Identification of a Novel Role for Foxo3 Isoform2 in Osteoclastic Inhibition Cheng Xu, Gregory J

Identification of a Novel Role for Foxo3 Isoform2 in Osteoclastic Inhibition Cheng Xu, Gregory J. Vitone, Kazuki Inoue, Courtney Ng and Baohong Zhao This information is current as of October 2, 2021. J Immunol 2019; 203:2141-2149; Prepublished online 20 September 2019; doi: 10.4049/jimmunol.1900707 http://www.jimmunol.org/content/203/8/2141 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2019/09/20/jimmunol.190070 Material 7.DCSupplemental References This article cites 32 articles, 5 of which you can access for free at: http://www.jimmunol.org/content/203/8/2141.full#ref-list-1 http://www.jimmunol.org/

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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 © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Identiﬁcation of a Novel Role for Foxo3 Isoform2 in Osteoclastic Inhibition

Cheng Xu,*,†,1 Gregory J. Vitone,*,†,1 Kazuki Inoue,*,†,‡ Courtney Ng,*,† and Baohong Zhao*,†,‡,x

Foxo3 acts as an important central regulator that integrates signaling pathways and coordinates cellular responses to environmental changes. Recent studies show the involvement of Foxo3 in osteoclastogenesis and rheumatoid arthritis, which prompted us to further investigate the FOXO3 locus. Several databases document FOXO3 isoform2, an N-terminal truncated mutation of the full-length FOXO3. However, the biological function of FOXO3 isoform2 is unclear. In this study, we established a conditional allele of Foxo3 in mice that deletes the full-length Foxo3 except isoform2, a close ortholog of the human FOXO3 isoform2. Expression of Foxo3 isoform2 specifically in macrophage/osteoclast lineage suppresses osteoclastogenesis and leads to the osteopetrotic phenotype in mice. Mechanistically, Foxo3 isoform2 enhances the expression of type I IFN response genes to RANKL stimulation and thus inhibits osteoclastogenesis via endogenous IFN-b–mediated feedback inhibition. Our findings identify, to our knowledge, the first known biological function of Foxo3 isoform2 that acts as a novel osteoclastic inhibitor in bone remodeling. The Journal Downloaded from of Immunology, 2019, 203: 2141–2149.

steoclasts, derived from monocyte/macrophage precur- signal binding protein for Ig k J region (RBP-J), and differen- sors, are the exclusive cell type responsible for bone tially expressed in FDCP 6 homolog (Def6), restrain osteoclas- O resorption in both bone homeostasis and pathological togenesis to prevent excessive bone resorption (5–9). Thus, the bone destruction. Bone loss is a major cause of morbidity and extent of osteoclastogenesis is delicately modulated and deter- http://www.jimmunol.org/ disability in many skeletal diseases, such as rheumatoid arthritis mined by the balance between these osteoclastogenic and anti- (RA), psoriatic arthritis, periodontitis, and periprosthetic loosening osteoclastogenic mechanisms. (1–3). Osteoclastogenesis is induced by the major osteoclastogenic Forkhead box class O (Foxo) proteins are a family of evolu- cytokine receptor activator of NF-kB ligand (RANKL). Binding of tionarily conserved transcription factors, which include Foxo1, RANKL to RANK receptors activates a broad range of signaling 3, 4, and 6 in mammals. Foxo proteins consist of four conserved cascades, including canonical and noncanonical NF-kB pathways, regions: a forkhead DNA-binding domain at the N terminus fol- MAPK pathways, and calcium signaling, which lead to the acti- lowed by a nuclear localization signal, a nuclear export signal, vation of an osteoclastic transcriptional network. The positive and a transactivation domain at the C terminus (10–12). Foxo regulators in this transcriptional network, such as the transcription proteins play important roles in diverse biological processes, such by guest on October 2, 2021 factors NFATc1, c-Fos, and Blimp1, drive osteoclast differentia- as metabolism, oxidative stress, cell cycle regulation, apoptosis, tion (1–4). In contrast, the process of osteoclast differentiation immunity, and inflammation. Foxo proteins are well known for is delicately controlled by a “braking system,” in which negative their cell type– and context-specific effects on cellular processes regulators, such as IFN regulatory factor (Irf) 8, recombination because of their variable posttranslational modifications, subcellular localization, and binding cofactors in different scenarios (10–15). Foxo1, 3, and 4 were reported to regulate RANKL-induced *Arthritis and Tissue Degeneration Program, Hospital for Special Surgery, osteoclast differentiation (16, 17). However, Foxo proteins seem to New York, NY 10021; †David Z. Rosensweig Genomics Research Center, Hos- pital for Special Surgery, New York, NY 10021; ‡Department of Medicine, exhibit different functions in osteoclastogenesis. For example, some Weill Cornell Medical College, New York, NY 10065; and xGraduate Program studies show that Foxo1, 3, and 4 proteins as a group are inhibitors in Cell and Development Biology, Weill Cornell Graduate School of Medical Sciences, New York, NY 10065 of osteoclastogenesis (16), whereas others found that Foxo1 is a positive regulator (17). These results indicate that Foxo family 1Equal contribution. plays an important but complex role in osteoclastogenesis. In ORCIDs: 0000-0001-9104-265X (G.J.V.); 0000-0002-1286-0919 (B.Z.). disease settings, FOXO3 activity is correlated with outcomes in Received for publication June 25, 2019. Accepted for publication August 20, 2019. infectious and inflammatory diseases, such as RA. Increased ex- This work was supported by Grants AR062047, AR071463, and AR068970 from the pression of FOXO3 in monocytes due to a single-nucleotide National Institutes of Health (to B.Z.) and by support for the David Z. Rosensweig Genomics Research Center from The Tow Foundation. polymorphism (FOXO3 [rs12212067: T.G]) is associated with The sequences presented in this article have been submitted to the Gene Expression reduced severity of RA (18, 19). Recently, we uncovered that Omnibus under accession number GSE 135479. Foxo3 is a target of miR-182 and plays an inhibitory role in in- Address correspondence and reprint requests to Dr. Baohong Zhao, Hospital for flammatory cytokine TNF-a–induced osteoclastogenesis and bone Special Surgery, Research Institute 8th Floor R804, 535 East 70th Street, New York, resorption (20). Thus, FOXO3 is closely involved in osteoclasto- NY 10021. E-mail address: [email protected] genesis and bone erosion in human RA. We further investigated The online version of this article contains supplemental material. the FOXO3 locus and found that there exists a short isoform of human Abbreviations used in this article: BMM, bone marrow–derived macrophage; CM, conditioned medium; Foxo, forkhead box class O; HEK, human embryonic kidney; FOXO3, named as isoform2 in contrast to the full-length isoform1. Irf, IFN regulatory factor; KO, knockout; RA, rheumatoid arthritis; RANKL, receptor The current database supports the presence of FOXO3 isoform2 in activator of NF-kB ligand; RNA-seq, RNA sequencing; siRNA, small interfering human cells and tissues, such as fibroblasts and skeletal muscles, in RNA; TRAP, tartrate-resistant acid phosphatase; WT, wild-type control. physiological conditions (https://gtexportal.org/home/transcriptPage). Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 The biological function of this FOXO3 isoform2 is unclear. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900707 2142 Foxo3 ISOFORM2 REGULATES BONE REMODELING

We primarily wished to take advantage of the conditional Foxo3 treated without or with RANKL (100 ng/ml, no. 310-01; PeproTech). knockout (KO) mice (Foxo3f/f; LysMcre) to provide genetic evidence Culture media were exchanged every 2 d. Tartrate-resistant acid phosphatase for the function of Foxo3 in vivo in osteoclastogenesis. However, to (TRAP) staining was performed with an acid phosphatase leukocyte diagnostic kit (Sigma-Aldrich), in accordance with the manufacturer’s instructions. our surprise, these conditional KO mice express a truncated Foxo3 protein in addition to the lack of full-length protein. Sequence Plasmids, cloning, and sequencing analysis demonstrated that this truncated Foxo3 is an ortholog of cDNA fragments encoding mouse full-length Foxo3 protein or exon 2 fused the human FOXO3 isoform2. Given this similarity, we named this with FLAG tag at the C terminus was amplified by PCR using the cDNA truncated Foxo3 as mouse Foxo3 isoform2. templates from WT BMMs and then subcloned into the XbalI/BamHI sites + + Over 90% of human genes are alternatively spliced to produce of pcDNA3.1 vector to construct the pcDNA3.1 full-length Foxo3-Flag plasmid or pcDNA3.1+-Foxo3 exon 2-Flag plasmid, respectively. Fur- mRNA and protein isoforms, which may have shared, related, thermore, cDNA fragment encoding mouse Foxo3 isoform2 fused with distinct, or even antagonistic functions. Alternative splicing is an FLAG tag at the C terminus was amplified by PCR using the cDNA essential biological process driving evolution and development. templates from Foxo3isoform2 BMMs, followed by subcloning into the The isoforms resulting from alternative splicing contribute to tran- XbalI/BamHI sites of pcDNA3.1+ vector to construct the pcDNA3.1+- scriptomic and proteomic diversity and complexity in physiologi- Foxo3 isoform2-Flag plasmid. The following primers were used for cloning: for Foxo3 full-length fragment, forward 59-ATTCTAGAGCCA- cal conditions (21, 22). Aberrant splicing or deregulated isoform CCATGGCAGAGGCACCAGCC-39, reverse 59-ATGGATCCTCACTTG- expression/function can lead to diseases, such as cancer and car- TCGTCATCGTCTTTGTAGTCGCCTGGTACCCAGCTTTGA-39;forexon diovascular and metabolic diseases (21–23). Recent efforts have 2ofFoxo3 fragment, forward 59-ATTCTAGAGCCACCATGGCAGAGGC- been made to investigate deregulated alternative splicing that could ACCAGCC-39, reverse 59-ATGGATCCTCACTTGTCGTCATCGTCTTTG- 9 be used as diagnostic markers or therapeutic targets for diseases. TAGTCCTTCCAGCCCGCAGAGCT-3 ; and for Foxo3 isoform2 fragment, forward 59-ATTCTAGAGCCACCATGCGCGTTCAGAATGAAGG-39, re- Downloaded from In this study, we identified Foxo3 isoform2 as a novel osteoclas- verse 59-ATGGATCCTCACTTGTCGTCATCGTCTTTGTAGTCGCCTG- togenic suppressor that leads to an osteopetrotic phenotype in mice. GTACCCAGCTTTGA-39. The sequence integrity of the inserted fragments Foxo3 inhibits osteoclast differentiation through type I IFN–mediated in each expression plasmid was verified by restriction enzyme digestion feedback inhibition. These Foxo3f/f mice could be used as a model and DNA sequencing at Cornell University Genomics Facility. to investigate the function of human FOXO3 isoform2 because of Transfection of human embryonic kidney 293 cells and the high protein sequence homology between human and mouse RAW264.7 cells Foxo3. To the best of our knowledge, this is the first report unveiling http://www.jimmunol.org/ the biological function of Foxo3 isoform2, which provides novel Lipofectamine 3000 reagent (L3000015; Thermo Fisher Scientific) was used for the transfection of the human embryonic kidney (HEK) 293 cells or knowledge and research tools and opens new avenues for studying RAW264.7 cells. Briefly, the cells were seeded (2.5 3 105 HEK cells/well the function of Foxo3 isoform2 in different scenarios and areas. and 1.2 3 105 RAW264.7 cells/well) and cultured with DMEM for HEK293 cells or a-MEM for RAW264.7 cells supplemented with 10% Materials and Methods FBS and 1% penicillin/streptomycin in a 24-well plate at 37˚C in a hu- Mice and analysis of bone phenotype midified atmosphere containing 5% CO2 overnight. The cells were then transfected with 500 ng plasmid DNAs using Lipofectamine 3000 reagent, We generated mice with myeloid-specific expression of mouse Foxo3 according to the manufacturer’s instructions. After 24 h, the medium was replaced with fresh completed DMEM for HEK293 cells or a-MEM for isoform2 (full-length Foxo3 is replaced by the isoform2) by crossing by guest on October 2, 2021 Foxo3flox/flox mice (stock no. 024668; The Jackson Laboratory) with a ly- RAW264.7 cells. The protein lysates from cell cultures were collected after sozyme M promoter-driven Cre transgene on the C57BL/6 background 48 h to assess plasmid expression. (known as LysMcre; The Jackson Laboratory). Gender- and age-matched Foxo3flox/floxLysMcre+ mice (referred to as Foxo3isoform2) and their litter- In vitro gene silencing by small interfering RNAs mates with Foxo3+/+LysMcre+ genotype as wild-type controls (WT) were 2/2 In vitro gene silencing by small interfering RNAs (siRNAs) was performed used for experiments. Global Foxo3 were purchased from The Jackson as previously described (20). Briefly, siRNAs targeting Foxo3 or their Laboratory (stock no. 022097). We maintained all mice under standard corresponding control oligos (80 nM) were transfected into murine BMMs 12 h light/dark cycles with ad libitum access to regular food and water. All using TransIT-TKO transfection reagent (Mirus Bio), in accordance with animal studies were approved by the Hospital for Special Surgery In- the manufacturer’s instructions. stitutional Animal Care and Use Committee and Weill Cornell Medical College Institutional Animal Care and Use Committee. RNA sequencing and bioinformatics analysis Microcomputed tomography analysis was conducted to evaluate bone volume and three-dimensional bone architecture using a SCANCO mCT-35 RNA sequencing (RNA-seq) and bioinformatics analysis were performed scanner (SCANCO Medical) as described (24). Twelve-week-old male as previously described (24). Briefly, total RNA was extracted using RNeasy mouse femora were fixed in 10% buffered formalin and scanned at 6 mm Mini Kit (QIAGEN) following the manufacturer’s instructions. TruSeq RNA + resolution. Proximal femoral trabecular bone parameters were analyzed Library preparation kits (Illumina) were used to purify poly-A transcripts using SCANCO software, according to the manufacturer’s instructions and and generate libraries with multiplexed barcode adaptors, following the the American Society of Bone and Mineral Research guidelines. manufacturer’s instructions. All samples passed quality control analysis using a Bioanalyzer 2100 (Agilent Technologies). RNA-seq libraries were Cell culture constructed per the Illumina TruSeq RNA sample preparation kit. High- throughput sequencing was performed using the Illumina HiSeq 4000 in Mouse bone marrow cells were harvested from tibiae and femora of age- and the Weill Cornell Medical College Genomics Resources Core Facility. RNA- gender-matched mutant and control mice and cultured for 3 d in Petri dishes seq reads were aligned to the mouse genome (mm10) using TopHat (25). (Corning Falcon, no. 351029) in a-MEM (Thermo Fisher Scientific) with Cufflinks (26) was subsequently used to assemble the aligned reads 10% FBS (S11550; Atlanta Biologicals), glutamine (2.4 mM, no. 25030164; into transcripts and then estimate the transcript abundances as reads per kilo Thermo Fisher Scientific), 1% penicillin/streptomycin (no. 15070063; base per million values. HTseq (27) was used to calculate raw reads counts, Thermo Fisher Scientific), and L929 cell supernatant (conditioned medium and edgeR (28) was used to calculate normalized counts as counts per million. [CM]), which contained the equivalent of 20 ng/ml of murine rM-CSF and Heatmaps were generated by pheatmap package in R. RNA-seq data (accession was used as a source of M-CSF. The attached bone marrow–derived no. GSE 135479) have been deposited in National Center for Biotechnology macrophages (BMMs) were scraped, seeded at a density of 4.5 3 104 cells 2 Information’s Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/ per cm , and cultured in a-MEM with 10% FBS, 1% penicillin/streptomycin, query/acc.cgi?acc=GSE 135479). 1% glutamine, and CM for overnight. The cells were then treated without or with an optimized concentration of RANKL (40 ng/ml, no. 310-01; Pepro- Reverse transcription and real-time PCR Tech) in the presence of CM for times indicated in the figure legends. Culture media were exchanged after 3 d. For RAW264.7 cell culture, 5 3 102 cells Reverse transcription and real-time PCR were performed as previously were seeded per well in 96-well plates in a-MEM with 10% FBS, 1% described (24). DNA-free RNA was obtained with the RNeasy Mini Kit penicillin/streptomycin, and 1% glutamine overnight. The cells were then (no. 74106; QIAGEN, Valencia, CA) with DNase treatment, and 1 mgof The Journal of Immunology 2143 total RNA was reverse transcribed using a First Strand cDNA Synthesis Kit protein is completely deleted. We first used BMMs as osteoclast (Thermo Fisher Scientific, Waltham, MA), according to the manufacturer’s precursors to examine in vitro osteoclast differentiation in re- instructions. Real-time PCR was done in triplicate with the QuantStudio sponse to RANKL, the master osteoclastogenic inducer. We found 5 Real-time PCR System and Fast SYBR Green Master Mix (Thermo Fisher Scientific). Gene expression was normalized relative to GAPDH. that Foxo3 KO–derived BMMs showed an increased responsive- The primers for real-time PCR were as follows: Acp5:59-ACGGCTAC- ness to RANKL, determined by more TRAP-positive multinu- TTGCGGTTTC-39 and 59-TCCTTGGGAGGCTGGTC-39; Dcstamp:59- cleated osteoclasts (Fig. 1A, 1B). Furthermore, we performed an 9 9 TTTGCCGCTGTGGACTATCTGC-3 and 5 -AGACGTGGTTTAGGAA- RNA-seq experiment using WT and Foxo3 KO BMMs to examine TGCAGCTC-39; Ctsk:59-AAGATATTGGTGGCTTTGG-39 and 59-ATC- GCTGCGTCCCTCT-39; Itgb3:59-CCGGGGGACTTAATGAGACCACTT- gene expression in response to RANKL. In parallel with increased 39 and 59-ACGCCCCAAATCCCACCCATACA-39; Calcr:59-ACATGAT- osteoclast formation, the expression of osteoclastic genes, such as CCAGTTCACCAGGCAGA-39 and 59-AGGTTCTTGGTGACCTCCC- Nfatc1 (encoding NFATc1), Prdm1 (encoding Blimp1), Acp (encoding AACTT-39; Foxo3-F3R3: 59-CTGTCCTATGCCGACCTGAT-39 and 59- TRAP), Oscar (encoding OSCAR), and Ctsk (encoding cathepsin K), 9 9 CTGTCGCCCTTATCCTTGAA-3 ; Foxo3-F4R4: 5 -ATGGGAGCTTG- was significantly enhanced by RANKL in Foxo3 KO BMM cultures GAATGTGAC-39 and 59-TTAAAATCCAACCCGTCAGC-39; Foxo3-F5R5: 59-AGGAGGAGGAATGTGGAAGG-39 and 59-CCGTGCCTTCATTCTG- compared with the BMMs cultured from WT controls (Fig. 1C). AAC-39; Ifnb1:59-TTACACTGCCTTTGCCATCC-39 and 59-AGAAA- These results indicate that Foxo3 functions as a negative regulator CACTGTCTGCTGGTG-39; Mx1: 59-GGCAGACACCACATACAACC-39 in RANKL-induced osteoclast differentiation. and 59-CCTCAGGCTAGATGGCAAG-39; Ifit1:59-CTCCACTTTCAGA- GCCTTCG-39 and 59-TGCTGAGATGGACTGTGAGG-39; Irf7:59-GTCT- Foxo3f/f;LysMcre mice express a truncated Foxo3 protein that CGGCTTGTGCTTGTCT-39 and 59-CCAGGTCCATGAGGAAGTGT-39; is an ortholog of human FOXO3 isoform2 Ifit2: 59-AAATGTCATGGGTACTGGAGTT-39 and 59-ATGGCAATTAT- CAAGTTTGTGG-39; Stat1: 59-CAGATATTATTCGCAACTACAA-39 and We next wished to examine the role of Foxo3 in vivo using con- 59-TGGGGTACAGATACTTCAGG-39;andGapdh:59-ATCAAGAAGGT- ditional Foxo3 KO mice. We deleted Foxo3 (encoding Foxo3) in Downloaded from GGTGAAGCA-39 and 59-AGACAACCTGGTCCTCAGTGT-39. myeloid lineage osteoclast precursors by crossing Foxo3flox/flox Immunoblot analysis mice (The Jackson Laboratory) with LysMcre mice that express Cre under the control of the myeloid-specific lysozyme M pro- Total cell extracts were obtained using lysis buffer containing 150 mM Tris-HCl moter. We used Foxo3flox/flox; LysMcre+ mice and littermate con- (pH 6.8), 6% SDS, 30% glycerol, and 0.03% bromophenol blue; 10% 2-ME +/+ + was added immediately before harvesting cells. Cell lysates were fractionated trols with a Foxo3 ; LysMcre genotype (hereafter referred to as on SDS-PAGE, transferred to Immobilon-P membranes (MilliporeSigma), and WT) in the experiments. The mouse Foxo3 gene has four exons, http://www.jimmunol.org/ incubated with specific Abs. Western Lightning Plus-ECL (PerkinElmer) was and the coding region within exons 2 and 3 produces a full-length used for detection. Foxo3 N-terminal (no. 2497, specifically recognizing the Foxo3 protein with 672 aa. The Foxo3flox/flox mice (The Jackson residues surrounding Glu50 in exon 2 of Foxo3) and C-terminal (no. 12829S, specifically recognizing the C terminus of Foxo3) Abs were purchased from Laboratory) possess loxP sites flanking exon 2 of the Foxo3 gene Cell Signaling Technology. Anti-Flag tag Ab (no. 637301) was purchased (Fig. 2A). To verify Foxo3 deletion, we first designed a series from BioLegend. p38a (sc-535) Ab was from Santa Cruz Biotechnology. of PCR primers that cover the coding region from exon 2 and exon Statistical analysis 3 (Fig. 2B, Table I). As shown in Fig. 2C, PCR products were detected in WT BMM cDNAs using all primer sets. As expected, Statistical analysis was performed using GraphPad Prism software. Two- the exon 2–3 primer set did not produce any PCR bands using the tailed Student t test was applied if there were only two groups of sam- Foxo3flox/flox; LysMcre+ BMM cDNAs. Surprisingly, other primer by guest on October 2, 2021 ples. In the case of more than two groups of samples, one-way ANOVA was used with one condition, and two-way ANOVA was used with more sets covering exon 3 or exon 3–4 generated the same PCR prod- than two conditions. ANOVA analysis was followed by post hoc Bonfer- ucts using BMM cDNAs obtained from either Foxo3flox/flox; roni correction for multiple comparisons. A p value ,0.05 was taken as LysMcre+ or WT mice (Fig. 2C). We further designed quantitative , , statistically significant: *p 0.05 and **p 0.01. Data are presented as PCR primer sets and found that the primers other than those lo- the mean 6 SD, as indicated in the figure legends. cated within exon 2 amplified the Foxo3 cDNAs in Foxo3flox/flox; LysMcre+ BMMs (Fig. 2D). These results indicate that there exists Results a truncated Foxo3 mRNA transcript in the Foxo3flox/flox; LysMcre+ Absence of Foxo3 enhances osteoclastogenesis mice. Interestingly, the primers located within exon 1 and exon 3 To provide genetic evidence for the role of Foxo3 in osteoclasts, we (F5 and R5 primers) were also able to generate PCR products first took advantage of Foxo3 global KO mice, in which the Foxo3 shorter than 300 bp, strongly implying that this truncated Foxo3

FIGURE 1. RANKL-induced osteoclast differentiation is enhanced by Foxo3 deﬁciency. BMMs derived from WT control and Foxo3 KO mice were stimulated with RANKL for 4 d. TRAP staining was performed (A), and the number of TRAP-positive multinucleated cells per well is shown in (B). TRAP- positive cells appear red in the photographs. Scale bar, 100 mm. Data are representative of three independent experiments. (C) Heat map of RANKL- induced osteoclastic gene expression enhanced by Foxo3 deﬁciency. Row z-scores of CPMs of osteoclast genes are shown in the heat map. **p , 0.01. 2144 Foxo3 ISOFORM2 REGULATES BONE REMODELING Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. Foxo3f/f;LysMcre (Foxo3isoform2) mice express a truncated Foxo3 protein that is an ortholog of human FOXO3 isoform2. (A) Molecular structure of mouse Foxo3 and Loxp sites. (B) PCR primer locations in Foxo3.(C) Foxo3 gene expression detected in WT and Foxo3 f/f;LysMcre BMMs by PCR using the indicated primer sets whose locations are shown in (B). n = 5 per group. (D) Foxo3 gene expression detected in WT and Foxo3f/f;LysMcre BMMs by quantitative PCR using the indicated primer sets whose locations are shown in (A). (E and F) DNA sequences of the cloned Foxo3 transcripts from WT or Foxo3f/f;LysMcre BMMs using a primer set (Exon 1F and Exon 3R) that covers WT Foxo3 mRNA starting from the transcription start site in exon 1 to the end of the coding sequence in exon 3. (G) Foxo3 protein expression detected in WT and Foxo3f/f;LysMcre BMMs by Western blot using Abs recognizing C terminus or exon 2 of Foxo3, respectively. p38 was used as a loading control. All the primer sequences are shown in Table I. The Journal of Immunology 2145 Downloaded from FIGURE 3. Mouse Foxo3 isoform2 suppresses osteoclastogenesis and leads to the osteopetrotic phenotype in mice. (A) BMMs derived from WT control and Foxo3isoform2 mice were stimulated with RANKL for 4 d. TRAP staining was performed (A), and the number of TRAP-positive multinucleated cells (MNCs) per well is shown in (B). Scale bar, 100 mm. Data http://www.jimmunol.org/ are representative of and statistical analysis was performed on three independent experiments. (C) mCT images and (D) bone morphometric analysis of trabecular bone of the distal femurs isolated from the WT and Foxo3isoform2 mice. n = 8 per group. (E) BMMs transfected with either control or Foxo3 siRNA (80 nM) were stimulated with RANKL for 5 d. The number of TRAP-positive MNCs ($3 nuclei per cell) per well was calculated. *p , 0.05, **p , 0.01. BV/TV, bone volume per tissue volume; Tb.N, trabecular number; by guest on October 2, 2021 Tb.Sp, trabecular separation; Tb.Th, trabecular thickness.

mRNA is transcribed from exon 1, skips exon 2, and is elongated novel exon 1 to exon 3 junction was present in Foxo3flox/flox; to exon 3. To directly demonstrate this, we cloned Foxo3 tran- LysMcre+ BMMs (Fig. 2F). These results confirm the presence of scripts from WT or Foxo3flox/flox; LysMcre+ BMMs using a primer a novel Foxo3 mRNA with exon 2 truncated in the Foxo3flox/flox; set (Exon 1F and Exon 3R in Fig. 2E, 2F) that covers WT Foxo3 LysMcre+ BMMs, resulting from an in-frame (nonframeshift) mRNA starting from the transcription start site in exon 1 to the deletion by the cre-lox recombination in these mice. We next end of the coding sequence in exon 3. As shown in Fig. 2E, we set off to detect the Foxo3 protein expression in the WT and detected the normal junction between exon 1 and exon 2 in WT Foxo3flox/flox; LysMcre+ BMMs. We used two Abs; one Ab rec- BMMs (Fig. 2E). However, the entire exon 2 was absent, and a ognizes the C-terminal region of Foxo3, whereas the other is an 2146 Foxo3 ISOFORM2 REGULATES BONE REMODELING mAb that specifically targets the exon 2 of Foxo3. As shown in osteoclast differentiation indicated by TRAP-positive multinu- Fig. 2G, the full length of WT Foxo3 proteins were detected by cleated osteoclast formation induced by RANKL was significantly both Abs in WT BMMs. In Foxo3flox/flox; LysMcre+ BMMs, the suppressed in Foxo3 isoform2 BMM cell cultures compared with the full length of Foxo3 proteins were deleted as expected. In contrast, WT littermate control cell cultures (Fig. 3A, 3B). a truncated Foxo3 protein (55 kDa) was detected by the C-terminal We next performed microcomputed tomographic (mCT) anal- Ab in Foxo3 flox/flox; LysMcre+ BMMs but not by the Ab specifically yses to examine the bone phenotype of Foxo3 isoform2 mice. The targeting exon 2. Furthermore, knockdown of Foxo3 by RNA in- Foxo3 isoform2 mice and their littermate controls exhibit similar terference completely deleted the truncated protein (55 kDa) in the body weight and body length (data not shown). As shown in Foxo3 flox/flox; LysMcre+ BMMs (Fig. 2G, top panel). Taken together Fig. 3C, 3D, Foxo3 isoform2 mice show an osteopetrotic phenotype with the cloning data in Fig. 2F, these results demonstrate that indicated by significantly increased trabecular bone volume and the full-length Foxo3 protein is absent, but there exists an exon number but decreased trabecular bone spacing. Taken together 2–truncated Foxo3 protein in Foxo3flox/flox; LysMcre+ BMMs. with the suppressed osteoclast differentiation in Foxo3 isoform2 When we investigated the human FOXO3 locus, we found an- cells, these data demonstrate that expression of Foxo3 isoform2 in notations for a short isoform of FOXO3 (Supplemental Fig. 1A), mice leads to an osteopetrotic bone phenotype. which is named as isoform2 (RefSeq gene database, Ensembl Consistent with the Foxo3 global KO data (Fig. 1), knockdown of genome database, and Uniprot Knowledgebase). The full length of Foxo3 using RNA interference in WT BMMs enhanced osteoclast FOXO3 is named as isoform1, which contains 673 aa. The human differentiation (Fig. 3E). Furthermore, knockdown of Foxo3 full-length FOXO3 isoform1 has two subisoforms (1a and 1b), isoform2 in Foxo3 isoform2 BMMs significantly elevated osteo- which have an identical coding sequence with variable 59 un- clastogenesis (Fig. 3E), supporting the inhibitory role of Foxo3 translated region. The isoform2, generated by alternative splicing isoform2 in osteoclast differentiation. Downloaded from with an alternate promoter, is a truncated FOXO3 protein with We next performed a structure-functional analysis of Foxo3 protein in 453 aa that are encoded by exon 2 (Supplemental Fig. 1B, 1C). osteoclast differentiation. We cloned and generated a series of plasmids The coding sequences of the mouse and human FOXO3 are highly that express full-length WT Foxo3 or recombinant Foxo3 peptides conserved, determined by 95% of identical amino acids (Supplemental encoded by the isoform2 or by exon 2 (hereafter referred to as Exon 2). Fig. 2). When comparing the coding and amino acid sequences of the We confirmed the protein expression of each plasmid in HEK293 human FOXO3 isoform2 with the mouse truncated Foxo3 in cells (Fig. 4A) and RAW264.7 cells (Fig. 4B) after transfection. As http://www.jimmunol.org/ Foxo3flox/flox; LysMcre+ BMMs, we found that 96% of the amino shown in Fig. 4C, RANKL induced osteoclast differentiation in the acids are identical (Supplemental Fig. 3). These new findings in- RAW264.7 cells transfected with empty vector. Overexpression of dicate that the mouse truncated Foxo3 in Foxo3flox/flox; LysMcre+ WT full-length Foxo3 or isoform2 drastically inhibited osteoclast BMMs is a mouse ortholog of human FOXO3 isoform2. We differentiation. The isoform2 seems to possess a stronger inhibitory therefore name this novel Foxo3 isoform as mouse Foxo3 isoform2. effect on osteoclast differentiation than the full-length protein. Inter- The biological function of the human FOXO3 isoform2 is unclear. estingly, expression of exon 2 significantly promoted osteoclast dif- Because the Foxo3flox/flox; LysMcre+ mice express Foxo3 isoform2 ferentiation (Fig. 4C). These data were further corroborated by the instead of the full-length protein, we hereafter refer to these mice corresponding changes in osteoclast marker gene expression, such as by guest on October 2, 2021 as Foxo3 isoform2 mice, which could be useful as a promising model TRAP and cathepsin K (Fig. 4D). Because isoform2 is encoded for studying the function of the newly identified Foxo3 isoform2. by exon 3, these results argue that exon 3 is mainly responsible for osteoclastic inhibition, whereas exon 2 likely counteracts this effect. Mouse Foxo3 isoform2 suppresses osteoclastogenesis and leads to the osteopetrotic phenotype in mice Foxo3 isoform2 represses osteoclast differentiation via To investigate the role of Foxo3 isoform2 in osteoclastogenesis, we endogenous type I IFN–mediated feedback inhibition used BMMs as osteoclast precursors to examine osteoclast dif- We next set off to explore the mechanisms by which Foxo3 iso- ferentiation in response to RANKL. As shown in Fig. 3A, 3B, the form2 inhibits osteoclastogenesis. In parallel with the suppressed

Table I. Sequences of regular PCR primers

Primer Name Sequence Product Size (bp) Exons 2–3 F: 59-TTCAAGGATAAGGGCGACAG-39 215 R: 59-CCTCGGCTCTTGGTGTACTT-39 Exons 3–4 F: 59-CGTTGTTGGTTTGAATGTGG-39 213 R: 59-CGTGGGAGTCTCAAAGGTGT-39 Primer set 1 F: 59-ATGCGCGTTCAGAATGAAG-39 207 R: 59-GGAGAGCTGGGAAGGACTGT-39 Primer set 2 F: 59-CCATGGACAACAGCAACAAG-39 389 R: 59-CAGCCCATCATTCAGATTCA-39 Primer set 3 F: 59-GATGATGATGGACCCCTGTC-39 416 R: 59-GAAGCAAGCAGGTCTTGGAG-39 Primer set 4 F: 59-GGGGAGTTTGGTCAATCAGA-39 348 R: 59-TTAAAATCCAACCCGTCAGC-39 F3 and R3 primers F: 59-CTGTCCTATGCCGACCTGAT-39 122 R: 59-CTGTCGCCCTTATCCTTGAA-39 F4 and R4 primers F: 59-ATGGGAGCTTGGAATGTGAC-39 73 R: 59-TTAAAATCCAACCCGTCAGC-39 F5 and R5 primers F: 59-AGGAGGAGGAATGTGGAAGG-39 221 R: 59-CCGTGCCTTCATTCTGAAC-39 Exon 1F F: 59-ATTCTAGACTAGGTTGAGGCTCCCTGT-39 2355 Exon 3R R: 59-ATTCCGGATCCGCCTGGTACCCAGCTTTGA-39 The Journal of Immunology 2147 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. Overexpression of Foxo3 isoform2 inhibits osteoclastogenesis. (A and B) Immunoblot analysis of the expression of full-length Foxo3, Foxo3 isoform2, and exon 2 in whole cell lysates of HEK293 cells (A) or RAW264.7 cells (B) transfected with corresponding pcDNA3.1+ plasmids containing specific Foxo3 fragments as indicated in the Materials and Methods. Anti-Flag Ab was used in (A). In (B), Foxo3 C-terminal Ab was used to detect full- length Foxo3 and Foxo3 isoform2. Foxo3 N-terminal exon 2 Ab was used to detect Foxo3 exon 2. (C) RAW264.7 cells transfected with the indicated plasmids were stimulated with RANKL for 6 d. TRAP staining was performed (left panel), and the number of TRAP-positive multinucleated cells per well is shown in the right panel. Scale bar, 100 mm. Data are representative of and statistical analysis was performed on three independent experiments. (D) Quantitaive PCR analysis of the relative expression of CtsK and Acp5 induced by RANKL for 6 d in the RAW264.7 cells transfected with the indicated plasmids. The induction folds of gene expression by RANKL relative to each basal condition was calculated and shown in the figure. Data are representative of three independent experiments. *p , 0.05, **p , 0.01. generation of TRAP-positive polykaryons, we found that the ex- produced by osteoclast precursors is a strong feedback mechanism pression of osteoclast marker genes Acp5 (encoding TRAP), Ctsk that restrains osteoclastogenesis (5, 30, 31). We therefore asked (encoding cathepsin K), Itgb3 (encoding b3 integrin), Dcstamp whether the inhibitory effects of Foxo3 isoform2 involves type I (encoding Dc-Stamp), Calcr (encoding calcintonin receptor), and IFN–mediated inhibition. Previous studies showed that RANKL Atp6V0d2 (encoding ATPase H+ Transporting V0 Subunit D2) was treatment can induce a low level of IFN-b expression in drastically decreased in RANKL-treated Foxo3 isoform2 cells rela- macrophages/osteoclast precursors. Although the magnitude of tive to the WT control cells (Fig. 5A). A previous study shows that type I IFN induction by RANKL is small (,10 pg/ml after 24 h Foxo3 targets catalase and Cyclin D1 to arrest cell cycle and stimulation) when compared with other stimuli such as TLR promote apoptosis in RANKL-induced osteoclastogenesis (16). stimulation, the high potency of type I IFN effects allow these Such Foxo3-mediated changes, however, were not detected in the small concentrations to inhibit osteoclast differentiation (30, 31). Foxo3 isoform2 osteoclastogenesis (data not shown). In contrast, we Consistent with these observations (30, 31), we found that RANKL found that the expression of Irf7, an IFN-responsive gene, was induced IFN-b expression in WT BMMs and Foxo3 isoform2 markedly elevated in RANKL-treated Foxo3 isoform2 cells relative significantly increased IFN-b induction (Fig. 5B). The enhancement to WT control cells (Fig. 5B). IRF7 has been identified as a Foxo3 of IFN expression by Foxo3 isoform2 was further corroborated target (29). It is also well established that endogenous IFN-b by the elevated expression of IFN-responsive genes, such as 2148 Foxo3 ISOFORM2 REGULATES BONE REMODELING

FIGURE 5. Mouse Foxo3 isoform2 suppresses osteoclastic gene expression but enhances type I IFN–responsive gene expression. BMMs derived from WT control and Foxo3 isoform2 mice were stimulated with RANKL for 3 d. The expression of osteoclastic marker genes (A) and type I IFN response genes (B) was examined by quantitative PCR. Data are representative of three independent experiments. **p , 0.01.

Mx1, Ifit1, Ifit2, Irf7, and Stat1 after RANKL treatment (Fig. 5B). inhibition. Further experiments are needed to elucidate the shared These results clearly demonstrate enhanced Ifnb expression and or distinct mechanisms mediated by full-length Foxo3 and the Downloaded from response by Foxo3 isoform2 during osteoclastogenesis and indi- isoform2. cate that Foxo3 isoform2 suppresses osteoclastogenesis via type I Protein isoforms from the exon skipping mode of alternative IFN–mediated feedback inhibition. splicing often end up with a lack of certain domains that distinguish the function of the isoforms from their original full-length proteins. Discussion For example, previous studies identify IRF7 as a critical direct Similarly to the other Foxo proteins, the function of Foxo3 is target of FOXO3, and FOXO3 negatively regulates IRF7 tran- http://www.jimmunol.org/ largely regulated through posttranslational modifications, such scription in the antiviral response (29). Our results show that as phosphorylation, acetylation, methylation, and ubiquitination. Foxo3 isoform2 expression elevates Irf7 transcription and corre- These posttranslational modifications are context dependent and sponding type I IFN response during osteoclastogenesis. Foxo3 create a complex set of codes, which affect the subcellular location isoform2 lacks the DNA-binding domain and thus may function of Foxo3 and give rise to the diverse functions of Foxo family as an activator to increase Irf7 expression in a DNA-binding proteins in response to different stimuli (10–14). For example, independent manner. Although Irf7 is a common target by both Foxo3 can be phosphorylated by various protein kinases at many full-length Foxo3 and the isoform2, they show distinct regula- phosphorylation sites from the N to C terminus of the protein. toryeffectsonIrf7expressionpresumablybecauseoftheirdif- by guest on October 2, 2021 Phosphorylation of specific sites by kinases, such as AKT, SGK1, ferent DNA binding capacity. CDK2, ERK, and IKK, induces cytoplasmic translocation and/or Our results revealed that Foxo3flox/flox;LysMcre+ mice are not degradation of Foxo3, leading to target gene inhibition. In con- fully conditional KO mice because of the existence of the iso- trast, phosphorylation of the activating sites by kinases MST1, form2. The position of the loxp sites caused an in-frame deletion JNK, and AMPK usually leads to nuclear localization of Foxo3 of exon 2 in this mouse line. This was not known at the time when and the activation of its target genes (10–14, 32). Foxo3 isoform2 previous loss-of-function studies used this Foxo3flox/flox line. The lacks most of the N-terminal DNA-binding domain while main- interpretation of the mutant phenotype in such studies might be taining the nuclear localization signal, the C-terminal nuclear now questionable, dependent on cell types. Therefore, future work export signal, and the transactivation domain at C terminus. This should pay close attention to the verification of frameshift deletion molecular structure implies that Foxo3 isoform2 is likely to lose by cre-loxp recombination as well as the presence of isoforms. the direct transcriptional regulation of the genes targeted by the Collectively, our findings in the current study identify the first, to full-length Foxo3 because of the lack of DNA-binding domain. our knowledge, known biological function of Foxo3 isoform2, However, Foxo3 isoform2 holds several activating phosphoryla- which acts as an important suppressor of osteoclast differentiation tion sites that usually contribute to gene activation. In addition to via endogenous type I IFN–mediated feedback inhibition. The the direct DNA-binding transcriptional activity, Foxo transcription Foxo3 floxed allele mice (Foxo3flox/flox) could be used as a mouse factors are able to regulate transcription in a DNA-binding- resource in various areas to investigate the function of Foxo3 independent manner, often by interaction with other transcrip- isoform2 that recapitulates human FOXO3 isoform2. Environmental tional activators or repressors. Hence, we cannot exclude the cues often affect gene transcription and alternative splicing. For possibility that Foxo3 isoform2 regulates gene transcription in the example, bone marrow macrophages/osteoclast precursors mainly nucleus together with other partners. In addition, Foxo3 isoform2 express full-length Foxo3 with a trace amount of isoform2 in a carries the nuclear localization signal as well as the nuclear export physiological condition. Upon RANKL stimulation, Foxo3 isoform2 signal that allow it to shuttle between the nucleus and cytoplasm expression is increased (Fig. 2), which contributes to osteoclastic in response to environmental cues. The overall impact from feedback inhibition. Thus, we speculate that the expression pat- these possibilities will determine the subcellular localization of terns and functions of Foxo3 isoform2 may be altered in response Foxo3 isoform2 and the mechanisms by which it inhibits osteo- to different environmental settings. It will be of particular interest clastogenesis. The exon 2 peptide is shown to promote osteoclast and clinical relevance to investigate the expression levels and differentiation. With the consideration that exon 2 contains an functions of FOXO3 isoform2 in human cells, for instance, in N-terminal DNA-binding domain, the direct DNA binding pre- human osteoclasts in healthy conditions versus disease settings, sumably results in the osteoclastogenic activity of exon 2, which such as in osteoporosis and RA. Future studies are expected to in turn attenuates the full-length Foxo39s ability in osteoclast uncover the expression profile of Foxo3 isoform2 in different cells The Journal of Immunology 2149 and tissues, the regulation of its expression/function, related bio- 15. Morris, B. J., D. C. Willcox, T. A. Donlon, and B. J. Willcox. 2015. FOXO3: a major gene for human longevity--A mini-review. Gerontology 61: 515–525. logical significance, and potential therapeutic implications. 16. Bartell, S. M., H. N. Kim, E. Ambrogini, L. Han, S. Iyer, S. Serra Ucer, P. Rabinovitch, R. L. Jilka, R. S. Weinstein, H. Zhao, et al. 2014. 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Human full length FOXO3 Human FOXO3 isoform2

Supplementary Figure 1. Molecular structure of human FOXO3 isoform2. (A) Human FOXO3 isoform2 from RefSeq gene database shown in UCSC genome browser. (B) Comparison of the molecular structures between full-length FOXO3 and FOXO3 isoform2. (C) Comparison of the coding sequences (upper lanes) and amino acid sequences (lower lanes) between full-length FOXO3 and FOXO3 isoform2. Blue labels: FH domain. Supplementary Fig. 2

mFoxo3 (672aa) hFOXO3 (673aa)

ATGGCAGAGGCACCAGCCTCCCCGGTCCCGCTCTCTCCGCTCGAAGTGGAGCTGGACCCA ATGGCAGAGGCACCGGCTTCCCCGGCCCCGCTCTCTCCGCTCGAAGTGGAGCTGGACCCG M A E A P A S P V P L S P L E V E L D P M A E A P A S P A P L S P L E V E L D P GAGTTCGAGCCACAGAGTCGGCCACGCTCCTGTACGTGGCCCCTGCAGAGGCCGGAGCTG GAGTTCGAGCCCCAGAGCCGTCCGCGATCCTGTACGTGGCCCCTGCAAAGGCCGGAGCTC E F E P Q S R P R S C T W P L Q R P E L E F E P Q S R P R S C T W P L Q R P E L CAGGCGAGCCCGGCCAAGCCCTCGGGGGAGACGGCCGCAGACTCCATGATCCCCGAGGAG CAAGCGAGCCCTGCCAAGCCCTCGGGGGAGACGGCCGCCGACTCCATGATCCCCGAGGAG Q A S P A K P S G E T A A D S M I P E E Q A S P A K P S G E T A A D S M I P E E GACGACGATGAAGACGACGAGGACGGCGGCGGCCGAGCCAGCTCGGCCATGGTGATCGGT GAGGACGATGAAGACGACGAGGACGGCGGGGGACGGGCCGGCTCGGCCATGGCGATCGGC D D D E D D E D G G G R A S S A M V I G E D D E D D E D G G G R A G S A M A I G GGCGGCGTGAGCAGCACGCTGGGTTCCGGGCTGCTCCTCGAGGATTCGGCCATGCTGCTG GGCGGCGGCGGGAGCGGCACGCTGGGCTCCGGGCTGCTCCTTGAGGACTCGGCCCGGGTG G G V S S T L G S G L L L E D S A M L L G G G G S G T L G S G L L L E D S A R V GCTCCAGGAGGGCAGGACCTCGGGTCGGGGCCAGCGTCCGCCGCAGGCGCTCTGAGTGGG CTGGCACCCGGAGGGCAAGACCCCGGGTCTGGGCCAGCCACCGCGGCGGGCGGGCTGAGC A P G G Q D L G S G P A S A A G A L S G xon1 L A P G G Q D P G S G P A T A A G G L S

GGCACGCCGACGCAGCTGCAGCCTCAGCAGCCACTGCCACAGCCGCAGCCGGGGGCGGCT hE GGGGGTACACAGGCGCTGCTGCAGCCTCAGCAACCGCTGCCACCGCCGCAGCCGGGGGCG mExon2 G T P T Q L Q P Q Q P L P Q P Q P G A A G G T Q A L L Q P Q Q P L P P P Q P G A GGGGGCTCTGGGCAACCAAGGAAATGCTCCTCGCGGCGGAATGCCTGGGGGAACCTGTCC GCTGGGGGCTCCGGGCAGCCGAGGAAATGTTCGTCGCGGCGGAACGCCTGGGGAAACCTG G G S G Q P R K C S S R R N A W G N L S A G G S G Q P R K C S S R R N A W G N L TATGCCGACCTGATCACCCGCGCCATCGAGAGCTCCCCGGACAAACGGCTCACTTTGTCC TCCTACGCGGACCTGATCACCCGCGCCATCGAGAGCTCCCCGGACAAACGGCTCACTCTG Y A D L I T R A I E S S P D K R L T L S S Y A D L I T R A I E S S P D K R L T L CAGATCTACGAGTGGATGGTGCGCTGTGTGCCCTACTTCAAGGATAAGGGCGACAGCAAC TCCCAGATCTACGAGTGGATGGTGCGTTGCGTGCCCTACTTCAAGGATAAGGGCGACAGC Q I Y E W M V R C V P Y F K D K G D S N S Q I Y E W M V R C V P Y F K D K G D S AGCTCTGCGGGCTGGAAG AACAGCTCTGCCGGCTGGAAG S S A G W K N S S A G W K

AACTCCATCCGGCACAACCTGTCCCTGCACAGCCGCTTCATGCGCGTTCAGAATGAAGGC AACTCCATCCGGCACAACCTGTCACTGCATAGTCGATTCATGCGGGTCCAGAATGAGGGA N S I R H N L S L H S R F M R V Q N E G N S I R H N L S L H S R F M R V Q N E G ACGGGCAAGAGCTCTTGGTGGATCATCAACCCCGATGGGGGAAAGAGCGGGAAGGCCCCC ACTGGCAAGAGCTCTTGGTGGATCATCAACCCTGATGGGGGGAAGAGCGGAAAAGCCCCC T G K S S W W I I N P D G G K S G K A P T G K S S W W I I N P D G G K S G K A P CGGCGGCGTGCGGTCTCCATGGACAACAGCAACAAGTACACCAAGAGCCGAGGCCGGGCA CGGCGGCGGGCTGTCTCCATGGACAATAGCAACAAGTATACCAAGAGCCGTGGCCGCGCA R R R A V S M D N S N K Y T K S R G R A R R R A V S M D N S N K Y T K S R G R A GCCAAGAAGAAGGCGGCCCTGCAGGCTGCCCCAGAGTCGGCAGACGACAGTCCTTCCCAG GCCAAGAAGAAGGCAGCCCTGCAGACAGCCCCCGAATCAGCTGACGACAGTCCCTCCCAG A K K K A A L Q A A P E S A D D S P S Q A K K K A A L Q T A P E S A D D S P S Q CTCTCCAAGTGGCCTGGCAGCCCCACGTCCCGCAGCAGCGACGAGCTGGATGCGTGGACC CTCTCCAAGTGGCCTGGCAGCCCCACGTCACGCAGCAGTGATGAGCTGGATGCGTGGACG L S K W P G S P T S R S S D E L D A W T L S K W P G S P T S R S S D E L D A W T GACTTCCGCTCGCGCACCAATTCCAACGCCAGCACCGTGAGCGGCCGCCTGTCGCCCATC GACTTCCGTTCACGCACCAATTCTAACGCCAGCACAGTCAGTGGCCGCCTGTCGCCCATC D F R S R T N S N A S T V S G R L S P I D F R S R T N S N A S T V S G R L S P I CTGGCAAGCACGGAGCTGGATGACGTCCAGGATGATGATGGACCCCTGTCCCCCATGCTG ATGGCAAGCACAGAGTTGGATGAAGTCCAGGACGATGATGCGCCTCTCTCGCCCATGCTC L A S T E L D D V Q D D D G P L S P M L M A S T E L D E V Q D D D A P L S P M L TACAGCAGCTCTGCCAGCCTGTCGCCCTCCGTGAGCAAGCCGTGTACTGTGGAGCTTCCG TACAGCAGCTCAGCCAGCCTGTCACCTTCAGTAAGCAAGCCGTGCACGGTGGAACTGCCA Y S S S A S L S P S V S K P C T V E L P Y S S S A S L S P S V S K P C T V E L P CGGCTGACGGACATGGCCGGCACCATGAATCTGAATGATGGGCTGGCCGAGAACCTCATG CGGCTGACTGATATGGCAGGCACCATGAATCTGAATGATGGGCTGACTGAAAACCTCATG R L T D M A G T M N L N D G L A E N L M R L T D M A G T M N L N D G L T E N L M GACGACCTGCTGGATAACATCGCGCTCCCGCCATCGCAGCCATCGCCTCCTGGCGGGCTT GACGACCTGCTGGATAACATCACGCTCCCGCCATCCCAGCCATCGCCCACTGGGGGACTC D D L L D N I A L P P S Q P S P P G G L D D L L D N I T L P P S Q P S P T G G L ATGCAGCGGGGCTCCAGCTTCCCATATACCGCCAAGAGCTCCGGCCTGGGCTCCCCAACC ATGCAGCGGAGCTCTAGCTTCCCGTATACCACCAAGGGCTCGGGCCTGGGCTCCCCAACC M Q R G S S F P Y T A K S S G L G S P T M Q R S S S F P Y T T K G S G L G S P T GGCTCCTTCAACAGTACCGTGTTTGGACCTTCGTCTCTGAACTCCTTGCGTCAGTCACCC AGCTCCTTTAACAGCACGGTGTTCGGACCTTCATCTCTGAACTCCCTACGCCAGTCTCCC G S F N S T V F G P S S L N S L R Q S P S S F N S T V F G P S S L N S L R Q S P ATGCAGACTATCCAGGAGAACAGACCAGCCACCTTCTCTTCCGTGTCACACTACGGCAAC xon2 ATGCAGACCATCCAAGAGAACAAGCCAGCTACCTTCTCTTCCATGTCACACTATGGTAAC

M Q T I Q E N R P A T F S S V S H Y G N hE M Q T I Q E N K P A T F S S M S H Y G N mExon3 CAGACACTCCAAGACCTGCTTGCTTCAGACTCACTCAGCCACAGCGACGTCATGATGACC CAGACACTCCAGGACCTGCTCACTTCGGACTCACTTAGCCACAGCGATGTCATGATGACA Q T L Q D L L A S D S L S H S D V M M T Q T L Q D L L T S D S L S H S D V M M T CAGTCGGACCCCTTGATGTCTCAGGCTAGCACCGCCGTGTCCGCCCAGAATGCCCGCCGG CAGTCGGACCCCTTGATGTCTCAGGCCAGCACCGCTGTGTCTGCCCAGAATTCCCGCCGG Q S D P L M S Q A S T A V S A Q N A R R Q S D P L M S Q A S T A V S A Q N S R R AACGTGATGCTTCGCAACGATCCAATGATGTCCTTTGCTGCCCAGCCTACCCAGGGGAGT AACGTGATGCTTCGCAATGATCCGATGATGTCCTTTGCTGCCCAGCCTAACCAGGGAAGT N V M L R N D P M M S F A A Q P T Q G S N V M L R N D P M M S F A A Q P N Q G S TTGGTCAATCAGAACTTGCTCCACCACCAGCACCAAACCCAGGGCGCTCTTGGTGGCAGC TTGGTCAATCAGAACTTGCTCCACCACCAGCACCAAACCCAGGGCGCTCTTGGTGGCAGC L V N Q N L L H H Q H Q T Q G A L G G S L V N Q N L L H H Q H Q T Q G A L G G S CGTGCCTTGTCAAATTCTGTCAGCAACATGGGCTTGAGTGACTCCAGCAGCCTTGGCTCA CGTGCCTTGTCGAATTCTGTCAGCAACATGGGCTTGAGTGAGTCCAGCAGCCTTGGGTCA R A L S N S V S N M G L S D S S S L G S R A L S N S V S N M G L S E S S S L G S GCCAAACACCAGCAGCAGTCTCCCGCCAGCCAGTCTATGCAAACCCTCTCGGACTCTCTC GCCAAACACCAGCAGCAGTCTCCTGTCAGCCAGTCTATGCAAACCCTCTCGGACTCTCTC A K H Q Q Q S P A S Q S M Q T L S D S L A K H Q Q Q S P V S Q S M Q T L S D S L TCAGGCTCCTCACTGTATTCAGCTAGTGCAAACCTTCCCGTCATGGGCCACGATAAGTTC TCAGGCTCCTCCTTGTACTCAACTAGTGCAAACCTGCCCGTCATGGGCCATGAGAAGTTC S G S S L Y S A S A N L P V M G H D K F S G S S L Y S T S A N L P V M G H E K F CCCAGTGACTTGGACCTGGACATGTTCAATGGGAGCTTGGAATGTGACATGGAGTCCATC CCCAGCGACTTGGACCTGGACATGTTCAATGGGAGCTTGGAATGTGACATGGAGTCCATT P S D L D L D M F N G S L E C D M E S I P S D L D L D M F N G S L E C D M E S I ATCCGTAGTGAACTCATGGATGCTGACGGGTTGGATTTTAACTTTGACTCCCTCATCTCC ATCCGTAGTGAACTCATGGATGCTGATGGGTTGGATTTTAACTTTGATTCCCTCATCTCC I R S E L M D A D G L D F N F D S L I S I R S E L M D A D G L D F N F D S L I S ACACAGAACGTTGTTGGTTTGAATGTGGGGAACTTCACTGGTGCTAAGCAGGCCTCATCT ACACAGAATGTTGTTGGTTTGAACGTGGGGAACTTCACTGGTGCTAAGCAGGCCTCATCT T Q N V V G L N V G N F T G A K Q A S S T Q N V V G L N V G N F T G A K Q A S S CAAAGCTGGGTACCAGGCTGA CAGAGCTGGGTGCCAGGCTGA Q S W V P G - Q S W V P G -

Supplementary Figure 2. Comparison of the coding sequences (upper lanes) and amino acid sequences (lower lanes) between mouse and human full-length FOXO3. Red labels indicate different amino acids in mouse Foxo3. Supplementary Fig. 3

Coding sequence and amino acids from Coding sequence and amino acids from hFOXO3 isoform2 (453aa) mFoxo3 isoform2 (453aa)

AACTCCATCCGGCACAACCTGTCACTGCATAGTCGATTCATGCGGGTCCAGAATGAGGGA AACTCCATCCGGCACAACCTGTCCCTGCACAGCCGCTTCATGCGCGTTCAGAATGAAGGC N S I R H N L S L H S R F M R V Q N E G N S I R H N L S L H S R F M R V Q N E G ACTGGCAAGAGCTCTTGGTGGATCATCAACCCTGATGGGGGGAAGAGCGGAAAAGCCCCC ACGGGCAAGAGCTCTTGGTGGATCATCAACCCCGATGGGGGAAAGAGCGGGAAGGCCCCC T G K S S W W I I N P D G G K S G K A P T G K S S W W I I N P D G G K S G K A P CGGCGGCGGGCTGTCTCCATGGACAATAGCAACAAGTATACCAAGAGCCGTGGCCGCGCA CGGCGGCGTGCGGTCTCCATGGACAACAGCAACAAGTACACCAAGAGCCGAGGCCGGGCA R R R A V S M D N S N K Y T K S R G R A R R R A V S M D N S N K Y T K S R G R A GCCAAGAAGAAGGCAGCCCTGCAGACAGCCCCCGAATCAGCTGACGACAGTCCCTCCCAG GCCAAGAAGAAGGCGGCCCTGCAGGCTGCCCCAGAGTCGGCAGACGACAGTCCTTCCCAG A K K K A A L Q T A P E S A D D S P S Q A K K K A A L Q A A P E S A D D S P S Q CTCTCCAAGTGGCCTGGCAGCCCCACGTCACGCAGCAGTGATGAGCTGGATGCGTGGACG CTCTCCAAGTGGCCTGGCAGCCCCACGTCCCGCAGCAGCGACGAGCTGGATGCGTGGACC L S K W P G S P T S R S S D E L D A W T L S K W P G S P T S R S S D E L D A W T GACTTCCGTTCACGCACCAATTCTAACGCCAGCACAGTCAGTGGCCGCCTGTCGCCCATC GACTTCCGCTCGCGCACCAATTCCAACGCCAGCACCGTGAGCGGCCGCCTGTCGCCCATC D F R S R T N S N A S T V S G R L S P I D F R S R T N S N A S T V S G R L S P I ATGGCAAGCACAGAGTTGGATGAAGTCCAGGACGATGATGCGCCTCTCTCGCCCATGCTC CTGGCAAGCACGGAGCTGGATGACGTCCAGGATGATGATGGACCCCTGTCCCCCATGCTG M A S T E L D E V Q D D D A P L S P M L L A S T E L D D V Q D D D G P L S P M L TACAGCAGCTCAGCCAGCCTGTCACCTTCAGTAAGCAAGCCGTGCACGGTGGAACTGCCA TACAGCAGCTCTGCCAGCCTGTCGCCCTCCGTGAGCAAGCCGTGTACTGTGGAGCTTCCG Y S S S A S L S P S V S K P C T V E L P Y S S S A S L S P S V S K P C T V E L P CGGCTGACTGATATGGCAGGCACCATGAATCTGAATGATGGGCTGACTGAAAACCTCATG CGGCTGACGGACATGGCCGGCACCATGAATCTGAATGATGGGCTGGCCGAGAACCTCATG R L T D M A G T M N L N D G L T E N L M R L T D M A G T M N L N D G L A E N L M GACGACCTGCTGGATAACATCACGCTCCCGCCATCCCAGCCATCGCCCACTGGGGGACTC GACGACCTGCTGGATAACATCGCGCTCCCGCCATCGCAGCCATCGCCTCCTGGCGGGCTT D D L L D N I T L P P S Q P S P T G G L D D L L D N I A L P P S Q P S P P G G L xon2 ATGCAGCGGAGCTCTAGCTTCCCGTATACCACCAAGGGCTCGGGCCTGGGCTCCCCAACC ATGCAGCGGGGCTCCAGCTTCCCATATACCGCCAAGAGCTCCGGCCTGGGCTCCCCAACC

hE M Q R S S S F P Y T T K G S G L G S P T M Q R G S S F P Y T A K S S G L G S P T mExon3 AGCTCCTTTAACAGCACGGTGTTCGGACCTTCATCTCTGAACTCCCTACGCCAGTCTCCC GGCTCCTTCAACAGTACCGTGTTTGGACCTTCGTCTCTGAACTCCTTGCGTCAGTCACCC S S F N S T V F G P S S L N S L R Q S P G S F N S T V F G P S S L N S L R Q S P ATGCAGACCATCCAAGAGAACAAGCCAGCTACCTTCTCTTCCATGTCACACTATGGTAAC ATGCAGACTATCCAGGAGAACAGACCAGCCACCTTCTCTTCCGTGTCACACTACGGCAAC M Q T I Q E N K P A T F S S M S H Y G N M Q T I Q E N R P A T F S S V S H Y G N CAGACACTCCAGGACCTGCTCACTTCGGACTCACTTAGCCACAGCGATGTCATGATGACA CAGACACTCCAAGACCTGCTTGCTTCAGACTCACTCAGCCACAGCGACGTCATGATGACC Q T L Q D L L T S D S L S H S D V M M T Q T L Q D L L A S D S L S H S D V M M T CAGTCGGACCCCTTGATGTCTCAGGCCAGCACCGCTGTGTCTGCCCAGAATTCCCGCCGG CAGTCGGACCCCTTGATGTCTCAGGCTAGCACCGCCGTGTCCGCCCAGAATGCCCGCCGG Q S D P L M S Q A S T A V S A Q N S R R Q S D P L M S Q A S T A V S A Q N A R R AACGTGATGCTTCGCAATGATCCGATGATGTCCTTTGCTGCCCAGCCTAACCAGGGAAGT AACGTGATGCTTCGCAACGATCCAATGATGTCCTTTGCTGCCCAGCCTACCCAGGGGAGT N V M L R N D P M M S F A A Q P N Q G S N V M L R N D P M M S F A A Q P T Q G S TTGGTCAATCAGAACTTGCTCCACCACCAGCACCAAACCCAGGGCGCTCTTGGTGGCAGC TTGGTCAATCAGAACTTGCTCCACCACCAGCACCAAACCCAGGGCGCTCTTGGTGGCAGC L V N Q N L L H H Q H Q T Q G A L G G S L V N Q N L L H H Q H Q T Q G A L G G S CGTGCCTTGTCGAATTCTGTCAGCAACATGGGCTTGAGTGAGTCCAGCAGCCTTGGGTCA CGTGCCTTGTCAAATTCTGTCAGCAACATGGGCTTGAGTGACTCCAGCAGCCTTGGCTCA R A L S N S V S N M G L S E S S S L G S R A L S N S V S N M G L S D S S S L G S GCCAAACACCAGCAGCAGTCTCCTGTCAGCCAGTCTATGCAAACCCTCTCGGACTCTCTC GCCAAACACCAGCAGCAGTCTCCCGCCAGCCAGTCTATGCAAACCCTCTCGGACTCTCTC A K H Q Q Q S P V S Q S M Q T L S D S L A K H Q Q Q S P A S Q S M Q T L S D S L TCAGGCTCCTCCTTGTACTCAACTAGTGCAAACCTGCCCGTCATGGGCCATGAGAAGTTC TCAGGCTCCTCACTGTATTCAGCTAGTGCAAACCTTCCCGTCATGGGCCACGATAAGTTC S G S S L Y S T S A N L P V M G H E K F S G S S L Y S A S A N L P V M G H D K F CCCAGCGACTTGGACCTGGACATGTTCAATGGGAGCTTGGAATGTGACATGGAGTCCATT CCCAGTGACTTGGACCTGGACATGTTCAATGGGAGCTTGGAATGTGACATGGAGTCCATC P S D L D L D M F N G S L E C D M E S I P S D L D L D M F N G S L E C D M E S I ATCCGTAGTGAACTCATGGATGCTGATGGGTTGGATTTTAACTTTGATTCCCTCATCTCC ATCCGTAGTGAACTCATGGATGCTGACGGGTTGGATTTTAACTTTGACTCCCTCATCTCC I R S E L M D A D G L D F N F D S L I S I R S E L M D A D G L D F N F D S L I S ACACAGAATGTTGTTGGTTTGAACGTGGGGAACTTCACTGGTGCTAAGCAGGCCTCATCT ACACAGAACGTTGTTGGTTTGAATGTGGGGAACTTCACTGGTGCTAAGCAGGCCTCATCT T Q N V V G L N V G N F T G A K Q A S S T Q N V V G L N V G N F T G A K Q A S S CAGAGCTGGGTGCCAGGCTGA CAAAGCTGGGTACCAGGCTGA Q S W V P G - Q S W V P G -

Supplementary Figure 3. Comparison of the coding sequences (upper lanes) and amino acid sequences (lower lanes) between mouse and human FOXO3 isoform2. Red labels indicate different amino acids in mouse Foxo3 isoform2.