Hyperphosphorylation of intermediate filament proteins is involved in microcystin-LR-induced toxicity in HL7702 cells

Toxicology Letters 214 (2012) 192–199

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Hyperphosphorylation of intermediate filament proteins is involved in microcystin-LR-induced toxicity in HL7702 cells Dong-ni Chen a,1 , Jing Zeng a,1 , Feng Wang b , Wei Zheng a , Wei-wei Tu a , Jin-shun Zhao a , Jin Xu a,∗ a b

Department of Preventive Medicine, School of Medicine, Ningbo University, Ningbo 315211, China Clinical Laboratory, Lihuili Hospital, Ningbo 315040, China

h i g h l i g h t s    

Microcystin-LR triggers accumulation of IFs around the nucleus and formation of dense bundles. MC-LR does not change the mRNA and protein levels of IF proteins. MC-LR causes hyperphosphorylation of K8/18 and vimentin. Hyperphosphorylation of IF proteins is regulated through MAPK pathway.

a r t i c l e

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Article history: Received 4 June 2012 Received in revised form 24 August 2012 Accepted 25 August 2012 Available online 1 September 2012 Keywords: Microcystin-LR Intermediate filament Keratin MAPK Hyperphosphorylation

a b s t r a c t Microcystin-LR (MC-LR) is commonly characterized as a hepatotoxin, which can cause disruption of keratin filaments. Keratins, however, account for only two types of intermediate filaments (IFs), and the potential involvement of other IF proteins in MC-LR-induced toxicity and the underlying mechanisms are still unclear. In this study, the human normal liver cell line HL7702 was used to investigate whether MC-LR can change the transcription, translation, and phosphorylation levels of major IF proteins and to elucidate the underlying mechanisms. The results showed that MC-LR triggered an accumulation of IFs around the nucleus and led to the formation of dense bundles. When the cells were treated with 10 ␮M MC-LR, cell proliferation significantly decreased with an increase in apoptosis and cell cycle arrest. Moreover, the mRNA and protein levels of keratin 18, vimentin and lamin A/C were not changed; however, the phosphorylation of K8/18 and vimentin was significantly increased. Furthermore, we found MC-LR exposure caused phosphoactivation of P38, JNK and ERK1/2 in a concentration-dependent manner, and P38 and ERK1/2 were involved in MC-LR-induced hyperphosphorylation of IF proteins. Taken together, the results of this study suggest that MC-LR exerts its potential hepatotoxicity through MAPK pathway activation, which cause hyperphosphorylation of IF proteins and result in cytoskeletal architecture remodeling and cell survival/death regulation. Since IFs serve as signaling platforms and dozens of IF proteins are involved in different signaling pathways, future studies focus on different IFs may provide helpful insights into the mechanisms of MC-LR toxicity. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Outbreaks of cyanobacterial (especially Microcystin aeruginosa) blooms caused by eutrophication have been a worldwide threat to aquatic ecosystems and human health (Dorr et al., 2010). Microcystins (MCs) are a family of monocyclic nonribosomal peptides that are produced by cyanobacteria, and microcystinLR (MC-LR), which is one of over 80 known toxic variants, is the most toxic and common member of the microcystin family.

∗ Corresponding author. Tel.: +86 574 87609603; fax: +86 574 87608638. E-mail address: [email protected] (J. Xu). 1 These authors contributed equally to this article. 0378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2012.08.024

Tritium-labeled MC-LR has been used to show that the liver is the prime target organ affected (Robinson et al., 1989), and in vivo studies have confirmed that hepatocytes are the target cells due to specific uptake of MC-LR via an organic anion transporter (Eriksson et al., 1990). Chronic exposure to a lower concentration of MC-LR has frequently been reported to cause acute liver hemorrhage, hepatic insufficiency and primary liver cancer (Nishiwaki-Matsushima et al., 1992; Runnegar et al., 1987; Svircev et al., 2010; Yoshida et al., 1998). Thus, MC-LR is thought to be a hepatotoxin. The cytoskeleton is a well-organized network of protein polymers that extends throughout the cytoplasm. The cytoskeleton not only provides cellular architecture but also plays a central role in signal transduction (Wickstead and Gull, 2011). Previous studies

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have indicated that MC-LR may exert hepatotoxic effects by causing cytoskeletal disruption (Wickstrom et al., 1995). Interestingly, an in vitro study of primary human hepatocytes revealed that MC-LR caused the actin mesh to collapse into the center of the cell (Batista et al., 2003), and similar results have been reported for primary rat hepatocytes (Ghosh et al., 1995). The typical filamentous pattern of microtubule (MT) distribution in rat hepatocytes has been shown to be disrupted after treatment with MC-LR. Indeed, one study showed that MTs appeared to retract from cell margins and progressively collapse toward the interior of affected cells (Wickstrom et al., 1995). Intermediate filaments (IFs) were also affected by MCLR through progressive perinuclear aggregation (Ohta et al., 1992; Wickstrom et al., 1995). Among the three cytoskeletal fibers, IFs are the most diverse and are composed of more than 70 unique gene products that make up a highly dynamic family of disease-associated cytoskeletal components (Omary et al., 2006; Pallari and Eriksson, 2006). IFs are classified into six major classes, which are expressed in cell-, tissue-, differentiation- and developmental-specific patterns (Eriksson et al., 2009; Godsel et al., 2008). In most vertebrate cells, cytoplasmic IFs are tethered to the nucleus and extend into the cytoplasm to form a scaffold for organelles and other cytoskeletal elements (Kim and Coulombe, 2007). Importantly, IF proteins are actively regulated by phosphorylation and other posttranslational modifications (Omary et al., 2006) and are involved in a wide range of metabolic, signaling, and regulatory processes (Pallari and Eriksson, 2006). The treatment of rat hepatocytes with MC-LR induced hyperphosphorylation of keratin 8/18 (K8/18) accompanied by increased keratin solubility and correlated with the observed morphological effects (Ohta et al., 1992; Toivola et al., 1997). Interestingly, a global quantitative analysis of the protein phosphorylation status in the livers of medaka fish showed variations in the phosphoryl content of keratin 18 after exposure of the fish to MC-LR (Mezhoud et al., 2008). Hyperphosphorylation of K8/18 may be involved in the toxicity of MC-LR through the inhibition of phosphatases. However, only K8/18 have been studied, and the roles of other IF proteins remain unknown. Furthermore, the related mechanisms and outcomes of hyperphosphorylation are still not clear. Therefore, this study aimed to investigate the transcription, translation, and phosphorylation levels of major IF proteins after treatment with MC-LR in the human liver cell line HL7702. Moreover, we investigated whether MAPK signaling is involved in the regulation of the MC-LRinduced hyperphosphorylation of IF proteins and the subsequent signaling pathways.

2. Materials and methods 2.1. Chemicals and reagents MC-LR was obtained from Alexis Biochemicals (Lausen, Switzerland). RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco (Scotland, UK). The RIPA cell lysis buffer, protein assay kit, and ECL chemiluminescence kit were purchased from Beyotime (Beijing, China). Anti-keratin 18 (ser33), antikeratin18 (ser52), and anti-keratin8 (ser74) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from KangChen Biotech (Shanghai, China). The other primary antibodies and horseradish peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). An Alexa Fluor 488-conjugated annexin V/PI apoptosis kit was obtained from Invitrogen (Eugene, USA). FITC-conjugated phalloidin, 4 ,6-diamidino-2-phenylindole (DAPI), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). Protease inhibitor cocktail tablets were purchased from Roche (Indianapolis, IN, USA). An RNA extraction kit and SYBR Green PCR master mix were purchased from Takara (Dalian, China). The P38-MAPK inhibitor SB203580 and JNK inhibitor SP600125 were purchased from Calbiochem (La Jolla, CA, USA). The ERK1/2 inhibitor U0126 was purchased from Cell Signaling Technology (Beverly, MA, USA).

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2.2. Cell culture and MC-LR exposure The human normal liver cell line HL7702 was obtained from the Cell Bank of the Chinese Academy of Science and cultured in RPMI 1640 medium supplemented with 10% FBS. The cells were incubated at 37 ◦ C in a humidified atmosphere containing 5% CO2 . After incubation for 24–36 h, the cells were exposed to 0.001–10 ␮M MC-LR for 24 h, and the control cells were treated with the same volume of Millipore water instead of MC-LR. For MAPK inhibition study, the cells were pretreated with MAPK inhibitors (25 ␮M SB203580 or 10 ␮M SP600125 for 12 h, 10 ␮M U0126 FOR 2 h) before MC-LR exposure. 2.3. MTT assay Cell proliferation was determined by an MTT assay (Mosmann, 1983). Approximately 2 × 103 cells were plated in each well of a 96-well plate and were allowed to attach to the plate for 24 h. The cells were exposed to 0, 0.001, 0.01, 0.1, 1 or 10 ␮M MC-LR for another 24 h. The exposure medium was subsequently removed, and 20 ␮L of 5 mg/mL MTT [dissolved in phosphate-buffered saline (PBS)] and 180 ␮L of fresh medium were added to each well and incubated at 37 ◦ C for an additional 4 h. After the medium was removed, the purple formazan crystals were dissolved in 150 ␮L of DMSO. The absorbance of each well was measured at 490 nm with a Thermo microplate reader. The reduction in cell proliferation was expressed as a percent of the nontreated cells. 2.4. Cell cycle analysis Approximately 1 × 105 cells were plated in each well of a 6-well plate and were allowed to attach to the plate for 24 h. After treatment with MC-LR for 24 h, the cells were washed twice with PBS and permeabilized with 70% ethanol in PBS overnight at 4 ◦ C. The cells were then incubated with 50 ␮g/mL PI solution containing 20 U/mL RNase A for 30 min and analyzed by BD flow cytometry. 2.5. Apoptosis analysis Apoptosis was detected with the annexin V-FITC/PI staining kit. Approximately 1 × 105 cells were plated in each well of a 6-well plate and were allowed to attach to the plate for 24 h. After treatment with MC-LR for 24 h, the cells were harvested and washed with PBS three times. The cells were then incubated with annexin V-FITC and PI in the dark for 15 min prior to analysis by BD flow cytometry. The cells that were positive for annexin V and negative for PI were considered to be early apoptotic cells. 2.6. Real-time PCR Approximately 1 × 105 cells were plated in 25 cm2 flasks and were allowed to attach to the bottom for 24 h. After treatment with MC-LR for 24 h, the cells were harvested. Total RNA was extracted with the RNA extraction kit and subsequently used to synthesize cDNA according to the manufacturer’s instructions. Real-time PCR was performed using SYBR Green PCR master mix with Roche LightCycler 480 real-time PCR. The comparative threshold cycle method was used to quantify the data using GAPDH as the normalization gene. 2.7. Protein extraction Approximately 1 × 105 cells were plated in 25 cm2 flasks and were allowed to attach to the bottom for 24 h. After MC-LR exposure for 24 h, the cells were washed twice with PBS. The cells were collected by scraping, and a cell pellet was obtained by centrifugation in microtubes. The samples were lysed in RIPA lysis buffer supplemented with protease inhibitor cocktail for 45 min on ice. The lysates were centrifuged, and the supernatant was carefully saved and stored at −80 ◦ C. The protein concentrations of the samples were determined by the Bradford method (Bradford, 1976) using a kit. 2.8. Western blotting analysis Aliquots containing 50 ␮g of protein were electrophoresed on an SDS-PAGE gel and transferred to a nitrocellulose membrane for 120 min. After the membrane was blocked with 5% nonfat dry milk for 3 h at room temperature, primary antibodies [p-ERK1/2 1:1000, p-JNK 1:500, p-P38 MAPK 1:1000, ERK1/2 1:1000, JNK 1:1000, P38 MAPK 1:1000, p-keratin18 (ser33) 1:200, p-keratin18 (ser52) 1:200, p-keratin8 (ser74) 1:200, pan-keratin 1:1000, keratin 8/18 1:1000, vimentin 1:1000, p-vimentin (ser56) 1:1000, p-vimentin (ser82) 1:1000, lamin A/C 1:1000, and p-lamin A/C(ser22) 1:1000] were added and incubated at 4 ◦ C overnight. The membranes were washed three times for 10 min each in 15 mL of Tris-Buffered Saline and Tween-20 (TBST) (50 mM Tris–HCl, 150 mM NaCl, pH 7.6, 0.1% Tween20) and incubated with the corresponding HRP-conjugated secondary antibodies (1:5000) for 2 h at room temperature. The membranes were washed three times in TBST and covered with ECL chemiluminescence reagents. The blots were exposed to X-ray film for detection of the bands. The autoradiograms were scanned, and the protein bands were quantified by Quantity One software (Bio-Rad). To verify equal

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1.2

70

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0.8 0.6 0.4 0.2 0

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80

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60 50

G0-G1

40

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20 10 0

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Fig. 1. MC-LR inhibits cell proliferation. HL7702 cells were exposed to different concentrations of MC-LR for 24 h, and cell proliferation was assessed by an MTT assay. The results are expressed as the mean ± SD from four independent experiments. The asterisks represent statistical significance (*P < 0.05 and **P < 0.01) compared with the control value. protein loading and transfer, the blots were probed for GAPDH using an anti-GAPDH antibody (1:5000). 2.9. Immunofluorescence Approximately 1 × 105 cells were plated in each well of a 6-well plate and were allowed to attach to the plate for 24 h. After MC-LR exposure for 24 h, the cells were washed twice with PBS, fixed in a 3.7% formaldehyde solution in PBS for 15 min at room temperature and blocked for 30 min in blocking buffer (PBS supplemented with 10% FBS). For visualization of the distribution of the IFs, anti-pan-keratin antibody (1:50) was added to fresh blocking buffer containing 0.1% saponin. The cells were incubated with anti-pan-keratin at 4 ◦ C overnight prior to incubation with Alexa Fluor 488-conjugated secondary antibody (1:100) for 1 h at room temperature in the dark. The nuclei were stained with 1 ␮g/mL DAPI for 10 min at room temperature. The images were acquired by an Olympus laser-scanning confocal microscope and analyzed using the Olympus Fluoview Viewer system. 2.10. Statistical analysis The data are presented as the mean ± SD from at least three sets of independent experiments. Statistical analyses were performed using SPSS (Statistical Package for the Social Sciences) 16.0 software. One-way analysis of variance (ANOVA) was used to analyze the differences between groups. A significant difference was considered to be P < 0.05.

0

0

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0.01 0.1 MC-LR (μM)

1

10

Fig. 2. MC-LR regulates cell cycle progression. After treatment with MC-LR for 24 h, the cell cycle stages were assessed by PI staining. The results are expressed as the mean ± SD from three independent experiments. The asterisks represent statistical significance (*P < 0.05 and **P < 0.01) compared with the control value.

cells in the G0–G1 phase significantly increased, whereas the number of cells in the S phase decreased (P < 0.05). Taken together with the cell proliferation assay, the results indicated that HL7702 cells that are exposed to a high concentration of MC-LR are prevented from proceeding past the G0–G1 phase. The cells subsequently stop growing and undergo apoptosis. The effects are not strong under current conditions, but when the concentration of MC-LR or exposure time increased, these effects may be more significant (Gan et al., 2010; Takumi et al., 2010). 3.3. MC-LR induces reorganization of IFs We further examined the effect of MC-LR on the structure and distribution of IFs using a pan-keratin antibody. In the control cells, the IFs were distributed in a regular filamentous pattern (Fig. 4). Treatment with MC-LR did not result in any alterations of cell shape or size. However, more IFs accumulated around the nucleus, and dense bundles were observed (Fig. 4), which implies that MC-LR induced the reorganization of IFs.

3. Results

3.4. qPCR analysis of mRNA levels of the IF family

3.1. MC-LR inhibits cell proliferation

After confirming that MC-LR could induce the reorganization of IFs, we further examined the mRNA levels of KRT8, KRT18, VIM, and LMNA, which belong to type I, II, III, and V of the IF family, respectively. As shown in Fig. 5, MC-LR did not change any of the

3.2. MC-LR regulates cell cycle progression and apoptosis Previous results have shown that MC-LR can induce apoptosis in rat hepatocytes and other cell lines via the mitochondrial apoptotic or nuclear factor ␬B (NF-␬B) pathways (Ding et al., 2000; Fu et al., 2004; Ji et al., 2011). After verification that MC-LR inhibited cell proliferation, the effects of MC-LR on the cell cycle and apoptosis were investigated. As shown in Figs. 2 and 3, treatment with 0.001–1 ␮M MC-LR for 24 h did not alter the cell cycle progression and apoptosis rate. When the concentration of MC-LR reached 10 ␮M, however, the apoptosis rate and the number of

4

*

3.5 Apoptosis rate (%)

To confirm the general toxicity of MC-LR in HL7702 cells, cell proliferation was measured by MTT assay. The proliferation of the control cells was designated as 100%, and the experimental groups were expressed as percentages of the control. After treatment with different concentrations of MC-LR (0, 0.001, 0.01, 0.1, 1 and 10 ␮M) for 24 h cell proliferation decreased in a concentration-dependent manner (Fig. 1). When exposed to 1 ␮M or higher concentration of MC-LR, cell proliferation significantly decreased (P < 0.05). Thus, 0.001–10 ␮M MC-LR were used in the following experiments.

3 2.5 2 1.5 1 0.5 0

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Fig. 3. MC-LR induces apoptosis in HL7702 cells. After treatment with MC-LR for 24 h, apoptosis was measured by annexin V/PI staining. The results are expressed as the mean ± SD from three independent experiments. The asterisk represent statistical significance (P < 0.05) compared with the control value.

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Fig. 4. The effects of MC-LR on intermediate filaments. HL7702 cells were exposed to different concentrations of MC-LR for 24 h, and intermediate filaments were stained with anti-pan-keratin antibody followed by a fluorescence-conjugated secondary antibody. For each sample, five randomly selected fields were visualized by confocal laser microscopy, and three independent replications were conducted. The arrows point to the obvious dense bundles. 2

mRNA levels, which indicated that the transcription of IF genes was not involved in MC-LR-induced toxicity under current study conditions.

1.8 1.6

We also investigated whether MC-LR could change the protein levels of the IF family proteins. As shown in Fig. 6, the protein levels assessed by pan-keratin, K18, vimentin, lamin A/C antibodies remained unchanged after MC-LR treatment. These results were consistent with the qPCR analysis and indicate that MC-LR does not induce toxic effects via alterations in the expression of IF family proteins. However, the K8 protein level decreased when the concentration of MC-LR reached 0.1 ␮M and above, which was inconsistent with qPCR analysis. This discrepancy may due to the effects on protein synthesis or degradation, since MC-LR can affect a cluster of proteins which are involved in protein degradation (Fu et al., 2009).

Relative fold

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3.5. Immunoblotting analysis of IF family protein levels

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KRT8

1

KRT18

0.8

VIM

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Fig. 5. MC-LR does not change the mRNA levels of the IF family members. After treatment with MC-LR for 24 h, the mRNA levels of the IF family members were measured by qPCR. The results are expressed as the mean ± SD from three independent experiments.

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Fig. 6. MC-LR upregulates the phosphorylation of IF proteins. After treatment with MC-LR for 24 h, the protein and phosphorylation levels of the IF family proteins were measured by western blotting. (A) Representative autoradiographs of IF proteins and their phosphorylation levels. (B)–(D) The intensity of each band was quantified by densitometry, and the data were normalized using the GAPDH signal. The protein expression from the control group was designated as 1, and the other groups were expressed as fold changes compared with the control. The results are expressed as the mean ± SD from three independent experiments. The asterisks represent statistical significance (P < 0.05) compared with the control value.

3.6. MC-LR induces hyperphosphorylation of intermediate filament proteins Because MC-LR did not change the mRNA or protein levels (except K8) of the IF family members, we investigated the phosphorylation levels of these IF proteins. Interestingly, treatment with MC-LR for 24 h significantly increased the phosphorylation levels of K8/18 and vimentin, however the p-lamin A/C remained unchanged

(Fig. 6). The p-K18 (ser52) level increased in a concentrationdependent manner, and the 10 ␮M group showed a 2.6-fold increase compared with the control group. 3.7. The MAPK pathway was activated after MC-LR exposure MC-LR has been shown to activate the MAPK pathway in OATP1B3-transfected HEK293 (Komatsu et al., 2007) and HeLa cells

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Fig. 7. MC-LR induces phosphoactivation of MAPKs. After treatment with MC-LR for 24 h, the protein levels and phosphorylation levels of MAPKs were measured by western blotting. (A) Representative autoradiographs of MAPKs and their phosphorylation levels. (B) The values were obtained by comparing the phosphorylated bands with the total protein bands. The results are expressed as the mean ± SD from three independent experiments. The asterisks represent statistical significance (P < 0.05) compared with the control value.

(Daily et al., 2010). The activation of the proteins in the MAPK superfamily was measured by immunoblot analysis. Consistent with previous studies (Meng et al., 2011; Sun et al., 2011), total ERK1/2, JNK, and P38 remained unchanged; however, phosphorylated ERK1/2, JNK and P38 were markedly upregulated by MC-LR treatment in a concentration-dependent manner (Fig. 7). 3.8. The MAPK pathway was involved in MC-LR-induced hyperphosphorylation of IF proteins Since MAPK pathway was activated by MC-LR, we further investigated the role of the MAPKs in the MC-LR-induced hyperphosphorylation of IF proteins in HL7702 cells. Pretreatment with P38 or ERK1/2 inhibitor, hyperphosphorylation of K8/18 and vimentin were diminished to normal level. However, JNK inhibitor has no significant effect (Fig. 8). 4. Discussion A wealth of data has shown that MC-LR-induced damage to hepatocytes is caused by cytoskeletal disorganization, including cell blebbing, cellular disruption, loss of membrane integrity, and apoptosis (Fischer et al., 2000; Wickstrom et al., 1995). Moreover, the disruption or rearrangement of all three cytoskeletal fibers has been observed after MC-LR exposure (Batista et al., 2003; Ghosh et al., 1995; Ohta et al., 1992; Toivola et al., 1997; Wickstrom et al., 1995). Although the toxic effects of cyanobacteria-derived MC-LR on the cytoskeleton have been extensively studied in vivo and in vitro, there has been little focus on the underlying mechanisms. Recently, Sun et al. (2011) reported that MC-LR induces reorganization of actin filaments in HL7702 cells through hyperphosphorylation of HSP27 via PP2A inhibition. In the present study, we also found that extensive F-actin depolymerization and aggregation of actin fibers around the cell periphery after 24 h exposure

to 10 ␮M MC-LR (data not shown). Moreover, Meng et al. (2011) indicated that MC-LR induces microtubule reorganization through hyperphosphorylation of tau and HSP27 via PP2A inhibition in PC12 cells. These studies provide an important signaling pathway for MC-LR-induced cytoskeleton reassembly. According to a study by Wickstrom et al. (1995) in fibroblasts and hepatocytes, IFs appeared to be affected prior to microtubules and microfilaments, which implies that the initial result of MC-LR-induced phosphatase inhibition may be progressive perinuclear aggregation of IFs. In primary rat hepatocyte cultures, hyperphosphorylation of K8/18 was accompanied by increased keratin solubility, which led to formation of condensed IFs and microfilaments (Toivola et al., 1997). However, IFs are composed of more than 70 different IF proteins that belong to 6 subgroups. Keratin 18 and 8 are type I and type II IF proteins, respectively, and it is unclear whether other members are involved in MC-LR-induced IF reorganization. Fu et al. (2005, 2009) performed a proteomic analysis of the cellular response to MC-RR (another form of microcystin) in human amnion FL cells and identified dozens of cytoskeleton-associated proteins that responded to MC-RR exposure (primarily keratins and lamins). Because IF proteins have been proposed to play an important role in MC-LRinduced toxicity, we studied the expression and phosphorylation of different types of IF proteins and the underlying mechanisms. We initially confirmed that MC-LR exposure could cause IFs reorganization around the nucleus and the formation of dense bundles. However, neither the mRNA nor protein levels of the IF family members were changed after MC-LR exposure, which implies that MC-LR does not induce cytoskeleton disruption via transcriptional or translational effects on IF family members. Thus, we assumed that the most possible mechanism is posttranslational modification, especially hyperphosphorylation of IF proteins. In the present study, the phosphorylated forms of keratin 8 (ser74), keratin 18 (ser52) and vimentin (ser56) were upregulated, however, keratin 18 (ser33) and lamin A/C (ser22) remained unchanged. Thus, we

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Fig. 8. Effects of MAPK inhibitors on MC-LR-induced IF phosphorylation. The cells were pre-incubated with MAPK inhibitors (25 ␮M SB203580 or 10 ␮M SP600125 for 12 h, 10 ␮M U0126 FOR 2 h) and then exposed with 10 ␮M MC-LR for 24 h (1 ␮M MC-LR for p-vimentin). The control was not exposed to MC-LR. (A) Representative autoradiographs of phosphorylated IF proteins. (B) The results are expressed as the mean ± SD from three independent experiments. The asterisks represent statistical significance (P < 0.05) compared with the values from MC-LR treated group.

move on to studied the mechanism by which MC-LR upregulates IF protein phosphorylation in HL7702 cells. Recent studies have demonstrated that MC-LR affects MAPK signaling through PP2A inactivation (Kins et al., 2003; Komatsu et al., 2007). In addition, studies have shown that MAPKs phosphorylate keratins (Menon et al., 2010; Woll et al., 2007). Therefore, we speculated that MC-LR-induced PP2A inhibition, and MAPK activation may lead to a change in the phosphorylated pool of IF proteins. Previous studies have shown that hyperphosphorylation of the cytoskeletal-associated proteins HSP27 and tau in PC12 cells is caused by MC-LR-induced PP2A inhibition and subsequent activation of the P38 MAPK signaling pathway (Meng et al., 2011). Likewise, Sun et al. (2011) found a dose-dependent and timedependent inhibition of PP2A activity in MC-LR treated HL7702 cells, whereas the p-P38, p-JNK and p-ERK levels increased in a dose-dependent and time-dependent manner, which suggested that the phosphorylation of HSP27 is regulated by MAPKs as a consequence of PP2A inhibition. Consistent with the findings of Sun et al., the present study showed that P38, JNK and ERK levels were unchanged after MC-LR treatment; however, p-P38, p-JNK and p-ERK levels were all increased in a concentration-dependent manner. Furthermore, through specific MAPK inhibition study, we found P38 MAPK and ERK 1/2 were involved in MC-LR-induced hyperphosphorylation of K8/18 and vimentin. According to the evidence mentioned above, we suggest that hyperphosphorylation of IF proteins results from MC-LR-induced PP2A inhibition and subsequent activation of the MAPK pathway. Phosphorylation is the major posttranslational modification of keratins, and site-specific phosphorylation plays a key role in regulating keratin filament organization and signaling pathways (Coulombe and Omary, 2002; Omary et al., 1998). Keratins are preferentially phosphorylated on serine residues in the head and tail region during different forms of cellular stress (Omary et al., 2006), and keratin phosphorylation could have a protective effect on stress-induced hepatotoxic injury (Ku et al., 1998b; Ku and Omary, 2006). In addition, K8/18 phosphorylation may exert other functions in a site-specific fashion, including modulating cell cycle progression via binding to 14-3-3 proteins (Ku et al., 1998a, 2002) and effecting keratin protein turnover by ubiquitination or during apoptosis (Ku and Omary, 2000, 2001). Many IF-interacting proteins are enzymes and receptors that are involved in the apoptotic pathway (Coulombe and Wong, 2004). For example, the IFs formed by K8/18 interact directly with the cytoplasmic domain

of tumor necrosis factor receptor 2 (TNFR2) and modulate JNK signaling and NF-␬B activation, thereby regulate epithelial resistance to tumor necrosis factor-induced apoptosis. Furthermore, K8/18 filaments regulate Fas receptor signaling, and modest overexpression of the phosphorylation mutant K8 S73A significantly enhances Fas-mediated apoptosis in transgenic mouse livers (Ku and Omary, 2006). In the current study, phosphorylation of keratin 8 (ser74) and keratin 18 (ser52) was significantly increased in a concentration-dependent manner; however, the cell cycle progression and apoptosis rates were changed only after exposure to the highest MC-LR concentration (10 ␮M). We hypothesize that keratins work as buffers of cellular stress signals to protect cells from cell cycle arrest and apoptosis. When MC-LR reaches a very high concentration, however, the balance between phosphatases and kinases is severely disturbed, and cells undergo apoptosis. Similar to keratins, vimentin and lamin A/C are also broadly involved in signal transduction. Similar to K8/18, phosphorylated vimentin can interact with 14-3-3 proteins to displace the association of Raf with these proteins so that they can no longer be activated by epidermal growth factor (Tzivion et al., 2000). Vimentin may also transport stress-induced signaling molecules, and soluble vimentin subunits enable the transport of p-ERK1/2 from the site of an axonal lesion to the nerve cell body (Perlson et al., 2005). Phosphorylation of lamin A/C at ser22 has been identified in vitro in several cell lines by mass spectrometry analysis in proteomic screens. The surrounding sequence of ser22 is a typical MAPK/CDK phosphorylation motif, which implicates a role for lamin A/C in the cell cycle and mitosis (Beausoleil et al., 2006; Lowery et al., 2007). Interestingly, the phosphorylation of vimentin was increased in the present study, while lamin A/C remained unchanged, which implies that PP2A inhibition and MAPK pathway activation may lead to hyperphosphorylation of different types of IF proteins. Because of the large number of IF family members and the complicated mechanical scaffold and signaling network that they form, further studies are needed to delineate the MC-LR-induced cellular response via IF-associated signaling pathways. The results of this study suggest that MC-LR exerts its potential hepatotoxicity through PP2A inhibition and subsequent MAPK pathway activation, which causes hyperphosphorylation of IF proteins and results in cytoskeletal architecture remodeling and cell survival/death regulation. These findings provide new insights into the mechanisms of MC-LR hepatotoxicity. Future studies that either investigate more IF proteins or focus on a specific IF protein may

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provide more valuable information on cyanobacterial toxin hepatotoxicity. Conflict of interest None. Acknowledgments This work was supported by the National Nature Science Foundation of China (No. 30901216), the Zhejiang Natural Science Foundation (No. LY12B07002), and K.C. Wong Magna Fund in Ningbo University. References Batista, T., de Sousa, G., Suput, J.S., Rahmani, R., Suput, D., 2003. Microcystin-LR causes the collapse of actin filaments in primary human hepatocytes. Aquatic Toxicology 65, 85–91. Beausoleil, S.A., Villen, J., Gerber, S.A., Rush, J., Gygi, S.P., 2006. A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nature Biotechnology 24, 1285–1292. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Coulombe, P.A., Omary, M.B., 2002. ‘Hard’ and ‘soft’ principles defining the structure, function and regulation of keratin intermediate filaments. Current Opinion in Cell Biology 14, 110–122. Coulombe, P.A., Wong, P., 2004. Cytoplasmic intermediate filaments revealed as dynamic and multipurpose scaffolds. Nature Cell Biology 6, 699–706. Daily, A., Monks, N.R., Leggas, M., Moscow, J.A., 2010. Abrogation of microcystin cytotoxicity by MAP kinase inhibitors and N-acetyl cysteine is confounded by OATPIB1 uptake activity inhibition. Toxicon 55, 827–837. Ding, W.X., Shen, H.M., Ong, C.N., 2000. Critical role of reactive oxygen species and mitochondrial permeability transition in microcystin-induced rapid apoptosis in rat hepatocytes. Hepatology 32, 547–555. Dorr, F.A., Pinto, E., Soares, R.M., Feliciano de Oliveira e Azevedo, S.M., 2010. Microcystins in South American aquatic ecosystems: occurrence, toxicity and toxicological assays. Toxicon 56, 1247–1256. Eriksson, J.E., Dechat, T., Grin, B., Helfand, B., Mendez, M., Pallari, H.M., Goldman, R.D., 2009. Introducing intermediate filaments: from discovery to disease. Journal of Clinical Investigation 119, 1763–1771. Eriksson, J.E., Gronberg, L., Nygard, S., Slotte, J.P., Meriluoto, J.A., 1990. Hepatocellular uptake of 3H-dihydromicrocystin-LR, a cyclic peptide toxin. Biochimica et Biophysica Acta 1025, 60–66. Fischer, W.J., Hitzfeld, B.C., Tencalla, F., Eriksson, J.E., Mikhailov, A., Dietrich, D.R., 2000. Microcystin-LR toxicodynamics, induced pathology, and immunohistochemical localization in livers of blue-green algae exposed rainbow trout (oncorhynchus mykiss). Toxicological Sciences 54, 365–373. Fu, W., Yu, Y., Xu, L., 2009. Identification of temporal differentially expressed protein responses to microcystin in human amniotic epithelial cells. Chemical Research in Toxicology 22, 41–51. Fu, W.Y., Li, M.W., Chen, J.P., Xu, L.H., 2004. Detection of apoptosis of renal cells of rat induced by microcystin-LR. Acta Hydrobiologica Sinica 6, 1099–1108. Fu, W.Y., Xu, L.H., Yu, Y.N., 2005. Proteomic analysis of cellular response to microcystin in human amnion FL cells. Journal of Proteome Research 4, 2207–2215. Gan, N., Sun, X., Song, L., 2010. Activation of Nrf2 by microcystin-LR provides advantages for liver cancer cell growth. Chemical Research in Toxicology 23, 1477–1484. Ghosh, S., Khan, S.A., Wickstrom, M., Beasley, V., 1995. Effects of microcystin-LR on actin and the actin-associated proteins alpha-actinin and talin in hepatocytes. Natural Toxins 3, 405–414. Godsel, L.M., Hobbs, R.P., Green, K.J., 2008. Intermediate filament assembly: dynamics to disease. Trends in Cell Biology 18, 28–37. Ji, Y., Lu, G., Chen, G., Huang, B., Zhang, X., Shen, K., Wu, S., 2011. Microcystin-LR induces apoptosis via NF-kappaB/iNOS pathway in INS-1 cells. International Journal of Molecular Sciences 12, 4722–4734. Kim, S., Coulombe, P.A., 2007. Intermediate filament scaffolds fulfill mechanical, organizational, and signaling functions in the cytoplasm. Genes and Development 21, 1581–1597. Kins, S., Kurosinski, P., Nitsch, R.M., Gotz, J., 2003. Activation of the ERK and JNK signaling pathways caused by neuron-specific inhibition of PP2A in transgenic mice. American Journal of Pathology 163, 833–843. Komatsu, M., Furukawa, T., Ikeda, R., Takumi, S., Nong, Q., Aoyama, K., Akiyama, S., Keppler, D., Takeuchi, T., 2007. Involvement of mitogen-activated protein kinase signaling pathways in microcystin-LR-induced apoptosis after its selective uptake mediated by OATP1B1 and OATP1B3. Toxicological Sciences 97, 407–416. Ku, N.O., Liao, J., Omary, M.B., 1998a. Phosphorylation of human keratin 18 serine 33 regulates binding to 14-3-3 proteins. EMBO Journal 17, 1892–1906.

199

Ku, N.O., Michie, S., Resurreccion, E.Z., Broome, R.L., Omary, M.B., 2002. Keratin binding to 14-3-3 proteins modulates keratin filaments and hepatocyte mitotic progression. Proceedings of the National Academy of Sciences of the United States of America 99, 4373–4378. Ku, N.O., Michie, S.A., Soetikno, R.M., Resurreccion, E.Z., Broome, R.L., Omary, M.B., 1998b. Mutation of a major keratin phosphorylation site predisposes to hepatotoxic injury in transgenic mice. Journal of Cell Biology 143, 2023–2032. Ku, N.O., Omary, M.B., 2000. Keratins turn over by ubiquitination in a phosphorylation-modulated fashion. Journal of Cell Biology 149, 547–552. Ku, N.O., Omary, M.B., 2001. Effect of mutation and phosphorylation of type I keratins on their caspase-mediated degradation. Journal of Biological Chemistry 276, 26792–26798. Ku, N.O., Omary, M.B., 2006. A disease- and phosphorylation-related nonmechanical function for keratin 8. Journal of Cell Biology 174, 115–125. Lowery, D.M., Clauser, K.R., Hjerrild, M., Lim, D., Alexander, J., Kishi, K., Ong, S.E., Gammeltoft, S., Carr, S.A., Yaffe, M.B., 2007. Proteomic screen defines the Polobox domain interactome and identifies Rock2 as a Plk1 substrate. EMBO Journal 26, 2262–2273. Meng, G., Sun, Y., Fu, W., Guo, Z., Xu, L., 2011. Microcystin-LR induces cytoskeleton system reorganization through hyperphosphorylation of tau and HSP27 via PP2A inhibition and subsequent activation of the p38 MAPK signaling pathway in neuroendocrine (PC12) cells. Toxicology 290, 218–229. Menon, M.B., Schwermann, J., Singh, A.K., Franz-Wachtel, M., Pabst, O., Seidler, U., Omary, M.B., Kotlyarov, A., Gaestel, M., 2010. p38 MAP kinase and MAPKAP kinases MK2/3 cooperatively phosphorylate epithelial keratins. Journal of Biological Chemistry 285, 33242–33251. Mezhoud, K., Praseuth, D., Francois, J.C., Bernard, C., Edery, M., 2008. Global quantitative analysis of protein phosphorylation status in fish exposed to microcystin. Advances in Experimental Medicine and Biology 617, 419–426. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 55–63. Nishiwaki-Matsushima, R., Ohta, T., Nishiwaki, S., Suganuma, M., Kohyama, K., Ishikawa, T., Carmichael, W.W., Fujiki, H., 1992. Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR. Journal of Cancer Research and Clinical Oncology 118, 420–424. Ohta, T., Nishiwaki, R., Yatsunami, J., Komori, A., Suganuma, M., Fujiki, H., 1992. Hyperphosphorylation of cytokeratins 8 and 18 by microcystin-LR, a new liver tumor promoter, in primary cultured rat hepatocytes. Carcinogenesis 13, 2443–2447. Omary, M.B., Ku, N.O., Liao, J., Price, D., 1998. Keratin modifications and solubility properties in epithelial cells and in vitro. Sub-cellular Biochemistry 31, 105–140. Omary, M.B., Ku, N.O., Tao, G.Z., Toivola, D.M., Liao, J., 2006. Heads and tails of intermediate filament phosphorylation: multiple sites and functional insights. Trends in Biochemical Sciences 31, 383–394. Pallari, H.M., Eriksson, J.E., 2006. Intermediate filaments as signaling platforms. Science’s STKE 366, e53. Perlson, E., Hanz, S., Ben-Yaakov, K., Segal-Ruder, Y., Seger, R., Fainzilber, M., 2005. Vimentin-dependent spatial translocation of an activated MAP kinase in injured nerve. Neuron 45, 715–726. Robinson, N.A., Miura, G.A., Matson, C.F., Dinterman, R.E., Pace, J.G., 1989. Characterization of chemically tritiated microcystin-LR and its distribution in mice. Toxicon 27, 1035–1042. Runnegar, M.T., Andrews, J., Gerdes, R.G., Falconer, I.R., 1987. Injury to hepatocytes induced by a peptide toxin from the cyanobacterium Microcystis aeruginosa. Toxicon 25, 1235–1239. Sun, Y., Meng, G.M., Guo, Z.L., Xu, L.H., 2011. Regulation of heat shock protein 27 phosphorylation during microcystin-LR-induced cytoskeletal reorganization in a human liver cell line. Toxicology Letters 207, 270–277. Svircev, Z., Baltic, V., Gantar, M., Jukovic, M., Stojanovic, D., Baltic, M., 2010. Molecular aspects of microcystin-induced hepatotoxicity and hepatocarcinogenesis. Journal of Environmental Science and Health C-Environmental Carcinogenesis Reviews 28, 39–59. Takumi, S., Komatsu, M., Furukawa, T., Ikeda, R., Sumizawa, T., Akenaga, H., Maeda, Y., Aoyama, K., Arizono, K., Ando, S., Takeuchi, T., 2010. p53 plays an important role in cell fate determination after exposure to microcystin-LR. Environmental Health Perspectives 118, 1292–1298. Toivola, D.M., Goldman, R.D., Garrod, D.R., Eriksson, J.E., 1997. Protein phosphatases maintain the organization and structural interactions of hepatic keratin intermediate filaments. Journal of Cell Science 110 (Pt 1), 23–33. Tzivion, G., Luo, Z.J., Avruch, J., 2000. Calyculin A-induced vimentin phosphorylation sequesters 14-3-3 and displaces other 14-3-3 partners in vivo. Journal of Biological Chemistry 275, 29772–29778. Wickstead, B., Gull, K., 2011. The evolution of the cytoskeleton. Journal of Cell Biology 194, 513–525. Wickstrom, M.L., Khan, S.A., Haschek, W.M., Wyman, J.F., Eriksson, J.E., Schaeffer, D.J., Beasley, V.R., 1995. Alterations in microtubules, intermediate filaments, and microfilaments induced by microcystin-LR in cultured cells. Toxicologic Pathology 23, 326–337. Woll, S., Windoffer, R., Leube, R.E., 2007. p38 MAPK-dependent shaping of the keratin cytoskeleton in cultured cells. Journal of Cell Biology 177, 795–807. Yoshida, T., Makita, Y., Tsutsumi, T., Nagata, S., Tashiro, F., Yoshida, F., Sekijima, M., Tamura, S., Harada, T., Maita, K., Ueno, Y., 1998. Immunohistochemical localization of microcystin-LR in the liver of mice: a study on the pathogenesis of microcystin-LR-induced hepatotoxicity. Toxicologic Pathology 26, 411–418.