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Liver-specific inactivation of the Nrf1
gene in adult mouse leads to nonalcoholic
steatohepatitis and hepatic neoplasia
Zhenrong Xu, Linyun Chen, Laura Leung, T. S. Benedict Yen, Candy Lee, and
Jefferson Y. Chan. Proc Natl Acad Sci U S A. 2005 March 15; 102(11): 4120–4125.
Introduction
 The term oxidative stress encompasses a broad spectrum of
circumstances that cause a change in the cellular redox status such as
 an increased production of free radical species within the cell or by
 pro-oxidant xenobiotics that are thiol reactive and mimic an oxidative insult.
 The antioxidant response element (ARE) is a cis-acting enhancer
sequence that mediates transcriptional activation of genes in cells
exposed to oxidative stress
 In keeping with this, genes that are regulated by the ARE encode
proteins that help control the cellular redox status and defend the cell
against oxidative damage
 Proteins that are encoded by the ARE gene battery include enzymes
associated with glutathione biosynthesis, redox proteins with active
sulfhydryl moieties, and drug-metabolizing enzymes
ARE
Annual Review of Pharmacology and Toxicology. 43: 233-260
Sequences of ARE Found in Different
Genes
 The consensus sequence for ARE is 5’-TGACnnnGC-3’
Annual Review of Pharmacology and Toxicology. 43: 233-260
Regulation of ARE
 Regulation of ARE function is mediated by various basic leucine
zipper (bZIP) transcription factors including members of the ‘‘cap n
collar’’ (CNC)-bZIP, such as Nrf1 and Nrf2, and small-Maf family of
proteins.
51qe.cn/pic/30/ 11/18/07707.jpg
Nrf1 and Nrf2
 Nrf1 and Nrf2 are CNC-bZIP proteins, and they function as obligate
heterodimers by complexing with small-Maf and other bZIP proteins.
 An important role for Nrf2 in xenobiotic metabolism and oxidative
stress response has been identified through knockout studies in mice
indicating that Nrf2 is an important activator of AREs.
 Analysis of nrf1 and nrf1::nrf2 mutant cells suggests that Nrf1 is also
involved in the oxidative stress response.
 The function of Nrf1 is not fully understood. Mice deficient in Nrf1
function die during development.
Nrf1 and Liver
 Chimeric mice generated with Nrf1-deficient embryonic stem cells
showed widespread apoptosis in fetal livers at late gestation,
demonstrating a cell autonomous role of Nrf1 in the survival of
hepatocytes.
 This finding suggests that Nrf1 is required for normal function of
hepatocytes.
Liver-specific inactivation of the Nrf1
gene in adult mouse leads to nonalcoholic
steatohepatitis and hepatic neoplasia
Zhenrong Xu, Linyun Chen, Laura Leung, T. S. Benedict Yen, Candy Lee, and
Jefferson Y. Chan. Proc Natl Acad Sci U S A. 2005 March 15; 102(11): 4120–4125.
Hypothesis
Nrf1 is critical to the oxidative stress response in the
adult liver, and it plays an important role in
oxidative stress-induced liver disease.
Approach
To bypass embryonic lethality, a Cre-lox system was
used to investigate the function of Nrf1 in adult
liver
Targeting DNA with the Cre/lox System
http://www.bioteach.ubc.ca/MolecularBiology/TargetingYourDNAWithTheCreloxSystem/
Targeting DNA with the Cre/lox System
http://www.bioteach.ubc.ca/MolecularBiology/TargetingYourDNAWithTheCreloxSystem/
Tissue (Liver)-Specific Knockout
Transactivator
Floxed Responder
X
Conventional Transgenic
ES-derived mouse
12 kb Albumin
Promoter & enhancer
Cre
Floxed allele: “normal” expression
Cre expression in liver
Liver
Liver-specific gene deletion
Elsewhere
“Normal” expression
http://med.umich.edu/hg/EDUCATION/COURSES/HG541/Camper3.ppt#337,15,Tissue-Specific Knockou
Targeting of the nrf1 Locus Using the CreloxP Strategy
 (a Top) Map of the targeting construct
containing loxP elements flanking the
terminal exon of nrf1 and the positive
(PGKNEO) selection cassette, IRES-GFP
cassette and a negative (DT) selection cassette
 (Middle) The terminal portion of the wildtype nrf1 gene, and the 5′ external probe used
to detect targeted clones.
 (Bottom) The targeted allele. The predicted
sizes of NsiI fragments of wild-type (14-kb)
and targeted allele (11.5-kb) are shown.
 (b) Southern blot analysis of ES clones
digested with NsiI and probed with the 5′
external probe.
Targeting of the nrf1 locus, contd.
 (c Top) The nrf1-neo knockout allele.
 (Middle) The floxed nrf1 allele.
 (Bottom) The Cre-mediated recombined
allele.
 (Right) Southern blot analysis of liver DNA
obtained from control and Nrf1LKO mice.
Top band: the 4kb EcoRI fragment of both the
nrf1-neo and floxed nrf1 alleles detected by
the indicated probe.
Lower band: the 2.1kb EcoRI fragment of the
recombined allele.
 (d) Analysis of recombined and
nonrecombined floxed nrf1 allele in liver
DNA.
Top band: the 550-bp product amplified from
the recombined floxed nrf1 allele.
Bottom band: the 250-bp product amplified
from the nonrecombined floxed nrf1 allele.
Conclusion
 Nrf1LKO Mice were generated.
 No increase in mortality or morbidity was observed early on in Nrf1LKO
mice compared with littermate controls (nrf1flox/– and AlbCre;nrf1+/–).
Steatohepatitis and Liver Cancer in
Nrf1LKO Mice
 (a) Serum concentration of alanine
aminotransferase (ALT) in control and
Nrf1LKO mice at 4 weeks of age.
 (b) Liver triglyceride levels in control and
Nrf1LKO mice at 6–8 weeks of age.
Steatohepatitis and Liver Cancer in
Nrf1LKO Mice
 (c) A representative Nrf1LKO liver at 4–8 weeks of age stained with hematoxylin/eosin showing apoptosis,
necrosis, inflammatory infiltrate, and vacuolated cells.
 (d) Immunohistochemical staining for active caspase-3 in Nrf1LKO liver showing multiple apoptotic
hepatocytes.
 (e) Frozen section of Nrf1LKO liver stained with oil red O showing increased number of lipid droplets.
 (f) Cultured primary Nrf1LKO hepatocytes stained with oil red O showing increased number of lipid droplets.
Steatohepatitis and Liver Cancer in
Nrf1LKO Mice
 (g) Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) showing increased
number of actively dividing hepatocytes in Nrf1LKO liver.
 (h) Masson's trichrome staining showing fibrosis in a 6-month-old Nrf1LKO animal.
 (i) Gross appearance of a liver from a 4-month-old Nrf1LKO mouse showing multiple small nodules.
 (j) Representative liver from a control mouse at 12 months of age.
Steatohepatitis and Liver Cancer in
Nrf1LKO Mice
 (k) Liver from an Nrf1LKO mouse at 12 months of age showing multiple large, vascularized nodules.
 (l) Hematoxylin/eosin-stained sections showing hepatocellular adenomas with distinctive borders
between tumors and parenchyma.
 (m) Hematoxylin/eosin stained sections showing HCC with trabecullar features.
 (n) Section showing HCC-containing clear cells.
Conclusion
 Loss of Nrf1 results in steatohepatitis and spontaneous liver cancer.
Question
 Whether oxidative stress was present before development of tumors?
Oxidative stress in Nrf1LKO livers
 (a) Liver TBARS levels in control and
Nrf1LKO mice.
 (b) Immunohistochemical staining for 8oxoG showing no reactivity in normal
liver.
 (c) Immunohistochemical detection of 8oxoG-positive cells in Nrf1LKO liver.
 Flow cytometric determination of
dichlorofluorescein (DCF) fluorescence
reporter DCF dye in untreated
hepatocytes (d)
 and in hepatocytes treated with 100 μM
tert-butylhydroperoxide (tBHP) (e).
Conclusion
 Loss of Nrf1 results in oxidative stress in hepatocytes, and oxidative
injury is present in Nrf1LKO livers before tumor formation.
 Mutant hepatocytes also showed a severe impairment in handling
additional oxidant stress.
 Nrf1 has been shown to regulate expression of both the catalytic and
regulatory subunits of glutamyl-cysteine ligase (Gclc and Gclm).
Question
 Whether oxidative stress resulted from altered regulation of these
genes (Gclc and Gclm).
Expression of ARE-Regulated Genes in
Nrf1LKO Livers
 (a) Western blot analysis
of control and Nrf1LKO
livers.
 No difference in the expression of Gclc and Gclm was detected.
 No difference in the expression of HO-1 and MnSOD and CuZn-SOD.
 Thus, oxidative stress does not seem to correlate with decreased
expression of these genes.
Question
 Whether expression of other known ARE-dependent genes was altered
in Nrf1LKO livers?
PCR Primers
Expression of ARE-Regulated Genes in
Nrf1LKO Livers
 (b) RT-PCR analysis of
mRNA encoding various
ARE-dependent genes and
lipid metabolism. 18s
levels were used as
control.
 No significant change in GSTA3, GSTA4, GSTM1, GSTM2, GSTM4, and GSTT1.
 Transcripts for GSTM3, GSTM6, and GSTP2 were clearly reduced in Nrf1LKO samples
 GSTA1 and metallothionein-1 and -2 were elevated in Nrf1LKO livers, indicating that
other counteractive mechanisms against oxidative stress were induced.
 A subset of ARE-containing genes was affected in Nrf1LKO livers.
 Fatty acid metabolism by peroxisomal and microsomal pathways
generates a significant source of ROS.
 The fatty liver phenotype in Nrf1LKO mice.
Question
 whether these pathways (peroxisomal and microsomal pathways )
were induced in Nrf1LKO mice.
Microsome Proliferation and Induction of
ω-fatty Acid Oxidation in Nrf1LKO Livers
 (a) RT-PCR analysis of mRNA
encoding various enzymes associated
with β- and ω-fatty acid oxidation.
 PPAR-α, peroxisome proliferatoractivated receptor α; Acox1, acyl-CoA
oxidase; Ehhadh, enoyl-CoA
hydratase/3-hydroxyacyl CoA
dehydrogenase; Acaa1, acetyl-CoA
acyltransferase 1
 No increase in the PPAR-α expression in Nrf1LKO.
 PPAR-α did not seem to be transcriptionally activated because
expression of its target genes, including Acox1, Ehhadh, and Acaa was
not altered.
 A significant induction of cytochrome P450 4a10 and 4a14 (CYP4a10
and CYP4a14)
Microsome Proliferation and Induction of
ω-fatty Acid Oxidation in Nrf1LKO Livers
 Representative electron
micrograph of control (b)
and Nrf1LKO (c) livers at
2 months. Note lipid
droplets, smaller and
darker mitochondria, and
proliferation of smooth
ER in the Nrf1LKO liver.
 Nrf1LKO livers showed proliferation of smooth endoplasmic
reticulum.
Conclusion
 β-oxidation pathway does not seem to be induced in Nrf1LKO livers.
 Nrf1LKO livers showed proliferation of smooth endoplasmic
reticulum.
 ω-oxidation of fatty acids by means of microsomal CYP4A enzymes
participates in the generation of oxidative stress in Nrf1LKO livers.
Summary and Conclusions
 No increase in mortality or morbidity was observed early on in Nrf1LKO
mice compared with littermate controls (nrf1flox/– and AlbCre;nrf1+/–).
 Loss of Nrf1 results in steatohepatitis and spontaneous development
of liver cancer.
 loss of Nrf1 results in oxidative stress in hepatocytes, and oxidative
injury is present in Nrf1LKO livers before tumor formation.
 A subset of ARE-containing genes was affected in Nrf1LKO livers.
 β-oxidation pathway does not seem to be induced in Nrf1LKO livers.
Summary and Conclusions
 Nrf1LKO livers showed proliferation of smooth endoplasmic
reticulum.
 ω-oxidation of fatty acids by means of microsomal CYP4A enzymes
participates in the generation of oxidative stress in Nrf1LKO livers.
 In Nrf1LKO livers, reactive oxygen species generated from CYP4Amediated fatty acid oxidation work synergistically with diminished
expression of ARE-responsive genes to cause oxidative stress in
mutant hepatocytes.
 The changes observed in Nrf1LKO livers characterized by the initial
presence of cell death, proliferation, and inflammation, followed by
the appearance of dysplastic cells and ultimately cancer, closely
mimic the salient features of NASH.
 These Nrf1LKO animals may prove useful as a model to study the
mechanisms in the development and progression of NASH and liver
cancer.
Critique

The article is well written. Results are consistent with conclusions

First report that provides genetic evidence identifying a critical role
for Nrf1 attenuating oxidative stress and neoplastic growth in livers
of adult mice.

“We cannot rule out that Nrf1 may function as a tumor suppressor in
hepatocytes, and it is also possible that Nrf1 may accelerate disease
progression by regulating yet to be identified pathways.”

Other ARE-containing target genes that are involved in the
oxidative stress response (e.g. GSTA2 and NQO1) may be also
affected by loss of Nrf1 in the liver.
Critique

Some discrepancies in text and figures.


“Wild-type and targeted alleles yield a 14-kb and a 16-kb fragment,
respectively” (Fig. 1b).
Lack of controls for Fig. 2
Future Directions
 Identify other ARE-containing target genes that are involved in the
oxidative stress response. They may be also affected by loss of Nrf1 in
the liver.
 Determine whether the level of expression of Nrf2 changes in
Nrf1LKO livers.
 it is possible that Nrf2 may functionally compensate for the loss of Nrf1, because several
of the ARE-regulated genes examined are also known targets of Nrf2
 Examine the role of other bZIP proteins and small-Maf proteins which
form heterodimers with Nrf1 in the oxidative stress response.
 Extend this study to other tissues like kidney, lung, ..etc
Thank You
http://web.indstate.edu/thcme/mwking/glutathione.gif
http://www.med.unibs.it/~marchesi/glu
tathione.gif
http://zoology.muohio.edu/oris/ZOO462/notes/images/06c_46204.jpg
Omega Oxidation
 While the main route of fatty acid
metabolism is through beta-oxidation, some
minor metabolic pathways such as omega
oxidation also contribute to the metabolism
of fatty acids and other molecules. Omega
oxidation occurs in the endoplasmic
reticulum rather than the mitochondria, the
site of beta-oxidation. The omega carbon in
a fatty acid is the carbon furthest in the alkyl
chain from the carboxylic acid. In the omega
oxidation pathway, this carbon is
progressively oxidized first to an alcohol
and then to a carboxylic acid, creating a
molecule with a carboxylic acid on both
ends. The first step in the pathway is
catalyzed by a cytochrome P450 mixed
function oxidase and requires both oxygen
and NADPH. Oxidation of the alcohol is
catalyzed by an alcohol dehydrogenase and
aldehyde dehydrogenase catalyzes the
formation of the dicarboxylic acid.
•
•
REGULATORY MECHANISMS CONTROLLING GENE EXPRESSION
MEDIATED BY THE ANTIOXIDANT RESPONSE ELEMENT
Truyen Nguyen, Philip J. Sherratt, and Cecil B. Pickett Schering-Plough Research
Institute, Kenilworth, New Jersey 07033; email: [email protected]
[email protected] [email protected]
•
The expression of genes encoding antioxidative and Phase II detoxification enzymes is
induced in cells exposed to electrophilic compounds and phenolic antioxidants.
Induction of these enzymes is regulated at the transcriptional level and is mediated by a
specific enhancer, the antioxidant response element or ARE, found in the promoter of
the enzyme's gene. The transcription factor Nrf2 has been implicated as the central
protein that interacts with the ARE to activate gene transcription constitutively or in
response to an oxidative stress signal. This review focuses on the molecular mechanisms
whereby the trancriptional activation mediated by the interaction between the ARE and
NF-E2-related factor 2 (Nrf2) is regulated. Recent studies suggest that the sequence
context of the ARE, the nature of the chemical inducers, and the cell type are important
for determining the activity of the enhancer in a particular gene.
http://mol-devserver.tara.tsukuba.ac.jp/official/Project_Introduction/E_Nrf2_project.html
http://mol-devserver.tara.tsukuba.ac.jp/official/Project_Introduction/E_Nrf2_project.html
dir.niehs.nih.gov/ dirlrb/eg/proj-nrf2.htm
Three major signaling pathways have been
implicated in the regulation of the AREmediated transcriptional response to chemical
stress.
In vitro data suggest that direct
phosphorylation by PKC may promote Nrf2
nuclear translocation as a mechanism leading
to transcriptional activation of the ARE. Nrf2
has been proposed to be retained in the
cytoplasm through an interaction with Keap1
and it is possible that phosphorylation of Nrf2
may also cause the disruption of this
interaction. As a bZIP protein, Nrf2 binds to
the ARE as a dimer. Although small Maf
proteins have been proposed to represent the
dimerizing partners for Nrf2 in the activation
complex, this has not been conclusively
demonstrated.
The molecular mechanisms controlling the
ARE-mediated transcription by the MAP
kinase and PI3 kinase pathways remain to be
determined
Introduction
 The antioxidant response element (ARE) is a cis-acting enhancer
sequence that mediates transcriptional activation of genes in cells
exposed to oxidative stress
 The term oxidative stress encompasses a broad spectrum of
circumstances that cause a change in the cellular redox status such as
 an increased production of free radical species within the cell or by
 pro-oxidant xenobiotics that are thiol reactive and mimic an oxidative insult.
 In keeping with this, genes that are regulated by the ARE encode
proteins that help control the cellular redox status and defend the cell
against oxidative damage
 Proteins that are encoded by the ARE gene battery include enzymes
associated with glutathione biosynthesis, redox proteins with active
sulfhydryl moieties, and drug-metabolizing enzymes
Introduction
 The expression of genes encoding antioxidative and Phase II
detoxification enzymes is induced in cells exposed to electrophilic
compounds and phenolic antioxidants.
 Induction of these enzymes is regulated at the transcriptional level and
is mediated by a specific enhancer, the antioxidant response element
or ARE, found in the promoter of the enzyme's gene.
 The transcription factor Nrf2 (NF-E2-related factor 2) has been
implicated as the central protein that interacts with the ARE to activate
gene transcription constitutively or in response to an oxidative stress
signal.
Introduction
 NF-E2 is a dimeric protein originally identified as involved in the
regulation of globin gene expression in hematopoietic cells.
 The NF-E2 protein activates gene transcription following binding to
its consensus DNA binding motif 5'-TGCTGAGTCAC-3' as a
heterodimer consisting of a 45-Kda and an 18-Kda subunit.
 The p45 subunit is a bZIP protein consisting of a transactivation
domain within the N-terminal region and a basic DNA binding
region/leucine zipper structure in the C-terminal region of the protein.
 The p18 subunit was subsequently identified as a member of the small
Maf proteins containing the basic region/leucine zipper but lacking
any apparent transactivation domain
Introduction
 Whereas expression of the p45 NF-E2 subunit is restricted to
hematopoietic tissues, two other related bZIP family members, Nrf1
and Nrf2, are ubiquitously expressed in a wide range of tissues and
cell types.
 The tissue distribution profile of these proteins combined with their
DNA binding motif being similar in comparison to that of the ARE led
to the suggestion that ARE-responsive genes may be regulated by
Nrf1 and/or Nrf2.
 Nrf2 was subsequently demonstrated to be involved in the
transcriptional activation of other ARE-responsive genes including
those encoding the human γ-GCSh and γ-GCSl subunits, mouse HO1, and rat NQO1 and GSTA2 subunits.
Introduction
 Nrf2 is a critical protein in regulating the expression of the Gsta1 and
Nqo1 genes. These data suggest that Nrf2 mediates both the basal and
inducible activity of the ARE. The loss of Nrf2 resulted in a profound
reduction in the expression and enzyme activities of NQO1, certain
GST isoenzymes, and the γ-GCSh subunit.
 Current data from in vitro DNA binding assays and transfection
experiments point to Nrf2 as the most important protein involved in
stimulating ARE-driven transcription.
 The reduced expression of Phase II drug-metabolizing enzymes in
Nrf2 (−/−) mice confers a sensitive phenotype to the toxic effects of
carcinogens and inflammatory drugs compared to wild-type animals.
Introduction
 Ectopic expression of Nrf1 activates the human NQO1 ARE reporter
gene in HepG2 cells. Nrf1 also activates a reporter gene linked to the
promoter of the γ-GCSh subunit gene and increases the intracellular
level of GSH.
 Although the loss of Nrf1 is lethal in early embryonic development,
an analysis of mouse fibroblasts derived from Nrf1 (−/−) mouse
embryonic tissue showed reduced levels of GSH and of γ-GCSl
subunit expression compared to wild-type cells.
 Interestingly, induction of this enzyme was not affected by the loss of
Nrf1, as treatment of the Nrf1-deficient fibroblasts with paraquat
resulted in an increased level of its mRNA. These data and those
obtained from studies involving Nrf2 (−/−) mice suggest that the
expression of γ-GCS genes is mediated by both Nrf1 and Nrf2, with
Nrf1 being involved in basal expression and Nrf2 in the inducible
expression.
Introduction
 Current data from in vitro DNA binding assays and transfection
experiments point to Nrf2 as the most important protein involved in
stimulating ARE-driven transcription.
 The reduced expression of Phase II drug-metabolizing enzymes in
Nrf2 (−/−) mice confers a sensitive phenotype to the toxic effects of
carcinogens and inflammatory drugs compared to wild-type animals.
Introduction
 Ectopic expression of Nrf1 activates the human NQO1 ARE reporter
gene in HepG2 cells. Nrf1 also activates a reporter gene linked to the
promoter of the γ-GCSh subunit gene and increases the intracellular
level of GSH.
 Although the loss of Nrf1 is lethal in early embryonic development,
an analysis of mouse fibroblasts derived from Nrf1 (−/−) mouse
embryonic tissue showed reduced levels of GSH and of γ-GCSl
subunit expression compared to wild-type cells.
 Interestingly, induction of this enzyme was not affected by the loss of
Nrf1, as treatment of the Nrf1-deficient fibroblasts with paraquat
resulted in an increased level of its mRNA. These data and those
obtained from studies involving Nrf2 (−/−) mice suggest that the
expression of γ-GCS genes is mediated by both Nrf1 and Nrf2, with
Nrf1 being involved in basal expression and Nrf2 in the inducible
expression.
Overview
• Transcription of many cytoprotective genes and phase II xenobiotic
metabolizing genes is regulated through cis-active sequences known
as antioxidant response elements (ARE).
• Regulation of ARE function is mediated by various basic leucine
zipper (bZIP) transcription factors including members of the ‘‘cap n
collar’’ (CNC)-bZIP and small-Maf family of proteins.
• Nrf1 and Nrf2 are CNC-bZIP proteins, and they function as obligate
heterodimers by complexing with small-Maf and other bZIP proteins.
• An important role for Nrf2 in xenobiotic metabolism and oxidative
stress response had been identified through knockout studies in mice
indicating that Nrf2 is an important activator of AREs.
Targeting of the nrf1 Locus Using the CreloxP Strategy
Exon-4 of nrf1 was flanked with loxP sequences
Targeted clones were injected into blastocyst
Germ-line transmission was achieved
F1 animals were obtained