Sphingosine-1-Phosphate Receptor 2 Controls Podosome Components Induced by RANKL Affecting Osteoclastogenesis and Bone Resorption

Abstract

Proinflammatory cytokine production, cell chemotaxis, and osteoclastogenesis can lead to inflammatory bone loss. Previously, we showed that sphingosine-1-phosphate receptor 2 (S1PR2), a G protein coupled receptor, regulates inflammatory cytokine production and osteoclastogenesis. However, the signaling pathways regulated by S1PR2 in modulating inflammatory bone loss have not been elucidated. Herein, we demonstrated that inhibition of S1PR2 by a specific S1PR2 antagonist (JTE013) suppressed phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinases (MAPKs), and nuclear factor kappa-B (NF-κB) induced by an oral bacterial pathogen, Aggregatibacter actinomycetemcomitans, and inhibited the release of IL-1β, IL-6, TNF-α, and S1P in murine bone marrow cells. In addition, shRNA knockdown of S1PR2 or treatment by JTE013 suppressed cell chemotaxis induced by bacteria-stimulated cell culture media. Furthermore, JTE013 suppressed osteoclastogenesis and bone resorption induced by RANKL in murine bone marrow cultures. ShRNA knockdown of S1PR2 or inhibition of S1PR2 by JTE013 suppressed podosome components, including PI3K, Src, Pyk2, integrin β_3, filamentous actin (F-actin), and paxillin levels induced by RANKL in murine bone marrow cells. We conclude that S1PR2 plays an essential role in modulating proinflammatory cytokine production, cell chemotaxis, osteoclastogenesis, and bone resorption. Inhibition of S1PR2 signaling could be a novel therapeutic strategy for bone loss associated with skeletal diseases.

Keywords: bone loss; chemotaxis; cytokines; osteoclast; podosome; sphingosine-1-phosphate receptor 2.

Figure 1

Inhibition of S1PR2 by its specific antagonist (JTE013) significantly decreased IL-1β, IL-6, TNF-a, and S1P levels induced by Aa in BMMs. Murine BMMs were treated either with vehicle or JTE013 for 30 min. Then cells were either uninfected or infected with Aa for 6 h. (A) IL-1β, (B) IL-6, and (C) TNF-α levels were quantified by ELISA. (D) S1P levels in cell protein lysates were quantified by mass spectrometry. Cytokine and S1P levels were normalized by protein levels in cell lysates. (E) Cell viability in BMMs treated with vehicle or JTE013 for 24 h. (* p < 0.05 *** p < 0.001).

Figure 2

Inhibition of S1PR2 by its specific antagonist (JTE013) attenuated p-PI3K, p-ERK, p-JNK, p-p38, and p-NF-kBp65 protein expressions induced by Aa in BMMs. Murine BMMs were treated with either vehicle or JTE013 for 30 min. Then cells were either uninfected or infected with Aa for 4 h. (A) p-PI3K, p-ERK, p-JNK, p-p38, p-NF-κBp65, and control GPADH protein expressions were evaluated by western blot. (B) p-PI3K protein density, (C) p-ERK protein density, (D) p-JNK protein density, (E) p-p38 protein density, and (F) p-NF-κBp65 protein density were analyzed and normalized by GAPDH protein expression. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

S1PR2 regulates cell chemotaxis induced by Aa-stimulated cell culture media. Murine BMMs, treated with either S1PR2 shRNA, control shRNA, JTE013, or vehicle, respectively, were loaded on the top inserts of transwell plates. The bottom chambers were filled with either serum-free (SF) media or Aa-stimulated cell culture media (Aa-media) as described in Methods. Cells were incubated for 6 h. (A,B) Representative images show crystal violet staining of cells on the bottom of inserts. (C,D) display migration index. (*** p < 0.001).

Figure 4

Inhibition of S1PR2 by its specific antagonist (JTE013) suppressed osteoclastogenesis and bone resorption induced by RANKL with or without co-culture with Aa-stimulated cell culture media. Murine BMs were treated with vehicle or JTE013. Cell were either unstimulated, cultured with Aa-stimulated cell culture media (Aa-media) alone, cultured with RANKL alone, or co-cultured with both RANKL and Aa-media as described in Methods. (A) Representative images show TRAP-stained cells at the 100× magnification view. (B) Number of TRAP⁺ multinucleated (more than three nuclei) osteoclasts/well (96-well) and (C) Total areas of osteoclasts/image were quantified. (D) Representative images show bone resorption pits at 100× magnification view. (E) Total areas of bone resorption pits /image were quantified. (*** p < 0.001).

Figure 5

Inhibition of S1PR2 by its specific antagonist (JTE013) attenuated Nfatc1, Ctsk, Acp5, Oscar, Dc-stamp, and Oc-stamp mRNA expressions induced by RANKL with or without co-culture with Aa-stimulated cell culture media. Murine BMs were treated with vehicle or JTE013. Cell were either unstimulated, cultured with Aa-stimulated cell culture media (Aa-media) alone, RANKL alone, or co-cultured with both RANKL and Aa-media as described in Methods. (A) Nfatc1 mRNA, (B) Ctsk mRNA, (C) Acp5 mRNA, (D) Oscar mRNA, (E) Dc-stamp mRNA, (F) Oc-stamp mRNA, (G) RANKL mRNA, and (H) OPG mRNA levels were quantified by real time PCR and normalized by GAPDH expression. (* p < 0.05, *** p < 0.001).

Figure 6

S1PR2 controls the activation of podosome protein kinases PI3K, Src, and Pyk2 induced by RANKL with or without co-stimulated by Aa-stimulated media. Murine BMMs were treated with control shRNA/S1PR2 shRNA for three days or treated with vehicle/ JTE013 for 30 min. Cells were unstimulated, stimulated with RANKL, or co-stimulated with both RANKL and Aa-stimulated media (Aa-media) for 1h. (A) S1PR2 mRNA levels were quantified by real time PCR and normalized by GAPDH expression. (B) and (F) p-Pyk2, p-Src, p-PI3K, and control GPADH protein expressions were evaluated by western blot. (C) and (G) p-Pyk2 protein density, (D) and (H) p-Src protein density, and (E) and (I) p-PI3K protein density were analyzed and normalized by GAPDH protein expression. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 7

S1PR2 regulates podosome adhesive protein F-actin, integrin β₃, and paxillin levels induced by RANKL with or without co-stimulated by Aa-stimulated media. Murine BMMs were treated with control shRNA/S1PR2 shRNA for one day or treated with vehicle/ JTE013 for 30 min. Then, cells were cultured in media containing M-CSF (50 ng/mL) alone or M-CSF with RANKL (250 ng/mL) for two days. On the third day, the media was changed with or without RANKL and/or JTE013. Some of the cells were co-cultured with both RANKL and Aa-stimulated media (Aa-media, 200 µL/mL) for another day. (A,E) F-actin, integrin β3, paxillin, and GAPDH protein expressions were evaluated by western blot. (B,F) F-actin protein density, (C,G) integrin β3 protein density, and (D,H) paxillin protein density were analyzed and normalized by GAPDH protein expression. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 8

Immunofluorescence staining of integrin β3, F-actin, and paxillin in BMMs. Murine BMMs were treated with control shRNA/S1PR2 shRNA for one day or treated with vehicle/ JTE013 for 30 min. Then, cells were cultured in media containing M-CSF (50 ng/mL) alone or M-CSF with RANKL (250 ng/mL) for two days. On the third day, the media was changed with or without RANKL and/or JTE013. Some of the cells were co-cultured with both RANKL and Aa-stimulated media (Aa-media, 200 µL/mL) for another day. (A,C) show representative images of integrin β3, F-actin, and DAPI staining in BMMs (four to six cells per image). (B,D) show representative images of paxillin, F-actin, and DAPI staining in BMMs (four to six cells per image). (E,H) show cell fluorescence intensity of F-actin. (F,I) show cell fluorescence intensity of integrin β3. (G,J) show cell fluorescence intensity of paxillin. (* p < 0.05, ** p < 0.01, *** p < 0.001).