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 NFκB 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 D₃ and prostaglandin E₂ 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 ¹²⁵I-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.

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.