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Suppressive effects of (-)-tubaic acid on RANKL-induced osteoclast differentiation and bone resorption

Suppressive effects of (-)-tubaic acid on RANKL-induced osteoclast differentiation and bone... ANIMAL CELLS AND SYSTEMS 2023, VOL. 27, NO. 1, 1–9 https://doi.org/10.1080/19768354.2023.2166107 Suppressive effects of (-)-tubaic acid on RANKL-induced osteoclast differentiation and bone resorption a, b, a c d e a Soomin Lim *, Hye Jung Ihn *, Ju Ang Kim , Jong-Sup Bae , Jung-Eun Kim , Yong Chul Bae , Hong-In Shin , f a Tae Hoon Kim and Eui Kyun Park Department of Oral Pathology and Regenerative Medicine, School of Dentistry, IHBR, Kyungpook National University, Daegu, Republic of b c Korea; Cell and Matrix Research Institute (CMRI), Kyungpook National University, Daegu, Republic of Korea; College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Republic of Korea; Department of Molecular Medicine, CMRI, School of Medicine, Kyungpook National University, Daegu, Republic of Korea; Department of Oral Anatomy and Neurobiology, School of Dentistry, Kyungpook National University, Daegu, Republic of Korea; Department of Food Science and Biotechnology, Daegu University, Gyeongsan, Republic of Korea ABSTRACT ARTICLE HISTORY Received 20 November 2022 Regulation of osteoclastogenesis and bone-resorbing activity can be an efficacious strategy for Revised 14 December 2022 treating bone loss diseases because excessive osteoclastic bone resorption leads to the Accepted 3 January 2023 development of such diseases. Here, we investigated the role of (-)-tubaic acid, a thermal degradation product of rotenone, in osteoclast formation and function in an attempt to identify KEYWORDS alternative natural compounds. (-)-Tubaic acid significantly inhibited receptor activator of (-)-tubaic acid; osteoclast; nuclear factor-κB ligand (RANKL)-mediated osteoclast differentiation at both the early and late bone resorption; nuclear stages, suggesting that (-)-tubaic acid affects the commitment and differentiation of osteoclast factor of activated T-cells progenitors as well as the cell-cell fusion of mononuclear osteoclasts. (-)-Tubaic acid attenuated cytoplasmic 1 (NFATc1) the activation of extracellular signal-regulated kinase (ERK) and expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and its target genes in response to RANKL. Furthermore, a pit-formation assay revealed that (-)-tubaic acid significantly impaired the bone- resorbing activity of osteoclasts. Our results demonstrated that (-)-tubaic acid exhibits anti- osteoclastogenic and anti-resorptive effects, indicating its therapeutic potential in the management of osteoclast-related bone diseases. Introduction proliferation and survival of osteoclast precursors and receptor activator of nuclear factor-κB ligand (RANKL) Osteoclastic bone resorption and osteoblastic bone promoting their differentiation into osteoclasts (Cappel- formation are balanced in physiological states that len et al. 2002). The interaction between RANKL and its sustain bone mass and mineral homeostasis (Siddiqui receptor RANK, expressed on osteoclast precursors, and Partridge 2016). However, bone-resorbing activity acts as a stimulating signal and activates multiple increases with aging and pathological conditions, and signal transduction pathways, including mitogen-acti- the imbalance in bone remodeling ultimately reduces vated protein kinases (MAPKs) and nuclear factor-κB the amount and quality of bones (Almeida and O’Brien (NF-κB). These ultimately stimulate the induction of the 2013). Several skeletal diseases are strongly associated essential transcription factor, the nuclear factor of acti- with enhanced osteoclast activation; therefore, osteo- vated T-cells cytoplasmic 1 (NFATc1), which regulates clasts have been considered promising targets for the the expression of genes required for osteoclast differen- development of therapeutic interventions to manage tiation and function (Takayanagi et al. 2002; Park et al. osteoclast-related bone diseases. 2017). Typically, osteoclasts are derived from monocyte/ Rotenoid compounds exhibit diverse biological and macrophage progenitors through several steps. The pharmacological functions, including antibacterial, anti- differentiation process is dependent on macrophage fungal, anticancer, and anti-inflammatory functions colony-stimulating factor (M-CSF) supporting the CONTACT Eui Kyun Park epark@knu.ac.kr Department of Oral Pathology and Regenerative Medicine, School of Dentistry, IHBR, Kyungpook National University, Daegu, Republic of Korea; Tae Hoon Kim skyey7@daegu.ac.kr Department of Food Science and Biotechnology, Daegu University, Gyeongsan, Republic of Korea *These authors contributed equally to this work. Supplemental data for this article can be accessed online at https://doi.org/10.1080/19768354.2023.2166107. © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 S. LIM ET AL. (Fang and Casida 1998; Takashima et al. 2002; Mathias Thermal transformation of rotenone and et al. 2005). Rotenone is a member of the rotenoid isolation of (-)-tubaic acid family, and attenuates osteoclast differentiation and A sample solution of rotenone (1.0 g) in H O (2.0 L) in suppresses inflammatory bone loss (Kwak et al. 2010). capped vials was autoclaved at 121°C for 12 h. The ′ ′ Rotenone derivative, 1 ,2 -dihydrorotenone, also exerts dried reactant was directly subjected to column chrom- a negative activity on osteoclastogenesis (Lee et al. atography over a YMC GEL ODS AQ 120-50S column 2011). One of degradation products of rotenone is a (1.1 cm i.d. × 36 cm) with aqueous MeOH to yield pure dihydrobenzofuran (coumaran) derivative, tubaic acid (-)-tubaic aid (16.6 mg). High-performance liquid chrom- (Figure 1(a), Fig. S1) (Cheng et al. 1972). Dihydrobenzo- atography (HPLC) analysis was performed on a YMC- furan is proposed as an important heterocyclic motif in Pack ODS A-302 column (4.6 mm i.d. × 150 mm; YMC biologically active natural products (Choi et al. 2015; Co., Ltd.), which consisted of a linear gradient that Sunden et al. 2016). Numerous natural bioactive com- started with 10% (v/v) MeCN in 0.1% HCOOH/H O pounds carrying a 2,3-dihydrobenzofuran scaffold play (detection: UV 280 nm; flow rate: 1.0 ml/min; at 40°C), beneficial roles in inflammation and HIV infection. increased to 80% MeCN over 23 min, and then to Lithospermic acid, containing the 2,3-dihydrobenzo- 100% MeCN over 5 min. The structure of the newly gen- furan skeleton isolated from Salvia miltiorrhiza roots, erated compound was determined based on the exhibits anti-HIV activity in H9 cells (Abd-Elazem et al. interpretation of the spectroscopic data. 2002). Inagaki et al. reported that the 2,3-dihydrobenzo- furan derivative, (E)-5-(7-tert-Butyl-3,3-dimethyl-2,3- dihydrobenzofuran-5-ylmethylene)-2-ethyl-1,2-isothia- Osteoclast differentiation and TRAP staining zolidine 1,1-dioxide, inhibited PGE2 production and Osteoclasts were generated from bone marrow-derived demonstrated anti-inflammatory activity in a carragee- macrophages (BMMs) as previously described (Ihn nan-induced footpad edema model (Inagaki et al. et al. 2019; Ihn et al. 2019; Ahn et al. 2021). Bone 2003). Furthermore, kadsurenone, a natural platelet-acti- marrow cells obtained from 6–8-week-old C57BL/6 vating factor (PAF) antagonist, has a 2,3-dihydrobenzo- mice (Dae Han Bio Link, Chungbuk, Korea) were incu- furan structure and attenuates breast cancer (BC) cell- bated in α-MEM containing 10% FBS for 24 h, and non- induced osteoclast formation as well as PAF-induced adherent cells were incubated in the presence of BC cell migration, supporting it as a potential thera- 30 ng/ml M-CSF for three days to generate BMMs. To peutic agent for BC-induced bone metastases (Hou induce osteoclastogenesis, BMMs were cultured with et al. 2018). Although tubaic acid is a pivotal intermedi- RANKL (20 ng/ml) and M-CSF (10 ng/ml) in the presence ate in rotenone synthesis and contains a 2,3-dihydro- of (-)-tubaic acid (0, 1, 2, or 5 μM). After four days, the cul- benzofuran skeleton, little is known about its biological tured cells were stained using the TRAP staining kit, and functions. Thus, we examined the therapeutic potential TRAP-positive cells containing over three nuclei were of tubaic acid in osteoclast-related bone diseases in quantified by manually counting under a Leica light this study. Furthermore, we investigated the effects of microscope (Leica, Germany). tubaic acid on osteoclastogenesis, bone resorption, and related action mechanisms. Cytotoxicity assay Materials and methods The cytotoxic effects were evaluated using the MTT assay (Jeong et al. 2021). BMMs were incubated with Fetal bovine serum (FBS) and α-minimum essential different doses of (-)-tubaic acid in the presence of medium (α-MEM) were obtained from Gibco BRL M-CSF (10 ng/ml). After three days, the MTT reagent (Grand Island, NY, USA). Recombinant murine M-CSF was added to each well, and the cells were incubated and RANKL were purchased from R&D Systems (Minnea- at 37°C for 2 h. The formazan crystals were solubilized polis, MN, USA). The acid phosphatase, leukocyte (tar- with DMSO, and cell viability was assessed by measuring trate-resistant acid phosphatase: TRAP) kit, the absorbance at 570 nm using a 96-well microplate methylthiazolyldiphenyl-tetrazolium bromide (MTT), reader (Bio-Rad, Hercules, CA, USA). dimethyl sulfoxide (DMSO), and all other reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA). (-)-Tubaic acid (4-hydroxy-2-(1-methylethenyl)- Real-time PCR 2,3-dihydrobenzofuran-5-carboxylic acid) was isolated by the thermal transformation of rotenone, and the iso- Total RNA was extracted using TRI-solution (Bio Science lation process is described later in this section. Technology, Daegu, Korea), according to the ANIMAL CELLS AND SYSTEMS 3 Figure 1. (-)-Tubaic acid suppresses RANKL-mediated osteoclastogenesis without inducing cytotoxicity. (A) Chemical structure of (-)-tubaic acid. (B) Bone marrow-derived macrophages (BMMs) were incubated with M-CSF (10 ng/ml) and various doses of (-)-tubaic acid, and cell viability was assessed using the MTT assay. (C) The BMMs were cultured with M-CSF (10 ng/ml) and RANKL (20 ng/ml) in the presence of (-)-tubaic acid or vehicle. After four days, the cells were stained to assess TRAP activity. Scale bar, 50 μm. (D) Quantification of TRAP-positive multinucleated cells (MNCs) with over three nuclei. **p < 0.01 versus vehicle- treated control, t-test. manufacturer’s instructions, and complementary DNA ml RANKL, with or without 5 μM (-)-tubaic acid. After four (cDNA) was synthesized using SuperScript II Reverse days, the cells were fixed with 4% paraformaldehyde, Transcriptase (Invitrogen, Carlsbad, CA, USA). Quantitat- treated with 0.25% Triton X-100, and stained with an ive real-time PCR was performed using a LightCycler 1.5 anti-NFATc1 antibody (Santa Cruz Biotechnology, Santa real-time PCR system (Roche Diagnostics, Basel, Switzer- Cruz, CA, USA), followed by an Alexa Fluor-488-conju- land) and SYBR Premix Ex Taq (Takara Bio Inc., Shiga, gated secondary antibody. Rhodamine-conjugated phal- Japan). Primer sequences for osteoclast-specific genes: loidin (Cytoskeleton, Denver, CO, USA) and 4 ,6- ′ ′ ′ TRAP (Acp5), 5 -TCCCCAATGCCCCATTC-3 and 5 -CGGTT diamidino-2-phenylindole dihydrochloride (DAPI; Santa ′ ′ CTGGCGATCTCTTTG-3 ; Ctsk,5 -GGCTGTGGAGGCGGCT Cruz Biotechnology, Santa Cruz, CA, USA) were used to ′ ′ ′ AT-3 and 5 -AGAGTCAATGCCTCCGTTCTG-3 ; Dcstamp, stain F-actin and cell nuclei, respectively. Cells with ′ ′ ′ 5 -CTTCCGTGGGCCAGAAGTT-3 and 5 -AGGCCAGTGCT actin rings or nuclear NFATc1 were counted from 10 ′ ′ ′ GACTAGGATGA-3 ; Nfatc1,5 -ACCACCTTTCCGCAACCA-3 random selected views using the Image J software ′ ′ and 5 -TTCCGTTTCCCGTTGCA-3 . (NIH, Bethesda, MA, USA). Immunofluorescence staining Western blotting BMMs were plated on glass coverslips and incubated in Cells were lysed in RIPA lysis buffer supplemented with α-MEM containing 10% FBS, 10 ng/ml M-CSF, and 20 ng/ protease and phosphatase inhibitors, and the total 4 S. LIM ET AL. protein concentration was measured using a BCA acid almost completely blocked osteoclast formation Protein Assay Kit (Pierce Biotechnology, Rockford, IL, (97.79% inhibition; Figure 1(d)). To further confirm the USA). Proteins (30 μg) were subjected to 10% SDS- stage at which (-)-tubaic acid impaired osteoclast for- PAGE and transferred onto a nitrocellulose membrane mation, (-)-tubaic acid (5 μM) was added to the culture (Whatman, Florham Park, NJ, USA). Membranes were medium at different periods, as previously reported immersed in 3% non-fat milk in Tris-buffered saline (Ihn et al. 2017; Lim et al. 2020). Addition of (-)-tubaic with 0.1% Tween 20 (TBS-T) for 1 h before incubation acid in the early (Period I: from days 0–2) or late with primary antibodies against p-p38, p-JNK, p-ERK, (Period II: from days 2–4) stages of osteoclast differen- p-MEK, p-AKT, p-IκBα (Cell Signaling Technology, tiation, the formation of TRAP-positive MNCs was signifi- Danvers, MA, USA), and β-actin (Sigma–Aldrich, cantly decreased (Figure 2(a)). The number of MNCs was St. Louis, MO, USA) at 4°C. Immunoreactive bands were reduced by 71.2% and 78.2% in the early and late treat- detected using a WesternBright ECL kit (Advansta, ments, respectively (Figure 2(b)), indicating that Menlo Park, CA, USA) and recorded using a chemilumi- (-)-tubaic acid can affect both stages, including the com- nescence imager (Azure Biosystems, Inc., Dublin, CA, mitment to osteoclastic differentiation and cell-cell USA). fusion. In vitro resorption assay (-)-Tubaic acid reduces osteoclast marker expression BMMs seeded on bone slices (IDS Nordic, Herlev, Denmark) were incubated in an osteoclastogenic To further determine the anti-osteoclastogenic potential medium for three days, followed by treatment with of (-)-tubaic acid, mRNA expression levels of NFATc1 and vehicle or 5 μM (-)-tubaic acid. After two days, the its target genes related to osteoclast differentiation and bone slices were washed and immersed in hematoxylin function were assessed. As shown in Figure 2(c), solution to visualize the resorption pits. The area of the (-)-tubaic acid (5 μM) suppressed the expression of pits was quantified by analyzing 10 randomly selected Nfatc1 and downstream osteoclast marker genes, such images using i-Solution image analysis software (IMT as TRAP (Acp5), Dcstamp, and cathepsin K (Ctsk). The i-Solution, Daejeon, Korea). protein levels of NFATc1 and cathepsin K were signifi- cantly down-regulated by (-)-tubaic acid treatment (Figure 2(d)). We also observed reduced nuclear localiz- Statistical analysis ation of NFATc1 in (-)-tubaic acid-treated cells (Figure 3 Experiments were conducted in triplicate and repeated (a,c)). three times, and the results are presented as the mean ± standard deviation (SD). A two-tailed student’s t-test was used to determine statistical significance. *P < 0.05 (-)-Tubaic acid suppresses the formation of actin or **P < 0.01 was considered statistically significant. rings and resorption pits As osteoclasts form actin rings around the cell periphery, Results which serves as an indicator of active osteoclasts and are vital for bone resorption (Boyce et al. 1992), the effects of (-)-Tubaic acid suppresses RANKL-mediated (-)-tubaic acid on actin ring formation and bone resorp- osteoclast differentiation in BMMs tion activity were examined. Phalloidin staining revealed To explore the effects of (-)-tubaic acid (Figure 1(a)) on that F-actin rings were present in the vehicle-treated RANKL-induced osteoclastogenesis, we first investigated control group, whereas the addition of 5 μM (-)-tubaic whether (-)-tubaic acid affects the viability of BMMs. The acid impaired actin ring formation (Figure 3(a,b)). Next, MTT assay results revealed that (-)-tubaic acid did not we performed a pit formation assay to explore the exhibit cytotoxicity, even at 5 μM, and increased the via- effects of (-)-tubaic acid on osteoclastic bone resorption. bility of BMMs (Figure 1(b)). When BMMs were cultured BMMs were cultured on bone slices in an osteoclast in an osteoclastogenic medium with different doses of induction medium for three days, and the cells were (-)-tubaic acid for four days, (-)-tubaic acid treatment sig- treated with either 5 μM (-)-tubaic acid or vehicle. nificantly suppressed the formation of TRAP-positive Numerous hematoxylin-stained resorption pits were multinucleated cells (MNCs) from BMMs in a dose- formed in the vehicle-treated control; however, dependent manner compared with that in the vehicle- (-)-tubaic acid treatment led to a significant decrease treated control (Figure 1(c)). Notably, 5 μM (-)-tubaic in the resorbed area (Figure 4(a,b)). ANIMAL CELLS AND SYSTEMS 5 Figure 2. (-)-Tubaic acid inhibits early and late stages of osteoclast differentiation and osteoclast-specific marker expression. (A) The BMMs were cultured in the presence of M-CSF (10 ng/ml) and RANKL (20 ng/ml), and the cells were treated with (-)-tubaic acid (5 μM) for the indicated period. Period I : from days 0–2, Period II : from days 2–4. Scale bar, 50 μm. (B) Quantification of TRAP-positive MNCs. (C, D) The BMMs were incubated in the presence of M-CSF (10 ng/ml) and RANKL (20 ng/ml) with or without (-)-tubaic acid (5 μM) for four days. The mRNA (C) and protein (D) expression levels of osteoclast markers were assessed by real-time PCR and immunoblotting, respectively. *p < 0.05, **p < 0.01 versus vehicle-treated control, t-test. (-)-Tubaic acid attenuates RANKL-mediated ERK (-)-tubaic acid reduced the RANKL-induced phosphoryl- activation ation of MEK, the upstream activator of ERK, and ERK without affecting the phosphorylation of JNK, p38, Akt, Mitogen-activated protein kinases [c-Jun-N-terminal and IκBα (Figure 4(c,d)). kinase (JNK), extracellular signal-regulated kinase (ERK), and p38] and NF-κB pathways are activated in response to RANKL stimulation, and they are essential for osteo- Discussion clast differentiation and function (Mizukami et al. 2002). As (-)-tubaic acid exhibits anti-osteoclastogenic Natural compounds and their derivatives have attracted and anti-resorptive potential, its effect on RANKL- attention as essential resources for the development of mediated signaling pathways was investigated to therapeutic agents for decades, owing to their potential better understand the molecular mechanism. After pre- medicinal properties. Phytoestrogens, including isofla- treatment with (-)-tubaic acid or vehicle, BMMs were vones, lignans, and coumestrol, are polyphenolic com- stimulated with RANKL, and the phosphorylation levels pounds, and several reports highlight their beneficial of signaling molecules were assessed by immunoblot- effects on bone health (Castelo-Branco and Cancelo ting. Upon RANKL treatment, phosphorylation of Hidalgo 2011; Abdi et al. 2016). Tubaic acid is produced MAPKs and IκBα increased within 15 min in control by the degradation of rotenone, a naturally occurring cells (Figure 4(c,d)). In contrast, pretreatment with isoflavone compound that exhibits antimicrobial activity 6 S. LIM ET AL. Figure 3. (-)-Tubaic acid impairs actin ring formation and decreases nuclear localization of NFATc1. (A) BMMs were incubated on glass coverslips in an osteoclastogenic medium containing M-CSF (10 ng/ml) and RANKL (20 ng/ml) with (-)-tubaic acid (5 μM) or vehicle. After four days, the cells were fixed and probed with anti-NFATc1 antibody (green), followed by staining with rhodamine-conjugated phalloidin (red) and DAPI (blue). Yellow dashed rectangles were magnified in the lower panels. Scale bar, 50 μm. Quantification of the percentages of (B) cells displaying actin rings and (C) cells with nuclear NFATc1. **p < 0.01 versus vehicle-treated control, t-test. (Obara et al. 1976). Tubaic acid contains a 2,3-dihydro- M-CSF plays essential roles in the survival, prolifer- benzofuran scaffold, which is considered a vital ation, and differentiation of osteoclast precursor cells, element in many biologically active natural compounds and binding of M-CSF to its receptor, c-Fms, activates (Qin et al. 2017). To date, the biological and pharmaco- various signaling molecules, including ERK and logical properties of tubaic acid have rarely been PI3 K/Akt (Richardson et al. 2015). BMMs are widely studied. In this study, we demonstrated that (-)-tubaic used as osteoclast precursors in an in vitro osteoclasto- acid inhibited RANKL-induced osteoclastogenesis and genesis system, and an increase in cell viability was suppressed bone-resorbing activity. observed when BMMs were treated with (-)-tubaic acid ANIMAL CELLS AND SYSTEMS 7 Figure 4. (-)-Tubaic acid reduces resorption pit formation and attenuates RANKL-induced activation of ERK. (A) The BMMs were cul- tured on bone slices with M-CSF (10 ng/ml) and RANKL (20 ng/ml) to induce osteoclast differentiation. After three days, the cells were treated with (-)-tubaic acid (5 μM) or vehicle for an additional two days. The bone slices were stained with hematoxylin. Scale bar, 100 μm. (B) Quantification of the resorbed area. (C, D) Serum-starved BMMs were pretreated with (-)-tubaic acid (5 μM) or vehicle for 1 h, followed by stimulation with 50 ng/ml RANKL for indicated times. The phosphorylation levels of (C) ERK, Akt, JNK, p38, and (D) IκBα were examined by western blotting. The graph (right panel) represents the relative band intensity of phosphorylated ERK. *p < 0.05, **p < 0.01, t-test. (Figure 1(b)), suggesting that tubaic acid may affect one osteoclast-related genes was attenuated in the presence of the c-Fms signaling pathways to induce cell survival or of (-)-tubaic acid (Figure 2(c,d)), which is consistent with proliferation. Osteoclast differentiation comprises the TRAP staining results, indicating that (-)-tubaic acid several steps, including commitment and differentiation has anti-osteoclastogenic properties. Cell-cell fusion to mononuclear preosteoclasts, cellular fusion to multi- required for the formation of multinucleated cells nuclear osteoclasts, and activation of bone-resorbing during osteoclastogenesis is crucial for efficient bone osteoclasts (Xing et al. 2012). (-)-Tubaic acid significantly resorption (Lee et al. 2009), and DC-STAMP is well estab- suppressed both the early and late stages of osteoclast lished as an essential regulator of this process (Yagi et al. differentiation, with over 70% inhibition of osteoclast 2005). Genetic ablation of Dcstamp results in the failure formation (Figure 2(b)), indicating that it impairs preos- to generate multinuclear osteoclasts, leading to a teoclast formation and cellular fusion in the early and reduction in bone-resorbing activity (Yagi et al. 2005). late stages, respectively. We also confirmed the inhibi- Although mononuclear osteoclastic cells in DC-STAMP tory effects of (-)-tubaic acid at the molecular level by knockout mice can resorb bone, their resorbing activity assessing changes in the expression of osteoclast- is lower than that of multinucleated osteoclasts (Yagi specific markers. The expression of NFATc1 and et al. 2005). Osteoclastic resorption is also affected by 8 S. LIM ET AL. the formation of an actin ring involved in creating a treatment and prevention of bone diseases associated tightly enclosed space, known as a resorption lacuna with excessive osteoclast formation and function. (Novack and Teitelbaum 2008). Switch-associated protein 70 (SWAP-70) deficiency leads to an osteopetro- Data availability tic phenotype caused by ineffective bone resorption owing to defects in actin ring formation (Roscher et al. The data underlying this article are available in the 2016). The formation of actin-based sealing rings is article. impaired in cortactin-depleted osteoclasts, which exhibit a loss of bone-resorbing function (Tehrani et al. Disclosure statement 2006). The presence of (-)-tubaic acid attenuated bone resorption (Figure 4(a)), which correlated with reduced No potential conflict of interest was reported by the author(s). actin ring formation (Figure 3(a)). It inhibited the for- mation of multinucleated osteoclasts (Figure 1(c)). Funding Osteoclast differentiation and bone resorptive function mainly rely on RANKL-RANK signaling, which activates This work was supported by a National Research Foundation of the MAPK pathways (Fuller et al. 1998). Various studies Korea (NRF) grant funded by the Korean government (MSIT): using a pharmacological inhibitor of MEK-ERK or [Grant Number 2017R1A5A2015391, 2020M3A9I4039539]. genetic disruption of Erk1 have demonstrated the importance of the ERK pathway in osteoclast formation ORCID and function (Yan et al. 2007; He et al. 2011). Nakamura et al. observed a significant role of ERK in the survival Jong-Sup Bae http://orcid.org/0000-0002-5756-9367 and polarity of osteoclasts (Nakamura et al. 2003). Additionally, ERK activation by the granulocyte-macro- References phage colony-stimulating factor (GM-SCF) induces DC- STAMP expression, which promotes cell fusion to gener- Abd-Elazem IS, Chen HS, Bates RB, Huang RC. 2002. 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Suppressive effects of (-)-tubaic acid on RANKL-induced osteoclast differentiation and bone resorption

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ANIMAL CELLS AND SYSTEMS 2023, VOL. 27, NO. 1, 1–9 https://doi.org/10.1080/19768354.2023.2166107 Suppressive effects of (-)-tubaic acid on RANKL-induced osteoclast differentiation and bone resorption a, b, a c d e a Soomin Lim *, Hye Jung Ihn *, Ju Ang Kim , Jong-Sup Bae , Jung-Eun Kim , Yong Chul Bae , Hong-In Shin , f a Tae Hoon Kim and Eui Kyun Park Department of Oral Pathology and Regenerative Medicine, School of Dentistry, IHBR, Kyungpook National University, Daegu, Republic of b c Korea; Cell and Matrix Research Institute (CMRI), Kyungpook National University, Daegu, Republic of Korea; College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Republic of Korea; Department of Molecular Medicine, CMRI, School of Medicine, Kyungpook National University, Daegu, Republic of Korea; Department of Oral Anatomy and Neurobiology, School of Dentistry, Kyungpook National University, Daegu, Republic of Korea; Department of Food Science and Biotechnology, Daegu University, Gyeongsan, Republic of Korea ABSTRACT ARTICLE HISTORY Received 20 November 2022 Regulation of osteoclastogenesis and bone-resorbing activity can be an efficacious strategy for Revised 14 December 2022 treating bone loss diseases because excessive osteoclastic bone resorption leads to the Accepted 3 January 2023 development of such diseases. Here, we investigated the role of (-)-tubaic acid, a thermal degradation product of rotenone, in osteoclast formation and function in an attempt to identify KEYWORDS alternative natural compounds. (-)-Tubaic acid significantly inhibited receptor activator of (-)-tubaic acid; osteoclast; nuclear factor-κB ligand (RANKL)-mediated osteoclast differentiation at both the early and late bone resorption; nuclear stages, suggesting that (-)-tubaic acid affects the commitment and differentiation of osteoclast factor of activated T-cells progenitors as well as the cell-cell fusion of mononuclear osteoclasts. (-)-Tubaic acid attenuated cytoplasmic 1 (NFATc1) the activation of extracellular signal-regulated kinase (ERK) and expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and its target genes in response to RANKL. Furthermore, a pit-formation assay revealed that (-)-tubaic acid significantly impaired the bone- resorbing activity of osteoclasts. Our results demonstrated that (-)-tubaic acid exhibits anti- osteoclastogenic and anti-resorptive effects, indicating its therapeutic potential in the management of osteoclast-related bone diseases. Introduction proliferation and survival of osteoclast precursors and receptor activator of nuclear factor-κB ligand (RANKL) Osteoclastic bone resorption and osteoblastic bone promoting their differentiation into osteoclasts (Cappel- formation are balanced in physiological states that len et al. 2002). The interaction between RANKL and its sustain bone mass and mineral homeostasis (Siddiqui receptor RANK, expressed on osteoclast precursors, and Partridge 2016). However, bone-resorbing activity acts as a stimulating signal and activates multiple increases with aging and pathological conditions, and signal transduction pathways, including mitogen-acti- the imbalance in bone remodeling ultimately reduces vated protein kinases (MAPKs) and nuclear factor-κB the amount and quality of bones (Almeida and O’Brien (NF-κB). These ultimately stimulate the induction of the 2013). Several skeletal diseases are strongly associated essential transcription factor, the nuclear factor of acti- with enhanced osteoclast activation; therefore, osteo- vated T-cells cytoplasmic 1 (NFATc1), which regulates clasts have been considered promising targets for the the expression of genes required for osteoclast differen- development of therapeutic interventions to manage tiation and function (Takayanagi et al. 2002; Park et al. osteoclast-related bone diseases. 2017). Typically, osteoclasts are derived from monocyte/ Rotenoid compounds exhibit diverse biological and macrophage progenitors through several steps. The pharmacological functions, including antibacterial, anti- differentiation process is dependent on macrophage fungal, anticancer, and anti-inflammatory functions colony-stimulating factor (M-CSF) supporting the CONTACT Eui Kyun Park epark@knu.ac.kr Department of Oral Pathology and Regenerative Medicine, School of Dentistry, IHBR, Kyungpook National University, Daegu, Republic of Korea; Tae Hoon Kim skyey7@daegu.ac.kr Department of Food Science and Biotechnology, Daegu University, Gyeongsan, Republic of Korea *These authors contributed equally to this work. Supplemental data for this article can be accessed online at https://doi.org/10.1080/19768354.2023.2166107. © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 S. LIM ET AL. (Fang and Casida 1998; Takashima et al. 2002; Mathias Thermal transformation of rotenone and et al. 2005). Rotenone is a member of the rotenoid isolation of (-)-tubaic acid family, and attenuates osteoclast differentiation and A sample solution of rotenone (1.0 g) in H O (2.0 L) in suppresses inflammatory bone loss (Kwak et al. 2010). capped vials was autoclaved at 121°C for 12 h. The ′ ′ Rotenone derivative, 1 ,2 -dihydrorotenone, also exerts dried reactant was directly subjected to column chrom- a negative activity on osteoclastogenesis (Lee et al. atography over a YMC GEL ODS AQ 120-50S column 2011). One of degradation products of rotenone is a (1.1 cm i.d. × 36 cm) with aqueous MeOH to yield pure dihydrobenzofuran (coumaran) derivative, tubaic acid (-)-tubaic aid (16.6 mg). High-performance liquid chrom- (Figure 1(a), Fig. S1) (Cheng et al. 1972). Dihydrobenzo- atography (HPLC) analysis was performed on a YMC- furan is proposed as an important heterocyclic motif in Pack ODS A-302 column (4.6 mm i.d. × 150 mm; YMC biologically active natural products (Choi et al. 2015; Co., Ltd.), which consisted of a linear gradient that Sunden et al. 2016). Numerous natural bioactive com- started with 10% (v/v) MeCN in 0.1% HCOOH/H O pounds carrying a 2,3-dihydrobenzofuran scaffold play (detection: UV 280 nm; flow rate: 1.0 ml/min; at 40°C), beneficial roles in inflammation and HIV infection. increased to 80% MeCN over 23 min, and then to Lithospermic acid, containing the 2,3-dihydrobenzo- 100% MeCN over 5 min. The structure of the newly gen- furan skeleton isolated from Salvia miltiorrhiza roots, erated compound was determined based on the exhibits anti-HIV activity in H9 cells (Abd-Elazem et al. interpretation of the spectroscopic data. 2002). Inagaki et al. reported that the 2,3-dihydrobenzo- furan derivative, (E)-5-(7-tert-Butyl-3,3-dimethyl-2,3- dihydrobenzofuran-5-ylmethylene)-2-ethyl-1,2-isothia- Osteoclast differentiation and TRAP staining zolidine 1,1-dioxide, inhibited PGE2 production and Osteoclasts were generated from bone marrow-derived demonstrated anti-inflammatory activity in a carragee- macrophages (BMMs) as previously described (Ihn nan-induced footpad edema model (Inagaki et al. et al. 2019; Ihn et al. 2019; Ahn et al. 2021). Bone 2003). Furthermore, kadsurenone, a natural platelet-acti- marrow cells obtained from 6–8-week-old C57BL/6 vating factor (PAF) antagonist, has a 2,3-dihydrobenzo- mice (Dae Han Bio Link, Chungbuk, Korea) were incu- furan structure and attenuates breast cancer (BC) cell- bated in α-MEM containing 10% FBS for 24 h, and non- induced osteoclast formation as well as PAF-induced adherent cells were incubated in the presence of BC cell migration, supporting it as a potential thera- 30 ng/ml M-CSF for three days to generate BMMs. To peutic agent for BC-induced bone metastases (Hou induce osteoclastogenesis, BMMs were cultured with et al. 2018). Although tubaic acid is a pivotal intermedi- RANKL (20 ng/ml) and M-CSF (10 ng/ml) in the presence ate in rotenone synthesis and contains a 2,3-dihydro- of (-)-tubaic acid (0, 1, 2, or 5 μM). After four days, the cul- benzofuran skeleton, little is known about its biological tured cells were stained using the TRAP staining kit, and functions. Thus, we examined the therapeutic potential TRAP-positive cells containing over three nuclei were of tubaic acid in osteoclast-related bone diseases in quantified by manually counting under a Leica light this study. Furthermore, we investigated the effects of microscope (Leica, Germany). tubaic acid on osteoclastogenesis, bone resorption, and related action mechanisms. Cytotoxicity assay Materials and methods The cytotoxic effects were evaluated using the MTT assay (Jeong et al. 2021). BMMs were incubated with Fetal bovine serum (FBS) and α-minimum essential different doses of (-)-tubaic acid in the presence of medium (α-MEM) were obtained from Gibco BRL M-CSF (10 ng/ml). After three days, the MTT reagent (Grand Island, NY, USA). Recombinant murine M-CSF was added to each well, and the cells were incubated and RANKL were purchased from R&D Systems (Minnea- at 37°C for 2 h. The formazan crystals were solubilized polis, MN, USA). The acid phosphatase, leukocyte (tar- with DMSO, and cell viability was assessed by measuring trate-resistant acid phosphatase: TRAP) kit, the absorbance at 570 nm using a 96-well microplate methylthiazolyldiphenyl-tetrazolium bromide (MTT), reader (Bio-Rad, Hercules, CA, USA). dimethyl sulfoxide (DMSO), and all other reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA). (-)-Tubaic acid (4-hydroxy-2-(1-methylethenyl)- Real-time PCR 2,3-dihydrobenzofuran-5-carboxylic acid) was isolated by the thermal transformation of rotenone, and the iso- Total RNA was extracted using TRI-solution (Bio Science lation process is described later in this section. Technology, Daegu, Korea), according to the ANIMAL CELLS AND SYSTEMS 3 Figure 1. (-)-Tubaic acid suppresses RANKL-mediated osteoclastogenesis without inducing cytotoxicity. (A) Chemical structure of (-)-tubaic acid. (B) Bone marrow-derived macrophages (BMMs) were incubated with M-CSF (10 ng/ml) and various doses of (-)-tubaic acid, and cell viability was assessed using the MTT assay. (C) The BMMs were cultured with M-CSF (10 ng/ml) and RANKL (20 ng/ml) in the presence of (-)-tubaic acid or vehicle. After four days, the cells were stained to assess TRAP activity. Scale bar, 50 μm. (D) Quantification of TRAP-positive multinucleated cells (MNCs) with over three nuclei. **p < 0.01 versus vehicle- treated control, t-test. manufacturer’s instructions, and complementary DNA ml RANKL, with or without 5 μM (-)-tubaic acid. After four (cDNA) was synthesized using SuperScript II Reverse days, the cells were fixed with 4% paraformaldehyde, Transcriptase (Invitrogen, Carlsbad, CA, USA). Quantitat- treated with 0.25% Triton X-100, and stained with an ive real-time PCR was performed using a LightCycler 1.5 anti-NFATc1 antibody (Santa Cruz Biotechnology, Santa real-time PCR system (Roche Diagnostics, Basel, Switzer- Cruz, CA, USA), followed by an Alexa Fluor-488-conju- land) and SYBR Premix Ex Taq (Takara Bio Inc., Shiga, gated secondary antibody. Rhodamine-conjugated phal- Japan). Primer sequences for osteoclast-specific genes: loidin (Cytoskeleton, Denver, CO, USA) and 4 ,6- ′ ′ ′ TRAP (Acp5), 5 -TCCCCAATGCCCCATTC-3 and 5 -CGGTT diamidino-2-phenylindole dihydrochloride (DAPI; Santa ′ ′ CTGGCGATCTCTTTG-3 ; Ctsk,5 -GGCTGTGGAGGCGGCT Cruz Biotechnology, Santa Cruz, CA, USA) were used to ′ ′ ′ AT-3 and 5 -AGAGTCAATGCCTCCGTTCTG-3 ; Dcstamp, stain F-actin and cell nuclei, respectively. Cells with ′ ′ ′ 5 -CTTCCGTGGGCCAGAAGTT-3 and 5 -AGGCCAGTGCT actin rings or nuclear NFATc1 were counted from 10 ′ ′ ′ GACTAGGATGA-3 ; Nfatc1,5 -ACCACCTTTCCGCAACCA-3 random selected views using the Image J software ′ ′ and 5 -TTCCGTTTCCCGTTGCA-3 . (NIH, Bethesda, MA, USA). Immunofluorescence staining Western blotting BMMs were plated on glass coverslips and incubated in Cells were lysed in RIPA lysis buffer supplemented with α-MEM containing 10% FBS, 10 ng/ml M-CSF, and 20 ng/ protease and phosphatase inhibitors, and the total 4 S. LIM ET AL. protein concentration was measured using a BCA acid almost completely blocked osteoclast formation Protein Assay Kit (Pierce Biotechnology, Rockford, IL, (97.79% inhibition; Figure 1(d)). To further confirm the USA). Proteins (30 μg) were subjected to 10% SDS- stage at which (-)-tubaic acid impaired osteoclast for- PAGE and transferred onto a nitrocellulose membrane mation, (-)-tubaic acid (5 μM) was added to the culture (Whatman, Florham Park, NJ, USA). Membranes were medium at different periods, as previously reported immersed in 3% non-fat milk in Tris-buffered saline (Ihn et al. 2017; Lim et al. 2020). Addition of (-)-tubaic with 0.1% Tween 20 (TBS-T) for 1 h before incubation acid in the early (Period I: from days 0–2) or late with primary antibodies against p-p38, p-JNK, p-ERK, (Period II: from days 2–4) stages of osteoclast differen- p-MEK, p-AKT, p-IκBα (Cell Signaling Technology, tiation, the formation of TRAP-positive MNCs was signifi- Danvers, MA, USA), and β-actin (Sigma–Aldrich, cantly decreased (Figure 2(a)). The number of MNCs was St. Louis, MO, USA) at 4°C. Immunoreactive bands were reduced by 71.2% and 78.2% in the early and late treat- detected using a WesternBright ECL kit (Advansta, ments, respectively (Figure 2(b)), indicating that Menlo Park, CA, USA) and recorded using a chemilumi- (-)-tubaic acid can affect both stages, including the com- nescence imager (Azure Biosystems, Inc., Dublin, CA, mitment to osteoclastic differentiation and cell-cell USA). fusion. In vitro resorption assay (-)-Tubaic acid reduces osteoclast marker expression BMMs seeded on bone slices (IDS Nordic, Herlev, Denmark) were incubated in an osteoclastogenic To further determine the anti-osteoclastogenic potential medium for three days, followed by treatment with of (-)-tubaic acid, mRNA expression levels of NFATc1 and vehicle or 5 μM (-)-tubaic acid. After two days, the its target genes related to osteoclast differentiation and bone slices were washed and immersed in hematoxylin function were assessed. As shown in Figure 2(c), solution to visualize the resorption pits. The area of the (-)-tubaic acid (5 μM) suppressed the expression of pits was quantified by analyzing 10 randomly selected Nfatc1 and downstream osteoclast marker genes, such images using i-Solution image analysis software (IMT as TRAP (Acp5), Dcstamp, and cathepsin K (Ctsk). The i-Solution, Daejeon, Korea). protein levels of NFATc1 and cathepsin K were signifi- cantly down-regulated by (-)-tubaic acid treatment (Figure 2(d)). We also observed reduced nuclear localiz- Statistical analysis ation of NFATc1 in (-)-tubaic acid-treated cells (Figure 3 Experiments were conducted in triplicate and repeated (a,c)). three times, and the results are presented as the mean ± standard deviation (SD). A two-tailed student’s t-test was used to determine statistical significance. *P < 0.05 (-)-Tubaic acid suppresses the formation of actin or **P < 0.01 was considered statistically significant. rings and resorption pits As osteoclasts form actin rings around the cell periphery, Results which serves as an indicator of active osteoclasts and are vital for bone resorption (Boyce et al. 1992), the effects of (-)-Tubaic acid suppresses RANKL-mediated (-)-tubaic acid on actin ring formation and bone resorp- osteoclast differentiation in BMMs tion activity were examined. Phalloidin staining revealed To explore the effects of (-)-tubaic acid (Figure 1(a)) on that F-actin rings were present in the vehicle-treated RANKL-induced osteoclastogenesis, we first investigated control group, whereas the addition of 5 μM (-)-tubaic whether (-)-tubaic acid affects the viability of BMMs. The acid impaired actin ring formation (Figure 3(a,b)). Next, MTT assay results revealed that (-)-tubaic acid did not we performed a pit formation assay to explore the exhibit cytotoxicity, even at 5 μM, and increased the via- effects of (-)-tubaic acid on osteoclastic bone resorption. bility of BMMs (Figure 1(b)). When BMMs were cultured BMMs were cultured on bone slices in an osteoclast in an osteoclastogenic medium with different doses of induction medium for three days, and the cells were (-)-tubaic acid for four days, (-)-tubaic acid treatment sig- treated with either 5 μM (-)-tubaic acid or vehicle. nificantly suppressed the formation of TRAP-positive Numerous hematoxylin-stained resorption pits were multinucleated cells (MNCs) from BMMs in a dose- formed in the vehicle-treated control; however, dependent manner compared with that in the vehicle- (-)-tubaic acid treatment led to a significant decrease treated control (Figure 1(c)). Notably, 5 μM (-)-tubaic in the resorbed area (Figure 4(a,b)). ANIMAL CELLS AND SYSTEMS 5 Figure 2. (-)-Tubaic acid inhibits early and late stages of osteoclast differentiation and osteoclast-specific marker expression. (A) The BMMs were cultured in the presence of M-CSF (10 ng/ml) and RANKL (20 ng/ml), and the cells were treated with (-)-tubaic acid (5 μM) for the indicated period. Period I : from days 0–2, Period II : from days 2–4. Scale bar, 50 μm. (B) Quantification of TRAP-positive MNCs. (C, D) The BMMs were incubated in the presence of M-CSF (10 ng/ml) and RANKL (20 ng/ml) with or without (-)-tubaic acid (5 μM) for four days. The mRNA (C) and protein (D) expression levels of osteoclast markers were assessed by real-time PCR and immunoblotting, respectively. *p < 0.05, **p < 0.01 versus vehicle-treated control, t-test. (-)-Tubaic acid attenuates RANKL-mediated ERK (-)-tubaic acid reduced the RANKL-induced phosphoryl- activation ation of MEK, the upstream activator of ERK, and ERK without affecting the phosphorylation of JNK, p38, Akt, Mitogen-activated protein kinases [c-Jun-N-terminal and IκBα (Figure 4(c,d)). kinase (JNK), extracellular signal-regulated kinase (ERK), and p38] and NF-κB pathways are activated in response to RANKL stimulation, and they are essential for osteo- Discussion clast differentiation and function (Mizukami et al. 2002). As (-)-tubaic acid exhibits anti-osteoclastogenic Natural compounds and their derivatives have attracted and anti-resorptive potential, its effect on RANKL- attention as essential resources for the development of mediated signaling pathways was investigated to therapeutic agents for decades, owing to their potential better understand the molecular mechanism. After pre- medicinal properties. Phytoestrogens, including isofla- treatment with (-)-tubaic acid or vehicle, BMMs were vones, lignans, and coumestrol, are polyphenolic com- stimulated with RANKL, and the phosphorylation levels pounds, and several reports highlight their beneficial of signaling molecules were assessed by immunoblot- effects on bone health (Castelo-Branco and Cancelo ting. Upon RANKL treatment, phosphorylation of Hidalgo 2011; Abdi et al. 2016). Tubaic acid is produced MAPKs and IκBα increased within 15 min in control by the degradation of rotenone, a naturally occurring cells (Figure 4(c,d)). In contrast, pretreatment with isoflavone compound that exhibits antimicrobial activity 6 S. LIM ET AL. Figure 3. (-)-Tubaic acid impairs actin ring formation and decreases nuclear localization of NFATc1. (A) BMMs were incubated on glass coverslips in an osteoclastogenic medium containing M-CSF (10 ng/ml) and RANKL (20 ng/ml) with (-)-tubaic acid (5 μM) or vehicle. After four days, the cells were fixed and probed with anti-NFATc1 antibody (green), followed by staining with rhodamine-conjugated phalloidin (red) and DAPI (blue). Yellow dashed rectangles were magnified in the lower panels. Scale bar, 50 μm. Quantification of the percentages of (B) cells displaying actin rings and (C) cells with nuclear NFATc1. **p < 0.01 versus vehicle-treated control, t-test. (Obara et al. 1976). Tubaic acid contains a 2,3-dihydro- M-CSF plays essential roles in the survival, prolifer- benzofuran scaffold, which is considered a vital ation, and differentiation of osteoclast precursor cells, element in many biologically active natural compounds and binding of M-CSF to its receptor, c-Fms, activates (Qin et al. 2017). To date, the biological and pharmaco- various signaling molecules, including ERK and logical properties of tubaic acid have rarely been PI3 K/Akt (Richardson et al. 2015). BMMs are widely studied. In this study, we demonstrated that (-)-tubaic used as osteoclast precursors in an in vitro osteoclasto- acid inhibited RANKL-induced osteoclastogenesis and genesis system, and an increase in cell viability was suppressed bone-resorbing activity. observed when BMMs were treated with (-)-tubaic acid ANIMAL CELLS AND SYSTEMS 7 Figure 4. (-)-Tubaic acid reduces resorption pit formation and attenuates RANKL-induced activation of ERK. (A) The BMMs were cul- tured on bone slices with M-CSF (10 ng/ml) and RANKL (20 ng/ml) to induce osteoclast differentiation. After three days, the cells were treated with (-)-tubaic acid (5 μM) or vehicle for an additional two days. The bone slices were stained with hematoxylin. Scale bar, 100 μm. (B) Quantification of the resorbed area. (C, D) Serum-starved BMMs were pretreated with (-)-tubaic acid (5 μM) or vehicle for 1 h, followed by stimulation with 50 ng/ml RANKL for indicated times. The phosphorylation levels of (C) ERK, Akt, JNK, p38, and (D) IκBα were examined by western blotting. The graph (right panel) represents the relative band intensity of phosphorylated ERK. *p < 0.05, **p < 0.01, t-test. (Figure 1(b)), suggesting that tubaic acid may affect one osteoclast-related genes was attenuated in the presence of the c-Fms signaling pathways to induce cell survival or of (-)-tubaic acid (Figure 2(c,d)), which is consistent with proliferation. Osteoclast differentiation comprises the TRAP staining results, indicating that (-)-tubaic acid several steps, including commitment and differentiation has anti-osteoclastogenic properties. Cell-cell fusion to mononuclear preosteoclasts, cellular fusion to multi- required for the formation of multinucleated cells nuclear osteoclasts, and activation of bone-resorbing during osteoclastogenesis is crucial for efficient bone osteoclasts (Xing et al. 2012). (-)-Tubaic acid significantly resorption (Lee et al. 2009), and DC-STAMP is well estab- suppressed both the early and late stages of osteoclast lished as an essential regulator of this process (Yagi et al. differentiation, with over 70% inhibition of osteoclast 2005). Genetic ablation of Dcstamp results in the failure formation (Figure 2(b)), indicating that it impairs preos- to generate multinuclear osteoclasts, leading to a teoclast formation and cellular fusion in the early and reduction in bone-resorbing activity (Yagi et al. 2005). late stages, respectively. We also confirmed the inhibi- Although mononuclear osteoclastic cells in DC-STAMP tory effects of (-)-tubaic acid at the molecular level by knockout mice can resorb bone, their resorbing activity assessing changes in the expression of osteoclast- is lower than that of multinucleated osteoclasts (Yagi specific markers. The expression of NFATc1 and et al. 2005). Osteoclastic resorption is also affected by 8 S. LIM ET AL. the formation of an actin ring involved in creating a treatment and prevention of bone diseases associated tightly enclosed space, known as a resorption lacuna with excessive osteoclast formation and function. (Novack and Teitelbaum 2008). Switch-associated protein 70 (SWAP-70) deficiency leads to an osteopetro- Data availability tic phenotype caused by ineffective bone resorption owing to defects in actin ring formation (Roscher et al. The data underlying this article are available in the 2016). The formation of actin-based sealing rings is article. impaired in cortactin-depleted osteoclasts, which exhibit a loss of bone-resorbing function (Tehrani et al. Disclosure statement 2006). The presence of (-)-tubaic acid attenuated bone resorption (Figure 4(a)), which correlated with reduced No potential conflict of interest was reported by the author(s). actin ring formation (Figure 3(a)). It inhibited the for- mation of multinucleated osteoclasts (Figure 1(c)). Funding Osteoclast differentiation and bone resorptive function mainly rely on RANKL-RANK signaling, which activates This work was supported by a National Research Foundation of the MAPK pathways (Fuller et al. 1998). Various studies Korea (NRF) grant funded by the Korean government (MSIT): using a pharmacological inhibitor of MEK-ERK or [Grant Number 2017R1A5A2015391, 2020M3A9I4039539]. genetic disruption of Erk1 have demonstrated the importance of the ERK pathway in osteoclast formation ORCID and function (Yan et al. 2007; He et al. 2011). Nakamura et al. observed a significant role of ERK in the survival Jong-Sup Bae http://orcid.org/0000-0002-5756-9367 and polarity of osteoclasts (Nakamura et al. 2003). Additionally, ERK activation by the granulocyte-macro- References phage colony-stimulating factor (GM-SCF) induces DC- STAMP expression, which promotes cell fusion to gener- Abd-Elazem IS, Chen HS, Bates RB, Huang RC. 2002. 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Journal

Animal Cells and Systems – Taylor & Francis

Published: Dec 11, 2023

Keywords: (-)-tubaic acid; osteoclast; bone resorption; nuclear factor of activated T-cells cytoplasmic 1 (NFATc1)

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