Trolox prevents osteoclastogenesis by suppressing RANKL expression and signaling

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vivo. In the present study, we have shown that RANKL is predominantly localized in lysosomal organelles, but little is found on the cell surface of osteoblastic cells. We have also shown that RANKL is relocated to the plasma membrane in response to stimulation with RANK-Fc coated beads, indicating that the lysosomal organelles where RANKL is localized function as secretory lysosomes. In addition, using a protein pull-down method, we have identified vacuolar protein sorting (Vps) 33a, a component of the Class C Vps protein complex, as interacting with the cytoplasmic tail of RANKL. Furthermore, knockdown of Vps33a expression reduced the lysosomal storage of RANKL and caused the accumulation of newly synthesized RANKL in the Golgi apparatus, indicating that Vps33a is involved in transporting RANKL from the Golgi apparatus to secretory lysosomes. We have also shown that suppression of Vps33a affects the cell surface expression level of RANKL and disrupts the regulated behavior of RANKL. These results suggest that RANKL storage in secretory lysosomes is important to control osteoclast activation and to maintain bone homeostasis. doi:10.1016/j.bone.2009.01.443

395 RANKL secretion from secretory lysosomes in osteoblastic cells — The involvement of small GTPase Rab27a/b Y. Kariya, M. Honma, H. Suzuki Department of Pharmacy, The University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan It has been established that Receptor Activator of NF-κB (RANK) Ligand (RANKL) expressed in osteoblasts induces osteoclast differentiation through binding to the receptor, RANK. This ligand–receptor interaction has been considered to occur by the cell-to-cell contact between osteoblasts and osteoclasts. On the other hand, our previous study revealed that RANKL is predominantly localized in secretory lysosomes but little in the plasma membrane. Therefore, the mechanism of RANKL translocation to the cell surface is considered important for osteoclastogenesis. By the analogy to the secretion mechanism of FasL, a member of TNF superfamily like RANKL, we examined the involvement of the small GTPase Rab27a in RANKL secretion in osteoblastic cell line, ST2 cells. To analyze the effect of Rab27a and other genes, we established stably knocking down ST2 cells using lenti virus encoding miRNA against target genes. When cell surface proteins were biotinylated, the amount of biotinylated RANKL was decreased by Rab27a suppression. We also performed coculture of stably knocked-down ST2 cells with bone marrow macrophages. TRAP activity, the marker of osteoclast maturation, was decreased by Rab27a suppression. These results suggest that Rab27a is involved in RANKL secretion in ST2 cells. We also examined the effect of Rab27b, the other isoform of Rab27 and Rab3a, closely-related to Rab27. Rab3a suppression did not alter the TRAP activity in the coculture, but Rab27b suppression decreased TRAP activity in coculture like Rab27a. These results suggest that Rab27a/b regulate RANKL secretion in osteoblastic cells. Next, we examined the effect of Rab27a/b on RANKL subcellular localization. While RANKL tagged with GFP were mostly localized in the secretory lysosomes in basal condition, the trend was observed by Rab27a/b suppression that RANKL slightly accumulated near the plasma membrane. This suggests that Rab27a/ b are involved in the secretion of RANKL containing vesicles, especially in the tethering and/or fusion step of the vesicles with the plasma membrane. In conclusion, Rab27a/b are involved in RANKL secretion, and Rab27a/b suppression leads to the reduction in the ability to support osteoclastogenesis in osteoblastic cells. doi:10.1016/j.bone.2009.01.444

396 Dose dependent anabolic and anti-catabolic response after local zoledronate treatment of cancellous bone grafts A bone chamber study in rats M. Tägil, O. Belfrage Dep of Orthopedics, Lund University Hospital, Lund, Sweden Bisphosphonates are strong inhibitors of bone resorption and normally administered systemically. Local application might be preferable in osteonecrosis, bone grafting or during callus formation. In some studies a decreased anabolic response has been noted. In the present study we evaluate how different doses, exposure time and modes of administration influence bone resorption as well as ingrowth of new bone into an allograft. Cancellous bone grafts were harvested from male SD rats and randomized into five groups. The grafts were placed in a bone conduction chamber and inserted into the proximal tibia of 50 female rats. Zoledronic acid (0.5 mg/ml) was used for local graft treatment. The grafts were soaked in excessive zoledronate solution for 5 s and 10 min respectively before being rinsed in saline. In the third group, 8 µL of the solution was adsorbed to the graft without rinsing. A saline group served as negative control and a systemically treated group as positive control. The grafts were harvested at 6 weeks and evaluated histomorphometrically. The relative amount of remaining bone in the remodeled part of the graft bone (BV/TV) was used to evaluate bone resorption. BV/TV was 11% in the controls, 41% in the group receiving systemic treatment (p = 0.001 vs control) and between 54 and 61% in the groups receiving local treatment (p < 0.007 vs control and p < 0.01 vs systemic). The ingrowth distance of new bone into the chamber was used to evaluate the anabolic response and was 2.6 mm in the saline and 2.5 mm in the systemic group. In the 10 min group, a decreased ingrowth was found (1.6 mm, p = 0.007 and p = 0.008). In the other two local treatment groups, ingrowth was 2.3 mm (5 s soaking, n.s.) and 2.2 mm (no rinsing, n.s.). We found a strong effect by a bisphosphonate on bone resorption but also a limited dose dependent inhibition of the ingrowth of new bone. Local treatment resulted in stronger inhibition of both resorption and bone formation compared to systemic treatment. Rinsing of the graft before implantation to remove any unbound zoledronic acid did not seem to make any marked difference. doi:10.1016/j.bone.2009.01.445

397 Trolox prevents osteoclastogenesis by suppressing RANKL expression and signaling Z. Lee, J. Lee, H. Kim, D. Yang, K. Jung, H. Ha Cell and Developmental Biology, Seoul National University School of Dentistry, Seoul, South Korea Excessive receptor of NF-κB ligand (RANKL) signaling causes enhanced osteoclast formation and bone resorption. Downregulation of RANKL expression or its downstream signals may be a therapeutic approach to the treatment of pathological bone loss. In this study, we investigated the effects of Trolox, a water-soluble vitamin E analogue, on osteoclastogenesis and RANKL signaling. Trolox potently inhibited IL-1-induced osteoclast formation in bone marrow cell-osteoblast coculture by abrogating RANKL induction in osteoblasts. This RANKL reduction was attributed to the reduced-prostaglandin E2 production via downregulating cyclooxyganase-2 activity. We also found that Trolox inhibits osteoclast formation from bone marrow macrophages (BMMs) induced by M-CSF plus RANKL in a reversible manner. Trolox was effective only when present during the early stage of culture,

Abstracts / Bone 44 (2009) S131–S141

implying that it targets early osteoclast precursors. Pretreatment with Trolox did not affect RANKL-induced early signaling pathways including MAPKs, NF-κB, and Akt. We found that Trolox downregulates RANKL induction of c-Fos protein by suppressing its translation. Ectopic overexpression of c-Fos rescued the Trolox inhibition of osteoclastogenesis in BMMs. Trolox also suppressed IL-1-induced osteoclast formation and bone loss in mouse calvarial bone. Taken together, our findings indicate that Trolox prevents osteoclast formation and bone loss by inhibiting both RANKL induction in osteoblasts and c-Fos expression in osteoclast precursors. doi:10.1016/j.bone.2009.01.446

398 Corning Bone Cell Assay Surface H. Rao, J. Tan, E.J. Fewkes Biochem. Techenologies, Corning Incorporated, Corning, New York, USA

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Bone cell assays are very important component in drug discovery for bone related diseases such as osteoporosis, multiple myeloma, Paget's disease and cancer bone metastasis. A newly developed inorganic crystal coating on polystyrene cell culture plates has been shown to be suitable for various bone cell assays. These inorganic coating materials have similar physical and chemical properties to the inorganic phase of natural bone. The Corning Bone Cell Assay Surface has been evaluated for osteoclast differentiation and bone resorption, osteoblast differentiation, co-culture of osteoclast and osteoblast. Measurement of the in vitro inhibition of bone resorption by alendronate was also assayed on Corning Bone Assay Surface. Corning Bone Cell Assay Surface provides: 1) a surface mimic bone inorganic phase; 2) a surface suitable to facilitate osteoclast differentiation and bone resorption, osteoblast differentiation and co-culture; and 3) a platform for bone related drug screening. doi:10.1016/j.bone.2009.01.447