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WSS25, a sulfated polysaccharide, inhibits RANKL-induced mouse osteoclast formation by blocking SMAD/ID1 signaling.

Chen C, Qin Y, Fang JP, Ni XY, Yao J, Wang HY, Ding K - Acta Pharmacol. Sin. (2015)

Bottom Line: In RAW264.7 cells and BMMs, WSS25 (2.5, 5, 10 μg/mL) did not affect the cell viability, but dose-dependently inhibited RANKL-induced osteoclastogenesis.In RAW264.7 cells, knockdown of Id1 attenuated RANKL-induced osteoclast differentiation, which was partially rescued by Id1 overexpression.WSS25 inhibits RANKL-induced osteoclast formation in RAW264.7 cells and BMMs by blocking the BMP-2/Smad/Id1 signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Glycochemistry and Glycobiology Laboratory, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

ABSTRACT

Aim: WSS25 is a sulfated polysaccharide extracted from the rhizome of Gastrodia elata BI, which has been found to bind to bone morphogenetic protein 2 (BMP-2) in hepatocellular cancer cells. Since BMP-2 may regulate both osteoclasts and osteoblasts, here we investigated the effects of WSS25 on osteoclastogenesis in vitro and bone loss in ovariectomized mice.

Methods: RAW264.7 cells or mouse bone marrow macrophages (BMMs) were treated with RANKL to induce osteoclastogenesis, which was assessed using TRAP staining, actin ring formation and pit formation assays, as well as bone resorption assay. Cell viability was detected with MTT assay. The mRNA levels of osteoclastogenesis-related genetic markers (TRAP, NFATc1, MMP-9 and cathepsin K) were detected using RT-PCR, while the protein levels of p-Smad1/5/8 and Id1 were measure with Western blotting. WSS25 was administered to ovariectomized mice (100 mg·kg(-1)·d(-1), po) for 3 months. After the mice were euthanized, total bone mineral density and cortical bone density were measured.

Results: In RAW264.7 cells and BMMs, WSS25 (2.5, 5, 10 μg/mL) did not affect the cell viability, but dose-dependently inhibited RANKL-induced osteoclastogenesis. Furthermore, WSS25 potently suppressed RANKL-induced expression of TRAP, NFATc1, MMP-9 and cathepsin K in RAW264.7 cells. Treatment of RAW264.7 cells with RANKL increased BMP-2 expression, Smad1/5/8 phosphorylation and Id1 expression, which triggered osteoclast differentiation, whereas co-treatment with WSS25 or the endogenous BMP-2 antagonist noggin suppressed the BMP-2/Smad/Id1 signaling pathway. In RAW264.7 cells, knockdown of Id1 attenuated RANKL-induced osteoclast differentiation, which was partially rescued by Id1 overexpression. In conformity to the in vitro experiments, chronic administration of WSS25 significantly reduced the bone loss in ovariectomized mice.

Conclusion: WSS25 inhibits RANKL-induced osteoclast formation in RAW264.7 cells and BMMs by blocking the BMP-2/Smad/Id1 signaling pathway. WSS25 administration reduces bone loss in ovariectomized mice, suggesting that it may be a promising therapeutic agent for osteoporosis.

No MeSH data available.


Related in: MedlinePlus

WSS25 reduces ovariectomy-induced bone loss in vivo. (A) Total bone mineral densities (BMD) of tibias from sham-operated mice, OVX mice, and mice treated with WSS25, strontium ranelate, or 17β-estradiol for 90 d were measured. (B) Cortical bone densities of tibias were also measured according to the above described method. (C) Each group of mice was orally administered 0.9% physiological saline, 100 mg/kg of WSS25, 500 mg/kg of SR or 25 μg/kg of 17β-estradiol every day for 90 d. Body weights were recorded every 10 d. n=6. Values are shown as the mean±SD. cP<0.01 vs sham group. eP<0.05, fP<0.01 vs OVX group.
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fig5: WSS25 reduces ovariectomy-induced bone loss in vivo. (A) Total bone mineral densities (BMD) of tibias from sham-operated mice, OVX mice, and mice treated with WSS25, strontium ranelate, or 17β-estradiol for 90 d were measured. (B) Cortical bone densities of tibias were also measured according to the above described method. (C) Each group of mice was orally administered 0.9% physiological saline, 100 mg/kg of WSS25, 500 mg/kg of SR or 25 μg/kg of 17β-estradiol every day for 90 d. Body weights were recorded every 10 d. n=6. Values are shown as the mean±SD. cP<0.01 vs sham group. eP<0.05, fP<0.01 vs OVX group.

Mentions: It has been suggested that the main reason for postmenopausal osteoporosis is bone loss caused by estrogen deficiency, which is accompanied by the production of osteoclastogenesis-related cytokines34,35. To explore whether WSS25 reduces OVX-induced bone loss, an OVX mouse model was employed to mimic postmenopausal bone loss in women36. Strontium ranelate (SR) decreased osteoclast bone resorption, increased bone formation, and prevented the bone loss normally induced by estrogen deficiency in the OVX model37. 17β-Estradiol also prevented OVX-induced bone loss in mice38. Therefore, SR and 17β-estradiol were used as positive controls. As shown in Figure 5A and 5B, OVX mice showed a significant decline in total bone mineral density (BMD) and cortical bone density (CBD) when analyzed using pQCT measurements. However, WSS25 markedly increased BMD and CBD in OVX mice, and SR and 17β-estradiol prevented OVX-induced bone loss (Figure 5A, 5B). Compared with the sham-operated group, WSS25 had no obvious effect on body weight over 90 days at the concentration tested (Figure 5C). These data suggest that WSS25 reduces ovariectomy-induced bone loss in vivo.


WSS25, a sulfated polysaccharide, inhibits RANKL-induced mouse osteoclast formation by blocking SMAD/ID1 signaling.

Chen C, Qin Y, Fang JP, Ni XY, Yao J, Wang HY, Ding K - Acta Pharmacol. Sin. (2015)

WSS25 reduces ovariectomy-induced bone loss in vivo. (A) Total bone mineral densities (BMD) of tibias from sham-operated mice, OVX mice, and mice treated with WSS25, strontium ranelate, or 17β-estradiol for 90 d were measured. (B) Cortical bone densities of tibias were also measured according to the above described method. (C) Each group of mice was orally administered 0.9% physiological saline, 100 mg/kg of WSS25, 500 mg/kg of SR or 25 μg/kg of 17β-estradiol every day for 90 d. Body weights were recorded every 10 d. n=6. Values are shown as the mean±SD. cP<0.01 vs sham group. eP<0.05, fP<0.01 vs OVX group.
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Related In: Results  -  Collection

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fig5: WSS25 reduces ovariectomy-induced bone loss in vivo. (A) Total bone mineral densities (BMD) of tibias from sham-operated mice, OVX mice, and mice treated with WSS25, strontium ranelate, or 17β-estradiol for 90 d were measured. (B) Cortical bone densities of tibias were also measured according to the above described method. (C) Each group of mice was orally administered 0.9% physiological saline, 100 mg/kg of WSS25, 500 mg/kg of SR or 25 μg/kg of 17β-estradiol every day for 90 d. Body weights were recorded every 10 d. n=6. Values are shown as the mean±SD. cP<0.01 vs sham group. eP<0.05, fP<0.01 vs OVX group.
Mentions: It has been suggested that the main reason for postmenopausal osteoporosis is bone loss caused by estrogen deficiency, which is accompanied by the production of osteoclastogenesis-related cytokines34,35. To explore whether WSS25 reduces OVX-induced bone loss, an OVX mouse model was employed to mimic postmenopausal bone loss in women36. Strontium ranelate (SR) decreased osteoclast bone resorption, increased bone formation, and prevented the bone loss normally induced by estrogen deficiency in the OVX model37. 17β-Estradiol also prevented OVX-induced bone loss in mice38. Therefore, SR and 17β-estradiol were used as positive controls. As shown in Figure 5A and 5B, OVX mice showed a significant decline in total bone mineral density (BMD) and cortical bone density (CBD) when analyzed using pQCT measurements. However, WSS25 markedly increased BMD and CBD in OVX mice, and SR and 17β-estradiol prevented OVX-induced bone loss (Figure 5A, 5B). Compared with the sham-operated group, WSS25 had no obvious effect on body weight over 90 days at the concentration tested (Figure 5C). These data suggest that WSS25 reduces ovariectomy-induced bone loss in vivo.

Bottom Line: In RAW264.7 cells and BMMs, WSS25 (2.5, 5, 10 μg/mL) did not affect the cell viability, but dose-dependently inhibited RANKL-induced osteoclastogenesis.In RAW264.7 cells, knockdown of Id1 attenuated RANKL-induced osteoclast differentiation, which was partially rescued by Id1 overexpression.WSS25 inhibits RANKL-induced osteoclast formation in RAW264.7 cells and BMMs by blocking the BMP-2/Smad/Id1 signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Glycochemistry and Glycobiology Laboratory, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

ABSTRACT

Aim: WSS25 is a sulfated polysaccharide extracted from the rhizome of Gastrodia elata BI, which has been found to bind to bone morphogenetic protein 2 (BMP-2) in hepatocellular cancer cells. Since BMP-2 may regulate both osteoclasts and osteoblasts, here we investigated the effects of WSS25 on osteoclastogenesis in vitro and bone loss in ovariectomized mice.

Methods: RAW264.7 cells or mouse bone marrow macrophages (BMMs) were treated with RANKL to induce osteoclastogenesis, which was assessed using TRAP staining, actin ring formation and pit formation assays, as well as bone resorption assay. Cell viability was detected with MTT assay. The mRNA levels of osteoclastogenesis-related genetic markers (TRAP, NFATc1, MMP-9 and cathepsin K) were detected using RT-PCR, while the protein levels of p-Smad1/5/8 and Id1 were measure with Western blotting. WSS25 was administered to ovariectomized mice (100 mg·kg(-1)·d(-1), po) for 3 months. After the mice were euthanized, total bone mineral density and cortical bone density were measured.

Results: In RAW264.7 cells and BMMs, WSS25 (2.5, 5, 10 μg/mL) did not affect the cell viability, but dose-dependently inhibited RANKL-induced osteoclastogenesis. Furthermore, WSS25 potently suppressed RANKL-induced expression of TRAP, NFATc1, MMP-9 and cathepsin K in RAW264.7 cells. Treatment of RAW264.7 cells with RANKL increased BMP-2 expression, Smad1/5/8 phosphorylation and Id1 expression, which triggered osteoclast differentiation, whereas co-treatment with WSS25 or the endogenous BMP-2 antagonist noggin suppressed the BMP-2/Smad/Id1 signaling pathway. In RAW264.7 cells, knockdown of Id1 attenuated RANKL-induced osteoclast differentiation, which was partially rescued by Id1 overexpression. In conformity to the in vitro experiments, chronic administration of WSS25 significantly reduced the bone loss in ovariectomized mice.

Conclusion: WSS25 inhibits RANKL-induced osteoclast formation in RAW264.7 cells and BMMs by blocking the BMP-2/Smad/Id1 signaling pathway. WSS25 administration reduces bone loss in ovariectomized mice, suggesting that it may be a promising therapeutic agent for osteoporosis.

No MeSH data available.


Related in: MedlinePlus