Volume 93, Pages 1-11 (December 2016) Sphingosine-1-phosphate/S1PR2-mediated signaling triggers Smad1/5/8 phosphorylation and thereby induces Runx2 expression in osteoblasts  Katsumasa Higashi, Etsuko Matsuzaki, Yoko Hashimoto, Fumi Takahashi-Yanaga, Aiko Takano, Hisashi Anan, Masato Hirata, Fusanori Nishimura  Bone  Volume 93, Pages 1-11 (December 2016) DOI: 10.1016/j.bone.2016.09.003 Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 1 S1P promotes the mRNA levels of osteoblast differentiation markers in MC3T3-E1 cells. Cells were cultured for 24h, and then incubated in a serum-free medium in the presence or absence of S1P (2μM) for the indicated times. mRNA levels of ALP (A), osteopontin (OPN) (B), osteocalcin (OCL) (C), and bone sialoprotein (BSP) (D) were determined by quantitative real-time RT-PCR and the fold values relative to the each control level at day 0 were determined. Values are expressed as means±SE of three independent experiments. *p<0.05 vs control cells at day 0, #p<0.05 vs control cells at day 7 by Student's t-test. Bone 2016 93, 1-11DOI: (10.1016/j.bone.2016.09.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 2 Both S1PR1 and S1PR2 contribute to S1P-induced ALP expression and osteoblast differentiation in MC3T3-E1 cells and primary osteoblasts. ALP mRNA (A) and the protein (B) in MC3T3-E1 cells following inhibition of S1PR1 and S1PR2 by RNAi. Cells were transfected with 50nM negative control (scramble), S1PR1, or S1PR2 siRNA and stimulated with 2μM S1P for 24 (A) or 48h (B). ALP mRNA levels were calculated as the percentages to the control (open column) level. ALP protein levels were determined by western blotting. Values are expressed as means±SE of three independent experiments. *p<0.05 vs control cells (open column), #p<0.05 vs S1P-treated cells (black column) by Student's t-test. (C) Calcified deposits were visualized by von Kossa staining (left). Cells at 50% confluency were transfected with negative control (scramble), S1PR1, or S1PR2 siRNA. After reaching confluence, osteogenesis was initiated with osteogenic differentiation medium in the presence or absence of S1P (2μM) for 14days. Magnified view (×200). von Kossa stained area were calculated as the fold to the control (open column) level (right). Values are expressed as means±SE of three independent experiments. *p<0.05 vs control cells, #p<0.05 vs S1P-treated cells (black column) by Student's t-test. (D) ALP staining in primary osteoblasts (upper). Cells were cultured in a serum-free medium for 24h, followed by pretreatment with 10μMW146 or JTE-013 for 30min, and then incubated in the presence or absence of S1P (2μM) for 4days. Values are expressed as means±SE of three independent experiments. *p<0.05 vs control cells, #p<0.05 vs S1P-treated cells (black column) by Student's t-test. Bone 2016 93, 1-11DOI: (10.1016/j.bone.2016.09.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 3 S1P increases the mRNA levels of S1PR1 and S1PR2. Cells were cultured for 24h, and then incubated in a serum-free medium in the presence or absence of S1P (2μM) for the indicated times. mRNA levels of S1PR1–S1PR5 were measured by quantitative real-time RT-PCR and represented in the fold values relative to each S1PRs level at day 0. Values are expressed as means±SE of three independent experiments. *p<0.05 vs control cells at day 0, #p<0.05 vs control cells at day 7 by Student's t-test. Bone 2016 93, 1-11DOI: (10.1016/j.bone.2016.09.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 4 S1PR2/Gi-independent signaling enhances S1P-induced RhoA activation. (A) Time course of S1P-induced RhoA activity. (B) Effect of W146 (S1PR1 antagonist), JTE-013 (S1PR2 antagonist), CAY10444 (S1PR3 antagonist), and PTX (Gi protein inhibitor; pertussis toxin) on S1P-induced RhoA activation. MC3T3-E1 Cells were incubated in a serum-free medium for 24h, and pretreated with 10μM of W146, JTE-013, or CAY10444 for 30min, or 100ng/mL PTX for 24h, and then incubated in the presence of S1P (2μM) for 3–30min (A) or 3min (B). RhoA activation was calculated as the fold value relative to control. Values are expressed as means±SE of three independent experiments. *p<0.05 vs control cells, #p<0.05 vs S1P-treated cells (black column) by Student's t-test. Bone 2016 93, 1-11DOI: (10.1016/j.bone.2016.09.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 5 S1PR2/RhoA/ROCK signaling induces Smad1/5/8 phosphorylation in MC3T3-E1 cells (A-D) and primary osteoblasts (E and F). (A) Effect of S1P on the phosphorylation of Smad1/5 at Ser463/465, and Smad8 at Ser426/428. Cells were cultured in a serum-free medium for 24h, and then incubated in the presence of S1P (2μM) for the indicated times. Phosphorylation of Ser463/465 on Smad1/5, and Ser426/428 on Smad8 was detected by western blotting normalized to the level of Smad1/5/8. Values are expressed as means±SE of three independent experiments. *p<0.05 vs control cells by Student's t-test. (B) Effects of inhibitors for S1PR1, S1PR2, and Gi on S1P-induced Smad1/5/8 phosphorylation. Cells were treated as described in Fig. 4B, and incubated in the presence of S1P (2μM) for 1.5h, and phosphorylation of Ser463/465 on Smad1/5, and Ser426/428 on Smad8 were then determined by western blotting. Values are expressed as means±SE of three independent experiments. *p<0.05 vs control cells, #p<0.05 vs S1P-treated cells (black column) by Student's t-test. (C, D) Effects of C3 toxin (RhoA inhibitor) and Y27632 (ROCK inhibitor) on S1P-induced Smad1/5/8 phosphorylation. Cells were cultured in serum-free medium for 24h and pretreated with indicated concentrations of C3 toxin for 2h (C), and Y27632 for 30min (D), followed by incubation with S1P (2μM) for 1.5h. Phosphorylation of Ser463/465 on Smad1/5, and Ser426/428 on Smad8 was detected by western blotting. Values are expressed as means±SE of three independent experiments. *p<0.05 vs control cells (open column), #p<0.05 vs C3 toxin-treated cells (checked column) (C) or Y27632-treated cells (checkboard column) (D) by Student's t-test. (E) Effect of S1P on phosphorylation of Smad1/5 at Ser463/465, and Smad8 at Ser426/428 in primary osteoblasts. Cells were cultured in a serum-free medium for 24h, and then incubated in the presence of S1P (2μM) for the indicated times. Values are expressed as means±SE of three independent experiments. *p<0.05 vs control cells by Student's t-test. (F) Effects of inhibitors for S1PR1, S1PR2, and RhoA on S1P-induced Smad1/5/8 phosphorylation in primary osteoblasts. Cells were cultured in a serum-free medium for 24h, and pretreated with 10μMW146, JTE-013 for 30min, or 1μg/mL C3 toxin for 2h, and then incubated in the presence of S1P (2μM) for 2h. Phosphorylation levels of Ser463/465 on Smad1/5, and Ser426/428 on Smad8 were determined by western blotting. Values are expressed as means±SE of three independent experiments. *p<0.05 vs control cells, #p<0.05 vs S1P-treated cells (black column) by Student's t-test. Bone 2016 93, 1-11DOI: (10.1016/j.bone.2016.09.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 6 S1PR2/RhoA/ROCK-dependent S1P signaling promotes nuclear translocation of Smad4 and nuclear expression of Smad6/7. (A) Effect of S1P on the nuclear translocation of Smad4 and the nuclear expression of Smad6/7. MC3T3-E1 cells were cultured in a serum-free medium for 24h, and then incubated in the presence of S1P (2μM) for the indicated times. Nuclear protein samples were subjected to western blot analysis for Smad4 (upper) and Smad6/7 (lower). The membrane was then reprobed with anti-Histone H3 antibody. Values are expressed as means±SE of three independent experiments. *p<0.05 vs control cells by Student's t-test. (B) Effect of W146, JTE-013, and C3 toxin on the nuclear translocation of Smad4 and the nuclear expression of Smad6/7. Cells were cultured in a serum-free medium for 24h, pretreated with 10μMW146 or JTE-013 for 30min, or 1μg/mL C3 toxin for 2h, and then incubated in the presence of S1P (2μM) for 6h. Nuclear protein samples were subjected to western blot analysis for Smad4 (upper) and Smad6/7 (lower). Values are expressed as means±SE of three independent experiments. *p<0.05 vs control cells, #p<0.05 vs S1P-treated cells (black column) by Student's t-test. Bone 2016 93, 1-11DOI: (10.1016/j.bone.2016.09.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 7 S1PR2/RhoA/ROCK-dependent S1P signaling increases Runx2 expression in MC3T3-E1 cells (A–D) and primary osteoblasts (E, F). (A) Effect of S1P on Runx2 mRNA (A) and protein (B) levels. MC3T3-E1 cells were cultured in a serum-free medium for 24h, and then incubated in the presence of S1P (2μM) for the indicated times. Runx2 mRNA levels (A) were determined by real-time RT-PCR and calculated as the percentages of the control level. Values are expressed as means±SE of three independent experiments. *p<0.05 vs control cells by Student's t-test. Runx2 protein levels (B) were determined by western blotting and normalized to the level of β-actin. Values are expressed as means±SE of three independent experiments. *p<0.05 vs control cells by Student's t-test. Effects of W146, JTE-013 (C), C3 toxin, Y27632, and PTX (D) on Runx2 protein levels. Cells were cultured in a serum-free medium for 24h, and pretreated with 10μMW146, JTE-013, or 1μg/mL C3 toxin for 2h, Y27632 for 30min, or 100ng/mL PTX for 24h and then incubated in the presence of S1P (2μM) for 24h. Runx2 protein levels were determined by western blotting. Values are expressed as means±SE of three independent experiments. *p<0.05 vs control cells, #p<0.05 vs S1P-treated cells (black column) by Student's t-test. (E) Effect of S1P on the Runx2 protein levels in primary osteoblasts. Cells were cultured in a serum-free medium for 24h, and then incubated in the presence of S1P (2μM) for the indicated times. Values are expressed as means±SE of three independent experiments. *p<0.05 vs control cells by Student's t-test. (F) Effects of inhibitors for S1PR1, S1PR2, and RhoA on S1P-induced Runx2 expression in primary osteoblasts. Cells were cultured in a serum-free medium for 24h, and pretreated with 10μMW146, JTE-013 for 30min, or 1μg/mL C3 toxin for 2h, and then incubated in the presence of S1P (2μM) for 48h. Runx2 protein levels were determined by western blotting. Values are expressed as means±SE of three independent experiments. *p<0.05 vs control cells, #p<0.05 vs S1P-treated cells (black column) by Student's t-test. Bone 2016 93, 1-11DOI: (10.1016/j.bone.2016.09.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 8 Effect of S1PR2/RhoA/ROCK signaling pathway in vivo. (A) Three-dimensional μCT images of metaphysis structure of proximal tibia. (B–D) Quantitative μCT data of trabecular bone volume relative to tissue volume (B.V./T.V.) (B), trabecular thickness (Tb.Th.) (C), and trabecular number (Tb.N.) (D) of tibia. Male mice at 8weeks of age were injected i.p. every day at 8am with CYM-5520 (1mg/kg; S1PR2 agonist) or FTY720 (1mg/kg; S1PR1, 3 agonist) alone, or along with W146 (1mg/kg), JTE-013 (1mg/kg) or Y27632 (1mg/kg) for 28days. Dissected right tibia was analyzed by μCT. Values are expressed as means±SE (n=4). *p<0.05 vs vehicle-treated mice, #p<0.05 vs CYM-5520-treated cells (black column), $p<0.05 vs FTY720-treated cells (gray column) by Student's t-test. Bone 2016 93, 1-11DOI: (10.1016/j.bone.2016.09.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 9 Model for the regulation of osteoblast differentiation by S1PR1/2 signaling pathway. S1PR1/Gi-mediated signaling pathway induces PI3K activity, leading to the phosphorylation of Akt, thereby promoting the translocation of β-catenin to nuclear and ALP expression. Gi-independent, S1PR2-mediated RhoA/ROCK signaling pathway induces phosphorylation of Smad1/5/8, resulting in the nuclear translocation of Smad4, thereby promoting Runx2 and ALP expression. This signaling pathway also increases Smad6/7 expression in the nucleus. The role of S1P-induced Smad6/7 on Runx2 expression and the complex formation with Smad1/5/8 and Smad4 was indicated by dashed line, as not addressed in our studies. Bone 2016 93, 1-11DOI: (10.1016/j.bone.2016.09.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Supplementary Fig. 1 S1PR1 and S1PR2 knock-down is assessed by quantitative real-time RT-PCR. MC3T3-E1 cells were transfected with 50nM negative control (scramble), S1PR1, or S1PR2 siRNA. S1PR1 and S1PR2 mRNA levels were determined by quantitative real-time RT-PCR. The mRNA levels were quantifies and calculated as the percentages of the control level. Values are expressed as means±SE of three independent experiments. *p<0.05 vs control cells by Student's t-test. Bone 2016 93, 1-11DOI: (10.1016/j.bone.2016.09.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Supplementary Fig. 2 S1P has no effect on BMP-2 secretion. MC3T3-E1 cells were cultured in a serum-free medium for 24h, and then incubated in the presence of S1P (2μM) for the indicated times. Protein levels of BMP-2 (R&D Systems; Minneapolis, MN) in culture supernatants were measured by enzyme-linked immunosorbent assay according to the manufacturers' protocols. Values are expressed as means±SE of three independent experiments. Bone 2016 93, 1-11DOI: (10.1016/j.bone.2016.09.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Supplementary Table 1 Primers used for quantitative real-time RT-PCR. Bone 2016 93, 1-11DOI: (10.1016/j.bone.2016.09.003) Copyright © 2016 Elsevier Inc. Terms and Conditions