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Mature bone-resorbing osteoclasts (OCs) mediate excessive bone loss seen in several bone disorders, including osteoporosis. Here, we showed that reveromycin A (RM-A), a small natural product with three carboxylic groups in its structure, induced apoptosis specifically in OCs, but not in OC progenitors, nonfunctional osteoclasts, or osteoblasts. RM-A inhibited protein synthesis in OCs by selectively blocking enzymatic activity of isoleucyl-tRNA synthetase. The proapoptotic effect of RM-A was inhibited by neutralization or disruption of the acidic microenvironment, a prominent characteristic of OCs. RM-A was incorporated in OCs but not in nonfunctional osteoclasts and OC progenitors in neutral culture medium. Effects of RM-A on OC apoptosis increased under acidic culture conditions. RM-A not only was incorporated, but also induced apoptosis in OC progenitors in acidic culture medium. RM-A inhibited osteoclastic pit formation, decreased prelabeled 45 Ca release in organ cultures, and antagonized increased bone resorption in ovariectomized mice. These results suggested that preventive effects of RM-A on bone resorption in vitro and in vivo were caused by apoptosis through inhibition of isoleucyl-tRNA synthetase in OCs and that specific sensitivity of OCs to RM-A was due to the acidic microenvironment, which increased cell permeability of RM-A by suppressing dissociation of protons from carboxylic acid moieties, making them less polar. This unique mechanism suggested that RM-A might represent a type of therapeutic agent for treating bone disorders associated with increased bone loss.isoleucyl-tRNA synthetase ͉ inhibitor ͉ antiresorptive agent M ature osteoclasts resorb bone and mediate excessive bone loss seen in several bone disorders, including osteoporosis, arthritis, periodontitis, bone metastasis, and corticosteroidinduced bone loss (1-4). Mature bone-resorbing osteoclasts (OCs) exhibit highly specialized morphological structures, such as actin rings, clear zones, and ruffled borders, which are functional markers of OCs (2-5). These structures permit OCs to establish an isolated acidic microenvironment between themselves and the bone surface. The acidic microenvironment formed by V-ATPase on the ruffled borders of OCs allows dissolution of bone minerals and degradation of the bone matrix by proteases (6, 7). Therefore, these specialized features in OCs represent major potential targets for reducing OC activity and, consequently, could be useful for treatment of bone disorders. In addition, recent studies showed that receptor activator of NF-B ligand (RANKL), which is an essential factor for OC differentiation and activation, was one of the major targets of antiresorptive agents (2).Two types of antiresorptive drugs targeting OCs, i.e., bisphosphonates (BPPs) and calcitonin (CT), are used for clinical treatment of osteoporosis. BPPs, the most effective class of antiresorptive drugs available, inhibit bone resorption by suppressing functions of OCs and by shortening their lifespan (8 -10). Because of targeting of ...

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Osteoclasts are multinucleated cells that differentiate from hematopoietic precursors 1) and possess characteristics to resorb mineralized bone. Osteoclast-like multinucleated cells (OCLs) can be differentiated in vitro from cocultures of mouse bone marrow cells and calvarial osteoblastic cells by treatment with osteotropic factors such as 1a,25-dihydroxyvitamin D 3 , prostaglandin E 2 , interleukin-1 (IL-1) or parathyroid hormone.2,3) Osteoblasts or stromal cells are the target cells for these factors in bone. Recently, an essential factor provided by osteoblasts or stromal cells has been identified and named osteoclast differentiation factor (ODF)/osteoprotegerin ligand (OPGL)/tumor necrosis factor-related activation-induced cytokine (TRANCE)/receptor activator of nuclear factor-kB (NF-kB) ligand (RANKL). 4,5) It has been shown that RANKL induces OCL formation in cultures of bone marrow cells in the presence of M-CSF without requiring osteoblasts or stromal cells.6) RAW cells are also known to differentiate into osteoclasts in the presence of RANKL. ; rats fed onions increased their bone mass. 8) Onion also inhibits bone resorption stimulated in ovariectomized rat, 9,10) and an extract from onion is known to prevent tibial cortical and cancellous bone loss induced by a combination of low protein intake and diet-mediated mild hyperparathyroidism in rats.11) Rutin (quercetin-3-O-glucose rhamnose) ( Fig. 1) has recently been reported to inhibit ovariectomy-stimulated bone resorption in rats.12) These facts taken together suggest that rutin is one of the principal components of onions that effectively facilitate bone resorption, and that its inhibitory effect on bone loss could in part be responsible for its effects on increasing bone mass.Quercetin (Fig. 1) is the major representative of the flavonoid subclass of flavonols commonly found in fruits and vegetables, 13,14) and is also abundant in onion extracts (200-600 mg quercetin/kg onion) and primarily in the form of glycoside (rutin).14) Dietary glycosides like rutin are thought to be converted into aglycone (like quercetin) in the large intestine by the glycosidase activity of intestinal bacteria.15) While these facts suggest that quercetin inhibits bone resorption in animals, its target cells for bone resorption and its mode of action has not been fully elucidated. We investigated the effects of quercetin on the differentiation and activation of osteoclasts, and on bone resorption in cultures. We show here that quercetin inhibits pOC formation induced by sRANKL Although quercetin has suppressed bone resorption in several animal studies, its target cells and the mechanism of its action related to bone resorption has not been fully elucidated. We investigated the effect of quercetin on the differentiation and activation of osteoclasts. We used cocultures of mouse spleen cells and ST2 cells, and cultures of osteoclast progenitor cells {M-CSF-dependent (MD) cells from mouse bone marrow and murine monocytic RAW 264 (RAW) cells}. Quercetin dose-dependently inhibite...