PRL2 serves as a negative regulator in cell adaptation to oxidative stress
High levels of ROS cause oxidative stress, which plays a critical role in cell death. As a ROS effector protein, PRL2 senses ROS and controls phagocyte bactericidal activity during infection. Here we report PRL2 regulates oxidative stress induced cell death. PRL2 senses oxidative stress via highly reactive cysteine residues at 46 and 101. The oxidation of PRL2 causes protein degradation and supports pro-survival PDK1/AKT signal which in turn to protect cells against oxidative stress. As a result, PRL2 levels have a high correlation with oxidative stress induced cell death. In vivo experiments showed PRL2 deficient cells survive better in inflammatory oxidative environment and resist to ionizing radiation. Our finding suggests PRL2 serves as a negative regulator in cell adaptation to oxidative stress. Therefore, PRL2 could be targeted to modulate cell viability in inflammation or irradiation associated therapy.
KeywordsPRL2 Oxidative stress Cell death Inflammation Ionizing radiation
phosphatases of regenerating liver 2
reactive oxygen species
mouse embryonic fibroblasts
conditional knockout mice
white blood cells
Reactive oxygen species (ROS) are generated during aerobic respiration in the mitochondria and in cellular response to xenobiotic, cytokines, and bacterial invasion [1, 2]. ROS include the superoxide anion (O2−), hydrogen peroxide (H2O2) and hydroxyl radicals (HO·) . In physiological condition, ROS function as signals to promote cell proliferation and survival . Severe increase of ROS, causing an imbalance between oxidants and antioxidants in favor of the oxidants, is referred as oxidative stress which results in damage of lipids, proteins and DNA. The accumulation of oxidative damage causes cell death and contributes to disease development .
The protein tyrosine phosphatase (PTP) family is a well-known ROS-effector protein family . In the catalytic pocket, PTP proteins commonly possess a highly reactive cysteine (Cys) residue, which is susceptible to oxidation by ROS . Phosphatases of regenerating liver (PRLs) belong to a subfamily of PTPs. They are dual specificity phosphatases, and have three members (PRL1, PRL2, and PRL3). PRLs are highly homologous, sharing at least 75% identity at the amino acid sequence level . Among PRLs, PRL2 is the most abundantly and ubiquitously expressed in adult human tissues, suggesting its various roles in physiological processes . Our previous work showed PRL2 is highly expressed in immune system . In innate phagocytes, PRL2 rapidly responds to the oxidative stress at the inflamed site by down regulating its own expression, leading to enhanced respiratory burst. This positive feedback mechanism promotes bactericidal activity of phagocytes [7, 8]. Here we report PRL2 levels are highly related to the sensitivity of cells to hydrogen peroxide in vitro. PRL2 senses oxidative stress via highly reactive cysteine residues, which in turn to diminish its protein and support cell survival via PDK1/AKT pathway. In vivo, PRL2 deficient cells survive better under inflammatory environment and resist to ionizing radiation. Our finding may reveal a basic signal in cell adaptation to oxidative stress and PRL2 could be targeted to modulate cell viability in inflammation or irradiation associated therapy.
Materials and methods
The generation of PRL2 myeloid conditional knockout (Ptp4a2fl/flLysMCre+ B6) mice, PRL2 total knockout (Ptp4a2fl/flE2aCre+ B6) mice and their wild-type littermates (Ptp4a2fl/fl B6) have been described previously [7, 8]. Wild-type C57BL/6 mice were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences. Wild-type C57BL/6 mice expressing CD45.1 were kindly provided from Junke Zheng’s Lab in Shanghai Jiao Tong University School of Medicine. Mice were housed in the Shanghai Jiao Tong University School of Medicine Animal Care Facilities under specific pathogen-free conditions.
Primary cell isolation
Bone marrow cells were harvested from mouse femurs and tibias using phosphate buffer saline (PBS). After RBC lysis, cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS), 2 mM l-glutamine, and 100 U/ml penicillin/streptomycin (D10) resting for 1 h before further experiment. Primary mouse embryonic fibroblasts (MEFs) were isolated from E14.5 embryos according to the standard protocols .
PRL2–pRK5 plasmid was generated as described previously . The mutant PRL2 C46S, PRL2 C101S and PRL2 C164S were generated by PCR-based site-directed mutagenesis. PRL2-MigR1 was generated by cloning amplified PRL2 fragment into MSCV-based retroviral vector MigR1. All constructs were confirmed by DNA sequencing.
Cell culture, transfection and infection
MEFs and HEK293T cells (ATCC) were cultured in D10. Transiently transfections were performed using Attractene transfection reagent according to manufacturer’s instruction (QIAGEN, Germany).
PRL2 overexpression stable cell lines were generated by retrovirus infection. To produce retroviruses, packaging cells (293T) were transfected with MSCV-based retroviral vector MigR1 containing gene of interest along with pVSVG and pCGP sequences using Attractene. Culture supernatants were collected and filtered 24 to 36 h post transfection. Target cells were infected with the filtered viral supernatants in the presence of 5 μg/ml polybrene (Sigma-Aldrich, USA) for 12 h, after which the medium was changed. Cells infected with MigR1 empty virus were used as control cell line. Following infection, cells were checked by fluorescence detection and western blotting.
Cell treatment with hydrogen peroxide (H2O2)
Bone marrow cells used for viability assay under oxidative stress were seeded in 96-well plates at a concentration of 1 × 105 cells in 100 μl medium. Used for immunoblotting were seeded in 12-well plates at a concentration of 3 × 106 cells in 1 ml medium. Then bone marrow cells were treated with H2O2 (Sigma-Aldrich, USA) immediately after resting for 1 h. MEFs and 293T cells were seeded in 96-well plates at a concentration of 1 × 104 cells in 100 μl medium or in 12-well plates at a concentration of 2 × 105 cells in 1 ml medium. 24 h later, the cells in the wells were treated with indicated concentrations of H2O2 for indicated times. In some experiments, AKT phosphorylation was activated by 10 μM SC79 (CSN pharm, USA) for 0.5 h prior to H2O2 treatment.
Cell viability assay
The cell viability was quantified by Cell Counting Kit-8 (YEASEN, China) according to the manufacturer’s instruction. In brief, the cells seeded in 96-well plates were incubated with the reagent 10 μl per well for 1 h under standard cell culture conditions and quantified by measuring absorbance at 450 nm. Data were normalized to control (100%) without stimulus, unless noted otherwise. Data of the growth curve were just shown in the form of absorbance at 450 nm.
Expression and purification of recombinant PRL2
A pair of primers: 5′-AATTGAATTCTATGAACCGTCCAGCCCCT-3′ (forward) and 5′-GATCGGATCCCTACTGAACACAGCAG-3′ (reverse) were designed to amplify the target PRL2 gene. PRL2 gene was subcloned into the pET302/NT-His plasmid (Invitrogen, USA). And then the plasmid was transformed into E. coli BL21 cells for protein expression. Protein expression was initiated by IPTG and bacteria were harvested after 4 h culture. And then they were lysed and sonicated. The recombinant fusion protein His-PRL2 from E. coli lysates was purified by Ni–NTA Superflow Cartridges according to the manufacturer’s instruction (QIAGEN, Germany). The molecular weight and purity of recombinant proteins were identified by SDS-PAGE.
In vitro H2O2 oxidation and DTT reduction
Recombinant protein His-PRL2 that dialyzed with PBS and adjusted to a final concentration of 4 mg/ml was incubated with various concentrations of H2O2 in a total volume of 20 μl for 20 min at room temperature. The reactions were stopped by adding 12.5 U of catalase (Sigma-Aldrich, USA) to consume H2O2. His-PRL2 reactions were further incubated with various concentrations of Dithiothreitol (DTT) for 20 min at room temperature. Following treatment, protein samples were denatured in non-reducing sample buffer (0.25 M Tris, pH 6.8, 10% SDS, 0.5% bromophenol blue, 50% glycerol). Protein oxidative and reductive states were separated on 12% SDS-PAGE by Coomassie Blue R-250 staining.
Protein extraction and immunoblotting
Cells were harvested after treatment. After being washed by PBS twice, cells were lysed in 100 μl RIPA lysis buffer (Beyotime, China) supplemented with 0.1 mM PMSF on ice for 15 min. The oxidation state of PRL2 was protected by N-ethylmaleimide (Sigma-Aldrich, USA, 40 mM in lysis buffer). Protein concentration was determined by BCA assay (Sangon Biotech, China). Equal quantities of proteins were separated by SDS-PAGE under reducing or non-reducing conditions, transferred to a nitrocellulose membrane, and blotted with specific antibodies. The membrane was developed using Thermo SuperSignal reagent (Thermo, USA) and detected by ImageQuant LAS 4000 mini (GE, USA). Antibodies were used as followed: mouse anti-PRL2 antibody (Millipore, USA), mouse anti-GAPDH antibody (Sigma, USA), rabbit anti-phospho-PDK1 antibody (CST, USA), rabbit anti-phospho-AKT (Thr308) antibody (CST, USA), rabbit anti-phospho-AKT (Ser473) antibody (CST, USA), HRP conjugated mouse anti-Myc-Tag (CST, USA), HRP linked anti mouse IgG (CST, USA) and HRP linked anti rabbit IgG (CST, USA).
The acute peritonitis model for studying myeloid cell survival at inflamed site in vivo
The acute peritonitis model was generated as described . In brief, C57BL/6 wild-type mice (recipient mice) were given intraperitoneal injection of 5% thioglycollate (1 ml/mouse) to induce acute peritonitis. 24 h later, the recipient mice were exposed to total-body X-ray irradiation at a dose of 6 Gy. And then, carboxyfluorescein succinimidyl ester (CFSE)-labeled bone marrow cells (2 × 107 per mouse) from PRL2−/− CD45.2+ and wild-type CD45.1+ mice, mixed at 1:1 ratio were injected into peritoneal cavity of recipient mice. Recipient mice were sacrificed 24 h later and their peritoneal cells were collected. The peritoneal cells were stained with anti-CD45.1-PE (eBioscience, USA), anti-CD45.2-PerCP/Cy5.5 (eBioscience, USA) and anti-CD11b-APC (eBioscience, USA), and the frequencies of PRL2−/−CD45.2+CFSE+CD11b+ and PRL2+/+CD45.1+CFSE+CD11b+ cells were determined by flow cytometry.
X-ray irradiation and blood cell count analyses
Female 6–8 week old PRL2 myeloid cell conditional knockout mice and their wild-type littermates were exposed to total-body X-ray irradiation at a dose of 9 Gy, which were administered at a dose rate of 1.89 Gy/min generated from RS-2000 Biological Irradiator (RadSource, USA). Circulating blood samples were collected from the mice tail for cell counting and peripheral blood smear at the indicated hours after irradiation.
Data were presented as the mean ± SEM and all the differences were analyzed by unpaired Student’s t test or Analysis of Variance using GraphPad Prism software.
PRL2 negatively regulates oxidative stress induced cell death
PRL2 senses oxidative stress via highly reactive cysteine residues
To determine whether the oxidative modulation of PRL2 also occurs in living cells, we examined endogenous PRL2 in 293T cells. While cells are treated with H2O2, there is the mobility shift in PRL2 corresponding to the transition between its reduced and oxidized forms (Fig. 2b, left panel). The oxidized form of PRL2 is initially detectable at 0.05 mM and becomes predominant at H2O2 concentrations exceeding 0.2 mM. Moreover, the total protein levels of PRL2 were significantly decreased with increasing concentration of H2O2. To examine whether this effect is associated with synthesis of new protein, we treated cells with 10 μg/ml cycloheximide 1 h prior to treatment with H2O2 and observed similar results (Fig. 2b, right panel).
To further test roles of cysteine residues in PRL2, we generated PRL2 mutants (C46S, C101S and C164S) in which individual cysteine residue was replaced with serine. We tested these mutant PRLs for their abilities to be modified by oxidation. WT PRL2, C46S, C101S and C164S PRL2 mutants were overexpressed in 293T cells respectively, and then cells were treated with different concentrations of H2O2. Unlike WT and C164S PRL2, the C46S and C101S PRL2 only showed single band following exposure to H2O2 (Fig. 2c). More importantly, C46S and C101S PRL2 mutants decreased less, as compared with WT and C164S PRL2. Above results suggest that C46 and C101 could participate in intramolecular disulfide bonding and are involved in protein degradation by oxidative stress.
PRL2 negatively regulates cell survival under oxidative stress via PDK1/AKT signaling
To further determine whether PRL2 regulates oxidative stress-induced cell death through PDK1/AKT pathway, we manipulated the AKT activation using chemical activator, SC79. As shown in Fig. 3c, SC79 increased cell survival in PRL2 overexpression cells and abolished the difference between cell with and without PRL2 overexpression. Taken together, these results demonstrate that PRL2 might be positioned upstream of, and signal through, the PDK1/AKT pathway in the regulation of cell death.
PRL2 deficient cells survive better in inflammatory environment
PRL2 deficient cells are resistant to ionizing radiation (IR)
High levels of ROS lead to oxidative stress, which cause severe damage to the cell and form the basis of a wide variety of diseases . Here we report PRL2 serves as a negative regulator in cell adaptation to oxidative stress. Our finding was supported by following evidences: (a) PRL2 deficient cells survive better in vivo and in vitro in oxidative environment; (b) PRL2 replenishment or overexpression promotes H2O2 induced cell death in vitro; (c) PRL2 negatively regulates ROS induced PI3K/PDK1/AKT signaling, a well-known pathway leading to cell survival.
Our previous work showed PRL2 is highly expressed in immune system. PRL2 senses ROS and controls phagocyte bactericidal activity by promoting respiratory burst . ROS are used by the immune system as weapons against pathogens, however, ROS may also cause cell damage . The finding in this study indicates that under inflammatory condition, decreased PRL2 in immune cells not only promotes ROS production but also protects cells against oxidative stress. Since PRL2 is expressed heavily in most of human tissues and cells , we speculate above mechanism exists wildly in human cells to survive in oxidative environment. It would be interesting to investigate the roles of PRL2 in ROS associated diseases, such as cancer, lung diseases or neural disorders.
Oxidative stress induced cell death is the result of the struggle between survival signaling and death signaling . Our results indicate PRL2 senses oxidative stress via highly reactive cysteine residues. The oxidation of PRL2 causes protein degradation and supports pro-survival PDK1/AKT signal which in turn to protect cells against oxidative stress. PDK1 and AKT are two direct downstream effectors of PI3K . Both of them possess a Pleckstrin Homology (PH) domain which interacts with phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3), and phosphatidylinositol-4,5-bisphosphate (PtdIns(3,4)P2) [18, 19]. PDK1 and AKT co-localize at the plasma membrane, hence enabling PDK1 to activate AKT by phosphorylating Thr308 on its activation loop and promote cell survival . In this study, we found AKT activator SC79 dramatically increased cell survival in PRL2 overexpression cells and abolished the difference between cells with and without PRL2 overexpression, suggesting PRL2 acts upstream of PIK3/PDK1/AKT. This observation is in line with our recent study that PRL2 negatively regulates Rac GTPase activation . Rac and PI3K are intracellular signal transducers able to regulate multiple signaling pathways fundamental for cell behavior. They are tightly connected due to the ability to regulate each other in a lipid-dependent (PI3K upstream) or -independent (Rac upstream) manner . It was reported that PI3K interacts with the GTP bound forms of Rac, via the p85 regulatory subunit, and serves as an effector of this GTPase .
Ionizing radiation induces direct generation of ROS in large quantities . In present study, we found PRL2 deficient cells survive better in the irradiated animal. PRL2 myeloid cell conditional knockout and wild-type mice were exposed to a lethal dose of X-ray irradiation. Lymphopenia occurs following whole-body IR exposure. Changes in the counts of lymphocytes of CKO mice were similar with WT mice for any of the time points after irradiation. This result is in line with expectations, since the lymphocytes in CKO mice have similar PRL2 expression as WT mice. IR exposure also causes the decline in neutrophil counts. But the decline is preceded by an initial phase of granulocytosis, an event that is believed to be due to demargination of granulocytes and release of mature and early precursor for the relatively large pool of granulocytic cells within the bone marrow compartment . 6–8 h after irradiation, more neutrophils appeared in the blood of CKO mice, suggesting without PRL2 neutrophils survive better under IR stress.
An increase of ROS can result in either cell survival or cell death [1, 25]. Preferential selection of survival signals lead to the protection of cells against damage induced by ROS, whereas preferential acceleration of death signals can be used to advantage in tumor therapy with oxidizing agents such as ionizing radiation and anticancer drugs . Ever since PRL3 was found to be overexpressed in metastatic lesions derived from colorectal cancer , the PRLs family of phosphatases has received much attention. PRLs are considered as oncogenes that promote cell proliferation, migration, invasion, tumor growth and metastasis [5, 28, 29]. As a member of PRLs, PRL2 is reported that upregulated in many cancer (pancreatic, breast and lung cancer) and drive cancer metastasis . However, there are also contradictory observations. PRL2 expression correlated with better survival in breast cancer patient  and high expression of PRL2 was a favorable prognostic marker in five independent breast cancer datasets . Our finding indicates high expression of PRL2 is associated with ROS induced cell death which might contribute to cancer patient survival and response to ROS based chemotherapy or radiotherapy. Whether PRL2 promotes tumor progression or cell death depends on the kind of cells and tissues, the location of PRL2 and the sensitivity of PRL2 to oxidation. The further studies will be needed to reconcile the roles of PRL2 in cancer.
Collectively, our study suggests PRL2 serves as a negative regulator in cell adaptation to oxidative stress via PDK1/AKT pathway. Thus PRL2 could be targeted to modulate cell viability in inflammation or irradiation associated therapy.
The authors thank Dr. Youhai Chen (University of Pennsylvania) for providing plasmid constructs. The authors also thank Dr. Junke Zheng (Shanghai Jiaotong University School of Medicine) for sharing C57BL/6 mice expressing CD45.1.
XD and ZW wrote the paper. ZW, XD, GC, YW designed the experiments. XD, YZ, XL, QL, CW performed and analyzed the data. YW contributed reagents. GC, XG and YW read the paper, and ZW oversaw the project. All authors read and approved the final manuscript.
This work was supported by grants from the National Natural Science Foundation of China (NSF-81971486), Natural Science Foundation of Shanghai (19ZR1428500) and innovative research team of high-level local universities in Shanghai. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Ethics approval and consent to participate
The conducts and procedures involving animal experiments were approved by the Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine (project number 2012008, A-2019-053,069) according to Regulations for the Administration of Affairs Concerning Experimental Animals (approved by the State Council of the People’s Republic of China) and Guide for the Care and Use of Laboratory Animals (Department of Laboratory Science, Shanghai Jiao Tong University School of Medicine, laboratory animal usage license number SYXK 2013-0050, certificated by Shanghai Committee of Science and Technology).
Consent for publication
The authors declare that they have no competing interests.
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