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Nuclear respiratory factor-1 regulation by PTEN signaling

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Nuclear respiratory factor-1 regulation by PTEN signaling

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NUCLEAR RESPIRATORY FACTOR-1 REGULATION BY PTEN SIGNALING

by

Yang Li

A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

August 2009

Copyright 2009 Yang Li

ii
Acknowledgements

I would like to thank my advisor, Dr. Bangyan Stiles, for being a very kind and
professional mentor, giving me advice on the thesis and providing valuable feedback
on all the experiments. I also want to thank Dr. Zoltan Tokes and Dr. Young Hong for
being my committee members and providing me with their support and advice.
Finally, I want to thank all my laboratory colleagues for helping me with experiments.

iii
Table of Contents

Acknowledgements ......................................................................................................ii

List of Figures..............................................................................................................iv

Abstract.........................................................................................................................v

Introduction..................................................................................................................1

Materials and Methods..............................................................................................11

Materials .....................................................................................................................11

Results .........................................................................................................................15

Discussion....................................................................................................................34

Future Direction.........................................................................................................38

Summary.....................................................................................................................39

References...................................................................................................................40

iv
List of Figures

Figure 1. Signaling pathways controlled by PTEN .......................................................3

Figure 2. PTEN loss induces mitochondrial intensity and mitochondrial respiration
level..............................................................................................................................16

Figure 3. PTEN knock-out induces mitochondrial biogenesis through regulation of
NRF-1 by PTEN/AKT signaling .................................................................................18

Figure 4. Decreased PTEN expression enhances NRF-1 expression in HeLa cells ....20

Figure 5. NRF-1 expression can be regulated by IGF-1 associated with the up-
regulation of AKT........................................................................................................22

Figure 6. The effects of PI3K downstream factor P-AKT on NRF-1 expression........24

Figure 7. NRF-1 expression can be regulated by cell culture confluence through
phosphorylation of AKT..............................................................................................26

Figure 8. NRF-1 expression is induced by constitutively active AKT construct
transfection...................................................................................................................27

Figure 9. Brief summary of conclusion implicated by above experiment...................28

Figure 10. Immunoprecipitation for GSK3 β and NRF-1/ β-Catenin............................30

Figure 11. Cloning of NRF-1 cDNA ...........................................................................32

v
Abstract

Based on many previous reports regarding cancer metabolism, it is believed
that tumor cells need to obtain metabolic changes to maintain the malignant
phenotype. Additionally, many PTEN-related studies revealed that PTEN mutation
within the liver will eventually lead to liver cancer. The aim of this paper is to
investigate the mechanism of how PTEN deletion gives rise to tumor phenotype and
what kind of biological metabolism is suffering dysregulation due to PTEN mutation.
In our results, mouse livers with PTEN mutation exhibited more lipid accumulation
compared to PTEN wild type livers. PTEN mutant hepatocytes showed more
mitochondrial biogenesis and enhanced respiration function than that in PTEN wild
type hepatocytes. By performing a series of experiments, such as siRNA transfection,
insulin-like growth factor-1 treatment, we concluded that NRF-1, which is an
important transcription factor for mitochondrial biogenesis, is up-regulated by PTEN
deletion through the activation of Phospo-AKT. This leads to more mitochondrial
biogenesis and mitochondrial dysfunction, which may further cause tumor. This
finding confirms previous studies and gives implication of the non-alcoholic fatty
liver diseases (NAFLD) pathogenesis.

1
Introduction

1.1 Introduction of PTEN
1.1.1. Signaling Pathway Regulated by PTEN
PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome 10) is a
phosphatase that was originally cloned in 1997 simultaneously by three groups (Laura
Simpson and Ramon Parsons, 2001; P L M Dahia, 2000; Bangyan Stiles et al., 2004).
PTEN regulates the Phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which plays
a key role in mitogenic signaling. PI3Ks are heterodimeric lipid kinases that catalyzes
the phosphorylation of PtdIns (4,5)P2 (PIP2) to form PtdIns (3,4,5)P3 (PIP3) (Claude
A. Piatadosi and Hagir B. Suliman, 2006; Dominique Pessayre, 2007; Fitzgerald PC
et al., 2007). PIP3 is an important second messenger that binds to the pleckstrin-
homology (PH) domain of downstream molecules. Binding of PIP3 leads to the
membrane localization of the PH domain containing proteins and initiate their
activation. For example, AKT, a serine/threonine kinase is activated by recruitment
to the plasma membrane through direct contact of its pleckstrin- homology (PH)
domain with PIP3 followed by phosphorylations at Thr308 and Ser473. The
biological consequences of AKT activation are cell survival, cell proliferation and cell
growth. There are many AKT-downstream targets involved in multiple cellular
processes, including NF-kB, P53, BAD for apoptosis, GSK-3 β for cell proliferation
and mTOR for cell growth (Igor Vivanco and Charles L. Sawyers, 2002).
PTEN acts as a lipid phosphatase that dephosphorylates PIP3 and PIP2 at the
D3 position of the inositol ring, resulting in PIP2 and PIP respectively. Therefore,
PTEN is the antagonist, working in opposition to PI3K. As a result, PTEN is
associated with cell death and arresting signals. PTEN allele is associated with
inheritable disorders, such as hamartomatous syndromes, including Cowden

2
syndrome (CS), an autosomal dominant disorder and Bannayan-Riley-Ruvalcaba
syndrome (BRR) (Laura Simpson and Ramon Parsons. 2001; Nick R. LESLIE and C.
Peter DOWNES, 2004). PTEN is also frequently mutated in sporadic cancers
including glioblastomas, prostate and breast cancer, endometrial neoplasms, liver
cancer and hematological malignancies (Igor Vivanco and Charles L. Sawyers, 2002;
Megan Cully et al., 2006). The signaling pathways controlled by PTEN are illustrated
in Figure 1.

3
Figure 1: Signaling pathways controlled by PTEN.

PTEN is a phosphatase that inhibits PI3 kinase signaling. One of PTEN ‘s enzymatic
activities is to dephosphorylate PIP3, the product of PI3 kinase and a major cellular second
messenger. By dephosphorylating PIP3, PTEN inhibits the activity of several downstream
molecules, of which, the most important one is AKT. Activation of AKT by growth factors
leads to increased cell cycle progression, suppressed cell death as well as increased translation.

4
1.1.2 The Biological Function of PTEN
The biological function of PTEN is evolutionarily conserved from C. elegans
to humans. In C. elegans, the important signaling axis regulating metabolism,
development and life span is the insulin-like signaling pathway, which is activated by
daf-2 that is an insulin receptor-like molecule. Age-1, a phosphoinositide 3-kinase
downstream of daf-2, further activates AKT-1/AKT-2 kinases. Daf-18 encodes a
homolog of PTEN dephosphorylating PIP3 and limiting AKT-1 and AKT-2 activities.
Previous reports showed that the daf-2 and age-1 signaling axis negatively regulates
the C. elegans life span and dauer formation (Scott Ogg and Gary Ruvkun, 1998),
which is an essential state of hibernation and growth arrest under starvation conditions.
By antagonizing the actions of daf-2 and age-1, daf-18 (PTEN homolog) regulates the
metabolism and longevity of C. elegans.
In mammalian tissues, the biological functions of PTEN rely on its enzymatic
activity as a lipid phosphatase. In response to growth factor stimulation, the activated
receptor tyrosine kinase leads to AKT activation through PI3K activation and PIP3
generation in mammalian tissues (Figure 1). The PI3K/AKT pathway is one of the
major signaling pathways induced by growth factor signaling to produce cell growth,
proliferation, survival and motility outcomes (Igor Vivanco and Charles L. Sawyers,
2002). By negatively regulating this signaling pathway, PTEN inhibits growth factor
signals transmitted by PI3K (Bangyan Stiles et al., 2004).
PTEN is ubiquitously expressed during the early stage of embryonic
development. Several groups using the PTEN mutant animal models to study PTEN
function concluded that PTEN is required for normal embryonic development. PTEN
null embryos died before birth due to abnormal development of multiple organs and
tissues (Bangyan Stiles et al., 2004; Di Cristofana et al., 1998). Tissue specific loss of

5
PTEN has been investigated. This analysis confirmed that PTEN is critical for tumor
growth and progression in multiple tissues such as prostate and lymphoma where
hereditary mutation/deletion of PTEN are linked to. In other tissues, PTEN loss may
or may not lead to tumor phenotype. In the liver, loss of PTEN leads to both
metabolic and tumor phenotypes, indicating potential role of PTEN in cancer
metabolism.

1.2 Cancer Metabolism
Cancer is a disease in which cells have lost their normal checks on cell cycle
progression. In addition, to meet the increased demands of proliferation, tumor cells
often suffer changes in fundamental pathways of metabolism and nutrition uptake.
The growing tumors face two major distinct metabolic challenges: 1) how to meet the
bioenergetic and biosynthetic demands of increased cell proliferation and 2) how to
engage strategies of metabolic adaptation to survive in external environments with
fluctuations of nutrients and oxygen. Mutations of many genes associated with
tumorigenesis are also linked to metabolic regulation. These mutations leading to
cancer development also drive the altered metabolism of tumor cells (Gatenby and
Gillies, 2004; Kroemer and Pouyssegur, 2008). Thus, it is believed that cell growth
and metabolism are intimately linked during tumor progression.
Evidence from the drosophila and C. elegans system suggests that the
PI3K/AKT signaling regulated by PTEN may control metabolism in addition to
growth potential (Scott Ogg and Gary Ruvkun, 1998). In mammalian tissues, the
PI3K/AKT signaling also mediate metabolic signals needed to clear glucose from the
blood stream in response to insulin (Bauer et al, 2004; Wullschleger et al, 2006).
AKT, the best characterized downstream factor of PI3K/AKT signaling, regulates the

6
rate of glucose uptake and glycolysis by facilitating cell surface Glut-1 expression
(Wullschleger et al, 2006). Enhanced PI3K/AKT signaling can promote metabolic
transformation through multiple pathways, including (1) increased surface expression
of nutrient transporters; (2) AKT-dependent stimulation of hexokinase and
phosphofructokinase to drive glycolysis; (3) enhanced transcription of genes involved
in glycolysis and lipogenesis. These evidences suggest that PTEN regulated
PI3K/AKT signaling may control metabolism in addition to growth and survival.
However, how and why PTEN may regulate metabolism and its contribution to tumor
progression is not clear.

Otto Warburg first proposed the theory that deficiency in energy metabolism,
especially in mitochondrial function, may be the basic cause of cancer. It was stated
that cancer cells prefer to use glycolysis over mitochondrial oxidative phosphorylation
(OXPHOS) for glucose-dependent ATP production (Warburg, 1956). In non-
proliferating tissues such as heart or muscle, cellular bioenergetics are directed toward
OXPHOS. Enzymes of the tricarboxylic acid (TCA) cycle, resident in the
mitochondrion, are facilitating the oxidation of pyruvate and other substrates for ATP
production through electron transport-coupled OXPHOS. The fundamental metabolic
switch in tumor cells mainly focuses on the induction of aerobic glycolysis. AKT, the
oncogenic protein is found to induce HKII to promote aerobic glycolysis. How
mitochondria are being regulated during this switch is not clear.

7
1.3 Mitochondria and Liver Diseases
1.3.1 Mitochondrial Genetic System and Biogenesis
Mitochondria are ubiquitous membrane-bound organelles that are a defining
and unique feature of the eukaryotic cell. Each mitochondrion has multiple copies of
its own covalently closed circular DNA (mtDNA), which is responsible for coding
and synthesizing 13 essential subunits of the inner membrane complexes of the
respiratory machineries. The mitochondrial genetic apparatus has features that are
distinct from the nuclear system. The mammalian cells generally have 103-104 copies
of mtDNA per organelle. The mitochondrial genome is presented as a closed circular
molecule of ~16.5 kb and is composed of heavy and light strands, which replicate in
opposing directions. Protein coding genes within the mtDNA include the following:
cytochrome oxidase (COX) subunits 1, 2, 3; NADH dehydrogenase subunits 1, 2, 3, 4,
4L, 5 and 6; ATP synthase (ATPS) subunits 6 and 8; cytochrome b and 22 tRNA-
coding genes (Richard C. Scarpulla, 2008).

1.3.2 Regulation of mitochondrial functions: Nuclear respiratory factor-1
The majority of protein subunits that comprise the five inner membrane
complexes of the electron transportation chain, oxidative phosphorylation system and
other gene products necessary for mitochondrial functions are derived from nuclear
genes. Therefore, mitochondria rely heavily on the expression of nuclear genes for its
biological functions (Hagir B. Suliman et al., 2003; Richard C. Scarpulla, 2008;
Claude A. Piatadosi and Hagir B Suliman, 2006; Lehman J. J et al., 2000; Virbasius J.
V et al., 1994). Nuclear factors, such as Nuclear Respiratory Factor-1 (NRF-1) and
PPARcoactivator-1 α (PGC-1 α) are important for mitochondrial components
synthesis and are found to be up-regulated when mitochondrial mass is increased

8
(Hagir B. Suliman et al., 2003). Nuclear Respiratory Factor-1 (NRF-1) plays an
important role in the interaction between the nucleus and mitochondria. (Richard C.
Scarpulla, 2008; Claude A. Piatadosi and Hagir B Suliman, 2006; Virbasius JV et al.,
1994). The NRF-1 binding sites are present in the promoters of several genes required
for mitochondrial respiratory function. The NRF-1 protein binds to its recognition site
as a homodimer through a unique DNA binding domain and regulates the expression
of genes, such as TFAM, TFB1M and TFB2M (Virbasius JV et al, 1994). These later
genes, when transcribed, translocate to the mitochondria and act as transcriptional
factors for mitochondrial encoded genes. In addition, NRF-1 also regulates several
genes whose functions are not directly linked to mitochondria. Most of these genes
are responsible for encoding metabolic enzymes, signaling pathway components and
nucleic maintenance regulators (Irfan A Asangani et al., 2007; Bindu Ramachandran
et al., 2008).

1.3.3 Mitochondrial Dysfunction in Non-Alcoholic Fatty Liver Disease
Mitochondria play a major role in regulate liver fatty acid metabolism. In
normal subjects, hepatic free fatty acids (FFAs) are derived from adipose tissue,
hydrolysis of chylomicrons from the intestine, or direct synthesis within hepatocytes
through lipogenesis. FFAs can either enter into mitochondria to undergo β-oxidation
or be synthesized into triglycerides, which is a storage form of three FFAs bound to
glycerol by ester bonds. The triglycerides will either accumulate within the cytoplasm
of hepatocytes as fat droplets or secrete as very low density lipoproteins (VLDL) in
plasma. After a carbohydrate meal, glucose and insulin levels are both increased,
which will in turn trigger the hepatic fatty acid synthesis. The enzyme Malonyl-
Coenzyme A (Malonyl-CoA), which is responsible for the first step in the synthesis of

9
fatty acids from acetyl-CoA, can inhibit the activity of carnitine palmitoyl transferase
I (CPT-I) whose function is transporting the long chain fatty acids into mitochondria
to undergo fat oxidation. Therefore, fatty acids will be used to synthesize triglycerides
instead of being degraded within the mitochondria. During fasting, hepatic FFA
synthesis and the Malonyl-CoA levels are low, allowing the import of FFA into
mitochondria, inducing β-oxidation to occur. In mitochondria, the β-oxidation of fatty
acids into acetyl-coenzyme A (acetyl-CoA), along with its subsequent oxidation by
the TCA cycle, could generate NADH and FADH
2
, which transfer their electrons to
the mitochondrial respiratory chain located in the inner mitochondrial membrane. As
a consequence, energy for cellular function is generated in the form of ATP.
In patients with hepatic steatosis and non-alcoholic steatohepatitis (NASH),
the mitochondria have a decreased ATP-synthesis rate and other alterations (Karima
Begriche et al., 2006; Pessayre D and Fromenty B, 2005). Although the mechanisms
of these mitochondrial abnormalities are unknown, several vicious cycles are believed
to be involved. Adipocytes, Kupffer cells and the hepatocytes secrete tumor necrosis
factor- α (TNF- α), which can trigger the caspase-8 activation. As a result, the
permeabilization of mitochondrial membrane occurs, causing the release of
cytochrome c, which partially blocks the electron flow within respiratory chain.
Concomitantly, the β-oxidation has the ability to increase the delivery of electrons
into the respiratory chain so that reactive oxygen species (ROS) formation is further
increased (Dominique, 2007). These reactive species may deplete mtDNA. Moreover,
mitochondrial ROS formation along with liver lipid accumulation could generate
biologically active lipid peroxidation products such as 4-hydroxynonenal (4-HNE).
The overproduction of ROS also increases expression of several cytokines including
Fas Ligand, TNF- α, transforming growth factor- β (TGF- β) and interleukin-8 (IL-8)

10
which are involved in development of different liver lesions via different mechanisms
(Pessayre D and Fromenty B, 2005; Leonarduzzi G et al., 1997; Parola M et al., 1993).
Hypothesis and Aims
In the PTEN null model, we observed fatty liver phenotype similar to NASH.
Since NASH is often linked to mitochondrial functional alterations, we suggest that
PTEN loss may also lead to mitochondrial function. Our preliminary analysis
suggests that PTEN may regulate mitochondrial function through NRF-1. Here, we
will test the hypothesis that PTEN regulates PI3K signaling may control NRF-1
stability and localization.
Aim 1: To determine how NRF-1 expression, mitochondrial biogenesis and
biological function are regulated by PTEN loss.
Aim 2: To identify what specific PI3K downstream factors are involved in
NRF-1 regulation and the mechanism of how NRF-1 is regulated.

11
Materials and Methods

Materials

The primary and secondary antibodies including anti-Actin, anti-Phospho-
AKT (Ser473) and anti-PTEN were obtained from Cell Signaling (Beverly, MA).
Anti-NRF-1 was purchased from Abcam (Cambridge, MA), Anti-GSK-3 β was
purchase from BD Biosciences (San Jose, CA). Secondary antibodies including anti-
rabbit lgG, anti-mouse lgG and protein G used for immunoprecipitation were
purchased from GE healthcare (Anaheim, CA). The Phosphoinositol 3-Kinases
inhibitor LY 294002 was obtained from Promega Corporation (Madison, WI) and was
used at the concentration of 10 μM diluted in DMEM. Lipofectamine 2000 used for
transfection, MitoTracker Mitochondrion-Selective Probes used for mitochondrial
staining and the pcDNA3.1/V5-His TOPO vector kit used for NRF-1 cDNA cloning
were bought from Invitrogen (Carlsbad, CA). The siRNA against PTEN was a
generous gift from Dr.Deborah Johnson’s laboratory.

Cell Lines and Culture Conditions
The mice hepatocytes cell lines 5019 (PTEN null) and 644 (PTEN wild type)
are generated by our laboratory. The human breast cancer cell line HeLa is also used
in this study. The 5019 and 644 cell lines were cultured in Dulbecco’s modified
Eagle’s medium (Mediatech Inc, Manassas, VA) supplemented with 10% fetal bovine
serum, 1% penicillin and streptomycin, 5 μg/ml insulin and 10 μg/ml Epidermal
growth factor. HeLa cells were cultured in DMEM supplemented with 10% fetal
bovine serum. All cells were incubated at 37
o
C and 5% CO
2
atmosphere.

12
Mitochondrial Staining
The mitochondria of PTEN wild type and PTEN mutant hepatocytes were
stained with MitoTracker Mitochondrion-Selective Probes (Invitrogen, Carlsbad, CA)
and observed under fluorescent microscope. Hepatocytes pellet was obtained by
centrifuging and the supernatant is aspirated. Cells were resuspended gently in
prewarmed (37
o
C) staining solution containing the MitoTracker probe (diluted at the
ratio of 1:100000 in DMEM) and incubated 30 minutes under normal growth
conditions. After staining, cells were re-pelleted and resuspended in fresh prewarmed
medium and analyzed by fluorescent microscope.

Total Cell Protein Lysate Preparation
After cells were cultured with specific drugs or reagents for various periods,
cells were harvested by adding cell lysis buffer to the plate, scraping directly on Petri
dish, vortexing every 5 minutes for 30 minutes and centrifuging for 15 minutes at the
speed of 14000 rpm. The amount of lysis buffer added to the plate depends on the size
of plates. After centrifuging, the liquid in the middle of tube is collected and
transferred into a new EP tube. Protein concentrations were measured using the Bio-
Rad Dc Protein Assay reagents (Bio-Rad Laboratories, Hercules, CA). For western
blot analysis, a combination of SDS loading buffer and 70 μg amount of protein were
prepared. The mixture was vortexed and heated in a water bath at 95
o
C for 2-5
minutes to allow complete disruption of three-dimensional structure of proteins. The
mixture was then spun down before loading onto the gel. Various proteins were
separated by SDS-PAGE gel.

13
SiRNA Silencing
The siRNA against PTEN was transfected into HeLa cells using reagent
Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Cells were seeded into 6-well plates
one day before transfection and 30-50% confluent was reached at the time of
transfection. For each well, 5 μL lipofactamine 2000 was diluted in DMEM and then
mixed with 100 pmol siRNA PTEN. After 20 minutes incubation at room temperature,
the complex was added to each well. Total cell lysate was obtained after 24 hours
incubation at normal cell culture condition.

Western Blotting
Primary antibodies were diluted 1:1000 in 1% BSA in TBS-Tween (0.1%).
Secondary antibodies were diluted at the concentration of 1:3000 (Mouse) or 1:5000
(Rabbit). Membranes were probed with specific diluted antibodies and were incubated
on shaker at 4
o
C overnight.
Fresh TBS-Tween was used for each wash. Primary-probed membranes were
washed 10 minutes for three times. Membranes were incubated with secondary
antibodies on shaker for 1 hour at room temperature. Membranes were washed three
times with TBS-Tween as described earlier.
To detect signals, the secondary antibodies were linked to horseradish
peroxidase. Signals were detected by chemiluminescence using the ECL Western
Blotting Substrate (Pierce, Rockford, IL). The membranes were incubated with ECL
substrate mixture solution for one minute before exposure. Kimwipes were used to
remove the excessive solution after one minute of incubation. Membranes were held
between two pieces of transparency and were placed in autoradiography cassette for
exposure. Blue X-Ray films from Bioland were placed on top of membranes and the

14
whole process was carried out in dark room. Both long and short exposure times were
needed in order to get a clear film. Several exposures were obtained to ensure
consistency of the results.

Immunoprecipitation
Cell lysates were collected by the way described above. Primary antibody for
pulling down was added into 200 μL cell lysate and the complex was rocked gently
overnight at 4
o
C. Protein A or G was added into the mixture and incubated for 1-3
hours at 4
o
C. Then pellet was obtained by centrifuging the complex and washing.
Pellet was resuspended and mixed with 3XSDS sample buffer. The samples were
heated and loaded on SDS-PAGE gel and then analyzed by western blotting described
above.

Plasmids Expressing NRF-1
NRF-1 expressing vectors were constructed by RT-PCR with platinum Taq
polymerase (Invitrogen, Carlsbad, CA). The NRF-1 coding sequence was amplified
with the NRF-1 primers sense, 5’-ATGGAGGAACACGGAGTGAC-3’, and
antisense, 5’-GCTTTTTGGGACAGTGAAAT-3’. The product was ligated into
pcDNA 3.1/V5-His TOPO TA expression vector to express wild type NRF-1 and
sequenced at USC Norris Cancer Center.

15
Results

The mouse models with liver-specific PTEN deletion were developed by our
laboratory (Rountree CB et al., 2009). Previous data from our lab discovered that
there is more lipid accumulation in the liver of PTEN mutant mice than that of PTEN
wild type at 3 months. As a result, fatty liver, which is believed to be the earliest and
most common stage of hepatocellular carcinoma (HCC), is formed in the liver of
PTEN mutant mice starting at 1 month and progresses through 6-month and 9-month
mice. At the age of 12 months, obvious malignant tumor phenotype could be seen in
PTEN mutant mice.
In this project, we wanted to investigate the pathogenesis of fatty liver found
in the mice with liver genetic PTEN mutation. It has been well established that
mitochondrial dysfunction is a main contributing factor of liver disease (Karima
Bergriche et al., 2006) We investigated the mitochondria status in the hepatocytes of
PTEN null and PTEN control mice. From the EM pictures of PTEN control and
mutant hepatocytes (Figure 2A), we found more fat accumulation in mutant
hepatocytes than that in wild type ones, consistent with our previous reports. In
control hepatocytes, mitochondria were sparsely and evenly distributed. The rarus
cristae, which is the specific mitochondrial structure formed by innermembrane and
containing many respiratory enzymes, are clear. In mutant hepatocytes, we noticed
that mitochondria were smaller than wild type and more densely distributed, highly
compact and contain condensed cristae inside (Figure 2A). These data suggest that
the function of mutant hepatocyte mitochondria might be enhanced since
innermembrane contains key enzymes for respiration. Additionally, MitoTracker
staining indicates the higher mitochondrial intensity in mutant compared to wild type
(Figure 2B). In order to prove that there is indeed a functional change between two

16
Figure 2: PTEN loss induces mitochondrial intensity and mitochondrial respiration level.

A: Electron microscope pictures of PTEN null and PTEN wild type hepatocytes. B:
Mitochondria staining of hepatocytes. PTEN null and PTEN wild type hepatocytes were
stained by MitoTracker and pictures were taken under fluorescence microscope. C, D: ATP
production and RCR ratio. ATP production and the relative control respiration ratio of PTEN
null and PTEN wild type were measured.
(The work in this figure was done by other laboratory colleagues).

17
genotypes, the ATP production and respiration control ratio(RCR) were measured and
results are showing in Figure 2C. The ATP production and RCR were both higher in
mutant than in wild type, indicating the mitochondrial function is indeed increased
between PTEN control and mutant hepatocytes.
To investigate the mechanism by which PTEN mutation induces
mitochondrial biogenesis, we decided to look at the NRF-1 expression level in PTEN
mutant and control hepatocytes. NRF-1 is a key transcription factor for mitochondrial
biogenesis (Richard C. Scarpulla, 2008). When we looked at the NRF-1 expression
status by western blot of cell lysates samples of PTEN mutant and control hepatocytes,
we found a dramatic induction of NRF-1 protein expression as a consequence of
PTEN mutation (Figure 3). This occurred at the same time as increased phospho-
AKT expression (Figure 3). HeLa cell lysates were serving as the positive control.
These findings above suggest that the increasing NRF-1 expression may play an
important role in the up-regulation of mitochondrial biogenesis by PTEN loss.

18
Figure 3: PTEN knock-out induces mitochondrial biogenesis through regulation of
NRF-1 by PTEN/AKT signaling.

A

B

A, PTEN null and PTEN wild type hepatocytes were cultured by conditions described in
materials and methods, total cell lysates were prepared for western blot analysis with specific
antibodies against Actin, which is loading control, Phospho-AKT (Ser 473), PTEN and NRF-
1. B, Quantification of the western blot results in A. Each bar indicates the density ratio of
NRF-1/ACTIN.

NRF-1/ACTIN
0
0.2
0.4
0.6
0.8
1
1.2
PTEN WT PTEN MUT
OD

19
To determine if the effects of PTEN loss on NRF-1 expression is direct and
not through secondary changes in the PTEN mutant cells, we transfected siRNA
against PTEN into HeLa cells to mimic the PTEN knock-out condition in mice liver
and analyzed the signaling pathway related to PTEN and NRF-1. When siRNA was
transfected into HeLa cells, we saw a significant drop of PTEN protein expression,
indicating siRNA was working properly (Figure 4). P-AKT and NRF-1 expression
both increased almost 2 or 3 folds as a result of PTEN silencing. In conclusion, the
siRNA PTEN experiment further confirmed that NRF-1 is up-regulated by
PTEN/PI3K/AKT axis.

20
Figure 4: Decreased PTEN expression enhances NRF-1 expression in HeLa cells.

A

B

A, HeLa cells were transfected with PTEN siRNA or control mmRNA. Total protein was
isolated, and immunoblot analysis was performed using antibodies against PTEN, Phospho-
AKT (Ser473), NRF-1 and Actin. B, Quantification of the western blot results in A. Each bar
indicates the density ratio of NRF-1/ACTIN.

SiRNA for PTEN
NRF-1/ACTIN

OD
0

0.4

0.2
0.6
0.8
1
Scrambled siRNA

21
The PI3K pathway is the main target of PTEN and AKT is a major
downstream target of PI3K signaling. AKT plays an important role in cell growth,
proliferative and survival. We hypothesized that PTEN loss is regulating NRF-1
expression through PI3K/AKT pathway.
To investigate how PI3K/AKT signaling affects NRF-1 expression, we
stimulated the PI3K/AKT signaling pathway by using the insulin-like growth factor-1
(IGF-1), a polypeptide protein hormone similar in molecular structure to insulin and
plays important role in cell growth. IGF-1 binds to specific IGF-1 receptors presented
on many cell types and it is also one of the most common activators of AKT signaling.
We found that P-AKT level was increased as a result of IGF-1 stimulation
both in PTEN control and PTEN mutant hepatocytes (Figure 5A). An induction of
NRF-1 expression was seen in PTEN mutant hepatocytes upon IGF-1 stimulation.
NRF-1 was hardly detectable in PTEN control liver cells even with IGF-1 stimulation
(Figure 5A). Then we did the IGF-1 treatment time course using HeLa cells and found
that P-AKT level was going down when HeLa cells were deplete of serum for 24
hours and P-AKT expression started going back from the 4 hours time point of IGF-1
treatment and became stronger at 8 hours. Correspondingly, there was also an obvious
stimulation of NRF-1 expression at 8 hours with IGF-1 treatment (Figure 5B).

22

Figure 5: NRF-1 expression can be regulated by IGF-1 associated with the up-regulation
of AKT.

A B
A: PTEN null and PTEN wild type hepatocytes were cultured in standard cell culture medium
with the presence of IGF-1 whose concentration was 100ng/ml. Cell lysates were collected for
western blot, which was conducted with specific antibodies against Phospho-Akt (Ser473),
NRF-1, PTEN, P-S6 and Actin. B: HeLa cells were cultured at the same condition as
described above. The cells were starved for 24 hours before the IGF-1 treatment and then
were given IGF-1. Cell lysastes were collected at 0, 4 hours, 8 hours respectively and western
blot was performed with antibodies against Phospho-AKT (Ser473), NRF-1 and Actin.

23
To inhibit PI3K signaling, we used LY294002, an effective PI3K inhibitor
(Hagir B. Suliman et al., 2003). As shown in Figure 6, both LY294002 short time
course and long time course were conducted with HeLa cells. Treatment with
LY294002 is able to inhibit p-AKT up to 2 hours. AKT phosphorylation recovered
after 2 hours (Figure 6A). Surprisingly, we did not observe obvious change of NRF-1
level (Figure 6A). The inability of LY294002 to produce sustained AKT
phosphorylation may be the cause for this ineffectiveness seen with NRF-1.

24
Figure 6: The effects of PI3K downstream factor P-AKT on NRF-1 expression.
A.

B

A: HeLa cells were cultured in medium described above with the presence of LY294002 with
the concentration of 10 μM. Total cell lysate was collected at different time points of 0 hour,
1 hour, 2 hours, 4 hours, 6 hours and 8 hours. Western blot was conducted using antibodies
against Phospho-AKT (Ser473), NRF-1 and Actin. B: HeLa cells was treated with LY294002
with the concentration of 10 μM, total cell lysates were collected at time points of 0 min, 5
mins, 15 mins, 30 mins and 60 mins. Western blot was performed with antibodies against
Phospho-AKT (Ser473), NRF-1 and Actin.

25
While LY294002 did not produce the expected suppression of NRF-1, IGF-1
treatment and siRNA PTEN experiment strongly suggest that PI3K signaling is
involved in the increased expression of NRF-1. Furthermore, we have observed an
association of NRF-1 expression with increased AKT phosphorylation previously.
When examining the influence of cell growth phase on NRF-1 expression by different
cell growth phases, we found that when cells reached 80% confluence, P-AKT level
increased dramatically compare to sparsely grown cells (approximately 30%
confluence). Similarly, the tendency of NRF-1 expression level was consistent with
the increase of P-AKT (Figure 7). To further explore the involvement of AKT, we
decided to test whether AKT activity is affecting NRF-1 expression. The AKT
transfection experiment was performed by other laboratory colleagues. We found
NRF-1 expression increased upon the expression of constitutive active AKT (Figure
8).

26

Figure 7: NRF-1 expression can be regulated by cell culture confluence through
phosphorylation of AKT.

PTEN null and PTEN wild type hepatocytes were cultured under standard conditions, cell
lysates were collected at log phase and subconfluent phase for western blot using antibodies
against NRF-1 and Phospoho-AKT(Ser473) and Actin.

27

Figure 8: NRF-1 expression is induced by constitutively active AKT construct
transfection.

PTEN wild type hepatocytes were transfected with constructs expressing GFP, wild type
AKT, constitutively active AKT (ca-AKT) and dominantly negative AKT (dn-AKT). Cell
lystates were collected and analyzed by western blot. Result is showing the NRF-1 and P-
AKT immunoblots.

28
Figure 9: Brief summary of conclusion implicated by above experiment.

PI3K involvement is confirmed by conditions of PTEN Loss, SiPTEN and IGF-1 treatment.
The association of P-AKT and NRF-1 is also found.

29
To explore the downstream factors of AKT, we evaluated the effects of
rapamycin, an inhibitor of mTOR and LiCl, an inhibitor of GSK3 β. We found that
rapamycin did not have any effect on NRF-1 expression. LiCl treatment induced a
moderate increase of NRF-1. To confirm this finding, we performed
immunoprecipitation experiment in order to determine whether GSK-3 β is directly
associated with NRF-1 regulation. GSK-3 β was first pulled down by primary antibody
against GSK-3 β. The pellet was loaded on SDS-PAGE gel and immunoblot with
NRF-1 antibody. We found there is an association between NRF-1 and GSK-3 β in
PTEN mutant hepatocytes (Figure 10). Since it has been proved that GSK-3 β binds to
β-catenin and targets for degradation, we immunoblot β-catenin to confirm the
specificity of the pulldown experiment. We found the GSK-3 β and β-catenin binding
from the results (Figure 10), suggesting that NRF-1 is likely bound to GSK3 β like β-
catenin. The wild type and mutant input were serving as control.

30

Figure 10: Immunoprecipitation for GSK3 β and NRF-1/ β-Catenin.

Immunoprecipitation of GSK-3 β using primary antibody against GSK-3 β and then
immunoblot with NRF-1 and β-catenin using samples of PTEN wild type and mutant
hepatocytes.

31
To investigate how NRF-1 may be regulated by PTEN signaling, we
performed qPCR analysis of NRF-1 expression. Our results indicate that there is no
difference in NRF-1 mRNA between PTEN mutant and control hepatocytes. Further,
our data in figure 5 showed that PI3K regulated NRF-1 expression change requires
long time course. Thus, we suggest that the stability of NRF-1 might be regulated by
GSK-3 β through degradation mechanisms, similar to the relationship of GSK-3 β with
β-catenin. In order to study the NRF-1 stability difference between PTEN control and
mutant, we cloned the NRF-1 gene using PCR cloning technique. First, we used NRF-
1 primers that have been reported before (Hagir B .Sliman et al., 2003) to amplify the
NRF-1 cDNA. Figure 11B shows the product after the reverse-transcription PCR. The
product length is 1.7 kb (Figure 11A). This product was inserted into pcDNA 3.1/V5-
His TOPO vector (Figure 11C). The vector contains poly A tail and His tag, allowing
expression and detection of NRF-1. The plasmids containing NRF-1 were extracted
by Mini Prep after transformation in E.Coli and confirmed with enzyme digestion for
colonies that contain NRF-1 cDNA insert (Figure 11D). After sequencing the
plasmids, we used Midi Prep to extract large amount of NRF-1 expressing plasmids
which can be used in our further NRF-1 research. Figure 11E is the enzyme digestion
analysis of plasmids extracted from Midi Prep.
The NRF-1 expressing constructs will be used in further experiments investigating
NRF-1 stability such as protein synthesis inhibitor cycloheximide (CHX) treatment.

32
Figure 11: Cloning of NRF-1 cDNA.
A

B

C

A. NRF-1 and primer information. B. Reverse-transcription PCR products using primer for
NRF-1 cDNA amplification described in materials and methods. C. pcDNA 3.1/V5-His
TOPO vector information. E. DNA gel of plasmid extracted by Mini Prep from E.Coli after
transformation (Left panel) and DNA gel after enzyme digestion (Right panel). E. DNA gel of
plasmid extracted by Midi Prep and DNA gel of enzyme digestion.

33
Figure 11, Continued.
D

E

34
Discussion

PTEN was first cloned in 1997 and has been studied for almost 20 years.
Many reports have demonstrated that there is high mutation frequency of PTEN in
several cancer types, proving that PTEN is a tumor suppressor gene (Bangyan Stiles
et al., 2004). Research on PTEN led to the discovery that the PI3K/AKT pathway is
involved in human cancers. Many surveys indicate that PTEN loss occurs in a wide
range of cancers, including HCC (Igor Vivanco and Charles L. Sawyers, 2002). From
our mice model, which has PTEN specifically deleted in the liver, we saw tumor
phenotype at 9 months (Xu et al 2006), which allow us to confirm the previous studies,
suggesting that PTEN is an important tumor suppressor gene.
To further study the features of liver disease that harbors PTEN mutation, we
applied electron microscope analysis on hepatocytes of PTEN wild type and mutant
and we saw a large difference from the electron microscope pictures between two
different genotypes. We noticed that mitochondria in mutant hepatocytes were highly
compact and have more cristae compared to PTEN wild type. The quantity of
mitochondria was also increased due to PTEN loss. The MitoTracker showed higher
mitochondrial intensity in PTEN mutant hepatocytes, indicating functional changes
may occur in PTEN mutant, which was further confirmed by ATP and RCR
measurement (Figure 2). When we went on and analyzed the expression of NRF-1 in
two different hepatocytes, we found the dramatic induction of NRF-1 in PTEN null
cells (Figure 3).
Liver is the major organ associates with many physiological functions, such as
energy storage, digestion, production and detoxification. It is easy for liver to suffer
the metabolic disorder (Karima Begriche et al., 2006). In addition, it is thought that
the non-alcoholic fatty liver disease (NAFLD) is actually a disease of mitochondria.

35
Our data showing that loss of Pten leads to fatty liver development and dysfunction in
mitochondria suggest that PTEN may regulate liver metabolism. Early in 1930, Otto
Warburg claimed that cancer is primarily caused by dysfunctionality in mitochondrial
metabolism, rather than because of uncontrolled growth of cells (Warburg, 1956).
Tumor cells need to obtain metabolic changes in order to fulfill the increased energy
demands for cell growth, replication and proliferation. Tumor cells have glycolysis
rates that are almost 200 times higher than those of normal tissues. A number of genes
regulating tumor development have been found to be associated with metabolic
regulation. However, how such regulation occur is not clear. Our model suggests
that PTEN, a tumor suppressor, may control metabolism through the mitochondria.
Our data suggest that PTEN deletion may enhance mitochondrial biogenesis and
mitochondrial biological function through increasing the levels of NRF-1.
Many oxidants, toxins and drugs, such as lipopolysaccharide (LPS), pyruvate,
thiazolidinediones (TZDs) have been reported that they could enhance mitochondrial
biogenesis through different mechanisms (Hagir B .Sliman et al., 2003; Leanne
Wilson et al., 2006; Kazuo Fujisawa et al., 2008). Hagir B .Suliman research group
reported that LPS could stimulate the mitochondrial biogenesis by activating NRF-1.
They found that LPS could induce NRF-1 expression and also NRF-1 DNA binding
ability. Additionally, they showed P-AKT level was enhanced by LPS treatment
(Hagir B .Sliman et al., 2003). We found NRF-1 protein levels was enhanced because
of PTEN deletion from our western analysis of PTEN wild type and PTEN mutant
hepatocytes cell lysates (Figure 3), indicating that the increased mitochondrial
biogenesis and biological functions due to PTEN deletion may be through NRF-1 up-
regulation. A series of experiments were also performed in order to further confirm
this phenomenon. SiRNA against PTEN was used in our experiments to decrease the

36
expression of PTEN in HeLa cells. After PTEN was significantly decreased, P-AKT
and NRF-1 expression levels were both going up which was the strong evidence to
support the previous finding (Figure 4). We have also used IGF-1 to stimulate the
PI3K/AKT pathway to mimic the same effect of PTEN knock-out since IGF-1
induces the PI3K/AKT signaling axis to transmit growth signals. From results in
figure 5, we found NRF-1 was induced in PTEN mutant hepatocytes while P-AKT
level was also up-regulated, indicating a strong connection between NRF-1
expression and the growth signaling PI3K/AKT axis. Taken together, all these
findings suggest that PTEN deletion gives rise to the activation of P-AKT, leading to
increased expression of NRF-1 and enhanced mitochondrial biogenesis. These
findings are collaborated with the expression of a constitutively active form of AKT.
A conflict in our analysis is the inability of LY294002 to inhibit NRF-1
expression. Within the LY294002 long term treatment experiments, P-AKT was
completely decreased at 1 hour and then increased due to feedback effect and P-AKT
expression was decreasing throughout the entire short term LY294002 treatment
(Figure 6). However, the NRF-1 level did not change significantly within either
treatment. The reason might be that the PI3K inhibition effect of LY294002 is not
very effective because of the feedback effect came out really soon after LY294002
treatment started, which could be seen from figure 6A. It is possible that the PI3K
inhibition time period is not long enough to reveal the NRF-1 regulation. Although P-
AKT level was indeed decreased in our LY short time treatment (Figure 6B), the time
required by NRF-1 change to appear is possibly still too short and that is possibly why
there were no significant NRF-1 expression change within our LY294002 treatments.
GSK-3 β is an important regulator of cellular metabolism. GSK-3 β is present
in a protein complex together with Axin, APC and β-cateinin. This complex

37
functions to target β-catenin for degradation. One of the signals that target β-catenin
for degradation is phosphorylation by GSK3β. We hypothesized that NRF-1 might be
regulated in the same way. Based on our preliminary data, the NRF-1 regulation by
PTEN loss is mainly on the translational rather than transcriptional level. Our
immunoprecipitation data with GSK-3 β and NRF-1 showed that there was an
association between GSK-3 β and NRF-1. The immunoblot of β-catenin confirmed
GSK-3 β was indeed pulled down by primary antibody (Figure 10). This finding
suggested GSK-3 β might be the PI3K downstream factor playing a role in regulating
the NRF-1 stability.
A few papers had investigated the connection between NRF-1 expression and
AKT pathway. The Suliman research group reported that the DNA binding ability of
NRF-1, NRF-1 phosphorylation by P-AKT and translocation to nucleus are enhanced
upon oxidant stimulation (Hagir B .Sliman et al., 2003; Claude A. Piantadosi and
Hagir B. Suliman, 2005). However, the exact mechanism of how NRF-1 is being
phosphorylated and translocated into nucleus is not clearly understood. We found that
NRF-1 level was up-regulated by PTEN deletion from our work. So far, based on the
work of mitochondrial staining, western analysis of mutant and wild type hepatocytes,
IGF-1 treatment, siRNA transfection, our evidence prove that NRF-1 expression,
mitochondrial biogenesis and mitochondrial function are induced due to PTEN
mutation and further AKT activation. Based on the experiment of
immunoprecipitation for GSK-3 β and NRF-1 blotting, we suggest that GSK-3 β might
be involving in NRF-1 regulation.

38
Future Direction

We have generated plasmids expressing NRF-1, we plan to study the
difference of stability of NRF-1 between PTEN control and mutant using this
construct. And applying different AKT constructs to investigate NRF-1 status might
be another choice. If this is confirmed, we will further dissect the NRF-1 molecule
and explore how phosphorylation by GSK3b and AKT or cellular localization affects
it stability.

39
Summary

In conclusion, this study provides evidence that the mitochondrial biogenesis is
up-regulated by PTEN signaling through P-AKT activation and increases in NRF-1
protein levels in both hepatocytes and HeLa cells. Since PI3K/AKT signaling is crucial
for cell growth, survival or apoptosis and mitochondria, this research provides
implication for the pathogenesis of non-alcoholic fatty liver disease.

40
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Abstract (if available)

Abstract Based on many previous reports regarding cancer metabolism, it is believed that tumor cells need to obtain metabolic changes to maintain the malignant phenotype. Additionally, many PTEN-related studies revealed that PTEN mutation within the liver will eventually lead to liver cancer. The aim of this paper is to investigate the mechanism of how PTEN deletion gives rise to tumor phenotype and what kind of biological metabolism is suffering dysregulation due to PTEN mutation. In our results, mouse livers with PTEN mutation exhibited more lipid accumulation compared to PTEN wild type livers. PTEN mutant hepatocytes showed more mitochondrial biogenesis and enhanced respiration function than that in PTEN wild type hepatocytes. By performing a series of experiments, such as siRNA transfection, insulin-like growth factor-1 treatment, we concluded that NRF-1, which is an important transcription factor for mitochondrial biogenesis, is up-regulated by PTEN deletion through the activation of Phospo-AKT. This leads to more mitochondrial biogenesis and mitochondrial dysfunction, which may further cause tumor. This finding confirms previous studies and gives implication of the non-alcoholic fatty liver diseases (NAFLD) pathogenesis.

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Asset Metadata

Creator Li, Yang (author)

Core Title Nuclear respiratory factor-1 regulation by PTEN signaling

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Degree Conferral Date 2009-08

Publication Date 08/07/2009

Defense Date 06/26/2009

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag fatty liver disease,mitochondria respiration,NRF-1,OAI-PMH Harvest,PTEN

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Tokes, Zoltan A. (committee chair), Hong, Young Kwon (committee member), Stiles, Bangyan L. (committee member)

Creator Email likesai123456@gmail.com,yli11@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2528

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Rights Li, Yang

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