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Endocrinology Vol. 145, No. 5 2148-2156
Copyright © 2004 by The Endocrine Society

Nitric Oxide Donor Increases Osteoprotegerin Production and Osteoclastogenesis Inhibitory Activity in Bone Marrow Stromal Cells from Ovariectomized Rats

Feng-Sheng Wang, Ching-Jen Wang, Yeung-Jen Chen, Yu-Ting Huang, Hui-Chen Huang, Per-Rong Chang, Yi-Chih Sun and Kuender D. Yang

Departments of Medical Research (F.-S.W., Y.-T.H., H.-C.H., Y.-C.S., K.D.Y.) and Orthopedic Surgery (C.-J.W.), Chang Gung Memorial Hospital; Department of Orthopedic Surgery, Chang Gung University (Y.-J.C.); and Fooyin University (P.-R.C.), Ta-Liau, Kaohsiung 833, Taiwan

Address all correspondence and requests for reprints to: Dr. Kuender D. Yang, Department of Medical Research, Chang Gung Memorial Hospital, 123 Ta-Pei Road, Niao-Sung, Kaohsiung 833, Taiwan. E-mail: wangfs{at}ms33.hinet.net.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide (NO) has emerged as a potent regulator useful in alleviating estrogen deficiency bone loss. Osteoprotegerin (OPG) and receptor activator of nuclear factor-{kappa}B ligand (RANKL) play important roles in regulating osteoclastogenesis. Although recent studies have reported NO donor attenuation of bone loss, the effect of NO donor on OPG and RANKL expression of osteogenic stromal cells and bone microenvironment in ovariectomized rats is not fully understood. Here, we showed that optimal NO donor treatment [2,2'-(hydroxynitrosohydrazino)bis-ethanamine; 15 µM] increased OPG, but not RANKL, levels in bone marrow stromal cells from ovariectomized rats. NO donor augmentation of OPG synthesis was transcriptionally mediated. The stimulatory action of NO donor on OPG expression appeared to be regulated by tyrosine kinase-dependent activation of Cbfa1/Runx2 binding to the OPG promoter, because cell cultures pretreated with tyrosine kinase inhibitor (herbimycin A), but not with protein kinase A inhibitor (calphostain C) or protein kinase C inhibitor [(Rp)-cAMP] significantly reduced NO-augmented Runx2 activation and OPG levels. Conditioned medium from NO donor-treated cells inhibited macrophage-colony-stimulating factor and RANKL-induced osteoclast formation of macrophage-colony-stimulating factor-dependent bone marrow macrophages. Neutralization with anti-OPG antibodies abolished the inhibitory effect of conditioned medium on osteoclastogenesis. Immunohistochemical observation also showed that 2,2'-(hydroxynitrosohydrazino)bis-ethanamine increased OPG expression of osteochondral cells located at metaphyseal endosteum and calcified cartilage of proximal femurs in ovariectomized rats. These findings suggest that NO donor can be an alternative pharmacological strategy for regulating bone resorption.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OSTEOPOROSIS IS ATTRIBUTED to an imbalance between bone formation and resorption, followed by bone mass loss and microarchitectural deterioration, leading to increased bone fragility and fracture risk. Estrogen deficiency, a major risk factor for osteoporosis, is characterized by high bone turnover with enhanced osteoclastic bone resorption (1, 2).

Bone resorption has been controlled by enhancing antiresorption molecule production from osteogenic cells (3). Osteoprotegerin (OPG) and receptor activator of nuclear factor-{kappa}B ligand (RANKL) produced by osteogenic stromal cells play important roles in regulating osteoclastogenesis. RANKL is involved in the differentiation and fusion of osteoclastic precursor cells, whereas OPG acts as a decoy receptor by blocking the interaction of RANKL with its functional receptor RANK, thereby inhibiting osteoclastogenesis (4). Deletion of the OPG gene results in increased osteoclast activity and severe osteoporosis. Administration of recombinant OPG increases bone mineral density in rats and alleviates bone loss in ovariectomized animals (5, 6). Several bone metabolic molecules, such as 17ß-estradiol and TGFß, have been reported to regulate OPG synthesis (7, 8).

Nitric oxide (NO) is produced from a guanidino-nitrogen of L-arginine and dioxygen by NO synthases. NO has been found to be a potent regulator of bone metabolism (9) and mediates 17ß-estradiol and mechanical stimulation of osteoblast proliferation and differentiation (10, 11). Inhibition of NO production by NO synthase inhibitors has been reported to stimulate osteoclastogenesis in bone tissue cultures and increase bone resorption in growing rats (12, 13). Recent studies have demonstrated that administration of NO donor attenuates estrogen deficiency bone loss in menopausal women and ovariectomized rats (14, 15, 16). NO donor attenuation of estrogen deficiency bone loss implies that NO donor may promote osteoclast inhibitory molecule production by osteogenic cells, which inhibits osteoclastogenesis and excess resorption in bone microenvironment. We hypothesize that NO donor may inhibit osteoclastogeneis through regulating OPG and RANKL synthesis by bone marrow stromal cells.

The purposes of this study were to investigate whether NO donor can attenuate osteoclastogenesis through modulating OPG and RANKL levels in bone marrow cells and whether NO donor regulates OPG expression in the bone microenvironment in ovariectomized rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Ovariectomized rats
All studies and protocols were approved by the institutional animal care and use committee of Chang Gung Memorial Hospital. Three-month-old female Sprague Dawley rats (National Experimental Animals Production Center, Taipei, Taiwan) were anesthetized by ip injection of pentobarbital sodium (50 mg/kg; nembutal sodium, Abbott Laboratories, Chicago, IL) and subjected to bilateral ovariectomy or sham operation. Animals were caged in pairs and maintained on rodent chow containing 1.05% calcium, 0.96% phosphate, 1.5 IU/g vitamin D3, and tap water ad libitum.

Primary bone marrow stromal cells
Ovariectomized or sham-operated rats were anesthetized with an overdose of pentobarbital sodium 2 months postoperatively. Bone marrow stromal cells in tibiae and femurs were harvested as previously described (17). Briefly, nucleated cells were harvested from the interface of the Ficoll-Paque density gradient (density, 1.007 g/ml; Pharmacia Biotech, Uppsala, Sweden) after centrifugation at 500 x g for 30 min. Cell number was determined using a hemocytometer after staining with 0.4% trypan blue. Nucleated cells were seeded in {alpha}MEM with 10% fetal bovine serum (FBS; Life Technologies, Gaithersburg, MD) for 30 min to separate adherent macrophages from nonadherent cells containing the stromal elements; nonadherent cells were collected and plated (5 x 105 cell/well; six-well plates). Twenty-four hours later, all nonadherent cells were discarded by two vigorous washing with PBS, and the remaining adherent stromal cells were then harvested by trypsinization. These stromal cells have osteoblastogenic capacity and can form mineralized nodules under appropriate conditions (18, 19).

NO donor treatment
Primary bone marrow stromal cells (1 x 105/well; 24-well plates) harvested form ovariectomized or sham-operated rats were cultured in {alpha}MEM containing 10% charcoal-treated FBS with or without 15 µM 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (NOC-18) or sodium nitroprusside or S-nitroso-N-acetylpenicillamine (SNAP) or 10 nM 17ß-estradiol (E2; Calbiochem-Novabiochem GmbH, Schwalbach, Germany) for 72 h. To investigate the role of NO in E2 promotion of OPG and RANKL expression, cells were pretreated with 100 µM N-nitro-L-arginine methyl ester (L-NAME) to inhibit NO production and followed with E2 treatment. To determine concentration and kinetic effects of NO donor on OPG and RANKL expression, cells were cultured in medium containing 0, 5, 15, 50, and 100 µM NOC-18 for 0, 12, 24, 48, and 72 h, respectively. In some experiments cells were pretreated with 10 µg/ml actinomycin D or 10 µg/ml cycloheximide (Sigma-Aldrich Corp., St. Louis, MO) and then cultured in medium containing 15 µM NOC-18. In the studies of signal pathways in NO-augmented OPG expression, cells were pretreated with 50 µM calphostain C (a protein kinase A inhibitor), 100 µM (Rp)-cAMP (a protein kinase C inhibitor), or 5 µM herbimycin A (a tyrosine kinase inhibitor; Calbiochem-Novabiochem GmbH) and then cultured in medium containing 15 µM NOC-18. Culture supernatants were harvested after centrifugation at 500 x g for 5 min and stored at –70 C until analysis.

Measurement of nitric oxide
The nitrite and nitrate levels in culture supernatants were measured using a NO analyzer (NOA280, Sievers, Inc., Denver, CO) as previously described (19). Results were normalized with protein concentration in each sample using a protein assay kit (Bio-Rad Laboratories, Hercules, CA).

Cell proliferation
Bone marrow stromal cells (5 x 104 cells/well; 96-well plates) with or without NO donors or E2 were cultured for 24 h before adding 1 µCi [3H]thymidine/well (Amersham Pharmacia Biotech, Little Chalfont, UK) for an additional 24-h culture. Cells in each well were released from the plates by trypsinization and processed for [3H]thymidine uptake determination by liquid scintillation counting (Tri-Crab 2100TR, Packard, Inc., Downers Grove, IL) (20).

RT-PCR
Total cellular RNA was extracted from bone marrow stromal cells (106 cells) by Tri-Reagent. Total RNA at 1 µg was reverse transcribed using 0.5 mg/ml oligo(deoxythymidine) primer, 10 mM deoxy-NTP mix, and avian myeloblastosis virus reverse transcriptase in the RT buffer (Promega Corp., Madison, WI), followed by PCR using rat gene-specific primers: OPG: forward, 5'-GTT CTT GCA CAG CTT CAC CA-3'; reverse, 5'-AAA CAG CCC AGT GAC CAT TC-3' (129-bp expected product); RANKL: forward, (5'-ACC AGC ATC AAA ATC CCA AG-3'; reverse, 5'-TTT GAA AGC CCC AAA GTA CG-3' (213-bp expected product); and ß-actin: forward, 5'-CGC CAA CCG CGA GAA GAT-3'; reverse, 5'-CGT CAC CGG AGT CCA TCA-3' (168 bp expected). The RT-PCR cycling parameters were set as follows: RT reaction at 50 C for 2 min and 60 C for 30 min, and PCR at 95 C for 5 min, followed 94 C for 20 sec and 60 C by 35 cycles of PCRs for 1 min. The PCR products were electrophoresed on a 1.5% agarose gel containing ethidium bromide and visualized by UV-induced fluorescence. All signals were quantified by scanning densitometry, and the OPG/ß-actin and RANKL/ß-actin ratios were calculated. Thirty-five cycles are within linear range (30–40 cycles) for generation of OPG and RANKL PCR products. The fold promotion was calculated as the increase over the value of its corresponding control sample.

Measurement of OPG production in the culture supernatants
The OPG levels in culture supernatants were determined using ELISA kits (Quantikine, R&D Systems, Inc., Minneapolis, MN) according to the manufacturer’s instructions. Results were calculated by interpolation from a standard curve made by a series of OPG concentrations.

Immunoblotting
Cytosolic and nuclear extracts of cell cultures were prepared as previously described (20). Nuclear extracts were immunoprecipitated with antibodies against Cbfa1/Runx2 (Runx2) and protein A agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immunoprecipitates were subjected to Western blot assay. The Runx2 bands on the blot were recognized by anti-Runx2 antibodies, followed by horseradish peroxidase-conjugated IgG secondary antibodies, and visualized with chemiluminescence agents (SuperSignal, Pierce Chemical Co., Rockford, IL). Phosphorylated Runx2 bands on the blot were recognized by stripping the membrane in a buffer containing 62.5 mM Tris-HCl (pH 6.7), 2% sodium dodecyl sulfate, and 100 mM mercaptoethanol for 30 min at 50 C and then were reprobed with mouse antiphosphotyrosine 4G10 antibodies using a similar procedure.

Electrophoretic mobility shift assay
A Runx2 consensus OPG promoter (underlined) oligonucleotide probe (5'-GCT CCC AAC CAC ATA TCC-3', 3'-CGA GGG TTG GTG TAG AGG-5') was 5' end-labeled with {gamma}-32P using T4 polynucleotide kinase and [{gamma}-32P]ATP (3000 Ci/mmol at 10 mCi/ml; Pharmacia Biotech). Nuclear extracts (10 µg) were incubated with a binding buffer containing 10 mM HEPES (pH 7.9), 1 mM dithiothreitol, 1 mM EDTA, 80 mM KCl, 20% glycerol, 0.25 mg/ml poly(dI-dC) (Pharmacia Biotech), and 1.75 pmol {gamma}-32P labeled oligonucleotide probe (30,000–40,000 cpm). To specify protein/DNA-binding reactions, 1 µl anti-Runx2 antibody or IgG was added to binding buffer and mixed with {gamma}-32P-labeled oligonucleotide probe. Samples were electrophoresed through a 6% polyacrylamide gel in 0.5% TBE (45 mM Tris, 45 mM boric acid, and 10 mM EDTA, pH 8.3). The gels were dried, and radioactive bands were visualized using Bio-Max films (Eastman Kodak Co., Rochester, NY) with an intensifying screen at –70 C.

In vitro osteoclastogenesis assay
Bone marrow nucleated cells (1 x 105 cells/well; 48-well plates) harvested from ovariectomized rats were cultured in {alpha}MEM containing 10% charcoal-treated FBS and 40 ng/ml macrophage-colony-stimulating factor (M-CSF) for 3 d. Nonadherent cells were removed from the cultures, and adherent cells were used as M-CSF-dependent bone marrow macrophages (M-BMMs). M-BMMs were further cultured for 4 d with osteoclast differentiation medium containing {alpha}MEM, 10% charcoal-treated FBS, 40 ng/ml M-CSF, and 20 ng/ml RANKL (R&D Systems, Inc.) as previously described (21). To assess osteoclast formation, M-BMMs were cultured for 4 d in a mixture (1:1) of sterile filtered conditioned medium and fresh osteoclast differentiation medium. To determine whether OPG were involved in inhibiting osteoclastogenesis, conditioned medium was neutralized with 20 ng/ml anti-OPG antibodies (R&D Systems, Inc.). In some experiments M-BMMs were cultured with osteoclast differentiation medium containing 10 nM E2. After incubation, cell cultures were subjected to tartrate-resistant acid phosphatase (TRAP) staining using a leukocyte acid phosphatase cytochemistry kit (Sigma-Aldrich Corp.). Total osteoclasts showing TRAP-positive multinucleated cells (with three or more nuclei) were counted under an inverted microscope.

OPG and RANKL expression in bone microenvironment
Twelve ovariectomized rats (2 months post operation) were randomly divided into three groups. Four rats in each group were sc given NOC-18 (500 µg/kg·d; dissolved in 200 µl normal saline), E2 (10 µg/kg·d; dissolved in 200 µl vehicle containing 95% corn oil and 5% ethanol), and vehicle treatment for 21 consecutive d, respectively. Four rats that received the sham operation were used as sham controls. On d 21, rats in each group were killed using an overdose of pentobarbital sodium, and femurs were dissected for histology. Femurs were fixed in 4% PBS-buffered paraformaldehyde, decalcified, embedded in paraffin, and then cut longitudinally into 5-µm thick sections. Antibodies against OPG and RANKL (Santa Cruz Biotechnology, Inc.) were used for immunohistochemistry. Immunoreactivity in sections was demonstrated using a horseradish peroxidase-3',3'-diaminobenzidine kit (R&D Systems, Inc.) according to the manufacturer’s instructions, followed by counterstaining with hematoxylin, dehydration, and mounting. Those without primary antibodies were enrolled as negative controls for the immunostaining.

Histomorphometry
Five areas within metaphyseal site in proximal femurs from three sections obtained from four rats were randomly selected using a microscope (Carl Zeiss, Inc., Gottingen, Germany). Three random images of 0.75 mm2 from each area (3 mm2) were then taken under x400 magnifications using a Cool CCD camera (SNAP-Pro Digital kit, Media Cybernetics, Silver Spring, MD). The number of positive immunolabeled cells was counted using an Image-Pro Plus image analysis software. The mean number of positive OPG- and RANKL-immunostained cells per high power field (0.75 mm2) in each section was then calculated. A professional pathologist, blinded to the treatment regimen, performed measurement on all sections.

Statistical analysis
All values were expressed as the mean ± SE. Wilcoxon test was used to evaluate differences between the sample of interest and its respective control. For analysis of concentration effect and time course, a multiple range of ANOVA and Bonferroni post hoc tests were used. P <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NO donors promoted NO levels and cell proliferation of bone marrow stromal cells
Bone marrow stromal cells from ovariectomized or sham-operated rats were cultured and subjected to assessment of NO production, cell growth, and OPG and RANKL levels. Compared with the sham-operated group, ovariectomy significantly decreased NO production by bone marrow stromal cells in 3 h (Fig. 1AGo). Ovariectomy did not alter cell proliferation as measured by [3H]thymidine uptake in 48 h (Fig. 1BGo). We investigated whether 15 µM NO donors and 10 nM E2 treatments could affect cell proliferation. NO donors and E2 treatments rescued ovariectomy-attenuated NO production (Fig. 1AGo) and cell proliferation (Fig. 1BGo). Inhibition of NO production by L-NAME significantly reduced E2-stimulated NO level and cell growth.



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  		FIG. 1. NO donor and E2 rescued ovariectomy-attenuated (A) NO levels in culture supernatants 3 h after culture. L-NAME pretreatment reduced E2 promotion of NO levels. B, NO donors and E2 increased [3H]thymidine uptake 48 h after culture. L-NAME pretreatment reduced E2-promoted cell growth. Bone marrow stromal cells were treated with or without 15 µM NO donors or 10 nM E2 for 72 h. Each primary culture harvested from each of 10 ovariectomized or sham-operated rats was assessed independently and then combined for statistical analysis using Wilcoxon test (*, difference from sham group; #, difference from vehicle group; +, shows difference from E2 group; P < 0.05). Shm, Sham-operated; OVX, ovariectomy; Veh, vehicle; SAP, SNAP; E, E2; L: L-NAME.

 
NO donors increased OPG, but not RANKL, levels
RT-PCR results indicated that ovariectomy reduced OPG mRNA (Fig. 2AGo) and increased RANKL mRNA expression (Fig. 2CGo). Down-regulation of OPG mRNA levels coincided with decreased OPG protein in culture supernatants (Fig. 2BGo). E2 rescued ovariectomy-attenuated OPG levels. L-NAME pretreatment reduced the promoting effect of E2 on OPG levels, but not RANKL levels. These findings suggested that NO was involved in E2 promotion of OPG levels in bone marrow stromal cells from ovariectomized rats. We determined whether NO donors could modulate OPG and RANKL levels of bone marrow stromal cells from ovariectomized rats. NO donors increased OPG levels (Fig. 2Go, A and B), but not RANKL mRNA expression (Fig. 2CGo), 72 h after culture. NO donor and E2 treatments alleviated the ovariectomy-induced increase ratio of RANKL mRNA/OPG mRNA (Fig. 2DGo). Of the NO donors, NOC-18 promoted OPG levels and decreased RANKL/OPG ratios best. It was then used in the proceeding experiments.



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  		FIG. 2. NO donor and E2 treatments rescued ovariectomy-attenuated OPG (A) mRNA and (B) protein levels. L-NAME pretreatment reduced E2-promoted OPG levels 72 h after culture. C, Ovariectomy and E2 increased RANKL mRNA expression. NO donors did not affect RANKL mRNA expression. D, NO donor and E2 treatments attenuated the ovariectomy-increased ratio of RANKL mRNA/OPG mRNA. After standardization of housekeeping gene expression, an equal amount of cDNA from each sample was subjected to 35 cycles of PCR to amplify OPG and RANKL mRNA expressions. Each primary culture harvested from each of 10 ovariectomized or sham-operated rats was assessed independently and then combined for statistical analysis using Wilcoxon test (*, difference from sham group; #, difference from vehicle group; +, difference from E2 group; P < 0.05). Shm, Sham-operated; OVX, ovariectomy; Veh, vehicle; SAP, SNAP; E, E2; L, L-NAME.

 
NO elicited dose- and time-dependent effects on OPG levels
We found that NOC-18 elicited a dose-dependent effect on NO levels (Fig. 3AGo), cell proliferation (Fig. 3BGo), OPG mRNA (Fig. 3CGo), and protein levels (Fig. 3DGo) in bone marrow stromal cells obtained from ovariectomized rats. NOC-18 (15 µM) had a maximum effect on OPG expression. NOC-18 treatments (50 and 100 µM) suppressed cell growth and OPG levels. NOC-18 treatment ranging from 5–100 µM did not affect RANKL mRNA expression. We also found that treatment with 15 µM NOC-18 significantly increased OPG mRNA (Fig. 4AGo) and protein levels (Fig. 4BGo) in 12 h. Treatment with 15 µM NOC-18 did not alter RANKL mRNA expression throughout the study period.



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  		FIG. 3. NOC-18 elicited a dose-dependent effect on NO levels in 3 h (A), [3H]thymidine uptake in 48 h (B), OPG mRNA (C), and OPG protein in 72 h (D). NOC-18 did not alter RANKL mRNA expression. NOC-18 treatment with 15 µM had best promotion effect, whereas treatments with 50 and 100 µM had an inhibitory effect on cell proliferation and OPG levels. Each primary culture harvested from each of 10 ovariectomized rats was assessed independently and then combined for statistical analysis using ANOVA and Bonferroni post hoc tests (*, difference from vehicle group; P < 0.05).

 


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  		FIG. 4. NOC-18 treatment elicited a time-dependent effect on OPG (A) mRNA and (B) protein levels. NOC-18 treatment did not affect RANKL mRNA expression. Bone marrow stromal cells were treated with 15 µM NOC-18 for 0–72 h. Each primary culture harvested from each of 10 ovariectomized rats was assessed independently and then combined for statistical analysis using ANOVA and Bonferroni post hoc tests.

 
NO donor augmentation of OPG mRNA expression is transcription-controlled
We further defined the action mechanism of NO donor-stimulated OPG mRNA expression in cell cultures. Blockade of transcription by 10 µg/ml actinomycin D decreased baseline and NOC-18-stimulated OPG mRNA expression (Fig. 5Go). Inhibition of translation by 10 µg/ml cycloheximide did not affect baseline or NOC-18-augmented OPG mRNA expression (Fig. 5Go). These findings suggest that NOC-18 modulation of OPG expression is transcriptionally mediated.



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  		FIG. 5. Pretreatment with actinomycin D, but not with cycloheximide, reduced NO donor-augmented OPG mRNA levels. Cells underwent 6-h exposure to 10 µg/ml actinomycin D or 10 µg/ml cycloheximide with or without 15 µM NOC-18. Each primary culture harvested from each of 10 ovariectomized rats was assessed independently and then combined for statistical analysis using Wilcoxon test (*, difference from vehicle group; #, difference from NOC-18 group; P < 0.05). ACT-D, actinomycin D; CHX, cycloheximide.

 
NO modulation of Runx2 activity and OPG expression was regulated by tyrosine kinase, but not by protein kinase A- or protein kinase C-dependent pathways
We investigated which signal pathway might be responsible for regulating NO-augmented OPG expression. Immunoblotting showed that NO donor elicited a time-dependent effect on promoting nuclear Runx2 phosphorylation (Fig. 6AGo). Autoradiograph of the gel-shift assay showed that NOC-18 treatment increased Runx2 binding to the OPG promoter sequence (Fig. 6BGo). We employed antibodies against Runx2 to confirm the DNA-protein binding activity. Autoradiograph showed that nuclear extracts harvested from NO donor-treated cell cultures were indeed supershifted by anti-Runx2 antibodies (Fig. 6CGo). Moreover, pretreatment with the tyrosine kinase inhibitor (herbimycin A), but not with the protein kinase A inhibitor (calphostain C) or the protein kinase C inhibitor [(Rp)-cAMP], reduced NO-enhanced Runx2 phosphorylation (Fig. 7AGo), Runx2 binding to the OPG promoter (Fig. 7BGo), and OPG levels (Fig. 7Go, C and D).



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  		FIG. 6. NOC-18 treatment promoted Runx2 phosphorylation (A) and nuclear Runx2 binding to the OPG promoter (B). The Runx2-immunopreicipates from nuclear extract of bone marrow stromal cells in ovariectomized rats were subjected to immunoblotting. After stripping, phosphorylated Runx2 on the blot was reprobed with antibodies against phosphotyrosine 4G10 and visualized using chemiluminescence agents. C, Supershift of Runx2. Nuclear extracts of were incubated with Runx2 probe in the presence or absence of anti-Runx2 antibodies or IgG and subjected to gel shift assay. SS, Supershift; NS, nonspecific binding; FP, free probe.

 


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  		FIG. 7. Pretreatment with herbimycin A, but not with calphostain C or (Rp)-cAMP, reduced NO donor-augmented Runx2 phosphorylation (A), Runx2 binding to the OPG promoter (B), OPG mRNA (C), and OPG protein levels (D). Cell cultures were pretreated with herbimycin A, calphostain C, and (Rp)-cAMP and then cultured for 72 h. Each primary culture harvested from each of 10 ovariectomized rats was assessed independently and then combined for statistical analysis using Wilcoxon test (*, difference from vehicle group; #, difference from NOC-18 group; P < 0.05). Herb A, Herbimycin A; Rp-C, (Rp)-cAMP; Calph, calphostain C, NS, nonspecific binding; FP, free probe.

 
Effects of culture supernatants from NO donor-treated cultures on osteoclast formation of M-BMMs
We examined whether NO-augmented OPG levels could attenuate M-CSF- and RANKL-induced osteoclastogenesis of M-BMMs from ovariectomized rats. Cytochemical results showed that multinuclear osteoclasts displayed strong TRAP staining in cytoplasm. M-BMMs cocultured with conditioned medium from NO-treated cultures significantly reduced M-CSF- and RANKL-induced osteoclast formation (Fig. 8AGo) compared with vehicle treatment (Fig. 8BGo). Conditioned medium neutralization with 20 ng/ml OPG antibodies significantly abrogated the inhibitory effect of conditioned medium on osteoclastogenesis (Fig. 8CGo). E2 treatment also significantly decreased M-CSF- and RANKL-induced osteoclast formation (Fig. 8DGo). The number of multinuclear TRAP-positive cells was counted (Fig. 8EGo). These results suggested that NO donor treatment inhibited osteoclast formation of M-BMMs from ovariectomized rats by inducing OPG levels.



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  		FIG. 8. Culture supernatants from NO donor-treated cell cultures (A) reduced M-CSF and RANKL-induced osteoclast formation of M-BMMs from ovariectomized rats in comparison with vehicle treatment (B). C, OPG antibody (20 ng/ml) neutralization reduced the osteoclastogenesis inhibitory effect of conditioned medium from NO donor-treated cell cultures. D, E2 inhibited M-CSF and RANKL-induced osteoclast formation. M-BMMs were cultured for 7 d and subjected to TRAP staining. Total osteoclasts showing TRAP-positive multinucleated cells (with three or more nuclei) were counted. E, Each primary culture harvested from each of 10 ovariectomized rats was assessed independently and then combined for statistical analysis using Wilcoxon test (*, differences from B group; P < 0.05).

 
Effect of NOC treatment on OPG and RANKL expression in femurs
Immunohistochemical observation showed that osteoblasts and bone marrow stromal cells lining the metaphyseal trabecular endosteum and chondrocytes at the growth plate displayed weak OPG and RANKL expression in the sham-operated group (Fig. 9Go). Osteoblasts at the junction of calcified cartilage expressed strong RANKL in ovariectomized rats that had received vehicle treatment. Osteoblasts and bone marrow stromal cells adjacent to trabecular endosteum and osteocytes at the trabecular bone expressed intense OPG after NOC-18 treatments (Fig. 9Go). Osteogenic cells located at trabecular endosteum expressed intensive OPG and RANKL after E2 treatment (Fig. 9Go). Table 1Go shows that NOC-18 treatment significantly increased the number of positive OPG-immunostained, but not RANKL-immunostained, cells. E2 treatment increased positive OPG- and RANKL-immunostained cells.



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  		FIG. 9. Immunohistochemical photograph of proximal femurs of ovariectomized rats. In the vehicle group, osteoblasts at the junction of calcified cartilage expressed strong RANKL and weak OPG. Osteoblasts and bone marrow stromal cells adjacent to trabecular endosteum and osteocytes at the trabecular bone expressed intense OPG after NOC-18 and E2 treatments. The positive OPG- and RANKL-immunostained cells are brown. Magnification, x400.

 

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  		TABLE 1. Effect of NOC-18 and E2 treatments on the number of positive OPG and RANKL immunostained cells at proximal femurs in ovariectomized and sham-operated rats

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study bone marrow stromal cells harvested from ovariectomized rats receiving NO donor elevated OPG mRNA expression and protein synthesis. Our findings first indicate that NO donor treatment increases OPG expression of bone microenvironment in ovariectomized rats. Moreover, bone marrow stromal cells from estrogen-deficient rats responded to NO donor treatment by increasing OPG expression through tyrosine kinase-dependent Runx2 pathways. We propose that it is the increased osteoclastogenesis inhibitory factor for suppressing osteoclast formation that brings about the clinical success of NO donor treatment in the attenuation of excess bone loss.

Down-regulation of OPG levels upon estrogen withdrawal has been found to increase osteoclastic bone resorption and contribute to postmenopausal bone loss (8). Hormone replacement therapy is reported to prevent estrogen deficiency bone loss by reducing bone turnover and increasing OPG and RANKL levels in osteoblastic cells (22, 23). NO has been found to mediate E2 stimulation of osteoblast proliferation and differentiation (24). Serum NO levels decrease in postmenopausal women and amenorrheic women and are increased by estrogen replacement (25, 26). In our study NO played an important role in regulating E2-promoted OPG levels in bone marrow stromal cells from ovariectomized rats because inhibition of NO production by L-NAME reduced the promoting effect of E2 on OPG levels. We examined whether NO donor treatment could modulate ovariectomy-induced alteration of osteoclast differentiation and inhibitory activities as effectively as E2 treatment has been reported to do. Our findings demonstrated that conditioned medium obtained from NO donor-treated cell cultures elicited an inhibitory effect on osteoclast formation, and the antiosteoclastic effect was diminished by anti-OPG antibody neutralization. Our research findings suggest that NO donor and E2 may share the same mechanism for regulating osteoclastogenesis. OPG is at least in part responsible for NO donor- and E2-attenuated osteoclast formation.

RANKL and OPG are potent regulators of bone homeostasis and act in opposite directions on osteoclastic differentiation. It has been suggested that decreasing the ratio of RANKL and OPG levels in bone microenvironment is critical in inhibiting osteoclast formation (27, 28). In our study, E2 reduction of RANKL/OPG ratio came about through increases in RANKL and OPG levels. Our present findings agree with previous studies that have reported that E2 treatment elevates RANKL and OPG levels in human osteoblasts (23) and mouse stromal cells (8). NO donor-attenuated RANKL/OPG ratio in current study was attributed to elevation of OPG, but not RANKL, levels. We speculate that different types of stimulation may affect OPG and RANKL expression in different ways.

Very few previous studies have focused on the effect of NO donor treatment on OPG and RANKL expression in estrogen deficiency bone microenvironment in vivo. Our current study provides the first immunohistochemical evidence that osteoblasts, bone marrow cells, and chondral cells in the areas of trabecular endosteum and calcified cartilage expressed intensive OPG immunoreactivity in ovariectomized rat femurs after NO donor and E2 treatments. The biological role of increased OPG expression of chondrocytes at the growth plate in ovariectomized rats is not clear. We suggest that osteochondral cells actively respond to NO and E2 treatments. Each cell lineage in the bone microenvironment may have a distinct role in regulating bone remodeling. The NO donor promotion of OPG expression in the bone microenvironment also indicates that a protective biological reaction against ovariectomy-induced excess bone resorption has occurred.

Previous studies have demonstrated that NO can induce apoptosis of osteoclast progenitors in inflammatory cytokine-stimulated bone marrow cell cultures (29). Holliday and co-workers (30) reported that a low concentration of NO (1 µM) inhibited osteoclastogenesis of osteoclastic precursors and that a high level of NO (100 µM) was required to suppress bone resorption of mature osteoclasts from mouse bone marrow cultures. In this study we verified that an optimal dose of NO donor (15 µM NOC-18) promoted cell proliferation and OPG mRNA and protein levels. A large amount of NO from a higher dose of NOC-18 (50 and 100 µM) suppressed OPG synthesis and cell proliferation. The results of our study support those of previous studies showing that NO has a biphasic effect on the proliferation and apoptosis of rat osteoblasts (31, 32). We cannot exclude the possibility that an NOC-18 concentration greater than 15 µM may elicit a cytotoxic or apoptotic effect on osteoclast formation. Nevertheless, the suppression of cell proliferation in bone marrow stromal cells by higher doses of NOC-18 (50 and 100 µM) is a significant concern. We speculate that inhibitory effect of NO on osteoclast formation may be dependent on the model system used, the NO concentration, and the developmental potential of osteoclastogenic cells. Our findings have suggested that multiple osteoclastogenesis inhibitory pathways are probably responsible for NO-modulated osteoclast formation in bone marrow cells.

NO donor control of OPG expression in estrogen deficiency bone marrow stromal cells has not, to our knowledge, been previously reported. We found NO donor-augmented OPG mRNA expression to be independent of protein synthesis and dependent on transcription. Although protein kinase A, protein kinase C, and tyrosine kinase have been found to regulate PTH- and platelet-derived growth factor-stimulated OPG expression in bone marrow stromal cells and osteoblasts (33, 34), it is unknown what intracellular signaling pathway may be responsible for NO donor-augmented OPG expression. Runx2 has been reported to be a critical regulator of mesenchymal stem cell differentiation toward osteogenic cell lineages (35). Recent studies have reported that Runx2 is involved in regulating OPG expression in bone marrow stromal cells (36), and down-regulation of OPG expression is found in Runx2–/– embryos (37). In our study, NO donor treatment activated Runx2 binding to the OPG promoter. Tyrosine kinase, but not protein kinase A or protein kinase C, was involved in NO-augmented Runx2 activation and OPG levels. These findings indicate that tyrosine kinases and Runx2 probably play regulatory roles in NO donor promotion of OPG production by bone marrow stromal cells from ovariectomized rats. Previous studies have reported that IL-6, IL-1, and TNF are candidate mediators of estrogen deficiency bone resorption (38, 39). We cannot exclude the possibility that the levels of these molecules in the bone microenvironment may be altered after NO donor treatment. Therefore, determination of signal transduction pathways that regulate OPG expression is important for a full understanding of NO donor attenuation of bone loss in future studies. Our observations reveal that NO donor treatment increases OPG levels and attenuates osteoclast formation. This study provides further evidence that NO donor can be used in the future as an alternative pharmacological strategy for the prevention of estrogen deficiency osteoporosis.


   Footnotes
 
This work was supported in part by a grant from National Health Research Institute, Taiwan (NHRI-EX-92-9128EI, to F.S.W.).

Abbreviations: E2, 17ß-Estradiol; FBS, fetal bovine serum; L-NAME, N-nitro-L-arginine methyl ester; M-BMM, macrophage-colony-stimulating factor-dependent bone marrow macrophage; M-CSF, macrophage-colony-stimulating factor; NO, nitric oxide; NOC-18, 2,2'-(hydroxynitrosohydrazino)bis-ethanamine; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor-{kappa}B ligand; SNAP, S-nitroso-N-acetylpenicillamine; TRAP, tartrate-resistant acid phosphatase.

Received August 18, 2003.

Accepted for publication February 4, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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