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Elevated Response to Type I IFN Enhances RANKL-Mediated Osteoclastogenesis in Usp18-Knockout Mice

This information is current as Hwa Young Yim, Cheolkyu Park, Yong Deok Lee, of October 2, 2021. Kei-ichiro Arimoto, Raok Jeon, Sung Hee Baek, Dong-Er Zhang, Hong-Hee Kim and Keun Il Kim J Immunol published online 25 March 2016 http://www.jimmunol.org/content/early/2016/03/24/jimmun ol.1501496 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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 25, 2016, doi:10.4049/jimmunol.1501496 The Journal of Immunology

Elevated Response to Type I IFN Enhances RANKL-Mediated Osteoclastogenesis in Usp18-Knockout Mice

Hwa Young Yim,*,†,1 Cheolkyu Park,‡,1 Yong Deok Lee,‡ Kei-ichiro Arimoto,x Raok Jeon,{ Sung Hee Baek,† Dong-Er Zhang,x Hong-Hee Kim,‡ and Keun Il Kim*

A balance between bone formation and bone resorption is critical for the maintenance of bone mass. In many pathological conditions, including chronic inflammation, uncontrolled activation of differentiation often causes excessive bone resorption that results in osteoporosis. In this study, we identified the osteopenia phenotype of mice lacking Usp18 (also called Ubp43), which is a deISGylating and is known as a negative regulator of type I IFN signaling. The expression of Usp18 was induced in preosteoclasts upon receptor activator of NF-kB (RANKL) treatment. In an in vitro osteoclast-differentiation assay, bone marrow from Usp18-deficient mice exhibited an enhanced differentiation to multinucleated cells, elevated activation of NFATc1, and an increased expression of osteoclast marker upon RANKL treatment. Furthermore, in vitro quantification of bone resorption Downloaded from revealed a great increase in osteoclastic activities in Usp18-deficient cells. Interestingly, proinflammatory genes, such as IP-10 (CXCL10), were highly expressed in Usp18-deficient bone marrow macrophages upon RANKL treatment compared with wild-type cells. In addition, serum cytokine levels, especially IP-10, were significantly high in Usp18-knockout mice. In sum, we suggest that, although type I IFN is known to restrict osteoclast differentiation, the exaggerated activation of the type I IFN response in Usp18- knockout mice causes an osteopenia phenotype in mice. The Journal of Immunology, 2016, 196: 000–000.

aintenance of bone mass is mediated by the comple- the RANK ligand (RANKL) on osteoblasts recruits the TNFR- http://www.jimmunol.org/ mentary processes of osteoclastic resorption and os- associated factor family, including TNFR-associated factor M teoblastic formation within local bone-remodeling sites 6, leading to activation of inhibitor of NF-kB kinase and JNK (12– (1–3). A loss of balance between osteoclastic bone resorption and 14). Consequently, transcription factors, such as NF-kB and AP-1, osteoblastic bone formation is often the cause of bone disease; for are activated (15–17), in addition to a key example, tipping the balance in favor of results in a NFATc1, which is for the expression of the multiple genes re- pathological bone resorption that leads to diseases such as auto- sponsible for osteoclastic functioning (16, 18, 19). In addition, the immune arthritis and postmenopausal osteoporosis (4–6). Osteo- RANK–RANKL interaction triggers –cell fusion and osteoclast

clasts are multinucleated cells (MNCs) generated from / differentiation. The multinucleated osteoclasts attach to the bone by guest on October 2, 2021 precursor cells through the stepwise differentiation surface using avb3 integrin, and the activated osteoclasts secrete processes that involve the regulation of and transcription protons and lytic , such as cathepsin K and tartrate-resistant factors. Bone marrow precursors are matured to preosteoclasts by acid phosphatase (TRAP), to degrade the organic bone matrix (12). k M-CSF, resulting in the expression of receptor activator of NF- B Although the RANK–RANKL interaction is crucial for osteo- (RANK) (7–11). The interaction of RANK on preosteoclasts with clastogenesis, the interaction induces another signal that nega- tively regulates osteoclast activation. It was shown that IFN-b2/2 and IFNAR12/2 mice exhibited osteopenia with enhanced osteo- *Department of Biological Sciences, Sookmyung Women’s University, Seoul 140- clastogenesis, indicating a negative effect of IFN signaling on 742, South Korea; †Department of Biological Sciences, Creative Research Initiative Center for Chromatin Dynamics, Seoul National University, Seoul 151-742, South osteoclast differentiation (20). The induction of c-Fos upon the Korea; ‡Department of Cell and Developmental Biology, Brain Korea 21 Program, RANK–RANKL interaction mediates the expression of the IFN-b Dental Research Institute, Seoul National University, Seoul 110-749, South Korea; b xDepartment of Pathology, Moores Cancer Center, University of California San in osteoclast precursor cells; interestingly, IFN- , in turn, Diego, La Jolla, CA 92093; and {Research Center for Cell Fate Control, College inhibits c-Fos, forming a type of autoregulatory circuit (20). of Pharmacy, Sookmyung Women’s University, Seoul 140-742, South Korea IFN-stimulated gene (ISG)15 is an ubiquitin-like protein whose 1H.Y.Y. and C.P. contributed equally to this work. expression and conjugation to target (ISGylation) increase Received for publication July 2, 2015. Accepted for publication February 25, 2016. greatly upon stimulation by type I IFN (21). Previous studies showed This work was supported by Grants NRF-2013R1A2A2A01067617 (to K.I.K.), that IFN-stimulated ISGylation exhibits antiviral activities against 2011-0030074 (to R.J.), and NRF-2014R1A2A1A10050406 (to H.-H.K.) from the several , including influenza A and B, lymphocytic chorio- National Research Foundation of Korea (funded by the Korean government). meningitis , and Sindbis virus (22, 23). USP18 (also known as Address correspondence and reprint requests to Dr. Keun Il Kim, Department of Biological Sciences, Sookmyung Women’s University, Cheongpa-ro 47-gil 100, UBP43) was originally identified as a deconjugating enzyme for Yongsan-gu, Seoul 140-742, South Korea. E-mail address: [email protected] ISGylation (24) and functions as a negative regulator of type I IFN The online version of this article contains supplemental material. signaling, independent of its enzyme activity (25). In the absence of Abbreviations used in this article: BMM, bone marrow–derived macrophage; 3D, USP18, cells exhibit an enhanced and prolonged STAT1 phosphor- three-dimensional; ISG, IFN-stimulated gene; microCT, microcomputed tomogra- ylation and an elevated expression of ISGs in response to IFN-a/b phy; MNC, multinucleated cell; RANK, receptor activator of NF-kB; RANKL, RANK ligand; shRNA, short hairpin RNA; siRNA, small interfering RNA; SOCS, (25). Although hypersensitivity to type I IFN was consistent in all suppressor of cytokine signaling; sRANKL, soluble RANKL; TRAP, tartrate- genetic backgrounds, phenotypes of Usp18-knockout mice were influ- resistant acid phosphatase. enced by genetic background; mice with mixed genetic background Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 of C57/B6 and 129 exhibited a decreased lifespan with growth

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501496 2 ENHANCED OSTEOCLASTOGENESIS IN Usp18-DEFICIENT MICE retardation primarily due to brain abnormalities (26). Although H&E and TRAP staining Usp18-knockout mice were embryonic lethal at embryonic day Femurs were isolated from 7-wk-old littermates of Usp18+/+ and Usp182/2 15.5 on the C57/B6 background, a majority of homozygous mice and fixed in 4% paraformaldehyde, followed by decalcification in knockout mice on the FVB background exhibited normal growth 14% EDTA for 2 wk. After decalcification, femurs were processed for and lifespan (21). Mice lacking Usp18 are more resistant to viral paraffin embedding and sectioned to 5 mm thickness. For H&E staining, and bacterial infections as a result of the elevated response to type rehydrated slides were stained with hematoxylin followed by eosin (Sigma-Aldrich). For TRAP staining, rehydrated slides were stained with I IFN (27, 28), and bone marrow cells from Usp18-deficient mice TRAP staining solution (1 mg Fast Red, 0.1 M sodium acetate, 50 mM are sensitive to type I IFN–induced (29). Analysis of the sodium tartrate, and 10 mg Naphthol AS-MX in distilled water) for 30 min underlying mechanism revealed that the negative regulation of at 37˚C. TRAP-stained slides were stained with hematoxylin (Sigma- type I IFN signaling is independent of its deISGylating activity Aldrich). The stained slides were observed using an IX71 microscope (Olympus). (30); instead, USP18 interacts directly with IFNAR2, a specific subunit of the type I IFNR, and excludes the association of JAK RT-PCR and quantitative real-time RT-PCR with IFNAR2. Because USP18 is one of the ISGs (25), it is be- Total RNA was isolated from 1 3 106 cells using TRIzol reagent (Life lieved that USP18 is a negative-feedback inhibitor specific to type Technologies), and 2.5 mg the total RNA was used to synthesize cDNA. A I IFN signaling. The knock-in mouse model expressing the en- reverse-transcription reaction was performed using a First-Strand cDNA zymatically inactive Usp18 further validated its function as a Synthesis Kit (Fermentas), according to the manufacturer’s instructions. negative regulator of type I IFN, independent of its activity (31). Sequences of the primers used for PCR are listed in Supplemental Table I. Interestingly, these mice were not hypersensitive to type I IFN; PCR was performed under the following conditions: after an initial ac- tivation at 95˚C for 5 min, 30 cycles of denaturation at 95˚C for 15 s, however, they showed increased resistance against virus

annealing at 58˚C for 30 s, and extension at 72˚C for 30 s were done. PCR Downloaded from and influenza B virus infections with increased cellular ISGylation products were electrophoresed on 1% agarose gel, followed by ethidium level. Other negative regulators of JAK/STAT signaling are protein bromide staining. For quantitative real-time RT-PCR, PCR amplification was inhibitor of activated STAT and suppressor of cytokine signaling performed with 23 Power SYBR Green PCR Master Mix with 1 pmol primers, 125 ng cDNA, and nuclease-free water, according to the manu- (SOCS). Intriguingly, the downregulation of protein inhibitor of facturer’s protocol (Applied Biosystems). PCR conditions included an initial activated STAT 3 significantly enhanced RANK-mediated osteo- activation at 95˚C for 15 min, denaturation at 95˚C for 15 s, annealing at clastogenesis in RAW264.7 cells (32). The deletion of SOCS3 in 60˚C for 30 s, and extension at 72˚C for 30 s. PCR reaction was repeated for mice (Socs32/Dvav) leads to exacerbated bone destruction, reduced 40 cycles. For relative quantification, the expressions of target genes were http://www.jimmunol.org/ b basal trabecular bone volume, and an enhanced number of oste- normalized to the expression of -actin, cyclophilin, or GAPDH. oclasts (33). Knockdown of Usp18 In the course of preparing bone marrow cells, we found that Bone marrow cells from the femurs of 6-wk-old ICR mice were differ- femurs from Usp18-deficient mice are more fragile than those entiated to BMMs by treatment with M-CSF (30 ng/ml) for 3 d. BMMs were from wild-type littermates; a morphological analysis of Usp18- transfected with 30 nM control small interfering RNA (siRNA) or Usp18- deficient femurs showed osteopenia phonotypes. Based on this specific siRNA using HiPerFect reagent (QIAGEN). One day later, cells finding, we examined the effect of a USP18 deficiency on RANKL- were treated with sRANKL (200 ng/ml) and M-CSF (30 ng/ml) and cul- mediated osteoclastogenesis. tured for 5 d until used for analyses. Control siRNA and Usp18-specific siRNA were purchased from Santa Cruz Biotechnology (sc-37007 for by guest on October 2, 2021 control and sc-60866 for Usp18). For the stable knockdown of Usp18, Materials and Methods RAW 264.7 cells were transfected with a plasmid construct containing Mice and cell culture control short hairpin RNA (shRNA) or Usp18-specific shRNA (34). Single colonies were isolated by culturing cells in a 96-well plate and tested for FVB.129-Usp18tm1Dzh mice were described previously (28). A majority of the knockdown of Usp18. Usp18-knockout mice on FVB and 129 mixed background showed normal growth, and ∼10% of knockout mice showed minor growth retardation. We In vitro bone-resorption assay used age- and size-matched mice for the analyses. All mouse experiments were 2 2 Bone marrow cells from the femurs of Usp18+/+ and Usp18 / mice were performed in accordance with the guidelines of the Institutional Animal Care differentiated to BMMs, as described above. BMMs were then seeded and and Use Committee at Sookmyung Women’s University. Mice were main- tained in a specific pathogen–free environment and used at 7 wk old. Bone cultured on dentine discs (Immunodiagnostic Systems) for 9 d in the marrow cells from the femurs of Usp18+/+ and Usp182/2 mice were harvested presence of 30 ng/ml M-CSF and 200 ng/ml RANKL. Cells were removed andculturedina-MEM (Welgene) supplemented with 10% FBS and 30 ng/ml by sonication, and resorption pits in the dentine discs were analyzed using M-CSF (PeproTech) for 1 d. Suspended cells were harvested and further confocal microscopy and ImageJ software. cultured with M-CSF (30 ng/ml) in a-MEM containing 10% FBS. After 3 d, Cytokine analysis differentiated bone marrow–derived macrophages (BMMs) were treated with 200 ng/ml recombinant soluble RANKL (sRANKL; PeproTech) and 30 ng/ml Sera were isolated from the blood of 7-wk-old littermates of Usp18+/+ and M-CSF for osteoclast differentiation. Usp182/2 mice. Cytokines in the serum samples were measured with a Mouse Magnetic Luminex Screening Assay (R&D Systems). Abs Statistical analysis The following Abs were used in this study: 3, cleaved caspase 3, phospho-ERK, ERK, phospho-JNK, JNK, phospho-p38, p38, phospho- Statistical differences between the test and control samples were determined Stat1, Stat1, and b-actin (all from ); c-Fos and NFATc1 nonparametrically by the Mann–Whitney U test using GraphPad Prism 5 (Santa Cruz Biotechnology); and a-tubulin (Sigma-Aldrich). Ab against software (GraphPad). Data are presented as mean 6 SD. USP18 was described previously (30). Microcomputed tomography Results Osteopenia bone phenotype of Usp18-deficient mice Femurs isolated from 7-wk-old littermates of Usp18+/+ and Usp182/2 mice were fixed in 4% paraformaldehyde at 4˚C and subjected to micro- Because we observed that the bones of Usp18-deficient mice were computed tomography (microCT) analysis. Scanned femurs were analyzed more fragile than those of the wild-type littermates, we suspected using a SkyScan 1172 scanner (SkyScan). One millimeter–thick images that the Usp18 deficiency caused the osteopenia phenotype. To ex- obtained from distal femoral area, starting from 1 mm below the growth +/+ 2/2 plate, were analyzed using the CT-Analyser program (SkyScan) to estimate amine this, we compared the femurs from Usp18 and Usp18 bone volume and bone parameters. A three-dimensional (3D) model of male littermates using microCT analysis. Usp18-deficient femurs images was constructed using CTVol software (SkyScan). showed a significantly reduced trabecular bone mass compared with The Journal of Immunology 3 the femurs of wild-type littermates (Fig. 1A). In addition, several bone tivation. The intensity of TRAP staining was increased in femurs parameters, such as bone volume/total volume, trabecular thickness, of Usp182/2 mice compared with those of Usp18+/+ mice and the numbers of trabeculae were decreased in Usp18-deficient (Fig. 1D). When TRAP/hematoxylin-stained sections were ana- femurs compared with wild-type femurs (Fig. 1B). The distances lyzed using OsteoMeasure histomorphometry software (Osteo- between trabeculae tended to be greater in the femurs of Usp182/2 Metrics), osteoclast activation was clearly enhanced in Usp18- mice compared with those of wild-type mice (Fig. 1B). The 3D deficient mice compared with wild-type controls. Several pa- models of femur images generated from the microCT images clearly rameters reflecting osteoclast activation, such as eroded surface revealed a reduced trabecular bone mass in Usp18-deficient femurs per bone surface, osteoclast surface per bone surface, number (Fig. 1C). The femurs of female Usp18-deficient mice also showed a of osteoclasts per bone perimeter, and number of osteoclasts significantly reduced trabecular bone mass (Supplemental Fig. 1A). per total area, were increased $2-fold in Usp18-deficient bones Consistently, microCT analyses revealed reduced cortical bone (Fig. 1E). Taken together, these results indicate that a Usp18 mass, shown by representative images, and reduced cortical volume deficiency in mice caused osteopenia with an increase in oste- and thickness in Usp18-deficient femurs compared with wild-type oclastic bone resorption. femurs from male and female mice (Fig. 1D, 1E, Supplemental Fig. 1B). Therefore, it is clear that Usp18 deficiency caused osteopenia Increased RANKL-mediated osteoclast differentiation of in male and female mice. Usp18-deficient bone marrow cells To examine whether the osteopenia phenotype of Usp18- Because Usp18-deficient bones exhibited osteopenia, we questioned deficient bones was the result of increased bone resorption, whether Usp18 is directly involved in osteoclastogenesis. We first we analyzed femurs from Usp18+/+ and Usp182/2 mice using evaluated the expression of Usp18 over the course of osteoclasto- TRAP-activity staining to measure the extent of osteoclast ac- genesis. Bone marrow cells from Usp18+/+ and Usp182/2 mice Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 1. Osteopenia phenotype of Usp18-deficient mice. (A) MicroCT analyses of coronal and axial sections of the distal femoral area from rep- resentative 7-wk-old littermates of Usp18+/+ and Usp182/2 mice. (B) Bone parameters analyzed from microCT images of femurs using the CTanalyzer program. BV/TV, percent of bone volume per total volume; Tb.Th, trabecular thickness; Tb.N, number of trabeculae; Tb.Sp, trabecular separation. Data are mean 6 SD (n = 4 each). +/+ and 2/2 represent femurs of Usp18+/+ and Usp182/2 mice, respectively. (C) A 3D model of images produced by CTVol software. (D) 3D reconstructed images for cortical bone of the femoral midshaft. (E) Evaluation of cortical bone density by measurement of cortical volume (Ct.V.) and average cortical thickness (Ct.Th). Data are mean 6 SD (n = 3 for each). (F) TRAP/hematoxylin and H&E staining of femurs from Usp18+/+ and Usp182/2 mice (original magnification 340). (G) Parameters for osteoclastic bone resorption: eroded surface per bone surface (ES/BS), osteoclast surface per bone surface (Oc.S/BS), osteoclast number per bone perimeter (N.Oc/B.Pm), and osteoclast number per total area (N.Oc/T.Ar) were measured using OsteoMeasure histomorphometry software from a representative slide. Data are mean 6 SD (n = 3 for each). +/+ and 2/2 represent femurs of Usp18+/+ and Usp182/2 mice, respectively. *p , 0.05 versus Usp18+/+. AU, arbitrary unit; n.s, not significant. 4 ENHANCED OSTEOCLASTOGENESIS IN Usp18-DEFICIENT MICE Downloaded from

FIGURE 2. Increased RANKL-mediated osteoclast differentiation of USP18-deficient bone marrow cells. (A) Bone marrow cells from femurs of Usp18+/+ and Usp182/2 mice were differentiated to BMMs by treatment with M-CSF (30 ng/ml) for 3 d. BMMs were then treated with sRANKL (150 ng/ml) and http://www.jimmunol.org/ M-CSF (30 ng/ml), and total were isolated from the cells at various times after treatment with sRANKL (0, 3, 6, 24, and 48 h). The expression of Usp18 mRNA was measured by quantitative RT-PCR. Data are mean 6 SD (n = 3). *p , 0.05. (B) The protein levels of Usp18 from the cells described in (A) were detected by immunoblotting with anti-USP18 Ab. Equal protein loading was confirmed with b-actin blotting. (C) BMMs were subjected to osteoclast-dif- ferentiation assays. After differentiation for 5 d, the cells were stained for TRAP activity and incubated with TRIC-conjugated phalloidin for the visualization of F-actin ring (original magnification 3100). (D) TRAP+ MNCs containing more than three nuclei were counted. Data are mean 6 SD (n = 3). *p , 0.05 versus Usp18+/+. were treated with M-CSF to generate macrophages. BMMs were examined RANKL-mediated osteoclast differentiation using the treated with RANKL, and the expression of Usp18 mRNA and pro- same in vitro system, whereby the differentiation was visualized by by guest on October 2, 2021 tein were measured over time. The basal expression of Usp18 was staining for TRAP activity and F-actin ring (Fig. 2C, 2D). When almost undetectable without RANKL treatment; however, mRNA TRAP+ MNCs containing three or more nuclei were counted, 5–6- and protein were induced 24 h after treatment with RANKL in fold more TRAP+ MNCs were detected from osteoclasts differen- wild-type BMMs (Fig. 2A, 2B), indicating that Usp18 is indeed tiated from Usp18-deficient BMMs compared with those from wild- expressed during RANKL-mediated osteoclastogenesis. We then type controls (Fig. 2D); furthermore, we obtained consistent results

FIGURE 3. Enhanced RANKL-induced signaling in Usp18-deficient osteoclast precursors. (A) Bone mar- row cells from femurs of Usp18+/+ and Usp182/2 mice were differentiated into BMMs by treatment with M-CSF for 3 d. BMMs were then treated with sRANKL and M-CSF and harvested at the indicated time points. Cell extracts were subjected to immunoblotting with Abs against phospho-ERK, ERK, phospho-p38, p38, phospho-JNK, and JNK. Equal protein loading was confirmed by b-actin blotting. (B)BMMsfromthe Usp18+/+ and Usp182/2 mice were prepared as indi- cated in (A). Cells were treated with sRANKL and M-CSF, harvested at the indicated time points, and subjected to immunoblotting with anti–c-Fos, anti- NFATc1, anti-iNOS, and anti-Usp18 Abs. Equal protein loading was confirmed by b-actin blotting. (C)BMMs from Usp18+/+ and Usp182/2 mice were stimulated as in (B). Total RNA was isolated from cells, and the ex- pression of genes was measured by quantitative RT- PCR. Data are mean 6 SD (n =3).*p , 0.05. The Journal of Immunology 5 with BMMs from female mice in the in vitro osteoclastogenesis cells lacking Usp18 to be differentiated to osteoclasts by RANKL, assay. The numbers of differentiated osteoclasts (TRAP+ MNCs) rather than an increase in osteoclast cell number, might be closely were greatly increased in BMMs that had been prepared from linked to the osteopenia phenotype of the mice. Usp182/2 female mice (Supplemental Fig. 2A). In our previous studies (27), as well as in the current experiment, we did not notice Enhanced RANKL signaling in Usp18-deficient osteoclast any difference in cell proliferation between BMMs derived from precursors wild-type and Usp18-deficient mice (data not shown). We also To examine whether a Usp18 deficiency enhances osteoclast checked for possible differences in cell death rate by measuring differentiation via RANKL signaling, we analyzed the RANKL- the conversion of caspase 3 to its shorter activated form. When induced activation of signaling cascades and gene expression in BMMs were treated with sRANKL, Usp18-deficient BMMs exhibited osteoclast precursors derived from the bone marrow of Usp18- slightly higher levels of caspase 3 and the activated form of cleaved deficient and wild-type mice by treating bone marrow cells with caspase 3 compared with wild-type BMMs (Supplemental Fig. 2B). M-CSF for 3 d prior to the RANKL treatment. The administration Thus, the osteopenia phenotype observed in Usp18-knockout mice is of RANKL to BMMs induced a rapid of ERK, not due to increased osteoclast cell number as a result of altered p38, and JNK in wild-type and Usp18-deficient BMMs; further- proliferation or lifespan of Usp18-deficient BMMs. Taken together, more, the patterns and intensities of the phosphorylation for all of these data indicate that an increased tendency of the bone marrow the kinases were similar in both genotypes (Fig. 3A). The protein Downloaded from http://www.jimmunol.org/

FIGURE 4. Increase in RANKL-mediated osteo- clastogenesis by Usp18 knockdown. (A) Bone marrow cells from femurs of ICR mice were differentiated to BMMs by treatment with M-CSF for 3 d. BMMs were transfected with control siRNA (siC) or Usp18-specific siRNA (siUsp18). One day later, cells were treated with sRANKL and M-CSF and differentiated for 5 d before by guest on October 2, 2021 counting of TRAP+ MNCs. Data are mean 6 SD (n = 3). *p , 0.05 versus siC. Original magnification 3200. (B) BMMs prepared from ICR mice, as described as in (A), were treated with sRANKL and M-CSF, and the expression of genes was measured by quantitative RT-PCR. (C) Stable RAW264.7 cells expressing control shRNA (shC) or Usp18-specific shRNA (shUsp18) were cultured in the media without serum for 4 h and then treated with sRANKL and M-CSF. The differen- tiated osteoclasts were stained for TRAP after 2 d of treatment and then incubated with TRIC-conjugated phalloidin for the visualization of F-actin ring (left panels). Original magnification 3200. TRAP+ MNCs containing more than three nuclei were counted (right panel). Data are mean 6 SD (n = 3). *p , 0.05 versus shC. (D) Control and Usp18-knockdown RAW264.7 cells were treated with sRANKL and M-CSF, as indi- cated in (C). Total RNA was isolated from the cells at the indicated time points, and the expression of genes was measured by quantitative RT-PCR. Data are mean 6 SD (n =3).*p , 0.05. 6 ENHANCED OSTEOCLASTOGENESIS IN Usp18-DEFICIENT MICE levels of c-Fos, which were increased at early time points (3–6 h) pits were analyzed. Consistent with the increased osteoclasto- after RANKL treatment, showed no significant difference between genesis, dentine discs with Usp18-deficient BMMs showed greatly wild-type and Usp18-deficient BMMs. However, the levels of increased resorbed areas (Fig. 5A). Image analyses revealed NFATc1, which are increased later (24–48 h) after RANKL ad- ∼2- and 1.8-fold increases in resorption depth and resorption area, ministration, were significantly elevated in Usp18-deficient respectively, from the dentine discs cultured with Usp18-deficient BMMs (Fig. 3B). In addition, the expression of marker genes BMMs compared with those cultured with wild-type BMMs for osteoclast differentiation, such as cathepsin K and TRAP, was (Fig. 5B). These results indicated that the osteopenia phenotype of clearly increased in Usp18-deficient BMMs compared with Usp18-deficient bones resulted from increased osteoclastogenesis BMMs from wild-type littermates (Fig. 3C). Interestingly, the and osteoclastic bone resorption. expression of iNOS, a well-known target gene of type I IFN, was Enhanced Stat1 activation and elevated cytokine expression in dramatically increased in Usp18-deficient BMMs (Fig. 3B), in- Usp18-deficient BMMs by RANKL dicating a hyperresponsiveness to type I IFN (most probably IFN-b) that is generated during RANKL-mediated osteoclast Because Usp18 is known to negatively regulate type I IFN sig- differentiation. The elevated expression of NFATc1, cathepsin K, naling, and Usp18-deficient cells are hypersensitive to IFN-a/b and TRAP was also clear in separate experiments in which BMMs (25, 30), we questioned whether increased RANKL-mediated from female wild-type and Usp18-deficient mice were treated with osteoclast differentiation in Usp18-deficient BMMs might be re- RANKL (Supplemental Fig. 2C). Taken together, these results lated to hypersensitivity to type I IFN. We examined RANKL- suggest that a Usp18 deficiency caused enhanced activation of induced activation of JAK/STAT signaling in osteoclast precursors RANKL signaling at late time points in osteoclast precursors, from Usp18-deficient mice and wild-type mice. When we ana- resulting in increased differentiation of Usp18-deficient BMMs to lyzed the tyrosine phosphorylation of Stat1 as an indicator of Downloaded from TRAP+ MNCs. the activation of JAK/STAT signaling during RANKL-mediated osteoclastogenesis, we detected a dramatic increase in phosphor- Depletion of Usp18 increased RANKL-mediated ylated Stat1 in Usp18-deficient BMMs, whereas it was very weak osteoclastogenesis in wild-type BMMs (Fig. 6A), indicating a hyperactivation of Although we obtained evidence that elevated RANKL-mediated JAK/STAT signaling in Usp18-deficient BMMs. Next, we mea- signaling and transcription might cause enhanced osteoclast dif- sured the expression of several cytokine genes (IP-10, IL-6, IL-15, http://www.jimmunol.org/ ferentiation of osteoclast precursor cells from Usp18-deficient TNF-a, and IFN-b) that are known to be associated with osteo- mice, we wanted to confirm whether this phenomenon is consis- clastogenesis (35). Among them, it was shown that the expression tent in other systems. We adapted two independent systems to of IP-10, IL-6 and IL-10 was highly elevated in Usp18-deficient deplete Usp18 expression, by the transient knockdown of Usp18 BMMs upon IFN-b treatment (36). Upon RANKL administration, in BMMs derived from femurs of ICR mice and by stable knock- the expression of IP-10, IL-6, and IL-15 was increased in Usp18- down of Usp18 in RAW264.7 mouse macrophage cells, and mea- deficient BMMs, and the expression levels were significantly sured RANKL-mediated osteoclast differentiation and expression of RANKL target genes. When we transfected control or Usp18 by guest on October 2, 2021 siRNA into BMMs from ICR mice and induced RANKL-mediated osteoclastogenesis, we detected a $2-fold increase in TRAP+ MNCs from the osteoclasts that were differentiated from Usp18- knockdown BMMs compared with the control (Fig. 4A); ac- cordingly, mRNA expression of NFATc1, TRAP, and cathepsin K was increased in osteoclasts from Usp18-knockdown BMMs (Fig. 4B). We achieved consistent results with the stable knock- down of Usp18 in RAW264.7 cells. We generated RAW264.7 cells in which control shRNA or Usp18 shRNA was stably expressed and validated the efficient removal of Usp18 mRNA (Supplemental Fig. 3); these cells were then used for a RANKL- mediated osteoclastogenesis assay. Compared with control cells, Usp18-depleted RAW264.7 cells showed a greatly increased num- ber of differentiated TRAP+ MNCs (Fig. 4C). Staining against F-actin ring showed a similar pattern to TRAP activity staining. mRNAs for TRAP, cathepsin K, and NFATc1 were also increased during RANKL-mediated osteoclast differentiation of Usp18- knockdown RAW264.7 cells (Fig. 4D). Together with the data from Usp18-knockout mice and cells, these results strongly sug- gest a causal relationship between Usp18 deficiency and increased RANKL-mediated osteoclastogenesis. Elevated bone-resorption ability of Usp18-deficient BMMs Because we detected increased osteoclastogenesis of bone mar- FIGURE 5. Increased bone-resorption ability of Usp18-deficient BMMs. (A) Representative image of five independent experiments. BMMs row cells in the absence of Usp18, we questioned whether this 2 2 generated from Usp18+/+ and Usp18 / mice were seeded on dentine mechanism is linked to the reduced bone mass in Usp18-deficient discs and cultured in the presence of 30 ng/ml M-CSF and 200 ng/ml mice, which might be caused by osteoclastic bone resorption. To RANKL for 9 d. After removal of the cells by sonication, resorption pits evaluate the outcome of increased osteoclastogenesis, BMMs were assessed by confocal microscopy. (B) Resorption depth and resorp- from wild-type and Usp18-deficient mice were cultured on dentine tion area were measured using ImageJ software. Data are mean 6 SD (n = discs in the presence of M-CSF and RANKL for 9 d, and resorption 5). *p , 0.05 versus Usp18+/+. The Journal of Immunology 7 higher in Usp18-deficient BMMs compared with wild-type BMMs matched wild-type and Usp18-deficient mice. TNF-a and IL-1b (Fig. 6B). TNF-a was not inducible by RANKL; however, mRNA were not detectable in the mice of either genotype. The median levels were significantly higher in Usp18-deficient BMMs com- serum levels of IL-6 and M-CSF in Usp18-deficient mice were pared with wild-type BMMs. Interestingly, the expression of ∼1.6- and 1.8-fold higher, respectively, than those of wild-type IFN-b was also elevated in Usp18-deficient BMMs upon RANKL controls (Fig. 6C). Of special significance, IP-10 levels of Usp18- treatment compared with wild-type BMMs. Thus, the elevated and deficient mice were 7-fold higher than those of wild-type controls prolonged activation of Stat1 in Usp18-deficient BMMs upon (Fig. 6C). Based on the sum of these results, elevated cytokine RANKL treatment (Fig. 6A) might be a combinatorial effect from levels in the serum might be an additional cause of the osteopenia the intrinsic hypersensitivity of Usp18-deficient cells and the in- phenotype of Usp18-knockout mice. creased autocrine/paracrine secretion of IFN-b. These findings suggest that several cytokine genes were considerably overex- Discussion pressed during RANKL-mediated osteoclastogenesis in Usp18- Osteoporosis is a pathological loss of bone matrix resulting from a deficient BMMs and that an abnormal increase in cytokines predominance of osteoclastic bone resorption over osteoblastic might be responsible for triggering enhanced osteoclast differen- bone formation (1). We observed a severe osteopenia phenotype in tiation. male and female Usp18-knockout mice and investigated its pos- Finally, we measured the amounts of five cytokines (TNF-a, IL- sible cause. We identified the following possible sources. Serum 1b, IP-10, IL-6, and M-CSF) in the blood sera collected from age- levels of inflammatory cytokines, such as IP-10, IL-6, and M-CSF, Downloaded from http://www.jimmunol.org/

FIGURE 6. Enhanced activation of JAK/STAT signaling during RANKL-mediated osteoclasto- genesis in Usp18-deficient BMMs and increased by guest on October 2, 2021 cytokine levels in the serum of Usp18-deficient mice. (A) Enhanced activation of JAK/STAT sig- naling in Usp18-deficient BMMs upon RANKL treatment. Bone marrow cells from femurs of Usp18+/+ and Usp182/2 mice were differentiated to BMMs by treatment with M-CSF for 3 d. BMMs were then treated with sRANKL and M-CSF, har- vested at the indicated time points, and subjected to immunoblotting with Abs against phospho-STAT1 (pSTAT1), STAT1, and Usp18. Equal protein loading was confirmed by a-tubulin blotting. (B)BMMs from Usp18+/+ and Usp182/2 mice were stimulated as in (A). Total RNA (2.5 mg)wasusedtomeasure the expression of genes by quantitative RT-PCR. Data are mean 6 SD (n = 3). *p , 0.05. (C)Serum samples from Usp18+/+ and Usp182/2 mice were measured by ELISA. Data are mean 6 SD (n =4). *p , 0.05 versus Usp18+/+. n.s, not significant. 8 ENHANCED OSTEOCLASTOGENESIS IN Usp18-DEFICIENT MICE were relatively higher in Usp18-deficient mice than in wild-type recruited into the synovium (39). In human circulating mice. Especially, median IP-10 levels were 7-fold higher in serving as early progenitors of osteoclasts, STAT1 and IP-10 the serum of Usp18-deficient mice. Most importantly, RANKL- were identified as candidate genes whose expression is responsible induced osteoclast differentiation (MNC formation) was mark- for the increase in the differentiation of peak bone mass at the edly increased in Usp18-deficient osteoclast precursor cells. In monocyte stage (39). In addition to increased expression during addition, bone-resorption activity was clearly higher in Usp18- RANKL-mediated osteoclastogenesis, serum levels of IP-10 were deficient BMMs compared with wild-type BMMs. The combina- abnormally high (over 7-fold) in Usp18-knockout mice compared torial effects of these causes could generate a severe osteopenia with normal littermates. Serum levels of M-CSF and IL-6 were phenotype in Usp18-knockout mice. Although we did not detect a also elevated in Usp18-knockout mice, although the increase critical difference in the proliferation rate of wild-type and Usp18- was less significant (,2-fold). Therefore, elevated levels of serum deficient BMMs in vitro, we cannot completely rule out the pos- cytokines, especially IP-10, may also contribute to the osteopenia sibility that an increase in BMM number might contribute, at least phenotype of Usp18-knockout mice. in part, to the osteopenia phenotype in vivo because Usp18- In summary, our analysis of the osteopenia phenotype of deficient mice showed a tendency toward increased levels of Usp18-knockout mice revealed some unexpected results. Although M-CSF compared with wild-type controls. IFN-b plays a negative-feedback regulatory role in RANKL- The most interesting observation from this study was enhanced induced osteoclast differentiation, the results show that an un- RANKL-mediated osteoclastogenesis in Usp18-knockout BMMs. controlled activation of IFN signaling in Usp18-knockout mice In our previous studies, Usp18 was identified as a negative regu- accelerated osteoclast differentiation with a high induction of lator of type I IFN signaling (25). The representative consequence osteoclastogenic cytokines. of Usp18 deficiency was an elevated and prolonged activation of Downloaded from JAK/STAT signaling upon IFN-a/b administration, which resulted Disclosures in a tremendous overexpression of ISGs in many of the cell types The authors have no financial conflicts of interest. tested, including BMMs (25). When BMMs were treated with the same dose of type I IFN, Usp18-deficient BMMs showed pro- longed and hyperphosphorylation of Stat1 (27). 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