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The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine Minghao Yan a, b, 1, Gu Shen a, b, c, 1, Yuan Zhou a, b, Xiannan Meng a, b, Xiaodong Han a, b, * a Immunology and Reproduction Biology Laboratory & State Key Laboratory of Analytical Chemistry for Life Science, Medical School, Nanjing University, Nanjing, Jiangsu, 210093, China b Jiangsu Key Laboratory of Molecular Medicine, Nanjing University, Nanjing, Jiangsu, 210093, China c Department of Hepatopancreatobiliary Surgery, Drum Tower Hospital, Medical School of Nanjing University, Nanjing, China
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Article history: Received 10 October 2019 Accepted 20 October 2019 Available online xxx
Microcystin-leucine arginine (MC-LR) is a potent specific hepatotoxin produced by cyanobacteria in diverse water systems, and it has been documented to induce liver injury and hepatocarcinogenesis. However, its toxic effects on intrahepatic biliary epithelial cells have not been invested in detail. In this study, we aimed to investigate the effects of MC-LR exposure on the intrahepatic biliary epithelial cells in the liver. MC-LR was orally administered to mice at 1 mg/L, 7.5 mg/L, 15 mg/L, or 30 mg/L for 180 consecutive days for histopathological and immunoblot analysis. We observed that MC-LR can enter intrahepatic bile duct tissue and induce hyperplasia of mice. Human primary intrahepatic biliary epithelial cells (HiBECs) were cultured with various concentrations of MC-LR for 24 h, meanwhile the cell viability and proteins level were detected. Western blotting analysis revealed that MC-LR increased RSK phosphorylation via ERK signaling. RSK participated in cell proliferation and cell cycle progression. Taken together, after chronic exposure, MC-LR-treated mice exhibited abnormal bile duct hyperplasia and thickened bile duct morphology through activating the ERK-RSK signaling. These data support the potential toxic effects of MC-LR on bile duct tissue of the liver. © 2019 Elsevier Inc. All rights reserved.
Keywords: MC-LR Intrahepatic biliary epithelial cells Proliferation ERK-RSK signaling
1. Introduction Microcystins (MCs) are a group of cyclic heptapeptides, produced by blue-green algae, which have been shown to pose a health threat to humans through the food chain [1]. So far, more than 90 MCs variants have been detected [2], among which microcystinleucine arginine (MC-LR) is the most common toxic variant [3]. To date, MC-LR has been identified to be able to bring about renal impairment, liver damage and reproductive system toxicity in mice [4,5]. It was reported that liver was one of the earliest target organs damaged by MC-LR, resulting in oxidative stress and hepatocarcinogenesis [6,7]. Zheng et al. demonstrated that serum MCLR was an independent risk factor for hepatocellular carcinoma in humans via a clinical case-control study [8]. It was also proved that
* Corresponding author. Immunology and Reproductive Biology Laboratory, Medical School, Nanjing University, Hankou Road 22, Nanjing, 210093, China. E-mail addresses: [email protected] (M. Yan), [email protected] (G. Shen), [email protected] (Y. Zhou), [email protected] (X. Meng), [email protected] (X. Han). 1 These authors contributed equally to this work.
persistent MC-LR exposure promoted proliferation, mobility, and colony formation of normal liver cells [9]. However, few studies focused on the effects of MC-LR on intrahepatic biliary epithelial cells. In the liver, the biliary system drains bile produced by hepatocytes to the gallbladder and intestine, which was consist of heterogeneous epithelial cells [10]. In addition to playing important roles in the regulation of ductal secretion, the biliary epithelial cells are the target cells of apoptotic, proliferative, and regenerative events leading to changes in biliary damage, hyperplasia, and regeneration [11,12]. Atypical ductules hyperplasia was found in the tissue specimens of long-standing biliary diseases such as primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC), ending up with jaundice and hepatic cirrhosis [13,14]. More importantly, studies have shown that intrahepatic bile duct hyperplasia is an important risk factor for intrahepatic cholangiocarcinoma [15]. Although it was reported that cholestasis and bacterial infection were associated with hyperplasia of cholangiocytes, the details of this interaction had not been described [16,17]. In this study, we firstly explore the relationship between MC-LR and proliferation in intrahepatic biliary epithelial cells.
https://doi.org/10.1016/j.bbrc.2019.10.143 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: M. Yan et al., The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.143
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The extracellular signal-regulated kinase (ERK) signaling has emerged as a central regulator in hepatoma cell proliferation [18]. Previous studies have shown that inhibition of protein phosphatase 2A (PP2A) could result in the activation of MAPK/ERK signaling via increasing the level of phosphorylated-MEK [19]. In addition, it was demonstrated that activation of the MAPK/ERK signaling could promote cell proliferation through increasing the level of phosphorylated RSK [18,20]. The MC-LR-induced cell toxicity was mainly mediated through inhibition of PP2A [21]. Taken together, the ERK/RSK signaling is supposed to be involved in MC-LR-induced biliary epithelial cell proliferation. In this study, we firstly investigated the effect of MC-LR on intrahepatic biliary epithelial cell proliferation with respect to its interaction with the ERK-RSK signaling in vitro and in vivo systems, which has helped us to gain insight into the mechanisms of MC-LRinduced proliferation in intrahepatic biliary epithelial cells.
2.4. PP2A activity
2. Materials and methods
This was determined as reported [26], by using a PP2A immunoprecipitation phosphatase assay (Millipore, Billerica, MA) that measures free phosphate with a malachite green dye. Total protein from the control and MC-LR-treated was extracted with lysis buffer. To immunoprecipitate PP2A, lysates containing 200 mg of protein were incubated with 4 mg of anti-PP2A, C subunit antibody and 40 ml of protein A-agarose slurry for 2 h at 4 C with constant rocking. The immunoprecipitates were washed three times in Tris-buffered saline and once with Ser/Thr assay buffer. The reaction was initiated by the addition of 60 ml of phosphopeptide substrate. Following incubation at 30 C for 10 min in a shaking incubator, the reaction mixture was centrifuged briefly, and the supernatant was transferred to a 96-well microtiter plate. The reaction was terminated by adding malachite green phosphate detection solution for 15 min at room temperature, and free phosphate was quantified by measuring the absorbance of the mixture at 650 nm using a microplate reader.
2.1. Main reagents and cell culture
2.5. EdU incorporation assay
MC-LR was purchased from Alexis Biochemicals (Lausen, Switzerland). MC-LR (1 mg) was dissolved in 100 ml DMSO and diluted to 1 mL with DMEM to prepare the stock solution (1 mM). PD98059 (the inhibitor for ERK1/2 MAP kinase pathway) and BID1870 (the inhibitor for RSK1/2/3/4) were purchased from selleckchem (Shanghai, China). Human primary intrahepatic biliary epithelial cells (HiBECs) were purchased from ScienCell Research Laboratories (San Diego, CA). HiBECs were cultured in high DMEM media (Hyclone) supplemented 10% FBS (Gibco), 1% L-glutamine, 1% penicillin and streptomycin. When HiBECs reached 80% confluence, they were routinely passaged using 0.25% trypsin and were diluted in a 6-well culture plate. After HiBECs reached 60e80% confluence, we discarded the medium and added the fresh high DMEM media containing various concentrations of MC-LR (0, 0.5, 5, 50 or 500 nM) for 24 h or added the fresh high DMEM media containing MC-LR (50 nM) for 24 h. In some experiments, HiBECs were pretreated for 30 min with various chemical inhibitors, such as ERK inhibitor or RSK inhibitor. HiBECs were characterized using a previously described immunofluorescence microscopic method with antibodies to cytokeratin 7 (Proteintech, Wuhan, China) and cytokeratin 19 (Boster, Wuhan, China), which labeled > 90% of the cells in culture [22e24].
Cell proliferation was assessed by 5-ethynyl-2-deoxyuridine (EdU) incorporation using the Click-iT EdU Alexa Fluor 488 cell proliferation assay kit (Invitrogen, Carlsbad, CA) as previously described [27]. 2.6. CCK-8 assay Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) was used to evaluate the cell viability according to the manufacturer’s instructions. HiBECs were treated with various concentrations of MC-LR (0, 0.5, 5, 50 or 500 nM) in 96-well culture plate and incubated for 24 h. Then CCK-8 solutions were added and incubated for another 2 h to measure optical density (OD) at 450 nm. 2.7. Histopathology The mouse livers were fixed in 10% formalin for 24 h at room temperature, dehydrated, transparentized and embedded in paraffin before sectioning into 5 mm-thick slices. Then the sections were staining with hematoxylin and eosin (H&E) for structured observation and immunohistochemistry/immunofluorescence analyses. 2.8. Immunohistochemical analyses and immunofluorescent staining
2.2. Animals and treatment Male specific pathogen-free (SPF) BALB/C mice (6e7 week old) weighing from 20 to 25 g were purchased from Experimental Animal Center of the Academy of Military Medical Science Institute, China. After 1 week of acclimation, mice were given drinking water containing 1, 7.5, 15 or 30 mg/L MC-LR for 180 consecutive days. For the control group, mice were provided with only the blank water. The actual daily exposure dose of MC-LR was performed as previously described [25]. All procedures carried out on animals were approved by the Animal Care and Use Committee of Nanjing University under the animal protocol number SYXK (Su) 2009-0017. 2.3. Western blotting The liver or cell extract protein was lysed with RIPA buffer (Beyotime, Shanghai, China) and the protein concentration was determined using Coomassie Brilliant Blue (Beyotime, Shanghai, China). We have provided a detailed description of the method in the Supplementary Methods.
We have provided a detailed description of the method in the Supplementary Methods. 2.9. Flow cytometric analysis Flow cytometry was performed to analyze the cell cycle distribution. HiBECs were washed with PBS and fixed in 75% cold ethanol overnight at 4 C. Before cell cycle analysis, the fixed cells were washed with PBS. Then the cells were resuspended in propidium iodide (PI)/DNA staining solution and incubated for 30 min at room temperature in the dark. Samples were analyzed for cell cycle with a BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data were analyzed using Flow Jo. Experiments were performed independently at least thrice in duplicates. 2.10. Statistical analyses Experimental results were expressed as means ± standard deviation (SD). All calculations and statistical analyses were
Please cite this article as: M. Yan et al., The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.143
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performed using Graph-Pad Prism 5 (USA). One-way analysis of variance (ANOVA) was used to analyze the difference between groups, followed by Tukey s Multiple Comparison test. P < 0.05 was regarded as statistically significant. 3. Results 3.1. MC-LR induced hyperplasia of biliary epithelial cell in the liver of mice To explore the influence of MC-LR on intrahepatic biliary
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epithelial cells, mice were treated with 1, 7.5, 15 or 30 mg/L MC-LR for 180 consecutive days. Under normal circumstances, there is no abnormal hyperplasia in the interlobular bile duct. In mice treated with 30 mg/L MC-LR, we found that the inflammatory cell infiltration and thickening of the bile duct in the portal area (Fig. 1A). To evaluate MC-LR-induced intrahepatic bile duct hyperplasia in mice, we examined biliary epithelial cell marker protein cytokeratin 19. It was shown that there were more cytokeratin 19 positive cells in the liver following exposure to 7.5, 15 or 30 mg/L MC-LR (Fig. 1B). Protein expression of cytokeratin 19 was also elevated and detected by western blotting (Fig. 1C). Moreover, we
Fig. 1. Mice were administered with microcystin-leucine-arginine (MC-LR) at 1 mg/kg, 7.5 mg/kg, 15 mg/kg and 30 mg/kg through drinking water for 180 consecutive days. (A) The structure of histological sections of intrahepatic bile duct was observed by H&E staining (scale bar ¼ 200 mm). (B) Liver tissue content of cytokeratin 19 was measured by immunohistochemical staining (scale bar ¼ 200 mm). Lower pictures correspond to magnified boxed areas (scale bar ¼ 100 mm). (C) Liver tissue content of cytokeratin 19 was measured by western blotting (left). The expression levels of the proteins were quantified by densitometry and normalized to the expression of GAPDH (right). Data are shown as means ± SD. *P < 0.05, vs control. (D) Expression of Ki67 in the liver was examined by immunohistochemistry (scale bar ¼ 200 mm). Lower pictures correspond to magnified boxed areas (scale bar ¼ 100 mm).
Please cite this article as: M. Yan et al., The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.143
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examined the expression of Ki67 in the intrahepatic bile duct and Ki67 increased significantly after MC-LR-treated in the mice liver (Fig. 1D).
3.2. MC-LR could be taken by biliary epithelial cell and promote cell proliferation In this study, we examined the distribution of MC-LR in liver by Immunofluorescence, and the results demonstrated that MC-LR could be transported into intrahepatic biliary epithelial cells (Fig. 2A). Besides, MC-LR could enter into HiBECs, as assessed by immunofluorescent staining (Fig. 2B) and western blotting (Fig. 2C). PP2A is emerging as a critical regulator of the microcystininduced molecular network. We measured the activity of PP2A in HiBECs after treated with MC-LR. PP2A activity was significantly attenuated compared to the control group (Fig. 2D). To evaluate the toxic effects of MC-LR on HiBECs, we examined the cell viability by CCK-8 assay after MC-LR treatment, and the results showed that the cell viability of HiBECs after MC-LR-treated was significantly enhanced (Fig. 2E). By the use of flow cytometry analysis, it was
shown that the cell percentage in S phase was markedly increased in HiBECs after exposure to 0.5, 5 or 50 nM MC-LR for 24 h (Fig. 2F). However, MC-LR treatment at 500 nM showed no significant change in S phase. We further examined the effect on MC-LRstimulated cell proliferation by EdU incorporation. The HiBECs treated with 0.5, 5 or 50 nM MC-LR heightened the rate of EdUpositive cells (Supplementary Fig. 1). Taken together, these results provided strong evidence to show that MC-LR promoted HiBECs proliferation.
3.3. The ERK-RSK signaling was activated during MC-LR-promoting HiBECs proliferation ERK-RSK signaling was found to be responsible for its proliferative action [20]. We found that high expression of phosphorylated MEK, phosphorylated ERK and phosphorylated RSK was observed in the intrahepatic portal area (Fig. 3A). In vitro, MC-LR exposure at 0.5, 50 or 500 nM could up-regulate phosphorylation of MEK, ERK and RSK (Fig. 3B). To further define the role of ERK-RSK signaling in the proliferation of HiBECs induced by MC-LR, we investigated
Fig. 2. MC-LR can be taken by biliary epithelial cell and promote cell proliferation. (A) The expression of MC-LR and cytokeratin 7 in mouse liver was detected by immunofluorescence assay (scale bar ¼ 50 mm). (B) Human intrahepatic biliary epithelial cells (HiBECs) were treated with 0.5 nM, 500 nM MC-LR for 24 h. Expression of MC-LR was determined by immunofluorescence staining (scale bar ¼ 50 mm). (C) Expression of MC-LR in HiBECs was detected by western blotting. (D) PP2A activity was measured using an immunoprecipitation assay kit. (E) Measurement of cell viability was carried out with Cell Counting Kit (CCK-8) assay. (F) HiBECs were stained with PI and then analyzed by flow cytometry. All data were expressed as means ± SD. *P < 0.05, vs control.
Please cite this article as: M. Yan et al., The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.143
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Fig. 3. MC-LR-induced phosphorylation of MEK, ERK and RSK in vivo and vitro. (A) The liver of MC-LR treated mice was analyzed by immunohistochemistry. The sections were stained with anti-p-MEK, anti-p-ERK and anti-p-RSK antibody. Images were obtained from 30 mg/L MC-LR treatment group and control group (scale bar ¼ 100 mm). (B) HiBECs were treated with or without MC-LR (0.5 nM, 5 nM, 50 nM, 500 nM) for 24 h. Protein levels of p-MEK, total MEK, p-ERK, total ERK, p-RSK and total RSK were examined by western blotting; the ratios of p-ERK/ERK and p-RSK/RSK were determined by densitometry and were expressed as means ± SD. *p < 0.05, vs control.
whether targeted inhibition of this signaling could attenuate the proliferative capacity. We used PD98059 almost completely abolished the phosphorylation of ERK caused by 50 nM MC-LR-treated. At the same time, the degree of phosphorylation of RSK was also suppressed (Fig. 4A). Besides, viability of HiBECs after treated with PD98059 in presence or absence of MC-LR was assessed by cell cycle experiment and the cell percentage in S phase was suppressed when cells were treated with MC-LR and PD98059 (Fig. 4B). CCK-8 assay showed that PD98059 attenuated cell viability in MC-LR treated cells (Fig. 4C). Thus, EdU-positive cells of MC-LR-treated were reduced by treating with PD98059 (Fig. 4D). We further examined the effect of RSK inhibitor BI-D1870 on MC-LRstimulated cell cycle, CCK-8 and EdU incorporation in HiBECs and confirmed the similar results (Supplementary Fig. 2). All above evidence indicated that MC-LR could promote the proliferation of HiBECs via the activation of ERK/RSK signaling. 4. Discussion MC-LR is a potent specific hepatotoxin and has been documented to induce hepatocyte apoptosis and liver injury [28]. Nevertheless, to our knowledge, there are few researches conducted to investigate the effects of prolonged MC-LR exposure on the proliferation of intrahepatic biliary epithelial cells. In this study, we have provided the initial evidence that ERK-RSK signaling is clearly associated with MC-LR-induced intrahepatic bile duct hyperplasia in vivo and in vitro. As no previous work was studied about the phenomenon, it is the first time we observed this effect in intrahepatic biliary epithelial cells and the proliferative effect.
A number of researches elucidated that MC-LR is highly liverspecific, causing irreversible hepatotoxicity in animals and humans. Previous studies showed that MC-LR could be up-taken by liver tissues [29,30]. In mouse chronic exposure models, we also found that MC-LR could be detected in the intrahepatic bile duct by immunofluorescence colocalization. The presence of MC-LR in human intrahepatic biliary epithelial cells treated with MC-LR at 0.5 nM or 500 nM for 24 h was observed by immunofluorescence staining and western blotting. These results indicated that MC-LR could enter HiBECs, which were similar to the phenomenon observed in human hepatocytes [9]. The intrahepatic bile duct is a highly organized tubular structure consisting of biliary epithelial cells, which drains bile produced by hepatocytes into the duodenum. Intrahepatic biliary epithelial cells are the target of biliary tract disease, such as PBC and PSC, which are associated with dysregulation of intrahepatic biliary epithelial cells proliferation/loss [31]. Normally, intrahepatic biliary epithelial cells are mitotically quiescent but following damage they begin to proliferate to repair the biliary tree to compensate for damage and loss of functionality [32,33]. In this study, we focused on the effects of MC-LR on intrahepatic biliary epithelial cells. Following exposure to MC-LR for 180 days, inflammatory infiltration and bile duct hyperplasia in the mouse portal were observed in the 15 and 30 mg/L MC-LR groups. HiBECs showed apparent proliferation after exposure to 50 nM MC-LR for 24 h via CCK-8 and cell cycle analysis. However, there was little difference compared with the control group after 500 nM treatment. Collectively, MC-LR could promote proliferation of biliary epithelial cells in the liver, which may increase the risks of biliary tract disease.
Please cite this article as: M. Yan et al., The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.143
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Fig. 4. Blocking ERK signaling with PD98059 inhibits proliferation of HiBECs. (A) Expressions of p-ERK, ERK, p-RSK, RSK were detected by western blotting. (B) Cell cycle was evaluated by flow cytometry measuring propidium iodide (PI) expression. (C) Cell proliferation was measured with the CCK-8 assay. (D) Culture medium were pulsed with EdU for 4 h of the 24-h incubation period and subsequently stained with EdU Click-iT reaction mixture (green) (scale bar ¼ 50 mm). (E) Schematic representation of roles of ERK-RSK signaling in MC-LR-induced cell proliferation. All data were expressed as means ± SD. *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Cell proliferation is the process by which cells increase their mass by upregulating the biosynthesis of macromolecules, membranes and organelles. In addition, many signals also play an important role in this process, as shown in Fig. 4E, ERK-RSK signaling was found to be responsible for proliferative action of various cell types. In the pathological process of the liver disease and hepatocarcinoma, extracellular signal-regulated kinase (ERK) signaling is an important participant [21,34]. The 90 kDa ribosomal S6 kinases (RSK) are a family of Ser/Thr kinases that lie downstream of the MAPK-ERK cascade. The RSK are directly activated by ERK in response to growth factors, many polypeptide hormones, neurotransmitters, chemokines and other stimuli [35]. The RSK phosphorylate many cytosolic and nuclear targets and they have been implicated in the regulation of diverse cellular processes, including cell proliferation, cell survival, cell growth and motility [36]. Our results confirmed that the ERK signaling was involved in the process of cell proliferation and the MC-LR-related effect could be inhibited by PD98059. Furthermore, we found that MC-LR-induced cell proliferation was attenuated after inhibiting RSK activation. Taken together, it is possible that the phosphorylation of ERK and
RSK followed treatment of MC-LR contributes to MC-LR-induced cell proliferation. In conclusion, our study indicates that MC-LR consistently accumulates in the mice liver intrahepatic bile duct within a long period of time. Meanwhile, the ERK-RSK signaling is activated and interacts to promote HiBECs proliferation. And these findings extend our knowledge about MC-LR-induced environmental liver diseases and the related mechanisms. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by National Natural Science Foundation of China (81470866, 31870492, and 31670519). Key Medical Subjects of Jiangsu Province (ZDRCA2016057).
Please cite this article as: M. Yan et al., The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.143
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The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine Minghao Yan a, b, 1, Gu Shen a, b, c, 1, Yuan Zhou a, b, Xiannan Meng a, b, Xiaodong Han a, b, * a Immunology and Reproduction Biology Laboratory & State Key Laboratory of Analytical Chemistry for Life Science, Medical School, Nanjing University, Nanjing, Jiangsu, 210093, China b Jiangsu Key Laboratory of Molecular Medicine, Nanjing University, Nanjing, Jiangsu, 210093, China c Department of Hepatopancreatobiliary Surgery, Drum Tower Hospital, Medical School of Nanjing University, Nanjing, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 October 2019 Accepted 20 October 2019 Available online xxx
Microcystin-leucine arginine (MC-LR) is a potent specific hepatotoxin produced by cyanobacteria in diverse water systems, and it has been documented to induce liver injury and hepatocarcinogenesis. However, its toxic effects on intrahepatic biliary epithelial cells have not been invested in detail. In this study, we aimed to investigate the effects of MC-LR exposure on the intrahepatic biliary epithelial cells in the liver. MC-LR was orally administered to mice at 1 mg/L, 7.5 mg/L, 15 mg/L, or 30 mg/L for 180 consecutive days for histopathological and immunoblot analysis. We observed that MC-LR can enter intrahepatic bile duct tissue and induce hyperplasia of mice. Human primary intrahepatic biliary epithelial cells (HiBECs) were cultured with various concentrations of MC-LR for 24 h, meanwhile the cell viability and proteins level were detected. Western blotting analysis revealed that MC-LR increased RSK phosphorylation via ERK signaling. RSK participated in cell proliferation and cell cycle progression. Taken together, after chronic exposure, MC-LR-treated mice exhibited abnormal bile duct hyperplasia and thickened bile duct morphology through activating the ERK-RSK signaling. These data support the potential toxic effects of MC-LR on bile duct tissue of the liver. © 2019 Elsevier Inc. All rights reserved.
Keywords: MC-LR Intrahepatic biliary epithelial cells Proliferation ERK-RSK signaling
1. Introduction Microcystins (MCs) are a group of cyclic heptapeptides, produced by blue-green algae, which have been shown to pose a health threat to humans through the food chain [1]. So far, more than 90 MCs variants have been detected [2], among which microcystinleucine arginine (MC-LR) is the most common toxic variant [3]. To date, MC-LR has been identified to be able to bring about renal impairment, liver damage and reproductive system toxicity in mice [4,5]. It was reported that liver was one of the earliest target organs damaged by MC-LR, resulting in oxidative stress and hepatocarcinogenesis [6,7]. Zheng et al. demonstrated that serum MCLR was an independent risk factor for hepatocellular carcinoma in humans via a clinical case-control study [8]. It was also proved that
* Corresponding author. Immunology and Reproductive Biology Laboratory, Medical School, Nanjing University, Hankou Road 22, Nanjing, 210093, China. E-mail addresses: [email protected] (M. Yan), [email protected] (G. Shen), [email protected] (Y. Zhou), [email protected] (X. Meng), [email protected] (X. Han). 1 These authors contributed equally to this work.
persistent MC-LR exposure promoted proliferation, mobility, and colony formation of normal liver cells [9]. However, few studies focused on the effects of MC-LR on intrahepatic biliary epithelial cells. In the liver, the biliary system drains bile produced by hepatocytes to the gallbladder and intestine, which was consist of heterogeneous epithelial cells [10]. In addition to playing important roles in the regulation of ductal secretion, the biliary epithelial cells are the target cells of apoptotic, proliferative, and regenerative events leading to changes in biliary damage, hyperplasia, and regeneration [11,12]. Atypical ductules hyperplasia was found in the tissue specimens of long-standing biliary diseases such as primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC), ending up with jaundice and hepatic cirrhosis [13,14]. More importantly, studies have shown that intrahepatic bile duct hyperplasia is an important risk factor for intrahepatic cholangiocarcinoma [15]. Although it was reported that cholestasis and bacterial infection were associated with hyperplasia of cholangiocytes, the details of this interaction had not been described [16,17]. In this study, we firstly explore the relationship between MC-LR and proliferation in intrahepatic biliary epithelial cells.
https://doi.org/10.1016/j.bbrc.2019.10.143 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: M. Yan et al., The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.143
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The extracellular signal-regulated kinase (ERK) signaling has emerged as a central regulator in hepatoma cell proliferation [18]. Previous studies have shown that inhibition of protein phosphatase 2A (PP2A) could result in the activation of MAPK/ERK signaling via increasing the level of phosphorylated-MEK [19]. In addition, it was demonstrated that activation of the MAPK/ERK signaling could promote cell proliferation through increasing the level of phosphorylated RSK [18,20]. The MC-LR-induced cell toxicity was mainly mediated through inhibition of PP2A [21]. Taken together, the ERK/RSK signaling is supposed to be involved in MC-LR-induced biliary epithelial cell proliferation. In this study, we firstly investigated the effect of MC-LR on intrahepatic biliary epithelial cell proliferation with respect to its interaction with the ERK-RSK signaling in vitro and in vivo systems, which has helped us to gain insight into the mechanisms of MC-LRinduced proliferation in intrahepatic biliary epithelial cells.
2.4. PP2A activity
2. Materials and methods
This was determined as reported [26], by using a PP2A immunoprecipitation phosphatase assay (Millipore, Billerica, MA) that measures free phosphate with a malachite green dye. Total protein from the control and MC-LR-treated was extracted with lysis buffer. To immunoprecipitate PP2A, lysates containing 200 mg of protein were incubated with 4 mg of anti-PP2A, C subunit antibody and 40 ml of protein A-agarose slurry for 2 h at 4 C with constant rocking. The immunoprecipitates were washed three times in Tris-buffered saline and once with Ser/Thr assay buffer. The reaction was initiated by the addition of 60 ml of phosphopeptide substrate. Following incubation at 30 C for 10 min in a shaking incubator, the reaction mixture was centrifuged briefly, and the supernatant was transferred to a 96-well microtiter plate. The reaction was terminated by adding malachite green phosphate detection solution for 15 min at room temperature, and free phosphate was quantified by measuring the absorbance of the mixture at 650 nm using a microplate reader.
2.1. Main reagents and cell culture
2.5. EdU incorporation assay
MC-LR was purchased from Alexis Biochemicals (Lausen, Switzerland). MC-LR (1 mg) was dissolved in 100 ml DMSO and diluted to 1 mL with DMEM to prepare the stock solution (1 mM). PD98059 (the inhibitor for ERK1/2 MAP kinase pathway) and BID1870 (the inhibitor for RSK1/2/3/4) were purchased from selleckchem (Shanghai, China). Human primary intrahepatic biliary epithelial cells (HiBECs) were purchased from ScienCell Research Laboratories (San Diego, CA). HiBECs were cultured in high DMEM media (Hyclone) supplemented 10% FBS (Gibco), 1% L-glutamine, 1% penicillin and streptomycin. When HiBECs reached 80% confluence, they were routinely passaged using 0.25% trypsin and were diluted in a 6-well culture plate. After HiBECs reached 60e80% confluence, we discarded the medium and added the fresh high DMEM media containing various concentrations of MC-LR (0, 0.5, 5, 50 or 500 nM) for 24 h or added the fresh high DMEM media containing MC-LR (50 nM) for 24 h. In some experiments, HiBECs were pretreated for 30 min with various chemical inhibitors, such as ERK inhibitor or RSK inhibitor. HiBECs were characterized using a previously described immunofluorescence microscopic method with antibodies to cytokeratin 7 (Proteintech, Wuhan, China) and cytokeratin 19 (Boster, Wuhan, China), which labeled > 90% of the cells in culture [22e24].
Cell proliferation was assessed by 5-ethynyl-2-deoxyuridine (EdU) incorporation using the Click-iT EdU Alexa Fluor 488 cell proliferation assay kit (Invitrogen, Carlsbad, CA) as previously described [27]. 2.6. CCK-8 assay Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) was used to evaluate the cell viability according to the manufacturer’s instructions. HiBECs were treated with various concentrations of MC-LR (0, 0.5, 5, 50 or 500 nM) in 96-well culture plate and incubated for 24 h. Then CCK-8 solutions were added and incubated for another 2 h to measure optical density (OD) at 450 nm. 2.7. Histopathology The mouse livers were fixed in 10% formalin for 24 h at room temperature, dehydrated, transparentized and embedded in paraffin before sectioning into 5 mm-thick slices. Then the sections were staining with hematoxylin and eosin (H&E) for structured observation and immunohistochemistry/immunofluorescence analyses. 2.8. Immunohistochemical analyses and immunofluorescent staining
2.2. Animals and treatment Male specific pathogen-free (SPF) BALB/C mice (6e7 week old) weighing from 20 to 25 g were purchased from Experimental Animal Center of the Academy of Military Medical Science Institute, China. After 1 week of acclimation, mice were given drinking water containing 1, 7.5, 15 or 30 mg/L MC-LR for 180 consecutive days. For the control group, mice were provided with only the blank water. The actual daily exposure dose of MC-LR was performed as previously described [25]. All procedures carried out on animals were approved by the Animal Care and Use Committee of Nanjing University under the animal protocol number SYXK (Su) 2009-0017. 2.3. Western blotting The liver or cell extract protein was lysed with RIPA buffer (Beyotime, Shanghai, China) and the protein concentration was determined using Coomassie Brilliant Blue (Beyotime, Shanghai, China). We have provided a detailed description of the method in the Supplementary Methods.
We have provided a detailed description of the method in the Supplementary Methods. 2.9. Flow cytometric analysis Flow cytometry was performed to analyze the cell cycle distribution. HiBECs were washed with PBS and fixed in 75% cold ethanol overnight at 4 C. Before cell cycle analysis, the fixed cells were washed with PBS. Then the cells were resuspended in propidium iodide (PI)/DNA staining solution and incubated for 30 min at room temperature in the dark. Samples were analyzed for cell cycle with a BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data were analyzed using Flow Jo. Experiments were performed independently at least thrice in duplicates. 2.10. Statistical analyses Experimental results were expressed as means ± standard deviation (SD). All calculations and statistical analyses were
Please cite this article as: M. Yan et al., The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.143
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performed using Graph-Pad Prism 5 (USA). One-way analysis of variance (ANOVA) was used to analyze the difference between groups, followed by Tukey s Multiple Comparison test. P < 0.05 was regarded as statistically significant. 3. Results 3.1. MC-LR induced hyperplasia of biliary epithelial cell in the liver of mice To explore the influence of MC-LR on intrahepatic biliary
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epithelial cells, mice were treated with 1, 7.5, 15 or 30 mg/L MC-LR for 180 consecutive days. Under normal circumstances, there is no abnormal hyperplasia in the interlobular bile duct. In mice treated with 30 mg/L MC-LR, we found that the inflammatory cell infiltration and thickening of the bile duct in the portal area (Fig. 1A). To evaluate MC-LR-induced intrahepatic bile duct hyperplasia in mice, we examined biliary epithelial cell marker protein cytokeratin 19. It was shown that there were more cytokeratin 19 positive cells in the liver following exposure to 7.5, 15 or 30 mg/L MC-LR (Fig. 1B). Protein expression of cytokeratin 19 was also elevated and detected by western blotting (Fig. 1C). Moreover, we
Fig. 1. Mice were administered with microcystin-leucine-arginine (MC-LR) at 1 mg/kg, 7.5 mg/kg, 15 mg/kg and 30 mg/kg through drinking water for 180 consecutive days. (A) The structure of histological sections of intrahepatic bile duct was observed by H&E staining (scale bar ¼ 200 mm). (B) Liver tissue content of cytokeratin 19 was measured by immunohistochemical staining (scale bar ¼ 200 mm). Lower pictures correspond to magnified boxed areas (scale bar ¼ 100 mm). (C) Liver tissue content of cytokeratin 19 was measured by western blotting (left). The expression levels of the proteins were quantified by densitometry and normalized to the expression of GAPDH (right). Data are shown as means ± SD. *P < 0.05, vs control. (D) Expression of Ki67 in the liver was examined by immunohistochemistry (scale bar ¼ 200 mm). Lower pictures correspond to magnified boxed areas (scale bar ¼ 100 mm).
Please cite this article as: M. Yan et al., The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.143
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examined the expression of Ki67 in the intrahepatic bile duct and Ki67 increased significantly after MC-LR-treated in the mice liver (Fig. 1D).
3.2. MC-LR could be taken by biliary epithelial cell and promote cell proliferation In this study, we examined the distribution of MC-LR in liver by Immunofluorescence, and the results demonstrated that MC-LR could be transported into intrahepatic biliary epithelial cells (Fig. 2A). Besides, MC-LR could enter into HiBECs, as assessed by immunofluorescent staining (Fig. 2B) and western blotting (Fig. 2C). PP2A is emerging as a critical regulator of the microcystininduced molecular network. We measured the activity of PP2A in HiBECs after treated with MC-LR. PP2A activity was significantly attenuated compared to the control group (Fig. 2D). To evaluate the toxic effects of MC-LR on HiBECs, we examined the cell viability by CCK-8 assay after MC-LR treatment, and the results showed that the cell viability of HiBECs after MC-LR-treated was significantly enhanced (Fig. 2E). By the use of flow cytometry analysis, it was
shown that the cell percentage in S phase was markedly increased in HiBECs after exposure to 0.5, 5 or 50 nM MC-LR for 24 h (Fig. 2F). However, MC-LR treatment at 500 nM showed no significant change in S phase. We further examined the effect on MC-LRstimulated cell proliferation by EdU incorporation. The HiBECs treated with 0.5, 5 or 50 nM MC-LR heightened the rate of EdUpositive cells (Supplementary Fig. 1). Taken together, these results provided strong evidence to show that MC-LR promoted HiBECs proliferation.
3.3. The ERK-RSK signaling was activated during MC-LR-promoting HiBECs proliferation ERK-RSK signaling was found to be responsible for its proliferative action [20]. We found that high expression of phosphorylated MEK, phosphorylated ERK and phosphorylated RSK was observed in the intrahepatic portal area (Fig. 3A). In vitro, MC-LR exposure at 0.5, 50 or 500 nM could up-regulate phosphorylation of MEK, ERK and RSK (Fig. 3B). To further define the role of ERK-RSK signaling in the proliferation of HiBECs induced by MC-LR, we investigated
Fig. 2. MC-LR can be taken by biliary epithelial cell and promote cell proliferation. (A) The expression of MC-LR and cytokeratin 7 in mouse liver was detected by immunofluorescence assay (scale bar ¼ 50 mm). (B) Human intrahepatic biliary epithelial cells (HiBECs) were treated with 0.5 nM, 500 nM MC-LR for 24 h. Expression of MC-LR was determined by immunofluorescence staining (scale bar ¼ 50 mm). (C) Expression of MC-LR in HiBECs was detected by western blotting. (D) PP2A activity was measured using an immunoprecipitation assay kit. (E) Measurement of cell viability was carried out with Cell Counting Kit (CCK-8) assay. (F) HiBECs were stained with PI and then analyzed by flow cytometry. All data were expressed as means ± SD. *P < 0.05, vs control.
Please cite this article as: M. Yan et al., The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.143
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Fig. 3. MC-LR-induced phosphorylation of MEK, ERK and RSK in vivo and vitro. (A) The liver of MC-LR treated mice was analyzed by immunohistochemistry. The sections were stained with anti-p-MEK, anti-p-ERK and anti-p-RSK antibody. Images were obtained from 30 mg/L MC-LR treatment group and control group (scale bar ¼ 100 mm). (B) HiBECs were treated with or without MC-LR (0.5 nM, 5 nM, 50 nM, 500 nM) for 24 h. Protein levels of p-MEK, total MEK, p-ERK, total ERK, p-RSK and total RSK were examined by western blotting; the ratios of p-ERK/ERK and p-RSK/RSK were determined by densitometry and were expressed as means ± SD. *p < 0.05, vs control.
whether targeted inhibition of this signaling could attenuate the proliferative capacity. We used PD98059 almost completely abolished the phosphorylation of ERK caused by 50 nM MC-LR-treated. At the same time, the degree of phosphorylation of RSK was also suppressed (Fig. 4A). Besides, viability of HiBECs after treated with PD98059 in presence or absence of MC-LR was assessed by cell cycle experiment and the cell percentage in S phase was suppressed when cells were treated with MC-LR and PD98059 (Fig. 4B). CCK-8 assay showed that PD98059 attenuated cell viability in MC-LR treated cells (Fig. 4C). Thus, EdU-positive cells of MC-LR-treated were reduced by treating with PD98059 (Fig. 4D). We further examined the effect of RSK inhibitor BI-D1870 on MC-LRstimulated cell cycle, CCK-8 and EdU incorporation in HiBECs and confirmed the similar results (Supplementary Fig. 2). All above evidence indicated that MC-LR could promote the proliferation of HiBECs via the activation of ERK/RSK signaling. 4. Discussion MC-LR is a potent specific hepatotoxin and has been documented to induce hepatocyte apoptosis and liver injury [28]. Nevertheless, to our knowledge, there are few researches conducted to investigate the effects of prolonged MC-LR exposure on the proliferation of intrahepatic biliary epithelial cells. In this study, we have provided the initial evidence that ERK-RSK signaling is clearly associated with MC-LR-induced intrahepatic bile duct hyperplasia in vivo and in vitro. As no previous work was studied about the phenomenon, it is the first time we observed this effect in intrahepatic biliary epithelial cells and the proliferative effect.
A number of researches elucidated that MC-LR is highly liverspecific, causing irreversible hepatotoxicity in animals and humans. Previous studies showed that MC-LR could be up-taken by liver tissues [29,30]. In mouse chronic exposure models, we also found that MC-LR could be detected in the intrahepatic bile duct by immunofluorescence colocalization. The presence of MC-LR in human intrahepatic biliary epithelial cells treated with MC-LR at 0.5 nM or 500 nM for 24 h was observed by immunofluorescence staining and western blotting. These results indicated that MC-LR could enter HiBECs, which were similar to the phenomenon observed in human hepatocytes [9]. The intrahepatic bile duct is a highly organized tubular structure consisting of biliary epithelial cells, which drains bile produced by hepatocytes into the duodenum. Intrahepatic biliary epithelial cells are the target of biliary tract disease, such as PBC and PSC, which are associated with dysregulation of intrahepatic biliary epithelial cells proliferation/loss [31]. Normally, intrahepatic biliary epithelial cells are mitotically quiescent but following damage they begin to proliferate to repair the biliary tree to compensate for damage and loss of functionality [32,33]. In this study, we focused on the effects of MC-LR on intrahepatic biliary epithelial cells. Following exposure to MC-LR for 180 days, inflammatory infiltration and bile duct hyperplasia in the mouse portal were observed in the 15 and 30 mg/L MC-LR groups. HiBECs showed apparent proliferation after exposure to 50 nM MC-LR for 24 h via CCK-8 and cell cycle analysis. However, there was little difference compared with the control group after 500 nM treatment. Collectively, MC-LR could promote proliferation of biliary epithelial cells in the liver, which may increase the risks of biliary tract disease.
Please cite this article as: M. Yan et al., The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.143
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Fig. 4. Blocking ERK signaling with PD98059 inhibits proliferation of HiBECs. (A) Expressions of p-ERK, ERK, p-RSK, RSK were detected by western blotting. (B) Cell cycle was evaluated by flow cytometry measuring propidium iodide (PI) expression. (C) Cell proliferation was measured with the CCK-8 assay. (D) Culture medium were pulsed with EdU for 4 h of the 24-h incubation period and subsequently stained with EdU Click-iT reaction mixture (green) (scale bar ¼ 50 mm). (E) Schematic representation of roles of ERK-RSK signaling in MC-LR-induced cell proliferation. All data were expressed as means ± SD. *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Cell proliferation is the process by which cells increase their mass by upregulating the biosynthesis of macromolecules, membranes and organelles. In addition, many signals also play an important role in this process, as shown in Fig. 4E, ERK-RSK signaling was found to be responsible for proliferative action of various cell types. In the pathological process of the liver disease and hepatocarcinoma, extracellular signal-regulated kinase (ERK) signaling is an important participant [21,34]. The 90 kDa ribosomal S6 kinases (RSK) are a family of Ser/Thr kinases that lie downstream of the MAPK-ERK cascade. The RSK are directly activated by ERK in response to growth factors, many polypeptide hormones, neurotransmitters, chemokines and other stimuli [35]. The RSK phosphorylate many cytosolic and nuclear targets and they have been implicated in the regulation of diverse cellular processes, including cell proliferation, cell survival, cell growth and motility [36]. Our results confirmed that the ERK signaling was involved in the process of cell proliferation and the MC-LR-related effect could be inhibited by PD98059. Furthermore, we found that MC-LR-induced cell proliferation was attenuated after inhibiting RSK activation. Taken together, it is possible that the phosphorylation of ERK and
RSK followed treatment of MC-LR contributes to MC-LR-induced cell proliferation. In conclusion, our study indicates that MC-LR consistently accumulates in the mice liver intrahepatic bile duct within a long period of time. Meanwhile, the ERK-RSK signaling is activated and interacts to promote HiBECs proliferation. And these findings extend our knowledge about MC-LR-induced environmental liver diseases and the related mechanisms. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by National Natural Science Foundation of China (81470866, 31870492, and 31670519). Key Medical Subjects of Jiangsu Province (ZDRCA2016057).
Please cite this article as: M. Yan et al., The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.143
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Please cite this article as: M. Yan et al., The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.143