Additional file 1: Figure S1. of Betulinic acid synergically enhances BMP2-induced bone formation via stimulating Smad 1/5/8 and p38 pathways

Radiographic study of the ectopic bone formation after treatment of BetA, low dose of BMP2 and BetA/BMP2. BetA (25 and 50 μg) with or without BMP2 (1 μg) was administered with absorbable collagen sponges into the subcutaneous spaces in the back of mice, as in Fig. 4. After 4 weeks, ectopic bone formation was analyzed by Soft X-ray (A), μ-CT (B), and quantified by using a CT-Analyzer program (C). Dotted circles in (A) indicate the new ectopic bones. ** , p < 0.01 compared to the control group (collagen sponge alone). NS, not significant. Representative data are shown. n = 5. (PDF 114 kb

FGF2 Stimulates COUP-TFII Expression via the MEK1/2 Pathway to Inhibit Osteoblast Differentiation in C3H10T1/2 Cells

<div><p>Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an orphan nuclear receptor that regulates many key biological processes, including organ development and cell fate determination. Although the biological functions of COUP-TFII have been studied extensively, little is known about what regulates its gene expression, especially the role of inducible extracellular factors in triggering it. Here we report that COUP-TFII expression is regulated specifically by fibroblast growth factor 2 (FGF2), which mediates activation of the MEK1/2 pathway in mesenchymal lineage C3H10T1/2 cells. Although FGF2 treatment increased cell proliferation, the induction of COUP-TFII expression was dispensable. Instead, FGF2-primed cells in which COUP-TFII expression was induced showed a low potential for osteoblast differentiation, as evidenced by decreases in alkaline phosphatase activity and osteogenic marker gene expression. Reducing COUP-TFII by U0126 or siRNA against COUP-TFII prevented the anti-osteogenic effect of FGF2, indicating that COUP-TFII plays a key role in the FGF2-mediated determination of osteoblast differentiation capability. This report is the first to suggest that FGF2 is an extracellular inducer of COUP-TFII expression and may suppress the osteogenic potential of mesenchymal cells by inducing COUP-TFII expression prior to the onset of osteogenic differentiation.</p></div

Repair of critical-sized cranial defects by combination of COMP-Ang1 and BMP2.

<p>The 5 mm diameter of critical-sized defect was created in the cranium of mice, and COMP-Ang1 (12 μg), BMP2 (4 μg), and COMP-Ang1 (12 μg) plus BMP2 (4 μg) with absorbable collagen sponges were implanted into the defects. Three weeks after surgery, newly formed cranial bone from each group were harvested and analyzed by μ-CT. A, Representative radiographic findings of cranial repairs were shown. Upper panel shows 2-dimensional sagittal views of cranial bone and lower panel shows the dorsal view of 3-dimensionally surface renderings. B, Volume and thickness of regenerated bone in the defects was quantified by CT-Analyzer program. *, <i>p <</i>0.05, and **, <i>p <</i>0.01 compared to control group, respectively. ^#, <i>p <</i>0.05 and ^##, <i>p <</i>0.01, compared to the indicated group. n = 4.</p

FGF2 induces COUP-TFII expression in C3H10T1/2 cells.

<p>(A) C3H10T1/2 cells were cultured in 0.1% FBS-containing DMEM for 24 h and then were treated with the indicated amounts of several extracellular factors. After 24 h, the cells were harvested, and the expression of COUP-TFII was analyzed by real-time RT-PCR. Relative COUP-TFII expression was calculated after normalization to β-actin. (B) C3H10T1/2 cells were cultured in 0.1% or 2% FBS-containing DMEM for 24 h with several extracellular factors, as in panel A. Cell lysates were applied to immunoblotting to analyze COUP-TFII protein levels. The level of β-actin was analyzed as a loading control. Numbers below gel images represent the normalized value of relative COUP-TFII levels. (C, E) After the cells were treated with the indicated amounts of FGF2 for 24 h in the same conditions as in panel A, the expression of COUP-TFII was analyzed by conventional RT-PCR analysis (upper panel) and real-time RT-PCR (lower panel), and by immunoblot analysis. (D, F) Cells were incubated with 10 ng/mL of FGF2 for the indicated time period, cell lysates were prepared, and the expression of COUP-TFII was analyzed as in panels C and E. (G) Effects of repeat treatment of FGF2 on COUP-TFII expression. C3H10T1/2 cells were incubated with 10 ng/mL of FGF2 and were then harvested at the indicated time points to undergo immunoblot analysis. The cells were pre-exposed to 10 ng/mL of FGF2 for first 72 h and then re-exposed (indicated as ball-nocks). COUP-TFII expression was analyzed by means of immunoblot analysis. (A-D) Values for the relative expression of the COUP-TFII gene are expressed as the mean ± SEM of a triplicate reaction of one representative experiment. All experiments were repeated three times. Statistical analysis was performed by ANOVA followed by the Tukey post hoc test. (E-G) Immunoblot bands were quantified by densitometry using Science Lab Image Gauge version 3.0 software (Fujifilm), and the ratio of COUP-TFII/β-actin was determined. Data shown are representative of three independent experiments, and the values are expressed as the mean ± SD of three independent experiments. Statistical analysis was performed by ANOVA with the Bonferroni post hoc test. * p<0.01; ** <i>p</i><0.01; *** <i>p</i><0.001 vs. control.</p

Effects of COMP-Ang1 and BMP2 on migration and osteogenic differentiation of pericytes.

<p>(A, B) Migration assay. Human microvascular pericytes were seeded in a transwell chamber with 8-μm pores and maintained with DMEM containing COMP-Ang1 (600 ng/ml) and/or BMP2 (200 ng/ml). After 6 hours, infiltrated cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution, and then counted under a light microscope. (C) Cells were cultured with DMEM containing 50 μg/ml of ascorbic acid and 5 mM of β-glycerophosphate in the presence of COMP-Ang1 (600 ng/ml) and/or BMP2 (200 ng/ml). After 3 days, cells were harvested and RT-PCR (upper panel) was performed with specific primers for bone sialoprotein, osteocalcin, osterix and β-actin. For Western blot analysis (lower panel), cells were harvested 1 hour after COMP-Ang1 and BMP2 treatment. (D) Calcium deposition assay. Human microvascular pericytes were cultured as in C. After 15 days, cells were stained with alizarin red solution, and then scanned (upper panel). For quantification, the stain was eluted with 10% cetylpyridinium and absorbance was measured by spectrophotometry. *, <i>p<</i>0.05, and **, <i>p <</i>0.01, as compared to control group, respectively. ^#, <i>p<</i>0.05 and ^##, <i>p <</i>0.01, as compared to the indicated group. n = 3.</p

COUP-TFII induction by FGF2 priming leads to a reduction in osteodifferentiation potential.

<p>(A, B) Pre-exposure to FGF2 prior to differentiation stimuli inhibits osteoblast differentiation of C3H10T1/2 cells. (A) Cells were treated with 10 ng/mL of FGF2 every other day for 4 days. After removing FGF2-containing media, cells were incubated with osteogenic media (OM) (50 μg/mL of ascorbic acid, 5 mM of β-glycerophosphate, and 100 ng/mL of BMP2). Cells were stained for alkaline phosphatase activity after 5 days of differentiation (left, ALP staining), and were subjected to alizarin red staining after 10 days of differentiation (right, AR staining). The bar graph shows the relative intensity of AR staining. Cells stained with AR were incubated in 10% cetylpyridinium chloride, and staining was quantified at 562 nm. The ratio of OM/GM was determined. (B) After cells were prepared as in panel A, cells were harvested on day 5 for COUP-TFII, ALP, and Osterix, and on day 10 after differentiation for BSP and osteocalcin (Oc). Total RNA was isolated and subjected to real-time RT-PCR. (C-F) Blocking of COUP-TFII induction abolished the anti-osteogenic effect of FGF2 priming. (C) After COUP-TFII–silenced cells were pretreated with FGF2 as in panel A, osteogenic differentiation was induced for 4 days. Alkaline phosphatase activity in the differentiated cells was analyzed by ALP staining. Control, non-FGF2 treated and control siRNAs-transfected cells; FGF2-primed control, FGF2-treated and control siRNA-transfected cell. (D) Osteogenic differentiated cells were harvested and subjected to real-time RT-PCR to analyze expression levels of Osterix (on day 4), ALP (on day 2), BSP and Oc (on day 10). The relative expression levels of COUP-TFII and Runx2 were analyzed on day 0 (that is, before the onset of differentiation). (E) Cells were pretreated with FGF2 as in panel A in the presence or absence of U0126, and the cells then underwent osteogenic differentiation for 4 days. Alkaline phosphatase activity was determined by ALP staining, and magnified images of the differentiated cells are representative of the relevant wells (left). (F) Before the onset of differentiation (day 0), the cells were harvested for analysis of COUP-TFII levels. The relative expression levels of ALP (on day 2), Osterix (on day 4), and Oc (on day 10) were determined. Representative data from three independent experiments are shown. Values for the relative expression of the indicated genes are expressed as the mean ± SEM of triplicate reactions in one representative experiment. Statistical analysis was performed by ANOVA followed by the Tukey post hoc test. * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001. (G) Working model for the role of overexpressed COUP-TFII in the FGF2-primed mesenchymal cells. FGF2 priming in uncommitted mesenchymal cells induces COUP-TFII expression via the MEK1/2 pathway and it might bring about low osteogenic potential and high pluripotency.</p

Immunofluorescent analysis for vascular formation and pericyte recruitment in defected regions.

<p>(A) Representative images of immunofluorescence staining with primary anti-CD31 and anti-NG2 antibodies. Immunoreactivities for CD31 (a marker of endothelial cells, red) or NG2 (a specific marker of pericyte, green) were visualized by Alexa Fluor 488-conjugated donkey anti-mouse IgG and Alexa Fluor 555-conjugated goat anti-rabbit IgG. For co-localization of endothelial cells and pericytes, the immunofluorescence images were merged (orange). DAPI (blue) was used as a nuclear counterstain. (B) Fold changes of immunofluorescence intensity. Immunoreactivities against anti-CD31 and anti-NG2 were quantified by using image analysis software (Carl Zeiss LSM software). *, <i>p<</i>0.05, and **, <i>p <</i>0.01 compared to control group, respectively. ^##, <i>p <</i>0.01, as compared to the indicated group. n = 4.</p

Microphotographs of regenerated cranial bone by COMP-Ang1 or/and BMP2.

<p>The samples of radiographic study (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140502#pone.0140502.g001" target="_blank">Fig 1</a>) were prepared for histology. Sagittal sections through the midline of defects are shown with hematoxylin and eosin stain. Arrowheads in left panel indicate margins of the trephine defect (X40; Bar, 1 mm). Right panel is the magnified images of box areas in left panel (X200; Bar, 0.1 mm).</p

FGF2-induced COUP-TFII expression mediates the MEK1/2 signaling pathway.

<p>(A, B) C3H10T1/2 cells were pretreated with DMSO, 5 μM of U0126, 10 μM of PD98059 (PD), 5 μM of LY294002 (LY), 10 μM of SP600125 (SP), or 10 μM of SB202190 (SB) for 30 min, and then FGF2 was added at a concentration of 10 ng/mL. After 24 h, the expression of COUP-TFII was analyzed by means of real-time RT-PCR (A) and immunoblot analysis (B). Relative COUP-TFII expression was calculated after normalization to β-actin (A). (C, D) FGF2 induction of COUP-TFII expression was abolished by U0126 but not by PD98059. Cells were pretreated with the indicated amounts of U0126 or PD98059 for 30 min, and then FGF2 was added. After 24 h, cell lysates were prepared, and COUP-TFII expression was analyzed by real-time RT-PCR and by immunoblot analysis. (E, F) MEK1 and MEK2 can induce COUP-TFII expression. C3H10T1/2 cells were transfected with the indicated siRNAs (E). After 24 h, the cells were incubated with FGF2 for 24 h. Cell lysates were analyzed by immunoblotting for the indicated proteins. Cells transfected with the indicated siRNAs were co-treated with FGF2 and compounds (10 μM of U0126 and 20 μM of PD98059) (F), as in (D). The levels of COUP-TFII and MEK2 were analyzed by immunoblot analysis. (G) Time course effect of FGF2 treatment on COUP-TFII, c-Fos, c-Jun, and Cyclin D1 expression. Cells were treated with FGF2 and then harvested at the indicated time points to analyze COUP-TFII, c-Jun, c-Fos, and Cyclin D1 protein levels by immunoblot analysis. The relative protein levels of the indicated proteins were calculated after normalization to β-actin. (H) Cells were prepared as in (D), and COUP-TFII, c-Jun, c-Fos, and Cyclin D1 protein levels were analyzed by immunoblotting, and their relative level was calculated after normalization to the β-actin level. All immunoblot data shown are representative of three independent experiments, and the values are expressed as the mean ± SD of three independent experiments. Statistical analysis was performed by ANOVA with the Bonferroni post hoc test. * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001 vs. control, # <i>p</i><0.05; ## <i>p</i><0.01; ### <i>p</i><0.001 vs. indicated group.</p

The extracellular matrix protein Edil3 stimulates osteoblast differentiation through the integrin α5β1/ERK/Runx2 pathway

<div><p>Epidermal growth factor-like repeats and discoidin I-like domain 3 (Edil3) is an extracellular matrix protein containing an Arg-Gly-Asp (RGD) motif that binds integrin. Recently, Edil3 has been implicated in various biological processes, including angiogenesis and cellular differentiation. It can inhibit inflammatory bone destruction. The objective of this study was to explore the role of Edil3 in osteoblast differentiation and its underlying molecular mechanisms. In wild-type mice, high expression levels of Edil3 mRNA were observed in isolated calvaria and tibia/femur bones. Immunohistochemical analysis showed that Edil3 protein was localized along periosteum and calcified regions surrounding bone tissues. When murine calvaria-derived MC3T3-E1 cells were cultured in osteogenic medium containing 50 μg/ml ascorbic acid and 5 mM β-glycerophosphate, Edil3 mRNA and protein expression levels were increased. Treatment with Edil3 protein in growth media increased expression levels of alkaline phosphatase and osteocalcin gene and phosphorylation level of extracellular signal-regulated kinase (ERK). Edil3 treatment with osteogenic medium induced mineralization. Treatment with a neutralizing antibody against α5β1 and MEK inhibitor U0126 inhibited Edil3-enhanced osteogenic marker gene expression and mineral deposition. Edil3 increased protein expression levels of transcription factor runt-related transcription factor2 (Runx2). Edil3-induced Runx2 protein expression was suppressed by pretreatment with U0126. Taken together, these results suggest that Edil3 may stimulate osteoblast differentiation and matrix mineralization by increasing expression of Runx2 through α5β1 integrin /ERK pathway.</p></div