Elsevier

Journal of Hepatology

Volume 72, Issue 6, June 2020, Pages 1182-1195
Journal of Hepatology

Research Article
NRF2 activates growth factor genes and downstream AKT signaling to induce mouse and human hepatomegaly

https://doi.org/10.1016/j.jhep.2020.01.023Get rights and content

Highlights

  • Liver p62 accumulation and constitutive NRF2 activation lead to liver lipid buildup, glycogen synthesis and hepatomegaly.

  • NRF2 activation mediates transcriptional induction of PDGF and EGF receptor ligands that activate AKT.

  • AKT and tyrosine kinase inhibitors block NRF2-mediated AKT activation and hepatomegaly.

  • NRF2-AKT signaling is elevated in HSOS- and AIH-related human hepatomegaly.

Background & Aims

Hepatomegaly can be triggered by insulin and insulin-unrelated etiologies. Insulin acts via AKT, but how other challenges cause hepatomegaly is unknown.

Methods

Since many hepatomegaly-inducing toxicants and stressors activate NRF2, we examined the effect of NRF2 activation on liver size and metabolism using a conditional allele encoding a constitutively active NRF2 variant to generate Nrf2Act-hep mice in which NRF2 is selectively activated in hepatocytes. We also used adenoviruses encoding variants of the autophagy adaptor p62/SQSTM1, which activates liver NRF2, as well as liver-specific ATG7-deficient mice (Atg7Δhep) and liver specimens from patients with hepatic sinusoidal obstruction syndrome (HSOS) and autoimmune hepatitis (AIH). RNA sequencing and cell signaling analyses were used to determine cellular consequences of NRF2 activation and diverse histological analyses were used to study effects of the different manipulations on liver and systemic pathophysiology.

Results

Hepatocyte-specific NRF2 activation, due to p62 accumulation or inhibition of KEAP1 binding, led to hepatomegaly associated with enhanced glycogenosis, steatosis and G2/M cell cycle arrest, fostering hyperplasia without cell division. Surprisingly, all manipulations that led to NRF2 activation also activated AKT, whose inhibition blocked NRF2-induced hepatomegaly and glycogenosis, but not NRF2-dependent antioxidant gene induction. AKT activation was linked to NRF2-mediated transcriptional induction of PDGF and EGF receptor ligands that signaled through their cognate receptors in an autocrine manner. Insulin and insulin-like growth factors were not involved. The NRF2-AKT signaling axis was also activated in human HSOS- and AIH-related hepatomegaly.

Conclusions

NRF2, a transcription factor readily activated by xenobiotics, oxidative stress and autophagy disruptors, may be a common mediator of hepatomegaly; its effects on hepatic metabolism can be reversed by AKT/tyrosine kinase inhibitors.

Lay summary

Hepatomegaly can be triggered by numerous etiological factors, including infections, liver cancer, metabolic disturbances, toxicant exposure, as well as alcohol abuse or drug-induced hepatitis. This study identified the oxidative stress response transcription factor NRF2 as a common mediator of hepatomegaly. NRF2 activation results in elevated expression of several growth factors. These growth factors are made by hepatocytes and activate their receptors in an autocrine fashion to stimulate the accumulation of glycogen and lipids that lead to hepatocyte and liver enlargement. The protein kinase AKT plays a key role in this process and its inhibition leads to reversal of hepatomegaly.

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.

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