Peptide drugs accelerate BMP-2-induced calvarial bone regeneration and stimulate osteoblast differentiation through mTORC1 signaling

Abstract

Both W9 and OP3-4 were known to bind the receptor activator of NF-κB ligand (RANKL), inhibiting osteoclastogenesis. Recently, both peptides were shown to stimulate osteoblast differentiation; however, the mechanism underlying the activity of these peptides remains to be clarified. A primary osteoblast culture showed that rapamycin, an mTORC1 inhibitor, which was recently demonstrated to be an important serine/threonine kinase for bone formation, inhibited the peptide-induced alkaline phosphatase activity. Furthermore, both peptides promoted the phosphorylation of Akt and S6K1, an upstream molecule of mTORC1 and the effector molecule of mTORC1, respectively. In the in vivo calvarial defect model, W9 and OP3-4 accelerated BMP-2-induced bone formation to a similar extent, which was confirmed by histomorphometric analyses using fluorescence images of undecalcified sections. Our data suggest that these RANKL-binding peptides could stimulate the mTORC1 activity, which might play a role in the acceleration of BMP-2-induced bone regeneration by the RANKL-binding peptides.

Keywords: BMP-2; bone regeneration; histomorphometry; mTORC1; osteoblast differentiation; peptide therapeutics; rapamycin.

Figure 1

RANKL‐binding peptides, W9 and OP3‐4, enhanced ALP activity and mineralization during osteoblast differentiation. A: Primary osteoblasts were cultured in differentiation medium for 6 days in the presence of RANKL‐binding peptides or a control peptide at 200 µM. The osteoblasts were fixed and ALP staining was performed. Images of the ALP‐stained cells are shown. The percentage of the ALP‐stained area in each well was calculated. B: Cells were cultured for 21 days and von Kossa staining was performed. Images of the von Kossa‐stained cells are shown. The percentage of the von Kossa‐stained area in each well was calculated. The data are expressed as the means ± standard deviation for each group (n = 5). Significant differences among the groups were assessed by ANOVA. When significant F‐values were detected, then Fisher's PLSD post hoc test was performed. *p < 0.05 versus vehicle, #p < 0.05 versus W9. We performed three‐independent experiments and obtained similar results. The data are the representative results of the three experiments.

Figure 2

RANKL‐binding peptides, W9 and OP3‐4, promoted bone regeneration induced by BMP‐2 in a murine calvarial defect model. A: Soft X‐ray photographic images of the calvarial defects in which gelatin hydrogel (GH) only, and GH containing BMP‐2 (0.3 µg), BMP‐2 (0.3 µg) plus W9 (0.4 µmol), or BMP‐2 (0.3 µg) plus OP3‐4 (0.4 µmol) were applied. B and C: µ‐CT images of the whole mount of calvariae (B) and a cross‐section (C) are shown for each group. Scale bars: 2 mm. D and E: The bone mineral content (BMC) and bone mineral density (BMD) were measured at the site of the calvarial defect using dual energy X‐ray absorptiometry. The data are expressed as the means ± standard deviation for each group (n = 5). Significant differences among the groups were assessed by ANOVA. When significant F‐values were detected, then Fisher's PLSD post hoc test was performed. *p < 0.05 versus BMP group. We performed two‐independent experiments and obtained similar results. The data are the representative results of the two experiments.

Figure 3

RANKL‐binding peptides, W9 and OP3‐4, promoted mineralization in vivo. A: Fluorescence images of undecalcified frozen sections of the right side of the calvaria are shown. The lower panels indicate higher magnification views of the box shown in white in the upper panels. The scale bar represents 0.5 mm. The green color shows calcein labeling, while the red color shows the alizarin labeling. B: Quantitative analyses of bone formation activity were performed using standard bone histomorphometric measurement techniques based on the calcein‐ and alizarin red‐labeled surface in the ROI (as described in Materials and methods section), centering on the area of regenerated bone. Local bone formation activity was calculated as (mineralizing surface) × (mineral apposition rate). The data are expressed as the means ± standard deviation for each group (n = 5). Significant differences among the groups were assessed by ANOVA. When significant F‐values were detected, then Fisher's PLSD post hoc test was performed.*p < 0.05 versus BMP, #p < 0.05 versus BMP + W9. We performed two‐independent experiments and obtained similar results. The data are the representative results of the two experiments.

Figure 4

mTORC1 inhibitor blunted the stimulated effects of osteoblast differentiation by RANKL‐binding peptides. A: Rapamycin inhibits osteoblast proliferation. The indicated concentration of rapamycin (0.1–20 nM) was added to cultures of primary osteoblasts in proliferation medium. Cell proliferation was assayed for 72 hours using Cell Count Reagent SF. B: RANKL‐binding peptides did not affect osteoblast proliferation. Primary osteoblasts were cultured with vehicle, W9, OP3‐4, or the control peptide in the presence or absence of rapamycin (10 nM), and cell proliferation was assayed. C: Rapamycin inhibited osteoblast differentiation. The differentiation of primary osteoblasts was induced during 6 days of culturing in differentiation medium in the absence or presence of rapamycin (Rapa) (10 nM) and ALP activity was measured. The data are expressed as the means ± standard deviation for each group (n = 5). Significant differences among the groups were assessed by ANOVA. When significant F‐values were detected, then Fisher's PLSD post hoc test was performed. **p < 0.01 versus vehicle without Rapa, ^## p < 0.01 versus W9 without Rapa, $$p < 0.01 versus OP3‐4 without Rapa. We performed two‐independent experiments and obtained similar results. The data are the representative results of the two experiments.

Figure 5

Both W9 and OP3‐4 enhanced the phosphorylation of Akt and S6K1, a downstream effector molecule of mTORC1 in the same manner. ST‐2 cells were seeded with 4 × 10⁴ cells/ per well in a 12‐well‐plate. The cells were stimulated by several concentrations of either W9 or OP3‐4 for 20 minutes after serum‐starvation for 12 hours, and then the cells were extracted and prepared for Western blotting to observe the phosphorylation of Akt and S6K1, which shows the upstream kinase of mTORC1 signals and the mTORC1 signal, respectively. We performed two‐independent experiments and obtained similar results. The data are the representative results of the two experiments.