Hepatocyte-specific NRF2 activation controls fibrogenesis and carcinogenesis in steatohepatitis

doi:10.1016/j.jhep.2020.09.037

Journal of Hepatology

Volume 74, Issue 3, March 2021, Pages 638-648

https://doi.org/10.1016/j.jhep.2020.09.037 Get rights and content

Highlights

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Patients with NAFLD display increased oxidative stress and high NRF2 activity.
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NRF2 activation correlates with the grade of inflammation.
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In mice, Keap1 deletion induces NRF2 target genes.
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NRF2 activation downregulates genes involved in cell cycle regulation.
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Consequently, Keap1 deletion reduces fibrosis and tumor development in mice.

Background & Aims

In chronic liver diseases, inflammation induces oxidative stress and thus may contribute to the progression of liver injury, fibrosis, and carcinogenesis. The KEAP1/NRF2 axis is a major regulator of cellular redox balance. In the present study, we investigated whether the KEAP1/NRF2 system is involved in liver disease progression in humans and mice.

Methods

The clinical relevance of oxidative stress was investigated by liver RNA sequencing in a well-characterized cohort of patients with non-alcoholic fatty liver disease (n = 63) and correlated with histological and clinical parameters. For functional analysis, hepatocyte-specific Nemo knockout (NEMO^Δhepa) mice were crossed with hepatocyte-specific Keap1 knockout (KEAP1^Δhepa) mice.

Results

Immunohistochemical analysis of human liver sections showed increased oxidative stress and high NRF2 expression in patients with chronic liver disease. RNA sequencing of liver samples in a human pediatric NAFLD cohort revealed a significant increase of NRF2 activation correlating with the grade of inflammation, but not with the grade of steatosis, which could be confirmed in a second adult NASH cohort. In mice, microarray analysis revealed that Keap1 deletion induces NRF2 target genes involved in glutathione metabolism and xenobiotic stress (e.g., Nqo1). Furthermore, deficiency of one of the most important antioxidants, glutathione (GSH), in NEMO^Δhepa livers was rescued after deleting Keap1. As a consequence, NEMO^Δhepa/KEAP1^Δhepa livers showed reduced apoptosis compared to NEMO^Δhepa livers as well as a dramatic downregulation of genes involved in cell cycle regulation and DNA replication. Consequently, NEMO^Δhepa/KEAP1^Δhepa compared to NEMO^Δhepa livers displayed decreased fibrogenesis, lower tumor incidence, reduced tumor number, and decreased tumor size.

Conclusions

NRF2 activation in patients with non-alcoholic steatohepatitis correlates with the grade of inflammation, but not steatosis. Functional analysis in mice demonstrated that NRF2 activation in chronic liver disease is protective by ameliorating fibrogenesis, initiation and progression of hepatocellular carcinogenesis.

Lay summary

The KEAP1 (Kelch-like ECH-associated protein-1)/NRF2 (erythroid 2-related factor 2) axis has a major role in regulating cellular redox balance. Herein, we show that NRF2 activity correlates with the grade of inflammation in patients with non-alcoholic steatohepatitis. Functional studies in mice actually show that NRF2 activation, resulting from KEAP1 deletion, protects against fibrosis and cancer.

Graphical abstract

Introduction

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and one of the leading causes of cancer-related deaths worldwide.¹ HCC occurs as a consequence of the malignant transformation of hepatocytes, which is frequently triggered by chronic inflammation and subsequent liver fibrosis.² Oxidative stress is considered as an associated pathological mechanism that contributes to the initiation and progression of liver injury.³

Two types of radicals are continuously generated from endogenous metabolic processes and from exogenous sources: oxygen-derived radicals (reactive oxygen species [ROS]) and nitrogen-based radicals (reactive nitrogen species [RNS]). Under healthy conditions, both types of radicals are produced and detoxified simultaneously.⁴ In a variety of chronic diseases, such as cancer,⁵ cardiovascular diseases,⁶ and neurodegenerative diseases,⁷ an insufficient anti-oxidative defense capacity leads to progressive ROS accumulation. The consequences are lipid-, protein-, and DNA-oxidation, which can lead to dysregulation of cellular pathways including adhesion, metabolism, cell cycle, and cell death.⁸

The nuclear factor erythroid 2-related factor 2 (NRF2)/Kelch-like ECH-associated protein 1 (KEAP1) system is essential to protect against oxidative and electrophilic stress, and to maintain cellular redox homeostasis. Under homeostatic conditions, KEAP1 retains NRF2 in the cytoplasm, operating as a negative regulator. Upon exposure to ROS or electrophiles, cysteine residues in KEAP1 are modified, resulting in NRF2 release and translocation into the nucleus, leading to the induction of NRF2 target gene expression.⁹ NRF2 target genes are involved in numerous signaling pathways including xenobiotic-, electrophilic- and oxidative- induced stress responses.

Nrf2^-/- mice are more susceptible to acetaminophen (APAP)-induced liver failure than KEAP1 knockdown mice, which are resistant to APAP-induced liver injury.¹⁰^,¹¹ In an acute ethanol intoxication model, as well as in a chronic ethanol model, the activation via KEAP1 or the inactivation via Nrf2 deletion either rescued or aggravated ethanol-induced liver damage.¹²^,¹³ Comparable results were obtained in models of non-alcoholic fatty liver disease (NAFLD) induced by methionine-choline deficient diet or high-fat feeding. In contrast to KEAP1 knockdown mice, Nrf2^-/- mice exhibit more pronounced steatosis, mainly due to enhanced oxidative stress and lipid peroxidation.¹⁴^,¹⁵

During liver carcinogenesis, the NRF2/KEAP1 pathway has the role of a double-edged sword. It is undisputed that free radicals react with DNA and may induce mutations that alter the expression of pro-inflammatory proteins, oncogenes, and tumor suppressors.² Moreover, studies on NRF2 activators revealed a beneficial therapeutic approach in HCC management.¹⁶ Nrf2^-/- mice are more susceptible to 2-amino-3-methylimidazo[4,5-f]quinolone-induced carcinogenesis,¹⁷ but are resistant to diethylnitrosamine (DEN)-induced carcinogenesis.¹⁸ Consistent with these observations, constitutive NRF2 activation has been reported in different tumor types, wherein it contributes to cancer cell survival/proliferation and chemotherapy resistance.¹⁹

Taken together, based on previous results, the role of NRF2 during HCC development is controversial. Consequently, the aim of the present study was to investigate the role of the oxidative stress response including NRF2 activation in humans and to functionally assess the relevance of these findings in an inflammation-driven murine model of liver carcinogenesis.

Section snippets

Patient cohort

Patients were recruited from the pediatric obesity outpatient clinic and pediatric gastroenterology outpatient clinic of Charité Universitätsmedizin Berlin between June 2014 and March 2017. The study protocol conformed to the guidelines of the Declaration of Helsinki and was approved by the institutional review board (EA2/059/14). For all patients included in the study, informed consent was obtained from parents or legal guardians. Overweight and obese children and adolescents (defined as body

Increased oxidative stress response in humans with chronic liver diseases

We first investigated the relevance and distribution of the oxidative stress response in patients with chronic liver disease (CLD). Human liver samples with CLD and respective controls were stained for 4-hydroxynonenal (4-HNE), a well-known product of lipid peroxidation. In contrast to healthy controls, hepatocytes from patients with CLD were 4-HNE positive, whereas areas of collagen and immune cell accumulation remained unstained (Fig. 1A). Next, we performed pNRF2 immunohistochemical

Discussion

ROS play an essential role in triggering inflammation, fibrosis, and carcinogenesis in the liver and other organs. Consistent with earlier data, we found that hepatocytes from patients suffering from CLD are positive for 4-HNE, a product of lipid peroxidation. Remarkably, areas of immune cell infiltration and collagen deposition were negative, suggesting that lipid peroxidation occurs specifically in hepatocytes. This aligns with a previous study demonstrating that reactive aldehydes

Financial support

This work was supported by the Deutsche Forschungsgemeinschaft (SFB/TRR57, TR285/10-2) and the Faculty of Medicine at the RWTH Aachen University (START program #120/17, IZKF - Interdisciplinary Center for Clinical Research #O3-6). C.H.H. is supported by the German Federal Ministry of Education and Research (BMBF)-funded project Systems Medicine of the Liver (LiSyM, FKZ: 031 L0049).

Conflict of interest

The authors have declared that no conflict of interest exists.

Please refer to the accompanying ICMJE disclosure forms for further details.

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Research ArticleHepatocyte-specific NRF2 activation controls fibrogenesis and carcinogenesis in steatohepatitis

Highlights

Background & Aims

Methods

Results

Conclusions

Lay summary

Graphical abstract

Introduction

Section snippets

Patient cohort

Increased oxidative stress response in humans with chronic liver diseases

Discussion

Financial support

Conflict of interest

Gastroenterology

Free Radic Biol Med

Biochem Biophys Res Commun

Toxicol Appl Pharmacol

Food Chem Toxicol

Free Radic Biol Med

Biochem Biophys Res Commun

Mol Cell

Gastroenterology

Free Radic Biol Med

Free Radic Biol Med

Cancer Cell

Am J Pathol

Cell Signal

J Thorac Oncol

Mol Cell

Cell Mol Gastroenterol Hepatol

Cancer Cell

The burden of primary liver cancer and underlying etiologies from 1990 to 2015 at the global, regional, and national level: results from the Global Burden of Disease Study 2015

JAMA Oncol

Oxidative stress as a crucial factor in liver diseases

World J Gastroenterol

Role of ROS and RNS sources in physiological and pathological conditions

Oxid Med Cell Longev

New insights on oxidative stress in cancer

Curr Opin Drug Discov Devel

Oxidative stress in cardiovascular disease

Int J Mol Sci

Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options

Curr Neuropharmacol

An important function of Nrf2 in combating oxidative stress: detoxification of acetaminophen

Proc Natl Acad Sci U S A

Increased Nrf2 activation in livers from Keap1-knockdown mice increases expression of cytoprotective genes that detoxify electrophiles more than those that detoxify reactive oxygen species

Toxicol Sci

Research Article
Hepatocyte-specific NRF2 activation controls fibrogenesis and carcinogenesis in steatohepatitis