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. Author manuscript; available in PMC: 2015 Mar 6.
Published in final edited form as: Mol Cancer Res. 2011 Oct 18;9(12):1708–1717. doi: 10.1158/1541-7786.MCR-11-0299

Adaptive basal phosphorylation of eIF2α is responsible for resistance to cellular stress induced cell death in Pten null hepatocytes

Ni Zeng 1, Yang Li 1, Lina He 1, Xiaoling Xu 2, Vivian Galicia 1, Chuxia Deng 2, Bangyan L Stiles 1,*
PMCID: PMC4351767  NIHMSID: NIHMS536864  PMID: 22009178

Abstract

The α-subunit of eukaryotic initiation factor 2 (eIF2α) is a key translation regulator that plays an important role in cellular stress responses. In the present study, we investigated how eIF2α phosphorylation can be regulated by a tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) and how such regulation is utilized by PTEN-deficient hepatocytes to adapt and cope with oxidative stress. We found that eIF2α was hyper-phosphorylated when Pten was deleted, and this process was AKT dependent. Consistent with this finding, we found that the Pten null cells developed resistance to oxidative glutamate and H2O2 induced cellular toxicity. We showed that the messenger level of CReP (constitutive repressor of eIF2 alpha phosphorylation), a constitutive phosphatase of eIF2α, was downregulated in Pten null hepatocytes, providing a possible mechanism through which PTEN/AKT pathway regulates eIF2α phosphorylation. Ectopic expression of CReP restored the sensitivity of the Pten mutant hepatocytes to oxidative stress, confirming the functional significance of the downregulated CReP and upregulated eIF2α in the resistance Pten mutant hepatocytes to cellular stress. In summary, our study suggested a novel role of PTEN in regulating stress response through modulating CReP/eIF2α pathway.

Keywords: PTEN, PI3K, eIF2α, CReP, Oxidative Stress

Introduction

The mitogenic signalling phosphatidylinositol-3-kinase (PI3K)/AKT signalling pathway is negatively regulated by a lipid phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10). Genetic studies have shown that loss of PTEN functions results in growth and survival phenotype and tumor development in multiple tissues and organ systems (1). In the liver, nearly 80% of hepatocytes carcinomas (HCC) are correlated with activation of the PI3K signalling pathway including loss of PTEN (2). We and others showed that PTEN loss in mouse models leads to lipid accumulation in the hepatocytes early and tumor development later in life (3–5). This two-stage progression of tumor development is similar to that observed with human HCC where underlying liver disease especially fatty liver disease is a common co-morbid factor. In human patients, the development of HCC is highly correlated with oxidative stress (6). In mouse liver where PTEN is lost and PI3K signalling is activated, accumulation of lipids is accompanied by high levels of hydrogen peroxide suggesting that the hepatocytes in the Pten null liver are under conditions of chronic oxidative stress (7). We used the hepatocytes isolated from this model to investigate whether and how PTEN/PI3K signal may provide the adaptation advantage for mutant cells to survive stress induced cell death.

Cancer cells are often “addictive” to their oncogenic events such as loss of a tumor suppressor or induction of an oncogene and resistant to stress-induced cell death. We investigated the response of the Pten null hepatocytes to stress and found that Pten null hepatocytes are resistant to various forms of stress including oxidative glutamate and H2O2 toxicity as well as ER stress. Phosphorylation of eukaryotic Initiation Factor 2 (eIF2) family of translation regulators is known for integrating various cellular stress responses including oxidative and ER stress (8). This mechanism may underlie the conditioned protection against more severe injury in cells growing in chronic low levels of stress conditions like the stressed conditions the Pten null cells are in (7). Under acute stress response, phosphorylation of eIF2α leads to shutdown of protein synthesis and mediation of global stress. We showed that PTEN, through its regulation of PI3K/AKT signalling, controls the basal phosphorylation of eIF2α. Chronic low level induction of eIF2α phosphorylation was reported to mediate an adaptive response of cells to chronic stress (9). Such mechanism may explain why tumor cells are more resistant to stress yet cannot survive when the oncogenic signals are lost. We further established that downregulation of CReP (constitutive repressor of eIF2α phosphorylation), a subunit of the PP1 phosphatase complex, is responsible for this basal phosphorylation of eIF2α. Overexpression of CReP restores the sensitivity of the Pten null hepatocytes to oxidative stress induced cytotoxicity. Together, our data suggest that basal phosphorylation of eIF2α induced by activation of PI3K may act as an adaptive response for the fast growing cells to cope with chronic stress.

Materials and Methods

Animals

PtenloxP/loxP; Alb-Cre+ (Mut, Pten null) and PtenloxP/loxP; Alb-Cre− (Con) mice were developed and characterized as previously described (4). All animals were kept in a 12 hour light/dark cycle controlled facility. All experimental procedures are performed following USC IACUC guidelines.

Cell culture and transfection

Immortalized hepatocyte cell lines were established from livers of CON and Pten null (Mut) mice (5). Briefly, freshly isolated hepatocytes were immortalized spontaneously with long term culturing using a 3T3 protocol and maintained in Dulbecco’s Modified Eagle Medium (DMEM, Mediatech, Manassas, VA) supplemented with 10% fetal bovine serum (US Scientific, Ocala, FL), 5 ug/ml insulin (Sigma, St. Louis, MO), 10 ng/ml epidermal growth factor (EGF, Invitrogen, Carlsbad, CA) (5). Mouse embryonic fibroblasts (kindly provided by Dr. Hong Wu, University of California at Los Angeles) (10), HepG2 (obtained from USC liver core facility), Huh-7 (a generous gift from Dr. James Ou, University of Southern California), and PLC.PRF/5 (provided by Dr. Aiwu Ruth He, Georgetown University) were cultured in DMEM supplemented with 10% fetal bovine serum. Hep3B cells were provided by Dr. Shelly Lu, University of Southern California and cultured in DMEM with 10% FBS and 1×non-essential amino acids (Invitrogen, Carlsbad, CA). SNU398, SNU449 and SNU475 (from Dr. Aiwu Ruth He, Georgetown University) were cultured in RPMI1640 (Mediatech, Manassas, VA) with 10% FBS.

Hepatocytes were transfected using the Lipofectamine 2000 system (Invitrogen, Carlsbad, CA) as described in the manufacturer’s instructions. Cells were culture in 6-well plates (1–2 × 105 cells/well) overnight to allow attachment. Four microgram DNA was delivered using 8μg Lipofectamine2000 in serum-free medium. Cells were harvested 24 hrs after transfection.

Xenograft

Nude mice 3–4 month of age were obtained from Jackson’s laboratory (Ann Harbor, Vt). Single cell suspensions of Con and Mut hepatocytes were obtained and prepared at four different concentrations (5×105, 1×106, 5×106, and 1×107). Each mouse was injected subcutaneously with 0.1 ml cell suspension. Tumor growth was observed and experiments were terminated 3 months later when the volume of the largest tumor reached 1.5 cm2.

Reagents and plasmids

L-glutamic acid and SIN-1 were obtained from Sigma (St. Louis, MO); H2O2 was provided by Fisher Scientifics (Pittsburgh, PA); LY294002 was purchased from Cell Signalling Technology (Danvers, MA). Caspase-12 Inhibitor Z-ATAD-FMK was from BioVision (Mountain View, CA).

pIRES CA-AKT and WT-AKT were constructed by inserting the coding sequence of the genes into the multiple cloning site of pIRES-GFP vector. The original constructs for PTEN, csPTEN, CA-AKT, DN-AKT and WT-AKT were obtained from Dr. Hong Wu (11). pIRE-GFP is used as control vector for all experiments where pIRES constructs are used. pSG5-wtPTEN was from Addgene (plasmid 10750) and provided by Dr. William Sellers (12). pFLAG-CReP (amino acid 24-698) was from Dr. David Ron (13). We subcloned the coding sequence into the p3xFLAG-myc-CMV™-26 vector from Sigma (St. Louis, MO).

Immunoblotting analysis

Cell lysate preparation and immunoblot analysis were performed as described (14). Antibodies against phospho-eIF2α, eIF2α, phospho-ERK, phospho-AKT, PTEN, PKR, caspase-8 and-9 were from Cell Signalling Technology (Danvers, MA); anti-AKT, anti-KDEL, anti-CHOP and anti-GRP78 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-actin antibody was from Sigma (St. Louis, MO). Anti-fatty acid syntheses (FAS) antibody was obtained from Millipore (Billerica, MA). Anti phospho-PKR antibody was from Abcam (Cambridge, MA). Anti caspase-3 antibody was from BD Biosciences (San Diego, CA).

Cell survival assay

Cells were seeded at a density of 3 × 103 cells/96-well plate, and then treated with H2O2, L-glutamic acid or SIN-1 at 37 °C, 5% CO2 with indicated doses. After 24 hours of treatment, 3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT, 50 μg/ml) was added into the culture and mixed by tapping gently on the side of the tray. After incubating at 37 °C for 4 h, the formazan crystals were dissolved by adding DMSO and then incubating at 37 °C for 30 min, and the absorbance at 570 nm was measured on a microplate reader. Each sample was assayed in pentad.

Propidium iodide (PI) staining and flow cytometry

Hepatocytes were plated in 12-well plates at the density of 0.5–1 × 105 cells/well. After treating with 10mM H2O2 for 24 hrs, cells were trypsinized and then collected by centrifuge. As described previously (15), cells re-suspended in PBS were stained with 1 ug/ml propidium iodide (PI) for 15 min at room temperature. Samples were then analyzed immediately using the BD LSR II flow cytometry system.

RNA isolation and quantitative real-time PCR

Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Reverse transcription and quantitative PCR were performed using M-MLV reverse transcriptase system (Promega, Madison, WI) and Maxima™ SYBR Green qPCR Master Mix (Fermentas, Glen Burnie, MD) following the manufacturer’s instructions.

Gene-specific primers for CReP: forward 5′-AGTCTCTGAGTTCACTGCGGC-3′, reverse 5′-GGCGCTGCAGAGTCTAAAGC-3′. GAPDH: forward 5′-GCACAGTCAA GGCCGAGAAT-3′, reverse 5′-GCCTTCTCCATGGTGGTGAA-3′. The cycling condition was 95°C for 5 min followed by amplification for 40 cycles at 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec in the Bio-Rad iCycler. Relative expression of mRNA levels was determined (using GAPDH as a standard) using the delta-delta Ct method (16, 17).

Statistical Analysis

The data are presented as means ± the standard error of the mean (SEM). Differences between individual groups were analyzed by Student’s t test, with two-tailed p values less than 0.05 considered statistically significant.

Results

Deletion of Pten in hepatocytes results in high oxidative stress

Oxidative stress occurring with inflammation is often implicated in promoting the development of tumors (18). Free radicals produced with oxidative stress can be both genotoxic and cytotoxic, causing some cells to acquire survival advantages while other cells to undergo cell death. We studied a gene that is commonly dysregulated in human liver cancers, PTEN and its role in oxidative stress response in the liver. We showed previously that mice lacking PTEN in the liver (PtenloxP/loxP;Alb-Cre+; Pten null) develops liver steatosis (Figure 1A and supplemental Figure 1) as well as liver cancer (4, 7). The steatosis in the liver is accompanied by high H2O2 contents indicating that the hepatocytes are experiencing high oxidative stress conditions (Figure 1B, left panel). We also investigated enzymes responsible for reducing oxidative stress in the cells. The expression of these enzymes often increases as a result of increased oxidative stress. The mRNA expressions of two of such enzymes, hydrogen peroxide scavenger glutathione peroxidase (GPx) and glutathione-s transferase (GST) are also significantly higher in Pten null mice compared to controls (Figure 1B, right two panels). Together, these data suggest that the hepatocytes in the Pten null mice are under high oxidative stress conditions. In addition, we analyzed liver tissues for trans-4-hydroxy-2-nonenal (4-HNE), a lipid peroxidation product (Figure 1C). Immunostaining with 4-HNE antibody identified dramatically more 4-HNE aggregates in the Pten null livers vs. the controls (Figure 1C), further supporting the presence of oxidative stress conditions in the Pten null livers.

Figure 1. High oxidative stress conditions in Pten null liver.

Figure 1

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(A) Deletion of Pten leads to lipid accumulation in liver hepatocytes. Images are H&E stained liver sections from Pten control (Con, Pten loxP/loxP;Alb-Cre−) and Pten null (Pten null, PtenloxP/loxP;Alb-Cre+). Pten null liver sections show lipid vacuoles. (B) High oxidative stress in Pten null liver. Left, hydrogen peroxide (H2O2) levels are significantly higher in Pten null livers vs. controls (Con). Expression of enzymes responsible for scavenging free radicals, glutathione peroxidase (GPx) and glutathione-S-transferase (GST) are both increased (Right two panels). n=5; * p≤0.05. (C) Immunohistostaining for lipid peroxidation indicates high oxidative stress in Pten null liver. Staining for 4-HNE (Green) indicates higher lipid peroxidation products in Pten null liver vs. controls. Blue indicates DAPI staining for nuclei.

Resistance of Pten null hepatocytes to cellular stress

To investigate how Pten null cells cope with the high oxidative stress conditions that they are exposed to in vivo, we established isogenic cell lines from the livers of control (PtenloxP/loxP;Alb-Cre−) and Pten null (PtenloxP/loxP;Alb-Cre+) mice using a standard 3T3 protocol (supplemental Figure 2). As expected, the Pten null (Mut) hepatocytes are transformed and capable of forming colonies on soft agar and in nude mice xenograft assays (supplemental Figure 3). The control cell lines (Con) are also capable of forming colonies and graft tumors (supplemental Figure 3A). The tumor graft derived from the control cell lines resembles the normal liver structure, suggesting that the control cell lines at least partially retain the properties of the wild type hepatocytes in vivo. The grafted tumors formed from the Pten null cell lines morphologically resemble the tumors observed in vivo in the PtenloxP/loxP;Alb-Cre+ mice. Significantly more colonies were formed in the Pten null cell cultures as compared to the controls when plated on soft agar (supplemental Figure 3B). This data suggests that the Pten null cells may have better survival potentials under the stressful condition of soft agar culturing than the control cells.

On the molecular level, the isogenic hepatic cell lines recapitulated the molecular signalling profiles observed in freshly isolated livers (supplemental Figure 3C). Both cell lines express albumin indicating that the origin of the cells is liver hepatocytes. The molecular signalling pathways such as mTOR were activated in the Pten null cell line as predicted. AKT is robustly induced when Pten is deleted. We also observed enhanced expression of fatty acid synthase (FAS) in the Pten null cell line vs. the controls, similar to what we have seen previously in mouse liver lysates (supplemental Figure 3C) (4).

The preliminary observation from the colony forming assay suggests that the Pten null hepatocytes are more resistance to the stressful culture conditions as more colonies were formed with the Pten null vs. Con cell lines. To determine whether loss of PTEN protects the hepatocytes from cell death induced by high oxidative stress condition, we treated the control and Pten null cells with three oxidative stressors: hydrogen peroxide (H2O2), 3-morpholinosydnonimine (SIN-1), and L-glutamic acid. Hydrogen peroxide is widely regarded as a cytotoxic agent that induces cell death through oxidative stress. Twenty-four hour exposure to H2O2 induced cell death in control (Con) hepatocytes in a dose dependent manner. The cell survival decreased from 88% to 12% as the doses increased from 1.25 mM to 10 mM (Figure 2A). The Pten null hepatocytes (Pten null), however, survived 1.25 mM H2O2 for 24 hours without detectable cell death. At high dose (10 mM), more than twice as much Pten null cells survived (29%) as compared to Con cells (12%). The improved survival of Pten null cells was also noted when cells were treated with SIN-1 (Figure 2B), a peroxinitrite donor used to generate nitric oxide and superoxide radical (19), as well as L-glutamic acid (Figure 2C), which depletes intracellular cysteine and glutathione, leading to oxidative stress (20, 21). Both treatments resulted in a dose dependent cytotoxicity in the Con cell line but a significantly diminished effect in the Pten null cell line.

Figure 2. Improved survival and decreased cell death in Pten null hepatocytes when exposed to oxidative stress.

Figure 2

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Control (Con) and Pten null hepatocytes are treated with H2O2 (A), SIN-1 (B) and L-glutamic acid (C) at the indicated concentrations. 100% survival is defined as the values in cells that were treated with vehicles only. n=3, * p < 0.05. (D) Cell death analysis via PI staining. Control (Con) and Pten null hepatocytes are treated with 10mM H2O2 or vehicle for 24 hours, harvested, stained and analyzed on flowcytometry. The Y axis is PI-area, the X axis is PI-width. Insets, PI-positive dead cells.

We further evaluated H2O2 induced cell death by staining the unpermeablized cells with Propidium Iodide (PI). Dead cells with compromised membranes incorporate PI and can be detected using flow cytometer. Treatment of Con hepatocytes with 10mM H2O2 for 24 hours resulted in the appearance of a distinct cell population that are high for PI staining (Figure 2D). This cell population was not present in the untreated Con cells, suggesting that this cell population is the dead cells induced by H2O2. Under the indicated treatment conditions, approximately 30% of Con hepatocytes became positive for PI labelling, indicating that they are dead cells with compromised membranes. Under the same conditions, only 1% of the Pten null hepatocytes incorporated PI. This represents a 30 folds decrease in cell death when PTEN is lost. Together with the cytotoxic analysis, this data suggests that PTEN loss protects hepatocytes from oxidative stress induced cell death.

Enhanced basal eIF2α phosphorylation in Pten deficient cells

The ISR response mechanism may underlie the conditioned protection against more severe injury in cells growing in chronic low levels of stress conditions like the stressed conditions the Pten null cells are in (7). We analyzed phosphorylation of eIF2α, the central regulator of the ISR response mechanism and found that p-eIF2α is induced in unstressed Pten null hepatocytes comparing to the control cells with intact PTEN (Figure 3A, left panel). Similarly, this basal level hyperphosphorylation of eIF2α is also observed in vivo in Pten null liver lysates vs. control lysate (Figure 3A, right panel). We observed a consistent two-fold increase of eIF2α phosphorylation in Pten null livers and hepatocytes vs. that of Controls. This difference in eIF2α phosphorylation is correlated with an increase of p-AKT but not pERK (Figure 3A, left panel).

Figure 3. Elevated phosphorylation of eIF2α in PTEN deficient hepatocytes and livers.

Figure 3

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(A) Immunobloting analysis of p-eIF2α, eIF2α, p-ERK, and p-AKT in hepatocytes (left) and mouse liver (right). Actin is detected as loading control. Bottom panels, quantification of western blots. * p < 0.05. (B) UPR stress is induced by treatment with 20nM TG in control (C) and Pten null (M) hepatocytes to assess the response of the cells to ER stress. In response to TG treatment, ER chaperone protein expressions (GRP78, 94 and PDI) increase. No consistent differences are detected between control (C) and Pten null (M) hepatocytes. Apoptotic factor CHOP is induced as a result of TG treatment. Loss of Pten significantly attenuates the induction of CHOP. At basal level, phospho-eIF2α is higher in Pten null (M) hepatocytes vs. controls (C). This difference diminishes in TG treated cells (at 1, 2 and 8 hours) and returns 16 hours after TG treatment. (C) p-eIF2α correlated with p-AKT but not GRP78 in mouse liver. No UPR stress is detected in Pten null livers. GRP78 levels are the same in liver lysates from Pten control (Con) and null mice. (D) Treatment of control and Pten null hepatocytes with caspase 12 inhibitor does not result in alteration in p-eIF2α.

To test whether the change in eIF2α phosphorylation also alters the cellular response to other stress, we tested the response of the Pten null hepatocytes to induced stress of the endoplasmic reticulum (ER) and compared this response to the control cells. Thapsigargin (TG) is a chemical used to induce stress of the ER and accumulation of GRP78 (22). In both Con and Pten null cell lines, TG treatment induced the expected increase of GRP78. No difference in GRP78 were observed between Con and Pten null cell lines at any time points after TG treatment (Figure 3B). Similarly, no changes were observed with other ER proteins such as GRP94 and PDI either, suggesting that the response of ER to unfolded protein (UPR) accumulation is not altered by PTEN loss. Similar to the response to H2O2 induced stress, the Pten null hepatocytes survived ER stress induced cell death much better as indicated by the attenuated CHOP induction in response to TG treatment. This data suggests that PTEN regulated signals may confer resistance to stress induced cell death regardless of the type of stress.

During TG induced ER stress response, the differences of p-eIF2α observed between control and Pten null cells disappeared as acute stress response in control cells led to enhanced phosphorylation of eIF2α (Figure 3B). Immediately following treatment with TG at 1 hour, we observe that the phosphorylation of eIF2α is induced to a similar level in Con and Pten null cell lines. This level of phospho-eIF2α remained for 8 hours before returning to baseline levels. By 24 hours, the phospho-eIF2α levels in Con cells are reduced back to control levels whereas phosphorylation of eIF2α is again higher in the Pten null cells vs. control ones. Thus, phosphorylation of eIF2α appears to respond to PTEN signal and ER stress independently. Basal phosphorylation of eIF2α is altered with PTEN status where phosphorylation of eIF2α is also robustly increased transiently (lasting 8 hours) when UPR is induced. The former event, basal phosphorylation of eIF2α changes with PTEN status, but is independent of ER stress signals. Consistent with this UPR independent role of PTEN regulated eIF2α phosphorylation, eIF2α phosphorylation correlates with induction of AKT but not GRP78 expression in vivo when PTEN is lost (Figure 3C). These data are consistent with the notion that PTEN and PI3K signal regulates all integrated stress response at the site of eIF2α regardless of the source of the stress.

The caspases are known targets of PTEN and PI3K signalling pathway. We evaluated the potential involvement of the caspase cascades in PTEN and PI3K signal regulated stress response. We are unable to observe any cleaved caspase 3, the final step of the caspase cascade, with H2O2 treatment (Supplemental Figure 4). Similarly, we also did not observe changes of caspases 8, which is involved in the extrinsic apoptotic pathway, or caspase 9, involved in the intrinsic apoptotic pathway with our H2O2 treatment conditions (data not shown). Thus, cell death induced by H2O2 in the hepatocytes is likely mediated by caspases independent pathways, such as the lysosomal protease pathways (23). Caspase 12 is reported to mediate ER stress induced cell death. We treated the control and Pten null cells with an inhibitor for caspase 12 (24). Inhibition of caspases 12 had no effect on the phosphorylation of eIF2α (Figure 3D), further confirming that ER mediated events are unlikely to be involved in ISR response regulation by PTEN loss.

PI3K/AKT signalling regulates the phosphorylation of eIF2α

To explore the signalling events leading to the accumulation of phosphor-eIF2α, we introduced PTEN into the Pten null cells to evaluate whether this introduction can diminish phosphorylation of eIF2α observed with PTEN loss. Compared to the GFP transfected cells, introduction of wtPTEN led to reduction in phospho-eIF2α (Figure 4A and supplemental Figure 5). The phosphatase dead mutant of Pten (csPTEN) did not induce phosphorylation of eIF2α, suggesting that this basal eIF2α phosphorylation depends on the phosphatase activity of PTEN. The enhanced phosphorylation of eIF2α is also observed in the isogenic mouse embryonic fibroblasts (mEFs) where PTEN is lost as well as Hep G2 cells where PTEN expression is relatively lower as compared to Huh 7 cells (Figure 4B).

Figure 4. PI3K/AKT signal regulates eIF2α phosphorylation.

Figure 4

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(A) Expression of wtPTEN in Pten null hepatocytes results in downregulation of eIF2α phosphorylation. GFP, wtPTEN and csPTEN containing plasmids are transfected into Pten null hepatocytes to evaluate their effects on p-eIF2α. wtPTEN induces downregulation of p-eIF2α as compared to GFP controls but not csPTEN. Actin and eIF2α were detected as loading controls. (B) Hyperphosphorylation of eIF-2α is observed in several cell lines lacking PTEN: hepatocyte cell lines isolated from control (C) and Pten null (M) mice, mouse embryonic fibroblasts (mEF) established from the Pten null (M) and control (C) mice and human hepatocytes cell lines with differential levels of PTEN expression (Huh 7 and HepG2). (C) Treatment of Pten null hepatocytes with 20μM PI3K inhibitor LY294002 leads to downregulation of p-eIF2α. Phospho-AKT is blotted to confirm the inhibition. Phospho-eIF2α and eIF2α are also analyzed on the same membrane. (D) AKT constructs (CA, constitutive active; WT, wild type; and DN, dominant negative) are transfected into Control (Con) and Pten null hepatocytes to assess the role of AKT in the regulation of p-eIF2α. The transfection efficiency is tested by examining p-AKT level. In both control (Con) and Pten null cell lines, transfection of CA-AKT (CA) results in induction of p-eIF2α. DN-AKT (DN) significantly downregulates p-AKT and leads to almost 2 fold decrease in p-eIF2α in control cells compared to vector transfected cells. In Pten null cells, AKT activity is moderately inhibited, leading to moderate attenuation of eIF2α phosphorylation.

To further interrogate the downstream signalling of PTEN, we manipulate the PI3K signalling pathway using chemical inhibitors. LY294002 is an inhibitor for PI3K and blocks the pathways downstream of PI3K signalling. We treated the Pten null cells with an activated PI3K signalling pathway with LY294002 (20mM). This treatment led to a time dependent reduction in the phosphorylation of eIF2α that follows the inhibition of p-AKT (Figure 4C). The phosphorylated AKT is dramatically decreased 30 min after LY294002 treatment and started to recover at 1 hour post treatment. Phospho-eIF2α started to reduce at 30 min but reached the lowest levels 2 hours after treatment with LY294002. This long delay suggests that the effect of PI3K/AKT on eIF2α phosphorylation is likely indirect. The level of phospho-eIF2α recovered 6 hours after treatment following the complete recovery of phospho-AKT. These data suggest that phosphorylation of eIF2α may be regulated by AKT signalling.

To substantiate the potential regulation of eIF2α by PI3K/AKT and determine if AKT is indeed involved in this regulation, we introduced constitutively active myristylated AKT (CA), dominant negative AKT (DN), wildtype (WT) AKT, and vector to the Con and Pten null hepatocytes (Figure 4D). In the control cells, introduction of caAKT moderately induced phospho-eIF2α. Inhibition of AKT activity with dominant negative AKT significantly reduced phospho-eIF2α, suggesting that this basal level of phospho-eIF2α is at least partially dependent on AKT activity. In Pten null cells, a similar trend is observed though more moderate, likely due to the already induced hyperphosphorylation of AKT and the inability of DN-AKT to significantly reduce the activity of this hyperactive AKT.

CReP downregulation mediates the hyper phosphorylation of eIF2α in Pten null cells

The delayed response of phospho-eIF2α to LY294002 treatment comparing to phospho-AKT suggests that the regulation of eIF2α phosphorylation by PI3K/AKT signalling is not direct. We found that the stress related kinases such as the ER stress induced PERK (8) are unlikely the mediators for the observed basal level increase of eIF2α since ER stress induced response on phospho-eIF2α dose not differ between the two cell lines (data not shown). RNA-dependent protein kinase (PKR) was previously reported to regulate PTEN mediated eIF2α independent of PI3K (25). We determined whether PKR may regulate the basal eIF2α phosphorylation we observed with the unstressed hepatocytes. We found that PKR is not detected in the untransfected and unstressed hepatocytes cell lines (data not shown), suggesting that this kinase is unlikely to be responsible for the enhanced basal phosphorylation of eIF2α observed with PTEN loss. We also were unable to observe an appreciated changed in the phosphatase GADD34 between Con and Pten null cells in the unstressed conditions (data not shown). GADD34 is induced under ER stress while not detectable in unstressed cells (13, 26, 27). The lack of response in GADD34 is consistent with the observation that UPR response did not differ between control and Pten null cells (Figure 3B). A homologue of GADD34, CReP is thought to regulate the phosphorylation of eIF2α at basal level (13) whereas GADD34 responds to stress induced recovery of phospho-eIF2α. CReP is also found to have a growth regulatory role (28), thus a more likely candidate to integrate the chronic stress with adaptive growth response. In the Pten null cells, we observed a significant decrease of CReP transcript level (Figure 5A). The mRNA levels of CReP were 40% less in the Pten null cells vs. the controls. Treatment of Pten control hepatocytes cell line with IGF-1 to induce PI3K activity led to a significant downregulation of mRNA expression of CReP (Figure 5B). Conversely, blocking PI3K activity in Pten null cell lines resulted in a significant induction of CReP expression (Figure 5B). Together, these data suggests that PI3K signals may regulate basal phosphorylation of eIF2α by downregulating the phosphatase CReP.

Figure 5. CReP is down-regulated in Pten null hepatocytes.

Figure 5

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(A) Total RNA from Con and Pten null hepatocytes is extracted and then reverse transcribed into cDNA. Quantitative PCR is performed to compare the mRNA levels of CReP in Con and Pten null cells. Each sample is assayed in triplicate and repeated three times. *, p≤0.05. (B) Control and Pten null (Mut) hepatocytes are treated with IGF-1 and LY294002 respectively to induce or inhibit PI3K/AKT signalling. Induction of PI3K/AKT with IGF-1 in control cells leads to attenuation of CReP mRNA expression. Inhibition of PI3K/AKT with LY294002 resulted in induction of CReP mRNA expression. (C) Transfection of CReP to Pten null (Mut) hepatocytes leads to downregulation of p-eIF2α. (D) Hepatocytes are transfected with CReP or vector as control and then treated with 5mM H2O2 for 72 hrs. Cell death is evaluated by using PI staining followed by FACS analysis. CReP transfection restores the sensitivity of Pten null hepatocytes H2O2 treatment induced cell death.

To evaluate the relationship between the upregulation of eIF2α and downregulation of CReP in Pten null cells and their response to cellular stresses, we sought to reduce p-eIF2α by overexpressing CReP in the Pten null cells. We found that the introduction of CReP is able to reduce the phosphorylation of eIF2α (Figure 5C). Consistently, we observed that 7.7% of the Pten null cells are stained for PI in vector transfected hepatocytes without H2O2 treatment, comparing to the 0.3% in the untransfected cells seen in Figure 3. When treated with H2O2, approximately 19% of the vector transfected cells underwent cell death. Reintroduction of CReP into Pten null hepatocytes partially restored their sensitivity to H2O2 (Figure 5D). When CReP was overexpressed, the amount of dead cells almost doubled (37%). Transfection alone also induced acute phosphorylation of eIF2α and hepatocytes death when cells were not treated with H2O2 likely due to activation of stress kinases. This data indicate that CReP downregulation and chronic low level of p-eIF2α is at least partially responsible for the stress resistance phenotype observed with the Pten null cells.

Discussion

The integrated stress response (ISR) is a defensive mechanism that cells developed to cope with the environmental insults they encounter (8). Phosphorylation/dephosphorylation of a translation initiation factor eIF2α lies at the centre of this integrated response. Phosphorylation of eIF2α induced by stress kinases coordinate a network of translation and transcription response, the ISR mechanism to lower cellular stress. When such stress is prolonged, the response switches from survival to apoptosis. In this paper, we found that activation of the mitogenic signalling pathway PI3K/AKT through deletion of Pten resulted in the upregulation of basal phosphorylation of eIF2α in unstressed hepatocytes. Our results indicate that chronic induction of low level eIF2α phosphorylation mediated by downregulation of the PP1 partner CReP protects the Pten null hepatocytes against stress-induced cell death.

Oxidative stress is one of the most common forms of stress that the cells need to cope with. Oxygen radicals accumulate as a by-product of normal cellular processes such as metabolism. A sophisticated anti-oxidant and oxygen radical scavenger systems are integrated with the ISR to reduce oxidative stress and allow cells to survive (29). Cancer cells with high growth and metabolic rates are often under higher oxidative stress than normal cells. Yet, cancer cells are able to survive the highly stressful conditions and develop into tumors. It is well established that cancer cells evoke survival mechanisms to evade growth/survival regulation (30). However, whether and how these mechanisms may interact with stress response to adapt to the stressful environment is not known. Our study here shows that the cell growth/survival signalling, PI3K/AKT pathway activation promotes the adaptive survival of hepatocytes to oxidative stress. In liver cancer model where PI3K signalling is upregulated due to loss of its negative regulator PTEN, the development of tumors requires the underlying fatty liver disease in the Pten null mice (7, 31). High levels of ROS accompany this fatty liver condition. Our data here clearly demonstrated that the Pten null hepatocytes are more advantage at being able to grow and survive in this high ROS (as well as other stress) environment.

Our data also indicated that the interaction of PI3K signalling with the ISR defense mechanism is at the level of eIF2α, the translation regulator that controls all stress related ISR. The unphosphorylated form of eIF2α is available to form the eIF2α-GTP-tRNAmet complex and initiates translation (8). In response to cellular stress, eIF2α can be transiently phosphorylated by various stress induced kinases (PERK, HRI, PKR, and GCN) responding to different stimuli. The phosphorylated eIF2α cannot participate in translation initiation, resulting in repression of global translation and activation of selective gene expression to alleviate stress conditions. The activation of PI3K signalling by chronic loss of PTEN resulted in hyperphosphorylation of eIF2α at basal conditions when cells are not under stress. This hyperphosphorylation is similar to conditions where phosphorylation of eIF2α is uncoupled from upstream stress kinases (9). When uncoupled with the stress kinases, the hyperphosphorylation of eIF2α at basal unstressed conditions enhanced the cytoprotection response to lethal stress. Consistent with this observation, eIF2α phosphorylation was found previously to protect cells against oxidative H2O2 and glutamate toxicity (3). Thus, chronic phosphorylation of eIF2α resulting from activation of PI3K signalling may represent an adaptive response of fast growing cells to stress.

The major contradictory to this observation is the report that PTEN, independently of PI3K upregulated eIF2α and control translation (25). Under doxycycline induction (likely high stress) conditions, PTEN was found to enhance phosphorylation of eIF2α by inducing the stress kinase PKR through its C2 domain. Through this phosphorylation, PTEN is thought to control translation independent of PI3K. This action, involves acute stress response at the stress sensing and not ISR response since PKR is induced in cells where PTEN levels are manipulated through transfection and doxycycline induction. Our observation with vector, GFP, PTEN and AKT construct transfected experiments supported this possibility. Regardless of the gene introduced, introduction of plasmids resulted in hyperphosphorylation of eIF2α in all cases. Thus, eIF2α phosphorylation may be differentially regulated at stressed and unstressed conditions.

Phosphorylation/dephosphorylation of eIF2α can be controlled by several enzymes including kinases and phosphatases (8). The four kinases responding to different stresses act as sensors to integrate stresses with cellular response by phosphorylating eIF2α. The major phosphatase involved in extinguishing this signal is GADD34 (27), the phosphatase that is induced by stress through selective translation by eIF2α. These kinases and GADD34 respond transiently to acute stress conditions but cannot explain the adaptation response under low chronic stress. CReP controls the basal level of eIF2α phosphorylation in unstressed cells and may be significant for the adaptive response of cells under chronic low levels of stress (28). Our study indicates that overexpression of CReP can reduce the basal phosphorylation of eIF2α and restore the sensitivity of PTEN deficient cells to hydrogen peroxide-induced cell death. Thus, CReP mediated phosphorylation of eIF2a likely acts as an adaptive response for the Pten null hepatocytes to cope with the high levels of cellular stress. Genetic studies targeting GADD34 and CReP respectively also support a role CReP and not GADD34 in integrating growth signal with stress response (28). Loss of CReP led to growth retardation (28) whereas mutants lacking GADD34 is phenotypically indistinguishable from the wild type controls (32, 33). We found a positive correlation between the expression of CReP and PTEN expression in several human HCC cell lines (Supplemental Figure 6). Together, these data suggest that CReP mediated basal phosphorylation of eIF2α may underlie the adaptive stress response observed in cancer cells.

Cellular adaptation to environmental stress is a major mechanism for tumor cells to respond to its stressful environment. The molecular mechanism for such response is not understood. Our study provided a novel molecular mechanism on how activation of PI3K/AKT signalling may allow cells to deal with the stress conditions and ultimately adapt to and survive the new environment. This adaptive response of the Pten null hepatocytes to oxidative (and other) stress may allow them to survive the stressful environment of fatty liver in vivo and play a role in the development of tumors. Our study uncovered a novel role of PTEN in regulating the adaptive response of cancer cells to chronic stress through modulating CReP/eIF2α pathway.

Supplementary Material

Acknowledgments

We acknowledge funding support from USC liver cancer seed fund (BLS). NZ is supported by the NIH-sponsored Predoctoral Research Training Program in Cellular, Biochemical and Molecular Sciences in USC.

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