Journal: Cell reports

Article Title: Osteocyte intrinsic TGFβ signaling regulates bone quality through perilacunar/canalicular remodeling

doi: 10.1016/j.celrep.2017.10.115

Figure Lengend Snippet: (A–D) qPCR analysis of PLR genes Mmp13, Mmp14 and Ctsk and Serpine1 upon TGFβ (5ng/mL) treatment in MLO-Y4 (A, B) and OCY454 (C, D) cells. (n=3 replicates/group). (E, F) Intracellular pH (pHi) of MLO-Y4 cells after 3 days of TGFβ (5ng/ml), TβRI inhibitor SB-431542 (10 μM), or recombinant sclerostin (rhSCL, 10 ng/ml). The representative image (E) shows the shift in the emission peak from 580 nm to 640 nm after TGFβ treatment of MLO-Y4 cells. Scale bar, 100 μm). TGFβ-induced acidification is blocked by SB-431542 (F) (n=4 replicates/group). Error bars indicate mean ± SD of 3 independent experiments, *p<0.05 different from control mRNA, a-p<0.05 different from control pHi, b-p<0.05 different from TGFβ pHi, and c-p<0.05 different from rhSCL pHi. Statistics calculated from Student’s t test.

Article Snippet: For treatment, cells were cultured in α-MEM containing 0.5–1% fetal bovine serum, supplemented with 5 ng/ml TGFβ1 (Humanzyme, HZ-1011), 10 μM SB431542 (Sigma, S4317) or 10 ng/ml recombinant human sclerostin (rhSCL, R&D Systems) for the indicated times.

Techniques: Recombinant, Control

Journal: Cell reports

Article Title: Osteocyte intrinsic TGFβ signaling regulates bone quality through perilacunar/canalicular remodeling

doi: 10.1016/j.celrep.2017.10.115

Figure Lengend Snippet: (A, B) TβRII-stained osteocytes (A) (arrow, scale bar, 50 μm) in the femoral cortical bone from WT and TβRIIocy−/− mice (8-week old males) were quantified as percentage of positively stained osteocytes normalized to total bone area (B) (n=5 mice/group) (C) qPCR analysis of TβRII and Serpine1 in WT and TβRIIocy−/− femoral bones. (n=8–10 mice/group). (D, E) Silver nitrate stained images of WT and TβRIIocy−/− femoral cortical bone shows the osteocyte lacuno-canalicular network (D) and canalicular length (E) (scale bar, 20 μm, n=5 mice/group). (F, G) qPCR analysis of PLR genes, Mmp2, Mmp13, Mmp14, Ctsk, and Acp5 (F) and OCY-specific genes, Sost, Dmp1 and Phex (G) in the WT and TβRIIocy−/− bones (n=8–10 mice/group) (H, I) IHC of MMP13, MMP14, CTSK and H&E staining of WT and TβRIIocy−/− femoral cortical bone. Arrows in the image indicate positively stained osteocytes (H) that were quantified and normalized to total bone area (I), (n=4 mice/group).(J–M) SRμT shows volume (J), degree of anisotropy (K), orientation (L) and mineralization (N) of osteocyte lacunae of WT and TβRIIocy−/− bone (n=3–4 mice/group). Error bars indicate mean ± SEM with *p<0.05 compared to WT from Student’s t test.

Article Snippet: For treatment, cells were cultured in α-MEM containing 0.5–1% fetal bovine serum, supplemented with 5 ng/ml TGFβ1 (Humanzyme, HZ-1011), 10 μM SB431542 (Sigma, S4317) or 10 ng/ml recombinant human sclerostin (rhSCL, R&D Systems) for the indicated times.

Techniques: Staining

Journal: BoneKEy Reports

Article Title: 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

doi: 10.1038/bonekey.2015.126

Figure Lengend Snippet: 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.

Article Snippet: Recombinant human sclerostin, DKK1 and BMP2 were generous gifts from Novartis (Basel, Switzerland), Prostrakan (Romainville, France) and Wyeth Research (Cambridge, MA, USA), respectively.

Techniques: Incubation, Activity Assay, Expressing, Marker, Western Blot, Phospho-proteomics, Control

Journal: BoneKEy Reports

Article Title: 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

doi: 10.1038/bonekey.2015.126

Figure Lengend Snippet: 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.

Article Snippet: Recombinant human sclerostin, DKK1 and BMP2 were generous gifts from Novartis (Basel, Switzerland), Prostrakan (Romainville, France) and Wyeth Research (Cambridge, MA, USA), respectively.

Techniques: Activity Assay, Incubation, Control, Western Blot, Phospho-proteomics, Recombinant

Journal: BoneKEy Reports

Article Title: 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

doi: 10.1038/bonekey.2015.126

Figure Lengend Snippet: 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.

Article Snippet: Recombinant human sclerostin, DKK1 and BMP2 were generous gifts from Novartis (Basel, Switzerland), Prostrakan (Romainville, France) and Wyeth Research (Cambridge, MA, USA), respectively.

Techniques: Inhibition, Staining, Incubation

Journal: BoneKEy Reports

Article Title: 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

doi: 10.1038/bonekey.2015.126

Figure Lengend Snippet: 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.

Article Snippet: Recombinant human sclerostin, DKK1 and BMP2 were generous gifts from Novartis (Basel, Switzerland), Prostrakan (Romainville, France) and Wyeth Research (Cambridge, MA, USA), respectively.

Techniques: Incubation, Western Blot

Journal: BoneKEy Reports

Article Title: 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

doi: 10.1038/bonekey.2015.126

Figure Lengend Snippet: 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.

Article Snippet: Recombinant human sclerostin, DKK1 and BMP2 were generous gifts from Novartis (Basel, Switzerland), Prostrakan (Romainville, France) and Wyeth Research (Cambridge, MA, USA), respectively.

Techniques: Incubation, Western Blot, Derivative Assay

Journal: BoneKEy Reports

Article Title: 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

doi: 10.1038/bonekey.2015.126

Figure Lengend Snippet: 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.

Article Snippet: Recombinant human sclerostin, DKK1 and BMP2 were generous gifts from Novartis (Basel, Switzerland), Prostrakan (Romainville, France) and Wyeth Research (Cambridge, MA, USA), respectively.

Techniques: Inhibition, Activity Assay, Incubation

Journal: BoneKEy Reports

Article Title: 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

doi: 10.1038/bonekey.2015.126

Figure Lengend Snippet: 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.

Article Snippet: Recombinant human sclerostin, DKK1 and BMP2 were generous gifts from Novartis (Basel, Switzerland), Prostrakan (Romainville, France) and Wyeth Research (Cambridge, MA, USA), respectively.

Techniques: Activity Assay, Western Blot, Incubation