Interleukin-17 inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing nuclear factor-κB p65 phosphorylation and nuclear factor of activated T cells c1 expression

Interleukin-17 inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing nuclear factor-κB p65 phosphorylation and nuclear factor of activated T cells c1 expression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Interleukin-17 inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing nuclear factor-κB p65 phosphorylation and nuclear factor of activated T cells c1 expression Shinzaburo Kobuchi, Hiroshi Inoue, Nagako Sougawa, Seiji Goda, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8121955/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Bone is constantly being regenerated while maintaining dynamic homeostasis through a delicate balance between bone destruction by osteoclasts and bone formation by osteoblasts, a process known as bone remodeling. Bone resorption on the compression side is closely related to the differentiation and activation of osteoclasts. Interleukin-17 (IL-17) is a pro-inflammatory cytokine secreted by Th17 cells and other cells. IL-17 has been shown to indirectly induce osteoclast differentiation by increasing the expression of receptor activator of NFκB ligand (RANKL) in osteoblasts. However, little has been reported about the effect of IL-17 directly acting on osteoclast precursor cells during osteoclast differentiation. This study aimed to investigate the effect of IL-17 on RANKL-induced osteoclast differentiation in RAW264.7 cells. Results Osteoclast differentiation and formation were assessed by measuring tartrate-resistant acid-phosphatase (TRAP) activity. RANKL stimulation enhanced TRAP activity in RAW264.7 cells, but co-stimulation with IL-17 attenuated it. RANKL stimulation activated the canonical NF-κB pathway, leading to increased phosphorylation of NF-κB p65 and its subsequent nuclear translocation, but IL-17 suppressed this increased phosphorylation and nuclear translocation of NF-κB p65. c-Fos and nuclear factor of activated T cells (NFATc1), nuclear transcription factors that play important roles in regulating the expression of many osteoclast-specific genes involved in osteoclast differentiation, were induced by RANKL stimulation. IL-17 reduced RANKL-stimulated c-Fos and NFATc1 expression. Conclusions IL-17 acts directly on RAW264.7 cells, inhibiting the RANKL/RANK signal-induced phosphorylation of NF-κB p65 and its subsequent nuclear translocation. This study provides evidence suggesting that the mechanism by which IL-17 suppresses RANKL-induced osteoclast differentiation may involve the suppression of c-Fos and NFATc1 expression, key transcription factors that control osteoclast formation, by inhibiting the activation of NF-κB p65. interleukin-17 osteoclast differentiation nuclear factor-κB p65 nuclear factor of activated T cells 1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Bone is continuously remodeled and tightly regulated to maintain homeostasis through bone formation and resorption by osteoblasts and osteoclasts, respectively [ 1 ]. The balance between osteoblasts and osteoclasts is important for maintaining bone mass, and disruption of this relationship leads to bone diseases such as osteoarthritis, rheumatoid arthritis, and periodontal disease. Osteoclasts are formed by the fusion of monocyte-macrophage osteoclast precursor cells as tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. These TRAP-positive multinucleated cells reorganize the actin cytoskeleton and become multinucleated osteoclasts that adhere to the bone surface and resorb bone [ 2 ]. Osteoclast differentiation is regulated by two essential cytokines, macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) [ 3 ]. M-CSF is important for the survival and proliferation of osteoclast precursor cells, and RANKL is required for inducing the fusion of mononuclear precursor cells and their differentiation into osteoclasts [ 2 ]. The binding of RANKL to receptor activator of NF-κB (RANK) induces the recruitment of adaptor molecules such as tumor necrosis factor receptor-associated factors (TRAFs). Recruitment of TRAF6 to RANK activates downstream signaling pathways, including mitogen-activated protein kinases (MAPKs) (extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38) and NF-κB, which then activate various transcription factors involved in osteoclast differentiation, such as c-Fos, AP-1, and nuclear factor of activated T cells (NFAT)c1 [ 4 ]. These signaling cascades regulate the transcription of target genes for many osteoclast-specific markers, including calcitonin receptor, TRAP, β3 integrin, cathepsin K, matrix metalloproteinase 9 (MMP-9), dendritic cell-specific transmembrane protein (DC-STAMP), calcitonin receptor, carbonic anhydrase II, and ATPase H + transporting V0 subunit d2 (ATP6V0D2), ultimately inducing the formation and activation of mature multinucleated osteoclasts [ 5 ]. Therefore, targeting these signaling pathways may enable control of osteoclast differentiation and activation, which may be an effective therapeutic strategy for bone remodeling-based treatments, such as for rheumatoid arthritis. Interleukin-17 (IL-17) is a pro-inflammatory cytokine produced by Th17 cells, a subset of helper T cells [ 6 ]. Th17 cells are known to induce inflammation by producing other pro-inflammatory cytokines, including IL-21, IL-22, and tumor necrosis factor (TNF)-α, in addition to IL-17 [ 7 ]. IL-17 transmits signals via a heterodimeric receptor complex consisting of IL-17RA and IL-17RC, activating downstream NF-kB pathways, MAPK pathways, and C/EBP pathways to produce pro-inflammatory cytokines and chemokines [ 8 ]. IL-17 has been shown to act indirectly on osteoclast precursor cells by inducing RANKL expression in osteoblasts, thereby inducing osteoclast formation [ 6 , 9 ]. RAW264.7 cells are a monocyte/macrophage-like cell line, and upon stimulation with RANKL, they differentiate into TRAP-positive multinucleated osteoclasts [ 10 ]. We previously confirmed the presence of IL-17RA and IL-17RC in RAW264.7 cells [ 11 , 12 ] and investigated the direct effect of IL-17 on osteoclast differentiation. The results obtained demonstrated that osteoclast differentiation is suppressed by the direct effects of IL-17. However, the details of the mechanism of action on osteoclast differentiation of RAW264.7 cells and the direct effect of IL-17 on osteoclast function have not been fully elucidated. Thus, this study aimed to investigate the effect of IL-17 on RANKL-induced osteoclast differentiation in RAW264.7 cells. We evaluated the effect of IL-17 on osteoclast differentiation by measuring TRAP enzyme activity. Western blotting was used to examine the effect of IL-17 on the intracellular signaling pathway involved in RANKL-induced osteoclast differentiation by examining the phosphorylation of NF-κBp65. Furthermore, we elucidated the involvement of IL-17 in the expression of the transcription factors, c-FOS and NFATc1, master transcription factors for osteoclast differentiation. Methods Cell culture The murine monocyte/macrophage cell line RAW264.7 was obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in 60 cm² culture dishes and maintained at 37°C in a humidified atmosphere containing 5% CO₂. The culture medium used was Dulbecco’s Modified Eagle Medium (D-MEM; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), supplemented with 10% fetal bovine serum (Cosmo Bio Co., Ltd., Tokyo, Japan), 100 µg/mL penicillin-streptomycin solution (Nacalai Tesque, Kyoto, Japan), and 2 mM L-alanyl-L-glutamine solution (Nacalai Tesque). Reagents and antibodies Recombinant RANKL was purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan), and IL-17 was obtained from PeproTech Inc. (Rocky Hill, NJ, USA). Anti-NFATc1 and anti-β-actin antibodies were acquired from Santa Cruz Biotechnology Inc. (Dallas, TX, USA), while anti-c-Fos and anti-NF-κB antibodies were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and goat anti-mouse IgG secondary antibodies were purchased from Jackson ImmunoResearch Inc. (West Grove, PA, USA). The kit for cytoplasmic and nuclear protein fractionation and extraction was obtained from FUJIFILM Wako Pure Chemical Corporation. TRAP activity assay RAW264.7 cells were seeded at a density of 8 × 10³ cells per well in 96-well plates and stimulated for 3 days with IL-17 (10 ng/mL) and RANKL (10 or 30 ng/mL) in D-MEM (FUJIFILM Wako Pure Chemical). On day 6, TRAP activity was assessed to evaluate osteoclast differentiation. Cells were fixed with 10% neutral-buffered formalin for 10 min, followed by fixation with an equal volume of acetone/ethanol (FUJIFILM Wako Pure Chemical). Subsequently, cells were incubated with 50 mM sodium citrate and 25 mM tartaric acid (pH 5.0) (FUJIFILM Wako Pure Chemical). The enzymatic reaction was terminated by adding an equal volume of 0.1 N sodium hydroxide (FUJIFILM Wako Pure Chemical Corporation), and absorbance was measured at 405 nm using a SpectraMax M5 multi-mode microplate reader (Molecular Devices, Sunnyvale, CA, USA). Western blot analysis Protein samples were extracted from RAW264.7 cells and subjected to electrophoresis for 90 min at 190 V, followed by transfer at 340 mV for 120 min. Membranes were blocked with Blocking One (Nacalai Tesque) at room temperature for 60 min. Primary antibodies against phospho-NF-κB p65, NF-κB p65, c-Fos, and NFATc1 were incubated overnight at 4°C. After washing, membranes were incubated with HRP-conjugated secondary antibodies (mouse and rabbit IgG) for 60 min. Protein bands were detected and visualized using the ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA). Statistical analysis Statistical analysis was performed using one-way analysis of variance with GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA). p < 0.05 was considered statistically significant. All experiments were conducted at least three times, and data are presented as mean ± standard deviation. Results Effect of IL-17 on TRAP activity in RANKL-stimulated RAW264.7 cells The effect of IL-17 on RANKL-induced osteoclast differentiation in RAW264.7 cells was examined by measuring TRAP activity. The enzymatic TRAP activity measured at the end of the differentiation process was similar to that of unstimulated controls when RAW264.7 cells were treated with IL-17 alone, but increased upon stimulation with RANKL (Fig. 1 ). The increased TRAP activity due to RANKL stimulation was suppressed by co-stimulation with IL-17 and RANKL (Fig. 1 ). Co-stimulation with RANKL and IL-17 reduced NF-κB p65 phosphorylation We examined whether IL-17 is involved in osteoclast differentiation via the NF-κB signaling pathway by analyzing NF-κB p65 phosphorylation using Western blotting. Phosphorylated NF-κB p65 protein levels were increased by stimulation with RANKL alone (30 ng/mL) compared to unstimulated controls (Fig. 2 a (upper panel), 2b). Co-stimulation with IL-17 and RANKL (30 ng/mL) reduced phosphorylated NF-κB p65 protein levels compared with stimulation with RANKL alone (Fig. 2 a (upper panel), 2b). To confirm that equal amounts of NF-κB p65 were obtained from the lysates, the Western blot membranes were stripped and re-probed with anti-NF-κB p65 antibodies. Equal amounts of NF-κB p65 were detected in the lysates obtained from each sample (Fig. 2 a [lower panel]). To determine whether IL-17 suppresses the RANKL-induced activity of transcription factors, we examined the effects of IL-17 on RANKL-induced nuclear translocation of NF-κBp65. NF-κB p65 translocated to the nucleus in response to RANKL, and IL-17 inhibited the nuclear translocation of NF-κB p65 (Fig. 2 c). Effect of IL-17 on c-Fos expression in RANKL-stimulated RAW264.7 cells c-Fos plays an important role in RANKL-induced NFATc1 expression by forming an AP-1 complex with c-Jun. Therefore, the effect of IL-17 on RANKL-induced c-Fos expression in RAW264.7 cells was examined by Western blot analysis. As shown in Fig. 3 a (upper panel), RANKL stimulation increased the expression of c-Fos in RAW 264.7 cells. Stimulation with RANKL alone increased c-Fos expression levels, whereas co-stimulation with IL-17 and RANKL suppressed c-Fos expression (Fig. 3 a, b). The lower panel of Fig. 3 a indicates that the total amount of β-actin was not affected by various stimuli. Effect of IL-17 on NFATc1 protein expression in RANKL-stimulated RAW264.7 cells Next, we investigated the expression of NFATc, which is essential for osteoclast formation and functions as a master transcriptional regulator of osteoclast differentiation. We confirmed that RANKL stimulation increased NFATc1 expression (Fig. 4 a, b). However, the increase in NFATc1 expression due to RANKL stimulation was suppressed by co-stimulation with IL-17 and RANKL (Fig. 4 a, b). The lower panel of Fig. 4 a indicates that the total amount of β-actin was not affected by various stimuli. Discussion In this study, we investigated the direct effect of IL-17 on RANKL-induced osteoclast differentiation in RAW264.7 cells. The process of osteoclast differentiation requires stimulation by M-CSF and RANKL, which are central regulators of osteoclast differentiation [ 13 ]. Osteoblasts produce M-CSF and RANKL, which are important promoters of osteoclastogenesis [ 2 ]. RAW264.7 cells are a mouse macrophage cell line that can differentiate into osteoclasts by RANKL stimulation alone, without M-CSF stimulation [ 14 ]. Osteoclast precursor cells undergo a multi-step process of proliferation, TRAP expression, and cell fusion upon stimulation with RANKL and M-CSF, and then degrade into osteoclasts [ 5 ]. TRAP-positive multinucleated cells can induce bone resorption [ 15 ]. Based on this theory, in this study, we evaluated osteoclast formation potential by assessing TRAP activity. We demonstrated that IL-17 stimulation, through co-stimulation with RANKL, could directly suppress RANKL-induced TRAP activity in RAW264.7 cells. IL-17 is a pro-inflammatory cytokine that produces various inflammatory cytokines and chemokines, such as IL-1β, IL-6, IL-8, GM-CSF, and TNF-α, in various types of cells, including Th17 and macrophages. IL-17 is known to indirectly promote osteoclast differentiation by inducing RANKL expression in osteoblasts in co-cultures of osteoclast precursors and osteoblasts [ 9 ]. However, Kitami et al. showed that the linear effect of IL-17 on osteoclast formation in osteoclast precursor cells is concentration-dependent, and that high concentrations (≥ 10 ng/mL) of IL-17 can suppress osteoclast formation in vitro [ 16 ]. The results of the study by Kitami et al. support the findings of this study, as well as those of our previous studies [ 11 , 17 , 18 ]. These findings suggest that IL-17 plays an important role in the progression of inflammation and the associated regulation of osteoclast differentiation. NF-κB signaling is a transcription factor involved in the transcription of many inflammatory genes, and NF-κB is activated in osteoclast precursor cells by RANKL stimulation [ 19 ]. RANKL-stimulated activation of the canonical NF-κB signaling pathway is essential for osteoclastogenesis [ 20 ]. In the absence of stimulation, the NF-κB p50 and p65 subunits are constitutively maintained in the cytoplasm in an inactive state by binding to the inhibitory molecule IκB [ 21 ]. RANKL stimulation induces IκB phosphorylation by IκB kinase and proteasomal degradation [ 22 ]. Degradation of IκBα leads to the phosphorylation and nuclear translocation of the NF-κB p65/p50 heterodimer, allowing it to activate target genes [ 23 ]. This is called the canonical NF-β signaling pathway, and it proceeds very rapidly, within minutes of stimulation [ 24 ]. We investigated whether NF-κB p65 is involved in the suppression of IL-17-mediated RANKL-induced osteoclast formation. To do this, we investigated the phosphorylation and nuclear translocation of NF-κB p65 upon stimulation using Western blotting. We found that RANKL stimulation induces both the phosphorylation of NF-κB p65 and its nuclear translocation, consistent with previous findings. However, IL-17 inhibited RANKL-induced phosphorylation of NF-κB p65 and its nuclear translocation. These results suggest that RANKL-induced osteoclast differentiation may be inhibited by the inhibitory role of IL-17 in the activation of the canonical NF-κB signaling pathway by RANKL/RANK signaling. Given that RANKL/RANK signaling is known to activate c-Fos expression [ 25 ], a transcription factor essential for osteoclast development, we next examined the effect of IL-17 on c-Fos expression. c-Fos is a proto-oncogene that is activated by RANKL/RANK signaling and is known to be involved in the regulation of osteoclastogenesis [ 26 , 27 ]. c-Fos and c-Jun are important components of the AP-1 transcription factor complex as heterodimers, and it has been shown that c-Fos-deficient mice develop osteopetrosis due to impaired osteoclast formation [ 26 , 27 ]. In this study, we confirmed that RANKL increased c-Fos levels in RAW264.7 cells and that co-stimulation of IL-17 and RANKL inhibited the RANKL-induced increase in c-Fos levels. This suggests that c-Fos plays an important role in the inhibition of RANKL-induced osteoclastogenesis by IL-17. RANKL/RANK signaling activates MAPKs and NF-κB, inducing the transcription factors NFATc1 and AP-1 (a complex of c-Jun and c-Fos), which are known to regulate the transcription of genes involved in osteoclast differentiation [ 28 ]. NFATc1 and AP-1 regulate the expression of osteoclast-specific markers, such as TRAP, cathepsin K, β-integrin, MMP-9, ATP6V0D2, and DC-STAMP, ultimately leading to osteoclast differentiation and maturation. NFATc1 and AP-1 regulate the expression of osteoclast-specific markers, such as TRAP, cathepsin K, β-integrin, MMP-9, ATP6V0D2, and DC-STAMP, ultimately leading to osteoclast differentiation and maturation [ 29 ]. NFATc1 is a master transcription factor that regulates bone formation, and NFATc1-deficient mice exhibit defects in osteoclast differentiation and osteopetrosis [ 20 , 30 ]. Loss of NFATc1 also completely inhibited RANKL-induced osteoclast formation in RAW264.7 cells [ 31 ]. Additionally, RANKL-stimulated NFATc1 expression is inhibited in c-Fos-deficient mice, resulting in the development of osteopetrosis due to impaired osteoclast formation [ 26 , 27 ]. This indicates that NFATc1 is downstream of c-Fos in RANKL-induced osteoclast differentiation. In this study, consistent with previous findings, RANKL stimulation induced NFATc1 expression. However, we confirmed that IL-17 suppressed RANKL-induced NFATc1 expression. These results suggest that IL-17 may be one of the factors that suppress RANKL-induced osteoclast differentiation by downregulating NFATc1 expression, a master transcription factor that regulates bone formation. IL-17 has been shown to indirectly stimulate osteoclast differentiation and function when mediated by osteoblasts [ 6 , 9 ]. Taken together with the results of this study, these findings suggest that the direct and indirect effects of IL-17 on osteoclastogenesis may differ. However, it is likely that IL-17 plays an important role in regulating bone metabolism. To address this discrepancy, we recognize the importance of further verifying these findings using mouse primary cells rather than the cell line RAW264.7. Future studies will include experiments using primary cells to more comprehensively examine the direct role of IL-17 in osteoclast formation. Conclusions Our results suggest that co-stimulation of IL-17 and RANKL inhibits RANKL/RANK signaling-induced NF-κBp65 phosphorylation and its associated nuclear translocation, thereby inhibiting the downstream induction of c-Fos and NFATc1. c-Fos and NFATc1 are key regulators of RANKL-induced osteoclastogenesis. IL-17 inhibited RANKL-induced c-Fos expression at the protein level, which may have resulted in the suppression of NFATc1 expression, a downstream transcription factor of c-Fos, and thus the inhibition of RANKL-induced osteoclast differentiation. These findings provide new insights into the mechanism of IL-17-induced inhibition of osteoclast differentiation. By advancing this research, we aim to eventually develop reagents that control osteoclast differentiation. Abbreviations ANOVA analysis of variance ATP6V0D2 ATPase H + transporting V0 subunit d2 DC-STAMP dendritic cell-specific transmembrane protein D-MEM Dulbecco’s modified Eagle’s medium IL interleukin MAPK mitogen-activated protein kinases M-CSF macrophage colony-stimulating factor MMP-9 matrix metalloproteinase 9 NFATc1 nuclear factor of activated T cells RANK receptor activator of NFκB RANKL receptor activator of NFκB ligand TNF- tumor necrosis factor-alpha TRAF tumor necrosis factor receptor-associated factors TRAP tartrate-resistant acid-phosphatase Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This study was supported by a grant from the Japan Society for the Promotion of Science KAKENHI, Grant Number JP24K12973 (Grant-in-Aid for Scientific Research (C)) and Grant Number JP24K13276 (Grant-in-Aid for Scientific Research (C)). Authors’ contributions SK and HI were involved in data collection and data analysis. NS, SG, and AN were involved in funding acquisition. SK, HI, and SG were involved in data interpretation, drafting the manuscript, and revising it critically. All authors read and approved the final manuscript. Acknowledgments We sincerely appreciate Dr. Fukawa and Dr. Sugimoto from the Department of Orthodontics at Osaka Dental University for their guidance and advice in conducting this research. 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Calycosin Suppresses RANKL-Mediated Osteoclastogenesis through Inhibition of MAPKs and NF-κB. Int J Mol Sci. 2015;16:29496–507. Takayanagi H. The role of NFAT in osteoclast formation. Ann N Y Acad Sci. 2007;1116:227–37. Hirotani H, Tuohy NA, Woo JT, Stern PH, Clipstone NA. The calcineurin/nuclear factor of activated T cells signaling pathway regulates osteoclastogenesis in RAW264.7 cells. J Biol Chem. 2004;279:13984–92. Additional Declarations No competing interests reported. Supplementary Files KobuchiFig2APhosphoNFkBp65.jpg KobuchiFig2ANFkBp65.jpg KobuchiFig2C.jpg KobuchiFig3AcFos.jpg KobuchiFig3ABetaActin.jpg KobuchiFig4ANFATc1.jpg KobuchiFig4ABetaActin.jpg Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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15:39:41","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":86620,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8121955/v1/ebb0ab7e50e1bd0843caca6b.html"},{"id":98812378,"identity":"03cca90a-781b-4f31-991f-4b667e03619c","added_by":"auto","created_at":"2025-12-22 15:39:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":81106,"visible":true,"origin":"","legend":"\u003cp\u003eIL-17 in combination with RANKL suppressed TRAP activity in RAW264.7 cells. RAW264.7 cells (8 × 10³ cells/well) were seeded in 96-well plates and cultured for 3 days with IL-17 (10 ng/mL) and RANKL (10 or 30 ng/mL). TRAP activity was then measured. The relative TRAP activity is presented as a bar graph. Statistical analysis was performed using one-way ANOVA with GraphPad Prism (*p \u0026lt; 0.05). Data represent the mean ± standard error of the mean (SEM) from three independent experiments.\u003c/p\u003e\n\u003cp\u003eTRAP, tartrate-resistant acid-phosphatase; RANKL, receptor activator of NFκB ligand; ANOVA, analysis of variance; IL, interleukin-17\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8121955/v1/755446a3b94ac50a624302ad.png"},{"id":98812392,"identity":"3cfbaa6d-a74b-428f-8560-589afad50d37","added_by":"auto","created_at":"2025-12-22 15:39:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":198956,"visible":true,"origin":"","legend":"\u003cp\u003eIL-17 suppresses RANKL-induced p-NF-κB expression when administered concurrently. RAW264.7 cells were treated with IL-17 (10 ng/mL) and RANKL (10 or 30 ng/mL) for 30 min, followed by Western blot analysis. (A) Immunoblotting was performed using antibodies against phosphorylated NF-κB (p-NF-κB; upper panel) and total NF-κB (lower panel). (B) Quantitative analysis of p-NF-κB expression normalized to total NF-κB is shown. Statistical significance was determined by one-way ANOVA using GraphPad Prism, with p \u0026lt; 0.05 indicating statistical significance. (C) RAW264.7 cells were treated with IL-17 (10 ng/mL) and RANKL (30 ng/mL) for 60 min. Cytoplasmic and soluble nuclear protein fractions were extracted, and NF-κB nuclear translocation was analyzed by Western blot analysis. Top panel: NF-κB in the cytoplasmic fraction; Middle panel: NF-κB in the soluble nuclear fraction; Bottom panel: β-actin (cytoplasmic marker).\u003c/p\u003e\n\u003cp\u003eRANKL, receptor activator of NFκB ligand; ANOVA, analysis of variance; IL-17, interleukin-17\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8121955/v1/3d2cec141c5d20717c70478d.png"},{"id":98812385,"identity":"51e95e82-7514-4f2b-816b-4efe40bd51f3","added_by":"auto","created_at":"2025-12-22 15:39:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":142123,"visible":true,"origin":"","legend":"\u003cp\u003eIL-17 suppresses RANKL-induced c-Fos expression. RAW264.7 cells were treated with IL-17 (10 ng/mL) and RANKL (10 or 30 ng/mL) for 2 days, followed by Western blot analysis. (A) Immunoblotting was performed using antibodies against c-Fos (upper panel) and β-actin (lower panel). (B) Quantitative analysis of c-Fos expression normalized to β-actin is shown. Statistical significance was determined by one-way ANOVA using GraphPad Prism, with p＜0.05 considered significant. The figure presents a representative result from three independent experiments.\u003c/p\u003e\n\u003cp\u003eRANKL, receptor activator of NFκB ligand; ANOVA, analysis of variance; IL, interleukin-17\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8121955/v1/c54f09745a28e1c18634d902.png"},{"id":98812363,"identity":"c6295d6a-a9cb-4c18-8d54-839f9e6445e5","added_by":"auto","created_at":"2025-12-22 15:39:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":128633,"visible":true,"origin":"","legend":"\u003cp\u003eIL-17 suppresses RANKL-induced NFATc1 expression. RAW264.7 cells were treated with IL-17 (10 ng/mL) and RANKL (10 or 30 ng/mL) for 3 days, followed by Western blot analysis. (A) Immunoblotting was performed using antibodies against NFATc1 (upper panel) and β-actin (lower panel). (B) Quantitative analysis of NFATc1 expression normalized to β-actin is shown. Statistical significance was determined by one-way ANOVA using GraphPad Prism, with p＜0.05 considered significant.\u003c/p\u003e\n\u003cp\u003eRANKL, receptor activator of NFκB ligand; NFATc1, nuclear factor of activated T cells; ANOVA, analysis of variance; IL-17, interleukin-17\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8121955/v1/6b3e371001d3f9a83ab428ed.png"},{"id":100548828,"identity":"d2ef34ad-42d3-4d4b-8811-5f21b551082a","added_by":"auto","created_at":"2026-01-19 08:21:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1168808,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8121955/v1/d8fc58be-f017-445c-b2b6-aef610f17620.pdf"},{"id":98812389,"identity":"fc1ac43f-2fec-44ca-bb3a-93a7e4131e1b","added_by":"auto","created_at":"2025-12-22 15:39:37","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":771426,"visible":true,"origin":"","legend":"","description":"","filename":"KobuchiFig2APhosphoNFkBp65.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8121955/v1/49f8d53d1e9489901eba53f9.jpg"},{"id":98812381,"identity":"731874d1-7b8e-4e36-ab0b-07e5f134e0ed","added_by":"auto","created_at":"2025-12-22 15:39:37","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":245951,"visible":true,"origin":"","legend":"","description":"","filename":"KobuchiFig2ANFkBp65.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8121955/v1/457d791c42ae546bbe7c4af5.jpg"},{"id":98812423,"identity":"bf232b73-4393-480b-a3e5-6c64b57d9b6c","added_by":"auto","created_at":"2025-12-22 15:39:38","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":436715,"visible":true,"origin":"","legend":"","description":"","filename":"KobuchiFig2C.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8121955/v1/361dafc613773837d33c17e1.jpg"},{"id":98812379,"identity":"99567f47-7b20-4da3-aeec-35512fe5d345","added_by":"auto","created_at":"2025-12-22 15:39:36","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":161221,"visible":true,"origin":"","legend":"","description":"","filename":"KobuchiFig3AcFos.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8121955/v1/118f22afdb7ad2adb0b1185a.jpg"},{"id":98812376,"identity":"94d778a5-9d3e-441a-b4ef-b625775f5a4b","added_by":"auto","created_at":"2025-12-22 15:39:36","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":201185,"visible":true,"origin":"","legend":"","description":"","filename":"KobuchiFig3ABetaActin.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8121955/v1/e573003268f9b5733aa98857.jpg"},{"id":98812383,"identity":"9abd976f-5975-4483-ad2f-bbc1560102a9","added_by":"auto","created_at":"2025-12-22 15:39:37","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":182533,"visible":true,"origin":"","legend":"","description":"","filename":"KobuchiFig4ANFATc1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8121955/v1/2d53298c62e3a42c49fb5b7f.jpg"},{"id":98812368,"identity":"e047edb3-9f83-4086-8755-c5e4cfde7ec6","added_by":"auto","created_at":"2025-12-22 15:39:36","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":204375,"visible":true,"origin":"","legend":"","description":"","filename":"KobuchiFig4ABetaActin.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8121955/v1/2acd822c1385e7811d20f0dc.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Interleukin-17 inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing nuclear factor-κB p65 phosphorylation and nuclear factor of activated T cells c1 expression","fulltext":[{"header":"Background","content":"\u003cp\u003eBone is continuously remodeled and tightly regulated to maintain homeostasis through bone formation and resorption by osteoblasts and osteoclasts, respectively [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The balance between osteoblasts and osteoclasts is important for maintaining bone mass, and disruption of this relationship leads to bone diseases such as osteoarthritis, rheumatoid arthritis, and periodontal disease.\u003c/p\u003e \u003cp\u003eOsteoclasts are formed by the fusion of monocyte-macrophage osteoclast precursor cells as tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. These TRAP-positive multinucleated cells reorganize the actin cytoskeleton and become multinucleated osteoclasts that adhere to the bone surface and resorb bone [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Osteoclast differentiation is regulated by two essential cytokines, macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. M-CSF is important for the survival and proliferation of osteoclast precursor cells, and RANKL is required for inducing the fusion of mononuclear precursor cells and their differentiation into osteoclasts [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The binding of RANKL to receptor activator of NF-κB (RANK) induces the recruitment of adaptor molecules such as tumor necrosis factor receptor-associated factors (TRAFs). Recruitment of TRAF6 to RANK activates downstream signaling pathways, including mitogen-activated protein kinases (MAPKs) (extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38) and NF-κB, which then activate various transcription factors involved in osteoclast differentiation, such as c-Fos, AP-1, and nuclear factor of activated T cells (NFAT)c1 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These signaling cascades regulate the transcription of target genes for many osteoclast-specific markers, including calcitonin receptor, TRAP, β3 integrin, cathepsin K, matrix metalloproteinase 9 (MMP-9), dendritic cell-specific transmembrane protein (DC-STAMP), calcitonin receptor, carbonic anhydrase II, and ATPase H\u003csup\u003e+\u003c/sup\u003e transporting V0 subunit d2 (ATP6V0D2), ultimately inducing the formation and activation of mature multinucleated osteoclasts [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, targeting these signaling pathways may enable control of osteoclast differentiation and activation, which may be an effective therapeutic strategy for bone remodeling-based treatments, such as for rheumatoid arthritis.\u003c/p\u003e \u003cp\u003eInterleukin-17 (IL-17) is a pro-inflammatory cytokine produced by Th17 cells, a subset of helper T cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Th17 cells are known to induce inflammation by producing other pro-inflammatory cytokines, including IL-21, IL-22, and tumor necrosis factor (TNF)-α, in addition to IL-17 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. IL-17 transmits signals via a heterodimeric receptor complex consisting of IL-17RA and IL-17RC, activating downstream NF-kB pathways, MAPK pathways, and C/EBP pathways to produce pro-inflammatory cytokines and chemokines [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. IL-17 has been shown to act indirectly on osteoclast precursor cells by inducing RANKL expression in osteoblasts, thereby inducing osteoclast formation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRAW264.7 cells are a monocyte/macrophage-like cell line, and upon stimulation with RANKL, they differentiate into TRAP-positive multinucleated osteoclasts [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. We previously confirmed the presence of IL-17RA and IL-17RC in RAW264.7 cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and investigated the direct effect of IL-17 on osteoclast differentiation. The results obtained demonstrated that osteoclast differentiation is suppressed by the direct effects of IL-17. However, the details of the mechanism of action on osteoclast differentiation of RAW264.7 cells and the direct effect of IL-17 on osteoclast function have not been fully elucidated.\u003c/p\u003e \u003cp\u003eThus, this study aimed to investigate the effect of IL-17 on RANKL-induced osteoclast differentiation in RAW264.7 cells. We evaluated the effect of IL-17 on osteoclast differentiation by measuring TRAP enzyme activity. Western blotting was used to examine the effect of IL-17 on the intracellular signaling pathway involved in RANKL-induced osteoclast differentiation by examining the phosphorylation of NF-κBp65. Furthermore, we elucidated the involvement of IL-17 in the expression of the transcription factors, c-FOS and NFATc1, master transcription factors for osteoclast differentiation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe murine monocyte/macrophage cell line RAW264.7 was obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in 60 cm\u0026sup2; culture dishes and maintained at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂. The culture medium used was Dulbecco\u0026rsquo;s Modified Eagle Medium (D-MEM; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), supplemented with 10% fetal bovine serum (Cosmo Bio Co., Ltd., Tokyo, Japan), 100 \u0026micro;g/mL penicillin-streptomycin solution (Nacalai Tesque, Kyoto, Japan), and 2 mM L-alanyl-L-glutamine solution (Nacalai Tesque).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReagents and antibodies\u003c/h3\u003e\n\u003cp\u003eRecombinant RANKL was purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan), and IL-17 was obtained from PeproTech Inc. (Rocky Hill, NJ, USA). Anti-NFATc1 and anti-β-actin antibodies were acquired from Santa Cruz Biotechnology Inc. (Dallas, TX, USA), while anti-c-Fos and anti-NF-κB antibodies were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and goat anti-mouse IgG secondary antibodies were purchased from Jackson ImmunoResearch Inc. (West Grove, PA, USA). The kit for cytoplasmic and nuclear protein fractionation and extraction was obtained from FUJIFILM Wako Pure Chemical Corporation.\u003c/p\u003e\n\u003ch3\u003eTRAP activity assay\u003c/h3\u003e\n\u003cp\u003eRAW264.7 cells were seeded at a density of 8 \u0026times; 10\u0026sup3; cells per well in 96-well plates and stimulated for 3 days with IL-17 (10 ng/mL) and RANKL (10 or 30 ng/mL) in D-MEM (FUJIFILM Wako Pure Chemical). On day 6, TRAP activity was assessed to evaluate osteoclast differentiation. Cells were fixed with 10% neutral-buffered formalin for 10 min, followed by fixation with an equal volume of acetone/ethanol (FUJIFILM Wako Pure Chemical). Subsequently, cells were incubated with 50 mM sodium citrate and 25 mM tartaric acid (pH 5.0) (FUJIFILM Wako Pure Chemical). The enzymatic reaction was terminated by adding an equal volume of 0.1 N sodium hydroxide (FUJIFILM Wako Pure Chemical Corporation), and absorbance was measured at 405 nm using a SpectraMax M5 multi-mode microplate reader (Molecular Devices, Sunnyvale, CA, USA).\u003c/p\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cp\u003eProtein samples were extracted from RAW264.7 cells and subjected to electrophoresis for 90 min at 190 V, followed by transfer at 340 mV for 120 min. Membranes were blocked with Blocking One (Nacalai Tesque) at room temperature for 60 min. Primary antibodies against phospho-NF-κB p65, NF-κB p65, c-Fos, and NFATc1 were incubated overnight at 4\u0026deg;C. After washing, membranes were incubated with HRP-conjugated secondary antibodies (mouse and rabbit IgG) for 60 min. Protein bands were detected and visualized using the ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA).\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using one-way analysis of variance with GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA). p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. All experiments were conducted at least three times, and data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEffect of IL-17 on TRAP activity in RANKL-stimulated RAW264.7 cells\u003c/h2\u003e \u003cp\u003eThe effect of IL-17 on RANKL-induced osteoclast differentiation in RAW264.7 cells was examined by measuring TRAP activity. The enzymatic TRAP activity measured at the end of the differentiation process was similar to that of unstimulated controls when RAW264.7 cells were treated with IL-17 alone, but increased upon stimulation with RANKL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The increased TRAP activity due to RANKL stimulation was suppressed by co-stimulation with IL-17 and RANKL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCo-stimulation with RANKL and IL-17 reduced NF-κB p65 phosphorylation\u003c/h3\u003e\n\u003cp\u003eWe examined whether IL-17 is involved in osteoclast differentiation via the NF-κB signaling pathway by analyzing NF-κB p65 phosphorylation using Western blotting. Phosphorylated NF-κB p65 protein levels were increased by stimulation with RANKL alone (30 ng/mL) compared to unstimulated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea (upper panel), 2b). Co-stimulation with IL-17 and RANKL (30 ng/mL) reduced phosphorylated NF-κB p65 protein levels compared with stimulation with RANKL alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea (upper panel), 2b). To confirm that equal amounts of NF-κB p65 were obtained from the lysates, the Western blot membranes were stripped and re-probed with anti-NF-κB p65 antibodies. Equal amounts of NF-κB p65 were detected in the lysates obtained from each sample (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea [lower panel]). To determine whether IL-17 suppresses the RANKL-induced activity of transcription factors, we examined the effects of IL-17 on RANKL-induced nuclear translocation of NF-κBp65. NF-κB p65 translocated to the nucleus in response to RANKL, and IL-17 inhibited the nuclear translocation of NF-κB p65 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEffect of IL-17 on c-Fos expression in RANKL-stimulated RAW264.7 cells\u003c/h2\u003e \u003cp\u003ec-Fos plays an important role in RANKL-induced NFATc1 expression by forming an AP-1 complex with c-Jun. Therefore, the effect of IL-17 on RANKL-induced c-Fos expression in RAW264.7 cells was examined by Western blot analysis. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea (upper panel), RANKL stimulation increased the expression of c-Fos in RAW 264.7 cells. Stimulation with RANKL alone increased c-Fos expression levels, whereas co-stimulation with IL-17 and RANKL suppressed c-Fos expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). The lower panel of Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea indicates that the total amount of β-actin was not affected by various stimuli.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEffect of IL-17 on NFATc1 protein expression in RANKL-stimulated RAW264.7 cells\u003c/h2\u003e \u003cp\u003eNext, we investigated the expression of NFATc, which is essential for osteoclast formation and functions as a master transcriptional regulator of osteoclast differentiation. We confirmed that RANKL stimulation increased NFATc1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). However, the increase in NFATc1 expression due to RANKL stimulation was suppressed by co-stimulation with IL-17 and RANKL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). The lower panel of Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea indicates that the total amount of β-actin was not affected by various stimuli.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated the direct effect of IL-17 on RANKL-induced osteoclast differentiation in RAW264.7 cells. The process of osteoclast differentiation requires stimulation by M-CSF and RANKL, which are central regulators of osteoclast differentiation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Osteoblasts produce M-CSF and RANKL, which are important promoters of osteoclastogenesis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. RAW264.7 cells are a mouse macrophage cell line that can differentiate into osteoclasts by RANKL stimulation alone, without M-CSF stimulation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Osteoclast precursor cells undergo a multi-step process of proliferation, TRAP expression, and cell fusion upon stimulation with RANKL and M-CSF, and then degrade into osteoclasts [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. TRAP-positive multinucleated cells can induce bone resorption [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Based on this theory, in this study, we evaluated osteoclast formation potential by assessing TRAP activity. We demonstrated that IL-17 stimulation, through co-stimulation with RANKL, could directly suppress RANKL-induced TRAP activity in RAW264.7 cells.\u003c/p\u003e \u003cp\u003eIL-17 is a pro-inflammatory cytokine that produces various inflammatory cytokines and chemokines, such as IL-1β, IL-6, IL-8, GM-CSF, and TNF-α, in various types of cells, including Th17 and macrophages. IL-17 is known to indirectly promote osteoclast differentiation by inducing RANKL expression in osteoblasts in co-cultures of osteoclast precursors and osteoblasts [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, Kitami et al. showed that the linear effect of IL-17 on osteoclast formation in osteoclast precursor cells is concentration-dependent, and that high concentrations (\u0026ge;\u0026thinsp;10 ng/mL) of IL-17 can suppress osteoclast formation in vitro [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The results of the study by Kitami et al. support the findings of this study, as well as those of our previous studies [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These findings suggest that IL-17 plays an important role in the progression of inflammation and the associated regulation of osteoclast differentiation.\u003c/p\u003e \u003cp\u003eNF-κB signaling is a transcription factor involved in the transcription of many inflammatory genes, and NF-κB is activated in osteoclast precursor cells by RANKL stimulation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. RANKL-stimulated activation of the canonical NF-κB signaling pathway is essential for osteoclastogenesis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In the absence of stimulation, the NF-κB p50 and p65 subunits are constitutively maintained in the cytoplasm in an inactive state by binding to the inhibitory molecule IκB [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. RANKL stimulation induces IκB phosphorylation by IκB kinase and proteasomal degradation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Degradation of IκBα leads to the phosphorylation and nuclear translocation of the NF-κB p65/p50 heterodimer, allowing it to activate target genes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This is called the canonical NF-β signaling pathway, and it proceeds very rapidly, within minutes of stimulation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe investigated whether NF-κB p65 is involved in the suppression of IL-17-mediated RANKL-induced osteoclast formation. To do this, we investigated the phosphorylation and nuclear translocation of NF-κB p65 upon stimulation using Western blotting. We found that RANKL stimulation induces both the phosphorylation of NF-κB p65 and its nuclear translocation, consistent with previous findings. However, IL-17 inhibited RANKL-induced phosphorylation of NF-κB p65 and its nuclear translocation. These results suggest that RANKL-induced osteoclast differentiation may be inhibited by the inhibitory role of IL-17 in the activation of the canonical NF-κB signaling pathway by RANKL/RANK signaling.\u003c/p\u003e \u003cp\u003eGiven that RANKL/RANK signaling is known to activate c-Fos expression [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], a transcription factor essential for osteoclast development, we next examined the effect of IL-17 on c-Fos expression. c-Fos is a proto-oncogene that is activated by RANKL/RANK signaling and is known to be involved in the regulation of osteoclastogenesis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. c-Fos and c-Jun are important components of the AP-1 transcription factor complex as heterodimers, and it has been shown that c-Fos-deficient mice develop osteopetrosis due to impaired osteoclast formation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In this study, we confirmed that RANKL increased c-Fos levels in RAW264.7 cells and that co-stimulation of IL-17 and RANKL inhibited the RANKL-induced increase in c-Fos levels. This suggests that c-Fos plays an important role in the inhibition of RANKL-induced osteoclastogenesis by IL-17.\u003c/p\u003e \u003cp\u003eRANKL/RANK signaling activates MAPKs and NF-κB, inducing the transcription factors NFATc1 and AP-1 (a complex of c-Jun and c-Fos), which are known to regulate the transcription of genes involved in osteoclast differentiation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. NFATc1 and AP-1 regulate the expression of osteoclast-specific markers, such as TRAP, cathepsin K, β-integrin, MMP-9, ATP6V0D2, and DC-STAMP, ultimately leading to osteoclast differentiation and maturation. NFATc1 and AP-1 regulate the expression of osteoclast-specific markers, such as TRAP, cathepsin K, β-integrin, MMP-9, ATP6V0D2, and DC-STAMP, ultimately leading to osteoclast differentiation and maturation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. NFATc1 is a master transcription factor that regulates bone formation, and NFATc1-deficient mice exhibit defects in osteoclast differentiation and osteopetrosis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Loss of NFATc1 also completely inhibited RANKL-induced osteoclast formation in RAW264.7 cells [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Additionally, RANKL-stimulated NFATc1 expression is inhibited in c-Fos-deficient mice, resulting in the development of osteopetrosis due to impaired osteoclast formation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This indicates that NFATc1 is downstream of c-Fos in RANKL-induced osteoclast differentiation. In this study, consistent with previous findings, RANKL stimulation induced NFATc1 expression. However, we confirmed that IL-17 suppressed RANKL-induced NFATc1 expression. These results suggest that IL-17 may be one of the factors that suppress RANKL-induced osteoclast differentiation by downregulating NFATc1 expression, a master transcription factor that regulates bone formation.\u003c/p\u003e \u003cp\u003eIL-17 has been shown to indirectly stimulate osteoclast differentiation and function when mediated by osteoblasts [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Taken together with the results of this study, these findings suggest that the direct and indirect effects of IL-17 on osteoclastogenesis may differ. However, it is likely that IL-17 plays an important role in regulating bone metabolism. To address this discrepancy, we recognize the importance of further verifying these findings using mouse primary cells rather than the cell line RAW264.7. Future studies will include experiments using primary cells to more comprehensively examine the direct role of IL-17 in osteoclast formation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur results suggest that co-stimulation of IL-17 and RANKL inhibits RANKL/RANK signaling-induced NF-κBp65 phosphorylation and its associated nuclear translocation, thereby inhibiting the downstream induction of c-Fos and NFATc1. c-Fos and NFATc1 are key regulators of RANKL-induced osteoclastogenesis. IL-17 inhibited RANKL-induced c-Fos expression at the protein level, which may have resulted in the suppression of NFATc1 expression, a downstream transcription factor of c-Fos, and thus the inhibition of RANKL-induced osteoclast differentiation. These findings provide new insights into the mechanism of IL-17-induced inhibition of osteoclast differentiation. By advancing this research, we aim to eventually develop reagents that control osteoclast differentiation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eANOVA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eanalysis of variance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eATP6V0D2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eATPase H\u003csup\u003e+\u003c/sup\u003e transporting V0 subunit d2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDC-STAMP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edendritic cell-specific transmembrane protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eD-MEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einterleukin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMAPK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emitogen-activated protein kinases\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eM-CSF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emacrophage colony-stimulating factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMMP-9\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ematrix metalloproteinase 9\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNFATc1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enuclear factor of activated T cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRANK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereceptor activator of NFκB\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRANKL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereceptor activator of NFκB ligand\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNF-\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etumor necrosis factor-alpha\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTRAF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etumor necrosis factor receptor-associated factors\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTRAP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etartrate-resistant acid-phosphatase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by a grant from the Japan Society for the Promotion of Science KAKENHI, Grant Number JP24K12973 (Grant-in-Aid for Scientific Research (C)) and Grant Number JP24K13276 (Grant-in-Aid for Scientific Research (C)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSK and HI were involved in data collection and data analysis. NS, SG,\u0026nbsp;and AN\u0026nbsp;were involved in funding acquisition. SK, HI, and SG were involved in data interpretation, drafting the manuscript, and revising it critically. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely appreciate Dr. Fukawa and Dr. Sugimoto from the Department of Orthodontics at Osaka Dental University for their guidance and advice in conducting this research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u0026nbsp;\u003c/sup\u003eGraduate School of Dentistry, Department of Orthodontics, Osaka Dental University, Osaka, Japan\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u0026nbsp;\u003c/sup\u003eDepartment of Physiology, Osaka Dental University, Osaka, Japan\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u0026nbsp;\u003c/sup\u003eDepartment of Orthodontics, Osaka Dental University, Osaka, Japan\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHotokezaka H, Sakai E, Kanaoka K, Saito K, Matsuo K, Kitaura H, et al. 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J Biol Chem. 2004;279:13984\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"interleukin-17, osteoclast differentiation, nuclear factor-κB p65, nuclear factor of activated T cells 1","lastPublishedDoi":"10.21203/rs.3.rs-8121955/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8121955/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBone is constantly being regenerated while maintaining dynamic homeostasis through a delicate balance between bone destruction by osteoclasts and bone formation by osteoblasts, a process known as bone remodeling. Bone resorption on the compression side is closely related to the differentiation and activation of osteoclasts. Interleukin-17 (IL-17) is a pro-inflammatory cytokine secreted by Th17 cells and other cells. IL-17 has been shown to indirectly induce osteoclast differentiation by increasing the expression of receptor activator of NFκB ligand (RANKL) in osteoblasts. However, little has been reported about the effect of IL-17 directly acting on osteoclast precursor cells during osteoclast differentiation. This study aimed to investigate the effect of IL-17 on RANKL-induced osteoclast differentiation in RAW264.7 cells.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOsteoclast differentiation and formation were assessed by measuring tartrate-resistant acid-phosphatase (TRAP) activity. RANKL stimulation enhanced TRAP activity in RAW264.7 cells, but co-stimulation with IL-17 attenuated it. RANKL stimulation activated the canonical NF-κB pathway, leading to increased phosphorylation of NF-κB p65 and its subsequent nuclear translocation, but IL-17 suppressed this increased phosphorylation and nuclear translocation of NF-κB p65. c-Fos and nuclear factor of activated T cells (NFATc1), nuclear transcription factors that play important roles in regulating the expression of many osteoclast-specific genes involved in osteoclast differentiation, were induced by RANKL stimulation. IL-17 reduced RANKL-stimulated c-Fos and NFATc1 expression.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIL-17 acts directly on RAW264.7 cells, inhibiting the RANKL/RANK signal-induced phosphorylation of NF-κB p65 and its subsequent nuclear translocation. This study provides evidence suggesting that the mechanism by which IL-17 suppresses RANKL-induced osteoclast differentiation may involve the suppression of c-Fos and NFATc1 expression, key transcription factors that control osteoclast formation, by inhibiting the activation of NF-κB p65.\u003c/p\u003e","manuscriptTitle":"Interleukin-17 inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing nuclear factor-κB p65 phosphorylation and nuclear factor of activated T cells c1 expression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-22 15:38:16","doi":"10.21203/rs.3.rs-8121955/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"33390fdb-c3fd-47b3-8826-9a5e51ccfa36","owner":[],"postedDate":"December 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T06:25:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-22 15:38:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8121955","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8121955","identity":"rs-8121955","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}