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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

BMP-2 enhances RANKL-induced osteoclast formation. However, WSS25 blocks this induction in RAW264.7 cells. (A) RAW264.7 cells (1×105 cells/mL) were incubated with BMP-2 (100 ng/mL), RANKL (20 ng/mL), or both, in the presence or absence of WSS25 (10 μg/mL) or noggin (1 μg/mL) for 4 d. Cells were then stained for TRAP expression detection and photographed (×1000). (B) The numbers of TRAP-positive, multinucleated (≥ 3 nuclei) osteoclasts were counted. (C) The average diameter of multinucleated osteoclasts was calculated. (D) RAW264.7 cells (1×105 cells/mL) were incubated with RANKL (50 ng/mL) and treated with noggin (1 μg/mL) or WSS25 (10 μg/mL). After 4 d, multinucleated osteoclasts were stained with TRAP solution. n=3. Values are shown as the mean±SD. bP<0.05 vs RANKL group. eP<0.05, fP<0.01 vs RANKL+BMP-2 group.
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fig7: BMP-2 enhances RANKL-induced osteoclast formation. However, WSS25 blocks this induction in RAW264.7 cells. (A) RAW264.7 cells (1×105 cells/mL) were incubated with BMP-2 (100 ng/mL), RANKL (20 ng/mL), or both, in the presence or absence of WSS25 (10 μg/mL) or noggin (1 μg/mL) for 4 d. Cells were then stained for TRAP expression detection and photographed (×1000). (B) The numbers of TRAP-positive, multinucleated (≥ 3 nuclei) osteoclasts were counted. (C) The average diameter of multinucleated osteoclasts was calculated. (D) RAW264.7 cells (1×105 cells/mL) were incubated with RANKL (50 ng/mL) and treated with noggin (1 μg/mL) or WSS25 (10 μg/mL). After 4 d, multinucleated osteoclasts were stained with TRAP solution. n=3. Values are shown as the mean±SD. bP<0.05 vs RANKL group. eP<0.05, fP<0.01 vs RANKL+BMP-2 group.

Mentions: To study the influence of BMP-2 on osteoclast differentiation, RAW264.7 cells were incubated with BMP-2, RANKL, or both in the presence or absence of WSS25 or noggin for 4 d. TRAP-positive, multinucleated, osteoclast-like cells were stained. RAW264.7 cells did not differentiate into multinucleated osteoclasts following stimulation by BMP-2 alone (Figure 7A). However, when cells were treated with suboptimal RANKL (20 ng/mL), administered at less than half of the effective dose, a modest number of relatively small, multinucleated osteoclasts were observed. The addition of BMP-2 markedly enhanced the number and average diameter of TRAP-positive osteoclasts that were induced by suboptimal RANKL (Figure 7A, 7B and 7C). The addition of WSS25 or the BMP-2-antagonist noggin significantly inhibited the osteoclast formation that was induced by BMP-2 and suboptimal RANKL (Figure 7A, 7B and 7C). To investigate whether endogenous BMP-2 signaling is a prerequisite for the differentiation of osteoclast precursor cells, RAW264.7 cells were incubated with RANKL (50 ng/mL) and treated with the endogenous BMP-2 antagonist noggin or WSS25. As shown in Figure 7D, noggin and WSS25 sharply blocked osteoclast formation, indicating that the endogenous BMP-2 signaling pathway also participates into 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)

BMP-2 enhances RANKL-induced osteoclast formation. However, WSS25 blocks this induction in RAW264.7 cells. (A) RAW264.7 cells (1×105 cells/mL) were incubated with BMP-2 (100 ng/mL), RANKL (20 ng/mL), or both, in the presence or absence of WSS25 (10 μg/mL) or noggin (1 μg/mL) for 4 d. Cells were then stained for TRAP expression detection and photographed (×1000). (B) The numbers of TRAP-positive, multinucleated (≥ 3 nuclei) osteoclasts were counted. (C) The average diameter of multinucleated osteoclasts was calculated. (D) RAW264.7 cells (1×105 cells/mL) were incubated with RANKL (50 ng/mL) and treated with noggin (1 μg/mL) or WSS25 (10 μg/mL). After 4 d, multinucleated osteoclasts were stained with TRAP solution. n=3. Values are shown as the mean±SD. bP<0.05 vs RANKL group. eP<0.05, fP<0.01 vs RANKL+BMP-2 group.
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fig7: BMP-2 enhances RANKL-induced osteoclast formation. However, WSS25 blocks this induction in RAW264.7 cells. (A) RAW264.7 cells (1×105 cells/mL) were incubated with BMP-2 (100 ng/mL), RANKL (20 ng/mL), or both, in the presence or absence of WSS25 (10 μg/mL) or noggin (1 μg/mL) for 4 d. Cells were then stained for TRAP expression detection and photographed (×1000). (B) The numbers of TRAP-positive, multinucleated (≥ 3 nuclei) osteoclasts were counted. (C) The average diameter of multinucleated osteoclasts was calculated. (D) RAW264.7 cells (1×105 cells/mL) were incubated with RANKL (50 ng/mL) and treated with noggin (1 μg/mL) or WSS25 (10 μg/mL). After 4 d, multinucleated osteoclasts were stained with TRAP solution. n=3. Values are shown as the mean±SD. bP<0.05 vs RANKL group. eP<0.05, fP<0.01 vs RANKL+BMP-2 group.
Mentions: To study the influence of BMP-2 on osteoclast differentiation, RAW264.7 cells were incubated with BMP-2, RANKL, or both in the presence or absence of WSS25 or noggin for 4 d. TRAP-positive, multinucleated, osteoclast-like cells were stained. RAW264.7 cells did not differentiate into multinucleated osteoclasts following stimulation by BMP-2 alone (Figure 7A). However, when cells were treated with suboptimal RANKL (20 ng/mL), administered at less than half of the effective dose, a modest number of relatively small, multinucleated osteoclasts were observed. The addition of BMP-2 markedly enhanced the number and average diameter of TRAP-positive osteoclasts that were induced by suboptimal RANKL (Figure 7A, 7B and 7C). The addition of WSS25 or the BMP-2-antagonist noggin significantly inhibited the osteoclast formation that was induced by BMP-2 and suboptimal RANKL (Figure 7A, 7B and 7C). To investigate whether endogenous BMP-2 signaling is a prerequisite for the differentiation of osteoclast precursor cells, RAW264.7 cells were incubated with RANKL (50 ng/mL) and treated with the endogenous BMP-2 antagonist noggin or WSS25. As shown in Figure 7D, noggin and WSS25 sharply blocked osteoclast formation, indicating that the endogenous BMP-2 signaling pathway also participates into 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