Sclerostin inhibits osteoblast differentiation without affecting BMP2/SMAD1/5 or Wnt3a/β-catenin signaling but through activation of platelet-derived growth factor receptor signaling in vitro

Abstract

Sclerostin inhibits bone formation mostly by antagonizing LRP5/6, thus inhibiting Wnt signaling. However, experiments with genetically modified mouse models suggest that a significant part of sclerostin-mediated inhibition of bone formation is due to interactions with other binding partners. The objective of the present work was to identify signaling pathways affected by sclerostin in relation with its inhibitory action on osteogenic differentiation of C3H10T1/2 cells, MC3T3-E1 cells and primary osteoblasts. Sclerostin inhibited BMP2-induced osteoblast differentiation without altering SMAD1/5 phosphorylation and transcriptional activity. Moreover, sclerostin prevented Wnt3a-mediated osteoblastogenesis without affecting LRP5/6 phosphorylation or β-catenin transcriptional activity. In addition, sclerostin inhibited mineralization promoted by GSK3 inhibition, which mimics canonical Wnt signaling without activation of LRP5/6, suggesting that sclerostin can prevent osteoblast differentiation without antagonizing LRP5/6. Finally, we found that sclerostin could activate platelet-derived growth factor receptor (PDGFR) and its downstream signaling pathways PLCγ, PKC, Akt and ERK1/2. PDGFR inhibition could reverse sclerostin-mediated inhibitory activity on BMP2-induced osteoblast differentiation. Therefore, our data suggest that sclerostin can activate PDGFR signaling by itself, and this functional interaction may be involved in the negative effect of sclerostin on osteoblast differentiation.

Figure 1

Sclerostin inhibits BMP2-induced osteoblast differentiation without affecting canonical BMP signaling in C3H10T1/2 cells. (a) C3H10T1/2 cells in osteogenic medium were pre-incubated with sclerostin (SOST, 5 μg ml⁻¹) or its vehicle (Veh) for 15 min and stimulated with BMP2 (50 ng ml⁻¹) or its vehicle (c) for 48 h before measurements of ALP activity. (b) Expression of osteoblast marker genes in the same conditions as for (a). (c) Evaluation of SMAD transcriptional activity in C3H10T1/2 cells pre-incubated with vehicle, 10 μg ml⁻¹ sclerostin or 250 ng ml⁻¹ noggin and stimulated with 25 ng ml⁻¹ BMP2 or its vehicle for 16 h. (d) Western blot analyses of SMAD1/5 phosphorylation in C3H10T1/2 cells pre-treated with 10 μg ml⁻¹ sclerostin or its vehicle and stimulated with 25 ng ml⁻¹ BMP2 or its vehicle for different incubation times (nsp, non-specific protein). *P<0.01 for BMP2 versus control; +P<0.01 for SOST versus vehicle treatment.

Figure 2

Sclerostin inhibits Wnt3a-induced osteoblast differentiation without affecting canonical Wnt signaling in C3H10T1/2 cells. (a) ALP activity of C3H10T1/2 cells pre-incubated with 10 μg ml⁻¹ sclerostin, 1 μg ml⁻¹ dikkopf 1 (DKK1) or vehicle and stimulated with 20% control-conditioned medium or Wnt3a-conditioned medium for 3 days. (b) Evaluation of β-catenin transcriptional activity in C3H10T1/2 cells pre-incubated with 10 μg ml⁻¹ sclerostin, 1 μg ml⁻¹ dikkopf 1 or vehicle and stimulated with 20% control-conditioned medium or Wnt3a-conditioned medium for 24 h. (c) Western blot analyses of LRP5/6 phosphorylation in C3H10T1/2 cells stimulated with Wnt3a-conditioned medium for different incubation times; and analysis of LRP5/6 phosphorylation in C3H10T1/2 cells pre-incubated with 10 μg ml⁻¹ sclerostin, 1 μg ml⁻¹ dikkopf 1 or vehicle and stimulated with 200 ng ml⁻¹ recombinant Wnt3a or its vehicle for 3 h. *P<0.01 for Wnt3a versus control; +P<0.01 for SOST or DKK1 versus vehicle treatment.

Figure 3

Sclerostin inhibits mineralization induced by GSK3 inhibition in C3H10T1/2 cells. (a) Quantification of matrix mineralization evaluated by Alizarin Red-S staining of C3H10T1/2 cells incubated with either vehicle, 20% Wnt3a-conditioned medium, 25 ng ml⁻¹ BMP2 or 10 μM SB216763 (a selective GSK3 inhibitor), with or without 10 μg ml⁻¹ sclerostin. (b) Quantification of matrix mineralization by C3H10T1/2 cells incubated with 10 μM SB216763 or its vehicle, in the presence of various doses of sclerostin. *P<0.01 for Wnt3a, BMP2 or SB216763 versus their vehicle; +P<0.01 for SOST versus its vehicle.

Figure 4

Sclerostin activates PLCγ, PKC, Akt and ERK1/2 signaling in C3H10T1/2 and MC3T3-E1 cells. (a) C3H10T1/2 and (b) MC3T3-E1 cells were exposed to 10 μg ml⁻¹ sclerostin for various incubation times before analysis of signaling proteins by western blotting. The arrows show a protein of approximately 130 kDa, which is phosphorylated on tyrosine residues in response to sclerostin. ERK, extracellular signal-regulated kinase; PKC, protein kinase C; PLCγ, phospholipase Cγ.

Figure 5

Sclerostin activates PDGFR signaling in C3H10T1/2 and MC3T3-E1 cells. (a) C3H10T1/2 and (b) MC3T3-E1 cells were treated with vehicle, 10 μg ml⁻¹ sclerostin or 2–10 ng ml⁻¹ PDGF-BB for various incubation times before analysis of signaling proteins by western blotting. PDGFR, platelet-derived growth factor receptor.

Figure 6

PDGFR inhibition reversed sclerostin-mediated inhibition of osteoblast differentiation. Alkaline phosphatase activity of C3H10T1/2 cells pre-incubated with 1 μM of the selective PDGFR inhibitor III or its vehicle, with sclerostin or its vehicle, and then stimulated with 25 ng ml⁻¹ BMP2 or its vehicle for 2 days. *P<0.01 for BMP2 versus its vehicle; +P<0.01 for SOST versus its vehicle.

Figure 7

Sclerostin activates PDGFR signaling and inhibits differentiation of primary osteoblasts. (a) Primary mouse osteoblasts were pre-treated with 10 μg ml⁻¹ sclerostin or its vehicle and stimulated or not with 100 ng ml⁻¹ BMP2 for 48 h before evaluation of ALP activity. (b) Western blot analyses of PDGFR signaling in primary mouse osteoblasts incubated with 10 μg ml⁻¹ sclerostin for different periods. *P< 0.01 for BMP2 versus its vehicle; +P<0.01 for SOST versus its vehicle.