title: Heparin enhances osteoclastic bone resorption by inhibiting osteoprotegerin activity.
abstract: Heparin is a highly sulfated glycosaminoglycan and has been shown to activate osteoclastic bone resorption though how is not yet clear. Here we investigate the molecule involved in heparin-induced activation of osteoclasts using an in vitro osteoclast culture assay. The formation and activation of osteoclasts are induced by receptor activator of NFkappaB ligand (RANKL) on osteoblasts, and inhibited by osteoprotegerin (OPG), a decoy receptor of RANKL, which is secreted from osteoblasts. In a coculture of mouse bone marrow cells and osteoblasts treated with 1,25-dihydroxyvitamin D(3) and prostaglandin E(2) on dentin slices, the bone marrow cells differentiate into osteoclasts, and resorption pits are formed on the dentin slices. Addition of heparin, various glycosaminoglycans, and chemically modified heparins to the coculture reveals that heparin enhances the pit-forming activity of osteoclasts, and this effect of heparin on the activation of osteoclasts is dependent on its sugar chain structure. By contrast, mRNA expression levels of RANKL, RANK, and OPG in the coculture are not altered by heparin treatment. Furthermore, neither RANK nor RANKL binds to heparin, suggesting that heparin does not directly interact with these proteins. Instead, heparin specifically binds to OPG and prevents OPG-mediated inhibition of osteoclastic bone resorption in the coculture. Heparin treatment does not enhance osteoclastic bone resorption in a monoculture of osteoclasts derived from bone marrow cells, and in the coculture using osteoblasts from OPG-deficient mice. A (125)I-OPG binding assay showed that OPG binds to osteoblasts and that this binding is inhibited by the addition of heparin, suggesting that OPG binds to RANKL on the osteoblast membrane and that heparin blocks this interaction. These results demonstrate that heparin enhances osteoclastic bone resorption by inhibiting OPG activity.
authors.affiliation: Biomembrane Signaling Project, The Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613, Japan. irie@rinshoken.or.jp
authors.forename: Atsushi
authors.initials: A
authors.lastname: Irie
authors.forename: Masamichi
authors.initials: M
authors.lastname: Takami
authors.forename: Hideo
authors.initials: H
authors.lastname: Kubo
authors.forename: Naoko
authors.initials: N
authors.lastname: Sekino-Suzuki
authors.forename: Kohji
authors.initials: K
authors.lastname: Kasahara
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Sanai
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D001862:Bone Resorption; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D018920:Coculture Techniques; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D015394:Molecular Structure; D010006:Osteoblasts / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D053244:Osteoprotegerin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D053245:RANK Ligand / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B / Q000378:metabolism
chemical_list: D053244:Osteoprotegerin; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D006493:Heparin