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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 the RANKL-induced expression of Id1 in RAW264.7 cells. (A) RAW264.7 cells (1×106 cells/mL) were treated with RANKL (50 ng/mL), in the presence or absence of WSS25 (10 μg/mL), for 22 h. The expression of Id1 was detected by Western blot assay. (B) RAW264.7 cells (1×106 cells/mL) were incubated with RANKL (50 ng/mL), in presence or absence of WSS25 (10 μg/mL) and noggin (250 ng/mL), for 22 h. The expression of Id1 was detected by immunoblotting. (C) The cells were treated with BMP-2 for the indicated times and the expression of Id1 was measured by Western blot assay. (D) RAW264.7 cells were treated with BMP-2 (100 ng/mL), in the presence or absence of WSS25 (10 μg/mL) or noggin (250 ng/mL), for 60 min. Then, the cells were lysed and the extracts were probed with anti-Id1 antibody. (E) The expression of Id1 was detected by reverse transcription PCR and Western blot analysis. RAW264.7 cells, negative control cells, and Id1 gene knockdown RAW264.7 cells (1×105 cells/mL) were treated with RANKL (50 ng/mL) for 4 d. Cells were then stained by TRAP and photographed. (F) The expression of Id1 was detected by reverse transcription PCR and Western blot analysis. In a final set of cells, Id1 was first genetically knocked down in RAW264.7 cells, and then overexpressed (1×105 cells/mL). These cells were treated with RANKL (50 ng/mL) for 4 d before they were stained with TRAP and photographs were taken (×100).
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fig8: WSS25 inhibits the RANKL-induced expression of Id1 in RAW264.7 cells. (A) RAW264.7 cells (1×106 cells/mL) were treated with RANKL (50 ng/mL), in the presence or absence of WSS25 (10 μg/mL), for 22 h. The expression of Id1 was detected by Western blot assay. (B) RAW264.7 cells (1×106 cells/mL) were incubated with RANKL (50 ng/mL), in presence or absence of WSS25 (10 μg/mL) and noggin (250 ng/mL), for 22 h. The expression of Id1 was detected by immunoblotting. (C) The cells were treated with BMP-2 for the indicated times and the expression of Id1 was measured by Western blot assay. (D) RAW264.7 cells were treated with BMP-2 (100 ng/mL), in the presence or absence of WSS25 (10 μg/mL) or noggin (250 ng/mL), for 60 min. Then, the cells were lysed and the extracts were probed with anti-Id1 antibody. (E) The expression of Id1 was detected by reverse transcription PCR and Western blot analysis. RAW264.7 cells, negative control cells, and Id1 gene knockdown RAW264.7 cells (1×105 cells/mL) were treated with RANKL (50 ng/mL) for 4 d. Cells were then stained by TRAP and photographed. (F) The expression of Id1 was detected by reverse transcription PCR and Western blot analysis. In a final set of cells, Id1 was first genetically knocked down in RAW264.7 cells, and then overexpressed (1×105 cells/mL). These cells were treated with RANKL (50 ng/mL) for 4 d before they were stained with TRAP and photographs were taken (×100).

Mentions: WSS25 downregulated the phosphorylation of Smad1/5/8, and Id1 is a downstream effector of the BMP2/Smad1/5/8 signaling pathway. To investigate the influence of WSS25 on Id1, RAW264.7 cells were cultured with RANKL and treated with WSS25 for 22 h, followed by measurement of Id1 expression by Western blot analysis. Indeed, RANKL promoted the expression of Id1, while WSS25 blocked RANKL-induced expression of Id1 (Figure 8A). When cells were treated with noggin for 22 h, the expression of Id1 was also inhibited. This inhibitory effect was similar to that of WSS25 (Figure 8B). In addition, the expression of Id1 was gradually increased by BMP-2 from 0 to 60 min of treatment in RAW264.7 cells (Figure 8C). However, after treatment with WSS25 or noggin for 60 min, BMP2-induced Id1 expression was significantly suppressed (Figure 8D).


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 the RANKL-induced expression of Id1 in RAW264.7 cells. (A) RAW264.7 cells (1×106 cells/mL) were treated with RANKL (50 ng/mL), in the presence or absence of WSS25 (10 μg/mL), for 22 h. The expression of Id1 was detected by Western blot assay. (B) RAW264.7 cells (1×106 cells/mL) were incubated with RANKL (50 ng/mL), in presence or absence of WSS25 (10 μg/mL) and noggin (250 ng/mL), for 22 h. The expression of Id1 was detected by immunoblotting. (C) The cells were treated with BMP-2 for the indicated times and the expression of Id1 was measured by Western blot assay. (D) RAW264.7 cells were treated with BMP-2 (100 ng/mL), in the presence or absence of WSS25 (10 μg/mL) or noggin (250 ng/mL), for 60 min. Then, the cells were lysed and the extracts were probed with anti-Id1 antibody. (E) The expression of Id1 was detected by reverse transcription PCR and Western blot analysis. RAW264.7 cells, negative control cells, and Id1 gene knockdown RAW264.7 cells (1×105 cells/mL) were treated with RANKL (50 ng/mL) for 4 d. Cells were then stained by TRAP and photographed. (F) The expression of Id1 was detected by reverse transcription PCR and Western blot analysis. In a final set of cells, Id1 was first genetically knocked down in RAW264.7 cells, and then overexpressed (1×105 cells/mL). These cells were treated with RANKL (50 ng/mL) for 4 d before they were stained with TRAP and photographs were taken (×100).
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Related In: Results  -  Collection

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fig8: WSS25 inhibits the RANKL-induced expression of Id1 in RAW264.7 cells. (A) RAW264.7 cells (1×106 cells/mL) were treated with RANKL (50 ng/mL), in the presence or absence of WSS25 (10 μg/mL), for 22 h. The expression of Id1 was detected by Western blot assay. (B) RAW264.7 cells (1×106 cells/mL) were incubated with RANKL (50 ng/mL), in presence or absence of WSS25 (10 μg/mL) and noggin (250 ng/mL), for 22 h. The expression of Id1 was detected by immunoblotting. (C) The cells were treated with BMP-2 for the indicated times and the expression of Id1 was measured by Western blot assay. (D) RAW264.7 cells were treated with BMP-2 (100 ng/mL), in the presence or absence of WSS25 (10 μg/mL) or noggin (250 ng/mL), for 60 min. Then, the cells were lysed and the extracts were probed with anti-Id1 antibody. (E) The expression of Id1 was detected by reverse transcription PCR and Western blot analysis. RAW264.7 cells, negative control cells, and Id1 gene knockdown RAW264.7 cells (1×105 cells/mL) were treated with RANKL (50 ng/mL) for 4 d. Cells were then stained by TRAP and photographed. (F) The expression of Id1 was detected by reverse transcription PCR and Western blot analysis. In a final set of cells, Id1 was first genetically knocked down in RAW264.7 cells, and then overexpressed (1×105 cells/mL). These cells were treated with RANKL (50 ng/mL) for 4 d before they were stained with TRAP and photographs were taken (×100).
Mentions: WSS25 downregulated the phosphorylation of Smad1/5/8, and Id1 is a downstream effector of the BMP2/Smad1/5/8 signaling pathway. To investigate the influence of WSS25 on Id1, RAW264.7 cells were cultured with RANKL and treated with WSS25 for 22 h, followed by measurement of Id1 expression by Western blot analysis. Indeed, RANKL promoted the expression of Id1, while WSS25 blocked RANKL-induced expression of Id1 (Figure 8A). When cells were treated with noggin for 22 h, the expression of Id1 was also inhibited. This inhibitory effect was similar to that of WSS25 (Figure 8B). In addition, the expression of Id1 was gradually increased by BMP-2 from 0 to 60 min of treatment in RAW264.7 cells (Figure 8C). However, after treatment with WSS25 or noggin for 60 min, BMP2-induced Id1 expression was significantly suppressed (Figure 8D).

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