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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 osteoclast differentiation in mouse bone marrow monocytes (BMMs) and RAW264.7 cells. (A) Mouse BMMs (5×104 cells/mL) were treated with M-CSF (20 ng/mL) and RANKL (50 ng/mL) in the presence or absence of WSS25 (2.5, 5 and 10 μg/mL). After 7 d of treatment, the cells were stained with a TRAP kit and photographed (×1000). (B) RAW264.7 cells (1×105 cells/mL) were incubated with RANKL (50 ng/mL) in the presence or absence of WSS25 (2.5, 5 and 10 μg/mL) for 4 d, followed by TRAP-staining. Then, the cells were photographed (×1000). (C) The numbers of TRAP-positive, multinucleated (≥3 nuclei) osteoclasts were counted in BMMs. n=3. Values are shown as the mean±SD. bP<0.05, cP<0.01 vs 0 μg/mL WSS25+RANKL+M-CSF group. (D) The numbers of TRAP-positive, multinucleated (≥3 nuclei) osteoclasts were counted in RAW264.7 cells. n=3. Values are shown as the mean±SD. bP<0.05, cP<0.01 vs 0 μg/mL WSS25+RANKL group. (E) BMMs (2×105 cells/mL) were incubated with M-CSF (20 ng/mL) and the indicated concentration of WSS25 or with M-CSF (20 ng/mL) plus RANKL (50 ng/mL), in the presence or absence of different concentrations of WSS25 for 7 d before cytotoxicity was tested by MTT assay. (F) RAW264.7 cells (2×105 cells/mL) were treated with various concentrations of WSS25 or with WSS25 plus RANKL (50 ng/mL) for 7 d before cytotoxicity was tested by MTT assay. n=3. Values are shown as the mean±SD. bP<0.05, cP<0.01 vs normal group.
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fig1: WSS25 inhibits RANKL-induced osteoclast differentiation in mouse bone marrow monocytes (BMMs) and RAW264.7 cells. (A) Mouse BMMs (5×104 cells/mL) were treated with M-CSF (20 ng/mL) and RANKL (50 ng/mL) in the presence or absence of WSS25 (2.5, 5 and 10 μg/mL). After 7 d of treatment, the cells were stained with a TRAP kit and photographed (×1000). (B) RAW264.7 cells (1×105 cells/mL) were incubated with RANKL (50 ng/mL) in the presence or absence of WSS25 (2.5, 5 and 10 μg/mL) for 4 d, followed by TRAP-staining. Then, the cells were photographed (×1000). (C) The numbers of TRAP-positive, multinucleated (≥3 nuclei) osteoclasts were counted in BMMs. n=3. Values are shown as the mean±SD. bP<0.05, cP<0.01 vs 0 μg/mL WSS25+RANKL+M-CSF group. (D) The numbers of TRAP-positive, multinucleated (≥3 nuclei) osteoclasts were counted in RAW264.7 cells. n=3. Values are shown as the mean±SD. bP<0.05, cP<0.01 vs 0 μg/mL WSS25+RANKL group. (E) BMMs (2×105 cells/mL) were incubated with M-CSF (20 ng/mL) and the indicated concentration of WSS25 or with M-CSF (20 ng/mL) plus RANKL (50 ng/mL), in the presence or absence of different concentrations of WSS25 for 7 d before cytotoxicity was tested by MTT assay. (F) RAW264.7 cells (2×105 cells/mL) were treated with various concentrations of WSS25 or with WSS25 plus RANKL (50 ng/mL) for 7 d before cytotoxicity was tested by MTT assay. n=3. Values are shown as the mean±SD. bP<0.05, cP<0.01 vs normal group.

Mentions: We used two osteoclastogenesis models to study the effect of WSS25 on osteoclast formation in vitro, including mouse bone marrow monocytes (BMMs) and RAW264.7 cells. Tartrate-resistant acid phosphate (TRAP) is a glycosylated monomeric metalloprotein enzyme that is a highly expressed, well-known marker of osteoclasts. TRAP staining revealed that TRAP-positive, multinucleated osteoclasts were induced by RANKL and M-CSF to form in BMMs within 7 d, and induced by RANKL to form in RAW264.7 cells within 4 d (Figure 1A and 1B). However, WSS25 significantly inhibited osteoclast differentiation in a dose-dependent manner in both BMMs and RAW264.7 cells (Figure 1A, 1B, 1C and 1D). Moreover, WSS25 had no obvious cytotoxic effects on BMMs or RAW264.7 cells at the concentration used to test the inhibitory effect of WSS25 on osteoclastogenesis (Figure 1E and 1F).


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 osteoclast differentiation in mouse bone marrow monocytes (BMMs) and RAW264.7 cells. (A) Mouse BMMs (5×104 cells/mL) were treated with M-CSF (20 ng/mL) and RANKL (50 ng/mL) in the presence or absence of WSS25 (2.5, 5 and 10 μg/mL). After 7 d of treatment, the cells were stained with a TRAP kit and photographed (×1000). (B) RAW264.7 cells (1×105 cells/mL) were incubated with RANKL (50 ng/mL) in the presence or absence of WSS25 (2.5, 5 and 10 μg/mL) for 4 d, followed by TRAP-staining. Then, the cells were photographed (×1000). (C) The numbers of TRAP-positive, multinucleated (≥3 nuclei) osteoclasts were counted in BMMs. n=3. Values are shown as the mean±SD. bP<0.05, cP<0.01 vs 0 μg/mL WSS25+RANKL+M-CSF group. (D) The numbers of TRAP-positive, multinucleated (≥3 nuclei) osteoclasts were counted in RAW264.7 cells. n=3. Values are shown as the mean±SD. bP<0.05, cP<0.01 vs 0 μg/mL WSS25+RANKL group. (E) BMMs (2×105 cells/mL) were incubated with M-CSF (20 ng/mL) and the indicated concentration of WSS25 or with M-CSF (20 ng/mL) plus RANKL (50 ng/mL), in the presence or absence of different concentrations of WSS25 for 7 d before cytotoxicity was tested by MTT assay. (F) RAW264.7 cells (2×105 cells/mL) were treated with various concentrations of WSS25 or with WSS25 plus RANKL (50 ng/mL) for 7 d before cytotoxicity was tested by MTT assay. n=3. Values are shown as the mean±SD. bP<0.05, cP<0.01 vs normal group.
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Related In: Results  -  Collection

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fig1: WSS25 inhibits RANKL-induced osteoclast differentiation in mouse bone marrow monocytes (BMMs) and RAW264.7 cells. (A) Mouse BMMs (5×104 cells/mL) were treated with M-CSF (20 ng/mL) and RANKL (50 ng/mL) in the presence or absence of WSS25 (2.5, 5 and 10 μg/mL). After 7 d of treatment, the cells were stained with a TRAP kit and photographed (×1000). (B) RAW264.7 cells (1×105 cells/mL) were incubated with RANKL (50 ng/mL) in the presence or absence of WSS25 (2.5, 5 and 10 μg/mL) for 4 d, followed by TRAP-staining. Then, the cells were photographed (×1000). (C) The numbers of TRAP-positive, multinucleated (≥3 nuclei) osteoclasts were counted in BMMs. n=3. Values are shown as the mean±SD. bP<0.05, cP<0.01 vs 0 μg/mL WSS25+RANKL+M-CSF group. (D) The numbers of TRAP-positive, multinucleated (≥3 nuclei) osteoclasts were counted in RAW264.7 cells. n=3. Values are shown as the mean±SD. bP<0.05, cP<0.01 vs 0 μg/mL WSS25+RANKL group. (E) BMMs (2×105 cells/mL) were incubated with M-CSF (20 ng/mL) and the indicated concentration of WSS25 or with M-CSF (20 ng/mL) plus RANKL (50 ng/mL), in the presence or absence of different concentrations of WSS25 for 7 d before cytotoxicity was tested by MTT assay. (F) RAW264.7 cells (2×105 cells/mL) were treated with various concentrations of WSS25 or with WSS25 plus RANKL (50 ng/mL) for 7 d before cytotoxicity was tested by MTT assay. n=3. Values are shown as the mean±SD. bP<0.05, cP<0.01 vs normal group.
Mentions: We used two osteoclastogenesis models to study the effect of WSS25 on osteoclast formation in vitro, including mouse bone marrow monocytes (BMMs) and RAW264.7 cells. Tartrate-resistant acid phosphate (TRAP) is a glycosylated monomeric metalloprotein enzyme that is a highly expressed, well-known marker of osteoclasts. TRAP staining revealed that TRAP-positive, multinucleated osteoclasts were induced by RANKL and M-CSF to form in BMMs within 7 d, and induced by RANKL to form in RAW264.7 cells within 4 d (Figure 1A and 1B). However, WSS25 significantly inhibited osteoclast differentiation in a dose-dependent manner in both BMMs and RAW264.7 cells (Figure 1A, 1B, 1C and 1D). Moreover, WSS25 had no obvious cytotoxic effects on BMMs or RAW264.7 cells at the concentration used to test the inhibitory effect of WSS25 on osteoclastogenesis (Figure 1E and 1F).

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