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Published as doi: 10.1096/fj.07-9209com.
 (The FASEB Journal. 2008;22:418-427.)
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(The FASEB Journal. 2008;22:418-427.)
© 2008 FASEB

The serotonin 5-HT2B receptor controls bone mass via osteoblast recruitment and proliferation

C. Collet*,1, C. Schiltz{dagger},1, V. Geoffroy{dagger}, L. Maroteaux{ddagger}, J.-M. Launay* and M.-C. de Vernejoul{dagger},2

* Service de Biochimie, Hôpital Lariboisière, Paris, France;

{dagger} INSERM U606, Hôpital Lariboisière, Paris, France; and

{ddagger} INSERM U839, Institut du Fer à Moulin, Paris, France.

2Correspondence: INSERM U606 Hôpital Lariboisière, 2 rue Ambroise Paré, 75475 Paris cedex 10, France. E-mail: christine.devernejoul{at}lrb.aphp.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The monoamine serotonin (5-HT), a well-known neurotransmitter, is also important in peripheral tissues. Several studies have suggested that 5-HT is involved in bone metabolism. Starting from our original observation of increased 5-HT2B receptor (5-HT2BR) expression during in vitro osteoblast differentiation, we investigated a putative bone phenotype in vivo in 5-HT2BR knockout mice. Of interest, 5-HT2BR mutant female mice displayed reduced bone density that was significant from age 4 months and had intensified by 12 and 18 months. This histomorphometrically confirmed osteopenia seems to be due to reduced bone formation because 1) the alkaline phosphatase-positive colony-forming unit capacity of bone marrow precursors was markedly reduced in the 5-HT2BR mutant mice from 4 to 12 months of age, 2) ex vivo primary osteoblasts from mutant mice exhibited reduced proliferation and delayed differentiation, and 3) calcium incorporation was markedly reduced in osteoblasts after 5-HT2BR depletion (produced genetically or by pharmacological inactivation). These findings support the hypothesis that the 5-HT2BR receptor facilitates osteoblast recruitment and proliferation and that its absence leads to osteopenia that worsens with age. We show here, for the first time, that the 5-HT2BR receptor is a physiological mediator of 5-HT in bone formation and, potentially, in the onset of osteoporosis in aging women.—Collet, C., Schiltz, C., Geoffroy, V., Maroteaux, L., Launay, J.-M., de Vernejoul, M.-C. The serotonin 5-HT2B receptor controls bone mass via osteoblast recruitment and proliferation.


Key Words: osteoporosis • aging • neurotransmitter


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OSTEOPOROSIS IS A DISORDER responsible for fragility fractures and is caused by an imbalance between bone formation and resorption, both of which are controlled by both systemic and local factors (1) . There is considerable current interest in neurohormonal regulators of bone metabolism, which have been shown to influence both the formation and resorption of bone. Bone cells can express receptors for vasoactive intestinal polypeptide (2) , calcitonin gene-related peptide (3) , catecholamines (4) , and glutamate (5) and leptin has been reported recently to control bone formation via a central mechanism (6) , implying the involvement of the sympathetic nervous system and cocaine amphetamine regulated transcript (CART), which have been shown to decrease bone formation dramatically (7) .

The monoamine serotonin [5-hydroxytryptamine (5-HT)] has mainly been investigated as a neurotransmitter principally involved in mood control. However, it also mediates a wide range of peripheral functions. 5-HT is synthesized by a two-step pathway in which tryptophan hydroxylase is the rate-limiting enzyme. Circulating 5-HT is principally stored in platelet-dense granules. Extracellular levels of 5-HT are determined by the 5-HT transporter (5-HTT) (8) . The diversity of actions of 5-HT results from the existence of multiple 5-HT receptors (5-HTRs), which have been divided into seven classes (5-HT1R–5-HT7R) on the basis of their signaling pathway (9) . The main targets of 5-HT in peripheral tissues are the cardiovascular system, the gastrointestinal tract, and platelets (8) .

Several studies have suggested that 5-HT is also involved in bone metabolism (10) . For instance, 5-HTT has been detected in osteoblasts, and its inactivation leads to reduced bone accrual (11 , 12) . Expression of various combinations of 5-HT1 (5-HT1A and/or 5-HT1D) and 5-HT2 (5-HT2A and/or 5-HT2B and/or 5-HT2C) receptors have been reported in vitro in chicken, rat, and mouse osteoblastic cell lines and primary osteoblast cultures, but their functional relationships to bone physiopathology have not been clearly established (11 , 13 14 15) .

In mouse embryogenesis, 5-HT appears to regulate epithelial/mesenchymal interactions during craniofacial development (16 , 17) . 5-HT2BR seems to be particularly important in mediating the effects of 5-HT on embryonic morphogenesis (18) and is involved during osteogenesis in an inducible mesoblastic murine cell line (19) .

We report here that during osteoblast differentiation, only 5-HT1ARs, 5-HT2ARs, and 5-HT2BRs are expressed and that only the expression of 5-HT2BRs is increased. We also show that targeted inactivation of the 5-HT2BR gene in mice (5-HT2BR–/–) leads to osteopenia and reduced bone formation in aging mice and that osteoblast proliferation and recruitment are reduced in 5-HT2BR-depleted primary cultures. The present study shows that 5-HT2BR plays a major role in bone formation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
{alpha}-Minimal essential medium ({alpha}-MEM) supplemented with L-glutamine (Invitrogen, Cergy-Pontoise, France), penicillin-streptomycin suspension (Invitrogen), and 10% FCS (Sigma-Aldrich Corp., St. Quentin Fallavier, France) depleted of 5-HT (20) was used. Ascorbic acid, β-glycerophosphate (β-GP), ritanserin, 5-HT binoxalate, and 1-[5(2-thienylmethoxy)-1H-3-indolyl]propan-2-amine hydrochloride (BW 723C86) were purchased from Sigma-Aldrich Corp. [45Ca]2+ (specific activity, 1.85 GBq/mg) was from GE Healthcare (Orsay, France). Radioligands [Perkin Elmer Life and Analytical Sciences (Boston, MA) or GE Healthcare] [3H](±)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (specific activity, 4.74 TBq/mmol), [125I](±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) (specific activity, 81.4 TBq/mmol), [125I]5-hydroxytryptamine-5-O-carboxymethylglycyltyrosinamide (GTI) (specific activity, 72.9 TBq/mmol), [3H]5-HT binoxalate (specific activity, 845 GBq/mmol), [3H]1-methyl-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)1H-indazole-3-carboxamide (LY 278584) (specific activity, 2.31 TBq/mmol), [125I]1-butyl-4-piperidinylmethyl-8-amino-7-iodo-1,4-benzodioxan-5-carboxylate (SB 207710) (specific activity, 21.2 TBq/mmol), [3H]lysergic acid diethylamide (LSD) (specific activity, 2.23 TBq/mmol), [3H](±)2,3-dimethoxyphenyl-1-[2-(4-piperidine)-methanol] (MDL) (specific activity, 2.63 TBq/mmol), [3H]1-(2-chloro-3,4-dimethoxybenzyl)-6-methyl-1,2,3,4-tetrahydro-9Hpyrido[3,4-b]indole hydrochloride (LY 266097) (specific activity, 925 GBq/mmol), [3H]mesulergine (specific activity, 2.83TBq/mmol), [3H]4-iodo-N-[4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-benzenesulfonamide (SB 258585) (specific activity, 2.32 TBq/mmol), and [3H](R)-3-(2-(2-(4-methylpiperidin-1-yl)ethyl)pyrrolidine-1-sulfonyl)phenol (SB 269970) (specific activity, 2.33 TBq/mmol) were used to assess the presence of 5-HTRs. 2-Amino-4-(4-fluoronaphthyl-1-yl)-6-isopropyl-pyrimidine (RS-127445) and [3H]LY 266097 were generous gifts of Drs. M. McNamara (Syntex, Palo-Alto, CA, USA) and J. Würch (Roche, Basel, Switzerland). All other chemicals were of the purest grade available and were from usual commercial sources.

Animal model
This study was performed using the 5-HT2BR knockout mouse model in which exon 2 of the 5-HT2BR locus had been substituted for the selective bacterial neo cassette (21) . Genotyping has been described previously (21) . In the mutant mice population, one third of embryonic mice die midgestation because of trabecular defects in the heart, and one third die at birth from cardiac failure. The mice that survive have a cardiac phenotype, but a normal life span (21) . The 5-HT2BR knockout mice are a pure 129sv/PAS background, and wild-type (WT) 129sv/PAS background mice used as controls were purchased from the Charles River Laboratories (L’arbresle, France). The mice were examined when they were aged 5 wk, 10 wk, 4 months, 12 months, and 18 months. Urine samples were collected individually 3 days before sacrifice. Mice were weighed and anesthetized by intraperitoneal injection of ketamine (45 mg/kg) and xylazine (5 mg/kg) (Sigma-Aldrich Corp.). Blood plasma (sodium heparinate) samples were collected by eye puncture. The mice were anesthetized and killed by cervical dislocation, and the femurs were harvested for histomorphometry and the tibiae for three-dimensional (3D) micro-computed tomography (CT) analysis. The animals were allowed free access to food and water in full compliance with French government and European Community animal welfare policy.

Radiography, dual-energy X-ray absorptiometry (DEXA), and micro-CT analysis
Femurs were analyzed by contact radiography using an X-ray cabinet (Faxitron X-ray Corp., Wheeling, IL, USA). 5-HT2BR–/– and WT mice at different ages were weighed (g) and anesthetized, and their bone mineral density (BMD) (g/cm2) was determined by DEXA, using a PIXImus II densitometer (Lunar; GE Healthcare, Lambesc, France). 3D micro-CT analysis was performed at the tibial metaphysis using a 3D micro-CT scanner (Scanco Medical, Bassersdorf, Switzerland) as described previously (22) .

Bone histomorphometry
To evaluate the dynamic bone formation parameters by histomorphometry, skeletons were doubly labeled by tetracycline and calcein as described previously. Before sacrifice, a tetracycline injection was administered first (20 mg/kg; Pfizer, Amboise, France), followed by a second injection of calcein (20 mg/kg; Sigma-Aldrich Corp.) 5 days later for the 4-month-old mice or 6 days later for the 18-month-old mice. Female mice were killed 24 h after the second injection.

For cortical and trabecular histomorphometry, the left femurs from 4- and 18-month-old mice were dissected and stored in 70% ethanol. They were dehydrated in ascending alcohol concentrations, defatted in xylene, and then embedded in methyl methacrylate. All histomorphometric analyses were performed according to the guidelines of the American Society of Bone and Mineral Research histomorphometry nomenclature committee (23) as described previously (24) .

Biochemical analysis
To quantify osteoclastic bone resorption, urinary samples were collected from 4- and 18-month-old female mice in the morning to measure the level of deoxypyridinoline cross-linkage using a chemiluminescent assay on an Immulite 2000 automated analyzer (DPC Siemens Medical Solutions Diagnostics, La Garenne-Colombes, France). Animals were fasted overnight, and urine specimens were collected during a 2- to 3-h period. Values are reported relative to creatinine concentrations as determined by a standardized colorimetric assay using alkaline picrate with a Advia 2400 automated analyzer (Siemens Medical Solutions Diagnostics, Puteaux, France).

To quantify osteoblast formation, osteocalcin (mouse osteocalcin IRMA kit; Immunotopics, San Clemente, CA, USA) and alkaline phosphatase (ALP) activity (Advia 2400 automated analyzer; Siemens Medical Solutions Diagnostics) were determined colorimetrically in the plasma (sodium heparinate) of 4- and 18-month-old female mice (at least 10 mice for each group).

Calvarial osteoblast primary culture
Primary osteoblasts were enzymatically isolated from calvariae of neonatal (2- to 3-day-old) WT and transgenic mice as described previously. Briefly, calvariae were dissected aseptically and sequentially digested for 70 min in a PBS collagenase solution containing 0.2% collagenase IV (Sigma-Aldrich Corp.) and 0.01% deoxyribonuclease (Sigma-Aldrich Corp.) at 37°C. Calvarial cells were collected by centrifuging and then washing and plating at 106 cells/75 cm2 flasks. Cells were expanded for 5 days in {alpha}-MEM supplemented with 10% FCS depleted of 5-HT, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were harvested with trypsin/EDTA (Invitrogen) and plated at a density of 105 cells/cm2 in the differentiation medium (50 µg/ml ascorbic acid with 10 mM β-GP).

To determine ALP activity, cells were washed twice in ice-cold PBS and then stored in purified water at –80°C until analysis. ALP activity in the lysate was measured as described previously for plasma. Total intracellular protein contents were measured using the BCA protein assay reagent kit (Pierce Perbio Science France, Brebières, France), and ALP activity was normalized in terms of protein content. The capacity of the osteoblasts to mineralize was assessed by alizarin staining on days 13 and 21. The number of mineralized nodules was measured using an image analyzer (Microvision, Les Ullis, France).

Radioligand binding assays
To prepare crude membranes for radioligand binding assays essentially as described previously (25) , calvarial osteoblast cells were washed twice in ice-cold PBS and then scraped off the plate into 1.5 ml of PBS containing 4 mM EDTA, 1 mM EGTA, and 10 mM imidazole (pH 7.30) supplemented with a set of protease inhibitors Complete (Roche Molecular Biochemicals, Meylan, France) (buffer 1) and then treated with 1 mg/ml collagenase and 4 mM EDTA. Samples were centrifuged, and pellets were frozen at –70°C. Frozen cell pellets were thawed at 4°C, homogenized, and resuspended in 10 ml of buffer 1. Extracts were centrifuged for 10 min at 5000 g. Supernatants were collected, poured onto a 20% sucrose cushion, and then centrifuged for 90 min at 100,000 g. The resulting membrane-containing pellets were resuspended in 75 mM KCl, 5 mM MgCl2, 1 mM EGTA, and 10 mM imidazole buffer (pH 7.30), for use in binding assays. Protein concentrations were determined by using the BCA protein assay reagent kit (cf. above).

Reverse transcriptase-polymerase chain reaction (PCR) and semiquantitative and quantitative PCR
Total RNA from calvarial osteoblasts was isolated using the Qiagen RNeasy Mini Kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s procedures using DNase. One microgram of each RNA was converted into cDNA using the High-Capacity cDNA Archive Kit (Applied Biosystems, Courtaboeuf, France), and amplifications were performed using standard protocols in a GeneAmp PCR 7500 system (Applied Biosystems).

Quantitative real-time PCR expression analysis was performed using a LightCycler (Roche) and ABsolute SYBR Green Capillary Mix (ABgene, Courtaboeuf, France). Primers with FAM-labeled TaqMan probes (Assays-on-Demand, Applied Biosystems) were used for detection of 5-HT1AR, 5-HT2AR, and 5-HT2BR genes and GADPH for normalization. The sets of primer used for the osteoblast differentiation markers were as follows: collagen type 1 (forward: 5'-CTTGGTGGTTTTGTATTCGATGAC-3'; reverse: 5'-GCGAAGGAACAGTCGCT-3'); ALP (forward: 5'-AAGGCTTCTTCTTGCTGGTG-3'; reverse: 5'-GCCTTACCCTCATGATGTCC-3'); osteocalcin (forward: 5'-CTCACAGATGCCAAGCCCA-3'; reverse: 5'-CCAAGGTAGCGCCGGAGTCT-3'); and endogenous Runx2 (forward: 5'-TTGACCTTTGTCCCAATGC-3'; reverse: 5'-AGGTTGGAGGCACACATAGG-3'). Aldolase A (forward: 5'-TGAAGCGCTGCCAGTATGTTA-3'; reverse: 5'-GGTCGCTCAGAGCCAGTATGGTTA-3') and 18S (forward: 5'-CGGCTACCAATCCAAGGAA-3'; reverse: 5'-GCTGGAATTACCGCGGCT-3') were used for normalization.

Proliferation and apoptosis assays
For proliferation, we used both 5-bromo-2'-deoxy-uridine (BrdU) labeling of osteoblast cells with the Cell Proliferation Kit (GE Healthcare, Orsay, France) and [3H]thymidine (GE Healthcare) incorporation. Briefly, cells were plated in 96-well plates at a density of 5 x 103/well and cultured in medium containing 10% 5-HT-depleted FCS until 80–90% confluence (48 h). Cells were cultured for an additional 18 h in a medium devoid of FCS. Then BrdU (final concentration: 10 mM) or [3H]thymidine (1 µCi/well) was added to the cultures for 24 h. Subsequently, BrdU labeling was measured as described by the manufacturer. The free radioactive thymidine was washed off using 5% trichloroacetic acid, and the incorporated radioactive thymidine was quantified by scintillation counting.

For apoptosis, caspase-3 activity was measured after 72 h of culture using the Caspase-3 Fluorometric Assay Kit (R&D Systems, Lille, France) according to the manufacturer’s protocol. Protein concentrations were measured by using the BCA protein assay reagent kit (cf. above).

Calcium incorporation
Cellular [45Ca]2+ uptake (19) by confluent calvarial osteoblasts from WT or mutated mice was measured in 24-well dishes after various culture times (days 6, 9, and 12) in the differentiation medium supplemented with 10% 5-HT-depleted FCS. Twenty-four hours before cellular [45Ca]2+ uptake, the initial medium was replaced by fresh medium containing 1% (v/v) 5-HT-depleted FCS. The cultures were then rinsed with PBS and incubated in calcium- and serum-free medium containing ascorbic acid, β-GP, and 5 µCi/ml [45Ca]2+ for 4 h. The medium was removed, and the cultures were incubated for 1 h in a medium containing serum and calcium. The monolayer was collected by scraping in PBS containing 0.01% sodium dodecyl sulfate and then extracted for 3 h in 0.5 N HCl before the radioactivity of the whole sample was determined. Quadruplicate determinations were done for each experimental point. Data were pooled from two independent experiments using [45Ca]2+ of similar specific activities.

Alkaline phosphatase-positive colony-forming unit (CFU-FALP+) assays
CFU-FALP+ cells were assayed as described previously (26) . Briefly, marrow stromal cells were collected from the femurs and tibiae of 4- and 12-month-old female mice. Bone marrow was flushed out from each bone with {alpha}-MEM supplemented with FCS (15%) using a syringe and a 26-gauge needle. After a short spin, the cell pellets were resuspended in the same culture medium. They were further filtered through a 0.45-µm-pore diameter nylon filter (Millipore, Molsheim, France). Cell suspensions were treated with a lysis solution (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA, pH 7.4) to remove the red blood cells. Cells were seeded at 3 x 106 cells/well in a six-well plate. After culturing for 4 days, 100 µg/ml ascorbic acid was added to the culture medium until the end of the experiment. After 11 days, cell colonies were fixed and stained for ALP by adding the Sigma fast substrate buffer bromochloroindoyl-phosphate/nitroblue tetrazolium chloride (Sigma-Aldrich Corp.). The number of CFU-FALP+ cells per dish was counted.

Statistical analysis
Statistical analysis was performed using StatView 4.5 software (Abacus Concepts Inc., Berkeley CA, USA). Statistical differences between experimental groups were assessed using analysis of variance (ANOVA). The significance threshold was set at P < 0.05. All values are shown as mean ± SE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
5-HT2BR was the only 5-HTR to display increased expression during osteoblast differentiation
We used various radioligands to identify 5-HT binding sites on WT calvarial osteoblasts. Before differentiation, the cells bound significant amounts of [3H]5-HT, indicating the presence of serotonergic receptors, and of [125I]DOI, suggesting the presence of 5-HT2-like binding sites. In contrast, [3H]LY 278584 and [125I]SB 207710 were not bound, demonstrating the absence of 5-HT3 and 5-HT4 binding sites, respectively. Moreover, the cells bound neither [3H]mesulergine nor [3H]LSD in the presence of 100 nM MDL 100907, indicating the absence of 5-HT2C, 5-HT5, 5-HT6, and 5-HT7 binding sites. The absence of 5-HT6 and 5-HT7 binding sites was reassessed by the absence of any specific binding of [3H]SB 258585 or [3H]SB 269970. Then specific labeling with [3H]8-OH-DPAT, [3H]MDL 100907, and [3H]LY 266097 indicated the presence of 5-HT1A, 5-HT2A, and 5-HT2B binding sites, respectively. Finally, the absence of labeling with [125I]GTI revealed the absence of 5-HT1B/1D binding sites.

To investigate the potential role of serotonergic receptors in osteogenesis, binding assays were performed during the differentiation of primary calvarial osteoblast cultures. The binding site profile (5-HT1A, 5-HT2A, and 5-HT2B binding sites) remained unchanged throughout differentiation, as did the numbers of 5-HT1A (Fig. 1 A) and 5-HT2A (Fig. 1B ) binding sites. In contrast, 5-HT2B binding sites increased for 10 days and then reached a plateau (Fig. 1C ). The level of 5-HT2B mRNA remained constant during osteoblast differentiation (data not shown), indicating that its regulation must occur post-transcriptionally. Taken together, these findings suggest that 5-HT2BRs may be important during osteoblast differentiation.


Figure 1
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  		Figure 1. Osteoblasts express several 5-HTRs; 5-HT2BRs increase during osteoblast differentiation. Binding assays in osteoblast primary cultures were carried out at plating (day 0) and after 7, 10, 13, and 21 days. 5-HT1A (A), 5-HT2A (B), and 5-HT2B (C) binding sites were determined in WT (black bars) and in 5HT2BR–/– osteoblastic cells (open bars). *Significant difference from WT at P < 0.001. $Significant difference from day 0 at P < 0.01. $$Significant difference from day 0 at P < 0.001.

Female 5-HT2BR–/– mice display decreased BMD that worsens as they age
To determine the role of 5-HT2BRs on bone in vivo, the BMDs of the 5-HT2BR–/– and WT mice were assessed at different times from 5 wk to 18 months. During this period, the increases in body weight and size were not statistically different in WT and 5-HT2BR–/– mice (data not shown). No difference was observed between 5-HT2BR–/– and WT mice with regard to basal locomotion (unpublished results). There was no difference in BMD in 5-HT2BR–/– and WT male mice at any time (data not shown). In contrast, aged 5-HT2BR–/– female mice (>4 months of age) had lower BMDs than equivalent WT mice. All subsequent in vivo analyses were therefore performed in females. The time-dependent increase in BMD observed in WT mice was blunted in 5-HT2BR–/– mice, both for the whole body (Fig. 2 A) and for the femur (Fig. 2B ). At 4 months of age, 5-HT2BR–/– mice had lower BMDs than WT mice, but only at the femur (–5.2%) (Fig. 2C ). At 18 months of age, the difference between WT and 5-HT2BR–/– BMD was more pronounced and was detected both for the whole body (–6.5%) (Fig. 2C ) and for the femur (–10.6%) (Fig. 2D ). Radiography of the femur did not reveal any obvious differences between WT and 5-HT2BR–/– 4-month-old females, whereas the radiopacity of the femur in 18-month-old 5-HT2BR–/– females was lower than that in WT mice (Fig. 2E ). These data show that 5-HT2BR knockout induces an age-dependent decrease in bone density in female mice.


Figure 2
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  		Figure 2. 5-HT2BR–/– mice display decreased BMD that worsens with aging. BMD was determined by DEXA for the whole body (A) and femurs (B) of 5HT2BR–/– and WT female mice at the times indicated (n=5/group). ANOVA for repeated measures revealed a statistically significant change related to genotype over time for both whole body and femoral BMDs (P<0.05). BMD was also evaluated in 4- and 18-month-old 5-HT2BR–/– and WT mice (n=15–20/group) for the whole body (C) and the femur (D). Radiographies were performed on the femurs (E). *Significant (P<0.05) difference between BMDs of 5-HT2BR–/– and WT mice. Mean values ± SE (bars) are shown.

Female 5-HT2BR–/– mice display trabecular and cortical osteopenia attributable to decreased bone formation
Histomorphometry and 3D micro-CT reconstruction were performed in 4- and 18-month-old WT and 5-HT2BR–/– mice to characterize changes in microarchitecture (Figs. 3 and 4 ). The ratio of trabecular bone volume to total bone volume (Fig. 3C ), trabecular number (Fig. 3D ), and trabecular and cortical thickness (Fig. 3F, G ) were all significantly lower in 5-HT2BR–/– mice. Accordingly, the trabecular separation (Fig. 3E ) was significantly greater in 5-HT2BR–/– mice. Moreover, both trabecular and cortical bone decreased with age. The mineral apposition rate (Fig. 4C ) was not affected, indicating that osteoblast function was normal in 5-HT2BR–/– female mice. In contrast, the mineralizing surface (Fig. 4B ) and bone formation rate (Fig. 4D ) were lower in 5-HT2BR–/– mice than in WT mice at both 4 and 18 months of age, suggesting that the number of osteoblasts might be reduced in 5-HT2BR–/– mice. This decrease in bone formation was confirmed by the low plasma levels of ALP activity (Fig. 4F ) and osteocalcin (Fig. 4G ). Finally, urinary deoxypyridinoline, a marker of osteoclast activity, was not significantly different in 5-HT2BR–/– and WT mice at 4 or 18 months of age (Fig. 4E ), and the osteoclast surfaces were not different between 5-HT2BR–/– and WT mice at 4 months of age (osteoclast surfaces: 14.3±2.4% for WT and 17.5±4.8% for 5-HT2BR–/–). Taken together, these findings indicate that low bone mass in 5-HT2BR–/– mice is associated with a decreased number of bone-forming cells that may account for the marked decrease in bone formation.


Figure 3
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  		Figure 3. 5-HT2BR–/– mice display trabecular and cortical osteopenia. 3D reconstruction by micro-CT of metaphyseal regions of the tibia (A) and the metaphyseal region of the distal femur (B) stained with toluidine blue in 4- and 12-month-old female mice illustrate the trabecular and cortical differences between 5-HT2BR–/– and WT mice. Histomorphometric structural parameters (C–G) in 4- and 18-month-old female mice were measured in the femoral distal metaphysis. Mean values ± SE (bars) are reported (n=10–12 sections/group). Significant differences between genotypes: *P < 0.05; **P < 0.01; and ***P < 0.001.


Figure 4
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  		Figure 4. Bone formation decreases in the absence of 5-HT2BR. Static and dynamic bone parameters and biochemical markers were measured in 4- and 18-month-old 5-HT2BR–/– (open bars) and WT female mice (black bars). Representative tetracycline/calcein double labeling in 4-month-old 5-HT2BR–/– and WT bone sections is shown (A). Dynamic formation parameters [MS/BS=mineralizing surface/bone surface (B)], mineralizing apposition rate (MAR) (C), and bone formation rate (BFR) (D) as well as biochemical markers of bone resorption (Dpd=urinary deoxypyridinoline cross-links) (E), biochemical markers of bone formation (ALP activity) (F), and plasma osteocalcin level (G) were determined. Data are shown as means ± SE (n=10–12 sections/sample). Significant differences between genotypes: *P < 0.05; **P < 0.01; and ***P < 0.001.

5-HT2BR knockout impacts osteoblast proliferation and recruitment
To investigate the cell defects that lead to low bone formation, we assessed the recruitment, proliferation, and survival of osteoblasts in WT and 5-HT2BR–/– mice. Caspase-3 activation appeared to be the same in WT and 5-HT2BR–/– osteoblasts (data not shown), indicating that there was no major change in apoptosis. In contrast, osteoblast proliferation was markedly lower in cultures of primary osteoblasts from 5-HT2BR–/– mice (–55%) (Fig. 5 A). Furthermore, RS-127445, a specific 5-HT2BR antagonist, decreased proliferation in WT osteoblasts (–45%) (Fig. 5A ) alone. Bone marrow adiposity was not affected by 5-HT2BR knockout (data not shown), but the number of CFU-FALP+ cells was dramatically lower in 5-HT2BR–/– mice than in WT mice at both 4 months (60±11 vs. 182±25/dish) and 12 months (26±6 vs. 114.0±16) of age (Fig. 5B ).


Figure 5
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  		Figure 5. 5-HT2BR affects osteoblast recruitment and proliferation. A) Proliferation was assessed after a 2 day-culture period by BrdU incorporation in WT and 5-HT2BR–/– calvarial osteoblasts and by [3H]thymidine incorporation in the absence or presence of RS-127445 (5 nM). Each value is the mean ± SE of 12 wells. Data from two independent experiments were pooled. Significant difference from WT cells: ***P < 0.001. B) CFU analysis was performed using bone marrow progenitors from the femurs of 4- and 12-month-old female mice. The numbers of CFU-FALP+ colonies/dish were significantly lower in 5-HT2BR–/– mice compared with WT mice. This graph is representative of two independent experiments with quadruplicate determinations. C) ALP activities were measured in WT and 5-HT2BR-deficient primary osteoblast cultures. Each value is the mean ± SE of 12 wells. Data were pooled from two independent experiments. Significant differences from WT are seen at days 7 (**P<0.001) and 10 (*P<0.03). D--F) At day 13 (D) and day 21 (F), the capacity of WT and HT2BR–/– primary osteoblasts to produced mineralized nodules was determined by alizarin staining; the mean area of the WT nodules was higher than that of the HT2BR–/– nodules (P<0.001). The nodules formed in culture appeared to be smaller in WT than in 5-HT2BR-deficient osteoblasts at day 21 (E).

To determine whether the 5-HT2BR-deficient osteoblasts had retained their ability to differentiate, the activity of ALP, a marker of osteoblast differentiation, was determined in primary osteoblast cultures on days 4, 7, 10, and 13. ALP activity was lower in 5-HT2BR–/– osteoblast cultures on days 7 (–50%) and 10 (–22%) but was no longer lower than that for WT on day 13 (Fig. 5C ). There were significantly fewer mineralized nodules in 5-HT2BR–/– than in WT osteoblast cultures only at day 13 (95±15 vs. 134±10) (Fig. 5D ), but not at day 21 (105±15 vs. 126±12) (Fig. 5F ), and they were also smaller in size (Fig. 5E ).

Taken together, these findings indicate that 5-HT2BR knockout induces a marked decrease in osteoblast proliferation and recruitment, without markedly impairing terminal differentiation.

5-HT2BR is the main 5-HTR affecting osteoblasts
As expected, we found that primary osteoblasts obtained from 5-HT2BR–/– mice expressed only 5-HT1AR and 5-HT2AR. Before differentiation, the number of 5-HT1AR binding sites in 5-HT2BR–/– mice was not significantly different from that in WT mice (Fig. 1A ), whereas the knockout mice had twice as many 5-HT2AR binding sites (Fig. 1B ). No change in this situation occurred during differentiation.

To investigate the role of the different 5-HT2Rs in osteoblasts, we measured [45Ca]2+ uptake (Fig. 6 ). In the absence of exogenous ligand, [45Ca]2+ uptake was lower for 5-HT2BR–/– primary cells than for WT cells (–23%), suggesting an intrinsic activity of the osteoblast 5-HT2BR. This hypothesis was supported by a higher [45Ca]2+ incorporation by WT osteoblasts treated with the selective 5-HT2BR antagonist RS-127445 (–10%) than in 5-HT2BR–/– osteoblasts. However, the osteoblast 5-HT2BR activity was principally agonist-dependent, and a similar increase in [45Ca]2+ incorporation was found when WT cells were treated with either 5-HT (+50%) or BW 723C86 (+44%), a preferential agonist of 5-HT2BR. Furthermore, exposing WT osteoblasts to RS-127445 led to a decrease (–20%) in [45Ca]2+ incorporation. However, treatment with ritanserin, an inverse 5-HT2R agonist, led to a further decrease (–37%) in [45Ca]2+ incorporation, suggesting that the 5-HT2ARs (as well as the 5-HT2BRs) also affect [45Ca]2+ incorporation. This point was confirmed by the finding that treating 5-HT2BR–/– osteoblasts with ritanserin produced a further decrease (–34%) in [45Ca]2+ incorporation and that treatment with 5-HT induced an increase in [45Ca]2+ incorporation only at day 13. Interestingly, at day 13, 5-HT doubled [45Ca]2+ incorporation in WT osteoblasts, whereas in 5-HT2BR-deficient cells the increase induced was only 25% (Fig. 6) , showing that the 5-HT2BR is responsible for most of the effect of 5-HT on osteoblasts.


Figure 6
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  		Figure 6. 5-HT2BR is the main 5-HTR affecting osteoblast [45Ca]2+ uptake. Incorporation of [45Ca]2+ after incubation for 24 h was measured after different times of primary osteoblast culture. Data were obtained at days 7, 10, and 13 for WT and 5-HT2BR–/– calvarial cells. WT calvarial cells were treated with the 5-HT2BR partial agonist BW 723C86 (10 nM), 5-HT (50 nM), the 5-HT2BR specific antagonist RS-127445 (20 nM) and the 5-HT2R inverse agonist ritanserin (100 nM). Results from two experiments performed in quadruplicate are shown as means ± SE. Significantly different from WT calvarial cells: *P < 0.05; ** P < 0.01; and *** P < 0.001. Significantly different from 5-HT2BR–/– calvarial cells: $$P < 0.01 and $$$P < 0.001.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
So far, no direct physiological role of 5-HT on bone has been identified (12 , 14 , 15 , 27) . We show here for the first time that 5-HT2BR has a key role among the various 5-HTRs expressed on bone cells. Female 5-HT2BR–/– mice exhibit an osteopenic phenotype (reduced BMD and trabecular and cortical bone volumes) that is observed at 4 months of age and progresses with senescence. We provide evidence that 5-HT2BRs affect recruitment and proliferation of osteoblasts in aging mice. The phenotype of low-turnover osteopenia is indicative of reduced bone formation, one of the hallmarks of human and murine models of osteoporosis (1 , 28 , 29) .

In vitro, we observed decreased osteoblast recruitment from mesenchymal stem cells from 5-HT2BR–/– mice. The 5-HT2BR has been reported to regulate embryonic mouse hind limb mesenchymal cell proliferation (17 , 30 , 31) and to increase the osteogenic differentiation of a mesoblastic cell line (19) . In our study, the recruitment of osteoblastic precursors (CFU-FALP+ colonies) from mesenchymal stem cells was reduced in bone marrow from 4- and 18-month-old 5-HT2BR–/– mice. Of interest, a significant decrease in bone density is first observed at the age of 4 months in these mice. Decreased bone formation in aging mice is associated with a decrease in the number of osteoblast colonies from the bone marrow (26 , 32) . We can, therefore, postulate that the reduced ability of 5-HT2BR–/– cells to produce alkaline-positive colonies might amplify the low osteoblast progenitor recruitment of aging mice. Age-dependent osteoporosis in humans has been associated with a decrease in the proliferative capacity of osteoprogenitor cells (33) and also with an increase in the number of adipocytes (34) . The same holds true in SAMP6 mice, a model of accelerated senescence (29 , 35) . In our 5-HT2BR–/– mice, decreased osteogenesis was not associated with a shift toward the adipocyte lineage in either bone sections or cell cultures. Our findings show that 5-HT2BRs have an impact on the recruitment of osteoblasts without producing a shift toward another pathway.

The 5-HT2BR appears to activate cell proliferation in several systems (18 , 36) : it activates the Ras pathway (20 , 37) and transactivates tyrosine kinase receptors, such as platelet-derived growth factor receptor (36) . Although mutant osteoblastic cells appear to have the same mineralizing rate as WT in vivo, in vitro we observed a decreased proliferation of 5-HT2BR–/– primary osteoblasts. In addition, the mineralized nodules generated by 5-HT2BR–/– primary cells presented a mineralization delay and were smaller in size than those generated by WT cells. [45Ca]2+ incorporation and ALP levels were lower in 5-HT2BR–/– than in WT cells between 7 and 13 days of culture. This transient delay in differentiation was probably due to decreased proliferation, as a reduction in the number of osteoblasts would reduce the intercellular contacts through cadherins that are important for acquisition of the osteoblast phenotype (38 , 39) . The decreased proliferation of osteoblasts is associated with reduced recruitment of progenitor cells from the bone marrow that may result in fewer bone-forming surfaces in vivo. Moreover, 5-HT may increase interleukin-6 (IL-6) production by stimulating 5-HT2Rs (40) . This cytokine is an important target of 5-HT2BR (41) and plays a role in osteoblast formation (42) . In our study, we observed a decrease of IL6 in 5-HT2BR–/– osteoblast cultures compared with WT osteoblasts (preliminary data). This down-regulation in 5-HT2BR–/– mice could affect osteoblast recruitment and proliferation.

The study of [45Ca]2+ incorporation in 5-HT2BR–/– osteoblast cultures demonstrates that 5-HT2BRs have a clear impact on osteoblast functions. The decrease in [45Ca]2+ uptake in the absence of 5-HT2BRs or the presence of a selective antagonist demonstrates the role of this receptor in calcium uptake. However, a further decrease in [45Ca]2+ incorporation was observed when the 5-HT2AR was inhibited by ritanserin, and 5-HT increased [45Ca]2+ incorporation in osteoblasts with the 5-HT2BR mutant. The mitogenic activity of 5-HT in other organs, such as the liver (43) and renal artery (44) , has been linked to both 5-HT2B and 5-HT2ARs. It is therefore likely that these two 5-HT2Rs have partially overlapping functions.

Our findings support the notion that 5-HT does indeed influence bone metabolism. So far, no data exclude the possibility that neuronal 5-HT could influence bone cell behavior. Also, platelets, which store serotonin, could release 5-HT in contact or near osteoblasts (10) . Finally, osteoblasts may also produce 5-HT that could act in an autocrine/paracrine fashion. Tryptophan hydroxylase-1 mRNA, the rate-limiting enzyme in 5-HT synthesis, has been detected in osteoblast and osteocyte cell lines (14) , and we detected significant levels of 5-HT in the supernatants of WT and 5-HT2BR–/– osteoblast cultures (preliminary data). In our study, the activity of 5-HT2BRs was mainly agonist-dependent, but a constitutive activity of 5-HT2BRs in osteoblast cannot be ruled out.

5-HT2BRs can control uptake activity of 5-HTT in different systems (45 , 46) . 5-HTT is expressed in osteoclast cell lines and decreases osteoclast differentiation. 5-HTT is also present and functional in primary osteoblasts and osteoblast and osteocyte cell lines (11 , 14 , 27) . Inhibition of 5-HTT by fluoxetine or gene invalidation results in low bone formation in mice (12) . The exact biological effect of 5-HTT on bone formation remains unclear; the regulatory mechanism seems to be complex, and further studies are required to clarify the relationship with the 5-HT2BR. Nevertheless, we have demonstrated that 5-HT via 5-HT2BRs is a peripheral modulator of osteoblast recruitment in bone formation in aging mice. These findings could provide new opportunities for treatments intended to increase bone formation in aging women.


   ACKNOWLEDGMENTS
 
This study was supported by grants from the "Rhumatisme et Travail" Association, INSERM, Université René Diderot, and the European Community 6 PCRD (ANABONOS consortium). The work of L.M. has been supported by funds from the Centre National de la Recherche Scientifique, INSERM, and Université Pierre et Marie Curie and by grants from the Fondation de France, the Fondation pour la Recherche Médicale, the Association pour la Recherche contre le Cancer, the French Ministry of Research (Agence Nationale pour la Recherche),and the European Community. The team of L.M. is an "Equipe Fondation pour la Recherche Médicale."


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication June 14, 2007. Accepted for publication August 9, 2007.


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 DISCUSSION
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