PP2Ac upregulates PI3K-Akt signaling and induces hepatocyte apoptosis in liver donor after brain death
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Multiple research groups have demonstrated that the outcome of patients receiving liver grafts from brain death donors (DBD) is poorer when compared with patients receiving grafts from living donors. This might be due to an increased hepatocyte apoptosis induced after brain death (BD). In this work, we found that the activity of PP2A-Akt pathway is significantly increased in clinical donor ex vivo hepatocytes after BD by iTRAQ protein quantification analysis. The same results were confirmed in animal models. A time-dependent promotion of apoptosis was also found in DBD rabbit liver, as demonstrated by the increased levels of cleaved Caspase 3 and the decreased of Bcl-2. To further investigate the roles of PP2A and Akt in regulating apoptosis of hepatocytes after BD, we cultivated human liver cell line L02 with serum deprivation and hypoxia, to simulate the ischemic and hypoxic conditions of hepatocytes in DBD. Increased apoptosis and decreased viability were observed during the time in this model. Meanwhile PP2A activity and Akt activity were respectively increased and decreased. Notably, the proportion of Akt phosphorylation at Ser473 decreased, while other known targets of PP2A (p38, JNK and ERK) were not affected in terms of protein levels or phosphorylation. These results suggested that PP2A is involved in apoptotic induction of hepatocytes after brain death by specific suppression of Akt. This discovery was further confirmed with pharmaceutical and genetic methods. Our work implied potential targets for reducing liver cell apoptosis and improving organ donor quality after BD.
KeywordsDonation after brain death Hepatocyte damage Apoptosis PP2A
There exists a vast disparity between the number of patients on transplant waiting list and that of available organs [1, 2, 3, 4] and the main source of donor organs are from brain death (DBD) and cardiac death (DCD). Several kidney-transplant studies have revealed a consistent and significant difference in the graft survival between grafts derived from brain-dead (BD) donors and the grafts from living donors. There are more incidents of primary graft non function and poor graft survival when grafts were from BD patients than that from living donors. Animal studies showed the same tendency: BD causes deteriorated organ quality and viability for both kidney and liver [5, 6, 7]. It was hypothesized that BD has a detrimental effect on donor organ viability [8, 9].
Even though a number of studies have assessed the effects of BD on kidney and liver grafts viability, the mechanism by which BD induces kidney and liver injuries has not been fully clarified. It has been suggested that apoptosis after BD contributes strongly to organ injury [10, 11, 12, 13]. But it is not clear yet how BD causes apoptosis.
PP2A, a serine/threonine protein phosphatases made up of three subunits A, B and C, is demonstrated to be involved in apoptosis. The subunit A is required for the formation of the trimeric complex, subunit C (PP2Ac) has catalytic domain  and B is the regulatory that binds with the AC core complex to form the heterotrimeric holoenzyme  and is responsible for the substrate specificity of PP2A . PP2A can act on a wide range of substrates through the holoenzyme structure that contains a distinct B subunit from the families of the proteins: B (PR55), Bʹ (B56 or PR56), B″ (PR72) and B‴ (PR93/PR110) . Some of the most common PP2A substrates include apoptotic protein Bax, DNA repair protein ATM, tumor suppressor protein pRb, receptor EGFR, MAPK cell signaling protein JNK and cell cycle-associated proteins CDK4, CDK9, CD16, CDC6 and CDC25 . PP2A can not only indirectly regulate the induction of apoptosis via the dephosphorylation of cell signaling and tumor suppressor proteins such as p38, JNK, Akt and Rb respectively [16, 18], but can also act directly on apoptotic and anti-apoptotic proteins [19, 20]. For example, it has been shown that protein PKR promotes mitochondrial localization of the PP2A–PR61α complex leading to Bcl-2 dephosphorylation . In addition, PP2A has been reported to dephosphorylate Bax and enhance its pro-apoptotic function via direct interaction with catalytic subunit PP2A/C .
Protein kinase B (PKB), also known as Akt, a serine/threonine-specific protein kinase that plays a key role in glucose metabolism, apoptosis, cell proliferation, transcription and cell migration, is mainly regulated by protein kinases and protein phosphatases [21, 22, 23]; both are highly expressed in the kidney, liver, and spleen. Akt activity has been shown to be regulated by PP2A. Overexpressed-eIF3I interacts with the activated form of oncogenic Akt1 via inhibition of PP2A phosphorylation in human hepatocellular carcinoma , whereas the protein REDD1 enhances PP2A-mediated dephosphorylation of Akt resulting in repression of mTORC1 signaling in 293T cells . In addition, it has been shown that PP2A regulates p53 and Akt cooperatively leading to apoptosis evasion by neuron cells . Lastly ceramide mediated vascular dysfunction in diet-induced obesity has been shown to be through dephosphorylation of the eNOS-Akt complex by PP2A . Although the actions of PP2A and Akt have been well defined in the aforementioned studies, the regulation of Akt signaling in the cellular apoptosis in the donor liver from BD patients has not yet been reported.
Based on these previous findings, we suspected that BD may induce Akt inactivation and hepatocyte apoptosis through PP2A activation in donor liver from BD individuals. In the present study, we explored the effects of BD on hepatocyte apoptosis and possible link of Akt and PP2A with the apoptosis.
Brain death (BD) inactivates Akt, activates PP2A and causes liver apoptosis both in animal model and donor liver
Hypoxia and ischemia (mimic BD) induced apoptosis is dependent on Akt inactivation and PP2A activation in vitro
Then we set up an in vitro model for further investigating the roles of PP2A and Akt in regulating apoptosis of hepatocytes after brain death. We cultivated human liver cell line L02 in starvation (with serum deprivation) and under hypoxia (low oxygen concentration), simulating the ischemic and hypoxic conditions of hepatocytes in BD patients .
We also found that the activity of PP2A in L02 cells was remarkably increased over time under serum deprivation and hypoxia, while the opposite was observed for the activity of Akt (Fig. 3c). Although western blotting failed to show change in total Akt and PP2A protein level, Akt phosphorylation at Ser473 decreased during the time of hypoxia exposure, consistent with the diminishing Akt activity (Fig. 3d). For other known targets of PP2A, such as p38, JNK and ERK, the content of total protein as well as phosphorylated forms seemed not to be affected (Fig. 8). These results implied that PP2A could be involved in the regulation of hepatocytes apoptosis after brain death, possibly through dephosphorylation and inhibition of Akt activity.
Hypoxia and ischemia (mimic BD) induced Akt inactivation is directly regulated by PP2A activation in vitro
In the present study, we revealed the involvement of PP2A in the induction of apoptosis in liver cells after brain death, by negatively regulating Akt activity. Suffering from undernourished and hypoxic conditions, the hepatocytes showed an increase in PP2A activity, which afterwards suppressed Akt activity but had little effect on the p38, JNK or ERK signaling. The inhibition of Akt activity may be the result of decreased phosphorylation at Ser473. Apoptosis level in the damaged hepatocytes were promoted as a consequence of such “PP2A activation-Akt inhibition” axis, and pharmaceutical targeting of PP2A or Akt could correspondingly worsen or attenuate the hepatocellular damage.
Brain death (BD) is defined as the irreversible cessation of all brain activities, including that in the brain stem. Clinical and experimental studies have shown that BD is a complex pathological process that damage organ morphology and function [34, 35, 36]. The physiological changes that occur after BD include hemodynamic changes, blood coagulation, endocrine and/or electrolyte disorders. Several studies have shown that grafts from BD donors have a lower short- and long-term posttranslational effectiveness and higher incidences of primary graft non-function  and higher incident of acute rejection , in comparison with that from living donors. Despite these crucial observations, the exact etiology of the poor quality of donor-liver function has not been fully examined.
Novitzky et al. reported that BD leads to hemodynamic changes, characterized by a dramatic increase in both systolic and diastolic blood pressure . These effects lead to tachycardia and the cardiac output is increased by 30%, whereas a 50% increase is noted in the peripheral vascular resistance . The blood flow in the portal vein and the hepatic tissue has been reported to be approximately 40% of that corresponding to the hypotensive state in the absence of brain death  leading to hepatic hypoxia. In the present study, hypoxia and ischemia were induced at the cellular level for the time periods of 0 h, 6 h, 12 h and 18 h, in order to mimic hepatic ischemia induced by brain death.
Several reports showed that the increased rate of apoptosis is one of the main factors that lead to poor liver graft quality [10, 12, 40, 41]. In our previous study, we confirmed that the increased apoptosis is the most important factor contributing to decreasing liver functions under the brain death conditions . Intervention at the level of the apoptotic pathway is likely to be a promising target for drug development, in terms of improving the quality of liver graft to decrease the incident of primary graft non function and acute rejection.
Previous studies have indicated that brain death induces liver donor cell apoptosis by various signaling pathways. Brain death can change the inflammation response, which releases inflammatory mediators and promotes apoptosis . In addition, it can contribute directly to the activation of caspase 3 leading to apoptosis . HO-1, which has strong anti-oxidant activity, can improve long-term brain dead graft survival . Thus, all evidences have pointed to brain dead induced cell apoptosis as the cause of poor graft quality.
In the current study, with the use of multiple biochemical and pharmacological approaches, we found that brain death activated PP2A and inactivated Akt in the liver. This finding was further verified with brain-death-mimic cell culture environment, in which hepatocyte apoptosis was induced. In this case PP2A activity increased and subsequently dephosphorylates Akt on ser473. PP2A is considered as a tumor suppressor enzyme that affects multiple signaling cascades such as the Ras, PI3 K, Akt and PKC pathways that have been implicated in various cancers . The activation of PP2A is a common event that is noted in several cancers that occurs as a result of the perturbed regulation of the cell growth and survival . Many reports showed that PP2A is activated with cAMP/PKA [44, 45]; hypoxia can induce cAMP/PKA activation through HIF transcription factor . Therefore, we speculated that enhanced PP2A activity in our brain death in vivo and in vitro model was due to the induction of hypoxia after brain death. With regard to Akt, PP2A has been shown to directly interact with this protein via the subunit B55 alpha . PP2A inactivates Akt by dephosphorylation and in hepatoma cancers the reduced expression of PP2A B55 alpha subunit results in the increased activation of Akt and consequently increased growth and proliferation . Previous studies demonstrated that the overexpression of eIF3I interacted with and activated the oncogenic Akt1 by preventing the PP2A-mediated dephosphorylation of Akt-1 in human hepatocellular carcinoma . REDD1 enhances protein phosphatase 2A-mediated dephosphorylation of Akt, in order to repress mTORC1 signaling in 293T cells . For the first time in the current study we demonstrated that the down regulation of Akt activity by PP2A results in apoptosis in liver cells in the brain-dead donors or in a similar in vivo brain death model.
In conclusion, the findings presented here disclose one of the underlying mechanisms for hepatocellular impairment after brain death, and imply potential targets for reducing hepatocyte apoptosis and improving hepatic graft quality, through PP2A inhibition or Akt activation. Studies in this field will help to prolong organ graft survival and lower the incidence of primary graft non-function or acute rejection.
Materials and methods
Clinical DBD samples
The study was approved by the ethics committee of the Zhongnan Hospital of Wuhan University. All patients or their legal representatives provided a written informed consent.
From February 2013 to March 2016, a total of 20 samples of DBD liver tissues were respectively collected from 20 donors at 2 h, 6 h or 12 h post brain death (Table S1) in the Zhongnan Hospital of Wuhan University, Wuhan, China. The DBD liver tissues were extracted without perfusion. The tissues were suitable for transplantation, i.e., without any lesions, cirrhosis, fatty liver disease, etc. The preservation solution was UW solution.
The liver tissues were homogenized in 10 volumes (mL/g wet tissue) homogenate buffer containing 50 mM Tris–HCl, pH 7.0, 0.5 mM Phenylmethylsulfonyl fluoride (PMSF), 2.5 mM ethylenediaminetetraacetic acid (EDTA), 2.5 mM ethylene glycol tetra acetic acid (EGTA), and 1:1000 protease inhibitor cocktail (Sigma-Aldrich Co., LLC., St. Louis, MO, USA). The tissue lysates were sonicated, and centrifuged at 16,000×g for 10 min. The supernatants were used for the evaluation of the PP2A and Akt activity assays.
Protein extraction and iTRAQ mass spectrometry analysis
Liver samples after brain death for 2 h and 12 h were collected. Each sample (100 mg of protein) was digested with SDT solution and labeled with iTRAQ reagents (Applied Biosystems) according to the manufacturer’s protocol. Subsequently, the labeled peptides were mixed equally and separated by 1260 Infinity HPLC (Agilent Technologies), followed by nano liquid chromatography tandem mass spectrometry using the Hybrid Quadrupole-Orbitrap mass spectrometer (Q-Exactive; Thermo Fisher Scientific) equipped with a nano-UPLC RSLC Ultimate 3000 (Dionex). Both peptide identification and quantitation were performed in an overall workflow in Proteome Discoverer software (version 1.4; Thermo Fisher Scientific) and searched against the UniProt human canonical sequence protein database (October 7, 2011; 56,869 entries) using Mascot search engine (version 2.4). For protein identification, 95% confidence was used. For quantitation and further validation experiments, all reported data were based on 95% confidence for protein identification as determined by Proteome Discoverer (Unique peptide > 1)
The study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee of the Ethics of Animal Experiments of Wuhan University 2014. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.
A total of 40 male New Zealand rabbits (weight 2800–3000 g, 3 months old) were obtained from the Center for Animal Experiments and ABSL-3 Laboratory of the Wuhan University (Wuhan, China). All animals were kept under standard laboratory conditions: 12 h of light and 12 h of darkness; lights were turned on at 6:00 a.m.; the temperature was kept at 22 ± 2.0 °C; and water and food were ad libitum.
The DBD model was established in 30 rabbits as described previously [28, 29]. The rabbits were deeply anesthetized with pentobarbital sodium at a dose of 40 mg/kg and then placed on the operating table. The femoral artery and vein cannulation, xiphoid separation and tracheal intubation, burr hole and catheter placement were carried out. The vital signs of the rabbits including electrocardiogram, blood pressure, respiratory functions, and electroencephalogram were monitored using a biological functional system, a rodent ventilator, and an intelligent temperature control instrument (Thai Union Technology, Co., Ltd., Chengdu, China). The intracranial pressure was increased as required until the occurrence of brain death. A total of ten rabbits were sacrificed respectively at 2 h, 4 h, and 8 h, following brain death.
The remaining ten rabbits received deep anesthesia without an increase in the intracranial pressure, and they were sacrificed 2 h following sham operation.
The liver tissues obtained from the animals were then processed as methods described for clinical samples, and used for the PP2A/Akt activity assays. Besides, after sonication, a part of tissue lysates was added with equal volume of phosphate inhibitor mixture (2.0 mM Na3VO4 and 100 mM NaF, pH 7.0) and stored at − 80 °C for Western blotting.
Human liver cell line L02 was obtained from Kunming Institute of Zoology (Chinese Academy of Sciences, Kunming, China). The cells were grown to 70–80% confluence in six-well culture plates in Dulbecco’s Modified Eagle’s medium (Gibco (Life Technologies), CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 0.1 mg/mL streptomycin.
The L02 cells were exposed to small molecule treatment or transfected with specific plasmids (see detailed description below). Then the cells were cultured in serum-free DMEM/low glucose medium (HyClone, USA) and placed in a tri-gas incubator (thermo fisher 3131, Waltham, MA USA) which can control the oxygen concentration at 1%, to make ischemic and hypoxic conditions mimicking adverse environment of liver in BD donors . The specific processing times were indicated in the corresponding figures.
After different treatments, some of cells were used for flow cytometry analysis, cell viability analysis and lactate dehydrogenase (LDH) measurement. The remaining cells were harvested and suspended in buffer containing 2.0 mM EGTA, 0.5 mM PMSF, 5 mM EDTA, 150 mM NaCl, 50 mM Tris–HCl (pH 7.4), 1.0% Triton X-100, and a protease inhibitor cocktail (1:200). The cell lysates were sonicated and divided into three parts. The first part was mixed with equal volume of phosphate inhibitor mixture (2.0 mM Na3VO4 and 100 mM NaF, pH 7.0) and stored at − 80°C for Western blotting. The rests were centrifuged at 16,000×g for 10 min, and the supernatants were used for PP2A and Akt activity assay.
Small molecule pretreatment
L02 cells were pretreated with specific concentrations of small molecules at 60 min time periods. The Akt inhibitor MK-2206 (Selleckchem, USA) and the PP2A inhibitor OA (Santa cruz, USA) were incubated with L02 cells at a concentration of 10 nM. The Akt activator SC79 (TOCRIS bioscience, Missouri, USA) and the PP2A activator DES (San Diego, CA, USA) were incubated with L02 cells at concentrations of 4 μg/mL and 5 μM, respectively.
The plasmids carrying the EGFP-siPP2Ac (a small interfering RNA construct designed specifically for pp2ac) and the DsRed-wild-type (wt) PP2Ac were kindly provided by Professor Jianzhi Wang (Tongji Medical College, HuaZhong University of Science and Technology, Wuhan, China). L02 cells were transfected with EGFP-siPP2A or DsRed-wtPP2Ac plasmids using Lipofectamine 2000 according to the manufacturer’s instruction (Invitrogen, San Diego, CA, USA). The plasmids that contained solely fluorescent proteins were used as the controls. Following 48 h of transfection, the cells were used for further analyses.
Flow cytometry analysis
The cellular apoptosis and necrosis were assessed using the Annexin V-FITC apoptosis kit (Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. The cells were digested with trypsin, centrifuged at 1000×g for 5 min, and incubated with 5 μL Annexin V and 5 μL PI for 10 min at room temperature in the dark. The cells from each sample were then identified using a FacsCalibur flow cytometer (BD Biosciences). The data were analyzed using CELLQuest software (BD). The experiments were repeated at least three times.
Cell viability analysis
The cell viability was assessed with the Cell Counting Kit-8 detection kit (CCK8) (Dojindo Laboratories, Kumamoto, Japan) according to the manufacture’s protocol. A total of 1 × 105 cells were cultured in 96-well plates. The cells were performed with different treatments, and then 100 μL 10% CCK8 solution was added in each well. The samples were incubated at 37 °C for 1 h. The absorbance was measured at 450 nm using a microplate reader (VersaMax, Molecular Devices, USA). The experiments were repeated at least three times.
Lactate dehydrogenase (LDH) measurement
The cell medium was collected for the LDH measurement using the AU5400 Clinical Chemistry System (beckman coulter, USA), and the results were expressed in the unit of U/L. The experiments were repeated at least three times.
The liver tissue lysates (from clinical samples or modeling rabbits) or the L02 cell lysates were electrophoresized in 10% SDS-polyacrylamide gel and the proteins were transferred to nitrocellulose membranes. The membranes were incubated with specific antibodies. Primary antibodies against: pSer473 Akt, Bcl-2, Caspase 3, Cleaved-Caspase 3, p38, p-p38, JNK, p-JNK, ERK, p-ERK were from cell signaling (Danvers, MA, USA); total Akt was from Proteintech (Chicago, UK); total PP2Ac was from Millipore (Billerica, MA, USA); β-actin was from Abcam (Cambridge, UK). Secondary antibodies conjugated to I IRDye™ 800CW were from Licor Biosciences (Lincoln, NE, USA). The results were visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).
PP2A/Akt activity assay
The supernatants of the liver tissue lysates (from clinical samples or modeling rabbits) or L02 cell lysates were prepared as described above. The activity of PP2A in the supernatants was detected using the phosphatase kit V2460 (Promega, Madison, WI, USA) according to the manufacturer’s protocol, with the results expressed as nmol phosphorus/mg/min. The activity of Akt was detected using the colorimetric method quantity detecting kit (Genmed Scientifics Inc., Arlington, MA, USA) according to the manufacturer’s protocol, with the results expressed as μmol NADH/mg/min.
The data are expressed as the mean ± standard error of measurement (SEM) and analyzed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). The one-way analysis of variance (ANOVA) procedure followed by the Fisher’s Least Significant Difference post hoc test (LSD) and Student’s t test were used to determine the differences among the groups. The statistical differences were considered significant if the p value was < 0.05.
This work was supported by the National Natural Science Foundation of China (Nos.: 81700574, U140322).
YX and JL conceived and planned the experiments. KH, LZ and YZ carried out the experiments. YW took the lead in writing the manuscript. All authors provided critical feedback and helped shape the research, analysis and manuscript.
Compliance with ethical standards
Conflict of interest
No conflicts of interest, financial or otherwise, are declared by the authors.
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