title: WSS25, a sulfated polysaccharide, inhibits RANKL-induced mouse osteoclast formation by blocking SMAD/ID1 signaling.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors: Glycochemistry and Glycobiology Laboratory, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.|Institute of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
authors: Cheng
authors: C
authors: Chen
authors: Glycochemistry and Glycobiology Laboratory, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors: Yi
authors: Y
authors: Qin
authors: Glycochemistry and Glycobiology Laboratory, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors: Jian-ping
authors: JP
authors: Fang
authors: Glycochemistry and Glycobiology Laboratory, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors: Xin-yan
authors: XY
authors: Ni
authors: Glycochemistry and Glycobiology Laboratory, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors: Jian
authors: J
authors: Yao
authors: Institute of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
authors: Hai-ying
authors: HY
authors: Wang
authors: Glycochemistry and Glycobiology Laboratory, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors: Kan
authors: K
authors: Ding
mesh_terms: D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism; D001862:Bone Resorption / Q000188:drug therapy* / Q000378:metabolism; D005260:Female; D005936:Glucans / Q000494:pharmacology* / Q000627:therapeutic use; D051798:Inhibitor of Differentiation Protein 1 / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D053245:RANK Ligand / Q000378:metabolism*; D000067996:RAW 264.7 Cells; D015398:Signal Transduction / Q000187:drug effects; D051785:Smad Proteins / Q000378:metabolism*
chemical_list: D055396:Bone Morphogenetic Protein 2; D005936:Glucans; D051798:Inhibitor of Differentiation Protein 1; D053245:RANK Ligand; D051785:Smad Proteins; C505689:Tnfsf11 protein, mouse; C553930:WSS25 glucan