NFR 2 Factor

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The Nrf2 transcription factor is a crucial regulator of the cellular redox homeostasis through its capacity to induce the expression of enzymes, which detoxify reactive oxygen species, and of other antioxidant proteins. Therefore, it plays an important role in the protection from carcinogenesis induced by various insults. In addi-tion, recent results identified a novel role of Nrf2 in tissue repair. In the liver, regeneration after partial hepatectomy was strongly delayed in the absence of Nrf2. This defect was shown to result from transient resistance to insulin and insulin-like growth factor 1 that was caused by chronic oxidative stress in hepatocytes. These results demonstrate a link between Nrf2 deficiency, oxidative stress and insulin resistance, and suggest that activation of this transcrip-tion factor could be a novel strategy to improve liver regeneration in patients with acute or chronic liver injury. In addition, it may help to alleviate oxidative stress-induced insulin resistance in the liver and potentially also in other organs.

Liver Regeneration

In contrast to other organs, the liver has a unique capability to fully regenerate after injury (reviewed in ref. 1). There are many conditions that require this regenerative potential, such as resection of liver tissue in patients with primary or metastatic liver tumors or cell loss caused by viruses, autoimmune diseases and toxins, including alcohol or commonly used anti-inflammatory, anticonvul-sant, or chemotherapeutic drugs. Due to the crucial function of the liver in metabolic regulation and detoxification of various substances, rapid and effective liver repair is essential. If the extent of injury is not too high, full regeneration of the liver can occur. However, the regenerative capacity is insufficient after chronic injury as observed for example in chronic viral hepatitis or after long-term alcohol abuse (reviewed in ref. 2). These conditions often cause liver cirrhosis, which is characterized by replacement of functional epithelial tissue by non-functional connective tissue. While such conditions affect an enormous part of the population world-wide, therapeutic approaches

are still unsatisfactory and mainly supportive. Therefore, the devel-opment of specific therapies to enhance the regenerative capacity of the liver is essential for the improvement of human health. This requires a thorough understanding of the mechanisms underlying liver homeostasis and regeneration and the identification of factors that control and accelerate this process.

A particularly useful model to study liver regeneration in rodents is partial hepatectomy. In this procedure, the large and median lobes of the liver, which comprise approximately two third of the organ, are surgically removed. As a consequence, the normally quiescent and highly differentiated liver cells proliferate, and the original liver mass is restored within a few days (reviewed in ref. 1).

The regeneration process after hepatectomy occurs in two phases: priming, and cell cycle progression (reviewed in ref. 1). The pro-inflammatory cytokines tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) play an important role in the priming step (reviewed in ref. 1). These cytokines activate nuclear factor κB (NFκB), activator protein 1 (AP-1), and signal transducer and acti-vator of transcription 3 (STAT3), resulting in G0 to G1 transition and survival of liver parenchymal cells.3-5 In the second phase of liver repair, growth factors are required for progression of hepatocytes through the cell cycle (reviewed in ref. 1). Mitogens involved in this process include hepatocyte growth factor (HGF),6,7 ligands of the epidermal growth factor receptor,8,9 and fibroblast growth factors.10 In addition, insulin-like growth factor 1 (IGF-1) was shown to be involved in this process.11 Upon completion of the repair process and restoration of the original liver mass, hepatocyte proliferation is inhibited and further growth of the liver does not occur. The mecha-nisms that are involved in this thorough growth control are not fully understood, but signaling through transforming growth factor β (TGFβ) and activin12 was shown to inhibit hepatocyte proliferation upon completion of repair.

Oxidative Stress Impairs Liver Regeneration

Although the regeneration process is highly efficient, it can be impaired by chronic oxidative stress caused by toxins, such as ethanol or haloalkanes. This causes severe damage of hepatocytes through lipid peroxidation and alkylation of cellular macromolecules (reviewed in refs. 13 and 14). In turn, apoptotic and in particular necrotic death of hepatocytes leads to activation of hepatic stellate cells (HSC). The latter are responsible for enhanced deposition of fibrillar collagen, and expression of various cytokines such as TGF-β,

*Correspondence to: Sabine Werner; Institute of Cell Biology; ETH Zurich; Honggerberg, Zurich CH-8093 Switzerland; Tel.: +41.44.633.3941; Fax: +41.44.633.1174; Email: [email protected]

Submitted: 01/15/08; Accepted: 01/18/08

Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/5617

Perspective

The cytoprotective Nrf2 transcription factor controls insulin receptor signaling in the regenerating liverTobias A. Beyer and Sabine Werner*

Institute of Cell Biology; Department of Biology; ETH Zurich; Zurich, Switzerland

Key words: Nrf2, insulin, IGF-1, liver regeneration, oxidative stress, PI3K, ROS

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Nrf2 and insulin resistance

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resulting in liver fibrosis and cirrhosis (reviewed in ref. 15). Oxidative stress is generated in the liver by reactive oxygen species (ROS). These harmful molecules are mainly produced by activated inflam-matory cells through NAD(P)H oxidases, but they are also metabolic intermediates of xenobiotics, and they are generated as by-products in several metabolic reactions, in particular in the respiratory chain (reviewed in ref. 16).

The Nrf2 transcription factor

To avoid damage by ROS, a tight regulation of the cellular redox balance is required, and this is achieved at least in part by the action of NF-E2 related factor 2 (Nrf2). Nrf2 is a member of the “cap‘n’ collar” family of proteins, which also includes the related Nrf1 and Nrf3 transcription factors, as well as p45 NF-E2, Bach1 and Bach2. These proteins, in particular Nrf2, bind to antioxidant response elements (ARE) in the promoters of their target genes. Expression of many antioxidant proteins and enzymes that detoxify ROS or various drugs are controlled by Nrf2, including glutathione S-transferases (GST), NAD(P)H quinone oxidoreductase 1 (NQO1), glutamate-cysteine ligase catalytic and modulatory subunits (GCLC and GCLM), peroxiredoxin-1, and heme oxygenase-1 (reviewed in refs. 17 and 18).

Under normal conditions, Nrf2 is retained in the cytoplasm via binding to the actin-binding protein Keap-1 (reviewed in ref. 19), which also mediates its degradation via the ubiquitin-protea-some pathway.20,21 Upon addition of electrophilic substances, which directly interact with Keap1 through Michael addition, Nrf2 becomes liberated and shuttles to the nucleus, where it activates its target genes. In addition, phosphorylation of Nrf2 by different kinases may also result in liberation from Keap1 and subsequent translocation to the nucleus. This mechanism has been suggested to regulate the activation of Nrf2 in response to oxidative stress (reviewed in refs. 18 and 19).

The important role of Nrf2 in the cellular stress response is reflected by the phenotype of Nrf2-deficient mice. Whereas young animals are healthy under normal laboratory conditions,22 older mice develop symptoms resembling those of patients with the auto-immune disease systemic lupus erythematosus, most likely due to enhanced oxidative tissue damage.23 Even young Nrf2 knockout mice are highly susceptible to treatment with butylated hydroxy-toluene, which caused death in these animals from acute respiratory distress syndrome.24 They are also highly sensitive to acetamino-phen-induced hepatotoxicity25,26 and to liver carcinogenesis induced by benz(a)pyrene.27

Expression and Function of Nrf2 during Tissue Repair

Although the roles of Nrf2 in toxin detoxification and cancer prevention have been well established, the functions of this tran-scription factor in tissue repair are still poorly characterized. In previous studies, our laboratory demonstrated a strong induction of Nrf2 expression in inflammatory cells and keratinocytes after skin injury. Most importantly, Nrf2 knockout mice were characterized by prolonged inflammation during wound healing, although the repair of the injured epidermis and wound closure in general were not affected by the loss of Nrf2.28 Following these studies on skin wounding, we now addressed the role of Nrf2 in liver regenera-tion. In contrast to the skin, upregulation of Nrf2 expression was

not observed at any stage of liver regeneration following partial hepatectomy. Since Nrf2 activity is predominantly regulated at the posttranslational level (see above), we used ARE reporter mice to monitor activation of this transcription factor during liver regenera-tion. These mice carry a transgene in their genome, which includes the ARE of the rat nqo1 gene, followed by a minimal promoter and the coding sequence for human placental alkaline phosphotase. Therefore, they allow detection of Nrf2 activation through alkaline phosphatase staining.29 Although a small population of hepato-cytes, in particular around blood vessels, clearly expressed the ARE reporter gene, most hepatocytes did not show activity of the reporter. Nevertheless, it is still possible that the basal activity of Nrf2 in these cells is required for liver regeneration. The important role of the basal Nrf2 activity in tissue homeostasis was demonstrated in a skin carcinogenesis study, which was recently performed in our labora-tory. Thus, mice expressing a dominant-negative mutant of Nrf2 in the epidermis showed a strongly enhanced incidence and multiplicity of chemically-induced skin tumors, although activation of the ARE reporter gene could not be observed at any time point during the development of the tumors.30 Therefore, the basal activity, which cannot be detected by the available reporter mice, is obviously important for skin cancer prevention and this may also be the case for liver regeneration.

Role of Nrf2 in Redox Homeostasis of the Liver

To determine the role of Nrf2 in liver regeneration, we first analyzed if the loss of Nrf2 disturbs the cellular redox balance in normal liver and after hepatectomy.31 Indeed, cultured primary hepatocytes from Nrf2 knockout mice had enhanced levels of intracellular ROS as determined biochemically. A similar finding was obtained in vivo using dihydroethidine, a dye that intercalates into DNA upon oxida-tion, and therefore allows monitoring of oxidative stress. Nuclear staining was observed in livers of Nrf2 knockout mice, in particular after hepatectomy, whereas livers of wild-type animals showed only weak fluorescence. These results identify Nrf2 as an important regu-lator of the cellular redox balance in the liver and demonstrate that deficiency in Nrf2 results in chronic oxidative stress in hepatocytes, which is further aggravated upon liver injury. Reduced expression of ROS-detoxifying Nrf2 target genes in normal and particularly in injured liver was identified as the underlying mechanism. However, this did not result in an obvious defect in liver function as revealed by the normal serum levels of aspartate and alanine aminotransferases. Excessive release of these liver-specific enzymes into the serum is a hallmark of liver dysfunction and damage (reviewed in ref. 2), and this was not observed in the Nr2 knockout mice.

Recent studies revealed that the related Nrf1 transcription factor is also involved in ROS detoxification in the liver. Cultured hepato-cytes of mice lacking Nrf1 in liver parenchymal cells had enhanced levels of ROS. These animals also developed severe steatosis and spontaneous liver cancer,32 a phenotype not observed in Nrf2-defi-cient mice. Therefore, Nrf1 and Nrf2 seem to regulate overlapping but also distinct target genes, and in particular the latter remain to be identified.

These results shed new light onto the regulation of the cellular redox balance in hepatocytes. They are complemented by studies on the role of NFκB in the liver. Luedde et al.,33 deleted the IκB kinase subunit NEMO/IKKγ, which is essential for activation of NFκB, in

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liver parenchymal cells. Interestingly, these mice developed sponta-neous steatosis and hepatocellular carcinoma. This phenotype was at least in part mediated by enhanced oxidative stress in the liver, since it could be alleviated by treatment of the animals with antioxidants. Enhanced oxidative stress was also seen in the absence of IKKβ, although phenotypic abnormalities were only seen in these mice upon challenging of the liver.34,35 The enhanced levels of ROS seen upon reduction of NFκB activity are consistent with the upregula-tion of antioxidative defenses by this transcription factor.36

Interestingly, our recent results suggest a cross-talk between Nrf2 and NFκB, since the activity of the latter was strongly enhanced in the regenerating liver of Nrf2 knockout mice.31 This is most likely due to the enhanced oxidative stress in Nrf2-deficient cells, since elevated levels of ROS cause NFκB activation.37 The latter in turn enhances survival of hepatocytes after hepatectomy.38 Therefore, its activation is most likely a compensatory effect to reduce the extent of oxidative stress and cell death in Nrf2 knockout mice.

Impaired Liver Regeneration in Nrf2 Knockout Mice

When partial hepatectomy was performed with Nrf2 knockout mice, we found a significant delay in liver regeneration.31 In mice of both genotypes only very few proliferating hepatocytes were detected in non-injured liver and 6–24 h after hepatectomy. However, a five-fold increase in the number of apoptotic cells was observed early (6 h) after injury in the knockout animals compared to wild-type controls, whereas necrosis could never be observed in mice of both genotypes. At later stages after hepatectomy, only extremely few apoptotic cells were detectable. Proliferation peaked 48 h after hepatectomy, but the peak was much less pronounced in Nrf2 knockout mice compared to wild-type controls. 1 day later, the number of proliferating cells remained lower in the knockout mice compared to wild-type animals. At day 5 after hepatectomy, proliferation was almost completed in the wild-type mice, whereas Nrf2-deficient hepatocytes continued to proliferate at this time point. In spite of these differences, liver repair was completed in mice of both genotypes at day 7 after injury as demonstrated by the almost complete absence of proliferating cells. Furthermore, the liver/body weight ratio had almost returned to the levels seen before injury. These findings demonstrate that repair can occur in the absence of Nrf2, but with a strong delay.

Role of Insulin/IGF-1 Signaling in the Liver Regeneration Phenotype of Nrf2 Knockout Mice

In a search for the mechanisms underlying the delayed regenera-tion of Nrf2-deficient liver, a defect in insulin/IGF-1 signaling in the regenerating liver was identified31 (Fig. 1). Consistent with data obtained with adipocytes,39 the chronic oxidative stress seen in hepa-tocytes of Nrf2-deficient mice resulted in resistance to exogenous insulin or IGF-1 in vitro. In vivo, tyrosine phosphorylation of the insulin receptor substrates 1 and 2 (IRS-1 and -2) in response to activation of the insulin receptor was strongly reduced in the regen-erating liver of Nrf2-deficient mice. The activation of the insulin receptor shortly after hepatectomy most likely occurs through release of IGF-1 from its binding proteins upon injury.

Previous studies had shown that the inhibitory effect of ROS on insulin/IGF-1 receptor signaling is mediated through activation of serine/threonine kinases by ROS, which in turn phosphory-late IRS-1 and IRS-2. This causes insulin/IGF-1 resistance, since

Ser/Thr-phosphorylated IRS proteins dissociate from the insulin receptor. Therefore, activation of IRS proteins through tyrosine phosphorylation does no longer occur. As a consequence, IRS-1 and IRS-2 can no longer associate with and activate phosphatidylinositide-3-kinase (PI3K) (reviewed in refs. 40 and 41). Consistent with these data, association of PI3K with IRS-1 in response to insulin was reduced in Nrf2-deficient hepatocytes in vitro, and reduced associa-tion of these proteins was also seen in hepatectomized liver of these mice in vivo. One of the inhibitory IRS serine/threonine kinases may be JNK, which is known to be activated by ROS35,42 and which showed enhanced and prolonged activation in the injured liver of Nrf2-deficient mice.31 The Ser phosphorylation of IRS-1 (at Ser307 in the mouse/Ser312 in human IRS-1) inhibits its phosphoryla-tion at Tyr608 (Tyr612 in human IRS-1) by the insulin or IGF-1 receptor,43 thereby causing insulin/IGF-1 resistance.44 The same effect can be achieved by other IRS kinases, including IKK-β45 or protein kinase Cζ,40 and the role of these kinases in the injured liver of Nrf2-deficient mice remains to be identified.

Loss of Nrf2 Impairs the Activation of Pro-Mitogenic and Anti-Apoptotic Signaling Pathways in the Regenerating Liver

Insulin/IGF-1 signaling is known to activate several pro-mitogenic and anti-apoptotic signaling pathways, including the mitogen-acti-vated kinase pathway and the PI3K-Akt pathway.46 These pathways were also activated in the liver of wild-type mice, but activation of the p38 MAPK pathway and in particular of the PI3K-Akt pathway was strongly impaired in the absence of Nrf2.31 Most importantly, the time-course of their activation correlated with the time-course of IRS activation, whereas activation of the epidermal growth factor receptor and the hepatocyte growth factor receptor, two major regulators of liver repair (see above) could not be observed at this time point, and there was also no difference in activation of these receptors between wild-type and Nrf2 knockout mice.

The reduced p38 activation may well be involved in the regen-eration defect of Nrf2-deficient mice, since cell cycle entry of hepatocytes after partial hepatectomy was strongly inhibited by systemic treatment of rats with a p38 inhibitor.47 However, enhanced and prolonged p38 activation was seen in c-Jun deficient mice and this caused reduced hepatocyte proliferation and enhanced mortality of these animals after hepatectomy.48 Thus, the role of this pathway in liver regeneration in general and in the phenotype of Nrf2 knockout mice in particular remains to be determined.

Of particular importance, however, is the strongly reduced activation of the PI3K/Akt signaling pathway in Nrf2-deficient hepa-tocytes after hepatectomy in vivo and after insulin treatment in vitro. Thus, the strong insulin-mediated activation of Akt, which was seen in cultured primary hepatocytes of wild-type mice, was no longer observed in cells from Nrf2-deficient mice. In vivo, phosphoryla-tion of Akt, which results in its activation and which occurs within 3–6 h after hepatectomy in the liver of wild-type mice, was strongly reduced in the absence of Nrf2. In turn, reduced phosphorylation of the Akt targets glycogen synthase kinase-3β, p70/S6K and Bad was observed. Since these proteins are crucial for cell proliferation and survival (reviewed in refs. 49 and 50), the defect in PI3K signaling is likely to be responsible for the delayed hepatocyte proliferation and for the increased apoptosis of these cells after hepatectomy of Nrf2-deficient mice.

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Possible Therapeutic Consequences

The results described above reveal a crucial role of Nrf2 in liver regeneration through its capacity to regulate the cellular redox balance and thus efficient insulin/IGF-1 signaling in the injured liver. The basal rather than the inducible activity of Nrf2 is likely to be responsible for this effect, since ARE activation was only observed in very few hepatocytes after hepatectomy, and since the defect in insulin/IGF-1 signaling occurred at an early stage, where upregulaton of Nrf2 target genes was not yet observed. Therefore, further enhancement of Nrf2 action may help to speed up the liver regeneration process. In addition, it may well be that activation of Nrf2 is insufficient in diseased liver. In this case pharmacologic activation of this transcription factor may be particularly useful and could possibly prevent the fibrosis, which frequently occurs after chronic injury. Therefore, activation of Nrf2 by natural substances,

such as sulforaphane or avicins, or by synthetic pharmaceuticals, e.g., by synthetic triterpenoids,51-54 could be a promising new strategy to improve regeneration in patients with acute or chronic liver damage. However, long-term activation may also be deleterious, in particular in patients with liver cancer, since this may enhance malignant progression and resistance to chemotherapy. This hypothesis is based on the finding that inactivating mutations in the Keap1 gene were found in non-small-cell lung cancer, resulting in Nrf2 hyper-activation. As a consequence, expression of antioxidant proteins, detoxification enzymes and drug transporters was enhanced, resulting in higher malignancy and resistance to chemotherapy.55 Thus, future studies will reveal if timely controlled activation of Nrf2 in the liver is beneficial. In addition, it will be interesting to determine if Nrf2 deficiency is also associated with insulin resistance in other organs.

Figure 1. Schematic representation of insulin/IGF-1 receptor signaling in the regenerating liver and its impairment in the absence of Nrf2. Nrf2-mediated expression of ROS-detoxifying enzymes prevents accumulation of ROS in the liver. Lack of Nrf2 causes oxidative stress and activation of serine-/threonine kinases, which phosphorylate IRS-1 and IRS-2. These kinases include JNK and possibly others, such as protein kinase Cζ. Ser/Thr phosphorylation of IRS-1 and IRS-2 causes dissociation of these proteins from the insulin receptor, resulting in reduced tyrosine phosphorylation of IRS-1 and IRS-2 by the receptor kinase. Since tyrosine phosphorylation of IRS-1 is required for PI3K activation, phosphorylation of Akt and downstream targets as well as p38 activation are reduced. The deficiency in these signaling pathways cause a delay in hepatocyte proliferation and enhanced apoptosis of these cells after liver injury. As a consequence, liver regeneration is delayed in the absence of Nrf2.

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Acknowledgements

The Nrf2 research in the laboratory of S.W. is supported by a grant from the Swiss National Science Foundation (3100A9-109340/1 to S.W.) and by the ETH Zurich.

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