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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 inhibits osteogenesis at an early stage. (A) BMMs (5×104 cells/mL) were incubated with RANKL (50 ng/mL) plus M-CSF (20 ng/mL) and then treated with WSS25 (10 μg/mL) on the indicated days. TRAP-positive, multinucleated osteoclasts were stained after 7 d of RANKL stimulation, when a photograph was taken (×1000). (B) RAW264.7 cells (1×105 cells/well) were stimulated with RANKL (50 ng/mL) and then treated with WSS25 (10 μg/mL) on the indicated days. On the 4th day after WSS25 treatment, TRAP-staining was performed and a photograph was taken (×1000). (C) Cells with at least 3 nuclei were counted as multinucleated osteoclasts in BMMs. (D) Cells with at least 3 nuclei were also counted as multinucleated osteoclasts in RAW264.7 cells. n=3. Values are shown as the mean±SD. bP<0.05 vs untreated group.
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fig3: WSS25 inhibits osteogenesis at an early stage. (A) BMMs (5×104 cells/mL) were incubated with RANKL (50 ng/mL) plus M-CSF (20 ng/mL) and then treated with WSS25 (10 μg/mL) on the indicated days. TRAP-positive, multinucleated osteoclasts were stained after 7 d of RANKL stimulation, when a photograph was taken (×1000). (B) RAW264.7 cells (1×105 cells/well) were stimulated with RANKL (50 ng/mL) and then treated with WSS25 (10 μg/mL) on the indicated days. On the 4th day after WSS25 treatment, TRAP-staining was performed and a photograph was taken (×1000). (C) Cells with at least 3 nuclei were counted as multinucleated osteoclasts in BMMs. (D) Cells with at least 3 nuclei were also counted as multinucleated osteoclasts in RAW264.7 cells. n=3. Values are shown as the mean±SD. bP<0.05 vs untreated group.

Mentions: Mature osteoclasts are large and multinucleated cells that develop through a series of processes, including proliferation, differentiation and mature osteoclast formation33. To identify at which stage WSS25 suppressed osteoclast formation, its inhibitory effect on osteoclastogenesis in BMMs and RAW264.7 cells were investigated by adding WSS25 at different time points after RANKL/M-CSF induction. In BMMs, the number and size of TRAP-positive osteoclasts clearly decreased when WSS25 treatment was administered between days 2-5, but there was no such effect when WSS25 treatment followed induction by more than 5 d (Figure 3A and 3C). Similarly, WSS25 significantly blocked osteoclast formation in RAW264.7 cells when treatment occurred between days 2-3, whereas there was no such effect by WSS25 treatment when it followed induction by more than 3 days (Figure 3B and 3D). These results suggest that WSS25 inhibits RANKL-induced osteoclast formation during an early stage of osteoclastogenesis.


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 inhibits osteogenesis at an early stage. (A) BMMs (5×104 cells/mL) were incubated with RANKL (50 ng/mL) plus M-CSF (20 ng/mL) and then treated with WSS25 (10 μg/mL) on the indicated days. TRAP-positive, multinucleated osteoclasts were stained after 7 d of RANKL stimulation, when a photograph was taken (×1000). (B) RAW264.7 cells (1×105 cells/well) were stimulated with RANKL (50 ng/mL) and then treated with WSS25 (10 μg/mL) on the indicated days. On the 4th day after WSS25 treatment, TRAP-staining was performed and a photograph was taken (×1000). (C) Cells with at least 3 nuclei were counted as multinucleated osteoclasts in BMMs. (D) Cells with at least 3 nuclei were also counted as multinucleated osteoclasts in RAW264.7 cells. n=3. Values are shown as the mean±SD. bP<0.05 vs untreated group.
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fig3: WSS25 inhibits osteogenesis at an early stage. (A) BMMs (5×104 cells/mL) were incubated with RANKL (50 ng/mL) plus M-CSF (20 ng/mL) and then treated with WSS25 (10 μg/mL) on the indicated days. TRAP-positive, multinucleated osteoclasts were stained after 7 d of RANKL stimulation, when a photograph was taken (×1000). (B) RAW264.7 cells (1×105 cells/well) were stimulated with RANKL (50 ng/mL) and then treated with WSS25 (10 μg/mL) on the indicated days. On the 4th day after WSS25 treatment, TRAP-staining was performed and a photograph was taken (×1000). (C) Cells with at least 3 nuclei were counted as multinucleated osteoclasts in BMMs. (D) Cells with at least 3 nuclei were also counted as multinucleated osteoclasts in RAW264.7 cells. n=3. Values are shown as the mean±SD. bP<0.05 vs untreated group.
Mentions: Mature osteoclasts are large and multinucleated cells that develop through a series of processes, including proliferation, differentiation and mature osteoclast formation33. To identify at which stage WSS25 suppressed osteoclast formation, its inhibitory effect on osteoclastogenesis in BMMs and RAW264.7 cells were investigated by adding WSS25 at different time points after RANKL/M-CSF induction. In BMMs, the number and size of TRAP-positive osteoclasts clearly decreased when WSS25 treatment was administered between days 2-5, but there was no such effect when WSS25 treatment followed induction by more than 5 d (Figure 3A and 3C). Similarly, WSS25 significantly blocked osteoclast formation in RAW264.7 cells when treatment occurred between days 2-3, whereas there was no such effect by WSS25 treatment when it followed induction by more than 3 days (Figure 3B and 3D). These results suggest that WSS25 inhibits RANKL-induced osteoclast formation during an early stage of osteoclastogenesis.

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