Decreased osteoclastogenesis in serotonin-deficient mice

Abstract

Peripheral serotonin, synthesized by tryptophan hydroxylase-1 (TPH(1)), has been shown to play a key role in several physiological functions. Recently, controversy has emerged about whether peripheral serotonin has any effect on bone density and remodeling.We therefore decided to investigate in detail bone remodeling in growing and mature TPH(1) knockout mice (TPH(1)(-/-)). Bone resorption in TPH(1)(-/-) mice, as assessed by biochemical markers and bone histomorphometry, was markedly decreased at both ages. Using bone marrow transplantation, we present evidence that the decrease in bone resorption in TPH(1)(-/-) mice is cell-autonomous. Cultures from TPH(1)(-/-) in the presence of macrophage colony-stimulating factor and receptor activator for NF-KB ligand (RANKL) displayed fewer osteoclasts, and the decreased differentiation could be rescued by adding serotonin. Our data also provide evidence that in the presence of RANKL, osteoclast precursors express TPH(1) and synthesize serotonin. Furthermore, pharmacological inhibition of serotonin receptor 1B with SB224289, and of receptor 2A with ketanserin, also reduced the number of osteoclasts. Our findings reveal that serotonin has an important local action in bone, as it can amplify the effect of RANKL on osteoclastogenesis.

Fig. 1.

Bone formation in WT and TPH₁^−/− mice during growth and maturity. Static and dynamic histomorphometric parameters were measured in 6- and 16-wk-old animals. (A) Bone formation rate/bone surface (BFR/BS) and (B) osteoblast surface / bone surface (OB/BS) were unchanged in both genotypes (WT, black bars; TPH₁^−/−, white bars) at 6 wk and were reduced at 16 wk. (C) Biochemical markers of bone formation. Serum osteocalcin levels were determined when the mice were 6 and 16 wk old. No significant difference was observed when the mice were 6 wk old, whereas a significant decrease in the osteocalcin level was observed when they were 16 wk old. (D) Proliferation was assessed after a 3-d culture period by BrdU incorporation in WT and TPH₁^−/− calvarial osteoblasts, and no difference was observed. (E) The capacity of WT and TPH₁^−/− primary osteoblasts to produce mineralized nodules was determined by alizarin staining after 18 d in culture, and no difference was observed. Data are shown as mean ± SEM; n = 8 mice per genotype. *P < 0.01 versus WT, **P < 0.001 versus WT, ***P < 0.0001 versus WT.

Fig. 2.

Lack of serotonin induces in vivo and in vitro decrease in bone resorption. Histomorphometric analysis was performed at 6 and 16 wk. TRAP staining of a section of the trabecular region of the distal femur was performed to identify osteoclasts. Bone resorption parameters were determined. (A) The number of osteoclasts per bone surface (OCs/BS) showed a marked decrease, and TPH₁^−/− mice had a lower osteoclast count than WT mice. (B) Osteoclast number/bone area (OCs number/BAr). (C) Biochemical markers of bone resorption. Urinary deoxypyridinoline cross-links normalized by the amount of creatinine present (DPD/creat) were measured in 6- and 16-wk-old WT and TPH₁^−/− mice. DPD levels were lower in 6- and 16-wk-old male TPH₁^−/− mice. WT, black bars; TPH₁^−/−, white bars. Results are reported as mean ± SEM; n = 8 mice per genotype. (D) TPH₁^−/− mice were treated twice a day with vehicle (white bars) or with 50 mg/kg body weight of 5-HTP (gray bars). DPD levels were significantly increased by 5-HTP administration to 4-wk-old mice after 1 or 2 wk of treatment; vehicle, n = 6; 5-HTP treatment, n = 9. (E) Osteoclastogenesis was assessed in WT and TPH₁^−/− spleen cells and bone marrow cells cultured with dialyzed serum without serotonin in the presence of M-CSF (25 ng/mL) for 4 d and of M-CSF and RANKL (30 ng/mL) for a further 5 d. Counts of OCLs in WT (black bars) and TPH₁^−/− (white bars) culture; osteoclast numbers were lower in TPH₁^−/− cultures than in WT cultures at the end of the differentiation of spleen cells and bone marrow cells. Representative pictures of WT and TPH₁^−/− spleen cell cultures (20× magnification). (F) OCL activity was assessed by pit assays on dentin slices. The resorbed area was stained with toluidine blue (10× magnification). The resorbed area was strongly decreased in TPH₁^−/− cultures, whereas the ratio pit area:OC number was unchanged. (G) WT spleen cells were cultured without any serotonin treatment, and TPH₁^−/− cells were treated with serotonin (2.5, 5, 10, 20, and 50 nM) throughout the culture in the presence of RANKL. From 5 nM 5-HT the number of OCLs increased to equal WT levels, without any dose effect. *P < 0.01 versus WT, **P < 0.001 versus WT, ***P < 0.0001 versus WT.

Fig. 3.

Synthesis, storage, and reuptake of serotonin in WT osteoclast precursors. (A) After 4 d of treatment with M-CSF (25 ng/mL), total RNAs were isolated at different times [day (D)1, 2, 3, and 5] in the presence (gray bar) and absence (black bar) of RANKL (30 ng/mL), and RT-PCR was performed. TPH₁ mRNA was expressed from day 1 to day 3. The presence of RANKL increased TPH₁ mRNA expression, but only at day 1. (B) Serotonin levels were measured by HPLC in WT cell lysates. Serotonin was detected from day 1 to day 5 in the presence or absence of RANKL. An increase in serotonin level in the presence of RANKL was observed during the culture. (C) WT cells were treated with several different doses of reserpine, an inhibitor of vesicular monoamine transporter (50,100, and 200 nM), which induced a dose-dependent decrease in OCL number in WT cultures. (D) WT and TPH₁^−/− cultures were treated with paroxetine (10 nM), an inhibitor of SERT. Paroxetine also induced a significant decrease in OCL number in WT culture but not in TPH₁^−/− culture. (E) Various different substances were added to the WT culture in the presence of M-CSF and RANKL. Ritanserin (100 nM), an inverse agonist of the 5-HT₂ family, induced a decrease in osteoclast number, whereas RS127445 (20 nM), a specific antagonist of 5-HT_2BR, did not induce any change. Several different doses of ketanserin (25 nM), an antagonist of 5-HT_2AR, were added to the WT culture. A significant dose-related decrease in osteoclast number was observed. SB224289 (10 nM) 5-HT_1BR antagonist treatments of WT cells decreased the OCL number, but without any dose effect. Data are reported as mean ± SEM of four independent experiments. *P < 0.01 versus WT, **P < 0.001 versus WT, ***P < 0.0001 versus WT.

Fig. 4.

In vivo decrease in bone resorption in TPH₁^−/− mice is due to an intrinsic osteoclast defect. Newborn WT and TPH₁^−/− mice were treated with busulfan (10 mg/kg) and transplanted with WT and TPH₁^−/− bone marrow cells. (A) At age 4 and 6 wk, urine samples were taken, and DPD was measured. We found that TPH₁^−/− mice transplanted with TPH₁^−/− cells had a lower DPD:creatinine ratio than WT mice transplanted with WT cells. Moreover, when WT mice were transplanted with TPH₁^−/− cells, the DPD ratio decreased, and the transplantation of WT cells into TPH₁^−/− mice led to a rescue of the DPD ratio. (B) Histomorphometric analysis was performed at 6 wk on the transplanted mice. TRAP staining of a section of the trabecular region of the distal femur was performed to identify osteoclast multinucleated and red cells (20× magnification): (a) WT mice transplanted with WT cells, (b) WT mice transplanted with TPH₁^−/− cells, (c) TPH₁^−/− mice transplanted with WT cells, and (d) TPH₁^−/− mice transplanted with TPH₁^−/− cells. (C) Bone resorption parameters were also determined: osteoclast number per bone surface (OCs/BS) and the osteoclast number. These parameters showed a similar pattern to the DPD ratio: When WT cells were transplanted into TPH₁^−/− mice, we observed an increase in the bone resorption parameter, and when TPH₁^−/− cells were transplanted into WT mice, we found that rescue of bone resorption occurred. Data are shown as mean ± SEM; n = 5–8 mice per genotype. *P < 0.05 versus WT, **P < 0.005 versus WT, ***P < 0.0005 versus WT.