Irie Atsushi, Takami Masamichi, Kubo Hideo, Sekino-Suzuki Naoko, Kasahara Kohji, Sanai Yutaka
Biomembrane Signaling Project, The Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613, Japan.
Bone. 2007 Aug;41(2):165-74. doi: 10.1016/j.bone.2007.04.190. Epub 2007 May 5.
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.
肝素是一种高度硫酸化的糖胺聚糖，已被证明可激活破骨细胞的骨吸收，但其具体机制尚不清楚。在此，我们使用体外破骨细胞培养试验研究参与肝素诱导破骨细胞激活的分子。破骨细胞的形成和激活由成骨细胞上的核因子κB受体活化因子配体（RANKL）诱导，并受到骨保护素（OPG）的抑制，OPG是RANKL的诱饵受体，由成骨细胞分泌。在牙本质切片上用1,25 - 二羟基维生素D(3)和前列腺素E(2)处理的小鼠骨髓细胞与成骨细胞共培养中，骨髓细胞分化为破骨细胞，并在牙本质切片上形成吸收陷窝。向共培养物中添加肝素、各种糖胺聚糖和化学修饰的肝素后发现，肝素增强了破骨细胞的陷窝形成活性，且肝素对破骨细胞激活的这种作用取决于其糖链结构。相比之下，共培养物中RANKL、RANK和OPG的mRNA表达水平不受肝素处理的影响。此外，RANK和RANKL均不与肝素结合，这表明肝素不直接与这些蛋白质相互作用。相反，肝素特异性结合OPG，并在共培养中阻止OPG介导的对破骨细胞骨吸收的抑制作用。肝素处理在源自骨髓细胞的破骨细胞单培养以及使用OPG缺陷小鼠的成骨细胞的共培养中均未增强破骨细胞的骨吸收。一项¹²⁵I - OPG结合试验表明，OPG与成骨细胞结合，且添加肝素可抑制这种结合，这表明OPG与成骨细胞膜上的RANKL结合，而肝素阻断了这种相互作用。这些结果表明，肝素通过抑制OPG活性增强破骨细胞的骨吸收。
