NRF2, not always friendly but perhaps misunderstood. NRF2 plays a key role in coordinating the responses to oxidative stress (1). It is expressed in multiple cell types, including hepatocytes. Liver, perhaps more than most organs, is exposed to oxidative stress generated in part from metabolic dysregulation and in part from enzymatic reactions related to drug metabolism and detoxification of xenobiotics. NRF2 is present in the cytoplasm bound to a protein partner known as Keap1. The latter targets NRF2 for proteasomal degradation. Small numbers of NRF2 escape and migrate to the nucleus where they induce basal levels of antioxidant enzyme expression. In the presence of a variety of oxidants and electrophiles that directly bind to Keap1, the NRF2-Keap1 binding is disrupted. NRF2 dissociates from Keap1 and migrates to the nucleus. There, in association with other transcription factors, it binds to DNA sequences known as “antioxidant response elements” (ARE) in the promoter/enhancer regions of target genes and initiates expression of a variety of genes involved in containing and eliminating free radicals derived from oxygen or electrophiles generated by the metabolism of xenobiotics. The functions and the variety of processes associated with NRF2 are multiple (1). NRF2 activation protects liver from ischemia reperfusion injury (2). NRF2 participates in several metabolic processes including oxidation of fatty acids in the mitochondria, crucial for regulation of the hepatocyte energy supply (3). Deletion of NRF2 impairs glucose tolerance and exacerbates hyperglycemia in mice with type I diabetes (4). In mice with genetic deletion of NRF2, high-fat diet progresses to nonalcoholic steatohepatitis, in contrast to a simple fatty change seen in the wild type mice (5). The cellular and gene expression adaptations of liver to pregnancy are also dependent on NRF2, with disruption of the AKT1/mTOR pathway seen in NRF2-null mice (6). There is an interesting relationship between NRF2 and Notch1. Among the many gene expression changes induced by Notch1 is the upregulation of NRF2 and its dependent enzymes via specific site recognized by the Notch1 intracellular domain (NICD) transcription factor complex on the NRF2 promoter (7). The Notch-NRF2 induced expression of anti-oxidant stress proteins is sufficient to protect mice from acetaminophen-induced liver injury (7). In addition, the well-known intrahepatic bile duct irregularities seen in NICD-null mice (8) are partially reversed by induced expression of NRF2. Equally interesting however is the presence of an NRF2responsive ARE site in the promoter of Notch1 (9), suggesting a bilateral and reciprocal activation of both pathways when one is initially activated. Genetic elimination of Keap1 results in activation of NRF2. Not surprisingly, there is also constitutive activation of Notch1 in Keap1 -/- mice (9). There are previous studies documenting involvement of Notch1 and dependent Hes1, 5 genes at the earliest stages of liver regeneration (10). The cross talk between the NRF2 and Notch1 pathways led to further studies, which showed that there is decreased activation of Notch1 and dependent genes in NRF2 -/- mice and delayed regeneration after partial hepatectomy (9). This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/hep.27090
Hepatology
Previous studies by from the laboratory of Dr. Werner had also shown that liver regeneration is impaired in NRF2 -/- mice (11). The regenerative response after 2/3 partial hepatectomy was prolonged, with decreased hepatocyte proliferation and enhanced hepatocyte apoptosis at all stages of regeneration. Proliferation of hepatocytes eventually rebounded at 120 hours, restoring liver mass. There was decreased activation of p38 mitogen-activated kinase, PI3 kinase and Akt and their downstream targets at the first 48 hours of liver regeneration, and decreased expression and phosphorylation of the insulin receptor. The effect on mitogenic receptors EGFR and MET was not investigated. Given the early activation of these two receptors within 30 minutes after partial hepatectomy (12), it is possible that expression or activation of these two receptors may also be an issue. Expression of the mitogenic receptor ligands HGF or TGFα was not affected in the NRF2 -/- mice, though this would be of no consequence since HGF is present as a reservoir protein in hepatic biomatrix and rapidly activated by urokinase whereas EGF is constantly supplied to the liver by the Brunner glands of the duodenum (13). It is also interesting that there was increased hepatocyte DNA oxidation in the NRF2 -/- mice. In addition to their relevance for liver regeneration, the observed effects on function of the insulin receptor may also related to the previously mentioned enhanced susceptibility of NRF2 -/- mice to high fat diet (5). In view of the above literature on impaired regeneration in NRF2 -/- mice, the findings of the paper by Dr. Werner and her co-workers in this issue of Hepatology (14) come as a mild surprise. One would expect that constitutive activation of NRF2 in hepatocytes would have the opposite effect to that of NRF2 -/-, i.e. enhanced and accelerated liver regeneration. The authors used mice with transgenic expression of a modified NRF2 protein missing the Keap1 binding site (Neh2 domain of NRF2), and thus not susceptible to Keap1 mediated degradation. The modified constitutively activated NRF2 (caNRF2) was targeted for expression in hepatocytes via a Cre-recombinase eliminating a STOPloxp/loxp site regulating expression of caNRF2 under control of the albumin promoter. There was no obvious histopathology or any impact of assessed indices of liver homeostasis in the caNRF2 transgenic mice. Acute and chronic administration of CCl4 showed only minor differences in number of necrotic hepatocytes, AST/ALT, long term fibrosis, etc. There were, however, measurable –and unexpected- differences, when liver regeneration was induced by partial hepatectomy. There was a 50% reduction in the number of proliferating hepatocytes at the peak of proliferation (48 hours in the mouse) in the caNRF2 transgenic mice. This was compensated by enhanced proliferation at 72 hours and eventual complete restoration of the liver mass. There were no differences in activation of insulin receptor and tyrosine phosphorylation of its IRS1,2 target proteins. There was also no difference in the Notch1 dependent expression of Hes1,5 genes, and no change in expression of p21 or p27. There was, however, upregulation of the cyclin-dependent kinase inhibitor p15, the proapoptotic gene Bcl2l11 (Bim) and the Growth arrest gene 1 (Gas1). The authors identified putative ARE sites upstream of the transcription initiation for p15 and Bim, demonstrated that these sites actually facilitate NRF2 binding by chromatin
Hepatology
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Hepatology
immunoprecipitation and ascribed the delay of liver regeneration of the caNRF2 transgenic mice to the upregulation of these genes. The findings are a fine example of the paradoxical peculiarities of the cybernetics of gene expression. Oftentimes, in the interest of providing intelligible paradigms of the complex functions of signaling proteins, stereotypes of function are ascribed in order to provide logical frameworks for mechanistic hypotheses. The internal logic of the cells, however, is more complicated. Unexpected and contradictory signaling functions are often discovered, which in retrospect are better understood as providing a fine balance for optimization of processes which the cell understands better than we do. Overexpression of some growth factors leads to enhanced apoptosis, as in the case of HGF disturbing the binding and lateral association of its receptor MET with the death receptor FAS (15). The findings of the paper by Kohler et al. definitely enhance the complexity of NRF2-related signaling. In retrospect, it is probably beneficial for hepatocytes in the throes of oxidant stress to have delayed proliferation, thus allowing more efficient removal of DNA adducts and damaged cellular proteins, decreasing the risk of genotoxicity or cell death and organ failure. Another theme running through many of the papers mentioned in this editorial is the compensating increase in hepatocyte proliferation at later stages of regeneration (72 hours and beyond) in all situations where there is decreased proliferation of hepatocytes at the early stages. So far, this is seen in all publications of models where early hepatocyte proliferation is delayed, with no exception. The hepatic Phoenix appears to be almost always capable of rising from its ashes…however, as the hundreds of annually reported cases of fulminant hepatic failure document, this is not always true, for reasons that we do not understand, but desperately need to do so. NRF2, as an umbrella protector for the hepatocytes, exercises a very important function in perhaps slowing things down, when needed, to prevent complete catastrophe. This may be one of its most important functions. George K. Michalopoulos Department of Pathology University of Pittsburgh REFERENCES 1. Ma Q, He X. Molecular basis of electrophilic and oxidative defense: promises and perils of Nrf2. Pharmacol Rev 2012;64:1055-1081. 2. Kudoh K, Uchinami H, Yoshioka M, Seki E, Yamamoto Y. Nrf2 Activation Protects the Liver From Ischemia/Reperfusion Injury in Mice. Ann Surg 2013. 3. Ludtmann MH, Angelova PR, Zhang Y, Abramov AY, Dinkova-Kostova AT. Nrf2 affects the efficiency of mitochondrial fatty acid oxidation. Biochem J 2014;457:415-424. 4. Aleksunes LM, Reisman SA, Yeager RL, Goedken MJ, Klaassen CD. Nuclear factor erythroid 2-related factor 2 deletion impairs glucose tolerance and
Hepatology
Hepatology
exacerbates hyperglycemia in type 1 diabetic mice. J Pharmacol Exp Ther 2010;333:140-151. 5. Wang C, Cui Y, Li C, Zhang Y, Xu S, Li X, Li H, et al. Nrf2 deletion causes "benign" simple steatosis to develop into nonalcoholic steatohepatitis in mice fed a high-fat diet. Lipids Health Dis 2013;12:165. 6. Zou Y, Hu M, Bao Q, Chan JY, Dai G. Nrf2 participates in regulating maternal hepatic adaptations to pregnancy. J Cell Sci 2013;126:1618-1625. 7. Wakabayashi N, Skoko JJ, Chartoumpekis DV, Kimura S, Slocum SL, Noda K, Palliyaguru DL, et al. Notch-nrf2 axis: regulation of nrf2 gene expression and cytoprotection by notch signaling. Mol Cell Biol 2014;34:653-663. 8. Sparks EE, Huppert KA, Brown MA, Washington MK, Huppert SS. Notch signaling regulates formation of the three-dimensional architecture of intrahepatic bile ducts in mice. Hepatology 2010;51:1391-1400. 9. Wakabayashi N, Shin S, Slocum SL, Agoston ES, Wakabayashi J, Kwak MK, Misra V, et al. Regulation of notch1 signaling by nrf2: implications for tissue regeneration. Sci Signal 2010;3:ra52. 10. Kohler C, Bell AW, Bowen WC, Monga SP, Fleig W, Michalopoulos GK. Expression of Notch-1 and its ligand Jagged-1 in rat liver during liver regeneration. Hepatology 2004;39:1056-1065. 11. Beyer TA, Xu W, Teupser D, auf dem Keller U, Bugnon P, Hildt E, Thiery J, et al. Impaired liver regeneration in Nrf2 knockout mice: role of ROS-mediated insulin/IGF-1 resistance. EMBO J 2008;27:212-223. 12. Stolz DB, Mars WM, Petersen BE, Kim TH, Michalopoulos GK. Growth factor signal transduction immediately after two-thirds partial hepatectomy in the rat. Cancer Res 1999;59:3954-3960. 13. Michalopoulos GK. Principles of liver regeneration and growth homeostasis. Compr Physiol 2013;3:485-513. 14. Kohler UA, Kurinna S, Schwitter D, Marti A, Schafer M, Hellerbrand C, Speicher T, et al. Activated Nrf2 impairs liver regeneration in mice by activation of genes involved in cell cycle control and apoptosis. Hepatology 2013. 15. Wang X, DeFrances MC, Dai Y, Pediaditakis P, Johnson C, Bell A, Michalopoulos GK, et al. A mechanism of cell survival: sequestration of Fas by the HGF receptor Met. Mol Cell 2002;9:411-421. Figure Legend Under situations of normal cytoplasmic conditions or low oxidant stress, NRF2 (activated in low levels) promotes liver regeneration. In situations of high oxidant stress causing damage to intracellular components, however, high levers of activated NRF2 delay regeneration, as shown in the manuscript by Kohler et al. This may serve as a protective function for hepatocyte survival, to ensure that damage can be repaired prior to allowing hepatocytes to enter into cell proliferation.
Hepatology
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Hepatology
170x120mm (300 x 300 DPI)
Hepatology
Hepatology
Previous studies by from the laboratory of Dr. Werner had also shown that liver regeneration is impaired in NRF2 -/- mice (11). The regenerative response after 2/3 partial hepatectomy was prolonged, with decreased hepatocyte proliferation and enhanced hepatocyte apoptosis at all stages of regeneration. Proliferation of hepatocytes eventually rebounded at 120 hours, restoring liver mass. There was decreased activation of p38 mitogen-activated kinase, PI3 kinase and Akt and their downstream targets at the first 48 hours of liver regeneration, and decreased expression and phosphorylation of the insulin receptor. The effect on mitogenic receptors EGFR and MET was not investigated. Given the early activation of these two receptors within 30 minutes after partial hepatectomy (12), it is possible that expression or activation of these two receptors may also be an issue. Expression of the mitogenic receptor ligands HGF or TGFα was not affected in the NRF2 -/- mice, though this would be of no consequence since HGF is present as a reservoir protein in hepatic biomatrix and rapidly activated by urokinase whereas EGF is constantly supplied to the liver by the Brunner glands of the duodenum (13). It is also interesting that there was increased hepatocyte DNA oxidation in the NRF2 -/- mice. In addition to their relevance for liver regeneration, the observed effects on function of the insulin receptor may also related to the previously mentioned enhanced susceptibility of NRF2 -/- mice to high fat diet (5). In view of the above literature on impaired regeneration in NRF2 -/- mice, the findings of the paper by Dr. Werner and her co-workers in this issue of Hepatology (14) come as a mild surprise. One would expect that constitutive activation of NRF2 in hepatocytes would have the opposite effect to that of NRF2 -/-, i.e. enhanced and accelerated liver regeneration. The authors used mice with transgenic expression of a modified NRF2 protein missing the Keap1 binding site (Neh2 domain of NRF2), and thus not susceptible to Keap1 mediated degradation. The modified constitutively activated NRF2 (caNRF2) was targeted for expression in hepatocytes via a Cre-recombinase eliminating a STOPloxp/loxp site regulating expression of caNRF2 under control of the albumin promoter. There was no obvious histopathology or any impact of assessed indices of liver homeostasis in the caNRF2 transgenic mice. Acute and chronic administration of CCl4 showed only minor differences in number of necrotic hepatocytes, AST/ALT, long term fibrosis, etc. There were, however, measurable –and unexpected- differences, when liver regeneration was induced by partial hepatectomy. There was a 50% reduction in the number of proliferating hepatocytes at the peak of proliferation (48 hours in the mouse) in the caNRF2 transgenic mice. This was compensated by enhanced proliferation at 72 hours and eventual complete restoration of the liver mass. There were no differences in activation of insulin receptor and tyrosine phosphorylation of its IRS1,2 target proteins. There was also no difference in the Notch1 dependent expression of Hes1,5 genes, and no change in expression of p21 or p27. There was, however, upregulation of the cyclin-dependent kinase inhibitor p15, the proapoptotic gene Bcl2l11 (Bim) and the Growth arrest gene 1 (Gas1). The authors identified putative ARE sites upstream of the transcription initiation for p15 and Bim, demonstrated that these sites actually facilitate NRF2 binding by chromatin
Hepatology
Page 2 of 5
Page 3 of 5
Hepatology
immunoprecipitation and ascribed the delay of liver regeneration of the caNRF2 transgenic mice to the upregulation of these genes. The findings are a fine example of the paradoxical peculiarities of the cybernetics of gene expression. Oftentimes, in the interest of providing intelligible paradigms of the complex functions of signaling proteins, stereotypes of function are ascribed in order to provide logical frameworks for mechanistic hypotheses. The internal logic of the cells, however, is more complicated. Unexpected and contradictory signaling functions are often discovered, which in retrospect are better understood as providing a fine balance for optimization of processes which the cell understands better than we do. Overexpression of some growth factors leads to enhanced apoptosis, as in the case of HGF disturbing the binding and lateral association of its receptor MET with the death receptor FAS (15). The findings of the paper by Kohler et al. definitely enhance the complexity of NRF2-related signaling. In retrospect, it is probably beneficial for hepatocytes in the throes of oxidant stress to have delayed proliferation, thus allowing more efficient removal of DNA adducts and damaged cellular proteins, decreasing the risk of genotoxicity or cell death and organ failure. Another theme running through many of the papers mentioned in this editorial is the compensating increase in hepatocyte proliferation at later stages of regeneration (72 hours and beyond) in all situations where there is decreased proliferation of hepatocytes at the early stages. So far, this is seen in all publications of models where early hepatocyte proliferation is delayed, with no exception. The hepatic Phoenix appears to be almost always capable of rising from its ashes…however, as the hundreds of annually reported cases of fulminant hepatic failure document, this is not always true, for reasons that we do not understand, but desperately need to do so. NRF2, as an umbrella protector for the hepatocytes, exercises a very important function in perhaps slowing things down, when needed, to prevent complete catastrophe. This may be one of its most important functions. George K. Michalopoulos Department of Pathology University of Pittsburgh REFERENCES 1. Ma Q, He X. Molecular basis of electrophilic and oxidative defense: promises and perils of Nrf2. Pharmacol Rev 2012;64:1055-1081. 2. Kudoh K, Uchinami H, Yoshioka M, Seki E, Yamamoto Y. Nrf2 Activation Protects the Liver From Ischemia/Reperfusion Injury in Mice. Ann Surg 2013. 3. Ludtmann MH, Angelova PR, Zhang Y, Abramov AY, Dinkova-Kostova AT. Nrf2 affects the efficiency of mitochondrial fatty acid oxidation. Biochem J 2014;457:415-424. 4. Aleksunes LM, Reisman SA, Yeager RL, Goedken MJ, Klaassen CD. Nuclear factor erythroid 2-related factor 2 deletion impairs glucose tolerance and
Hepatology
Hepatology
exacerbates hyperglycemia in type 1 diabetic mice. J Pharmacol Exp Ther 2010;333:140-151. 5. Wang C, Cui Y, Li C, Zhang Y, Xu S, Li X, Li H, et al. Nrf2 deletion causes "benign" simple steatosis to develop into nonalcoholic steatohepatitis in mice fed a high-fat diet. Lipids Health Dis 2013;12:165. 6. Zou Y, Hu M, Bao Q, Chan JY, Dai G. Nrf2 participates in regulating maternal hepatic adaptations to pregnancy. J Cell Sci 2013;126:1618-1625. 7. Wakabayashi N, Skoko JJ, Chartoumpekis DV, Kimura S, Slocum SL, Noda K, Palliyaguru DL, et al. Notch-nrf2 axis: regulation of nrf2 gene expression and cytoprotection by notch signaling. Mol Cell Biol 2014;34:653-663. 8. Sparks EE, Huppert KA, Brown MA, Washington MK, Huppert SS. Notch signaling regulates formation of the three-dimensional architecture of intrahepatic bile ducts in mice. Hepatology 2010;51:1391-1400. 9. Wakabayashi N, Shin S, Slocum SL, Agoston ES, Wakabayashi J, Kwak MK, Misra V, et al. Regulation of notch1 signaling by nrf2: implications for tissue regeneration. Sci Signal 2010;3:ra52. 10. Kohler C, Bell AW, Bowen WC, Monga SP, Fleig W, Michalopoulos GK. Expression of Notch-1 and its ligand Jagged-1 in rat liver during liver regeneration. Hepatology 2004;39:1056-1065. 11. Beyer TA, Xu W, Teupser D, auf dem Keller U, Bugnon P, Hildt E, Thiery J, et al. Impaired liver regeneration in Nrf2 knockout mice: role of ROS-mediated insulin/IGF-1 resistance. EMBO J 2008;27:212-223. 12. Stolz DB, Mars WM, Petersen BE, Kim TH, Michalopoulos GK. Growth factor signal transduction immediately after two-thirds partial hepatectomy in the rat. Cancer Res 1999;59:3954-3960. 13. Michalopoulos GK. Principles of liver regeneration and growth homeostasis. Compr Physiol 2013;3:485-513. 14. Kohler UA, Kurinna S, Schwitter D, Marti A, Schafer M, Hellerbrand C, Speicher T, et al. Activated Nrf2 impairs liver regeneration in mice by activation of genes involved in cell cycle control and apoptosis. Hepatology 2013. 15. Wang X, DeFrances MC, Dai Y, Pediaditakis P, Johnson C, Bell A, Michalopoulos GK, et al. A mechanism of cell survival: sequestration of Fas by the HGF receptor Met. Mol Cell 2002;9:411-421. Figure Legend Under situations of normal cytoplasmic conditions or low oxidant stress, NRF2 (activated in low levels) promotes liver regeneration. In situations of high oxidant stress causing damage to intracellular components, however, high levers of activated NRF2 delay regeneration, as shown in the manuscript by Kohler et al. This may serve as a protective function for hepatocyte survival, to ensure that damage can be repaired prior to allowing hepatocytes to enter into cell proliferation.
Hepatology
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Page 5 of 5
Hepatology
170x120mm (300 x 300 DPI)
Hepatology
