Nuclear Nrf2 Induction by Protein Transduction Attenuates Osteoclastogenesis

Free Radical Biology and Medicine 77 (2014) 239–248

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Original Contribution

Nuclear Nrf2 Induction by Protein Transduction Attenuates Osteoclastogenesis Hiroyuki Kanzaki a,b,n, Fumiaki Shinohara c, Mikihito Kajiya d, Sari Fukaya b, Yutaka Miyamoto b, Yoshiki Nakamura b a

Tohoku University Hospital, Maxillo-Oral Disorders Department of orthodontics, School of Dental Medicine, Tsurumi University 2-1-3 Tsurumi, Tsurumi-ku, Yokohama, Kanagawa 230-8501, Japan c Tohoku University Graduate School of Dentistry, Oral MicrobiologySendai, Miyagi 980-8577, Japan d Department of Periodontal Medicine, Division of Applied Life Science, Hiroshima University Graduate School of Biomedical & Health Sciences Hiroshima University, Hiroshima, Japan b

art ic l e i nf o

a b s t r a c t

Article history: Received 28 April 2014 Received in revised form 26 August 2014 Accepted 5 September 2014 Available online 16 September 2014

It has been reported that reactive oxygen species (ROS) play a role as intracellular signaling molecules in RANKL stimulation. Previously we demonstrated that induction of cytoprotective enzyme expression by Nrf2gene transfer successfully ameliorated RANKL-dependent osteoclastogenesis. In the present study, we hypothesized that Nrf2 activation by inhibiting ubiquitination and degradation of Nrf2 by ETGE-peptide would induce Nrf2-dependent cytoprotective enzyme expression, attenuate ROS signaling, and thereby inhibit RANKL-dependent osteoclastogenesis. ETGE-peptide containing a cell-permeable sequence (seven consecutive arginine; 7R-ETGE) was applied to a mouse macrophage cell-line RAW 264.7 cell or a primary macrophage culture. ETGE-peptide prevents Keap1 from binding to Nrf2. Nrf2 nuclear translocation and Nrf2-dependent cytoprotective enzyme induction was observed. The effects of 7R-ETGE on RANKL-dependent induction of intracellular ROS levels and osteoclastogenesis were examined. Finally, the protective effect of 7R-ETGE on RANKL-mediated bone destruction was investigated in mice. 7R-ETGE dose-dependently induced nuclear Nrf2, followed by the induction of cytoprotective enzyme expression at both the gene and protein level. 7R-ETGE inhibited upregulation of intracellular ROS levels by RANKL stimulation, and osteoclastogenesis was attenuated. Of particular interest was that local injection of 7R-ETGE ameliorated RANKL-mediated bone destruction. Local induction of nuclear Nrf2 by protein transduction is a potential novel therapeutic target for bone destruction diseases such as periodontitis and rheumatoid arthritis. & 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Keywords: Oxidative stress Nrf2 Cytoprotective enzyme Osteoclast RANKL ROS

\ Introduction

Oxidative stress is thought to be one of the drivers of bone destructive disease such as periodontitis [1], and reactive oxygen species (ROS) play a role in osteoclastogenesis as intracellular signaling molecules following RANKL signaling [2]. It is well known that ROS act not only as intracellular signaling molecules, but also exert cytotoxic effects such as peroxidation of lipids and phospholipids [3], and oxidative damage to proteins and DNA [4]. Therefore, cells have several protective mechanisms against these oxidative stressors [5]. Transcriptional factor nuclear factor E2-related factor 2 (Nrf2) transcriptionally controls the gene expression of many cytoprotective enzymes, such as heme oxygenase-1 (HO-1) [6], NAD(P)H: quinone reductase (NQO1) [7], gamma-glutamylcysteine synthetase n Corresponding author at: Department of orthodontics, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama, Kanagawa pref., 230–8501, Japan. Fax: þ 81 45 573 9599. E-mail address: [email protected] (H. Kanzaki).

(GCS) [8], and glucose 6-phosphate dehydrogenase (G6PD) [9]. These cytoprotective enzymes exhibit ROS scavenging ability [10–13]. Previously we reported that the Keap1/Nrf2 axis regulates RANKLdependent osteoclastogenesis through modulation of intracellular ROS signaling by the expression of cytoprotective enzymes [14]. More importantly, in vivo local Nrf2-gene transfer significantly inhibited RANKL-mediated osteoclastogenesis. These results stimulated the experimental idea to attenuate RANKL-mediated osteoclastogenesis by inhibiting ROS signaling with the activation of Nrf2-dependent cytoprotective enzyme expression. The fate of Nrf2 is strictly regulated by kelch-like ECH-associated protein 1 (Keap1), and Nrf2-dependent transcription of cytoprotective enzymes is negatively regulated by inhibition of Nrf2 nuclear translocation, with cytoplasmic ubiquitination and degradation of Nrf2 [15]. During this step, Keap1 binds to the ETGE sequence (LQLDEETGEFLPIQ) of Nrf2, and ubiquitinate Nrf2 [16]. In the present study, we hypothesized that Nrf2 activation, by protecting Nrf2 from Keap1-mediated ubiquitination and degradation, would increase Nrf2-dependent expression of cytoprotective enzymes,

http://dx.doi.org/10.1016/j.freeradbiomed.2014.09.006 0891-5849/& 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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and they would scavenge ROS, and therefore attenuate RANKL signaling and osteoclastogenesis. To examine this hypothesis, we used a synthesized peptide whose sequence (ETGE sequence) is identical to the binding domain of Nrf2 to Keap1, and prevents Keap1 from binding to Nrf2, thereby protecting Nrf2 from ubiquitination and degradation [16]. We were particularly interested in examining whether the peptide can attenuate RANKL-mediated osteoclastogenesis both in vitro and in vivo.

Material and Methods Cells Mouse cells from the RAW 264.7 monocytic cell line RAW 264.7 cells were obtained from the Riken BioResource Center (Tsukuba, Japan). Mouse primary peritoneal macrophages Three C57/B6 mice (Japan SLC Inc., Hamamatsu, Japan) were sacrificed, and cold phosphate-buffered saline solution (PBS) was injected into their peritoneal cavities. Peritoneal lavage fluid was collected and centrifuged to harvest cells. The cells recovered from the precipitated pellet were pre-cultured with 50 ng/ml of recombinant macrophage colony-stimulating factor (M-CSF; Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 4 days and the attached cells were used as peritoneal macrophages [17]. Recombinant M-CSF (20 ng/ml) was added to the culture medium. Cell culture Cells were cultured in alpha-modified Eagle Medium (α-MEM; Wako Pure Chemical Industries, Ltd., Osaka, Japan) containing 10% (V/V) fetal bovine serum (HyClone; Thermo Scientific, South Logan, UT) supplemented with antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). All cells were cultured at 37 1C in a 5% CO2 incubator. Construction of ETGE-peptide The ETGE-peptide (LQLDEETGEFLPIQ) sequence is identical to the binding domain of Nrf2 to ubiquitination facilitator protein Keap1, and inhibits Keap1 from binding to Nrf2 [16]. ETGE-peptide and ETGEpeptide containing the cell-permeable sequence (seven consecutive arginine; 7R-ETGE: RRRRRRRRLQLDEETGEFLPIQ) were synthesized (Medical & Biological Laboratories Co., Ltd, Nagoya, Japan). Also, 7R peptide (RRRRRRR) was synthesized (PH Japan, Hiroshima, Japan). These peptides were dissolved in N,N-dimethylformamide (DMF) at a concentration of 50 mM and stored in a freezer. In the preliminary experiments, we compared control cells and DMF treated cells and observed no prominent difference including ROS production in vitro experiments (data not shown), and set no DMF control group in the series of in vitro experiments. Osteoclastogenesis assay RAW 264.7 cells were plated onto 96-well plates at a density of 103 cells/well, in the presence or absence of recombinant sRANKL (100 ng/ml final concentration; Wako Pure Chemical Industries, Ltd.) with or without ETGE-peptides or 7R peptide (50 μM final, each). Mouse primary peritoneal macrophages (104 cells/well) were plated onto 96-well plates, in the presence or absence of recombinant sRANKL (100 ng/ml final concentration; Wako Pure Chemical Industries, Ltd.) and M-CSF (20 ng/ml) with or without ETGE-peptides (50 μM final). After 4days culture, cells were stained for tartrateresistant acid phosphatase (TRAP) using an acid phosphatase kit

(Sigma Chemical Co., St Louis, MO) according to the manufacturer's instructions. Dark-red multi-nucleated cells (Z3 nuclei) were counted as TRAP-positive multi-nucleated cells. Real-time PCR analysis In some experiments, RNA was extracted using the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich Co., St Louis, MO) with on-column DNA digestion according to the manufacturer's instructions. After measurement of the RNA concentration, isolated RNA (100 ng each) was reverse transcribed with iScript cDNA Supermix (Bio-Rad Laboratories, Hercules, CA), and cDNA stock was diluted (  5) with Tris-EDTA (TE) buffer. Real-time RTPCR was performed with SsoFast EvaGreen Supermix (Bio-Rad Laboratories) using a CFX96 instrument (Bio-Rad Laboratories). The PCR primers used in the experiments have been described elsewhere [14]. Fold changes of gene of interest were calculated with ΔΔCt method using ribosomal protein S18 (RPS18) as reference gene [19]. Resorption assay RAW 264.7 cells were seeded on synthesized calcium phosphate substrate (bone resorption assay plate; PG Research, Tokyo, Japan) and stimulated with RANKL (100 ng/ml) in the presence or absence of ETGE peptides (50 μM final). After 7 days' cultivation, cells were removed with bleach, and the calcium phosphate substrate was washed with distilled water and then dried. Photographs were taken, and the average of the resorbed area per field was calculated from twelve images of each sample, using Image-J software (National Institutes of Health, Bethesda, MD). Intracellular ROS detection Intracellular ROS levels were detected using the Total ROS/ Superoxide Detection Kit (Enzo Life Sciences Inc., Farmingdale, NY, USA), according to the manufacturer's instructions. The ROS detection dye in the kit reacts directly with a wide range of reactive species, such as hydrogen peroxide (H2O2), peroxynitrite (ONOO-), hydroxyl radicals (HO  ), nitric oxide (NO), and peroxy radical (ROO  ). Cells were pre-cultured with or without ETGEpeptide (50 μM final) for 24 h, and then washed with PBS. Cells were stimulated with sRANKL for 6 h, washed and collected in PBS supplemented with 2% fetal bovine serum (FBS). The cell suspension was incubated with oxidative stress detection reagent for 30 min on ice. After washing with PBS, intracellular ROS were detected using an Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ). The viable monocyte/macrophage fraction was gated on an FSC/SSC plot, and ROS levels were monitored in the FL-1 channel. Additionally, we used the cell-permeable specific H2O2 probe (BES-H2O2-Ac; Wako) [20] and specific superoxide probe (BES-SoAM; Wako) [21] to detect each reactive oxygen species, respectively. Briefly, these probe were dissolved in dimethylformamide, and used at the final concentration of 5 μM, and fluorescence were monitored in the FL-1 channel. Preparation of nuclear protein lysate Nuclear protein lysate was prepared from RAW 264.7 cells using the DUALXtract cytoplasmic and nuclear protein extraction kit (Dualsystems Biotech AG, Schlieren, Switzerland) according to the manufacturer's instructions. Cultured cells were washed with PBS and treated with cell lysis buffer. After centrifugation, supernatant of this step was used as cytoplasmic protein samples, and the nuclear pellet was washed twice and lysed with nuclear lysis

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reagent. After centrifugation, the cleared supernatant was used as the nuclear protein extract. The protein concentrations of each of the nuclear lysates were measured with the Quick Start Protein Assay Kit (Bio-Rad Laboratories), and the concentrations were adjusted to be the same. After mixing with  4 sample buffer containing beta-mercaptoethanol, the samples were heatdenatured at 70 1C for 10 min. Western blot analysis for Nrf2 The prepared nuclear lysates, containing equal amounts of protein, were electrophoresed on a TGX Precast gel (BioRad Laboratories), and the proteins transferred to a polyvinylidene difluoride (PVDF) membrane using an iBlot blotting system (Life Technologies Japan Ltd., Tokyo, Japan). The transferred membrane was treated with Qentix Western Blot Signal Enhancer (Thermo Fisher Scientific Inc., Rockford, IL) according to the manufacturer's instructions. After washing with deionized water, the membrane was blocked with BlockAce (DS Pharma Biomedical Inc., Osaka, Japan) for 30 min, and then incubated for 1 h with rabbit IgG antiNrf2 antibody (Santa Cruz Biotechnology Inc.) in Can Get Signal Solution-1 (Toyobo Co. Ltd, Tokyo, Japan). After thorough washing with PBS containing 0.5% Tween-20 (PBS-T), the membrane was incubated for 1 h with horseradish peroxidase (HRP)-conjugated protein A/G (Thermo Fisher Scientific Inc.) in Can Get Signal Solution-2 (Toyobo Co. Ltd), and washed with PBS-T. Chemiluminescence was produced using Luminata Forte (EMD Millipore Corporation, Billerica, MA) and detected with an ImageQuant LAS-4000 digital imaging system (GE Healthcare Japan, Tokyo, Japan). To confirm the equivalence of loaded nuclear protein, the membrane was re-probed with Restore Plus Western Blot Stripping Buffer (Thermo Fisher Scientific Inc.) for 15 min, washed, blocked, and then blotted in anti-histone H3 antibody (Cell Signaling Technology Japan, Tokyo, Japan) followed by HRP-conjugated protein A/G. Western blot analysis for HO-1, NQO1, and GCS. Cytoplasmic protein samples were used for the detection of HO-1, NQO1, and GCS using different membranes. The antibodies used in these experiments were anti HO-1 antibody (StressMarq Biosciences Inc, Victoria, BC), anti-NQO1 antibody (Abcam plc, Cambridge, MA) and anti-GCS antibody (Thermo Fisher Scientific Inc.). To confirm the equivalence of loaded cytoplasmic protein, TGX Stain-Free Precast Gels (BioRad Laboratories) were used for cytoplasmic protein samples. Proteins in the electrophoresed gel were visualized with ultra violet (UV) treatment before transfer to a PVDF membrane. Visualized protein on the membrane was imaged with an ImageQuant LAS4000 under UV transillumination, and the value of the total protein band density was used for calibration. Immunoprecipitation against Keap1 RAW 264 cells were preloaded 7R-ETGE (50 μM final) for 1 h, and then stimulated with RANKL (100 ng/mL final) for 3 h, and cytoplasmic and nuclear protein samples were prepared as described above. Cytoplasmic protein samples of RANKL-treated cells and 7R-ETGE/RANKL-treated cells were immunoprecipitated against Keap1 using anti Keap1 antibody (AVIVA systems biology, San Diego, CA). Briefly, 50 ng of cytoplasmic protein samples and 2 μg of antibody were incubated on ice with gentle shaking for 9 hours, and further incubated with antibody purification magnetic beads (recombinant protein A-derivatives conjugated magnetic beads: Ab-Capcher Mag; ProteNova Inc., Tokushima, Japan) for 2 hours. Then magnetic beads were separated, washed with PBS-T, and bound protein samples were eluted with tricine sample buffer (BioRad Laboratories) containing 2% β-mercaptoethanol

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(Sigma) with heat treatment (90 1C 15 min). Eluates were then used for western blot analysis for Nrf2 as described above with slight modification of using the EasyBlot anti Rabbit IgG-HRP conjugated (GeneTex, Irvine, CA) as secondary antibody for the purpose of decreasing the interference caused by the heavy and light chains of the IgG used for immunoprecipitation. Detection of phospho-NF-κB RAW 264 cells were preloaded 7R-ETGE (50 μM final) for 1 h, and then stimulated with RANKL (100 ng/mL final) for 3 h, and whole cell lysate were prepared using lysis buffer (EDTA (5 mM), Glycerol (10% v/w), Triton X-100 (1% v/w), SDS (0.1% w/w), and NP40 (1% v/w) in PBS) containing proteinase inhibitor cocktail (Roche Applied science, Tokyo, Japan). Protein concentration of samples were measured with Micro BCA protein assay kit (Thermo Scientific inc., Rockford, IL), and the concentrations were adjusted to be the same. After mixing with  4 sample buffer containing betamercaptoethanol, the samples were heat-denatured at 70 1C for 10 min. Samples were then used for western blot analysis for phospho-NF-κB as described above using anti phospho-NF-κB p65 antibody (Cell Signaling Technology) for primary antibody. In addition, to detect phospho-NF-κB by flow cytometry, stimulated cells were scraped and resuspended into PBS. Then cells were fixed and permeabilized using FOXP3 Fix/Perm Buffer (Biolegend, San Diego, CA), washed with FOXP3 Perm Buffer (Biolegend), stained with alexa Fluor 647-conjugated anti phospho-NF-κB p65 antibody (Beckman coulter, Brea, CA), and examined by an Accuri C6 flow cytometer. In vivo bone destruction model All animal experiment protocols were reviewed and approved by the Institutional Animal Care and Use Committee (Committee) of the Tohoku University Environmental & Safety Committee, Japan. Animal experiments were performed in complying with the Regulations for Animal Experiments and Related Activities at Tohoku University. We utilized repeat injections of LPS for our in vivo bone destruction model [22]. This bone destruction model induces RANKL-dependent osteoclastogenesis and extensive bone destruction. Thirty-two, 8-week-old C57B/6 male mice (Japan SLC Inc., Hamamatsu, Japan) were used in the experiments. The mice were randomized into four equal groups: PBS injection group (control group); 7R-ETGE peptide injected group (7R-ETGE group); LPSinduced bone resorption group (LPS group); and LPS-induced bone resorption and 7R-ETGE peptide injected group (LPS þ 7R-ETGE group). Purified LPS from Escherichia coli O111:B4 (Sigma Chemical Co.) was dissolved in PBS at a concentration of 1 mg/mL. 10 mL of LPS solution with 2 mL of DMF or 7R-ETGE peptide (50 mM) were injected into the animals of LPS group and LPS þ 7RETGE group, respectively. 10 mL of PBS with 2 mL of DMF or 7R-ETGE peptide (50 mM) were injected into animals in the control and 7RETGE groups, respectively. Injections were performed under anesthesia with a 30-gauge needle at a point on the midline of the skull located between the eyes, on days 1, 3, 5, 7 and 9. On day 11, the mice were sacrificed by cervical dislocation. RNA from the excised cranial tissue around the injected site (three RNA samples per group) was extracted and reverse transcribed (vide supra). Gene expression was examined by real-time PCR. Other cranial tissue samples (5 samples per group) were fixed overnight with 4% paraformaldehyde in PBS, and the specimen was subsequently scanned with an X-ray micro tomography (microCT) system (ScanXmate-E090; Comscan Tecno Co., Ltd., Kanagawa, Japan). The scanned microCT images were reconstituted using ConeCTexpress software (Comscan Tecno Co., Ltd.). After reconstitution and obtaining the DICOM files, 3D-volume rendering was performed using Pluto software (http://pluto.newves.org/trac).

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Statistical analysis All data are presented as the mean 7 standard deviation (SD). All in vitro data are the means of 3 independent experiments.

Multiple comparisons were performed using Tukey's test (MEPHAS; http://www.gen-info.osaka-u.ac.jp/testdocs/tomocom/ tukey-e.html). p o0.05 was considered to be statistically significant.

Fig. 1. 7R-ETGE peptide inhibited osteoclastogenesis. RAW 264.7 cells were stimulated with RANKL for 4 days and TRAP staining was performed. (A) RAW 264.7 cells stimulated with RANKL (100 ng/ml). (B) RAW 264.7 cells cultured with RANKL and 7R-ETGE peptide. (C) RAW 264.7 cells cultured with RANKL and ETGE peptide. (D) RAW 264.7 cells cultured with RANKL and 7R peptide. Arrowhead indicates TRAP þ multi-nucleated cells. Bar ¼ 100 μm. Representative photographs are shown. (E) The number of TRAPþ multi-nucleated cells per well was counted. The data shown are representative of three independent experiments performed in triplicate. np o 0.05 between groups. NS: no significant difference between groups. Mouse primary peritoneal macrophage were stimulated with M-CSF and RANKL for 4 days and TRAP staining was performed. (F) Control. (G) Stimulated with RANKL (100 ng/ml). (H) Stimulated with RANKL (100 ng/ml) in the presence of 7R-ETGE peptide. Arrowhead indicates TRAPþ multinucleated cells. Bar ¼ 100 μm. Representative photographs were shown. (I) Number of TRAP þ multi-nucleated cells per well was counted. The data shown are representative of three independent experiments performed in triplicate. np o 0.05 between groups.

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Results ETGE peptide inhibited osteoclastogenesis and resorption activity First, we examined whether ETGE peptide had an inhibitory effect on RANKL-mediated osteoclastogenesis by using RAW 264.7 cells at day¼4. TRAP staining revealed that 7R-ETGE peptide (Fig. 1B) inhibited RANKL-mediated osteoclastogenesis (Fig. 1A). The number of TRAP positive multi-nucleated cells was statistically significantly different between RANKL alone and RANKL with 7R-ETGE peptide (Fig. 1E). There was no statistically significant difference between RANKL alone, RANKL with ETGE peptide (non cell permeable peptide; Fig. 1C), signifying cell permeable property is necessary for the inhibition of RANKL-mediated osteoclastogenesis by ETGE peptide. In addition, no significant difference was observed between RANKL alone

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and RANKL with 7R peptide (Fig. 1D), signifying cell permeable peptide sequence, 7R had nothing to do with osteoclastogenesis. Similar results were obtained using mouse primary peritoneal macrophages (Fig. 1F-I). 7R-ETGE peptide (Fig. 1H) inhibited RANKLmediated osteoclastogenesis (Fig. 1G). The number of TRAP positive multi-nucleated cells was statistically different between RANKL alone and RANKL with 7R-ETGE peptide (Fig. 1I). We further examined the effect of ETGE peptide on RANKLmediated osteoclast marker gene induction. 7R-ETGE peptide inhibited RANKL-mediated inductions of ATP6v0d2, DC-STAMP and OSCAR, respectively (Fig. 2). These results suggest that 7R-ETGE might inhibit not only osteoclastogenesis but also osteoclast function. Next, we examined whether 7R-ETGE peptide shows inhibitory effect on RANKL-mediated resorption activity. Compared to RANKL alone (Fig. 3A), 7R-ETGE peptide inhibited RANKL-mediated resorption activity (Fig. 3B). Non cell permeable ETGE peptide failed to inhibit RANKL-mediated resorption activity (Fig. 3C). Fig. 3D shows the percentage area of the assay plate resorbed, and statistical significance was observed between RANKL alone and RANKL with 7R-ETGE. No statistical difference was observed between RANKL alone and RANKL with non cell permeable ETGE peptide. These results suggest that cell permeable 7R-ETGE peptide, but not ETGE peptide can inhibit RANKLmediated osteoclastogenesis and osteoclast activation. 7R-ETGE peptide attenuated RANKL-mediated intracellular ROS Next, we investigated whether 7R-ETGE peptide could interfere with RANKL-mediated intracellular ROS signaling by using flow

Fig. 2. Osteoclast marker gene expressions were reduced by 7R-ETGE peptide. The expression of the osteoclast marker genes, ATP6v0d2, DC-STAMP and OSCAR in RAW 264.7 cells was examined by real-time PCR. The fold increase from the control is shown. The displayed data are representative of three independent experiments performed in triplicate. np o 0.05 versus stimulation with RANKL alone.

Fig. 3. 7R-ETGE peptide attenuated osteoclastic resorption activity. RAW 264.7 cells were stimulated with RANKL (100 ng/ml) on synthesized calcium phosphate substrate for 7 days, and resorption area was analysed. (A) Stimulated with RANKL. (B) Stimulated with RANKL in the presence of 7R-ETGE peptide. (C) Stimulated with RANKL in the presence of ETGE peptide. Arrowhead indicates resorbed lacunae. Bar ¼100 μm. Representative photographs are shown. (D) The percentage of the resorbed area was calculated. The data shown are representative of three independent experiments performed in triplicate. np o0.05 versus stimulation with RANKL alone. NS: no significant difference between groups.

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Fig. 4. Flowcytometry analysis for intracellular ROS. Intracellular ROS levels in RAW 264.7 cells were examined using flow cytometry. (A) RANKL induced intracellular ROS. The red line indicates the control, and the blue line indicates stimulation with RANKL. The data shown are representative of three independent experiments performed in triplicate. Vertical black line indicates threshold. (B) 7R-ETGE peptide but not ETGE peptide reduced RANKL-induced intracellular ROS in RAW 264.7 cells. The blue line, red line, and orange line indicate RANKL alone, RANKL with 7R-ETGE peptide, and RANKL with ETGE peptide, respectively. The data shown are representative of three independent experiments performed in triplicate. Vertical black line indicates threshold. (C) Quantitative analysis for percent of ROS positive in control and RANKL samples. Percent of ROS positive population judged by the threshold in Fig. 4A were calculated from triplicated samples. nnp o0.01 between samples. (D) Quantitative analysis for percent of ROS positive in RANKL alone, RANKL with 7R-ETGE peptide, and RANKL with ETGE peptide samples. Percent of ROS positive population judged by the threshold in Fig. 4B were calculated from triplicated samples. np o 0.05 versus RANKL alone. NS: no significant difference between groups. (E) RANKL induced intracellular superoxide. Intracellular superoxide was observed using specific superoxide probe, BES-So-AM. The the blue line indicates stimulation with RANKL, and red line indicates the RANKL with 7R-ETGE peptide. The data shown are representative of three independent experiments performed in triplicate. Vertical black line indicates threshold. (F) RANKL induced intracellular H2O2. Intracellular H2O2 was observed using specific superoxide probe, BES-H2O2-Ac. The the blue line indicates stimulation with RANKL, and red line indicates the RANKL with 7R-ETGE peptide. The data shown are representative of three independent experiments performed in triplicate. Vertical black line indicates threshold. (G) Quantitative analysis for percent of superoxide positive in the samples. Percent of superoxide positive population judged by the threshold in Fig. 4E were calculated from triplicated samples. np o0.05 between samples. (H) Quantitative analysis for percent of H2O2 positive in the samples. Percent of H2O2 positive population judged by the threshold in Fig. 4F were calculated from triplicated samples. np o 0.05 between samples.

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cytometry. The addition of RANKL induced the formation of intracellular ROS at 6 h (Fig. 4A and C). This RANKL-mediated ROS induction was attenuated by 24 h-pretreatment of 7R-ETGE peptide though that of ETGE peptide had no discernable effects

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(Fig. 4B and D). These results suggest that cell permeable 7R-ETGE peptide attenuates RANKL-mediated intracellular ROS signaling. To further explore intracellular ROS signaling, we used specifc superoxide probe and H2O2 probe, and found that 7R-ETGE attenuated

Fig. 5. 7R-ETGE peptide induced Nrf2-mediated upregulation of cytoprotective enzyme expression at both the mRNA and protein level. (A) Western blot analysis for nuclear Nrf2. Equal amounts of extracted nuclear protein were electrophoresed, transferred onto PDVF membranes, and detected Nrf2 (Top panel). To confirm the equivalence of loaded nuclear protein, stripped membranes were subjected to western blot analysis using anti Histone H3 antibody (Bottom panel). Densitometry analysis was used to calculate the ratio relative to the control, and the mean values of three experiments are shown above each panel. (B) 7R-ETGE peptide inhibited Keap1 from the binding to Nrf2, and increased nuclear translocation of Nrf2. Western blot analysis for Nrf2 of keap1-immunoprecipitated samples from cytoplasm were shown (Top panel). Nuclear Nrf2 were examined by western blot (Bottom panel). (C) mRNA expression of HO1, NQO1, and GCS were examined by real-time PCR. Fold change of each gene expression from control were shown. Open bar: control, Close bar: 7R-ETGE treated cells.np o 0.05 versus control. The data shown are representative of three independent experiments performed in triplicate. (D) Western blot analysis for HO1, NQO1, and GCS. Equal amount of cytoplasmic protein were electrophoresed, transferred onto PDVF membrane, and detected HO1, NQO1, and GCS, respectively. The data shown are representative of three independent experiments. Separate lanes from the same blots had been spliced together, and line indicate the places at which the lanes were joined. (E) Densitometry analysis of each band as shown in Fig. 5D were used to calculate each relative expression ratio of HO1, NQO1 and GCS compared to the control. These values were normalized with respect to the amount of whole protein. The mean values from three experiments are shown. Open bar: control, Close bar: 7R-ETGE treated cells. np o 0.05 versus control. (F) Western blot analysis for phosphorylated NF-κB. Equal amount of whole protein were electrophoresed, transferred onto PDVF membrane, and detected phosphorylated NF-κB using phosphorylated NF-κB specific antibody. Densitometry analysis was used to calculate the ratio relative to the control, and the mean values of three experiments are shown above each panel. (G) Flowcytometry analysis for phosphorylated NF-κB. Each cells were stained with fluorophore labeled different anti phospho-NF-κB p65 antibody and observed by flow cytometer. The blue line, red line, and orange line indicate control, RANKL alone, and RANKL with 7R-ETGE peptide, respectively. The data shown are representative of three independent experiments performed in triplicate. Vertical black line indicates threshold. (H) Quantitative analysis for percent of phospho-NF-κB positive in the samples. Percent of phospho-NF-κB positive population judged by the threshold in Fig. 5G were calculated from triplicated samples. npo 0.05 between samples.

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both RANKL-mediated superoxide (Fig. 4E and G) and H2O2 induction (Fig. 4F and H).

7R-ETGE peptide dose-dependently induced nuclear Nrf2 To understand further the mechanism of the inhibition of RANKL-mediated osteoclastogenesis by 7R-ETGE peptide, we examined nuclear Nrf2 by western blot analysis (Fig. 5A). Treatment with 7R-ETGE peptide for 24 h dose-dependently induced nuclear Nrf2. Then because we presumed 7R-ETGE peptide would inhibit the binding of Nrf2 and Keap1 and thereby translocate into nuclear, the binding of Nrf2 to keap1 in cytoplasm were examined. Immunoprecipitation using same amount of protein for input revealed that 7R-ETGE treatment significantly attenuated the formation of Keap1-Nrf2 complex in cytoplasm (Fig. 5B top panel). Oppositely, nuclear Nrf2 was increased by 7R-ETGE treatment. These results suggest that 7R-ETGE peptide would mimic ETGE sequence of Nrf2, bind to Keap1 instead of Nrf2, augment the translocation of Nrf2 to nucleus, and favor Nrf2-dependent transcription of cytoprotective enzymes.

7R-ETGE peptide upregulated cytoprotective enzyme expression at both the mRNA and protein level Next, the effect of 7R-ETGE peptide on the expression of cytoprotective enzymes were examined. Real-time PCR analysis revealed that all three genes, HO1, NQO1, and GCS, were upregulated by 7R-ETGE peptide treatment (Fig. 5C). Consistent with the mRNA upregulation, protein level upregulation were unequivocally detected using western blot analysis (Fig. 5D). Relative band intensity normalized with total protein indicated 2.1-, 7.2-, and 6.8-fold increases in HO1, NQO1, and GCS expression after 7R-ETGE treatment, respectively (Fig. 5E).

These results suggest that 7R-ETGE peptide can upregulate cytoprotective enzyme expression via Nrf2-dependent transcription. Interference on RANKL-mediated NF-κB signaling by 7R-ETGE peptide To further explore the interference on intracellular RANKL signaling by 7R-ETGE, we examined the essential signaling pathway, NF-κB. Western blot analysis revealed that RANKL-mediated phosphorylation of NF-κB was attenuated by 7R-ETGE treatment (Fig. 5F). Similar results were obtained by flowcytometry using fluorophore-labeled different anti phospho-NF-κB antibody (Fig. 5G and H). These results suggest that the NF-κB signaling could be the downstream of RANKLmediated ROS signaling. 7R-ETGE peptide inhibited bone destruction in experimental animals Finally, we determined whether 7R-ETGE peptide ameliorates RANKL-mediated bone destruction in vivo. Repeat LPS injection upregulated osteoclastogenic cytokine mRNA, RANKL and TNF-α (Fig. 6A). This RANKL and TNF-α augmentation led to the upregulation of osteoclast marker genes such as MMP9 and TRAP, signifying the induction of osteoclastic bone destruction. 7RETGE peptide significantly reduced both osteoclastogenic cytokine and osteoclast marker gene expression. These results suggest that 7R-ETGE peptide attenuated osteoclastogenesis even in vivo. MicroCT analysis revealed that repeated LPS injections induced extensive resorption of cranial bone (Fig. 6B). 7R-ETGE peptide ameliorated LPS-induced RANKL-mediated bone destruction. The calculated resorbed area in the cranial bone revealed that there is a statistically significant difference between LPS alone and LPS with 7R-ETGE peptide (Fig. 6C). In addition, there is no statistically significant difference between the control, and LPS with 7R-ETGE peptide, signifying 7R-ETGE peptide almost completely inhibited

Fig. 6. ETGE peptide inhibited bone destruction in experimental animals. LPS (10 μg/site) or PBS were injected 5 times into each mouse every other day, with or without 7RETGE peptide. (A) Real-time PCR analysis was performed using three RNA samples per group. Fold change from control are shown. np o 0.05 between groups. (B) MicroCT images were taken using 5 samples per group, and representative photographs are shown. Arrowheads indicate resorbed lacunae or holes formed in the cranial bone. Bar ¼1 mm. (C) The resorbed area in the cranial bone was calculated using Image-J software. Mean values are shown for the percentage resorbed (calculated from the ratio of the number of pixels in the resorbed area in the cranial bone to the number of pixels in the cranial bone in the analyzed image). npo 0.05 versus control. NS: no significant difference versus control. †:p o0.05 between groups.

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LPS-induced RANKL-mediated bone destruction. These data suggest that 7R-ETGE peptide could inhibit RANKL-mediated osteoclastogenesis not only in vitro but also in vivo.

Discussion In the present study, we discovered that 7R-ETGE peptide could inhibit osteoclastogenesis in vitro and in vivo. Additionally, we clarified the inhibitory mechanism of 7R-ETGE peptide on RANKLmediated osteoclastogenesis as follows; 1) 7R-ETGE peptide mimics the ETGE sequence of Nrf2, and Keap1 binds to 7R-ETGE peptide instead of Nrf2. This leads the attenuation of Nrf2 degradatation, and induces nuclear translocation of Nrf2. 2) Nuclear translocated Nrf2 transcriptionally increases the Nrf2-dependent expression of cytoprotective enzymes such as HO1, GCS and NQO1. 3) Upregulated cytoprotective enzymes scavenge ROS, intracellular signaling molecules of RANKL. 4) Attenuated ROS signaling gives the reduction of RANKL-mediated osteoclastogenesis. We also tested the effect of Nrf2 activation on osteoclastogenesis using chemical Nrf2 activator sulforaphane and Epigallocatechin-3-gallate, and found that both Nrf2 activator attenuated osteoclastogenesis (Data not shown). Additionally, sulforaphane was reported as osteoclastogenesis inhibitor via Nrf2 activation [23] and via another pathway [24]. Taken together, Nrf2 activation and thereby upregulation of cytoprotective enzyme would attenuate ROS signaling in RANKL-mediated osteoclastogenesis. Among cytoprotective enzymes, HO-1 is known to be able to catalyze heme, and produces biliverdin, iron, and carbon monoxide, that have anti-oxidative functions [25]. We assume that the upregulated cytoprotective enzymes decrease intracellular ROS levels, thereby attenuating ROS-mediated RANKL signaling. Regarding the intracellular signaling of RANKL, various signaling pathway for osteoclast differentiation, function, and survival were reported. Which includes Akt, NFATc1, NF-κB, JNK, ERK, and p38 [26]. Of them, as the name of RANKL suggests (receptor activator of NF-κB ligand), RANKL mediates NF-κB signaling during osteoclastogenesis [27]. Our results revealed the interference of NF-κB signaling by 7RETGE with the attenuation of intracellular ROS signaling via induction of cytoprotective enzymes. Consistent with our results, Bharti et al. reported that curcumin, which has ROS-scavenging property, inhibits RANKL-induced NF-κB activation [28]. Taken together, the NF-κB signaling could be the downstream of RANKL-mediated ROS signaling, or ROS signaling could be the modulator of NF-κB signaling. ETGE peptide (LQLDEETGEFLPIQ), whose sequence is identical to the binding domain of Nrf2 to Keap1, inhibits Keap1 from binding to Nrf2 by working as decoy molecule against Keap1, thereby protecting Nrf2 from ubiquitination and degradation [16]. The 7R-ETGE peptide showed anti-osteoclastogenic activity, though ETGE peptide had no effects. The seven consecutive arginines sequence (7R) we used is reported that it confers cell permeability [29], and ETGE peptide without 7R has no cell permeability and fail to inhibit Nrf2 degradation. Consistent with our results, Steel and colleagues also reported that a cell-penetrating peptide targeting the Nrf2/Keap1 interaction induced HO-1 in a dose-dependent manner, and had an anti-inflammatory effect [30]. The results of our in vitro assay showed a direct inhibitory effect of 7R-ETGE peptide on RANKL-mediated osteoclastogenesis. In addition, in vivo evaluation of inhibitory effect of 7R-ETGE peptide on bone resorption revealed that osteoclastic bone resorption was attenuated both in microCT analysis and osteoclast marker gene expression measured by realtime PCR. These results indicate that 7R-ETGE peptide directly acts on osteoclast/precursor cells even in vivo. However, real-time PCR analysis of RANKL and TNFα gene expression, those are two major osteoclastogenic cytokines, in RNA samples obtained from tissue specimen indicated 7R-ETGE peptide can inhibit LPSmediated upregulation of osteoclastogenic cytokine expression. As

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Kawai et al. reported in inflammatory bone destructive diseases such as periodontitis, a major source of RANKL is from the infiltrating lymphocytes [31]. Although we have not tested the effects of 7R-ETGE peptide on RANKL expression in lymphocytes, some reports mentioned the possibility of developing anti-inflammatory therapeutic targeting of Nrf2 [32,33]. These reports suggest the possibility of the attenuation of RANKL expression in osteoclastogenesis supporting cells by 7R-ETGE peptide. Additionally, osteoblasts or other stromal cells could induce RANKL expression by LPS stimulation [34], and ROS play a role in RANKL upregulation [35]. Rana et al. reported downregulation of RANKL by Nrf2 activation [36]. Furthermore, another famous osteoclastogenic cytokine TNF-α is downregulated by Nrf2 activation [37]. Therefore, further investigations, especially of potential indirect effects of 7R-ETGE peptide on osteoclastogenesis – i.e. downregulation of osteoclastogenic cytokine expression – will be necessary. In conclusion, 7R-ETGE peptide can inhibit osteoclastogenesis via augmentation of Nrf2-dependent cytoprotective enzyme expression and intracellular ROS scavenging. 7R-ETGE peptide could be useful for therapeutic approach to the treatment of destructive bone diseases such as periodontitis and rheumatoid arthritis.

Acknowledgements This research was supported by the Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan (23689081 and 25670841), and the Nestle Nutrition Council, Japan. The authors acknowledge the Center of Research Instruments, Institute of Development, Aging and Cancer, Tohoku University for generous permission to use the experimental instruments. Finally, the authors give their heartfelt appreciation to the experimental reagent companies and instrument companies for their various support in the rehabilitation from the damage caused by the Tohoku earthquake on March 11, 2011.

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