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Endocrinology Vol. 145, No. 11 5068-5074
Copyright © 2004 by The Endocrine Society

Regulation of Bone Mass and Bone Turnover by Neuronal Nitric Oxide Synthase

Robert J. van’t Hof, Jeny MacPhee, Helene Libouban, Miep H. Helfrich and Stuart H. Ralston

Bone Research Group, Institute of Medical Sciences, Foresterhill, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom

Address all correspondence and requests for reprints to: Dr. R. J. van’t Hof, Bone Research Group, Institute of Medical Sciences, Department of Medicine and Therapeutics, Foresterhill, Aberdeen AB25 2ZD, United Kingdom. E-mail: r.hof{at}abdn.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide (NO) is produced by NO synthase (NOS) and plays an important role in the regulation of bone cell function. The endothelial NOS isoform is essential for normal osteoblast function, whereas the inducible NOS isoform acts as a mediator of cytokine effects in bone. The role of the neuronal isoform of NOS (nNOS) in bone has been studied little thus far. Therefore, we investigated the role of nNOS in bone metabolism by studying mice with targeted inactivation of the nNOS gene. Bone mineral density (BMD) was significantly higher in nNOS knockout (KO) mice compared with wild-type controls, particularly the trabecular BMD (P < 0.01). The difference in BMD between nNOS KO and control mice was confirmed by histomorphometric analysis, which showed a 67% increase in trabecular bone volume in nNOS KO mice when compared with controls (P < 0.001). This was accompanied by reduced bone remodeling, with a significant reduction in osteoblast numbers and bone formation surfaces and a reduction in osteoclast numbers and bone resorption surfaces. Osteoblasts from nNOS KO mice, however, showed increased levels of alkaline phosphatase and no defects in proliferation or bone nodule formation in vitro, whereas osteoclastogenesis was increased in nNOS KO bone marrow cultures. These studies indicate that nNOS plays a hitherto unrecognized but important physiological role as a stimulator of bone turnover. The low level of nNOS expression in bone and the in vitro behavior of nNOS KO bone cells indicate that these actions are indirect and possibly mediated by a neurogenic relay.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NITRIC OXIDE (NO) is a pleiotropic signaling molecule with important regulatory effects on bone cell function. Previous studies have shown that bone cells produce NO in response to various stimuli including proinflammatory cytokines, mechanical loading, and estrogen. Studies of mice with targeted inactivation of the NO synthase (NOS) genes have shown that NO derived from the endothelial NOS (eNOS) pathway appears to be important for normal osteoblast function, the anabolic response of bone to exogenous estrogen (1, 2, 3), and the response of bone to mechanical loading (4, 5). In contrast, NO derived from the inducible NOS (iNOS) pathway has been shown to regulate the effects of proinflammatory cytokines on bone and to be essential for the stimulatory effects of IL-1 on bone resorption both in vitro and in vivo (6, 7, 8, 9). The neuronal NOS isoform (nNOS) enzyme is generally considered to account for the largest proportion of tissue NOS activity because it is highly expressed in the brain and in other tissues such as skeletal muscle, the intestine, the kidney, and the heart (10). Although nNOS knockout (KO) mice are viable and of normal appearance, they have enlarged stomachs due to hypertrophy of the pyloric muscle, exhibit behavioral disturbances and insulin resistance, and develop left ventricular hypertrophy with ageing (11). Also, nNOS KO mice have been found to be resistant to neural damage as the result of stroke induced by middle cerebral artery ligation (12).

Most investigators have failed to detect evidence of nNOS expression in normal adult bone or bone cell cultures (13, 14), although the nNOS isoform has been detected during skeletal development (15) and during fracture healing, where nNOS is present in chondral and fibrochondral regions within fracture callus (16).

Until now, the role of nNOS in bone metabolism has been little studied. The importance of NO as a regulator of bone cell function, however (17), coupled with recent evidence that has shown that the central nervous system plays a role in regulating bone turnover and bone mass (18), led us to investigate the role of nNOS in bone metabolism by studying various aspects of bone metabolism in mice with targeted inactivation of the nNOS gene (12).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Human recombinant receptor activator of nuclear factor {kappa}B ligand (RANKL) was obtained from Insight Biotechnology (Middlesex, UK); human recombinant macrophage colony-stimulating factor (M-CSF) was obtained from R&D Systems (Abingdon, UK); and human recombinant TNF{alpha} was obtained from Peprotech (London, UK). {alpha}-MEM, fetal calf serum (FCS), penicillin, and streptomycin were obtained from Life Technologies, Inc. (Paisley, UK), and tissue culture plates were obtained from Costar (Cambridge, MA). All other reagents were from Sigma (Poole, Dorset, UK), unless otherwise indicated in the text.

Animals
Mice with targeted inactivation of the nNOS gene and wild-type controls were obtained from the Jackson Laboratories (Bar Harbor, ME) (strain name: B6;129S4-Nos1tm1Plh; stock no. 002633). The targeting strategy used to generate these mice has been described previously (12, 19). The animals were housed in a designated animal facility and routinely maintained on a 12-h light, 12-h dark cycle and given ad libitum access to food and water. The tibial bones from 10-wk-old female mice (nine nNOS wild-type mice and 12 nNOS KO mice) were dissected and used for bone mineral and histomorphometric analysis (see Bone mineral measurements and Bone histomorphometry).

Bone mineral measurements
Full-body bone mineral density (BMD) and bone mineral content (BMC) were determined by dual-energy x-ray absorptiometry scanning using a PIXImus small animal scanner (GE Medical, Bedford, UK) according to the manufacturer’s instructions.

In addition, volumetric BMC and BMD were measured ex vivo using peripheral quantitative computed tomography at the left proximal tibial metaphysis using an XCT Research M bone densitometer with a voxel size of 70 µm and analysis software version 5.1.4. (Stratec Medizintechnik, Pforzheim, Germany). Daily quality assurance measurements were performed using a plexicoated polyvinyl chloride-fluorinated hydrocarbon phantom (Stratec) according to the manufacturer’s instructions. Scans were performed at the proximal tibial metaphysis 0.9 mm distal to the growth plate, as previously described (2). The precision was 1.19% for total BMD, 3.53% for trabecular BMD, and 1.04% for cortical BMD.

Bone histomorphometry
Bone histomorphometry was performed on left tibiae. The bones were dissected free of soft tissues, fixed in 4% buffered formalin/saline (pH 7.4), and embedded in methyl methacrylate. Longitudinal sections (4 µm) were then prepared and stained with von Kossa and counterstained with Paragon. Histomorphometric measurements were made on sections of the proximal metaphysis distal to the epiphyseal growth plate using a x10 objective lens on a Zeiss Axioskop (Carl Zeiss, Welwyn Garden City, UK) coupled to a Progress C14 digital camera (ISS, Manchester, UK) using a 1 x C-mount adapter. The camera was connected to an image analysis system running in-house-designed software developed using Aphelion ActiveX Objects (Adcis SA, Hérouville-Saint-Clair, France). Bone histomorphometric variables were expressed according to the guidelines of the American Society of Bone and Mineral Research Nomenclature Committee (20).

The mineral apposition rate of bone was assessed on unstained sections using fluorescent microscopy in tibiae obtained from 10-wk-old mice that had received ip injections of 20 mg/kg calcein green (Sigma) 7 and 2 d before they were killed.

Immunostaining for nNOS
Immunostaining was performed on primary osteoblasts grown on 13-mm-diameter collagen-coated (0.15 mg/ml) coverslips and on 5-µm cryosections of fresh mouse brain. Specimens were fixed with 1:1 acetone-methanol for 1 min, washed in PBS, and permeabilized for 5 min with Triton X-100 (0.5% in PBS) at room temperature. Nonspecific binding was blocked by a 20-min incubation with 20% FCS in PBS before incubation with anti-nNOS primary antibody (2 µg/ml, anti-nNOS Clone 16; BD Transduction Laboratories, Mississauga, Ontario, Canada) at room temperature for 30 min. Samples were washed three times in PBS for 5 min and subsequently incubated with Alexa Fluor 594 goat antimouse (Molecular Probes, Eugene, OR) at room temperature for 30 min, followed by three more washes in PBS, and mounted in 80% glycerol in PBS. Specimens were examined using a Zeiss Axioskop fluorescence microscope (Carl Zeiss) with a x40 oil objective lens, and images were captured using an Optronics CCD camera (Optronics, Goleta, CA).

Detection of nNOS by RT-PCR
Total RNA was isolated from primary osteoblast cultures and whole mouse brain homogenates using Trizol as described by Helfrich et al. (14). Subsequently, 5 µg of RNA was reverse transcribed using Superscript Reverse Transcriptase (Life Technologies, Inc., Gaithersburg, MD) using a random hexamer as primer, and the resulting cDNA was taken up in 100 µl of sterile water and used to detect nNOS and ß-actin mRNA by PCR. PCRs were performed using a MJ Research Opticon2 Real Time PCR machine (MJ Research, South San Francisco, CA) using the Finnzymes DyNAmo SYBR Green kit (GRI, Braintree, UK) according to the manufacturer’s instructions. The primers used were as follows: nNOS forward, AGC ACC TAC CAG CTC AAG GA; nNOS reverse, ATA GTG ATG GCC GAC CTG AG; ß-actin forward, TCG TGG GGC GCC CCA GGA CC; and ß-actin reverse, GAA ATC GTG CGT GAC ATT AAG GAG. The cycling protocol consisted of an initial step of 94 C for 10 min, followed by 40 cycles of 30 sec at 94 C, 30 sec annealing, 1 min at 72 C, 10 sec at 82 C to melt any primer dimers, and a read of the SYBR Green levels. The annealing temperatures for the nNOS primers and ß-actin primers were 65 and 60 C, respectively. A serial dilution of the whole-brain cDNA was used as a standard curve. The expression of nNOS was calculated relative to this standard and corrected using the ß-actin levels as an internal control. For experiments investigating the effects of cytokines and calciotropic hormones on nNOS expression, osteoblast cultures were treated overnight with 1,25-dihydroxyvitamin D3 (10 nM), parathyroid hormone (1–34 fragment), IL-1 (105 U/ml), or TNF{alpha} (50 ng/ml) before isolating RNA.

Osteoblast isolation and culture
Osteoblasts were isolated from the calvarial bones of 2-d-old mice by sequential collagenase digestion as described by van’t Hof (21) and cultured in {alpha}-MEM supplemented with 10% FCS and penicillin/streptomycin cocktail at 37 C in 5% CO2. Osteoblasts were grown overnight, harvested by trypsin digestion, seeded in 96-well plates at 4 x 103 cells/well in 100 µl {alpha}-MEM supplemented with 10% FCS, penicillin, and streptomycin, and incubated for up to 72 h. After 24, 48, and 72 h of culture, cell numbers were determined by adding 10 µl Alamar Blue reagent (Biosource, Camarillo, CA) per well, incubating the cells for a further 3 h, and measuring viability as fluorescence (excitation 530 nm; emission 590 nm) using a Labtech FL600 plate fluorometer (Biotech Instruments, Winooski, VT).

Alkaline phosphatase (ALP) activity was assessed by measuring the conversion of p-nitrophenyl phosphate to p-nitrophenyl using a Labtech FL600 plate spectrometer. Briefly, after assessing the cell number by the Alamar Blue assay, the cell layer was resuspended in 150 µl of lysis buffer (1 M diethanolamine, 1 mM MgCl2, and 0.05% Triton X-100), and the lysates were stored at –20 C. Fifty microliters of sample and 50 µl of substrate solution (20 mM paranitrophenol phosphate in lysis buffer) were pipetted into 96-well plates, and absorption was measured at 414 nm every 3 min at 37 C.

For mineralization assays, osteoblasts were seeded in six-well plates at 1 x 105 cells/well in 2 ml of {alpha}-MEM supplemented with 10% FCS, penicillin, streptomycin, 2 mM glutamine, 10 mM ß-glycerophosphate, and 50 µg/ml L-ascorbic acid. Medium was changed every 3 d, whereas L-ascorbic acid was added daily because of its short half-life. Osteoblasts were cultured for 10, 14, and 17 d. At the end of the culture, osteoblasts were washed five times with PBS and fixed in 70% cold ethanol for 1 h, and mineralized nodules were detected by Alizarin red staining. Briefly, the cultures were washed in deionized water, stained with 40 mM Alizarin red S (pH 4.2) for 20 min at room temperature on an orbital rotator, washed four times with deionized water, and analyzed.

Osteoclast generation
Bone marrow was flushed out of the femora and tibiae of adult mice using Hanks’ balanced salt solution. The resulting cell suspension was spun down at 300 x g for 3 min, resuspended in 10 ml of culture medium ({alpha}-MEM supplemented with 10% FCS and penicillin/streptomycin), and cultured in one 10-cm petri dish per mouse for 3 d at 37 C in the presence of 100 ng/ml M-CSF. Subsequently, the petri dish was washed three times with PBS, and the adhered cells were harvested using trypsin digestion. The cells were resuspended in culture medium supplemented with 100 ng/ml RANKL and 25 ng/ml M-CSF at a density of 4 x 104 cells/ml, and 125 µl/well of this suspension was seeded onto dentine slices in a 96-well plate. The cells were cultured at 37 C in 5% CO2 for 7 more days, with medium changes after 2 and 4 d. In experiments where cultures were stimulated by TNF{alpha}, 50 ng/ml of human recombinant TNF{alpha} (Peprotech) or vehicle (PBS) was present during the last 3 d of culture. At the end of the culture period, osteoclasts were identified by staining for tartrate-resistant acid phosphatase essentially as described by van’t Hof et al. (22). Briefly, dentine slices with adherent cells were fixed in 4% paraformaldehyde, washed with PBS, and incubated with naphthol-ASBI phosphate, pararosanilin, and 30 mM sodium tartrate in acetate buffer at 37 C for 45 min. Tartrate-resistant acid phosphatase-positive cells with three or more nuclei were considered to be osteoclasts. After counting adherent osteoclasts, dentine slices were immersed in 20% sodium hypochlorite to remove the cell layer. Resorption pits were visualized by reflected light microscopy and a Diagnostic Instruments Insight camera (Diagnostic Instruments, Inc., Sterling Heights, MI) coupled to an Image Analysis system (ADCIS, Herouville-Saint-Clair, France). The area resorbed was quantified by Image Analysis using custom software developed using Aphelion ActiveX objects (Adcis) (21).

Statistical analyses
Statistical analyses were performed using SPSS for Windows version 11 (SPSS Inc., Chicago, IL). Significant differences between groups were determined by ANOVA. If more than two groups were compared, significant differences were determined using the Bonferroni correction. All data are presented as means ± SEM, unless stated otherwise. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Bone mineral measurements
Total-body BMD and BMC, as assessed by dual-energy x-ray absorptiometry, were significantly higher (13 and 26%, respectively) in nNOS KO mice compared with wild-type controls (Fig. 1AGo). A similar increase was observed in the proximal tibia using peripheral quantitative computed tomography (9.6% in nNOS KO mice and 8.4% in wild-type controls; Fig. 1BGo). The difference in BMD was particularly pronounced in the trabecular compartment, where BMD was 29% higher in nNOS KO mice than in wild-type controls, with a smaller (3%) increase in the cortical BMD (Fig. 1BGo). The nNOS KO mice also displayed significant increases in cortical area (7.3 ± 1.6%, P < 0.01), cortical content (10.4 ± 1.5%, P < 0.001), and cortical width (8.4 ± 0.2%, P < 0.01) when compared with control animals. There were no differences in overall length and thickness of the bones (data not shown).



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  		FIG. 1. BMD is increased in nNOS KO mice. A, Whole-body BMD and BMC were measured using a Lunar PIXImus dual-energy x-ray absorptiometry scanner. Average BMD and BMC for wild-type (WT) mice were 0.0505 ± 0.0006 g/cm2 and 0.451 ± 0.009 g, respectively. B, BMD of the proximal tibia was measured ex vivo using a Stratec peripheral quantitative computed tomography scanner. TOT_DEN, Total BMD (average in WT, 704 mg/cm3); TRAB_DEN, trabecular BMD; CRT_DEN, cortical BMD. Values are expressed as % WT ± SEM (n = 10). *, P < 0.05 from WT; **, P < 0.01 from WT; ***, P < 0.001 from WT.

 
Bone histomorphometry
Bone histomorphometric analysis showed that nNOS KO mice had a 67% increase in trabecular bone volume compared with controls and an increase of about 10% in cortical and trabecular thickness (Table 1Go). Indices of bone formation and bone resorption were also significantly lower in the nNOS KO mice compared with controls. Osteoblast surface and osteoid surface were significantly reduced to between 20 and 30% of the values noted in wild-type mice, whereas indices of bone resorption were reduced by between 30 and 70% (Table 1Go). No significant differences were observed for osteoid width and mineral apposition rate (Table 1Go). These differences in BMD and bone histomorphometry were readily apparent on low-power photomicrographs of the tibial metaphysis, where we observed also that the trabecular bone tended to extend deeper into the metaphysis in the nNOS KO mice than in the controls (Fig. 2Go).


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  		TABLE 1. Bone histomorphometric parameters of wild-type and nNOS KO mice

 


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  		FIG. 2. Histology of wild-type and nNOS KO bone. The micrographs show sections of the proximal tibia (von Kossa and Paragon stain) of a wild-type (A) and a nNOS KO (B) mouse. Note the marked increase in trabecular bone and cortical width in the nNOS KO mouse compared with the wild-type mouse.

 
nNOS expression in osteoblasts
We found weak staining for nNOS in primary osteoblasts (Fig. 3AGo) and were also able to detect nNOS mRNA in osteoblasts using RT-PCR (Fig. 3EGo). However, both mRNA and immunolabeling intensities appeared to be much less than those observed in brain cells. When we quantified the levels of nNOS mRNA using real-time PCR, we found the levels in osteoblasts to be 1.4 ± 1.1% of a whole-brain RNA sample used as positive control, and these levels were not affected by overnight treatment of the cells with PTH, vitamin D3, or IL-1 (data not shown).



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  		FIG. 3. Osteoblasts express low levels of nNOS. Osteoblasts and brain cryosections were stained for nNOS as described in Materials and Methods and visualized using fluorescence microscopy (A–D). A, Osteoblasts, 0.5-sec exposure; B, phase contrast image of same field; C, brain cryosection at 0.067 sec; D, brain cryosection, no primary antibody, 0.067-sec exposure; E, nNOS and ß-actin mRNA expression as detected by RT-PCR. Lane 1, Whole-brain homogenate; lane 2, primary mouse osteoblasts.

 
Osteoblast proliferation and osteoclast formation in vitro
Because we found evidence for expression of nNOS in bone cells, albeit at a very low level, we wanted to determine whether the reduction in numbers of osteoblasts and osteoclasts observed in nNOS KO mice in vivo was accompanied by any abnormalities of osteoblast function or osteoclast formation in vitro. We found no qualitative difference in proliferation of nNOS KO osteoblasts compared with wild-type osteoblasts (Fig. 4AGo) or in the ability of these cells to form mineralized bone nodules (Fig. 5Go). However, ALP levels were significantly higher in nNOS KO cells under basal and PTH-stimulated conditions. Surprisingly, we also found that RANKL- and M-CSF-induced osteoclast formation was increased in bone marrow cultures derived from nNOS KO mice, both in the absence and presence of TNF{alpha} stimulation (Fig. 4CGo).



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  		FIG. 4. Comparison of osteoblast function and osteoclast formation and activity in bone cell cultures derived from nNOS KO and wild-type (WT) mice. A, Osteoblast proliferation as measured by the Alamar Blue assay. B, ALP activity of primary osteoblasts, cultured for 48 h in the presence or absence of 5 x 10–8 M PTH. Values are corrected for cell number using the Alamar Blue assay. C, Osteoclast formation in murine bone marrow cultures in vitro. D, Resorption area as percentage of unstimulated WT cultures. Control resorption areas in the three experiments performed were 0.15, 0.46, and 0.22 mm2. Control: no stimulation; TNF: stimulated with 50 ng/ml recombinant human TNF{alpha} during the last 3 d of the culture. All values represent overall mean ± SD from three independent experiments (n = 5 in each experiment). **, P < 0.01; ***, P < 0.001 from WT control; ++, P < 0.01; +++, P < 0.001 from no PTH or TNF.

 


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  		FIG. 5. Bone nodule formation in osteoblast cultures from nNOS KO and wild-type mice. Osteoblasts were cultured in the presence of ß-glycerophosphate and ascorbic acid for the times indicated, and mineralized bone nodules were identified by Alizarin red staining. We observed no difference in the timing of onset of mineralization or the level of mineralization between nNOS KO and wild-type mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies from our group and others have shown that NO is an important regulator of bone metabolism, and studies in mice with targeted inactivation of the eNOS and iNOS genes have shown that these isoforms serve different functions in bone (17). To determine whether NO derived from the nNOS isoform also plays a role in bone metabolism, we investigated BMD and bone histomorphometry in mice with targeted inactivation of the nNOS gene. We found that BMD values in nNOS KO mice were significantly increased compared with wild-type controls and that this particularly affected the trabecular bone compartment. The differences in BMD were accompanied by a marked reduction in histomorphometric indices of bone turnover. These findings are quite different from those observed in eNOS KO mice, which exhibit low bone mass and evidence of decreased bone formation but show no abnormalities of bone resorption (1, 2). The skeletal phenotype in nNOS KO mice also differs from that in iNOS KO mice, which show no abnormalities of bone mass or bone turnover under normal conditions but are partially protected from ovariectomy-induced bone loss (23), inflammation-induced bone loss (8, 24), and bone loss resulting from immobilization (25).

The mechanisms responsible for the reduction in bone turnover and increased bone mass in nNOS KO mice are unclear at present. Expression of nNOS has been detected in osteoblasts and osteoclasts during bone development in rats (15) and during endochondral bone formation in healing fracture callus (16), which raises the possibility that expression of nNOS during fetal life could play a role in regulating skeletal development. Although this could partly explain the difference in BMD between nNOS KO mice and controls, it is unlikely to explain the reduction in bone turnover in adult mice. Furthermore, a local effect of nNOS on bone cell activity seems unlikely because the nNOS isoform is expressed at very low levels by bone cells. The in vitro studies of osteoblast proliferation and bone nodule formation that we conducted also argue against a cell-autonomous effect of nNOS because cell growth was almost identical in nNOS KO and wild-type cultures, and no qualitative differences in bone nodule formation were observed. Surprisingly, however, ALP activity was increased in nNOS KO osteoblasts when compared with wild-type osteoblasts, as was RANKL- and TNF{alpha}-stimulated osteoclast formation. A possible explanation for the suppressed bone turnover that we observed in vivo and the normal or increased activity of nNOS KO cells that we observed in vitro is that nNOS acts through a systemic endocrine or neurogenic pathway to regulate bone turnover. Two scenarios can be envisaged, one in which nNOS stimulates the systemic release of factors that enhance bone turnover and another in which nNOS blocks the release of factors that inhibit bone turnover. The observation that ALP activity was increased and osteoclast formation enhanced in nNOS KO-derived cells in vitro fits best with the notion that there is a non-cell-autonomous, systemic inhibition of bone turnover in nNOS KO mice and that the cells are released from that inhibition when cultured in vitro. Of course, we cannot exclude the possibility that the low levels of nNOS that are expressed by bone cells exert direct effects on osteoclast and osteoblast activity, in an opposite direction to the effects that are observed in vivo.

Because nNOS is strongly expressed in the central nervous system, it is tempting to speculate that nNOS might influence bone metabolism by a neurogenic relay, as has been suggested for the effects of leptin (26, 27, 28) and neuropeptide Y (29) on bone metabolism. It is unlikely that the leptin signaling pathway is involved because leptin inhibits nNOS mRNA expression in the hypothalamus (30) and, at the same time, reduces bone mass (26). Alterations in circulating levels of sex hormones are also unlikely because the strain of mice we studied has been reported to have normal fertility (11). However, a different strain of nNOS KO mice created by deletion of the heme-binding region in exon 6 showed evidence of hypogonadism (31), which if anything, would be expected to reduce bone mass. High levels of nNOS expression are also observed in skeletal muscle, but an effect mediated through skeletal muscle seems unlikely because previous investigators have found no evidence of muscle pathology in the same strain of nNOS KO mice as we studied here (32).

In conclusion, this study has shown that nNOS KO mice have increased bone mass and decreased bone turnover, indicating that the nNOS isoform plays an important role in the regulation of bone mass and bone turnover in mice. The low levels of nNOS expression in bone and the contrast between the suppressed bone turnover in vivo and the normal or increased activity of nNOS KO-derived bone cells in vitro indicate that the actions of nNOS on bone remodeling are not cell autonomous and probably are mediated by a hitherto unidentified endocrine pathway or neurogenic relay that regulates bone turnover. Further studies are now warranted to identify and characterize this pathway and to define the underlying mechanisms by which nNOS regulates bone turnover.


   Acknowledgments
 
We thank Ms. Wendy Spinks and Dr. Ken Armour for technical assistance.


   Footnotes
 
This work was supported in part by grants from the Arthritis Research Campaign, United Kingdom (Grant V01510), and the Wellcome Trust (Grant 068454).

Abbreviations: ALP, Alkaline phosphatase; BMC, bone mineral content; BMD, bone mineral density; eNOS, endothelial nitric oxide synthase; FCS, fetal calf serum; iNOS, inducible nitric oxide synthase; KO, knockout; M-CSF, macrophage colony-stimulating factor; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NOS, nitric oxide synthase; RANKL, receptor activator of nuclear factor {kappa}B ligand.

Received February 17, 2004.

Accepted for publication July 30, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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