Limits...
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 osteoclastic bone resorption in both RAW264.7 cells and BMMs. (A) RAW264.7 cells (1×105 cells/mL) were cultured on bone slices and stimulated by RANKL (50 ng/mL), with or without WSS25 (10 μg/mL) for 5 d. Bone slices were stained with hematoxylin after cells were removed. Formed resorption pits were photographed using a microscope (×1000). (B) The numbers of resorption pits were counted and statistically analyzed. (C) The areas of resorption pits were calculated using Image-Pro Plus 6.0. n=3. Values are shown as the mean±SD. cP<0.01 vs RANKL group. (D) BMMs (5×104 cells/mL) were cultured with M-CSF (20 ng/mL) and RANKL (50 ng/mL), in the presence or absence of WSS25 (10 μg/mL), on bone slices for 7 d. Bone slices were then stained with hematoxylin and photographed (×1000). (E) The numbers of resorption pits were counted and statistically analyzed. (F) The areas of resorption pits were calculated by Image-Pro Plus 6.0. n=3. Values are shown as the mean±SD. cP<0.01 vs M-CSF+RANKL group.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4561976&req=5

fig4: WSS25 inhibits osteoclastic bone resorption in both RAW264.7 cells and BMMs. (A) RAW264.7 cells (1×105 cells/mL) were cultured on bone slices and stimulated by RANKL (50 ng/mL), with or without WSS25 (10 μg/mL) for 5 d. Bone slices were stained with hematoxylin after cells were removed. Formed resorption pits were photographed using a microscope (×1000). (B) The numbers of resorption pits were counted and statistically analyzed. (C) The areas of resorption pits were calculated using Image-Pro Plus 6.0. n=3. Values are shown as the mean±SD. cP<0.01 vs RANKL group. (D) BMMs (5×104 cells/mL) were cultured with M-CSF (20 ng/mL) and RANKL (50 ng/mL), in the presence or absence of WSS25 (10 μg/mL), on bone slices for 7 d. Bone slices were then stained with hematoxylin and photographed (×1000). (E) The numbers of resorption pits were counted and statistically analyzed. (F) The areas of resorption pits were calculated by Image-Pro Plus 6.0. n=3. Values are shown as the mean±SD. cP<0.01 vs M-CSF+RANKL group.

Mentions: To further investigate whether WSS25 suppresses the bone resorption function of osteoclasts, bone resorption assays were performed on RAW264.7 cells and BMMs. RAW264.7 cells were cultured on a bovine bone slice and induced by RANKL, in the presence or absence of WSS25, for 5 d. Mature osteoclasts resorbed the bone and consequently formed resorbing pits on the slices after RANKL induction (Figure 4A). However, the number of pits and the resorption area on the surface were decreased after WSS25 treatment (Figure 4A, 4B and 4C). Moreover, when BMMs were cultured on a bone slice stimulated by M-CSF and RANKL, in the presence or absence of WSS25, for 7 d, the number of pits and the resorption area on the surface of the bone slices were also notably decreased (Figure 4D, 4E and 4F).


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 osteoclastic bone resorption in both RAW264.7 cells and BMMs. (A) RAW264.7 cells (1×105 cells/mL) were cultured on bone slices and stimulated by RANKL (50 ng/mL), with or without WSS25 (10 μg/mL) for 5 d. Bone slices were stained with hematoxylin after cells were removed. Formed resorption pits were photographed using a microscope (×1000). (B) The numbers of resorption pits were counted and statistically analyzed. (C) The areas of resorption pits were calculated using Image-Pro Plus 6.0. n=3. Values are shown as the mean±SD. cP<0.01 vs RANKL group. (D) BMMs (5×104 cells/mL) were cultured with M-CSF (20 ng/mL) and RANKL (50 ng/mL), in the presence or absence of WSS25 (10 μg/mL), on bone slices for 7 d. Bone slices were then stained with hematoxylin and photographed (×1000). (E) The numbers of resorption pits were counted and statistically analyzed. (F) The areas of resorption pits were calculated by Image-Pro Plus 6.0. n=3. Values are shown as the mean±SD. cP<0.01 vs M-CSF+RANKL group.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4561976&req=5

fig4: WSS25 inhibits osteoclastic bone resorption in both RAW264.7 cells and BMMs. (A) RAW264.7 cells (1×105 cells/mL) were cultured on bone slices and stimulated by RANKL (50 ng/mL), with or without WSS25 (10 μg/mL) for 5 d. Bone slices were stained with hematoxylin after cells were removed. Formed resorption pits were photographed using a microscope (×1000). (B) The numbers of resorption pits were counted and statistically analyzed. (C) The areas of resorption pits were calculated using Image-Pro Plus 6.0. n=3. Values are shown as the mean±SD. cP<0.01 vs RANKL group. (D) BMMs (5×104 cells/mL) were cultured with M-CSF (20 ng/mL) and RANKL (50 ng/mL), in the presence or absence of WSS25 (10 μg/mL), on bone slices for 7 d. Bone slices were then stained with hematoxylin and photographed (×1000). (E) The numbers of resorption pits were counted and statistically analyzed. (F) The areas of resorption pits were calculated by Image-Pro Plus 6.0. n=3. Values are shown as the mean±SD. cP<0.01 vs M-CSF+RANKL group.
Mentions: To further investigate whether WSS25 suppresses the bone resorption function of osteoclasts, bone resorption assays were performed on RAW264.7 cells and BMMs. RAW264.7 cells were cultured on a bovine bone slice and induced by RANKL, in the presence or absence of WSS25, for 5 d. Mature osteoclasts resorbed the bone and consequently formed resorbing pits on the slices after RANKL induction (Figure 4A). However, the number of pits and the resorption area on the surface were decreased after WSS25 treatment (Figure 4A, 4B and 4C). Moreover, when BMMs were cultured on a bone slice stimulated by M-CSF and RANKL, in the presence or absence of WSS25, for 7 d, the number of pits and the resorption area on the surface of the bone slices were also notably decreased (Figure 4D, 4E and 4F).

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