Heparin inhibits osteoclastic differentiation and function

Wataru Ariyoshi, Tetsu Takahashi, Takahiro Kanno, Hisashi Ichimiya, Kouhei Shinmyouzu, Hiroshi Takano, Takeyoshi Koseki, Tatsuji Nishihara

Research output: Contribution to journalArticlepeer-review

44 Citations (Scopus)

Abstract

We investigated the effects of Glycosaminoglycans (GAGs) on mouse monocytic cell line in regard to their differentiation, proliferation, and function in vitro. RAW 264.7 cells were cultured with receptor activator of NF-κB ligand (RANKL) and various GAGs. Osteoclastic cells were visualized by staining for tartrate-resistant acid phosphatase (TRAP) and detected using a phenyl-phosphate substrate method. RAW 264.7 cells were also cultured with stimulants contained in BD BioCoat OSTEOLOCIC™ kit, and bone resorption activity was assessed by counting the numbers of resorption pits. We also examined the effect of heparin on cell growth using MTT assay, while the expression level of c-Src protein was determined by immunoblot analysis. Heparin suppressed TRAP-positive multinucleated cell formation and TRAP activity induced by RANKL, whereas the other GAGs showed no effects on osteoclast differentiation. Heparin also inhibited the formation of resorption pits, while the others did not. In the MTT assay, none of the tested GAGs had an influence on RAW 264.7 cell proliferation. However, heparin reduced the level of c-Src protein in RAW 264.7 cells stimulated with RANKL. To determine the affinity of heparin and RANKL, they were subjected by HiTrap heparin column chromatography and each fraction was collected. Western blotting analysis revealed the expression of RANKL in the fraction bound to heparin. The binding of RANKL and heparin was confirmed by quartz-crystal microbalance. These results indicate that the inhibitory effect of heparin toward osteoclastogenesis induced by RANKL is due to the binding of heparin to RANKL.

Original languageEnglish
Pages (from-to)1707-1717
Number of pages11
JournalJournal of Cellular Biochemistry
Volume103
Issue number6
DOIs
Publication statusPublished - 2008 Apr 15

Keywords

  • Binding
  • Glycosaminoglycan
  • Heparin
  • Osteoclastogenesis
  • Receptor activator of NF-κB ligand

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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