2. ABSTRACT___________________________________________
Nuclear Erythroid 2-related factor (Nrf2) is a master transcription factor, and thereby is a
major regulator of cytoprotective responses to oxidative and electrophilic stress. This is
accomplished by recognition and binding to antioxidant response elements (ARE) in the
promoter of target genes, which triggers activation of genes encoding proteins that range
from drug metabolizing enzymes II family to drug efflux pumps. Numerous studies have
shown direct and indirect interactions between Nrf2 and different signaling pathways
including components of the Pi3K/AKT/mTOR signaling pathway.
The potential for a role for Nrf2 in cancer metabolism directed our study towards its
impact on mTOR, the metabolic maestro of this pathway. We observed that modulation
of Nrf2 levels in lung cancer cell lines regulates mTOR protein levels. In order to verify
if this regulation is present at the transcriptional level, we performed both RT-qPCR
analysis and a luciferase assay to functionally analyze the promoter region of this gene
for the presence of functional ARE motifs. We found that transcription of the Mtor
protein was directly modulated by Nrf2 levels in the non small cell lung cancer cell line
A549, as well as in the non-transformed human cell line HEK293. Mutation of the ARE
sequence in the promoter of the mTOR gene, decreased the effect of Nrf2 on an ARE-
luciferase construct’s activity by more than 50%. The physical binding of Nrf2 with the
ARE sequence in mTOR promoter was further confirmed in vitro via DNA pull-down
and EMSA and in vivo via in a ChIP assay. Additional studies show intimate interactions
between other components of the PI3K pathway and Nrf2.
3. RÉSUMÉ_____________________________________________
Nuclear Erythroid 2-related factor (Nrf2) est un facteur de transcription qui joue un rôle
primordial dans la défense cellulaire contre les stress oxydatif et électrophile. Il régule la
transcription en se fixant sur les éléments de réponse antioxidative (ARE) impliqués dans
la résistance et le métabolisme des médicaments. En outre, plusieurs études montrent des
intercactions directes ou indirectes de Nrf2 avec la voie de signalisation
Pi3K/AKT/mTOR
En se basant sur le rôle de Nrf2 dans le métabolisme du cancer et son interaction avec la
voie de signalisation mTOR, nous avons formulé l'hypothèse selon laquelle Nrf2
régulerait les niveaux de mTOR. Tout D'abord, nous avons observé que la modulation
des niveaux de Nrf2 dans les cellules du cancer du poumon régule mTOR au niveau
protéique. Ensuite, l'utilisation de la PCR quantitative à temps réel et l'essai de
transactivation sur un vecteur rapporteur luciférase contenant le promoteur de mTOR
nous a permis de montrer que Nrf2 régule mTOR au niveau transcriptionnel dans les
cellules HEK293 et A549.
D'autre part, l'introduction des mutations au sein de la séquence de l'ARE du promoteur
de mTOR réduit l'activité luciférase par plus de 50%. Ceci confirme que malgré sa
séquence différente de la séquence consensus, cet ARE est requis pour la liaison et la
régulation de l'expression de mTOR.
l'interaction physique de Nrf2 avec l'ARE du promoteur de mTOR a été confirmé in vitro
par DNA pull down et par retard sur gel (EMSA) et in vivo par immunoprécipitation de la
chromatine. En conclusion, nos résultats suggèrent que le rôle de Nrf2 dans la sensibilité
aux traitements cytotoxiques pourrait découler de sa capacité à réguler l'expression de
mTOR.
4. TABLE OF CONTENTS_________________________________
Abstract........................................................................................................................... 2
Table of Contents......................................................................................................... 4
1. Introduction......................................................................................................... 7
1.1 Nrf2 and the cap ‘n’ collar (Cnc) family........................................................... 7
1.1.1 Discovering Nrf2………………………................................................... 7
1.1.2 Nrf2 molecular structure ……................................................................... 8
1.2.3 Nrf2 regulation………............................................................................... 9
1.2 Cytoprotective apparatus of cellular detoxification ...................................... 11
1.3 Antioxidant Response Element (ARE)………………………………………12
1.3.1 Discovering the ARE ………………………....…..………....................12
1.4 Nrf2 clinical relevance..………………………………………...................... 13
1.4.1 Nrf2 and carcinogenesis ………………................................................. 14
1.5 Nrf2 cross talk with various pathways involved in cancer………...……….. 15
1.6 The PI3K/Akt/mTOR pathway……………………………………………... 16
1.6.1 Nrf2 interactions with the PI3K pathway….…….……...……..…..….. 18
1.6.2 Clinical relevance of the interaction between Nrf2 and the Pi3K/AKT
pathway…………………………………………………………………………. 18
1.7 Nrf2 enhance the PI3K pathway in systems with high metabolic state.……. 19
1.8 mTOR………………………………………………….………………….... 20
1.8.1 mTORC1………………………………………...………..…………... 20
1.8.2 mTORC2……………………………………………..……………….. 21
1.9 Role of Nrf2 on mTOR expression ……..………...…...…………………... 22
5. 2. Hypothesis.......................................................................................................... 22
3. Materials and Methods.................................................................................. 23
3.1 Cell Lines and Tissue Culture/ Transient Transfection…….......................... 23
3.2 Western blot …………………………………………..…..………..………. 24
3.3 Quantitative RT-PCR…………………………………………………………........ 25
3.4 Bioinformatic Analysis………………….…………………………………. 25
3.5 Molecular Cloning and Vector Construction………….……………………. 25
3.6 Nrf2 modulation …………………………………………………..…........... 26
3.7 Luciferase assay constructs……………………………………………...….. 26
3.8 Luciferase Assay…………………………………………….……………… 27
3.9 Electrophoretic Mobility Shift Assay (EMSA)……………………............... 28
3.10 DNA Pull-Down Assay ………….………………………………………... 28
3.11 Chromatin immunoprecipitation ………………………………………..… 29
4. Results................................................................................................................. 30
4.1 Nrf2 modulates mtor expression in A549 cells……..………….….………... 31
4.1.1 mTOR expression when Nrf2 is up-regulated………..……………… 31
4.1.2 mTOR expression when Nrf2 is down-regulated…………………….. 33
4.2 Functional ARE present on mTOR promoter activates its transcription in Nrf2
inducible condition……………………………………………………………… 34
4.3 Nrf2 binds to mTOR promoter region at basal conditions in vitro…………. 36
4.3.1 Nrf2 binding to mTOR promoter region decreases in Nrf2 silencing
conditions……………………………………………………………………….. 39
4.3.2 Nrf2 binds to mTOR promoter in vivo at inducible conditions…........ 40
4.4 Expression analyses of the other elements of PI3K pathway due to Nrf2
modulation ……………………………………………………………………... 41
6. 4.4.1 TSC2, S6K and AKT expression when Nrf2 is up-regulated….…… 42
4.4.1.1 TSC2 is a potential indirect Nrf2 transcriptional target at inducible
conditions on H460 cells ……………………………………………….. 42
4.4.1.2 At Nrf2 inducible conditions AKT is a possible indirect Nrf2
transcriptional target on H460 cells and posttranslational target on A549
cells……………………………………………………………………... 43
4.4.2 TSC2, S6K and AKT expression when silencing Nrf2…...……..…… 49
4.4.2.1 TSC2, S6K and AKT may be affected post translationally, when
Nrf2 is silenced ………………………………………………………… 49
5. Discussion & Conclusions................................................................................. 53
6. Future Directions................................................................................................. 63
7. Acknowledges........................................................................................................ 66
8. References.............................................................................................................. 66
9. Appendix................................................................................................................ 77
7. 1. INTRODUCTION_____________________________________________________
1.1 Nrf2 and the cap ‘n’ collar (Cnc) family
Nrf2 is a basic leucine zipper (bZIP) transcription factor from the cap ‘n’ collar (Cnc)
family. The Cnc domain has 43 conserved amino acids located N-terminal to the DNA
binding domain. Prior to interaction with their target genes, the Cnc family of
transcription factors binds to Maf-recognition elements (MAREs), also known as the
erythroid transcription factor NF-E2 binding sequence(1)
. Maf (musculo-aponeurotic
fibrosarcoma oncogene) are a family of proteins that lack transcriptional activation
domains. In the nucleus CNC factors function via heterodimerizing with small Maf
proteins, which provide high affinity, sequence-specific DNA-binding activity of the
CNC factors to the MARE element(2)
.
The Cnc protein family is composed of SKN-1 (Skinhead family member 1) in
Caenorhabditis elegans and Cnc in Drosophila. In vertebrates this family is represented
by, p45 NFE2 subunit(3)
and the NFE2-related factors, known as “Nrf” proteins,
Nrf1(NFE2L1/LCRF1/TCF11)(4)
, Nrf2(NFE2L2) (Itoh et al., 1995)(5)
, and Nrf3
(NFE2L3)(6)
. Bach1 and Bach2 (7)
are other members of this family, witch however have
no transactivation capacity and instead function as transcriptional repressors. Bach1 is a
truncated isoform of Nrf1, while Bach2 is a caspase-cleaved form of Nrf2. The p45 NFE2
acts during development and is present only in hematopoietic progenitor cells. Besides
their role in early development, the Nrf proteins have a broad and sometimes overlapping
function as stress-activated transcription factors.
1.1.1 Discovering Nrf2
Nrf2 was first isolated and characterized in 1994 by Moi et al,(8)
who identified closely
regulated proteins of erythroid-derived 2 (NF-E2). NF-E2, a member of the family of
bZIP transcription factors is a dimeric protein involved in the regulation of the β- globin
gene expression in hematopoietic cells. Nrf2 was named for its ability to bind to the
nuclear factor, NF-E2/ activating protein 1 (AP-1) repeat in the promoter of the β -globin
8. gene. Tandem Binding of Nrf2 to NF-E2/ AP1 was achieved via expression cloning of
the consensus sequence (5'GCACAGCAATGCTGAGTCATGATGAGTCATGCTG-3')
in K562 erythroid cell line. This repeat sequence is a oligonucleotide containing double-
strand concatemers of the tandem NF-E2/ AP1 repeat of the β- globin locus control
region, DNase I-hypersensitive site 2 (HS2).
1.2.2 Nrf2 molecular structure
The Nrf2 protein, has a molecular weight ranging from 95 to 110 kDa(9)
, and is
composed of 605 amino acids with 6 functional domains called Neh1-6 (Nrf2-ECH
<chicken Nrf2> homologous domain). The Neh1 holds the CNC homology region and a
basic-leucine zipper domain. It is responsible for heterodimerisation between Nrf2 and
small Maf proteins .The C terminal Neh3 motif is also responsible for Nrf2
transactivation activity (10)
The Neh4 and Neh5 are conserved acidic domains that interact
with CBP [CREB cyclic AMP- response element binding protein (CREB) binding
protein], and are responsible for Nrf2 transcription activation strengths(11)
. Neh6 is a
serine-rich conserved region and serves as a target for a GSK 3 mediated phosphorylation
and consequently proteasomal degradation via ubiquitination(12)
.
Neh2 is a composite domain that is structurally divisible into two subregions. The
carboxy-terminal of Neh2 (amino acid residues 33–73) is hydrophilic and with no present
functional importance, while the amino-terminal region of Neh2 has 32 amino acids,
which are rich in hydrophobic residues, and shows conservation with Nrf1 and the C.
elegans Skn-1. It is an important functional domain, working as a negative regulator of
Nrf2, proved via domain deletion by Itoh et al(13)
. They also identified Kelch-like ECH-
associated protein1 (Keap1) responsible for post translational control of Nrf2.
Keap1 is an actin-binding cytoplasmic protein with four main domains, a intervening
region (IVR), double glycine repeat (DGR), C-terminal region (CTR) and broad
complex–tramtrack–bric-a-brac (BTB) domain. The DGR domain, also called Kelch
domain owing to its homology with Drosophila Kelch protein, is important for the
interaction with Nrf2 and for binding to actin. The BTB domain, present in Keap1 C-
terminus, is required for Nrf2 cytoplasmic sequestration and is involved in dimer
9. formation(14)
. The IVR domain, which is cysteine-rich protein with 27 cysteine residues,
is important for its reactivity to electrophilic and oxidative stimuli. In the presence of
oxidative stress 10 of these cysteines are activated by positively charged amino acids(15)
,
which leads to conformational changes in Keap1.
1.2.3 Nrf2 regulation
Keap1 is an important interacting protein of Nrf2 and they form a “hinge and latch”
structure with one another as shown by X-ray crystallography(16)
. The “hinge” structure is
formed due to a high-affinity interaction of ETGE motif, a stretch of four amino acids
present in the Neh2 domain of Nrf2, with keap1 kelch domain. While the “latch”
structure is generated via low-affinity interaction of DLG motif of nrf2-neh2 domain with
other keap1 monomers(17)
.
Under basal conditions, the redox–sensitive protein, Keap1 binds Nrf2 to form a
Keap1/Nrf2 complex, and anchors it in the cytoplasm. This cytoplasmic localization was
proved by confocal laser microscopic immunohistochemical analysis, where Keap1 was
shown to be tethered to the actin cytoskeleton(18)
. As others broad complex–tramtrack–
bric-a-brac (BTB)-containing proteins, Keap1 is an adaptor protein for the Cullin 3
ubiquitin E3 ligase (Cul3) which is a scaffold protein in the E3 ligase complex and forms
a catalytic core complex together with roc1/rbx1/Hrt1.The cognate E2 enzyme is then
recruited by Roc 1. This way, Nrf2 is specifically targeted (Lawah Zellers) for
degradation by the ubiquitin-proteasome pathway by 26 S proteasome(14).
In situations of oxidative stress, Keap1 undergoes conformational changes, which result
in the breakdown of the Nrf2-Keap1 complex. This occurs due to the difference in
affinity of “hinge” and “latch,” interactions, which have a difference of 2 orders of
magnitude, caused by the variance in the number of electrostatic interactions between
each domain and Keap1. This difference in affinity, weakens the interaction of the DLG
motif leading to the Nrf2-Keap1 complex disruption(17,19)
. This culminates in the release
of Nrf2 and its translocation to the nucleus, where it accumulates and activates the
cytoprotective program. Prior binding to its target genes, Nrf2 forms a heterodimer with
members of the small Maf family. This hetero-dimerization happen in the Nrf2 Neh1
10. domain. The complex Nrf2/small Maf then binds antioxidant response elements (AREs)
localized in the promoter region of its target genes(20)
.
Apart from the Keap1 mechanism of post-translational regulation of Nrf2, it is known
that some kinases, such as p38 kinase (21)
and PTEN, can inhibit Nrf2. Kensuke Sakamoto
et al(22)
showed via chromatin immunoprecipitation of Jurkat human leukemia, baring a
PTEN mutation, that the PI3K inhibitor LY294002 blocks CBP and Nrf2 recruitment to
ARE while it releases Bach1 to ARE. Glycogen synthetase kinase 3 (GSK-3ß) is also a
Kinase that can inhibit Nrf2.(12,23,24)
The serine/threonine GSK-3ß protein regulates glycolytic metabolism and directs the
ubiquitination and proteasomal of a variety of transcription factors(24)
. GSK-3ß is
involved in metabolic processes such as glycogen metabolism, Wnt signaling and
sensitization to oxidative-stress-mediated apoptosis. GSK-3ß is negatively regulated by
the Ser/Thr kinase Akt(25)
. AKT phosphorylates GSK-3ß’s Ser-9 in its pseudosubstrate
domain which inactivates GSK-3ß and consequently inhibits apoptosis. In order to
understand the mechanistic connection between the phase II genes’ cyto protection
against oxidative stress and the PI3K survival pathway, Salazar et al (23)
focused on
control of nuclear Nrf2 accumulation. They suggested that Nrf2 was negatively regulated
via GSK-3ß phosphorylation in the nucleus post-translation. This study found that Nrf2
contains a consensus sequence for GSK-3ß phosphorylation (S/T)XXX(S/T) which was
confirmed by both immunocytochemistry and subcellular fractionation analyses. In a
following study by Rada P et al(24)
, it was demonstrated in mouse, that GSK-3ß acts as an
adapter protein for Nrf2 by phosphorylating a group of Ser residues in its Neh6 domain
and consequently targeting it to the SCF/ ß -TrCP SCF protein.
There is thus evidence for interaction between elements of the PI3Kinase pathway and
Nrf2 transcription factor. To date that data demonstrates regulation of Nrf2 by proteins
such as p38 Kinase, PTEN and GSK-3ß
11. 1.2 Cytoprotective apparatus of cellular detoxification
In normal physiological conditions, nuclear factor NRF2 is essential for cell homeostsis
against endogenous and exogenous redox stress. This master cytoprotective transcription
factor is responsible for the activation of phase II detoxifying enzymes, antioxidants,
phase III drug efflux pumps and transporters(26)
.
The cytoprotective apparatus of cellular detoxification has been stratified into 3
categories phase I, II and III drug metaboling enzymes (DMEs). The phase I and II
enzyme systems are localized in the endoplasmic reticulum (ER) while the phaseIII is
present in the cytoplasmic membrane.(27)
Phase I is composed of cytochrome
P450s(CYPs) gene superfamily. These large hydrophobic organic molecules are
responsible for oxidation and reduction by introducing polar functional groups into
nonpolar molecules. This group of enzymes are regulated by, ligand activated, Aryl
hydrocarbon receptor (AHR) transcription factor. DNA sequences called xenobiotic
response elements (XREs) are present in the promoter region of Phase I DMEs and are
essential for the regulation of these classes of enzymes. XREs are the target regions for
AHR binding, which activate transcription, after chaperoning with a nuclear transporter
called ARNT. There are growing evidences that Nrf2 regulates AHR, thus also phase I
DMEs.(27,28)
The phase II DMEs are Nrf2-dependent gene battery that includes enzymes acting on
cellular redox status and cell protection against oxidative damage, cytotoxicity,
mutagenicity and carcinogenicity. Phase II DMEs works synergistically with phase III
DMEs transporters in various metabolic reactions. Together, their functions involves
disposition of xenobiotics, and endogenous substances (26)
. Some of the phase II DMEs
are glutathione S-transferases (GSTs), sulfotransferases (SULTs) UDP-glucuronosyl
transferases (UGTs) FAD containing flavoprotein NAD(P)H:Quinone
Oxidoreductase(NQO1), Heme oxygenase (HO-1). These are involved in catalyzing
conjugation reactions through covalent linkage of xenobiotics or phase reaction products,
to groups that are more functionally polar (glucuronate, sulfate, amino acids and
glutathione) which occurs via nucleophilic trapping. In this context, GSTs assign
12. glutathione, a cellular nucleophile, to electrophilic xenobiotics (9)
. Similar mechanism is
also seen in SULTs and methyltransferases(29,30)
. The other category of enzymes present
in the DME phase II is represented by UGTs. These conjugate adenosine-containing
cofactors with nucleophilic xenobiotics. Superoxide dismutases, glutathione peroxidase,
and catalase such as the NQO1 function in a similar manner. The detoxification
mechanism of NQO1 involves catalyzing quinone to hydroquinones via two electron
reduction, bypassing the formation of highly reactive semiquinone(30)
. Phase II DMEs are
also represented by thiol-containing molecules, such as, glutathione and thioredoxin and
HO-1. HO-1 is an essential enzyme in heme catabolism and is responsible for cleaving
heme to form biliverdin, which is ultimately converted to bilirubin. (27)
The third category is composed of membrane efflux transporters such as the multidrug
resistance associated proteins (MRPs 1,2,3 and 4).). The MRPs are adenosine
triphosphate-dependent drug transporters. They are responsible for the excretion of
endogenous substances, such as bilirubin and xenobiotis, together their conjugated
metabolites products from the DME phase II enzymes.
1.3 Antioxidant Response Element (ARE).
The phase II and III DMEs reach their highest level of expression primarily through
activation of a specific enhancer in their respective promoter region. These enhancers are
cis-acting regulatory elements, called antioxidant response element (ARE). Present in
phase II and III enzymes, ARE regulate the expression of genes involved in the cellular
redox status and are present as a single or multiple copies(27)
.
1.3.1 Discovering the AREs
The ARE pathway was originally observed by Talalay et al(31)
, when analyzing the
different ways by which some xenobiotics regulate Phase I and Phase II drug-
metabolizing enzymes. This was the first evidence of a Phase II enzyme induction.
Further studies were done(32)
in order to identify trans-acting proteins that interact with
these cis-acting regulatory elements. These were classified and characterized by
Rushmore and Pickett(33)
after identification of oxidative responsive elements and basal
promoter elements in the rat GST Ya subunit (Gsta2) gene. This novel Cis-acting element
13. in the 5'-flanking region element, when used in a reporter construct was shown to induce
the activity of the phenolic antioxidant tert-Butylhydroquinone(tBHQ), hence the name
antioxidant response element. The ARE core sequence (cARE), 5′-TGACnnnGC-3′ was
determined via deletion and mutational analysis. Jaiswal el al (34)
established the role of
Nrf2 as a transcription factor for genes containing ARE in their promoter region, hence
regulating expression of genes affecting xenobiotic metabolism. The NQO1 induction by
Nrf2 and Nrf1 was shown via supershift assay after transient transfection of these
transcription factors into human hepatoblastoma HepG2 cells. More experiments(35)
involving a broad spectrum of Nrf2 inducers demonstrated the activation of various phase
II DMEs by Nrf2. Sternberg et al 2006 (36)
used high-performance liquid chromatography
(HPLC) to show that retinal pigment epithelium (RPE) cells, when treated with zinc,
increased the levels of glutathione synthesis through Nrf2. The cARE motif was further
confirmed as a binding site for Nrf2 via numerous ChIP-seq methodologies followed by
global transcriptional profiling, which demonstrated the variety of Nrf2 proteins
interactions. In recent literature, Biswal et al 2010(37)
performed a global Nrf2 ChIP-seq
analysis of mouse embryonic fibroblasts (MEF) with either constitutive nuclear
accumulation (Keap1-/-) or depletion (Nrf2-/-) of Nrf2. Integrating ChIP-Seq and
microarray analyses, they identified 645 basal and 654 inducible direct targets of Nrf2,
with 244 genes overlapping a microarray datasets used to identify Nrf2 direct
transcriptional targets. Also, Chorley et al 2012(38)
performed another ChIP-seq analysis
of NRF2-regulated genes utilizing the same cARE motif. Utilizing lymphoid cells with
Nrf2 induced by isothiocyanate, sulforaphane (SFN) they were able to identify 242 high
confidence genomic regions to which Nrf2 binds.
1.4 Nrf2 clinical relevance
There is abundant evidence of Nrf2 involvement in direct protein interactions and
pathway cross talk. This complex regulatory system generated by Nrf2 interactions is
reflected in the clinic by the vast variety of pathologies in which it is involved. In mice
Nrf2 was shown to play a role in carcinogenesis, chronic obstructive pulmonary disease,
obesogenesis, and neurodegeneration(39)
. Although, Nrf2 knock out was shown to be
nonessential for the normal development in mice(40)
, the Nrf2/ARE interaction is vital in
14. humans for normal cell homeostasis promoting cellular antioxidant defenses and
increased capacity to detoxify drugs. Previous studies with Nrf2
_ / _
mouse models(41)
have shown a high sensitivity of mice to chemical and physical insults. As previously
mentioned, these insults have a strong correlation with the incidence of cancer via
oxidative and electrophilic stressors, or drugs that induce the production of free radicals.
It was also shown that Nrf2-deficient mice seemed to be more sensitive to
carcinogenesis,(42,43)
and are at an enhanced risk of metastasis(44),(45)
.Consequently, Nrf2
was considered to work only as tumor suppressor and so the benefits of Nrf2 signaling in
cancer chemoprevention were largely explored (46)
.
However, this increase in cellular protection, via high Nrf2 levels, leads to unwanted side
effects in some cancer types(47)
, as constitutive activation or augmented signaling of the
Nrf2 pathway may promote tumorigenesis and be involved in resistance to chemo- and
radiotherapeutic treatments, showing that the transcription factor could have a proto-
oncogenic role(48)
.
1.4.1 Nrf2 and carcinogenesis
The role of Nrf2 in cancer promotion was first found in an hepatocellular carcinoma
model by Ikeda et al in 2004(49)
. In this study both levels of Nrf2 and GSTP1, a neoplastic
marker, were elevated. It was also found that Nrf2 was regulating GSTP1 through an
ARE, present in the promoter region of the gene. Additional studies have proven Nrf2
relation to tumorigenesis, chemoresistance, increased cell survival, metastasis, and cell
growth (47)-(49,50)-(51)
.
While much focus remains on enhancing Nrf2 as a cancer chemoprevention strategy
against genotoxic agents(52), (53)
or inflammation(54)
, participation of Nrf2 in the process of
carcinogenesis is also strongly demonstrated in many papers in the literature. Nrf2
together with its downstream genes, is elevated in many cancers cell lines and human
cancer tissues, resulting in chemoresistance(50)
and a poor prognosis in patients (55,56, 59,60)
thus providing the cancer cells an advantage for survival and growth.
One of the principal reasons for the constitutively high levels of active Nrf2 in cancer is
15. due to loss-of-function mutations in Keap1.(16,55)
which causes its inactivation or reduced
expression. This results in increased Nrf2 stability and its translocation to the nucleus and
consequently transcriptional activation of its target genes. Constitutive stabilization of
NRF2, due to Keap1 mutations, was found in various human cancers, with increased Nrf2
activity in lung (~40%), head and neck (~20%), gallbladder (~30%), liver, and breast
cancers(56)
. There are also some cell lines in which gain-of-function mutations in the Nrf2
gene is observed, (56-58)
like in advance Esophageal squamous cancer (ESC) with
occurrence of (18/82, 22%)(50)
In both, in-vivo and in clinical specimens of non-small cell lung cancer (NSCLC)(55)
,
loss-of-function Keap1 mutations resulted in constitutively high levels of active Nrf2 and
subsequent resistance to chemotherapeutic drugs (taxanes, platinums) and radiotherapy.
Keap1 mutations are reported in up to 60% of papillary lung adenocarcinoma, as well as
in other cancers including ovarian, gall bladder and others(59)
.
The inverse of the abovementioned is also the case. A low level of Nrf2 within the cancer
cells is responsible for chemo sensitisation. Batist et al 2009(51)
found very low Nrf2
levels in breast cancer cell lines and in the majority of a 200-sample tissue microarray,
which is consistent with the high response rates of breast cancer to many cytotoxic
therapies.
1.5. Nrf2 cross talk with various pathways involved in cancer
As mentioned before Nrf2 can block cell damage induced by oxidative and electrophilic
drugs and also reduce their accumulation in the cell via MDR protein. However, Nrf2
chemoresistance can also occur, due to its interaction with other pathways present in
cancer, which are related to metastasis, increase in cell survival and cell growth.
Some Nrf2 target genes, such as HO-1, were shown to be related to cellular metastatic
potential. HO-1 is overexpressed in various solid tumors(60)
and is related with
angiogenesis and acceleration of prostate cancer progression(54)
. The HO-1 protein is also
related with increased cell survival via apoptosis inhibition in chronic myelogenous
leukemia (CML). Nrf2, also, regulates proteins from the Bcl-2 family through
16. transcriptional control of the antiapoptotic proteins Bcl-2 and Bcl-XL. Additionally, Nrf2
was shown to increase cell survival via inhibition of p53-dependent apoptosis(61).
In
response to stress stimuli, the tumor suppressor p53, control the expression of the
cycling-dependent kinase inhibitor p21 via cell cycle G1 arrest(62)
. Nrf2 is stabilized by
p21 via direct interaction of the DLG and the ETGE Nrf2’s motifs with the KRR motif in
p21, which displaces the Nrf2-Keap1 interaction(63)
. In a ROS-dependent mechanism,
p53 induces apoptosis via a two-phase Nrf2 response. Under conditions where ROS
levels are low, in a phase called induction, p53 is also low and it enhances the protein
level of Nrf2 transcriptionally via the target gene p21. The other side of this biphasic
regulation is called the repression phase, and it is present when ROS, and consequently
p53 levels, are high. In this phase p53 binds to a sequence near the ARE which repress
Nrf2 transcription by displacing it from the ARE(61,64).
P53 was also showed to negatively
regulate TSC2, PTEN, consequently inhibiting the IGF-1-AKT-mTOR axis. (65)
This
suggest at least an indirect relationship between Nrf2 biding to its cognate sequence
(ARE) and elements of the PI3K pathway, including mTOR.
1.6 The PI3K/Akt/mTOR pathway
The phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway is important for cell
survival and is involved in metabolism, apoptosis, cell growth, differentiation, calcium
signaling, and insulin signaling(66)
. In addition to those cited above, a variety of recent
studies suggest that this pathway interacts with Nrf2(67,68)
. PI3K/AKT pathway has a role
in tumor development and has shown potential in tumor treatment, through the PI3K
pathway inhibitor Wortmannin(66) (69)
. Multiple molecules that target this pathway are
currently in clinical development.
PI3Ks are part of a lipid Kinase family with main distinctive feature is its capability to
phosphorylate inositol ring 3’-OH group in inositol phospholipids. The mechanism of
action of this signaling pathway starts with PI3K activation. One mode of activation is
through binding of an extracellular growth factor to the RPTK (Receptor Protein
Tyrosine Kinase). Binding of this receptor by growth factors lead to dimerization of
RPTK monomers along with heterologous auto phosphorylation of this receptor
17. monomers, the IRS-1 (insulin receptor substrate I) then binds to a phosphorylated IGF
receptor. This complex function as binding and activation site for PI3K. Another mode of
activation is via direct binding to a phosphorylated receptor Tyrosine Kinase. This
pathway can also be activated by binding of PI3K to a small membrane bound, active
GTP-Ras(66)
.
The next step of this pathway involves activation of the second messenger
phosphatidylinositol-3,4,5-trisphosphate (PIP3) and AKT (a serine/treonine kinase
protein also known as protein kinase B). Migration of PI3K to the inner membrane and
binding to PIP2 (Phosphatidylinositol 4,5-bisphosphate) leads to phosphorylation of PIP2
to PIP3 which then activates AKT. The Pi3K pathway is negatively regulated by the
presence of phosphatases capable of dephosphorylating PIP3 back to PIP2. Inhibition of
this pathway can be achieved via chromosome 10 (PTEN) barring a homologue deletion
of phosphatase and tensin. Decrease in PTEN expression indirectly stimulates PI3K
activity and is largely seen in cancer(66)
.
There are at least four main downstream effects of AKT activation. The first one is the
inhibition of apoptosis via binding with BAX (BCL2-associated X protein) which in-turn
stops BAX from creating holes in the mitochondria inner membrane, responsible for
generating apoptosis by the Caspase cascade. The second effect is the phosphorylation of
Forkhead box O (FoxO) which serves as a substrate for the enzyme ubiquitin ligase,
resulting in its degradation in the proteasome. In the absence of this process FoxO
inhibits cell proliferation. The third effect is the inhibition of Glycogen synthase kinase-
3ß (GSK-3ß). The fourth effect is its role in translation by a multi step protein cascade.
This cascade begins with the activation of Rheb by AKT, which activates the protein
kinase mechanistic target of rapamycin (mTOR; formerly known as mammalian TOR)(70)
.
Another mechanism of mTOR activation via AKT is by phosphorylation o the mTOR
inhibitor PRAS40 (proline-rich Akt/PKB substrate 40 kDa)(71)
.
18. 1.6.1 NRF2 interactions with the PI3K pathway
As noted, several studies showed evidence for interactions between the PI3K pathway
and NRF2 using different techniques and models. In the previously mentioned global
mapping of Nrf2 biding sites(37)
, TSC2 was shown to be a basal target for Nrf2; since the
cells were not “stimulated” in any way with respect to Nrf2 function or nuclear
accumulation, this type of study is mute on Nrf2’s potential role in the transcription of
these proteins in conditions of redox stress.
In an in silico analyses of Nrf2 interactome and regulome, that includes 289 protein–
protein, 7469 TF–DNA and 85 miRNA interactions, shown in a manually curated
network of Nrf2, it was observed that AKT functions as an indirect activator of Nrf2 (67)
.
Biological evidence of this interaction was also observed in previous studies where
human dopaminergic neuroblastoma SH-SY5Y cells(72)
showed PI3K involvement in the
Nrf2 regulation of antioxidative proteins HO-1, Trx, and PrxI, According to the paper,
after treating the cells with hemin, a dose dependent nuclear translocation of Nrf2 was
observed together with PI3K phosphorylation. Also, PI3K inhibitors, wortmannin and
LY294002, lead to inhibition of Nrf2 nuclear translocation. In another study(68)
, Nrf2 up
regulation via the PI3K and the Extracellular Regulated Kinase (Erk) pathways was
observed after cell treatment with eckol, which is a phlorotannin component of brown
algae such as Ecklonia cava (Laminariaceae), and is known to upregulate ERK and AKT
individually. In this paper it was also shown that treatments with any of the drugs (
U0126, an Erk kinase inhibitor, or LY294002) or short interfering RNAs (Erk1 siRNA,
and Akt siRNA) suppressed Nrf2 activity, which was observed by decrease of HO-1
levels.
1.6.2 Clinical relevance of the interactions between Nrf2 and the Pi3K/AKT
pathway
Interaction between the PI3K/AKT pathway and Nrf2 might well be clinically relevant,
as the pharmacological inhibition of this pathway suppresses the nuclear translocation of
Nrf2 in cancer cells (73,74)
. This was also shown by Ling Wang et al,(75)
who working on
age-related macular degeneration (AMD) caused by accumulated oxidative injury, found
19. that cultured human retinal pigment epithelium (RPE) cells treated with PI3K inhibitors
were able to decrease Nrf2 levels. Additionally, a study by Papaiahgari et al 2006(76)
showed that PI3K/Akt signaling regulates Nrf2 activation by hyperoxia. Lung injury due
oxygen supplementation (hyperoxia) is currently used in the treatment of pulmonary
diseases such as respiratory distress syndrome (ARDS) and emphysema. PI3K inhibition
blocked hyperoxia-stimulated Akt and ERK1/2 kinase activation, which activate Nrf2
transcriptional activity. Nrf2 regulation by AKT was later shown to occur via inactivation
of GSK-3b(12)
.
1.7 Nrf2 enhances the PI3K pathway in systems with high metabolic state
There is growing evidence that Nrf2 also enhance the PI3K pathway in systems with a
high metabolic state (74-77)
. A hyperproliferative phenotype is a fundamental feature of
tumor growth, and this depends on the metabolic reorganization of elements involved in
bioenergetics, macromolecular synthesis, and cell division(77)
. Besides Nrf2’s role in
cancer cell resistance to cytotoxic agents, it also cross-talks with other pathways
responsible for modulating metabolism and cell growth, including PI3K/AKT/mTOR and
MAP/ERK pathways. In this context, Nrf2 was observed to mediate NSCLC cell
proliferation via activation of the epidermal growth factor receptor EGFR/MEK1-2/ERK
axis. In the NSCLC H292 cell line, which expresses both wild-type EGFR and Keap1,
EGFR ligand was shown to increase Nrf2 levels in a dose-dependent manner via the
MAP/ERK pathway(78)
. Also, when EGFR is constitutively active, due to gain of function
mutations, Nrf2 is permanently active(78)
.
Nrf2 was shown to reinforce the metabolic reprogramming triggered by proliferative
signals. Mitsuishi, Y et al(79)
has shown that in the presence of active PI3K-Akt signaling,
combined with high Nrf2 levels in the cell, higher than the ones required for the
transcription of antioxidant target genes, Nrf2 redirects glucose and glutamine into
anabolic pathways. Direct Nrf2 transcriptional targets are associated with de novo
nucleotide synthesis via the pentose phosphate pathway (PPP). AKT activation via Nrf2
was observed in another study of liver repair in mice NRF2 KO mice(80)
. As expected,
Mitsuishi, Y et al(79)
also found AKT to be phosphorylated in a Nrf2 dependent manner,
20. thus activating the AKT/mTORC1/Sterol Regulatory Element-Binding Proteins (SREBP)
axis. SREBP is a transcription factor known to induce the PPP genes when mTORC1 is
activated (81)
.
1.8 mTOR
mTor (also known as RAFT1 or FRAP) is a vital cell metabolic regulatory component of
the PI3K pathway, indirectly activated by AKT via Rheb. mTOR plays a central role in
various signaling pathways, is responsible for the intra and extra cellular detection of
nutrients levels, and functions as a metabolic regulator of cellular anabolic and catabolic
processes coupling growth signals to nutrient availability via ribosome biogenesis and
autophagy(82-84)
The mTOR protein has a molecular weight, of 289 kDa and contains 2549 amino acids
with several conserved structural domains. The N terminus possesses 20 tandem
Huntington, EF3, A subunit of PP2A, TOR1 (HEAT) repeats, forming two α helices of
40 amino acids with hydrophobic and hydrophilic residues. These HEAT repeats are
responsible for protein-protein interactions. The kinase domain of mTOR is located in the
C-terminal. The FKBP12-rapamycin-binding (FRB) domain is located upstream of its
catalytic domain and is, responsible for the formation of the rapamycin inhibitory
complex. Near FRB domain a large FRAP, ATM, TRAP (FAT) domain is present. This
FAT domain is essential for mTOR activity because of its interaction with another FAT
domain, present in the end of the C terminal domain, called FATC. The interaction
between those two domains produces a configuration that exposes the catalytic domain.
Between the FATC and the catalytic domain there is a putative negative regulatory
domain (NRD)(82)
.
1.8.1 mTORC1
mTOR is part of two functionally and structurally distinct complexes, namely,
rapamycin-sensitive mTOR complex 1 (mTORC1) and rapamycin-insensitive mTOR
complex 2 (mTORC2). mTORC1 is related to regulation of translation, autophagy, cell
growth, lipid biosynthesis, mitochondria biogenesis, and ribosome biogenesis. The
21. downstream effects of mTORC1 are initiated by its interaction with the accessory protein
regulatory-associated protein of mTOR (Raptor). This interaction mediates the
association of this complex to a conserved short sequence called the TOS motif of S6K
and the eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E–BP1 and 2). Once
bound, the raptor–mTORC1 complex phosphorylates S6K, and 4E–BP, which are
markers for mTORC1 activity. S6K is phosphorylated on its Thr389 site, and functions to
enhance the translation of 5′-terminal oligopolypyrimidine (5′-TOP) mRNA’s via
activation of 40S ribosomal subunit. These activated mRNA’s encode anabolic elements
such as, ribosomal proteins, elongation factors and insulin growth factor 2(83,84)
.
In its non-phosphorylated form 4E-BP binds to eIF4E at the 5 ́-cap of mRNAs, inhibiting
the interaction of eIF4E with eIF-4G protein, consequently arresting initiation of
translation. The 4E-BP/ eIF4E complex is released after 4E-BP phosphorylation by the
raptor–mTOR complex. Therefore enhancing cap-dependent protein translation via eIF4E
activation, resulting in a global boost of cellular protein synthesis and ribosome
biogenesis. Anabolic processes generated by mTORC1 also involve stimulation of
glucose uptake, glycolysis and NADPH production. One of the mechanisms that generate
these effects is the increase in translation of hypoxia-inducible factor 1α (HIF1α),
resulting in higher levels of glucose transporters and glycolytic enzymes(83,84)
.
1.8.2 mTORC2
The second mTOR complex, mTORC2, interacts with rapamycin-insensitive companion
of mTOR (RICTOR) which is a hydrophobic motif kinase for Akt/PKB activation. Akt is
a vital element of the insulin/PI3K signaling pathway and regulates the influx of nutrients
that activate the raptor–mTOR pathway. The role of mTORC2 in cancer is well
documented (79,80)
. This complex is hyper activated in cancers via inactivation of the
tumor suppressor PTEN. mTORC2 is known to control cell survival and proliferation by
enhancing the p53-regulator mdm2 and transcription factors from the FOXO family(83,84).
There are a myriad of known mTOR regulators such as growth factors, amino acids,
glucose, energy status, stress (e.g. osmotic stress, DNA damage) and, the tumor
suppressors TSC1 (hamartin) and TSC2 (tuberin). The TSC1/2 complex indirectly
22. inhibits raptor–mTOR by working as a GTPase-activating protein (GAP) for rheb, a
GTP-binding protein from the ras-family that activates raptor–mTOR by direct biding(69)
.
mTOR complex 1 activity is also regulated by Rheb via RagD. This member of the
small G-protein family binds directly to the mTOR complex, recruiting it to the
endosomal fraction where mTOR is activated(85)
.
Using the UCSC genome browser we identified an extended list of ubiquitous
transcription factors acting on mTOR including SP-1, C-MYC and C-FOS. From this list,
the activating factor (ATF-5) was mentioned in the literature. ATF-5 is a member of the
cAMP response element binding (CREB)/ATF subfamily of basic leucine zipper
transcription factors(86)
. It was shown that the oncoprotein BCR-ABL suppresses
authophagy by up regulating ATF-5 via PI3K/AKT/FOXO4 signaling(87)
. ATF-5 then
activates mTOR by a direct binding to its promoter, which is in a region between 1560-
2227 bp upstream of the transcription start site, as demonstrated via ChIP assay(87)
.
Interestingly, a member of the same group of transcription factors, ATF3, is known to
inhibit Nrf2 via direct ATF3-Nrf2 protein-protein interactions(88).
Nrf2 belongs to the
same family of transcription factors as ATF and has already been shown to indirectly
interact with mTOR via TSC2 and AKT.
1.9 Role of Nrf2 on mTOR expression
Due to the multi-level interaction of Nrf2 with the PI3K pathway we were interested to
know if Nrf2 could directly act on different components of this pathway. Recent studies
of Nrf2 participation on translation and in cancer anabolism focused our attention to the
metabolic regulator of this pathway, mTOR.
2. HYPOTHESIS________________________________________________________
From literature it is observed that Nrf2 interacts with different components of the PI3K
pathway and regulate specific processes. Recently, Nrf2 has been shown to be involved in
the regulation of metabolic processes in the cell(78) - (80)
and hence, we hypothesized that
Nrf2 might also be interacting directly with mTOR, which has not previously been
shown. If demonstrated, this would be one of the possible pathways in which Nrf2
23. directly regulates the metabolic processes of the cell, positioning it as a link between cell
metabolism and cytoprotection.
To examine this hypothesis, mTOR expression was analyzed using western blot and RT-
PCR in conditions where Nrf2 levels are modulated. Our experiments were focused in
three different cell lines, selected according to the mutations present in them. We used
the non-tranformed Human Embryonic Kidney (HEK293) cells, as well as two human
non-small-cell lung cancer (NSCLC) cell lines. A549 cells have a Kras mutation in
addition to mutations in keap1. Another NSCLC cell line H460, contains a loss of
function mutation on keap1(55)
and gain of function mutation on PIK3CA (E545K) and
Kras(89)
. To further study the Nrf2/mTOR interaction we performed mutation analysis in
dual luciferase assay, as well as DNA pulldown, electroctrophoretic mobility shift assay
(EMSA) and ChIP assay. Additionally, we analyzed the expression of the other elements
of the PI3K pathway (TSC2, S6K and AKT), under Nrf2 silencing and inducing
conditions, via western blot, RT PCR and luciferase assay.
3. MATERIALS & METHODS____________________________________________
3.1 Cell Lines and Tissue Culture/ Transient Transfection
The cell lines A549, HEK293 and H460 (Sigma) were cultured in RPMI (Sigma) media,
supplemented with 10% fetal bovine serum (Sigma), 5% antibiotic/antimycotic (Life
Technologies) and grown in 5% CO2 at 37°C. The cell lines were storage at -80o
C in
cryogenic vials containing 106
cell in 1 ml solution of 90% FBS plus 10% DMSO.
Twenty-four hours prior to transfection, 9 X 104 cells were plated in 6 well dish plates
and were transfected when they were approximately 60% confluent. The cells were
incubated with fresh media 1 hour before transfection. The transfections, except for the
ones utilized on ChIP assay, were carried out using Lipofectamine LTX Reagent PLUS™
(Life Technologies) as per manufacturer protocol, utilizing Opti-MEM with a 1:5 ratio of
plasmid to LTX and Plus reagent. The transfection mix was vortexed thoroughly and
incubated for 30 min before addition to cells. Cells were incubated 24 hours before
24. collection. The internal control used for Luciferase assay, pRL Vector, was co transfected
with the modulatory reagents (pCDNA_Nrf2 or siNrf2 with their respective controls
pCDNA 4.0 or scrambled RNA) and the construct containing the sequence of interest, in
1:1 ratio. 24 hours after transfection the cells were harvested and split for Western blot,
qPCR and luceferace applications.
3.2 Western blot
Protein expression analysis of the cells A549, H460 and HEK293 were performed by
Western blot. Cells were disrupted with lysis buffer (20mM Tris pH 7.5, 420mM NaCl,
2mM MgCl2, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.5% Triton, 1x P8340 (Sigma),
1mM PMSF, 1mM DTT, 2mM NaF, 10mM BGP) for 30 min on ice followed by a 20
min spin at 13000rpm to pellet debris. The supernatant was then removed and quantified
using the Bradford reagent. The OD595 of each sample was then measured using a
spectrophotometer and compared to a standard curve prepared with bovine serum
albumin. An equal concentration of sample was then separated using standard Sodium
Dodecyl Sulfate-Polyacrilamide Gel Electrophoresis (SDS-PAGE) techniques. 40 µg of
cell protein/lysate per each sample was loaded and run through a 10% SDS-PAGE gel
before transferring electrophoretically at 400mA for 2 hours onto a BioRad
nitrocellulose membrane. For the incubation with antibodies, the membrane was first
blocked with 10% fat-free milk solution in 1x Tris Buffered Saline and 0.1% Tween
(TBS-T) for 1 hour at room temperature and probed overnight at 4
o
C with the antibodies
listed below at the dilutions provided by the manufactor. The day after, membranes were
washed three times in TBS-T and were then incubated with secondary anti-mouse or anti-
rabbit horseradish-peroxidase for 1hour at room temperature. This was followed by three
additional washes with TBS-T.
The results were documented on x-ray film with ECL detection and autophotography to
capture the differences in protein levels in the cells between samples. The antibodies used
as probes for Western were as follows; Nrf2 (abcam) all the others antibodies, beta-Actin,
TSC2, AKT, S6K and Nqo1 were purchased from Cell signaling.
25. 3.3 Quantitative RT-PCR
Total RNA was isolated from, HEK293, A549 and H460 using EZ-10 DNAaway RNA
Mini-Preps Kit (Bio Basic Canada INC.) according to the manufacturer's protocol.
cDNAs were synthesized from total RNA (1 µg) of each sample using , SuperScript® II
Reverse Transcriptase (Invitrogen™)), diluted 4 times with water. The cDNA was used
as the template for quantitative PCR detection using the GoTaq® qPCR Master Mix
(Promega). The real-time PCR conditions were optimized as 95 °C for 7 min and 40
cycles of 95°C for 10 s, 61°C for 5 s, and 72°C for 20 s followed by melting curve cycle.
The amplification reactions were carried out with the AB Applied Biosystems 7500 Fast
Real-Time PCR System. The primers for amplifying human genes (Nrf2, mTOR,Nqo1,
HMOX1, TSC2, AKT, S6K and Gapdh)appendix(Table 1)
. The comparative ΔΔCt method was
used for relative quantification of the amount of mRNA in each sample normalized to
GAPDH transcript levels. Fold induction is expressed as the ratio of induction from
treated cells versus untreated. Values represent the mean +- S.E. of three independent
measurements. Statistical analysis (Student’s t test) was performed by comparison of
treated and untreated cells (*, p < 0.05).
3.4 Bioinformatic Analysis
We screened for the presence of the core ARE sequence (TGAxxxxGC) up to 5kb
upstream of the transcription start site of the target genes. This ARE motif analysis was
performed using BlAST, SCOPE and InSilicase algorithms.
3.5 Molecular Cloning and Vector Construction
Primers were designed using the Primer3 software (http://fokker.wi.mit.edu)2
,
synthesized by Integrated DNA Technologies, Montreal, QC. PCR was done according to
the Phusion® High-Fidelity DNA Polymerase protocol (Thermo). Sanger DNA
sequencing at the Innovation Centre, located at McGill University, confirmed the
presence of the desired promoters. The restriction enzymes used on molecular cloning
were purchased from Invitrogen™
26. 3.6 Nrf2 modulation
Inducible Nrf2 construct – The inducible construct PC_Nrf2 appendix (figure 1A)
containing
1925bp of the Nrf2 coding sequence was obtained by amplifying the coding sequence of
Nrf2 from A549 RNA (cDNA). Restriction sites for BamHI and XbaI were included in
the primers used for Nrf2 amplification, and enabled the insertion of Nrf2 cDNA into the
pCDNA 4.0 plasmid (Life Technologies). The resulting construct, PC_Nrf2, was
sequenced to validate the plasmid identity. Nrf2 induction was generated via transient
transfection of inducible PC_Nrf2 plasmid. pcDNA 4.0 was used as a negative control for
the cells transfected with inducible Nrf2.
siNrf2 – Nrf2 silencing was generated via transient transfection of Small interfering RNA
targeting Nrf2 (siNrf2) NFE2L2HSS181505 (Invitrogen). Scramble RNA (Invitrogen)
was used as a negative control.
3.7 Luciferase assay constructs
pRL – The internal control used was pRL Vector, which is wildtype Renilla luciferase
(Rluc) control reporter vectors that is used for the purpose of normalizing the luciferase
values.
PCR cloning was used to amplify the target regions and clone into PGL3 basic vector. In
short, the constructs were digested with the restriction enzymes Kpn1 and Xho1 with the
exception of Mtor, which was digested by SacI and MluI.
For site directed mutagenesis the TGA portion of the ARE’s analyzed were deleted using
the Quickchange II XL Stie-directed mutagenesis Kit. The primers sequence for Nqo1,
mTOR, TSC2, and S6K mutations are listed at appendix (Table 1).
Molecular Cloning of Nqo1 Promoter – The ARE site at 550bp upstream of start of
transcription is shown to be active on Nqo1(90)
. This region was cloned on the
PGL3_basic vector used as positive control (Nqo1_PGL3) Appendix (figure 1B)
. Nqo1_Pgl3
with the deleted TGA sequence (Nqo1_Pgl3 mut) was used as negative control.
Molecular Cloning of mTOR Promoter –The screened mTOR promoter region contained
27. eight ARE binding sites. I studied the closest ARE site present at 723bp upstream of the
TSS. The promoter region of mTOR, 1231 bp upstream from TSS, was cloned into the
Pgl3 basic vector (mTOR_Pgl3) Appendix (figure 1C)
and used in subsequent functional
analyses. For site-directed deletion analyses the mTOR_Pgl3 mut was created.
Molecular Cloning of TSC2 Promoter –The screened TSC2 promoter region contained 6
ARE binding sites. I studied the closest ARE site present at 756bp upstream of the TSS.
The promoter region of TSC2, 1079 bp upstream from TSS, was cloned into the pgl3
basic vector (TSC2 _Pgl3) Appendix (figure 1D)
and used in subsequent functional analyses.
For site-directed deletion analyses the TSC2_Pgl3 mut was created.
Molecular Cloning of S6K Promoter –The screened S6K promoter region contained 12
ARE binding sites. I studied the firsts 5 closest ARE sites, present at 255bp, 285bp,
324bp, 432bp and 2543bp upstream of the TSS. The promoter region of S6K, 2660bp
upstream from TSS, was cloned into the pgl3 basic vector (S6K_Pgl3) Appendix (figure 1E)
and
used in subsequent functional analyses. For site-directed deletion analyses the two
closest ARE’s to TSS were mutated at the TGA (S6K_Pgl3 mut).
Molecular Cloning of AKT Promoter –The screened AKT promoter region contained 3
ARE binding sites. I studied those Are’s were present at 1191 bp, 1403 bp and 1681 bp
upstream of the TSS. The promoter region of AKT, 2200 bp upstream from TSS, was
cloned into the pgl3 basic vector (AKT _Pgl3) Appendix (figure 1F)
and used in subsequent
functional analyses.
3.8 Luciferase Assay
Cells were lysed with Passive Lysis Buffer, and kept at -80ºC overnight. Luciferase
activities were analyzed in 20-µl cell extracts with the dual luciferase assay kit
(Promega).
Firefly and Renilla luciferase activities were then determined in triplicates for each
sample on the EnSpire multimode plate reader (PerKinElmer). The luciferase activities
reported were expressed as a ratio of the pGL3 reporter activity to that of the control
plasmid pRL. -Fold induction (Relative Luciferase activity) is expressed as the ratio of
28. induction from treated cells(PC_Nrf2 and siNrf2) versus untreated (pcDNA 4.0 and
Scramble RNA) respectively. Values represent the mean +- S.E. of three independent
measurements. Statistical analysis (Student’s t test) was performed by comparing treated
and non-treated cells (*, p < 0.05).
3.9 Electrophoretic Mobility Shift Assay (EMSA)
A549 cells (4 x106
), were plated in four 175cm2
flasks with RPMI for 24 hours. The cells,
were transfected with Nrf2 siRNA or scrambled SiRNA and were harvested 24 hours
later. Nuclear extracts of A549 cells were prepared using 1M tris ph 7.5, 100mM Mgcl2,
3M Kcl, 500mM EDTA, 1M sucrose, 100% Glycerol, 1MDTT, 1M orthvanadate, 0.5M
BGlyc-phos, 100mM PMSF and 100x protease cocktail. The annealed primers for Nqo1
wild type, Nqo1 mutant, mTOR wild type, mTOR mutant 1, mTOR mutant 2, and mTOR
mutant 3 composed the probes used for the experiment appendix (table1)
. The primers were
annealed by heating at 95°C for 10 minutes followed by overnight incubation at 4 °C.
The probes were then labeled with the radioactive isotope g-[32P]ATP at 30°C for 30
minutes following 10 minutes incubation at 65C. For DNA-protein binding reactions, 10
µg of nuclear extract was incubated at room temperature for 30 min with 20 mM HEPES-
KOH (pH 7.9), 60 mM KCL, 1 mM MgCL2, 1 mM EDTA, 1 µg poly(dI-dC)
dithiothreitol, 10% glycerol, 0.2 mM ZnSo4 and 10,000 cpm g-[32P]ATP-labeled probe.
Protein-DNA complexes were resolved through a 4% polyacrylamide gel. The gel was
then dried and subjected to autoradiography with an intensifying screen at -80°C.
3.10 DNA Pull-Down Assay
Tissue culture, transient transfection and the nuclear extraction were performed for both
the DNA pull down as it was for the EMSA assay. This assay was performed via a
modified protocol described by Benoit Grondin et all 2006(91).
The biotinylated primers
Nqo1 wild type, NQO1 mutant, Mtor wild type, Mtor mutant appendix (table1)
were generated
at IDT (Integrated DNA Technologies). Annealing reaction of the primers was performed
as described for EMSA experiment. For DNA-protein binding reactions, 200 µg of
29. nuclear protein extraction was incubated over night at – 10C on a shaker with 10 µg of
biotinylated probes on 1 ml of biding/washing buffer (20 mM Tris [pH 8.0], 10%
glycerol, 6.25 mM MgCl2, 5 mM dithiothreitol, 0.1 mM EDTA, 0.01% NP-40) in a final
concentration of 200 mM NaCl. After 1 hour incubation with 50 µl of the magnetic beads
(Dynabeads® MyOne™ Streptavidin C1), immobilized templates were washed three
times with 0.5 ml of binding buffer, dried and resuspended on SDS and loading dye. The
samples were than boiled and resolved on a 10% SDS-PAGE gel for immunoblot
analysis with Nrf2 antibody (abcam).
3.11 Chromatin immunoprecipitation
This experiment was carried with as a modified protocol previously described by Donner
et al 2007(92)
, 2010(93)
. Briefly, A549 cells were grown until 80% confluence in 15 cm
plates and were transfected with 15µg of PC_Nrf2 using GenJet Plus transfection reagent
(SignaGen Laboratories). Before harvesting, the cells were cross-linked with 1%
formaldehyde for 10 mins at room temperature on a rocker. The cross-linking reaction
was quenched using 125mM glycine and washed twice with ice-cold phosphate-buffered
saline. The cells were harvested by scraping in RIPA buffer (150mM NaCl;1% v/v
Nonidet P-40;0.5% w/v deoxycholate; 0.1% w/v SDS;50mM Tris pH 8.0;5mM EDTA)
supplemented with protease inhibitor cocktail(Fisher), phosphatase inhibitors and PMSF.
These cells were sonicated on ice with 15 pulses of 15 seconds(20% amplitude) with
30second intervals to obtain an average chromatin length of 500 to 1,000 bps using a
Sonic Dismembrator (Fisher Scientific, Pittsburgh, Pa.) and centrifuged. The supernatant,
containing the chromatin, was collected and quantified alongside BSA standards and
equalized to a final concentration of 1mg/ml. The chromatin (1mg/ml) was pre-cleared
using 25μl of IgG magnetic beads (Dynabeads Invitrogen), previously washed with
RIPA, for 2 hrs at 4°C on a rocker. 10μl of pre-cleared chromatin was reserved as input
sample. The rest was immunoprecipitated with 25μl IgG magnetic beads, blocked with
salmon sperm DNA(0.3mg/ml) and BSA(1mg/ml), and with either anti-Nrf2 antibody
(Santa Cruz, Santa Cruz, Calif.), anti-RNA pol II antibody (Active Motif), or no antibody
overnight at 4°C with rotation. The next day, the beads were washed with RIPA and wash
buffer (100mM TrisHCl pH 8.8;500mM LiCl;1% v/v Nonidet P-40; 1% w/v deoxycholic
30. acid) and were resuspended in 100μl of 1X TE buffer. To elute the imunocomplexes,
200μl of elution buffer (70mM Tris HCl pH8.0;1mM EDTA;1.5% w/v SDS) was added
and the samples were incubated for 10min at 65°C with occasional vortexing. To reverse
cross-linked chromatin, 200mM NaCl is added to the eluted complexes and input samples
and incubated at 65°C for 6hrs. All the samples were then treated with 20 mg/ml
proteinase K (Fisher) and extracted with phenol-chloroform-isoamyl alcohol (25:24:1).
DNA was precipitated with ethanol and 3M sodium acetate and re-suspended in 100μl of
water. 2μl of purified DNA was used for qPCR appendix (table1)
.
4. RESULTS__________________________________________
Evidence from the literature shows that Nrf2 interacts with PI3K pathway at different
locations and regulates various functions of the cell(23, (37), (67)-80)
. The aim of this study
was to determine if Nrf2 transcriptionally controls the expression of the mTOR gene and
to illustrate whether this regulation is through direct or indirect binding of Nrf2 to the
mTOR promoter. To achieve these goals, western blot and qPCR analysis in conditions of
induced and silenced Nrf2 protein levels were performed. This was followed by
luciferase assays to confirm the presence of functionally active AREs in the mTOR
promoter. Lastly, we performed DNA pull down, EMSA and ChIP assays to confirm
direct binding of Nrf2 to elements in the mTOR promoter. The possibility of an Nrf2
impact on the other elements of the PI3K pathway (TSC2, S6K and AKT), was also
analyzed via western blot, qPCR and luciferase assay.
31. 4.1 Nrf2 modulates mTOR expression in A549 cells
4.1.1 mTOR expression when Nrf2 is up-regulated
Expression analysis of mTOR was performed in A549, H460 and HEK293 cell lines.
Induction of Nrf2 was carried out by transiently transfecting Nrf2 cDNA (PC_Nrf2)
appendix (figure 1A)
for 24h. pcDNA 4.0 was used as a negative control for the cells .
The transiently transfected cell lines (figure 1)
have significant increase in Nrf2 mRNA and
protein level, however the basal levels differ amongst the three cell lines. A549 cells have
the lowest basal Nrf2 protein levels such that the effect of transfection was most dramatic
in these cells. In A549 cells, mTOR expression was significantly increased, by
approximately five folds at both transcriptional and protein levels. In HEK 293 cells, an
increase in mTOR transcription was observed while protein levels showed no change. In
H460 cell lines there was 1.6 fold increase in mTOR protein, although thre was no
observable increase in transcriptional activity.
32. Figure 1. mTOR (Nrf2 inducible) expression analysis- A. mTOR protein levels were
not increased in HEK293 cells. B and C. mTOR protein levels were increased five fold
in A549 cells and 1.6 folds increased on H460 cells, respectively. D and E. mTOR
transcription was increased two folds in HEK 293 cells and four folds in A549 cells. F.
No increase in mTOR transcription was observed in H460 cells. G. The relative
Luciferase activity of mTOR-WT in HEK293 cells was 20 folds increased and five folds
increased in mTOR- mut. H. The A549 cells presented three folds increase of mTOR-WT
relative luciferase activity with no change in mTOR-mut. I. The H460 cell lines did not
present a significant change of relative Luciferase activity in both mTOR-WT and
mTOR-mut. The Relative luciferase activity was expressed as a ratio of the pGL3
reporter activity to that of the control plasmid pRL. Relative Luciferase activity and
mRNA expression levels were represented as the fold change of the ratio of induction
from treated cells (PC_Nrf2) versus Control (pcDNA 4.0). Values represent the mean +-
S.E. of three independent measurements. Statistical analysis (Student’s t test) was
performed by comparison of treated and Control cells (*, p < 0.05).
Nrf2 Inducible
(mTOR)
Western blot
mTOR
β-Actin
mTOR
mTOR
β-Actin
1 : 0.96
1 : 1.6
β-Actin
HEK293
A549
H460
A)
B)
C)
Nrf21 : 1.73
Nqo11 : 1.3
Control Pc_Nrf2
1 : 5.3
Nrf21 : 1.63
Control Pc_Nrf2
Nqo11 : 60
Nrf21: 1.5
Control Pc_Nrf2
Nqo11 : 1.6
qPCR
D)
E)
F)
Control Nrf2 Nqo1 mTOR
0.0
0.5
1.0
1.5
2.0
2.5
mRNAexpression
levels
*
Control Nrf2 Nqo1 mTOR
0.0
0.5
1.0
1.5
2.0
2
3
4
5
6
7
8
mRNAexpression
levels
Control Nrf2 Nqo1 mTOR
0.0
0.5
1.0
1.5
2.0
2
3
4
5
6
7
8
mRNAexpression
levels
Luciferase
Control Nqo1 -WT Nqo1-mut mTOR - WT mTOR-mut
0.0
0.5
1.0
1.5
2.0
5
10
15
20
25
RelativeLuciferase
Activity
Control Nqo1 -WT Nqo1-mut mTOR - WT mTOR-mut
0.0
0.5
1.0
1.5
2.0
2
3
4
5
6
7
8
RelativeLuciferase
Activity
Control Nqo1 -WT Nqo1-mut mTOR - WT mTOR-mut
0.0
0.5
1.0
1.5
2.0
2
3
4
5
6
7
8
RelativeLuciferase
Activity
G)
*
*
*
*
H)
*
*
*
*
*
**
*
*
*
*
*
I)
33. 4.1.2 mTOR expression when Nrf2 is down-regulated
Figure 2. mTOR (Nrf2 silencing) expression analysis. A, B and C. mTOR protein
levels were significantly transiently decreased in the three cell lines. D and E. mTOR
transcription was decreased proximately 1.5 folds on HEK293 cells and 2 folds in A549
cells. F. No change was observed on mTOR transcription in H460 cell lines. G, H and I.
No change in the luciferase activity was observed for Mtor-WT and mtor mut in all the
three cell lines. The Relative luciferase activity was expressed as a ratio of the pGL3
reporter activity to that of the control plasmid pRL. Relative Luciferase activity and
mRNA expression levels were represented as the fold change of the ratio of silencing
from treated cells (siNrf2) versus Control (Scramble RNA). Values represent the mean +-
S.E. of three independent measurements. Statistical analysis (Student’s t test) was
performed by comparison of treated and Control cells (*, p < 0.05)
Nrf2 Silencing
(mTOR)
Western blot
mTOR
β-Actin
mTOR
mTOR
β-Actin
1 : 0.03
1 : 0.65
β-Actin
HEK293
A549
H460
A)
B)
C)
Nrf21 : 0.03
Nqo11 : 0.07
Control Si_Nrf2
1 : 0.51
Nrf21 : 0.02
Control Si_Nrf2
Nqo11 : 0.53
Nrf21 : 0.77
Control Si_Nrf2
Nqo11 : 0.43
qPCR
D)
E)
F)
Luciferase
G)
H)
I)
Control Nrf2 Nqo1 mTOR
0.0
0.5
1.0
1.5
mRNAexpression
levels
Control Nrf2 Nqo1 mTOR
0.0
0.5
1.0
1.5
mRNAexpression
levels
Control Nrf2 Nqo1 mTOR
0.0
0.5
1.0
1.5
mRNAexpression
levels
Control Nqo1 -WT Nqo1-mut mTOR - WT mTOR-mut
0.0
0.5
1.0
1.5
RelativeLuciferase
Activity
Control Nqo1 -WT Nqo1-mut mTOR - WT mTOR-mut
0.0
0.5
1.0
1.5
RelativeLuciferase
Activity
Control Nqo1 -WT Nqo1-mut mTOR - WT mTOR-mut
0.0
0.5
1.0
1.5
RelativeLuciferase
Activity
*
*
*
*
*
**
*
* *
*
*
34. In conditions where Nrf2 is silenced (figure 2)
, all the three cell lines presented a significant
decrease of Nrf2 at both transcriptional and protein levels, with the most significant
effects seen at protein levels in HEK 293 and A549 cells. Silencing Nrf2 transcription
resulted in a two-fold decrease in mTOR, both its transcription and protein levels. In
HEK293 cells, a small decrease in mTOR transcription was observed.
4.2 Functional ARE present on mTOR promoter activates its transcription in Nrf2
inducible conditions.
Luciferase assay was performed in order to verify if the regulation of mTOR gene
expression was due to the presence of a functional ARE binding site in the mTOR
promoter region. Biswal et al(37)
, performed ChIP-Seq experiment to explore the network
of Nrf2 regulated genes and in this work they used the consensus core ARE sequence
TGANNNNGC. Here the mTOR promoter region was screened for ARE sites that had
the same motif sequence. Biswal et al, also screened 5225 background sequences relative
to the closest gene transcription starting site (TSS) in order to identify ARE sites. They
identified the highest peaks at AREs closest to the genes’ TSS. Similarly, in another Nrf2
ChIP-seq study performed by Chorley BN et al (38)
, based on 39 currently known
functional human AREs, NRF2-binding sites were found to be cis-acting elements more
commonly located at an average distance of ~1800 bp from the gene TSS. For these
reasons, in this study, from the eight ARE’s found within 5000bp of mTOR promoter
region, the “TGACCAGGC” ARE, located closest to mTOR TSS (723 bp upstream from
TSS), was cloned into an expression vector. The PRL-mTOR vector contained 1231 bp
of the mTOR promoter was then used on Luciferase assay (mTOR WT) appendix (figure1C)
.
As shown by Biswal et al(37)
via alignment of 20 known ARE binding sites and MEME
motif discovery algorithm on their Nrf2 ChIP-Seq dataset, the “TGA” portion of the ARE
is the most recurrent portion of the sequence. For this reason, in this study, site-directed
deletion was performed in the mTOR WT construct where the “TGA” of the ARE biding
site was deleted (mTOR Mut). Both mTOR WT and mTOR Mut constructs were
analyzed by luciferase activity assay at inducible and silencing conditions. Promoter of
the Nqo1 gene, a known target of Nrf2, was used as a positive control for this assay
b
35. (Nqo1 WT) appendix (figure 1B)
.
When transfected with the inducible PC_Nrf2 construct Nqo1 was substantially increased
at the protein and transcription level on all the cell lines (figure 1)
. In Nrf2 inducible
conditions, A549 cell line showed a 60 fold increase in the Nqo1 protein and a three fold
increase in the transcription of Nqo1 gene, compared to basal conditions. Whereas, in
Nrf2 silencing conditions (figure 2)
, Nqo1 expression was reduced in all the three cell lines.
Both transcription and protein levels of the control were decreased two fold in A549
cells.
The negative control consisted of the same Nqo1 promoter region with a mutated ARE
(Nqo1 Mut). At the basal level (Graph 1)
, the luciferase assay showed that the negative
control, when compared with Nqo1 WT activity, decreased five fold in A549 cells and
two fold in both of HEK293 and H460 cells. In this same condition, the activity of the
mTOR Mut was two folds lower than the mTOR WT in A549 and HEK293 cells while
no change was recorded on H460 cells.
Analysis of Nrf2 modulation was performed by comparing the fold change of the
luciferase activity of the Pgl3 constructs at basal Nrf2 levels (control) with cells
transfected with the same construct and Pc_Nrf2(figure 1)
or Si_Nrf2 (figure 2)
. Induction or
silencing of Nrf2 was validated with Nqo1 WT activity following Nrf2 up and down
patterns of expression in the three cell lines, with three folds increase and 7 folds
decrease on A549 cells. The negative control was not affected by Nrf2 variations in the
cells. The one exception was HEK293 cells in Nrf2 inducible condition, where there was
a four folds increase. Nevertheless, Nqo1 Mut activity was 6 fold lower than Nqo1 WT
in these conditions in HEK293 cells, so the Nrf2 is playing a regulatory role through its
interaction with ARE. When transfecting the cells with the inducible construct (figure 1)
it
was observed that the luciferase activity of the mTOR wild type (mTOR WT) construct
was increase 20 folds in HEK293 and four folds on A549 cells, but there is no change on
H460 cells. mTOR Mut activity remained unchanged during Nrf2 up regulation in A549
but not in HEK293 cells. In silencing conditions (figure 2)
no change in activity for the wild
type and mutant mTOR constructs were observed in any of the cell lines. From the cell
lines analyzed, A549 cells presented a clearer correlation between Nrf2 levels and mTOR
36. expression. For this reason, additional analyses of the Nrf2/mTOR interaction were
performed in this cell line.
Graph1. Nqo1 and mTOR (Nrf2 basal levels) Luciferase activity. A. Nqo1 Mut
presented a 2 fold decrease in HEK293 and H460 cells and 4 fold decrease in A549 cells.
B. mTOR Mut presented 2 fold decrease in HEK293 and A549 cell and no change on
HEK293 cells. The Relative luciferase activity was expressed as a ratio of the pGL3
reporter activity to that of the control plasmid pRL. Relative Luciferase activity was
represented as the fold change of the ratio from cells transfected with mutant constructs
(Nqo1-Mut and mTOR-Mut) versus cells transfected with wild type constructs (Nqo1-
WT and mTOR-WT). Values represent the mean +- S.E. of three independent
measurements. Statistical analysis (Student’s t test) was performed by comparison of
mutant and wild type constructs expression (*, p < 0.05).
4.3 Nrf2 binds to mTOR promoter region at basal conditions in vitro
Nrf2 binding to the mTOR promoter was demonstrated in vitro using DNA pull-down
and EMSA experiments. In the DNA pull down assay the mTOR promoter region was
used as a probe to selectively obtain a protein-DNA complex from an A549 nuclear
extract. The high affinity tag, biotin, was present in both extremities of the probe and the
complex purification was performed with streptavidin magnetic beads. The proteins were
eluted from DNA and detected via western blot (figure 3)
. Assessment of the biding capacity
of the ARE sequence present in this promoter region was performed via a mTOR probe
with a scrambled ARE site appendix (table 1)
. Nqo1 promoter region was used as a positive
control, and scrambled ARE site was used as a negative control
N
qo1-W
TN
qo1-M
ut
N
qo1-W
TN
qo1-M
ut
N
qo1-W
TN
qo1-M
ut
0.0
0.5
1.0
1.5
2.0
HEK A549 H460
*
*
*
RelativeLuciferase
Activity
A)
m
TO
R
-W
Tm
TO
R
-M
ut
m
TO
R
-W
Tm
TO
R
-M
ut
m
TO
R
-W
Tm
TO
R
-M
ut
0.0
0.5
1.0
1.5
2.0
HEK A549 H460
* *
RelativeLuciferase
Activity
B)
37. Figure 3. Western blot from DNA pull-down samples using Nrf2 antibody - Blot
analysis of (Input) nuclear extract from A549 cells, (No Probes) negative control
comprising of reaction mix alone incubated with magnetic beads and probed samples.
The probed samples consisted of (Nqo1 wt) Nqo1 promoter region containing functional
ARE which was used as a positive control, scramble ARE from Nqo1 promoter region
which was used as a negative control (Nqo1mutant), mTOR promoter region containing
ARE (mTOR WT) and scramble ARE from mTOR promoter region which was used as a
negative control (mTor mutant). It was observed an 2 folds decrease of Nrf2 protein
pulled down with mTOR mutant probe when compered with the amount of protein pulled
down with mTOR WT probe, as it was for the controls, Nqo1 mutant and Nqo1 WT.
On western blot analysis, our results suggest that Nrf2 binds to an element(s) in mTOR
promoter region. The fact that the amount of Nrf2 protein pulled down with the WT
mTOR probe was 2 folds higher than the amount pulled down with mutant mTOR probe
and with negative control (no probe), adds our speculation that the ARE is the biding site
of the Nrf2.
EMSA was carried out in order to further verify the Nrf2 biding is at the mTOR’s ARE
located 1231 bp upstream from the TSS (mTOR wild type). For this experiment a
mutation was done by removing the entire ARE sequence TGACCAGGC and adding 5
bp in both 5’ and 3’ prime extremities (mTOR Mut) (figure 4). The mTOR wt, mTOR
mutants as well as the positive(Nqo1 wild type) and the negative control (Nqo1 mutant)
were end labeled with [32
P] ATP and incubated with nuclear extract isolated from A549
cells.
It was observed that the predicted Nrf2 site was present in the sample incubated with
mTOR wild type and not in mTOR mutant. It was also observed that an additional biding
was present in the mutated mTOR probe at an adjacent site (figure 5)
.
38. Figure 4. mTOR probes used on EMSA assay. mTOR WT sequence containing the “
TGACCAGGC” ARE and mTOR Mut with deleted ARE sequence and 5 bp extension at
5’and 3’ ends. The primers were annealed, with it respective reverse complementary
sequence, end labeled with [32
P] ATP and used on EMSA experiments.
5’-TTCACCATGTTGACCAGGCTGGTCTCGAC-3’
5’-GGGAATTTCACCATGT********* TGGTCTCGACTCCTC-3’
Figure 5. ARE dependent biding of nuclear
components to mTOR-WT- EMSA was
performed using labeled promoter fragment of
Nqo1-WT (positive control), Nqo1-Mut (negative
control), mTOR –WT (mTOR promoter region
containg ARE site) and mTOR –Mut (mTOR
promoter region containg deleted ARE site plus
addiction of 5bp on 5’ and 3’ ends) incubated with
nuclear extracts (10 µg per lane ) from A549 cells.
Top red arrow indicate shift of predicted Nrf2
biding site and bottom black arrows indicate new
and unknown biding appeared on cells incubated
with labeled mTOR –Mut probes. Predicted Nrf2
biding site (red arrow) was presented on samples
incubated with mTOR-WT and Nqo1-WT and not
on samples incubated with Nqo1-Mut and mTOR
–Mut
39. 4.3.1 Nrf2 binding to mTOR promoter region decreases in Nrf2 silencing conditions
EMSA assay was also performed in A549 cells in which Nrf2 was silencing (figure 6)
. After
incubation nuclear extract of the Nrf2 down regulated A549 cell with radioactive labeled
mTOR WT probe a significant decrease in bound protein was observed. Intensity of the
blots present on samples incubated with mTOR WT probes suggests that in basal
conditions the Nrf2-mTOR biding is weak.
Figure 6. Biding of nuclear components to mTOR-
WT at Nrf2 silencing conditions. A. EMSA was done
on nuclear extract (NE) of transiently transfect A549
cell with SiNrf2 or scrambled RNA (control). SiNrf2
A549cells NE and Scramble A549cells NE were
incubated with labeled promoter fragment of Nqo1-WT
(positive control), Nqo1-mut (negative control ). The
films containing shift of predicted Nrf2 biding site (red
arrow) were developed after over nigh or four days gel
incubation at -80o
C. Once incubated over nigh the
SiNrf2 A549cells NE samples that contained Nqo1-WT
probes presented decreased blot intensity when
compared with Scramble A549 cells NE. After four days
incubation, the SiNrf2 A549cells NE samples that
contained mTOR-WT probes presented decreased blot
40. 4.3.2 Nrf2 binds to mTOR promoter in vivo at inducible conditions
In order to clarify the in vitro results of the Nrf2/mTOR binding, this interaction was
analyzed in vivo. One of the factors that can influence the assays performed in vitro
assays is the lack of the natural DNA conformational topology on those assays(94)
. In
order for genomic regulation and recombination to occur, these processes require DNA
bending, twisting, and looping as well as wrapping around histone octamers in order to
occur. Thus, in vitro assays, such as the ones performed in this study, may not give the
precise representation of the actual intracellular processes. Also, in addition to DNA
structure, molecular crowding caused by the presence of particles on the cytoplasmic
microenviroment may influence local and distal interactions(95)
. Biochemical reactions in
vivo occur at crowding conditions with high concentrations of biomacromolecules. While
the majority of the biochemical reactions in vitro are performed in solutions containing
low concentrations of biomacromolecules.
ChIP assay followed by qPCR amplification enables the capture of protein–DNA
interactions in vivo and is considered a definitive confirmatory method when analyzing
Nrf2 transcriptional targets(96)
. This assay was used in the past to identify important Nrf2
targets such as antiapoptotic protein Bcl-2, catalytic subunit of glutamylcysteine ligase
(GCLC) and Aldose reductase (AR)(97-99)
among others. Nrf2/mTOR biding in vivo
Chromatin ImmunoPrecipitation (ChIP) coupled to detection by quantitative real-time
PCR was performed on A549 cells (Graph 2)
. The samples were immunoprecipitated with
either anti-Nrf2 antibody, anti-RNA pol II antibody or no antibody. The experiment
compared the fold enrichment, with respect to no antibody control, of crosslinked
protein-DNA complexes in two Nrf2 conditions, basal and inducible. At the basal levels,
the ChIP performed using anti-Nrf2 antibody, showed a 2.5 fold enrichment compered to
no antibody control, of the mTOR promoter, which denotes a weak binding at basal
levels. Whereas in Nrf2 inducible conditions, the enrichment of the same mTOR
promoter was seen to increase to 13 folds. Anti-RNA polII antibody was used as a
41. positive control antibody to confirm successfulness of the ChIP assay. Nqo1 promoter
region was used as a positive control for the anti-nrf2 antibody, while GAPDH served as
a positive control for anti RNA Pol II antibody and as a negative control for anti Nrf2
antibody.
Graph2. ChIP assay.
Crosslinked protein-DNA
complexes were
immunoprecipitated using
either anti-RNA polymerase II
antibody (Pol II, positive
control), anti-Nrf2 antibody or
no antibody in A549 cells
transfected with inducible or
basal (empty vector) constructs
(Nrf2 cDNA containing
plasmid). Enrichment was
measured as fold increase of
antibody vs the no antibody
control by q-PCR.
4.4 Expression analyses of the other elements of PI3K pathway due to Nrf2
modulation
Expression of other components of the PI3K pathway elements including TSC2, S6K and
AKT as well as luciferase assay on promoters of these genes, in which ARE core
sequence were identified, were also analyzed in conditions of Nrf2 modulation. The Nrf2
inducible and silencing conditions as well as the control were the same as the ones
ChIP Assay A549 cells
N
rf2-basal
N
rf2-inducible
PolII-basal
PolII-inducible
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
200
400
600
800
1000
1200
1400
Mtor
Nqo1
Gapdh
Antibody used for ChIP
FoldEnrichmenttoNoab
42. performed for mTOR. The presence of Nrf2 affected the expression of the targeted
proteins in a very heterogenous fashion across the three cell lines. Also, for some of the
above-mentioned genes, protein expression and transcriptional activity did not followed
the same pattern in all the three cell lines.
Luciferase assay was performed on the promoter regions containing the ARE sites. As
was the case for mTOR, 5000bps upstream from the TSS of each of the respective genes
were screened for the presence of AREs. TSC2 promoter region contained 6 ARE
binding sites. Luciferase assay was performed on the closest ARE present at 756bp
upstream of the TSS (TSC2 WT)(figure1Dappendix
). S6K promoter region contained 12
ARE binding sites. The firsts 5 closest AREs, present at 255bp, 285bp, 324bp, 432bp and
2543bp upstream of the TSS were used in this assay (S6K WT) (figure1Eappendix
). AKT
promoter region contained 3 ARE’s present at 1191 bp, 1403 bp and 1681 bp upstream of
the TSS witch were cloned and also used for this assay (AKT WT) (figure1Fappendix
). For
site-directed deletion analyses the TGA site of the TSC2 ARE was mutated (TSC2 Mut),
and on S6K the two closest ARE’s to TSS were mutated as well ( S6K Mut )(table
1appendix
). The activity of the abovementioned AREs showed great variation amongst the
three cell lines and in many cases did not followed the same pattern of the transcription
levels observed via qPCR.
4.4.1 TSC2, S6K and AKT expression when Nrf2 is up-regulated
4.4.1.1 TSC2 is a potential indirect Nrf2 transcriptional target at inducible
conditions on H460 cells
When upregulating Nrf2 (figure 7)
, TSC2 protein expression was induced only in H460 cells
while transcription was increase in all the three cell lines. The ARE present on TSC2
promoter region (graph 3)
showed, in basal conditions, a small decrease in activity for TSC2
mut (A549 and H460). When Nrf2 is induced (figure 7)
, this ARE driven construct had
increased activity for when the ARE was WT (TSC2 WT) in A549 cells and HEK cells
and also in TSC2 mut in A549 cells. This could indicate that TSC2 is potentially an
43. indirect Nrf2 transcriptional target of increased Nrf2 as opposed to at basal conditions. In
H460 cells where TSC2 protein levels and transcription were increased. Although TSC2
transcription levels where increased by 10 fold in A549 cells no change was observed at
the protein level, perhaps suggesting a post-translational level of regulation of TSC2 in
these cells.
As observed for TSC2, when Nrf2 is increased (figure 8)
, S6K transcription is increased in
A549 and H460 cells. However, although, at basal Nrf2 levels (graph 4)
, luciferase activity
of S6K-mut was decreased 2 fold in the two cell lines, at Nrf2 inducible conditions, both
luciferase activity of S6K-WT and S6K-mut were increased in the A549 cells. This
implies that, while the ARE present on S6K promoter region is important for
transcription at Nrf2 basal levels, it is probable not induced by increased Nrf2 levels.
4.4.1.2 At Nrf2 inducible conditions AKT is a possible indirect Nrf2 transcriptional
target on H460 cells and posttranslational target on A549 cells
In H460 cell lines, at Nrf2 inducible conditions (figure 9)
, AKT transcription was increased
2 fold and proteins levels by over 5 fold. Since, no change was observed on the AKT
luciferase activity in this cell line, the results suggest that Nrf2 regulates this gene
indirectly, probably at the protein level. The increase of AKT luciferase activity on
HEK293 and A549 cells were also deceptive, since no significant changes were observed
at the transcription and protein levels in HEK293 cells and at the transcription level in
A549 cells. At protein level however, AKT was proximately 2 folds decreased in A549
cells. Hence, high Nrf2 levels affect some post-translational regulation of AKT protein
expression in A549 cells.
44. Figure 7. TSC2 (Nrf2 inducible) expression analysis. A and B. No significant change
on Tsc2 protein levels were observed in HEK293 cells and A549 cells. C. Tsc2 protein
levels were 6.78 fold increased in H460 cells. D, E and F. Tsc2 transcription was
increased proximately two fold in HEK293 cells, 10 fold on A549 cells and two fold in
H460 cells. G. The relative Luciferase activity of TSC2-WT in HEK293 cells was 1.5
fold increased with no change in activity on TSC2- mut. H. The A549 cell lines shown
proximately two fold increase of TSC2-WT relative Luciferase activity with 1.5 folds
increase on TSC2-mut. I. No significant change was observed in H460 cells for the
relative Luciferase activities of TSC2-WT and TSC2-mut. The Relative luciferase
activity was expressed as a ratio of the pGL3 reporter activity to that of the control
plasmid pRL. Relative Luciferase activity and mRNA expression levels were represented
as the fold change of the ratio of induction from treated cells (PC_Nrf2) versus Control
(pcDNA 4.0). Values represent the mean +- S.E. of three independent measurements.
Statistical analysis (Student’s t test) was performed by comparison of treated and Control
cells (*, p < 0.05).
Nrf2 Inducible
(TSC2)
Western blot
TSC2
β-Actin
TSC2
TSC2
β-Actin
1 : 1.16
1 : 6.78
β-Actin
HEK293
A549
H460
A)
B)
C)
Nrf21 : 1.73
Nqo11 : 1.3
Control Pc_Nrf2
1 : 0.91
Nrf21 : 1.63
Control Pc_Nrf2
Nqo11 : 60
Nrf21: 1.5
Control Pc_Nrf2
Nqo11 : 1.6
qPCR
D)
E)
F)
Luciferase
G)
*
*
*
H)
*
I)
Control Nrf2 Nqo1 TSC2
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
mRNAexpression
levels
Control Nrf2 Nqo1 TSC2
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
6
9
12
15
mRNAexpression
levels
Control Nrf2 Nqo1 TSC2
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
6
9
12
15
mRNAexpression
levels
Control Nqo1 -WT Nqo1-mut TSC2-WT TSC2-mut
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
6
9
12
15
RelativeLuciferase
Activity
Control Nqo1 -WT Nqo1-mut TSC2-WT TSC2-mut
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
RelativeLuciferase
Activity
Control Nqo1 -WT Nqo1-mut TSC2-WT TSC2-mut
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
RelativeLuciferase
Activity
*
*
*
*
***
*
*
*
* *
45. Graph 3. TSC2 (Nrf2 basal) Luciferase activity. TSC2 Mut presented a small decrease
on A549 and H460 cells and no change on HEK cells. The Relative luciferase activity
was expressed as a ratio of the pGL3 reporter activity to that of the control plasmid pRL.
Relative Luciferase activity was represented as the fold change of the ratio from cells
transfected with mutant construct (TSC2-Mut) versus cells transfected with wild type
constructs (TSC2-WT). Values represent the mean +- S.E. of three independent
measurements. Statistical analysis (Student’s t test) was performed by comparison of
mutant and wild type constructs expression (*, p < 0.05).
TSC
2-W
TTSC
2-M
ut
TSC
2-W
TTSC
2-M
ut
TSC
2-W
TTSC
2-M
ut
0.0
0.5
1.0
1.5
2.0
HEK A549 H460
*
*
RelativeLuciferase
Activity
46. Figure 8. S6K (Nrf2 inducible) expression analysis. A, B and C. No significant change
was observed on S6K protein levels on the three cell lines D, E and F. S6K transcription
did not changed in HEK293 cells and it was 2 fold increased in A549 and H460 cells. G.
The relative Luciferase activity of S6K –WT and S6K-mut were 1.5 fold increased in
HEK293 cells H.The relative Luciferase activity of S6K –WT and S6K-mut were 2 fold
increased in A549 cells I. No change was observed on the relative Luciferase activity of
S6K –WT and S6K-mut in H460 cells. The Relative luciferase activity was expressed as
a ratio of the pGL3 reporter activity to that of the control plasmid pRL. Relative
Luciferase activity and mRNA expression levels were represented as the fold change of
the ratio of induction from treated cells (PC_Nrf2) versus Control (pcDNA 4.0). Values
represent the mean +- S.E. of three independent measurements. Statistical analysis
(Student’s t test) was performed by comparison of treated and Control cells (*, p < 0.05).
Nrf2 Inducible
(S6K)
Western blot
S6K
β-Actin
S6K
S6K
β-Actin
1 : 0.95
1 : 0.85
β-Actin
HEK293
A549
H460
A)
B)
C)
Nrf21 : 1.73
Nqo11 : 1.3
Control Pc_Nrf2
1 : 1.32
Nrf21 : 1.63
Control Pc_Nrf2
Nqo11 : 60
Nrf21: 1.5
Control Pc_Nrf2
Nqo11 : 1.6
qPCR
D)
E)
F)
Luciferase
G)
*
*
*
H)
*
*
I)
Control Nrf2 Nqo1 S6K
0.0
0.5
1.0
1.5
2.0
2.5
3.0
mRNAexpression
levels
Control Nrf2 Nqo1 S6K
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3
6
9
12
15
mRNAexpression
levels
Control Nrf2 Nqo1 S6K
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3
6
9
12
15mRNAexpression
levels
Control Nqo1 -WT Nqo1-mut S6K-WT S6K-mut
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3
6
9
12
15
RelativeLuciferase
Activity
Control Nqo1 -WT Nqo1-mut S6K-WT S6K-mut
0.0
0.5
1.0
1.5
2.0
2.5
3.0
RelativeLuciferase
Activity
Control Nqo1 -WT Nqo1-mut S6K-WT S6K-mut
0.0
0.5
1.0
1.5
2.0
2.5
3.0
RelativeLuciferase
Activity
*
*
*
*
*
*
*
* *
47. Graph 4. S6K (Nrf2 basal ) Luciferase activity. S6K Mut presented 2 fold decrease in
A549 and H460 cells and no change in HEK293 cells. The Relative luciferase activity
was expressed as a ratio of the pGL3 reporter activity to that of the control plasmid pRL.
Relative Luciferase activity was represented as the fold change of the ratio from cells
transfected with mutant construct (S6K-Mut) versus cells transfected with wild type
constructs (S6K-WT). Values represent the mean +- S.E. of three independent
measurements. Statistical analysis (Student’s t test) was performed by comparison of
mutant and wild type constructs expression (*, p < 0.05).
S6K-W
TS6K-M
ut
S6K-W
TS6K-M
ut
S6K-W
TS6K-M
ut
0.0
0.5
1.0
1.5
2.0
HEK A549 H460
* *
RelativeLuciferase
Activity
48. Figure 9. AKT (Nrf2 inducible) expression analysis. A, B and C. AKT protein levels
were 5.15 fold increased in H460 cells, proximately 2 fold decreased in A549 cells and
no significant change was observed in HEK293 cells. D, E and F. No significant change
in AKT transcription was observed in HEK293 and A549 cells and it was two folds
increased in H460 cell lines. G. Luciferase activity of AKT-WT was 1.5 fold increased in
HEK293 cells, proximately 2 fold increased in A549 cells and no change was observed in
H460 cells. The Relative luciferase activity was expressed as a ratio of the pGL3 reporter
activity to that of the control plasmid pRL. Relative Luciferase activity and mRNA
expression levels were represented as the fold change of the ratio of induction from
treated cells (PC_Nrf2) versus Control (pcDNA 4.0). Values represent the mean +- S.E.
of three independent measurements. Statistical analysis (Student’s t test) was performed
by comparison of treated and Control cells (*, p < 0.05).
Nrf2 Inducible
(AKT)
Western blot
AKT
β-Actin
AKT
AKT
β-Actin
1 : 0.85
1 : 5.15
β-Actin
HEK293
A549
H460
A)
B)
C)
Nrf21 : 1.73
Nqo11 : 1.3
Control Pc_Nrf2
1 : 0.56
Nrf21 : 1.63
Control Pc_Nrf2
Nqo11 : 60
Nrf21: 1.5
Control Pc_Nrf2
Nqo11 : 1.6
qPCR
D)
E)
F)
Luciferase
G)
*
*
*
H)
*
I)
Control Nrf2 Nqo1 AKT
0.0
0.5
1.0
1.5
2.0
2.5
3.0
mRNAexpression
levels
Control Nrf2 Nqo1 AKT
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3
6
9
12
15
mRNAexpression
levels
Control Nrf2 Nqo1 AKT
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3
6
9
12
15
mRNAexpression
levels
Control Nqo1 -WT Nqo1-mut AKT-WT
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3
6
9
12
15
RelativeLuciferase
Activity
Control Nqo1 -WT Nqo1-mut AKT-WT
0.0
0.5
1.0
1.5
2.0
2.5
3.0
RelativeLuciferase
Activity
Control Nqo1 -WT Nqo1-mut AKT-WT
0.0
0.5
1.0
1.5
2.0
2.5
3.0
RelativeLuciferase
Activity
*
*
***
*
*
*
*
49. 4.4.2 TSC2, S6K and AKT expression when silencing Nrf2
4.4.2.1 TSC2, S6K and AKT may be affected post translationally, when Nrf2 is
silenced.
Decreasing Nrf2 has no significant effect on the observed on TSC2 (figure 10)
and S6K
(figure 11)
transcription and luciferase activity. However, Tsc2 protein levels where
decreased 2.64 folds in HEK293 cells and S6K was 5.84 folds increased on A549 cells.
This suggests that at low cellular Nrf2 levels, TSC2 (HEK293 cells) and S6K (A549
cells) protein levels are in some way affected.
When silencing Nrf2 in A549 cells (figure 12)
, luciferase activity of AKT WT decreased
four folds alongside with two folds decrease in AKT transcription. These findings imply
that AKT could be a direct Nrf2 transcriptional target. However, the small increase in
AKT protein levels suggests that those changes in transcription and luciferase activity
may not be biological relevant. In both H460 and HEK293 cell, the changes in AKT
transcription was also probably misleading since they did not followed the same pattern
observed in the AKT Western blot. However, because AKT protein levels were
proximately two fold decreased in HEK293 cells, we believe that AKT may be a potential
Nrf2 post-translational target.
50. Figure 10. TSC2 (Nrf2 silencing) expression analysis. A, B and C. TSC2 protein
levels were 2.64 fold decreased in HEK293 cells with no significant change observed in
A549 and H460 cells. D-I.In all three cell line, no significant change was observed on
TSC2 transcription and Luciferase activity of TSC2-WT and TSC2-mut. The Relative
luciferase activity was expressed as a ratio of the pGL3 reporter activity to that of the
control plasmid pRL. Relative Luciferase activity and mRNA expression levels were
represented as the fold change of the ratio of silencing from treated cells (siNrf2) versus
Control (Scramble RNA). Values represent the mean +- S.E. of three independent
measurements. Statistical analysis (Student’s t test) was performed by comparison of
treated and Control cells (*, p < 0.05)
Nrf2 Silencing
(TSC2)
Western blot
TSC2
β-Actin
TSC2
TSC2
β-Actin
1 : 0.34
1 : 0.78
β-Actin
HEK293
A549
H460
A)
B)
C)
Nrf21 : 0.03
Nqo11 : 0.07
Control Si_Nrf2
1 : 1.01
Nrf21 : 0.02
Control Si_Nrf2
Nqo11 : 0.53
Nrf21 : 0.77
Control Si_Nrf2
Nqo11 : 0.43
qPCR
D)
E)
F)
Luciferase
G)
H)
I)
*
*
Control Nrf2 Nqo1 TSC2
0.0
0.5
1.0
1.5
2.0
mRNAexpression
levels
Control Nrf2 Nqo1 TSC2
0.0
0.5
1.0
1.5
2.0mRNAexpression
levels
Control Nrf2 Nqo1 TSC2
0.0
0.5
1.0
1.5
2.0
mRNAexpression
levels
Control Nqo1 -WT Nqo1-mut TSC2-WT TSC2-mut
0.0
0.5
1.0
1.5
2.0
RelativeLuciferase
Activity
Control Nqo1 -WT Nqo1-mut TSC2-WT TSC2-mut
0.0
0.5
1.0
1.5
2.0
RelativeLuciferase
Activity
Control Nqo1 -WT Nqo1-mut TSC2-WT TSC2-mut
0.0
0.5
1.0
1.5
2.0
RelativeLuciferase
Activity
*
*
*
*
*
*
*
51. Figure 11. S6K (Nrf2 silencing) expression analysis. A, B and C. S6K protein levels
were 5.84 fold increased in A549 and no significant change was observed in HEK293 and
H460 cells D, E and F. no significant change was detected in S6K transcription on the
three cell lines G, H and I. Relative Luciferase activity of both wild type and mutant
S6K constructs were 1.7 fold increased in HEK293 and H460 cells, no significant
change was observed on A549 cells. The Relative luciferase activity was expressed as a
ratio of the pGL3 reporter activity to that of the control plasmid pRL. Relative Luciferase
activity and mRNA expression levels were represented as the fold change of the ratio of
silencing from treated cells (siNrf2) versus Control (Scramble RNA). Values represent
the mean +- S.E. of three independent measurements. Statistical analysis (Student’s t
test) was performed by comparison of treated and Control cells (*, p < 0.05)
Nrf2 Silencing
(S6K)
Western blot
S6K
β-Actin
S6K
S6K
β-Actin
1 : 1.09
1 : 1.35
β-Actin
HEK293
A549
H460
A)
B)
C)
Nrf21 : 0.03
Nqo11 : 0.07
Control Si_Nrf2
1 : 5.84
Nrf21 : 0.02
Control Si_Nrf2
Nqo11 : 0.53
Nrf21 : 0.77
Control Si_Nrf2
Nqo11 : 0.43
qPCR
D)
E)
F)
Luciferase
G)
H)
I)
*
*
*
Control Nrf2 Nqo1 S6K
0.0
0.5
1.0
1.5
2.0
mRNAexpression
levels
Control Nrf2 Nqo1 S6K
0.0
0.5
1.0
1.5
2.0mRNAexpression
levels
Control Nrf2 Nqo1 S6K
0.0
0.5
1.0
1.5
2.0
mRNAexpression
levels
Control Nqo1 -WT Nqo1-mut S6K-WT S6K-mut
0.0
0.5
1.0
1.5
2.0
RelativeLuciferase
Activity
Control Nqo1 -WT Nqo1-mut S6K-WT S6K-mut
0.0
0.5
1.0
1.5
2.0
RelativeLuciferase
Activity
Control Nqo1 -WT Nqo1-mut S6K-WT S6K-mut
0.0
0.5
1.0
1.5
2.0
RelativeLuciferase
Activity
*
*
**
*
*
**
**
52. Figure 12. AKT (Nrf2 silencing) expression analysis. A, B and C. AKT protein levels
were proximately two fold decreased in HEK293 cells and no significant change was
observed in A549 and H460 cells. D, E and F. AKT transcription was proximately 1.5
fold increased in HEK293 cells, 2 fold decreased in A549 cells and proximately 2 fold
decreased in H460 cells. G, H and I. The Relative Luciferase activity for AKT-WT was
four fold decreased on A549 cells and no significant change was observed in HEK293
and H460 cells. The Relative luciferase activity was expressed as a ratio of the pGL3
reporter activity to that of the control plasmid pRL. Relative Luciferase activity and
mRNA expression levels were represented as the fold change of the ratio of silencing
from treated cells (siNrf2) versus Control (Scramble RNA). Values represent the mean +-
S.E. of three independent measurements. Statistical analysis (Student’s t test) was
performed by comparison of treated and Control cells (*, p < 0.05)
Nrf2 Silencing
(AKT)
Western blot
AKT
β-Actin
AKT
AKT
β-Actin
1 : 0.54
1 : 1.26
β-Actin
HEK293
A549
H460
A)
B)
C)
Nrf21 : 0.03
Nqo11 : 0.07
Control Si_Nrf2
1 : 1.38
Nrf21 : 0.02
Control Si_Nrf2
Nqo11 : 0.53
Nrf21 : 0.77
Control Si_Nrf2
Nqo11 : 0.43
qPCR
D)
E)
F)
Luciferase
G)
H)
I)
**
*
Control Nrf2 Nqo1 AKT
0.0
0.5
1.0
1.5
mRNAexpression
levels
Control Nrf2 Nqo1 AKT
0.0
0.5
1.0
1.5
mRNAexpression
levels
Control Nrf2 Nqo1 AKT
0.0
0.5
1.0
1.5
mRNAexpression
levels Control Nqo1 -WT Nqo1-mut AKT-WT
0.0
0.5
1.0
1.5
RelativeLuciferase
Activity
Control Nqo1 -WT Nqo1-mut AKT-WT
0.0
0.5
1.0
1.5
RelativeLuciferase
Activity
Control Nqo1 -WT Nqo1-mut AKT-WT
0.0
0.5
1.0
1.5RelativeLuciferase
Activity
*
***
**
*
*
**
