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

Abstract

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-HT(2B) receptor (5-HT(2B)R) expression during in vitro osteoblast differentiation, we investigated a putative bone phenotype in vivo in 5-HT(2B)R knockout mice. Of interest, 5-HT(2B)R 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-HT(2B)R 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-HT(2B)R depletion (produced genetically or by pharmacological inactivation). These findings support the hypothesis that the 5-HT(2B)R 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-HT(2B)R receptor is a physiological mediator of 5-HT in bone formation and, potentially, in the onset of osteoporosis in aging women.

Figure 1. Osteoblasts express several 5-HTRs; 5-HT_2BRs 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-HT_1A (A), 5-HT_2A (B), and 5-HT_2B (C) binding sites were determined in WT (black bars) and in 5HT_2BR^−/− 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.

Figure 2. 5-HT_2BR^−/− mice display decreased BMD that worsens with aging

BMD was determined for the whole body (A) and femurs (B) of 5HT_2BR^−/− and wild-type (WT) female mice at the times indicated (n=5 per 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-HT_2BR^−/− and WT mice (n = 15 to 20 per 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-HT_2BR^−/− and WT mice. Mean values ± SEM (bars) are shown.

Figure 3. 5-HT_2BR^−/− mice display trabecular and cortical osteopenia

3D reconstruction by μCT of metaphyseal regions of tibia (A) and the metaphyseal region of distal femur (B) stained with toluidine blue in 4- and 12-month-old female mice illustrate the trabecular and cortical differences between 5-HT_2BR^−/− 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 ± SEM (bars) are reported (n = 10–12 sections per group). Asterisks indicate a significant difference between genotypes (* = P < 0.05, ** = P < 0.01, *** = P < 0.001).

Figure 4. Bone formation decreases in the absence of 5-HT_2BR

Static and dynamic bone parameters and biochemical markers were measured in 4- and 18-month-old 5-HT_2BR^−/− (open bars) and WT female mice (black bars). Representative tetracycline/calcein double-labeling in 4-month-old 5-HT_2BR^−/− and WT bone sections are shown (A). Dynamic formation parameters (mineralizing surface/bone surface = MS/BS) (B), mineralizing apposition rate = MAR) (C) and bone formation rate = BFR) (D), as well, as biochemical markers of bone resorption (urinary deoxypyridinoline cross-links = Dpd) (E), biochemical markers of bone formation, plasma alkaline phosphatase activity = ALP activity) (F) and plasma osteocalcin level (G) were determined. Data are shown as means ± SEM (n=10–12 sections per sample). Asterisks indicate significant difference between genotypes (* = P < 0.05, ** = P < 0.01, *** = P < 0.001).

Figure 5. 5-HT_2BR impacts on osteoblast recruitment and proliferation

(A) Proliferation was assessed after a 2 day-culture period by BrdU incorporation in WT and 5-HT_2BR^−/− calvarial osteoblasts, and by [³H] thymidine incorporation in the absence or presence of RS-127445 (5 nM). Each value is the mean ± SEM 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-F_ALP+ colonies/dish were significantly lower in 5-HT_2BR^−/− mice as compared to WT mice. This graph is representative of two independent experiments with quadruplicate determinations. (C) ALP activities were measured in WT and 5-HT_2BR-deficient primary osteoblast cultures. Each value is the mean ± SEM of 12 wells. Data were pooled from two independent experiments. Difference from WT at day 7 (** = p < 0.001) and 10 (* = p <0.03). (D–F) At day 13 (D) and day 21 (F), the capacity of WT and HT_2BR^−/− primary osteoblast to produced mineralized nodules was determined by alizarin staining; the mean area of the WT nodules was higher than that of the HT_2BR^−/− nodules (p<0.001). The nodules formed in culture appeared to be smaller in WT than in 5-HT_2BR-deficient osteoblasts (E).

Figure 6. 5-HT_2BR is the main 5-HTR impacting on osteoblasts

[⁴⁵Ca]²⁺ uptake Incorporation of [⁴⁵Ca]²⁺ after incubating for 24 hours was measured after different times of primary osteoblast culture. Data were obtained at day 7, 10 and 13 for WT and 5-HT_2BR^−/− calvarial cells. WT calvarial cells were treated with the 5-HT_2BR partial agonist BW 723C86 (10 nM), 5-HT (50 nM), the 5-HT_2BR specific antagonist RS-127445 (20 nM) and the 5-HT2R inverse agonist ritanserin (100 nM). Results from 2 experiments performed in quadruplicate are shown as means ± SEM.* indicates p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from WT calvarial cells. $$ indicates p < 0.01, $$$ p < 0.001 significantly different from 5-HT_2BR^−/− calvarial cells.