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ISSN : 1226-7155(Print)
ISSN : 2287-6618(Online)
International Journal of Oral Biology Vol.50 No.1 pp.17-25
DOI : https://doi.org/10.11620/IJOB.2025.50.1.17

FK866 attenuates receptor activator of nuclear factor kappa-B ligand-induced osteoclastogenesis

Chang Youp Ok1,2, Hye-Ock Jang1, Moon-Kyoung Bae3, Soo-Kyung Bae1,2*
1Department of Dental Pharmacology, School of Dentistry, Pusan National University, Yangsan 50612, Republic of Korea
2Dental and Life Science Institute, School of Dentistry, Pusan National University, Yangsan 50612, Republic of Korea
3Department of Oral Physiology, School of Dentistry, Pusan National University, Yangsan 50612, Republic of Korea
*Correspondence to: Soo-Kyung Bae, E-mail: skbae@pusan.ac.krhttps://orcid.org/0000-0002-1106-9260
December 24, 2024 February 3, 2025 February 13, 2024

Abstract


Visfatin, an adipokine secreted by cells, is crucial for intracellular nicotinamide adenine dinucleotide+ biosynthesis. Extracellularly, visfatin plays diverse roles in inflammatory conditions, including obesity, which is closely linked to osteoclastogenesis. We previously showed that visfatin enhances receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis in bone marrow-derived macrophages. However, its enzymatic activity during this process is poorly understood. Here, we investigated visfatin’s effects on RANKL-induced osteoclast differentiation. Our results demonstrate that visfatin promotes this differentiation, an effect inhibited by FK866, an inhibitor of visfatin’s enzymatic activity. Furthermore, FK866 also inhibited RANKL-induced osteoclast differentiation. These findings suggest that inhibiting visfatin’s enzymatic activity modulates osteoclast differentiation. Thus, visfatin plays an important role in osteoclastogenesis, both intracellularly and extracellularly, and FK866 has therapeutic potential for diseases characterized by imbalanced osteoclast formation, such as osteoporosis and periodontitis.


Visfatin , Nicotinamide phosphoribosyltransferase , FK866 , receptor activator of nuclear factor-kappa B ligand , Osteoclastogenesis

초록

    © The Korean Academy of Oral Biology. All rights reserved.

    This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Introduction

    Bone homeostasis is the process by which bone tissue maintains a healthy state by balancing bone formation and resorption [1,2]. Bone tissue is primarily composed of osteoblasts [2]. Osteoblasts are responsible for the formation and mineralization of new bone tissue [2]. These cells produce proteins such as collagen, which creates the structural framework of bone tissue. Osteoclasts induce bone resorption [2,3]. These cells break down minerals and other inorganic components within bone tissue and release them into the bloodstream [3]. Under normal physiological conditions, the activities of osteoclasts and osteoblasts are closely interconnected, ensuring that eroded bone is completely replaced with fresh bone [3]. When this homeostatic balance shifts towards excessive osteoblast activity, an osteosclerotic phenotype can occur [4]. Conversely, decreased osteoblastic activity can lead to osteomalacia and rickets. In contrast, increased osteoclast activity can result in bone pathologies such as osteoporosis, Paget’s disease, rheumatoid arthritis, osteoarthritis, and autoimmune arthritis [4,5]. However, osteopetrosis can occur if osteoclast differentiation and/or function are impaired [6]. Understanding the mechanisms underlying bone homeostasis is crucial for understanding the pathogenesis of bone-related diseases and developing new therapies for bone disorders.

    Osteoclast differentiation is the process by which precursor cells, typically derived from the monocyte/macrophage lineage, transform into mature osteoclasts, mono-nuclear osteoclast precursors fuse into large and mature multinucleated cells through the expression of cell-cell fusion genes such as dendritic cell-specific transmembrane protein (DC-STAMP), ATP6v0d2, and Gα13 [7-11]. Osteoclast formation is tightly regulated and maintains a balance between osteoclast and anti-osteoclast formation mechanisms [7,8]. In the canonical pathway, osteoclast differentiation is induced by the receptor activator of nuclear factor kappa-B ligand (RANKL), which is primarily expressed in bone tissue [8,12,13]. The RANKLRANK- OPG signaling pathway plays a crucial role in bone metabolism [12,13]. RANKL is mainly expressed by osteoblasts and certain cells in the bone tissue [12,13]. In addition, lymphocytes, activated T cells, and activated macrophages express RANKL [8,12,13]. RANKL induces various signaling pathways, including NF-κB pathways, mitogen-activated protein kinase (MAPK) pathways, and calcium signaling [14]. These pathways activate downstream transcription factors, such as c-Fos, nuclear factor of activated T cells c1 (NFATc1), and B lymphocyte-induced maturation protein-1 (Blimp1), promoting osteoclast differentiation [14,15]. Conversely, negative regulators mediating intrinsic anti-osteoclast formation mechanisms, such as the FDCP 6 homologous gene (Def6), interferon regulatory factor 8 (IRF8), and v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MafB), play roles in inhibit excessive osteoclast formation and bone resorption [16-18]. In non-canonical pathways, cytokines/growth factors that can substitute RANKL can induce osteoclast formation [19,20]. Substitutes for RANKL include LIGHT, TNF-α, interleukins 6, 11, and 8 [20]. These growth factors also influence the canonical (RANKL-induced) osteoclast formation [20]. Reports indicate that osteoclast formation increases with aging and is also increased by inflammatory cytokines [21]. Recent studies have shown that adipokines influence osteoclastogenesis [22]. Omentin and resistin inhibit osteoclast formation [23,24]. In contrast, leptin, chemerin, and adiponectin promote osteoclast formation by regulating osteoblast formation [25-27].

    Visfatin (pre-B-cell colony-enhancing factor [PBEF] or nicotinamide phosphoribosyltransferase [NAMPT]) is an adipokine that is primarily secreted by cells but can also be found intracellularly [28,29]. Visfatin (iNAMPT) performs a predominant intracellular enzymatic function by catalyzing the rate-limiting step in nicotinamide adenine dinucleotide (NAD) biosynthesis, namely the conversion of nicotinamide to nicotinamide mononucleotide [29,30]. Through this effect, visfatin can regulate the intracellular levels of NAD and, therefore, cellular energy metabolism [29,30]. Visfatin (eNAMPT) is produced by various cell types, including amniotic epithelial cells, monocytes/ macrophages, and neutrophils, and is associated with various inflammatory conditions, including cancer, obesity, type 2 diabetes, and cardiovascular diseases [29,31-33]. In a previous study, we demonstrated that visfatin induces cellular senescence under H2O2-induced conditions [34]. Additionally, we showed that visfatin mediated the production of SASP and inflammatory cytokines through toll-like receptor 4 (TLR4) [35]. However, there are conflicting reports on the effects of visfatin on osteoclast formation. One report suggested that visfatin inhibits osteoclast formation similarly to omentin and resistin, while another report indicated that osteoclast formation is inhibited in osteoclast precursors (bone marrow-derived macrophages [BMDM]) with visfatin knockdown and promoted in osteoclast precursors with visfatin overexpression [36-38]. Recently, we confirmed that visfatin induces osteoclast differentiation and promotes RANKL-induced osteoclastogenesis [39]. However, the mechanism through which visfatin affects osteoclast differentiation remains unclear.

    In this study, we investigated the molecular mechanism by which visfatin enhances RANKL-induced osteoclast differentiation in mouse BMDM, and examined whether RANKLinduced osteoclast differentiation is influenced by visfatin enzymatic activity using the visfatin-specific enzyme inhibitor FK866.

    Materials and Methods

    1. Antibodies and reagents

    The following antibodies were used in this study: β-Actin (Abcam); DC-STAMP (Novus); Cathepsin K (Biovision); visfatin and NFATc1 (Santa Cruz Biotechnology); Integrin-β3 (Cell Signaling Technology); and horseradish peroxidase-conjugated IgG (ENZO). Visfatin was purchased from AdipoGen. Macrophage colony-stimulating factor (M-CSF) and RANKL were purchased from PeproTech.

    2. Mouse bone marrow-derived macrophage preparation

    All animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication no. 85-23, revised 1996) and approved by the Institutional Animal Care and Use Committee at Pusan National University, Korea (PNU-IACUC; approval code: PNU-2023-0401; approval date: 11 December 2023). 6-week-old C57BL/6 mice (Koatech) were used in this study. BMDMs were isolated from the whole bone marrow of mice as described previously. The harvested bone marrow cells were cultured in alpha minimum essential medium (α-MEM) (Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL), 1% penicillin/streptomycin (Gibco BRL), and M-CSF (20 ng/mL). After 3 days of incubation, the medium was replaced under the same conditions. Adherent cells cultured for 7 days were used as BMDMs for further analysis.

    3. Osteoclast differentiation and tartrate-resistant acid phosphatase staining

    Mouse BMDMs were cultured in 24-well plates at 37℃ and 5% CO2 in α-MEM supplemented with 10% FBS, 20 ng/mL M-CSF, and 100 ng/mL RANKL for 6 days, with the medium being replaced every 3 days. Control cells were treated with M-CSF only, without RANKL supplementation. Subsequently, the cells were stained for tartrate-resistant acid phosphatase (TRAP) using a TRACP and ALP double-staining kit (Takara), following the manufacturer’s protocol. The area of TRAP (+) cells was measured using the ImageJ software.

    4. Western blot analysis

    The harvested cells were lysed in radioimmunoprecipitation assay buffer (iNtRON Biotechnology) containing a protease inhibitor cocktail (Roche). Protein extracts (30 µg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech). Subsequently, the membranes were blocked with 5% skim milk in phosphate-buffered saline containing 0.1% Tween 20 for 1 hour at room temperature and probed with the appropriate antibodies. Protein blots were visualized using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech).

    5. Cell viability assay

    Mouse BMDMs were plated in a 96-well plate and cultured with 20 ng/mL M-CSF and 1, 2, 5, 10, 100, 1,000, and 10,000 nM FK866 for 24 hours. Then, 5 μg/mL of 3-(4,5-Dimethylthiazol- 2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) (Invitrogen) was added into each well and incubated at 37℃ for 90 minutes. Absorbance was measured at 450 nm using a Victor X3 P Multimode Plate Reader (Perkin Elmer).

    6. Statistical analysis

    Data are presented as the mean ± standard deviation from at least three independent experiments. For comparisons among multiple groups, a one-way analysis of variance followed by Tukey’s honest significant difference post-hoc test was used. Statistical significance was considered at a p-value of less than 0.05, with significance levels indicated as **p ＜ 0.01, and ***p ＜ 0.001.

    Results

    1. Visfatin induces osteoclast differentiation in BMDM

    We recently reported that visfatin enhanced RANKL-induced osteoclastogenesis [39]. In the present study, we confirmed that visfatin increases osteoclast differentiation. We first investigated the effects of visfatin on osteoclast differentiation in BMDMs at various concentrations (0, 50, 250, and 500 ng/ mL). Since 500 ng/mL of visfatin exhibited an osteoclastogenic effect on BMDMs, we selected this concentration for subsequent experiments. The other concentrations did not significantly affect osteoclast differentiation and were therefore excluded from further analysis. As shown in Fig. 1A, visfatin induced osteoclast differentiation on its own, although to a lesser extent than in the RANKL-treated group. Osteoclasts induced by visfatin were approximately 2–2.5 times fewer numbers and smaller than those induced by RANKL (Fig. 1B and 1C).

    2. Visfatin upregulates the expression of osteoclastassociated molecules

    To investigate the molecular mechanism by which visfatin affects osteoclast formation, BMDM were treated with visfatin and the expression of NFATc1, a key transcription factor in osteoclastogenesis, was examined. The RANKL-treated group was used as a control to compare expression levels. On day 1, NFATc1 was upregulated in both RANKL-treated and visfatintreated groups. On day 3, the visfatin-treated group showed a higher expression of NFATc1 than the RANKL-treated group (Fig. 2A and 2B). Then, on day 6, when osteoclast differentiation was expected to occur, the expression levels of NFATc1, DC-STAMP (associated with osteoclast fusion), Cathepsin K, and Integrin-β3 (osteoclast markers) proteins were examined. These markers were expressed at lower levels in visfatintreated groups than in RANKL-induced differentiation but were clearly upregulated in comparison to the control (Fig. 2C).

    3. Visfatin is upregulated during RANKL-induced osteoclast differentiation

    We examined the expression of visfatin during RANKLinduced osteoclast differentiation using western blot analysis. BMDMs were stimulated with 100 ng/mL RANKL or 500 ng/ mL visfatin, and visfatin protein expression was examined on 1 day and 3 days (Fig. 3). The results showed that visfatin levels increased on day 1 and decreased on day 3 after the treatment of visfatin. In the RANKL-treated group, visfatin expression was not prominent on day 1 but was expressed on day 3.

    4. FK866 attenuated visfatin-induced osteoclastogenensis

    To confirm whether FK866, a well-known enzyme inhibitor of visfatin, inhibited RANKL-induced osteoclast differentiation, we performed an MTT assay to determine the viability of BMA DMs (Fig. 4A). BMDMs treated with FK866 at concentrations of 1, 2, and 5 nM showed cell viability of approximately 80% or higher. Thus, based on this, we pretreated BMDM with 5 nM FK866 and found that visfatin-induced osteoclast differentiation was completely inhibited by FK866 (Fig. 4B).

    5. FK866 attenuated RANKL-induced osteoclastogenensis

    As visfatin expression was induced by RANKL, we also confirmed whether RANKL-induced osteoclast differentiation was regulated by FK866 (Fig. 5A and 5B). After pretreatment of BMDM with FK866 at concentrations of 1, 2, and 5 nM and stimulation with RANKL, there was no effect at 1 and 2 nM; however, at 5 nM, osteoclast differentiation was entirely inhibited (Fig. 5A). Upon examining the protein expression of osteoclast differentiation markers, it was found that NFATc1, DCSTAMP, Cathepsin K, and integrin-β3, which were increased by RANKL, were also reduced by FK866 (Fig. 5B).

    Discussion

    This study demonstrates that visfatin regulates osteoclast differentiation through its enzymatic activity. Previous studies on visfatin and bone metabolism have shown that the expression and activity of visfatin in osteoblasts influences osteoclast recruitment during alveolar bone remodeling [40]. In addition, visfatin enzymatic activity affects osteoclast activity [41]. Our recent studies reported that extracellular visfatin induces osteoclast differentiation in BMDMs and accelerates RANKL-induced osteoclast differentiation [39]. Based on these findings, visfatin appears to play an important role in bone metabolism.

    Visfatin plays important roles in various biological processes, including osteoclast differentiation; however, its mechanism of action remains unclear. Although visfatin possesses enzymatic activity and acts as an adipokine and extracellular ligand, its receptor is still not well understood. Previous studies have suggested potential receptors such as the insulin receptor and TLR4, but the exact mechanism of action of visfatin remains unclear [35,42-44]. In addition to receptor-mediated signaling, visfatin may increase NAD+ biosynthesis through its enzymatic activity [29,30].

    Understanding the mechanism by which visfatin regulates bone metabolism is essential for clarifying its role in bone health and developing targeted therapies to alleviate bone loss-related disorders caused by uncontrolled bone resorption, such as osteoporosis and periodontitis [1,4,5]. Visfatin mediates various cellular responses, such as inflammation, through signaling pathways like NF-κB and MAPK, but it is also known to promote NAD+ biosynthesis through its enzymatic activity, acting both intracellularly (iNAMPT) and extracellularly (eNAMPT) [28-30]. Previous reports have shown that extracellular NAD+ regulates osteoclast formation in mouse BMDM and controls Ca2+ metabolism in human osteoblasts, inducing apoptosis [45,46]. In this study, visfatin, which was upregulated by RANKL treatment, may have been released both intracellularly and extracellularly to regulate NAD+ levels or act as a ligand. Therefore, the inhibition of osteoclast differentiation by FK866 treatment may be due to either the direct inhibition of enzymatic activity or the antioxidant properties of FK866, which have been shown to inhibit ROS in previous studies [47].

    NAD+ metabolism plays an important role in macrophage growth and function [48,49]. Macrophages with depleted NAD+ levels undergo necroptosis [49]. Therefore, in this study, it was necessary to determine the appropriate concentration of FK866 required to maintain cell activity in BMDMs using cytotoxicity assays. BMDMs treated with 1, 2, or 5 nM FK866 showed approximately 80% cell viability. However, BMDMs pretreated with 1 or 2 nM FK866, followed by RANKL stimulation, successfully differentiated into osteoclasts. Therefore, the inhibition of RANKL-induced osteoclast differentiation in BMDMs treated with 5 nM FK866 showed a similar level of cell viability to those treated with 1 or 2 nM FK866, suggesting that the regulation was due to the inhibition of visfatin enzyme activity or function by FK866 rather than the cytotoxicity caused by the higher FK866 concentration.

    Understanding the mechanism of visfatin in bone metabolism will play a crucial role in understanding its function in bone health and in the development of therapies aimed at preventing and alleviating bone loss caused by the uncontrolled differentiation and activation of osteoclasts, such as periodontitis and osteoporosis. Therefore, based on this study, further research on the mechanisms of visfatin in bone metabolism and the discovery of new drugs targeting visfatin regulation is expected.

    Funding

    This work was supported by a 2-Year Research Grant of Pusan National University.

    Conflicts of Interest

    No potential conflict of interest relevant to this article was reported.

    Figure

    IJOB-50-1-17_F1.gif

    Effects of visfatin on osteoclast formation in BMDMs. BMDMs were treated with 100 ng/mL RANKL or 500 ng/mL visfatin for 6 days. (A) TRAP staining was performed to determine osteoclasts generation (arrow; magnification, ×200). (B) The number of TRAPpositive and multinucleated cells (≥ 3 nuclei) was counted. (C) The area of TRAP-positive and multinucleated cells (≥ 3 nuclei).

    BMDM, bone marrow-derived macrophages; RANKL, receptor activator of nuclear factor kappa-B ligand; TRAP, tartrate-resistant acid phosphatase.

    **p < 0.01, ***p < 0.001.

    IJOB-50-1-17_F2.gif

    Effects of visfatin on osteoclast differentiation in BMDMs. BMDMs were treated with 100 ng/mL RANKL or 500 ng/mL visfatin for 1 day, 3 days, and 6 days. (A, B) Western blotting for measure protein levels of NFATc1, with β-Actin used as a control. (C) Western blotting for measure protein levels of NFATc1, DC-STAMP, Cathepsin K, and Integrin-β3, with β-Actin used as a control.

    BMDM, bone marrow-derived macrophages; RANKL, receptor activator of nuclear factor kappa-B ligand; NFATc1, nuclear factor of activated T cells c1; DC-STAMP, dendritic cellspecific transmembrane protein.

    IJOB-50-1-17_F3.gif

    Expression of visfatin in RANKL-stimulated BMDMs. BMDMs were treated with 100 ng/mL RANKL or 500 ng/mL visfatin for 1 day and 3 days. Western blotting for measure protein levels of visfatin, with β-Actin used as a control.

    RANKL, receptor activator of nuclear factor kappa-B ligand; BMDM, bone marrow-derived macrophages.

    IJOB-50-1-17_F4.gif

    Effects of FK866 on visfatin-induced osteoclast formation in BMDMs. (A) BMDMs were treated 1, 2, 5, 10, 100, 1,000, 10,000 nM FK866 for 1 day. MTT assay performed to measure cytotoxicity of FK866 on BMDMs. (B) BMDMs were pretreated 5nM FK866 for 2 hours and then stimulated with 500 ng/mL visfatin for 6 days. TRAP staining was performed to determine osteoclasts generation (arrow; magnification, ×200).

    BMDM, bone marrow-derived macrophages; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide; TRAP, tartrate-resistant acid phosphatase.

    IJOB-50-1-17_F5.gif

    Effects of FK866 on RANKL-induced osteoclast differentiation in BMDMs. BMDMs were pretreated 1, 2, and 5 nM FK866 for 2 hours and then stimulated with 100 ng/mL RANKL for 6 days. (A) TRAP staining was performed to determine osteoclasts generation (magnification, ×200). (B) Western blotting for measure protein levels of NFATc1, DC-STAMP, Cathepsin K, and Integrin-β3, with β-Actin used as a control.

    RANKL, receptor activator of nuclear factor kappa-B ligand; BMDM, bone marrow-derived macrophages; TRAP, tartrate-resistant acid phosphatase; NFATc1, nuclear factor of activated T cells c1; DC-STAMP, dendritic cell-specific transmembrane protein.

    Table

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