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a b s t r a c tOsteolytic bone diseases including osteoporosis are commonly accompanied with enhanced osteoclast formation and bone resorption. Naringin, a natural occurring flavonoid has been found to protect against retinoic acid-induced osteoporosis and improve bone quality in rats. Here, we showed that naringin perturbs osteoclast formation and bone resorption by inhibiting RANK-mediated NFjB and ERK signaling. Naringin suppressed gene expression of key osteoclast marker genes. Naringin was found to inhibit RANKL-induced activation of NF-jB by suppressing RANKL-mediated IjB-a degradation. In addition, naringin inhibited RANKL-induced phosphorylation of ERK. This study identifies naringin as an inhibitor for osteoclast formation and bone resorption, and provides evidence that natural compounds such as naringin might be beneficial as an alternative medicine for the prevention and treatment of osteolysis.

Osteolytic bone diseases such as osteoporosis have a common pathological feature in which osteoclastic bone resorption outstrips bone synthesis. Osteoclast formation and activation are regulated by receptor activator of nuclear factor κB ligand (RANKL). The induction of RANKL-signaling pathways occurs following the interaction of RANKL to its cognate receptor, RANK. This specific binding drives the activation of downstream signaling pathways; which ultimately induce the formation and activation of osteoclasts. In this study, we showed that a natural immunomodulator, mangiferin, inhibits osteoclast formation and bone resorption by attenuating RANKL-induced signaling. Mangiferin diminished the expression of osteoclast marker genes, including cathepsin K, calcitonin receptor, DC-STAMP, and V-ATPase d2. Mechanistic studies revealed that mangiferin inhibits RANKL-induced activation of NF-κB, concomitant with the inhibition of IκB-α degradation, and p65 nuclear translocation. In addition, mangiferin also exhibited an inhibitory effect on RANKL-induced ERK phosphorylation. Collectively, our data demonstrates that mangiferin exhibits anti-resorptive properties, suggesting the potential application of mangiferin for the treatment and prevention of bone diseases involving excessive osteoclastic bone resorption.

Vesicular transport along microtubules must be strictly regulated to sustain the unique structural and functional polarization of bone-resorbing osteoclasts. However, the molecular mechanisms bridging these vesicle-microtubule interactions remain largely obscure. Rab3D, a member of the Rab3 subfamily (Rab3A/B/ C/D) of small exocytotic GTPases, represents a core component of the osteoclastic vesicle transport machinery. Here, we identify a new Rab3D-interacting partner, Tctex-1, a light chain of the cytoplasmic dynein microtubule motor complex, by a yeast two-hybrid screen. We demonstrate that Tctex-1 binds specifically to Rab3D in a GTP-dependent manner and co-occupies Rab3D-bearing vesicles in bone-resorbing osteoclasts. Furthermore, we provide evidence that Tctex-1 and Rab3D intimately associate with the dynein motor complex and microtubules in osteoclasts. Finally, targeted disruption of Tctex-1 by RNA interference significantly impairs bone resorption capacity and mislocalizes Rab3D vesicles in osteoclasts, attesting to the notion that components of the Rab3D-trafficking pathway contribute to the maintenance of osteoclastic resorptive function.Osteoclasts are terminally differentiated polykaryons whose exclusive function is the degradation of mineralized bone matrix (45). Excessive osteoclast numbers and/or activity manifests in many pathological osteolytic disorders, including Paget's disease, multiple myeloma, and osteoporosis (55). These multinucleated cells mature from the asynchronous fusion of mononuclear precursors of the monocyte/macrophage lineage, a process orchestrated by two principal osteoclastogenic cytokines, namely, macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kB (RANK) ligand (RANKL).Upon attachment to bone, osteoclasts undergo a defined program of cytoskeletal and membrane reorganizations which culminate in the segregation of their plasmalemma into four distinct domains: (i) the ruffled border, (ii) the sealing zone, (iii) the basolateral domain, and (iv) the functional secretory domain (5,30,53,57). The sealing zone is circumscribed by a tight ring of a filamentous actin which serves as a site of osteoclast attachment and seals off the underlying resorptive space. Adjacent to the bone surface and limited by the sealing zone, the ruffled border membrane presents the resorptive organelle and serves as an exit site for protons and osteolytic enzymes (e.g., cathepsin K) as well as an uptake zone for the removal of degraded osseous tissue. Thus, vesicular trafficking must be tightly coupled to the osteoclastic cytoskeleton in order to sustain the specialized structural and functional polarization of the ruffled border and basolateral domains. In recent years, several components of the osteoclast vesicle transport machinery have been unveiled (1,32,41,50,58,59). Among these, small Ras-related Rab GTPases (40, 56) have emerged as key regulators of ruffled border formation and function.We have previously shown that Rab3D, a member of the exocytotic subfamil...