Serotonin 2B receptor (5-HT2B R) signals through prostacyclin and PPAR-ß/δ in osteoblasts

Abstract

Osteoporosis is due to an imbalance between decreased bone formation by osteoblasts and increased resorption by osteoclasts. Deciphering factors controlling bone formation is therefore of utmost importance for the understanding and the treatment of osteoporosis. Our previous in vivo results showed that bone formation is reduced in the absence of the serotonin receptor 5-HT2B, causing impaired osteoblast proliferation, recruitment, and matrix mineralization. In this study, we investigated the signaling pathways responsible for the osteoblast defect in 5-HT2BR(-/-) mice. Notably, we investigated the phospholipase A2 pathway and synthesis of eicosanoids in 5-HT2BR(-/-) compared to wild type (WT) osteoblasts. Compared to control osteoblasts, the lack of 5-HT2B receptors was only associated with a 10-fold over-production of prostacyclin (PGI2). Also, a specific prostacyclin synthase inhibitor (U51605) rescued totally osteoblast aggregation and matrix mineralization in the 5-HT2BR(-/-) osteoblasts without having any effect on WT osteoblasts. Prostacyclin is the endogenous ligand of the nuclear peroxisome proliferator activated receptor ß/δ (PPAR-ß/δ), and its inhibition in 5-HT2BR(-/-) cells rescued totally the alkaline phosphatase and osteopontin mRNA levels, cell-cell adhesion, and matrix mineralization. We conclude that the absence of 5-HT2B receptors leads to the overproduction of prostacyclin, inducing reduced osteoblast differentiation due to PPAR-ß/δ -dependent target regulation and defective cell-cell adhesion and matrix mineralization. This study thus reveals a previously unrecognized cell autonomous osteoblast defect in the absence of 5-HT2BR and highlights a new pathway linking 5-HT2B receptors and nuclear PPAR- ß/δ via prostacyclin.

Figure 1. Cell-cell adhesion and expression profile of osteoblastic markers of 5-HT_2BR-/- osteoblasts.

A: Representative pictures of aggregation assays are shown at Day 1 (D1) after the induction of 5-HT_2BR^−/− and WT primary cultures. Counts of cell aggregates were assessed in WT, and 5-HT_2BR^−/− cells, and in WT cells treated with either ritanserin (an inverse agonist of 5-HT₂Rs) or RS127445 (a selective 5-HT_2BR antagonist). The 5-HT_2BR^−/− and WT-treated cells displayed a much lower level of cell adhesion than the untreated WT cells. B: the main markers of osteoblast differentiation were measured by RTqPCR. Runx2, osteocalcin expression levels were not modified by the absence of 5-HT_2BR, whereas cyclin D1, alkaline phosphatase and osteopontin expression levels were decreased. Data are presented as means ± SEM of three independent experiments performed in triplicate. ** p<0.005 vs WT, * p<0.05 vs WT.

Figure 2. Prostacyclin over-produced in 5-HT_2BR^−/− osteoblasts.

A: To determine the role of the PLA₂ pathway in the 5-HT_2BR^−/− osteoblasts, the expression of PLA₂ and of COX₁ and COX₂ were analyzed by Western blotting. The expression of COX₂ was markedly lower in the 5-HT_2BR^−/− cells than in the WT cells, whereas no difference was observed in the expression of PLA₂ or COX₁. B: Measurement of the main eicosanoids in both genotypes: Prostaglandin E2, Thromboxane B2, Leukotriene B4 and 6-keto prostaglandin F1α, a stable metabolite of prostacyclin, at D1 and D4. Prostacyclin production was greater in 5-HT_2BR^−/− primary cultures than in WT cultures. C: It was also greater in WT cultures treated with RS 127445 at D4. D: Prostacyclin synthase expression was evaluated by Western blotting. Levels of prostacyclin synthase were slightly higher in 5-HT_2BR^−/− than in WT cultures. E: Prostacyclin synthase activity was markedly higher in 5-HT_2BR^−/− than WT cultures. Data are presented as means ± SEM of three experiments performed in triplicate. *** p<0.001 vs. WT.

Figure 3. 5-HT_2BR−/− osteoblasts phenotype is dependent on prostacyclin overexpression.

A specific inhibitor of prostacyclin synthase (U51605) was used to evaluate the role of the prostacyclin overproduction. A: a cell aggregation assay was performed with this inhibitor on WT and 5-HT_2BR^−/− cells. U51605 had no effect on WT aggregation and restored 5-HT_2BR^−/− cell aggregation. B: mineralization assay: U51605 had no effect on WT cells, but did rescue 5-HT_2BR^−/− mineralization. C: The expressions of the alkaline phosphatase, osteopontin and cyclin D1 level markers modified by the absence of 5-HT_2BR were restored by adding U51605 to 5-HT_2BR−/− osteoblasts. Data are presented as means ± SEM of three independent experiments performed in triplicate. ** p<0.05 vs. WT, *** p<0.001 vs. WT. ^$$$ p<0.005 vs 5-HT_2BR−/−.

Figure 4. Pharmacological inhibition of PPAR-ß/δ restores the phenotype of 5-HT2BR−/− osteoblasts.

PGI₂ is an endogenous ligand of nuclear PPAR ß/δ, and also can act via the membrane IP receptor. We evaluated the involvement of these two potential prostacyclin targets in 5-HT_2BR^−/− cells. A: For the membrane IP receptor: the cAMP level and the binding parameters of the IP receptors were measured in both cell types. None of these parameters was modified by the absence of the 5-HT_2B receptor. B: PPAR-ß/δ and PPAR-γ expressions were analyzed by Western blotting, and found to be unchanged in both cell types. The involvement of PPAR ß/δ was determined using specific antagonist of this receptor. C: An aggregation assay was carried out in the presence of GSK0660 (a specific antagonist of PPAR-ß/δ) for 5-HT_2BR^−/− cultures. The GSK0660 treatment restored cell aggregation in 5-HT_2BR^−/− cultures. D: A mineralization assay (red alizarin staining) was performed under the various different conditions: GSK0660 treatment rescued the phenotype in 5-HT_2BR^−/− osteoblasts. E: Similar results were obtained for the transcriptional expressions of the alkaline phosphatase, osteopontin, and cyclin D1 level markers. Data are presented as means ± SEM of three independent experiments performed in triplicate. *** p<0.005 vs. WT and ^$$$ p<0.005 vs 5-HT_2BR−/−.