NAC (30 mM) for 2 hours, before readdition of RANKL. Cells were harvested by scraping 30 minutes later. Nuclear extracts were prepared as described previously (29), except that the cysteine protease inhibitor _trans_-epoxysuccinyl-l-leucylamido(4-guanidino)-butane (Sigma-Aldrich), was included in the LS buffer (20 nM HEPES pH 7.9, 2 mM MgCl2) at a final concentration of 3 nM, and, in addition, microcystin-LR (Qbiogene-Alexis, Bingham, Nottinghamshire, United Kingdom) was included at a final concentration of 4 nM. The cells were washed in situ twice with cold PBS, once with cold LS buffer, and lysed by the addition of LS buffer containing 0.1% Triton X-100 with gentle scraping with a cell scraper.

The protein concentration in nuclear extracts was determined using the bicinchoninic acid protein assay reagent (Pierce.), and an electrophoretic mobility shift assay (EMSA) was carried out using aliquots containing equal amounts of protein (4 μg per assay). The NF-κB p50 rabbit polyclonal Ab was supplied by Active Motif LLC (Rixensart, Belgium). The following modifications were made to the previously published EMSA procedure (29). For the Supershift assay the nuclear extracts were incubated for 30 minutes at 4° with the Ab (2 μl per assay) prior to the addition of the probe and poly(dIdC). The protein-DNA complexes were resolved on a 4% polyacrylamide gel in 0.5× tris-glycine-EDTA (TGE) buffer for 2 hours at 4°C at 12.5 V/cm following the protocol recommended by Active Motif LLC. The NF-κB oligonucleotide probe 5′-AGTTGAGGGGACTTTCCCAGG was supplied by Promega UK Ltd. (Southampton, United Kingdom). The mutant NF-κB oligonucleotide 5′-GCCATGGGCCGATCCCCGAAGTCC was supplied by Active Motif.

For assessment of the effect of 17-β estradiol on osteoclastic TNF-α mRNA expression, osteoclasts were generated as above in 75-cm2 flasks. After 5 days, the medium was discarded and replaced with phenol red-free MEM containing 10% charcoal-stripped serum and M-CSF (50 ng/ml), together with 17-β estradiol (10–9 M) or vehicle. After incubation for 18 hours, RANKL (30 ng/ml) was added. Cells were incubated for a further 3 hours and then harvested for analysis of RNA. For this, total RNA was extracted from cultures on plastic using TRIzol (Invitrogen Ltd., Paisley, United Kingdom). Total RNA (25–40 μg) was size separated in a 1.2% agarose gel and blotted as described previously (30). Probes were labeled by Megaprime DNA-labeling system (Amersham International, Amersham, United Kingdom) with α-32 P (ATP) (Amersham International). Blots were hybridized with probes for murine TNF-α and β-actin. The murine TNF-α probe was obtained by PCR using sense primer 5′-CCCCAAAGGGATGAGAAGTT and antisense primer 5′-CTTAGACTTTGCGGAGTCCG (Genbank accession no. XM_110221); murine β-actin was obtained using the sense primer 5′-GTCATCACTATTGGCAACGA and antisense primer 5′-CCTGTCAGCAATGCCTGGGT (Genbank accession no. M12481).

For assessment of the effect of the effect of NAC on osteoclastic TNF-α expression, osteoclasts were generated as above in 75-cm2 flasks. After 5 days, the medium was discarded and replaced with fresh medium containing M-CSF (50 ng/ml) and RANKL (30 ng/ml) with or without NAC (30 mM). The cells were harvested after 3 more hours for analysis of TNF-α mRNA expression as above.

Statistical analysis was by ANOVA (Fisher’s protected least-squares difference [PLSD] test) for multiple comparisons and the Student t test for paired comparisons.