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Endocrinology Vol. 143, No. 11 4304-4309
Copyright © 2002 by The Endocrine Society

ARTICLE

The Increased Bone Mass in {Delta}FosB Transgenic Mice Is Independent of Circulating Leptin Levels

M. Kveiborg, R. Chiusaroli, N. A. Sims, M. Wu, G. Sabatakos, W. C. Horne and R. Baron

Departments of Cell Biology and Orthopedics, Yale University School of Medicine, New Haven, Connecticut 06510

Address all correspondence and requests for reprints to: Dr. Roland Baron, Department of Orthopedics and Cell Biology, Yale University, School of Medicine, 333 Cedar Street, SHM IE-55, New Haven, Connecticut 06510. E-mail: roland.baron{at}yale.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transgenic mice overexpressing {Delta}FosB, a naturally occurring splice variant of FosB, develop an osteosclerotic phenotype. The increased bone formation has been shown to be due, at least in part, to autonomous effects of {Delta}FosB isoforms on cells of the osteoblast lineage. However, abdominal fat and marrow adipocytes are also markedly decreased in {Delta}FosB mice, leading to low serum leptin levels. Increased bone mass has been linked to the absence of leptin and leptin receptor signaling in ob/ob and db/db mice. Thus, in addition to affecting directly osteoblastogenesis and bone formation, {Delta}FosB isoforms might increase bone mass indirectly via a decrease in leptin. To test this hypothesis, we restored normal circulating levels of leptin in {Delta}FosB mice via sc implanted osmotic pumps. Complete histomorphometric analysis demonstrated that trabecular bone volume as well as dynamic parameters of bone formation was unchanged by this treatment in both {Delta}FosB transgenic mice and control littermates. This demonstration that restoring circulating levels of leptin in {Delta}FosB transgenic mice failed to rescue the bone phenotype further indicates that the marked increase in bone formation is autonomous to the osteoblast lineage.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE FOS (C-FOS, FosB, Fra-1, Fra-2) and Jun (c-Jun, JunB, JunD) proteins are components of the activator protein-1 family of basic leucine zipper transcription factors that are involved in the immediate response of a variety of cells to transmembrane signaling agents (1). The activator protein-1 factors bind as either Jun/Jun homodimers or Jun/Fos heterodimers to specific sequences in the promoter regions of target genes, thereby regulating transcription. {Delta}FosB is a naturally occurring truncated isoform of FosB, arising from alternative splicing of the fosB transcript (2). {Delta}FosB retains the dimerization and DNA-binding properties of FosB but lacks a potent proline-rich transactivation domain located in the C terminus.

We have reported previously that transgenic mice overexpressing {Delta}FosB, using the TetOff, tetracycline-regulated system under control of the neuron-specific enolase (NSE) promoter, have a dramatically increased bone mass throughout the skeleton (3). Histomorphometric and biochemical analysis demonstrated that the increased bone density results from increased bone formation, rather than impaired bone resorption. Furthermore, the increase in bone formation is reversible, as revealed by switching off {Delta}FosB overexpression after osteosclerosis had developed, resulting in almost complete cessation of bone formation and loss of bone mass (4). Ex vivo and in vitro experiments revealed that the increase in bone formation is due, at least in part, to a cell-autonomous effect of {Delta}FosB isoforms on cells of the osteoblast lineage. However, {Delta}FosB is overexpressed in a variety of other tissues, and it is possible that endocrine effects also contribute to the bone phenotype.

In addition to the osteosclerotic phenotype, {Delta}FosB transgenic mice have a dramatic decrease in body fat, and marrow smears demonstrated a reduced number of adipocytes (3). Because several studies have indicated an inverse relationship between osteoblastogenesis and adipogenesis (5, 6), the observation of decreased fat in the presence of increased bone formation raised the possibility that the two events are linked. Such a link could be at several levels. For example, osteoblast and adipocyte differentiation could be mutually exclusive or events downstream of adipocyte formation could affect osteoblasts. Indeed, the adipocyte-secreted polypeptide hormone, leptin, has been proposed to be involved in a specific molecular mechanism linking bone mass and body weight (7), independent of its major role in controlling energy metabolism (8). Both leptin-deficient ob/ob mice and leptin receptor-deficient db/db mice have increased bone formation rates and high bone mass, leading to the conclusion that leptin inhibits bone formation, possibly via a central hypothalamic effect (7). Other in vivo and in vitro studies have, however, suggested a stimulatory effect of leptin on bone formation (9, 10, 11, 12).

Consistent with the decreased fat of the {Delta}FosB transgenic mice, circulating levels of leptin were significantly lower in both female and male {Delta}FosB transgenic mice (3), raising the possibility that, in addition to affecting osteoblastogenesis and bone formation directly, {Delta}FosB might increase bone mass indirectly through a decrease in leptin levels. To test this hypothesis, we restored physiological levels of leptin in {Delta}FosB transgenic mice by infusing recombinant leptin via sc implanted osmotic pumps. Subsequent histomorphometric analysis demonstrated that restored leptin levels failed to rescue the osteosclerotic phenotype, indicating that the decreased circulating leptin level does not contribute to the increased bone formation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Transgenic mice
{Delta}FosB transgenic mice were generated using the TetOff system, as previously described (3, 13, 14). In short, mice expressing the tetracycline transactivator (tTA) under control of the NSE promoter were cross-bred with mice carrying the {Delta}FosB gene under control of the tetracycline-responsive promoter (TetOp), giving rise to animals expressing elevated {Delta}FosB levels in a variety of different tissues, including bone and fat. As in previous publications using these transgenic mice (3, 13, 14), bitransgenic (NSE-tTA/TetOp-{Delta}FosB) offspring from breeding between homozygous NSE-tTA mice and heterozygous TetOp-{Delta}FosB mice, referred to as {Delta}FosB transgenic mice, were used in all described experiments and compared with NSE-tTA littermates, referred to as control mice.

Animal treatment
At the age of 6 wk, {Delta}FosB transgenic and control mice were anesthetized using methoxyflurane (Medical Developments, Springvale, Australia) and microosmotic pumps (Alzet model 1002, Alza Corp., Palo Alto, CA), infusing saline or recombinant mouse leptin (R\|[amp ]\|D Systems, Inc., Minneapolis, MN) at a rate of 200 ng/h and were placed in the dorsal sc space. Osmotic pumps were replaced after 2 wk, and mice were treated for a total continuous period of 4 wk. All mice used for analysis were injected with calcein (20 mg/kg, Sigma, St. Louis, MO) and demeclocycline (20 mg/kg, Sigma) 10 and 3 d before being killed to label bone mineralization fronts (15). At 10 wk of age, animals were anesthetized using methoxyflurane and bled by cardiac puncture to determine serum levels of leptin by the QuantikineM RIA (R\|[amp ]\|D Systems, Inc.). Abdominal fat weight was determined, and femur and tibia were collected for histomorphometric analysis. All animal protocols were approved by the Yale University Institutional Animal Care and Use Committee.

Histomorphometric analysis
Bone samples were fixed in 3.7% formaldehyde/PBS and embedded by standard procedures in methylmethacrylate resin (15). Five-micrometer toluidine blue-stained and 10-µm unstained sections were used for bone histomorphometry by standard procedures using the Osteomeasure system (OsteoMetrics, Atlanta, GA). Von Kossa staining was performed by standard procedure (16). Data were expressed as mean ± SEM. Statistical differences were calculated using t test or ANOVA for multiple comparison. P less than 0.05 was considered statistically significant.

Western blot analysis of Stat3 phosphorylation
{Delta}FosB transgenic and control mice received ip injection of recombinant mouse leptin (R&D Systems, Inc.) at a dose of 10 µg/g body weight, lipopolysaccharide (LPS) (Salmonella minnesota R595, Sigma) at a dose of 12.5 µg/g body weight or PBS. After 30 min, extracts were prepared from abdominal fat pads, liver, or brain tissue by homogenization (1:10, wt/vol) in lysis buffer containing 62.5 mM Tris (pH 6.8), 2% SDS, 10% glycerol, 2 mM sodium orthovanadate, 40 mM ß-glycerophosphate, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Homogenates were boiled for 5 min and cleared by centrifugation at 8000 x g for 10 min at 4 C (17). Protein amounts were determined using the Micro BCA protein assay reagent kit (Pierce Chemical Co., Rockford, IL), as described by the manufacturer. Extracts (60 µg protein) were subjected to electrophoresis on 8% Tris-glycine gels (Novex, Invitrogen, Carlsbad, CA) and transferred to Protran nitrocellulose membranes (Schleicher & Schuell Inc., Keene, NH). Immunoblot analysis of signal transducer and activator of transcription 3 (Stat3) phosphorylated on tyrosine 705 and total Stat3 was performed using the PhosphoPlus Stat3 (Tyr705) antibody kit (Cell Signaling Technology, Inc., Beverly, MA). Immunoreactive bands were detected by the ECL method (Amersham Pharmacia Biotech, Piscataway, NJ).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Restored physiological serum leptin levels in {Delta}FosB transgenic mice
As reported previously, in addition to exhibiting an osteosclerotic phenotype, {Delta}FosB transgenic mice have a substantially decreased amount of body fat (Ref. 3 and Fig. 1AGo). As could be expected from the decreased amount of fat tissue, the concentration of the adipocyte-secreted peptide hormone leptin in serum was also reduced in {Delta}FosB transgenic mice, compared with control littermates (Fig. 1BGo).



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  		Figure 1. Abdominal fat weight (A) and immunoassay quantification (B) of serum leptin levels in vehicle- (black bars) or leptin-treated (white bars) control (C) or {Delta}FosB transgenic mice at 10 wk of age. Data are shown as mean ± SEM, n = 9–11. *, P < 0.01 relative to vehicle-treated control mice.

 
To test the possibility that the decreased serum leptin level in the {Delta}FosB-expressing mice could contribute to the observed increase in bone formation over and above the cell autonomous change in osteoblasts, circulating leptin in {Delta}FosB transgenic mice was restored to a normal physiological level. After preliminary time-course and dose-response experiments, osmotic pumps infusing mouse recombinant leptin or vehicle were implanted sc in {Delta}FosB transgenic mice and control littermates at 6 wk of age. The pumps were replaced after 2 wk, and after a continuous period of 4 wk of treatment, mice were killed, serum leptin levels were measured, and the bone and fat phenotypes determined.

As shown in Fig. 1BGo, leptin treatment fully restored the level of circulating leptin in {Delta}FosB transgenic mice to that measured in untreated control littermates. In contrast, leptin treatment did not significantly change serum leptin levels in control mice, probably because of the negative feedback mechanism whereby increased leptin levels cause a decrease in the amount of fat and consequently in endogenous serum leptin (8). A decreased amount of abdominal fat tissue was in fact observed in leptin-treated control mice, compared with vehicle-treated control mice, whereas the already low fat levels in {Delta}FosB transgenic mice were unchanged by leptin infusion (Fig. 1AGo).

Restored leptin levels fail to alter bone formation and rescue the osteosclerotic phenotype
Having restored normal circulating levels of leptin in {Delta}FosB transgenic mice, the bone phenotype was determined. As seen from the Von Kossa-stained proximal tibia in Fig. 2Go, leptin-treated {Delta}FosB transgenic mice were still characterized by an extremely high trabecular bone volume, despite the physiological hormone level. A complete histomorphometric analysis (Fig. 3Go) revealed no statistically significant differences in the trabecular bone parameters, such as bone volume, trabecular thickness, and trabecular number between leptin-treated and vehicle-treated {Delta}FosB transgenic mice (Fig. 3Go, A–C). Nor was there any difference in the bone formation rate between leptin- and vehicle-treated {Delta}FosB mice (Fig. 3DGo). Also, consistent with the unchanged serum leptin levels in control mice, no differences in either static or dynamic bone parameters were detected in leptin-treated, compared with vehicle-treated, control mice (Fig. 3Go).



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  		Figure 2. Von Kossa-stained proximal tibia from vehicle- or leptin-treated control or {Delta}FosB transgenic mice at 10 wk of age.

 


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  		Figure 3. Histomorphometric analysis of vehicle- (black bars) and leptin-treated (white bars) control (C) and {Delta}FosB transgenic mice at 10 wk of age. A, Trabecular bone volume (BV/TV); B, trabecular thickness (TbTh); C, trabecular number (Tb N); and D, bone formation rate (BFR). Data are shown as mean ± SEM, n = 9–11. *, P < 0.05 vs. vehicle-treated control; **, P < 0.01 vs. vehicle-treated control; #, P < 0.05 vs. leptin-treated control; ##, P < 0.01 vs. leptin-treated control.

 
{Delta}FosB transgenic mice exhibit a normal response to leptin signaling
The inability of normal leptin levels to correct the bone phenotype suggested that the decreased leptin levels of the {Delta}FosB mice were not contributing to the increase in bone formation. However, another possibility could be that the transgenic animals were resistant to leptin and thus unresponsive to the increase in hormone level. To address this question, a proximal signaling response to leptin was measured. Under normal circumstances, binding of leptin to its receptor triggers phosphorylation of the Stats (18). To examine whether {Delta}FosB transgenic mice exhibit a normal response to leptin signaling, Stat3 activation was assayed in several tissues, including fat and brain (Fig. 4Go). Extracts were isolated from {Delta}FosB transgenic mice and wild-type littermates 30 min after ip injection with recombinant mouse leptin at a single dose of 10 µg/g body weight, which has been shown previously to be sufficient to induce Stat phosphorylation (18, 19). LPS, which is known to activate Stats in various tissues in an indirect manner via cytokine release (20), was used as a positive control. As can be seen in Fig. 4Go, injection of recombinant leptin caused a prompt increase in the tyrosine phosphorylation of Stat3 in both {Delta}FosB transgenic mice and control littermates, demonstrating that the transgenic animals have retained a normal response to leptin signaling. Leptin failed to induce any increase in Stat3 phosphorylation in primary calvarial osteoblast cultures from either control or {Delta}FosB transgenic mice (data not shown).



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  		Figure 4. Expression and tyrosine phosphorylation of Stat3 in adipose (A) and brain tissue (B) of control and {Delta}FosB transgenic mice injected ip with (+) or without (-) recombinant murine leptin at a dose of 10 µg/g body weight or vehicle. LPS was used as a positive control. Extracts from different animals were prepared 30 min after injection, separated by SDS-PAGE, transferred to nitrocellulose membranes, and analyzed by immunoblotting with anti-Stat3 and anti-phosphotyrosine 705 Stat3 antibodies.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Bone is a highly dynamic tissue that is constantly being renewed in a process involving resorption of existing bone by osteoclasts and de novo bone formation by osteoblasts. Increased bone mass can arise from either impaired bone resorption, resulting in osteopetrosis or from increased bone formation, leading to osteosclerosis. Until recently, only very mild osteosclerotic phenotypes have been reported, as in osteocalcin-deficient mice (21). However, within the last few years, two major findings regarding regulation of bone formation have been published. The first relates to the effect of Fos-related proteins in osteoblast function, exemplified by the overexpression of either Fra-1 (22) or {Delta}FosB (3), giving rise to severe osteosclerosis in mice. The second finding is an increased bone formation in leptin-signaling-deficient ob/ob and db/db mice and the observation that intracerebroventricular infusion of leptin caused bone loss in these mice as well as in control animals (7).

The reported effects of leptin on bone metabolism are, however, inconsistent because other reports have shown a positive effect of leptin on bone mass in vivo (9, 10) as well as stimulatory effects on osteoblast differentiation in vitro (12, 23). It is difficult to compare directly the results of the studies, though, because of differences in experimental models, animal age, treatment regiments, and different end points as a basis for evaluating leptin’s effects. We consequently tested whether the osteosclerotic phenotype in transgenic mice that overexpress {Delta}FosB is dependent on reduced circulating leptin levels and thus secondary to decreased adipose tissue in these mice.

By continuous infusion of recombinant leptin at concentrations 10–20 times lower than the pharmacological doses shown in some other studies to affect bone formation (9, 10), we were able to fully restore a normal level of circulating leptin in the transgenic mice without changing the level in wild-type littermates, thereby allowing us to test whether the decreased leptin levels contribute to the increased bone mass. Four weeks of leptin treatment resulted in a slight reduction in food intake by both the control and transgenic animals, indicating that the peptide was effective in the central nervous system. The reduced food intake was associated with a decrease in adipose tissue in wild-type mice but not in transgenic animals, which are characterized by already very low amount of adipose tissue. Despite the restoration of normal leptin levels, bone formation parameters did not change significantly in transgenic or wild-type mice, as demonstrated by complete histomorphometric analysis. These results strongly indicate that the osteosclerotic phenotype in {Delta}FosB mice is independent of the reduced serum leptin levels.

There are, however, possible alternative explanations of the failure of infused leptin to reverse the increased bone mass of the {Delta}FosB mice. Leptin receptors have been reported to be expressed in a variety of tissues and cell types (24), including bone cells (9, 12), but the relative importance of the effects of leptin on central vs. peripheral sites remain unclear. Still, leptin is able to cross the blood-brain barrier and because there is no reason to think that this transport is affected in the {Delta}FosB mice, the cerebrospinal fluid leptin level is expected to reflect the changes in the serum level because it has been demonstrated in humans (25, 26). Also, the possibility that the transgenic mice have become resistant to leptin signaling was ruled out by demonstrating that both control and {Delta}FosB mice responded to ip injection of recombinant leptin with a substantial increase in Stat3 activation in all tissues tested, including brain. This strongly indicates normal leptin signaling and a response of the central nervous system to increased leptin in these animals. Finally, a 4-wk period of restored leptin levels might not have been sufficient to reverse the phenotype. Yet previous experiments, involving switching off {Delta}FosB expression by doxycycline treatment, showed that 2 wk were sufficient to cause a significant decrease in bone mass and bone formation as well as a correction of the fat phenotype (4). Thus, if the decrease in leptin levels is a major contributor to the increased bone mass in {Delta}FosB mice, 4 wk of normal hormone levels should at least partly rescue the phenotype.

Although the present findings show that the low level of circulating leptin in {Delta}FosB mice is not likely to contribute to the osteosclerotic phenotype, it remains to be determined whether the bone and fat phenotypes are related through other ways or whether the increased osteoblastogenesis and decreased adipogenesis are mediated by separate and independent mechanisms. In this regard, it has recently been reported that treating mice with troglitazone, a potent stimulator of adipogenesis, increased the amount of adipose tissue but did not affect bone volume, demonstrating that the two processes can be regulated independently in skeletally mature mice (27). Also supporting this view is the observation that overexpression of Fra-1 gives rise to an osteosclerotic phenotype very similar to that of {Delta}FosB transgenic mice without any reported changes in fat and adipocyte differentiation (22). (A direct comparison of the {Delta}FosB and Fra-1 transgenic mice is, however, problematic because of the usage of different promoters driving transgene expression.)

In conclusion, this study demonstrates that restoring a physiological level of circulating leptin did not significantly alter bone formation parameters in {Delta}FosB transgenic mice, thereby strongly suggesting that the osteosclerotic phenotype is independent of the reduced serum leptin levels in these mice. These findings therefore further support our previous conclusions that the ability of {Delta}FosB to increase bone formation is an autonomous effect on cells of the osteoblast lineage.


   Acknowledgments
 
The authors thank Jennifer Juel for technical assistance in histology and Karen Ford for animal care. We also thank Dr. Eric Nestler for providing us with the transgenic mice.


   Footnotes
 
This work was supported by a postdoctoral fellowship from the Danish Natural Research Council (to M. K.) and by grants from Aventis and NIH/National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR48218.01; to R.B.).

Current address for N.A.S.: St. Vincent’s Institute of Medical Research, Melbourne, Australia.

Abbreviations: LPS, Lipopolysaccharide; NSE, neuron-specific enolase; Stat, signal transducer and activator of transcription; TetOp, tetracycline-responsive promoter; tTA, tetracycline transactivator.

Received April 19, 2002.

Accepted for publication July 17, 2002.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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