Mariko Takahashi

A TNF receptor loop peptide mimic blocks RANK ligand–induced signaling, bone resorption, and bone loss

Activating receptor activator of NF-kappaB (RANK) and TNF receptor (TNFR) promote osteoclast differentiation. A critical ligand contact site on the TNFR is partly conserved in RANK. Surface plasmon resonance studies showed that a peptide (WP9QY) that mimics this TNFR contact site and inhibits TNF-alpha-induced activity bound to RANK ligand (RANKL). Changing a single residue predicted to play an important role in the interaction reduced the binding significantly. WP9QY, but not the altered control peptide, inhibited the RANKL-induced activation of RANK-dependent signaling in RAW 264.7 cells but had no effect on M-CSF-induced activation of some of the same signaling events. WP9QY but not the control peptide also prevented RANKL-induced bone resorption and osteoclastogenesis, even when TNFRs were absent or blocked. In vivo, where both RANKL and TNF-alpha promote osteoclastogenesis, osteoclast activity, and bone loss, WP9QY prevented the increased osteoclastogenesis and bone loss induced in mice by ovariectomy or low dietary calcium, in the latter case in both wild-type and TNFR double-knockout mice. These results suggest that a peptide that mimics a TNFR ligand contact site blocks bone resorption by interfering with recruitment and activation of osteoclasts by both RANKL and TNF.

Tool-use learning induces BDNF expression in a selective portion of monkey anterior parietal cortex

Learning but not execution of tool-use induced expression of brain-derived neurotrophic factor (BDNF). The expression was highest in the anterior bank of the intraparietal sulcus, especially in the region posteriorly adjacent to the somatosensory shoulder and forearm region in area 3b, suggesting that BDNF plays a role in altering the body image of the hand to include the repeatedly used tool as its extension.

Tool-use learning selectively induces expression of brain-derived neurotrophic factor, its receptor trkB, and neurotrophin 3 in the intraparietal multisensorycortex of monkeys.

When humans repeatedly use a tool, our body image alters until the tool finally becomes a part or an extension of the body. This alteration of body image perhaps results from re-integration of somatosensory and visual signals. We trained Japanese monkeys to use a rake-shaped tool to retrieve a distant food pellet, then used a novel tissue-sampling method to suction brain tissue from the anterior bank of their intraparietal sulcus, where somatosensory and visual signals converge. Examination of the messenger RNA expression levels of neurotrophins and their receptors using real-time quantitative polymerase chain reaction revealed learning-selective induction in the expression of brain-derived neurotrophic factor, its receptor trkB, and NT-3 during, but not after, the learning. These results suggest that these factors are involved in the reorganization of the somatosensory and visual signals in the anterior bank of the intraparietal sulcus when monkeys are learning the use of the tool.

P130Cas, Crk-associated substrate, plays important roles in osteoclastic bone Resorption

p130Cas, Crk‐associated substrate (Cas), is an adaptor/scaffold protein that plays a central role in actin cytoskeletal reorganization. We previously reported that p130Cas is not tyrosine‐phosphorylated in osteoclasts derived from Src‐deficient mice, which are congenitally osteopetrotic, suggesting that p130Cas serves as a downstream molecule of c‐Src and is involved in osteoclastic bone resorption. However, the physiological role of p130Cas in osteoclasts has not yet been confirmed because the p130Cas‐deficient mice displayed embryonic lethality. Osteoclast‐specific p130Cas conditional knockout (p130CasΔOCL–) mice exhibit a high bone mass phenotype caused by defect in multinucleation and cytoskeleton organization causing bone resorption deficiency. Bone marrow cells from p130CasΔOCL– mice were able to differentiate into osteoclasts and wild‐type cells in vitro. However, osteoclasts from p130CasΔOCL– mice failed to form actin rings and resorb pits on dentine slices. Although the initial events of osteoclast attachment, such as β3‐integrin or Src phosphorylation, were intact, the Rac1 activity that organizes the actin cytoskeleton was reduced, and its distribution was disrupted in p130CasΔOCL– osteoclasts. Dedicator of cytokinesis 5 (Dock5), a Rho family guanine nucleotide exchanger, failed to associate with Src or Pyk2 in osteoclasts in the absence of p130Cas. These results strongly indicate that p130Cas plays pivotal roles in osteoclastic bone resorption.

Defective nuclear factor‐κB‐inducing kinase in aly/aly mice prevents bone resorption induced by local injection of lipopolysaccharide

BACKGROUND AND OBJECTIVE Nuclear factor-κB (NF-κB) is activated at sites of inflammation in many diseases, including periodontitis. Nuclear factor-κB induces the transcription of proinflammatory cytokines, resulting in increased osteoclastogenesis and bone resorption. Recently, it has been shown that the NF-κB alternative pathway is important for maintainance of physiological bone homeostasis. Activation of this pathway is by processing of the inhibitor p100 into the active subunit p52 by nuclear factor-κB-inducing kinase (NIK). Defective NIK in aly/aly mice (NIK(aly)) causes mild osteopetrosis and blunted RANKL-stimulated osteoclastogenesis in vivo and in vitro, suggesting that NIK is necessary for basal and stimulated osteoclastogenesis. Nevertheless, the role of NIK in pathological bone resorption is not well investigated. The present study was undertaken to investigate the role of NIK in lipopolysaccharide (LPS)-induced inflammatory bone resorption using aly/aly mice. MATERIAL AND METHODS Mice were injected with LPS over the calvariae and killed 5 d later. Calvariae were subjected to radiological analysis. Histological sections were stained for tartrate-resistant acid phosphatase, and histomorphometric analysis was performed to quantify the number of osteoclasts and the area of bone resorption. RESULTS Lipopolysaccharide-induced inflammation was observed in wild-type and aly/+ mice but not in aly/aly mice. Lipopolysaccharide significantly reduced the calvarial bone mineral density in wild-type and aly/+ mice, whereas bone mineral density was comparable in LPS- and vehicle-injected aly/aly mice. In addition, aly/aly mice were resistant to LPS-induced bone resorption and osteoclastogenesis. CONCLUSION Taken together, these data show that NIK is important in the bone-destructive components of inflammation and represents a possible therapeutic target.

The inhibitory effects of a RANKL-binding peptide on articular and periarticular bone loss in a murine model of collagen-induced arthritis: a bone histomorphometric study

IntroductionWe designed OP3-4 (YCEIEFCYLIR), a cyclic peptide, to mimic the soluble osteoprotegerin (OPG), and was proven to bind to RANKL (receptor activator of NF-κB ligand), thereby inhibiting osteoclastogenesis. We recently found that another RANKL binding peptide, W9, could accelerate bone formation by affecting RANKL signaling in osteoblasts. We herein demonstrate the effects of OP3-4 on bone formation and bone loss in a murine model of rheumatoid arthritis.MethodsTwenty-four seven-week-old male DBA/1J mice were used to generate a murine model of collagen-induced arthritis (CIA). Then, vehicle or OP3-4 (9 mg/kg/day or 18 mg/kg/day) was subcutaneously infused using infusion pumps for three weeks beginning seven days after the second immunization. The arthritis score was assessed, and the mice were sacrificed on day 49. Thereafter, radiographic, histological and biochemical analyses were performed.ResultsThe OP3-4 treatment did not significantly inhibit the CIA-induced arthritis, but limited bone loss. Micro-CT images and quantitative measurements of the bone mineral density revealed that 18 mg/kg/day OP3-4 prevented the CIA-induced bone loss at both articular and periarticular sites of tibiae. As expected, OP3-4 significantly reduced the CIA-induced serum CTX levels, a marker of bone resorption. Interestingly, the bone histomorphometric analyses using undecalcified sections showed that OP3-4 prevented the CIA-induced reduction of bone formation-related parameters at the periarticular sites.ConclusionThe peptide that mimicked OPG prevented inflammatory bone loss by inhibiting bone resorption and stimulating bone formation. It could therefore be a useful template for the development of small molecule drugs for inflammatory bone loss.

Nanogel-crosslinked nanoparticles increase the inhibitory effects of W9 synthetic peptide on bone loss in a murine bone resorption model

We investigated the biological activity of W9, a bone resorption inhibitor peptide, using NanoClik nanoparticles as an injectable carrier, where acryloyl group-modified cholesterol-bearing pullulan (CHPOA) nanogels were crosslinked by pentaerythritol tetra (mercaptoethyl) polyoxyethylene. Thirty 5-week-old male C57BL/6J mice were fed a low calcium diet and received once-daily subcutaneous injections of the carrier alone, W9 24 mg/kg/day alone, W9 24 mg/kg/day incorporated in cholesterol bearing pullulan (CHP) nanogels, or W9 (8 and 24 mg/kg/day) incorporated in NanoClik nanoparticles for 4 days (n=5). Mice that received a normal calcium diet with NanoClik nanoparticle injections without W9 were used as a control group. Radiological analyses showed that administration of W9 24 mg/kg/day significantly prevented low calcium-induced reduction of bone mineral density in the long bones and lumbar vertebrae, but only when the NanoClik nanoparticles were used as a carrier. Histomorphometric analyses of the proximal tibiae revealed that W9 24 mg/kg/day incorporated in NanoClik nanoparticles prevented the increase in bone resorption indices induced by a low calcium diet, which was confirmed by measurement of serum bone resorption markers. These data suggest that NanoClik nanoparticles could be a useful carrier for peptide therapeutics, and also demonstrate that daily subcutaneous injections of the W9 peptide with the nanoparticles were able to inhibit bone loss in vivo. An osteoclastogenesis inhibition assay performed in vitro confirmed a slower release profile of W9 from NanoClik nanoparticles compared with conventional CHP nanogels.