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Endocrinology Vol. 145, No. 2 751-759
Copyright © 2004 by The Endocrine Society

Nitric Oxide Regulates Receptor Activator of Nuclear Factor-{kappa}B Ligand and Osteoprotegerin Expression in Bone Marrow Stromal Cells

Xian Fan, Eileen Roy, Liping Zhu, Tamara C. Murphy, Cheryl Ackert-Bicknell, C. Michael Hart, Clifford Rosen, Mark S. Nanes and Janet Rubin

Department of Medicine (X.F., E.R., L.Z., T.C.M., C.M.H., M.S.N., J.R.), Veterans Affairs Medical Center and Emory University, Atlanta, Georgia 30033; and Jackson Laboratory (C.A.-B., C.R.), Bar Harbor, Maine 04609

Address all correspondence and requests for reprints to: Xian Fan, M.D., VAMC-151, 1670 Clairmont Road, Decatur, Georgia 30033. E-mail: xfan{at}emory.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bone remodeling reflects an equilibrium between bone resorption and formation. The local expression of receptor activator of nuclear factor-{kappa}B ligand (RANKL) and osteoprotegerin (OPG) in bone determines the entry of monoblastic precursors into the osteoclast lineage and subsequent bone resorption. Nitric oxide (NO) inhibits osteoclastic bone resorption in vitro and regulates bone remodeling in vivo. An interaction of NO with RANKL and OPG has not been studied. Here, we show that treatment of ST-2 murine stromal cells with the NO donor sodium nitroprusside (100 µM) for 24 h inhibited 1,25 dihydroxyvitamin D3-induced RANKL mRNA to less than 33 ± 7% of control level, whereas OPG mRNA increased to 204 ± 19% of control. NOR-4 replicated these NO effects. The effects of NO were dose dependent and associated with changes in protein levels: RANKL protein decreased and OPG protein increased after treatment with NO. PTH-induced RANKL expression in primary stromal cells was inhibited by sodium nitroprusside, indicating that the NO effect did not require vitamin D. NO donor did not change the stability of RANKL or OPG mRNAs, suggesting that NO affected transcription. Finally, cGMP, which can function as a second messenger for NO, did not reproduce the NO effect, nor did inhibition of endogenous guanylate cyclase prevent the NO effect on these osteoactive genes. The effect of NO to decrease the RANKL/OPG equilibrium should lead to decreased recruitment of osteoclasts and positive bone formation. Thus, drugs and conditions that cause local increase in NO formation in bone may have positive effects on bone remodeling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NITRIC OXIDE (NO), an intracellular messenger stimulated by PTH and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], has complex effects on bone cells. One of the earliest reports linking NO to bone remodeling showed that inhibition of NO synthase (NOS), the enzyme that synthesizes NO from L-arginine, potentiated ovariectomized bone resorption in rats (1). Since then, studies have shown that NO can slow bone loss in animals and humans (2, 3, 4). More recently, Armour et al. (5) have shown that mice lacking endothelial NOS were unable to respond to estrogen rechallenge after ovariectomy with an anabolic response.

Effects of NO reported in vitro indicate that it may enhance osteoblast function. In ST-2 murine stromal cells, NO promotes differentiation as measured by increased osteocalcin and nodule formation (6). Human osteoblasts respond to PTH or 1,25(OH) 2D3 treatment with increased NO production accompanied by decreased cell proliferation (7). NO also alters osteoclast indices in vitro: Osteoclastic resorption is potentiated during NOS inhibition (1). This could result from effects on osteoclast activity as suggested by an action of NO to produce a shape change in the osteoclast associated with a reduction in bone resorption as well as by decreased osteoclast recruitment (8). Both Holliday et al. (9) and Collin-Osdoby et al. (10) have shown that reduced NO levels increase osteoclast recruitment and bone resorption.

Receptor activator of nuclear factor-{kappa}B (NF{kappa}B) ligand (RANKL) is up-regulated in response to hormones and factors that are known to promote bone resorption, such as PTH (11) and vitamin D (12). RANKL binds to RANK receptors on osteoclast precursors and stimulates differentiation toward the osteoclast lineage (12, 13). Another component of the RANKL-RANK equilibrium is osteoprotegerin (OPG), a TNF-family soluble receptor. As such, OPG is a key factor in the remodeling environment, acting as a negative regulator of osteoclastogenesis and bone resorption (14, 15, 16, 17). Hence, the established effect of NO on osteoclastogenesis operates through the local RANKL/OPG equilibrium generated by stromal cells.

Here, we show that nitric oxide potently suppresses RANKL expression in both primary murine stromal cells and a stromal cell line. The effect of NO on RANKL expression prevails whether RANKL is induced by either vitamin D or PTH. Concurrently, NO donors up-regulate OPG expression in concert with the down-regulation of RANKL. Our results suggest a specific mechanism by which NO, and agents that alter NO, might impact skeletal remodeling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The ST-2 bone stromal cell line (Riken Cell Bank, Tsukuba Science City, Japan) was plated in {alpha}-MEM with 10% fetal bovine serum and antibiotics at 50,000 cells/6-well plates. For experiments using primary stromal cells, bone marrow was obtained from tibiae and femurs of 3- to 5-wk-old male C57BL/6 mice and plated at 15 million cells per 6-well plates, as described previously (18). After a 1-h incubation to remove macrophages, nonadherent cells containing stromal cells were transferred to new 6-well plates. Twenty-four hours later, all nonadherent cells were discarded, and the remaining stromal cells were cultured for 1 wk for experiments. Animal protocols were approved by the Institutional Animal Review Board.

When specified, 10 nM 1,25(OH)2D3 (vitamin D; Biomol, Plymouth Meeting, PA) or 100 ng/ml of PTH (Sigma Chemical Co., St. Louis, MO) was added to stimulate RANKL expression. Sodium nitroprusside (SNP; Sigma Chemical Co.) and NOR-4 (Sigma Chemical Co.) were used to deliver NO to cells. 2-(4-Carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) was a NO scavenger to reverse the SNP effect. 1H-(1,2,4-oxadiazolo(4,3-{alpha})quinoxalin-1-one (ODQ) was used to inhibit guanylate cyclase (Sigma Chemical Co.).

Real-time PCR
Analysis of RANKL, OPG, and 18S mRNA was performed using the iCycler (Bio-Rad Laboratories, Hercules, CA), as described previously (19, 20). Reverse transcription (RT) of 1 µg total RNA in 20 µl of reaction was performed with random decamers (Ambion, Austin, TX) and superscript II reverse transcriptase (Invitrogen, Carlsbad, CA). Amplification reactions were performed in 25 µl containing primers at 0.5 µM and deoxynucleotide triphosphates (0.2 mM each) in PCR buffer and 0.03 U Taq polymerase (Invitrogen) along with SYBR-green (Molecular Probes, Eugene, OR) at 1:150,000. Aliquots of cDNA were diluted 10- to 10,000-fold for 18S and 5- to 625-fold for RANKL and OPG to generate relative standard curves with which sample cDNA was compared (21). For RANKL, forward and reverse primers were 5'-CCAAGATCTCTAACATGACG-3' and 5'-CACCATCAGCTGAAGATAGT-3', respectively, creating a product of 140 bp. A 369-bp product of OPG amplicon was generated from forward primer 5'-AACAGCACTGCACAGTGAG-3' and reverse primer 5'-AATTSGCAGGAGGCCAAATG-3'. For 18S, an amplicon of 345 was generated with forward primer 5'-GAACGTCTGCCCTATCAACT-3'and reverse 5'-CCAAGATCCAACTACGAGCT-3'. Standards and samples were run in triplicate. Dilution curves showed that PCR efficiency was more than 95% for all species studied. RANKL and OPG were normalized for the amount of 18S in the same RT sample, which was also standardized on a dilution curve from RT sample (21).

OPG ELISA
ST-2 cell-conditioned media were collected and cell numbers were counted after 24 h treatment of SNP. Following the protocol from R&D Systems (Minneapolis, MN), 50 µl of assay diluent and 50 µl of standard or sample were added to each well precoated with a monoclonal antibody specific for mouse OPG and incubated 2 h at room temperature. A 100-µl antimouse OPG conjugated polyclonal antibody was added and incubated for 2 h, followed by five washes. A 100-µl substrate solution was incubated for 30 min before adding 100 µl stop solution. ODs were read at 450 nm, and reference wavelength set at 570 nm. The sensitivity of this assay is approximately 4.5 pg/ml. The intraassay coefficient of variation in experiments was 3.7%.

RANKL Western
As described in the protocol (Santa Cruz Biotechnology, Santa Cruz, CA), cells were lysed with radioimmunoprecipitation assay buffer for 30 min on ice after treatment with vitamin D for 5 d and SNP for 24 h. Cell lysates were removed by centrifuging at 10,000 x g for 10 min at 4 C and followed by concentration with Microcon YM-30 (Millipore Co., Bedford, MA). Then, 100 µg cell protein were loaded on 15% SDS-PAGE and after electrophoresis, transferred to polyvinyl difluoride membrane. After the membrane was blocked (5% milk in PBS), rabbit antihuman RANKL polyclonal antibody (1:200; Santa Cruz Biotechnology) was incubated overnight at 4 C, followed by donkey antirabbit IgG, horseradish peroxidase-linked F(ab)2 fragment (1:1000; Amersham Life Science, Arlington Heights, IL) for 1 h. X-ray film was exposed to membranes developed by ECL plus (Amersham Biosciences, Piscataway, NJ). The membrane-associated RANKL band appeared at 40 kDa (22, 23).

Cell viability assay
ST-2 cells were treated with vitamin D for 48 h and SNP for 24–48 h and mitochondrial dehydrogenase activity assayed with the substrate, methylthiazoletetrazolium (MTT). Briefly, MTT solution (5 mg/ml) added to each well to 10% volume and cultured at 37 C for 4 h. An equal amount of isopropanol in 0.1 N HCl was added to dissolve MTT formazan crystals. Absorbances were measured at 570 nm, and background absorbance at 630 nm was subtracted.

Statistical analysis
Results are expressed as the mean ± SEM. Statistical significance was evaluated by one-way ANOVA with intergroup significantly determined by the methods of Dunnett or Bonferroni (GraphPad Prism, GraphPad, Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NO decreases the ratio of RANKL/OPG in ST-2 cells
Using a sensitive real-time PCR assay (19, 20), we showed that 1,25(OH)2D3 dose-dependently induced RANKL mRNA expression in murine stromal ST-2 cells (Fig. 1AGo). To evaluate the effect of NO on RANKL, cells were treated with the NO donor SNP for 24 h. As shown in Fig. 1AGo, 100 µM SNP reduced stimulated RANKL mRNA by 50%. The RANKL mRNA response to SNP was dose dependent, as shown in Fig. 1BGo; doses of 1 µM and greater significantly reduced the vitamin D stimulation of RANKL. The response of vitamin D-induced RANKL to SNP inhibition was also time dependent, as shown in Fig. 1CGo. Cultures treated with SNP for only 6 h had significantly attenuated RANKL mRNA expression and reached maximum inhibitory effect after 14 h of treatment. Furthermore, SNP inhibited RANKL expression stimulated by either 1 or 10 nM vitamin D in a similar proportion (Fig. 1CGo).



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  		FIG. 1. SNP inhibits vitamin D-induced RANKL mRNA expression in ST-2 cells. A, ST-2 cells were treated with varying doses of vitamin D for 48 h and RANKL mRNA expression assessed with real-time PCR. In the absence of vitamin D, RANKL amplicons reached threshold at 28.5 cycles. RANKL mRNA rose slightly but not significantly with 0.1 nM vitamin D and continued to rise until at least 10 nM vitamin D treatment. Addition of 100 µM SNP during the last 24 h decreased the 10 nM 1,25(OH) 2D3-stimulated RANKL to levels achieved with a 10-fold lower dose of the hormone. Significant differences between groups are shown by use of different letters (P < 0.05, a != b or c, b != c). B, ST-2 cells were treated with 10 nM vitamin D for 48 h, and the NO donor SNP was then added during the last 24 h at the doses indicated. SNP inhibited RANKL mRNA (analyzed with real-time PCR) with an ED50 less than 100 µM. The graph combines three experiments. a, Significant difference from control cells at P < 0.001; b, significant difference between SNP treatment at 1 µM and 100 µM at P < 0.001. C, SNP attenuation of RANKL expression is time dependent and present at different concentrations of vitamin D. ST-2 cells were treated with 1 or 10 nM vitamin D for 48 h of the experiment. 100 µM SNP was added for 6, 14, or 24 h before the end of culture, as indicated. SNP significantly inhibited vitamin D-induced RANKL expression in the presence of both 1 and 10 nM vitamin D. Results are combined three experiments and expressed such that RANKL expression stimulated by 10 µM vitamin D was set at 100%. *, Significant difference from cells not treated with SNP (P < 0.01).

 
Concurrently, NO donor significantly increased OPG mRNA expression after 24 h of 100 µM SNP treatment (204 ± 19%). There was a trend to an increase seen at 6 h in cells treated with 10 nM vitamin D (Fig. 2Go). Note that OPG mRNA decreased with increasing vitamin D concentration (Fig. 2BGo), as expected (24). At a lower dose of vitamin D (1 nM), the effect of SNP to increase OPG mRNA expression was statistically significant at 14 h (Fig. 2BGo). Combining data to generate a RANKL/OPG ratio, Fig. 3Go shows that NO dramatically and dose-dependently reduced the ratio of RANKL/OPG, a mRNA change associated with a decrease of osteoclastic recruiting potential.



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  		FIG. 2. SNP increases OPG expression in ST-2 cells. A, ST-2 cells were treated with vitamin D for 48 h and SNP for the last 24 h of culture. SNP increased OPG mRNA expression. The figure combines four experiments. An asterisk represents a significant difference from control cells at P < 0.001. B, ST-2 cells were treated with vitamin D (1 or 10 nM) for the entirety of the 48-h experimental culture period and 100 µM SNP added at 6, 14, and 24 h before the end of culture as indicated. OPG mRNA levels were less in cultures treated with 10 nM vitamin D, compared with 1 nM. SNP significantly induced OPG mRNA expression in both 1 and 10 nM vitamin D conditions. The figure combines three experiments. *, Significant differences from control cells, i.e. in the absence of SNP (P < 0.05).

 


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  		FIG. 3. SNP decreases the ratio of RANKL/OPG mRNA. ST-2 cells were treated with vitamin D (48 h) and SNP (24 h), and total RNA was extracted for RT-PCR to measure RANKL and OPG mRNA expression. Figure shows that ratio of RANKL/OPG mRNA decreased in a dose-dependent fashion. Data are from three experiments. Asterisks represent a significant difference comparing with control (P < 0.01).

 
To determine whether NO regulated the protein levels of RANKL and OPG, we used Western analysis for membrane-associated RANKL protein and ELISA to measure secreted OPG protein in cell culture-conditioned media. Although Western analysis did reveal cell membrane-associated RANKL protein expression in vitamin D-treated ST-2 cells, it is currently neither a very quantitative nor sensitive method for measurement of RANKL. However, we were able to show a decreased RANKL protein in response to 24-h treatment with SNP: 300 µM decreased RANKL protein expression to 70 ± 23% treatment, and 100 µM SNP showed a slight response of 82 ± 6% compared with control cells assessed by densitometry. These results indicate that NO does have an inhibitory effect on RANKL protein. Meanwhile, OPG protein levels significantly increased to 194 ± 3% of control when cells were treated with 50 µM SNP or greater, as quantitatively assessed with an ELISA measuring secreted OPG. Thus, RANKL/OPG mRNA is reflected in the respective protein levels as expected (Fig. 4Go).



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  		FIG. 4. SNP effects on OPG protein expression in ST-2 cells. ST-2 cells were treated as in Fig. 2AGo. Conditioned media were collected and OPG measured by ELISA. Cell numbers were also counted and the results expressed as OPG/cell number. SNP increased OPG secretion by 2-fold in cells treated with 50 and 100 µM SNP. The asterisk represents a significant difference from control cells at P < 0.001. Similar results were obtained in two replicate experiments.

 
To assure that SNP effects on RANKL/OPG were not due to cytotoxicity, we performed two assays of cell viability: cell number comparisons and the MTT assay, which measures the activity of living cells via mitochondrial dehydrogenase activity. As shown in Fig. 5AGo, cell numbers in groups treated with SNP did not vary from control cell numbers at 14 or 24 h. As well, MTT was not different between control cells and those exposed to SNP for 24 or 48 h (Fig. 5BGo).



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  		FIG. 5. SNP treatment does not affect cell viability. A, ST-2 cell numbers were counted after SNP (100 µM) treatment for last 14 and 24 h of cultures (vitamin D was present for a total of 48 h). There were no significant changes in cell number between control and SNP-treated cells. B, MTT assay shows no difference in cells treated with SNP. There were no significant differences between control and SNP-treated (100 µM) cells at either 24 or 48 h.

 
SNP effect on RANKL/OPG requires NO delivery
To test the question of whether SNP effect on RANKL/OPG was specific for NO, we used two supporting approaches. First, we used a second NO donor, NOR-4, that spontaneously releases NO and has a short half-life (60 min). As shown in Fig. 6Go, 100 µM NOR-4 significantly inhibited RANKL expression and induced OPG levels, effects similar to those due to treatment with SNP. Second, the NO scavenger cPTIO was added to cultures; 100 µM cPTIO completely reversed SNP (100 µM) effects to increase RANKL levels (cPTIO alone = 100 ± 23%, cPTIO + SNP = 94 ± 10%) and decrease OPG levels (cPTIO alone = 100 ± 2%, cPTIO + SNP = 101 ± 16%).



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  		FIG. 6. NOR-4 duplicates SNP effects on RANKL and OPG. A, ST-2 cells were treated with 10 nM vitamin D for 48 h, and the NO donors SNP and NOR-4 (both at 100 µM) were added during the last 24 h at the doses indicated. Both donors inhibited RANKL mRNA expression to less than 50%. Asterisk is significantly different from control at P < 0.01. B, ST-2 cells were treated as above and mRNA assayed for OPG mRNA expression. As shown in the figure, both SNP and NOR-4 increased OPG mRNA, significant at P < 0.05.

 
NO effect on PTH-induced RANKL in bone primary stromal cells
To ascertain whether the NO effect on RANKL was specific to vitamin D stimulation, we used PTH to stimulate RANKL expression. Because ST-2 cells possess little, if any, PTH receptor, primary bone stromal cells were used for study. We first showed that NO donor inhibition of 1,25(OH)2D3-induced RANKL mRNA was present in primary murine bone stromal cells: RANKL mRNA decreased to 68 ± 9% that of control cells after 100 µM SNP treatment as assessed by real-time PCR (Fig. 7AGo). RANKL mRNA was robustly stimulated in primary bone stromal cells by 2 d treatment with PTH at 100 ng/ml. The effect of NO to repress RANKL mRNA expression was also seen after PTH induction of RANKL. As shown in Fig. 7BGo, 100 µM SNP decreased PTH-induced RANKL mRNA expression by more than 50%, consistent with the inhibitory effect on vitamin D-induced RANKL expression. PTH has been shown to effectively block OPG mRNA expression in primary bone cell cultures (11). When SNP was added to cultures treated with PTH, we did not observe a significant change in the OPG mRNA (data not shown), perhaps due to a predominant inhibitory effect of PTH.



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  		FIG. 7. NO decreases 1,25(OH)2D3 and PTH stimulated RANKL mRNA in primary bone stromal cells. A, Real-time PCR was performed on total RNA collected from primary murine bone stromal cells treated with 10 nM vitamin D (48 h) and the NO donor SNP at doses indicated during the last 24 h. The graph shows a compilation of two experiments. A significant difference from control (P < 0.05) is shown by the asterisk. B, Primary stromal cells were treated with PTH (100 ng/ml) for 48 h ± SNP in the last 24 h. Total RNA was collected for RT-PCR. The real-time PCR data were compiled from three experiments. SNP inhibited PTH-stimulated RANKL mRNA expression. The asterisk represents a significant difference from control cells at P < 0.05.

 
NO does not change the mRNA stability of RANKL or OPG
To understand how NO might regulate both RANKL and OPG expression, we measured RANKL and OPG mRNA half-life using real-time PCR after treatment of cells with actinomycin D to prevent additional mRNA production. As shown in Fig. 8AGo, the half-life of RANKL mRNA in ST-2 cells treated with 100 µM SNP was about 4 h, unchanged from those cells treated with vitamin D alone. The RANKL mRNA half-life reported here is similar to that we found previously using Northern gel densitometry (20). We also measured OPG mRNA stability using real-time PCR. There was a rapid initial fall-off in OPG mRNA level as shown in Fig. 8BGo, with degradation to nearly half within the first hour. The curve became flat during the next several hours. The picture of a steep slope, followed by a flat degradation pattern after 1 h, was replicated in three separate OPG mRNA half-life assessments. We previously reported a half-life for OPG of more than 4 h using Northern analysis (19); an inability to discern two slopes with Northern analysis might explain our findings of a more stable message in that work. In any case, there was no difference in the degradation slopes for OPG mRNA taken from control and SNP-treated cells, as shown in Fig. 8BGo. These data predict that NO donor effect on RANKL and OPG is likely to be at the level of transcriptional control.



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  		FIG. 8. NO donor does not change the decay of RANKL and OPG mRNA. ST-2 cells were treated with vitamin D for 48 h and SNP (100 µM) in the last 2 h before addition of actinomycin D to measure message decay. Actinomycin D (25 µg/ml) was added to cultures for 0, 1, 2, or 4 h. Real-time PCR shows half-life of RANKL mRNA was approximately 4 h in both control and SNP-treated cells (A) and half-life of OPG mRNA was approximately 1 h in both control and SNP-treated cells (B).

 
The NO effect is cGMP independent
NO is known to use cGMP signaling to achieve many of its intracellular actions (25). To define whether NO-mediated RANKL repression and OPG induction required NO stimulation of endogenous guanylate cyclase, ST-2 cells were treated with 1,25(OH)2D3 for 48 h to raise RANKL. During the last 24 h, SNP and dibutyryl cGMP, a cell-permeable cGMP analog, or ODQ, an inhibitor of endogenous guanylate cyclase, were added. RANKL and OPG mRNA were assessed 24 h later. As shown in Fig. 9Go, cGMP at 200 µM was not able to reproduce SNP effects on either RANKL or OPG. Furthermore, 20 µM ODQ, a dose known to inhibit endogenous cGMP production (25), did not prevent the decrease in RANKL mRNA or increase in OPG mRNA due to SNP treatment. Higher doses of both cGMP (400 µM) and ODQ (40 µM) were also ineffective, i.e. did not reproduce or block SNP effect, respectively (data not shown). These results indicate that NO uses signaling pathways other than cGMP to down-regulate RANKL and up-regulate OPG.



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  		FIG. 9. Mechanism of NO effect is cGMP independent pathway. ST-2 cells were treated with vitamin D for 48 h. During the second 24 h, SNP (100 µM) and cGMP (200 µM) or ODQ (20 µM) were added as indicated. A, SNP reduced RANKL mRNA by half, whereas cGMP had no significant effect. The asterisk represents a significant difference from control value (P < 0.01). ODQ was not able to block the SNP effect. B, SNP increased OPG mRNA by 60%, compared with control. cGMP did not reproduce the SNP effect, whereas ODQ was not able to reverse the SNP effect. Data are combined from three experiments. The asterisk is representative of a significant difference from control value (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
NO has pleotropic effects in almost every cell and tissue thus far studied. Increasing appreciation of a role for NO has developed in skeletal physiology, in particular the promising therapeutic effects of nitroglycerin, a NO donor, on estrogen deficiency bone loss (2, 3). The ability of NO to support anabolic actions of estrogen in bone is further confirmed by the inability of ovariectomized mice lacking an endothelial NOS, the major NOS expressed in mature bone (26), to respond to estrogen with positive bone formation. Other studies have suggested that NO may exert its effects on bone remodeling through inhibition of bone resorption (1, 8). Most pointedly, reducing NO levels increases osteoclast recruitment and subsequent bone resorption (9, 10). These effects suggest that NO might directly regulate two molecules, RANKL and OPG, produced by stromal bone cells that control entry of precursors into the osteoclast lineage. The potent inhibition of NO on the RANKL/OPG ratio might indeed underlie aspects of NO action in the adult skeleton.

Low concentrations of NO donors have significant effects on both mRNA and protein levels of RANKL and OPG. RANKL expression induced by either vitamin D or PTH falls after treatment with 1 µM SNP. Other osteoactive factors are known to effect similar coordinate changes in RANKL and OPG expression. PTH stimulates RANKL mRNA expression, as we have confirmed here, while also decreasing OPG expression (27). We have shown previously that the remodeling factor IGF-I up-regulates RANKL expression while decreasing that of OPG (19). TGFß1, similar to NO, decreases RANKL while increasing OPG (28), as does the isoflavonoid genistein, shown to decrease the osteoclastogenic potential by decreasing RANKL and increasing OPG (29). Because of the universality of NO signaling, it may be that some of these factors regulate RANKL/OPG ratios through modulating NOS activity in bone cells.

The possibility that SNP regulated RANKL/OPG through causing cell cytotoxicity or via non-NO-mediated effects was ruled out. First, cells treated with SNP did not decrease cell number or viability. Second, another NO donor, NOR-4, had similar effects on RANKL and OPG expression. Finally, the presence of a specific NO scavenger, cPTIO (30, 31, 32), completely abrogated the ability of SNP to regulate RANKL and OPG expression. These results confirm that RANKL and OPG are regulated through NO-mediated pathways.

Some NO actions are known to require activation of cGMP-dependent signaling via activation of soluble guanylate cyclase (25). Soluble guanylate cyclase has been described in murine primary osteoblasts (33), and a stromal cell line expresses both A and B forms of guanylate cyclase (34). In our experiments, cGMP was unable to reproduce the regulatory effects of NO on RANKL and OPG expression. Furthermore, ODQ, an agent that inhibits endogenous guanylate cyclase, was unable to block NO effects. Thus, our results indicate that the actions of NO on both RANKL and OPG are cGMP independent. In contrast, Holliday et al. (9) suggested that cGMP was involved in inhibition of osteoclast formation by NO: they found that SNP increased cGMP in marrow cultures and that cGMP analogs reduced osteoclast formation. As well, cGMP has biphasic effects on primary osteoblasts including both stimulation and inhibition of apoptosis (35). Thus, it is probable that cGMP has other effects in culture that lead to reduced osteoclastogenesis separate from control of the RANKL/OPG equilibrium.

Many effects of NO are, however, known to be independent of cGMP action (36). NO can influence transcription through structural modification of transcription factors, for instance changing the binding affinity of c-fos, c-jun, or NF{kappa}B by S-nitrosylation of cysteines near their DNA-binding domains (37, 38, 39). Interestingly, a recent study suggests that nitrosylation may be a broad-based mechanism for regulating interactions between proteins, including the interaction of NOSs with their substrates as well as transcription factors with their coactivators and repressors (40). Finally, NO has been shown to both inhibit (41) and activate NF{kappa}B (37), which is thought to be critical to RANKL expression (42).

NO is also known to be released by macrophages and osteoblasts during inflammation and can be associated with inflammatory bone resorption (43). It is also associated with the response to fracture (44). The source and timing of NO release may be important to determine whether the effect of NO is anabolic or catabolic. For instance, NO generated from inflammatory NOS may be associated with resorption (45). Alternatively, endothelial NOS is associated with anabolic bone responses (46, 47), having known effects to increase osteoblast proliferation (48). Our work here supports a role for NO in promoting a positive balance in bone remodeling.

The mechanics of transcriptional control of both RANKL and OPG are still to be discovered. Many investigations have underscored the induction of RANKL expression by skeletally active agents such as vitamin D (12), PTH (11, 27), and IGF-I (19), to name only a few. As well, mechanical stimulation, which represses osteoclastogenesis in vitro and in vivo, inhibits RANKL expression (20, 49). The mechanism by which all these osteoactive factors regulate RANKL appears to be transcriptional, because no changes in mRNA half-life have been reported, as we have shown in this study for NO. However, control through the RANKL promoter has not been so easily elucidated. Seven kilobases of the promoter appear to lack both tissue specificity and hormone responsivity (50, 51). We have studied the RANKL promoter with both luciferase and chloramphenicol acetyl transferase reporters and found it to be unresponsive to vitamin D (52). However, we did find that NO had an effect on the murine RANKL promoter in a luciferase reporter, causing a dose-dependent decrease in measured luciferase activity. The effect of NO on luciferase activity turned out to be promoter independent and was most likely due to a separate effect of NO to decrease stability of the luciferase mRNA (52). In an effort to rule out a change on the half-lives of mRNA for RANKL and OPG, we demonstrated here that NO does not alter the stability of either message. In summary, the exact mechanism by which NO decreases the expression of these important factors will require further study.

Thus, our experiments, taken together with published effects of NO to inhibit osteoclast activation and bone turnover, suggest that NO has a previously unrecognized ability to modulate RANKL and OPG, resulting in a reduced osteoclastic potential of the targeted bone cells. Recognition of this potent activity should help investigators sort out the pleotropic effects that NO has on bone both in vitro and in the whole animal.


   Footnotes
 
This work was supported by a RO1 grant from the National Institute of Arthritis and Musculoskeletal and Skin Diseases and Merit and Research Enhancement Award Program grants from Department of Veterans Affairs.

Abbreviations: cPTIO, 2-(4-Carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide; MTT, methylthiazoletetrazolium; NF{kappa}B, nuclear factor-{kappa}B; NO, nitric oxide; NOS, NO synthase; ODQ, 1H-(1,2,4-oxadiazolo(4,3-{alpha})quinoxalin-1-one; 1,25(OH)2D3, 1,25 dihydroxyvitamin D3; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor-{kappa}B ligand; RT, reverse transcription; SNP, sodium nitroprusside.

Received June 10, 2003.

Accepted for publication October 10, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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