Research ArticleNRF2 activates growth factor genes and downstream AKT signaling to induce mouse and human hepatomegaly
Graphical abstract
Introduction
Hepatomegaly, or liver enlargement, is a non-specific pathological reaction triggered by infections, liver cancer, metabolic disturbances, toxicant exposure, as well as alcohol abuse or drug-induced hepatitis.1 Congenital disorders, including hemolytic anemia, polycystic liver disease, sickle cell disease, hereditary fructose intolerance or carnitine palmitoyltransferase deficiency can also elicit hepatomegaly.[2], [3], [4], [5], [6] Hepatomegaly has been frequently observed in patients with type I and type II diabetes with poor glycemic control.7,8 In such cases, hepatomegaly has been attributed to hepatocyte glycogen accumulation driven by insulin-induced hepatic glycogenosis, especially in pediatric patients.8 By contrast, the mechanisms underlying hepatomegaly caused by other etiologies are poorly understood. Histopathological analysis suggests the involvement of vascular swelling and inflammation, as well as increased accumulation of iron, glycogen, fat or insoluble proteins within hepatocytes.1 Mimicking insulin overdosing, adenovirus-mediated hepatic overexpression of a constitutively active form of AKT, the key effector of insulin signaling,9 resulted in pronounced hepatomegaly accompanied by hypoglycemia and hypertriglyceridemia.10
Autophagy defects can also induce hepatomegaly. Indeed, both liver-specific Atg5 (Atg5Δhep) and Atg7 (Atg7Δhep) knockout mice exhibit marked liver enlargement.[11], [12], [13], [14], [15], [16] Autophagy-deficient livers show accumulation of the autophagy substrate and signaling scaffold p62/SQSTM1 (hereafter referred to p62), which sequesters Kelch-like ECH-associated protein 1 (KEAP1) away from the oxidant-responsive transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), resulting in nuclear accumulation and activation of NRF2.13,16,17 Nuclear NRF2 also accumulates in response to oxidative stress and KEAP1 inactivation.18,19 Normally, NRF2 plays a critical role in the induction of genes that code for cytoprotective redox-active proteins and enzymes that detoxify reactive oxygen species (ROS) and xenobiotics.18 But NRF2 can also undergo persistent activation in lung, liver and colon cancers due to gain-of-function mutations in the NFE2L2 gene that prevent binding to KEAP1 or loss-of-function mutations in the KEAP1 gene.[20], [21], [22], [23] In addition to NRF2, several other factors may contribute to hepatomegaly in the autophagy-deficient liver, including YAP,24 mTORC1,11 and FXR.25 Thus, it is not clear whether NRF2 activation alone is sufficient for induction of hepatomegaly in oxidatively stressed or autophagy-deficient livers. Also, the relationship between the NRF2-induced protective response and hepatomegaly remains unknown.
NRF2-deficient mice are susceptible to numerous electrophiles and oxidants,18,26 and also exhibit defective liver regeneration due to oxidative stress-mediated insulin/insulin-like growth factor (IGF) resistance.27 Conversely, NRF2 activation redirects glucose and glutamine into the anabolic pentose phosphate pathway when superimposed on persistently activated AKT in cancer cells that exhibit chronic upregulation of phosphatidylinositol 3-kinase (PI3K) signaling.28 Although in cancer cells NRF2 activation confers survival and growth advantage,[29], [30], [31], [32] in autophagy-defective livers NRF2 activation was reported to provoke hepatocyte death and liver injury.12,13,16,25,33 How NRF2 activation promotes liver injury in the absence of autophagy is unknown. Moreover, persistent activation of NRF2 in an autophagy competent liver does not cause liver injury as shown by liver-specific Keap1 knockout (Keap1Δhep) mice, which exhibit hepatomegaly without liver damage.12 Evidently, the effects of persistent NRF2 activation are highly context dependent.
We previously found that in the unstressed liver, p62 controls basal NRF2 activity32 and that p62 accumulation due to autophagy disruption contributes to liver and pancreatic tumorigenesis by activating NRF2.30,32,34 To further understand the role of persistent NRF2 activation in hepatocytes and whether it is sufficient to induce hepatomegaly, we generated transgenic mice that express a KEAP1-resistant form of NRF2 in their hepatocytes (Nrf2Act-hep). We also used p62 adenovirus vectors to selectively overexpress p62 in hepatocytes. Here, we demonstrate that hepatocyte-specific activation of NRF2 by either method causes marked hepatomegaly, glycogenosis, hypoglycemia and hypertriglyceridemia. Unexpectedly, we found that persistent NRF2 activation leads to hepatomegaly via upregulation of AKT signaling and that the latter depends on autocrine epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) signaling. The NRF2-AKT axis is also activated in human hepatomegaly.
Section snippets
Mouse experimentation
Nfe2l2iE79Q/+ mice were generated at Genentech using C57BL/6N embryonic stem cells and standard methodology. A targeting cassette containing the loxP sequence, a duplicated copy of the last 130 bp of Nfe2l2 intron 1, wild-type (WT) Nfe2l2 cDNA (exons 2-5), a human growth hormone 3' UTR followed by a 4× polyadenylation signal, an FRT-Neo-FRT selection marker and a second loxP sequence was inserted into the Nfe2l2 locus 130 bp 5' to a mutated exon 2 encoding the E79Q (GAA to CAA) mutation. The Neo
p62 induces NRF2-dependent hepatomegaly
Overexpression of p62 in mouse liver causes hepatocellular carcinoma.32 To investigate early effects of p62 overexpression that may shed new light on its tumorigenic activity, we used adenovirus (Adv) to transiently express GFP, p62 WT or a p62 variant, p62KIR- (that no longer binds KEAP1), in mouse livers.13 Transduction of p62 WT, but not p62KIR- or GFP, led to pronounced hepatomegaly, resulting in a 2.5-fold increase in liver to body weight ratio within 7 days of Adv infection (Fig.1A,B).
Discussion
Hepatomegaly can be triggered by insulin overdosing and many other etiologies that cause dysregulated glycogen or lipid accumulation in hepatocytes.8 Other than insulin overdosing, the mechanisms underlying hepatomegaly were unknown, although hepatomegaly in the autophagy-deficient liver was attributed to NRF2 activation.12,13 Originally recognized as the master activator of the anti-oxidant response,19 NRF2 is also an important metabolic regulator that redirects glucose and glutamine into the
Financial support
This research was supported by the Superfund Basic Research Program (P42-ES010337), NIH (P01-DK098108, 5R01DK120714-02, R01-CA118165, R01CA198103, R37-AI043477, P01-CA128814, R01-CA211794) and a C3 Pedal the Cause grant to M.K., who holds the Ben and Wanda Hildyard Chair for Mitochondrial and Metabolic Diseases; The NIH also supported M.T.D.-M. (R01-CA192642) and J.M. (R01-DK108743 and R01-CA211794). Research at Nanjing University was supported by the National Key Research and Development
Authors' contributions
F.H., L.A., and S.Y. designed and performed the main experiments and wrote the paper with M.K., who conceived and supervised the project. K.T., A.U., G.H. and M.G. participated in the main experiments. G.H. and M.G. provided mutant NRF2 mice used by L.A. to generate Nrf2Act mice. F.H. and S.Y. generated p62 adenovirus. Z.Z. and B.S. provided and analyzed human samples. M.R.C., A.M., M.T.D.-M. and J.M. performed the RNA-seq analyses. All authors discussed and interpreted the results and revised
Conflict of interest
G.H and M.G. are full time employees of Genentech/Roche and hold company shares. Remaining authors declare no competing interests.
Please refer to the accompanying ICMJE disclosure forms for further details.
Acknowledgment
We thank eBioscience, Cell Signaling Technologies, Santa Cruz Technologies and Promega for gifts of reagents. We thank Randal Kaufman, Su Hua and Elsa Sanchez-Lopez for advice and helpful suggestions. We also thank Elijah Hatfield for editorial assistance.
References (54)
- et al.
Gastrointestinal and hepatic complications of sickle cell disease
Clin Gastroenterol Hepatol
(2010) - et al.
Hereditary fructose intolerance: an inborn defect of hepatic fructose-1-phosphate splitting aldolase
Am J Med
(1963) - et al.
Carnitine palmitoyltransferase deficiencies
Mol Genet Metab
(1999) Interplay between FOXO, TOR, and Akt
Biochim Biophys Acta
(2011)- et al.
Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice
Cell
(2007) - et al.
Nrf2 promotes the development of fibrosis and tumorigenesis in mice with defective hepatic autophagy
J Hepatol
(2014) - et al.
p62 in cancer: signaling adaptor beyond autophagy
Cell
(2016) - et al.
The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress
J Biol Chem
(2009) - et al.
Nrf2 redirects glucose and glutamine into anabolic pathways in metabolic reprogramming
Cancer Cell
(2012) - et al.
NRF2 and the hallmarks of cancer
Cancer Cell
(2018)
Stress-activated NRF2-MDM2 cascade controls neoplastic progression in pancreas
Cancer Cell
NRF2 promotes tumor maintenance by modulating mRNA translation in pancreatic cancer
Cell
p62, upregulated during preneoplasia, induces hepatocellular carcinogenesis by maintaining survival of stressed HCC-initiating cells
Cancer Cell
Skeletal muscle-specific ablation of raptor, but not of rictor, causes metabolic changes and results in muscle dystrophy
Cell Metab
NF-κB restricts inflammasome activation via elimination of damaged mitochondria
Cell
Akt-mediated liver growth promotes induction of cyclin E through a novel translational mechanism and a p21-mediated cell cycle arrest
J Biol Chem
Modulation of proteostasis by transcription factor NRF2 and impact in neurodegenerative diseases
Redox Biol
AKT/PKB signaling: navigating the network
Cell
Chemical proteomics identifies druggable vulnerabilities in a genetically defined cancer
Cell
Autophagy inhibition compromises degradation of ubiquitin-proteasome pathway substrates
Mol Cell
Hepatocyte-specific deletion of the keap1 gene activates Nrf2 and confers potent resistance against acute drug toxicity
Biochem Biophysical Res Commun
Hepatic sinusoidal-obstruction syndrome: toxicity of pyrrolizidine alkaloids
J Hepatol
Hepatomegaly. Encyclopedia of Molecular Mechanisms of Disease
Massive hepatomegaly in adult polycystic liver disease
Am J Surg Pathol
Hepatic manifestations in hematological disorders
Int J Hepatol
Hepatomegaly due to self-induced hyperinsulinism
Arch Dis Child
Hepatomegaly and abnormal liver tests due to glycogenosis in adults with diabetes
Medicine
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