Mol. Cells 2020; 43(3): 264-275
Published online March 9, 2020
https://doi.org/10.14348/molcells.2020.2197
© The Korean Society for Molecular and Cellular Biology
Correspondence to : shback@ulsan.ac.kr
Reactive oxygen species (ROS) play a significant role in intracellular signaling and regulation, particularly when they are maintained at physiologic levels. However, excess ROS can cause cell damage and induce cell death. We recently reported that eIF2α phosphorylation protects hepatocytes from oxidative stress and liver fibrosis induced by fructose metabolism. Here, we found that hepatocyte-specific eIF2α phosphorylation-deficient mice have significantly reduced expression of the epidermal growth factor receptor (EGFR) and altered EGFR-mediated signaling pathways. EGFR-mediated signaling pathways are important for cell proliferation, differentiation, and survival in many tissues and cell types. Therefore, we studied whether the reduced amount of EGFR is responsible for the eIF2α phosphorylation-deficient hepatocytes’ vulnerability to oxidative stress. ROS such as hydrogen peroxide and superoxides induce both EGFR tyrosine phosphorylation and eIF2α phosphorylation. eIF2α phosphorylation-deficient primary hepatocytes, or EGFR knockdown cells, have decreased ROS scavenging ability compared to normal cells. Therefore, these cells are particularly susceptible to oxidative stress. However, overexpression of EGFR in these eIF2α phosphorylation-deficient primary hepatocytes increased ROS scavenging ability and alleviated ROS-mediated cell death. Therefore, we hypothesize that the reduced EGFR level in eIF2α phosphorylation-deficient hepatocytes is one of critical factors responsible for their susceptibility to oxidative stress.
Keywords eIF2α phos phorylation, epidermal growth factor receptor, hydrogen peroxide, menadione, reactive oxygen species
The epidermal growth factor receptor (EGFR), or ErbB1 receptor (ErbB1), is one of four members of the ErbB family of tyrosine kinase receptors. ErbB1 is expressed in various tissues and mediates cell proliferation, differentiation, migration and survival through various signaling pathways (Berasain and Avila, 2014). When a ligand such as epidermal growth factor (EGF) (Schneider and Wolf, 2009) binds to the EGFR, it forms homo- and hetero-dimers with all four family members (Berasain and Avila, 2014; Schneider and Wolf, 2009). Ligand-activated EGFR proteins are auto-phosphorylated and cross-phosphorylated at multiple tyrosine residues (such as Y1068), which are located in the C-terminal non-catalytic sequence (Sato, 2013). The autophosphorylated tyrosine residues are believed to serve as docking sites for a variety of signaling molecules (such as Src homology 2) that contain phosphotyrosine-binding domains (Carpenter and Cohen, 1990; Sato, 2013). Subsequently, the activated EGFR triggers the following four main downstream signaling pathways: the PI3K/Akt pathway, the phospholipase C-gamma (PLC-γ)/protein kinase C (PKC) pathway, the ras/raf/MEK/MAPK pathway (comprised of the activation of extracellular signal-regulated kinase [ERK] and JUN N-terminal kinase [JNK]), and the signal transducers and activators of transcription (STATs) pathway (Citri and Yarden, 2006; Jorissen et al., 2003; Liebmann, 2011). The four signaling pathways collectively control cell proliferation, differentiation, migration, and survival.
Apart from ligand-dependent EGFR tyrosine activation, accumulating evidence has shown that reactive oxygen species (ROS) are involved in another mechanism of EGFR transactivation (Abdelmohsen et al., 2003; Chiarugi and Buricchi, 2007; Filosto et al., 2011; Gamou and Shimizu, 1995; Kim et al., 2015; Weng et al., 2018). Diverse modes of EGFR activation have been suggested in which ROS are directly or indirectly involved in EGFR transactivation (Heppner and van der Vliet, 2016; Weng et al., 2018). First, ROS directly modulate a specific cysteine residue (Cys797) within the EGFR kinase domain that is associated with increased tyrosine kinase activity (Paulsen et al., 2011). Next, the ROS induce oxidation of reduction-oxidation targets, such as protein tyrosine (Tyr) phosphatases (PTPs), to enhance EGFR Tyr phosphorylation (Lee et al., 1998). Finally, ROS induce cysteine oxidation within Src and ADAM17, which results in their activation and further EGFR transactivation (Sham et al., 2013).
ROS-involved EGFR transactivation can initiate multiple signaling pathways that are similar to ligand-dependent EGFR tyrosine activation. Ultimately, this signaling helps to protect cells against oxidative stress. Oxidative stress activates AKT via the EGFR/PI3K-dependent pathway in a number of cell types (Wang et al., 2000). After activation of the EGFR/PI3K-dependent pathway, the PI3K/Akt pathway inhibits glycogen synthase kinase 3β (GSK-3β) and thereby induces nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) (Chowdhry et al., 2013; Ma, 2013). Nuclear factor erythroid 2-related factor 2 subsequently induces the expression of an array of ROS-detoxifying enzymes and stimulates the production of antioxidants, including GSH (Ma, 2013). Activated AKT also phosphorylates and inactivates components of the cell death machinery including Bax, Bad, and caspase 9 (Brunet et al., 1999; Sadidi et al., 2009). Activated AKT also regulates the activity of FOXO3, which is a member of the Forkhead family of transcription factors that induces genes necessary for cell death (Brunet et al., 1999; Hagenbuchner et al., 2012). Therefore, AKT increases the cell’s antioxidant capacity and thereby promotes cell survival under oxidative stress in transcription-independent and/or dependent manners. Next, it has been previously shown that H2O2-induced EGFR transactivation increases PLC-γ1-mediated pro-survival function. Genetic ablation and pharmacological inhibition of PLC-γ1 enhances a cell’s susceptibility to hydrogen peroxide-induced cell death; however, it is not clear how activated PLC-γ1 mitigated the cellular oxidative damage (Goldkorn et al., 1998; Morita et al., 2008; Wang et al., 2001). Oxidative stress also triggers the phosphorylation of STAT proteins (STAT1, STAT3, and STAT5), which can induce genes that control cell growth and survival (Carballo et al., 1999; Miklossy et al., 2013; Rodriguez-Fragoso et al., 2009). During oxidative stress, EGFR transactivation is transferred by Janus kinases 1 and 2 (JAK1/2) or c-Src to STAT-1/3 and STAT-5. JAK1/2 and c-Src kinases modulate STATs activation in at least the following two ways: (1) by direct phosphorylation of STATs and (2) by phosphorylating the EGFR at Y845.
Eukaryotic translation initiation factor 2 alpha (eIF2α) is a subunit of the trimeric eIF2 complex that is involved in the initiation step of cap-dependent mRNA translation (Sonenberg and Hinnebusch, 2009). The eIF2 complex delivers the initiator methionyl-tRNA (Met-tRNAi) to the ribosome during translation initiation of cytoplasmic mRNAs in eukaryotic cells. Mammalian cells respond to various forms of physiological or pathological stress (such as hypoxia, amino acid deprivation, glucose deprivation, viral infection, endoplasmic reticulum [ER] stress, and oxidative stress) by blocking the translational initiation process through phosphorylation of eIF2α at Serine 51. Such stresses are sensed by the four mammalian eIF2α protein kinases: PERK, GCN2, PKR, and HRI, which respond to distinct stimuli (Proud, 2005; Wek et al., 2006). However, it has been reported that oxidative stress also activates multiple eIF2α kinases (including PERK, PKR, and GCN) and regulates the eIF2 complex through eIF2α phosphorylation (Liu et al., 2008; Rajesh et al., 2015). Therefore, several groups, including ours, have reported that genetic loss of the eIF2α kinases or eIF2α phosphorylation make cells susceptible to death by oxidative stress (Choi et al., 2017; Han et al., 2013; Lewerenz and Maher, 2009; Liu et al., 2008; Rajesh et al., 2015). In addition, our group recently suggested that fructose diet-induced hepatocyte death results from a diminished antioxidant capacity in hepatocyte-specific eIF2α phosphorylation deficient mice (Choi et al., 2017). However, we did not identify the upstream signaling pathways whose activation in response to oxidative stress may play important roles in maintaining the antioxidant capacity and alleviating oxidative hepatocyte damage. Here, we report that hepatocyte-specific eIF2α phosphorylation-deficient mice have significantly reduced expression of the EGFR and altered EGFR-mediated signaling pathways. Both the EGFR and EGFR-mediated signaling pathways are required for a cell to appropriately defend against oxidative stress. Therefore, EGFR knockdown cells have decreased ROS scavenging ability and aggravated oxidative stress-mediated cell death. Conversely, enhanced expression of EGFR increased ROS scavenging ability and alleviated ROS-mediated cell death in eIF2α phosphorylation-deficient primary hepatocytes. Therefore, our results suggest that a decreased EGFR level partially explains the susceptibility of eIF2α-phosphorylation-deficient hepatocytes to ROS.
The
Total RNAs were isolated from liver tissues using the Trizol reagent (Life Technologies, USA). cDNA was prepared with a High Capacity cDNA RT kit (Ambion, USA) for quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR was carried out using the SYBR green PCR master mix (Bio-Rad, USA) with the appropriate primers on a StepOnePlus Real Time System (Applied Biosystems, USA). The specificity of each primer pair was confirmed using melting curve analysis. The housekeeping
Liver tissues and cells were homogenized in Nonidet P40 lysis buffer (1% NP40, 50 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.05% SDS, 0.5 mM Na-vanadate, 100 mM NaF, 50 mM β-glycerophosphate, 1 mM PMSF) that was supplemented with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, USA). The homogenates were centrifuged at 12,000
Dichlorodihydrofluorescein diacetate (DCF-DA) was purchased from Thermo Fisher Scientific. The following were purchased from Sigma-Aldrich: crystal violet, parformaldehyde, polybrene (hexadimethrine bromide), hydrogen peroxide, menadione, propidium iodide (PI), Hoechst 33258, 4′,6-diamidino-2-phenylindole (DAPI), dihydroethidine hydrochloride (DHE), and N-acetyl-L-cysteine (NAC).
The primary mouse hepatocytes were obtained as has been described previously (Choi et al., 2017). The isolated primary hepatocytes were inoculated on collagen-coated plates with/without round coverslips (5 × 105 cells/well in 6-well plates), and cultured in high glucose-DMEM (WelGENE, Korea) medium containing 10% FBS (WelGENE), and 1% penicillin/ streptomycin (WelGENE). The medium was replaced with FBS-free DMEM media 2 h after plating. The hepatocytes were incubated for another 12 h before the experimental treatment.
Immortalized embryonic hepatocytes were cultured in Medium 199 (WelGENE) that was supplemented with 10% fetal bovine serum (WelGENE) and 1% penicillin-streptomycin (WelGENE), as previously described (Back et al., 2009). AML12 muse normal hepatocytes were obtained from the American Type Culture Collection (ATCC, USA). The cells were cultured in DMEM/F12 (WelGENE) that was supplemented with 10% fetal bovine serum (WelGENE), 100 nM dexamethason (Sigma-Aldrich), 1% insulin-transferrin-selenium-pyruvate supplement (ITSP; WelGENE), and 1% penicillin-streptomycin (WelGENE). The Lenti-X 293T Cell Line (Clontech, USA) was cultured in DMEM (WelGENE) containing 10% fetal bovine serum (WelGENE) and 1% penicillin-streptomycin (WelGENE).
The five MISSION shRNA clones of mouse
The C-terminal Flag tagged mouse EGFR (mEGFR-Flag) expression vector pCMV3-mEGFR-Flag was purchased from Sino Biological. The pShuttle-CMV-mEGFR-Flag vector was then constructed by inserting the cDNA fragment encoding mEGFR-Flag from the pCMV3-mEGFR-Flag treated with
Cell viability analysis using crystal violet was performed as has been previously described with slight modifications (Oh et al., 2006). In brief, the drug-treated cells were washed twice with cold phosphate-buffered saline (PBS). The cells were stained with crystal violet solution (0.625 g of crystal violet dissolved in a solution containing 50 ml of 37% paraformaldehyde and 450 ml of methanol) for 4 min at room temperature. The stained cells were washed three times in tap water, and the plates were allowed to dry. Microscopic images of the stained cells were taken with a Primovert inverted microscope (Zeiss, Germany) equipped with an Axiocam ERc 5s camera. The cells were then lysed with 1% SDS solution, and dye uptake was measured at 550 nm using a microplate reader (SpectraMax iD3; Molecular Devices, USA).
Cell death analysis using PI and Hoechst 33258 were performed as previously described (Choi et al., 2017). The drug-treated cells on the coverslips were double-stained with PI (1 μg/ml) and Hoechst 33258 (1 μg/ml), and fixed with 3.5% (w/v) paraformaldehyde at room temperature for 15 min. The coverslips were then mounted on glass slides for observation under fluorescence microscopy (Olympus microscope; Olympus, Japan). At least 500 cells were counted. A quantitation of dead cells (apoptotic and necrotic cells) was expressed as a percentage of total cells counted.
Intracellular ROS levels were measured using confocal microscopy using the fluorescent probes DHE or DCF-DA, as described previously (Choi et al., 2017). The drug-treated primary hepatocytes and immortalized hepatocytes were plated on collagen coated 35 mm coverglass bottom dishes (SPL, Korea) and cultured overnight. The next day, the cells were treated with the indicated chemicals. After treatment, the cells were stained with DHE (15 μM) or DCF-DA (15 μM) in phenol red-free culture medium for 30 min. Fluorescence images of the living cells were obtained using an FV1200-OSR confocal laser microscope (Olympus).
For the immunofluorescence detection of mEGFR-Flag proteins, immortalized embryonic hepatocytes (2 × 105) were plated on 6-well plates coated with 0.01% collagen in PBS and cultured overnight. The next day, the cells were infected with adenovirus encoding mEGFR-Flag (Ad-mEGFR-Flag) at a multiplicity of infection of 100 for 12 h. The cells were then fixed with 4% paraformaldehyde in PBS for 15 min, and permeabilized with 0.1% Triton X-100 in PBS for 4 min. The cells were blocked with 3% bovine serum albumin in PBS for 30 min, and incubated with the indicated primary antibody overnight at 4°C. The cells were further incubated with a Tetramethylrhodamine (TRITC)-conjugated secondary antibodies at room temperature for 1 h. The nuclei were stained with DAPI (Sigma-Aldrich). Finally, the cells were observed by confocal laser microscopy using an FV1200-OSR microscope.
The scale bars (μm) have been inserted into the microscopic images.
Experiments were repeated three times in each case. The data were analyzed using GraphPad Prism 5 (GraphPad Software, USA). Unpaired 2-tailed Student’s
The mRNA levels of EGFR (or the ErbB1 receptor) were diminished to ~40% in the liver tissues of hepatocyte-specific eIF2α phosphorylation (p-eIF2α)-deficient mice (Fig. 1A). Similarly, the levels of the EGFR (~60%) and phosphorylated EGFR (PY1068-EGFR, ~30%) proteins were significantly reduced in p-eIF2α deficient liver tissues (Figs. 1B and 1C).
In order to examine the effect of decreased EGFR in intact livers, starved control and
Several groups have reported that EGFR can be activated by oxidative stress (Abdelmohsen et al., 2003; Chiarugi and Buricchi, 2007; Filosto et al., 2011; Gamou and Shimizu, 1995; Kim et al., 2015; Wang et al., 2000; Weng et al., 2018) (Supplementary Fig. S1). In contrast, the inhibition or genetic disruption of EGFR is involved in the induction of oxidative stress and cytotoxicity
We previously showed that vulnerability to oxidative stress in
Next, in order to examine whether the level of EGFR expression can affect a cell’s antioxidant capacity, we established EGFR knockdown cell lines. These knockdown cell lines were created by transducing EGFR targeting shRNA-expressing lentiviruses and following puromycin selection in both immortalized mouse embryonic hepatocytes (Fig. 4A) and AML12 cells (Supplementary Fig. S2A). Among the established EGFR knockdown cell lines, the cell lines with the lowest expression of EGFR protein were chosen for further experiments. These included the shEGFR-83 cell line for immortalized hepatocytes (Fig. 4A) and the shEGFR-81 cell line for AML12 cells (Supplementary Fig. S2A). In order to assess the ROS-scavenging activity of the EGFR knockdown cell lines, the levels of accumulated ROS were compared to the fluorescence intensity of an oxidant-sensing probe DCF-DA in H2O2-treated shNC control or shEGFR-83 cell lines. Under H2O2-treated conditions, the fluorescence intensity of shEGFR-83 cells was significantly higher than that of shNC control cells (Fig. 4B). However, there was no significant difference in the fluorescence intensity between them before H2O2 treatment or after H2O2 and N-acetyl-L-cysteine (NAC) co-treatment. In addition, the fluorescent microscopic examination of menadione-treated cells (using a superoxide indicator DHE) revealed that DHE fluorescence in the EGFR knockdown cells (shEGFR) was stronger than that in shNC control cells (Fig. 4C). These results indicate that EGFR protein levels are important to maintain a cell’s hydrogen peroxide- or superoxide-scavenging activity.
Having observed that EGFR knockdown cells have reduced ROS-scavenging activity, we next examined whether knockdown of EGFR increases a cell’s vulnerability to oxidative stress. To do so, we applied the crystal violet assay and Hoechst 33258/PI staining analysis to compare cell death between EGFR knockdown cells and controls (Figs. 4D and 4E, Supplementary Figs. S2A, S2B, and S3). As expected, the crystal violet assay showed that the shEGFR-83 cells had poorer cell viability than did shNC control cells under menadione or H2O2 treatment although the cell viability of shNC control cells was also reduced in a dose-dependent manner in these treatments. However, the levels in the shEGFR-83 cells were similar to those in shNC control cells in NAC-treated conditions. The cell death analysis (using the Hoechst 33258/PI staining procedure) also revealed that shEGFR-83 cells had a higher rate of cell death than did the shNC control cells in menadione or H2O2 treated conditions (Fig. 4E). However, the levels in the shEGFR-83 cells were similar to those in shNC control cells in NAC-treated conditions. We obtained similar results in the shEGFR-81 cell line of AML12 cells (Supplementary Figs. S3B and S3C). The shEGFR-81 cells were more susceptible to menadione- or H2O2-mediated oxidative stress than were shNC control cells. These cell death analyses suggested that the EGFR protein is important to alleviate oxidative stress-mediated cell death. Collectively, our experiments suggest that attenuated EGFR expression reduces cellular ROS-scavenging activity, which then increases cells’ vulnerability to oxidative stress.
We next investigated whether transient enforced expression of EGFR in p-eIF2α deficient primary hepatocytes can increase cellular ROS-scavenging activity and therefore reduce their sensitivity to oxidative stress. Primary hepatocytes purified from livers of hepatocyte specific p-eIF2α deficient (
In this study, we showed that hepatocyte-specific eIF2α phosphorylation-deficient mice have significantly reduced expression of the EGFR and altered EGFR-mediated signaling pathways. Moreover, the eIF2α phosphorylation-deficiency or reduced expression of the EGFR decreased ROS-scavenging activity and increased ROS-mediated cell death in hepatocytes. However, the drawbacks were alleviated by enforced expression of EGFR in p-eIF2α deficient primary hepatocytes. Therefore, we concluded that reduced EGFR level in eIF2α phosphorylation-deficient hepatocytes is one of the critical factors responsible for a cell’s susceptibility to oxidative stress. Furthermore, vulnerability to oxidative stress in hepatocytes that have reduced EGFR level or EGFR activity might be the underlying mechanism of hepatotoxicity induced by several EGFR inhibitors (e.g., erlotinib, lapatinib, and gefitinib) used for the treatment of non-small cell lung cancer and pancreatic cancer (Kim et al., 2018; Paech et al., 2017), since most EGFR inhibitors undergo intense metabolism by cytochrome P450 enzymes in hepatocytes (Li et al., 2007; Rakhit et al., 2008) which produces reactive metabolites (Haouzi et al., 2000). However, further studies are required to verify whether the hepatotoxicity is related to oxidative stress susceptibility in EGFR inhibitor-treated hepatocytes.
The authors have no potential conflicts of interest to disclose.
This work was supported by the Basic Science Research Program (2014R1A1A4A01004329 and 2017R1D1A1B0 3028229), the Bio & Medical Technology Development Program (2017M3A9G7072745), and the Priority Research Centers Program (2014R1A6A1030318) of the National Research Foundation of Korea (NRF) funded by the Korean government.
Mol. Cells 2020; 43(3): 264-275
Published online March 31, 2020 https://doi.org/10.14348/molcells.2020.2197
Copyright © The Korean Society for Molecular and Cellular Biology.
Mi-Jeong Kim1,3 , Woo-Gyun Choi1,3
, Kyung-Ju Ahn1
, In Gyeong Chae1
, Rina Yu2
, and Sung Hoon Back1,*
1School of Biological Sciences and 2Department of Food Science and Nutrition, University of Ulsan, Ulsan 44610, Korea, 3These authors contributed equally to this work.
Correspondence to:shback@ulsan.ac.kr
Reactive oxygen species (ROS) play a significant role in intracellular signaling and regulation, particularly when they are maintained at physiologic levels. However, excess ROS can cause cell damage and induce cell death. We recently reported that eIF2α phosphorylation protects hepatocytes from oxidative stress and liver fibrosis induced by fructose metabolism. Here, we found that hepatocyte-specific eIF2α phosphorylation-deficient mice have significantly reduced expression of the epidermal growth factor receptor (EGFR) and altered EGFR-mediated signaling pathways. EGFR-mediated signaling pathways are important for cell proliferation, differentiation, and survival in many tissues and cell types. Therefore, we studied whether the reduced amount of EGFR is responsible for the eIF2α phosphorylation-deficient hepatocytes’ vulnerability to oxidative stress. ROS such as hydrogen peroxide and superoxides induce both EGFR tyrosine phosphorylation and eIF2α phosphorylation. eIF2α phosphorylation-deficient primary hepatocytes, or EGFR knockdown cells, have decreased ROS scavenging ability compared to normal cells. Therefore, these cells are particularly susceptible to oxidative stress. However, overexpression of EGFR in these eIF2α phosphorylation-deficient primary hepatocytes increased ROS scavenging ability and alleviated ROS-mediated cell death. Therefore, we hypothesize that the reduced EGFR level in eIF2α phosphorylation-deficient hepatocytes is one of critical factors responsible for their susceptibility to oxidative stress.
Keywords: eIF2α phos phorylation, epidermal growth factor receptor, hydrogen peroxide, menadione, reactive oxygen species
The epidermal growth factor receptor (EGFR), or ErbB1 receptor (ErbB1), is one of four members of the ErbB family of tyrosine kinase receptors. ErbB1 is expressed in various tissues and mediates cell proliferation, differentiation, migration and survival through various signaling pathways (Berasain and Avila, 2014). When a ligand such as epidermal growth factor (EGF) (Schneider and Wolf, 2009) binds to the EGFR, it forms homo- and hetero-dimers with all four family members (Berasain and Avila, 2014; Schneider and Wolf, 2009). Ligand-activated EGFR proteins are auto-phosphorylated and cross-phosphorylated at multiple tyrosine residues (such as Y1068), which are located in the C-terminal non-catalytic sequence (Sato, 2013). The autophosphorylated tyrosine residues are believed to serve as docking sites for a variety of signaling molecules (such as Src homology 2) that contain phosphotyrosine-binding domains (Carpenter and Cohen, 1990; Sato, 2013). Subsequently, the activated EGFR triggers the following four main downstream signaling pathways: the PI3K/Akt pathway, the phospholipase C-gamma (PLC-γ)/protein kinase C (PKC) pathway, the ras/raf/MEK/MAPK pathway (comprised of the activation of extracellular signal-regulated kinase [ERK] and JUN N-terminal kinase [JNK]), and the signal transducers and activators of transcription (STATs) pathway (Citri and Yarden, 2006; Jorissen et al., 2003; Liebmann, 2011). The four signaling pathways collectively control cell proliferation, differentiation, migration, and survival.
Apart from ligand-dependent EGFR tyrosine activation, accumulating evidence has shown that reactive oxygen species (ROS) are involved in another mechanism of EGFR transactivation (Abdelmohsen et al., 2003; Chiarugi and Buricchi, 2007; Filosto et al., 2011; Gamou and Shimizu, 1995; Kim et al., 2015; Weng et al., 2018). Diverse modes of EGFR activation have been suggested in which ROS are directly or indirectly involved in EGFR transactivation (Heppner and van der Vliet, 2016; Weng et al., 2018). First, ROS directly modulate a specific cysteine residue (Cys797) within the EGFR kinase domain that is associated with increased tyrosine kinase activity (Paulsen et al., 2011). Next, the ROS induce oxidation of reduction-oxidation targets, such as protein tyrosine (Tyr) phosphatases (PTPs), to enhance EGFR Tyr phosphorylation (Lee et al., 1998). Finally, ROS induce cysteine oxidation within Src and ADAM17, which results in their activation and further EGFR transactivation (Sham et al., 2013).
ROS-involved EGFR transactivation can initiate multiple signaling pathways that are similar to ligand-dependent EGFR tyrosine activation. Ultimately, this signaling helps to protect cells against oxidative stress. Oxidative stress activates AKT via the EGFR/PI3K-dependent pathway in a number of cell types (Wang et al., 2000). After activation of the EGFR/PI3K-dependent pathway, the PI3K/Akt pathway inhibits glycogen synthase kinase 3β (GSK-3β) and thereby induces nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) (Chowdhry et al., 2013; Ma, 2013). Nuclear factor erythroid 2-related factor 2 subsequently induces the expression of an array of ROS-detoxifying enzymes and stimulates the production of antioxidants, including GSH (Ma, 2013). Activated AKT also phosphorylates and inactivates components of the cell death machinery including Bax, Bad, and caspase 9 (Brunet et al., 1999; Sadidi et al., 2009). Activated AKT also regulates the activity of FOXO3, which is a member of the Forkhead family of transcription factors that induces genes necessary for cell death (Brunet et al., 1999; Hagenbuchner et al., 2012). Therefore, AKT increases the cell’s antioxidant capacity and thereby promotes cell survival under oxidative stress in transcription-independent and/or dependent manners. Next, it has been previously shown that H2O2-induced EGFR transactivation increases PLC-γ1-mediated pro-survival function. Genetic ablation and pharmacological inhibition of PLC-γ1 enhances a cell’s susceptibility to hydrogen peroxide-induced cell death; however, it is not clear how activated PLC-γ1 mitigated the cellular oxidative damage (Goldkorn et al., 1998; Morita et al., 2008; Wang et al., 2001). Oxidative stress also triggers the phosphorylation of STAT proteins (STAT1, STAT3, and STAT5), which can induce genes that control cell growth and survival (Carballo et al., 1999; Miklossy et al., 2013; Rodriguez-Fragoso et al., 2009). During oxidative stress, EGFR transactivation is transferred by Janus kinases 1 and 2 (JAK1/2) or c-Src to STAT-1/3 and STAT-5. JAK1/2 and c-Src kinases modulate STATs activation in at least the following two ways: (1) by direct phosphorylation of STATs and (2) by phosphorylating the EGFR at Y845.
Eukaryotic translation initiation factor 2 alpha (eIF2α) is a subunit of the trimeric eIF2 complex that is involved in the initiation step of cap-dependent mRNA translation (Sonenberg and Hinnebusch, 2009). The eIF2 complex delivers the initiator methionyl-tRNA (Met-tRNAi) to the ribosome during translation initiation of cytoplasmic mRNAs in eukaryotic cells. Mammalian cells respond to various forms of physiological or pathological stress (such as hypoxia, amino acid deprivation, glucose deprivation, viral infection, endoplasmic reticulum [ER] stress, and oxidative stress) by blocking the translational initiation process through phosphorylation of eIF2α at Serine 51. Such stresses are sensed by the four mammalian eIF2α protein kinases: PERK, GCN2, PKR, and HRI, which respond to distinct stimuli (Proud, 2005; Wek et al., 2006). However, it has been reported that oxidative stress also activates multiple eIF2α kinases (including PERK, PKR, and GCN) and regulates the eIF2 complex through eIF2α phosphorylation (Liu et al., 2008; Rajesh et al., 2015). Therefore, several groups, including ours, have reported that genetic loss of the eIF2α kinases or eIF2α phosphorylation make cells susceptible to death by oxidative stress (Choi et al., 2017; Han et al., 2013; Lewerenz and Maher, 2009; Liu et al., 2008; Rajesh et al., 2015). In addition, our group recently suggested that fructose diet-induced hepatocyte death results from a diminished antioxidant capacity in hepatocyte-specific eIF2α phosphorylation deficient mice (Choi et al., 2017). However, we did not identify the upstream signaling pathways whose activation in response to oxidative stress may play important roles in maintaining the antioxidant capacity and alleviating oxidative hepatocyte damage. Here, we report that hepatocyte-specific eIF2α phosphorylation-deficient mice have significantly reduced expression of the EGFR and altered EGFR-mediated signaling pathways. Both the EGFR and EGFR-mediated signaling pathways are required for a cell to appropriately defend against oxidative stress. Therefore, EGFR knockdown cells have decreased ROS scavenging ability and aggravated oxidative stress-mediated cell death. Conversely, enhanced expression of EGFR increased ROS scavenging ability and alleviated ROS-mediated cell death in eIF2α phosphorylation-deficient primary hepatocytes. Therefore, our results suggest that a decreased EGFR level partially explains the susceptibility of eIF2α-phosphorylation-deficient hepatocytes to ROS.
The
Total RNAs were isolated from liver tissues using the Trizol reagent (Life Technologies, USA). cDNA was prepared with a High Capacity cDNA RT kit (Ambion, USA) for quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR was carried out using the SYBR green PCR master mix (Bio-Rad, USA) with the appropriate primers on a StepOnePlus Real Time System (Applied Biosystems, USA). The specificity of each primer pair was confirmed using melting curve analysis. The housekeeping
Liver tissues and cells were homogenized in Nonidet P40 lysis buffer (1% NP40, 50 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.05% SDS, 0.5 mM Na-vanadate, 100 mM NaF, 50 mM β-glycerophosphate, 1 mM PMSF) that was supplemented with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, USA). The homogenates were centrifuged at 12,000
Dichlorodihydrofluorescein diacetate (DCF-DA) was purchased from Thermo Fisher Scientific. The following were purchased from Sigma-Aldrich: crystal violet, parformaldehyde, polybrene (hexadimethrine bromide), hydrogen peroxide, menadione, propidium iodide (PI), Hoechst 33258, 4′,6-diamidino-2-phenylindole (DAPI), dihydroethidine hydrochloride (DHE), and N-acetyl-L-cysteine (NAC).
The primary mouse hepatocytes were obtained as has been described previously (Choi et al., 2017). The isolated primary hepatocytes were inoculated on collagen-coated plates with/without round coverslips (5 × 105 cells/well in 6-well plates), and cultured in high glucose-DMEM (WelGENE, Korea) medium containing 10% FBS (WelGENE), and 1% penicillin/ streptomycin (WelGENE). The medium was replaced with FBS-free DMEM media 2 h after plating. The hepatocytes were incubated for another 12 h before the experimental treatment.
Immortalized embryonic hepatocytes were cultured in Medium 199 (WelGENE) that was supplemented with 10% fetal bovine serum (WelGENE) and 1% penicillin-streptomycin (WelGENE), as previously described (Back et al., 2009). AML12 muse normal hepatocytes were obtained from the American Type Culture Collection (ATCC, USA). The cells were cultured in DMEM/F12 (WelGENE) that was supplemented with 10% fetal bovine serum (WelGENE), 100 nM dexamethason (Sigma-Aldrich), 1% insulin-transferrin-selenium-pyruvate supplement (ITSP; WelGENE), and 1% penicillin-streptomycin (WelGENE). The Lenti-X 293T Cell Line (Clontech, USA) was cultured in DMEM (WelGENE) containing 10% fetal bovine serum (WelGENE) and 1% penicillin-streptomycin (WelGENE).
The five MISSION shRNA clones of mouse
The C-terminal Flag tagged mouse EGFR (mEGFR-Flag) expression vector pCMV3-mEGFR-Flag was purchased from Sino Biological. The pShuttle-CMV-mEGFR-Flag vector was then constructed by inserting the cDNA fragment encoding mEGFR-Flag from the pCMV3-mEGFR-Flag treated with
Cell viability analysis using crystal violet was performed as has been previously described with slight modifications (Oh et al., 2006). In brief, the drug-treated cells were washed twice with cold phosphate-buffered saline (PBS). The cells were stained with crystal violet solution (0.625 g of crystal violet dissolved in a solution containing 50 ml of 37% paraformaldehyde and 450 ml of methanol) for 4 min at room temperature. The stained cells were washed three times in tap water, and the plates were allowed to dry. Microscopic images of the stained cells were taken with a Primovert inverted microscope (Zeiss, Germany) equipped with an Axiocam ERc 5s camera. The cells were then lysed with 1% SDS solution, and dye uptake was measured at 550 nm using a microplate reader (SpectraMax iD3; Molecular Devices, USA).
Cell death analysis using PI and Hoechst 33258 were performed as previously described (Choi et al., 2017). The drug-treated cells on the coverslips were double-stained with PI (1 μg/ml) and Hoechst 33258 (1 μg/ml), and fixed with 3.5% (w/v) paraformaldehyde at room temperature for 15 min. The coverslips were then mounted on glass slides for observation under fluorescence microscopy (Olympus microscope; Olympus, Japan). At least 500 cells were counted. A quantitation of dead cells (apoptotic and necrotic cells) was expressed as a percentage of total cells counted.
Intracellular ROS levels were measured using confocal microscopy using the fluorescent probes DHE or DCF-DA, as described previously (Choi et al., 2017). The drug-treated primary hepatocytes and immortalized hepatocytes were plated on collagen coated 35 mm coverglass bottom dishes (SPL, Korea) and cultured overnight. The next day, the cells were treated with the indicated chemicals. After treatment, the cells were stained with DHE (15 μM) or DCF-DA (15 μM) in phenol red-free culture medium for 30 min. Fluorescence images of the living cells were obtained using an FV1200-OSR confocal laser microscope (Olympus).
For the immunofluorescence detection of mEGFR-Flag proteins, immortalized embryonic hepatocytes (2 × 105) were plated on 6-well plates coated with 0.01% collagen in PBS and cultured overnight. The next day, the cells were infected with adenovirus encoding mEGFR-Flag (Ad-mEGFR-Flag) at a multiplicity of infection of 100 for 12 h. The cells were then fixed with 4% paraformaldehyde in PBS for 15 min, and permeabilized with 0.1% Triton X-100 in PBS for 4 min. The cells were blocked with 3% bovine serum albumin in PBS for 30 min, and incubated with the indicated primary antibody overnight at 4°C. The cells were further incubated with a Tetramethylrhodamine (TRITC)-conjugated secondary antibodies at room temperature for 1 h. The nuclei were stained with DAPI (Sigma-Aldrich). Finally, the cells were observed by confocal laser microscopy using an FV1200-OSR microscope.
The scale bars (μm) have been inserted into the microscopic images.
Experiments were repeated three times in each case. The data were analyzed using GraphPad Prism 5 (GraphPad Software, USA). Unpaired 2-tailed Student’s
The mRNA levels of EGFR (or the ErbB1 receptor) were diminished to ~40% in the liver tissues of hepatocyte-specific eIF2α phosphorylation (p-eIF2α)-deficient mice (Fig. 1A). Similarly, the levels of the EGFR (~60%) and phosphorylated EGFR (PY1068-EGFR, ~30%) proteins were significantly reduced in p-eIF2α deficient liver tissues (Figs. 1B and 1C).
In order to examine the effect of decreased EGFR in intact livers, starved control and
Several groups have reported that EGFR can be activated by oxidative stress (Abdelmohsen et al., 2003; Chiarugi and Buricchi, 2007; Filosto et al., 2011; Gamou and Shimizu, 1995; Kim et al., 2015; Wang et al., 2000; Weng et al., 2018) (Supplementary Fig. S1). In contrast, the inhibition or genetic disruption of EGFR is involved in the induction of oxidative stress and cytotoxicity
We previously showed that vulnerability to oxidative stress in
Next, in order to examine whether the level of EGFR expression can affect a cell’s antioxidant capacity, we established EGFR knockdown cell lines. These knockdown cell lines were created by transducing EGFR targeting shRNA-expressing lentiviruses and following puromycin selection in both immortalized mouse embryonic hepatocytes (Fig. 4A) and AML12 cells (Supplementary Fig. S2A). Among the established EGFR knockdown cell lines, the cell lines with the lowest expression of EGFR protein were chosen for further experiments. These included the shEGFR-83 cell line for immortalized hepatocytes (Fig. 4A) and the shEGFR-81 cell line for AML12 cells (Supplementary Fig. S2A). In order to assess the ROS-scavenging activity of the EGFR knockdown cell lines, the levels of accumulated ROS were compared to the fluorescence intensity of an oxidant-sensing probe DCF-DA in H2O2-treated shNC control or shEGFR-83 cell lines. Under H2O2-treated conditions, the fluorescence intensity of shEGFR-83 cells was significantly higher than that of shNC control cells (Fig. 4B). However, there was no significant difference in the fluorescence intensity between them before H2O2 treatment or after H2O2 and N-acetyl-L-cysteine (NAC) co-treatment. In addition, the fluorescent microscopic examination of menadione-treated cells (using a superoxide indicator DHE) revealed that DHE fluorescence in the EGFR knockdown cells (shEGFR) was stronger than that in shNC control cells (Fig. 4C). These results indicate that EGFR protein levels are important to maintain a cell’s hydrogen peroxide- or superoxide-scavenging activity.
Having observed that EGFR knockdown cells have reduced ROS-scavenging activity, we next examined whether knockdown of EGFR increases a cell’s vulnerability to oxidative stress. To do so, we applied the crystal violet assay and Hoechst 33258/PI staining analysis to compare cell death between EGFR knockdown cells and controls (Figs. 4D and 4E, Supplementary Figs. S2A, S2B, and S3). As expected, the crystal violet assay showed that the shEGFR-83 cells had poorer cell viability than did shNC control cells under menadione or H2O2 treatment although the cell viability of shNC control cells was also reduced in a dose-dependent manner in these treatments. However, the levels in the shEGFR-83 cells were similar to those in shNC control cells in NAC-treated conditions. The cell death analysis (using the Hoechst 33258/PI staining procedure) also revealed that shEGFR-83 cells had a higher rate of cell death than did the shNC control cells in menadione or H2O2 treated conditions (Fig. 4E). However, the levels in the shEGFR-83 cells were similar to those in shNC control cells in NAC-treated conditions. We obtained similar results in the shEGFR-81 cell line of AML12 cells (Supplementary Figs. S3B and S3C). The shEGFR-81 cells were more susceptible to menadione- or H2O2-mediated oxidative stress than were shNC control cells. These cell death analyses suggested that the EGFR protein is important to alleviate oxidative stress-mediated cell death. Collectively, our experiments suggest that attenuated EGFR expression reduces cellular ROS-scavenging activity, which then increases cells’ vulnerability to oxidative stress.
We next investigated whether transient enforced expression of EGFR in p-eIF2α deficient primary hepatocytes can increase cellular ROS-scavenging activity and therefore reduce their sensitivity to oxidative stress. Primary hepatocytes purified from livers of hepatocyte specific p-eIF2α deficient (
In this study, we showed that hepatocyte-specific eIF2α phosphorylation-deficient mice have significantly reduced expression of the EGFR and altered EGFR-mediated signaling pathways. Moreover, the eIF2α phosphorylation-deficiency or reduced expression of the EGFR decreased ROS-scavenging activity and increased ROS-mediated cell death in hepatocytes. However, the drawbacks were alleviated by enforced expression of EGFR in p-eIF2α deficient primary hepatocytes. Therefore, we concluded that reduced EGFR level in eIF2α phosphorylation-deficient hepatocytes is one of the critical factors responsible for a cell’s susceptibility to oxidative stress. Furthermore, vulnerability to oxidative stress in hepatocytes that have reduced EGFR level or EGFR activity might be the underlying mechanism of hepatotoxicity induced by several EGFR inhibitors (e.g., erlotinib, lapatinib, and gefitinib) used for the treatment of non-small cell lung cancer and pancreatic cancer (Kim et al., 2018; Paech et al., 2017), since most EGFR inhibitors undergo intense metabolism by cytochrome P450 enzymes in hepatocytes (Li et al., 2007; Rakhit et al., 2008) which produces reactive metabolites (Haouzi et al., 2000). However, further studies are required to verify whether the hepatotoxicity is related to oxidative stress susceptibility in EGFR inhibitor-treated hepatocytes.
The authors have no potential conflicts of interest to disclose.
This work was supported by the Basic Science Research Program (2014R1A1A4A01004329 and 2017R1D1A1B0 3028229), the Bio & Medical Technology Development Program (2017M3A9G7072745), and the Priority Research Centers Program (2014R1A6A1030318) of the National Research Foundation of Korea (NRF) funded by the Korean government.
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