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Endocrinology Vol. 147, No. 7 3196-3202
Copyright © 2006 by The Endocrine Society

Cart Overexpression Is the Only Identifiable Cause of High Bone Mass in Melanocortin 4 Receptor Deficiency

Jong Deok Ahn, Beatrice Dubern, Cecile Lubrano-Berthelier, Karine Clement and Gerard Karsenty

Department of Molecular and Human Genetics and Children’s Nutrition Research Center (J.D.A., G.K.), Baylor College of Medicine, Houston, Texas 77030; Institut National de la Santé et de la Recherche Médicale (B.D., C.L.-B., K.C.), Unité 755 Nutriomique, University Pierre and Marie Curie-Paris 6, Faculty of Medicine, Les Cordeliers, Assistance Publique Hôpitaux de Paris, Hôtel-Dieu Hospital, Nutrition Department, 75004 Paris, France; and Department of Pediatric Gastroenterology and Nutrition (B.D.), Armand-Trousseau Teaching Hospital, 75012 Paris, France

Address all correspondence and requests for reprints to: Gerard Karsenty, M.D., Ph.D., Department of Molecular and Human Genetics, One Baylor Plaza, Baylor College of Medicine, Houston, Texas 77030. E-mail: karsenty{at}bcm.tmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The neural regulation of bone remodeling has proven to be increasingly complex at the molecular level because it involves both positive and negative mediators of bone formation and resorption. One of the mediators expressed in hypothalamic neurons that leptin uses to inhibit osteoclast differentiation and thereby bone resorption is cocaine- and amphetamine-regulated transcript (CART). CART expression in the hypothalamus is increased in mice lacking melanocortin 4 receptor (Mc4r–/– mice). Moreover, we show here that humans or mice lacking only one allele of Mc4r display a decrease in bone resorption parameters, high bone mass, and an increase in CART serum levels and/or hypothalamic expression. To demonstrate that the Cart overexpression is the only identifiable cause for the high bone mass observed upon Mc4r inactivation, we removed one allele of Cart from mice either heterozygous or homozygous for Mc4r inactivation. This manipulation sufficed to either significantly improve or normalize bone resorption parameters, without improving the energy metabolism disturbance that characterizes Mc4r-deficient mice. These results identify CART signaling as the main if not only molecular pathway accounting for the decrease in bone resorption leading to high bone mass in mice and humans deficient in Mc4r. As importantly, they also indicate that CART regulates bone resorption independently of the role it may exert in energy metabolism, suggesting that the neural control of appetite and bone remodeling are independent of each other.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A RECENT ADVANCE in our understanding of the regulation of bone remodeling has been the realization that it is controlled, in part, by neuroendocrine mechanisms (1). This type of regulation was first demonstrated by showing that, after its binding to its receptor on hypothalamic neurons, the hormone leptin inhibits bone formation (2, 3). Subsequently, the existence of this type of regulation was confirmed by showing that mice lacking, in the hypothalamus only, the Y2 receptor that can bind proteins of the neuropeptide Y family display bone remodeling abnormalities (4, 5, 6). Likewise, patients lacking the melanocortin 4 receptor (Mc4R), a receptor expressed in hypothalamic neurons, were reported to have high bone mineral density (7).

Of all these pathways, the leptin-dependent regulation of bone mass is the one for which more molecular information is available. Briefly, the antiosteogenic function of leptin requires neurons and/or neuronal networks sensitive to gold-thioglucose, which are located mainly in the ventromedial hypothalamic nuclei (3). At the peripheral level, the mediator of leptin antiosteogenic function is the sympathetic nervous system acting, in osteoblasts, via the adrenergic receptor ß2 (Adrß2) and the molecular clock to regulate cell proliferation (3, 8, 9). In addition to its inhibition of bone formation, leptin exerts a dual influence on bone resorption; it favors it through the sympathetic nervous system, and it inhibits it through another molecular relay, cocaine- and amphetamine-regulated transcript (CART) (8).

CART is encoded by a gene expressed in hypothalamic neurons, in other parts of the nervous system, and in peripheral organs such as the pancreas and the adrenal glands (10, 11, 12, 13). Cart expression is regulated by leptin and is virtually absent in hypothalamic neurons of ob/ob mice (14, 15). The absence of Cart expression in ob/ob mice explains, at least in part, their increase in bone resorption (8). This conclusion is based on three observations. First, gonadectomized Adrß2-deficient mice, which should have the same bone phenotype as ob/ob mice that are hypogonadic and have low sympathetic tone, do not share with the ob/ob mice an increase in bone resorption. Second, Cart-deficient mice display a low-bone-mass phenotype because of a marked and isolated increase in osteoclast number and function. Third, Mc4r–/– mice, which display an increase in hypothalamic Cart expression, have a high bone mass because of an isolated decrease in osteoclast number and function; the same decrease in bone resorption activity was also noted in Mc4r-deficient patients.

This latter observation, albeit of correlative nature, is of importance because it potentially provides a molecular explanation for the otherwise unexplained increase in bone mass noted in Mc4r-deficient mice and patients. However, Mc4r inactivation results in a myriad of metabolic abnormalities (16, 17), the effect of which on bone remodeling is unknown. Thus, to address more directly whether or not the increase in Cart expression is the main, if not the only, identifiable cause of the high bone mass observed in the absence of Mc4R we analyzed Mc4r+/– mice as well as generated Mc4r-deficient mice lacking either one or two copies of Cart and analyzed bone remodeling and energy metabolism in these mice. These mouse studies were accompanied by a more complete analysis of bone remodeling parameters in a larger group of Mc4r-deficient patients.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human patients
Genomic DNA was extracted from peripheral leukocytes for all subjects. Two primers, MC4R-AF (5'-ATCAATTCAGGGGGACACTG-3') and MC4R-ER (5'-TGCATGTTCCTATATTGCGTG-3') were used in a PCR to amplify the entire coding region of the Mc4r gene as described (16). The sequencing reaction was performed with the BigDye terminator kit (Applied Biosystems, Foster City, CA). PCR products were sequenced using MC4R-AF, MC4R-ER, and two internal primers, MC4R-CF (5'-TGTAGCTCCTTGCTTGCATC-3') and MC4R-CR (5'-GGCCATCAGGAACATGTGGA-3'). Sequencing was performed on an ABI Prism 3700 automated DNA sequencer (Applied Biosystems). Human subjects were followed both at Hotel-Dieu and Trousseau Hospital, Paris, France. Agreement for human genetic study was obtained by Institutional Review Board (IRB) of Hotel-Dieu Hospital (18).

Animals
Mice lacking Mc4r (C57BL/6J) have been described previously (19). Cart-deficient mice (C57BL/6J) were obtained from Amgen (Thousand Oaks, CA) (20). Compound heterozygous mice (Mc4r+/–;Cart+/–) were produced by mating Mc4r–/– and Cart–/– animals. These compound heterozygote mice were subsequently interbred or backcrossed to produce offspring of all nine possible genotypes. Mc4r and Cart genotypes were determined by PCR analysis. All mice were analyzed at 6 months of age. Figures present data from female animals. All procedures were approved by Baylor College of Medicine Institutional Animal Care and Use Committee.

Bone analyses
Histological analyses were performed as previously described (2, 21, 22). Briefly, lumbar vertebrae were dissected, fixed 12 h in 4% paraformaldehyde/PBS (pH 7.3), dehydrated in graded ethanol series, and embedded in methyl methacrylate resin according to standard protocols (22). Seven-micrometer frontal sections were stained by von Kossa and counterstained by von Gieson for bone volume measurement. Unstained 12-µm sections were analyzed for bone formation rate (BFR) measurements. BFR was measured by the calcein double-labeling method, which enables the measurement of the amount of new mineralized bone laid down per micrometer of bone surface per year (23). Calcein (Sigma Chemical Co., St. Louis, MO) was administered twice ip at 0.125 mg/g body weight dissolved in calcein buffer (0.15 M NaCl, 2% NaHCO3) on d 1 and 8, and mice were killed on d 10. Osteoclast and osteoblast surfaces were calculated after tartrate-resistant acid phosphatase and toluidine blue staining, respectively (24, 25). The 12-µm resolution microcomputed tomography (µCT) measurements were performed on distal femurs (Scanco Medical, Bassersdorf, Switzerland). Static and dynamic histomorphometric analyses were performed in accordance with standard nomenclature (23) using the Osteomeasure Analysis System (Osteometrics, Atlanta, GA). Five to 10 mice were analyzed for each group.

Biochemistry
Leptin (Crystal Chem Inc., Downer’s Grove, IL), osteocalcin (Immutopics Inc., San Clemente, CA), insulin (Crystal Chem), and CART (RK-003-62; range, 0–128 pg/tube) (Phoenix Pharmaceuticals Inc., Belmont, CA) serum levels were measured using commercially available ELISA or RIA kits. Glucose was measured using the Accu-Check glucometer (Roche, Indianapolis, IN). Deoxypyridinoline cross-links, creatinine urinary values, and C-terminal telopeptides of type I collagen (CTX) (Nordic Biosciences Inc., Walkersville, MD) (26) were measured using commercial kits.

Molecular studies
Real-time PCR analyses of Cart gene expression level were carried out using cDNAs derived from mouse hypothalamus RNA. RNA was extracted and reverse transcribed with random primers using the Superscript II first-strand cDNA synthesis kit (Invitrogen, Carlsbad, CA). cDNA samples were then used as templates for analysis of gene expression levels by fluorescent analysis using Applied Biosystems predesigned TaqMan gene expression assays and a Prism 7000 (Applied Biosystems) real-time PCR machine. The relative amounts of transcripts were calculated using the standard curve method. Expression levels for the studied gene were normalized using the 18S rRNA levels in each sample.

Statistical analyses
Data are expressed as the mean ± SE. Statistical significance was assessed by Student’s or Bonferroni’s multiple comparision test. Values were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Low bone resorption and high bone mass in Mc4r+/– mice
One goal of this study was to dissociate the pathogenesis of bone mass abnormalities from that of energy metabolism abnormalities in Mc4r-deficient mice. We had shown earlier that inactivating mutations in molecules involved in the leptin-dependent regulation of bone mass result in bone mass phenotypes that are dominant, i.e. present in heterozygous mutant mice (8). In particular, this is the case for haploinsufficiency of Adrß2, the only adrenergic receptor expressed on osteoblasts (3), and of CART (8). Because we suspected that it is through an increase in Cart expression that their bone phenotype develops, we asked whether the high bone mass observed in Mc4r–/– mice was also present in Mc4r+/– mice.

To address this question, we performed histological and histomorphometric analysis of vertebrae in 6-month-old Mc4r+/– and wild-type (WT) mice. We also performed µCT analysis of long bones. As shown in Fig. 1Go, female Mc4r+/– mice had a significantly higher bone mass than WT littermates. This was observed in vertebrae and in long bones (Fig. 1Go, A and B). Histomorphometric analysis ruled out that this high bone mass was a result of an increase in osteoblast number or in bone formation parameters (Fig. 1CGo). In contrast, we noted a significant decrease in osteoclast number and in urinary elimination of deoxypyridinoline (Fig. 1DGo), a collagen degradation byproduct and a biomarker of osteoclast activity (27). To determine whether this decrease in bone resorption parameters could be correlated with a change in Cart expression, we compared hypothalamic Cart expression and CART level in blood between WT and Mc4r+/– mice. Indeed, Cart expression in hypothalamus and CART serum level were both significantly increased in Mc4r+/– compared with WT mice (Fig. 1EGo). Taken together, these data demonstrate that the high-bone-mass phenotype caused by Mc4r inactivation is dominant and that it is correlated with a significant increase in hypothalamic Cart expression and CART serum levels.


Figure 1
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  		FIG. 1. Cart as the main regulator of the high-bone-mass phenotype observed in Mc4r-deficient mice. A, Mc4r mutation exert a dominant influence on bone mass. Increased bone volume over tissue volume (BV/TV) in vertebrae of Mc4r+/– mice; B, µCT analysis of distal femurs in 6-month-old mice; C, bone formation parameters, showing that BFR (µm3/µm2·yr) and osteoblast number over bone perimeter (N.Ob/B.Pm) are normal in Mc4r+/– mice; D, bone resorption parameters, showing that osteoclast surface over bone surface (Oc.S/BS) and urinary elimination of deoxypyridinoline (Dpd) are decreased in Mc4r+/– mice; E, increased serum CART level and Cart expression in Mc4r+/– hypothalamus; F, vertebral sections, showing that Mc4R mutant mice lacking one or two copies of Cart show a significantly lower bone mass; G, µCT analysis of distal femurs in 6-month-old mice; H, BFR and osteoblast number are normal in Mc4r-deficient mice lacking one or two alleles of Cart; I, bone resorption parameters, showing that removing one or two copies of Cart restores decreased bone resorption parameters in Mc4R mutant mice; J, increased serum CART level and Cart expression and decreased Rankl expression in Mc4R mutant hypothalami or bones were normalized by removing one or two copies of Cart. n = 6 per group; *, P < 0.05.

 
Cart expression affects bone mass in Mc4r-deficient mice
As mentioned earlier, the suspicion that the high-bone-mass phenotype observed in the absence of Mc4R signaling is because of an increase in Cart expression is based solely on correlative arguments (8). To address this question more directly, we took advantage of fact that Cart inactivation results in a low-bone-mass phenotype that is dominant. We reasoned that removing one allele of Cart should be enough to correct the high-bone-mass phenotype observed in Mc4r+/– or Mc4r–/– mice whether this phenotype is indeed caused by Cart overexpression. We thus generated and analyzed Mc4r+/– and Mc4r–/– mice lacking one or two alleles of Cart.

As shown in Fig. 1Go, F–I, removing one allele of Cart sufficed to normalize bone resorption parameters and bone mass in Mc4r+/– mice whether we analyzed vertebrae histologically or long bones by µCT. The same was true in Mc4r–/– mice lacking either one or two copies of Cart. Next we tried to correlate the correction of the bone remodeling abnormalities observed in Mc4r+/–; Cart+/– and Mc4r–/–; Cart+/– mice with Cart hypothalamic expression and CART serum levels. Cart expression in hypothalamus was significantly decreased in Mc4r+/–; Cart+/– mice compared with Mc4r+/– mice, as well as in Mc4r–/–; Cart+/– mice compared with Mc4r–/– mice (Fig. 1JGo). We also noted that CART serum level was significantly decreased and/or normalized in Mc4r+/– and Mc4r–/– mice lacking one allele of Cart (Fig. 1JGo). Taken together, these results suggest that a high level of Cart expression, and thereby of CART signaling, is the main molecular event accounting for the low-bone-resorption/high-bone-mass phenotype observed in Mc4r-deficient mice. Consistent with this hypothesis, Rankl expression was high in Cart–/– bones, as previously reported (8), and was lower in Mc4r–/– than in WT bones and normal in compound homozygote mice (Fig. 1JGo).

Cart expression affects bone mass without improving energy metabolism in Mc4r-deficient mice
Both Mc4r+/– and Mc4r–/– mice are obese and display multiple endocrine abnormalities such as increased insulin and leptin serum levels (7, 19). It has been suggested that an increase in insulin serum level could explain the bone mass abnormalities sometimes observed in mutant mouse strains characterized by obesity and high bone mass (7). Thus, to determine whether Cart inactivation normalizes bone resorption parameters in Mc4r-deficient mice with or without normalizing insulin serum levels and other energy metabolism parameters, we measured body weight, fat pad weight, food intake, and serum leptin and insulin levels in WT, Mc4r+/–, Mc4r–/–, Mc4r+/–; Cart+/–, Mc4r–/–; Cart+/–, and Mc4r–/–; Cart–/– mice.

As shown in Table 1Go, removing either one or two alleles of Cart from Mc4r+/– or Mc4r–/– mice did not correct their obesity and their endocrine abnormalities. Instead, Cart deletion worsened some of the metabolic parameters such as body weight, fat pad weight, and more importantly serum leptin and insulin. These observations are important for at least two reasons. First, they rule out that CART regulation of bone resorption is secondary to a role it may have in regulating energy metabolism. Second, they also rule out that increased serum insulin level is the cause of the low bone resorption/high bone mass observed in Mc4r-deficient mice.


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  		TABLE 1. Measurement of energy metabolism parameters in Mc4r-deficient mice lacking one or two copies of Cart

 
Increased CART serum levels and bone resorption activity in Mc4r-deficient patients
The notion that Mc4r deficiency exerts a CART-dependent dominant effect on bone resorption activity and bone mass led us to test whether patients lacking one allele of Mc4r also had bone remodeling abnormality. To that end, we studied serum bone remodeling parameters and serum CART levels in six prepubertal Mc4r+/– patients and control subjects of the same sex and age showing similar metabolic parameters (Table 2Go). All Mc4r mutations detected in these patients (Phe202Leu, Thr150Ile, Ala175thr, Gly252Ser, and Ser58Cys) had been shown earlier to result in a loss of function (18, 28, 29, 30, 31, 32, 33, 34).


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  		TABLE 2. Clinical and metabolic data in subjects with or without Mc4r mutations

 
We first compared serum levels of bone formation markers such as osteocalcin in subjects matched for age and body mass index with or without Mc4R mutations. As shown in Fig. 2AGo, serum osteocalcin levels were normal in patients heterozygous for Mc4R inactivation. In contrast, all these patients displayed a significant decrease in the serum level of CTX, a biomarker of bone resorption similar to the one previously reported in two patients homozygous for this type of mutation (Fig. 2BGo) (8).


Figure 2
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  		FIG. 2. Mc4R mutation exerts a dominant effect on bone resorption parameters in human patients. A, Serum osteocalcin, a bone formation marker, is normal in Mc4R patients; B, Crosslaps CTX, a bone resorption marker, is increased in Mc4r-deficient patients; C, serum CART level is increased in Mc4r-deficient patients.

 
Next, we measured serum CART level and found that it was significantly increased in patients heterozygous for Mc4r. Although this study could not rely, for obvious reasons, on the invasive techniques we used to analyze our mouse models, it provides nevertheless strong suggestive evidence that Mc4R mutation exerts a dominant effect on bone resorption parameters in humans and that their bone phenotype correlates with CART serum levels as it does in mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study provides in vivo direct evidence that the high-bone-mass phenotype observed in Mc4r-deficient mice is secondary to an increase in Cart expression and that it can be corrected without improving serum insulin levels and/or other energy metabolism parameters. Remarkably, as is the case in Mc4r-deficient mice, the high-bone-mass phenotype of Mc4r-deficient patients appears to be secondary only to abnormality of bone resorption and correlates also with an increase in the serum level of CART, a leptin-dependent regulator of bone resorption (8).

The finding that Mc4r-deficient patients had increased bone density provided further credence to the concept of a neuronal control of bone mass originally revealed by the analyses of the high bone mass observed in ob/ob mice (7). However, given the obesity and energy metabolism abnormalities marring these patients, a question was, as it had been previously for ob/ob mice, whether these bone abnormalities were secondary to energy metabolism perturbations or not. In the case of leptin regulation of bone remodeling, the analysis of the bone phenotype of Adrß2-deficient mice demonstrated formally that leptin regulates bone mass independently of its regulation of energy metabolism (8, 35). In the case of the effect of Mc4R deletion, this question had not been addressed until recently. One possible explanation for the high bone mass observed in Mc4r-deficient mice and humans was provided by the observation that hypothalamic expression of Cart, a gene regulated by leptin and itself inhibiting bone resorption through a yet unknown mechanism, was increased in Mc4r-deficient mice (8). The importance of this observation stemmed in large part from the fact that the high bone mass caused by the absence of Mc4r was a result only of a decrease in bone resorption parameters with normal bone formation activity. These observations, however, were only of correlative nature.

To go beyond correlative observations, we used here Mc4r- and Cart-deficient mice as an experimental genetic tool. Specifically, we removed one or both alleles of Cart from Mc4r-deficient mice and then analyzed bone mass and energy metabolism in these compound mutant mice. These genetic manipulations show that removing only one allele of Cart was sufficient to correct fully the high-bone-mass phenotype observed in mice lacking either one or two copies of Mc4r. Most importantly, this normalization of bone mass occurred without improving any metabolic abnormalities and in particular without affecting serum insulin levels. The correction of the Mc4r-deficient mice bone phenotype by decreasing Cart expression establishes that the high bone mass caused by Mc4r inactivation is mediated by an increase in CART signaling. This experiment also establishes a link between the bone phenotype caused by Mc4r inactivation and leptin regulation of bone mass. Indeed, Cart regulates bone resorption under the control of leptin (8). Furthermore and as importantly, showing that deletion of one or both Cart alleles corrects the bone phenotype of Mc4r-deficient mice without normalizing serum insulin level and other metabolic abnormalities also demonstrates that the dysregulation of bone mass occurs independently of the dysregulation of energy metabolism in Mc4r-deficient mice.

How does CART act to regulate bone resorption? Cart is expressed in hypothalamic neurons, in other parts of the nervous system, and in peripheral organs such as the pancreas and the adrenal glands. Therefore, CART could act either as a classical neuropeptide or as a hormone. We observed in Mc4r-deficient mice that Cart hypothalamic expression as well as CART serum levels were increased, thus making it difficult to favor one mode of action over another. Clearly, to address rigorously this question, one will have to wait for the cloning of a specific receptor for CART. Regardless of the mode of action of CART, the facts that Cart inactivation does not affect energy metabolism in unchallenged animals and that it worsens slightly energy metabolism parameters in Mc4r-deficient mice while correcting their bone phenotype indicate that CART uses different mechanisms to regulate bone resorption and to affect energy metabolism.

Lastly, and with all the limitations inherent to bone remodeling studies performed in humans, results presented here indicate that, in humans like in mice, the low-bone-resorption/high-bone-mass phenotype caused by Mc4R inactivation is dominant and is associated with an increase in CART serum level. This conservation of mechanism is important because it underscores the notion that CART fulfills the same function in bone remodeling in mice and humans.


   Acknowledgments
 
We are indebted to Dr. P. Ducy for critical reading of the manuscript and M. Starbuck and X. Liu for superb technical assistance.


   Footnotes
 
This work was supported by National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK58883 and a research grant from ORGANON N.V. (to G.K.) and a Fellowship from the Children’s Nutrition Research Center (to J.A.). The human study was supported by AFERO (French association for the study of obesity) (to B.D.) and by GIP-ANR of the Center of Research for Human Nutrition (to Institut National de la Santé et de la Recherche Médicale Unité 755).

Disclosure Statement: J.A., B.D., C.L.-B., and K.C. have nothing to declare. G.K. received grant support (2005–2006) from ORGANON.

First Published Online April 13, 2006

Abbreviations: BFR, Bone formation rate; CART, cocaine- and amphetamine-regulated transcript; µCT, microcomputed tomography; CTX, C-terminal telopeptides of type I collagen; Mc4R, melanocortin 4 receptor; WT, wild type.

Received March 3, 2006.

Accepted for publication April 3, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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M. K. Singh, F. Elefteriou, and G. Karsenty
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