Inhibition of GSK-3β ameliorates hepatic ischemia-reperfusion injury through GSK-3β/ β-catenin signaling pathway in mice

Nanjing, China

Inhibition of GSK-3β ameliorates hepatic ischemia-reperfusion injury through GSK-3β/ β-catenin signaling pathway in mice

Yong-Xiang Xia, Ling Lu, Zheng-Shan Wu, Li-Yong Pu, Bei-Cheng Sun and Xue-Hao Wang

Nanjing, China

BACKGROUND: Glycogen synthase kinase (GSK)-3β/β-catenin signaling regulates ischemia-reperfusion (I/R)-induced apoptosis and proliferation, and inhibition of GSK-3β has beneficial effects on I/R injury in the heart and the central nervous system. However, the role of this signaling in hepatic I/R injury remains unclear. The present study aimed to investigate the effects and mechanism of GSK-3β/β-catenin signaling in hepatic I/R injury.

METHODS: Male C57BL/6 mice (weighing 22-25 g) were pretreated with either SB216763, an inhibitor of GSK-3β, or vehicle. These mice were subjected to partial hepatic I/R. Blood was collected for test of alanine aminotransferase (ALT), and liver specimen for assays of phosphorylation at the Ser9 residue of GSK-3β, GSK-3β activity, axin 2 and the antiapoptotic factors Bcl-2 and survivin, as well as the proliferative factors cyclin D1 and proliferating cell nuclear antigen, and apoptotic index (TUNEL). Real-time PCR, Western blotting and immunohistochemical staining were used.

RESULTS: SB216763 increased phospho-GSK-3β levels and suppressed GSK-3β activity (1880±229 vs 3280±272 cpm,P＜0.01). ALT peaked at 6 hours after reperfusion. Compared with control, SB216763 decreased ALT after 6 hours of reperfusion (4451±424 vs 7868±845 IU/L,P＜0.01), and alleviated hepatocyte necrosis and vacuolization. GSK-3β inhibition led to the accumulation of β-catenin in the cytosol (0.40±0.05 vs 1.31±0.11,P＜0.05) and nucleus (0.62±0.14 vs 1.73±0.12,P＜0.05), β-catenin further upregulated the expression of axin 2. Upregulation of GSK-3β/β-catenin signaling increased Bcl-2, survivin and cyclin D1. Serological and histological analyses showed that SB216763 alleviated hepatic I/R-induced injury by reducing apoptosis (1.4±0.2% vs 3.6±0.4%,P＜0.05) and enhanced liver proliferation (56±8% vs 19±4%,P＜0.05).

CONCLUSION: Inhibition of GSK-3β ameliorates hepatic I/R injury through the GSK-3β/β-catenin signaling pathway.

(Hepatobiliary Pancreat Dis Int 2012;11:278-284)

glycogen synthase kinase 3; beta-catenin; reperfusion injury; signal transduction

Introduction

Hepatic ischemia-reperfusion (I/R) injury is a common occurrence in hepatectomy surgery, transplantation, and trauma.[1-3]I/R liver damage is of great clinical importance because it causes primary graft dysfunction after liver transplantation and compromises the function of the remaining liver after major hepatectomy.[4]Recent studies[5,6]suggest that apoptosis is the central mechanism of the major death processes for hepatocytes and sinusoidal endothelial cells following reperfusion of the ischemic liver. Apoptosis is a regulated process, and is determined by a balance between pro- and anti-apoptotic factors. Promoting hepatocyte proliferation is also an important strategy to protect the liver from I/R injury.[7]Alleviating apoptosis and enhancing liver proliferation are therefore the major strategies for the treatment of ischemic liver diseases. Many molecules and signaling pathways are involved in the process of apoptosis, and glycogen synthase kinase 3 (GSK-3) is one of them.

GSK-3, a serine/threonine protein kinase, was discovered more than two decades ago and is considered to be a constitutively active enzyme. The regulatory mechanisms consist of controlling its activity andsubcellular distribution. GSK-3 phosphorylates and inactivates glycogen synthase.[8]There are two highly homologous isoforms, GSK-3α and GSK-3β; both share substrate specificityin vitro.[9]Beyond its function in glycogen metabolism, GSK-3β plays an important role in liver physiology and pathology by regulating various basic cellular events, including apoptosis, proliferation, differentiation, and oxidative stress.[10]Targeting GSK-3β ameliorates hepatic I/R injury. Varela and colleagues[11]claimed that inhibition of GSK-3β protects the liver against I/R injury by maintaining mitochondrial function and hepatic energy-balance, while Ren and coworkers[12]declared the involvement of inflammatory cytokines. However, studies[13-15]in the heart and central nervous system have shown that it is the β-catenin signaling pathway that mediates the protection against I/R injury via anti-apoptosis. This mechanism has not been investigated in hepatic I/R injury.

The present study aimed to clarify the role of the β-catenin signaling pathway in the liver after I/R injury. SB216763 was used as a GSK-3β inhibitor. The cytosolic and nuclear β-catenin content was measured, and expression of the anti-apoptotic factors Bcl-2 and survivin and the proliferative factor cyclin D1 were also quantified. TUNEL, an apoptotic index, was evaluated.

Methods

Animals

Male C57BL/6 mice weighing 22-25 g were purchased from the Animal Center of Nanjing University (Nanjing, China). The mice were maintained in a 12:12-hour light/dark cycle and hadad libitumaccess to standard rodent chow and tap water. The experiments were carried out in accordance with the guidelines approved by the Institutional Animal Care and Use Committee at Nanjing Medical University (Protocol Number NJMU08-092).

Experimental design and surgical procedures

Seventy-eight mice were randomly assigned to the following experimental groups: 1) Sham-operated group: mice were subjected to the surgical procedure with the omission of vascular occlusion (n=6); 2) Vehicle control group: mice were treated with DMSO 2 hours before I/R surgery and then subjected to 90 minutes of ischemia followed by 1, 3, 6, 12, 24 or 36 hours of reperfusion (n=6 per group); 3) SB216763 group: mice were treated with SB216763 (25 mg/kg, i.p.; Sigma) 2 hours before I/R surgery.

Mice were anesthetized by intraperitoneal injection of 50-60 mg/kg sodium pentobarbital. A midline laparotomy incision was made to expose the liver, extending from the xiphisternum to the pubis. The artery and the portal vein supply to the left lateral and median lobes was microclamped as described previously.[16]Then, reperfusion was initiated by removing the clamp. This model leads to segmental (70%) hepatic ischemia. Since the blood supply to the right and caudate lobes was not interrupted, there was no mesenteric congestion. Body temperature was monitored with a rectal probe and maintained at 37 ℃ by a heating pad during the procedure. The abdomen was then closed with 4-0 silk sutures, and the mice were allowed to recover for the required reperfusion period. Mice were sacrificed by bleeding from the vena cava at different times after reperfusion for tissue and blood sample collection.

GSK-3β kinase assay

GSK-3β activity was measured in control and ischemic liver lobes 1 hour after reperfusion. Frozen livers were homogenized and immunoprecipitated using anti-GSK-3β antibody (Sigma). The activity of the enzyme was assessed by a kinase assay using32P-[ATP] and glycogen synthase peptide-2 (Sigma) as substrate, as described by Haq et al.[17]

Serum alanine aminotransferase (ALT)

Blood samples for ALT assessment were obtained after 6-hour reperfusion, and analyzed using a serum analyzer (Hitachi 7600-10, Hitachi High-Technologies Corp., Japan).

Western blotting

Proteins were extracted from the liver lobes subjected to ischemia, and their concentrations were determined by the Bradford assay (Bio-Rad, CA). Cytosolic and nuclear protein extracts were prepared according to the manufacturer's protocol (NE-PER nuclear and cytoplasmic extraction reagents; Thermo Scientific, IL). Membranes were then incubated with antibodies to GAPDH (1:2000), β-catenin (1:1000), survivin (1:1000), Bcl-2 (1:1000), cyclin D1 (1:1000), histone H3 (1:2000) and cleaved caspase-3 (1:1000) (Cell Signaling Technology, Danvers, MA). Secondary antibodies (anti-rabbit) conjugated with horseradish peroxidase were from Santa Cruz Biotechnology (1:3000). The results were visualized with a chemiluminescent detection system (ECL, Pierce, IL., USA) and exposed to X-ray film. GAPDH was used as the internal control for all proteins examined. The relative expression of each protein was quantified by computerized densitometric scanning of the images using Image J software. Results are expressed in densitometric units normalized to internal control expression.

Quantitative real-time PCR

Reverse transcription reactions were performed using the Super-Script First-Strand Synthesis System (Invitrogen, CA). To determine the relative number of cDNA molecules in the reverse-transcribed samples, real-time PCR analyses were performed using the Light-Cycler system (Roche, Indianapolis, IN). PCR was performed according to the procedure as previously described.[18]Primers (sense sequence and antisense sequence, respectively) for the following genes were: Bcl-2 (5'-GGA ATT GCC AAG AAA CGT GTG-3' and 5'-GGG TCC TGT GCC ACT TGC-3'); survivin (5'-GCG GAG GCT GGC TTC A-3' and 5'-TGC TCC TCT ATC GGG TTG TCA-3'); GAPDH (5'-GGT CAC CAG GGC TGC CAT TTG-3' and 5'-CTG GTA CTC CAT ACA CTG GCT-3'); cyclin D1 (5'-GGA TGT CCA CAC ACG CAT TCA-3' and 5'-CGG TGG TGC GAG AAC AGA GTT-3'); axin 2 (5'-ATG AGT AGC GCC GTG TTA GTG-3' and 5'-GGG CAT AGG TTT GGT GGA CT-3').

Histology and immunohistochemistry

Liver tissues fixed in 4% paraformaldehyde were embedded in paraffin, sectioned at 5 μm and stained with hematoxylin and eosin. The sectioned slides were stained immunohistochemically for β-catenin (Cell Signaling Technology) and proliferating cell nuclear antigen (PCNA) (ImmunoCruz Staining System, Santa Cruz Biotechnology) as described previously.[19]

TUNEL assay

To detect apoptotic cells, sections were stained by TUNEL using a kit (in situcell death detection kit, Roche-Boehringer Mannheim, Germany).

Statistical analysis

All data are presented as mean±SEM. Differences between groups were analyzed with Student'sttest. APvalue ＜0.05 was considered statistically significant.

Results

Inhibition of GSK-3β ameliorates hepatic I/R injury

Fig. 1. Amelioration of hepatic I/R injury by inhibition of GSK-3β. A: Mice were treated with vehicle control (DMSO) or SB216763. GSK-3β activity: DMSO-treated and SB216763-treated livers. GSK-3β activity was assessed in liver samples harvested 1 hour later (*:P＜0.01, versus vehicle control). B: Mice were treated with SB216763 and/or subjected to I/R. Liver samples, harvested 1 hour later, were subjected to Western blotting analysis of phosphorylation at the Ser9 residue of GSK-3β (*:P＜0.05, #:P＜0.01, versus sham-operated). C: ALT reached a peak at 6 hours after reperfusion in control and SB216763+I/R groups. D: At 6 hours of reperfusion, serum ALT was decreased in the I/R+SB216763 compared with I/R+vehicle control (*:P＜0.01, versus vehicle control). E: Liver histopathology is normal in sham-operated; I/R+vehicle control: hemorrhage and hepatocyte necrosis; I/R+SB216763: hepatocyte necrosis significantly alleviated (HE staining, original magnification ×400).

To confirm the effect of SB216763 on GSK-3β, we performed an immune complex kinase assay and found that, compared with vehicle control, SB216763 decreased GSK-3β activity within 1 hour after treatment (1880±229 vs 3280±272 cpm,P＜0.01, Fig. 1A). GSK-3β is a special enzyme that is inactivated after phosphorylation; therefore, we assessed the phosphorylated residue Ser9 on GSK-3β by Western blotting. In the sham-operatedgroup, phospho-GSK-3β was just detectable, while the total GSK-3β expression was strong, and the expression of phospho-GSK-3β increased with I/R or SB216763 treatment. However, treatment with SB216763 alone or followed by I/R, increased the phospho-GSK-3β level in comparison to the sham-operated group or I/R alone, while the total GSK-3β remained unchanged (0.18± 0.03 in sham-operated, 0.34±0.05 in I/R, 1.26±0.27 in SB216763 and 1.33±0.29 in SB216763+I/R, allP＜0.05) (Fig. 1B). We assessed serum ALT after 1, 3, 6, 12, 24 and 36 hours of reperfusion. Similar to previous studies,[20,21]we found that ALT peaked at 6 hours after the onset of reperfusion (Fig. 1C), and SB216763 decreased ALT after 6 hours of reperfusion (4451±424 vs 7868±845 IU/L,P＜0.01, Fig. 1D). Furthermore, in parallel with the ALT changes, hematoxylin and eosin (HE) staining showed that SB216763 significantly alleviated hepatocyte necrosis and vacuolization in I/R liver after 90 minutes of ischemia followed by 6 hours of reperfusion (Fig. 1E).

Inhibition of GSK-3β activates GSK-3β/β-catenin signaling during hepatic I/R

β-catenin significantly accumulated in the cytosol and nucleus in SB216763-treated mice at 1 hour of reperfusion compared with the vehicle control (0.40±0.05 vs 1.31±0.11 and 0.62±0.14 vs 1.73±0.12, bothP＜0.05) (Fig. 2A). The results of immunohistochemical staining showed the same pattern: there was scant staining in the liver from shamoperated, and the density of β-catenin was significantly increased in the liver of SB216763-treated I/R mice at 1 hour of reperfusion compared with the vehicle control (2.4±0.4% vs 23.7±4.6%,P＜0.01, Fig. 2B). To further verify whether GSK-3β/β-catenin signaling was activated, we tested the expression of axin 2, a ubiquitous target gene of this signaling pathway,[22]and found that axin 2 expression was significantly increased within 3 hours after reperfusion in SB216763-treated mice compared with both sham-operated and vehicle control groups (Fig. 2C). These results indicated that the inhibition of GSK-3β activated GSK-3β/β-catenin signaling in hepatic I/R.

SB216763 upregulates Bcl-2 and survivin mRNA expression and attenuates apoptosis

Quantitative real-time PCR showed that SB216763 significantly increased the expression of Bcl-2 and survivin at 3 hours of reperfusion compared to the vehicle control (Fig. 3A). Moreover, the protein expression of Bcl-2 and survivin also significantly increased in the SB216763-treated liver compared to the vehicle control (0.58±0.09 vs 1.70±0.11 and 0.45±0.06 vs 1.48±0.19, bothP＜0.05). In parallel with Bcl-2 and survivin, the expression of cleaved caspase-3 (1.23±0.30 vs 0.39±0.04,P＜0.05) (Fig. 3B) and the TUNEL assay showed that hepatic apoptosis was decreased in the SB216763-treated liver compared with the vehicle control (1.4±0.2% vs 3.6±0.4%,P＜0.05, Fig. 3C).

Fig. 2. Inhibition of GSK-3β activates GSK-3β/β-catenin signaling during hepatic I/R (*:P＜0.05, #:P＜0.01, verus vehicle control group). A: Western blotting showed that at 1 hour of reperfusion, β-catenin was increased in cytosolic and nuclear extracts from I/R+SB216763 compared with I/R+vehicle control. B: Representative immunohistochemical staining for β-catenin 1 hour after I/R (original magnification ×400). Nuclear β-cateninpositive cells were increased in I/R+SB216763 compared with vehicle control. C: Expression of axin 2 detected by real-time PCR after 3 hours of reperfusion.

SB216763 upregulates cyclin D1 mRNA expression and enhances liver proliferation

Fig. 3. SB216763 upregulates Bcl-2 and survivin mRNA expression and attenuates apoptosis (*:P＜0.05, versus vehicle control). A: Real-time PCR showed that the transcription of both Bcl-2 and survivin increased in I/R+SB216763 (3 hours reperfusion) compared with I/R+vehicle control. B: Western blotting showed that the expression of both Bcl-2 and survivin increased in I/R+SB216763 (6 hours reperfusion) compared with I/R+vehicle control, and cleaved caspase-3 decreased. C: Apoptotic cells were quantified in six high-power fields at a microscope magnification of ×400, and expressed as percentages of apoptotic cells among total hepatocytes. Apoptosis-positive cells decreased in I/R+SB216763 (6 hours reperfusion) compared with vehicle control.

Cyclin D1 mRNA expression was significantly increased in the SB216763-treated liver at 3 hours of reperfusion compared with sham-operated and vehicle control groups (3.03±0.12 vs 0.74±0.03,P＜0.05) (Fig. 4A); at 6 hours of reperfusion, the amount of protein evaluated by Western blotting showed the same tendency as real-time PCR (0.51± 0.06 vs 1.67±0.13,P＜0.05) (Fig. 4B). Immunohistochemical staining showed that PCNA was elevated in the SB216763-treated liver at 24 hours of reperfusion compared with sham-operated and vehicle control groups (56±8% vs 19± 4%,P＜0.05, Fig. 4C).

Fig. 4. SB216763 upregulates cyclin D1 mRNA expression and enhances proliferation (*:P＜0.05, versus vehicle control). A: Realtime PCR showed that the transcription of cyclin D1 increased in I/R+SB216763 (3 hours reperfusion) compared with vehicle control. B: Western blotting showed that the expression of cyclin D1 increased in I/R+SB216763 (6 hours reperfusion) compared with vehicle control. C: Liver complementary proliferation was assessed by immunohistochemical staining of PCNA. The number of PCNA-positive cells was quantified from five fields at high-power magnification of ×400. PCNA-positive cells increased in I/R+SB216763 (24 hours reperfusion) compared with vehicle control.

Discussion

GSK-3β is involved in I/R-induced liver injury,[11,12]but the mechanism is not clear. The present study is the first to demonstrate that anti-apoptosis plays an important role in GSK-3β inhibitor-mediated protection against I/R-induced liver injury and this effect is via the GSK-3β/β-catenin signaling pathway.

GSK-3β is constitutively active in dephosphorylated form and has pleiotropic functions including regulating cell activation, differentiation and survival. GSK-3β inhibition has been successfully tested as a cardioprotectionstrategy in myocardial infarction. Our results clearly showed that inhibition of GSK-3β ameliorated hepatic I/R injury, which differred from that phosphorylation and inactivation of GSK-3β contribute to hepatocyte apoptosis and severe ischemic injury in liver grafts during cold preservation[23]. We speculate that warm I/R injury might have mechanisms different from cold I/R injury. However, our results are in agreement with those of two recent reports.[11,12]Using indirubin-3'-oxime as an inhibitor, Varela and colleagues[11]found that GSK-3β inhibition protects the liver from damage by maintaining mitochondrial function and hepatic energy-balance, while Ren and coworkers[12]suggested that GSK-3β inhibition decreases pro-inflammatory (IL-12, TNF-α), increases anti-(IL-10) inflammatory cytokines, and reduces local inflammation, thus protecting the liver from I/R injury. Besides inflammation, apoptosis is also very important in I/R-induced liver damage. Rudiger's study indicated that apoptosis plays a central role in the major death process for hepatocytes and sinusoidal endothelial cells after reperfusion of the ischemic liver.[6]

Most of the cardiac and neuronal studies showed that the protective effects of GSK-3β inhibition are through anti-apoptosis. Kaga et al[13]clarified that SB216763 increases β-catenin accumulation in both cytosol and nucleus and activates GSK-3β/β-catenin; this pathway further enhances anti-apoptotic signaling through the induction of Bcl-2 and survivin expression. The antiapoptotic effects of GSK-3β/β-catenin play an important role in rat ischemic preconditioned myocardium.[13]Interestingly, using adeno-shRNA, the same research group found that β-catenin knockdown abolishes ischemic preconditioning-mediated cardioprotection by downregulating its target genes Bcl-2 and survivin in ischemic rat myocardium.[14]Using a transient middle cerebral artery occlusion model, Koh et al[24]also verified that GSK-3β inhibition protects neuronal tissue from occlusion-induced injury via anti-apoptosis.

Studies of different types of liver damage also support the hypothesis that the protective effects of GSK-3β inhibition are through anti-apoptosis. Using β-catenin antisense phosphorodiamidate morpholino oligomer, Monga et al[25]found that β-catenin inhibition increases hepatocyte apoptosis in embryonic liver culture. Others[26]found that palmitate-induced apoptosis in both primary hepatocytes and cell lines is via GSK-3 activation. These cells resist palmitate-induced apoptosis when GSK-3β is either pharmacologically inhibited or selectively knocked down by shRNA.[26]GSK-3β inhibition also protects against cholestatic liver injury by alleviating antiapoptosis.[27]Our data showed that GSK-3β inhibition enhanced the accumulation of β-catenin in the cytosol and nucleus and increased anti-apoptotic factors such as Bcl-2 and survivin in the I/R liver, while decreasing the apoptosis of hepatic and endothelial cells (from 3.6% to 1.4%). The anti-apoptotic effects of GSK-3β inhibition in our I/R injury model are consistent with those of other studies in different models.[26,27]

Beside anti-apoptosis, SB216763 also augmented the expression of the proliferative factors cyclin D1 and PCNA in the I/R liver, indicating that SB216763 also promoted liver proliferation. Complementary proliferation contributes to the maintenance of normal liver function during I/R injury.[28]Cyclin D1, involved in cell cycle progression through the G1 phase, is an important canonical β-catenin target. Our data showed that cyclin D1 expression was upregulated when GSK-3β activity was inhibited. Meanwhile, PCNA, a protein involved in the DNA repair pathway, was markedly increased after β-catenin signaling was activated. These results are consistent with those of Sekine's study.[22]Using β-catenin knockout mice, Sekine found that β-catenin is critical for the proper regulation of hepatocyte proliferation during liver regeneration, and the β-catenin downstream gene, cyclin D1, is significantly impaired after hepatectomy in β-catenin-knockout mice. We therefore propose that inhibition of GSK-3β promotes hepatocyte proliferation via a β-catenin-dependent mechanism during hepatic I/R. Liver proliferation is another mechanism to overcome the cellular loss induced by apoptosis. Overall, SB216763 improved liver histology and decreased serum ALT.

In conclusion, we present evidence that GSK-3β inhibition activated the β-catenin signaling pathway which elevated both anti-apoptotic and proliferative factors. To our knowledge, hepatic I/R induces death of hepatocytes and nonparenchymal cells via apoptosis and necrosis.[29]Since we did not assess whether GSK-3β inhibition affects liver necrosis, we will further study it. Moreover we also did not detect whether SB216763 affects other GSK-3β targets, such as NF-κB, that might help to explain our observations. In the present study we partly explained how the inhibition of GSK-3β protects the liver against I/R injury by ameliorating apoptosis and enhancing proliferation.

Contributors: XYX, LL and PLY proposed the study. XYX wrote the first draft. WZS and SBC analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. WXH is the guarantor.

Funding: This work was supported by grants from the Key Projects in the National Science & Technology Pillar Program during the Eleventh Five-Year Plan of China (2008BAI60B02) and the Natural Science Foundation of China (30872390).

Ethical approval: The protocol was approved by the Ethics Committee of Nanjing Medical University, Nanjing, China.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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October 25, 2011

Accepted after revision February 15, 2012

Author Affiliations: Liver Transplantation Center, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China (Xia YX, Lu L, Wu ZS, Pu LY, Sun BC and Wang XH)

Xue-Hao Wang, MD, Liver Transplantation Center, First Affiliated Hospital, Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, China (Tel: 86-25-83718836ext6476; Fax: 86-25-83672106; Email: wangxh@njmu.edu.cn).

? 2012, Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872(12)60161-1