Heparin enhances osteoclastic bone resorption by inhibiting osteoprotegerin activity
Introduction
Heparin is a member of the glycosaminoglycan (GAG) family and is produced by mast cells in the connective tissues and peritoneal cavity [1]. Heparin sugar chains are highly sulfated and bind to various extracellular molecules including growth factors, adhesion molecules, and receptors, and can modulate the functions of these heparin-binding molecules [2], [3]. Heparin shows anticoagulant activity by binding to antithrombin III [4] and is widely used for anticoagulant therapy. Administration of heparin is used for prophylaxis and treatment of thrombosis during pregnancy because, unlike coumarin derivatives, heparin does not pass through the placenta and does not affect the fetus [5]. For several decades, however, it has been reported that treatment with heparin can lead to a reduction in bone density and the development of osteoporosis [6], [7].
Bone metabolism is a continuous remodeling process that is normally maintained as a tightly coupled balance between bone formation by osteoblasts and bone resorption by osteoclasts. Heparin has been implicated in the activation of bone resorption in vivo and in vitro. For example, administration of heparin to rats promotes the activation of osteoclasts and decreases bone volume within several days of treatment [8]. Furthermore, heparin enhances bone resorption in both mouse tissue culture [9] and rat osteoclasts in vitro [10], [11]. Despite these lines of evidence, little is known about the molecules involved in the heparin-mediated activation of osteoclasts.
Osteoclasts are multinucleated cells that differentiate from hematopoietic precursors of the monocyte–macrophage lineage [12], [13]. Osteoclast precursors differentiate into osteoclasts upon cell–cell interaction with osteoblasts. Osteoblasts express receptor activator of NFκB ligand (RANKL; also called TRANCE, ODF, and OPGL), which is the most important factor in osteoclast differentiation [12], [13], whereas osteoclast precursors express RANK, the receptor for RANKL. Furthermore, RANK–RANKL interaction is critical for the bone-resorbing activity as well as differentiation of osteoclasts. Mature osteoclasts are activated by RANKL, and release cathepsins and H+-ATPase, resulting in the formation of resorption pits on the bone matrix [6].
In addition to RANKL, osteoblasts also express osteoprotegerin (OPG; also called OCIF), a negative regulator of the differentiation and activation of osteoclasts [13], [14]. OPG is a soluble decoy receptor for RANKL and blocks RANKL–RANK interaction, resulting in an inhibition of osteoclastogenesis and osteoclast-mediated bone resorption [15], [16]. The expression of OPG is regulated by a number of growth factors, hormones, and cytokines and the ratio of RANKL to OPG is critical for maintaining bone mass [14], [17]. Mice lacking OPG show a decrease in bone density and develop severe osteoporosis [18], whereas bone density is increased in mice over-expressing OPG [15]. Although OPG has a highly basic domain in its C-terminus that can bind to heparin [19], it is not known whether heparin plays a role in the OPG-mediated inhibition of osteoclast activity.
Here we have investigated the effect of heparin on the activity of osteoclasts using a coculture system of mouse bone marrow cells and osteoblasts, and demonstrate that heparin augments osteoclastic bone resorption by inhibiting OPG activity.
Section snippets
Materials
Anti-OPG, anti-RANK, and anti-RANKL antibodies, recombinant human OPG, and recombinant human transforming growth factor β1 (TGF-β1) were purchased from R&D Systems (Minneapolis). Recombinant human RANKL and RANK were from Peprotech EC (London). Recombinant human macrophage colony-stimulating factor (M-CSF) was from Kyowa Hakko Kogyo Co. (Tokyo). Chondroitin sulfate A (CS-A), CS-B, CS-C, and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) were from Sigma. Bovine kidney heparan sulfate (HS), keratan
Heparin stimulates osteoclastic bone resorption
In cocultures of osteoblasts and bone marrow cells, treatment with 1,25(OH)2D3 and PGE2 induces the differentiation and activation of osteoclasts by stimulating the expression of RANKL in osteoblasts [22]. Differentiated osteoclasts are multinucleated giant cells that express tartrate-resistant acid phosphatase (TRAP), a marker enzyme of osteoclastic differentiation [21]. To examine the effect of heparin on osteoclastogenesis, we cocultured mouse osteoblasts and bone marrow cells for 6 days and
Discussion
Abnormal activation of osteoclasts mediates bone loss in several bone diseases, including osteoporosis. Since RANKL–RANK as well as RANKL–OPG interaction is critical for osteoclastic bone-resorbing activity, the coculture of osteoblasts and bone marrow cells used in this study has particular advantages for understanding the mechanism of heparin-induced activation of osteoclasts. Using this system, we demonstrated here that heparin enhances osteoclastic activity by inhibiting OPG. Although it
Acknowledgments
We would like to thank Drs. Shuhei Yamada and Hidenori Suzuki for technical advice. We also thank Drs. Masaharu Kotani and Hiromi Ashino for encouragement.
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