Stimulation of bone formation in cortical bone of mice treated with a receptor activator of nuclear factor-κB ligand (RANKL)-binding peptide that possesses osteoclastogenesis inhibitory activity

Abstract

To date, parathyroid hormone is the only clinically available bone anabolic drug. The major difficulty in the development of such drugs is the lack of clarification of the mechanisms regulating osteoblast differentiation and bone formation. Here, we report a peptide (W9) known to abrogate osteoclast differentiation in vivo via blocking receptor activator of nuclear factor-κB ligand (RANKL)-RANK signaling that we surprisingly found exhibits a bone anabolic effect in vivo. Subcutaneous administration of W9 three times/day for 5 days significantly augmented bone mineral density in mouse cortical bone. Histomorphometric analysis showed a decrease in osteoclastogenesis in the distal femoral metaphysis and a significant increase in bone formation in the femoral diaphysis. Our findings suggest that W9 exerts bone anabolic activity. To clarify the mechanisms involved in this activity, we investigated the effects of W9 on osteoblast differentiation/mineralization in MC3T3-E1 (E1) cells. W9 markedly increased alkaline phosphatase (a marker enzyme of osteoblasts) activity and mineralization as shown by alizarin red staining. Gene expression of several osteogenesis-related factors was increased in W9-treated E1 cells. Addition of W9 activated p38 MAPK and Smad1/5/8 in E1 cells, and W9 showed osteogenesis stimulatory activity synergistically with BMP-2 in vitro and ectopic bone formation. Knockdown of RANKL expression in E1 cells reduced the effect of W9. Furthermore, W9 showed a weak effect on RANKL-deficient osteoblasts in alkaline phosphatase assay. Taken together, our findings suggest that this peptide may be useful for the treatment of bone diseases, and W9 achieves its bone anabolic activity through RANKL on osteoblasts accompanied by production of several autocrine factors.

FIGURE 1.

Anabolic effect of W9 peptide on cortical bone in normal mice. W9 (10 mg/kg) or vehicle (PBS) was administered subcutaneously to 6-week-old female mice for 5 days (n = 8). A, soft x-ray images of femurs of treated mice. B, calcein labeling in mice treated with W9 or vehicle was carried out on days 1 and 4. After 12 h from the last administration of W9 on day 5, both femurs were extirpated and fixed with 70% ethanol. The inner and outer lines show the labeled bones on days 1 and 4, respectively. Bar, 10 μm.

FIGURE 2.

Effects of W9 on ALP activity and mineralization in mouse preosteoblastic cells and human mesenchymal stem cells. A, E1 cells were cultured with 50–200 μm W9, 200 μm W5 (negative control peptide), or 10 ng/ml BMP-2 for 5 days. ALP activity was measured by the pNPP assay and expressed as the mean ± S.D. a, p < 0.05; b, p < 0.01 versus control. Significant difference was determined using ANOVA with Dunnett's test. Mineralization of E1 cells was evaluated by alizarin red staining. They were cultured in osteoblastic differential media with 200 μm W9 or W5 for 21 days. B, hMSCs were cultured in the osteoblastic differential media with 100 and 200 μm W9 or 200 μm W5 for 4 days. Data were measured as described above and expressed as the mean ± S.D. a, p < 0.05; b, p < 0.01 versus control. Significant difference was determined using ANOVA with Dunnett's test. Mineralization of hMSCs was evaluated by alizarin red staining. They were cultured in osteoblastic differential media with 200 μm W9 for 21 days. C, mouse bone marrow cells were cultured in α-minimal essential medium containing 10% FBS in the presence of 10 nm sRANKL and 50 ng/ml M-CSF with or without 100 μm W9. After 7 days, cells were fixed and stained for TRAP and ALP. Bar, 500 μm. D, RAW264 cells were cultured in the presence of 10 nm sRANKL with or without 100 μm W9 for 5 days, and the cells were fixed and stained for TRAP. Bar, 500 μm. E, suppressive effect of W9 on osteoclastogenesis was examined in RAW264 cells cultured in the presence of 10 nm sRANKL with 0–200 μm W9 and 200 μm W5 for 4 days. TRAP activity was detected by TRAP solution assay. b, p < 0.01 versus sRANKL. Significant difference was determined using ANOVA with Dunnett's test.

FIGURE 3.

Signaling pathways involved in W9 stimulation in E1 cells. A, E1 cells were cultured in the presence of 100 μm W9 or 5 ng/ml BMP-2 with or without 2–10 μm p38 inhibitor (SB203580) for 5 days. ALP activity was measured by the pNPP method and expressed as the mean ± S.D. b, p < 0.01 versus W9; c, p < 0.05; d, p < 0.01 versus BMP-2. Significant difference was determined using ANOVA with Dunnett's test. B, postconfluent E1 cells were stimulated with 100 μm W9 peptide for 10–1080 min. Each whole cell lysate was prepared from the cells using RIPA buffer. Phosphorylated p38, total p38, and β-actin were detected by Western blotting. N, no treatment. C, E1 cells were cultured in the presence or absence of 50 ng/ml Wnt3a and 100 μm W9 for 5 days. Data were measured as described above and expressed as the mean ± S.D. c, p < 0.001 versus control; d, p < 0.01 versus W9. Significant difference was determined using Student's t test. D, postconfluent cells were stimulated with 100 μm W9 or 100 ng/ml Wnt3a for 10–1440 min, and each whole cell lysate was prepared as described above. β-Catenin and β-actin were detected by Western blotting. E, E1 cells were cultured in the presence or absence of 100 μm W9 with or without 0.08–2.0 μg/ml Dkk-1 for 5 days. F and G, E1 cells were cultured with 0.1–1.0 μg/ml Wnt5a or 0.2 and 2.0 μg/ml Wnt10b for 5 days. Wnt3a (50 ng/ml) and W9 (50 μm) were used as positive controls. Data were measured as described above and expressed as the mean ± S.D. c, p < 0.001 versus control. Significant difference was determined using Student's t test.

FIGURE 4.

Participation of BMPs in W9 stimulation in E1 cells. A, E1 cells were cultured in the presence of 50 μm W9 or 5 ng/ml BMP-2 with or without 0.25 and 1.0 μg/ml sBMPR-1A for 5 days. ALP activity was measured by the pNPP method and expressed as the mean ± S.D. b, p < 0.01 versus W9; d, p < 0.01 versus BMP-2. Significant difference was determined using ANOVA with Dunnett's test. B, postconfluent E1 cells were stimulated with 100 μm W9 for 10–360 min, and each whole cell lysate was prepared from the cells using RIPA buffer. Phosphorylated Smad1/5/8, total Smad1, and β-actin were detected by Western blotting. C, E1 cells were cultured in the presence or absence of 100 μm W9 with or without 0.4 and 2.0 μg/ml anti-BMP-2/4 neutralizing antibody (αBMP-2/4) for 5 days. Data were measured as described above and expressed as the mean ± S.D. b, p < 0.01 versus W9. Significant difference was determined using ANOVA with Dunnett's test.

FIGURE 5.

Synergistic effect of W9 with BMP-2 on osteoblast differentiation/mineralization and ectopic bone formation. A, E1 cells were cultured in the presence or absence of 25 μm W9 with or without 0.1–5 ng/ml BMP-2 for 5 days. Data were expressed as the mean ± S.D. a, p < 0.05; b, p < 0.01 versus control; c, p < 0.05; d, p < 0.01 versus W9. Significant difference was determined using ANOVA with Dunnett's test. B, mineralization of E1 cells cultured in the presence or absence of 10 ng/ml BMP-2 with or without 50 and 100 μm W9 for 10 days was evaluated by alizarin red staining. C, experimental protocol. Collagen sponge disc was used as a carrier of BMP-2. These collagen discs were freeze-dried with BMP-2 and then surgically implanted into dorsal muscle pouches. After a week, 5 or 10 mg/kg W9, 10 mg/kg W5, or vehicle (PBS) was administered subcutaneously to the implanted mice three times/day for 5 days. D, bone mineral content (BMC) of ectopic bone was measured by dual-energy x-ray absorptiometry and expressed as the mean ± S.D. b, p < 0.01 versus W5. Significant difference was determined using ANOVA with Dunnett's test. E, three-dimensional reconstruction images of ectopic bones were obtained by micro-focal computed tomography. Bar, 1 mm.

FIGURE 6.

A signaling through RANKL is involved in part in W9-induced osteoblast differentiation. A, E1 cells were cultured in the presence of 25–200 μm W9 or Y6 for 5 days. ALP activity was measured by the pNPP method. Data were expressed as the mean ± S.D. b, p < 0.01 versus each concentration of Y6. Significant difference was determined using Student's t test. B, RANKL and TNF-α gene expression was suppressed by siRNA-mediated knockdown with 10 nm stealth RNAi in E1 cells. The cells were transfected with stealth RNAi for 48 h. Upper panel, expression of RANKL, TNF-α, and GAPDH was measured by RT-PCR. Lower panel, treated cells were further cultured with 100 μm W9 or 10 ng/ml BMP-2 (B2) for 5 days. Data were measured as described above and expressed as the mean ± S.D. The ratios of A₄₀₅/each control are shown in parentheses. c, p < 0.001 versus each control. C, L929 cells were cultured with 1 μg/ml actinomycin D in the presence or absence of 25 pg/ml TNF-α with or without 25 and 100 μm W9 and W5 for 24 h. The viable cells were detected with WST-1 assay kit. b, p < 0.01 versus control. Significant difference was determined using Student's t test. D, osteoblasts from RANKL-deficient (KO) mice and wild-type mice were cultured in the presence or absence of 100 μm W9 or 40 ng/ml BMP-2 (B2) for 5 days. Data were measured as described above and expressed as the mean ± S.D. The ratios of A₄₀₅/each control are shown in parentheses. a, p < 0.05; b, p < 0.01 versus each control.

FIGURE 7.

A model illustrating a mechanism by which W9 differentiates osteoblasts; a hypothesis that a bidirectional signaling through RANKL and RANK regulates osteoblastogenesis and osteoclastogenesis. W9 binds RANKL on osteoblasts and stimulates production of autocrine factors like BMPs, IGFs, and connective tissue growth factor (CTGF) for osteoblast differentiation. The p38 and Smad1/5/8 signaling pathways are involved in W9-induced osteoblastogenesis. W9 may bind other receptors on osteoblasts to exert its activity in part. In contrast, W9 binds RANKL and sRANKL to inhibit osteoclastogenesis. In the hypothesis RANK is an endogenous ligand for RANKL. The bidirectional signaling between RANKL and RANK could regulate the coupling between bone formation and resorption. RANKL and RANK could be pivotal coupling factors.