FOXO1 Mediates RANKL-Induced Osteoclast Formation and Activity Yu Wang, Guangyu Dong, Hyeran Helen Jeon, Mohamad Elazizi, Lan B. La, Alhassan Hameedaldeen, E Xiao, Chen This information is current as Tian, Sarah Alsadun, Yongwon Choi and Dana T. Graves of October 5, 2021. J Immunol published online 18 February 2015 http://www.jimmunol.org/content/early/2015/02/13/jimmun ol.1402211 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 18, 2015, doi:10.4049/jimmunol.1402211 The Journal of Immunology

FOXO1 Mediates RANKL-Induced Osteoclast Formation and Activity

Yu Wang,*,† Guangyu Dong,† Hyeran Helen Jeon,‡ Mohamad Elazizi,† Lan B. La,† Alhassan Hameedaldeen,† E Xiao,†,x Chen Tian,† Sarah Alsadun,† Yongwon Choi,{ and Dana T. Graves†

We have previously shown that the transcription factor FOXO1 is elevated in conditions with high levels of bone resorption. To investigate the role of FOXO1 in the formation of osteoclasts, we examined mice with lineage-specific deletion of FOXO1 in os- teoclast precursors and by knockdown of FOXO1 with small interfering RNA. The receptor activator for NF-kB ligand (RANKL), a principal bone-resorbing factor, induced FOXO1 expression and nuclear localization 2 d after stimulation in bone marrow and RAW264.7 osteoclast precursors. RANKL-induced osteoclast formation and osteoclast activity was reduced + L/L

in half in vivo and in vitro with lineage-specific FOXO1 deletion (LyzM.Cre FOXO1 ) compared with matched controls Downloaded from (LyzM.Cre2FOXO1L/L). Similar results were obtained by knockdown of FOXO1 in RAW264.7 cells. Moreover, FOXO1-mediated osteoclast formation was linked to regulation of NFATc1 nuclear localization and expression as well as a number of downstream

factors, including dendritic cell–specific transmembrane protein, ATP6vod2, cathepsin K, and integrin av. Lastly, FOXO1 deletion reduced M-CSF–induced RANK expression and migration of osteoclast precursors. In the present study, we provide evidence that FOXO1 plays a direct role in osteoclast formation by mediating the effect of RANKL on NFATc1 and several downstream

effectors. This is likely to be significant because FOXO1 and RANKL are elevated in osteolytic conditions. The Journal of http://www.jimmunol.org/ Immunology, 2015, 194: 000–000.

steoclasts play a critical role in bone development, naling stimulates formation of single-cell TRAP+ osteoclast pre- normal remodeling, and pathologic conditions such as cursors and induces cell fusion to form multinucleated TRAP+ O periodontitis, rheumatoid arthritis, and osteoporosis. Os- cells. M-CSF and RANKL stimulate several signaling inter- teoclasts are generated from myeloid progenitors that are pro- mediates and transcription factors such as DAP12, TRAF6, NF-kB duced in bone marrow and released into the vasculature (1). The components (relA, p50, p52), and potential downstream effector formation of osteoclasts is governed by ligands, particularly molecules needed for cell fusion, osteoclast activity, and survival, M-CSF, receptor activator for NF-kB ligand (RANKL), and TNF-a including CD9, dendritic cell–specific transmembrane protein by guest on October 5, 2021

as well as several other factors that directly or indirectly con- (DC-STAMP), NFATc1, Atp6v0d2, OC-STAMP, integrin avb3, tribute to their formation and activity (2). Osteoclast formation cathepsin K, and others (5, 6). involves several steps, including differentiation of myeloid pre- Cell fusion is a characteristic feature of osteoclasts. It involves cursors to preosteoclasts, fusion of mononuclear preosteoclasts to cell migration, chemotaxis, intercellular adhesion, and fusion of multinucleated osteoclasts, and maturation and activation of osteo- membranes. DC-STAMP, a transmembrane protein, plays an im- clasts to resorbing bone. portant role in cell fusion (7). It plays a central role in cell fusion Several cytokines have been shown to play a key role in oste- and is induced by two transcription factors, NFATc1 and c-Fos (8). oclast formation. M-CSF stimulates progenitors to upregulate NFATc1 is involved in a number of cellular functions in various RANK expression, the receptor for RANKL (3, 4). RANKL sig- cell types. RANKL induces NFATc1 in osteoclast precursors through activation of NF-kB, which is further enhanced by Ca+ oscillation regulated by Tmem64 (9). Overexpression of NFATc1 *Department of Implantology, School of Stomatology, Jilin University, Changchun 130021, China; †Department of Periodontics, School of Dental Medicine, University can stimulate osteoclast differentiation without RANKL signaling, of Pennsylvania, Philadelphia, PA 19104; ‡Department of Orthodontics, School of suggesting that NFATc1 is a master switch that regulates osteoclast Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104; xSchool and Hospital of Stomatology, Peking University, Beijing 100081, China; and {Depart- differentiation downstream of RANKL (8). Once formed, osteo- ment of Pathology and Laboratory Medicine, University of Pennsylvania, Philadel- clasts are polarized so that the cellular constituents needed for phia, PA 19104 resorption are localized to the bone surface. This process involves

Received for publication August 29, 2014. Accepted for publication January 19, the formation of actin rings and the integrin avb3 (10). Resorption 2015. of the bone surface requires the action of vacuolar ATPase This work was supported by National Institutes of Health Grant AR-060055. (Atp6v1c1) proton pump located in the osteoclast cell membrane Address correspondence and reprint requests to Dr. Dana T. Graves, University of and the release of cathepsin K, a lysosomal cysteine protease that Pennsylvania, School of Dental Medicine, 240 South 40th Street, Evans F1B, Phil- adelphia, PA 19104. E-mail address: [email protected] degrades bone matrix proteins, including collagen (11, 12). The online version of this article contains supplemental material. FOXO1 is a transcription factor that has been linked to protection Abbreviations used in this article: BMM, bone marrow ; DC-STAMP, of osteoblasts from oxidative stress (13, 14). FOXO1 is a member dendritic cell–specific transmembrane protein; NOS, NO synthase; RANK, receptor of the forkhead transcription factors in the O-box subfamily. There activator for NF-kB; RANKL, receptor activator for NF-kB ligand; ROS, reactive are four members, FOXO-1, -3, -4, and -6 (15). FOXO1 regulates oxygen species; siRNA, small interfering RNA. transcription of many different classes of depending on the Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 cell type and nature of the stimulus (16). We have recently shown

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402211 2 FOXO1 MEDIATES OSTEOCLASTOGENESIS that FOXO1 mediates inflammatory expression in dendritic at 37˚C overnight. Then, nonadherent cells were harvested and transferred cells (17) and that FOXO1 is elevated in conditions with high to media with 30 ng/ml M-CSF (PeproTech). Seventy-two hours later, levels of osteoclastogenesis (18, 19). Similarly FOXO1 mediates floating cells were removed and attached BMMs were obtained. Spleens were harvested from experimental and control littermate mice in macrophages (20). A recent report indicated (26), and single cells were obtained with a cell strainer (70 mm). Eryth- that long-term combined deletion of FOXO1, -3, and -4 decreases rocytes were removed by RBC lysis buffer (Sigma-Aldrich, St. Louis, MO) physical bone mass by increasing osteoclast numbers and activity and splenocytes were cultured in DMEM supplemented with 10% FBS, (21). Although that report examined the indirect effects of FOXO 1% penicillin, and 0.05% streptomycin (Life Technologies) at 37˚C overnight. The next day, nonadherent cells were harvested and used for deletion associated with aging and focused on simultaneous de- immunofluorescence with Ab specific for FOXO1. letion of three FOXO members in vivo, we took an alternative RAW264.7 cells were cultured in complete DMEM supplemented with approach and examined the direct effect of FOXO1 in mediating 10% FBS, 0.05% 2-ME, 1% penicillin, and streptomycin. In some exper- short-term RANKL-stimulated osteoclastogenesis. The results iments RAW264.7 cells were transfected with FOXO1 siRNA, FOXO3 indicate that FOXO1 is activated 2 d after RANKL stimulation in siRNA, or control scrambled siRNA (Santa Cruz Biotechnology) with Lipofectin (Invitrogen, Carlsbad, CA) or GeneMute siRNA transfection bone marrow macrophages (BMMs) and RAW264.7 cells. To reagents (Signagen Laboratories, Rockville, MD). determine whether FOXO1 mediates the effects of RANKL In some experiments, freshly isolated single-cell suspensions of bone stimulation in vivo, a calvarial model was used (2, 6). FOXO1 marrow cells were obtained from bone marrow and centrifuged at 500 rpm deletion significantly reduced osteoclastogenesis and osteoclast for 5 min in a cytocentrifuge (Thermo Scientific, Rockford, IL). Attached cells were fixed with 4% paraformaldehyde, washed, and subjected to activity stimulated by RANKL injection. In vitro experiments immunofluorescence with Ab specific for FOXO1 or control IgG. No signal agreed well with in vivo experiments and demonstrated that was detected with matched control. Results were obtained by measuring FOXO1 deletion reduced osteoclastogenesis and osteoclast ac- mean fluorescence intensity or counting immunopositive cells. Downloaded from tivity induced by RANKL in both BMMs derived from experi- In vitro formation of TRAP+ multinucleated cells and mental mice and in RAW264.7 cells with FOXO1 knockdown by osteoclast activity assays small interfering RNA (siRNA). Moreover, FOXO1 deletion re- duced the expression of several proteins that are involved in os- BMMs were stimulated with 100 ng/ml RANKL and 30 ng/ml M-CSF or teoclast formation or function. These results point to FOXO1 100 ng/ml RANKL alone for 4 d and fixed with 4% paraformaldehyde, whereas RAW264.7 cells were stimulated with 100 ng/ml RANKL and then having a role in mediating RANKL-stimulated osteoclast forma- fixed with 4% paraformaldehyde. The number of multinucleated TRAP+ tion, which is distinct from its long-term effect associated with (more than three nuclei) cells, average area, and average number of nuclei http://www.jimmunol.org/ aging (21). per multinucleated TRAP+ cell were counted after staining for TRAP with a kit from Sigma-Aldrich or F-actin with FITC-labeled phalloidin from Sigma-Aldrich. Materials and Methods Osteoclast bone resorption activity was measured with the OsteoLyse Reagents and mice assay kit (Lonza, Allendale, NJ) according to the manufacturer’s protocol. Briefly, BMMs from experimental and control mice or RAW264.7 cells Abs were obtained from Santa Cruz Biotechnology (Dallas, Texas) unless noted transfected with FOXO1 siRNA or control scrambled siRNA were cultured otherwise. Mice that express Cre recombinase under control of the lysozyme M in 96-well OsteoLyse plates (Lonza) at 50,000 cells/well in complete + promoter (LyzM .Cre) were purchased from The Jackson Laboratory (Bar a-MEM. BMMs were incubated with 30 ng/ml M-CSF and 100 ng/ml Harbor,ME).FOXO1L/L mice were provided by Dr. Ronald De Pinho (Uni- soluble RANKL, and RAW265.7 cells incubated with 100 ng/ml soluble by guest on October 5, 2021 L/L versity of Texas MD Anderson Cancer Center, Houston, TX) (22). FOXO1 RANKL. Four days later culture medium was renewed and samples were + mice were bred with LyzM.Cre mice to generate experimental mice (LyzM.Cre collected after an additional 24 h of culture, and calcium release was L/L 2 L/L FOXO1 ) and the control littermates (LyzM.Cre FOXO1 ) (17, 23). measured by fluorescence. Homozygous LyzM.Cre mice were crossed with FOXO1L/L to generate mice that were heterozygous for FOXO1 conditional alleles (LyzM.Cre+FOXO1L/W) Treatment by inhibitors and then backcrossed with the FOXO1L/L mice to obtain the experimental LyzM.Cre+FOXO1L/L and the FOXO1L/L control littermates. Genotypes were RAW264.7 cells were stimulated with RANKL for 48 h. Inhibitors such as 9 BML-257 (12.5 mM), triciribine (1 mM), SB203580 (10 mM), JNK inhibitor I determined by PCR using primers specific for LyzM.Cre (5 -ATCCGAAA- m m G AGAAAACGTTGA-39 and 59-ATCCAGGTTACGGATATAGT-39)andspe- (10 M), PD098059 (20 M), N-acetyl-L-cysteine (5mM), L-N -nitroarginine- 9 9 9 methylester (100 mM), sirtinol (10 mM), and DON (50 mM) were added for cific for FOXO1 (5 -GCTTAGAGCAGAGATGTTCTCACATT-3 ,5- 2 CCAGAGTCTTTGTATCAG GCAAATAA-39,and59-CAAGTCCATTAAT- the last 24 h. Cells were then fixed in 100% methanol at 20˚C for 10 min and TCAGCACATTG A-39). We chose both male and female 10-wk-old mice for incubated with primary Ab to FOXO1 or matched nonimmune primary Ab RANKL injection experiment. Anesthesia was achieved with ketamine overnight at 4˚C. Ab was then detected by a biotinylated secondary Ab and (80 mg/kg of body weight) and xylazine (10 mg/kg) in sterile PBS. All pro- visualized using Alexa Fluor 546–conjugated streptavidin and counterstained cedures were approved by the Institutional Animal Care and Use Committee of with DAPI. Images were acquired using a Nikon 90i fluorescent microscope, the University of Pennsylvania. Soluble RANKL (3 mg/injection) (PeproTech, and image analysis was performed using NIS-Elements software (Nikon, Rocky Hill, NJ) or vehicle (PBS) was injected at the midline of the calvaria of Melville, NY). Nuclear localization with standard immunofluorescence was + L/L 2 L/L confirmed with deconvolution immunofluorescent microscopy. Cells were LyzM.Cre FOXO1 and littermate control LyzM.Cre FOXO1 mice daily m for 5 consecutive days (24). One week after the final injection, mice were imaged in five 1.7- m stacks. The middle stack representing the center of the euthanized and calvarial bone was examined by mCT-40 (Scanco Medical, cell was then further analyzed for FOXO1 nuclear localization using NIS- Bassersdorf, Switzerland). RANKL was injected at the midline between first Elements AR software (Nikon). and second coronal sutures and was compared with mice injected with vehicle Quantitative real-time PCR alone, PBS groups. Specimens were fixed in paraformaldehyde, and small pits on the surface of the calvaria were quantified in reconstructed images fol- Total RNA was extracted and quantitative real-time PCR was performed lowing mCT scanning with NIS-Elements AR software (Nikon). Specimens with primers designed using the Universal ProbeLibrary Assay Design were then decalcified in EDTA and osteoclasts were counted as TRAP+ Center (Roche Applied Science) and labeled probes (Roche Applied Sci- multinucleated cells in sutures and bone marrow cavities by TRAP histostain ence). Each value was normalized to ribosomal protein L32 and represents (24) and immunofluorescence with Ab specific for TRAP. the mean of three independent experiments. Cell culture Migration assay BMMs were cultured from bone marrow cells isolated from femurs and Chemotaxis of BMMs and RAW264.7 cells was measured using trans- tibiae of experimental and control littermate mice (25). After the marrow wells with polycarbonate filters (8-mm pores) (Corning, Corning, NY) was obtained, single-cell suspensions were prepared by mechanically with or without M-CSF added to the bottom chamber for 6 h at 37˚C. dispersing the bone marrow through a 70-mm cell strainer. Then bone Cells on the top of the filter were removed and those that had migrated to marrow cells were cultured in a-MEM supplemented with 10% FBS, 1% the bottom chamber were counted after DAPI staining by fluorescence penicillin, and 0.05% streptomycin (Life Technologies, Grand Island, NY) microscopy. The Journal of Immunology 3

Western blot analysis Luciferase activity was measured 2 d later using a Dual-Luciferase assay system (Promega) and reported as the firefly/Renilla luciferase ratio. Cytoplasmic and nuclear extractions were prepared by using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Scientific). The Statistical analysis cytosolic and nuclear fractions were resolved by SDS-PAGE. Primary Ab against FOXO1 (Cell Signaling Technology, Boston, MA), laminin Most in vitro experiments were carried out three times to obtain mean and A (Sigma-Aldrich), and actin (Cell Signaling Technology) were used SE from independent experiments. In some cases experiments were carried according to the manufacturers’ directions. Bands were visualized by out in triplicate to provide a mean and SEM, with experiments having been biotinylated species-specific secondary Ab and chemiluminescence using performed three times to confirm results. In vivo experiments were carried a kit from Thermo Scientific. No signal was detected with matched control. out with five to seven animals per group. Statistical significance was de- termined by one-way ANOVA with a Tukey post hoc test for comparisons Immunofluorescence between multiple groups at the p , 0.05 level. In some cases, significance between experimental and control groups was directly compared at a given 2 For in vitro assays, cells were fixed in 100% methanol (10 min, 20˚C) followed time point by a Student t test to establish significance at the p , 0.05 level. by incubation with primary Ab specific for FOXO1, CD11b, RANK, and the integrin av or matched control Ab. Ab was then detected by a biotinylated secondary Ab and visualized using Alexa Fluor 546–conjugated streptavidin Results and counterstained with DAPI. Images were acquired using a Nikon 90i fluo- FOXO1-specific deletion in osteoclast precursors rescent microscope, and image analysis was performed using NIS-Elements + L/L software (Nikon). No signal was detected with matched control. For most BMMs were isolated from experimental (LyzM.Cre FOXO1 ) 2 L/L assays data are presented as the mean fluorescence intensity for each group. In and matched control (LyzM.Cre FOXO1 ) mice. LyzM-driven some experiments nuclear localization was determined by colocalization of Cre recombinase reduced FOXO1 mRNA levels (Fig. 1A) by 95% FOXO1 immunostaining and DAPI nuclear staining. For in vivo assays, paraffin and protein levels by 90% in BMMs (Fig. 1B). Most bone marrow sections were subjected to Ag retrieval in citrate buffer at 95˚C. Sections were incubated with Ab specific to TRAP, FOXO1, or NFATc1. Abs were localized cells and splenocytes directly isolated from experimental and Downloaded from with biotinylated secondary Ab followed by avidin–biotin HRP complex control mice did not exhibit FOXO1 deletion as expected because (Vector Laboratories, Burlingame, CA). Abs were visualized using streptavidin most are not CD11c+ (Fig. 1C, 1D, Supplemental Fig. 1). Alexa Fluor 546 (Invitrogen) for TRAP and fluorescein–avidin DCS (Vector Laboratories) for FOXO1 or NFATc1 and counterstained with DAPI. Tyramide signal amplification (PerkinElmer, Waltham, MA) was used to enhance the signal. Fluorescent immunostaining of TRAP+ and FOXO1+ or NFATc1+ cells were observed under 320 magnification of images captured with a Nikon http://www.jimmunol.org/ Eclipse 90i microscope (Nikon) and analyzed with NIS-Elements AR software (Nikon). Luciferase reporter assay RAW264.7 cells were transfected with FOXO1 WT, FOXO1 AAA construct or pcDNA empty vector alone, as we have described (27), by electro- poration (Neon transfection system; Life Technologies) and cotransfected with an NFATc1–luciferase reporter (provided by Dr. Yihong Wan) (28). by guest on October 5, 2021

FIGURE 2. FOXO1 deletion reduces osteoclastogenesis in vivo. (A)Sol- uble RANKL (3 mg/injection) or vehicle (PBS) was injected at the midline of FIGURE 1. LyzM deletes FOXO1 in BMMs. (A) FOXO1 mRNA levels the calvaria of LyzM.Cre+FOXO1L/L and littermate control LyzM.Cre2 were measured in BMMs by quantitative real-time PCR. (B)FOXO1ex- FOXO1L/L mice daily for 5 consecutive days. After 12 d, calvarial bone was pression was measured by immunofluorescence as mean fluorescence intensity examined by mCT. (B and C) Pit number and total pit area per calvaria were in BMMs. (C) Percentage FOXO1 immunopositive cells in single-cell sus- analyzed with mCT. (D and E)TRAP+ multinucleated bone-lining osteoclasts pension of bone marrow cells was measured by immunofluorescence. (D) were identified by histostain in calvarial sutures or bone marrow cavities. (F) FOXO1 expression was measured by immunofluorescence as mean fluores- Osteoclasts were counted as TRAP+ multinucleated cells lining bone in bone cence intensity in splenic cells. Each value represents the mean 6 SEM of marrow cavity and detected by immunofluorescence. Each value is the mean 6 triplicate specimens. *p , 0.05 compared with matched Cre2 group. SEM for n = 6 mice/group. *p , 0.05 compared with matched Cre2 group. 4 FOXO1 MEDIATES OSTEOCLASTOGENESIS

FOXO1 deletion reduces osteoclastogenesis in vivo was completely blocked in bone marrow cavities (Fig. 2D–F). There . Studies were carried out in which RANKL was injected over the was no difference in osteoclast numbers in PBS-injected groups (p calvarial bone of experimental and control mice. The formation of 0.05). The effect of FOXO1 deletion on cortical bone architecture and resorption pits was measured on the surface of calvarial bone in three- cancellous bone volume, mineral density, number, thickness, and dimensional mCT images. The number of resorption pits decreased spacing of trabeculae associated with remodeling in young mice was by 60% and total pit area was reduced by 64% in experimental mice examined (Supplemental Fig. 2A–E). No significant difference was . compared with control mice (p , 0.05) (Fig. 2A–C). The number of observed between experimental and control mice (p 0.1). osteoclasts was measured in the sutures and bone marrow cavities of calvarial bone by TRAP histostain (Fig. 2D, 2E) or by immunoflu- Loss of FOXO1 leads to reduced osteoclast formation and orescence with Ab to TRAP (Fig. 2F). RANKL stimulated a 3- to 10- activity in vitro fold increase in osteoclasts. RANKL-induced osteoclast formation BMMs from FOXO1 deleted or matched control mice were was reduced by 67% in sutures of LyzM.Cre+FOXO1L/L mice stimulated with RANKL in vitro to induce osteoclastogenesis compared with control LyzM.Cre2FOXO1L/L mice (p , 0.05) and (Fig. 3A, Supplemental Fig. 2F). Deletion of FOXO1 resulted in Downloaded from http://www.jimmunol.org/ by guest on October 5, 2021

FIGURE 3. FOXO1 deletion or FOXO1 silencing reduces osteoclastogenesis and osteoclast activity in vitro. (A–D) BMMs from LyzM.Cre+FOXO1L/L and littermate control LyzM.Cre2FOXO1L/L mice were incubated with RANKL (100 ng/ml) and M-CSF (30 ng/ml). After 4 d cells were fixed and stained as indicated. (A)TRAP+ multinucleated osteoclasts (original magnification, 310). (B)TRAP+ multinucleated osteoclasts with .30 nuclei per osteoclast. (C) Mean area per osteoclast. (D)Mean number of nuclei per osteoclast. (E–H) RAW264.7 cells were transfected with FOXO1siRNAorscrambledsiRNAandincubated with RANKL (100 ng/ml). After 4 d cells were fixed and stained as indicated. (E) FOXO1 mRNA levels were measured in RAW264.7 cells by quantitative real-time PCR after siRNA transfection. (F) TRAP+ multinucleated osteoclasts with more than three nuclei per osteoclast (original magnification, 310). (G) Mean area per osteoclast. (H)Meannumberofnucleiper osteoclast. Each value represents the mean 6 SEM of three independent measurements. (I) BMMs from experimental and control mice were stimulated with RANKL (100 ng/ml) and M-CSF (30 ng/ml) or incubated with vehicle alone. Osteoclast activity was measured by release of europium-labeled collagen measured by florescence. (J) RAW264.7 cells were transfected with FOXO1 siRNA or scramble siRNA and were stimulated with RANKL (100 ng/ml) for 4 d. Osteoclast activity was measured as in (I). (K) RAW264.7 cells were transfected with siFOXO1, siFOXO3, or scrambled siRNA and then incubated with RANKL (100 ng/ml) for 4 d and fixed and stained for osteoclasts with TRAP histostain. (L) RAW264.7 cells were transfected with siFOXO3 or scrambled siRNA and FOXO3 mRNA levels were measured by quantitative real-time PCR after 2 d. Each value represents the mean 6 SEM of three or four independent experiments. *p , 0.05 compared with matched Cre2 or scrambled siRNA group, +p , 0.05 compared with matched Cre2 or scrambled siRNA group stimulated with RANKL and M-CSF or RANKL alone. The Journal of Immunology 5 a 65% reduction in the number of osteoclasts formed (p , 0.05) FOXO1 is downregulated by RANKL through different (Fig. 3B). Additionally, there was a 58% reduction in the average pathways area per osteoclast and a 59% reduction in the average number of Both BMMs and RAW264.7 cells were examined for RANKL- , nuclei per osteoclast (p 0.05) (Fig. 3C, 3D). Experiments were induced FOXO1 by both quantitative real-time PCR and immu- also carried out with RAW264.7 cells transfected with FOXO1 nofluorescence (Fig. 4A–C). At the protein level little to no siRNA or scrambled siRNA followed by RANKL stimulation FOXO1 was detected in BMMs or RAW264.7 cells at baseline. (Supplemental Fig. 2G). FOXO1 siRNA reduced FOXO1 levels RANKL stimulated an increase in FOXO1 levels that could be by 70% (Fig. 3E) and reduced the number of osteoclasts induced detected within 1 d and that increased further on days 2 and 4. by RANKL in RAW264.7 cells by 49%, the average area per RANKL stimulated a maximum 7.9-fold increase in FOXO1 in osteoclast by 27%, and the average nuclei per osteoclast by BMMs and a 4.5-fold increase in RAW264.7 cells (p , 0.05) 35% (p , 0.05) (Fig. 3F–H). Scrambled siRNA had no effect (Fig. 4C–E). To confirm that FOXO1 expression was associated (p . 0.05). with osteoclastogenesis in vivo, double immunofluorescence was Osteoclast activity in vitro was measured by release of labeled performed to identify FOXO1+, TRAP+ multinucleated osteo- collagen. RANKL stimulated a 16-fold increase in resorptive clastic cells (Fig. 4F). activity in osteoclasts from BMMs of control mice, which was FOXO1 is active in the nucleus, and FOXO1 activation can be blocked by FOXO1 deletion (p , 0.05) (Fig. 3I). FOXO1 followed by nuclear localization. FOXO1 nuclear localization knockdown by siRNA also blocked RANKL-induced resorptive induced by RANKL could also be detected after 1 d and peaked activity from RAW264.7 cells (p , 0.05) (Fig. 3J). with a 10-fold increase on day 2 in both BMMs and RAW264.7

To compare the role of FOXO1 and FOXO3, each was knocked cells by immunofluorescence (Fig. 5A, 5B). Interestingly, the Downloaded from down separately in RAW264.7 cells and then stimulated with pattern of FOXO1 nuclear localization differed from the expres- RANKL. FOXO1 knockdown reduced osteoclast formation by sion pattern by declining substantially between days 2 and 4 with 66%, and FOXO3 knockdown had no significant impact on oste- a 70% reduction in FOXO1 nuclear localization in BMMs and oclastogenesis even though FOXO3 siRNA effectively reduced a 64% decline in RAW264.7 cells (p , 0.05) (Fig. 5A, 5B). FOXO3 levels (Fig. 3K, 3L). Nuclear localization was confirmed by deconvolution immuno- http://www.jimmunol.org/ by guest on October 5, 2021

FIGURE 4. RANKL induces FOXO1 in BMMs and RAW264.7 cells. (A and B) BMMs from FOXOL/L mice were incubated with M-CSF (30 ng/ml) and RANKL (100 ng/ml) or RAW264.7 cells were incu- bated with RANKL (100 ng/ml). Cells were fixed 0, 1, 2, and 4 d later. FOXO1 (red) was examined by im- munofluorescence with specific Ab, and cells were counterstained with DAPI (blue) (original magnifica- tion 3400 (A), 3200 (B). No signal was detected with matched control Ab (data not shown). (C) FOXO1 mRNA levels were measured by quantitative real-time PCR in BMMs following stimulation with M-CSF (30 ng/ml) and RANKL (100 ng/ml). (D and E) FOXO1 expression was measured by immunofluorescence as mean fluorescence intensity in BMMs and RAW264.7 cells. (F) RANKL was injected into the calvaria as described in Fig. 2. FOXO1+, TRAP+ double-immu- nopositive cells in close proximity to the bone surface werecountedinthesuturearea(n = 6 mice/group). (A–E) Each value represents mean 6 SEM of three independent measurements. *p , 0.05 compared with day 0 or with matched Cre2 group. 6 FOXO1 MEDIATES OSTEOCLASTOGENESIS

FIGURE 5. RANKL stimulates FOXO1 nuclear localization. (A and B) BMMs were incubated with M-CSF (30 ng/ml) and RANKL (100 ng/ml) or RAW264.7 cells were incubated with RANKL (100 ng/ml) for 0–4 d. FOXO1 nuclear localization was determined by FOXO1 immunofluorescence and colocalization with DAPI nuclear stain. (C and D) Immunofluoresence was repeated and colocalization of FOXO1 and DAPI nuclear stain was determined by Z-stack analysis in the center plane of BMMs and RAW264.7 cells. (E) RAW264.7 cells were incubated with RANKL (100 ng/ml). Cells were fixed after 0, 4, and 48 h. Cytoplasmic and nuclear extracts were Downloaded from prepared, and FOXO1 cytoplasmic and nuclear levels were measured by immunoblot analysis. (F) RAW264.7 cells were stimulated with RANKL (100 ng/ml) for 48 h. Inhibitors of Akt, BML-257 (12.5 mM), triciribine (1 mM); p38, SB203580 (10 mM); JNK inhibitor I (10 mM); ERK, PD098059 (20 mM); G ROS, N-acetyl-L-cysteine (5 mM); NOS, L-N -nitro- http://www.jimmunol.org/ arginine-methylester (100 mM); -1, sirtinol (10 mM); and phosphate-activated , DON (50 mM) were added for the last 24 h. Cells were then fixed and FOXO1 was measured as described in (A). Each value represents mean 6 SEM of three inde- pendent measurements. *p , 0.05 compared with BMMs on day 0 or with matched control group, +p , 0.05 compared with BMMs on day 2 after RANKL and M-CSF stimulation or with matched control group. by guest on October 5, 2021

fluorescent microscopy, which examined FOXO1 colocalization Effect of FOXO1 on expression of target genes with DAPI at the center of each cell. By this analysis, RANKL Several potential target genes were examined at the mRNA level to stimulated a 7.4-fold increase in FOXO1 nuclear localization in investigate the role of FOXO1 in mediating RANKL stimulation. BMMs from WT mice and a 5.6-fold increase in RAW264.7 cells This was done in BMMs obtained from experimental mice with (p , 0.05) (Fig. 5C, 5D), consistent with results obtained by FOXO1 deletion compared with matched controls. RANKL in- standard immunofluorescence microscopy (Fig. 5A, 5B). RANKL- creased FOXO1 mRNA levels, which were blocked by Cre induced FOXO1 nuclear localization was also examined by recombinase (Fig. 6A) (p , 0.05). Deletion of FOXO1 reduced by Western blot analysis. FOXO1 protein accumulated in the cyto- ∼60% the increase in NFATc1 mRNA levels stimulated by plasm within 4 h of RANKL stimulation (Fig. 5E). Nuclear lo- RANKL in vitro (Fig. 6B, 6C, Supplemental Fig. 3A) and reduced calization was strongly detected 2 d after RANKL stimulation , (Fig. 5E), which was consistent with the immunofluorescence data. RANKL-stimulated NFATc1 nuclear localization by 53% (p 0.05) (Fig. 6D). In vivo RANKL simulated a 2.1-fold increase in To investigate RANKL-stimulated FOXO1 nuclear localization + + further, experiments were performed examining pathways that may NFATc1 TRAP multinucleated cells measured by double im- + mediate RANKL-induced FOXO1 nuclear localization. Inhibitors munofluorescence staining. This was reduced 70% in LyzM.Cre L/L 2 L/L , of p38 and JNK but not Erk MAPK inhibitors reduced FOXO1 FOXO1 mice compared with LyzM.Cre FOXO1 mice (p nuclear localization by ∼50%. A reactive oxygen species (ROS) 0.05) (Fig. 6E). However, cotransfection of WT (FOXO1 WT) or inhibitor, sirtuin-1 inhibitor, and NO synthase (NOS) inhibitor a constitutively active FOXO1 (FOXO1 AAA) expression vector also reduced FOXO1 nuclear localization by 39–65% (p , 0.05) with an NFATc1 reporter did not enhance NFATc1 transcriptional (Fig. 5F). Thus, a number of known pathways stimulated by activity compared with cotransfection with empty vector (PcDNS) RANKL induce FOXO1 nuclear localization. However, an Akt (Fig. 6F). However, deletion of FOXO1 reduced the capacity of inhibitor did not have a significant effect on RANKL-induced RANKL to stimulate increased mRNA levels of factors that are FOXO1 nuclear localization. involved in osteoclast formation and activity including ATP6v0d2 The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 6. FOXO1 deletion inhibits expression of genes involved in osteoclastogenesis. BMMs from LyzM.Cre+FOXO1L/L or littermate control LyzM.Cre2FOXO1L/L mice were stimulated with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 0 or 2 d. (A and B) FOXO1 or NFATc1 mRNA levels were by guest on October 5, 2021 assessed by real-time PCR. (C) NFATc1 was examined by immunofluorescence with Ab specific for NFATc1. Mean fluorescence intensity was measured. (D) NFATc1 was detected by immunofluorescence and nuclear colocalization with DAPI measured. (E) RANKL was applied to experimental and matched control mice described in Fig. 2. NFATc1 and TRAP were examined by double immunofluorescence, and sections were counterstained with DAPI. The percentage NFATc1+, TRAP+ double-immunopositive cells in close proximity to bone were measured in the calvarial sutures. (F) Luciferase assays were performed in RAW264.7 cells cotransfected with FOXO1 WT expression vector, FOXO1 AAA constitutively active or PcDNS empty vector alone, and an NFATc1 promoter–luciferase construct. Luciferase activity was measured as described in Materials and Methods.(G–I) mRNA levels of ATP6v0d2, cathepsin K, and DC-STAMP were measured in BMMs from LyzM.Cre+FOXO1L/L and littermate control LyzM.Cre2FOXO1L/L mice stimulated with M-CSF and RANKL as described in (A). Each value represents the mean 6 SEM of three independent measurements. *p , 0.05 compared with matched control group. by 49%, cathepsin K by 63%, and DC-STAMP by 55% (p , 0.05) FOXO1 knockdown in RAW264.7 cells reduced migration in (Fig. 6G–I). these cells up to 56% (p , 0.05) (Fig. 7E). To determine how FOXO1 may affect BMM migration, integrin a was examined in FOXO1 regulates RANK and osteoclast precursor migration v BMMs based on reports that it plays an important role in migra- + Osteoclast precursors (CD11b BMMs) are induced by M-CSF tion of these cells by immunofluorescence (31, 32). FOXO1 de- from myeloid progenitor cells (6). The effect of FOXO1 dele- letion resulted in a 73% decrease of integrin av at the protein level + tion on production of CD11b BMMs was examined in vitro by (p , 0.05) (Fig. 7F). immunofluorescence. Neither the percentage CD11b+ bone mar- row cells or the number of M-CSF–induced BMMs in vitro was affected by FOXO1 deletion, indicating that the effects of FOXO1 Discussion on osteoclastogenesis are downstream of BMM formation (p . The role of FOXO1 in regulating monocytic cells has received 0.05) (Fig. 7A, 7B, Supplemental Fig. 4). RANK expression was relatively little attention. FOXO1 has been shown to modulate TLR examined in bone marrow cells and BMMs from LyzM.Cre+ expression and cytokine production in monocytes and dendritic FOXO1L/L mice and matched control LyzM.Cre2FOXO1L/L mice cells, which share a common precursor with osteoclasts (17, 20). following stimulation with M-CSF (Supplemental Fig. 3B). In the present study, we report that FOXO1 has an important role BMMs from FOXO1-deleted mice had an ∼40% reduction in in mediating induced osteoclastogenesis. RANKL stimulated RANK+ cells compared with BMMs from matched control mice FOXO1 expression and FOXO1 nuclear localization in vivo and (p , 0.05) (Fig. 7C). We also examined M-CSF stimulation of in vitro. FOXO1 deletion or knockdown reduced RANKL- BMM migration (29, 30). M-CSF induced a dose-dependent 16- stimulated osteoclast formation in vivo and reduced osteoclast fold increase in BMM migration. This increase was reduced by up formation from BMMs and RAW264.7 cells in vitro. Additionally, to 72% when FOXO1 was deleted (p , 0.05) (Fig. 7D). Similarly, FOXO1 deletion in myeloid cells reduced M-CSF–induced RANK 8 FOXO1 MEDIATES OSTEOCLASTOGENESIS

FIGURE 7. FOXO1 deletion or knockdown reduces M-CSF–induced RANK, integrin expression, and BMM mi- gration. (A) Bone marrow cells and BMMs were prepared from LyzM.Cre+FOXO1L/L and littermate control LyzM. Cre2FOXO1L/L mice as described in Materials and Methods. The cells characterized as BMMs were determined by im- munofluorescence with Ab specific for CD11b and DAPI counterstain. (B) CD11b expression was measured by im- munofluorescence as mean fluorescence intensity. (C) Bone marrow cells and BMMs from LyzM.Cre+FOXO1L/L and littermate control LyzM.Cre2FOXO1L/L mice were fixed and RANK was visualized by immunofluorescence and DAPI counterstained. (D) BMM migration was measured in a Downloaded from transwell assay in response to M-CSF (0–30 ng/ml) placed in the lower chamber. (E) RAW264.7 cells were transfected with scrambled or FOXO1 siRNA and cell migration was measured in response to M-CSF (0–30 ng/ml) placed in the lower chamber. (F) The protein level of the integrin av was measured in BMMs from LyzM.Cre+FOXO1L/L or littermate control LyzM.Cre2FOXO1L/L mice by immunofluorescence http://www.jimmunol.org/ with specific Ab and the results are expressed as mean fluorescence intensity. Each value represents the mean 6 SEM of three independent measurements. *p , 0.05 com- pared with matched Cre2 or matched control group. by guest on October 5, 2021

expression and migration of BMMs. However, FOXO1 deletion assays was carried out with primary Abs to FOXO1 from different did not affect formation of BMMs in vivo or in vitro. sources and gave consistent results. The time frame for induction To investigate the role of FOXO1 on RANKL-stimulated of FOXO1 nuclear localization by RANKL is considerably slower osteoclastogenesis, mice that expressed lysozyme M promoter- than the 1–2 h induction that is observed for TNF stimulation. driven Cre recombinase (33) were bred with floxed FOXO1 However, it is similar to the time frame of RANKL-induced mice (34). This led to substantial deletion of FOXO1 in osteoclast NFATc1 (8). Interestingly, both the protein expression and nu- precursor cells, BMMs. BMMs from experimental mice had clear localization of NFATc1 are continuously increased during a 95% reduction of FOXO1 mRNA and 90% reduction of FOXO1 RANKL-induced differentiation of BMMs to osteoclasts. Because at the protein level compared with control mice. This was not FOXO1 activity is directly related to its nuclear localization, the a general reduction of FOXO1 in hematopoietic cells because functional significance of high FOXO1 expression but exclusion there was no significant difference in FOXO1 levels in bone from the nucleus on day 4 requires further investigation. Pathways marrow cells or splenocytes from experimental and control mice. that are known to mediate RANKL-induced NFATc1 also were Several lines of evidence establish that RANKL stimulates shown to be involved in FOXO1 nuclear localization (35–38). FOXO1 in osteoclast precursors. RANKL induced a time- Thus, FOXO1 nuclear localization was significantly reduced by dependent increase in FOXO1 mRNA and protein expression in inhibitors of MAPK, ROS, NOS, and deacetylase. In contrast, Akt, both BMMs and the osteoclast precursor cell line, RAW264.7, which is known to negatively regulate FOXO1, did not have which increased 5- to 8-fold after stimulation. In vivo RANKL a significant effect on RANKL-induced FOXO1 nuclear locali- stimulated an increase in FOXO1 in osteoclasts as determined by zation 2 d after stimulation. the number of FOXO1+TRAP+ double-positive multinucleated That FOXO1 had a significant effect in mediating RANKL cells. Interestingly, RANKL also stimulated an increase in FOXO1 stimulation was shown by significantly reduced osteoclast for- nuclear localization, which peaked at day 2 and declined on day 4, mation in vivo and reduced total resorption pit area in experimental although expression was still high on day 4. This pattern was mice compared with matched control mice. The effect of FOXO1 observed in both BMMs and RAW264.7 cells. FOXO1 nuclear deletion on reducing RANKL-stimulated osteoclast formation localization 2 d after RANKL stimulation was shown by both from BMMs or RAW264.7 cells in vitro was similar to in vivo immunofluorescence and Western blot assays. Each of these results. Additionally, the size and number of nuclei per osteo- The Journal of Immunology 9 clast was significantly smaller when FOXO1 was deleted or si- Disclosures lenced, suggesting that the amount of cell fusion was diminished. The authors have no financial conflicts of interest. Osteoclast resorbing activity was also reduced when FOXO1 was deleted in BMMs or silenced in RAW264.7 cells in vitro. We also compared cancellous bone volume and several aspects of bone References + L/L 1. Miyamoto, T. 2011. Regulators of osteoclast differentiation and cell-cell fusion. architecture in vertebrae of LyzM.Cre FOXO1 mice versus Keio J. Med. 60: 101–105. control littermates. However, there was no significant difference 2. Xing, L., Y. Xiu, and B. 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