Metallothionein 3 promotes osteoclast differentiation and survival by regulating the intracellular Zn2+ concentration and NRF2 pathway

Metallothionein 3 promotes osteoclast differentiation and survival by regulating the intracellular Zn2+ concentration and NRF2 pathway | 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 Article Metallothionein 3 promotes osteoclast differentiation and survival by regulating the intracellular Zn2+ concentration and NRF2 pathway Toshifumi Fujiwara, Shinkichi Arisumi, Keitaro Yasumoto, Tomoko Tsutsui, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3262822/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Dec, 2023 Read the published version in Cell Death Discovery → Version 1 posted 10 You are reading this latest preprint version Abstract In osteoclastogenesis, the metabolism of metal ions plays an essential role in controlling reactive oxygen species (ROS) production, mitochondrial biogenesis, and survival, and differentiation. However, the mechanism regulating metal ions during osteoclast differentiation remains unclear. The metal-binding protein metallothionein (MT) detoxifies heavy metals, maintains metal ion homeostasis, especially zinc, and manages cellular redox levels. We carried out tests using murine osteoclast precursors to examine the function of MT in osteoclastogenesis and evaluated their potential as targets for future osteoporosis treatments. MT genes were significantly upregulated upon differentiation from osteoclast precursors to mature osteoclasts in response to receptor activators of nuclear factor-κB (NF-κB) ligand (RANKL) stimulation, and MT3 expression was particularly pronounced in mature osteoclasts among MT genes. The knockdown of MT3 in osteoclast precursors demonstrated a remarkable inhibition of differentiation into mature osteoclasts. In preosteoclasts, MT3 knockdown suppressed the activity of mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways upon RANKL stimulation, leading to increased apoptosis through elevated cleaved Caspase 3 and poly (ADP-ribose) polymerase (PARP) levels. Additionally, ROS levels were decreased, and nuclear factor erythroid 2-related factor 2 (NRF2) (a suppressor of ROS) and the downstream antioxidant proteins, such as catalase (CAT) and heme oxygenase 1 (HO-1), were more highly expressed in the MT3 preosteoclast knockdowns. mitochondrial ROS, which is involved in mitochondrial biogenesis and the production of reactive oxygen species, were similarly decreased because cAMP response element-binding (CREB) and peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β) were less activated due to MT3 depletion. Thus, by modulating ROS through the NRF2 pathway, MT3 plays a crucial role in regulating osteoclast growth and survival, acting as a metabolic modulator of intracellular zinc ions. Biological sciences/Developmental biology/Differentiation Biological sciences/Molecular biology/RNAi Biological sciences/Physiology/Bone Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Osteoclast- and osteoblast-mediated bone formation is critical for skeletal development, homeostasis, and repair [ 1 ]. In adults, bone development and bone loss are intricately linked and balanced through a continuous self-renewal process known as bone remodeling that ensures that bone mass and strength are maintained throughout life [ 2 – 3 ]. Increased osteoclast activity and numbers have been attributed to pathological bone loss and fragility fractures in metabolic bone illnesses such as osteoporosis, Paget's disease, and osteolytic tumor [ 4 – 6 ]. The cellular and molecular mechanisms governing osteoclast differentiation and operation in normal and pathological circumstances must be clarified. This understanding is essential for creating novel therapeutic approaches to cure bone loss in various skeletal illnesses efficiently. Mononuclear progenitor cells are produced from the monocyte/macrophage lineage of hematopoietic stem cells, which fuse to form multinucleated cells called osteoclasts. The two cytokine receptor activators of nuclear factor-κB (NF-κB) ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) are essential for osteoclastogenesis in vitro and in vivo [ 1 , 7 ]. M-CSF primarily controls macrophage proliferation and osteoclast survival by stimulating the phosphoinositide-3-kinase/AKT (PI3K/AKT) and extracellular signal-regulated kinase (ERK) pathways [ 8 – 9 ], while NF-κB, PI3K/AKT, and mitogen-activated protein kinase (MAPK) pathways are all activated by RANKL to control osteoclast differentiation [ 1 , 4 ]. Furthermore, the main transcription factor for osteoclast differentiation, nuclear factor of activated T cells 1 (NFATc1), is induced by RANKL signaling [ 10 ], regulating calcium oscillation [ 7 ]. Metal ions such as calcium, iron, and zinc play pivotal roles in osteoclast differentiation and function. Primarily, iron homeostasis controls osteoclastogenesis and function via intracellular and mitochondrial reactive oxidative species (ROS). Moreover, zinc is essential for fosteཌྷring the mineralization and development of bones by stimulating osteoblasts, and it exerts a suppressive effect on bone resorption by inhibiting osteoclast formation. Thus, the metabolism of metal ions remains unknown mainly due to their complex roles. The metal-binding protein, metallothionein (MT), which comprises high cysteine content (30%) and low molecular weight (6–7 kDa), has four primary isoforms (MT1-MT4). MT1 and MT2 are ubiquitously expressed in various soft tissues, while MT3 is primarily found in brain tissue, heart, kidney, and genital organs [ 22 – 23 ]. MT4 is specifically expressed in certain squamous epithelia [ 24 ]. MT has a wide range of physiological functions, including heavy metal detoxification, maintenance of metal ion homeostasis (especially zinc and copper), regulation of cellular redox balance, and facilitation of cell proliferation [ 25 – 29 ]. In addition, the MT protein family has been implicated in pathological conditions such as cancer and neurodegenerative diseases [ 30 – 31 ]. In mouse bone marrow stromal cells, MT serves a protective function against oxidative stress-induced suppression of osteoblast development. Additionally, a zinc-rich diet, which stimulates endogenous MT production, affects both osteoblast differentiation and postnatal bone growth [ 34 ]. However, MT function in osteoclastogenesis is still unclear. In this study, we identified MT3 as the dominant MT isoform expressed in osteoclasts, which was upregulated in murine and human osteoclasts after RANKL stimulation. Apoptosis and a reduction of osteoclast differentiation were caused by the loss of MT3, which downregulated RANKL-stimulated MAPK/NF-κB signaling. Furthermore, loss of MT3 raised intracellular zinc concentrations, which in turn increased nuclear factor erythroid 2-related factor 2 (NRF2), an antioxidant defense mechanism, and decreased ROS in osteoclastogenesis. Our research suggests that MT3 is a new osteoclastogenesis regulator that controls ROS and the NRF2 pathway. Results Osteoclast development and bone resorption are inhibited in vitro by deleting MT3, which is abundantly expressed in murine and human osteoclasts. We first assessed the mRNA levels of Mt1-3 with osteoclast formation using real-time quantitative PCR to determine the function of MT in osteoclast lineage cells. To achieve this, bone marrow macrophages (BMM) were cultivated with M-CSF alone or in combination with RANKL for 2 and 4 days for differentiation into preosteoclasts (pOC) and mature osteoclasts (mOC), respectively, each of which has a different function. Figure 1 A demonstrates that during osteoclast development, the mRNA levels of Mt1-3 increased, and Mt3 had the highest expression among the Mt osteoclast isoforms, showing that Mt3 was the most prevalent isoform within the MT family during osteoclastogenesis. We also examined the expression of Mt3 in human peripheral blood mononuclear cell-derived osteoclasts and found that its expression increased during osteoclastogenesis (Fig. 1 B). Our next goal was to define MT3's cell-autonomous roles in osteoclast differentiation and function. To achieve this, we investigated if two short hairpin (sh) RNAs (MT3-sh1 and MT3-sh2) that targeted various murine Mt3 mRNA locations could knock down MT3 expression in BMM via lentiviral transduction. The negative control was a shRNA directed against firefly luciferase. BMMs that had been positively transduced were either cultivated with M-CSF alone or for three days with M-CSF and RANKL. Both MT3-specific shRNAs, but not the control, significantly decreased Mt3 mRNA expression in RANKL-induced mOC (Fig. 1 C). Tartrate-resistant acid phosphatase (TRAP), a marker of osteoclast development, revealed a decrease in the number of multinucleated osteoclasts in the bone. Knockdown of MT3 in BMM inhibited osteoclast formation and significantly decreased the total osteoclast number and spreading area (Fig. 1 D). Reduced mRNA expression of osteoclast marker genes, including NFATc1 (encoded by Nfatc1 ), cathepsin K (encoded by Ctsk ), TRAP (encoded by Acp5 ), DC-STAMP (encoded by Dcstamp ), and calcitonin receptor (encoded by Calcr ), compared to control cells in MT3-depleted mOC (Fig. 1 E) confirmed the reduction. Furthermore, MT3-depleted cells markedly decreased the protein expressions of NFATc1 and CTSK (Fig. 1 F). We stained actin filaments using Alexa- 488- conjugated phalloidin in control and MT3 knockdown osteoclasts cultivated on plastic dishes because actin cytoskeleton organization is crucial for osteoclast spreading and the production of podosome belts on plastic plates. Depleting MT3 in osteoclasts may impair their function because osteoclast activation and function are required for actin ring formation. In support of this hypothesis, MT3 knockdown osteoclasts exhibited less bone resorption than control osteoclasts, as shown by the staining of resorption pits on cortical bovine bone slices (Fig. 1 H). These data suggest that MT3 is upregulated by RANKL and positively regulates osteoclast differentiation and function in vitro . MT3 is critical for osteoclast survival. To investigate the potential involvement of MT3 in BMM cell growth, we conducted a comprehensive analysis of cell growth over 96 hours. MT3 knockdown by shRNA rescued the increased number of BMM compared with the control (Fig. 2 A). Next, we attempted to determine which cellular processes were involved in the cell growth of osteoclast lineage cells after MT3 depletion. Figure 2 B compares the cell cycle progression (S and G2/M) in BMM between control and MT3 knockdown groups using flow cytometry and reveals no discernible differences. The cell population in the G0-1 phase in control BMM or MT3 knockdown was also unchanged, suggesting that MT3 knockdown did not affect the BMM cell cycle progression. Western blotting of the apoptosis markers poly (ADP-ribose) polymerase (PARP) and cleaved (active) caspase 3 revealed that ablation of MT3 in BMM had a negligible impact on apoptosis triggered by cytokine or serum deprivation (Fig. 2 C). Furthermore, knockdown of MT3 in pOC promoted apoptosis under the same circumstances (Fig. 2 C). These results indicate that MT3 in osteoclast lineage cells accelerated osteoclastogenesis by affecting osteoclast survival. MT3 regulates osteoclastogenesis via activation of RANKL-induced JNK and NF-κB. M-CSF and RANKL signaling activation are essential for the proliferation, survival, and differentiation of osteoclastogenesis. Control and MT3 knockdown preosteoclasts were starved with serum and cytokines and then stimulated with M-CSF or RANKL for the designated amount of time to determine which signaling in M-CSF or RANKL affected preosteoclasts of MT3 loss [ 35 – 36 ]. Western blots were used to analyze the activation of c-Jun N-terminal kinase (JNK), NF-κB, and ERK, which are all induced by RANKL and M-CSF, respectively. AKT, also known as protein kinase B, and AK strain transformation are induced by M-CSF. Both MT3 shRNAs that knocked down MT3 in preosteoclasts had no impact on ERK and AKT phosphorylation in response to M-CSF stimulation (Fig. 3 B). In contrast, JNK and NF-κB activation caused by RANKL was consistently reduced by MT3 deletion by both shRNAs, evidenced by lower levels of phospho-JNK and phospho-IκB (Fig. 3 A). Given that MT3 regulates the JNK and NF-κB pathways, which are essential for osteoclast differentiation and survival, these findings imply that MT3 modulates RANKL-induced activation of JNK and NF-κB, which controls osteoclast differentiation and survival [ 5 ]. The Loss of MT3 triggers the activation of the NRF2 pathway, leading to the inhibition of RANKL-induced ROS production. In osteoclastogenesis, RANKL-induced activation of MAPKs (ERK, JNK, and p38), NF-κ-B, and the PI3K/AKT pathways depends on ROS generated by RANKL and MT has a protective role against oxidative stress [ 37 – 40 ]. So, we examined the production of ROS during osteoclastogenesis by DCFH-DA staining. As shown in Fig. 4 A, deletion of MT3 in preosteoclasts dramatically decreased intracellular ROS production by assessing the positivity rate of DCF fluorescence, indicating that depletion of MT3 reduced ROS production induced by RANKL during osteoclastogenesis. ROS act as mediators of intracellular signaling involved in the development and activation of osteoclasts in response to RANKL stimulation [ 37 , 41 – 42 ]. Additionally, nuclear factor erythroid 2-related factor 2 (NRF2) is a defensive mechanism against oxidative stress that regulates several antioxidant enzymes, such as NAD(P)H quinone dehydrogenase 1 (NQO1), catalase (CAT), haemoxygenase-1 (HO-1), and γ-glutamyl cysteine synthetase (GCS) [ 43 – 46 ]. The overexpression of NRF2 suppresses osteoclastogenesis in vitro and in vivo due to the reduction of oxidative stress. To investigate the association with ROS-related enzymes in MT3 loss, the expression of ROS-related enzymes, including CAT, HO-1, and NRF2, with osteoclast differentiation is presented in Fig. 4 B by western blotting. In comparison to the control, MT3 knockdown considerably boosted the expression levels of NRf2 and its downstream antioxidant factors, HO-1 and CAT, in BMM and pOC (Fig. 4 B), leading to less ROS production during osteoclast differentiation. These findings may provide mechanistic evidence that RANKL-induced intracellular ROS production is effectively attenuated by the loss of MT3, which upregulates NRF2, and the associated downstream antioxidant enzymes. Depletion of MT3 decreased mitochondrial ROS levels in osteoclast precursors. ROS activates the downstream mediator peroxisome proliferator-activated receptor coactivator 1β (PGC-1β) via phosphorylating the cAMP response element-binding protein (CREB) during osteoclastogenesis, stimulating mitochondrial biogenesis, and the generating of mitochondrial ROS, which in a positively feedback-induced manner increases CREB and PGC-1β activity and facilitates NFATc1 activation. The deletion of MT3 in preosteoclasts also diminished mitochondrial ROS levels, similarly to intracellular ROS production (Fig. 4 C). We then analyzed CREB phosphorylation and PGC-1β expression in cells treated with MT3 shRNA to promote osteoclastogenesis, which resulted in a reduction of CREB phosphorylation and PGC-1β expression in the depletion of MT3 osteoclasts (Fig. 4 D). These data suggest that MT3 regulates mitochondrial ROS levels via the activation of CREB and PGC-1β. MT3 loss led to the accumulation of intracellular Zn 2+ , potentially leading to the upregulation of NRF2 gene expression. MT acts as a biochemical regulator of intracellular levels of free Zn 2+ by capturing and subsequently releasing Zn 2+ in response to various biochemical events, such as oxidative signaling. Visualization using FluoZin-3 fluorescence microscopy verified that preosteoclasts without MT3 resulted in an elevation of intracellular Zn 2+ levels at baseline conditions. Furthermore, intracellular levels of Zn 2+ were enhanced one hour after the administration of 50 µM ZnSO 4 in the MTs-depleted cells (Fig. 5 ). The change in expression of NRF2 in preosteoclasts after treatment with Zn 2+ was examined with or without MT3 because it has been reported that Zn 2+ is essential for NRF2 expression and transcriptional function [ 53 – 57 ]. As shown in Fig. 6 , MT3 depletion in preosteoclasts showed higher intracellular NRF2 expression, consistent with western blotting (Fig. 4 B). After administration of ZnSO 4 , NRF2 expression was increased in MT3 knockdown cells than controls. However, this effect was treated with N, N, N', N'-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), a cell-permeable zinc chelator, and was successfully removed (Fig. 6 ), suggesting that MT3 loss elevated NRF2 expression by the accumulation of intracellular Zn 2+ . Discussion Increased intracellular ROS production in osteoclastogenesis encourages cell survival and differentiation through the activation of MAPK and NF-κB. It is also implicated in the generation of mitochondrial ROS, which is mediated by pCREB and PGC1β. In particular, the metabolism of cellular metal ions, such as iron and copper, regulates ROS production and mitochondrial biogenesis. However, the molecular processes governing cellular metal ions in osteoclastogenesis are still unclear. This study elucidated that the metal-binding protein MTs, particularly MT3, were upregulated during osteoclast differentiation of murine and human osteoclasts. MTs have various physiological functions, such as detoxification of heavy metals, maintenance of metal ion homeostasis (particularly Zn 2+ ), and regulation of cellular redox balance [ 25 – 29 ]; However, MT3 suppression prevented osteoclast development and induced apoptosis in osteoclast precursor cells. This effect was linked to increased free intracellular Zn 2+ that was not bound to MT3 and led to the downregulation of ROS production by increasing the expression of NRF2, HO-1, and CAT. The decreased ROS resulted in reduced activation of MAPK, NF-κB, pCREB, and PGC1β, resulting in decreased survival and differentiation of osteoclasts (Fig. 7 ). MT controls the redox reactions and forms the redox complex with Zn 2+ , which scavenges and neutralizes free radicals via cysteine sulfur ligands and donates Zn 2+ in a redox-dependent manner [ 38 – 40 ]. Although MT3 is specifically expressed in the central nervous system, it has been recently observed to have various functions in other tissues [ 20 ]. MT3 regulates ROS by controlling metal metabolism such as zinc and cooper. The suppression of ROS could be effective in inhibiting RANKL-induced osteoclastogenesis given that RANKL activates MAPK and NF-κB, causing ROS to enhance osteoclastogenesis and associate with the pathogenesis of osteoporosis. MT promotes osteoblastic differentiation due to the suppression of ROS in osteoblasts [ 32 , 34 ]. In this study, MT3 knockdown in osteoclast progenitor cells decreased JNK activation, and ROS production, and RANKL induced NF-κB stimulation, suggesting that MT3 may affect osteoclast survival and differentiation by regulating ROS and downstream MAPK and NF-κB signals. Therefore, to investigate the association with MT3, ROS, and osteoclastogenesis, we focused on the metabolism of metals, especially zinc. The presence of zinc is essential for the antioxidant action of MT. After exposure to oxidative stress, glutathione (GSH) neutralizes free radicals by donating hydrogen to form glutathione disulfide (GSSG). Glutathione reductase can reconvert GSSG to GSH to continue the redox cycle in the presence of intracellular free Zn 2+ -bound MT [ 61 ]. Zinc is known to promote bone formation and suppress bone resorption [ 15 – 18 ] via inhibition of osteoclast differentiation induced by phosphorylation of calcineurin [ 62 ], inhibition of NF-κB signaling [ 63 ], and enhancement of apoptosis [ 64 ]. MTs bind and release Zn 2+ under physiological conditions and are part of a network that tightly regulates intracellular Zn 2+ in concert with the cell-specific Zn 2+ importer, Zrt- and Irt-like proteins (ZIP) and Zn 2+ exporter, Zn transporters (ZnT) [ 52 ]. When the free intracellular Zn 2+ concentration reaches a threshold, the activation of metal-responsive transcription factor 1 induces MT expression, which sequesters Zn 2+ and releases it in response to other biochemical events, such as oxidative signals, leading to the maintenance of homeostasis of free Zn 2+ . In this study, the knockdown of MT3 increased intracellular Zn 2+ and the production of ROS and failed to sequester Zn 2+ , leading to enhanced ROS. In support of this result, Malaiyandi LM et al. [ 66 ] and Habel N et al . [ 67 ] demonstrated that MT overexpression reduced the concentration of intracellular Zn 2+ . NRF2, which is induced by Zn 2+ , plays a central role as a part of the antioxidant defense system of osteoclasts in reducing ROS [ 68 – 69 ]. NRF2, as a redox-sensitive transcription factor, expresses antioxidant enzymes against oxidative stress and inflammatory responses and suppresses RANKL-induced osteoclastogenesis [ 45 – 46 ]. NRF2 suppresses ROS and NFATc1 and PGC1β directly [ 70 ]; the NRF2 activator is a powerful therapeutic target [ 5 , 47 , 71 ]. In our study, MT3 knockdown activated NRF2 at the stage of BMM and preosteoclasts compared with control cells. HO-1 and CAT, which are downstream factors, were also activated, indicating that NRF2 signals suppressed ROS production. Furthermore, as described above, zinc has been reported to be involved in activating NRF2 [ 53 – 57 ]. We also demonstrated that NRF2 was upregulated in MT3 knockdown, with elevated Zn 2+ in preosteoclasts, and inhibition of intracellular Zn 2+ by TPEN decreased NRF2 expression. These results suggest that the increased concentration of intracellular Zn 2+ may be involved in the upregulation of NRF2 in preosteoclasts. During osteoclastogenesis, RANKL-induced ROS induced PGC-1β via activation of CREB, and PGC-1β increased the production of reactive oxygen species and mitochondrial biogenesis, leading to the induction of osteoclastogenesis through a positive feedback mechanism [ 11 – 12 , 49 – 50 ]. In addition, NRF2 activity suppresses ROS production and directly reduces PGC-1β, resulting in a lower expression level of mitochondrial genes [ 11 ]. Subsequently, the expression of IRF8, an NFATc1 antagonist, is upregulated and inhibits NFATc1 activation and osteoclast differentiation [ 70 , 72 ]. Our results also showed that the deletion of MT3 reduced the activation of CREB, PGC-1β, and mitochondrial ROS, suggesting that the reduction of NRF2 activity and ROS by MT3 knockdown led to the inhibition of osteoclast differentiation through this mechanism. We suggest that RANKL elevates MT3 with osteoclast development based on the findings of this investigation and those published in the literature. MT3 regulates intracellular Zn 2+ activity, leading to ROS production regulation via the NRF2, HO-1, and CAT pathways. Subsequently, ROS activation promotes RANKL-induced MAPK and NF-κB, enhancing osteoclastogenesis. In addition, MT3 regulates mitochondrial ROS by activating PGC-1β and CREB (Fig. 7 ). Materials and Methods Reagents and antibodies The following antibodies were used: Nfatcl (#sc-7294) and catalase (#sc-271803), all of which were bought from Santa Cruz Biotechnology (Dallas, TX, USA); Actin (#A00702), from GeneScript (Piscataway, NJ, USA); cathepsin K (#MAB3324), purchased from EMD Millipore Corporation (Temucula, CA, USA); PARP (#9542), cleaved Caspase 3 (#9664), JNK/SAPK (#9252), phosphor-JNK/SAPK (#9251), IκBα (#9242), phosphor -IκBα (#9246), AKT (#2920), phosphor-AKT (#4058), phosphor-ERK1/2 (#9106), ERK1/ 2(#9102), CREB (#4820), phosphor-CREB (#9191) were bought from Cell Signaling Technology (Beverly, MA, USA); NRF2 (#A0674) were bought from ABclonal (Wuhan, Hunan, China); HO-1 (#ab189491), PGC-1β (#ab176328) were purchased from Abcam (Cambridge, MA, USA). α-Minimum Essential Medium (α-MEM) and Penicillin-Streptomycin Solution (×100) were bought from FUJIFILM Wako (Osaka, Japan), and fetal bovine serum (FBS) was purchased from Gibco (Billings, MT, USA). Animal use approval The Institutional Animal Care and Use Committee at Kyushu University approved the study's research techniques, which used mice. Furthermore, the National Institutes of Health's recommendations for the moral treatment and use of animals were strictly followed in all the research that was carried out. BMM and osteoclast cultures BMMs were prepared following the previously described method [ 35 ]. The tibia and femurs of 8–10-week-old C57/BL6J mice were extracted for the entire bone marrow. A lysis buffer (150 mM NH4Cl, 10 mM KNCO3, 0.1 mM EDTA, pH 7.4) was used for 5 min at room temperature to remove red blood cells. Then, for the next four-five days, 5 × 10 6 bone marrow cells were plated onto a 100 mm petri dish and cultured in α-10 medium (α-MEM, 10% heat-inactivated FBS, 1 penicillin-streptomycin) with an addition of 1/10 volume of CMG 14 − 12 (conditioned medium supernatant containing recombinant M-CSF at 1 µg/ml) [ 73 ]. Every other day, the CMG 14 − 12 supernatant, and culture media were replaced. After three and five days of BMM culture (at a density of 160/mm), preosteoclasts and osteoclasts were produced by adding 1/100 volume of CMG 14 − 12 culture supernatant and 100 ng/ml of recombinant RANKL (Oriental yeast, Tokyo, Japan), respectively. human osteoclast cultures Using Ficoll-PaqueTM PLUS (Cytiva, Uppsala, Sweden), human peripheral blood monocytes (PBMCs) were separated from the blood of a healthy volunteer. Monocytes were purified from PBMCs using CD14 MicroBeads, human (Miltenyi Biotec, North Rhine-Westphalia, Germany) as per the guidelines provided by the manufacturer. To differentiate human osteoclasts, monocytes of human PBMC origin were cultured for seven days (at a density of 3000/mm) in α-10 media containing 50 ng/ml of recombinant human M-CSF (Abcam, #ab259396) and 100 ng/ml of recombinant RANKL. Every three days, new media, and cytokines were replenished. Lentivirus mediated shRNA expression ShRNA expression is mediated by lentiviruses. ShRNA targeting the mRNA of murine MT3 is expressed by the LKO.1 lentiviral vector [TRCN0000257921/NM_013603.1-200s21c1 (MT3-sh1) and TRCN0000249518/NM_013603.1-93s21c1 were purchased from Sigma-Aldrich. The control was pLKO.1 puro nontarget shRNA control transduction particles (07181827MN). Using the TransIT transfection reagent (Mirus), 293-T cells were co-transfected with an LKO.1 gene transfer vector and the virus packaging vectors ΔH8.2 and VSVG. After 48 hours of transfection, virus supernatants were collected. The virus supernatant was used to transduce bone marrow-derived macrophages (BMMs), including M-CSF and 20 g/ml of protamine (Sigma-Aldrich). The transduced cells were subsequently chosen for 3 days in an α -10 medium supplemented with M-CSF and 60 µg/ml of puromycin (Sigma-Aldrich) [ 74 – 75 ]. TRAP staining On a 48-well tissue culture plate, TRAP stains, BMMs were grown for 4–5 days in an α-10 medium containing M-CSF and RANKL. Following the culture, the cells were fixed using a solution of 4% paraformaldehyde (Wako) and phosphate-buffered saline (PBS). According to the previously reported procedure, TRAP staining was carried out using NaK tartrate and Naphthol AS-BI phosphoric acid (Sigma-Aldrich) [ 35 ]. Quantitative real-time RT-PCR and RNA isolation Quantitative real-time RT-PCR and RNA isolation were performed following the instructions provided by the manufacturer. Total RNA was purified using the RNeasy mini kit (Qiagen, Hilden, Germany). According to the manufacturer's instructions, the Prime-ScriptTM RT reagent kit (Takara Bio, Kusatsu, Japan) was used with 0.5–1 µg of total RNA for synthesizing first-strand cDNAs. Using the following: primers from Thermo-Fisher Scientific, TaqMan quantitative real-time PCR was carried out: Mt-1 (Mm00496660_g1), Mt-2 (Mm00809556_s1), Mt-3 (Mm00496661_g1, Hs00359394_g1), Ctsk (Mm00484039_m1, Nfatc1 (Mm00479445_m1), Hs00166156_m1), Acp5 (Mm00475698_m1), Dcstamp (Mm01168058_m1), Calcr (Mm00432282_m1), Mrps2 (Mm03991065_g1 Hs00211443_m1). Thermo-Fisher Scientific ABI QuantStudio3 equipment was utilized for the amplification of the samples. Denaturation at 95°C for 10 min was the first stage in the amplification process. This was followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. Normalization was performed using the ΔCt method [ 76 ] to determine the relative cDNA amount, with the expression level of mitochondrial gene Mrps2 serving as the reference, whereby both BMMs, and osteoclasts show steady expression. The delta ΔCt technique was used to analyze the relative amounts of MT3 cDNAs in BMMs. Every test was run in triplicate. Immunoblotting After two ice-cold PBS washes, cultured cells were lysed using Cell Lytic M (Sigma-Aldrich), which contained Phosphatase Inhibitor Cocktail (ab201112, Abcam) and Protease Inhibitor (cOmplete Mini, EDTA-free, Sigma-Aldrich). The cell lysates were centrifuged at 14,000 rpm for 15 min at 4°C to remove cellular debris after being incubated on ice for 30 min. Polyacrylamide gels with a 4–12% gradient were loaded with a total of 10–30 micrograms of total protein (Invitrogen, Carlsbad, CA, USA) and electrophoretically transferred onto a polyvinylidene difluoride membrane (Amersham Biosciences, Arlington Heights, IL, USA) using a semi-dry blotting system (Bio-Rad, Hercules, CA, USA). The membrane was next blocked in 5% fat-free milk/Tris-buffered saline for an h before being treated with primary antibodies overnight at 4°C (Santa Cruz Biotechnology). Finally, secondary antibodies conjugated with horseradish peroxidase were applied to the membrane. Immunoreactivity was detected using ECL Prime (Amersham Biosciences) and photographed using an Ez Capture MG (ATTO, Tokyo, Japan) after three washings with Tris-buffered saline containing 0.1% Tween 20. Fluorescent staining of actin filament and nuclei Actin filament and nuclear fluorescent staining osteoclasts cultivated on glass coverslips were permeabilized with 0.2% Triton X-100/PBS for 10 min at room temperature and fixed with 4% paraformaldehyde (Wako) in PBS for 20 min. For 15 minutes at room temperature, Alexa-488 conjugated phalloidin (1:100 from a 1 mg/ml stock) was used to label filament actin. Slow Fade Diamond Antifade Mountant with DAPI (Invitrogen) was used to stain nuclei following two 5-minute PBS washes [ 77 ]. A fluorescence microscope (BZ-X810; Keyence, Osaka, Japan) was used to take pictures of the samples. The mean number of active osteoclasts (podosome-belt bearing osteoclasts on glass coverslips and actin-ring bearing osteoclasts on bone slices) with different nuclei and the percentage of spreading osteoclasts were calculated by Hybrid Cell Count Software using images of five randomly selected areas on each glass coverslip or bone slice. Resorption pit staining 4 percent paraformaldehyde (Wako)/PBS was used to fix mature osteoclasts cultured on cortical bovine bone slices for 20 min cells were eliminated from bone slices using a soft brush after PBS was washed twice for 5 min. The slices were then treated for 60 min at room temperature with 20 µg/ml peroxidase-conjugated wheat germ agglutinin lectin. After washing twice in PBS, bone chips were treated with 0.03% H 2 O 2 and 0.52 mg/ml 3,3-diaminobenzidine for 30 min. Samples were imaged using a fluorescent microscope (BZ-X810; Keyence, Osaka, Japan) and mounted in 80% glycerol/PBS [ 78 ]. Hybrid Cell Count Software was used to determine the mean percentage of resorbed area in each bone slice after collecting blinded photographs of five randomly selected locations per bone slice. Viability Assay In 96-well plates, cells were seeded at 5.0 × 10 3 per well. The vitality of cells in each well was assessed as a reference value after four h of seeding. The CellTiter-Glo Luminescent Cell vitality kit (Promega, Madison, WI, USA) was then used to check the relative cell vitality every 24 h for a total of 96 h. Flow cytometric study of the cell cycle Trypsin was used to collect the cells, which were then washed with PBS before being fixed in ice-cold 70% ethanol at 4°C for 30 min. The cells were fixed, washed with PBS, and then treated with propidium iodide and RNase (Immunostep S.L., Salamanca, Spain) for 15 min. Flow cytometry was conducted using BD FACS Verse (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), and the data were analyzed using BD FlowJo Software. ROS detection 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) was used to measure the production of ROS (Cayman, Michigan, USA) as a fluorescent probe. Briefly, in an FBS-free medium with DCFH-DA (final concentration of 50 µM), cells from various groups were grown and incubated for 30 min. Subsequently, to remove extra DCFH-DA, cells were washed three times in serum-free media. A BZ-X800 microscope was used to take the fluorescence images, and the percentage of cells exhibiting positive fluorescence and the fluorescence intensity were quantified and analyzed using Hybrid Cell Count Software. Mitochondrial ROS production measurements We worked with (Thermo-Fisher Scientific's) MitoSOX Red Mitochondrial Superoxide Indicator to examine the generation of mitochondria-derived ROS. In brief, MitoSOX was incubated with cells in glass-bottom imaging dishes for 15 min at 37°C at a concentration of 5 µM. Following this, Hanks' buffer was used to wash the cells three times to eliminate any remaining probe, and then the cells were examined using a fluorescent microscope. Hybrid Cell Count Software examined the percentage of positive cells and the fluorescence intensity. The levels of intracellular Zn 2+ measurements In 24-well plates, 3 × 10 4 cells were planted, and the cells were then treated with M-CSF and RANKL for two days. Subsequently, the cells were stained with FluoZin-3 (Invitrogen) at a concentration of 10 µM for 30 min. Following staining, Hanks' buffer was used to wash the cells twice and fix them with a 4% paraformaldehyde (Wako) solution. We used Slow Fade Diamond Antifade Mountant for nuclear staining with DAPI (Invitrogen). The immunostained samples were visualized using fluorescence microscopy. The Hybrid Cell Count Software was utilized to analyze positive cells' fluorescence intensity and percentage. Immunocytochemistry In 24-well plates, 3 × 10 4 cells were planted, and the cells were then treated with M-CSF and RANKL for two days. In the following incubation, the cells were fixed for 10 min at room temperature using a 4% paraformaldehyde (PFA) solution (Wako). Subsequently, 0.2% bovine serum albumin (BSA) and 0.3% Triton X-100 (Sigma-Aldrich) were used to block the cells for an h at room temperature. Antibodies against NRF2 (#Sc-365949, Santa Cruz Biotechnology) were treated with the cells for 3 h at a dilution 1:200 in 0.2% BSA. The samples were then exposed for an h at room temperature to Alexa Fluor 546 goat anti-mouse IgG2a cross-adsorbed secondary antibody (#A21133, Invitrogen). Slow Fade Diamond Antifade Mountant with DAPI (Invitrogen) was used to stain nuclei. Immunostaining was visualized using fluorescence microscopy. Blinded, five randomly chosen areas per glass coverslip were imaged. The fluorescence intensity and the percentage of positive cells on cells were quantified using Hybrid Cell Count Software, and the mean values were calculated. Statistics All graphs show the data as the mean ± standard deviation. The Shapiro-Wilk test was conducted to determine whether the data distribution was normal. A two-tailed Student's t-test was used to investigate two-group comparisons when the distribution was normal. A one-way or two-way analysis of variance was carried out for comparisons involving more than two groups, followed by Tukey’s post hoc test. The Kruskal–Wallis test was used for comparisons involving more than two groups when the distribution was non-normal, and Dunn's post hoc test was used for two-group comparisons in those situations. Prism 9 software from GraphPad Software, La Jolla, CA, was used for all statistical calculations. A p < 0.05 was considered statistically significant. Declarations Acknowledgements This study was supported by JSPS KAKENHI (Grant Number: JP18K16626 and 22K09359). Conflict of interest The authors declare no competing interests. Author contributions SA, TF, and HZ designed research; SA, KY, and TT performed research; SA and TF analyzed data and wrote the paper; HS, KK, OS, and YN supervised the study. Ethics approval and consent to participate All animal protocols and procedures used in animal studies were approved by the Institutional Animal Care and Use Committees of Kyushu university (approval number: A23-004-0). The protocols for generation and use of shRNAs and lentivirus were approved by Institutional Biosafety Committee of Kyushu university (approval number: 4-93) Funding Statement The authors received no specific funding for this work. References Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature. 2003;423(6937):337–42. Crockett JC, Rogers MJ, Coxon FP, Hocking LJ, Helfrich MH. Bone remodelling at a glance. J Cell Sci. 2011;124(Pt 7):991–8. Zaidi M. Skeletal remodeling in health and disease. Nat Med. 2007;13(7):791–801. Boyce BF. Advances in the regulation of osteoclasts and osteoclast functions. J Dent Res. 2013;92(10):860–7. Novack DV, Teitelbaum SL. The osteoclast: friend or foe? Annu Rev Pathol. 2008;3:457–84. Tsutsui T, Fujiwara T, Matsumoto Y, Kimura A, Kanahori M, Arisumi S, et al. Geriatric nutritional risk index as the prognostic factor in older patients with fragility hip fractures. Osteoporos Int. 2023;34(7):1207–21. Teitelbaum SL, Ross FP. Genetic regulation of osteoclast development and function. Nat Rev Genet. 2003;4(8):638–49. Ross FP, Teitelbaum SL. alphavbeta3 and macrophage colony-stimulating factor: partners in osteoclast biology. Immunol Rev. 2005;208:88–105. Takeshita S, Faccio R, Chappel J, Zheng L, Feng X, Weber JD, et al. c-Fms tyrosine 559 is a major mediator of M-CSF-induced proliferation of primary macrophages. J Biol Chem. 2007;282(26):18980–90. Takayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H, et al. Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell. 2002;3(6):889–901. Ishii KA, Fumoto T, Iwai K, Takeshita S, Ito M, Shimohata N, et al. Coordination of PGC-1beta and iron uptake in mitochondrial biogenesis and osteoclast activation. Nat Med. 2009;15(3):259–66. Zhou J, Ye S, Fujiwara T, Manolagas SC, Zhao H. Steap4 plays a critical role in osteoclastogenesis in vitro by regulating cellular iron/reactive oxygen species (ROS) levels and cAMP response element-binding protein (CREB) activation. J Biol Chem. 2013;288(42):30064–74. Wang L, Fang B, Fujiwara T, Krager K, Gorantla A, Li C, et al. Deletion of ferroportin in murine myeloid cells increases iron accumulation and stimulates osteoclastogenesis in vitro and in vivo. J Biol Chem. 2018;293(24):9248–64. Das BK, Wang L, Fujiwara T, Zhou J, Aykin-Burns N, Krager KJ, et al. Transferrin receptor 1-mediated iron uptake regulates bone mass in mice via osteoclast mitochondria and cytoskeleton. Elife. 2022;11. Seo HJ, Cho YE, Kim T, Shin HI, Kwun IS. Zinc may increase bone formation through stimulating cell proliferation, alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells. Nutr Res Pract. 2010;4(5):356–61. Moonga BS, Dempster DW. Zinc is a potent inhibitor of osteoclastic bone resorption in vitro. J Bone Miner Res. 1995;10(3):453–7. Togari A, Arakawa S, Arai M, Matsumoto S. Alteration of in vitro bone metabolism and tooth formation by zinc. Gen Pharmacol. 1993;24(5):1133–40. Holloway WR, Collier FM, Herbst RE, Hodge JM, Nicholson GC. Osteoblast-mediated effects of zinc on isolated rat osteoclasts: inhibition of bone resorption and enhancement of osteoclast number. Bone. 1996;19(2):137–42. Coyle P, Philcox JC, Carey LC, Rofe AM. Metallothionein: the multipurpose protein. Cell Mol Life Sci. 2002;59(4):627–47. Moleirinho A, Carneiro J, Matthiesen R, Silva RM, Amorim A, Azevedo L. Gains, losses and changes of function after gene duplication: study of the metallothionein family. PLoS One. 2011;6(4):e18487. Albrecht AL, Singh RK, Somji S, Sens MA, Sens DA, Garrett SH. Basal and metal-induced expression of metallothionein isoform 1 and 2 genes in the RWPE-1 human prostate epithelial cell line. J Appl Toxicol. 2008;28(3):283–93. Uchida Y, Takio K, Titani K, Ihara Y, Tomonaga M. The growth inhibitory factor that is deficient in the Alzheimer's disease brain is a 68 amino acid metallothionein-like protein. Neuron. 1991;7(2):337–47. Moffatt P, Séguin C. Expression of the gene encoding metallothionein-3 in organs of the reproductive system. DNA Cell Biol. 1998;17(6):501–10. Quaife CJ, Findley SD, Erickson JC, Froelick GJ, Kelly EJ, Zambrowicz BP, et al. Induction of a new metallothionein isoform (MT-IV) occurs during differentiation of stratified squamous epithelia. Biochemistry. 1994;33(23):7250–9. Palmiter RD. The elusive function of metallothioneins. Proc Natl Acad Sci U S A. 1998;95(15):8428–30. Maret W. The function of zinc metallothionein: a link between cellular zinc and redox state. J Nutr. 2000;130(5S Suppl):1455s-8s. Lynes MA, Hidalgo J, Manso Y, Devisscher L, Laukens D, Lawrence DA. Metallothionein and stress combine to affect multiple organ systems. Cell Stress Chaperones. 2014;19(5):605–11. Klaassen CD, Liu J, Diwan BA. Metallothionein protection of cadmium toxicity. Toxicol Appl Pharmacol. 2009;238(3):215–20. Bhandari S, Melchiorre C, Dostie K, Laukens D, Devisscher L, Louwrier A, et al. Detection and Manipulation of the Stress Response Protein Metallothionein. Curr Protoc Toxicol. 2017;71:17.9.1-.9.28. Si M, Lang J. The roles of metallothioneins in carcinogenesis. J Hematol Oncol. 2018;11(1):107. Adam P, Křížková S, Heger Z, Babula P, Pekařík V, Vaculovičoá M, et al. Metallothioneins in Prion- and Amyloid-Related Diseases. J Alzheimers Dis. 2016;51(3):637–56. Liu A-L, Zhang Z-M, Zhu B-F, Liao Z-H, Liu Z. Metallothionein protects bone marrow stromal cells against hydrogen peroxide-induced inhibition of osteoblastic differentiation. Cell Biol Int. 2004;28(12):905–11. Trost Z, Trebse R, Prezelj J, Komadina R, Logar DB, Marc J. A microarray based identification of osteoporosis-related genes in primary culture of human osteoblasts. Bone. 2010;46(1):72–80. Fong L, Tan K, Tran C, Cool J, Scherer MA, Elovaris R, et al. Interaction of dietary zinc and intracellular binding protein metallothionein in postnatal bone growth. Bone. 2009;44(6):1151–62. Fujiwara T, Zhou J, Ye S, Zhao H. RNA-binding protein Musashi2 induced by RANKL is critical for osteoclast survival. Cell Death Dis. 2016;7(7):e2300. Ye S, Fujiwara T, Zhou J, Varughese KI, Zhao H. LIS1 Regulates Osteoclastogenesis through Modulation of M-SCF and RANKL Signaling Pathways and CDC42. Int J Biol Sci. 2016;12(12):1488–99. Lee NK, Choi YG, Baik JY, Han SY, Jeong DW, Bae YS, et al. A crucial role for reactive oxygen species in RANKL-induced osteoclast differentiation. Blood. 2005;106(3):852–9. Ruttkay-Nedecky B, Nejdl L, Gumulec J, Zitka O, Masarik M, Eckschlager T, et al. The role of metallothionein in oxidative stress. Int J Mol Sci. 2013;14(3):6044–66. Maret W, Vallee BL. Thiolate ligands in metallothionein confer redox activity on zinc clusters. Proc Natl Acad Sci U S A. 1998;95(7):3478–82. Kang YJ. Metallothionein redox cycle and function. Exp Biol Med (Maywood). 2006;231(9):1459–67. Bhatt NY, Kelley TW, Khramtsov VV, Wang Y, Lam GK, Clanton TL, et al. Macrophage-colony-stimulating factor-induced activation of extracellular-regulated kinase involves phosphatidylinositol 3-kinase and reactive oxygen species in human monocytes. J Immunol. 2002;169(11):6427–34. Ha H, Kwak HB, Lee SW, Jin HM, Kim HM, Kim HH, et al. Reactive oxygen species mediate RANK signaling in osteoclasts. Exp Cell Res. 2004;301(2):119–27. Kang TC. Nuclear Factor-Erythroid 2-Related Factor 2 (Nrf2) and Mitochondrial Dynamics/Mitophagy in Neurological Diseases. Antioxidants (Basel). 2020;9(7). Li Z, Chen C, Zhu X, Li Y, Yu R, Xu W. Glycyrrhizin Suppresses RANKL-Induced Osteoclastogenesis and Oxidative Stress Through Inhibiting NF-κB and MAPK and Activating AMPK/Nrf2. Calcif Tissue Int. 2018;103(3):324–37. Ishii T, Itoh K, Takahashi S, Sato H, Yanagawa T, Katoh Y, et al. Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J Biol Chem. 2000;275(21):16023–9. Hyeon S, Lee H, Yang Y, Jeong W. Nrf2 deficiency induces oxidative stress and promotes RANKL-induced osteoclast differentiation. Free Radic Biol Med. 2013;65:789–99. Sun X, Xie Z, Hu B, Zhang B, Ma Y, Pan X, et al. The Nrf2 activator RTA-408 attenuates osteoclastogenesis by inhibiting STING dependent NF-κb signaling. Redox Biol. 2020;28:101309. Kanzaki H, Shinohara F, Itohiya K, Yamaguchi Y, Katsumata Y, Matsuzawa M, et al. RANKL induces Bach1 nuclear import and attenuates Nrf2-mediated antioxidant enzymes, thereby augmenting intracellular reactive oxygen species signaling and osteoclastogenesis in mice. FASEB J. 2017;31(2):781–92. Wei W, Wang X, Yang M, Smith LC, Dechow PC, Sonoda J, et al. PGC1beta mediates PPARgamma activation of osteoclastogenesis and rosiglitazone-induced bone loss. Cell Metab. 2010;11(6):503–16. Callaway DA, Jiang JX. Reactive oxygen species and oxidative stress in osteoclastogenesis, skeletal aging and bone diseases. J Bone Miner Metab. 2015;33(4):359–70. Sugiura T, Kuroda E, Yamashita U. Dysfunction of macrophages in metallothionein-knock out mice. J uoeh. 2004;26(2):193–205. Murakami M, Hirano T. Intracellular zinc homeostasis and zinc signaling. Cancer Sci. 2008;99(8):1515–22. Li B, Cui W, Tan Y, Luo P, Chen Q, Zhang C, et al. Zinc is essential for the transcription function of Nrf2 in human renal tubule cells in vitro and mouse kidney in vivo under the diabetic condition. J Cell Mol Med. 2014;18(5):895–906. Ha KN, Chen Y, Cai J, Sternberg P, Jr. Increased glutathione synthesis through an ARE-Nrf2-dependent pathway by zinc in the RPE: implication for protection against oxidative stress. Invest Ophthalmol Vis Sci. 2006;47(6):2709–15. Cortese MM, Suschek CV, Wetzel W, Kroncke KD, Kolb-Bachofen V. Zinc protects endothelial cells from hydrogen peroxide via Nrf2-dependent stimulation of glutathione biosynthesis. Free Radic Biol Med. 2008;44(12):2002–12. Ge MH, Tian H, Mao L, Li DY, Lin JQ, Hu HS, et al. Zinc attenuates ferroptosis and promotes functional recovery in contusion spinal cord injury by activating Nrf2/GPX4 defense pathway. CNS Neurosci Ther. 2021;27(9):1023–40. Li D, Tian H, Li X, Mao L, Zhao X, Lin J, et al. Zinc promotes functional recovery after spinal cord injury by activating Nrf2/HO-1 defense pathway and inhibiting inflammation of NLRP3 in nerve cells. Life Sci. 2020;245:117351. Gaetke LM, Chow CK. Copper toxicity, oxidative stress, and antioxidant nutrients. Toxicology. 2003;189(1–2):147–63. Jomova K, Baros S, Valko M. Redox active metal-induced oxidative stress in biological systems. Transition Metal Chemistry. 2012;37(2):127–34. Ni S, Qian Z, Yuan Y, Li D, Zhong Z, Ghorbani F, et al. Schisandrin A restrains osteoclastogenesis by inhibiting reactive oxygen species and activating Nrf2 signalling. Cell Prolif. 2020;53(10):e12882. Sridhar V, Narnaware S, Kumar P, Kale SB, Majumdar AS. Co-treatment with sulforaphane–zein microparticles enhances the chemopreventive potential of zinc in a 1,2-dimethylhydrazine induced colon carcinogenesis rat model. RSC Advances. 2016;6(41):34233–44. Park KH, Park B, Yoon DS, Kwon SH, Shin DM, Lee JW, et al. Zinc inhibits osteoclast differentiation by suppression of Ca2+-Calcineurin-NFATc1 signaling pathway. Cell Commun Signal. 2013;11:74. Yamaguchi M, Weitzmann MN. Zinc stimulates osteoblastogenesis and suppresses osteoclastogenesis by antagonizing NF-κB activation. Mol Cell Biochem. 2011;355(1–2):179–86. Li X, Senda K, Ito A, Sogo Y, Yamazaki A. Effect of Zn and Mg in tricalcium phosphate and in culture medium on apoptosis and actin ring formation of mature osteoclasts. Biomed Mater. 2008;3(4):045002. Lichtlen P, Schaffner W. The "metal transcription factor" MTF-1: biological facts and medical implications. Swiss Med Wkly. 2001;131(45–46):647–52. Malaiyandi LM, Dineley KE, Reynolds IJ. Divergent consequences arise from metallothionein overexpression in astrocytes: zinc buffering and oxidant-induced zinc release. Glia. 2004;45(4):346–53. Habel N, Hamidouche Z, Girault I, Patino-Garcia A, Lecanda F, Marie PJ, et al. Zinc chelation: a metallothionein 2A's mechanism of action involved in osteosarcoma cell death and chemotherapy resistance. Cell Death Dis. 2013;4(10):e874. Sun YX, Xu AH, Yang Y, Li J. Role of Nrf2 in bone metabolism. J Biomed Sci. 2015;22:101. Sánchez-de-Diego C, Pedrazza L, Pimenta-Lopes C, Martinez-Martinez A, Dahdah N, Valer JA, et al. NRF2 function in osteocytes is required for bone homeostasis and drives osteocytic gene expression. Redox Biol. 2021;40:101845. Sakai E, Morita M, Ohuchi M, Kido MA, Fukuma Y, Nishishita K, et al. Effects of deficiency of Kelch-like ECH-associated protein 1 on skeletal organization: a mechanism for diminished nuclear factor of activated T cells cytoplasmic 1 during osteoclastogenesis. FASEB J. 2017;31(9):4011–22. Xue P, Hu X, Powers J, Nay N, Chang E, Kwon J, et al. CDDO-Me, Sulforaphane and tBHQ attenuate the RANKL-induced osteoclast differentiation via activating the NRF2-mediated antioxidant response. Biochem Biophys Res Commun. 2019;511(3):637–43. Han J, Yang K, An J, Jiang N, Fu S, Tang X. The Role of NRF2 in Bone Metabolism - Friend or Foe? Front Endocrinol (Lausanne). 2022;13:813057. Takeshita S, Kaji K, Kudo A. Identification and characterization of the new osteoclast progenitor with macrophage phenotypes being able to differentiate into mature osteoclasts. J Bone Miner Res. 2000;15(8):1477–88. Zhou J, Fujiwara T, Ye S, Li X, Zhao H. Downregulation of Notch modulators, tetraspanin 5 and 10, inhibits osteoclastogenesis in vitro. Calcif Tissue Int. 2014;95(3):209–17. Zhou J, Fujiwara T, Ye S, Li X, Zhao H. Ubiquitin E3 Ligase LNX2 is Critical for Osteoclastogenesis In Vitro by Regulating M-CSF/RANKL Signaling and Notch2. Calcif Tissue Int. 2015;96(5):465–75. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008;3(6):1101–8. Ito Y, Teitelbaum SL, Zou W, Zheng Y, Johnson JF, Chappel J, et al. Cdc42 regulates bone modeling and remodeling in mice by modulating RANKL/M-CSF signaling and osteoclast polarization. J Clin Invest. 2010;120(6):1981–93. Fujiwara T, Ye S, Castro-Gomes T, Winchell CG, Andrews NW, Voth DE, et al. PLEKHM1/DEF8/RAB7 complex regulates lysosome positioning and bone homeostasis. JCI Insight. 2016;1(17):e86330. Additional Declarations (Not answered) Supplementary Files Originalwesternblots.tif Cite Share Download PDF Status: Published Journal Publication published 01 Dec, 2023 Read the published version in Cell Death Discovery → Version 1 posted Unknown event 22 Sep, 2023 Editorial decision: Reject after peer review 21 Sep, 2023 Review # 2 received at journal 19 Sep, 2023 Reviewer # 2 agreed at journal 13 Sep, 2023 Review # 1 received at journal 10 Sep, 2023 Reviewer # 1 agreed at journal 10 Sep, 2023 Reviewers invited by journal 29 Aug, 2023 Submission checks completed at journal 15 Aug, 2023 Editor assigned by journal 14 Aug, 2023 First submitted to journal 14 Aug, 2023 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3262822","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":232018105,"identity":"2fb6adc8-ebca-4bef-9b8d-4797adf62143","order_by":0,"name":"Toshifumi Fujiwara","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-9381-9696","institution":"Kyushu University Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Toshifumi","middleName":"","lastName":"Fujiwara","suffix":""},{"id":232018106,"identity":"beb294d6-ebbd-45ee-bd50-03e4b77a0bd3","order_by":1,"name":"Shinkichi Arisumi","email":"","orcid":"","institution":"Japan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shinkichi","middleName":"","lastName":"Arisumi","suffix":""},{"id":232018107,"identity":"e7d80dc3-cc8b-4055-9bbc-fae2ff009255","order_by":2,"name":"Keitaro Yasumoto","email":"","orcid":"","institution":"Kyushu University Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Keitaro","middleName":"","lastName":"Yasumoto","suffix":""},{"id":232018108,"identity":"0a4154fc-19ce-4685-b515-3ffe73064f6c","order_by":3,"name":"Tomoko Tsutsui","email":"","orcid":"","institution":"Kyushu University Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tomoko","middleName":"","lastName":"Tsutsui","suffix":""},{"id":232018109,"identity":"b0ca5a5e-0f8f-4d94-83d3-2d8680109550","order_by":4,"name":"Hirokazu Saiwai","email":"","orcid":"","institution":"Kyushu University Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hirokazu","middleName":"","lastName":"Saiwai","suffix":""},{"id":232018110,"identity":"cbfa9420-e5ec-4f92-9a02-0e7025b1f8ee","order_by":5,"name":"Kazu Kobayakawa","email":"","orcid":"","institution":"Kyushu University Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kazu","middleName":"","lastName":"Kobayakawa","suffix":""},{"id":232018111,"identity":"c46dec36-bcbc-4b8b-8e95-c582acbc58cc","order_by":6,"name":"Seiji Okada","email":"","orcid":"","institution":"Osaka University Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Seiji","middleName":"","lastName":"Okada","suffix":""},{"id":232018112,"identity":"85fa271b-5af6-4f88-8598-3421ee2da7f0","order_by":7,"name":"Haibo Zhao","email":"","orcid":"","institution":"Tibor Rubin VA Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haibo","middleName":"","lastName":"Zhao","suffix":""},{"id":232018113,"identity":"d3aac708-c43d-4015-a27c-07994131e34d","order_by":8,"name":"Yasuharu Nakashima","email":"","orcid":"","institution":"Kyushu University Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yasuharu","middleName":"","lastName":"Nakashima","suffix":""}],"badges":[],"createdAt":"2023-08-14 13:16:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3262822/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3262822/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41420-023-01729-y","type":"published","date":"2023-12-01T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":43141526,"identity":"0b5fc0c8-02ef-42d0-b4d2-063ec83a680b","added_by":"auto","created_at":"2023-09-14 15:58:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1656466,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn osteoclast, MT3 is the most highly expressed in MT isoforms and is critical for osteoclast differentiation \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Bone marrow macrophages (BMM) were cultured with M-CSF, or with both M-CSF and RANKL for 2 and 4 days to generate pre-osteoclasts (pOC) and mature osteoclasts (OC), respectively. The mRNA expression levels of \u003cem\u003eMt1-3\u003c/em\u003ewere measured using real-time PCR (n=3). \u003cstrong\u003eB\u003c/strong\u003e Human peripheral blood monocytes (PBMC) were cultured with M-CSF, or with both M-CSF and RANKL for 3 and 7 days to generate pOCs and OCs. TRAP staining of OC (scale bar = 500 μm) and the mRNA expression levels of human\u003cem\u003e Mt3\u003c/em\u003e and osteoclast marker, cathepsin K (\u003cem\u003eCtsk\u003c/em\u003e), were examined (n=3). \u003cstrong\u003eC\u003c/strong\u003e After knocking down BMM by MT3-shRNAs (MT-sh1 or -sh2) or a control shRNA (Control), the cells were induced to differentiate into osteoclasts for a duration of 4 days. The levels of \u003cem\u003eMt3\u003c/em\u003e expression were quantified using real-time PCR (n=3). \u003cstrong\u003eD\u003c/strong\u003e TRAP staining and quantification of numbers of total and spreading OC cultured on plastic 96-well dishes (scale bar = 500 μm) (n=6). \u003cstrong\u003eE\u003c/strong\u003e The mRNA expression levels of osteoclast markers, NFATc1 (\u003cem\u003eNfatc1\u003c/em\u003e); cathepsin K (\u003cem\u003eCtsk\u003c/em\u003e); TRAP (\u003cem\u003eAcp5\u003c/em\u003e); DC-STAMP (\u003cem\u003eDcstamp\u003c/em\u003e); and calcitonin receptor (\u003cem\u003eCalcr\u003c/em\u003e) were measured by real-time PCR (n=3). \u003cstrong\u003eF\u003c/strong\u003e Protein expressions of osteoclast markers were detected by western blotting. Actin served as a loading control. \u003cstrong\u003eG\u003c/strong\u003e Actin filaments and nuclei were stained with Alexa-488-conjugated phalloidin and DAPI in osteoclasts cultured on glass coverslips (scale bar = 100 μm). The number of osteoclasts with different nuclei and the percentage of spreading osteoclasts with a peripheral podosome-belt were quantified (n=5). \u003cstrong\u003eH\u003c/strong\u003e Actin filaments and nuclei were stained with Alexa-488 conjugated Phalloidin and DAPI in osteoclasts cultured on cortical bovine bone slices (scale bar = 50 μm). The number and the percentage of osteoclasts exhibiting an actin ring per bone slice were calculated (n=3). \u003cstrong\u003eI\u003c/strong\u003e Resorption pits were stained by horseradish peroxidase conjugated wheat-germ agglutinin (scale bar = 100 μm). The percentage of resorbed area per bone slice was calculated by Hybrid Cell Count Software (n=3). The data are presented as mean ± SD. * p\u0026lt;0.05; ** p\u0026lt;0.01; *** p\u0026lt;0.001; **** p\u0026lt;0.0001 vs control.\u003c/p\u003e","description":"","filename":"Figure.1.png","url":"https://assets-eu.researchsquare.com/files/rs-3262822/v1/ea77e59c8480c14a31575237.png"},{"id":43141522,"identity":"e04056a6-a224-45f4-83d8-e1c87180422c","added_by":"auto","created_at":"2023-09-14 15:58:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":616608,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of MT3-induced apoptosis in pre-osteoclasts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Relative cell number of Control and MT3-sh1/sh2 BMMs was presented up to 96 hours in growth curve. The data are presented as mean (n=3). * p\u0026lt;0.001 vs control. \u003cstrong\u003eB\u003c/strong\u003e Representative histograms of cell cycle distribution in BMMs were measured by flow cytometry using Propidium iodide. The average percentage of cell numbers in each phase of the cell cycle is shown in the graphs. The data are presented as mean ± SD (n=4). ns: no significance, vs control. \u003cstrong\u003eC\u003c/strong\u003e Control and MT3-sh1/sh2 of BMM or pOC were either untreated or serum/cytokine starved for 4 h. The levels of PARP and Cleaved Caspase 3, which are markers of apoptosis, were assessed using Western blot analysis. Actin served as a loading control.\u003c/p\u003e","description":"","filename":"Figure.2.png","url":"https://assets-eu.researchsquare.com/files/rs-3262822/v1/979333d43f2f3c690d34f7e7.png"},{"id":43141524,"identity":"8ef18c00-2a0a-46a3-8a5f-7d8d2df37d3c","added_by":"auto","created_at":"2023-09-14 15:58:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1039219,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMT3 is required for RANKL-induced JNK and NF-κB activation in pre-osteoclasts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWestern blot analysis detected the levels of phosphorylated and total JNK, IκBα, AKT, and ERK in pOC treated with RANKL (100 ng/mL) (\u003cstrong\u003eA\u003c/strong\u003e) or M-CSF (100 ng/mL) (\u003cstrong\u003eB\u003c/strong\u003e) for the indicated time. Actin served as loading controls.\u003c/p\u003e","description":"","filename":"Figure.3.png","url":"https://assets-eu.researchsquare.com/files/rs-3262822/v1/bb61359fc45ead344fb36cf3.png"},{"id":43142553,"identity":"cf95173b-c613-4797-96b6-1f83bd7bc865","added_by":"auto","created_at":"2023-09-14 16:06:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1262025,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChange of RANKL-induced ROS and mitochondrial ROS levels by depletion of MT3 expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Representative images of intracellular ROS levels in pOCs were measured by DCFH-DA assay followed by RANKL stimulation for 48 h (scale bar = 100 μm) and the percentage of positive cells and the fluorescence intensity on cells of each well were calculated by Hybrid Cell Count Software (Keyence) (n=5). \u003cstrong\u003eB \u003c/strong\u003eThe expressions of NRF2, HO-1, and CAT were detected by western blots. Actin served as a loading control. \u003cstrong\u003eC\u003c/strong\u003eRepresentative images of mitochondrial ROS levels in pOCs were measured by MitoSOX Red Mitochondrial Superoxide Indicator, followed by RANKL stimulation for 48 h (scale bar = 100 μm) and the percentage of positive cells and the fluorescence intensity on cells of each well were calculated, respectively (n=5). \u003cstrong\u003eD\u003c/strong\u003e The expressions of p-CREB and PGC-1β were detected by western blots. t-CREB and Actin served as loading controls, respectively. The data are presented as mean ± SD. * p\u0026lt;0.05; ** p\u0026lt;0.01; *** p\u0026lt;0.001; **** p\u0026lt;0.0001 vs control.\u003c/p\u003e","description":"","filename":"Figure.4.png","url":"https://assets-eu.researchsquare.com/files/rs-3262822/v1/2032bc74683a2566b284397e.png"},{"id":43142554,"identity":"d0878a0b-23ce-4cb2-8c6a-8fda61ab8e22","added_by":"auto","created_at":"2023-09-14 16:06:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1304919,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe levels of intracellular Zn\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e were increased in the MT3 knock down pOC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConfocal fluorescence microscopic images of pOC of Control and MT3-sh1/sh2 stained with FluoZin-3 (green) and DAPI (blue). Samples were treated and untreated with 50 μM ZnSO\u003csub\u003e4\u003c/sub\u003e for 1 hour (scale bar = 100 μm). The percentage of positive cells and the fluorescence intensity on cells were calculated by Hybrid Cell Count Software (n=6). The data are presented as mean ± SD. * p\u0026lt;0.05; ** p\u0026lt;0.01 vs control.\u003c/p\u003e","description":"","filename":"Figure.5.png","url":"https://assets-eu.researchsquare.com/files/rs-3262822/v1/6a808997764fe787abd5fd90.png"},{"id":43141528,"identity":"47bdcf1f-db50-4775-bc5a-3af3d543bbae","added_by":"auto","created_at":"2023-09-14 15:58:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":725378,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of NRF2 was upregulated by MT3 knock down, increased by zinc administration, and suppressed by zinc chelation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunofluorescence assay of NRF2 (red) and DAPI-labeled nuclei (blue) treated and untreated with 50 μM ZnSO\u003csub\u003e4\u003c/sub\u003e and 5 μM TPEN in pOc (scale bar = 100 μm). The percentage of positive cells and the fluorescence intensity were calculated by Hybrid Cell Count Software (n=3). The data are presented as mean ± SD. * p\u0026lt;0.05 vs control.\u003c/p\u003e","description":"","filename":"Figure.6.png","url":"https://assets-eu.researchsquare.com/files/rs-3262822/v1/afcf6a5732bcad1fed45ff96.png"},{"id":43141525,"identity":"433e15d7-6155-4879-b322-9f93ba4b4371","added_by":"auto","created_at":"2023-09-14 15:58:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":415507,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematics of signaling pathways and a model of MT3 function in osteoclastogenesis.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure.7.png","url":"https://assets-eu.researchsquare.com/files/rs-3262822/v1/182961b5151a71b553787156.png"},{"id":47477528,"identity":"97ba6498-548c-4308-9714-2f601a22da86","added_by":"auto","created_at":"2023-12-02 08:33:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3180735,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3262822/v1/0beb6125-d686-4d2d-8912-72490d277dbd.pdf"},{"id":43141553,"identity":"c2cd7411-bfa5-42c7-ba3e-61596d77c45e","added_by":"auto","created_at":"2023-09-14 15:59:05","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":138732192,"visible":true,"origin":"","legend":"","description":"","filename":"Originalwesternblots.tif","url":"https://assets-eu.researchsquare.com/files/rs-3262822/v1/4d337f146ab9f6cac073dbfb.tif"}],"financialInterests":"(Not answered)","formattedTitle":"Metallothionein 3 promotes osteoclast differentiation and survival by regulating the intracellular Zn2+ concentration and NRF2 pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteoclast- and osteoblast-mediated bone formation is critical for skeletal development, homeostasis, and repair [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In adults, bone development and bone loss are intricately linked and balanced through a continuous self-renewal process known as bone remodeling that ensures that bone mass and strength are maintained throughout life [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Increased osteoclast activity and numbers have been attributed to pathological bone loss and fragility fractures in metabolic bone illnesses such as osteoporosis, Paget's disease, and osteolytic tumor [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The cellular and molecular mechanisms governing osteoclast differentiation and operation in normal and pathological circumstances must be clarified. This understanding is essential for creating novel therapeutic approaches to cure bone loss in various skeletal illnesses efficiently.\u003c/p\u003e \u003cp\u003eMononuclear progenitor cells are produced from the monocyte/macrophage lineage of hematopoietic stem cells, which fuse to form multinucleated cells called osteoclasts. The two cytokine receptor activators of nuclear factor-κB (NF-κB) ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) are essential for osteoclastogenesis \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. M-CSF primarily controls macrophage proliferation and osteoclast survival by stimulating the phosphoinositide-3-kinase/AKT (PI3K/AKT) and extracellular signal-regulated kinase (ERK) pathways [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], while NF-κB, PI3K/AKT, and mitogen-activated protein kinase (MAPK) pathways are all activated by RANKL to control osteoclast differentiation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Furthermore, the main transcription factor for osteoclast differentiation, nuclear factor of activated T cells 1 (NFATc1), is induced by RANKL signaling [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], regulating calcium oscillation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Metal ions such as calcium, iron, and zinc play pivotal roles in osteoclast differentiation and function. Primarily, iron homeostasis controls osteoclastogenesis and function via intracellular and mitochondrial reactive oxidative species (ROS). Moreover, zinc is essential for fosteཌྷring the mineralization and development of bones by stimulating osteoblasts, and it exerts a suppressive effect on bone resorption by inhibiting osteoclast formation. Thus, the metabolism of metal ions remains unknown mainly due to their complex roles.\u003c/p\u003e \u003cp\u003eThe metal-binding protein, metallothionein (MT), which comprises high cysteine content (30%) and low molecular weight (6\u0026ndash;7 kDa), has four primary isoforms (MT1-MT4). MT1 and MT2 are ubiquitously expressed in various soft tissues, while MT3 is primarily found in brain tissue, heart, kidney, and genital organs [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. MT4 is specifically expressed in certain squamous epithelia [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. MT has a wide range of physiological functions, including heavy metal detoxification, maintenance of metal ion homeostasis (especially zinc and copper), regulation of cellular redox balance, and facilitation of cell proliferation [\u003cspan additionalcitationids=\"CR26 CR27 CR28\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In addition, the MT protein family has been implicated in pathological conditions such as cancer and neurodegenerative diseases [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In mouse bone marrow stromal cells, MT serves a protective function against oxidative stress-induced suppression of osteoblast development. Additionally, a zinc-rich diet, which stimulates endogenous MT production, affects both osteoblast differentiation and postnatal bone growth [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, MT function in osteoclastogenesis is still unclear.\u003c/p\u003e \u003cp\u003eIn this study, we identified MT3 as the dominant MT isoform expressed in osteoclasts, which was upregulated in murine and human osteoclasts after RANKL stimulation. Apoptosis and a reduction of osteoclast differentiation were caused by the loss of MT3, which downregulated RANKL-stimulated MAPK/NF-κB signaling. Furthermore, loss of MT3 raised intracellular zinc concentrations, which in turn increased nuclear factor erythroid 2-related factor 2 (NRF2), an antioxidant defense mechanism, and decreased ROS in osteoclastogenesis. Our research suggests that MT3 is a new osteoclastogenesis regulator that controls ROS and the NRF2 pathway.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eOsteoclast development and bone resorption are inhibited in vitro by deleting MT3, which is abundantly expressed in murine and human osteoclasts.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe first assessed the mRNA levels of \u003cem\u003eMt1-3\u003c/em\u003e with osteoclast formation using real-time quantitative PCR to determine the function of MT in osteoclast lineage cells. To achieve this, bone marrow macrophages (BMM) were cultivated with M-CSF alone or in combination with RANKL for 2 and 4 days for differentiation into preosteoclasts (pOC) and mature osteoclasts (mOC), respectively, each of which has a different function. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA demonstrates that during osteoclast development, the mRNA levels of \u003cem\u003eMt1-3\u003c/em\u003e increased, and \u003cem\u003eMt3\u003c/em\u003e had the highest expression among the \u003cem\u003eMt\u003c/em\u003e osteoclast isoforms, showing that Mt3 was the most prevalent isoform within the MT family during osteoclastogenesis. We also examined the expression of \u003cem\u003eMt3\u003c/em\u003e in human peripheral blood mononuclear cell-derived osteoclasts and found that its expression increased during osteoclastogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur next goal was to define MT3's cell-autonomous roles in osteoclast differentiation and function. To achieve this, we investigated if two short hairpin (sh) RNAs (MT3-sh1 and MT3-sh2) that targeted various murine Mt3 mRNA locations could knock down MT3 expression in BMM via lentiviral transduction. The negative control was a shRNA directed against firefly luciferase. BMMs that had been positively transduced were either cultivated with M-CSF alone or for three days with M-CSF and RANKL. Both MT3-specific shRNAs, but not the control, significantly decreased \u003cem\u003eMt3\u003c/em\u003e mRNA expression in RANKL-induced mOC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Tartrate-resistant acid phosphatase (TRAP), a marker of osteoclast development, revealed a decrease in the number of multinucleated osteoclasts in the bone. Knockdown of MT3 in BMM inhibited osteoclast formation and significantly decreased the total osteoclast number and spreading area (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Reduced mRNA expression of osteoclast marker genes, including NFATc1 (encoded by \u003cem\u003eNfatc1\u003c/em\u003e), cathepsin K (encoded by \u003cem\u003eCtsk\u003c/em\u003e), TRAP (encoded by \u003cem\u003eAcp5\u003c/em\u003e), DC-STAMP (encoded by \u003cem\u003eDcstamp\u003c/em\u003e), and calcitonin receptor (encoded by \u003cem\u003eCalcr\u003c/em\u003e), compared to control cells in MT3-depleted mOC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) confirmed the reduction. Furthermore, MT3-depleted cells markedly decreased the protein expressions of NFATc1 and CTSK (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). We stained actin filaments using Alexa- 488- conjugated phalloidin in control and MT3 knockdown osteoclasts cultivated on plastic dishes because actin cytoskeleton organization is crucial for osteoclast spreading and the production of podosome belts on plastic plates. Depleting MT3 in osteoclasts may impair their function because osteoclast activation and function are required for actin ring formation. In support of this hypothesis, MT3 knockdown osteoclasts exhibited less bone resorption than control osteoclasts, as shown by the staining of resorption pits on cortical bovine bone slices (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). These data suggest that MT3 is upregulated by RANKL and positively regulates osteoclast differentiation and function \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMT3 is critical for osteoclast survival.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the potential involvement of MT3 in BMM cell growth, we conducted a comprehensive analysis of cell growth over 96 hours. MT3 knockdown by shRNA rescued the increased number of BMM compared with the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Next, we attempted to determine which cellular processes were involved in the cell growth of osteoclast lineage cells after MT3 depletion. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB compares the cell cycle progression (S and G2/M) in BMM between control and MT3 knockdown groups using flow cytometry and reveals no discernible differences. The cell population in the G0-1 phase in control BMM or MT3 knockdown was also unchanged, suggesting that MT3 knockdown did not affect the BMM cell cycle progression. Western blotting of the apoptosis markers poly (ADP-ribose) polymerase (PARP) and cleaved (active) caspase 3 revealed that ablation of MT3 in BMM had a negligible impact on apoptosis triggered by cytokine or serum deprivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Furthermore, knockdown of MT3 in pOC promoted apoptosis under the same circumstances (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These results indicate that MT3 in osteoclast lineage cells accelerated osteoclastogenesis by affecting osteoclast survival.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMT3 regulates osteoclastogenesis via activation of RANKL-induced JNK and NF-κB.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eM-CSF and RANKL signaling activation are essential for the proliferation, survival, and differentiation of osteoclastogenesis. Control and MT3 knockdown preosteoclasts were starved with serum and cytokines and then stimulated with M-CSF or RANKL for the designated amount of time to determine which signaling in M-CSF or RANKL affected preosteoclasts of MT3 loss [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Western blots were used to analyze the activation of c-Jun N-terminal kinase (JNK), NF-κB, and ERK, which are all induced by RANKL and M-CSF, respectively. AKT, also known as protein kinase B, and AK strain transformation are induced by M-CSF. Both MT3 shRNAs that knocked down MT3 in preosteoclasts had no impact on ERK and AKT phosphorylation in response to M-CSF stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In contrast, JNK and NF-κB activation caused by RANKL was consistently reduced by MT3 deletion by both shRNAs, evidenced by lower levels of phospho-JNK and phospho-IκB (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Given that MT3 regulates the JNK and NF-κB pathways, which are essential for osteoclast differentiation and survival, these findings imply that MT3 modulates RANKL-induced activation of JNK and NF-κB, which controls osteoclast differentiation and survival [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe Loss of MT3 triggers the activation of the NRF2 pathway, leading to the inhibition of RANKL-induced ROS production.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn osteoclastogenesis, RANKL-induced activation of MAPKs (ERK, JNK, and p38), NF-κ-B, and the PI3K/AKT pathways depends on ROS generated by RANKL and MT has a protective role against oxidative stress [\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. So, we examined the production of ROS during osteoclastogenesis by DCFH-DA staining. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, deletion of MT3 in preosteoclasts dramatically decreased intracellular ROS production by assessing the positivity rate of DCF fluorescence, indicating that depletion of MT3 reduced ROS production induced by RANKL during osteoclastogenesis. ROS act as mediators of intracellular signaling involved in the development and activation of osteoclasts in response to RANKL stimulation [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Additionally, nuclear factor erythroid 2-related factor 2 (NRF2) is a defensive mechanism against oxidative stress that regulates several antioxidant enzymes, such as NAD(P)H quinone dehydrogenase 1 (NQO1), catalase (CAT), haemoxygenase-1 (HO-1), and γ-glutamyl cysteine synthetase (GCS) [\u003cspan additionalcitationids=\"CR44 CR45\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The overexpression of NRF2 suppresses osteoclastogenesis \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e due to the reduction of oxidative stress. To investigate the association with ROS-related enzymes in MT3 loss, the expression of ROS-related enzymes, including CAT, HO-1, and NRF2, with osteoclast differentiation is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB by western blotting. In comparison to the control, MT3 knockdown considerably boosted the expression levels of NRf2 and its downstream antioxidant factors, HO-1 and CAT, in BMM and pOC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), leading to less ROS production during osteoclast differentiation. These findings may provide mechanistic evidence that RANKL-induced intracellular ROS production is effectively attenuated by the loss of MT3, which upregulates NRF2, and the associated downstream antioxidant enzymes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDepletion of MT3 decreased mitochondrial ROS levels in osteoclast precursors.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eROS activates the downstream mediator peroxisome proliferator-activated receptor coactivator 1β (PGC-1β) via phosphorylating the cAMP response element-binding protein (CREB) during osteoclastogenesis, stimulating mitochondrial biogenesis, and the generating of mitochondrial ROS, which in a positively feedback-induced manner increases CREB and PGC-1β activity and facilitates NFATc1 activation. The deletion of MT3 in preosteoclasts also diminished mitochondrial ROS levels, similarly to intracellular ROS production (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). We then analyzed CREB phosphorylation and PGC-1β expression in cells treated with MT3 shRNA to promote osteoclastogenesis, which resulted in a reduction of CREB phosphorylation and PGC-1β expression in the depletion of MT3 osteoclasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These data suggest that MT3 regulates mitochondrial ROS levels via the activation of CREB and PGC-1β.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMT3 loss led to the accumulation of intracellular Zn\u003c/b\u003e \u003csup\u003e \u003cb\u003e2+\u003c/b\u003e \u003c/sup\u003e, \u003cb\u003epotentially leading to the upregulation of NRF2 gene expression.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMT acts as a biochemical regulator of intracellular levels of free Zn\u003csup\u003e2+\u003c/sup\u003e by capturing and subsequently releasing Zn\u003csup\u003e2+\u003c/sup\u003e in response to various biochemical events, such as oxidative signaling. Visualization using FluoZin-3 fluorescence microscopy verified that preosteoclasts without MT3 resulted in an elevation of intracellular Zn\u003csup\u003e2+\u003c/sup\u003e levels at baseline conditions. Furthermore, intracellular levels of Zn\u003csup\u003e2+\u003c/sup\u003e were enhanced one hour after the administration of 50 \u0026micro;M ZnSO\u003csub\u003e4\u003c/sub\u003e in the MTs-depleted cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The change in expression of NRF2 in preosteoclasts after treatment with Zn\u003csup\u003e2+\u003c/sup\u003e was examined with or without MT3 because it has been reported that Zn\u003csup\u003e2+\u003c/sup\u003e is essential for NRF2 expression and transcriptional function [\u003cspan additionalcitationids=\"CR54 CR55 CR56\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, MT3 depletion in preosteoclasts showed higher intracellular NRF2 expression, consistent with western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). After administration of ZnSO\u003csub\u003e4\u003c/sub\u003e, NRF2 expression was increased in MT3 knockdown cells than controls. However, this effect was treated with N, N, N', N'-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), a cell-permeable zinc chelator, and was successfully removed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), suggesting that MT3 loss elevated NRF2 expression by the accumulation of intracellular Zn\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIncreased intracellular ROS production in osteoclastogenesis encourages cell survival and differentiation through the activation of MAPK and NF-κB. It is also implicated in the generation of mitochondrial ROS, which is mediated by pCREB and PGC1β. In particular, the metabolism of cellular metal ions, such as iron and copper, regulates ROS production and mitochondrial biogenesis. However, the molecular processes governing cellular metal ions in osteoclastogenesis are still unclear. This study elucidated that the metal-binding protein MTs, particularly MT3, were upregulated during osteoclast differentiation of murine and human osteoclasts. MTs have various physiological functions, such as detoxification of heavy metals, maintenance of metal ion homeostasis (particularly Zn\u003csup\u003e2+\u003c/sup\u003e), and regulation of cellular redox balance [\u003cspan additionalcitationids=\"CR26 CR27 CR28\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]; However, MT3 suppression prevented osteoclast development and induced apoptosis in osteoclast precursor cells. This effect was linked to increased free intracellular Zn\u003csup\u003e2+\u003c/sup\u003e that was not bound to MT3 and led to the downregulation of ROS production by increasing the expression of NRF2, HO-1, and CAT. The decreased ROS resulted in reduced activation of MAPK, NF-κB, pCREB, and PGC1β, resulting in decreased survival and differentiation of osteoclasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMT controls the redox reactions and forms the redox complex with Zn\u003csup\u003e2+\u003c/sup\u003e, which scavenges and neutralizes free radicals via cysteine sulfur ligands and donates Zn\u003csup\u003e2+\u003c/sup\u003e in a redox-dependent manner [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Although MT3 is specifically expressed in the central nervous system, it has been recently observed to have various functions in other tissues [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. MT3 regulates ROS by controlling metal metabolism such as zinc and cooper. The suppression of ROS could be effective in inhibiting RANKL-induced osteoclastogenesis given that RANKL activates MAPK and NF-κB, causing ROS to enhance osteoclastogenesis and associate with the pathogenesis of osteoporosis. MT promotes osteoblastic differentiation due to the suppression of ROS in osteoblasts [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In this study, MT3 knockdown in osteoclast progenitor cells decreased JNK activation, and ROS production, and RANKL induced NF-κB stimulation, suggesting that MT3 may affect osteoclast survival and differentiation by regulating ROS and downstream MAPK and NF-κB signals. Therefore, to investigate the association with MT3, ROS, and osteoclastogenesis, we focused on the metabolism of metals, especially zinc.\u003c/p\u003e \u003cp\u003eThe presence of zinc is essential for the antioxidant action of MT. After exposure to oxidative stress, glutathione (GSH) neutralizes free radicals by donating hydrogen to form glutathione disulfide (GSSG). Glutathione reductase can reconvert GSSG to GSH to continue the redox cycle in the presence of intracellular free Zn\u003csup\u003e2+\u003c/sup\u003e-bound MT [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Zinc is known to promote bone formation and suppress bone resorption [\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] via inhibition of osteoclast differentiation induced by phosphorylation of calcineurin [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], inhibition of NF-κB signaling [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], and enhancement of apoptosis [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. MTs bind and release Zn\u003csup\u003e2+\u003c/sup\u003e under physiological conditions and are part of a network that tightly regulates intracellular Zn\u003csup\u003e2+\u003c/sup\u003e in concert with the cell-specific Zn\u003csup\u003e2+\u003c/sup\u003e importer, Zrt- and Irt-like proteins (ZIP) and Zn\u003csup\u003e2+\u003c/sup\u003e exporter, Zn transporters (ZnT) [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. When the free intracellular Zn\u003csup\u003e2+\u003c/sup\u003e concentration reaches a threshold, the activation of metal-responsive transcription factor 1 induces MT expression, which sequesters Zn\u003csup\u003e2+\u003c/sup\u003e and releases it in response to other biochemical events, such as oxidative signals, leading to the maintenance of homeostasis of free Zn\u003csup\u003e2+\u003c/sup\u003e. In this study, the knockdown of MT3 increased intracellular Zn\u003csup\u003e2+\u003c/sup\u003e and the production of ROS and failed to sequester Zn\u003csup\u003e2+\u003c/sup\u003e, leading to enhanced ROS. In support of this result, Malaiyandi LM \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] and Habel N \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e] demonstrated that MT overexpression reduced the concentration of intracellular Zn\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNRF2, which is induced by Zn\u003csup\u003e2+\u003c/sup\u003e, plays a central role as a part of the antioxidant defense system of osteoclasts in reducing ROS [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. NRF2, as a redox-sensitive transcription factor, expresses antioxidant enzymes against oxidative stress and inflammatory responses and suppresses RANKL-induced osteoclastogenesis [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. NRF2 suppresses ROS and NFATc1 and PGC1β directly [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]; the NRF2 activator is a powerful therapeutic target [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. In our study, MT3 knockdown activated NRF2 at the stage of BMM and preosteoclasts compared with control cells. HO-1 and CAT, which are downstream factors, were also activated, indicating that NRF2 signals suppressed ROS production. Furthermore, as described above, zinc has been reported to be involved in activating NRF2 [\u003cspan additionalcitationids=\"CR54 CR55 CR56\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. We also demonstrated that NRF2 was upregulated in MT3 knockdown, with elevated Zn\u003csup\u003e2+\u003c/sup\u003e in preosteoclasts, and inhibition of intracellular Zn\u003csup\u003e2+\u003c/sup\u003e by TPEN decreased NRF2 expression. These results suggest that the increased concentration of intracellular Zn\u003csup\u003e2+\u003c/sup\u003e may be involved in the upregulation of NRF2 in preosteoclasts.\u003c/p\u003e \u003cp\u003eDuring osteoclastogenesis, RANKL-induced ROS induced PGC-1β via activation of CREB, and PGC-1β increased the production of reactive oxygen species and mitochondrial biogenesis, leading to the induction of osteoclastogenesis through a positive feedback mechanism [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In addition, NRF2 activity suppresses ROS production and directly reduces PGC-1β, resulting in a lower expression level of mitochondrial genes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Subsequently, the expression of IRF8, an NFATc1 antagonist, is upregulated and inhibits NFATc1 activation and osteoclast differentiation [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Our results also showed that the deletion of MT3 reduced the activation of CREB, PGC-1β, and mitochondrial ROS, suggesting that the reduction of NRF2 activity and ROS by MT3 knockdown led to the inhibition of osteoclast differentiation through this mechanism.\u003c/p\u003e \u003cp\u003eWe suggest that RANKL elevates MT3 with osteoclast development based on the findings of this investigation and those published in the literature. MT3 regulates intracellular Zn\u003csup\u003e2+\u003c/sup\u003e activity, leading to ROS production regulation via the NRF2, HO-1, and CAT pathways. Subsequently, ROS activation promotes RANKL-induced MAPK and NF-κB, enhancing osteoclastogenesis. In addition, MT3 regulates mitochondrial ROS by activating PGC-1β and CREB (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eReagents and antibodies\u003c/h2\u003e \u003cp\u003eThe following antibodies were used: Nfatcl (#sc-7294) and catalase (#sc-271803), all of which were bought from Santa Cruz Biotechnology (Dallas, TX, USA); Actin (#A00702), from GeneScript (Piscataway, NJ, USA); cathepsin K (#MAB3324), purchased from EMD Millipore Corporation (Temucula, CA, USA); PARP (#9542), cleaved Caspase 3 (#9664), JNK/SAPK (#9252), phosphor-JNK/SAPK (#9251), IκBα (#9242), phosphor -IκBα (#9246), AKT (#2920), phosphor-AKT (#4058), phosphor-ERK1/2 (#9106), ERK1/ 2(#9102), CREB (#4820), phosphor-CREB (#9191) were bought from Cell Signaling Technology (Beverly, MA, USA); NRF2 (#A0674) were bought from ABclonal (Wuhan, Hunan, China); HO-1 (#ab189491), PGC-1β (#ab176328) were purchased from Abcam (Cambridge, MA, USA). α-Minimum Essential Medium (α-MEM) and Penicillin-Streptomycin Solution (\u0026times;100) were bought from FUJIFILM Wako (Osaka, Japan), and fetal bovine serum (FBS) was purchased from Gibco (Billings, MT, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAnimal use approval\u003c/h2\u003e \u003cp\u003e The Institutional Animal Care and Use Committee at Kyushu University approved the study's research techniques, which used mice. Furthermore, the National Institutes of Health's recommendations for the moral treatment and use of animals were strictly followed in all the research that was carried out.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBMM and osteoclast cultures\u003c/h2\u003e \u003cp\u003eBMMs were prepared following the previously described method [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The tibia and femurs of 8\u0026ndash;10-week-old C57/BL6J mice were extracted for the entire bone marrow. A lysis buffer (150 mM NH4Cl, 10 mM KNCO3, 0.1 mM EDTA, pH 7.4) was used for 5 min at room temperature to remove red blood cells. Then, for the next four-five days, 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e bone marrow cells were plated onto a 100 mm petri dish and cultured in α-10 medium (α-MEM, 10% heat-inactivated FBS, 1 penicillin-streptomycin) with an addition of 1/10 volume of CMG 14\u0026thinsp;\u0026minus;\u0026thinsp;12 (conditioned medium supernatant containing recombinant M-CSF at 1 \u0026micro;g/ml) [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Every other day, the CMG 14\u0026thinsp;\u0026minus;\u0026thinsp;12 supernatant, and culture media were replaced. After three and five days of BMM culture (at a density of 160/mm), preosteoclasts and osteoclasts were produced by adding 1/100 volume of CMG 14\u0026thinsp;\u0026minus;\u0026thinsp;12 culture supernatant and 100 ng/ml of recombinant RANKL (Oriental yeast, Tokyo, Japan), respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ehuman osteoclast cultures\u003c/h2\u003e \u003cp\u003eUsing Ficoll-PaqueTM PLUS (Cytiva, Uppsala, Sweden), human peripheral blood monocytes (PBMCs) were separated from the blood of a healthy volunteer. Monocytes were purified from PBMCs using CD14 MicroBeads, human (Miltenyi Biotec, North Rhine-Westphalia, Germany) as per the guidelines provided by the manufacturer. To differentiate human osteoclasts, monocytes of human PBMC origin were cultured for seven days (at a density of 3000/mm) in α-10 media containing 50 ng/ml of recombinant human M-CSF (Abcam, #ab259396) and 100 ng/ml of recombinant RANKL. Every three days, new media, and cytokines were replenished.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eLentivirus mediated shRNA expression\u003c/h2\u003e \u003cp\u003eShRNA expression is mediated by lentiviruses. ShRNA targeting the mRNA of murine MT3 is expressed by the LKO.1 lentiviral vector [TRCN0000257921/NM_013603.1-200s21c1 (MT3-sh1) and TRCN0000249518/NM_013603.1-93s21c1 were purchased from Sigma-Aldrich. The control was pLKO.1 puro nontarget shRNA control transduction particles (07181827MN). Using the TransIT transfection reagent (Mirus), 293-T cells were co-transfected with an LKO.1 gene transfer vector and the virus packaging vectors ΔH8.2 and VSVG. After 48 hours of transfection, virus supernatants were collected. The virus supernatant was used to transduce bone marrow-derived macrophages (BMMs), including M-CSF and 20 g/ml of protamine (Sigma-Aldrich). The transduced cells were subsequently chosen for 3 days in an α -10 medium supplemented with M-CSF and 60 \u0026micro;g/ml of puromycin (Sigma-Aldrich) [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eTRAP staining\u003c/h2\u003e \u003cp\u003eOn a 48-well tissue culture plate, TRAP stains, BMMs were grown for 4\u0026ndash;5 days in an α-10 medium containing M-CSF and RANKL. Following the culture, the cells were fixed using a solution of 4% paraformaldehyde (Wako) and phosphate-buffered saline (PBS). According to the previously reported procedure, TRAP staining was carried out using NaK tartrate and Naphthol AS-BI phosphoric acid (Sigma-Aldrich) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time RT-PCR and RNA isolation\u003c/h2\u003e \u003cp\u003eQuantitative real-time RT-PCR and RNA isolation were performed following the instructions provided by the manufacturer. Total RNA was purified using the RNeasy mini kit (Qiagen, Hilden, Germany). According to the manufacturer's instructions, the Prime-ScriptTM RT reagent kit (Takara Bio, Kusatsu, Japan) was used with 0.5\u0026ndash;1 \u0026micro;g of total RNA for synthesizing first-strand cDNAs. Using the following: primers from Thermo-Fisher Scientific, TaqMan quantitative real-time PCR was carried out: Mt-1 (Mm00496660_g1), Mt-2 (Mm00809556_s1), Mt-3 (Mm00496661_g1, Hs00359394_g1), Ctsk (Mm00484039_m1, Nfatc1 (Mm00479445_m1), Hs00166156_m1), Acp5 (Mm00475698_m1), Dcstamp (Mm01168058_m1), Calcr (Mm00432282_m1), Mrps2 (Mm03991065_g1 Hs00211443_m1). Thermo-Fisher Scientific ABI QuantStudio3 equipment was utilized for the amplification of the samples. Denaturation at 95\u0026deg;C for 10 min was the first stage in the amplification process. This was followed by 40 cycles of denaturation at 95\u0026deg;C for 15 s and annealing/extension at 60\u0026deg;C for 1 min. Normalization was performed using the ΔCt method [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e] to determine the relative cDNA amount, with the expression level of mitochondrial gene Mrps2 serving as the reference, whereby both BMMs, and osteoclasts show steady expression. The delta ΔCt technique was used to analyze the relative amounts of MT3 cDNAs in BMMs. Every test was run in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblotting\u003c/h2\u003e \u003cp\u003eAfter two ice-cold PBS washes, cultured cells were lysed using Cell Lytic M (Sigma-Aldrich), which contained Phosphatase Inhibitor Cocktail (ab201112, Abcam) and Protease Inhibitor (cOmplete Mini, EDTA-free, Sigma-Aldrich). The cell lysates were centrifuged at 14,000 rpm for 15 min at 4\u0026deg;C to remove cellular debris after being incubated on ice for 30 min. Polyacrylamide gels with a 4\u0026ndash;12% gradient were loaded with a total of 10\u0026ndash;30 micrograms of total protein (Invitrogen, Carlsbad, CA, USA) and electrophoretically transferred onto a polyvinylidene difluoride membrane (Amersham Biosciences, Arlington Heights, IL, USA) using a semi-dry blotting system (Bio-Rad, Hercules, CA, USA). The membrane was next blocked in 5% fat-free milk/Tris-buffered saline for an h before being treated with primary antibodies overnight at 4\u0026deg;C (Santa Cruz Biotechnology). Finally, secondary antibodies conjugated with horseradish peroxidase were applied to the membrane. Immunoreactivity was detected using ECL Prime (Amersham Biosciences) and photographed using an Ez Capture MG (ATTO, Tokyo, Japan) after three washings with Tris-buffered saline containing 0.1% Tween 20.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFluorescent staining of actin filament and nuclei\u003c/h2\u003e \u003cp\u003eActin filament and nuclear fluorescent staining osteoclasts cultivated on glass coverslips were permeabilized with 0.2% Triton X-100/PBS for 10 min at room temperature and fixed with 4% paraformaldehyde (Wako) in PBS for 20 min. For 15 minutes at room temperature, Alexa-488 conjugated phalloidin (1:100 from a 1 mg/ml stock) was used to label filament actin. Slow Fade Diamond Antifade Mountant with DAPI (Invitrogen) was used to stain nuclei following two 5-minute PBS washes [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA fluorescence microscope (BZ-X810; Keyence, Osaka, Japan) was used to take pictures of the samples. The mean number of active osteoclasts (podosome-belt bearing osteoclasts on glass coverslips and actin-ring bearing osteoclasts on bone slices) with different nuclei and the percentage of spreading osteoclasts were calculated by Hybrid Cell Count Software using images of five randomly selected areas on each glass coverslip or bone slice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eResorption pit staining\u003c/h2\u003e \u003cp\u003e4 percent paraformaldehyde (Wako)/PBS was used to fix mature osteoclasts cultured on cortical bovine bone slices for 20 min cells were eliminated from bone slices using a soft brush after PBS was washed twice for 5 min. The slices were then treated for 60 min at room temperature with 20 \u0026micro;g/ml peroxidase-conjugated wheat germ agglutinin lectin. After washing twice in PBS, bone chips were treated with 0.03% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 0.52 mg/ml 3,3-diaminobenzidine for 30 min. Samples were imaged using a fluorescent microscope (BZ-X810; Keyence, Osaka, Japan) and mounted in 80% glycerol/PBS [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. Hybrid Cell Count Software was used to determine the mean percentage of resorbed area in each bone slice after collecting blinded photographs of five randomly selected locations per bone slice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eViability Assay\u003c/h2\u003e \u003cp\u003eIn 96-well plates, cells were seeded at 5.0 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e per well. The vitality of cells in each well was assessed as a reference value after four h of seeding. The CellTiter-Glo Luminescent Cell vitality kit (Promega, Madison, WI, USA) was then used to check the relative cell vitality every 24 h for a total of 96 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometric study of the cell cycle\u003c/h2\u003e \u003cp\u003eTrypsin was used to collect the cells, which were then washed with PBS before being fixed in ice-cold 70% ethanol at 4\u0026deg;C for 30 min. The cells were fixed, washed with PBS, and then treated with propidium iodide and RNase (Immunostep S.L., Salamanca, Spain) for 15 min. Flow cytometry was conducted using BD FACS Verse (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), and the data were analyzed using BD FlowJo Software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eROS detection\u003c/h2\u003e \u003cp\u003e2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) was used to measure the production of ROS (Cayman, Michigan, USA) as a fluorescent probe. Briefly, in an FBS-free medium with DCFH-DA (final concentration of 50 \u0026micro;M), cells from various groups were grown and incubated for 30 min. Subsequently, to remove extra DCFH-DA, cells were washed three times in serum-free media. A BZ-X800 microscope was used to take the fluorescence images, and the percentage of cells exhibiting positive fluorescence and the fluorescence intensity were quantified and analyzed using Hybrid Cell Count Software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial ROS production measurements\u003c/h2\u003e \u003cp\u003eWe worked with (Thermo-Fisher Scientific's) MitoSOX Red Mitochondrial Superoxide Indicator to examine the generation of mitochondria-derived ROS. In brief, MitoSOX was incubated with cells in glass-bottom imaging dishes for 15 min at 37\u0026deg;C at a concentration of 5 \u0026micro;M. Following this, Hanks' buffer was used to wash the cells three times to eliminate any remaining probe, and then the cells were examined using a fluorescent microscope. Hybrid Cell Count Software examined the percentage of positive cells and the fluorescence intensity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eThe levels of intracellular Zn\u003csup\u003e2+\u003c/sup\u003e measurements\u003c/h2\u003e \u003cp\u003eIn 24-well plates, 3 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells were planted, and the cells were then treated with M-CSF and RANKL for two days. Subsequently, the cells were stained with FluoZin-3 (Invitrogen) at a concentration of 10 \u0026micro;M for 30 min. Following staining, Hanks' buffer was used to wash the cells twice and fix them with a 4% paraformaldehyde (Wako) solution. We used Slow Fade Diamond Antifade Mountant for nuclear staining with DAPI (Invitrogen). The immunostained samples were visualized using fluorescence microscopy. The Hybrid Cell Count Software was utilized to analyze positive cells' fluorescence intensity and percentage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eImmunocytochemistry\u003c/h2\u003e \u003cp\u003eIn 24-well plates, 3 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells were planted, and the cells were then treated with M-CSF and RANKL for two days. In the following incubation, the cells were fixed for 10 min at room temperature using a 4% paraformaldehyde (PFA) solution (Wako). Subsequently, 0.2% bovine serum albumin (BSA) and 0.3% Triton X-100 (Sigma-Aldrich) were used to block the cells for an h at room temperature. Antibodies against NRF2 (#Sc-365949, Santa Cruz Biotechnology) were treated with the cells for 3 h at a dilution 1:200 in 0.2% BSA. The samples were then exposed for an h at room temperature to Alexa Fluor 546 goat anti-mouse IgG2a cross-adsorbed secondary antibody (#A21133, Invitrogen). Slow Fade Diamond Antifade Mountant with DAPI (Invitrogen) was used to stain nuclei. Immunostaining was visualized using fluorescence microscopy. Blinded, five randomly chosen areas per glass coverslip were imaged. The fluorescence intensity and the percentage of positive cells on cells were quantified using Hybrid Cell Count Software, and the mean values were calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eAll graphs show the data as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The Shapiro-Wilk test was conducted to determine whether the data distribution was normal. A two-tailed Student's t-test was used to investigate two-group comparisons when the distribution was normal. A one-way or two-way analysis of variance was carried out for comparisons involving more than two groups, followed by Tukey\u0026rsquo;s post hoc test. The Kruskal\u0026ndash;Wallis test was used for comparisons involving more than two groups when the distribution was non-normal, and Dunn's post hoc test was used for two-group comparisons in those situations. Prism 9 software from GraphPad Software, La Jolla, CA, was used for all statistical calculations. A p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by JSPS KAKENHI (Grant Number: JP18K16626 and 22K09359).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSA, TF, and HZ designed research; SA, KY, and TT performed research; SA and TF analyzed data and wrote the paper; HS, KK, OS, and YN supervised the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal protocols and procedures used in animal studies were approved by the Institutional Animal\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCare and Use Committees of Kyushu university (approval number: A23-004-0). The protocols for generation and use of shRNAs and lentivirus were approved by Institutional Biosafety Committee of Kyushu university (approval number: 4-93)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors received no specific funding for this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBoyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature. 2003;423(6937):337\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrockett JC, Rogers MJ, Coxon FP, Hocking LJ, Helfrich MH. Bone remodelling at a glance. J Cell Sci. 2011;124(Pt 7):991\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZaidi M. Skeletal remodeling in health and disease. Nat Med. 2007;13(7):791\u0026ndash;801.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoyce BF. Advances in the regulation of osteoclasts and osteoclast functions. J Dent Res. 2013;92(10):860\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNovack DV, Teitelbaum SL. The osteoclast: friend or foe? Annu Rev Pathol. 2008;3:457\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsutsui T, Fujiwara T, Matsumoto Y, Kimura A, Kanahori M, Arisumi S, et al. Geriatric nutritional risk index as the prognostic factor in older patients with fragility hip fractures. Osteoporos Int. 2023;34(7):1207\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeitelbaum SL, Ross FP. Genetic regulation of osteoclast development and function. Nat Rev Genet. 2003;4(8):638\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoss FP, Teitelbaum SL. alphavbeta3 and macrophage colony-stimulating factor: partners in osteoclast biology. Immunol Rev. 2005;208:88\u0026ndash;105.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakeshita S, Faccio R, Chappel J, Zheng L, Feng X, Weber JD, et al. c-Fms tyrosine 559 is a major mediator of M-CSF-induced proliferation of primary macrophages. J Biol Chem. 2007;282(26):18980\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H, et al. Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell. 2002;3(6):889\u0026ndash;901.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIshii KA, Fumoto T, Iwai K, Takeshita S, Ito M, Shimohata N, et al. Coordination of PGC-1beta and iron uptake in mitochondrial biogenesis and osteoclast activation. Nat Med. 2009;15(3):259\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou J, Ye S, Fujiwara T, Manolagas SC, Zhao H. Steap4 plays a critical role in osteoclastogenesis in vitro by regulating cellular iron/reactive oxygen species (ROS) levels and cAMP response element-binding protein (CREB) activation. J Biol Chem. 2013;288(42):30064\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Fang B, Fujiwara T, Krager K, Gorantla A, Li C, et al. Deletion of ferroportin in murine myeloid cells increases iron accumulation and stimulates osteoclastogenesis in vitro and in vivo. J Biol Chem. 2018;293(24):9248\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDas BK, Wang L, Fujiwara T, Zhou J, Aykin-Burns N, Krager KJ, et al. Transferrin receptor 1-mediated iron uptake regulates bone mass in mice via osteoclast mitochondria and cytoskeleton. Elife. 2022;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeo HJ, Cho YE, Kim T, Shin HI, Kwun IS. Zinc may increase bone formation through stimulating cell proliferation, alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells. Nutr Res Pract. 2010;4(5):356\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoonga BS, Dempster DW. Zinc is a potent inhibitor of osteoclastic bone resorption in vitro. J Bone Miner Res. 1995;10(3):453\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTogari A, Arakawa S, Arai M, Matsumoto S. Alteration of in vitro bone metabolism and tooth formation by zinc. Gen Pharmacol. 1993;24(5):1133\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHolloway WR, Collier FM, Herbst RE, Hodge JM, Nicholson GC. Osteoblast-mediated effects of zinc on isolated rat osteoclasts: inhibition of bone resorption and enhancement of osteoclast number. Bone. 1996;19(2):137\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoyle P, Philcox JC, Carey LC, Rofe AM. Metallothionein: the multipurpose protein. Cell Mol Life Sci. 2002;59(4):627\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoleirinho A, Carneiro J, Matthiesen R, Silva RM, Amorim A, Azevedo L. Gains, losses and changes of function after gene duplication: study of the metallothionein family. PLoS One. 2011;6(4):e18487.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlbrecht AL, Singh RK, Somji S, Sens MA, Sens DA, Garrett SH. Basal and metal-induced expression of metallothionein isoform 1 and 2 genes in the RWPE-1 human prostate epithelial cell line. J Appl Toxicol. 2008;28(3):283\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUchida Y, Takio K, Titani K, Ihara Y, Tomonaga M. The growth inhibitory factor that is deficient in the Alzheimer's disease brain is a 68 amino acid metallothionein-like protein. Neuron. 1991;7(2):337\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoffatt P, S\u0026eacute;guin C. Expression of the gene encoding metallothionein-3 in organs of the reproductive system. DNA Cell Biol. 1998;17(6):501\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuaife CJ, Findley SD, Erickson JC, Froelick GJ, Kelly EJ, Zambrowicz BP, et al. Induction of a new metallothionein isoform (MT-IV) occurs during differentiation of stratified squamous epithelia. Biochemistry. 1994;33(23):7250\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePalmiter RD. The elusive function of metallothioneins. Proc Natl Acad Sci U S A. 1998;95(15):8428\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaret W. The function of zinc metallothionein: a link between cellular zinc and redox state. J Nutr. 2000;130(5S Suppl):1455s-8s.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLynes MA, Hidalgo J, Manso Y, Devisscher L, Laukens D, Lawrence DA. Metallothionein and stress combine to affect multiple organ systems. Cell Stress Chaperones. 2014;19(5):605\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKlaassen CD, Liu J, Diwan BA. Metallothionein protection of cadmium toxicity. Toxicol Appl Pharmacol. 2009;238(3):215\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhandari S, Melchiorre C, Dostie K, Laukens D, Devisscher L, Louwrier A, et al. Detection and Manipulation of the Stress Response Protein Metallothionein. Curr Protoc Toxicol. 2017;71:17.9.1-.9.28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSi M, Lang J. The roles of metallothioneins in carcinogenesis. J Hematol Oncol. 2018;11(1):107.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdam P, Kř\u0026iacute;žkov\u0026aacute; S, Heger Z, Babula P, Pekař\u0026iacute;k V, Vaculovičo\u0026aacute; M, et al. Metallothioneins in Prion- and Amyloid-Related Diseases. J Alzheimers Dis. 2016;51(3):637\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu A-L, Zhang Z-M, Zhu B-F, Liao Z-H, Liu Z. Metallothionein protects bone marrow stromal cells against hydrogen peroxide-induced inhibition of osteoblastic differentiation. Cell Biol Int. 2004;28(12):905\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrost Z, Trebse R, Prezelj J, Komadina R, Logar DB, Marc J. A microarray based identification of osteoporosis-related genes in primary culture of human osteoblasts. Bone. 2010;46(1):72\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFong L, Tan K, Tran C, Cool J, Scherer MA, Elovaris R, et al. Interaction of dietary zinc and intracellular binding protein metallothionein in postnatal bone growth. Bone. 2009;44(6):1151\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFujiwara T, Zhou J, Ye S, Zhao H. RNA-binding protein Musashi2 induced by RANKL is critical for osteoclast survival. Cell Death Dis. 2016;7(7):e2300.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYe S, Fujiwara T, Zhou J, Varughese KI, Zhao H. LIS1 Regulates Osteoclastogenesis through Modulation of M-SCF and RANKL Signaling Pathways and CDC42. Int J Biol Sci. 2016;12(12):1488\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee NK, Choi YG, Baik JY, Han SY, Jeong DW, Bae YS, et al. A crucial role for reactive oxygen species in RANKL-induced osteoclast differentiation. Blood. 2005;106(3):852\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuttkay-Nedecky B, Nejdl L, Gumulec J, Zitka O, Masarik M, Eckschlager T, et al. The role of metallothionein in oxidative stress. Int J Mol Sci. 2013;14(3):6044\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaret W, Vallee BL. Thiolate ligands in metallothionein confer redox activity on zinc clusters. Proc Natl Acad Sci U S A. 1998;95(7):3478\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang YJ. Metallothionein redox cycle and function. Exp Biol Med (Maywood). 2006;231(9):1459\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhatt NY, Kelley TW, Khramtsov VV, Wang Y, Lam GK, Clanton TL, et al. Macrophage-colony-stimulating factor-induced activation of extracellular-regulated kinase involves phosphatidylinositol 3-kinase and reactive oxygen species in human monocytes. J Immunol. 2002;169(11):6427\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHa H, Kwak HB, Lee SW, Jin HM, Kim HM, Kim HH, et al. Reactive oxygen species mediate RANK signaling in osteoclasts. Exp Cell Res. 2004;301(2):119\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang TC. Nuclear Factor-Erythroid 2-Related Factor 2 (Nrf2) and Mitochondrial Dynamics/Mitophagy in Neurological Diseases. Antioxidants (Basel). 2020;9(7).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Z, Chen C, Zhu X, Li Y, Yu R, Xu W. Glycyrrhizin Suppresses RANKL-Induced Osteoclastogenesis and Oxidative Stress Through Inhibiting NF-κB and MAPK and Activating AMPK/Nrf2. Calcif Tissue Int. 2018;103(3):324\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIshii T, Itoh K, Takahashi S, Sato H, Yanagawa T, Katoh Y, et al. Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J Biol Chem. 2000;275(21):16023\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHyeon S, Lee H, Yang Y, Jeong W. Nrf2 deficiency induces oxidative stress and promotes RANKL-induced osteoclast differentiation. Free Radic Biol Med. 2013;65:789\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun X, Xie Z, Hu B, Zhang B, Ma Y, Pan X, et al. The Nrf2 activator RTA-408 attenuates osteoclastogenesis by inhibiting STING dependent NF-κb signaling. Redox Biol. 2020;28:101309.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKanzaki H, Shinohara F, Itohiya K, Yamaguchi Y, Katsumata Y, Matsuzawa M, et al. RANKL induces Bach1 nuclear import and attenuates Nrf2-mediated antioxidant enzymes, thereby augmenting intracellular reactive oxygen species signaling and osteoclastogenesis in mice. FASEB J. 2017;31(2):781\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei W, Wang X, Yang M, Smith LC, Dechow PC, Sonoda J, et al. PGC1beta mediates PPARgamma activation of osteoclastogenesis and rosiglitazone-induced bone loss. Cell Metab. 2010;11(6):503\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCallaway DA, Jiang JX. Reactive oxygen species and oxidative stress in osteoclastogenesis, skeletal aging and bone diseases. J Bone Miner Metab. 2015;33(4):359\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSugiura T, Kuroda E, Yamashita U. Dysfunction of macrophages in metallothionein-knock out mice. J uoeh. 2004;26(2):193\u0026ndash;205.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurakami M, Hirano T. Intracellular zinc homeostasis and zinc signaling. Cancer Sci. 2008;99(8):1515\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi B, Cui W, Tan Y, Luo P, Chen Q, Zhang C, et al. Zinc is essential for the transcription function of Nrf2 in human renal tubule cells in vitro and mouse kidney in vivo under the diabetic condition. J Cell Mol Med. 2014;18(5):895\u0026ndash;906.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHa KN, Chen Y, Cai J, Sternberg P, Jr. Increased glutathione synthesis through an ARE-Nrf2-dependent pathway by zinc in the RPE: implication for protection against oxidative stress. Invest Ophthalmol Vis Sci. 2006;47(6):2709\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCortese MM, Suschek CV, Wetzel W, Kroncke KD, Kolb-Bachofen V. Zinc protects endothelial cells from hydrogen peroxide via Nrf2-dependent stimulation of glutathione biosynthesis. Free Radic Biol Med. 2008;44(12):2002\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGe MH, Tian H, Mao L, Li DY, Lin JQ, Hu HS, et al. Zinc attenuates ferroptosis and promotes functional recovery in contusion spinal cord injury by activating Nrf2/GPX4 defense pathway. CNS Neurosci Ther. 2021;27(9):1023\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi D, Tian H, Li X, Mao L, Zhao X, Lin J, et al. Zinc promotes functional recovery after spinal cord injury by activating Nrf2/HO-1 defense pathway and inhibiting inflammation of NLRP3 in nerve cells. Life Sci. 2020;245:117351.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGaetke LM, Chow CK. Copper toxicity, oxidative stress, and antioxidant nutrients. Toxicology. 2003;189(1\u0026ndash;2):147\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJomova K, Baros S, Valko M. Redox active metal-induced oxidative stress in biological systems. Transition Metal Chemistry. 2012;37(2):127\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNi S, Qian Z, Yuan Y, Li D, Zhong Z, Ghorbani F, et al. Schisandrin A restrains osteoclastogenesis by inhibiting reactive oxygen species and activating Nrf2 signalling. Cell Prolif. 2020;53(10):e12882.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSridhar V, Narnaware S, Kumar P, Kale SB, Majumdar AS. Co-treatment with sulforaphane\u0026ndash;zein microparticles enhances the chemopreventive potential of zinc in a 1,2-dimethylhydrazine induced colon carcinogenesis rat model. RSC Advances. 2016;6(41):34233\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark KH, Park B, Yoon DS, Kwon SH, Shin DM, Lee JW, et al. Zinc inhibits osteoclast differentiation by suppression of Ca2+-Calcineurin-NFATc1 signaling pathway. Cell Commun Signal. 2013;11:74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamaguchi M, Weitzmann MN. Zinc stimulates osteoblastogenesis and suppresses osteoclastogenesis by antagonizing NF-κB activation. Mol Cell Biochem. 2011;355(1\u0026ndash;2):179\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi X, Senda K, Ito A, Sogo Y, Yamazaki A. Effect of Zn and Mg in tricalcium phosphate and in culture medium on apoptosis and actin ring formation of mature osteoclasts. Biomed Mater. 2008;3(4):045002.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLichtlen P, Schaffner W. The \"metal transcription factor\" MTF-1: biological facts and medical implications. Swiss Med Wkly. 2001;131(45\u0026ndash;46):647\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalaiyandi LM, Dineley KE, Reynolds IJ. Divergent consequences arise from metallothionein overexpression in astrocytes: zinc buffering and oxidant-induced zinc release. Glia. 2004;45(4):346\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHabel N, Hamidouche Z, Girault I, Patino-Garcia A, Lecanda F, Marie PJ, et al. Zinc chelation: a metallothionein 2A's mechanism of action involved in osteosarcoma cell death and chemotherapy resistance. Cell Death Dis. 2013;4(10):e874.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun YX, Xu AH, Yang Y, Li J. Role of Nrf2 in bone metabolism. J Biomed Sci. 2015;22:101.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS\u0026aacute;nchez-de-Diego C, Pedrazza L, Pimenta-Lopes C, Martinez-Martinez A, Dahdah N, Valer JA, et al. NRF2 function in osteocytes is required for bone homeostasis and drives osteocytic gene expression. Redox Biol. 2021;40:101845.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSakai E, Morita M, Ohuchi M, Kido MA, Fukuma Y, Nishishita K, et al. Effects of deficiency of Kelch-like ECH-associated protein 1 on skeletal organization: a mechanism for diminished nuclear factor of activated T cells cytoplasmic 1 during osteoclastogenesis. FASEB J. 2017;31(9):4011\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXue P, Hu X, Powers J, Nay N, Chang E, Kwon J, et al. CDDO-Me, Sulforaphane and tBHQ attenuate the RANKL-induced osteoclast differentiation via activating the NRF2-mediated antioxidant response. Biochem Biophys Res Commun. 2019;511(3):637\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan J, Yang K, An J, Jiang N, Fu S, Tang X. The Role of NRF2 in Bone Metabolism - Friend or Foe? Front Endocrinol (Lausanne). 2022;13:813057.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakeshita S, Kaji K, Kudo A. Identification and characterization of the new osteoclast progenitor with macrophage phenotypes being able to differentiate into mature osteoclasts. J Bone Miner Res. 2000;15(8):1477\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou J, Fujiwara T, Ye S, Li X, Zhao H. Downregulation of Notch modulators, tetraspanin 5 and 10, inhibits osteoclastogenesis in vitro. Calcif Tissue Int. 2014;95(3):209\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou J, Fujiwara T, Ye S, Li X, Zhao H. Ubiquitin E3 Ligase LNX2 is Critical for Osteoclastogenesis In Vitro by Regulating M-CSF/RANKL Signaling and Notch2. Calcif Tissue Int. 2015;96(5):465\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008;3(6):1101\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIto Y, Teitelbaum SL, Zou W, Zheng Y, Johnson JF, Chappel J, et al. Cdc42 regulates bone modeling and remodeling in mice by modulating RANKL/M-CSF signaling and osteoclast polarization. J Clin Invest. 2010;120(6):1981\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFujiwara T, Ye S, Castro-Gomes T, Winchell CG, Andrews NW, Voth DE, et al. PLEKHM1/DEF8/RAB7 complex regulates lysosome positioning and bone homeostasis. JCI Insight. 2016;1(17):e86330.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3262822/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3262822/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn osteoclastogenesis, the metabolism of metal ions plays an essential role in controlling reactive oxygen species (ROS) production, mitochondrial biogenesis, and survival, and differentiation. However, the mechanism regulating metal ions during osteoclast differentiation remains unclear. The metal-binding protein metallothionein (MT) detoxifies heavy metals, maintains metal ion homeostasis, especially zinc, and manages cellular redox levels. We carried out tests using murine osteoclast precursors to examine the function of MT in osteoclastogenesis and evaluated their potential as targets for future osteoporosis treatments. MT genes were significantly upregulated upon differentiation from osteoclast precursors to mature osteoclasts in response to receptor activators of nuclear factor-κB (NF-κB) ligand (RANKL) stimulation, and MT3 expression was particularly pronounced in mature osteoclasts among MT genes. The knockdown of MT3 in osteoclast precursors demonstrated a remarkable inhibition of differentiation into mature osteoclasts. In preosteoclasts, MT3 knockdown suppressed the activity of mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways upon RANKL stimulation, leading to increased apoptosis through elevated cleaved Caspase 3 and poly (ADP-ribose) polymerase (PARP) levels. Additionally, ROS levels were decreased, and nuclear factor erythroid 2-related factor 2 (NRF2) (a suppressor of ROS) and the downstream antioxidant proteins, such as catalase (CAT) and heme oxygenase 1 (HO-1), were more highly expressed in the MT3 preosteoclast knockdowns. mitochondrial ROS, which is involved in mitochondrial biogenesis and the production of reactive oxygen species, were similarly decreased because cAMP response element-binding (CREB) and peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β) were less activated due to MT3 depletion. Thus, by modulating ROS through the NRF2 pathway, MT3 plays a crucial role in regulating osteoclast growth and survival, acting as a metabolic modulator of intracellular zinc ions.\u003c/p\u003e","manuscriptTitle":"Metallothionein 3 promotes osteoclast differentiation and survival by regulating the intracellular Zn2+ concentration and NRF2 pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-14 15:58:51","doi":"10.21203/rs.3.rs-3262822/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"transferred","content":"Cell Death Discovery","date":"2023-09-22T12:10:22+00:00","index":"","fulltext":""},{"type":"decision","content":"Reject after peer review","date":"2023-09-21T13:41:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-09-20T01:34:42+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-09-14T00:30:32+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-09-10T07:10:34+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-09-10T06:10:34+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2023-08-29T09:08:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-08-15T09:29:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-08-14T13:11:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2023-08-14T13:11:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"651c9e16-f935-4da8-a78f-2af8fe22951d","owner":[],"postedDate":"September 14th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":24521421,"name":"Biological sciences/Developmental biology/Differentiation"},{"id":24521422,"name":"Biological sciences/Molecular biology/RNAi"},{"id":24521423,"name":"Biological sciences/Physiology/Bone"}],"tags":[],"updatedAt":"2023-12-02T08:33:05+00:00","versionOfRecord":{"articleIdentity":"rs-3262822","link":"https://doi.org/10.1038/s41420-023-01729-y","journal":{"identity":"cell-death-discovery","isVorOnly":false,"title":"Cell Death Discovery"},"publishedOn":"2023-12-01 05:00:00","publishedOnDateReadable":"December 1st, 2023"},"versionCreatedAt":"2023-09-14 15:58:51","video":"","vorDoi":"10.1038/s41420-023-01729-y","vorDoiUrl":"https://doi.org/10.1038/s41420-023-01729-y","workflowStages":[]},"version":"v1","identity":"rs-3262822","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3262822","identity":"rs-3262822","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}