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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 RANKL-induced actin ring structure formation and the expression of osteoclastogenesis-related gene markers. (A) Actin ring structures were stained red by phalloidin, and nuclei were stained blue by DAPI. Images of actin rings and nuclei were merged. (B) RAW264.7 cells (1×105 cells/well) were incubated with RANKL (50 ng/mL) in the presence or absence of various concentrations of WSS25 (2.5, 5 and 10 μg/mL) for 4 d. Next, the cells were stained with phalloidin to detect actin rings and with DAPI to detect nuclei. (C) Osteoclasts with actin ring structures were counted. n=3. Values are shown as the mean±SD. bP<0.05, cP<0.01 vs 0 μg/mL WSS25+RANKL group. (D) RAW264.7 cells (1×105 cells/mL) were treated with RANKL (50 ng/mL) and various concentrations of WSS25. The total RNA was extracted and the mRNA expression levels of TRAP, NFATc1, cathepsin K, and MMP-9 were measured by reverse transcription PCR. (E) The expression levels of NFATc1, cathepsin K, MMP-9 and TRAP were calculated by Image J. 1–5 on the x-axis refers to panel 1–5 in (D). n=3. Values are shown as the mean±SD. bP<0.05, cP<0.01 vs 0 μg/mL WSS25+RANKL group.
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fig2: WSS25 inhibits RANKL-induced actin ring structure formation and the expression of osteoclastogenesis-related gene markers. (A) Actin ring structures were stained red by phalloidin, and nuclei were stained blue by DAPI. Images of actin rings and nuclei were merged. (B) RAW264.7 cells (1×105 cells/well) were incubated with RANKL (50 ng/mL) in the presence or absence of various concentrations of WSS25 (2.5, 5 and 10 μg/mL) for 4 d. Next, the cells were stained with phalloidin to detect actin rings and with DAPI to detect nuclei. (C) Osteoclasts with actin ring structures were counted. n=3. Values are shown as the mean±SD. bP<0.05, cP<0.01 vs 0 μg/mL WSS25+RANKL group. (D) RAW264.7 cells (1×105 cells/mL) were treated with RANKL (50 ng/mL) and various concentrations of WSS25. The total RNA was extracted and the mRNA expression levels of TRAP, NFATc1, cathepsin K, and MMP-9 were measured by reverse transcription PCR. (E) The expression levels of NFATc1, cathepsin K, MMP-9 and TRAP were calculated by Image J. 1–5 on the x-axis refers to panel 1–5 in (D). n=3. Values are shown as the mean±SD. bP<0.05, cP<0.01 vs 0 μg/mL WSS25+RANKL group.

Mentions: Actin ring formation is a prerequisite for the resorption of osteoclasts and is the most distinct marker of mature osteoclasts during osteoclast differentiation32. To further investigate the effect of WSS25 on osteoclast differentiation, we examined whether WSS25 blocks RANKL-induced actin ring formation in osteoclasts. After stimulation by RANKL, RAW264.7 cells differentiated into mature osteoclasts and formed obvious actin ring structures (Figure 2A). However, the number and size of actin rings gradually decreased as WSS25 concentrations increased (2.5, 5 and 10 μg/mL) (Figure 2B and 2C). This indicated that WSS25 inhibited the formation of actin rings in a dose-dependent manner.


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 RANKL-induced actin ring structure formation and the expression of osteoclastogenesis-related gene markers. (A) Actin ring structures were stained red by phalloidin, and nuclei were stained blue by DAPI. Images of actin rings and nuclei were merged. (B) RAW264.7 cells (1×105 cells/well) were incubated with RANKL (50 ng/mL) in the presence or absence of various concentrations of WSS25 (2.5, 5 and 10 μg/mL) for 4 d. Next, the cells were stained with phalloidin to detect actin rings and with DAPI to detect nuclei. (C) Osteoclasts with actin ring structures were counted. n=3. Values are shown as the mean±SD. bP<0.05, cP<0.01 vs 0 μg/mL WSS25+RANKL group. (D) RAW264.7 cells (1×105 cells/mL) were treated with RANKL (50 ng/mL) and various concentrations of WSS25. The total RNA was extracted and the mRNA expression levels of TRAP, NFATc1, cathepsin K, and MMP-9 were measured by reverse transcription PCR. (E) The expression levels of NFATc1, cathepsin K, MMP-9 and TRAP were calculated by Image J. 1–5 on the x-axis refers to panel 1–5 in (D). n=3. Values are shown as the mean±SD. bP<0.05, cP<0.01 vs 0 μg/mL WSS25+RANKL group.
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Related In: Results  -  Collection

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fig2: WSS25 inhibits RANKL-induced actin ring structure formation and the expression of osteoclastogenesis-related gene markers. (A) Actin ring structures were stained red by phalloidin, and nuclei were stained blue by DAPI. Images of actin rings and nuclei were merged. (B) RAW264.7 cells (1×105 cells/well) were incubated with RANKL (50 ng/mL) in the presence or absence of various concentrations of WSS25 (2.5, 5 and 10 μg/mL) for 4 d. Next, the cells were stained with phalloidin to detect actin rings and with DAPI to detect nuclei. (C) Osteoclasts with actin ring structures were counted. n=3. Values are shown as the mean±SD. bP<0.05, cP<0.01 vs 0 μg/mL WSS25+RANKL group. (D) RAW264.7 cells (1×105 cells/mL) were treated with RANKL (50 ng/mL) and various concentrations of WSS25. The total RNA was extracted and the mRNA expression levels of TRAP, NFATc1, cathepsin K, and MMP-9 were measured by reverse transcription PCR. (E) The expression levels of NFATc1, cathepsin K, MMP-9 and TRAP were calculated by Image J. 1–5 on the x-axis refers to panel 1–5 in (D). n=3. Values are shown as the mean±SD. bP<0.05, cP<0.01 vs 0 μg/mL WSS25+RANKL group.
Mentions: Actin ring formation is a prerequisite for the resorption of osteoclasts and is the most distinct marker of mature osteoclasts during osteoclast differentiation32. To further investigate the effect of WSS25 on osteoclast differentiation, we examined whether WSS25 blocks RANKL-induced actin ring formation in osteoclasts. After stimulation by RANKL, RAW264.7 cells differentiated into mature osteoclasts and formed obvious actin ring structures (Figure 2A). However, the number and size of actin rings gradually decreased as WSS25 concentrations increased (2.5, 5 and 10 μg/mL) (Figure 2B and 2C). This indicated that WSS25 inhibited the formation of actin rings in a dose-dependent manner.

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