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: Journal of Biological Chemistry

Article Title: Sclerostin Is a Novel Secreted Osteoclast-derived Bone Morphogenetic Protein Antagonist with Unique Ligand Specificity

doi: 10.1074/jbc.m301716200

Figure Lengend Snippet: FIG. 1. Expression of sclerostin during mouse development. A, frontal section of E11 head. The only tissue expressing sclerostin mRNA is the endothelium of the pharyngeal artery (arrow). B, intense punc- tuated expression is seen at the sites of mandibular and maxillary bones at E15 (arrows). Meckel’s cartilage is negative. C, Bsp mRNA expression in osteoblasts marks the extent of bone formation at E16. D, scattered sclerostin mRNA-expressing cells are present on the surfaces of all bones in the head of a newborn mouse. The cells are abundant in the bone surrounding the growing tooth germs (arrows). E, in develop- ing long bones of E16 and E18 mouse embryos, sclerostin mRNA ex- pression is seen in the perichondrium and periosteum as well as in trabecular bone but not in the cartilage. White grains in dark field images indicate the expression of sclerostin mRNA. F, in a section through the ribs of E17 embryo, sclerostin mRNA is expressed in iso- lated large cells in the cartilage perichondrium. G, in the liver of E12 embryo, sclerostin mRNA expression is intense in hematopoietic cells. M, molar tooth germ; T, tongue; MC, Meckel’s cartilage; R, resting chondrocytes; P, proliferating chondrocytes; H, hypertrophic chondro- cytes; TB, trabecular bone; PC, perichondrium; PO, periosteum; RC, rib cartilage.

Article Snippet: After the cells had reached confluence, the medium was replaced with -minimum essential medium containing 100 units/ml penicillin G, 100 g/ml streptomycin, 10 mM -glycerophosphate, and 50 g/ml ascorbic acid, and cells were cultured for 24 h. The cells were then cultured in -minimum essential medium containing 1% fetal bovine serum, 100 units/ml penicillin G, 100 g/ml streptomycin, 10 mM -glycerophosphate, 50 g/ml ascorbic acid, 10 ng/ml recombinant human BMP2 (25 ng/ml), BMP4 (10 ng/ml), BMP6 (10 ng/ml), or BMP7 (25 ng/ml) protein (R&D Systems), either and 0–100 ng/ml recombinant mouse sclerostin protein or 100 ng/ml recombinant mouse noggin/Fc chimera (R&D Systems) for 72 h. Cells were washed twice with ice-cold PBS and scraped in 10 mM Tris-HCl-containing 2 mM MgCl2 and 0.05% Triton X-100, pH 8.2.

Techniques: Expressing

Journal: Journal of Biological Chemistry

Article Title: Sclerostin Is a Novel Secreted Osteoclast-derived Bone Morphogenetic Protein Antagonist with Unique Ligand Specificity

doi: 10.1074/jbc.m301716200

Figure Lengend Snippet: FIG. 2. Codistribution of sclerostin and MMP-9. The patterns of cells expressing sclerostin mRNA and MMP-9 (a marker of osteoclasts) mRNA are similar in the mandibular bone at E15 (A and B), in calvarial bone in the newborn mouse (NB) (C and D), and in the mandibular bone around the tooth germ in the newborn mouse (E and F).

Article Snippet: After the cells had reached confluence, the medium was replaced with -minimum essential medium containing 100 units/ml penicillin G, 100 g/ml streptomycin, 10 mM -glycerophosphate, and 50 g/ml ascorbic acid, and cells were cultured for 24 h. The cells were then cultured in -minimum essential medium containing 1% fetal bovine serum, 100 units/ml penicillin G, 100 g/ml streptomycin, 10 mM -glycerophosphate, 50 g/ml ascorbic acid, 10 ng/ml recombinant human BMP2 (25 ng/ml), BMP4 (10 ng/ml), BMP6 (10 ng/ml), or BMP7 (25 ng/ml) protein (R&D Systems), either and 0–100 ng/ml recombinant mouse sclerostin protein or 100 ng/ml recombinant mouse noggin/Fc chimera (R&D Systems) for 72 h. Cells were washed twice with ice-cold PBS and scraped in 10 mM Tris-HCl-containing 2 mM MgCl2 and 0.05% Triton X-100, pH 8.2.

Techniques: Expressing, Marker

Journal: Journal of Biological Chemistry

Article Title: Sclerostin Is a Novel Secreted Osteoclast-derived Bone Morphogenetic Protein Antagonist with Unique Ligand Specificity

doi: 10.1074/jbc.m301716200

Figure Lengend Snippet: FIG. 3. Detection of recombinant mouse sclerostin protein. A, the cell lysate and culture medium of cells expressing recombinant mouse sclerostin protein were separated by SDS-polyacrylamide gel electrophoresis under reducing (with 1,4-dithiothreitol; DTT) or non- reducing (without 1,4-dithiothreitol; DTT) conditions followed by Western blotting analysis with anti-E tag antibodies. B, purified recom- binant mouse sclerostin (0.35 g) was separated by SDS-polyacryl- amide gel electrophoresis under reducing conditions and subjected to protein staining.

Article Snippet: After the cells had reached confluence, the medium was replaced with -minimum essential medium containing 100 units/ml penicillin G, 100 g/ml streptomycin, 10 mM -glycerophosphate, and 50 g/ml ascorbic acid, and cells were cultured for 24 h. The cells were then cultured in -minimum essential medium containing 1% fetal bovine serum, 100 units/ml penicillin G, 100 g/ml streptomycin, 10 mM -glycerophosphate, 50 g/ml ascorbic acid, 10 ng/ml recombinant human BMP2 (25 ng/ml), BMP4 (10 ng/ml), BMP6 (10 ng/ml), or BMP7 (25 ng/ml) protein (R&D Systems), either and 0–100 ng/ml recombinant mouse sclerostin protein or 100 ng/ml recombinant mouse noggin/Fc chimera (R&D Systems) for 72 h. Cells were washed twice with ice-cold PBS and scraped in 10 mM Tris-HCl-containing 2 mM MgCl2 and 0.05% Triton X-100, pH 8.2.

Techniques: Recombinant, Expressing, Polyacrylamide Gel Electrophoresis, Western Blot, Purification, Nucleic Acid Electrophoresis, Staining

Journal: Journal of Biological Chemistry

Article Title: Sclerostin Is a Novel Secreted Osteoclast-derived Bone Morphogenetic Protein Antagonist with Unique Ligand Specificity

doi: 10.1074/jbc.m301716200

Figure Lengend Snippet: FIG. 4. Effects of sclerostin and nog- gin on alkaline phosphatase activity in MC3T3-E1 cells induced by BMPs. MC3T3-E1 cells were treated with BMP6 (10 ng/ml) (A), BMP7 (25 ng/ml) (B), BMP2 (25 ng/ml) (C), or BMP4 (10 ng/ml) (D) and different concentrations of mouse recombinant sclerostin or 100 ng/ml nog- gin for 72 h. After treatment, alkaline phosphatase activity in MC3T3-E1 cells was determined. Results are the means S.D. for five independent wells.

Article Snippet: After the cells had reached confluence, the medium was replaced with -minimum essential medium containing 100 units/ml penicillin G, 100 g/ml streptomycin, 10 mM -glycerophosphate, and 50 g/ml ascorbic acid, and cells were cultured for 24 h. The cells were then cultured in -minimum essential medium containing 1% fetal bovine serum, 100 units/ml penicillin G, 100 g/ml streptomycin, 10 mM -glycerophosphate, 50 g/ml ascorbic acid, 10 ng/ml recombinant human BMP2 (25 ng/ml), BMP4 (10 ng/ml), BMP6 (10 ng/ml), or BMP7 (25 ng/ml) protein (R&D Systems), either and 0–100 ng/ml recombinant mouse sclerostin protein or 100 ng/ml recombinant mouse noggin/Fc chimera (R&D Systems) for 72 h. Cells were washed twice with ice-cold PBS and scraped in 10 mM Tris-HCl-containing 2 mM MgCl2 and 0.05% Triton X-100, pH 8.2.

Techniques: Activity Assay, Recombinant

Journal: Journal of Biological Chemistry

Article Title: Sclerostin Is a Novel Secreted Osteoclast-derived Bone Morphogenetic Protein Antagonist with Unique Ligand Specificity

doi: 10.1074/jbc.m301716200

Figure Lengend Snippet: FIG. 5. Binding of sclerostin to BMP6. Mouse recombinant sclerostin was fixed on the carboxylmethyl sensor tip. The binding of different concentrations of BMP6 on the tip was analyzed using the BIAcore 2000 system.

Article Snippet: After the cells had reached confluence, the medium was replaced with -minimum essential medium containing 100 units/ml penicillin G, 100 g/ml streptomycin, 10 mM -glycerophosphate, and 50 g/ml ascorbic acid, and cells were cultured for 24 h. The cells were then cultured in -minimum essential medium containing 1% fetal bovine serum, 100 units/ml penicillin G, 100 g/ml streptomycin, 10 mM -glycerophosphate, 50 g/ml ascorbic acid, 10 ng/ml recombinant human BMP2 (25 ng/ml), BMP4 (10 ng/ml), BMP6 (10 ng/ml), or BMP7 (25 ng/ml) protein (R&D Systems), either and 0–100 ng/ml recombinant mouse sclerostin protein or 100 ng/ml recombinant mouse noggin/Fc chimera (R&D Systems) for 72 h. Cells were washed twice with ice-cold PBS and scraped in 10 mM Tris-HCl-containing 2 mM MgCl2 and 0.05% Triton X-100, pH 8.2.

Techniques: Binding Assay, Recombinant

Journal: Journal of cellular physiology

Article Title: The Skeletal Cell-Derived Molecule Sclerostin Drives Bone Marrow Adipogenesis

doi: 10.1002/jcp.25976

Figure Lengend Snippet: SOST is able to induce adipogenesis in primary stem-like progenitors. Recombinant mouse sclerostin may induce lipid accumulation in ear mesenchymal stromal cells (eMSCs) (A). 10 ng/mL SOST induced an increase in adipogenic transcription factors Pparg (B) and Cebpa (C) after 7 days of differentiation.

Article Snippet: Recombinant mouse and human sclerostin were obtained from R&D Systems.

Techniques: Recombinant