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Silver nanoparticle-induced phosphorylation of histone H3 at serine 10 is due to dynamic changes in actin filaments and the activation of Aurora kinases
Toxicology Letters 276 (2017) 39–47
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Silver nanoparticle-induced phosphorylation of histone H3 at serine 10 is due to dynamic changes in actin filaments and the activation of Aurora kinases Xiaoxu Zhaoa, Tatsushi Toyookab, Yuko Ibukia, a b
MARK
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Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, Shizuoka, Japan Industrial Toxicology and Health Effects Research Group, National Institute of Occupational Safety and Health, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords: Silver nanoparticles Actin Phosphorylation Histone Aurora kinases
The phosphorylation of histone H3 at serine 10 (p-H3S10) has been closely correlated with mitotic chromosome condensation. We previously reported that silver nanoparticles (AgNPs) significantly induced p-H3S10 independent of mitosis. In the present study, we examined the mechanisms underlying the induction of pH3S10 by AgNPs. A treatment with AgNPs markedly induced p-H3S10 in a dose-dependent manner in three types of cell lines, and this was dependent on the cellular incorporation of AgNPs. The immunofluorescent staining of AgNP-induced p-H3S10 was thin and solid throughout the nucleus, and differed from that normally associated with mitosis. AgNPs induced the formation of globular actin in a dose-dependent manner. Latrunculin B (LatB) and phalloidin, inhibitors of actin polymerization and depolymerization, respectively, inhibited pH3S10, suggesting that dynamic changes in actin filaments are related to AgNP-induced p-H3S10. Furthermore, p-H3S10 was mediated by Aurora kinase (AURK) pathways, which were suppressed by LatB and siRNA for cofilin 1, an actin-depolymerizing protein. AgNO3 (Ag ions) exerted similar effects to those of AgNPs. These results suggest that Ag ions released from AgNPs incorporated into inner cells changed the dynamics of actin filaments, and this was followed by the activation of AURKs, leading to the induction of p-H3S10.
1. Introduction Nanoparticles (NPs) are ultra-fine materials (length or diameter of 1–100 nm) that are being increasingly used in modern technology, medical health care, and commercial products. Among metal NPs, silver NPs (AgNPs), which exhibit potent antibacterial and antifungal activities, have been widely used in biomedical devices and applications such as food and beverage containers, textiles, and room sprays (Nowack et al., 2011; Washington, 2011). Although humans frequently make contact with AgNPs, their toxic effects on human health have not yet been examined in detail. Ag is regarded as a safe metal and Ag products such as silverware have been used for a long time. Recent studies have indicated that exposure to AgNPs has negative effects on human health. In vitro studies demonstrated multiple abnormal physiological effects such as inhibited cell proliferation, membrane damage, mitochondrial dysfunc-
tion, and apoptosis, following an exposure to AgNPs (Guo et al., 2016; Jiang et al., 2013; Kim et al., 2009; Singh and Ramarao, 2012). AgNPs have been shown to contribute to the excessive generation of reactive oxygen species (ROS) and the resultant oxidative stress may eventually lead to cell injury (Guo et al., 2016; Jiang et al., 2013; Kim et al., 2009). The release of Ag ions from AgNPs is another important mechanism underlying AgNP-induced toxicity. Ag ions easily bind to the thiol groups of proteins in cells, leading to the inactivation of biological functions (Singh and Ramarao, 2012). We previously demonstrated that AgNPs significantly induced the phosphorylation of histone H3 at serine 10 (p-H3S10) (Zhao and Ibuki, 2015). p-H3S10 has been associated with mitotic chromosome condensation (Hendzel et al., 1997; Wei et al., 1999), but has also been reported to play a role in gene transcription related to tumor promotion (Choi et al., 2005; Kim et al., 2008). Epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA), which are related to
Abbreviations: NPs, nanoparticles; AgNPs, silver nanoparticles; ROS, reactive oxygen species; p-H3S10, phosphorylation of histone H3 at serine 10; EGF, epidermal growth factor; TPA, 12-O-tetradecanoylphorbol 13-acetate; CSS, cigarette sidestream smoke; UVB, ultraviolet B; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; JNK, cJun N-terminal protein kinase; AURKs, Aurora kinases; siRNA, small interfering RNA; DMEM, Dulbecco's Modified Eagle's Medium; FBS, fetal bovine serum; CCB, cytochalasin B; LatB, latrunculin B; Pha, phalloidin; NAC, N-acetylcysteine; ZM, ZM447439; G-actin, globular actin; FITC, fluorescein isothiocyanate; DAPI, 4′,6-diamidino-2-phenylindole; PI, propidium iodide; F-actin, filament actin; FCM, flow cytometry; SS, side-scattered light; HP1β, heterochromatin protein 1β; GSH, glutathione; CBB, Coomassie brilliant blue ⁎ Corresponding author. E-mail address: [email protected] (Y. Ibuki). http://dx.doi.org/10.1016/j.toxlet.2017.05.009 Received 26 October 2016; Received in revised form 8 April 2017; Accepted 5 May 2017 Available online 10 May 2017 0378-4274/ © 2017 Elsevier B.V. All rights reserved.
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2.2. Cells and cell culture conditions
tumor promotion, enhance the induction of p-H3S10. Several environmental factors such as cigarette sidestream smoke (CSS), ultraviolet B (UVB), arsenite, and nickel have also been shown to induce p-H3S10 (Ibuki et al., 2014; Ke et al., 2008; Keum et al., 2013; Li et al., 2003). Arsenite-induced p-H3S10 has been attributed to the activation of the extracellular signal-regulated kinase (ERK) pathway in the mitogenactivated protein kinase (MAPK) cascade (Li et al., 2003). Nickel was found to phosphorylate H3S10 via the c-Jun N-terminal protein kinase (JNK) pathway (Ke et al., 2008). CSS also activated the JNK and Akt pathways, leading to p-H3S10 (Ibuki et al., 2014). p-H3S10 induced by CSS, UVB, EGF, TPA, and arsenite has been associated with the induction of immediate-early genes, including the proto-oncogenes, cfos and c-jun (Choi et al., 2005; Ibuki et al., 2014; Keum et al., 2013; Kim et al., 2008; Li et al., 2003). This induction is also regulated downstream of the activation of the MAPK pathway. However, the molecular mechanisms responsible for AgNP-induced p-H3S10 have not yet been elucidated in detail. Aurora kinases (AURKs) belong to the serine/threonine kinase family and are known to phosphorylate H3S10 in mitotic budding yeasts, nematodes, and mammals (Crosio et al., 2002; Hsu et al., 2000; Le et al., 2013). Many low species have one or two AURKs, whereas mammals have at least three: AURKA, B, and C (Nigg, 2001). Mitotic pH3S10 is catalyzed by AURKA and/or AURKB in mammals (Crosio et al., 2002; Le et al., 2013). AURKs have essential functions in mitotic processes such as chromosome condensation and spindle dynamics (Carmena and Earnshaw, 2003), in which p-H3S10 plays some roles as an initiator of chromosome condensation and proper chromosome segregation (Adams et al., 2001; Giet and Glover, 2001). AURKs have also been implicated in the regulation of the actin cytoskeleton. The activation of Drosophila AURKs has been suggested to be involved in actin-dependent asymmetric protein localization during mitosis (Berdnik and Knoblich, 2002). The overexpression of AURKA increases the expression of phosphatase Slingshot-1, leading to the dephosphorylation and activation of the actin-depolymerizing protein, cofilin (Wang et al., 2010). AURKs also interact with LIM kinase 1, which is involved in the reorganization of the actin cytoskeleton (Bernard, 2007; Ritchey et al., 2012). Cells mainly take up metal NPs by endocytosis and phagocytosis (Panariti et al., 2012), and the actin cytoskeleton has been suggested to play a role in these processes (Castellano et al., 2001; Deschamps et al., 2013; Jeng and Welch, 2001). However, p-H3S10 induced by AgNPs and its relationships with AURKs and the actin cytoskeleton have not yet been investigated. In the present study, we elucidated the mechanisms responsible for AgNP-induced p-H3S10. We used several inhibitors or small interfering RNA (siRNA)-knockdown cells to investigate the relationships between p-H3S10 and the actin cytoskeleton and AURK pathways. The results obtained demonstrated the induction of p-H3S10 by AgNPs via the activation of AURK pathways, which occurred downstream of the changes induced in actin filaments by Ag ions released from AgNPs.
Human skin keratinocytes (HaCaT; provided by Dr. Norbert Fusening, German Cancer Research Center, Heidelberg, Germany) as well as human lung and breast adenocarcinoma cells (A549 and MCF-7; provided by Japanese Collection of Research Bioresources, Osaka, Japan) were cultured in DMEM supplemented with 10% FBS and 100 U/mL of penicillin-streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. Cells were grown in adherent cultures and, when they reached the logarithmic phase of growth, were used in experiments. 2.3. Treatment with AgNPs or Ag ions When cell density reached 70–80% confluence, medium was changed to DMEM with 0.5% FBS. After being cultured for 24 h, cells were treated with various concentrations of AgNPs (∼1 mg/mL) and AgNO3 (∼50 μM) for ∼24 h. Cytochalasin B (CCB; ∼20 μM) or latrunculin B (LatB; ∼100 μM) was added 0.5 h before the treatment to inhibit actin polymerization. Phalloidin (Pha; ∼100 μM) was added 0.5 h before the treatment to inhibit actin depolymerization. N-acetylcysteine (NAC; 20 mM) was added 0.5 h before the treatment to remove the Ag ions released into the medium. The inhibitor ZM447439 (ZM; 5 μM) was added 0.5 h before the treatment to inhibit AURKs. 2.4. Western blot analysis Cells treated with AgNPs or AgNO3 were lysed in lysis buffer and Western blotting was performed as described previously (Zhao and Ibuki, 2015). Primary antibodies against p-H3S10 (Millipore Co., Billerica, MA), actin (Santa Cruz Biotechnology Inc., CA), phosphoAurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr98) and Aurora B (Cell Signaling Technology, Inc., MA, USA) (1:1000) were used, followed by secondary antibodies conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories, PA) (1:1000). In the analysis of globular actin (G-actin), cells treated with AgNPs or AgNO3 were suspended in ice-cold hypertonic buffer (210 mM mannitol, 70 mM sucrose, 5 mM Tris-HCl (pH 7.5), and 1 mM EDTA (pH 7.5)). After being suspended for 30 min, cells were centrifuged at 5000 × g for 5 min and supernatants were subjected to Western blotting in order to detect G-actin. 2.5. Immunofluorescence staining Cells treated with AgNPs were fixed and stained as described previously (Zhao and Ibuki, 2015). Fixed cells were incubated with the primary antibody against p-H3S10 (1:200) for 2 h, followed by the secondary antibody conjugated with FITC (Jackson ImmunoResearch Laboratories, 1:200). Actin was stained with Acti-stain™ 555 fluorescent Pha (Cytoskeleton Inc., Denver, CO). Nuclei were stained with 4′,6diamidino-2-phenylindole (DAPI) or propidium iodide (PI) (1 μg/mL) in order to confirm the distribution of p-H3S10 foci and filament actin (F-actin). Images were acquired on a fluorescence microscope (BX51, Olympus Co., Tokyo, Japan).
2. Materials and methods 2.1. Preparation of AgNPs AgNPs, the primary (listed) sizes of which were < 0.1 μm, were purchased from Sigma–Aldrich (Cat. No. 576832, St. Louis, MO, USA). The mean diameter of AgNPs in distilled water (pH 7.0) was approximately 200 nm (Supplementary Figure 1A). ζ-potential exhibited negative values. ζ-potential in pH 5.0–12.0 was −20 to −30 mV (Supplementary Figure 1B). AgNPs suspended in Dulbecco's Modified Eagle's Medium (DMEM; Sigma–Aldrich) containing 0.5% (v/v) fetal bovine serum (FBS; Life Technologies, Grand Island, NY, USA) at a final concentration of 10 mg/mL were immediately sonicated in a bath-type sonicator (Bioruptor UCW-310; Cosmo Bio, Tokyo, Japan) for 1 min before being applied to cells.
2.6. Flow cytometric analysis of actin The conformation of F-actin was analyzed using flow cytometry (FCM). Cells treated with AgNPs were fixed with 3.7% formalin. F-actin was stained with Acti-stain™ 555 fluorescent Pha. Fluorescence intensity was analyzed using FCM (FACSCanto™ II; Becton Dickinson, Franklin Lakes, NJ). 2.7. Evaluation of the uptake of AgNPs Cells treated with AgNPs were washed three times with PBS to 40
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remove free AgNPs. Cells were then resuspended in DMEM and the amount of particles taken up was analyzed with side-scattered light (SS) in FCM. Light scattered at a 90° angle to the axis of the laser beam was measured as SS, and is related to intracellular density. Changes in cellular SS after the treatment with AgNPs reflect the uptake potential of the particles (Suzuki et al., 2007; Toduka et al., 2012). 2.8. Knockdown of cofilin 1 using siRNA transfection Stealth siRNA target human cofilin 1 (oligo ID: HSS141560; HSS141561; HSS173851) and negative control siRNA were purchased from Life Technologies Co., MA, USA. A549 cells seeded on a 35-mm dish were transfected with 100 pmol of siRNA using lipofectamine RNAi-MAX reagent (Life Technologies Co., CA, USA) as recommended by the manufacturer. Following further cultivation for 4 days, cells were treated with AgNPs for 1 h or AgNO3 for 0.5 h, and p-H3S10, Gactin, and AURKs were then detected by Western blotting. 2.9. RNA isolation and real-time reverse-transcription PCR The experimental method has been described in a previous study (Zhao and Ibuki, 2015). Briefly, using a total RNA isolation kit (Macherey-Nagel GmbH & Co. KG, Germany), we isolated total RNA from A549 cells transfected with siRNA. RNA was converted into complementary DNA using the PrimeScript® RT reagent kit (Takara, Shiga, Japan) and real-time PCR reactions were performed on the LightCycler® Nano System using SYBR Green PCR Master Mix (Roche Applied Science, Mannheim, Germany). The primer pairs for cofilin 1 and GAPDH were as follows: cofilin 1: Forward, 5′-GGT GCT CTT CTG CCT GAG TG-3′; Reverse, 5′-TCT TGA CAA AGG TGG CGT AG-3′. GAPDH: Forward, 5′-GAG TCA ACG GAT TTG GTC GT-3′; Reverse, 5′TTG ATT TTG GAG GGA TCT CG-3′. Fold changes were normalized to GAPDH levels and the ratio to untreated samples was calculated.
Fig. 1. p-H3S10 after a treatment with AgNPs. (A) p-H3S10 after a treatment with AgNPs. HaCaT, A549, and MCF-7 cells were treated with AgNPs (∼0.3 mg/mL) for 1 h. H3 (Coomassie brilliant blue (CBB) staining) was used as a standard for the equal loading of proteins for SDS-PAGE. (B) Images of p-H3S10 after a treatment with AgNPs. A549 cells treated with AgNPs (1 mg/mL) for 1 h were stained with the antibody for p-H3S10 and PI. Left side images: p-H3S10; middle images: PI staining; right side images: merged images of p-H3S10 and PI. Numbered cells ((1)–(4) in left panels) are shown as magnified images in bottom panels. The scale bars indicate 20 μm.
manner. Similar results were obtained in the analysis of polymerized actin (F-actin) (Fig. 2B). The treatment with AgNPs decreased F-actin attached to fluorescent Pha. Fig. 2C shows images of F-actin. Actin filaments clearly visible in untreated cells were degraded by the treatment with AgNPs. These results suggest that AgNPs induced the depolymerization of F-actin.
3. Results 3.1. Generation of p-H3S10 after the treatment with AgNPs AgNPs dose-dependently induced p-H3S10 in three types of cell lines (Fig. 1A). Its phosphorylation was more pronounced in MCF-7 cells than in A549 cells, and was weak in HaCaT cells. We previously reported that the incorporation of AgNPs into cells is dependent on cell lines (Zhao et al., 2016). The cell line that took up the greatest amount of AgNPs generated more p-H3S10. Immunofluorescence staining revealed the clear phosphorylation of H3S10 in nuclei (Fig. 1B). p-H3S10 has been strongly correlated with chromosome condensation during cell division in all eukaryotes (Hendzel et al., 1997; Wei et al., 1999). Cells during premature chromosome condensation also show p-H3S10 (Ajiro and Nishimoto, 1985). We detected this phosphorylation with clear and bright staining (arrows 1 and 2). AgNPs induced p-H3S10 throughout the nucleus, in which staining was thin and solid (arrows 3 and 4). Since the phosphorylation of H3S10 was induced 10 min after the treatment with AgNPs (data not shown) and observed in all cells independent of the cell cycle, p-H3S10 induced by AgNPs differed from that normally associated with mitosis and premature chromosome condensation.
3.3. Relationship between dynamic changes in actin filaments and p-H3S10 induced by AgNPs The relationship between p-H3S10 and dynamic changes in actin filaments was examined in more detail. LatB, a specific inhibitor of the polymerization of actin, generated G-actin in a dose-dependent manner (Fig. 3A). p-H3S10 induced by AgNPs was suppressed by LatB and CCB, a similar inhibitor of the polymerization of actin (Cooper, 1987) (Fig. 3B). On the other hand, Pha, a specific inhibitor of the depolymerization of actin, decreased G-actin (Fig. 3A). Pha partially inhibited p-H3S10 in a dose-dependent manner (Fig. 3C). Immunofluorescence staining also revealed similar results (Fig. 3D). LatB completely inhibited, whereas Pha partially suppressed the phosphorylation of H3S10. We previously showed that the intercellular uptake of AgNPs is related to the induction of p-H3S10 (Zhao and Ibuki, 2015). We attributed the inhibition of p-H3S10 by the treatment with actin inhibitors to the suppressed uptake of AgNPs. Therefore, changes in the uptake of AgNPs after the treatment with inhibitors were examined (Fig. 3E). SS intensity detected by FCM increased after the treatment with AgNPs, and was not affected by LatB, CCB, or Pha (Fig. 3E). These results suggest that the accelerated cycle between the polymerization and depolymerization of actin was due to p-H3S10.
3.2. Depolymerization of actin after the treatment with AgNPs We investigated the dynamics of actin when AgNPs were incorporated. Actin always repeats dynamic structural changes: polymerization to F-actin from G-actin and depolymerization to G-actin from F-actin (Lee and Dominguez, 2010). In order to analyze the depolymerization of actin, G-actin was separated using hypertonic buffer and centrifugation, and then detected by Western blotting (Fig. 2A). The treatment with AgNPs released G-actin into the cytoplasm in a dose-dependent 41
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Fig. 2. Depolymerization of actin after a treatment with AgNPs. (A) G-actin after a treatment with AgNPs. A549 cells were treated with AgNPs (∼1 mg/mL) for ∼24 h and suspended in hypertonic buffer. Cells were centrifuged, and the supernatant was subjected to Western blotting in order to detect G-actin. CBB staining was used as a standard for the equal loading of proteins for SDS-PAGE. (B) F-actin after a treatment with AgNPs. A549 cells were treated with AgNPs (∼1 mg/mL) for 1 h, and stained F-actin with Acti-stain™ 555 fluorescent phalloidin. The fluorescence intensity of F-actin was analyzed using FCM. (C) Images of F-actin after a treatment with AgNPs. A549 or HaCaT cells treated with AgNPs (∼1 mg/mL) for 1 h were stained with the Acti-stain™ 555 fluorescent phalloidin and DAPI. Left side images: F-actin; middle images: DAPI staining; right side images: merged images of F-actin and DAPI. The scale bars indicate 20 μm.
depolymerization of F-actin. The stealth RNAi used for cofilin 1 in this study mostly suppressed the expression of cofilin 1 mRNA (Fig. 6A). The knockdown of cofilin 1 decreased the amount of p-H3S10 and release of G-actin induced by AgNPs and AgNO3 (Fig. 6B). The knockdown of cofilin 1 did not affect the uptake of AgNPs into cells (Fig. 6C). Furthermore, the knockdown of cofilin 1 decreased the amount of p-AURKB induced by AgNPs and AgNO3 (Fig. 6D). These results indicate that p-H3S10 induced by Ag ions released from AgNPs incorporated into inner cells occurred via the activation of AURK pathways, mainly the AURKB pathway, which occurred downstream of dynamic changes in actin filaments.
3.4. H3S10 was phosphorylated via AURK pathways AURKs are required for the induction of p-H3S10 in mammals (Crosio et al., 2002; Le et al., 2013). AgNPs phosphorylated AURKA, B, and C, with the phosphorylation of AURKB being the most prominent (Fig. 4A). ZM, an inhibitor of AURKs, was applied before the treatment with AgNPs. p-H3S10 induced by AgNPs was completely inhibited by ZM (Fig. 4B). Images of immunofluorescence staining are shown in Fig. 4C. AgNP-induced p-H3S10 was significantly inhibited by ZM. The AURK inhibitor did not affect the uptake of AgNPs into cells (Fig. 4D). These results indicate that p-H3S10 induced by AgNPs occurred via the activation of AURK pathways, mainly the AURKB pathway. Furthermore, the phosphorylation of AURKB after the treatment with AgNPs was significantly inhibited by LatB (Fig. 4E), and ZM did not affect the release of G-actin induced by AgNPs (Fig. 4F), suggesting that changes in actin filaments occur upstream of the activation of AURKs.
4. Discussion In the present study, we showed the rapid induction of p-H3S10 in all cells treated with AgNPs, which was clearly different from that during mitosis. The induction of p-H3S10 has been correlated with mitotic chromosome condensation (Hendzel et al., 1997; Wei et al., 1999). During prophase to metaphase, p-H3S10 was reported to become concentrated in a few nuclear foci and on condensed chromosomes, and eventually dispersed when daughter nuclei formed (Hayashi-Takanaka et al., 2009). We also identified some mitotic cells with condensed p-H3S10 (Fig. 1B: arrows 1 and 2). On the other hand, previous studies demonstrated that p-H3S10 mediated by another mechanism plays a role in tumor promotion (Choi et al., 2005; Kim et al., 2008). Some substances referred to as carcinogens significantly induce p-H3S10, and this has been associated with the induction of the proto-oncogenes c-fos and c-jun genes (Choi et al., 2005; Ibuki et al.,
3.5. Release of Ag ions from AgNPs and depolymerization of actin The depolymerization of actin was also induced after the treatment with AgNO3 (Fig. 5A). G-actin was detected in a dose-dependent manner. The release of G-actin induced by AgNPs was inhibited by NAC, the thiol group of which has high affinity for Ag ion (Fig. 5B). AgNO3 also induced p-H3S10, and this was suppressed by LatB and CCB (Fig. 5C). In order to further confirm the relationships between p-H3S10 and the actin cytoskeleton and activation of AURKB, we performed a series of experiments using siRNA cofilin 1, which plays a role in the 42
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Fig. 3. Dynamic changes in actin after a treatment with AgNPs and p-H3S10. (A) Depolymerization and polymerization of actin after a treatment with actin inhibitors. A549 cells were treated with LatB (∼100 μM) or Pha (∼100 μM) for 1 h and suspended in hypertonic buffer. After centrifugation, the supernatant was subjected to Western blotting in order to detect Gactin. CBB staining was used as a standard for the equal loading of proteins for SDS-PAGE. (B) p-H3S10 after a treatment with AgNPs in the presence of actin polymerization inhibitors. A549 cells were treated with LatB (∼10 μM) or CCB (∼20 μM) for 0.5 h and treated with AgNPs (∼1 mg/mL) for 1 h. (C) p-H3S10 after a treatment with AgNPs in the presence of an actin depolymerization inhibitor. A549 cells were treated with Pha (∼100 μM) for 0.5 h and then treated with AgNPs (∼1 mg/mL) for 1 h. (D) Images of p-H3S10 after a treatment with AgNPs in the presence of actin inhibitors. A549 cells were treated with LatB (5 μM), CCB (20 μM), or Pha (100 μM) for 0.5 h, treated with AgNPs (1 mg/mL) for 1 h, and then stained with the antibody for p-H3S10 and PI. Upper side images: p-H3S10; middle images: PI staining; lower side images: merged images of p-H3S10 and PI. The scale bars indicate 20 μm. (E) Intercellular uptake of AgNPs in the presence of actin inhibitors. A549 cells were treated with LatB (10 μM), CCB (20 μM), or Pha (100 μM) for 0.5 h and then treated with AgNPs (∼1 mg/mL) for 1 h. Cells were analyzed using SS in FCM.
that the pathways that mediated the phosphorylation of H3S10 were those of AURKs. In general, AURKs are a family of cell cycle-regulated kinases. In dividing cells, AURKB is responsible for H3S10 phosphorylation (Fischle et al., 2005; Hirota et al., 2005). On the other hand, thyroid hormone phosphorylated H3S10, which was associated with gene transcription (Tardáguila et al., 2011). p-H3S10 by AURKB enhanced double modifications with H3K9me3, indicating that AURKB has a role in marking silent chromatin independently of the cell cycle (Sabbattini et al., 2007). These findings together with the present results provide evidence for the roles of AURKs not being restricted to mitosis. The phosphorylation of AURKB at Thr232 is essential for kinase activity in mitosis (Yang et al., 2009; Yasui et al., 2004). The AgNPinduced phosphorylation site of AURKB was also at Thr232, as detected by Western blotting in this study. Thr232 of AURKB is a common site of phosphorylation that may function in mitosis and under other conditions. The upstream event that mediates the activation of AURKB was examined. We previously reported that the incorporation of AgNPs into
2014; Kim et al., 2008; Li et al., 2003). Cell-transforming activity was previously reported to be weaker in histone H3S10A mutant cells than in H3 wild-type cells (Choi et al., 2005). AgNP-induced p-H3S10 was observed in all cells independent of the cell cycle. Ke et al. (2008) also reported that the increased levels of p-H3S10 induced by nickel chloride were not simply due to a larger number of nickel-exposed cells undergoing mitosis. Images of p-H3S10 after the treatment with AgNPs revealed thin staining throughout the nuclei (Fig. 1B: arrows 3 and 4). Based on its rapid phosphorylation after the treatment with AgNPs, p-H3S10 may have been induced via the activation of any signal transduction pathway. The contribution of signal transduction via MAPK and Akt pathways has been reported for some carcinogenic substances (Ibuki et al., 2014; Ke et al., 2008; Keum et al., 2013). We have preliminary data that AgNPs activated MAPK (data no shown), and meanwhile we found that AURK pathways were also significantly activated by AgNPs. AgNPs phosphorylated AURKA, B, and C and AgNP-induced pH3S10 was inhibited by ZM, an inhibitor of AURKs (Fig. 4), suggesting 43
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Fig. 4. Phosphorylation of Aurora kinases after a treatment with AgNPs and p-H3S10. (A) Phosphorylation of AURKA, B, and C after a treatment with AgNPs. A549 cells were treated with AgNPs (1 mg/mL) for ∼6 h. The band intensity of p-AURKs detected by Western blotting was assessed using an analysis tool in Adobe Photoshop CS5, and the ratio to the untreated control was plotted. (B) p-H3S10 after a treatment with AgNPs in the presence of an AURK inhibitor. A549 cells were treated with ZM (5 μM) for 0.5 h and then treated with AgNPs (∼1 mg/mL) for ∼10 h. H3 (CBB staining) was used as a standard for the equal loading of proteins for SDS-PAGE. (C) Images of p-H3S10 after a treatment with AgNPs in the presence of an AURK inhibitor. A549 cells were treated with ZM (5 μM) for 0.5 h, treated with AgNPs (1 mg/mL) for 1 h, and then stained with the antibody for p-H3S10 and PI. Upper side images: pH3S10; middle images: PI staining; lower side images: merged images of p-H3S10 and PI. The scale bars indicate 20 μm. (D) Intercellular uptake of AgNPs in the presence of an AURK inhibitor. A549 cells were treated with ZM (5 μM) for 0.5 h and then treated with AgNPs (∼1 mg/mL) for 1 h. Cells were analyzed using SS in FCM. (E) p-AURKB after a treatment with AgNPs in the presence of an actin polymerization inhibitor. A549 cells were treated with LatB (∼10 μM) for 0.5 h and then treated with AgNPs (1 mg/mL) for 1 h. (F) Depolymerization of F-actin after a treatment with AgNPs in the presence of an AURK inhibitor. A549 cells were treated with ZM (5 μM) for 0.5 h and then treated with AgNPs (∼1 mg/mL) for 1 h. G-actin was separated using hypertonic buffer and centrifugation.
Knoblich, 2002; Bernard, 2007; Ritchey et al., 2012; Wang et al., 2010), we expected changes in actin filaments during the process of the incorporation of AgNPs to lead to the induction of p-H3S10. The polymerization and depolymerization of actin filaments are essential
cells was dependent on cell lines (Zhao et al., 2016), and this was related to p-H3S10. Since cells have the ability to take up metal NPs by endocytosis and phagocytosis (Panariti et al., 2012), and AURKs have been reported to associate with the actin cytoskeleton (Berdnik and
Fig. 5. Ag ion-induced depolymerization of actin and p-H3S10. (A) Depolymerization of F-actin after a treatment with AgNO3. A549 cells were treated with AgNO3 (∼100 μM) for 0.5 h. G-actin was separated using hypertonic buffer and centrifugation, and detected by Western blotting. CBB staining was used as a standard for the equal loading of proteins for SDS-PAGE. (B) Depolymerization of F-actin after a treatment with AgNPs in the presence of an Ag ion inhibitor. A549 cells were treated with NAC (20 mM) for 0.5 h and then treated with AgNPs (∼1 mg/mL) for 1 h. (C) p-H3S10 after a treatment with AgNO3 in the presence of actin polymerization inhibitors. A549 cells were treated with LatB (5 μM) or CCB (20 μM) for 0.5 h and then treated with AgNO3 (∼50 μM) for 1 h.
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Fig. 6. p-H3S10 and p-AURKs in cofilin 1-knockdown cells. (A) Expression of cofilin 1 mRNA in cells knocked down using siRNA. A549 cells were transfected with siRNA (oligo ID: HSS141560; HSS141561 and HSS173851) or negative control siRNA as described in the experimental procedures. Fold changes were normalized to GAPDH levels and the ratio to untreated samples was calculated. Stealth siRNA oligo ID: HSS141561 was used in subsequent experiments. (B) p-H3S10 and G-actin in cofilin 1-knockdown cells after a treatment with AgNPs or AgNO3. Cells in which cofilin 1 was knocked down using siRNA were treated with AgNPs (1 mg/mL) for 1 h or AgNO3 (50 μM) for 0.5 h. G-actin was separated using hypertonic buffer and centrifugation. CBB staining was used as a standard for the equal loading of proteins for SDS-PAGE. Con: control; siC: control siRNA; siR: cofilin 1 siRNA. (C) Intercellular uptake of AgNPs in cofilin 1-knockdown cells after a treatment with AgNPs or AgNO3. Cells in which cofilin 1 was knocked down using siRNA were treated with AgNPs (1 mg/mL) for 1 h or AgNO3 (50 μM) for 0.5 h. Cells were analyzed using SS in FCM. (D) p-AURKs in cofilin 1-knockdown cells after a treatment with AgNPs or AgNO3. Cells in which cofilin 1 was knocked down using siRNA were treated with AgNPs (1 mg/mL) for 1 h or AgNO3 (50 μM) for 0.5 h.
The interaction of metal ions with the cytoskeleton has been extensively studied (DalleDonne et al., 1999; Fagotti et al., 1996; Li et al., 1993; Macdonald et al., 1987). Aluminum ions compete with magnesium ions at GTP-binding sites on tubulin, and, thus, affect microtubule polymerization (Macdonald et al., 1987). Nickel ions alter the conformation of G-actin, which may affect dynamic changes in actin filament polymerization or depolymerization (DalleDonne et al., 1999). Another mechanism involves the nickel-mediated oxidation of SH groups in cytoskeletal proteins as a result of intracellular glutathione (GSH) depletion and an increase in ROS. Decreases in the number of SH groups and the formation of disulphide bonds may promote actin filament aggregation (Li et al., 1993). Ag ions may also bind to SH groups (Singh and Ramarao, 2012; Zhao et al., 2016). Exposure to copper may interfere with the tyrosine phosphorylation of actin molecules, which is one of the mechanisms underlying actin filament depolymerization (Fagotti et al., 1996). Further studies are needed in order to clarify the mechanisms responsible for Ag ion-induced changes in the actin cytoskeleton. In the present study, we used one kind of AgNPs. Several parameters such as particle sizes, surface charges and shapes affect the uptake of AgNPs and release of Ag ions (Powers et al., 2011; Tak et al., 2015), which may change the AgNPs-induced p-H3S10 and its mechanism. In Fig. 3, AgNP-induced p-H3S10 was suppressed by the inhibitors of actin polymerization (LatB or CBB) and depolymerization (Pha), and LatB and CBB exerted stronger inhibitory effects than Pha. Which process, actin polymerization or depolymerization, is more important for AgNP-induced p-H3S10, is under consideration.
for endocytosis, phagocytosis, and cell movement (Castellano et al., 2001; Deschamps et al., 2013; Jeng and Welch, 2001). In phagocytosis, deformation of the plasma membrane relies on transient changes in Factin and the microtubule cytoskeleton (Deschamps et al., 2013). Specific F-actin ring-like structures are formed by the internalization of particles, which is controlled by the polymerization of G-actin (Castellano et al., 2001; Jeng and Welch, 2001). F-actin is rapidly depolymerized immediately after the uptake of particles has begun (Kuhn et al., 2014). AgNPs induced the depolymerization of F-actin and polymerization of G-actin, and AgNP-induced p-AURKB and p-H3S10 levels were suppressed by actin inhibitors. The knockdown of cofilin 1 decreased the amount of p-H3S10 and p-AURKs as well as the release of G-actin (Fig. 6B and D). Therefore, we first considered dynamic changes in actin filaments induced by ‘the incorporation of AgNPs’ to occur upstream of p-AURKB and p-H3S10. On the other hand, we observed similar actin depolymerization after the treatment with AgNO3. AgNO3-induced p-H3S10 was inhibited by actin inhibitors and the AgNP-induced depolymerization of F-actin was inhibited by the depletion of Ag ions. These results indicate that the trigger for p-H3S10 is a change in actin filaments caused by Ag ions released from AgNPs incorporated into inner cells, and not the process of the incorporation of AgNPs. The amount of Ag ions released from AgNPs (1 mg/mL) was 0.024 μg/mL (0.22 μM) during incubation for 0.5 h (Zhao et al., 2013). The concentrations of AgNO3 which remarkably induced p-H3S10 were 5–50 μM (Fig. 5C) and AgNO3-induced pH3S10 was transient (Zhao and Ibuki, 2015). Since p-H3S10 is dependent on the uptake of AgNPs, the persistent release of Ag ions from AgNPs incorporated into inner cells, not outer cells is important. 45
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Hayashi-Takanaka, Y., Yamagata, K., Nozaki, N., Kimura, H., 2009. Visualizing histone modifications in living cells: spatiotemporal dynamics of H3 phosphorylation during interphase. J. Cell Biol. 187, 781–790. Hendzel, M.J., Wei, Y., Mancini, M.A., Van Hooser, A., Ranalli, T., Brinkley, B.R., BazettJones, D.P., Allis, C.D., 1997. Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106, 348–360. Hirota, T., Lipp, J.J., Toh, B.H., Peters, J.M., 2005. Histone H3 serine 10 phosphorylation by Aurora B causes HP1 dissociation from heterochromatin. Nature 438, 1176–1180. Hsu, J.Y., Sun, Z.W., Li, X., Reuben, M., Tatchell, K., Bishop, D.K., Grushcow, J.M., Brame, C.J., Caldwell, J.A., Hunt, D.F., Lin, R., Smith, M.M., Allis, C.D., 2000. Mitotic phosphorylation of histone H3 is governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes. Cell 102, 279–291. Ibuki, Y., Toyooka, T., Zhao, X., Yoshida, I., 2014. Cigarette sidestream smoke induces histone H3 phosphorylation via JNK and PI3K/Akt pathways, leading to the expression of proto-oncogenes. Carcinogenesis 35, 1228–1237. Jeng, R.L., Welch, M.D., 2001. Cytoskeleton: actin and endocytosis – no longer the weakest link. Curr. Biol. 11, R691–R694. Jiang, X., Foldbjerg, R., Miclaus, T., Wang, L., Singh, R., Hayashi, Y., Sutherland, D., Chen, C., Autrup, H., Beer, C., 2013. Multi-platform genotoxicity analysis of silver nanoparticles in the model cell line CHO-K1. Toxicol. Lett. 222, 55–63. Ke, Q., Li, Q., Ellen, T.P., Sun, H., Costa, M., 2008. Nickel compounds induce phosphorylation of histone H3 at serine 10 by activating JNK-MAPK pathway. Carcinogenesis 29, 1276–1281. Keum, Y.S., Kim, H.G., Bode, A.M., Surh, Y.J., Dong, Z., 2013. UVB-induced COX-2 expression requires histone H3 phosphorylation at Ser10 and Ser28. Oncogene 32, 444–452. Kim, H.G., Lee, K.W., Cho, Y.Y., Kang, N.J., Oh, S.M., Bode, A.M., Dong, Z., 2008. Mitogen- and stress-activated kinase 1-mediated histone H3 phosphorylation is crucial for cell transformation. Cancer Res. 68, 2538–2547. Kim, S., Choi, J.E., Choi, J., Chung, K.H., Park, K., Yi, J., Ryu, D.Y., 2009. Oxidative stressdependent toxicity of silver nanoparticles in human hepatoma cells. Toxicol. In Vitro 23, 1076–1084. Kuhn, D.A., Vanhecke, D., Michen, B., Blank, F., Gehr, P., Petri-Fink, A., RothenRutishauser, B., 2014. Different endocytotic uptake mechanisms for nanoparticles in epithelial cells and macrophages. Beilstein J. Nanotechnol. 5, 1625–1636. Le, L.T., Vu, H.L., Nguyen, C.H., Molla, A., 2013. Basal aurora kinase B activity is sufficient for histone H3 phosphorylation in prophase. Biol. Open 2, 379–386. Lee, S.H., Dominguez, R., 2010. Regulation of actin cytoskeleton dynamics in cells. Mol. Cells 29, 311–325. Li, J., Gorospe, M., Barnes, J., Liu, Y., 2003. Tumor promoter arsenite stimulates histone H3 phosphoacetylation of proto-oncogenes c-fos and c-jun chromatin in human diploid fibroblasts. J. Biol. Chem. 278, 13183–13191. Li, W., Zhao, Y., Chou, I.N., 1993. Alterations in cytoskeletal protein sulfhydryls and cellular glutathione in cultured cells exposed to cadmium and nickel ions. Toxicology 77, 65–79. Macdonald, T.L., Humphreys, W.G., Martin, R.B., 1987. Promotion of tubulin assembly by aluminum ion in vitro. Science 236, 183–186. Nigg, E.A., 2001. Mitotic kinases as regulators of cell division and its checkpoints. Nat. Rev. Mol. Cell Biol. 2, 21–32. Nowack, B., Krug, H.F., Height, M., 2011. 120 years of nanosilver history: implications for policy makers. Environ. Sci. Technol. 45, 1177–1183. Panariti, A., Miserocchi, G., Rivolta, I., 2012. The effect of nanoparticle uptake on cellular behavior: disrupting or enabling functions? Nanotechnol. Sci. Appl. 5, 87–100. Powers, C.M., Badireddy, A.R., Ryde, I.T., Seidler, F.J., Slotkin, T.A., 2011. Silver nanoparticles compromise neurodevelopment in PC12 cells: critical contributions of silver ion, particle size, coating, and composition. Environ. Health Perspect. 119, 37–44. Ritchey, L., Ottman, R., Roumanos, M., Chakrabarti, R., 2012. A functional cooperativity between Aurora A kinase and LIM kinase1: implication in the mitotic process. Cell Cycle 11, 296–309. Sabbattini, P., Canzonetta, C., Sjoberg, M., Nikic, S., Georgiou, A., Kemball-Cook, G., Auner, H.W., Dillon, N., 2007. A novel role for the Aurora B kinase in epigenetic marking of silent chromatin in differentiated postmitotic cells. EMBO J. 26, 4657–4669. Singh, R.P., Ramarao, P., 2012. Cellular uptake, intracellular trafficking and cytotoxicity of silver nanoparticles. Toxicol. Lett. 213, 249–259. Suzuki, H., Toyooka, T., Ibuki, Y., 2007. Simple and easy method to evaluate uptake potential of nanoparticles in mammalian cells using a flow cytometric light scatter analysis. Environ. Sci. Technol. 41, 3018–3024. Tak, Y.K., Pal, S., Naoghare, P.K., Rangasamy, S., Song, J.M., 2015. Shape-dependent skin penetration of silver nanoparticles: does it really matter? Sci. Rep. 5, 16908. Tardáguila, M., González-Gugel, E., Sánchez-Pacheco, A., 2011. Aurora kinase B activity is modulated by thyroid hormone during transcriptional activation of pituitary genes. Mol. Endocrinol. 25, 385–393. Toduka, Y., Toyooka, T., Ibuki, Y., 2012. Flow cytometric evaluation of nanoparticles using side-scattered light and reactive oxygen species-mediated fluorescencecorrelation with genotoxicity. Environ. Sci. Technol. 46, 7629–7636. Wang, L.H., Xiang, J., Yan, M., Zhang, Y., Zhao, Y., Yue, C.F., Xu, J., Zheng, F.M., Chen, J.N., Kang, Z., Chen, T.S., Xing, D., Liu, Q., 2010. The mitotic kinase Aurora-A induces mammary cell migration and breast cancer metastasis by activating the Cofilin-F-actin pathway. Cancer Res. 70, 9118–9128. Washington, D.C., 2011. The Silver Institute. The Future Demand of Silver Industrial Demand. The Silver Institute. http://www.silverinstitute.org/images/stories/silver/ PDF/futuresilverindustrialdemand.pdf.
5. Conclusion The present study described a possible pathway for the induction of p-H3S10 by AgNPs (Supplementary Figure 2). AgNPs incorporated into cells released Ag ions, which changed the actin polymerization cycle. The dynamic change in actin filaments activated AURKs and induced pH3S10 independent of the cell cycle. We need to study the mechanisms responsible and the meaning of the phosphorylation of H3S10 in more detail. Conflict of interest The authors of this manuscript have no conflict of interest to disclose. Transparency document The http://Transparency%20document(http://dx.doi.org/10.1016/ j.toxlet.2017.05.009) associated with this article can be found in the online version. Acknowledgments This work was supported in part by a Grant-in-Aid for Research on Risk of Chemical Substances from the Ministry of Health, Labour and Welfare, Japan, and JSPS KAKENHI 15H02828. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.toxlet.2017.05.009. References Adams, R.R., Maiato, H., Earnshaw, W.C., Carmena, M., 2001. Essential roles of Drosophila inner centromere protein (INCENP) and aurora B in histone H3 phosphorylation, metaphase chromosome alignment, kinetochore disjunction, and chromosome segregation. J. Cell Biol. 153, 865–880. Ajiro, K., Nishimoto, T., 1985. Specific site of histone H3 phosphorylation related to the maintenance of premature chromosome condensation. Evidence for catalytically induced interchange of the subunits. J. Biol. Chem. 260, 15379–15381. Berdnik, D., Knoblich, J.A., 2002. Drosophila Aurora-A is required for centrosome maturation and actin-dependent asymmetric protein localization during mitosis. Curr. Biol. 12, 640–647. Bernard, O., 2007. Lim kinases, regulators of actin dynamics. Int. J. Biochem. Cell Biol. 39, 1071–1076. Carmena, M., Earnshaw, W.C., 2003. The cellular geography of aurora kinases. Nat. Rev. Mol. Cell Biol. 4, 842–854. Castellano, F., Chavrier, P., Caron, E., 2001. Actin dynamics during phagocytosis. Semin. Immunol. 13, 347–355. Choi, H.S., Choi, B.Y., Cho, Y.Y., Mizuno, H., Kang, B.S., Bode, A.M., Dong, Z., 2005. Phosphorylation of histone H3 at serine 10 is indispensable for neoplastic cell transformation. Cancer Res. 65, 5818–5827. Cooper, J.A., 1987. Effects of cytochalasin and phalloidin on actin. J. Cell Biol. 105, 1473–1478. Crosio, C., Fimia, G.M., Loury, R., Kimura, M., Okano, Y., Zhou, H., Sen, S., Allis, C.D., Sassone-Corsi, P., 2002. Mitotic phosphorylation of histone H3: spatio-temporal regulation by mammalian Aurora kinases. Mol. Cell. Biol. 22, 874–885. DalleDonne, I., Milzani, A., Ciapparelli, C., Comazzi, M., Gioria, M.R., Colombo, R., 1999. The assembly of Ni2+-actin: some peculiarities. Biochim. Biophys. Acta 1426, 32–42. Deschamps, C., Echard, A., Niedergang, F., 2013. Phagocytosis and cytokinesis: do cells use common tools to cut and to eat? Highlights on common themes and differences. Traffic 14, 355–364. Fagotti, A., Di Rosa, I., Simoncelli, F., Pipe, R.K., Panara, F., Pascolini, R., 1996. The effects of copper on actin and fibronectin organization in Mytilus galloprovincialis haemocytes. Dev. Comp. Immunol. 20, 383–391. Fischle, W., Tseng, B.S., Dormann, H.L., Ueberheide, B.M., Garcia, B.A., Shabanowitz, J., Hunt, D.F., Funabiki, H., Allis, C.D., 2005. Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation. Nature 438, 1116–1122. Giet, R., Glover, D.M., 2001. Drosophila aurora B kinase is required for histone H3 phosphorylation and condensin recruitment during chromosome condensation and to organize the central spindle during cytokinesis. J. Cell Biol. 152, 669–682. Guo, H., Zhang, J., Boudreau, M., Meng, J., Yin, J.J., Liu, J., Xu, H., 2016. Intravenous administration of silver nanoparticles causes organ toxicity through intracellular ROS-related loss of inter-endothelial junction. Part. Fibre Toxicol. 13, 21.
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Zhao, X., Ibuki, Y., 2015. Evaluating the toxicity of silver nanoparticles by detecting phosphorylation of histone H3 in combination with flow cytometry side-scattered light. Environ. Sci. Technol. 49, 5003–5012. Zhao, X., Takabayashi, F., Ibuki, Y., 2016. Coexposure to silver nanoparticles and ultraviolet A synergistically enhances the phosphorylation of histone H2AX. J. Photochem. Photobiol. B 162, 213–222. Zhao, X., Toyooka, T., Ibuki, Y., 2013. Synergistic bactericidal effect by combined exposure to Ag nanoparticles and UVA. Sci. Total Environ. 458–460, 54–62.
Wei, Y., Yu, L., Bowen, J., Gorovsky, M.A., Allis, C.D., 1999. Phosphorylation of histone H3 is required for proper chromosome condensation and segregation. Cell 97, 99–109. Yang, J., Zappacosta, F., Annan, R.S., Nurse, K., Tummino, P.J., Copeland, R.A., Lai, Z., 2009. The catalytic role of INCENP in Aurora B activation and the kinetic mechanism of Aurora B/INCENP. Biochem. J. 417, 355–360. Yasui, Y., Urano, T., Kawajiri, A., Nagata, K., Tatsuka, M., Saya, H., Furukawa, K., Takahashi, T., Izawa, I., Inagaki, M., 2004. Autophosphorylation of a newly identified site of Aurora-B is indispensable for cytokinesis. J. Biol. Chem. 279, 12997–13003.
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Silver nanoparticle-induced phosphorylation of histone H3 at serine 10 is due to dynamic changes in actin filaments and the activation of Aurora kinases Xiaoxu Zhaoa, Tatsushi Toyookab, Yuko Ibukia, a b
MARK
⁎
Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, Shizuoka, Japan Industrial Toxicology and Health Effects Research Group, National Institute of Occupational Safety and Health, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords: Silver nanoparticles Actin Phosphorylation Histone Aurora kinases
The phosphorylation of histone H3 at serine 10 (p-H3S10) has been closely correlated with mitotic chromosome condensation. We previously reported that silver nanoparticles (AgNPs) significantly induced p-H3S10 independent of mitosis. In the present study, we examined the mechanisms underlying the induction of pH3S10 by AgNPs. A treatment with AgNPs markedly induced p-H3S10 in a dose-dependent manner in three types of cell lines, and this was dependent on the cellular incorporation of AgNPs. The immunofluorescent staining of AgNP-induced p-H3S10 was thin and solid throughout the nucleus, and differed from that normally associated with mitosis. AgNPs induced the formation of globular actin in a dose-dependent manner. Latrunculin B (LatB) and phalloidin, inhibitors of actin polymerization and depolymerization, respectively, inhibited pH3S10, suggesting that dynamic changes in actin filaments are related to AgNP-induced p-H3S10. Furthermore, p-H3S10 was mediated by Aurora kinase (AURK) pathways, which were suppressed by LatB and siRNA for cofilin 1, an actin-depolymerizing protein. AgNO3 (Ag ions) exerted similar effects to those of AgNPs. These results suggest that Ag ions released from AgNPs incorporated into inner cells changed the dynamics of actin filaments, and this was followed by the activation of AURKs, leading to the induction of p-H3S10.
1. Introduction Nanoparticles (NPs) are ultra-fine materials (length or diameter of 1–100 nm) that are being increasingly used in modern technology, medical health care, and commercial products. Among metal NPs, silver NPs (AgNPs), which exhibit potent antibacterial and antifungal activities, have been widely used in biomedical devices and applications such as food and beverage containers, textiles, and room sprays (Nowack et al., 2011; Washington, 2011). Although humans frequently make contact with AgNPs, their toxic effects on human health have not yet been examined in detail. Ag is regarded as a safe metal and Ag products such as silverware have been used for a long time. Recent studies have indicated that exposure to AgNPs has negative effects on human health. In vitro studies demonstrated multiple abnormal physiological effects such as inhibited cell proliferation, membrane damage, mitochondrial dysfunc-
tion, and apoptosis, following an exposure to AgNPs (Guo et al., 2016; Jiang et al., 2013; Kim et al., 2009; Singh and Ramarao, 2012). AgNPs have been shown to contribute to the excessive generation of reactive oxygen species (ROS) and the resultant oxidative stress may eventually lead to cell injury (Guo et al., 2016; Jiang et al., 2013; Kim et al., 2009). The release of Ag ions from AgNPs is another important mechanism underlying AgNP-induced toxicity. Ag ions easily bind to the thiol groups of proteins in cells, leading to the inactivation of biological functions (Singh and Ramarao, 2012). We previously demonstrated that AgNPs significantly induced the phosphorylation of histone H3 at serine 10 (p-H3S10) (Zhao and Ibuki, 2015). p-H3S10 has been associated with mitotic chromosome condensation (Hendzel et al., 1997; Wei et al., 1999), but has also been reported to play a role in gene transcription related to tumor promotion (Choi et al., 2005; Kim et al., 2008). Epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA), which are related to
Abbreviations: NPs, nanoparticles; AgNPs, silver nanoparticles; ROS, reactive oxygen species; p-H3S10, phosphorylation of histone H3 at serine 10; EGF, epidermal growth factor; TPA, 12-O-tetradecanoylphorbol 13-acetate; CSS, cigarette sidestream smoke; UVB, ultraviolet B; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; JNK, cJun N-terminal protein kinase; AURKs, Aurora kinases; siRNA, small interfering RNA; DMEM, Dulbecco's Modified Eagle's Medium; FBS, fetal bovine serum; CCB, cytochalasin B; LatB, latrunculin B; Pha, phalloidin; NAC, N-acetylcysteine; ZM, ZM447439; G-actin, globular actin; FITC, fluorescein isothiocyanate; DAPI, 4′,6-diamidino-2-phenylindole; PI, propidium iodide; F-actin, filament actin; FCM, flow cytometry; SS, side-scattered light; HP1β, heterochromatin protein 1β; GSH, glutathione; CBB, Coomassie brilliant blue ⁎ Corresponding author. E-mail address: [email protected] (Y. Ibuki). http://dx.doi.org/10.1016/j.toxlet.2017.05.009 Received 26 October 2016; Received in revised form 8 April 2017; Accepted 5 May 2017 Available online 10 May 2017 0378-4274/ © 2017 Elsevier B.V. All rights reserved.
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2.2. Cells and cell culture conditions
tumor promotion, enhance the induction of p-H3S10. Several environmental factors such as cigarette sidestream smoke (CSS), ultraviolet B (UVB), arsenite, and nickel have also been shown to induce p-H3S10 (Ibuki et al., 2014; Ke et al., 2008; Keum et al., 2013; Li et al., 2003). Arsenite-induced p-H3S10 has been attributed to the activation of the extracellular signal-regulated kinase (ERK) pathway in the mitogenactivated protein kinase (MAPK) cascade (Li et al., 2003). Nickel was found to phosphorylate H3S10 via the c-Jun N-terminal protein kinase (JNK) pathway (Ke et al., 2008). CSS also activated the JNK and Akt pathways, leading to p-H3S10 (Ibuki et al., 2014). p-H3S10 induced by CSS, UVB, EGF, TPA, and arsenite has been associated with the induction of immediate-early genes, including the proto-oncogenes, cfos and c-jun (Choi et al., 2005; Ibuki et al., 2014; Keum et al., 2013; Kim et al., 2008; Li et al., 2003). This induction is also regulated downstream of the activation of the MAPK pathway. However, the molecular mechanisms responsible for AgNP-induced p-H3S10 have not yet been elucidated in detail. Aurora kinases (AURKs) belong to the serine/threonine kinase family and are known to phosphorylate H3S10 in mitotic budding yeasts, nematodes, and mammals (Crosio et al., 2002; Hsu et al., 2000; Le et al., 2013). Many low species have one or two AURKs, whereas mammals have at least three: AURKA, B, and C (Nigg, 2001). Mitotic pH3S10 is catalyzed by AURKA and/or AURKB in mammals (Crosio et al., 2002; Le et al., 2013). AURKs have essential functions in mitotic processes such as chromosome condensation and spindle dynamics (Carmena and Earnshaw, 2003), in which p-H3S10 plays some roles as an initiator of chromosome condensation and proper chromosome segregation (Adams et al., 2001; Giet and Glover, 2001). AURKs have also been implicated in the regulation of the actin cytoskeleton. The activation of Drosophila AURKs has been suggested to be involved in actin-dependent asymmetric protein localization during mitosis (Berdnik and Knoblich, 2002). The overexpression of AURKA increases the expression of phosphatase Slingshot-1, leading to the dephosphorylation and activation of the actin-depolymerizing protein, cofilin (Wang et al., 2010). AURKs also interact with LIM kinase 1, which is involved in the reorganization of the actin cytoskeleton (Bernard, 2007; Ritchey et al., 2012). Cells mainly take up metal NPs by endocytosis and phagocytosis (Panariti et al., 2012), and the actin cytoskeleton has been suggested to play a role in these processes (Castellano et al., 2001; Deschamps et al., 2013; Jeng and Welch, 2001). However, p-H3S10 induced by AgNPs and its relationships with AURKs and the actin cytoskeleton have not yet been investigated. In the present study, we elucidated the mechanisms responsible for AgNP-induced p-H3S10. We used several inhibitors or small interfering RNA (siRNA)-knockdown cells to investigate the relationships between p-H3S10 and the actin cytoskeleton and AURK pathways. The results obtained demonstrated the induction of p-H3S10 by AgNPs via the activation of AURK pathways, which occurred downstream of the changes induced in actin filaments by Ag ions released from AgNPs.
Human skin keratinocytes (HaCaT; provided by Dr. Norbert Fusening, German Cancer Research Center, Heidelberg, Germany) as well as human lung and breast adenocarcinoma cells (A549 and MCF-7; provided by Japanese Collection of Research Bioresources, Osaka, Japan) were cultured in DMEM supplemented with 10% FBS and 100 U/mL of penicillin-streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. Cells were grown in adherent cultures and, when they reached the logarithmic phase of growth, were used in experiments. 2.3. Treatment with AgNPs or Ag ions When cell density reached 70–80% confluence, medium was changed to DMEM with 0.5% FBS. After being cultured for 24 h, cells were treated with various concentrations of AgNPs (∼1 mg/mL) and AgNO3 (∼50 μM) for ∼24 h. Cytochalasin B (CCB; ∼20 μM) or latrunculin B (LatB; ∼100 μM) was added 0.5 h before the treatment to inhibit actin polymerization. Phalloidin (Pha; ∼100 μM) was added 0.5 h before the treatment to inhibit actin depolymerization. N-acetylcysteine (NAC; 20 mM) was added 0.5 h before the treatment to remove the Ag ions released into the medium. The inhibitor ZM447439 (ZM; 5 μM) was added 0.5 h before the treatment to inhibit AURKs. 2.4. Western blot analysis Cells treated with AgNPs or AgNO3 were lysed in lysis buffer and Western blotting was performed as described previously (Zhao and Ibuki, 2015). Primary antibodies against p-H3S10 (Millipore Co., Billerica, MA), actin (Santa Cruz Biotechnology Inc., CA), phosphoAurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr98) and Aurora B (Cell Signaling Technology, Inc., MA, USA) (1:1000) were used, followed by secondary antibodies conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories, PA) (1:1000). In the analysis of globular actin (G-actin), cells treated with AgNPs or AgNO3 were suspended in ice-cold hypertonic buffer (210 mM mannitol, 70 mM sucrose, 5 mM Tris-HCl (pH 7.5), and 1 mM EDTA (pH 7.5)). After being suspended for 30 min, cells were centrifuged at 5000 × g for 5 min and supernatants were subjected to Western blotting in order to detect G-actin. 2.5. Immunofluorescence staining Cells treated with AgNPs were fixed and stained as described previously (Zhao and Ibuki, 2015). Fixed cells were incubated with the primary antibody against p-H3S10 (1:200) for 2 h, followed by the secondary antibody conjugated with FITC (Jackson ImmunoResearch Laboratories, 1:200). Actin was stained with Acti-stain™ 555 fluorescent Pha (Cytoskeleton Inc., Denver, CO). Nuclei were stained with 4′,6diamidino-2-phenylindole (DAPI) or propidium iodide (PI) (1 μg/mL) in order to confirm the distribution of p-H3S10 foci and filament actin (F-actin). Images were acquired on a fluorescence microscope (BX51, Olympus Co., Tokyo, Japan).
2. Materials and methods 2.1. Preparation of AgNPs AgNPs, the primary (listed) sizes of which were < 0.1 μm, were purchased from Sigma–Aldrich (Cat. No. 576832, St. Louis, MO, USA). The mean diameter of AgNPs in distilled water (pH 7.0) was approximately 200 nm (Supplementary Figure 1A). ζ-potential exhibited negative values. ζ-potential in pH 5.0–12.0 was −20 to −30 mV (Supplementary Figure 1B). AgNPs suspended in Dulbecco's Modified Eagle's Medium (DMEM; Sigma–Aldrich) containing 0.5% (v/v) fetal bovine serum (FBS; Life Technologies, Grand Island, NY, USA) at a final concentration of 10 mg/mL were immediately sonicated in a bath-type sonicator (Bioruptor UCW-310; Cosmo Bio, Tokyo, Japan) for 1 min before being applied to cells.
2.6. Flow cytometric analysis of actin The conformation of F-actin was analyzed using flow cytometry (FCM). Cells treated with AgNPs were fixed with 3.7% formalin. F-actin was stained with Acti-stain™ 555 fluorescent Pha. Fluorescence intensity was analyzed using FCM (FACSCanto™ II; Becton Dickinson, Franklin Lakes, NJ). 2.7. Evaluation of the uptake of AgNPs Cells treated with AgNPs were washed three times with PBS to 40
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remove free AgNPs. Cells were then resuspended in DMEM and the amount of particles taken up was analyzed with side-scattered light (SS) in FCM. Light scattered at a 90° angle to the axis of the laser beam was measured as SS, and is related to intracellular density. Changes in cellular SS after the treatment with AgNPs reflect the uptake potential of the particles (Suzuki et al., 2007; Toduka et al., 2012). 2.8. Knockdown of cofilin 1 using siRNA transfection Stealth siRNA target human cofilin 1 (oligo ID: HSS141560; HSS141561; HSS173851) and negative control siRNA were purchased from Life Technologies Co., MA, USA. A549 cells seeded on a 35-mm dish were transfected with 100 pmol of siRNA using lipofectamine RNAi-MAX reagent (Life Technologies Co., CA, USA) as recommended by the manufacturer. Following further cultivation for 4 days, cells were treated with AgNPs for 1 h or AgNO3 for 0.5 h, and p-H3S10, Gactin, and AURKs were then detected by Western blotting. 2.9. RNA isolation and real-time reverse-transcription PCR The experimental method has been described in a previous study (Zhao and Ibuki, 2015). Briefly, using a total RNA isolation kit (Macherey-Nagel GmbH & Co. KG, Germany), we isolated total RNA from A549 cells transfected with siRNA. RNA was converted into complementary DNA using the PrimeScript® RT reagent kit (Takara, Shiga, Japan) and real-time PCR reactions were performed on the LightCycler® Nano System using SYBR Green PCR Master Mix (Roche Applied Science, Mannheim, Germany). The primer pairs for cofilin 1 and GAPDH were as follows: cofilin 1: Forward, 5′-GGT GCT CTT CTG CCT GAG TG-3′; Reverse, 5′-TCT TGA CAA AGG TGG CGT AG-3′. GAPDH: Forward, 5′-GAG TCA ACG GAT TTG GTC GT-3′; Reverse, 5′TTG ATT TTG GAG GGA TCT CG-3′. Fold changes were normalized to GAPDH levels and the ratio to untreated samples was calculated.
Fig. 1. p-H3S10 after a treatment with AgNPs. (A) p-H3S10 after a treatment with AgNPs. HaCaT, A549, and MCF-7 cells were treated with AgNPs (∼0.3 mg/mL) for 1 h. H3 (Coomassie brilliant blue (CBB) staining) was used as a standard for the equal loading of proteins for SDS-PAGE. (B) Images of p-H3S10 after a treatment with AgNPs. A549 cells treated with AgNPs (1 mg/mL) for 1 h were stained with the antibody for p-H3S10 and PI. Left side images: p-H3S10; middle images: PI staining; right side images: merged images of p-H3S10 and PI. Numbered cells ((1)–(4) in left panels) are shown as magnified images in bottom panels. The scale bars indicate 20 μm.
manner. Similar results were obtained in the analysis of polymerized actin (F-actin) (Fig. 2B). The treatment with AgNPs decreased F-actin attached to fluorescent Pha. Fig. 2C shows images of F-actin. Actin filaments clearly visible in untreated cells were degraded by the treatment with AgNPs. These results suggest that AgNPs induced the depolymerization of F-actin.
3. Results 3.1. Generation of p-H3S10 after the treatment with AgNPs AgNPs dose-dependently induced p-H3S10 in three types of cell lines (Fig. 1A). Its phosphorylation was more pronounced in MCF-7 cells than in A549 cells, and was weak in HaCaT cells. We previously reported that the incorporation of AgNPs into cells is dependent on cell lines (Zhao et al., 2016). The cell line that took up the greatest amount of AgNPs generated more p-H3S10. Immunofluorescence staining revealed the clear phosphorylation of H3S10 in nuclei (Fig. 1B). p-H3S10 has been strongly correlated with chromosome condensation during cell division in all eukaryotes (Hendzel et al., 1997; Wei et al., 1999). Cells during premature chromosome condensation also show p-H3S10 (Ajiro and Nishimoto, 1985). We detected this phosphorylation with clear and bright staining (arrows 1 and 2). AgNPs induced p-H3S10 throughout the nucleus, in which staining was thin and solid (arrows 3 and 4). Since the phosphorylation of H3S10 was induced 10 min after the treatment with AgNPs (data not shown) and observed in all cells independent of the cell cycle, p-H3S10 induced by AgNPs differed from that normally associated with mitosis and premature chromosome condensation.
3.3. Relationship between dynamic changes in actin filaments and p-H3S10 induced by AgNPs The relationship between p-H3S10 and dynamic changes in actin filaments was examined in more detail. LatB, a specific inhibitor of the polymerization of actin, generated G-actin in a dose-dependent manner (Fig. 3A). p-H3S10 induced by AgNPs was suppressed by LatB and CCB, a similar inhibitor of the polymerization of actin (Cooper, 1987) (Fig. 3B). On the other hand, Pha, a specific inhibitor of the depolymerization of actin, decreased G-actin (Fig. 3A). Pha partially inhibited p-H3S10 in a dose-dependent manner (Fig. 3C). Immunofluorescence staining also revealed similar results (Fig. 3D). LatB completely inhibited, whereas Pha partially suppressed the phosphorylation of H3S10. We previously showed that the intercellular uptake of AgNPs is related to the induction of p-H3S10 (Zhao and Ibuki, 2015). We attributed the inhibition of p-H3S10 by the treatment with actin inhibitors to the suppressed uptake of AgNPs. Therefore, changes in the uptake of AgNPs after the treatment with inhibitors were examined (Fig. 3E). SS intensity detected by FCM increased after the treatment with AgNPs, and was not affected by LatB, CCB, or Pha (Fig. 3E). These results suggest that the accelerated cycle between the polymerization and depolymerization of actin was due to p-H3S10.
3.2. Depolymerization of actin after the treatment with AgNPs We investigated the dynamics of actin when AgNPs were incorporated. Actin always repeats dynamic structural changes: polymerization to F-actin from G-actin and depolymerization to G-actin from F-actin (Lee and Dominguez, 2010). In order to analyze the depolymerization of actin, G-actin was separated using hypertonic buffer and centrifugation, and then detected by Western blotting (Fig. 2A). The treatment with AgNPs released G-actin into the cytoplasm in a dose-dependent 41
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Fig. 2. Depolymerization of actin after a treatment with AgNPs. (A) G-actin after a treatment with AgNPs. A549 cells were treated with AgNPs (∼1 mg/mL) for ∼24 h and suspended in hypertonic buffer. Cells were centrifuged, and the supernatant was subjected to Western blotting in order to detect G-actin. CBB staining was used as a standard for the equal loading of proteins for SDS-PAGE. (B) F-actin after a treatment with AgNPs. A549 cells were treated with AgNPs (∼1 mg/mL) for 1 h, and stained F-actin with Acti-stain™ 555 fluorescent phalloidin. The fluorescence intensity of F-actin was analyzed using FCM. (C) Images of F-actin after a treatment with AgNPs. A549 or HaCaT cells treated with AgNPs (∼1 mg/mL) for 1 h were stained with the Acti-stain™ 555 fluorescent phalloidin and DAPI. Left side images: F-actin; middle images: DAPI staining; right side images: merged images of F-actin and DAPI. The scale bars indicate 20 μm.
depolymerization of F-actin. The stealth RNAi used for cofilin 1 in this study mostly suppressed the expression of cofilin 1 mRNA (Fig. 6A). The knockdown of cofilin 1 decreased the amount of p-H3S10 and release of G-actin induced by AgNPs and AgNO3 (Fig. 6B). The knockdown of cofilin 1 did not affect the uptake of AgNPs into cells (Fig. 6C). Furthermore, the knockdown of cofilin 1 decreased the amount of p-AURKB induced by AgNPs and AgNO3 (Fig. 6D). These results indicate that p-H3S10 induced by Ag ions released from AgNPs incorporated into inner cells occurred via the activation of AURK pathways, mainly the AURKB pathway, which occurred downstream of dynamic changes in actin filaments.
3.4. H3S10 was phosphorylated via AURK pathways AURKs are required for the induction of p-H3S10 in mammals (Crosio et al., 2002; Le et al., 2013). AgNPs phosphorylated AURKA, B, and C, with the phosphorylation of AURKB being the most prominent (Fig. 4A). ZM, an inhibitor of AURKs, was applied before the treatment with AgNPs. p-H3S10 induced by AgNPs was completely inhibited by ZM (Fig. 4B). Images of immunofluorescence staining are shown in Fig. 4C. AgNP-induced p-H3S10 was significantly inhibited by ZM. The AURK inhibitor did not affect the uptake of AgNPs into cells (Fig. 4D). These results indicate that p-H3S10 induced by AgNPs occurred via the activation of AURK pathways, mainly the AURKB pathway. Furthermore, the phosphorylation of AURKB after the treatment with AgNPs was significantly inhibited by LatB (Fig. 4E), and ZM did not affect the release of G-actin induced by AgNPs (Fig. 4F), suggesting that changes in actin filaments occur upstream of the activation of AURKs.
4. Discussion In the present study, we showed the rapid induction of p-H3S10 in all cells treated with AgNPs, which was clearly different from that during mitosis. The induction of p-H3S10 has been correlated with mitotic chromosome condensation (Hendzel et al., 1997; Wei et al., 1999). During prophase to metaphase, p-H3S10 was reported to become concentrated in a few nuclear foci and on condensed chromosomes, and eventually dispersed when daughter nuclei formed (Hayashi-Takanaka et al., 2009). We also identified some mitotic cells with condensed p-H3S10 (Fig. 1B: arrows 1 and 2). On the other hand, previous studies demonstrated that p-H3S10 mediated by another mechanism plays a role in tumor promotion (Choi et al., 2005; Kim et al., 2008). Some substances referred to as carcinogens significantly induce p-H3S10, and this has been associated with the induction of the proto-oncogenes c-fos and c-jun genes (Choi et al., 2005; Ibuki et al.,
3.5. Release of Ag ions from AgNPs and depolymerization of actin The depolymerization of actin was also induced after the treatment with AgNO3 (Fig. 5A). G-actin was detected in a dose-dependent manner. The release of G-actin induced by AgNPs was inhibited by NAC, the thiol group of which has high affinity for Ag ion (Fig. 5B). AgNO3 also induced p-H3S10, and this was suppressed by LatB and CCB (Fig. 5C). In order to further confirm the relationships between p-H3S10 and the actin cytoskeleton and activation of AURKB, we performed a series of experiments using siRNA cofilin 1, which plays a role in the 42
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Fig. 3. Dynamic changes in actin after a treatment with AgNPs and p-H3S10. (A) Depolymerization and polymerization of actin after a treatment with actin inhibitors. A549 cells were treated with LatB (∼100 μM) or Pha (∼100 μM) for 1 h and suspended in hypertonic buffer. After centrifugation, the supernatant was subjected to Western blotting in order to detect Gactin. CBB staining was used as a standard for the equal loading of proteins for SDS-PAGE. (B) p-H3S10 after a treatment with AgNPs in the presence of actin polymerization inhibitors. A549 cells were treated with LatB (∼10 μM) or CCB (∼20 μM) for 0.5 h and treated with AgNPs (∼1 mg/mL) for 1 h. (C) p-H3S10 after a treatment with AgNPs in the presence of an actin depolymerization inhibitor. A549 cells were treated with Pha (∼100 μM) for 0.5 h and then treated with AgNPs (∼1 mg/mL) for 1 h. (D) Images of p-H3S10 after a treatment with AgNPs in the presence of actin inhibitors. A549 cells were treated with LatB (5 μM), CCB (20 μM), or Pha (100 μM) for 0.5 h, treated with AgNPs (1 mg/mL) for 1 h, and then stained with the antibody for p-H3S10 and PI. Upper side images: p-H3S10; middle images: PI staining; lower side images: merged images of p-H3S10 and PI. The scale bars indicate 20 μm. (E) Intercellular uptake of AgNPs in the presence of actin inhibitors. A549 cells were treated with LatB (10 μM), CCB (20 μM), or Pha (100 μM) for 0.5 h and then treated with AgNPs (∼1 mg/mL) for 1 h. Cells were analyzed using SS in FCM.
that the pathways that mediated the phosphorylation of H3S10 were those of AURKs. In general, AURKs are a family of cell cycle-regulated kinases. In dividing cells, AURKB is responsible for H3S10 phosphorylation (Fischle et al., 2005; Hirota et al., 2005). On the other hand, thyroid hormone phosphorylated H3S10, which was associated with gene transcription (Tardáguila et al., 2011). p-H3S10 by AURKB enhanced double modifications with H3K9me3, indicating that AURKB has a role in marking silent chromatin independently of the cell cycle (Sabbattini et al., 2007). These findings together with the present results provide evidence for the roles of AURKs not being restricted to mitosis. The phosphorylation of AURKB at Thr232 is essential for kinase activity in mitosis (Yang et al., 2009; Yasui et al., 2004). The AgNPinduced phosphorylation site of AURKB was also at Thr232, as detected by Western blotting in this study. Thr232 of AURKB is a common site of phosphorylation that may function in mitosis and under other conditions. The upstream event that mediates the activation of AURKB was examined. We previously reported that the incorporation of AgNPs into
2014; Kim et al., 2008; Li et al., 2003). Cell-transforming activity was previously reported to be weaker in histone H3S10A mutant cells than in H3 wild-type cells (Choi et al., 2005). AgNP-induced p-H3S10 was observed in all cells independent of the cell cycle. Ke et al. (2008) also reported that the increased levels of p-H3S10 induced by nickel chloride were not simply due to a larger number of nickel-exposed cells undergoing mitosis. Images of p-H3S10 after the treatment with AgNPs revealed thin staining throughout the nuclei (Fig. 1B: arrows 3 and 4). Based on its rapid phosphorylation after the treatment with AgNPs, p-H3S10 may have been induced via the activation of any signal transduction pathway. The contribution of signal transduction via MAPK and Akt pathways has been reported for some carcinogenic substances (Ibuki et al., 2014; Ke et al., 2008; Keum et al., 2013). We have preliminary data that AgNPs activated MAPK (data no shown), and meanwhile we found that AURK pathways were also significantly activated by AgNPs. AgNPs phosphorylated AURKA, B, and C and AgNP-induced pH3S10 was inhibited by ZM, an inhibitor of AURKs (Fig. 4), suggesting 43
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Fig. 4. Phosphorylation of Aurora kinases after a treatment with AgNPs and p-H3S10. (A) Phosphorylation of AURKA, B, and C after a treatment with AgNPs. A549 cells were treated with AgNPs (1 mg/mL) for ∼6 h. The band intensity of p-AURKs detected by Western blotting was assessed using an analysis tool in Adobe Photoshop CS5, and the ratio to the untreated control was plotted. (B) p-H3S10 after a treatment with AgNPs in the presence of an AURK inhibitor. A549 cells were treated with ZM (5 μM) for 0.5 h and then treated with AgNPs (∼1 mg/mL) for ∼10 h. H3 (CBB staining) was used as a standard for the equal loading of proteins for SDS-PAGE. (C) Images of p-H3S10 after a treatment with AgNPs in the presence of an AURK inhibitor. A549 cells were treated with ZM (5 μM) for 0.5 h, treated with AgNPs (1 mg/mL) for 1 h, and then stained with the antibody for p-H3S10 and PI. Upper side images: pH3S10; middle images: PI staining; lower side images: merged images of p-H3S10 and PI. The scale bars indicate 20 μm. (D) Intercellular uptake of AgNPs in the presence of an AURK inhibitor. A549 cells were treated with ZM (5 μM) for 0.5 h and then treated with AgNPs (∼1 mg/mL) for 1 h. Cells were analyzed using SS in FCM. (E) p-AURKB after a treatment with AgNPs in the presence of an actin polymerization inhibitor. A549 cells were treated with LatB (∼10 μM) for 0.5 h and then treated with AgNPs (1 mg/mL) for 1 h. (F) Depolymerization of F-actin after a treatment with AgNPs in the presence of an AURK inhibitor. A549 cells were treated with ZM (5 μM) for 0.5 h and then treated with AgNPs (∼1 mg/mL) for 1 h. G-actin was separated using hypertonic buffer and centrifugation.
Knoblich, 2002; Bernard, 2007; Ritchey et al., 2012; Wang et al., 2010), we expected changes in actin filaments during the process of the incorporation of AgNPs to lead to the induction of p-H3S10. The polymerization and depolymerization of actin filaments are essential
cells was dependent on cell lines (Zhao et al., 2016), and this was related to p-H3S10. Since cells have the ability to take up metal NPs by endocytosis and phagocytosis (Panariti et al., 2012), and AURKs have been reported to associate with the actin cytoskeleton (Berdnik and
Fig. 5. Ag ion-induced depolymerization of actin and p-H3S10. (A) Depolymerization of F-actin after a treatment with AgNO3. A549 cells were treated with AgNO3 (∼100 μM) for 0.5 h. G-actin was separated using hypertonic buffer and centrifugation, and detected by Western blotting. CBB staining was used as a standard for the equal loading of proteins for SDS-PAGE. (B) Depolymerization of F-actin after a treatment with AgNPs in the presence of an Ag ion inhibitor. A549 cells were treated with NAC (20 mM) for 0.5 h and then treated with AgNPs (∼1 mg/mL) for 1 h. (C) p-H3S10 after a treatment with AgNO3 in the presence of actin polymerization inhibitors. A549 cells were treated with LatB (5 μM) or CCB (20 μM) for 0.5 h and then treated with AgNO3 (∼50 μM) for 1 h.
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Fig. 6. p-H3S10 and p-AURKs in cofilin 1-knockdown cells. (A) Expression of cofilin 1 mRNA in cells knocked down using siRNA. A549 cells were transfected with siRNA (oligo ID: HSS141560; HSS141561 and HSS173851) or negative control siRNA as described in the experimental procedures. Fold changes were normalized to GAPDH levels and the ratio to untreated samples was calculated. Stealth siRNA oligo ID: HSS141561 was used in subsequent experiments. (B) p-H3S10 and G-actin in cofilin 1-knockdown cells after a treatment with AgNPs or AgNO3. Cells in which cofilin 1 was knocked down using siRNA were treated with AgNPs (1 mg/mL) for 1 h or AgNO3 (50 μM) for 0.5 h. G-actin was separated using hypertonic buffer and centrifugation. CBB staining was used as a standard for the equal loading of proteins for SDS-PAGE. Con: control; siC: control siRNA; siR: cofilin 1 siRNA. (C) Intercellular uptake of AgNPs in cofilin 1-knockdown cells after a treatment with AgNPs or AgNO3. Cells in which cofilin 1 was knocked down using siRNA were treated with AgNPs (1 mg/mL) for 1 h or AgNO3 (50 μM) for 0.5 h. Cells were analyzed using SS in FCM. (D) p-AURKs in cofilin 1-knockdown cells after a treatment with AgNPs or AgNO3. Cells in which cofilin 1 was knocked down using siRNA were treated with AgNPs (1 mg/mL) for 1 h or AgNO3 (50 μM) for 0.5 h.
The interaction of metal ions with the cytoskeleton has been extensively studied (DalleDonne et al., 1999; Fagotti et al., 1996; Li et al., 1993; Macdonald et al., 1987). Aluminum ions compete with magnesium ions at GTP-binding sites on tubulin, and, thus, affect microtubule polymerization (Macdonald et al., 1987). Nickel ions alter the conformation of G-actin, which may affect dynamic changes in actin filament polymerization or depolymerization (DalleDonne et al., 1999). Another mechanism involves the nickel-mediated oxidation of SH groups in cytoskeletal proteins as a result of intracellular glutathione (GSH) depletion and an increase in ROS. Decreases in the number of SH groups and the formation of disulphide bonds may promote actin filament aggregation (Li et al., 1993). Ag ions may also bind to SH groups (Singh and Ramarao, 2012; Zhao et al., 2016). Exposure to copper may interfere with the tyrosine phosphorylation of actin molecules, which is one of the mechanisms underlying actin filament depolymerization (Fagotti et al., 1996). Further studies are needed in order to clarify the mechanisms responsible for Ag ion-induced changes in the actin cytoskeleton. In the present study, we used one kind of AgNPs. Several parameters such as particle sizes, surface charges and shapes affect the uptake of AgNPs and release of Ag ions (Powers et al., 2011; Tak et al., 2015), which may change the AgNPs-induced p-H3S10 and its mechanism. In Fig. 3, AgNP-induced p-H3S10 was suppressed by the inhibitors of actin polymerization (LatB or CBB) and depolymerization (Pha), and LatB and CBB exerted stronger inhibitory effects than Pha. Which process, actin polymerization or depolymerization, is more important for AgNP-induced p-H3S10, is under consideration.
for endocytosis, phagocytosis, and cell movement (Castellano et al., 2001; Deschamps et al., 2013; Jeng and Welch, 2001). In phagocytosis, deformation of the plasma membrane relies on transient changes in Factin and the microtubule cytoskeleton (Deschamps et al., 2013). Specific F-actin ring-like structures are formed by the internalization of particles, which is controlled by the polymerization of G-actin (Castellano et al., 2001; Jeng and Welch, 2001). F-actin is rapidly depolymerized immediately after the uptake of particles has begun (Kuhn et al., 2014). AgNPs induced the depolymerization of F-actin and polymerization of G-actin, and AgNP-induced p-AURKB and p-H3S10 levels were suppressed by actin inhibitors. The knockdown of cofilin 1 decreased the amount of p-H3S10 and p-AURKs as well as the release of G-actin (Fig. 6B and D). Therefore, we first considered dynamic changes in actin filaments induced by ‘the incorporation of AgNPs’ to occur upstream of p-AURKB and p-H3S10. On the other hand, we observed similar actin depolymerization after the treatment with AgNO3. AgNO3-induced p-H3S10 was inhibited by actin inhibitors and the AgNP-induced depolymerization of F-actin was inhibited by the depletion of Ag ions. These results indicate that the trigger for p-H3S10 is a change in actin filaments caused by Ag ions released from AgNPs incorporated into inner cells, and not the process of the incorporation of AgNPs. The amount of Ag ions released from AgNPs (1 mg/mL) was 0.024 μg/mL (0.22 μM) during incubation for 0.5 h (Zhao et al., 2013). The concentrations of AgNO3 which remarkably induced p-H3S10 were 5–50 μM (Fig. 5C) and AgNO3-induced pH3S10 was transient (Zhao and Ibuki, 2015). Since p-H3S10 is dependent on the uptake of AgNPs, the persistent release of Ag ions from AgNPs incorporated into inner cells, not outer cells is important. 45
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Hayashi-Takanaka, Y., Yamagata, K., Nozaki, N., Kimura, H., 2009. Visualizing histone modifications in living cells: spatiotemporal dynamics of H3 phosphorylation during interphase. J. Cell Biol. 187, 781–790. Hendzel, M.J., Wei, Y., Mancini, M.A., Van Hooser, A., Ranalli, T., Brinkley, B.R., BazettJones, D.P., Allis, C.D., 1997. Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106, 348–360. Hirota, T., Lipp, J.J., Toh, B.H., Peters, J.M., 2005. Histone H3 serine 10 phosphorylation by Aurora B causes HP1 dissociation from heterochromatin. Nature 438, 1176–1180. Hsu, J.Y., Sun, Z.W., Li, X., Reuben, M., Tatchell, K., Bishop, D.K., Grushcow, J.M., Brame, C.J., Caldwell, J.A., Hunt, D.F., Lin, R., Smith, M.M., Allis, C.D., 2000. Mitotic phosphorylation of histone H3 is governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes. Cell 102, 279–291. Ibuki, Y., Toyooka, T., Zhao, X., Yoshida, I., 2014. Cigarette sidestream smoke induces histone H3 phosphorylation via JNK and PI3K/Akt pathways, leading to the expression of proto-oncogenes. Carcinogenesis 35, 1228–1237. Jeng, R.L., Welch, M.D., 2001. Cytoskeleton: actin and endocytosis – no longer the weakest link. Curr. Biol. 11, R691–R694. Jiang, X., Foldbjerg, R., Miclaus, T., Wang, L., Singh, R., Hayashi, Y., Sutherland, D., Chen, C., Autrup, H., Beer, C., 2013. Multi-platform genotoxicity analysis of silver nanoparticles in the model cell line CHO-K1. Toxicol. Lett. 222, 55–63. Ke, Q., Li, Q., Ellen, T.P., Sun, H., Costa, M., 2008. Nickel compounds induce phosphorylation of histone H3 at serine 10 by activating JNK-MAPK pathway. Carcinogenesis 29, 1276–1281. Keum, Y.S., Kim, H.G., Bode, A.M., Surh, Y.J., Dong, Z., 2013. UVB-induced COX-2 expression requires histone H3 phosphorylation at Ser10 and Ser28. Oncogene 32, 444–452. Kim, H.G., Lee, K.W., Cho, Y.Y., Kang, N.J., Oh, S.M., Bode, A.M., Dong, Z., 2008. Mitogen- and stress-activated kinase 1-mediated histone H3 phosphorylation is crucial for cell transformation. Cancer Res. 68, 2538–2547. Kim, S., Choi, J.E., Choi, J., Chung, K.H., Park, K., Yi, J., Ryu, D.Y., 2009. Oxidative stressdependent toxicity of silver nanoparticles in human hepatoma cells. Toxicol. In Vitro 23, 1076–1084. Kuhn, D.A., Vanhecke, D., Michen, B., Blank, F., Gehr, P., Petri-Fink, A., RothenRutishauser, B., 2014. Different endocytotic uptake mechanisms for nanoparticles in epithelial cells and macrophages. Beilstein J. Nanotechnol. 5, 1625–1636. Le, L.T., Vu, H.L., Nguyen, C.H., Molla, A., 2013. Basal aurora kinase B activity is sufficient for histone H3 phosphorylation in prophase. Biol. Open 2, 379–386. Lee, S.H., Dominguez, R., 2010. Regulation of actin cytoskeleton dynamics in cells. Mol. Cells 29, 311–325. Li, J., Gorospe, M., Barnes, J., Liu, Y., 2003. Tumor promoter arsenite stimulates histone H3 phosphoacetylation of proto-oncogenes c-fos and c-jun chromatin in human diploid fibroblasts. J. Biol. Chem. 278, 13183–13191. Li, W., Zhao, Y., Chou, I.N., 1993. Alterations in cytoskeletal protein sulfhydryls and cellular glutathione in cultured cells exposed to cadmium and nickel ions. Toxicology 77, 65–79. Macdonald, T.L., Humphreys, W.G., Martin, R.B., 1987. Promotion of tubulin assembly by aluminum ion in vitro. Science 236, 183–186. Nigg, E.A., 2001. Mitotic kinases as regulators of cell division and its checkpoints. Nat. Rev. Mol. Cell Biol. 2, 21–32. Nowack, B., Krug, H.F., Height, M., 2011. 120 years of nanosilver history: implications for policy makers. Environ. Sci. Technol. 45, 1177–1183. Panariti, A., Miserocchi, G., Rivolta, I., 2012. The effect of nanoparticle uptake on cellular behavior: disrupting or enabling functions? Nanotechnol. Sci. Appl. 5, 87–100. Powers, C.M., Badireddy, A.R., Ryde, I.T., Seidler, F.J., Slotkin, T.A., 2011. Silver nanoparticles compromise neurodevelopment in PC12 cells: critical contributions of silver ion, particle size, coating, and composition. Environ. Health Perspect. 119, 37–44. Ritchey, L., Ottman, R., Roumanos, M., Chakrabarti, R., 2012. A functional cooperativity between Aurora A kinase and LIM kinase1: implication in the mitotic process. Cell Cycle 11, 296–309. Sabbattini, P., Canzonetta, C., Sjoberg, M., Nikic, S., Georgiou, A., Kemball-Cook, G., Auner, H.W., Dillon, N., 2007. A novel role for the Aurora B kinase in epigenetic marking of silent chromatin in differentiated postmitotic cells. EMBO J. 26, 4657–4669. Singh, R.P., Ramarao, P., 2012. Cellular uptake, intracellular trafficking and cytotoxicity of silver nanoparticles. Toxicol. Lett. 213, 249–259. Suzuki, H., Toyooka, T., Ibuki, Y., 2007. Simple and easy method to evaluate uptake potential of nanoparticles in mammalian cells using a flow cytometric light scatter analysis. Environ. Sci. Technol. 41, 3018–3024. Tak, Y.K., Pal, S., Naoghare, P.K., Rangasamy, S., Song, J.M., 2015. Shape-dependent skin penetration of silver nanoparticles: does it really matter? Sci. Rep. 5, 16908. Tardáguila, M., González-Gugel, E., Sánchez-Pacheco, A., 2011. Aurora kinase B activity is modulated by thyroid hormone during transcriptional activation of pituitary genes. Mol. Endocrinol. 25, 385–393. Toduka, Y., Toyooka, T., Ibuki, Y., 2012. Flow cytometric evaluation of nanoparticles using side-scattered light and reactive oxygen species-mediated fluorescencecorrelation with genotoxicity. Environ. Sci. Technol. 46, 7629–7636. Wang, L.H., Xiang, J., Yan, M., Zhang, Y., Zhao, Y., Yue, C.F., Xu, J., Zheng, F.M., Chen, J.N., Kang, Z., Chen, T.S., Xing, D., Liu, Q., 2010. The mitotic kinase Aurora-A induces mammary cell migration and breast cancer metastasis by activating the Cofilin-F-actin pathway. Cancer Res. 70, 9118–9128. Washington, D.C., 2011. The Silver Institute. The Future Demand of Silver Industrial Demand. The Silver Institute. http://www.silverinstitute.org/images/stories/silver/ PDF/futuresilverindustrialdemand.pdf.
5. Conclusion The present study described a possible pathway for the induction of p-H3S10 by AgNPs (Supplementary Figure 2). AgNPs incorporated into cells released Ag ions, which changed the actin polymerization cycle. The dynamic change in actin filaments activated AURKs and induced pH3S10 independent of the cell cycle. We need to study the mechanisms responsible and the meaning of the phosphorylation of H3S10 in more detail. Conflict of interest The authors of this manuscript have no conflict of interest to disclose. Transparency document The http://Transparency%20document(http://dx.doi.org/10.1016/ j.toxlet.2017.05.009) associated with this article can be found in the online version. Acknowledgments This work was supported in part by a Grant-in-Aid for Research on Risk of Chemical Substances from the Ministry of Health, Labour and Welfare, Japan, and JSPS KAKENHI 15H02828. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.toxlet.2017.05.009. References Adams, R.R., Maiato, H., Earnshaw, W.C., Carmena, M., 2001. Essential roles of Drosophila inner centromere protein (INCENP) and aurora B in histone H3 phosphorylation, metaphase chromosome alignment, kinetochore disjunction, and chromosome segregation. J. Cell Biol. 153, 865–880. Ajiro, K., Nishimoto, T., 1985. Specific site of histone H3 phosphorylation related to the maintenance of premature chromosome condensation. Evidence for catalytically induced interchange of the subunits. J. Biol. Chem. 260, 15379–15381. Berdnik, D., Knoblich, J.A., 2002. Drosophila Aurora-A is required for centrosome maturation and actin-dependent asymmetric protein localization during mitosis. Curr. Biol. 12, 640–647. Bernard, O., 2007. Lim kinases, regulators of actin dynamics. Int. J. Biochem. Cell Biol. 39, 1071–1076. Carmena, M., Earnshaw, W.C., 2003. The cellular geography of aurora kinases. Nat. Rev. Mol. Cell Biol. 4, 842–854. Castellano, F., Chavrier, P., Caron, E., 2001. Actin dynamics during phagocytosis. Semin. Immunol. 13, 347–355. Choi, H.S., Choi, B.Y., Cho, Y.Y., Mizuno, H., Kang, B.S., Bode, A.M., Dong, Z., 2005. Phosphorylation of histone H3 at serine 10 is indispensable for neoplastic cell transformation. Cancer Res. 65, 5818–5827. Cooper, J.A., 1987. Effects of cytochalasin and phalloidin on actin. J. Cell Biol. 105, 1473–1478. Crosio, C., Fimia, G.M., Loury, R., Kimura, M., Okano, Y., Zhou, H., Sen, S., Allis, C.D., Sassone-Corsi, P., 2002. Mitotic phosphorylation of histone H3: spatio-temporal regulation by mammalian Aurora kinases. Mol. Cell. Biol. 22, 874–885. DalleDonne, I., Milzani, A., Ciapparelli, C., Comazzi, M., Gioria, M.R., Colombo, R., 1999. The assembly of Ni2+-actin: some peculiarities. Biochim. Biophys. Acta 1426, 32–42. Deschamps, C., Echard, A., Niedergang, F., 2013. Phagocytosis and cytokinesis: do cells use common tools to cut and to eat? Highlights on common themes and differences. Traffic 14, 355–364. Fagotti, A., Di Rosa, I., Simoncelli, F., Pipe, R.K., Panara, F., Pascolini, R., 1996. The effects of copper on actin and fibronectin organization in Mytilus galloprovincialis haemocytes. Dev. Comp. Immunol. 20, 383–391. Fischle, W., Tseng, B.S., Dormann, H.L., Ueberheide, B.M., Garcia, B.A., Shabanowitz, J., Hunt, D.F., Funabiki, H., Allis, C.D., 2005. Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation. Nature 438, 1116–1122. Giet, R., Glover, D.M., 2001. Drosophila aurora B kinase is required for histone H3 phosphorylation and condensin recruitment during chromosome condensation and to organize the central spindle during cytokinesis. J. Cell Biol. 152, 669–682. Guo, H., Zhang, J., Boudreau, M., Meng, J., Yin, J.J., Liu, J., Xu, H., 2016. Intravenous administration of silver nanoparticles causes organ toxicity through intracellular ROS-related loss of inter-endothelial junction. Part. Fibre Toxicol. 13, 21.
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X. Zhao et al.
Zhao, X., Ibuki, Y., 2015. Evaluating the toxicity of silver nanoparticles by detecting phosphorylation of histone H3 in combination with flow cytometry side-scattered light. Environ. Sci. Technol. 49, 5003–5012. Zhao, X., Takabayashi, F., Ibuki, Y., 2016. Coexposure to silver nanoparticles and ultraviolet A synergistically enhances the phosphorylation of histone H2AX. J. Photochem. Photobiol. B 162, 213–222. Zhao, X., Toyooka, T., Ibuki, Y., 2013. Synergistic bactericidal effect by combined exposure to Ag nanoparticles and UVA. Sci. Total Environ. 458–460, 54–62.
Wei, Y., Yu, L., Bowen, J., Gorovsky, M.A., Allis, C.D., 1999. Phosphorylation of histone H3 is required for proper chromosome condensation and segregation. Cell 97, 99–109. Yang, J., Zappacosta, F., Annan, R.S., Nurse, K., Tummino, P.J., Copeland, R.A., Lai, Z., 2009. The catalytic role of INCENP in Aurora B activation and the kinetic mechanism of Aurora B/INCENP. Biochem. J. 417, 355–360. Yasui, Y., Urano, T., Kawajiri, A., Nagata, K., Tatsuka, M., Saya, H., Furukawa, K., Takahashi, T., Izawa, I., Inagaki, M., 2004. Autophosphorylation of a newly identified site of Aurora-B is indispensable for cytokinesis. J. Biol. Chem. 279, 12997–13003.
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