Impaired liver regeneration in Nrf2 knockout mice: role of ROS-mediated insulin/IGF-1 resistance
2007; Springer Nature; Volume: 27; Issue: 1 Linguagem: Inglês
10.1038/sj.emboj.7601950
ISSN1460-2075
AutoresTobias A. Beyer, Weihua Xu, Daniel Teupser, Ulrich auf dem Keller, Philippe Bugnon, Eberhard Hildt, Joachim Thiery, Yuet Wai Kan, Sabine Werner,
Tópico(s)Macrophage Migration Inhibitory Factor
ResumoArticle6 December 2007free access Impaired liver regeneration in Nrf2 knockout mice: role of ROS-mediated insulin/IGF-1 resistance Tobias A Beyer Tobias A Beyer Department of Biology, Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Weihua Xu Weihua Xu Department of Biology, Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Daniel Teupser Daniel Teupser Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Department of Medicine, University of Leipzig, Leipzig, Germany Search for more papers by this author Ulrich auf dem Keller Ulrich auf dem Keller Department of Biology, Institute of Cell Biology, ETH Zurich, Zurich, SwitzerlandPresent address: Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada V6T 1Z3 Search for more papers by this author Philippe Bugnon Philippe Bugnon Department of Biology, Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Eberhard Hildt Eberhard Hildt Department of Internal Medicine II, University of Freiburg, Freiburg, Germany Search for more papers by this author Joachim Thiery Joachim Thiery Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Department of Medicine, University of Leipzig, Leipzig, Germany Search for more papers by this author Yuet Wai Kan Yuet Wai Kan Department of Medicine, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Sabine Werner Corresponding Author Sabine Werner Department of Biology, Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Tobias A Beyer Tobias A Beyer Department of Biology, Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Weihua Xu Weihua Xu Department of Biology, Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Daniel Teupser Daniel Teupser Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Department of Medicine, University of Leipzig, Leipzig, Germany Search for more papers by this author Ulrich auf dem Keller Ulrich auf dem Keller Department of Biology, Institute of Cell Biology, ETH Zurich, Zurich, SwitzerlandPresent address: Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada V6T 1Z3 Search for more papers by this author Philippe Bugnon Philippe Bugnon Department of Biology, Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Eberhard Hildt Eberhard Hildt Department of Internal Medicine II, University of Freiburg, Freiburg, Germany Search for more papers by this author Joachim Thiery Joachim Thiery Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Department of Medicine, University of Leipzig, Leipzig, Germany Search for more papers by this author Yuet Wai Kan Yuet Wai Kan Department of Medicine, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Sabine Werner Corresponding Author Sabine Werner Department of Biology, Institute of Cell Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Author Information Tobias A Beyer1, Weihua Xu1, Daniel Teupser2, Ulrich auf dem Keller1, Philippe Bugnon1, Eberhard Hildt3, Joachim Thiery2, Yuet Wai Kan4 and Sabine Werner 1 1Department of Biology, Institute of Cell Biology, ETH Zurich, Zurich, Switzerland 2Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Department of Medicine, University of Leipzig, Leipzig, Germany 3Department of Internal Medicine II, University of Freiburg, Freiburg, Germany 4Department of Medicine, University of California San Francisco, San Francisco, CA, USA *Corresponding author. Department of Biology, Institute of Cell Biology, ETH Zurich, Honggerberg, HPM D42, Zurich 8093, Switzerland. Tel.: +41 44 633 3941; Fax: +41 44 633 1174; E-mail: [email protected] The EMBO Journal (2008)27:212-223https://doi.org/10.1038/sj.emboj.7601950 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The liver is frequently challenged by surgery-induced metabolic overload, viruses or toxins, which induce the formation of reactive oxygen species. To determine the effect of oxidative stress on liver regeneration and to identify the underlying signaling pathways, we studied liver repair in mice lacking the Nrf2 transcription factor. In these animals, expression of several cytoprotective enzymes was reduced in hepatocytes, resulting in oxidative stress. After partial hepatectomy, liver regeneration was significantly delayed. Using in vitro and in vivo studies, we identified oxidative stress-mediated insulin/insulin-like growth factor resistance as an underlying mechanism. This deficiency impaired the activation of p38 mitogen-activated kinase, Akt kinase and downstream targets after hepatectomy, resulting in enhanced death and delayed proliferation of hepatocytes. Our results reveal novel roles of Nrf2 in the regulation of growth factor signaling and in tissue repair. In addition, they provide new insight into the mechanisms underlying oxidative stress-induced defects in liver regeneration. These findings may provide the basis for the development of new strategies to improve regeneration in patients with acute or chronic liver damage. Introduction The liver is the only organ in the mammalian body that can fully regenerate after injury. Upon loss of functional liver tissue, for example, by surgery, toxin-induced necrosis or viral infections, quiescent hepatocytes re-enter the cell cycle and proliferate to restore the original liver mass. This is essential to the body, as the liver fulfills central roles in metabolic homeostasis, detoxification of various compounds, and in the synthesis, storage and secretion of nutrients. Impairment of regeneration contributes to the pathogenesis of liver failure or development of fibrosis/cirrhosis (Diehl, 2002). Liver regeneration after partial hepatectomy can be divided into three phases: priming, proliferation and cessation (Fausto, 2000; Diehl, 2002; Taub, 2004). Priming is mediated by pro-inflammatory cytokines like tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) (Fausto, 2000; Diehl, 2002; Taub, 2004). The subsequent activation of nuclear factor κB (NF-κB), activator protein 1 (AP-1), and signal transducer and activator of transcription 3 (STAT3) is responsible for G0 to G1 transition and survival of hepatocytes (Cressman et al, 1995; FitzGerald et al, 1995; Heim et al, 1997). Hepatocyte proliferation is regulated by different mitogens, including hepatocyte growth factor (HGF), and ligands of the epidermal growth factor (EGF) and fibroblast growth factor receptors (Mead and Fausto, 1989; Steiling et al, 2003; Borowiak et al, 2004; Huh et al, 2004; Mitchell et al, 2005). In addition, recent studies revealed a role of insulin-like growth factor 1 (IGF-1) and its receptor in hepatocyte proliferation after hepatectomy (Pennisi et al, 2004; Desbois-Mouthon et al, 2006). Once the original liver mass is restored, hepatocytes are rendered quiescent, most likely through transforming growth factor β (TGF-β) and activin signaling (Oe et al, 2004). The liver often encounters oxidative stress, which affects liver function, induces hepatocyte cell death and disturbs the regeneration process after injury (Fausto, 2000; Kamata et al, 2005; Schwabe and Brenner, 2006). Therefore, a tight regulation of the cellular redox balance in the liver is essential. A crucial player in the defense against oxidative stress is the NF-E2-related factor 2 (Nrf2). This transcription factor controls the expression of antioxidant proteins and enzymes involved in the detoxification of harmful compounds, such as glutathione S-transferase (GST)-ya, GST-π, glutamate-cysteine ligase catalytic subunit (GCLC) and NAD(P)H quinone oxidoreductase 1 (NQO1) (reviewed by Jaiswal, 2004). Nrf2 is a member of the ‘cap“n”collar’ family of transcription factors, which also includes the related Nrf1 and Nrf3 proteins, as well as p45 NF-E2, Bach1 and Bach2 (reviewed by Motohashi et al, 2002). These transcription factors bind to cis-acting elements in the promoters of their target genes, called antioxidant response element (ARE) (reviewed by Nguyen et al, 2003). The crucial role of Nrf2 in the cellular stress response is reflected by the phenotype of Nrf2 knockout mice. Upon ageing they develop an autoimmune disorder resembling lupus erythematosus (Li et al, 2004). Even young animals are more susceptible to various toxins (Chan and Kan, 1999; Chan et al, 2001), and the lack of Nrf2-mediated gene expression enhances the susceptibility to cancer as shown for the liver (Ramos-Gomez et al, 2001) and the skin (auf dem Keller et al, 2006). Nrf2 was also identified as a regulator of inflammation in cutaneous wound repair, although the lack of Nrf2 did not result in impaired healing (Braun et al, 2002). Here, we demonstrate a novel role of Nrf2 in liver regeneration and insulin resistance. Results Reduced liver/body weight ratio but lack of liver damage in Nrf2 knockout mice A histological analysis of the livers of Nrf2-deficient mice at 8–10 weeks of age did not reveal apparent abnormalities (Supplementary Figure 1A). The liver/body weight ratio of these animals was reduced (Supplementary Figure 1B), possibly as a result of reduced hepatocyte proliferation during development. To test whether this affects general liver functions, a serum analysis was performed. Levels of total protein and albumin were comparable between animals of both genotypes, and glucose levels were also unaltered. An increase in total bilirubin (normalized to liver weight) was observed in the Nrf2 knockout mice (Supplementary Figure 1C), but the activities of aspartate aminotransferase and alanine aminotransferase in the serum (normalized to the liver weight) were similar in mice of both genotypes (Supplementary Figure 1D and E). Since excessive release of these liver-specific enzymes into the serum is a hallmark of liver dysfunction and damage, it seems likely that liver function is not severely impaired in the knockout animals. Nrf2 is crucial for liver regeneration To determine the role of Nrf2 in liver regeneration, we applied the model of two-third hepatectomy to Nrf2 knockout mice and wild-type littermates. As shown by RNase protection assay (RPA), Nrf2 mRNA levels were similar in normal and injured livers of wild-type mice and undetectable in the knockout mice. Nrf1 was also expressed in normal liver and after hepatectomy, but was not upregulated in the absence of Nrf2. Nrf3 mRNA was undetectable in mice of both genotypes (Supplementary Table 1). A histological analysis as well as staining for adipophilin revealed that the transient steatosis, which is normally observed after hepatectomy and which is important for regeneration (Shteyer et al, 2004), was aggravated in the Nrf2 knockout mice 48 h after hepatectomy (Figure 1A). Using 5-bromo-2′-deoxyuridine (BrdU) incorporation studies, we found a significant delay in liver cell proliferation in the Nrf2 knockout animals (Figure 1B and C). In mice of both genotypes, only very few proliferating hepatocytes were detected in non-injured liver and 6 h after hepatectomy. Even 24 h after injury, less than 1% of the hepatocytes had incorporated BrdU (data not shown). Proliferation then peaked 48 h after hepatectomy, but the peak was much lower in Nrf2 knockout mice compared to wild-type animals (21.3% BrdU-positive cells versus 13.36% in the knockout mice; Figure 1B and C; P=0.0057, n⩾8). At 72 h after injury, the number of BrdU-labeled cells remained lower in the knockout mice compared to wild-type animals (Figure 1C; P=0.0931, n=6), although the difference was no longer statistically significant. At day 5, proliferation was almost completed in the wild-type mice, whereas Nrf2-deficient hepatocytes continued to proliferate at this time point (Figure 1C; P=0.0111, n=7). The percentage of BrdU-positive hepatocytes was less than 1% at day 7 after injury in mice of both genotypes (data not shown). Consistent with this finding, the liver/body weight ratio had almost returned to the levels seen before injury in both wild-type and knockout mice at day 7 post-hepatectomy. These findings demonstrate that livers of Nrf2-deficient mice can regenerate, albeit with a significant delay. Figure 1.Liver regeneration is impaired in Nrf2-deficient mice. (A) Liver sections of Nrf2 knockout mice (ko) and wild-type (wt) littermates 48 h after hepatectomy were stained with hematoxylin/eosin. Arrows indicate lipid droplets in hepatocytes. (B) Proliferation was assessed by BrdU incorporation. Representative sections from injured liver (48 h after hepatectomy) are shown. (C) The percentage of proliferating cells was determined by counting 3–5 independent microscopic fields per liver at × 200 magnification, n (number of mice) >6 per genotype and time point. (D) Cryosections were stained for cleaved caspase-3. The percentage of stained cells 6 h after hepatectomy was determined by counting 3–5 independent microscopic fields ( × 200 magnification, n=4 per genotype). Bars represent mean±s.e.m.; *P<0.05; **P<0.01. Download figure Download PowerPoint Impaired liver regeneration in Nrf2 knockout mice is not due to reduced expression of major hepatocyte mitogens To determine if the delayed hepatocyte proliferation in Nrf2 knockout mice results from reduced expression of growth factors and cytokines involved in liver repair, we analyzed their expression by RPA. HGF, TGF-α and TGF-β1 mRNA levels increased transiently during the regeneration process with different kinetics, but no difference was observed between knockout and wild-type mice (Supplementary Table 1). Consistent with these findings, phosphorylation of the EGF and the HGF receptor (c-Met), which reflects their activation in response to ligand binding, was comparable in wild-type and knockout mice at all stages after hepatectomy (Supplementary Figure 2A and B). IGF-1 and vascular endothelial growth factor were highly expressed in normal and injured livers, but the mRNA levels of these growth factors were not affected by hepatectomy or genotype (Supplementary Figure 1). IGF-1 levels in the serum were generally high in wild-type and knockout mice at all time points. Serum levels of knockout mice were slightly lower compared to wild-type mice, but this difference was not statistically significant and may also reflect the lower liver weight (Supplementary Figure 2C). Finally, IL-6 and TNF-α mRNA levels were slightly induced after injury in mice of both genotypes (Supplementary Table 1). Enhanced hepatocyte apoptosis in injured liver of Nrf2 knockout mice Under pathological conditions, enhanced hepatocyte apoptosis can occur after hepatectomy (Malhi et al, 2006; Schwabe and Brenner, 2006). Staining for cleaved caspase-3 revealed only few apoptotic cells in non-injured liver and in the liver of sham-operated mice of both genotypes (data not shown). However, 6 h after hepatectomy, the number of apoptotic cells was five-fold increased in Nrf2 knockout mice (P=0.0159, n=4) compared to wild-type controls (Figure 1D). Necrotic areas were never detected in hepatectomized liver, independent of the genotype (data not shown). Reduced expression of Nrf2 target genes in normal and hepatectomized livers of Nrf2 knockout mice We next analyzed the mRNA levels of major Nrf2 target genes in normal and injured livers of Nrf2 knockout mice. Expression of GST-ya, NQO1 and to a lesser extent GST-π was reduced in resting livers of Nrf2 knockout mice. Induction of GST-ya and NQO1 expression, and a slight increase in GST-π mRNA levels were observed within 48–120 h after hepatectomy in livers of wild-type animals, but to a much lesser extent in Nrf2-deficient mice (Figure 2A and Supplementary Table 1). RNA levels encoding the catalytic subunit of the rate-limiting enzyme for glutathione biosynthesis, GCLC, were slightly induced in wild-type livers 48 h after hepatectomy, but not in Nrf2 knockout mice (Figure 2A and Supplementary Table 1). Expression of heme oxygenase-1 (HO-1), and of peroxiredoxins (Prdx) 1 and 6 was not affected by the loss of Nrf2 (Supplementary Table 1). Figure 2.Reduced mRNA levels of Nrf2 target genes and enhanced oxidative stress in normal and hepatectomized livers of Nrf2 knockout mice. (A) Total cellular RNA (10 μg from pooled livers of at least four mice per time point and genotype) was analyzed by RPA for transcripts encoding Nrf2 target genes. The time after hepatectomy or sham surgery is indicated on top of each lane; 0 h indicates resting liver, which was removed during hepatectomy; 20 μg tRNA served as a negative control and 1000 c.p.m. of the hybridization probes were used as a size marker. Hybridization with a GAPDH riboprobe served as a loading control. Representative autoradiograms of two independent experiments are shown. (B) Total lysates from resting and injured livers 72 h after partial hepatectomy were assayed for GST activity. The formation of glutathione/1-chloro-2,4-dinitrobenzene conjugates was measured spectrophotometrically. Bars represent mean±s.e.m.; **P<0.01 (n=6). (C) Hepatocytes were analyzed for GST activity. Bars represent mean±s.e.m.; *P<0.05; **P<0.01 (n=6). (D) Freshly prepared cryosections (7 μm) from non-injured and injured livers (72 h after hepatectomy) were stained with DEH and analyzed by fluorescence microscopy (n⩾5). (E) Primary hepatocytes from Nrf2 knockout and wild-type mice were treated with H2DCFH-DA and analyzed by flow cytometry for the levels of intracellular ROS. Download figure Download PowerPoint To identify the full spectrum of Nrf2 target genes in the liver, we performed microarray analysis of RNAs from non-injured livers of young wild-type and knockout mice. In the liver of Nrf2 knockout mice, we found reduced expression of several GSTs and NQO1, whereas GCLC, HO-1, and Prdx 1 and 6 were expressed at similar levels. Therefore, the differential expression of known Nrf2 target genes was shown by two independent methods. In addition, the microarray analysis identified the differential expression of several enzymes involved in drug metabolism and detoxification in the liver of Nrf2 knockout mice (Supplementary Table 2). The complete microarray data can be downloaded from the GEO repository: (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=tvwphcsycaqgcti&acc=GSE8969). Consistent with the mRNA data, GST activity was reduced by 36% in resting livers of Nrf2 knockout mice compared to controls and by 26% 72 h after hepatectomy (Figure 2B). Reduced GST activity was also observed in cultured primary hepatocytes from Nrf2-deficient mice (Figure 2C), demonstrating that hepatocytes are directly affected. Increased oxidative stress in the liver of Nrf2-deficient mice We subsequently stained the livers with dihydroethidine, a dye that intercalates into DNA upon oxidation, and therefore allows monitoring of oxidative stress in vivo. Nuclear staining was observed in livers of Nrf2 knockout mice, whereas livers of wild-type animals showed only weak fluorescence (Figure 2D). Numbers of stained nuclei increased 72 h after hepatectomy in animals of both genotypes (Figure 2D lower panels), with stronger staining in Nrf2-deficient mice. This finding was verified biochemically with freshly isolated hepatocytes. Cells were incubated with H2DCFH-DA that is converted by intracellular ROS to the fluorescent derivative DCF. The latter was detected by flow cytometry. Hepatocytes from Nrf2 knockout mice displayed strongly increased fluorescence, reflecting an increase in intracellular ROS levels (Figure 2E). These results identify Nrf2 as an important regulator of the cellular redox balance in normal and hepatectomized livers and demonstrate that deficiency in Nrf2 results in chronic oxidative stress in hepatocytes, which is further aggravated upon liver injury. MAPK signaling is altered in regenerating livers of Nrf2 knockout mice To elucidate the mechanisms underlying the impaired liver regeneration in Nrf2-deficient mice, we first analyzed the injury-induced activation of NF-κB, STAT3 and AP-1 transcription factor complexes. In resting liver, cytoplasmic staining for the NF-κB p65 subunit was observed in animals of both genotypes (Supplementary Figure 3A). At 3 h after hepatectomy NF-κB was predominantly localized in the nuclei of hepatocytes, and no difference was observed between wild-type and Nrf2-deficient mice (Supplementary Figure 3A). In addition, IKK-β, which is at least in part responsible for NF-κB activation, was expressed at similar levels in wild-type and knockout mice (Supplementary Figure 3B). However, EMSA experiments revealed that the extent of NF-κB activation was stronger in the liver of Nrf2-deficient mice 3 and 6 h after hepatectomy (Supplementary Figure 3C). This finding is consistent with the enhanced oxidative stress, which was shown to result in NF-κB activation (Schreck et al, 1991). Since NF-κB enhances survival of hepatocytes after hepatectomy (Luedde et al, 2006), its activation is most likely a compensatory effect to reduce the extent of cell death in Nrf2 knockout mice. A strong increase in Tyr705 phosphorylation of STAT3, which reflects its activation, was observed 3 and 6 h after hepatectomy in animals of both genotypes in comparison to resting liver, which was removed upon hepatectomy (Supplementary Figure 4). In contrast, total STAT3 levels were not affected (Supplementary Figure 4). Thus, STAT3 activity is apparently not altered in Nrf2 knockout mice after hepatectomy. As an additional control, we analyzed the activation of STAT3 in sham-operated mice. The latter underwent abdominal surgery and manipulation of the liver, but not hepatectomy. In sham-operated mice, activation of STAT3 was also observed to a similar extent in mice of both genotypes, most likely reflecting the stress response (Supplementary Figure 4). Phosphorylation of p38 was apparent 3 and 6 h after hepatectomy in wild-type mice. Interestingly, the levels of phosphorylated p38 were significantly lower in hepatectomized liver of Nrf2 knockout mice compared to wild-type mice within 3 and 6 h after injury in three independent experiments (Figure 3A) (average reduction 39% (3 h after injury) and 43% (6 h after injury)). No difference in Erk phosphorylation was detectable (Figure 3A). Using an antibody that recognizes the phosphorylated forms of the 54 and 46 kDa variants of both JNK1 and JNK2, we found hyperphosphorylation of both variants within 3 and 6 h after hepatectomy compared to control littermates (Figure 3A). However, this did not result in abnormal AP-1 activation, as suggested by the similar levels of phosphorylated c-Jun in mice of both genotypes (Figure 3B) and by the similar induction of AP-1 DNA-binding activity within 3 and 6 h after hepatectomy as demonstrated by EMSA (Figure 3C). This may be due to differential activities of MAPK phosphatases in the nucleus and the cytoplasm (Wu et al, 2006). Sham operation did not affect the phosphorylation status of Erk, JNK and c-Jun. Although p38 was also activated by sham surgery, the activation was independent of the genotype. Figure 3.Altered JNK and p38 activation but normal AP-1 activity in injured liver of Nrf2 knockout mice. (A, B) Total protein (60 μg) from liver lysates of Nrf2 knockout and wild-type mice (from pooled livers of at least four mice per time point after hepatectomy and genotype) was analyzed by immunoblotting for the levels of phosphorylated and total Erk, p38 and JNK (A), and phosphorylated c-Jun (B). Staining of the membrane with antibodies to GAPDH or Lamin A was used as a loading control. Representative blots from two independent experiments with lysates from different hepatectomy experiments are shown. (C) Radiolabeled oligonucleotides containing AP-1-binding sites were incubated with total protein lysates (20 μg) from normal and hepatectomized livers, and EMSAs were performed. Addition of an excess of non-labeled oligonucleotides (lanes labeled: cold comp.) inhibited mobility shifts, whereas addition of non-labeled oligonucleotides with mutated AP-1-binding sites had no effect (lanes labeled: mut. cold comp.). Download figure Download PowerPoint Reduced PI3 kinase/Akt signaling in Nrf2 knockout mice after partial hepatectomy Expression of the regulatory subunit of phosphoinositide 3-kinase (PI3K), p85α, and its target Akt was unaltered in Nrf2-deficient mice. However, phosphorylation of Akt (Ser473), which results in its activation, occurred within 3 h after hepatectomy in wild-type mice. At this time point, however, levels of phosphorylated Akt were significantly lower in the knockout mice (average reduction 48% in four independent experiments) (Figures 4A, 5A and 7D). Consistent with these findings, phosphorylation of the Akt target glycogen synthase kinase-3β (GSK-3β) at Ser9 was reduced (56% reduction 3 h after hepatectomy; 63% reduction 6 h after hepatectomy) (Figure 4A). In addition, p70/S6K and Bad, two major targets of PI3K signaling, displayed reduced phosphorylation in injured liver of the Nrf2 knockout mice (Figure 4A and B) (pS6K: 34 and 60% reduction after 3 or 6 h, respectively; pBad: 56% reduction after 3 h). Since PI3K, Akt and their downstream targets are crucial for cell survival and proliferation (Lawlor and Alessi, 2001; Song et al, 2005), the reduced activation of this pathway provides a likely explanation for the observed defect in liver regeneration in Nrf2-deficient mice and could also account for the increased cell death after hepatectomy. Figure 4.Reduced activation of the PI3K/Akt signaling pathway in injured liver of Nrf2 knockout mice. Total protein (60 μg) from liver lysates of Nrf2 knockout and wild-type mice (from pooled livers of at least four mice per time point after hepatectomy and genotype) was analyzed by immunoblotting for the levels of the PI3K p85α subunit, phosphorylated and total Akt, GSK-3β and S6 kinase (A), non-phosphorylated and phosphorylated Bad, GAPDH and Lamin A (B). Representative blots from two to four experiments with lysates from different hepatectomy experiments are shown. Download figure Download PowerPoint Figure 5.Reduced IGF-1R/IR signaling in injured liver of Nrf2 knockout mice. (A) Liver lysates (60 μg protein) of Nrf2 knockout and wild-type mice at different time points after hepatectomy were analyzed by immunoblotting for the levels of the total IGF-1R and IR, phosphorylated IGF-1R/IR, phosphorylated and total Akt, phosphorylated IRS-1/2 (Tyr608) and total IRS-1, and GAPDH. Representative blots from two to three experiments with lysates from different hepatectomy experiments are shown. (B) Total liver lysates from injured liver (3 h after hepatectomy) of Nrf2-deficient and wild-type mice were subjected to immunoprecipitation using antibodies against IR. Precipitates were analyzed by immunoblotting using antibodies against IR or pIR/IGF-1R. Download figure Download PowerPoint Insulin/IGF-I receptor signaling is impaired in injured liver of Nrf2-deficient mice We next wondered about the upstream signals that are responsible for the reduced activation of PI3K and p38. Since most hepatocyte mitogens are only expressed at later stages of regeneration (Supplementary Table 1), we speculated that IGF-1 expression or signaling might be affected. IGF-1 is known for its potent activation of the PI3K/Akt signaling pathway, and it can also activate p38 (Cheng and Feldman, 1998). Using an antibody, which recognizes the phosphorylated forms of the IGF-1 receptor (IGF-1R) and the insulin receptor (IR), we found strongly enhanced levels of the phosphorylated form(s) within 3 h after hepatectomy of wild-type mice and to a much lesser extent in Nrf2 knockout mice (71 and 73% reduction in two independent experiments). Phosphorylation of IGF-1R/IR strongly correlated with the levels of pAkt. High levels of IR and much lower levels of IGF-1R were detected in normal and hepatectomized livers, and their levels were similar at all stages of the repair process (Figure 5A). This indicates that either ligand-dependent activation or dephosphorylation, in particular of the IR, is affected by the lack of Nrf2 (Figure 5A). Consistent with this assumption, immunoprecipitation with antibodies against the IR or the IGF-1R, respectively, and subsequent western blotting with an antibody against pIR/IGF-1R revealed that it is predominantly the IR, which is expressed in the liver and activated in response to hepatectomy, and that the activation of this receptor is reduced in the absence of Nrf2 (Figure 5B). In contrast, only very low levels of IGF-1R could be precipitated, although the same antibody efficiently precipitated the IGF-1R from cultured human hepatoma cells (data not shown). Consistent with the reduced IR activation, levels of phosphorylated IR substrate 1 (IRS-1) (Tyr608 corresponding to Tyr612 in human IRS-1) and pIRS-2 were reduced in knockout animals (Figure 5A). Phosphorylation of Tyr608 in IRS-1 and the corresponding residue in IRS-2 reflects their activation (Bloch-Damti et al, 2006). These findings suggest that IR signaling is impaired, resulting in reduced PI3K activity. Oxidative stress reduces insulin responsiveness in liver cells To determine if the abnormalities in IGF-1
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