Citation
Regulation of Manganese Superoxide Dismutase via Amino Acid Deprivation

Material Information

Title:
Regulation of Manganese Superoxide Dismutase via Amino Acid Deprivation
Creator:
AIKEN, KIMBERLY JEAN ( Author, Primary )
Copyright Date:
2008

Subjects

Subjects / Keywords:
Amino acids ( jstor )
Cell growth ( jstor )
Gene expression ( jstor )
Hep G2 cells ( jstor )
Hormones ( jstor )
Manganese ( jstor )
Messenger RNA ( jstor )
Nutrients ( jstor )
Phosphorylation ( jstor )
Superoxides ( jstor )

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Source Institution:
University of Florida
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University of Florida
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Copyright Kimberly Jean Aiken. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
8/31/2006

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Full Text












REGULATION OF MANGANESE SUPEROXIDE DISMUTASE VIA AMINO ACID
DEPRIVATION















By

KIMBERLY JEAN AIKEN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006

































Copyright 2006

by

Kimberly Jean Aiken


































To my mother, the strongest person I have ever known, always inspiring me to do the
best that I can.















ACKNOWLEDGMENTS

I would like to thank my mentor, Dr. Harry Nick. He has been a great teacher to

me in science and life. I am grateful for all of the support he has always offered to me

and for the opportunity to work in his lab. I would also like thank all of my committee

members (Drs. Bloom, Kilberg and Swanson). They have always been great to work

with and instrumental in the completion of my thesis.

My time spent in the lab would not have been the same without all of my lab mates;

especially Ann Chokas and JD Herlihy, my partners in crime. I have also enjoyed

meeting all of the colorful people in Gainesville; all of my different teammates, the

foreign contingency, and of course all of my fellow classmates. I would also like to

thank my family, who have known me all my life and are still my friends.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES .................................................... ............ ............. .. viii

LIST OF FIGURES ......... ......................... ...... ........ ............ ix

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

Manganese Superoxide Dismutase.............................. ....................2
Superoxide Dismutases and Reactive Oxygen Species......................................2
Cytoprotective R oles of M nSO D .......................................................................4
Transcriptional Regulation of M nSOD ...............................................................5
Nutrient Regulation of Gene Expression ....................................... ...............7
Glucose Control of Gene Regulation ....................................... ............... 7
Am ino A cid Control of Gene Regulation................................... .....................9
Amino Acids as Signaling M olecules .......................... ...... ....... ........... 11

2 CHARACTERIZATION OF MANGANESE SUPEROXIDE DISMUTASE BY
AM INO ACID CONTROL ......................................................... ............... 18

In tro d u ctio n ........................................................................................1 8
M materials and M methods ....................................................................... .................. 19
C e ll C u ltu re ................................................................................................... 1 9
Isolation of total R N A .............................................. ..... ............. ............... 20
Electro-Transfer and Northern Analysis .................................. ............... 21
Protein Isolation and Immunoblot Analysis....... .... ..................................... 21
D ensitom etry and Statistical Analysis...................................... ............... 22
R e su lts .................. .. ......... ...... ..... ........................... ....................... ........... 2 3
Amino Acid Deprivation Induces MnSOD Steady State Messenger RNA
L levels ...................................................... .. ... .. .. .. .. .... ........... 23
Induction of MnSOD is Specific to Amino Acid Deprivation............................24
Induction of MnSOD Messenger RNA is Specific to Essential Amino Acid
D deprivation ................. .. ...................... .....................................27
Cellular Specificity of Amino Acid Deprivation for MnSOD Induction ............28
Transcriptional control of MnSOD in response to amino acid deprivation ........30
D isc u ssio n .................................................................................. 3 1


v









3 METABOLIC CONTROL OF MANGANESE SUPEROXIDE DISMUTASE IN
RESPONSE TO AMINO ACID DEPRIVATION .......................... .....................34

In tro du ctio n ...................................... ................................................ 3 4
M materials and M methods .............................................. .................. ............... 36
Isolation of T otal R N A .......................................................................... .... ... 36
M easurem ents of ATP Levels .................................................. ...................36
R e ag en ts U se d ............................................................................................... 3 6
R esu lts ...................... ............................................ ........ ... ... ... ............... 3 7
Glutamine is Required for the Induction of MnSOD by Amino Acid
D e p riv atio n .......................................................... .................. ............... 3 7
Inhibition of the TCA Cycle Blocks MnSOD Induction by Histidine
D epriv action .............. ................................... ... ............ .... ............... 4 3
Both a Functional Electron Transport Chain and F1-Fo ATP Synthase
Complex are Required for MnSOD Induction in Response to Amino Acid
D epriv action ..................................... ...... ........ .... ........ ................ 49
An Intact Electrochemical Gradient but not ATP Synthesis is Required for
M n SO D Induction ......... .............. ...................................... ...... .... .... .. 52
D iscu ssio n ...................................... ................................................. 5 5

4 SIGNAL TRANSDUCTION PATHWAYS ASSOCIATED WITH
MANGANESE SUPEROXIDE DISMUTASE INDUCTION IN RESPONSE TO
AM IN O ACID DEPRIVATION .............. ................................. .....................59

Introduction ............... ........... ........................ ............................59
M materials and M methods .............................................. .................. ............... 63
Isolation of total RN A .....................................................................................63
Protein Isolation and Immunoblot Analysis.................................................63
R e ag en ts U se d ............................................................................................... 6 3
R results ..............................................................3
Requirem ent of ERK 1/2 Signaling.................................. ........................ 63
Amino Acid Deprivation and mTOR Signaling...............................................66
D isc u ssio n ............. ....... ..... ... ................. ................................. 6 9

5 TRANSCRIPTIONAL REGULATION OF MANGANESE SUPEROXIDE
DISMUTASE BY FORKHEAD BINDING PROTEINS IN RESPONSE TO
AM IN O ACID DEPRIVATION .............. ................................. .....................74

Introduction .............. ....... ............. ...... ............................... 74
M materials and M ethods ............................................. .................. ............... 77
Growth Hormone Reporter Constructs............................................ ..........77
O verexpression Plasm ids.......................................................... ............... 78
Quick Change PCR ... ........... ........ .... .. .. .............. .. ................ ........ ......79
Transient Transfection of Reporter Constructs ........... ...............................79
Transfection of FOXO Expression Constructs............. ................................80
Cell Culture and Transfection of siRNAs ............... ............ .....................80
G en eration of cD N A .................................................................. .............. 8 1









R eal-Tim e PCR ..................... ................ ... ................................. 81
Protein Isolation and Immunoblot Analysis .....................................................82
Isolation of N u clei .............................. ......................... ... ...... .. .... ............82
R e ag en ts U se d ............................................................................................... 8 3
R results ............................................................... .. .. ............. ........ 83
Endogenous Regulation of MnSOD by FOXO Transcription Factors ..............83
Transcriptional Regulation of MnSOD in Response to Amino Acid
D ep riv atio n ............... ... ....... ... .... ... ..... .... ... .... ................ 84
FOXO Messenger RNA Levels in Response to Amino Acid Deprivation .........91
Localization of FOX O Proteins ................................. ....................94
D iscu ssio n ...................................... ................................................. 9 5

6 DISCUSSION AND FUTURE DIRECTIONS...................................................98

D iscu ssio n ...................................... ................................................. 9 8
Future D directions .................. ........................................ ............ .. 101

L IST O F R E FE R E N C E S ....................................................................... .................... 105

BIOGRAPH ICAL SKETCH .............................................................. ............... 121
















LIST OF TABLES


Table page

1-1 List of amino acids and their normal cellular functions and conditions of disease
when these amino acids are found in excess or in a depleted state..........................17















LIST OF FIGURES


Figure page

1-1 Catalysis of superoxide to oxygen and hydrogen peroxide through the action of
M n SO D .............................................................................. 3

1-2 Production of free radicals at complex I and III of the electron transport chain........3

1-3 Rat MnSOD genomic clone, with identified sites of regulation. ............................6

2-1 Northern blot analysis of MnSOD in response to various stimuli ........................24

2-2 Northern blot analysis of total RNA isolated from HepG2 cells incubated in
complete medium (FED), or medium lacking either histidine (-HIS) or glucose
(-G L C ) ........................................................................... 2 5

2-3 Densitometric quantification of replicate experiments as done in 2-2 of the
M nSO D 4kb m message ......... ................. ...................................... ............... 26

2-4 Immunotblot analysis of MnSOD in response to histidine deprivation. ................26

2-5 Representative northern blots of HepG2 cells incubated for the indicated times
in medium lacking a single amino acid ........................................ ............... 27

2-6 Representative bar graph of at least three independent experiments as in Figure
2 -5 ............... ............... ............... ................................................ 2 8

2-7 Northern analysis from HUH7 (human hepatoma) cells, incubated for the
indicated amount of time, in various medium conditions ....................................29

2-8 Northern blot analysis of CCDLU, A549 and L2 cells incubated for 12 h, in the
in dictated m ediu m .................. .... .............................. ............... .......... ... .. 30

2-9 Northern blot analysis of cells treated with actinomycin-D or cyclohexamide. ......31

3-1 Northern analysis of HepG2 cells incubated in medium lacking all amino acids
(EBSS), or in EBSS with the inclusion of the indicated amino acid at a
concentration of 5 m M .. .............................. ... ........................................ 38

3-2 HepG2 cells were incubated in various medium conditions with the addition of 5
m M glutam ine for 12 h .............................................................................. .... .......39









3-3 HepG2 cells were incubated in complete medium (FED), medium lacking
glutamine (-GLN), histidine (-HIS) or both amino acids (-HIS/-GLN)...................40

3-4 Northern analysis of HepG2 cells incubated for 12 h in the indicated medium
conditions with increasing concentrations of glutamine. .......................................41

3-5 HepG2 cells were incubated in the presence or absence of vitamins under the
indicated conditions......... ............................................................ ...... .... .... 42

3-6 Northern analysis of total RNA isolated from HepG2 cells incubated with or
without 10% dFBS, or two components of serum, IGF or EGF............................43

3-7 Tricarboxylic acid (TCA ) cycle ........................................ .......................... 44

3-8 Representative northern blot of HepG2 cells incubated for 12 h in complete
medium (FED) or complete medium lacking histidine (-HIS), treated with
increasing concentrations of fluoroacetate. .... .................................................45

3-9 D ensitometric data from three independent experiments following treatment
with fluoroacetate (as done in Figure 3-9). ................................... ............... 45

3-10 Representative northern blot of HepG2 cells incubated for 12 h in complete
medium (FED) or complete medium lacking histidine (-HIS) and treated with
increasing concentrations of a-keto-P-methyl-n-valeric acid (KMV) ...... ........ 46

3-11 Densitometry data collected from three independent experiments following
treatment with KM V (as done in Figure 3-13)............................................. 47

3-12 Representative northern blot of HepG2 cells incubated for 12 h in complete
medium (FED) or complete medium lacking histidine (-HIS) and treated with
increasing concentrations of 3-nitropropionic acid (3-NPA). ...............................48

3-13 Densitometry data collected from three independent experiments following
treatment with 3-NPA (as done in Figure 3-13)............................... ............... 48

3-14 Northern blot of HepG2 cells incubated for 12 h in complete medium (FED) or
complete medium lacking histidine (-HIS) with increasing concentrations of
m alonate. ............................................................................49

3-15 The electron transport chain and its relevant points.............................. .............50

3-16 Northern blot analysis of HepG2 cells incubated for 12 h in complete medium
(FED) or complete medium lacking histidine (-HIS) with increasing
concentrations of antim ycin A .......................... ........ ................................. 51

3-17 Northern blot analysis of HepG2 cells incubated for 12 h in complete medium
(FED) or complete medium lacking histidine (-HIS) with increasing
concentrations of oligomycin. ..... ......................................................................52









3-18 Northern blot analysis of HepG2 cells incubated for 12 h in the indicated
medium for 12 h with the addition of 5 mM glutamine (EBSS +GLN), with
increasing concentrations of the glucose analogue 2-deoxy-D-glucose (2-DOG)...53

3-19 ATP levels from four independent experiments in which HepG2 cells were
incubated in the various conditions ........................................ ....... ............... 54

3-20 Northern blot analysis of HepG2 cells incubated for 12 h in complete medium
(FED) or complete medium lacking histidine (-HIS) with increasing
concentrations of 2,4 dinitrophenol................. ...................... ............... 55

4-1 Northern analysis of HepG2 cells treated for 12 h in FED, -HIS, EBSS or EBSS
+GLN with the indicated inhibitor; SB202 (10 tM), SB203 (10 lM), or a JNK
inhib itor (2 0 .M)..................................................................................... 64

4-2 Evaluation of HepG2 cells treated for 12 h in the indicated medium and the
indicated inhibitor. .................................................. ................. 65

4-3 Northern analysis of HepG2 cells treated for 12 h in FED, -HIS, EBSS or EBSS
+GLN with increasing concentrations of rapamycin. ..................... ............67

4-4 Densitometry of Northern analysis of HepG2 cells treated for 12 h in FED, -
HIS, EBSS or EBSS +GLN with increasing concentrations of rapamycin (as in
Figure 4-3)............................................................................ ......... 67

4-5 Immunoblot analyses of HepG2 cells incubated for 12 h in FED, -HIS, EBSS or
EBSS +GLN medium with the indicated inhibitor and immunoblotted for the
respective proteins............ ..... .... ....................... ........ ........... ..............68

4-6 Relevant signal transduction pathways involved in the induction of MnSOD in
response to amino acid deprivation. ........................................ ....... ............... 70

5-1 Northern blot of HepG2 cells incubated in FED or -HIS medium, with
increasing concentrations of insulin ............................................... ............... 85

5-2 Northern blot of HepG2 cells incubated in EBSS, FED, -HIS or +GLN medium
with or without the PKB inhibitor, LY294002. .................................. ...............85

5-3 Representative northern blot of HepG2 cells transfected with a growth hormone
construct containing the indicated promoter fragments. ........................................87

5-4 Human growth hormone constructs used to evaluate the human MnSOD
prom other deletions .................. ....................................... .. .......... 87

5-5 Representative northern blot of HepG2 cells transfected with a growth hormone
construct containing the respective promoter fragments and the human MnSOD
enhancer. ............................................................................88









5-6 Northern blot of HepG2 cells transfected with a growth hormone construct
containing the TK promoter and the human MnSOD enhancer.............................89

5-7 Human MnSOD promoter constructs generated and tested by histidine
deprivation ...................... ......... ..... ................................. ......... 90

5-8 Northern analysis of growth hormone constructs evaluating the contribution of
the FOXO binding site to M nSOD induction............................... ............... 90

5-9 Densitometric analysis of northern blots as shown in the top panel of Figure 5-9..91

5-10 Overexpression of FOXO proteins with MnSOD growth hormone reporter
plasm id .............................................................................9 1

5-11 Real time PCR analysis from total mRNA isolated from HepG2 cells incubated
for 12 h in the indicated m edium ........................................ ........................ 92

5-12 Real time PCR analysis from total mRNA isolated from HepG2 cells incubated
for 12 h in of FOXO3a mRNA levels in FED or -HIS medium.............................93

5-13 Immunoblot analysis of FOXO3a protein in FED or -HIS medium for the
indicate am ount of tim e ............................. ................... ........ ............... .93

5-14 Immunoblot analysis of FOXO3a protein from total protein isolated from
HepG2 cells transfected with the indicated siRNA.......................... .........93

5-15 Northern blot analysis of HepG2 cells in FED or -HIS MEM, transfected with
the indicated siRNA. A gene specific probe for MnSOD was used to determine
the relative am ounts of m R N A .................................................................... ....... 94

5-16 Immunoblot of cytoplasmic and nuclear fractions isolated from HepG2 cells
incubated for 6 h in the indicated m edium ........................................ ...................95















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

REGULATION OF MANGANESE SUPEROXIDE DISMUTASE VIA AMINO ACID
DEPRIVATION

By

Kimberly Jean Aiken

August 2006

Chair: Harry S. Nick
Major Department: Biochemistry and Molecular Biology

Amino acids play an intricate role in protein synthesis, gene regulation, and overall

cellular homeostasis. The extent of this control is exerted at many levels and is just

beginning to be understood. The focus of this dissertation is on the gene regulation of the

cytoprotective enzyme manganese superoxide dismutase (MnSOD). This gene has

previously been demonstrated to be regulated, through increased transcription by a

number of pro-inflammatory cytokines. However this gene has not been previously

demonstrated to be under the regulation of nutrient levels. The research presented here

identifies and characterizes the regulation of MnSOD in response to several different

nutrients. Also described is a level of metabolic control previously not demonstrated for

any other amino acid regulated gene. Relevant amino acid signal transduction pathways

were also evaluated and provide evidence for the orchestration of several nutrient

sensitive pathways. Furthermore, identification of potential transcription factors involved

in this regulation were also evaluated.














CHAPTER 1
INTRODUCTION

How nutrients and their subsequent metabolism affect gene expression is becoming

an increasingly important area of research. Amino acid and glucose levels are constantly

changing under normal physiological conditions. These changes are even more

pronounced during times of stress or disease. How the body orchestrates these metabolic

cues are only just beginning to be understood. Alterations in nutrient levels involve the

regulation of many genes through chromatin remodeling, RNA splicing and stabilization,

transcription and translation [1]. Gaining an understanding of the mechanisms

underlying the regulation of gene transcription in response to the complex and ever

changing environment, will help to further our knowledge of cellular mechanisms in

general and how the cell responds during stress.

A critical consequence of nutrient availability and subsequent metabolism is the

generation of reactive oxygen species (ROS) [2]. It has been estimated that 1-3% of

consumed oxygen is released as superoxide radicals from mitochondrial electron

transport [3]. All aerobic organisms, through energy generating metabolism, produce

harmful ROS such as superoxides, hydroxides and nitric oxide. External sources

including ultraviolet radiation, cigarette smoke and numerous inflammatory mediators

contributing to the production of ROS also affect the cell [4,5]. These free radicals are

highly reactive and cause extensive damage to cellular components including lipids,

DNA and proteins. As a defense mechanism, organisms have adapted and developed

several means of protection against the harmful effects of ROS. These include:









antioxidant enzymes such as superoxide dismutases (SOD), heme oxygenases, catalase,

glutathione peroxidase, DNA damage repair enzymes as well as small molecular

antioxidants such as P-carotene and vitamins E and C [2]. Despite evolutionary

adaptations, free radicals are implicated in a number of diseases such as cancer [6],

asthma, rheumatoid arthritis, stroke, Parkinsons [7] and Alzheimers [8] disease.

A key ROS scavenging enzyme is MnSOD, a cytoprotective mitochondrial enzyme

that converts free radicals formed during normal cellular respiration, as well as a

consequence of the inflammatory response, into the less reactive products, hydrogen

peroxide and oxygen. Given its central importance to cellular homeostasis, the regulation

of the MnSOD gene in response to nutrient deprivation was evaluated. The research

presented in this dissertation identifies components involved in the regulation of MnSOD

in response to nutrient deprivation, giving insight into the regulatory mechanisms and

providing a greater understanding of nutrient control.

Manganese Superoxide Dismutase

Superoxide Dismutases and Reactive Oxygen Species

As already indicated, the cell's front line of defense against ROS comes from the

family of superoxide dismutases (SODs) which, in eukaryotes, consists of the

cytoplasmic and extracellular localized copper/zinc SOD (Cu/Zn-SOD), and the

mitochondrial localized manganese SOD(MnSOD) [9]. These enzymes all catalyze the

dismutation of the superoxide radical to hydrogen peroxide and oxygen (Figure 1-1) [10].

Hydrogen peroxide is then detoxified by either catalase or glutathione peroxidase to

water and oxygen. Although all three of the SODs catalyze the same reaction, the

generation of null mice for the three different isoforms demonstrated that only the

mitochondrial localized MnSOD had a significant phenotype [11].










202. + 2H+ 02 + H202

Figure 1-1. Catalysis of superoxide to oxygen and hydrogen peroxide through the action
of MnSOD.

MnSOD is a nuclear-encoded, mitochondrial localized protein serving as the cells'

primary defense against ROS derived as a normal byproduct of cellular respiration. The

electron transport chain accounts for 1 to 3% of total cellular superoxide production

within the mitochondria alone [3]. Superoxides are a byproduct of both complex I and

III, presumably due to the inability of electron carriers to efficiently transport the electron

to the next complex (Figure 1-2). In order for the cell to continue to produce energy, the

reactive/toxic products of this pathway must be removed. Through the action of the

nuclear-encoded, mitochondrial-localized MnSOD, free radicals are efficiently

eliminated, thus protecting the mitochondrial DNA as well as the remainder of the cell by

preventing free radicals from leaving the mitochondria.


2e- NADH 02
NADH -> Dehydrogenase >* 02.
(5Fe-S)
COMPLEX I \e
COMPLEX III COMPLEX IV
4e- 0-2
2e- Succinate IQc_ y cyt. bl cyt. c a 02
Succinate Dehydrogenase e-20
(3Fe-S)
COMPLEX II 0 O -.

Figure 1-2. Production of free radicals at complex I and III of the electron transport
chain.

The critical cellular importance of MnSOD was demonstrated when two

independent groups produced transgenic mice lacking MnSOD activity. Li et al [12]

deleted exon 3 of the Sod2 gene on a CD-1 strain background (Sod2m""""). All mice died

within 10 days of birth due to dilated cardiomyopathy, with an accumulation of lipid in









liver and skeletal muscle and metabolic acidosis [12]. Lebovitz et al. [13] deleted exons

1 and 2 of the Sod2 gene on a mixed mouse background (Sod2mlbcm). These mice

survived for up to 18 days and exhibited severe anemia; degeneration of neurons in the

basal ganglia and brain stem; and progressive motor disturbances characterized by

weakness, rapid fatigue, and cycling behavior [13]. The mice also exhibited extensive

mitochondrial injury within the degenerative neurons and cardiac myocytes.

Heterozygous mice (Sod2mlcsf and Sod2mlbcm) with a 50% reduction of MnSOD activity

also showed chronic oxidative stress with increased mitochondrial permeability and

premature induction of apoptosis [13,14].

Cytoprotective Roles of MnSOD

The generation of ROS has been associated with a number of disease states [15-18]

and as such, the cell has evolved protective measures to aid in the survival of the cell by

inducing MnSOD [19]. MnSOD gene expression is up-regulated by a wide variety of

pro-inflammatory stimuli including IL-1, TNF, IL-6, and LPS, as well as down-regulated

by glucocorticoids [20-24]. The generation of Sod2 null and heterozygous mice

established the critical need for this antioxidant enzyme. Additionally, overexpression, in

cell culture and in transgenic mice, has demonstrated a protective role against oxidative

mediated insults. Extensive studies in cell culture have demonstrated the protective role

of MnSOD in response to radiation, cytotoxic effects of IL-1 and TNF and several

chemotherapeutic agents [25-29]. MnSOD over expression has also been shown to

suppress or cause partial reversal of the malignant phenotype seen in breast cancer cells

and SV40-transformed human lung fibroblasts. [30,31]. In transgenic mice

overexpressing MnSOD, protection was conferred against the damaging effects of ROS









production following ischemia/ reperfusion in the heart [32] and ionizing radiation of the

lung [33].

Transcriptional Regulation of MnSOD

The molecular regulation of the MnSOD gene by cytokine induction has been

extensively studied by our lab and others, demonstrating increased MnSOD steady state

mRNA levels in response to a variety of pro-inflammatory stimuli [21,22,34-36]. The

stimulus dependent induction of the gene is blocked when co-treated with the

transcriptional inhibitor actinomycin-D, suggesting de novo transcription. Nuclear run on

studies were subsequently performed, confirming transcriptional control by cytokine

induction of MnSOD [37].

The human SOD2 gene encodes a 22 kDa protein, which forms a 88.6 kDa tetramer

consisting of a dimer of dimers with approximately 90% homology to the rat and mouse

proteins [38]. The human MnSOD gene is localized to chromosome 6q25, and like the

rat, consists of 5 exons and 4 introns. The promoter is consistent with that of a

housekeeping gene in that it is TATA- and CAAT-less, with approximately 80% GC

content, and containing several putative SP1 and AP2 sites [39]. The human gene

produces two mRNA messages whereas in the rat there are five, all of which arise from

the same gene, differing only in their 3' untranslated region due to alternative

polyadenylation [40,41].

Although the MnSOD promoter has the elements of a housekeeping gene, this gene

is highly inducible by cytokines and therefore, studies in our laboratory sought to further

analyze the chromatin structure utilizing DNase I and Dimethyl sulfate (DMS) in vivo

footprinting. DNase I hypersensitive studies identified seven constitutive hypersensitive









Northern analysis
Rat MnSOD Rat Human
Genomic Clone


5' 3'

enhancer

10 constitutive factors = DNase I hypersensitive site
1 inducible

= Alternative
Polyadenylation site

Figure 1-3. Rat MnSOD genomic clone, with identified sites of regulation.

sites and are represented as an asterisk (*) (Figure 1-3) [37]. High resolution DMS in

vivo footprinting of the promoter region identified 10 putative constitutive protein-DNA

binding sites and one inducible site. In vitro footprinting of this region linked Spl and

gut-enriched Kruppel-like factor (GKLF) binding to 5 of the 10 constitutive sites [37].

Most recently, ChIP and PIN*POINT analysis confirmed the presence of Spl on the

promoter and, through site-directed mutagenesis, showed the functional importance of

two of the Spl binding sites [42].

Reporter constructs containing genomic elements 5' to exon 1 of MnSOD could not

fully recapitulate the stimulus-dependent induction observed from studies on the

endogenous gene [43]. Further analysis of the remaining hypersensitive sites led to the

identification of an inducible enhancer element within intron two [43]. This enhancer

element, in conjunction with the MnSOD minimal promoter, was found to mimic the

stimulus-dependent endogenous gene levels in an orientation and position independent

manner. The enhancer element is highly conserved between mouse, rat and human and

our laboratory's current efforts have focused on identifying the cognate transcription









factors responsible for the induction by pro-inflammatory stimuli. Further description of

this element is limited because evidence presented in this thesis will demonstrate that this

enhancer is not involved in nutrient control of MnSOD gene expression.

Nutrient Regulation of Gene Expression

Organisms have evolved many mechanisms to adapt to changing environmental

conditions. In times of nutrient deprivation when glucose and amino acids levels are

limited, several mechanisms regulating gene expression are induced and aid in cell

survival. In yeast, these mechanisms are well understood, but in more complex

mammalian systems these mechanisms have yet to be fully defined. As such, the yeast

systems for glucose and amino acid control will be described and compared to their

mammalian counterparts.

Glucose Control of Gene Regulation

Glucose is a primary source of metabolic energy for the cell and also plays an

important role in protein glycosylation. Glucose starvation leads to the improper folding

of newly made glycoproteins in the endoplasmic reticulum (ER) and subsequent

accumulation of these proteins, causing a decrease in overall protein synthesis with a

concomitant increase in the expression of genes such as the ER chaperonin protein

glucose regulated protein (GRP78) [44-46]. This signaling of the ER to the nucleus is

known in yeast as the (unfolded protein response) UPR pathway and in mammalian cells,

the ER stress response (ERSR) [47-49].

The UPR pathway is basically conserved from yeast to mammalian cells. In yeast,

a bZIP transcription factor homologue ATF/CREB (HAC1), is the unfolded protein

response element (UPRE) binding protein. Under normal conditions, HAC 1 is

constitutively transcribed within the nucleus, but not translated due to a non-classical









untranslated region at the 3' end of the mRNA. When there is an accumulation of

unfolded proteins in the ER, Irelp dimerizes and splices out the intron within the HAC 1

mRNA. Irelp is a transmembrane protein with its N-terminal domain in the ER lumen

and its kinase/C-terminal domain residing in the cytosol/nucleus. The N-terminal domain

acts as a sensor within the ER and dimerizes when there is an accumulation of unfolded

proteins [50,51]. The C-terminal domain, upon dimerization, auto-trans-phosphorylates

and removes the intron from HAC1 through site specific cleavage [52]. Once the intron

is spliced out, tRNA ligase Rlglp, rejoins the message, which can now be translated [53].

The Haclp protein can now bind to a UPRE and initiate transcription of various target

genes.

In mammalian cells, there are two pathways working in concert to monitor protein

folding. When low levels of proteins are unfolded, a proteolytic cleavage of activating

transcription factor 6 (ATF6) occurs. This releases the nuclear half of the protein into the

cytosol of the nucleus. ATF6 is a constitutively expressed type II transmembrane protein

which, in response to unfolded proteins, is sequentially cleaved by site 1 and 2 proteases,

S1P and S2P, respectively [54]. The soluble nuclear form, ATF6(N), binds to an ER

stress element (ERSE) causing the transcription of various genes, such as, ER

chaperones, to aid in protein folding, and also the mammalian homologue of HAC 1, X-

box binding protein-1 (XBP1). As stated above, transcription of XBP1, also a bZIP

protein, is induced by low levels of unfolded proteins due to ATF6(N) binding. XBP1,

like HAC1, also contains an intron that is spliced out, in the same manner as in yeast, by

the mammalian homologue of Irelp, IRE1. The protein generated from the spliced

mRNA, pXBP (S), also binds to ERSE sequences inducing transcription of the same









target genes as ATF6(N). In addition to ERSE elements, pXBP1(S) also binds to specific

target genes which contain a UPRE sequence. When a low level of ER stress is

encountered, ATF6 can quickly react by inducing ER chaperones and then, through the

induction of XBP1, continued ER stress is met by both UPR systems. By utilizing this

dual control, mammalian cells have developed a fine tuned mechanism in response to

glucose deprivation.

Amino Acid Control of Gene Regulation

Amino acid availability plays an important role in cell viability and protein

synthesis. Amino acid deprivation in yeast induces the general amino acid control

(GAAC) response which causes a global decrease in general protein synthesis while

increasing the transcription of over 40 genes encoding amino acid biosynthetic enzymes

[55,56]. This is achieved through the activation of the protein kinase general control

nonderepressible protein (Gcn2) in which uncharged tRNA accumulates and binds to the

regulatory domain of Gcn2p. When Gcn2p is bound by an uncharged tRNA it

phosphorylates and inactivates elongation initiation factor (eIF2). Phosphorylation of the

F subunit of elF2 causes it to bind and sequester eIF2B, a guanyl nucleotide exchange

factor [57]. eIF2B is responsible for the regeneration of GDP-eIF2 to GTP-eIF2, which

is necessary for the binding of Met-tRNAiMet. The phosphorylation of the a subunit of

GDP-eIF2 also inhibits its conversion to its active GTP form. When this process is

inhibited overall protein synthesis slows due to a lack of ternary complexes required for

the base pairing of Met-tRNAiMt to AUG [58]. It is this mechanism that ultimately leads

to an increase in the transcription of the general nutritional/transcriptional control

element (GCN4) which causes increased transcription of amino acid biosynthetic

enzymes [55,56,59]. GCN4 mRNA contains four short upstream open reading frames









(uORF) and when Gcn2p is activated, Met-tRNAiMet is slow to associate with the 40S

complex allowing initiation to occur at the proper GCN4 ORF and produces a full length

mRNA [60,61]. Following transcriptional activation of Gcn2p target genes, the balance

of amino acids is restored, uncharged tRNAs return to normal levels, Met-tRNAiMet

binding to the 40S subunit is no longer inhibited and translation of GCN4 no longer

occurs [1,56,62,63]. Mammalian cells have a similar system but they can not synthesize

all of the amino acids needed for protein synthesis. However, they do elicit a response

that causes the transcription of many genes that help to maintain cellular homeostasis

until amino acids become available [1].

Nutrient deprivation, in the form of glucose or amino acids, converges to activate a

number of genes. In mammalian cells, deprivation of essential amino acids activates the

amino acid response pathway (AAR) which leads to the translation of activating

transcription factor (ATF4), the mammalian homologuelog to the yeast GCN4, through

the same the mechanism as described above for yeast. ATF4 has been demonstrated to

regulate a number of genes including membrane transporters, transcription factors,

growth factors, and metabolic enzymes through binding to the consensus sequence 5'-

CATGATG-3'[1,57,64,65].

The mechanisms described thus far have demonstrated cellular mechanisms in

response to nutrient deprivation. However, amino acid sufficiency is also detected

through the kinase, target of rapamycin (TOR) (mTOR in mammalian cells). This level

of control is exerted at the level of the mRNA cap binding protein, eIF-4E. 4E binds to

the mRNA cap and then 4A and 4G bind allowing for the 43 S pre-initiation complex to

bind and scan the mRNA for an AUG start site. When there are adequate amounts of









amino acids mTOR is active and phosphorylates 4E-BP1, inhibiting its binding to 4E,

and translation is maintained [66]. When amino acids are limited, especially leucine,

mTOR can not phosphorylate 4E-BP1, the 4E protein is sequestered and the assembly of

the pre-initiation complex is prevented, ultimately leading to the inhibition of translation.

Amino Acids as Signaling Molecules

As described above, amino acids play an important role in mediating a number of

cellular responses. The advantage to maintaining a proper balance of amino acids is

clear; amino acids are the essential building blocks of life, providing the primary

composition of all proteins. There are 20 major amino acids which can be subdivided as

essential or nonessential. The essential amino acids are phenylalanine, valine, threonine,

tryptophan, isoleucine, methionine, histidine, arginine, leucine and lysine. These amino

acids are defined as essential because our body cannot make them on its own, and thus it

is essential that they be provided from the diet. However, depending on diet or other

circumstances of stress, some non-essential amino acids may also become conditionally

essential. For example, glutamine is a non-essential amino acid and the most abundant

amino acid in the blood stream but during times of metabolic stress its levels are reduced

and it is considered conditionally essential [67,68].

Typically, amino acid levels are in a balanced state and play a vital role in overall

homeostasis of the body by acting as neurotransmitters, hormones, mineral transportation

and in energy production. However, poor nutrition and disease can cause alterations in

these levels, which may lead to fluctuations in amino acid levels. Under these

circumstances, amino acids are imbalanced and as a consequence their levels can be

found either in excess or in a depleted state, leading to the activation of the cellular

responses described above. Table 1-1 lists the 20 principle amino acids and some of their









functions in the body, with the essential amino acids shown in bold. Also provided in

Table 1-1 is the status of these amino acids during times of the indicated stress or disease,

demonstrating conditions where these amino acids will be found in excess or in a

depleted state demonstrating the wide variety of functions and instances that will cause

amino acid imbalances in the cell. For example, depletion of branched chain amino acids

(BCAA) has been associated with muscle protein turnover and as such have been

supplemented in cases of burn sepsis and other forms of trauma to help prevent protein

loss [69-71]. Another example is the genetic disease phenlyketonuria (PKU) in which

amino acid imbalance can lead to mental retardation due to excess phenylalanine levels in

the brain [72]. Additionally, the manipulation of gene regulation in response to amino

acid deprivation is also currently involved in the treatment of acute lymphoblastic

leukemia (ALL), a cancer of the white blood cells. In ALL, the bone marrow makes

precursor cells (blasts) that never form into lymphocytes, but undergo several mutations,

eventually leading to unchecked growth [73]. These leukemia cells cannot synthesize

asparagine, and are therefore sensitive to asparaginase (ASNase), an enzyme that rapidly

depletes the extracellular pools of asparagine. ASNase treatment is responsible for

complete remission of 40-60% of these cases [74,75]. When used in combination with

vincristine and predonisone, the remission is increased to 95% [76].














Table 1-1. List of amino acids and their normal cellular functions and conditions of disease when these amino acids will be found in
excess or in a depleted state (Adapted from http://www.aminoacidpower.com/aboutAmino/aminoTour20)
Amino Acid Main functions Excess seen in Deficiencies seen in
Alanine Important source of energy for muscle Low insulin and glucagon levels Hypoglycemia
The primary amino acid in sugar metabolism Diabetes mellitus Muscle breakdown
Boosts immune system by producing antibodies Kwashiorkor (starvation) Fatigue
Major part of connective tissue Viral infections
Elevated insulin and
glucagons levels
Arginine Essential for normal immune system activity AIDS
Necessary for wound healing Immune deficiency
Decreases size of tumors syndromes
Assists with regeneration of damaged liver Candidiasis
Increases release of insulin and glucagons
Precursor to GABA, an important inhibitory neurotransmitter
Aspartic Acid Increases stamina Amytrophic Lateral Sclerosis Calcium and magnesium
Involved in DNA and RNA metabolism Epilepsy deficiencies
Excitatory amino acids
Helps protect the liver by aiding the removal of ammonia
Enhances immunoglobulin production and anti-body
formation
Asparagine Excitatory neurotransmitters
Aids in removing ammonia from the body
May increase endurance and decrease fatigue
Detoxifies harmful chemicals
Involved in DNA synthesis
Cysteine Protective against causes of increased free radical production Chemical Sensitivity
Natural detoxifier Food Allergy
Essential in growth, maintenance, and repair of skin
Key ingredient in hair
Precursor to Chondroitin Sulfate, the main component of
cartilage
Glutamic Excitatory neurotransmitter Excesses in brain tissue can cause cell
Acid Increases energy damage
Accelerates wound healing and ulcer healing
Plays major role in DNA synthesis














Table 1-1. Continued
Amino Acid Main functions Excess seen in Deficiencies seen in
Glutamine The highest blood concentration of all the amino acids Use of some anti-convulsant Chronic Fatigue Syndrome
Involved in DNA synthesis medications Alcoholism
Precursor to the neurotransmitter GABA Anxiety and Panic Disorders
Important glycogenic amino acid
Essential to gastrointestinal function
Involved with muscle strength and insurance
Precursor to the neurotransmitter GABA
Glycine Part of the stucture of hemoglobin Starvation Chronic Fatigue Syndrome
Involved in glucagon production, which assists in glycogen Viral Infections
Metabolism Candidiasis
Inhibitory neurotransmitter Hypoglycemia
Part of cytochromes, which are enzymes involved in energy Anemia
production
Glycogenic amino acids
Histidine Found in high concentrations in hemoglobin Pregnancy Rheumatoid arthritis
Useful in treating anemia due to relationship to hemoglobin Anemia
Has been used to treat rheumatoid arthritis Dysbiosis
Precursor to histamine
Associated with allergic response and has been used to treat
allergy
Assists in maintaining proper blood Ph
Isoleucine Involved in muscle strength, endurance, and stamina Diabetes Mellitus with ketotic Obesity
BCAA levels are significantly decreased by insulin hypoglycemia Kwashiorkor (starvation)
Muscle tissue uses Isoleucine as an energy source Hyperinsulinemia
Required in the formation of hemoglobin Panic Disorder
Chronic Fatigue Syndrome
Acute hunger














Table 1-1. Continued
Amino Acid Main functions Excess seen in Deficiencies seen in
Leucine Involved in muscle strength, endurance, and stamina Ketosis Hyperinsulinemia
Potent stimulator of insulin Depression
Helps with bone healing Chronic Fatigue Syndrome
Helps promote skin healing Kwashiorkor (starvation)
Vitamin B-12 deficiency in
pernicious anemia
Acute hunger
Lysine Inhibits viral growth Excess of ammonia in the blood Herpes
Invovled in the formation of L-Carnitine Epstein-Barr Virus
Helps form collagen Chronic Fatigue Syndrome
Assists in the absorption of calcium AIDS
Essential for children, as it is critical for bone formation Anemia
Involved in hormone production Hair loss
Lowers serum triglyceride levels Weight loss
Irritability
Methionine Assists in breakdown of fats Severe liver disease Chemical Exposure
Helps reduce blood cholesterol levels Multiple Chemical
Required for synthesis of RNA and DNA Sensitivity (MCS)
Antioxidant Vegan Vegetarians
Assists in the removal of toxic wastes from the liver
Involved in the breakdown of Epinephrine, Histamine, and
Nicotinic Acid
Natural chelating agent for heavy metals, such as lead and
mercury
Phenylalanine Precursor to the hormone thyroxine Depression
Major part of collagen formation Obesity
DL-Phenylalanine is useful in reducing arthritic pain Cancer
Used in the treatment of Parkinson's Disease AIDS
Parkinson's Disease














Table 1-1. Continued

Amino Acid Main functions Excess seen in Deficiencies seen in
Proline Critical component of cartilage Chronic Liver Disease
Involved in keeping heart muscle strong Sepsis (infection of the blood)
Works in conjunction with Vitamin C in keeping skin and Acute alcohol intake
joints healthy
Serine Glycogenic amino acid Vitamin B-6 Deficiency Total body gamma and
Critical in maintaining blood sugar levels neutron irradiation
Aids in the production of antibodies and immunoglobulins Hypoglycemia
Required for growth and maintenance of muscle Candidiasis
Threonine Required for formation of collagen Alcohol ingestion Depression
Helps prevent fatty deposits in the liver Vitamin B6 deficiency AIDS
Aids in production of antibodies Pregnancy Muscle Spasticity
Can be converted to Glycine Liver cirrhosis ALS (Amyotrophic Lateral
(a neurotransmitter) Sclerosis)
Acts as detoxifier Vegetarianism
Needed by the GI (gastrointensinal) tract for normal Epilepsy
functioning
Provides symptomatic relief in Amyotrophic Lateral Sclerosis
(ALS)
Tryptophan Precursor to the key neurotransmitter, serotonin Increased intake of salicylates (aspirin) Depression
Effective sleep aid, due to conversion to serotonin Increased blood levels of free fatty Insomnia
Lowers risk of arterial spasms acids ALS
Reduces anxiety Sleep deprivation Chronic Fatigue Syndrome
The only plasma amino acid that is bound to protein Niacin intake
Can be converted into niacin (Vitamin B3).
Treatment for migraine headaches
Stimulates growth hormone














Table 1-1. Continued

Amino Acid Main functions Excess seen in Deficiencies seen in
Tyrosine Precursor to neurotransmitters dopamine, norepinephrine, Hyperthyroidism Depression
epinephrine and melanin Chronic liver disease; cirrhosis Chronic Fatigue Syndrome
Precursor to thyroxine and growth hormone Gulf War Syndrome
Increases energy, improves mental clarity and concentration Hypothyroidism
Parkinson's Disease
Drug addiction and
dependency
Valine Involved with muscle strength, endurance, and muscle Ketotic Hypoglycemia Kwashiorkor
stamina Visual and tactile hallucinations Obesity
Not processed by the liver before entering the blood stream Neurological deficit
Actively absorbed and used directly by muscle as an energy Elevated insulin levels
source
Valine deficiency decreases absorbtion by the GI tract of all
other amino acids














CHAPTER 2
CHARACTERIZATION OF MANGANESE SUPEROXIDE DISMUTASE BY AMINO
ACID CONTROL

Introduction

Organisms have adapted metabolic strategies to accommodate changes in the

availability of nutrients. Amino acid deprivation causes a global decrease in protein

synthesis and an increase in a subset of specific proteins, through several different

mechanisms. Deprivation of essential amino acids has been demonstrated to evoke

responses at both the transcriptional and post-transcriptional levels for genes such as

asparagine synthetase (ASNS), CCAAT/enhancer binding protein (C/EBP) homologous

protein (CHOP), cationic amino acid transporter (Cat-1), sodium-coupled neutral amino

acid transporter system A (SNAT2) and insulin-like growth factor binding protein-1

(IGFBP-1) [1]. Fernandez et al. [77] have also demonstrated the existence of an internal

ribosome entry site within the 5' UTR of the Cat-1 gene that controls translation of this

transport protein under conditions of amino acid depletion.

As described in Chapter one, all aerobic organisms metabolize nutrients to provide

energy for the cell and, as consequence, also produce reactive oxygen species (ROS) [2].

Through normal respiration, approximately 1-3% of consumed oxygen is released as

superoxide radicals from mitochondrial electron transport [3]. As a protective measure,

cells have also evolved a mechanism to relieve the cell of these harmful superoxides, the

mitochondrial localized, nuclear encoded protein MnSOD.









Given the wide variety of responses elicited by nutrient deprivation, initial studies

were aimed at characterizing the regulation of MnSOD in response to amino acid and

glucose deprivation. MnSOD has previously been demonstrated to be cytoprotective

during times of stress mediated by inflammation and cytokine production. Therefore,

nutrient availability, in the form of either amino acid deprivation or glucose, may have

relevant metabolic and cell survival benefits mediated through the elevation of MnSOD

levels.

Materials and Methods

Cell Culture

HepG2 (human hepatoma), HUH7 (human hepatoma), FAO (rat hepatoma),

CaCO2 (human intestinal epithelial) and CCDLU (human adenocarcinoma) cells were

maintained in minimal essential medium (MEM) (Sigma, St. Louis, MO) at pH 7.4

supplemented with 25 mM NaHCO3, 4 mM glutamine, antibiotic/antimycotic (ABAM)

(Gibco, Grand Island, NY #15240-062) and 10% FBS (Gibco) at 370C with 5% CO2. L2

(rat pulmonary epithelial-like, ATCC CCL 149) and A549 (human epithelial-like) cells

were maintained in Ham's modified F-12K medium supplemented with 4 mM glutamine,

ABAM and 10% FBS at 370C with 5% CO2. Cells were grown to 50-65% confluency on

10 cm dishes then split 1:8 into 60 mm dishes. After 24 h, with a fresh media change 12

h before the start of the experiment to ensure the amino acid levels in general are not

depleted, the cells were washed three times with PBS and incubated in 3 mL of the

appropriate media and collected at the indicated times for either northern or western

analysis.









To examine the amino acids individually, a base medium containing no amino

acids, Earl's Balanced Salt Solution (EBSS Sigma #E888), was used. The EBSS medium

was then reconstituted to contain the nutrients found in MEM. This includes the addition

of vitamins, 10% dialyzed FBS, ABAM, and amino acids. Each amino acid was prepared

as a 100X stock (from Sigma) and added individually to the media to the same levels

found in MEM medium but omitting the amino acid being tested. For add back

experiments, the same EBSS base and supplements was used and only the indicated

amino acid was added at a 5 mM concentration. The pH of the medium was maintained

at 7.2-7.4 and adjusted (when necessary) with 0.1M NaOH or HCL.

Isolation of total RNA

Total RNA was isolated from cells as described by the Chomczynski and Sacchi

method with modifications [20]. Medium was removed and the cells were washed two

times with PBS. The cells were then lysed in 1 mL of GTC denaturing solution

consisting of 4 M Guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5%

Sarcosyl, and 0.1 M P-mercaptoethanol. After vortexing briefly to help lyse the cells, 50

[tL of 2 M sodium acetate, pH 4.0, and 500 [tL of water-saturated phenol was added. The

solution was incubated at room temperature for 5 min. 110 [L of a 49:1

chloroform:isoamyl alcohol mixture was added to the lysate, vortexed and centrifuged at

14,000 rpm for 10 min. The aqueous phase was then removed, an equal amount of

isopropanol added and then incubated at -200C for 30 min. The lysate was then

centrifuged at 14,000 rpm for 10 min. at 40C and the RNA pellet was resuspended in 75

[tL diethyl pyrocarbonate (DEPC) treated double distilled water. 25 [tL of 8 M lithium

chloride (LiC1) was then added, mixed and incubated at -20C for 30 min. The RNA was









pelleted by centrifugation at 14,000 rpm for 20 min. at 40C, washed one time with 70%

ethanol and resuspended in 100 [L DEPC water. RNA concentrations were determined

by absorbance at 260 nm.

Electro-Transfer and Northern Analysis

Five to ten [tg of total RNA was denatured and fractionated on 1% agarose, 6%

formaldehyde gels, electrotransferred to a Zeta-Probe blotting membrane from Bio-

RAD #162-0159 and UV cross-linked. Membranes were then incubated for 1 h in a

prehybridization buffer [78] consisting of 0.45 M sodium phosphate, 6% sodium dodecyl

sulfate (SDS), 1 mM EDTA, and 1% bovine serum albumin (BSA). The membranes

were then incubated overnight at 620C in the same hybridization buffer with a 32P

radiolabeled gene specific probe for MnSOD, asparagine synthetase, or GRP 78,

generated by random primer extension (Invitrogen, Carlsbad, CA #18187-013). The

membrane was then washed three times for 10 min. at 660C in a high stringency buffer

composed of 0.04 M sodium phosphate, 2 mM EDTA, and 1%SDS and then exposed to

film (Amersham, Piscataway, NJ #RNP 1677K,RNP30H).

Protein Isolation and Immunoblot Analysis

HepG2 cells were incubated in complete medium (FED) or complete medium

lacing histidine (-HIS) for the indicated times. The cells were then washed two times

with ice cold PBS and lysed with a buffer containing 50 mM Tris HCL (pH 7.5), 100 mM

NaC1, 5 mM EDTA (pH 8.0), 1% Triton X-100 with a protease inhibitor cocktail tablet

(Roche, Pleasanton, CA # 11836153001) and just before use, a phosphatase inhibitor

cocktail diluted 1:100 (Roche #1697498) was added. Protein concentrations were

determined by bicinchoninic acid (BCA) assay in triplicate (Pierce, Rockford, IL









#23227). 20 [g of total cellular protein was diluted in a loading buffer containing 2%

SDS, 100 mM DTT, 60 mM Tris HCL (pH 7.5). Loading dye from a 3X stock

containing 6% SDS, 180 mM Tris HCL (pH 7.5), 30% glycerol, and 0.03% bromophenol

blue (BMB) with 0.2 M DTT and BME were added fresh just before use was added at a

1X concentration to the diluted sample and boiled for five min. Samples were then

centrifuged for 10 min. at room temperature, separated on a 12% SDS/polyacrylamide gel

and transferred to a hybond ECL nitrocellulose membrane (Amersham). The membranes

were then blocked overnight with 5% non-fat milk dissolved in a TTBS buffer containing

0.137 M NaC1, 2.7 mM KC1, 25 mM Tris HCL (pH 7.5), and 0.005% Tween 20 at 40C.

The membranes were then incubated with rabbit anti-MnSOD polyclonal antibody

(Stressgen, San Diego, CA #sod-110) diluted 1:5,000 in TBTS with 5% BSA for 1.5 h,

washed three times with TBST, incubated with an anti-rabbit antibody diluted 1:10,000 in

5% non-fat milk for 2 h, washed again three times, and subjected to ECL

chemiluminescence (Amersham).

Densitometry and Statistical Analysis

All densitometry was quantified from autoradiography films using a Microtek scan

maker 9600XL and analyzed with the UN-SCAN-IT program, Silk Scientific

Corporation, version 5.1. Densitometric quantification of the autoradiographs for

MnSOD employed the intensity of the 4 kb mRNA. The relative fold-induction was

determined from FED or EBSS levels, normalized to the internal control, ribosomal

protein L7a. Data points are the means from independent experiments. An asterisk (*)

denotes significance as determined by a Student's t-test to a value of p 0.05.









Results

Amino Acid Deprivation Induces MnSOD Steady State Messenger RNA Levels

Nutrient availability in the mammalian diet has a potentially critical impact on

metabolic flux, the generation of ATP and as a consequence, the generation of

mitochondrial-derived ROS. Given the role of MnSOD in the detoxification of

mitochondrial-derived ROS, nutrient availability could have direct affects on the levels of

MnSOD gene expression. To test this hypothesis, northern analysis on human hepatoma

cell line (HepG2) incubated in cell culture, with the exclusion of a single essential amino

acid, histidine was used. As a positive control for MnSOD gene induction, cells were

also treated with the pro-inflammatory mediators lipopolysaccharide (LPS), interleukins

1 and 6 (IL-1 and IL-6), tumor necrosis factor-alpha (TNF-a) and interferon-y (IFN-y)

which has previously been demonstrated to induce MnSOD at the mRNA and protein

level [21,22,34-36]. It has also been previously demonstrated that two mRNA species are

produced from the human MnSOD gene due to alternative polyadenylation [40,41]. It

was also shown that different cell lines vary in the relative expression of the two

messages [40,41]. The mRNA levels of MnSOD are increased in response IL-13, IL-6

and TNF-a, with IL-10 being the only stimulus to induce the 1kb message equal to that of

the 4kb message (Figure 2-1). Also established (Figure 2-1), is the increase of steady

state MnSOD mRNA levels in response to -HIS, relative to FED. Furthermore, the

addition of 5mM histidine to FED conditions (first lane of Figure 2-1, (+HIS)) reduces

basal levels, demonstrating that depletion of histidine over the 12 h incubation time may

contribute to an increase in the basal levels of MnSOD (+HIS compared to zero and Fed).

In order to ensure that all experiments have similar MnSOD basal levels and amino acid

levels in general are not depleted, fresh medium was give to the cells 12 h before the start








of all experiments. Because HepG2 cells preferentially produce the 4kb message in

response to -HIS, this is the species referred to and utilized in densitometry for all

experiments. In all northern analysis, the large ribosomal subunit 7a (L7a) was used as

an internal control.





kB
4


MnSOD







L7a

Figure 2-1. Northern blot analysis of MnSOD in response to various stimuli. Total RNA
was isolated from HepG2 cells incubated in complete medium (FED) with or
without cytokines (LPS (lipopolysaccharide), TNF-a (tumor necrosis factor-
alpha) IL-1 and IL-6 interleukinss 1 and 6)), complete medium lacking
histidine (-HIS) or with the addition of 5mM histidine (+HIS) for 12 h. Gene
specific probes for MnSOD and L7a (loading control) were used to determine
the relative amounts of messenger RNA (mRNA).

Induction of MnSOD is Specific to Amino Acid Deprivation

Many other genes known to be regulated by amino acid deprivation also respond to

glucose starvation [1]. In addition, glucose metabolism may have profound downstream

affects on the production of mitochondrial derived ROS. To evaluate the specificity of

changes in MnSOD mRNA levels, cells were starved for glucose (-GLC) and compared

to -HIS. MnSOD mRNA levels are induced only in response to amino acid deprivation









(Figure 2-2). As a positive control for glucose deprivation, the membranes were re-

probed for glucose regulated protein (GRP78). GRP78 (also known as BiP) is an

endoplasmic reticulum (ER) chaperone that, during ER stress, increases gene expression

[44,79]. A summary of densitometric data from three independent experiments as in

Figure 2-2 is also shown (Figure 2-3). Furthermore, two other compounds known to

activate the ER stress response (ERSR) pathway, thapsigargin and tunicamycin were also

tested [80]. Thapsigargin is a calcium ATPase inhibitor whereas tunicamycin disrupts

the glycosylation of newly synthesized proteins [81]. These compounds were also tested

and, similar to glucose deprivation, showed no induction of MnSOD mRNA (data not

shown).

FED -HIS -GLC
Hrs-- 0 2 6 12 18 2 6 12 18 2 6 12 18
e. 4. .. .. 204Ml~eu -4 i f p



MnSOD







GRP78 fo



L7a -*

Figure 2-2. Northern blot analysis of total RNA isolated from HepG2 cells incubated in
complete medium (FED), or medium lacking either histidine (-HIS) or
glucose (-GLC). At the indicated times, the cells were lysed and subjected to
northern analysis. Gene specific probes for MnSOD, L7a (loading control)
and GRP78 (glucose regulated protein).were used to determine the relative
amounts of mRNA.








Immunoblot analysis was also employed to determine if the MnSOD mRNA

induction was translated to the protein level. HepG2 cells were incubated in the absence

of histidine and at the indicated times total protein was isolated. After 48 h the MnSOD

protein levels are increased in response to histidine deprivation (Figure 2-4).


o 4.5 *
4.0
3.5
7
S3.0 -
2.5 -
0 2.0
a 1.5

0.5
S0 2 6 12 18 2 6 12 182 6 12 18 -Hrs
FED -HIS -GLC
Figure 2-3. Densitometric quantification of replicate experiments as done in 2-2 of the
MnSOD 4kb message. Northern blots were evaluated using a Microtek scan
maker 9600XL and analyzed with the UN-SCAN-IT program. Data points are
represented as relative fold induction, as compared to the FED condition; all samples
were normalized to the internal control, L7a. Data points are the means +/- SEM (n >
3). An asterisk (*) denotes significance as determined by a Student's t-test to a value
ofp < 0.05.

-HIS

Hrs-* 0 12 24 48 72

MnSOD -
Figure 2-4. Immunotblot analysis of MnSOD in response to histidine deprivation. Total
protein was isolated from HepG2 cells incubated in complete medium lacking
histidine for the indicated amount of time. 20 tg of total cellular protein was
fractionated on a 12% SDS/polyacrylamide gel, transferred to a nitrocellulose
membrane and incubated with rabbit anti-MnSOD polyclonal antibody.









Induction of MnSOD Messenger RNA is Specific to Essential Amino Acid
Deprivation

To further characterize the MnSOD gene induction in response to amino acid

deprivation, mRNA levels were evaluated when each individual amino acid was omitted

from complete medium and incubated for 2, 6 and 12 h. As shown in the representative

northern blots (Figure 2-5), depletion of essential amino acids other than histidine also

causes a similar increase in MnSOD mRNA levels, with the exception oftryptophan. On

the other hand, depletion of non-essential amino acids from culture medium had no

effect. A densitometric and statistical summary is also shown (Figure 2-6). The fact that

MnSOD is induced in response to deprivation of only essential amino acids indicates an

important role for the utilization of these amino acids. A possible explanation for these

results is that non-essential amino acids and glucose can be made from within the cell and

their depletion may not be as stressful to the cell. However, depletion of essential amino

acids, a non-replenishable resource, places a demanding strain on the cell, and as a

protective measure, MnSOD is induced.

FED -HIS -ARG -LYS -TRP -GLY -SER -PHE
Hrs-- 0 2 6 12 2 6 12 2 612 2 6 12 2 6 12 2 6 12 2 6 12 2 6 12
--^- -- a_ .me


MnSOD



L7a UUS S -f

Figure 2-5. Representative northern blots of HepG2 cells incubated for the indicated
times in medium lacking a single amino acid. Gene specific probes for
MnSOD and L7a (loading control) were used to determine the relative
amounts of mRNA.










= 5 ,

4 *


r--
12 *

O I
2 -









Figure 2-6. Representative bar graph of at least three independent experiments as in
Figure 2-5. Densitometry was quantitated from multiple experiments as in 2-5
using a Microtek scan maker 9600XL and analyzed with the UN-SCAN-IT
program. Relative fold induction of the MnSOD 4kb message, as compared to
the FED condition was determined for each experiment; all samples were normalized
to the internal control, L7a. Data points are the means +/- SEM (n > 3). An asterisk
(*) denotes significance as determined by a Student's t-test to a value ofp < 0.05.

Cellular Specificity of Amino Acid Deprivation for MnSOD Induction

Due to the important role of the liver in amino acid and nitrogen metabolism, initial

studies of MnSOD and amino acid deprivation utilized human liver derived cell lines. In

another human hepatoma cell line, HuH7, there was an identical response to histidine

deprivation (Figure 2-7). To evaluate the cellular specificity of this metabolic response,

other cells lines were also evaluated.

Cell lines derived from the lung and intestine were also tested. These cell lines

were chosen because the lung is a well studied system in our laboratory and the

involvement of the intestine with nutrient uptake into the body. In these cells lines, the

asparagine synthetase (ASNS) gene was used as a positive control for both -HIS and

-GLC.








Several cell lines were tested including human lung fibroblasts cell line, CCDLU, a

lung adenocarcinoma cell line, A549, and a Caco-2 (not shown) cell line derived from a

primary colonic tumor were tested to see if MnSOD mRNA levels were increased in

response to amino acid deprivation (Figure 2-8). There was only a minor response in the

human lung fibroblasts with no obvious changes occurring in the other cell types, as

compared to the significant induction observed in mRNA levels when these membranes

were re-probed for ASNS. Similarly, this response was evaluated in a rat cell line, a

normal lung epithelial line, L2 (Figure 2-8), and again found no induction following

histidine deprivation.


FED
Hrs-- 2 6 12
MnSOD '



MnSOD


HUH7

-HIS -GLC
2 6 12 2 6 12



1


4


ASNS


L7a

Figure 2-7. Northern analysis from HUH7 (human hepatoma) cells, incubated for the
indicated amount of time, in various medium conditions. Conditions tested
included complete medium (FED), complete medium lacking either histidine
(-HIS) or glucose (-GLC). Gene specific probes for MnSOD, ASNS and L7a
(loading control) were used to determine the relative amounts of mRNA.


I


!









CCDLU A549 L2






MnSOD
MnSOD MnSOD





AASNS ASNS
ASNS


L7a L7a L7a

Figure 2-8. Northern blot analysis of CCDLU, A549 and L2 cells incubated for 12 h, in
the indicated medium. Conditions tested included complete medium (FED) or
in complete medium lacking histidine (-HIS). Also included in the A549 and
L2 cells is complete medium lacking glucose (-GLC). Gene specific probes
for MnSOD, ASNS and L7a (loading control) were used to determine the
relative amounts of mRNA.

Transcriptional control of MnSOD in response to amino acid deprivation

As previously discussed, amino acid gene control has been described at many

levels including transcriptional, translational and through mRNA stability [1,57]. To help

elucidate the mechanism of increased MnSOD mRNA levels, in response to amino acid

deprivation, inhibitors of transcription and translation, actinomycin-D and cyclohexamide

were used. Cells were first co-treated with the transcriptional inhibitor actinomycin-D

(Figure 2-9) and the induction of MnSOD caused by histidine starvation is completely

blocked. This indicates that de novo transcription is necessary for this induction. When

cells are co-treated with the protein synthesis inhibitor cyclohexamide, the steady state

levels of the MnSOD mRNA levels are equal to that of the FED condition. This implies

that inhibition of protein synthesis potentially stabilizes the MnSOD mRNA. This









suggests that the induction due to amino acid depletion is blocked by inhibition of protein

synthesis since the mRNA levels in the -HIS condition are not higher than in the FED

state. Treatment of cells with cyclohexamide or actinomycin-D blocks the induction of

MnSOD, indicating that there may be a transcriptional component to this induction.

Although these experiments alone cannot support a transcriptionally based induction, this

is addressed further in chapter five.



Control Actino Cyclo








MnSOD






L7a


Figure 2-9. Northern blot analysis of cells treated with actinomycin-D or cyclohexamide.
HepG2 cells were incubated for 12 hours in complete media (FED), complete
media lacking histidine (-HIS). Where indicated, co-treated with either
actinomycin or cyclohexamide. Gene specific probes for MnSOD and L7a
were used to determine the relative amounts of mRNA.

Discussion

Amino acid dependent regulation of gene expression plays a significant role in

cellular growth and metabolism. This has been shown to be regulated at many levels

including transcription, translation and mRNA stability [1]. The data presented here









demonstrate that MnSOD mRNA and protein levels are induced by amino acid

deprivation. Specifically, MnSOD mRNA levels are induced in response to deprivation

of the essential amino acids, with the exception of tryptophan. Several different cell lines

were evaluated, with the most significant responses occurring in the liver cell lines tested,

HepG2 and HUH7, inducing in response to amino acid deprivation and not glucose

deprivation. Furthermore, treatment of cells with transcriptional and translational

inhibitors block the induction of MnSOD, indicating that there may be a transcriptional

component to this induction, which will be addressed further in Chapter 5.

Amino acid deprivation has been demonstrated to cause a wide variety of responses

including increases in membrane transporters, amino acid synthetases, and other

metabolically relevant enzymes. However, it has also been demonstrated that other

proteins not related to the restoration of amino acid levels are induced in response to

amino acid deprivation. One example is the insulin-like growth factor binding protein

(IGFBP-1) [82]. This protein binds to insulin-like growth factors (IGF) I and II and

functions to extend their half-life and alter their interactions with cell surface receptors.

IGF I promotes cell proliferation and inhibition of apoptosis whereas IGF II has been

associated with early development. These genes are all induced to promote cell survival

during stressed caused by amino acid deprivation.

The data presented here establish a novel mechanism of induction for the

cytoprotective enzyme MnSOD. This protein functions to scavenge free radicals

produced in the mitochondria as a consequence of respiration. The gene has been

demonstrated to be regulated by a number of stimuli including TNF, IL-1 and ionizing

radiation [21,22,34-36]. However, the link between nutrient deprivation and the









induction of MnSOD has not previously been established. The focus of this dissertation

is to characterize this induction by evaluating the nutrient requirements, signal

transduction pathways involved and the regulatory elements involved.














CHAPTER 3
METABOLIC CONTROL OF MANGANESE SUPEROXIDE DISMUTASE IN
RESPONSE TO AMINO ACID DEPRIVATION

Introduction

Nutrient availability relative to both carbohydrates and amino acids, in the

mammalian diet, has potentially critical impacts on metabolic flux and ultimately the

generation of ATP and its equivalents. With constantly changing constituents associated

with the mammalian diet, organisms have adapted metabolic strategies to efficiently

accommodate changes in the availability of critical nutrients. Extensive studies have

addressed the importance of glucose excess [83] and deprivation [46] as well as amino

acid availability on metabolic and nuclear events [1] As established in Chapter two,

HepG2 cells, in response to single amino acid deprivation, induces MnSOD at the mRNA

and protein levels. A critical consequence of nutrient availability and subsequent

metabolism is the generation of reactive oxygen species (ROS), the target of MnSOD

[66,84]. Furthermore, the connection between nutrient levels and the generation of ROS

is underscored when considering that caloric restriction can significantly delay the aging

process [85], an observation in line with the free radical theory of aging [86,87]. One

contributing factor is the importance of the mitochondrial localized anti-oxidant enzyme,

MnSOD, to energy/redox metabolism, aging and disease pathologies [88]. The results

demonstrated in Chapter two established that depletion of individual essential amino

acids and not glucose causes induction of human MnSOD mRNA and protein levels.









Nutrient uptake, in the form of carbohydrates, lipids and protein, and their

subsequent breakdown to glucose, amino acids, fatty acids and glycerol is the basis for

metabolism. Metabolism is the modification of compounds to maintain cellular

homeostasis through the synthesis (anabolic pathways) or breakdown (catabolic

pathways) of nutrients. The flux of these metabolites within the cell is important to

maintain the balance of anabolic and catabolic functions. The major function of these

metabolites is to feed into the citric acid cycle, ultimately leading to the production of

energy for the cell. One major metabolite that feeds into the TCA cycle, after the

conversion of pyruvate to acetyl-CoA, is glucose. However, as previously established,

glucose does not affect the induction of MnSOD and the main focus of this section will

be on the catabolism of amino acids into the TCA cycle.

The major functions occurring in the mitochondria are the citric acid cycle, electron

transport, oxidative phosphorylation, fatty acid oxidation, pyruvate oxidation and amino

acid catabolism. Following the conversion to acetyl Co-A, the metabolites pyruvate,

fatty acids, and amino acids, feed into the TCA cycle. Amino acids can also feed

directly, or through transamination reactions, into the TCA cycle. The TCA cycle

generates reducing units in the form ofNADH and FADH2 that drive electron transport,

ultimately generating ATP, and providing energy for the cell. As a byproduct of normal

respiration, superoxide radicals are produced at complex I and III. In order to ensure the

cells survival, the mitochondrial localized MnSOD protein converts the superoxide

products into hydrogen peroxide and water. The hydrogen peroxide is then detoxified, in

the mitochondria, by glutathione peroxidase. Given the close connection between

cellular nutrients and free radical production, perturbations in the flux of amino acids into









the TCA may be the basis for the amino acid regulation of MnSOD gene expression. As

a nuclear-encoded gene, but mitochondrial-localized protein, the activity of this enzyme

is important to a wide variety of metabolic cellular events including the oxidation state of

the cell, cellular respiration, ATP synthesis and overall cell viability. Given the

importance of overall mitochondrial homeostasis, cell survival and the localization of

MnSOD, the importance of several mitochondrial pathways were evaluated.

Materials and Methods

Isolation of Total RNA

RNA isolation was performed as described in Chapter 2.

Measurements of ATP Levels

ATP levels were measured from HepG2 cells utilizing the Adenosine

5'-triphosphate (ATP) bioluminescent kit #FLASC from Sigma. After 12 h of treatment

cells were trypsinized, 3 mL of medium was added, the cells were mixed well, and 1 mL

was transferred to a 1.5 mL microfuge tube. A 100 [L aliquot of ATP assay solution

(diluted 1:25) was added to a fresh tube and incubated at room temperature for 3 min.,

allowing for endogenous ATP to be hydrolyzed and decreasing the background. In a

separate 1.5 mL microfuge tube, the following was mixed: 100 [iL releasing agent (FL-

SAR), 50 iL water and 50 [L of the cell sample. A 100 [L aliquot of this mix was

added to the 100 [L ATP assay solution and relative light units were measured using a

Berthold SIRUS luminometer V3.0.

Reagents Used

KMV #K7125, 3-nitropropionic acid #N5636, malonate #1750, antimycin #A8674,

oligomycin #08476 and 2,4-dinitrophenol #D198501 were purchased from Sigma.

Fluoroacetate was from Fluka, St. Louis, MO #71520.









Results

Glutamine is Required for the Induction of MnSOD by Amino Acid Deprivation

Given that deprivation of a single essential amino acid resulted in the induction of

MnSOD mRNA, total amino acid deprivation was also evaluated to determine if this

would cause a similar induction. Notably, MnSOD mRNA levels were not elevated in

HepG2 cells incubated in medium lacking all amino acids (EBSS) (Figure 3-1). This is

completely contrary to the response detected for other genes regulated by depletion of

essential amino acids, in that these genes can respond to single or complete amino acid

deprivation [89,90]. In fact, there are no other genes currently known to be regulated by

single amino acid deprivation that are not also induced in response to complete amino

acid depletion.

To address the loss of induction of MnSOD by total amino acid deprivation, several

components of the medium were tested to evaluate their contribution to the induction of

MnSOD through amino acid deprivation. First, the individual amino acids were tested to

determine if the induction could be restored. To address the amino acid specificity of the

"add back" experiment, cells were incubated in EBSS or in EBSS with the addition of a

single amino acid. The effects of each amino acid were analyzed by northern analysis

and a subset of this data is shown (Figure 3-1). Analysis of these data demonstrate that

glutamine is the only amino acid when added back to EBSS, is able to re-establish the

induction of MnSOD mRNA levels. To further address the specificity of this response,

asparagine synthetase (ASNS) [1,89,90], a gene known to be induced by both single and

complete amino acid deprivation was also evaluated (Figure 3-1). These data

demonstrate MnSOD is clearly not regulated in a manner identical to ASNS and

moreover, the addition of GLN to EBSS is sufficient to re-establish the induction of









MnSOD with no effects on ASNS. A separate experiment directly comparing MnSOD

and ASNS levels in -HIS, EBSS and EBSS+GLN as well as a quantitative summary of

GLN "add back" as compared to MnSOD mRNA levels in EBSS at 12 h is also shown

(Figure 3-2).


CZ


Hrs-- 6 12 6 12 6 12 6 12


6 12 6 12 6 12 6 12


we. m --


MnSOD


ASNS g 0 *


m


L7a


Figure 3-1. Northern analysis of HepG2 cells incubated in medium lacking all amino
acids (EBSS), or in EBSS with the inclusion of the indicated amino acid at a
concentration of 5 mM. Total RNA was collected at the indicated times and
analyzed by northern analysis. Gene specific probes for MnSOD, ASNS and
L7a (loading control) were used to determine the relative amounts of mRNA.


-77


*'.4 -t 40 40C





















MnSOD

4 4.5
4.0
= 3.5



1.5
> 1.0
L7a
0.0
EBSS EBSS +GLN
Figure 3-2. HepG2 cells were incubated in various medium conditions with the addition
of 5 mM glutamine for 12 h. Conditions tested included complete medium
(FED), complete medium lacking histidine (-HIS), medium lacking all amino
acids (EBSS) or in medium lacking all amino acids with the addition of 5mM
glutamine (EBSS +GLN) for 12 h. Gene specific probes for MnSOD, ASNS
and L7a (loading control) were used to determine the relative amounts of
mRNA. Densitometry was quantitated from multiple experiments using a
Microtek scan maker 9600XL and analyzed with the UN-SCAN-IT program.
Data points are represented as relative fold induction, as compared to the EBSS
condition; all samples were normalized to the internal control, L7a. Data points are
the means +/- SEM (n > 3). An asterisk (*) denotes significance as determined by a
Student's t-test to a value ofp < 0.05.

To further define the importance of glutamine in the induction of MnSOD mRNA

by amino acid deprivation, HepG2 cells were incubated in -HIS, or in -HIS/-GLN. As

demonstrated (Figure 3-3), the absence of GLN completely abolishes the -HIS induction

(-HIS/-GLN). As previously established, glucose deprivation did not cause an induction

of MnSOD. However, removal of glutamine from the medium does not induce MnSOD









mRNA levels, as it is a required component for the induction. Given the potential use of

glutamine and/or glucose as a carbon/fuel source, glucose was also evaluated to

determine if this component was required for MnSOD induction by amino acid

deprivation. Contrary to the effects of -GLN, glucose (-GLC) deprivation did not alter

the -HIS response (Figure 3-3).





FED -GLN -HIS -HIS/-GLN
Hrs-- 0 2 6 12 2 6 12 2 6 12 2 6 12




MnSOD







L7a

Figure 3-3. HepG2 cells were incubated in complete medium (FED), medium lacking
glutamine (-GLN), histidine (-HIS) or both amino acids (-HIS/-GLN). Cells
were also evaluated for the effect of glucose deprivation in conjunction with
histidine deprivation (-HIS/-GLC). At the indicated times, total RNA was
collected and subjected to northern analysis. Gene specific probes for
MnSOD and L7a (loading control) were used to determine the relative
amounts of mRNA.

To further evaluate the requirement of glutamine and to determine the levels of

glutamine required for the induction of MnSOD, either in response to single amino acid

deprivation (-HIS) or total amino acid deprivation (EBSS) cells were incubated in these

conditions with increasing amounts of glutamine (Figure 3-4) demonstrating the addition

of glutamine to FED cells has no effect. However, in single amino acid deprivation









(-HIS) or in total amino acid deprivation (EBSS), the addition of 1 mM glutamine is

permissive to MnSOD mRNA induction in response to amino acid deprivation.

FED -HIS EBSS
mMGLN-- 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5

.3^HF^ ''

MnSOD .
.- -..^- ,



L7a

Figure 3-4. Northern analysis of HepG2 cells incubated for 12 h in the indicated medium
conditions with increasing concentrations of glutamine. Conditions tested
included complete medium (FED), complete medium lacking histidine (-HIS)
or medium lacking all amino acids (EBSS) with increasing concentrations of
glutamine. Gene specific probes for MnSOD and L7a (loading control) were
used to determine the relative amounts of mRNA.

With glutamine established as an obligatory component for the induction of

MnSOD by amino acid deprivation, other components of the medium were evaluated.

One such component of the complete medium which could be, in part, responsible for the

increases in mRNA levels is the presence of the vitamin supplements, and as shown

(Figure 3-5), are not necessary for the observed induction. Additionally, other amino

acids were evaluated to further evaluate the specificity of glutamine as a requirement for

MnSOD induction in response to amino acid deprivation. Specifically, the induction of

MnSOD in cells depleted with both histidine and glutamine to growth conditions lacking

histidine in conjunction with depletion of tryptophan or methionine were tested. The lack

of glutamine is the only condition that can inhibit the induction of MnSOD by essential

amino acid deprivation (Figure 3-5).









Another major component of the culture medium is serum. Previous reports on

another amino acid regulated gene, C/EBP homologous protein (CHOP), demonstrated

the requirement of serum, specifically IGF, in the induction of this gene in response to

amino acid deprivation [91]. The presence of serum, as with vitamins, did not alter the

level of MnSOD mRNA induction (Figure 3-6). Also evaluated was the contribution of

two important growth factors found in serum IGF and EGF and the induction of MnSOD

by amino acid deprivation is not dependent on either any of these growth factors (Figure

3-6).


+VITAMINS

g^f

I<"<'


-VITAMINS


I I I
K
q< "<"


L7a __._n -

Figure 3-5. HepG2 cells were incubated in the presence or absence of vitamins under the
indicated conditions. Complete medium (FED), medium lacking glutamine (-
GLN), histidine (-HIS) or lacking a combination of two amino acids (-HIS/-
GLN), (-HIS/-TRP) or (-HIS/-MET) were tested. After 12 h, total RNA was
collected and subjected to northern analysis. Gene specific probes for
MnSOD and L7a (loading control) were used to determine the relative
amounts of mRNA.


MnSOD









10% dFBS 0% dFBS

FED -HIS FED -HIS FED -HIS FED -HIS
IGF (ng/ml) EGF (ng/ml) IGF (ng/ml) EGF (ng/ml)
S0 1100 0 1100 0 50 0 i 50 0 1100 0 100 0 50 10 150





MnSOD





L7a (g

Figure 3-6. Northern analysis of total RNA isolated from HepG2 cells incubated with or
without 10% dFBS, or two components of serum, IGF or EGF. Under these
conditions, cells were then incubated in FED or -HIS medium for 12 h. Gene
specific probes for MnSOD and L7a (loading control) were used to determine
the relative amounts of mRNA.

Inhibition of the TCA Cycle Blocks MnSOD Induction by Histidine Deprivation

With glutamine established as the only required component for MnSOD induction

by amino acid deprivation, the role of this amino acid was further evaluated. To identify

a mechanistic link between glutamine and MnSOD gene activation, the contribution of

glutamine metabolism and its potential connection to increases in MnSOD mRNA levels

was tested. The primary pathway for the utilization of glutamine, as both an energy

source and a possible signaling molecule is catabolism through the tricarboxylic acid

(TCA) cycle. Glutamine enters the TCA cycle through glutamate and c-ketoglutarate

(Figure 3-7 (adapted from [92])). Therefore, the contribution of the TCA cycle to the

induction of MnSOD was evaluated by utilizing selective inhibitors to several different

key enzymes. Aconitase was first evaluated by inhibition with 3-fluoroacetate [93,94].










PyruvateFatty Acds
Glucose J CO' Malonl CoA
S /AcetylCoA CoA-SH Fluoracetate
co.
PEP CS AcetylCoA

\I / itt S s ~Citrate
Aspartate Citrate Synthase

Asparagine estate Cis-Aconitase

Malate NADH Acitase
Dehydrogenase

Malate TCA Isocitrate
NADH Isocitrate
|Fumarase|\ CO, Dehydrogenase
c a-Ketoglutarate f Glutamate 4- Glutamine
Fumarate
A FADH a-Ketogluterate
Phenylalanine NADH ehydroenase Histidine
Tyrosine Succinate I Proline
Dehdeyonase Succinate Succinyl-CoA o Arginine
Succinate I
Thiokinase Valine
Isoleucine KMV
Methionine
3-NPA Threonine

Figure 3-7. Tricarboxylic acid (TCA) cycle. The major metabolites and the reducing
units generated by the TCA cycle, NADH and FADH2, are shown in bold.
The enzymes of the TCA cycle are shown in boxes. The applicable TCA
cycle inhibitors are shown in italics.

Aconitase is an important enzyme involved in the conversion of citrate to isocitrate

through a dehydration-rehydration rearrangement, with the inhibition by fluoroacetate

occurring through the intracellular conversion to the substrate inhibitor, fluorocitrate.

Fluoracetate can inhibit the induction, in a concentration dependent manner, of MnSOD

mRNA levels by histidine deprivation (Figure 3-8). A quantitative summary of three

independent experiments is also shown (Figure 3-9), with an approximately 65 to 70%

inhibition.

Given that glutamine is required for MnSOD gene activation, the entry point for

glutamine into the TCA cycle, ca-ketoglutarate dehydrogenase, may be another relevant










FED -HIS


0


Fluoroacetate
(mM)


5 10 15 20 25 30 0 5 10 15 20 25 30


MnSOD





ASNS .n.a .



L7a

Figure 3-8. Representative northern blot of HepG2 cells incubated for 12 h in complete
medium (FED) or complete medium lacking histidine (-HIS), treated with
increasing concentrations of fluoroacetate. Gene specific probes for MnSOD,
ASNS and L7a (loading control) were used to determine the relative amounts
of mRNA.


100-
a 90
80-
C-
70
S60-
0 50-
P 40-
* '30-
20-
1 10
0-


---HIS
-- FED





0 5 10 15 20 25 30
Fluroacetate (mM)
Figure 3-9. D ensitometric data from three independent experiments following treatment
with fluoroacetate (as done in Figure 3-9). Data points are represented as
relative expression (percent) relative to the induced message (-HIS, no
fluoroacetate); all samples were normalized to the internal control, L7a. Data
points are the means, +/- SEM.


-L -* 'C --


FED


-HIS









step in the signaling pathway for MnSOD gene activation. Furthermore, the lack of

MnSOD induction in the absence of glutamine can also be viewed essentially as an

inhibition of the TCA cycle. As a direct assessment of the importance of this entry point

for glutamine, a structural analogue of a-ketoglutarate and a competitive inhibitor of the

a-ketoglutarate dehydrogenase complex, a-keto-P-methyl-n-valeric acid (KMV) was

used [95]. The results show that, similar to fluoroacetate, KMV causes an -60%

reduction of the MnSOD induction by histidine depletion, again with no effect on ASNS

expression (Figure 3-10 and 3-11).

FED -HIS
KMV 0 0.10.5 1 10 20 0 0.1 0.5 1 10 20
(mM)




MnSOD






ASNS



L7a .

Figure 3-10. Representative northern blot of HepG2 cells incubated for 12 h in complete
medium (FED) or complete medium lacking histidine (-HIS) and treated with
increasing concentrations of a-keto-P-methyl-n-valeric acid (KMV). Gene
specific probes for MnSOD, ASNS and L7a (loading control) were used to
determine the relative amounts of mRNA.










100 -HIS
90 -- FED
1 80
570 -
S60
S 05 40 -

30
20 -
10-
0
0 0.1 0.5 1 10 20

KMV (mM)
Figure 3-11. Densitometry data collected from three independent experiments following
treatment with KMV (as done in Figure 3-13). Data points are represented as
relative expression (percent) relative to the induced message (-HIS, no
KMV); all samples were normalized to the internal control, L7a. Data points
are the means, +/- SEM.
Two inhibitory compounds, which have been shown to selectively inhibit the

succinate dehydrogenase complex, were used. 3-nitropropionic acid (3-NPA) inactivates

succinate dehydrogenase by covalently and irreversibly binding to its active site [96]

whereas malonate is a competitive inhibitor of succinate dehydrogenase [97,98]. The

inhibitor 3-NPA caused a significant inhibition of the -HIS induction (Figures 3-12 and

3-13), with no response to treatment with increasing concentrations of malonate (Figure

3-14). This is consistent with the biochemical argument that as a competitive inhibitor

malonate can be displaced when cellular succinate concentrations are elevated,

presumably the case when adequate levels of glutamine are available. Interestingly, 3-

NPA did cause the inhibition of the ASNS induction by -HIS, whereas fluoroacetate and

KMV had no effect.









FED
3-NPA--- 0 1 5


-HIS
10 0 1 5


(mM)


MnSOD






ASNS



L7a


GaNP='s


Figure 3-12. Representative northern blot of HepG2 cells incubated for 12 h in complete
medium (FED) or complete medium lacking histidine (-HIS) and treated with
increasing concentrations of 3-nitropropionic acid (3-NPA). Gene specific
probes for MnSOD, ASNS and L7a (loading control) were used to determine
the relative amounts of mRNA.


100
90
80
.70
60
50
40
'30
20
10


-- -HIS
-U- FED


0 1 5 10
3-NPA (mM)
Figure 3-13. Densitometry data collected from three independent experiments following
treatment with 3-NPA (as done in Figure 3-13). Data points are represented
as relative expression (percent) relative to the induced message (-HIS, no 3-
NPA); all samples were normalized to the internal control, L7a. Data points
are the means, +/- SEM.









FED -HIS
Malonate 0 1 5 15 0 1 5 15
(mM)





MnSOD







ASNS


L7a


w rn


Figure 3-14. Northern blot of HepG2 cells incubated for 12 h in complete medium (FED)
or complete medium lacking histidine (-HIS) with increasing concentrations
of malonate. Gene specific probes for MnSOD, ASNS and L7a (loading
control) were used to determine the relative amounts of mRNA.

Both a Functional Electron Transport Chain and F1-Fo ATP Synthase Complex are
Required for MnSOD Induction in Response to Amino Acid Deprivation

Given the importance of the TCA cycle in MnSOD gene activation by amino acid

deprivation and the metabolic role of this cycle in the generation of reducing units and

the accompanying electrons for consumption through oxidative phosphorylation, the

contribution of events from the electron transport chain was also evaluated. The electron

transport chain is responsible for the production of ATP via oxidative phosphorylation,

resulting from the flow of electrons from NADH (or FADH2) to molecular oxygen thus

establishing a proton gradient (Figure 3-15) that drives phosphorylation of ADP to ATP








by ATP synthase. Of particular note is that superoxide radicals, the substrates for

MnSOD enzymatic activity, are formed as byproducts at both complexes I and III of the

electron transport chain (Figure 3-15).


H20


TCA
Cycle


H (matrix) H (outside)


B COMPLEX I 02 -
2e- NADH
NADH -- Dehydrogenase
(5Fe-S)

\COMPLEX III COMPLEX IV
UQ- cyt. b--cyt. c y- 0
/" -\ H20
COMPLEX II /e o Antimycin A
2e- Succinate 02
Succinate Dehydrogenase
(3Fe -S)
Figure 3-15. The electron transport chain and its relevant points. A) The flow of
protons, through the electron transport chain, establishes a proton gradient
between the mitochondrial matrix and the intermembrane space. B)
Production of superoxides at complex I and III in the electron transport chain.
Complex III is inhibited by antimycin A.

In order to determine the potential contribution of the electron transport chain to

MnSOD gene induction, several inhibitors were evaluated. An inhibitor of complex III,

antimycin A, which our lab previously demonstrated inhibits the induction of MnSOD by

TNF-a, but not the induction by IL-13 or LPS was used [16]. Exposure to increasing









concentrations of antimycin A inhibited the -HIS induction of MnSOD with effective

concentrations in the low nM range (Figure 3-16). These results demonstrate the

importance on MnSOD induction of continued flow from the metabolism of glutamine in

the TCA cycle through the transfer of resulting reducing units and electrons towards the

ultimate goal of energy production through ATP formation.

FED -HIS

Antimycin A--- o 0 o 6 ; < 6 6 6 6 c -
(nM)




MnSOD..






ASNS


L7a

Figure 3-16. Northern blot analysis of HepG2 cells incubated for 12 h in complete
medium (FED) or complete medium lacking histidine (-HIS) with increasing
concentrations of antimycin A. Gene specific probes for MnSOD, ASNS and
L7a (loading control) were used to determine the relative amounts of mRNA.

The importance of the electrochemical gradient in the generation of ATP through

an inhibitor of the FiFo ATP synthase complex, oligomycin [16,99,100] was also

evaluated. The F1Fo ATP synthase complex utilizes the proton gradient across the inter-

mitochondrial membrane to drive ATP synthesis. Oligomycin blocks the Fo portion of

ATP synthase, inhibiting its activity and blocking ATP synthesis coupled to the proton

gradient established across the inter-membrane space and the mitochiondrial matrix. The









induction of MnSOD is inhibited in the low nM range of oligomyicn, implying that the

underlying signaling mechanism requires adequate ATP levels or an intact

electrochemical gradient (Figure 3-17).

FED -HIS
Oligomycin-- 0 0.1 1 10 0 0.1 1 10
(nM)



MnSOD







ASNS

L7a

Figure 3-17. Northern blot analysis of HepG2 cells incubated for 12 h in complete
medium (FED) or complete medium lacking histidine (-HIS) with increasing
concentrations of oligomycin. Gene specific probes for MnSOD, ASNS and
L7a (loading control) were used to determine the relative amounts of mRNA.

An Intact Electrochemical Gradient but not ATP Synthesis is Required for MnSOD
Induction

Since both inhibitors of the electron transport chain blocked MnSOD induction,

one possible explanation is that this is due to a decrease in ATP levels. To examine this

possibility, ATP levels were measured under each of our growth conditions. To establish

a growth condition which would definitively reduce cellular ATP levels as a positive

control, cells were treated with 2-deoxy-D-glucose (2-DOG), a non-metabolizable form

of glucose, creating a cellular state analogous to glucose starvation [101]. As previously

demonstrated (Figures 2-2 and 2-3), conditions of limiting glucose did not induce









MnSOD levels nor affect the induction by -HIS (Figure 3-3). To confirm that 2-DOG

does not affect MnSOD levels in FED, -HIS, or EBSS -/+ GLN, HepG2 cells were

incubated in these conditions and evaluated by northern analysis (Figure 3-18). ATP

levels were then measured under a variety of experimental conditions (Figure 3-19).

FED -HIS EBSS +GLN

2-DOG-k g NV 'g V N rV ~N.> '\" 4, '\. % g
(mM)

MnSOD




L7a

Figure 3-18. Northern blot analysis of HepG2 cells incubated for 12 h in the indicated
medium for 12 h with the addition of 5 mM glutamine (EBSS +GLN), with
increasing concentrations of the glucose analogue 2-deoxy-D-glucose (2-
DOG). Conditions tested included complete medium (FED), complete
medium lacking histidine (-HIS), medium lacking all amino acids (EBSS) or
in medium lacking all amino acids with the addition of 5mM glutamine
(EBSS +GLN), with increasing concentrations of the glucose analogue 2-
deoxy-D-glucose (2-DOG). Gene specific probes for MnSOD and L7a
(loading control) were used to determine the relative amounts of mRNA.

Comparable reductions in ATP levels were observed for all conditions relative to

complete medium (FED). These data indicate that the induction of MnSOD levels by -

HIS conditions is not dependent on alterations in ATP levels, because 2-DOG and -HIS

both result in reduced ATP levels, yet the former condition has no effect on MnSOD

mRNA levels (Figure 3-18). In addition, these results also demonstrate that the -HIS

condition only reduces ATP levels by -25% as compared to reductions of more than 50%

in either EBSS or any of the conditions with 2-DOG. Of equal importance is that the re-

addition of GLN to EBSS results in a recovery of ATP levels.









Since ATP levels do not affect MnSOD mRNA levels, another possibility is that an

intact proton gradient is required for the amino acid induction of MnSOD mRNA. In

order to test this theory, an uncoupler of electron transport and oxidative phosphorylation,

2,4 dinitrophenol, was used [102]. 2,4 dinitrophenol is hydrophobic and can freely pass

through the inner mitochondrial membrane due to its ability to bind free protons and

transports them into the mitochondrial matrix bypassing the F1Fo ATPase production of

ATP, effectively uncouples electron transport. Increasing concentrations of 2,4-

dinitrophenol inhibited the induction of MnSOD mRNA levels by -HIS (Figure 3-20),

strongly implicating the importance of an intact proton gradient.


100
= 90
80
"- 70
560
F2 50
S40
30
20
P 10
FED -HIS EBSS EBSS FED -HIS EBSS EBSS
+GLN +GLN
+2-DOG
Figure 3-19. ATP levels from four independent experiments in which HepG2 cells were
incubated in the various conditions. Conditions included complete medium
(FED) or complete medium lacking histidine (-HIS), medium lacking all
amino acids (EBSS) or in medium lacking all amino acids with the addition of
5mM glutamine (EBSS +GLN), with or without the addition of 10 mM 2-
DOG for 12 h. Cells were then lysed and ATP levels were measured using the
bioluminescent somatic cell assay kit from Sigma/Aldrich. Relative light
units were measured using a Berthold SIRUS luminometer V3.0. Data points
are represented as relative expression (percent), as compared to FED. Data
points are means +/- SEM.









FED -HIS
2,4 dinitrophenol-. 0 0.1 0.5 1 0 0.1 0.5 1
(mM)





MnSOD








L7a

Figure 3-20. Northern blot analysis of HepG2 cells incubated for 12 h in complete
medium (FED) or complete medium lacking histidine (-HIS) with increasing
concentrations of 2,4 dinitrophenol. Gene specific probes for MnSOD and
L7a (loading control) were used to determine the relative amounts of mRNA.

Discussion

MnSOD gene expression is induced in response to a wide variety of pro-

inflammatory stimuli including IL-1, TNF, IL-6, and LPS [22] as a protective measure

against the harmful effects of reactive oxygen species (ROS). As a nuclear-encoded

gene, but mitochondrial-localized protein, the activity of this enzyme is important to a

wide variety of metabolic cellular events including the oxidation state of the cell, cellular

respiration, ATP synthesis and overall cell viability. Tissue and cellular adaptation to

nutrient availability also affects carbon and nitrogen utilization through glycolysis, the

TCA cycle and ultimately the aerobic generation of ATP via electron transport. A critical

consequence of nutrient availability and subsequent metabolism is the generation of ROS

as byproducts of normal metabolism [2]. Previous estimates have indicated that under









normal aerobic and nutrient conditions, 1-3% of consumed oxygen is released as

superoxide radicals from mitochondrial electron transport [3]. Therefore, nutrient

availability, may have relevant metabolic and cell survival benefits mediated through the

elevation of MnSOD levels.

The results presented here provide evidence for a level of metabolic control for

MnSOD gene expression in response to amino acid deprivation. Experimental data

established that the induction of MnSOD in response to amino acid depletion is

dependent on the presence of glutamine. This is contrary to effects on other amino acid

regulated genes, such as ASNS [1,103], which are regulated by depletion of a single or

total amino acid depletion. Furthermore, no other components tested, including other

amino acids, vitamins, glucose or serum, were determined to be required for the induction

of MnSOD.

A review by Bode et al. [104] offers a potential explanation for the dependence of

glutamine to the induction of MnSOD by amino acid deprivation in HepG2 cells. Recent

studies by this group have established the importance of glutamine as a critical carbon

and nitrogen source in human hepatomas which may supersede the metabolic importance

of glucose in these tumor cells [104]. These investigators also demonstrate that human

hepatoma cells uptake and potentially utilize glutamine at a much higher rate than normal

human hepatocytes, most likely dependent on a switch between the system N (neutral) to

system ASC amino acid transporter [104]. These studies thus provide a potential

explanation for the importance of glutamine to hepatoma metabolism and in the context

of MnSOD gene regulation.









One path of utilization for glutamine is through the TCA cycle. When HepG2 cells

are starved for glutamine, the formation of a-ketoglutarate is inhibited and may cause an

inhibition of the TCA cycle. The TCA, electron transport chain and MnSOD are closely

linked through the production of and protection against, harmful free radicals produced

from the electron transport chain as a by-product of normal respiration. To determine if

inhibition of the TCA cycle contributes to the inhibition of MnSOD, specific inhibitors of

the TCA cycle were utilized. Inhibitors of three different TCA cycle enzymes were

targeted and like glutamine deprivation, blocked the induction of MnSOD mRNA levels

by amino acid deprivation. These data provide a potential link, in HepG2 cells, for the

requirement of glutamine to help drive the TCA cycle, contributing to the electron

transport chain, the generation of superoxide radicals and ultimately the generation of

ATP. The results presented here suggest that the induction of MnSOD is dependent on

glutamine because of its contribution to the TCA cycle.

The function of the TCA cycle is to provide reducing units to the electron transport

chain. Inhibition of the TCA cycle may block the induction of MnSOD as a consequence

of the prevention of the downstream effects of the TCA. To examine this possibility,

components of the electron transport chain were targeted with inhibitors and MnSOD

mRNA levels were evaluated. Inhibition of both complex III and the F1Fo subunits

resulted in the inhibition of MnSOD by histidine deprivation.

The electron transport chain functions to establish a proton gradient to drive the

synthesis of ATP. Therefore, the inhibition of the electron transport chain could be

blocking MnSOD induction due a decrease in ATP levels. To determine if ATP levels

were affecting the induction of MnSOD by amino acid deprivation, ATP levels were









measured and compared to northern analysis of MnSOD. ATP measurements did reveal

that when there are no amino acids present (EBSS), ATP levels are at their lowest,

indicating that the lack of induction for MnSOD mRNA levels could be a result of

decreased ATP levels. However, reducing ATP levels to that below seen with EBSS, by

using a glucose analogue 2-deoxy-D-glucose, established that MnSOD mRNA could still

be induced under the various medium conditions. These results demonstrate that

although EBSS, as expected, does result in low ATP levels, it is not ultimately the cause

for the inhibition of MnSOD mRNA levels in response to amino acid deprivation.

Given that reduced ATP levels did not effect the induction of MnSOD, the

contribution of an intact proton gradient was also tested. To test this, an uncoupler of the

electron transport chain was utilized. Use of the inhibitor blocked the induction of

MnSOD, demonstrating a requirement for an intact proton gradient. In summary, these

data demonstrate that induction of MnSOD by histidine depletion requires signals

dependent on the TCA cycle, the electron transport chain, and is mediated through a

functional mitochondrial membrane potential, but not ATP levels.














CHAPTER 4
SIGNAL TRANSDUCTION PATHWAYS ASSOCIATED WITH MANGANESE
SUPEROXIDE DISMUTASE INDUCTION IN RESPONSE TO AMINO ACID
DEPRIVATION

Introduction

The classic cellular sensing mechanism for detecting essential amino acid

deprivation, referred to as the Amino Acid Response (AAR) pathway, is associated with

an increase in the concentration of uncharged tRNA leading to the activation of the

GCN2 kinase [63]. The activation of GCN2 kinase leads to phosphorylation of

translation initiation factor eIF-2a, causing a decline in global protein synthesis [63]. The

GCN2-dependent pathway can be activated by either deprivation of a single essential

amino acid or complete amino acid deprivation (EBSS) [1]. As previously established in

Chapters two and three, the induction of MnSOD occurs in response to depletion of a

single essential amino acid but not complete amino acid deprivation, implying that the

GCN2 pathway may not be utilized as the sensor for MnSOD induction.

Given that the classic GCN2 pathway does not seem to be solely responsible for the

signal transduction leading to the transcriptional activation of MnSOD in response to

amino acid deprivation, other relevant signaling pathways potentially linking the

induction of MnSOD to either -HIS or EBSS +GLN were evaluated. Recently, mitogen

activated protein (MAP) kinase signaling pathways have been implicated in amino acid

dependent signaling. Franchi-Gazzola et al. [105] have previously demonstrated that the

MAP kinase pathway, through an increase in extracellular regulated kinase (ERK1/2)

phosphorylation and activity, was involved in the induction of fibroblast system A









transport activity following incubation in amino acid free medium. It has more recently

been demonstrated that histidine deprivation causes an increase in the phosphorylation of

ERK1/2 and that inhibition of the ERK signal transduction pathway causes a reduction in

the -HIS dependent increase in p21 mRNA [106].

MAP kinase signaling has been linked to a number of stimuli including amino

acids, growth factors and stress. MAP kinases, a family of serine/threonine protein

kinases are ubiquitously expressed and form a network of signaling cascades that

mediate, thru extracellular cues, a number of cellular responses such as proliferation,

apoptosis and cell survival [107-109]. Each core cascade is comprised of at least three

protein kinases, each one activating the next, with the final result being the activation of

the terminal kinase in the cascade, a specific MAP kinase. In some cases there may be up

to six protein kinases within a cascade, ultimately leading to the activation of a MAP

kinase.

Extracellular cues initiate a cascade of phosphorylation events leading to the

activation of a MAP/ERK kinase kinase (MEKK). This leads to the activation of a MEK

which then phosphorylates and activates the terminal MAP kinase. There are four main

MAP kinases, ERK1/2, JNK, p38MAPK and ERK5, all differing in their physiological

activities that are cell type and stimulus specific [108,109]. The activation of specific

isoforms within each cascade provides the necessary specificity for activation of a

specific MAP kinase. Some level of crosstalk occurs between the signaling cascades,

leading to the activation of the terminal MAP kinase. However, there is a level of control

within each signaling cascade at the level of substrate recognition. MEKKs are serine

threonine kinases that recognize the Ser-X-X-X-Ser/Thr motif. This activates MEKs









which phosphorylate serine, threonine and tyrosine, recognizing the Thr-X-Tyr, on the

terminal MAP kinase. Activation of the MAP kinase requires dual phosphorylation. The

substrate specificities of MEKs are narrow, with each phosphorylating one or only a few

MAP kinases. The amino acid located between the threonine and tyrosine confers the

level of substrate specificity for the activation of each kinase, being glutamine, glycine,

or proline for the ERK1/2, p38 or JNK pathways, respectively. The ERK1/2 MAPK is

typically activated in response to the availability of growth factors whereas JNK and p38

MAPK are activated by stresses such as UV light, cytokines and osmotic shock. ERK5

has been shown to be regulated by both mitogenic and stress signals. Once a MAP

kinase is activated it then phosphorylates a number of effector molecules such as

transcription factors, phosphatases and other protein kinases. This level of regulation

provides the cell with an exquisite level of control that allows for a number of cellular

responses to a variety of extracellular stimuli.

Another protein kinase, also dependent on amino acid availability, that plays a

central role in monitoring and regulating downstream translational events is the

mammalian target of rapamycin (mTOR) [110-117]. As the name implies, mTOR is a

target of the drug rapamycin, which potently inhibits its activity. Rapamycin is a

lipophilic macrolide, which was identified from a soil screen taken from the island Rapa

Nui, more commonly known as Easter Island [118]. It was found to inhibit yeast growth

and subsequently, mutations conferring resistance to the drug led to the identification of

the drugs target protein and name, target of rapamycin (TOR) [119,120]. Rapamycin

functions by forming a complex with a peptidyl-prolyl cis/trans isomerase, FKBP12, that

binds to and inhibits the activity of TOR [120,121]. mTOR is a large (=280 kda)









serine/threonine kinase, in the phosphatidylinositol kinase related kinase (PIKK) family

of proteins, with a number of regulatory domains [122]. mTOR forms two different

complexes, both associating with G3L, through a TOS (TOR signaling) motif [123], and

then either raptor or rictor which are rapamycin sensitive and insensitive, respectively

[124-126]. mTOR is regulated by a number of extracellular cues, including growth

factors such as insulin and amino acids, mediating cell growth, metabolism and

proliferation [127].

The role of mTOR in the cell, when proper nutrients are available, is to maintain

ribosomal biogenesis, translation initiation and nutrient import [121]. Specifically,

mTOR activity is regulated by the availability of branch chain amino acids, where the

lack of, for example leucine, leads to inhibition of mTOR-dependent downstream events.

The primary downstream effectors of mTOR are 4EBP1 and P70 S6K. The role of

4EBP1 is to regulate cap dependent translation of mRNA. When 4EBP1 is

phosphorylated by mTOR, it causes dissociation from the cap binding protein eIF4E,

allowing for its binding to mRNA and increasing eIF4E dependent translation. P70 S6K

functions to phosphorylate S6, a major ribosomal protein [128]. Phosphorylation of the

S6 ribosomal protein functions to selectively increase the translation of a subset of

mRNA that contains a tract of pyrimidines [129]. These mRNAs account for

approximately 30% of the total mRNA in the cell and encode for ribosomal proteins, as

well as, translation initiation and elongation factors [130]. Through the regulation of the

translational components 4EBP1 and p70 S6K mTOR regulates cell growth and

proliferation, mediated by the availability of growth factors and nutrients.









Materials and Methods

Isolation of Total RNA

RNA isolation was performed as described in Chapter 2.

Protein Isolation and Immunoblot Analysis

Protein isolation was performed as described in Chapter 2 with the following

modifications for the indicated antibody which were obtained from Cell signaling,

Danvers, MA: phospho-p70S6 kinase (Thr389) (#9205), phospho-4E-BP1 (Thr37/46)

(#9459); phospho-p70S6 kinase (Th421/Ser424) (#9204).

Samples immunoblotted for p70S6 kinase (Thr389) and p70S6 kinase

(Th421/Ser424) were run on a 7.5% gel. Samples immunoblotted for phospho-4E-BP1

(Thr37/46) were run on a 12.5% gel. The gels were then transferred to a nitrocellulose

membrane and blocked for 1 h at room temperature in 5% non-fat milk. The primary

antibodies were diluted 1:1000 in 5% BSA in TBST incubated with the membrane

overnight at 40C. Anti-rabbit secondary antibody was used at 1:2000 diluted in 5% non-

fat milk for 1 h at room temperature.

Reagents Used

From Calbiochem, San Diego, CA: SB202190 (#559388); SB203580 (#559389);

PD98059 (#513000); JNK (c-Jun N-terminal kinase) inhibitor II (#SP600125); U0126

(#662005). Rapamycin was obtained from LC laboratories, Woburn, MA #ASW-104.

Results

Requirement of ERK1/2 Signaling

To investigate the signaling mechanisms involved in the regulation of MnSOD in

response to amino acid deprivation, two regulatory pathways known to be regulated by

amino acid availability were evaluated, MAP kinase signaling and the protein kinase









mTOR. Previous data has linked the availability of amino acids to the phosphorylation of

the MAP kinase ERK1/2 [105,106]. However, with the unique nature of the MnSOD

induction, relative to other amino acid regulated genes, other members of the MAPK and

JNK pathways were also tested. In order to study the potential signal transduction

pathways involved in the regulation of MnSOD, inhibitors of the MAP kinase pathways

were used. Specifically, SB203580 (10 [tM) and SB202190 (10 [tM) were employed to

inhibit p38 kinase and SP600125 (20 [tM) as a JNK inhibitor. Two different inhibitors of

the ERK1/2 kinase, PD98059 (50 VM) and U0126 (50 [tM), were also utilized. PD98059

and U0126 are both selective and cell-permeable inhibitors of MEK kinase. However,

PD98059 inhibits the active form of MEK whereas U0126 inhibits both the active and

inactive MEK [131,132]. The concentrations utilized for these inhibitors have been

demonstrated previously to inhibit the activation of these kinases in HepG2 cells

[99,133,134]. Inhibitors for p38 MAPK and JNK had no effect on the induction by -HIS

or EBSS +GLN (Figure 4-1). Inhibitors targeting ERK1/2 activity, PD98059 and U0126,

blocked both the -HIS and EBSS +GLN inductions (Figure 4-2).

No Treatment SB 202 SB 203 JNK inhibitor






MnSOD




L7a

Figure 4-1. Northern analysis of HepG2 cells treated for 12 h in FED, -HIS, EBSS or
EBSS +GLN with the indicated inhibitor; SB202 (10 tM), SB203 (10 tM), or
a JNK inhibitor (20 [tM). Gene specific probes for MnSOD and L7a (loading
control) were used to determine the relative amounts of mRNA.









No
Treatment PD 98059


3

2

1 -

0- -
No
Treatment


0 FED
U-HIS
O EBSS
E+GLN


PD 98059


U0126


Figure 4-2. Evaluation of HepG2 cells treated for 12 h in the indicated medium and the
indicated inhibitor. A) Representative northern blot of HepG2 cells treated for
12 h in FED, -HIS, EBSS or EBSS +GLN with the indicated inhibitor;
PD98059 (50 gM) or U0126 (50 tM). B) The corresponding densitometry.
Gene specific probes for MnSOD and L7a (loading control) were used to
determine the relative amounts of mRNA. Densitometry was quantitated from
multiple experiments using a Microtek scan maker 9600XL and analyzed with
the UN-SCAN-IT program. Data points are represented as relative fold
induction, as compared to the FED or EBSS condition; all samples were
normalized to the internal control, L7a. Data points are the means +/- SEM (n
> 3). An asterisk (*) denotes significance as determined by a Student's t-test
to a value ofp < 0.05.


U0126


MnSOD




L7a


a~h r-->,









Amino Acid Deprivation and mTOR Signaling

mTOR is a key regulator of cell growth, predominantly through the regulation of

translation events. The availability of nutrients, such as amino acids and other growth

factors, are required for mTOR to remain active. Unlike GCN2, which senses uncharged

tRNA, mTOR senses the levels of intracellular pools of amino acids (although the

mechanism by which this occurs is still unknown) [111,135]. The inhibition of mTOR,

through nutrient withdrawal or the use of rapamycin, causes a global decrease in general

protein synthesis due to a loss of phosphorylation of key translational components [121].

With mTOR at the center of regulation by growth factors and amino acids, this

pathway was evaluated by use of the potent mTOR inhibitor rapamycin. To determine

the involvement of mTOR in the regulation of MnSOD, HepG2 cells were treated in the

FED condition with increasing concentrations of rapamycin, with the hypothesis that this

would mimic amino acid deprivation and cause an induction of MnSOD mRNA levels.

Interestingly, rapamycin treatment in FED medium did not induce MnSOD mRNA levels

(Figure 4-3). However, rapamycin treatment did cause a concentration dependent

inhibition of the MnSOD induction in -HIS conditions (Figure 4-3). Furthermore,

rapamycin had no effect on the induction of MnSOD mRNA levels when cells were

incubated in EBSS +GLN conditions (Figure 4-3). A quantitative summary of this data is

also shown (Figure 4-4). The ability of rapamycin to distinguish between the effects of -

HIS and EBSS +GLN conditions is in contrast to the ERK1/2 inhibitors ability to block

both modes of MnSOD induction. Furthermore, the ability of rapamycin to distinguish

between the two conditions strongly suggests that, although necessary for the induction

by -HIS, the addition of GLN to EBSS may constitute a distinct but interdependent

pathway for induction of MnSOD.










FED -HIS EBSS EBSS + GLN
Rapamycin- 0 0 10 0 1 5 10 0 10 0 1 5 10
(LIM)


- _, _


n~-nfl


MnSOD


a-4


L7a


Figure 4-3. Northern analysis of HepG2 cells treated for 12 h in FED, -HIS, EBSS or
EBSS +GLN with increasing concentrations of rapamycin. Gene specific
probes for MnSOD and L7a (loading control) were used to determine the
relative amounts of mRNA.


4

0
-3
I 2
-e
"o 1
F4


FED 0 1 5 10
Rapamycin ([M)
-HIS


EBSS 0 1 5 10
Rapamycin ([M)
EBSS+GLN


Figure 4-4. Densitometry of Northern analysis of HepG2 cells treated for 12 h in FED, -
HIS, EBSS or EBSS +GLN with increasing concentrations of rapamycin (as
in Figure 4-3). Data points are represented as relative fold induction, as compared
to the FED or EBSS condition; all samples were normalized to the internal control,
L7a. Data points are the means +/- SEM (n > 3). An asterisk (*) denotes significance
as determined by a Student's t-test to a value of p < 0.05.

Given the unexpected results obtained with rapamycin treatment, additional

experiments to fully understand the mechanism underlying this phenomenon was

employed. To further define mTOR's role in the amino acid dependent gene regulation

of MnSOD, two downstream effectors of mTOR, p70S6K and 4EBP-1 [136,137] were


4
3

2-

F4


ALC-~I- Wa~~~~Y .)~_


,.~


ww ww









evaluated. As previously discussed, both mTOR and ERK1/2 lead to the phosphorylation

of p70S6K. mTOR phosphorylates p70S6K at threonine 389, an event that is rapamycin

sensitive [130,138]. A p70S6K immunoblot for cells in a variety of growth conditions in

conjunction with exposure to the inhibitors rapamycin, PD98059 or U0126. The FED

condition demonstrates mTOR-dependent phosphorylation of threonine 389 (T389)

(Figure 4-5), a relevant mTOR target [138]. The antibody for p70S6K also recognizes an

85kD isoform of p70S6K which is marked by an asterisk (*) [139]. Interestingly,

deprivation of a single essential amino acid (-HIS) did not block this phosphorylation at

position T389, but, as expected, total amino acid deprivation or rapamycin treatment

blocked this phosphorylation. GLN add back to EBSS did not restore phosphorylation at

T389, further implicating two distinct but interconnected pathways for MnSOD

induction. Inhibition of ERK1/2 caused an -50% reduction in the phosphorylation of

T389 as compared to untreated FED or -HIS conditions.

Rapamycin PD98059 U0126




___ P70 S6K .
----.. -(421/424)-" i- m '-e m. -' -

3-S) } 4EBPl-p --
Figure 4-5. Immunoblot analyses of HepG2 cells incubated for 12 h in FED, -HIS, EBSS
or EBSS +GLN medium with the indicated inhibitor and immunoblotted for
the respective proteins. An asterisk (*) marks the 85kD isoform of the p70S6
kinase protein.

The phosphorylation at sites T421 and S424 in the autoinhibitory domain of

p70S6K, which are believed to be important as the first sites leading to kinase activation

as well as potential sites for phosphorylation by ERK1/2 [140,141] were also evaluated.









Conditions for both -HIS and EBSS +GLN increased the phosphorylation at these sites

and inhibition of either mTOR or ERK1/2 activity by rapamycin or PD98059/U0126

[142], respectively, blocks the p70S6K phosphorylation at T421 and S424 (Figure 4-5).

This correlates with the importance of mTOR and ERK1/2 in the amino acid dependent

induction of MnSOD.

Another translational regulatory protein phosphorylated by mTOR, eukaryotic

initiation factor 4E-binding protein (4EBP1), was also evaluated. When mTOR is active,

4EBP1 is phosphorylated at four sites, threonines 37 and 46, serine 65 and threonine 70

[143]. The phosphorylation of threonines 37 and 46 is required for the subsequent

phosphorylation of the other two sites [144]. Furthermore, in vitro studies have

demonstrated that mTOR can directly phosphorylate threonines 37 and 46 [145,146] A

phospho-peptide antibody for positions T37/S46 of 4EBP-1 shows that the

phosphorylation of these sites is unchanged in FED versus -HIS, whereas EBSS

significantly reduces the phosphorylation at these sites (Figure 4-5). The addition of

GLN to EBSS stimulates the partial re-phosphorylation at these sites, with no effect in

any condition when rapamycin or PD98059/U0126 is included. This observation argues

for a glutamine dependent phosphorylation of 4EBP-1 that is independent of both

mTOR/raptor and ERK1/2. However, the +GLN response could be explained by

regulation through a known alternative rapamycin insensitive pathway, mTOR/rictor

[126,147-149].

Discussion

While organisms have adapted metabolic strategies to accommodate changes in the

availability of critical nutrients, how these changes are sensed by the cell still remains to

be fully understood. However, two pathways known to mediate downstream effects in









response to amino acid availability are through ERK1/2 signaling and the protein kinase

mTOR. The data presented here, and summarized (Figure 4-6), implicates the

importance of MEK/ERK and mTOR/raptor signaling pathways in the regulation of

MnSOD mRNA in response to amino acid deprivation.



Cytoplasm
MEK 1/2
TSC2 GAP
TSC2 GAP
TSC I SC Erk12


-- \ -... Thr421/
-Rheb-GDP .-Rheb-GTP TaSer 424
(inactive) (active) L -

Thr 37/ 40 T Thr 389
Ser 46

elpF4E 4EBP-1 4B
TranslationTranslation OFFON
Translation OFF MnSOD gene q
Figure 4-6. Relevant signal transduction pathways involved in the induction of MnSOD
in response to amino acid deprivation.

MAP kinase signaling is a signal transduction pathway orchestrating a large

number of extracellular cues to mediate a number of intracellular responses. In

particular, it has been demonstrated that ERK1/2 activation occurs in response to amino

acid deprivation [105,106]. To evaluate the contribution of MAP kinase signaling in the

regulation of MnSOD, several inhibitors were used. As determined by the MAP kinase

inhibitors tested, only the MEK/ERK1/2 pathway was required for MnSOD mRNA

induction by amino acid deprivation. As discussed, crosstalk between the MAP kinase

pathways can occur. However, at this time, ERK1/2 is the only known target for









MEK1/2. Utilization of inhibitors specific for MEK1/2 blocked MnSOD, induction,

supporting the importance of ERK1/2 activity. Furthermore, evaluation of the

downstream effectors of ERK1/2 signaling, by immunoblot analysis, confirmed the

activity of this enzyme. The phosphorylation of T421 and S424 of p70S6K demonstrates

that, through both means of MnSOD induction (-HIS and EBSS +GLN), ERK1/2 activity

is increased. Additionally, phosphorylation at these sites was also blocked by both of the

MEK1/2 inhibitors, as well as rapamycin. This strongly implicates a role for ERK1/2

signaling in the regulation of MnSOD in response to amino acid deprivation.

mTOR, a central mediator of growth, metabolism and proliferation [127], was also

evaluated as a potential contributor to the induction of MnSOD. mTOR regulation is

mediated by amino acid availability and is active when amino acid levels are sufficient.

Initial studies to determine the involvement of mTOR in the regulation of MnSOD

utilized the inhibitor rapamycin, which should mimic amino acid deprivation. However,

inhibition of mTOR alone did not induce MnSOD mRNA levels. Furthermore, the

induction by histidine deprivation was blocked by rapamycin treatment, whereas the

EBSS +GLN induction was not affected, indicating an interdependent regulation for this

pathway. These data led to the hypothesis that mTOR activity and signaling may be

required for MnSOD induction, but not involved in the activation of signaling pathways

regulating this induction. To further understand the role of mTOR in the induction of

MnSOD, the downstream effectors p70S6K and 4EBP-1 were evaluated to determine the

signaling status of mTOR.

A major rapamycin sensitive target of mTOR is p70S6K T389. Immunoblot

analysis with a phospho-specific antibody to this protein was used to determine the status









of mTOR activity. As expected, T389 phosphorylation did occur when HepG2 cells were

incubated in FED medium. Additionally, as hypothesized, histidine deprivation still

resulted in the phosphorylation of T389, indicating that mTOR signaling was occurring.

Equally important, inhibition of mTOR with rapamycin blocks both the phosphorylation

of T389 and mRNA levels of MnSOD in response to histidine deprivation. On the other

hand, EBSS or EBSS +GLN medium did not result in the phosphorylation of T389, nor

did rapamycin treatment block the induction of MnSOD mRNA in EBSS +GLN medium.

These data suggest a requirement of mTOR activity for the induction of MnSOD by

histidine deprivation and a separate rapamycin insensitive pathway for the induction

occurring in response to EBSS +GLN.

Recent reports have associated ERK1/2 signaling and the regulation of mTOR,

through the regulation of TSC1 (tuberin) and TSC2 (hamartin) [150-153]. TSC1 and

TSC2 are tumor suppressor genes that normally participate in cell growth and

proliferation. However, mutations in these genes cause an autosomal dominant genetic

disease, tuberous sclerosis complex (TSC), characterized by benign tumors (hamartomas

and hamartias) affecting many organs. TSC1 and TSC2 form a heterodimer and, through

the GTPase activity of TSC2, function to inhibit Rheb (Ras homologue enriched in

brain), whose activity is required for mTOR signaling to occur [151]. Ma et al. [150]

recently published a paper demonstrating the phosphorylation of TSC2, through

treatment with phorbol 12-myristate 13-acetate (PMA), which was blocked by the

ERK1/2 inhibitor U0126. Activation of ERK1/2 leads to the phosphorylation of TSC2

and inhibition of its ability to deactivate Rheb and thus mTOR activity is maintained

(Figure 4-6) [150]. Therefore, inhibition of ERK1/2 signaling would also block mTOR






73


activity and ultimately the induction of MnSOD by amino acid deprivation. The

connection of ERK1/2 signaling to mTOR fits well with the data presented here and

establishes an amino acid dependent regulation pathway for MnSOD that is completely

novel when compared to the cellular mechanisms currently proposed for the induction of

gene expression via amino acid deprivation.














CHAPTER 5
TRANSCRIPTIONAL REGULATION OF MANGANESE SUPEROXIDE
DISMUTASE BY FORKHEAD BINDING PROTEINS IN RESPONSE TO AMINO
ACID DEPRIVATION

Introduction

The transcriptional regulation of MnSOD gene has been extensively studied in the

human, mouse and rat species in response to a number of stimuli [18,23,24,39,43,154-

158]. Given that the characterization of MnSOD induction in response to amino acid

deprivation has been limited to the human species, the transcriptional regulation of the

human MnSOD will be discussed, although many comparisons can be made between

species. The human MnSOD gene has a typical housekeeping basal promoter that it is

GC rich and contains no TATA or CATT box [159]. In vivo footprinting with purified

SP1 and AP2 proteins identified eight SP1 sites and nine AP2 sites within an =250 bp

proximal promoter [159]. Further studies demonstrated a requirement of SP1 binding for

the activation of MnSOD gene transcription to occur with AP2 functioning to block SP1

binding [39,159]. As previously discussed, MnSOD is inducible in response to several

pro-inflammatory stimuli including lipopolysaccharide (LPS), interleukins 1 and 6 (IL-1

and IL-6), tumor necrosis factor alpha (TNF-a) and interferon-y [15,20,21,154]. The 5'

region of the MnSOD promoter does contain several NF-KB and API elements, however,

this region alone is not inducible with these stimuli, requiring an enhancer element

located within intron two [160].

The significant induction of MnSOD gene expression by pro-inflammatory

cytokines [15,20,21] has been proposed to contribute to cell survival by reducing the









increased levels of reactive oxygen species (ROS) associated with the inflammatory

response [4,5,15,16]. ROS are highly reactive and cause extensive damage to cellular

components including lipids, DNA and proteins, contributing to the pathogenesis of

disease [6-8] and aging [87]. A critical consequence of nutrient availability and

subsequent metabolism is the generation of ROS with previous estimates indicating 1-3%

of consumed oxygen in the basal state is released as superoxide radicals from

mitochondrial electron transport [3]. The connection between nutrient levels and the

generation of ROS is also thought to help delay the aging process through caloric

restriction [85], which may be explained by a reduction in metabolic flux and a

concomitant decline in ROS production [87]. This observation is also consistent with the

mitochondrial theory of aging which implicates continuous generation of ROS as a

critical factor for damage to mitochondrial DNA as well as oxidative reactions with

components of the cytosol and nucleus [86,161].

In C. elegans, a model organism for aging studies due to ease of genetic

manipulation and short life span (=19 days), two genes have been identified in the

regulation of aging, age-], a homologue of the mammalian phosphatidylinositol-3-OH

(PI3) kinase, and daf-2, a homologue of the insulin or insulin-like growth factor receptor

family [162]. The age-] mutation was identified in a screen for long lived mutants and

conferred an approximately 65% increase in life span [163]. Mutations affecting dauer

formation, a developmentally arrested stage occurring in C. elegans when there is a

limited amount of food, led to the identification of the daf-2 gene [164]. Mutations in the

daf-2 gene caused an increase in dauer formation marked by long life span, decreased

growth, increased resistance to starvation and reproductive immaturity causing an









increase in life span twice that of the wild type counterpart [164,165]. Subsequently,

Honda et al. [166] demonstrated that mutations in the age-] and daf-2 genes caused a life

span extension phenotype by conferring an increase resistance to oxidative stress

corresponding with an increase in MnSOD levels. The mutant phenotypes of age-] and

daf-2 were demonstrated to require the activity of the downstream target daf-16

[165,167] and, soon after, daf-16 were established to directly regulate the transcription of

genes involved in metabolism and development leading to the dauer formation and

increased lifespan [168].

Work by Furuyama et al. [169] determined the canonical binding site

(TTGTTTAC) for daf-16 and, through computer analysis, demonstrated that the C.

elegans sod3 promoter contained a daf-16 binding element (DBE). Subsequently, Kops

et al. demonstrated that overexpression of the daf-16 human homologue, FOXO3a, could

protect human colon carcinoma cells from oxidative stress through increases in MnSOD

mRNA and protein levels [170]. Furthermore, overexpression of FOXO3a caused an

increase in activity of a luciferase reporter construct, and mutational analysis led to the

identification of one functional inverse DBE at position -1249 of the MnSOD gene [170].

FOXO proteins belong to the FOX (forkhead box) family of winged helix/forkhead

transcription factors consisting of over 100 family members ranging from FOXA to

FOXS [171-173]. Classification of these transcription factors is based on the conserve

DNA binding domain which binds as a monomer forming three alpha-helices and two

characteristic large loops or "wings" [171,174]. The forkhead member of the class O

(other), commonly referred to as FOXO, comprises a group of the following functionally

related proteins: FOXO1 (forkhead in rhabdosarcoma or FKHR), FOXO3a (FKHR-like









lor FKHR-L1), FOXO4 (acute lymphocytic leukemia-1 fused gene from chromosome X

or AFX) and FOXO6 [172,173]. The FOXO class of proteins are implicated in the

regulation of a variety of cellular processes, including the cell cycle, apoptosis, DNA

repair, stress resistance, and metabolism [170,173,175-178].

Although FOXO proteins share a high level of homology, knock out studies in

mice have demonstrated different functions for each isoform [179]. Foxol homozygous

null mutants die before birth [179], whereas haplo-insufficiency rescues the diabetic

phenotype in insulin resistant mice (Irs2 -/-) by reducing hepatic expression of

glucogenetic genes and increasing p-cell proliferation [179-181]. Foxo3a-/- female mice

have age dependent abnormal ovarian follicular development, mild anemia and decreased

glucose uptake in glucose-tolerance tests [179,182]. Foxo4 -/- mice have no phenotype

and are indistinguishable from their littermates [179].

FOXO binding proteins are negatively regulated by the presence of growth/survival

signals through protein kinase B (PKB) [175]. When nutrients are available, PKB is in

its active state and phosphorylates FOXO proteins, facilitating the binding of the

chaperonin 14-3-3, which can then be exported out of the nucleus and thus causing

retention of the FOXO proteins in the cytostol [183-185]. On the other hand, removal of

growth factors impairs PKB signaling and the phosphorylation of the FOXO protein,

resulting in its localization to the nucleus and activation of its target genes [183-185].

Materials and Methods

Growth Hormone Reporter Constructs

Regions of the MnSOD gene were cloned into a human growth hormone reporter

plasmid [186]. A PAC (phage artificial chromosome) clone, obtained from the Sanger

center (RP1-56L9), containing the entire human MnSOD gene was used to clone regions









of interest into the growth hormone vector. A 3.6 kb BamHI fragment of the human

promoter was digested from the PAC. This fragment was then run on a 0.7% agarose gel,

gel purified and cloned into the BamHI site of a promoterless growth hormone reporter

plasmid pPGH. This construct was sequenced and analyzed by restriction digest for

confirmation of the correct sequence and orientation. The unique restriction enzyme sites

were used to generate a 1.4, 1.3, 1.1 and 0.83 kb promoter fragments. Briefly, the

constructs were generated using an Xbal site at the 5' end of the promoter fragment along

with the following 3' restriction enzymes Nrul (1.4), SexAI (1.3), XmnI (1.1) and Spel

(0.83). To generate the promoter constructs in conjunction with the human enhancer a

488 bp fragment was digested from a construct previously generated in the lab and

described in [43]. The 488 bp human enhancer fragment was digested from the HindIII

site of the pPGH contract and religated into the HindIII site of the GH constructs

containing the indicated human MnSOD promoter. The TKGH with the human enhancer

construct was already generated and is described in [43].

Overexpression Plasmids

The FOXO3a and FOXO1 plasmids were generously provided. The FOXO3a

plasmid was from Dr. Burrenger and is described in [170]. The FOXO1 plasmid was

from Dr. Tang and described in [185]. Briefly, the FOXO3a plasmid is an estrogen

receptor (ER) fusion protein that remains inhibited (localized to the cytosol) until treated

with 4-Hydroxytamoxifen (4-OHT), a modified ligand for the ER receptor. The protein

is constitutively active due to the mutations of the PKB sites to alanines, leaving this

protein unable to be phosphorylated. The FOXO1 plasmid is not an ER fusion protein

but it is constitutively active due to similar mutations.









Quick Change PCR

To delete the FOXO site from the growth hormone promoter constructs the

Stratagene QuickChange Site-Directed Mutagenesis Kit #200518 was used. Briefly, 25

ng of either the human MnSOD 3.4 promoter GH construct or the human MnSOD 3.4

promoter + enhancer fragment GH construct used as the template. 125 ng of each of the

following primers Forward: ATT CTT CTG ACG TCT GCC CAG CCC TTC CTG

Reverse: CAG GAA GGG CTG GGC AGA CGT CAG AAG AAT. Also added to the

reaction was a 1X final concentration of reaction buffer, 8 mM DNTP mixture, water and

PFU enzyme. A PTC 100 peltier thermal cycler was use with the following parameters:

Cycle 1 (95C for 30 seconds) X 1 cycle. Cycle 2 (95C for 30 seconds, 55C for 1

minute, 68C for 14 minutes) X 18 cycles. The reaction was then incubated on ice for

two minutes and 1 tL of DPNI was added to remove the parental strand, leaving only the

mutated desired product which was subsequently transformed into XL-10 gold competent

cells and incubated on a plate with ampicilin. From the resulting colonies, the plasmids

were isolated and sequenced for verification.

Transient Transfection of Reporter Constructs

HepG2 cells were cultured as described previously and transfected at

approximately 50% confluency in a 10 cm dish. The reporter plasmid containing

different regions of the MnSOD gene were transiently transfected using a FUGENE 6

transfection reagent (Roche #11 814 443 001). For each 10 cm dish, 15 utL of the

FUGENE 6 transfection reagent was diluted to a final volume of 600 utL in serum free

MEM. 5 [tg of reporter plasmid was then added and the reaction was incubated at room

temperature for 30 minutes and then transferred to HepG2 cells. After 24 h, the cells

were split 1:10 into 35 mm dishes and incubated for another 12 h after which the cells









were then incubated in either FED or -HIS media. The promoterless hGH was also

transfected to ensure that the transfection itself or the hGH plasmid does not have an

effect on the MnSOD message. Northern analysis was used evaluate the effect of

histidine starvation on the hGH message. A fragment from the hGH cDNA was used to

create a probe for northern analysis.

Transfection of FOXO Expression Constructs

HepG2 cells were grown to 60 % confluency in 35 mm dishes and transfected with

0.5 [tg of growth hormone construct containing the 1.3 kb promoter fragment and

increasing concentrations of either the FOXO1 or FOXO3a plasmid (0, 0.5, 1 or 5 tg)

using FUGENE6 transfection reagent. 24 h post transfection, 500 nM of 4-OHT was

added to the FOXO3a series of transfections, including the "zero ag" of plasmid to

control for effects caused by this treatment. 48 h post transfection, cells were collected

and total RNA was isolated as described in Chapter two.

Cell Culture and Transfection of siRNAs

Human hepatoma (HepG2) cells were maintained in MEM supplemented with 25

mM NaHCO3, 2 mM glutamine, antibiotic/antimycotic (ABAM) and 10% FBS at 370C

with 5% C02. The cells were grown to 70-85% confluency on 10cm dishes and then split

1:12 into 35 mm dishes and grown to 40% confluency. The cells were then transfected

with a final concentration of 100 nM SMARTpool FOXO3a siRNA (Dharmacon) using

DharmaFECTTm 4 siRNA Transfection Reagent (Dharmacon). To control for off target

effects of siRNA, a cyclophilin siRNA (Dharmacon) was also transfected. After 60 h,

cells were washed three times with PBS to ensure all histidine containing medium is

removed. Cells were then incubated in MEM (FED), MEM-HIS, EBSS or EBSS+GLN

medium. To ensure the amino acid levels in general are not depleted, the cells received









fresh medium 12 h before the start of all experiments (48 h post transfection). After 12 h

of amino acid deprivation, cells were collected and MnSOD expression was determined

by northern blot, immunoblot or real time.

Generation of cDNA

To generate cDNA for real-time PCR analysis SuperScriptTM first strand sythesis

kit from invitrogen #12371-019 was used. 1 ptg of total RNA isolated as described in

Chapter two was used as the template to which the following components were added: 1

pL of a 10 mM dNTP mix, 0.5 pg of Oligo(dT), and water. This reaction was incubated

at 65C for 5 minutes then placed on ice for 2 minutes. To this reaction the following

was added, 2 pL of 10 RT buffer, 25 mM MgCl2, 2 pL of 0.1 M DTT, and 1 pL of

RNAseOUTTM recombinant RNAase inhibitor. The reaction was then incubated for 2

min. at 420C, 50 units of SuperScriptTM II RT was added to each reaction and then

incubated for an additional 50 min. at 420C. The reaction was then terminated by

incubation at 700C for 15 min. and then incubated on ice for for at least 5 min.. 2 units of

Rnase H was then added and incubated at 370C for 20 min.. The sample was then diluted

with 79 pL of water and stored at -200C.

Real-Time PCR

We used 2 pL of cDNA generated from first strand synthesis (as described above)

was used as the template for real time PCR. To this, 0.3 pM of each primer was added,

12.5 pL of iTaqTM SYBER Green Supermix with ROX (Bio Rad, Hercules, CA #170-

8851 and water to a final volume of 25 pL. The Applied Biosystems, Foster City, CA

7000 sequence detection system was used with the following parameters: Cycle 1 (95C

for 10 minutes) X 1, Cycle 2 (95C for 15 seconds, 60C for 1 minute) X 40 cycles. The









AACT method was used to determine the relative fold changes, normalized to the

cyclophilin A gene, and is described in [187]

Real time primers: FOXO3A: F: (5'-TGG ATG CTG ATG GGT TGG A-3') R:

(5'-ATG GCG TGG GAT TCA CAAAG-3'). FOXO4: F: (5'-AGC GAC TGA CAC

TTG CCC A -3') R: 5'-GCC TCG TTG TGA ACC TTG ATG-3'). FOXOl: (F: 5'-TGG

TCA AGA GCG TGC CCT AC-3') R: (5'-GCT CGG CTT CGG CTC TTA G-3').

Cyclophilin A: F: (5'-CAT CCT AAA GCA TAC GGG TCC-3') R (5'-GCT GGT CTT

GCC ATT CCT G).

Protein Isolation and Immunoblot Analysis

Protein isolation and immunoblot analysis was performed as in Chapter two with

modifications for the indicated antibody. Samples immunoblotted for FOXO1 were run

on a 7.5% gel and transferred to a nitrocellulose membrane and blocked for 1 h at room

temperature in 7% non-fat milk. 1.25 tg/mL of Anti-FOXO1 (Chemicon #Ab3130) was

diluted in 5% BSA in TBST and the membrane was incubated overnight at 40C. Anti-

rabbit secondary antibody was used at 1:5000 diluted in 5% non-fat milk for one h at

room temperature. Samples immunoblotted for FOXO3a (Upstate 07-702) were run on a

7.5% gel and transferred to a nitrocellulose membrane and blocked for 1 h at room

temperature in 7% non-fat milk. 1.25 [tg/mL of Anti-FOXO3a was diluted in 5% BSA in

TBST and the membrane was incubated overnight at 40C. Anti-rabbit secondary

antibody was used at 1:5000 diluted in 5% non-fat milk for one h at room temperature.

Isolation of Nuclei

HepG2 cells were cultured as described in Chapter two. Following incubation for 6

h in the indicated medium, cells from two 150 mm dishes (per condition) were placed on

ice and rinsed two times with ice cold PBS. 5 mL of PBS was then added to each plate









and cells were scraped into a 15 mL tube. The cells were then centrifuged at 514g for 10

minutes. The PBS was aspirated and the pellet was transferred to a 1.5 mL eppendorph

tube and resuspended by pipetting up and down until there were no clumps in 900 [L of

lysis buffer containing 20 mM HEPES (pH 7.6), 10 mM NaC1, 1.5 mM MgC12, 0.2 mM

EDTA, 20% glycerol, and added just prior to use, 0.1 M DTT and IX protease inhibitor

tablet (from complete mini pellet dissolved in 1 mL water) and incubated on ice for 15

min.. 100 [L of a 10% Triton X-100 solution (final concentration is 0.1%) was added,

vortexed briefly and the cells were centrifuged at 5200 rpm for 10 minutes at 40C. The

supernatant containing the cytosolic fraction was removed and stored at -800C for later

use. To each sample 500 tL of nuclear extraction buffer containing 20 mM HEPES (pH

7.6), 400 mM NaC1, 1.5 mM MgC12, 0.2 mM EDTA, 20% glycerol, and added just prior

to use, 0.1 M DTT and IX protease inhibitor tablet (from complete mini pellet dissolved

in 1 mL water). The samples were then gently rocked for two h at 40C and then

centrifuged for 10 min. at 14,000 rpm. The supernatant containing the nuclear extract

was collected and stored for later use.

Reagents Used

4-Hydroxytamoxifen (4-OHT) (#H7904) and insulin (#1-9278) were obtained from

Sigma. LY294002 was obtained from Calbiochem (#440204).

Results

Endogenous Regulation of MnSOD by FOXO Transcription Factors

Extensive studies in C. elegans have demonstrated that limitation of food supply

causes an increase in life span which is dependent on the transcription factor daf-16,

conferring increased resistance to oxidative stress and an increase in MnSOD levels

[162,165-168,188]. Studies in humans demonstrated that MnSOD levels can be









increased by overexpression of the daf-16, homologue FOXO [170,189]. Given that in

C. elegans the FOXO homologue daf-16 is regulated by the limitation of food, the

regulation of this protein in response to amino acid deprivation was evaluated. To

determine if FOXO proteins may play a part in the regulation of MnSOD, modulators of

the FOXO intracellular signaling pathways were used in combination with histidine

deprivation. Protein kinase B (PKB) regulates the cytoplasmic localization of the FOXO

proteins by phosphorylation. Insulin is a known activator of the PKB pathway and thus

proposed to inhibit of the transcriptional activity of the FOXO proteins [175,176]. To

evaluate this possibility, HepG2 cells were incubated in FED or -HIS medium and

treated with increasing amounts of insulin. Insulin does decrease the induction of

MnSOD by amino acid deprivation indicating that the FOXO proteins may have a role in

this regulation (Figure 5-1).

Activation of PKB with insulin causes localization of the FOXO proteins to the

cytoplasm, while, on the other hand, blocking PKB activity inhibits the phosphorylation

of the FOXO proteins and causes translocation of the FOXO proteins to the nucleus to

activate transcription of target genes. LY294002, an inhibitor of PKB [190], was tested

on HepG2 cells in FED, -HIS, EBSS and +GLN conditions. The inhibitor LY294002, in

all medium conditions tested, caused an increase in MnSOD mRNA levels, further

implicating the role FOXO proteins may play in the regulation of MnSOD (Figure 5-2).

Transcriptional Regulation of MnSOD in Response to Amino Acid Deprivation

To determine DNA regulatory elements involved in MnSOD gene induction in

response to histidine, a human growth hormone (hGH) reporter plasmid was used. An

advantage to using the hGH plasmid is that it is an intact eukaryotic gene, complete with

introns and exons. Thus, the expressed message will undergo splicing and maturation in










Insulin gM 0 0.01 0.05 0.1 0.25 0.5 1 0 0.01 0.05 0.1 0.25 0.5 1


4002WO


MnSOD


Figure 5-1. Northern blot of HepG2 cells incubated in FED or -HIS medium, with
increasing concentrations of insulin. After 12 h, cells were collected and total
RNA was isolated. A gene specific probe for MnSOD was used to determine
the relative amounts of mRNA.





EBSS FED -HIS +GLN


LY294002 -






MnSOD


- +


- +


- +


- +


Figure 5-2. Northern blot of HepG2 cells incubated in EBSS, FED, -HIS or +GLN
medium with or without the PKB inhibitor, LY294002. After 12 h, cells were
collected and total RNA was isolated. A gene specific probe for MnSOD was
used to determine the relative amounts of mRNA.


FED


-HIS


53 'q T' 3 ** "-. .0 % Z









a manner identical to endogenous mRNAs. Another advantage is that growth hormone

expression is usually confined to the pituitary gland, eliminating any background in the

HepG2 cells. In order to confirm this, cells which were not transfected with hGH

plasmid were also probed with a hGH cDNA probe confirming that there was no

endogenous growth hormone mRNA in any cell lines used. By placing MnSOD gene

elements in front of the growth hormone message, in conjunction with the MnSOD

promoter, the contributions of these regions to the induction seen by histidine starvation

were evaluated.

As previously demonstrated, the MnSOD promoter contains a FOXO binding site

at 1249 bp upstream of the transcriptional start site [170]. To determine if histidine

deprivation caused an increase in the MnSOD promoter activity, a 3.4 kb promoter

fragment was cloned into the BamHI site of the hGH reporter plasmid. To further define

the promoter, this plasmid was then used to create two more constructs using convenient

restriction sites within the MnSOD promoter, creating 1.4kb and 0.83kb fragments.

These constructs were then transfected into HepG2 cells as described in the materials and

methods section. To ensure that there was little to no amino acid deprivation before the

start of the experiment, fresh MEM was given to the cells 12 h before the start of the

experiment. There is a small, but reproducible, induction with the 3.4 and 1.4kb

constructs and no effect with the 0.83kb promoter fragment in response to histidine

deprivation (Figure 5-3). A summary of the constructs generated and the corresponding

results from the promoter deletion analysis are also shown (Figure 5-4).

Previous studies have demonstrated that, in the rat, the MnSOD promoter has an

approximately 2 fold induction by IL-1. Initially, this was included as a positive control,









3.4 kb


FED -HIS


1.4 kb


FED -HIS


0.83 kb


FED -HIS


GH a m Uf 1


Figure 5-3. Representative northern blot of HepG2 cells transfected with a growth
hormone construct containing the indicated promoter fragments. 36 h post
transfection, cells were cultured in FED, -HIS medium or treated with IL-1
for 12 h. A gene specific probe for human growth hormone message (GH)
was used to determine the relative amounts of messenger RNA (mRNA).

Human growth hormone


I Human MnSODogig l, a


^yw +1 -His

IlIII +


-1.3 t
-1.1
-0.83
Figure 5-4. Human growth hormone constructs used to evaluate the human MnSOD
promoter deletions. The white boxes represent the growth hormone exons,
and the striped boxes represent the untranslated message of growth hormone.
Below the reporter constructs is a summary of results of the corresponding
deletions of the human MnSOD promoter following histidine deprivation.

treatment. Given that the enhancer element is known to regulate MnSOD induction by

cytokines, this region in conjunction with each of the previously described promoter

constructs, were also evaluated following histidine deprivation. The addition of the

enhancer element does not seem to add the relative fold induction through amino acid

deprivation (Figure 5-5). However, the addition of the enhancer element did, as


expected, induce as a result of IL-1 treatment.




Full Text

PAGE 1

REGULATION OF MANGANESE SUPEROXI DE DISMUTASE VIA AMINO ACID DEPRIVATION By KIMBERLY JEAN AIKEN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

PAGE 2

Copyright 2006 by Kimberly Jean Aiken

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To my mother, the strongest person I have ever known, always inspiring me to do the best that I can.

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ACKNOWLEDGMENTS I would like to thank my mentor, Dr. Harry Nick. He has been a great teacher to me in science and life. I am grateful for all of the support he has always offered to me and for the opportunity to work in his lab. I would also like thank all of my committee members (Drs. Bloom, Kilberg and Swanson). They have always been great to work with and instrumental in the completion of my thesis. My time spent in the lab would not have been the same without all of my lab mates; especially Ann Chokas and JD Herlihy, my partners in crime. I have also enjoyed meeting all of the colorful people in Gainesville; all of my different teammates, the foreign contingency, and of course all of my fellow classmates. I would also like to thank my family, who have known me all my life and are still my friends. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv LIST OF TABLES ...........................................................................................................viii LIST OF FIGURES ...........................................................................................................ix CHAPTER 1 INTRODUCTION........................................................................................................1 Manganese Superoxide Dismutase...............................................................................2 Superoxide Dismutases and Reactive Oxygen Species.........................................2 Cytoprotective Roles of MnSOD..........................................................................4 Transcriptional Regulation of MnSOD.................................................................5 Nutrient Regulation of Gene Expression......................................................................7 Glucose Control of Gene Regulation....................................................................7 Amino Acid Control of Gene Regulation..............................................................9 Amino Acids as Signaling Molecules.................................................................11 2 CHARACTERIZATION OF MANGANESE SUPEROXIDE DISMUTASE BY AMINO ACID CONTROL........................................................................................18 Introduction.................................................................................................................18 Materials and Methods...............................................................................................19 Cell Culture.........................................................................................................19 Isolation of total RNA.........................................................................................20 Electro-Transfer and Northern Analysis.............................................................21 Protein Isolation and Immunoblot Analysis........................................................21 Densitometry and Statistical Analysis.................................................................22 Results.........................................................................................................................23 Amino Acid Deprivation Induces MnSOD Steady State Messenger RNA Levels...............................................................................................................23 Induction of MnSOD is Specific to Amino Acid Deprivation............................24 Induction of MnSOD Messenger RNA is Specific to Essential Amino Acid Deprivation......................................................................................................27 Cellular Specificity of Amino Acid Deprivation for MnSOD Induction............28 Transcriptional control of MnSOD in response to amino acid deprivation........30 Discussion...................................................................................................................31 v

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3 METABOLIC CONTROL OF MANGANESE SUPEROXIDE DISMUTASE IN RESPONSE TO AMINO ACID DEPRIVATION.....................................................34 Introduction.................................................................................................................34 Materials and Methods...............................................................................................36 Isolation of Total RNA........................................................................................36 Measurements of ATP Levels.............................................................................36 Reagents Used.....................................................................................................36 Results.........................................................................................................................37 Glutamine is Required for the Induction of MnSOD by Amino Acid Deprivation......................................................................................................37 Inhibition of the TCA Cycle Blocks MnSOD Induction by Histidine Deprivation......................................................................................................43 Both a Functional Electron Transport Chain and F 1 -F 0 ATP Synthase Complex are Required for MnSOD Induction in Response to Amino Acid Deprivation......................................................................................................49 An Intact Electrochemical Gradient but not ATP Synthesis is Required for MnSOD Induction............................................................................................52 Discussion...................................................................................................................55 4 SIGNAL TRANSDUCTION PATHWAYS ASSOCIATED WITH MANGANESE SUPEROXIDE DISMUTASE INDUCTION IN RESPONSE TO AMINO ACID DEPRIVATION................................................................................59 Introduction.................................................................................................................59 Materials and Methods...............................................................................................63 Isolation of total RNA.........................................................................................63 Protein Isolation and Immunoblot Analysis........................................................63 Reagents Used.....................................................................................................63 Results.........................................................................................................................63 Requirement of ERK1/2 Signaling......................................................................63 Amino Acid Deprivation and mTOR Signaling..................................................66 Discussion...................................................................................................................69 5 TRANSCRIPTIONAL REGULATION OF MANGANESE SUPEROXIDE DISMUTASE BY FORKHEAD BINDING PROTEINS IN RESPONSE TO AMINO ACID DEPRIVATION................................................................................74 Introduction.................................................................................................................74 Materials and Methods...............................................................................................77 Growth Hormone Reporter Constructs................................................................77 Overexpression Plasmids.....................................................................................78 Quick Change PCR..............................................................................................79 Transient Transfection of Reporter Constructs...................................................79 Transfection of FOXO Expression Constructs....................................................80 Cell Culture and Transfection of siRNAs...........................................................80 Generation of cDNA............................................................................................81 vi

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Real-Time PCR...................................................................................................81 Protein Isolation and Immunoblot Analysis........................................................82 Isolation of Nuclei...............................................................................................82 Reagents Used.....................................................................................................83 Results.........................................................................................................................83 Endogenous Regulation of MnSOD by FOXO Transcription Factors................83 Transcriptional Regulation of MnSOD in Response to Amino Acid Deprivation......................................................................................................84 FOXO Messenger RNA Levels in Response to Amino Acid Deprivation.........91 Localization of FOXO Proteins...........................................................................94 Discussion...................................................................................................................95 6 DISCUSSION AND FUTURE DIRECTIONS..........................................................98 Discussion...................................................................................................................98 Future Directions......................................................................................................101 LIST OF REFERENCES.................................................................................................105 BIOGRAPHICAL SKETCH...........................................................................................121 vii

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LIST OF TABLES Table page 1-1 List of amino acids and their normal cellular functions and conditions of disease when these amino acids are found in excess or in a depleted state..........................17 viii

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LIST OF FIGURES Figure page 1-1 Catalysis of superoxide to oxygen and hydrogen peroxide through the action of MnSOD......................................................................................................................3 1-2 Production of free radicals at complex I and III of the electron transport chain........3 1-3 Rat MnSOD genomic clone, with identified sites of regulation................................6 2-1 Northern blot analysis of MnSOD in response to various stimuli...........................24 2-2 Northern blot analysis of total RNA isolated from HepG2 cells incubated in complete medium (FED), or medium lacking either histidine (-HIS) or glucose (-GLC)......................................................................................................................25 2-3 Densitometric quantification of replicate experiments as done in 2-2 of the MnSOD 4kb message...............................................................................................26 2-4 Immunotblot analysis of MnSOD in response to histidine deprivation. ................26 2-5 Representative northern blots of HepG2 cells incubated for the indicated times in medium lacking a single amino acid....................................................................27 2-6 Representative bar graph of at least three independent experiments as in Figure 2-5.............................................................................................................................28 2-7 Northern analysis from HUH7 (human hepatoma) cells, incubated for the indicated amount of time, in various medium conditions........................................29 2-8 Northern blot analysis of CCDLU, A549 and L2 cells incubated for 12 h, in the indicated medium.....................................................................................................30 2-9 Northern blot analysis of cells treated with actinomycin-D or cyclohexamide.......31 3-1 Northern analysis of HepG2 cells incubated in medium lacking all amino acids (EBSS), or in EBSS with the inclusion of the indicated amino acid at a concentration of 5 mM.............................................................................................38 3-2 HepG2 cells were incubated in various medium conditions with the addition of 5 mM glutamine for 12 h.............................................................................................39 ix

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3-3 HepG2 cells were incubated in complete medium (FED), medium lacking glutamine (-GLN), histidine (-HIS) or both amino acids (-HIS/-GLN)...................40 3-4 Northern analysis of HepG2 cells incubated for 12 h in the indicated medium conditions with increasing concentrations of glutamine..........................................41 3-5 HepG2 cells were incubated in the presence or absence of vitamins under the indicated conditions..................................................................................................42 3-6 Northern analysis of total RNA isolated from HepG2 cells incubated with or without 10% dFBS, or two components of serum, IGF or EGF..............................43 3-7 Tricarboxylic acid (TCA) cycle...............................................................................44 3-8 Representative northern blot of HepG2 cells incubated for 12 h in complete medium (FED) or complete medium lacking histidine (HIS), treated with increasing concentrations of fluoroacetate...............................................................45 3-9 D ensitometric data from three independent experiments following treatment with fluoroacetate (as done in Figure 3-9)...............................................................45 3-10 Representative northern blot of HepG2 cells incubated for 12 h in complete medium (FED) or complete medium lacking histidine (HIS) and treated with increasing concentrations of -keto--methyl-n-valeric acid (KMV).....................46 3-11 Densitometry data collected from three independent experiments following treatment with KMV (as done in Figure 3-13).........................................................47 3-12 Representative northern blot of HepG2 cells incubated for 12 h in complete medium (FED) or complete medium lacking histidine (HIS) and treated with increasing concentrations of 3-nitropropionic acid (3-NPA)...................................48 3-13 Densitometry data collected from three independent experiments following treatment with 3-NPA (as done in Figure 3-13).......................................................48 3-14 Northern blot of HepG2 cells incubated for 12 h in complete medium (FED) or complete medium lacking histidine (HIS) with increasing concentrations of malonate...................................................................................................................49 3-15 The electron transport chain and its relevant points.................................................50 3-16 Northern blot analysis of HepG2 cells incubated for 12 h in complete medium (FED) or complete medium lacking histidine (-HIS) with increasing concentrations of antimycin A.................................................................................51 3-17 Northern blot analysis of HepG2 cells incubated for 12 h in complete medium (FED) or complete medium lacking histidine (-HIS) with increasing concentrations of oligomycin...................................................................................52 x

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3-18 Northern blot analysis of HepG2 cells incubated for 12 h in the indicated medium for 12 h with the addition of 5 mM glutamine (EBSS +GLN), with increasing concentrations of the glucose analogue 2-deoxy-D-glucose (2-DOG)...53 3-19 ATP levels from four independent experiments in which HepG2 cells were incubated in the various conditions..........................................................................54 3-20 Northern blot analysis of HepG2 cells incubated for 12 h in complete medium (FED) or complete medium lacking histidine (-HIS) with increasing concentrations of 2,4 dinitrophenol..........................................................................55 4-1 Northern analysis of HepG2 cells treated for 12 h in FED, -HIS, EBSS or EBSS +GLN with the indicated inhibitor; SB202 (10 M), SB203 (10 M), or a JNK inhibitor (20 M)......................................................................................................64 4-2 Evaluation of HepG2 cells treated for 12 h in the indicated medium and the indicated inhibitor....................................................................................................65 4-3 Northern analysis of HepG2 cells treated for 12 h in FED, -HIS, EBSS or EBSS +GLN with increasing concentrations of rapamycin...............................................67 4-4 Densitometry of Northern analysis of HepG2 cells treated for 12 h in FED, -HIS, EBSS or EBSS +GLN with increasing concentrations of rapamycin (as in Figure 4-3)................................................................................................................67 4-5 Immunoblot analyses of HepG2 cells incubated for 12 h in FED, -HIS, EBSS or EBSS +GLN medium with the indicated inhibitor and immunoblotted for the respective proteins....................................................................................................68 4-6 Relevant signal transduction pathways involved in the induction of MnSOD in response to amino acid deprivation..........................................................................70 5-1 Northern blot of HepG2 cells incubated in FED or HIS medium, with increasing concentrations of insulin.........................................................................85 5-2 Northern blot of HepG2 cells incubated in EBSS, FED, -HIS or +GLN medium with or without the PKB inhibitor, LY294002........................................................85 5-3 Representative northern blot of HepG2 cells transfected with a growth hormone construct containing the indicated promoter fragments...........................................87 5-4 Human growth hormone constructs used to evaluate the human MnSOD promoter deletions....................................................................................................87 5-5 Representative northern blot of HepG2 cells transfected with a growth hormone construct containing the respective promoter fragments and the human MnSOD enhancer...................................................................................................................88 xi

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5-6 Northern blot of HepG2 cells transfected with a growth hormone construct containing the TK promoter and the human MnSOD enhancer...............................89 5-7 Human MnSOD promoter constructs generated and tested by histidine deprivation................................................................................................................90 5-8 Northern analysis of growth hormone constructs evaluating the contribution of the FOXO binding site to MnSOD induction...........................................................90 5-9 Densitometric analysis of northern blots as shown in the top panel of Figure 5-9..91 5-10 Overexpression of FOXO proteins with MnSOD growth hormone reporter plasmid.....................................................................................................................91 5-11 Real time PCR analysis from total mRNA isolated from HepG2 cells incubated for 12 h in the indicated medium.............................................................................92 5-12 Real time PCR analysis from total mRNA isolated from HepG2 cells incubated for 12 h in of FOXO3a mRNA levels in FED or HIS medium..............................93 5-13 Immunoblot analysis of FOXO3a protein in FED or HIS medium for the indicate amount of time............................................................................................93 5-14 Immunoblot analysis of FOXO3a protein from total protein isolated from HepG2 cells transfected with the indicated siRNA..................................................93 5-15 Northern blot analysis of HepG2 cells in FED or HIS MEM, transfected with the indicated siRNA. A gene specific probe for MnSOD was used to determine the relative amounts of mRNA.................................................................................94 5-16 Immunoblot of cytoplasmic and nuclear fractions isolated from HepG2 cells incubated for 6 h in the indicated medium...............................................................95 xii

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy REGULATION OF MANGANESE SUPEROXIDE DISMUTASE VIA AMINO ACID DEPRIVATION By Kimberly Jean Aiken August 2006 Chair: Harry S. Nick Major Department: Biochemistry and Molecular Biology Amino acids play an intricate role in protein synthesis, gene regulation, and overall cellular homeostasis. The extent of this control is exerted at many levels and is just beginning to be understood. The focus of this dissertation is on the gene regulation of the cytoprotective enzyme manganese superoxide dismutase (MnSOD). This gene has previously been demonstrated to be regulated, through increased transcription by a number of pro-inflammatory cytokines. However this gene has not been previously demonstrated to be under the regulation of nutrient levels. The research presented here identifies and characterizes the regulation of MnSOD in response to several different nutrients. Also described is a level of metabolic control previously not demonstrated for any other amino acid regulated gene. Relevant amino acid signal transduction pathways were also evaluated and provide evidence for the orchestration of several nutrient sensitive pathways. Furthermore, identification of potential transcription factors involved in this regulation were also evaluated. xiii

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CHAPTER 1 INTRODUCTION How nutrients and their subsequent metabolism affect gene expression is becoming an increasingly important area of research. Amino acid and glucose levels are constantly changing under normal physiological conditions. These changes are even more pronounced during times of stress or disease. How the body orchestrates these metabolic cues are only just beginning to be understood. Alterations in nutrient levels involve the regulation of many genes through chromatin remodeling, RNA splicing and stabilization, transcription and translation [1]. Gaining an understanding of the mechanisms underlying the regulation of gene transcription in response to the complex and ever changing environment, will help to further our knowledge of cellular mechanisms in general and how the cell responds during stress. A critical consequence of nutrient availability and subsequent metabolism is the generation of reactive oxygen species (ROS) [2]. It has been estimated that 1-3% of consumed oxygen is released as superoxide radicals from mitochondrial electron transport [3]. All aerobic organisms, through energy generating metabolism, produce harmful ROS such as superoxides, hydroxides and nitric oxide. External sources including ultraviolet radiation, cigarette smoke and numerous inflammatory mediators contributing to the production of ROS also affect the cell [4,5]. These free radicals are highly reactive and cause extensive damage to cellular components including lipids, DNA and proteins. As a defense mechanism, organisms have adapted and developed several means of protection against the harmful effects of ROS. These include: 1

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2 antioxidant enzymes such as superoxide dismutases (SOD), heme oxygenases, catalase, glutathione peroxidase, DNA damage repair enzymes as well as small molecular antioxidants such as -carotene and vitamins E and C [2]. Despite evolutionary adaptations, free radicals are implicated in a number of diseases such as cancer [6], asthma, rheumatoid arthritis, stroke, Parkinsons [7] and Alzheimers [8] disease. A key ROS scavenging enzyme is MnSOD, a cytoprotective mitochondrial enzyme that converts free radicals formed during normal cellular respiration, as well as a consequence of the inflammatory response, into the less reactive products, hydrogen peroxide and oxygen. Given its central importance to cellular homeostasis, the regulation of the MnSOD gene in response to nutrient deprivation was evaluated. The research presented in this dissertation identifies components involved in the regulation of MnSOD in response to nutrient deprivation, giving insight into the regulatory mechanisms and providing a greater understanding of nutrient control. Manganese Superoxide Dismutase Superoxide Dismutases and Reactive Oxygen Species As already indicated, the cells front line of defense against ROS comes from the family of superoxide dismutases (SODs) which, in eukaryotes, consists of the cytoplasmic and extracellular localized copper/zinc SOD (Cu/Zn-SOD), and the mitochondrial localized manganese SOD(MnSOD) [9]. These enzymes all catalyze the dismutation of the superoxide radical to hydrogen peroxide and oxygen (Figure 1-1) [10]. Hydrogen peroxide is then detoxified by either catalase or glutathione peroxidase to water and oxygen. Although all three of the SODs catalyze the same reaction, the generation of null mice for the three different isoforms demonstrated that only the mitochondrial localized MnSOD had a significant phenotype [11].

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3 2O2+ 2H+ O2+ H2O2 2O2+ 2H+ O2+ H2O2 Figure 1-1. Catalysis of superoxide to oxygen and hydrogen peroxide through the action of MnSOD. MnSOD is a nuclear-encoded, mitochondrial localized protein serving as the cells primary defense against ROS derived as a normal byproduct of cellular respiration. The electron transport chain accounts for 1 to 3% of total cellular superoxide production within the mitochondria alone [3]. Superoxides are a byproduct of both complex I and III, presumably due to the inability of electron carriers to efficiently transport the electron to the next complex (Figure 1-2). In order for the cell to continue to produce energy, the reactive/toxic products of this pathway must be removed. Through the action of the nuclear-encoded, mitochondrial-localized MnSOD, free radicals are efficiently eliminated, thus protecting the mitochondrial DNA as well as the remainder of the cell by preventing free radicals from leaving the mitochondria. NADH Succinate 2eSuccinate Dehydrogenase(3Fe-S)COMPLEX II NADHDehydrogenase(5Fe-S)COMPLEX I UQcyt. bcyt. c1 COMPLEX III cyt. c cyt. a-a3 e-e-COMPLEX IV O2O2-. e-e4e-O2H2O O2O2-.2eFigure 1-2. Production of free radicals at complex I and III of the electron transport chain. The critical cellular importance of MnSOD was demonstrated when two independent groups produced transgenic mice lacking MnSOD activity. Li et al [12] deleted exon 3 of the Sod2 gene on a CD-1 strain background (Sod2 mlucsf ). All mice died within 10 days of birth due to dilated cardiomyopathy, with an accumulation of lipid in

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4 liver and skeletal muscle and metabolic acidosis [12]. Lebovitz et al. [13] deleted exons 1 and 2 of the Sod2 gene on a mixed mouse background (Sod2 mlbcm ). These mice survived for up to 18 days and exhibited severe anemia; degeneration of neurons in the basal ganglia and brain stem; and progressive motor disturbances characterized by weakness, rapid fatigue, and cycling behavior [13]. The mice also exhibited extensive mitochondrial injury within the degenerative neurons and cardiac myocytes. Heterozygous mice (Sod2 mlucsf and Sod2 mlbcm ) with a 50% reduction of MnSOD activity also showed chronic oxidative stress with increased mitochondrial permeability and premature induction of apoptosis [13,14]. Cytoprotective Roles of MnSOD The generation of ROS has been associated with a number of disease states [15-18] and as such, the cell has evolved protective measures to aid in the survival of the cell by inducing MnSOD [19]. MnSOD gene expression is up-regulated by a wide variety of pro-inflammatory stimuli including IL-1, TNF, IL-6, and LPS, as well as down-regulated by glucocorticoids [20-24]. The generation of Sod2 null and heterozygous mice established the critical need for this antioxidant enzyme. Additionally, overexpression, in cell culture and in transgenic mice, has demonstrated a protective role against oxidative mediated insults. Extensive studies in cell culture have demonstrated the protective role of MnSOD in response to radiation, cytotoxic effects of IL-1 and TNF and several chemotherapeutic agents [25-29]. MnSOD over expression has also been shown to suppress or cause partial reversal of the malignant phenotype seen in breast cancer cells and SV40-transformed human lung fibroblasts. [30,31]. In transgenic mice overexpressing MnSOD, protection was conferred against the damaging effects of ROS

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5 production following ischemia/ reperfusion in the heart [32] and ionizing radiation of the lung [33]. Transcriptional Regulation of MnSOD The molecular regulation of the MnSOD gene by cytokine induction has been extensively studied by our lab and others, demonstrating increased MnSOD steady state mRNA levels in response to a variety of pro-inflammatory stimuli [21,22,34-36]. The stimulus dependent induction of the gene is blocked when co-treated with the transcriptional inhibitor actinomycin-D, suggesting de novo transcription. Nuclear run on studies were subsequently performed, confirming transcriptional control by cytokine induction of MnSOD [37]. The human SOD2 gene encodes a 22 kDa protein, which forms a 88.6 kDa tetramer consisting of a dimer of dimers with approximately 90% homology to the rat and mouse proteins [38]. The human MnSOD gene is localized to chromosome 6q25, and like the rat, consists of 5 exons and 4 introns. The promoter is consistent with that of a housekeeping gene in that it is TATAand CAAT-less, with approximately 80% GC content, and containing several putative SP1 and AP2 sites [39]. The human gene produces two mRNA messages whereas in the rat there are five, all of which arise from the same gene, differing only in their 3 untranslated region due to alternative polyadenylation [40,41]. Although the MnSOD promoter has the elements of a housekeeping gene, this gene is highly inducible by cytokines and therefore, studies in our laboratory sought to further analyze the chromatin structure utilizing DNase I and Dimethyl sulfate (DMS) in vivo footprinting. DNase I hypersensitive studies identified seven constitutive hypersensitive

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6 Rat MnSOD Genomic Clone = exons = Alternative Polyadenylation site 10 constitutive factors1 inducible enhancer 3'5' = DNase I hypersensitive site******** Northern analysisRatHumanRat MnSOD Genomic Clone = exons = Alternative Polyadenylation site 10 constitutive factors1 inducible enhancer 3'5' = DNase I hypersensitive site******** Northern analysisRatHuman Figure 1-3. Rat MnSOD genomic clone, with identified sites of regulation. sites and are represented as an asterisk (*) (Figure 1-3) [37]. High resolution DMS in vivo footprinting of the promoter region identified 10 putative constitutive protein-DNA binding sites and one inducible site. In vitro footprinting of this region linked Sp1 and gut-enriched Krppel-like factor (GKLF) binding to 5 of the 10 constitutive sites [37]. Most recently, ChIP and PIN*POINT analysis confirmed the presence of Sp1 on the promoter and, through site-directed mutagenesis, showed the functional importance of two of the Sp1 binding sites [42]. Reporter constructs containing genomic elements 5 to exon 1 of MnSOD could not fully recapitulate the stimulus-dependent induction observed from studies on the endogenous gene [43]. Further analysis of the remaining hypersensitive sites led to the identification of an inducible enhancer element within intron two [43]. This enhancer element, in conjunction with the MnSOD minimal promoter, was found to mimic the stimulus-dependent endogenous gene levels in an orientation and position independent manner. The enhancer element is highly conserved between mouse, rat and human and our laboratorys current efforts have focused on identifying the cognate transcription

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7 factors responsible for the induction by pro-inflammatory stimuli. Further description of this element is limited because evidence presented in this thesis will demonstrate that this enhancer is not involved in nutrient control of MnSOD gene expression. Nutrient Regulation of Gene Expression Organisms have evolved many mechanisms to adapt to changing environmental conditions. In times of nutrient deprivation when glucose and amino acids levels are limited, several mechanisms regulating gene expression are induced and aid in cell survival. In yeast, these mechanisms are well understood, but in more complex mammalian systems these mechanisms have yet to be fully defined. As such, the yeast systems for glucose and amino acid control will be described and compared to their mammalian counterparts. Glucose Control of Gene Regulation Glucose is a primary source of metabolic energy for the cell and also plays an important role in protein glycosylation. Glucose starvation leads to the improper folding of newly made glycoproteins in the endoplasmic reticulum (ER) and subsequent accumulation of these proteins, causing a decrease in overall protein synthesis with a concomitant increase in the expression of genes such as the ER chaperonin protein glucose regulated protein (GRP78) [44-46]. This signaling of the ER to the nucleus is known in yeast as the (unfolded protein response) UPR pathway and in mammalian cells, the ER stress response (ERSR) [47-49]. The UPR pathway is basically conserved from yeast to mammalian cells. In yeast, a bZIP transcription factor homologue ATF/CREB (HAC1), is the unfolded protein response element (UPRE) binding protein. Under normal conditions, HAC1 is constitutively transcribed within the nucleus, but not translated due to a non-classical

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8 untranslated region at the 3 end of the mRNA. When there is an accumulation of unfolded proteins in the ER, Ire1p dimerizes and splices out the intron within the HAC 1 mRNA. IreIp is a transmembrane protein with its N-terminal domain in the ER lumen and its kinase/C-terminal domain residing in the cytosol/nucleus. The N-terminal domain acts as a sensor within the ER and dimerizes when there is an accumulation of unfolded proteins [50,51]. The C-terminal domain, upon dimerization, auto-trans-phosphorylates and removes the intron from HAC1 through site specific cleavage [52]. Once the intron is spliced out, tRNA ligase Rlg1p, rejoins the message, which can now be translated [53]. The Hac1p protein can now bind to a UPRE and initiate transcription of various target genes. In mammalian cells, there are two pathways working in concert to monitor protein folding. When low levels of proteins are unfolded, a proteolytic cleavage of activating transcription factor 6 (ATF6) occurs. This releases the nuclear half of the protein into the cytosol of the nucleus. ATF6 is a constitutively expressed type II transmembrane protein which, in response to unfolded proteins, is sequentially cleaved by site 1 and 2 proteases, S1P and S2P, respectively [54]. The soluble nuclear form, ATF6(N), binds to an ER stress element (ERSE) causing the transcription of various genes, such as, ER chaperones, to aid in protein folding, and also the mammalian homologue of HAC1, X-box binding protein-1 (XBP1). As stated above, transcription of XBP1, also a bZIP protein, is induced by low levels of unfolded proteins due to ATF6(N) binding. XBP1, like HAC1, also contains an intron that is spliced out, in the same manner as in yeast, by the mammalian homologue of Ire1p, IRE1. The protein generated from the spliced mRNA, pXBP1(S), also binds to ERSE sequences inducing transcription of the same

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9 target genes as ATF6(N). In addition to ERSE elements, pXBP1(S) also binds to specific target genes which contain a UPRE sequence. When a low level of ER stress is encountered, ATF6 can quickly react by inducing ER chaperones and then, through the induction of XBP1, continued ER stress is met by both UPR systems. By utilizing this dual control, mammalian cells have developed a fine tuned mechanism in response to glucose deprivation. Amino Acid Control of Gene Regulation Amino acid availability plays an important role in cell viability and protein synthesis. Amino acid deprivation in yeast induces the general amino acid control (GAAC) response which causes a global decrease in general protein synthesis while increasing the transcription of over 40 genes encoding amino acid biosynthetic enzymes [55,56]. This is achieved through the activation of the protein kinase general control nonderepressible protein (Gcn2) in which uncharged tRNA accumulates and binds to the regulatory domain of Gcn2p. When Gcn2p is bound by an uncharged tRNA it phosphorylates and inactivates elongation initiation factor (eIF2). Phosphorylation of the subunit of eIF2 causes it to bind and sequester eIF2B, a guanyl nucleotide exchange factor [57]. eIF2B is responsible for the regeneration of GDP-eIF2 to GTP-eIF2, which is necessary for the binding of Met-tRNA i Met The phosphorylation of the subunit of GDP-eIF2 also inhibits its conversion to its active GTP form. When this process is inhibited overall protein synthesis slows due to a lack of ternary complexes required for the base pairing of Met-tRNA i Met to AUG [58]. It is this mechanism that ultimately leads to an increase in the transcription of the general nutritional/transcriptional control element (GCN4) which causes increased transcription of amino acid biosynthetic enzymes [55,56,59]. GCN4 mRNA contains four short upstream open reading frames

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10 (uORF) and when Gcn2p is activated, Met-tRNA i Met is slow to associate with the 40S complex allowing initiation to occur at the proper GCN4 ORF and produces a full length mRNA [60,61]. Following transcriptional activation of Gcn2p target genes, the balance of amino acids is restored, uncharged tRNAs return to normal levels, Met-tRNA i Met binding to the 40S subunit is no longer inhibited and translation of GCN4 no longer occurs [1,56,62,63]. Mammalian cells have a similar system but they can not synthesize all of the amino acids needed for protein synthesis. However, they do elicit a response that causes the transcription of many genes that help to maintain cellular homeostasis until amino acids become available [1]. Nutrient deprivation, in the form of glucose or amino acids, converges to activate a number of genes. In mammalian cells, deprivation of essential amino acids activates the amino acid response pathway (AAR) which leads to the translation of activating transcription factor (ATF4), the mammalian homologuelog to the yeast GCN4, through the same the mechanism as described above for yeast. ATF4 has been demonstrated to regulate a number of genes including membrane transporters, transcription factors, growth factors, and metabolic enzymes through binding to the consensus sequence 5-CATGATG-3[1,57,64,65]. The mechanisms described thus far have demonstrated cellular mechanisms in response to nutrient deprivation. However, amino acid sufficiency is also detected through the kinase, target of rapamycin (TOR) (mTOR in mammalian cells). This level of control is exerted at the level of the mRNA cap binding protein, eIF-4E. 4E binds to the mRNA cap and then 4A and 4G bind allowing for the 43 S pre-initiation complex to bind and scan the mRNA for an AUG start site. When there are adequate amounts of

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11 amino acids mTOR is active and phosphorylates 4E-BP1, inhibiting its binding to 4E, and translation is maintained [66]. When amino acids are limited, especially leucine, mTOR can not phosphorylate 4E-BP1, the 4E protein is sequestered and the assembly of the pre-initiation complex is prevented, ultimately leading to the inhibition of translation. Amino Acids as Signaling Molecules As described above, amino acids play an important role in mediating a number of cellular responses. The advantage to maintaining a proper balance of amino acids is clear; amino acids are the essential building blocks of life, providing the primary composition of all proteins. There are 20 major amino acids which can be subdivided as essential or nonessential. The essential amino acids are phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, histidine, arginine, leucine and lysine. These amino acids are defined as essential because our body cannot make them on its own, and thus it is essential that they be provided from the diet. However, depending on diet or other circumstances of stress, some non-essential amino acids may also become conditionally essential. For example, glutamine is a non-essential amino acid and the most abundant amino acid in the blood stream but during times of metabolic stress its levels are reduced and it is considered conditionally essential [67,68]. Typically, amino acid levels are in a balanced state and play a vital role in overall homeostasis of the body by acting as neurotransmitters, hormones, mineral transportation and in energy production. However, poor nutrition and disease can cause alterations in these levels, which may lead to fluctuations in amino acid levels. Under these circumstances, amino acids are imbalanced and as a consequence their levels can be found either in excess or in a depleted state, leading to the activation of the cellular responses described above. Table 1-1 lists the 20 principle amino acids and some of their

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12 functions in the body, with the essential amino acids shown in bold. Also provided in Table 1-1 is the status of thes e amino acids during times of the indicated stress or disease, demonstrating conditions where these amino acids will be found in excess or in a depleted state demonstrating the wide variety of functions and instances that will cause amino acid imbalances in the cell. For example, depletion of branched chain amino acids (BCAA) has been associated with muscle protein turnover and as such have been supplemented in cases of burn sepsis and othe r forms of trauma to help prevent protein loss [69-71]. Another example is the gene tic disease phenlyketonuria (PKU) in which amino acid imbalance can lead to mental reta rdation due to excess phe nylalanine levels in the brain [72]. Additionally, the manipulation of gene regulation in response to amino acid deprivation is also currently involved in the treatment of acute lymphoblastic leukemia (ALL), a cancer of the white blood cells. In ALL, the bone marrow makes precursor cells (blasts) that never form into lymphocytes but undergo several mutations, eventually leading to unchecked growth [73] These leukemia cells cannot synthesize asparagine, and are therefore sensitive to aspa raginase (ASNase), an enzyme that rapidly depletes the extracellular pool s of asparagine. ASNase treatment is responsible for complete remission of 40-60% of these cases [74,75] When used in combination with vincristine and predonisone, the remi ssion is increased to 95% [76].

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Table 1-1. List of amino acids and their normal cellular functions and conditions of disease when these amino acids will be found in excess or in a depleted state (Adapted from http://www.aminoacidpower.com/aboutAmino/aminoTour20 ) Amino Acid Main functions Excess seen in Deficiencies seen in Alanine Important source of energy for muscle Low insulin and glucagon levels Hypoglycemia The primary amino acid in sugar metabolism Diabetes mellitus Muscle breakdown Boosts immune system by producing antibodies Kwashiorkor (starvation) Fatigue Major part of connective tissue Viral infections Elevated insulin and glucagons levels Arginine Essential for normal immune system activity AIDS Necessary for wound healing Decreases size of tumors Immune deficiency syndromes Assists with regeneration of damaged liver Candidiasis Increases release of insulin and glucagons Precursor to GABA, an important inhibitory neurotransmitter Aspartic Acid Increases stamina Involved in DNA and RNA metabolism Amytrophic Lateral Sclerosis Epilepsy Calcium and magnesium deficiencies Excitatory amino acids Helps protect the liver by aiding the removal of ammonia Enhances immunoglobulin production and anti-body formation Asparagine Excitatory neurotransmitters Aids in removing ammonia from the body May increase endurance and decrease fatigue Detoxifies harmful chemicals Involved in DNA synthesis Cysteine Protective against causes of increased free radical production Chemical Sensitivity Natural detoxifier Food Allergy Essential in growth, maintenance, and repair of skin Key ingredient in hair Precursor to Chondroitin Sulfate, the main component of cartilage Glutamic Acid Excitatory neurotransmitter Increases energy Excesses in brain tissue can cause cell damage Accelerates wound healing and ulcer healing Plays major role in DNA synthesis 13

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14 Table 1-1. Continued Amino Acid Main functions Excess seen in Deficiencies seen in Glutamine The highest blood concentration of all the amino acids Involved in DNA synthesis Use of some anti-convulsant medications Chronic Fatigue Syndrome Alcoholism Precursor to the neurotransmitter GABA Anxiety and Panic Disorders Important glycogenic amino acid Essential to gastrointestinal function Involved with muscle strength and indurance Precursor to the neurotransmitter GABA Glycine Part of the stucture of hemoglobin Involved in glucagon production, which assists in glycogen Metabolism Starvation Chronic Fatigue Syndrome Viral Infections Candidiasis Inhibitory neurotransmitter Hypoglycemia Part of cytochromes, which are enzymes involved in energy production Anemia Glycogenic amino acids Histidine Found in high concentrations in hemoglobin Pregnancy Rheumatoid arthritis Useful in treating anemia due to relationship to hemoglobin Anemia Has been used to treat rheumatoid arthritis Dysbiosis Precursor to histamine Associated with allergic response and has been used to treat allergy Assists in maintaining proper blood Ph Isoleucine Involved in muscle strength, endurance, and stamina BCAA levels are significantly decreased by insulin Diabetes Mellitus with ketotic hypoglycemia Obesity Kwashiorkor (starvation) Muscle tissue uses Isoleucine as an energy source Hyperinsulinemia Required in the formation of hemoglobin Panic Disorder Chronic Fatigue Syndrome Acute hunger

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15 Table 1-1. Continued Amino Acid Main functions Excess seen in Deficiencies seen in Leucine Involved in muscle strength, endurance, and stamina Potent stimulator of insulin Ketosis Hyperinsulinemia Depression Helps with bone healing Helps promote skin healing Chronic Fatigue Syndrome Kwashiorkor (starvation) Vitamin B-12 deficiency in pernicious anemia Acute hunger Lysine Inhibits viral growth Excess of ammonia in the blood Herpes Invovled in the formation of L-Carnitine Epstein-Barr Virus Helps form collagen Chronic Fatigue Syndrome Assists in the absorption of calcium AIDS Essential for children, as it is critical for bone formation Anemia Involved in hormone production Hair loss Lowers serum triglyceride levels Weight loss Irritability Methionine Assists in breakdown of fats Severe liver disease Chemical Exposure Helps reduce blood cholesterol levels Required for synthesis of RNA and DNA Multiple Chemical Sensitivity (MCS) Antioxidant Vegan Vegetarians Assists in the removal of toxic wastes from the liver Involved in the breakdown of Epinephrine, Histamine, and Nicotinic Acid Natural chelating agent for heavy metals, such as lead and mercury Phenylalanine Precursor to the hormone thyroxine Major part of collagen formation DL-Phenylalanine is useful in reducing arthritic pain Used in the treatment of Parkinson's Disease Depression Obesity Cancer AIDS Parkinson's Disease

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16 Table 1-1. Continued Amino Acid Main functions Excess seen in Deficiencies seen in Proline Critical component of cartilage Involved in keeping heart muscle strong Works in conjunction with Vitamin C in keeping skin and Chronic Liver Disease Sepsis (infection of the blood) Acute alcohol intake joints healthy Serine Glycogenic amino acid Critical in maintaining blood sugar levels Aids in the production of antibodies and immunoglobulins Vitamin B-6 Deficiency Total body gamma and neutron irradiation Hypoglycemia Required for growth and maintenance of muscle Candidiasis Threonine Required for formation of collagen Alcohol ingestion Depression Helps prevent fatty deposits in the liver Vitamin B6 deficiency AIDS Aids in production of antibodies Pregnancy Muscle Spasticity Can be converted to Glycine (a neurotransmitter) Liver cirrhosis ALS (Amyotrophic Lateral Sclerosis) Acts as detoxifier Vegetarianism Needed by the GI (gastrointensinal) tract for normal functioning Epilepsy Provides symptomatic relief in Amyotrophic Lateral Sclerosis (ALS) Tryptophan Precursor to the key neurotransmitter, serotonin Effective sleep aid, due to conversion to serotonin Lowers risk of arterial spasms Increased intake of salicylates (aspirin) Increased blood levels of free fatty acids Depression Insomnia ALS Reduces anxiety Sleep deprivation Chronic Fatigue Syndrome The only plasma amino acid that is bound to protein Effective Can be converted into niacin (Vitamin B3).in some forms of n Niacin intake Treatment for migraine headaches Stimulates growth hormone

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17 Table 1-1. Continued Amino Acid Main functions Excess seen in Deficiencies seen in Tyrosine Precursor to neurotransmitters dopamine, norepinephrine, epinephrine and melanin Precursor to thyroxine and growth hormone Increases energy, improves mental clarity and concentration Hyperthyroidism Chronic liver disease; cirrhosis Depression Chronic Fatigue Syndrome Gulf War Syndrome Hypothyroidism Parkinson's Disease Drug addiction and dependency Valine Involved with muscle strength, endurance, and muscle stamina Not processed by the liver befo re entering the blood stream Ketotic Hypoglycemia Visual and tactile hallucinations Kwashiorkor Obesity Neurological deficit Actively absorbed and used directly by muscle as an energy source Elevated insulin levels Valine deficiency decreases abso rbtion by the GI tract of all other amino acids

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CHAPTER 2 CHARACTERIZATION OF MANGANESE SUPEROXIDE DISMUTASE BY AMINO ACID CONTROL Introduction Organisms have adapted metabolic strategies to accommodate changes in the availability of nutrients. Amino acid deprivation causes a global decrease in protein synthesis and an increase in a subset of specific proteins, through several different mechanisms. Deprivation of essential amino acids has been demonstrated to evoke responses at both the transcriptional and post-transcriptional levels for genes such as asparagine synthetase (ASNS), CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), cationic amino acid transporter (Cat-1), sodium-coupled neutral amino acid transporter system A (SNAT2) and insulin-like growth factor binding protein-1 (IGFBP-1) [1]. Fernandez et al. [77] have also demonstrated the existence of an internal ribosome entry site within the 5 UTR of the Cat-1 gene that controls translation of this transport protein under conditions of amino acid depletion. As described in Chapter one, all aerobic organisms metabolize nutrients to provide energy for the cell and, as consequence, also produce reactive oxygen species (ROS) [2]. Through normal respiration, approximately 1-3% of consumed oxygen is released as superoxide radicals from mitochondrial electron transport [3]. As a protective measure, cells have also evolved a mechanism to relieve the cell of these harmful superoxides, the mitochondrial localized, nuclear encoded protein MnSOD. 18

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19 Given the wide variety of responses elicited by nutrient deprivation, initial studies were aimed at characterizing the regulation of MnSOD in response to amino acid and glucose deprivation. MnSOD has prevously been demonstrated to be cytoprotective durring times of stress mediated by inflamation and cytokine production. Therefore, nutrient availability, in the form of either amino acid deprivation or glucose, may have relevant metabolic and cell survival benefits mediated through the elevation of MnSOD levels. Materials and Methods Cell Culture HepG2 (human hepatoma), HUH7 (human hepatoma), FAO (rat hepatoma), CaCO2 (human intestinal epithelial) and CCDLU (human adenocarcinoma) cells were maintained in minimal essential medium (MEM) (Sigma, St. Louis, MO) at pH 7.4 supplemented with 25 mM NaHCO3, 4 mM glutamine, antibiotic/antimycotic (ABAM) (Gibco, Grand Island, NY #15240-062) and 10% FBS (Gibco) at 37C with 5% CO2. L2 (rat pulmonary epithelial-like, ATCC CCL 149) and A549 (human epithelial-like) cells were maintained in Hams modified F-12K medium supplemented with 4 mM glutamine, ABAM and 10% FBS at 37C with 5% CO2. Cells were grown to 50-65% confluency on 10 cm dishes then split 1:8 into 60 mm dishes. After 24 h, with a fresh media change 12 h before the start of the experiment to ensure the amino acid levels in general are not depleted, the cells were washed three times with PBS and incubated in 3 mL of the appropriate media and collected at the indicated times for either northern or western analysis.

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20 To examine the amino acids individually, a base medium containing no amino acids, Earls Balanced Salt Solution (EBSS Sigma #E888), was used. The EBSS medium was then reconstituted to contain the nutrients found in MEM. This includes the addition of vitamins, 10% dialyzed FBS, ABAM, and amino acids. Each amino acid was prepared as a 100X stock (from Sigma) and added individually to the media to the same levels found in MEM medium but omitting the amino acid being tested. For add back experiments, the same EBSS base and supplements was used and only the indicated amino acid was added at a 5 mM concentration. The pH of the medium was maintained at 7.2-7.4 and adjusted (when necessary) with 0.1M NaOH or HCL. Isolation of total RNA Total RNA was isolated from cells as described by the Chomczynski and Sacchi method with modifications [20]. Medium was removed and the cells were washed two times with PBS. The cells were then lysed in 1 mL of GTC denaturing solution consisting of 4 M Guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% Sarcosyl, and 0.1 M -mercaptoethanol. After vortexing briefly to help lyse the cells, 50 L of 2 M sodium acetate, pH 4.0, and 500 L of water-saturated phenol was added. The solution was incubated at room temperature for 5 min. 110 L of a 49:1 chloroform:isoamyl alcohol mixture was added to the lysate, vortexed and centrifuged at 14,000 rpm for 10 min. The aqueous phase was then removed, an equal amount of isopropanol added and then incubated at -20C for 30 min. The lysate was then centrifuged at 14,000 rpm for 10 min. at 4 o C and the RNA pellet was resuspended in 75 L diethyl pyrocarbonate (DEPC) treated double distilled water. 25 L of 8 M lithium chloride (LiCl) was then added, mixed and incubated at -20 o C for 30 min. The RNA was

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21 pelleted by centrifugation at 14,000 rpm for 20 min. at 4 o C, washed one time with 70% ethanol and resuspended in 100 L DEPC water. RNA concentrations were determined by absorbance at 260 nm. Electro-Transfer and Northern Analysis Five to ten g of total RNA was denatured and fractionated on 1% agarose, 6% formaldehyde gels, electrotransferred to a Zeta-Probe blotting membrane from Bio-RAD #162-0159 and UV cross-linked. Membranes were then incubated for 1 h in a prehybridization buffer [78] consisting of 0.45 M sodium phosphate, 6% sodium dodecyl sulfate (SDS), 1 mM EDTA, and 1% bovine serum albumin (BSA). The membranes were then incubated overnight at 62C in the same hybridization buffer with a 32 P radiolabeled gene specific probe for MnSOD, asparagine synthetase, or GRP 78, generated by random primer extension (Invitrogen, Carlsbad, CA #18187-013). The membrane was then washed three times for 10 min. at 66C in a high stringency buffer composed of 0.04 M sodium phosphate, 2 mM EDTA, and 1%SDS and then exposed to film (Amersham, Piscataway, NJ #RNP 1677K,RNP30H). Protein Isolation and Immunoblot Analysis HepG2 cells were incubated in complete medium (FED) or complete medium lacing histidine (-HIS) for the indicated times. The cells were then washed two times with ice cold PBS and lysed with a buffer containing 50 mM Tris HCL (pH 7.5), 100 mM NaCl, 5 mM EDTA (pH 8.0), 1% Triton X-100 with a protease inhibitor cocktail tablet (Roche, Pleasanton, CA # 11836153001) and just before use, a phosphatase inhibitor cocktail diluted 1:100 (Roche #1697498) was added. Protein concentrations were determined by bicinchoninic acid (BCA) assay in triplicate (Pierce, Rockford, IL

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22 #23227). 20 g of total cellular protein was diluted in a loading buffer containing 2% SDS, 100 mM DTT, 60 mM Tris HCL (pH 7.5). Loading dye from a 3X stock containing 6% SDS, 180 mM Tris HCL (pH 7.5), 30% glycerol, and 0.03% bromophenol blue (BMB) with 0.2 M DTT and BME were added fresh just before use was added at a 1X concentration to the diluted sample and boiled for five min. Samples were then centrifuged for 10 min. at room temperature, separated on a 12% SDS/polyacrylamide gel and transferred to a hybond ECL nitrocellulose membrane (Amersham). The membranes were then blocked overnight with 5% non-fat milk dissolved in a TTBS buffer containing 0.137 M NaCl, 2.7 mM KCl, 25 mM Tris HCL (pH 7.5), and 0.005% Tween 20 at 4C. The membranes were then incubated with rabbit anti-MnSOD polyclonal antibody (Stressgen, San Diego, CA #sod-110) diluted 1:5,000 in TBTS with 5% BSA for 1.5 h, washed three times with TBST, incubated with an anti-rabbit antibody diluted 1:10,000 in 5% non-fat milk for 2 h, washed again three times, and subjected to ECL chemiluminescence (Amersham). Densitometry and Statistical Analysis All densitometry was quantified from autoradiography films using a Microtek scan maker 9600XL and analyzed with the UN-SCAN-IT program, Silk Scientific Corporation, version 5.1. Densitometric quantification of the autoradiographs for MnSOD employed the intensity of the 4 kb mRNA. The relative fold-induction was determined from FED or EBSS levels, normalized to the internal control, ribosomal protein L7a. Data points are the means from independent experiments. An asterisk (*) denotes significance as determined by a Students t-test to a value of p 0.05.

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23 Results Amino Acid Deprivation Induces MnSOD Steady State Messenger RNA Levels Nutrient availability in the mammalian diet has a potentially critical impact on metabolic flux, the generation of ATP and as a consequence, the generation of mitochondrial-derived ROS. Given the role of MnSOD in the detoxification of mitochondrial-derived ROS, nutrient availability could have direct affects on the levels of MnSOD gene expression. To test this hypothesis, northern analysis on human hepatoma cell line (HepG2) incubated in cell culture, with the exclusion of a single essential amino acid, histidine was used. As a positive control for MnSOD gene induction, cells were also treated with the pro-inflammatory mediators lipopolysaccharide (LPS), interleukins 1 and 6 (IL-1 and IL-6), tumor necrosis factor-alpha (TNF-) and interferon(IFN-) which has previously been demonstrated to induce MnSOD at the mRNA and protein level [21,22,34-36]. It has also been previously demonstrated that two mRNA species are produced from the human MnSOD gene due to alternative polyadenylation [40,41]. It was also shown that different cell lines vary in the relative expression of the two messages [40,41]. The mRNA levels of MnSOD are increased in response IL-1, IL-6 and TNF-, with IL-1 being the only stimulus to induce the 1kb message equal to that of the 4kb message (Figure 2-1). Also established (Figure 2-1), is the increase of steady state MnSOD mRNA levels in response to HIS, relative to FED. Furthermore, the addition of 5mM histidine to FED conditions (first lane of Figure 2-1, (+HIS)) reduces basal levels, demonstrating that depletion of histidine over the 12 h incubation time may contribute to an increase in the basal levels of MnSOD (+HIS compared to zero and Fed). In order to ensure that all experiments have similar MnSOD basal levels and amino acid levels in general are not depleted, fresh medium was give to the cells 12 h before the start

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24 of all experiments. Because HepG2 cells preferentially produce the 4kb message in response to HIS, this is the species referred to and utilized in densitometry for all experiments. In all northern analysis, the large ribosomal subunit 7a (L7a) was used as an internal control. +HISMnSOD L7aZeroFed-HISIL-1LPSIL-6TNF-kB 41 +HISMnSOD L7aZeroFed-HISIL-1LPSIL-6TNF-kB 41 Figure 2-1. Northern blot analysis of MnSOD in response to various stimuli. Total RNA was isolated from HepG2 cells incubated in complete medium (FED) with or without cytokines (LPS (lipopolysaccharide), TNF(tumor necrosis factor-alpha) IL-1 and IL-6 (interleukins 1 and 6)), complete medium lacking histidine (-HIS) or with the addition of 5mM histidine (+HIS) for 12 h. Gene specific probes for MnSOD and L7a (loading control) were used to determine the relative amounts of messenger RNA (mRNA). Induction of MnSOD is Specific to Amino Acid Deprivation Many other genes known to be regulated by amino acid deprivation also respond to glucose starvation [1]. In addition, glucose metabolism may have profound downstream affects on the production of mitochondrial derived ROS. To evaluate the specificity of changes in MnSOD mRNA levels, cells were starved for glucose (-GLC) and compared to HIS. MnSOD mRNA levels are induced only in response to amino acid deprivation

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25 (Figure 2-2). As a positive control for glucose deprivation, the membranes were re-probed for glucose regulated protein (GRP78). GRP78 (also known as BiP) is an endoplasmic reticulum (ER) chaperone that, during ER stress, increases gene expression [44,79]. A summary of densitometric data from three independent experiments as in Figure 2-2 is also shown (Figure 2-3). Furthermore, two other compounds known to activate the ER stress response (ERSR) pathway, thapsigargin and tunicamycin were also tested [80]. Thapsigargin is a calcium ATPase inhibitor whereas tunicamycin disrupts the glycosylation of newly synthesized proteins [81]. These compounds were also tested and, similar to glucose deprivation, showed no induction of MnSOD mRNA (data not shown). FED-GLC-HISHrs 2061218MnSODGRP78L7a261218261218 FED-GLC-HISHrs 2061218MnSODGRP78L7a261218261218 Figure 2-2. Northern blot analysis of total RNA isolated from HepG2 cells incubated in complete medium (FED), or medium lacking either histidine (-HIS) or glucose (-GLC). At the indicated times, the cells were lysed and subjected to northern analysis. Gene specific probes for MnSOD, L7a (loading control) and GRP78 (glucose regulated protein).were used to determine the relative amounts of mRNA.

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26 Immunoblot analysis was also employed to determine if the MnSOD mRNA induction was translated to the protein level. HepG2 cells were incubated in the absence of histidine and at the indicated times total protein was isolated. After 48 h the MnSOD protein levels are increased in response to histidine deprivation (Figure 2-4). FED-HIS-GLC 0261218261218261218Hrs Relative fold induction0.00.51.01.52.02.53.03.54.04.5*** FED-HIS-GLC 0261218261218261218Hrs Relative fold induction0.00.51.01.52.02.53.03.54.04.5*** Figure 2-3. Densitometric quantification of replicate experiments as done in 2-2 of the MnSOD 4kb message. Northern blots were evaluated using a Microtek scan maker 9600XL and analyzed with the UN-SCAN-IT program. Data points are represented as relative fold induction, as compared to the FED condition; all samples were normalized to the internal control, L7a. Data points are the means +/SEM (n 3). An asterisk (*) denotes significance as determined by a Students t-test to a value of p 0.05. 012244872 -HISHrs MnSOD 012244872 -HISHrs MnSOD Figure 2-4. Immunotblot analysis of MnSOD in response to histidine deprivation. Total protein was isolated from HepG2 cells incubated in complete medium lacking histidine for the indicated amount of time. 20 g of total cellular protein was fractionated on a 12% SDS/polyacrylamide gel, transferred to a nitrocellulose membrane and incubated with rabbit anti-MnSOD polyclonal antibody.

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27 Induction of MnSOD Messenger RNA is Specific to Essential Amino Acid Deprivation To further characterize the MnSOD gene induction in response to amino acid deprivation, mRNA levels were evaluated when each individual amino acid was omitted from complete medium and incubated for 2, 6 and 12 h. As shown in the representative northern blots (Figure 2-5), depletion of essential amino acids other than histidine also causes a similar increase in MnSOD mRNA levels, with the exception of tryptophan. On the other hand, depletion of non-essential amino acids from culture medium had no effect. A densitometric and statistical summary is also shown (Figure 2-6). The fact that MnSOD is induced in response to deprivation of only essential amino acids indicates an important role for the utilization of these amino acids. A possible explanation for these results is that non-essential amino acids and glucose can be made from within the cell and their depletion may not be as stressful to the cell. However, depletion of essential amino acids, a non-replenishable resource, places a demanding strain on the cell, and as a protective measure, MnSOD is induced. L7a02612261226122612 FED-HIS-ARG-LYSHrs MnSOD L7a02612261226122612 FED-HIS-ARG-LYSHrs MnSOD 2612261226122612-TRP-GLY-SER-PHE 2612261226122612-TRP-GLY-SER-PHE L7a02612261226122612 FED-HIS-ARG-LYSHrs MnSOD L7a02612261226122612 FED-HIS-ARG-LYSHrs MnSOD 2612261226122612-TRP-GLY-SER-PHE 2612261226122612-TRP-GLY-SER-PHE Figure 2-5. Representative northern blots of HepG2 cells incubated for the indicated times in medium lacking a single amino acid. Gene specific probes for MnSOD and L7a (loading control) were used to determine the relative amounts of mRNA.

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28 *******FED-TRP-VAL-ILE-THR-LEU-LYS-PHE-ARG-MET-HISRelative fold induction012345* *******FED-TRP-VAL-ILE-THR-LEU-LYS-PHE-ARG-MET-HISRelative fold induction012345* Figure 2-6. Representative bar graph of at least three independent experiments as in Figure 2-5. Densitometry was quantitated from multiple experiments as in 2-5 using a Microtek scan maker 9600XL and analyzed with the UN-SCAN-IT program. Relative fold induction of the MnSOD 4kb message, as compared to the FED condition was determined for each experiment; all samples were normalized to the internal control, L7a. Data points are the means +/SEM (n 3). An asterisk (*) denotes significance as determined by a Students t-test to a value of p 0.05. Cellular Specificity of Amino Acid Deprivation for MnSOD Induction Due to the important role of the liver in amino acid and nitrogen metabolism, initial studies of MnSOD and amino acid deprivation utilized human liver derived cell lines. In another human hepatoma cell line, HuH7, there was an identical response to histidine deprivation (Figure 2-7). To evaluate the cellular specificity of this metabolic response, other cells lines were also evaluated. Cell lines derived from the lung and intestine were also tested. These cell lines were chosen because the lung is a well studied system in our laboratory and the involvement of the intestine with nutrient uptake into the body. In these cells lines, the asparagine synthetase (ASNS) gene was used as a positive control for both HIS and GLC.

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29 Several cell lines were tested including human lung fibroblasts cell line, CCDLU, a lung adenocarcinoma cell line, A549, and a Caco-2 (not shown) cell line derived from a primary colonic tumor were tested to see if MnSOD mRNA levels were increased in response to amino acid deprivation (Figure 2-8). There was only a minor response in the human lung fibroblasts with no obvious changes occurring in the other cell types, as compared to the significant induction observed in mRNA levels when these membranes were re-probed for ASNS. Similarly, this response was evaluated in a rat cell line, a normal lung epithelial line, L2 (Figure 2-8), and again found no induction following histidine deprivation. Hrs 261226122612 FED-HIS-GLCHUH7 ASNSMnSODL7a --Hrs 261226122612 FED-HIS-GLCHUH7 ASNSMnSODL7a -Figure 2-7. Northern analysis from HUH7 (human hepatoma) cells, incubated for the indicated amount of time, in various medium conditions. Conditions tested included complete medium (FED), complete medium lacking either histidine (-HIS) or glucose (-GLC). Gene specific probes for MnSOD, ASNS and L7a (loading control) were used to determine the relative amounts of mRNA.

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30 ASNSL7a L2 -GLC-HISFED L2 -GLC-HISFED ASNSL7a -GLC-HISFED A549 -GLC-HISFED A549 ASNSL7a -HIS-HISFEDFED CCDLU -HIS-HISFEDFED CCDLU MnSODMnSODMnSODASNSL7a L2 -GLC-HISFED L2 -GLC-HISFED ASNSL7a -GLC-HISFED A549 -GLC-HISFED A549 ASNSL7a -HIS-HISFEDFED CCDLU -HIS-HISFEDFED CCDLU MnSODMnSODMnSOD Figure 2-8. Northern blot analysis of CCDLU, A549 and L2 cells incubated for 12 h, in the indicated medium. Conditions tested included complete medium (FED) or in complete medium lacking histidine (-HIS). Also included in the A549 and L2 cells is complete medium lacking glucose (-GLC). Gene specific probes for MnSOD, ASNS and L7a (loading control) were used to determine the relative amounts of mRNA. Transcriptional control of MnSOD in response to amino acid deprivation As previously discussed, amino acid gene control has been described at many levels including transcriptional, translational and through mRNA stability [1,57]. To help elucidate the mechanism of increased MnSOD mRNA levels, in response to amino acid deprivation, inhibitors of transcription and translation, actinomycin-D and cyclohexamide were used. Cells were first co-treated with the transcriptional inhibitor actinomycin-D (Figure 2-9) and the induction of MnSOD caused by histidine starvation is completely blocked. This indicates that de novo transcription is necessary for this induction. When cells are co-treated with the protein synthesis inhibitor cyclohexamide, the steady state levels of the MnSOD mRNA levels are equal to that of the FED condition. This implies that inhibition of protein synthesis potentially stabilizes the MnSOD mRNA. This

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31 suggests that the induction due to amino acid depletion is blocked by inhibition of protein synthesis since the mRNA levels in the HIS condition are not higher than in the FED state. Treatment of cells with cyclohexamide or actinomycin-D blocks the induction of MnSOD, indicating that there may be a transcriptional component to this induction. Although these experiments alone cannot support a transcriptionally based induction, this is addressed further in chapter five. Control Actino Cyclo Fed-HisFed-HisFed-His MnSODL7a Control Actino Cyclo Fed-HisFed-HisFed-His MnSODL7aControl Actino Cyclo Fed-HisFed-HisFed-His MnSODL7a Figure 2-9. Northern blot analysis of cells treated with actinomycin-D or cyclohexamide. HepG2 cells were incubated for 12 hours in complete media (FED), complete media lacking histidine (-HIS). Where indicated, co-treated with either actinomycin or cyclohexamide. Gene specific probes for MnSOD and L7a were used to determine the relative amounts of mRNA. Discussion Amino acid dependent regulation of gene expression plays a significant role in cellular growth and metabolism. This has been shown to be regulated at many levels including transcription, translation and mRNA stability [1]. The data presented here

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32 demonstrate that MnSOD mRNA and protein levels are induced by amino acid deprivation. Specifically, MnSOD mRNA levels are induced in response to deprivation of the essential amino acids, with the exception of tryptophan. Several different cell lines were evaluated, with the most significant responses occurring in the liver cell lines tested, HepG2 and HUH7, inducing in response to amino acid deprivation and not glucose deprivation. Furthermore, treatment of cells with transcriptional and translational inhibitors block the induction of MnSOD, indicating that there may be a transcriptional component to this induction, which will be addressed further in Chapter 5. Amino acid deprivation has been demonstrated to cause a wide variety of responses including increases in membrane transporters, amino acid synthetases, and other metabolically relevant enzymes. However, it has also been demonstrated that other proteins not related to the restoration of amino acid levels are induced in response to amino acid deprivation. One example is the insulin-like growth factor binding protein (IGFBP-1) [82]. This protein binds to insulin-like growth factors (IGF) I and II and functions to extend their half-life and alter their interactions with cell surface receptors. IGF I promotes cell proliferation and inhibition of apoptosis whereas IGF II has been associated with early development. These genes are all induced to promote cell survival durring stressed caused by amino acid deprivation. The data presented here establish a novel mechanism of induction for the cytoprotective enzyme MnSOD. This protein functions to scavenge free radicals produced in the mitochondria as a consequence of respiration. The gene has been demonstrated to be regulated by a number of stimuli including TNF, IL-1 and ionizing radiation [21,22,34-36]. However, the link between nutrient deprivation and the

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33 induction of MnSOD has not previously been established. The focus of this dissertation is to characterize this induction by evaluating the nutrient requirements, signal transduction pathways involved and the regulatory elements involved.

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CHAPTER 3 METABOLIC CONTROL OF MANGANESE SUPEROXIDE DISMUTASE IN RESPONSE TO AMINO ACID DEPRIVATION Introduction Nutrient availability relative to both carbohydrates and amino acids, in the mammalian diet, has potentially critical impacts on metabolic flux and ultimately the generation of ATP and its equivalents. With constantly changing constituents associated with the mammalian diet, organisms have adapted metabolic strategies to efficiently accommodate changes in the availability of critical nutrients. Extensive studies have addressed the importance of glucose excess [83] and deprivation [46] as well as amino acid availability on metabolic and nuclear events [1] As established in Chapter two, HepG2 cells, in response to single amino acid deprivation, induces MnSOD at the mRNA and protein levels. A critical consequence of nutrient availability and subsequent metabolism is the generation of reactive oxygen species (ROS), the target of MnSOD [66,84]. Furthermore, the connection between nutrient levels and the generation of ROS is underscored when considering that caloric restriction can significantly delay the aging process [85], an observation in line with the free radical theory of aging [86,87]. One contributing factor is the importance of the mitochondrial localized anti-oxidant enzyme, MnSOD, to energy/redox metabolism, aging and disease pathologies [88]. The results demonstrated in Chapter two established that depletion of individual essential amino acids and not glucose causes induction of human MnSOD mRNA and protein levels. 34

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35 Nutrient uptake, in the form of carbohydrates, lipids and protein, and their subsequent breakdown to glucose, amino acids, fatty acids and glycerol is the basis for metabolism. Metabolism is the modification of compounds to maintain cellular homeostasis through the synthesis (anabolic pathways) or breakdown (catabolic pathways) of nutrients. The flux of these metabolites within the cell is important to maintain the balance of anabolic and catabolic functions. The major function of these metabolites is to feed into the citric acid cycle, ultimately leading to the production of energy for the cell. One major metabolite that feeds into the TCA cycle, after the conversion of pyruvate to acetyl-CoA, is glucose. However, as previously established, glucose does not affect the induction of MnSOD and the main focus of this section will be on the catabolism of amino acids into the TCA cycle. The major functions occurring in the mitochondria are the citric acid cycle, electron transport, oxidative phosphorylation, fatty acid oxidation, pyruvate oxidation and amino acid catabolism. Following the conversion to acetyl Co-A, the metabolites pyruvate, fatty acids, and amino acids, feed into the TCA cycle. Amino acids can also feed directly, or through transamination reactions, into the TCA cycle. The TCA cycle generates reducing units in the form of NADH and FADH 2 that drive electron transport, ultimately generating ATP, and providing energy for the cell. As a byproduct of normal respiration, superoxide radicals are produced at complex I and III. In order to ensure the cells survival, the mitochondrial localized MnSOD protein converts the superoxide products into hydrogen peroxide and water. The hydrogen peroxide is then detoxified, in the mitochondria, by glutathione peroxidase. Given the close connection between cellular nutrients and free radical production, perturbations in the flux of amino acids into

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36 the TCA may be the basis for the amino acid regulation of MnSOD gene expression. As a nuclear-encoded gene, but mitochondrial-localized protein, the activity of this enzyme is important to a wide variety of metabolic cellular events including the oxidation state of the cell, cellular respiration, ATP synthesis and overall cell viability. Given the importance of overall mitochondrial homeostasis, cell survival and the localization of MnSOD, the importance of several mitochondrial pathways were evaluated. Materials and Methods Isolation of Total RNA RNA isolation was performed as described in Chapter 2. Measurements of ATP Levels ATP levels were measured from HepG2 cells utilizing the Adenosine 5-triphosphate (ATP) bioluminescent kit #FLASC from Sigma. After 12 h of treatment cells were trypsinized, 3 mL of medium was added, the cells were mixed well, and 1 mL was transferred to a 1.5 mL microfuge tube. A 100 L aliquot of ATP assay solution (diluted 1:25) was added to a fresh tube and incubated at room temperature for 3 min., allowing for endogenous ATP to be hydrolyzed and decreasing the background. In a separate 1.5 mL microfuge tube, the following was mixed: 100 L releasing agent (FL-SAR), 50 L water and 50 L of the cell sample. A 100 L aliquot of this mix was added to the 100 L ATP assay solution and relative light units were measured using a Berthold SIRUS luminometer V3.0. Reagents Used KMV #K7125, 3-nitropropionic acid #N5636, malonate #1750, antimycin #A8674, oligomycin #O8476 and 2,4-dinitrophenol #D198501 were purchased from Sigma. Fluoroacetate was from Fluka, St. Louis, MO #71520.

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37 Results Glutamine is Required for the Induction of MnSOD by Amino Acid Deprivation Given that deprivation of a single essential amino acid resulted in the induction of MnSOD mRNA, total amino acid deprivation was also evaluated to determine if this would cause a similar induction. Notably, MnSOD mRNA levels were not elevated in HepG2 cells incubated in medium lacking all amino acids (EBSS) (Figure 3-1). This is completely contrary to the response detected for other genes regulated by depletion of essential amino acids, in that these genes can respond to single or complete amino acid deprivation [89,90]. In fact, there are no other genes currently known to be regulated by single amino acid deprivation that are not also induced in response to complete amino acid depletion. To address the loss of induction of MnSOD by total amino acid deprivation, several components of the medium were tested to evaluate their contribution to the induction of MnSOD through amino acid deprivation. First, the individual amino acids were tested to determine if the induction could be restored. To address the amino acid specificity of the add back experiment, cells were incubated in EBSS or in EBSS with the addition of a single amino acid. The effects of each amino acid were analyzed by northern analysis and a subset of this data is shown (Figure 3-1). Analysis of these data demonstrate that glutamine is the only amino acid when added back to EBSS, is able to re-establish the induction of MnSOD mRNA levels. To further address the specificity of this response, asparagine synthetase (ASNS) [1,89,90], a gene known to be induced by both single and complete amino acid deprivation was also evaluated (Figure 3-1). These data demonstrate MnSOD is clearly not regulated in a manner identical to ASNS and moreover, the addition of GLN to EBSS is sufficient to re-establish the induction of

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38 MnSOD with no effects on ASNS. A separate experiment directly comparing MnSOD and ASNS levels in HIS, EBSS and EBSS+GLN as well as a quantitative summary of GLN add back as compared to MnSOD mRNA levels in EBSS at 12 h is also shown (Figure 3-2). 612612612612EBSS EBSS +HISHrs L7aMnSOD ASNS 612612612612EBSS EBSS +GLNEBSS +GLYEBSS +ARGEBSS +ALAEBSS +SER 612612612612EBSS EBSS +HISHrs L7aMnSOD ASNS 612612612612EBSS EBSS +GLNEBSS +GLYEBSS +ARGEBSS +ALAEBSS +SER Figure 3-1. Northern analysis of HepG2 cells incubated in medium lacking all amino acids (EBSS), or in EBSS with the inclusion of the indicated amino acid at a concentration of 5 mM. Total RNA was collected at the indicated times and analyzed by northern analysis. Gene specific probes for MnSOD, ASNS and L7a (loading control) were used to determine the relative amounts of mRNA.

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39 -HISFEDEBSS MnSODL7aEBSS +GLN ASNS -HISFEDEBSS MnSODL7aEBSS +GLN ASNS *Relative fold induction0.00.51.01.52.02.53.03.54.04.5EBSSEBSS +GLN *Relative fold induction0.00.51.01.52.02.53.03.54.04.5EBSSEBSS +GLN Figure 3-2. HepG2 cells were incubated in various medium conditions with the addition of 5 mM glutamine for 12 h. Conditions tested included complete medium (FED), complete medium lacking histidine (-HIS), medium lacking all amino acids (EBSS) or in medium lacking all amino acids with the addition of 5mM glutamine (EBSS +GLN) for 12 h. Gene specific probes for MnSOD, ASNS and L7a (loading control) were used to determine the relative amounts of mRNA. Densitometry was quantitated from multiple experiments using a Microtek scan maker 9600XL and analyzed with the UN-SCAN-IT program. Data points are represented as relative fold induction, as compared to the EBSS condition; all samples were normalized to the internal control, L7a. Data points are the means +/SEM (n 3). An asterisk (*) denotes significance as determined by a Students t-test to a value of p 0.05. To further define the importance of glutamine in the induction of MnSOD mRNA by amino acid deprivation, HepG2 cells were incubated in HIS, or in HIS/-GLN. As demonstrated (Figure 3-3), the absence of GLN completely abolishes the HIS induction (HIS/-GLN). As previously established, glucose deprivation did not cause an induction of MnSOD. However, removal of glutamine from the medium does not induce MnSOD

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40 mRNA levels, as it is a required component for the induction. Given the potential use of glutamine and/or glucose as a carbon/fuel source, glucose was also evaluated to determine if this component was required for MnSOD induction by amino acid deprivation. Contrary to the effects of -GLN, glucose (-GLC) deprivation did not alter the -HIS response (Figure 3-3). 02612261226122612FED-GLN-HIS-HIS/-GLN Hrs L7aMnSOD FED-HIS-HIS / -GLCEBSS + GLC 02612261226122612FED-GLN-HIS-HIS/-GLN Hrs L7aMnSOD FED-HIS-HIS / -GLCEBSS + GLC 02612261226122612FED-GLN-HIS-HIS/-GLN Hrs L7aMnSOD FED-HIS-HIS / -GLCEBSS + GLC Figure 3-3. HepG2 cells were incubated in complete medium (FED), medium lacking glutamine (-GLN), histidine (-HIS) or both amino acids (-HIS/-GLN). Cells were also evaluated for the effect of glucose deprivation in conjunction with histidine deprivation (-HIS/-GLC). At the indicated times, total RNA was collected and subjected to northern analysis. Gene specific probes for MnSOD and L7a (loading control) were used to determine the relative amounts of mRNA. To further evaluate the requirement of glutamine and to determine the levels of glutamine required for the induction of MnSOD, either in response to single amino acid deprivation (-HIS) or total amino acid deprivation (EBSS) cells were incubated in these conditions with increasing amounts of glutamine (Figure 3-4) demonstrating the addition of glutamine to FED cells has no effect. However, in single amino acid deprivation

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41 (HIS) or in total amino acid deprivation (EBSS), the addition of 1 mM glutamine is permissive to MnSOD mRNA induction in response to amino acid deprivation. 012345012345-HISEBSS 012345mM GLN FED MnSODL7a 012345012345-HISEBSS 012345mM GLN FED MnSODL7a Figure 3-4. Northern analysis of HepG2 cells incubated for 12 h in the indicated medium conditions with increasing concentrations of glutamine. Conditions tested included complete medium (FED), complete medium lacking histidine (-HIS) or medium lacking all amino acids (EBSS) with increasing concentrations of glutamine. Gene specific probes for MnSOD and L7a (loading control) were used to determine the relative amounts of mRNA. With glutamine established as an obligatory component for the induction of MnSOD by amino acid deprivation, other components of the medium were evaluated. One such component of the complete medium which could be, in part, responsible for the increases in mRNA levels is the presence of the vitamin supplements, and as shown (Figure 3-5), are not necessary for the observed induction. Additionally, other amino acids were evaluated to further evaluate the specificity of glutamine as a requirement for MnSOD induction in response to amino acid deprivation. Specifically, the induction of MnSOD in cells depleted with both histidine and glutamine to growth conditions lacking histidine in conjunction with depletion of tryptophan or methionine were tested. The lack of glutamine is the only condition that can inhibit the induction of MnSOD by essential amino acid deprivation (Figure 3-5).

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42 Another major component of the culture medium is serum. Previous reports on another amino acid regulated gene, C/EBP homologous protein (CHOP), demonstrated the requirement of serum, specifically IGF, in the induction of this gene in response to amino acid deprivation [91]. The presence of serum, as with vitamins, did not alter the level of MnSOD mRNA induction (Figure 3-6). Also evaluated was the contribution of two important growth factors found in serum IGF and EGF and the induction of MnSOD by amino acid deprivation is not dependent on either any of these growth factors (Figure 3-6). L7aMnSODFED-HIS-HIS/-GLN-HIS/-TRP-HIS/-MET+VITAMINS FED-HIS-HIS/-GLN-HIS/-TRP-HIS/-MET-VITAMINS L7aMnSODFED-HIS-HIS/-GLN-HIS/-TRP-HIS/-MET+VITAMINS FED-HIS-HIS/-GLN-HIS/-TRP-HIS/-MET-VITAMINS Figure 3-5. HepG2 cells were incubated in the presence or absence of vitamins under the indicated conditions. Complete medium (FED), medium lacking glutamine (-GLN), histidine (-HIS) or lacking a combination of two amino acids (-HIS/-GLN), (-HIS/-TRP) or (-HIS/-MET) were tested. After 12 h, total RNA was collected and subjected to northern analysis. Gene specific probes for MnSOD and L7a (loading control) were used to determine the relative amounts of mRNA.

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43 0100010005005001000100050050 IGF (ng/ml)IGF (ng/ml)EGF (ng/ml)EGF (ng/ml) FED -HIS FED -HIS FED -HIS FED -HIS 10% dFBS0% dFBSMnSOD L7a 0100010005005001000100050050 IGF (ng/ml)IGF (ng/ml)EGF (ng/ml)EGF (ng/ml) FED -HIS FED -HIS FED -HIS FED -HIS 10% dFBS0% dFBS 0100010005005001000100050050 IGF (ng/ml)IGF (ng/ml)EGF (ng/ml)EGF (ng/ml) FED -HIS FED -HIS FED -HIS FED -HIS 10% dFBS0% dFBSMnSOD L7a Figure 3-6. Northern analysis of total RNA isolated from HepG2 cells incubated with or without 10% dFBS, or two components of serum, IGF or EGF. Under these conditions, cells were then incubated in FED or HIS medium for 12 h. Gene specific probes for MnSOD and L7a (loading control) were used to determine the relative amounts of mRNA. Inhibition of the TCA Cycle Blocks MnSOD Induction by Histidine Deprivation With glutamine established as the only required component for MnSOD induction by amino acid deprivation, the role of this amino acid was further evaluated. To identify a mechanistic link between glutamine and MnSOD gene activation, the contribution of glutamine metabolism and its potential connection to increases in MnSOD mRNA levels was tested. The primary pathway for the utilization of glutamine, as both an energy source and a possible signaling molecule is catabolism through the tricarboxylic acid (TCA) cycle. Glutamine enters the TCA cycle through glutamate and -ketoglutarate (Figure 3-7 (adapted from [92])). Therefore, the contribution of the TCA cycle to the induction of MnSOD was evaluated by utilizing selective inhibitors to several different key enzymes. Aconitase was first evaluated by inhibition with 3-fluoroacetate [93,94].

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44 Figure 3-7. Tricarboxylic acid (TCA) cycle. The major metabolites and the reducing units generated by the TCA cycle, NADH and FADH 2 are shown in bold. The enzymes of the TCA cycle are shown in boxes. The applicable TCA cycle inhibitors are shown in italics. Aconitase is an important enzyme involved in the conversion of citrate to isocitrate through a dehydration-rehydration rearrangement, with the inhibition by fluoroacetate occurring through the intracellular conversion to the substrate inhibitor, fluorocitrate. Fluoracetate can inhibit the induction, in a concentration dependent manner, of MnSOD mRNA levels by histidine deprivation (Figure 3-8). A quantitative summary of three independent experiments is also shown (Figure 3-9), with an approximately 65 to 70% inhibition. Given that glutamine is required for MnSOD gene activation, the entry point for glutamine into the TCA cycle, -ketoglutarate dehydrogenase, may be another relevant

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45 051015202530FED-HISFluoroacetate(mM) L7aMnSODASNS051015202530 051015202530FED-HISFluoroacetate(mM) L7aMnSODASNS051015202530 Figure 3-8. Representative northern blot of HepG2 cells incubated for 12 h in complete medium (FED) or complete medium lacking histidine (HIS), treated with increasing concentrations of fluoroacetate. Gene specific probes for MnSOD, ASNS and L7a (loading control) were used to determine the relative amounts of mRNA. -HIS FED1009080706050403020100051015202530Fluroacetate(mM)Relative Expression(Percent) -HIS FED1009080706050403020100051015202530Fluroacetate(mM)Relative Expression(Percent)Relative Expression(Percent) Figure 3-9. D ensitometric data from three independent experiments following treatment with fluoroacetate (as done in Figure 3-9). Data points are represented as relative expression (percent) relative to the induced message (HIS, no fluoroacetate); all samples were normalized to the internal control, L7a. Data points are the means, +/SEM.

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46 step in the signaling pathway for MnSOD gene activation. Furthermore, the lack of MnSOD induction in the absence of glutamine can also be viewed essentially as an inhibition of the TCA cycle. As a direct assessment of the importance of this entry point for glutamine, a structural analogue of -ketoglutarate and a competitive inhibitor of the -ketoglutarate dehydrogenase complex, -keto--methyl-n-valeric acid (KMV) was used [95]. The results show that, similar to fluoroacetate, KMV causes an ~60% reduction of the MnSOD induction by histidine depletion, again with no effect on ASNS expression (Figure 3-10 and 3-11). KMV(mM) L7aFED-HISMnSOD 00.10.51102000.10.511020ASNS KMV(mM) L7aFED-HISMnSOD 00.10.51102000.10.511020ASNS Figure 3-10. Representative northern blot of HepG2 cells incubated for 12 h in complete medium (FED) or complete medium lacking histidine (HIS) and treated with increasing concentrations of -keto--methyl-n-valeric acid (KMV). Gene specific probes for MnSOD, ASNS and L7a (loading control) were used to determine the relative amounts of mRNA.

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47 -HIS FED1009080706050403020100KMV (mM)00.10.511020Relative Expression(Percent) -HIS FED1009080706050403020100KMV (mM)00.10.511020Relative Expression(Percent)Relative Expression(Percent) Figure 3-11. Densitometry data collected from three independent experiments following treatment with KMV (as done in Figure 3-13). Data points are represented as relative expression (percent) relative to the induced message (HIS, no KMV); all samples were normalized to the internal control, L7a. Data points are the means, +/SEM. Two inhibitory compounds, which have been shown to selectively inhibit the succinate dehydrogenase complex, were used. 3-nitropropionic acid (3-NPA) inactivates succinate dehydrogenase by covalently and irreversibly binding to its active site [96] whereas malonate is a competitive inhibitor of succinate dehydrogenase [97,98]. The inhibitor 3-NPA caused a significant inhibition of the HIS induction (Figures 3-12 and 3-13), with no response to treatment with increasing concentrations of malonate (Figure 3-14). This is consistent with the biochemical argument that as a competitive inhibitor malonate can be displaced when cellular succinate concentrations are elevated, presumably the case when adequate levels of glutamine are available. Interestingly, 3-NPA did cause the inhibition of the ASNS induction by HIS, whereas fluoroacetate and KMV had no effect.

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48 3-NPA(mM) 01510 FED-HIS01510MnSODL7a ASNS 3-NPA(mM) 01510 FED-HIS01510MnSODL7a ASNS Figure 3-12. Representative northern blot of HepG2 cells incubated for 12 h in complete medium (FED) or complete medium lacking histidine (HIS) and treated with increasing concentrations of 3-nitropropionic acid (3-NPA). Gene specific probes for MnSOD, ASNS and L7a (loading control) were used to determine the relative amounts of mRNA. -HIS FED1009080706050403020100015103-NPA (mM)Relative Expression(Percent) -HIS FED1009080706050403020100015103-NPA (mM)Relative Expression(Percent)Relative Expression(Percent) Figure 3-13. Densitometry data collected from three independent experiments following treatment with 3-NPA (as done in Figure 3-13). Data points are represented as relative expression (percent) relative to the induced message (HIS, no 3-NPA); all samples were normalized to the internal control, L7a. Data points are the means, +/SEM.

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49 FED-HIS151 05105Malonate (mM) 15 MnSOD L7aASNS FED-HIS151 05105Malonate (mM) 15 MnSOD L7aASNS Figure 3-14. Northern blot of HepG2 cells incubated for 12 h in complete medium (FED) or complete medium lacking histidine (HIS) with increasing concentrations of malonate. Gene specific probes for MnSOD, ASNS and L7a (loading control) were used to determine the relative amounts of mRNA. Both a Functional Electron Transport Chain and F 1 -F 0 ATP Synthase Complex are Required for MnSOD Induction in Response to Amino Acid Deprivation Given the importance of the TCA cycle in MnSOD gene activation by amino acid deprivation and the metabolic role of this cycle in the generation of reducing units and the accompanying electrons for consumption through oxidative phosphorylation, the contribution of events from the electron transport chain was also evaluated. The electron transport chain is responsible for the production of ATP via oxidative phosphorylation, resulting from the flow of electrons from NADH (or FADH 2 ) to molecular oxygen thus establishing a proton gradient (Figure 3-15) that drives phosphorylation of ADP to ATP

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50 by ATP synthase. Of particular note is that superoxide radicals, the substrates for MnSOD enzymatic activity, are formed as byproducts at both complexes I and III of the electron transport chain (Figure 3-15). NADNADHQH2 Q CytoCCytoCreduced H2OO2 H+ (matrix)H+ (outside) TCACycleNADNADHQH2 Q CytoCCytoCreduced H2OO2 H+ (matrix)H+ (outside) TCACycleNADNADHQH2 Q CytoCCytoCreduced H2OO2 H+ (matrix)H+ (outside) TCACycleANADH Succinate 2eSuccinate Dehydrogenase(3Fe-S)COMPLEX II NADHDehydrogenase(5Fe-S)COMPLEX I UQcyt. bcyt. c1 COMPLEX III cyt. c cyt. a-a3 e-e-COMPLEX IVO2O2-. e-e4e-O2H2O O2O2-.Antimycin A2eNADH Succinate 2eSuccinate Dehydrogenase(3Fe-S)COMPLEX II NADHDehydrogenase(5Fe-S)COMPLEX I UQcyt. bcyt. c1 COMPLEX III cyt. c cyt. a-a3 e-e-COMPLEX IVO2O2-. e-e4e-O2H2O O2O2-.Antimycin A2eBNADNADHQH2 Q CytoCCytoCreduced H2OO2 H+ (matrix)H+ (outside) TCACycleNADNADHQH2 Q CytoCCytoCreduced H2OO2 H+ (matrix)H+ (outside) TCACycleNADNADHQH2 Q CytoCCytoCreduced H2OO2 H+ (matrix)H+ (outside) TCACycleANADH Succinate 2eSuccinate Dehydrogenase(3Fe-S)COMPLEX II NADHDehydrogenase(5Fe-S)COMPLEX I UQcyt. bcyt. c1 COMPLEX III cyt. c cyt. a-a3 e-e-COMPLEX IVO2O2-. e-e4e-O2H2O O2O2-.Antimycin A2eNADH Succinate 2eSuccinate Dehydrogenase(3Fe-S)COMPLEX II NADHDehydrogenase(5Fe-S)COMPLEX I UQcyt. bcyt. c1 COMPLEX III cyt. c cyt. a-a3 e-e-COMPLEX IVO2O2-. e-e4e-O2H2O O2O2-.Antimycin A2eB Figure 3-15. The electron transport chain and its relevant points. A) The flow of protons, through the electron transport chain, establishes a proton gradient between the mitochondrial matrix and the intermembrane space. B) Production of superoxides at complex I and III in the electron transport chain. Complex III is inhibited by antimycin A. In order to determine the potential contribution of the electron transport chain to MnSOD gene induction, several inhibitors were evaluated. An inhibitor of complex III, antimycin A, which our lab previously demonstrated inhibits the induction of MnSOD by TNF-, but not the induction by IL-1 or LPS was used [16]. Exposure to increasing

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51 concentrations of antimycin A inhibited the HIS induction of MnSOD with effective concentrations in the low nM range (Figure 3-16). These results demonstrate the importance on MnSOD induction of continued flow from the metabolism of glutamine in the TCA cycle through the transfer of resulting reducing units and electrons towards the ultimate goal of energy production through ATP formation. Antimycin A(nM)0.05 FED-HIS MnSOD00.10.250.515100.0500.10.250.51510 L7aASNS Antimycin A(nM)0.05 FED-HIS MnSOD00.10.250.515100.0500.10.250.51510 L7aASNS Figure 3-16. Northern blot analysis of HepG2 cells incubated for 12 h in complete medium (FED) or complete medium lacking histidine (-HIS) with increasing concentrations of antimycin A. Gene specific probes for MnSOD, ASNS and L7a (loading control) were used to determine the relative amounts of mRNA. The importance of the electrochemical gradient in the generation of ATP through an inhibitor of the F 1 F o ATP synthase complex, oligomycin [16,99,100] was also evaluated. The F 1 F o ATP synthase complex utilizes the proton gradient across the inter-mitochondrial membrane to drive ATP synthesis. Oligomycin blocks the F o portion of ATP synthase, inhibiting its activity and blocking ATP synthesis coupled to the proton gradient established across the inter-membrane space and the mitochiondrial matrix. The

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52 induction of MnSOD is inhibited in the low nM range of oligomyicn, implying that the underlying signaling mechanism requires adequate ATP levels or an intact electrochemical gradient (Figure 3-17). 00.1110 FED-HIS00.1110 Oligomycin(nM)MnSODL7a ASNS 00.1110 FED-HIS00.1110 Oligomycin(nM)MnSODL7a ASNS Figure 3-17. Northern blot analysis of HepG2 cells incubated for 12 h in complete medium (FED) or complete medium lacking histidine (-HIS) with increasing concentrations of oligomycin. Gene specific probes for MnSOD, ASNS and L7a (loading control) were used to determine the relative amounts of mRNA. An Intact Electrochemical Gradient but not ATP Synthesis is Required for MnSOD Induction Since both inhibitors of the electron transport chain blocked MnSOD induction, one possible explanation is that this is due to a decrease in ATP levels. To examine this possibility, ATP levels were measured under each of our growth conditions. To establish a growth condition which would definitively reduce cellular ATP levels as a positive control, cells were treated with 2-deoxy-D-glucose (2-DOG), a non-metabolizable form of glucose, creating a cellular state analogous to glucose starvation [101]. As previously demonstrated (Figures 2-2 and 2-3), conditions of limiting glucose did not induce

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53 MnSOD levels nor affect the induction by HIS (Figure 3-3). To confirm that 2-DOG does not affect MnSOD levels in FED, -HIS, or EBSS -/+ GLN, HepG2 cells were incubated in these conditions and evaluated by northern analysis (Figure 3-18). ATP levels were then measured under a variety of experimental conditions (Figure 3-19). FEDL7aMnSOD 00.112.551000.112.5510-HIS2-DOG(mM) FEDL7aMnSOD 00.112.551000.112.5510-HIS2-DOG(mM) EBSS00.112.551000.112.5510+GLN EBSS00.112.551000.112.5510+GLN Figure 3-18. Northern blot analysis of HepG2 cells incubated for 12 h in the indicated medium for 12 h with the addition of 5 mM glutamine (EBSS +GLN), with increasing concentrations of the glucose analogue 2-deoxy-D-glucose (2-DOG). Conditions tested included complete medium (FED), complete medium lacking histidine (-HIS), medium lacking all amino acids (EBSS) or in medium lacking all amino acids with the addition of 5mM glutamine (EBSS +GLN), with increasing concentrations of the glucose analogue 2-deoxy-D-glucose (2-DOG). Gene specific probes for MnSOD and L7a (loading control) were used to determine the relative amounts of mRNA. Comparable reductions in ATP levels were observed for all conditions relative to complete medium (FED). These data indicate that the induction of MnSOD levels by HIS conditions is not dependent on alterations in ATP levels, because 2-DOG and -HIS both result in reduced ATP levels, yet the former condition has no effect on MnSOD mRNA levels (Figure 3-18). In addition, these results also demonstrate that the HIS condition only reduces ATP levels by ~25% as compared to reductions of more than 50% in either EBSS or any of the conditions with 2-DOG. Of equal importance is that the re-addition of GLN to EBSS results in a recovery of ATP levels.

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54 Since ATP levels do not affect MnSOD mRNA levels, another possibility is that an intact proton gradient is required for the amino acid induction of MnSOD mRNA. In order to test this theory, an uncoupler of electron transport and oxidative phosphorylation, 2,4 dinitrophenol, was used [102]. 2,4 dinitrophenol is hydrophobic and can freely pass through the inner mitochondrial membrane due to its ability to bind free protons and transports them into the mitochondrial matrix bypassing the F 1 Fo ATPase production of ATP, effectively uncouples electron transport. Increasing concentrations of 2,4-dinitrophenol inhibited the induction of MnSOD mRNA levels by HIS (Figure 3-20), strongly implicating the importance of an intact proton gradient. (Percent)1009080706050403020100FED-HISEBSSEBSS+GLNFED-HISEBSSEBSS+GLN +2-DOGRelative Expression (Percent)1009080706050403020100FED-HISEBSSEBSS+GLNFED-HISEBSSEBSS+GLN +2-DOGRelative Expression Figure 3-19. ATP levels from four independent experiments in which HepG2 cells were incubated in the various conditions. Conditions included complete medium (FED) or complete medium lacking histidine (-HIS), medium lacking all amino acids (EBSS) or in medium lacking all amino acids with the addition of 5mM glutamine (EBSS +GLN), with or without the addition of 10 mM 2-DOG for 12 h. Cells were then lysed and ATP levels were measured using the bioluminescent somatic cell assay kit from Sigma/Aldrich. Relative light units were measured using a Berthold SIRUS luminometer V3.0. Data points are represented as relative expression (percent), as compared to FED. Data points are means +/SEM.

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55 2,4 dinitrophenol(mM)00.110.500.110.5 MnSOD FED-HIS L7a 2,4 dinitrophenol(mM)00.110.500.110.5 MnSOD FED-HIS L7a Figure 3-20. Northern blot analysis of HepG2 cells incubated for 12 h in complete medium (FED) or complete medium lacking histidine (-HIS) with increasing concentrations of 2,4 dinitrophenol. Gene specific probes for MnSOD and L7a (loading control) were used to determine the relative amounts of mRNA. Discussion MnSOD gene expression is induced in response to a wide variety of pro-inflammatory stimuli including IL-1, TNF, IL-6, and LPS [22] as a protective measure against the harmful effects of reactive oxygen species (ROS). As a nuclear-encoded gene, but mitochondrial-localized protein, the activity of this enzyme is important to a wide variety of metabolic cellular events including the oxidation state of the cell, cellular respiration, ATP synthesis and overall cell viability. Tissue and cellular adaptation to nutrient availability also affects carbon and nitrogen utilization through glycolysis, the TCA cycle and ultimately the aerobic generation of ATP via electron transport. A critical consequence of nutrient availability and subsequent metabolism is the generation of ROS as byproducts of normal metabolism [2]. Previous estimates have indicated that under

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56 normal aerobic and nutrient conditions, 1-3% of consumed oxygen is released as superoxide radicals from mitochondrial electron transport [3]. Therefore, nutrient availability, may have relevant metabolic and cell survival benefits mediated through the elevation of MnSOD levels. The results presented here provide evidence for a level of metabolic control for MnSOD gene expression in response to amino acid deprivation. Experimental data established that the induction of MnSOD in response to amino acid depletion is dependent on the presence of glutamine. This is contrary to effects on other amino acid regulated genes, such as ASNS [1,103], which are regulated by depletion of a single or total amino acid depletion. Furthermore, no other components tested, including other amino acids, vitamins, glucose or serum, were determined to be required for the induction of MnSOD. A review by Bode et al.[104] offers a potential explanation for the dependence of glutamine to the induction of MnSOD by amino acid deprivation in HepG2 cells. Recent studies by this group have established the importance of glutamine as a critical carbon and nitrogen source in human hepatomas which may supersede the metabolic importance of glucose in these tumor cells [104]. These investigators also demonstrate that human hepatoma cells uptake and potentially utilize glutamine at a much higher rate than normal human hepatocytes, most likely dependent on a switch between the system N (neutral) to system ASC amino acid transporter [104]. These studies thus provide a potential explanation for the importance of glutamine to hepatoma metabolism and in the context of MnSOD gene regulation.

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57 One path of utilization for glutamine is through the TCA cycle. When HepG2 cells are starved for glutamine, the formation of -ketoglutarate is inhibited and may cause an inhibition of the TCA cycle. The TCA, electron transport chain and MnSOD are closely linked through the production of and protection against, harmful free radicals produced from the electron transport chain as a by-product of normal respiration. To determine if inhibition of the TCA cycle contributes to the inhibition of MnSOD, specific inhibitors of the TCA cycle were utilized. Inhibitors of three different TCA cycle enzymes were targeted and like glutamine deprivation, blocked the induction of MnSOD mRNA levels by amino acid deprivation. These data provide a potential link, in HepG2 cells, for the requirement of glutamine to help drive the TCA cycle, contributing to the electron transport chain, the generation of superoxide radicals and ultimately the generation of ATP. The results presented here suggest that the induction of MnSOD is dependent on glutamine because of its contribution to the TCA cycle. The function of the TCA cycle is to provide reducing units to the electron transport chain. Inhibition of the TCA cycle may block the induction of MnSOD as a consequence of the prevention of the downstream effects of the TCA. To examine this possibility, components of the electron transport chain were targeted with inhibitors and MnSOD mRNA levels were evaluated. Inhibition of both complex III and the F 1 F o subunits resulted in the inhibition of MnSOD by histidine deprivation. The electron transport chain functions to establish a proton gradient to drive the synthesis of ATP. Therefore, the inhibition of the electron transport chain could be blocking MnSOD induction due a decrease in ATP levels. To determine if ATP levels were affecting the induction of MnSOD by amino acid deprivation, ATP levels were

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58 measured and compared to northern analysis of MnSOD. ATP measurements did reveal that when there are no amino acids present (EBSS), ATP levels are at their lowest, indicating that the lack of induction for MnSOD mRNA levels could be a result of decreased ATP levels. However, reducing ATP levels to that below seen with EBSS, by using a glucose analogue 2-deoxy-D-glucose, established that MnSOD mRNA could still be induced under the various medium conditions. These results demonstrate that although EBSS, as expected, does result in low ATP levels, it is not ultimately the cause for the inhibition of MnSOD mRNA levels in response to amino acid deprivation. Given that reduced ATP levels did not effect the induction of MnSOD, the contribution of an intact proton gradient was also tested. To test this, an uncoupler of the electron transport chain was utilized. Use of the inhibitor blocked the induction of MnSOD, demonstrating a requirement for an intact proton gradient. In summary, these data demonstrate that induction of MnSOD by histidine depletion requires signals dependent on the TCA cycle, the electron transport chain, and is mediated through a functional mitochondrial membrane potential, but not ATP levels.

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CHAPTER 4 SIGNAL TRANSDUCTION PATHWAYS ASSOCIATED WITH MANGANESE SUPEROXIDE DISMUTASE INDUCTION IN RESPONSE TO AMINO ACID DEPRIVATION Introduction The classic cellular sensing mechanism for detecting essential amino acid deprivation, referred to as the Amino Acid Response (AAR) pathway, is associated with an increase in the concentration of uncharged tRNA leading to the activation of the GCN2 kinase [63]. The activation of GCN2 kinase leads to phosphorylation of translation initiation factor eIF-2, causing a decline in global protein synthesis [63]. The GCN2-dependent pathway can be activated by either deprivation of a single essential amino acid or complete amino acid deprivation (EBSS) [1]. As previously established in Chapters two and three, the induction of MnSOD occurs in response to depletion of a single essential amino acid but not complete amino acid deprivation, implying that the GCN2 pathway may not be utilized as the sensor for MnSOD induction. Given that the classic GCN2 pathway does not seem to be solely responsible for the signal transduction leading to the transcriptional activation of MnSOD in response to amino acid deprivation, other relevant signaling pathways potentially linking the induction of MnSOD to either HIS or EBSS +GLN were evaluated. Recently, mitogen activated protein (MAP) kinase signaling pathways have been implicated in amino acid dependent signaling. Franchi-Gazzola et al. [105] have previously demonstrated that the MAP kinase pathway, through an increase in extracellular regulated kinase (ERK1/2) phosphorylation and activity, was involved in the induction of fibroblast system A 59

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60 transport activity following incubation in amino acid free medium. It has more recently been demonstrated that histidine deprivation causes an increase in the phosphorylation of ERK1/2 and that inhibition of the ERK signal transduction pathway causes a reduction in the HIS dependent increase in p21 mRNA [106]. MAP kinase signaling has been linked to a number of stimuli including amino acids, growth factors and stress. MAP kinases, a family of serine/threonine protein kinases are ubiquitously expressed and form a network of signaling cascades that mediate, thru extracellular cues, a number of cellular responses such as proliferation, apoptosis and cell survival [107-109]. Each core cascade is comprised of at least three protein kinases, each one activating the next, with the final result being the activation of the terminal kinase in the cascade, a specific MAP kinase. In some cases there may be up to six protein kinases within a cascade, ultimately leading to the activation of a MAP kinase. Extracellular cues initiate a cascade of phosphorylation events leading to the activation of a MAP/ERK kinase kinase (MEKK). This leads to the activation of a MEK which then phosphorylates and activates the terminal MAP kinase. There are four main MAP kinases, ERK1/2, JNK, p38MAPK and ERK5, all differing in their physiological activities that are cell type and stimulus specific [108,109]. The activation of specific isoforms within each cascade provides the necessary specificity for activation of a specific MAP kinase. Some level of crosstalk occurs between the signaling cascades, leading to the activation of the terminal MAP kinase. However, there is a level of control within each signaling cascade at the level of substrate recognition. MEKKs are serine threonine kinases that recognize the Ser-X-X-X-Ser/Thr motif. This activates MEKs

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61 which phosphorylate serine, threonine and tyrosine, recognizing the Thr-X-Tyr, on the terminal MAP kinase. Activation of the MAP kinase requires dual phosphorylation. The substrate specificities of MEKs are narrow, with each phosphorylating one or only a few MAP kinases. The amino acid located between the threonine and tyrosine confers the level of substrate specificity for the activation of each kinase, being glutamine, glycine, or proline for the ERK1/2, p38 or JNK pathways, respectively. The ERK1/2 MAPK is typically activated in response to the availability of growth factors whereas JNK and p38 MAPK are activated by stresses such as UV light, cytokines and osmotic shock. ERK5 has been shown to be regulated by both mitogenic and stress signals. Once a MAP kinase is activated it then phosphorylates a number of effector molecules such as transcription factors, phosphatases and other protein kinases. This level of regulation provides the cell with an exquisite level of control that allows for a number of cellular responses to a variety of extracellular stimuli. Another protein kinase, also dependent on amino acid availability, that plays a central role in monitoring and regulating downstream translational events is the mammalian target of rapamycin (mTOR) [110-117]. As the name implies, mTOR is a target of the drug rapamycin, which potently inhibits its activity. Rapamycin is a lipophilic macrolide, which was identified from a soil screen taken from the island Rapa Nui, more commonly known as Easter Island [118]. It was found to inhibit yeast growth and subsequently, mutations conferring resistance to the drug led to the identification of the drugs target protein and name, target of rapamycin (TOR) [119,120]. Rapamycin functions by forming a complex with a peptidyl-prolyl cis/trans isomerase, FKBP12, that binds to and inhibits the activity of TOR [120,121]. mTOR is a large (280 kda)

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62 serine/threonine kinase, in the phosphatidylinositol kinase related kinase (PIKK) family of proteins, with a number of regulatory domains [122]. mTOR forms two different complexes, both associating with GL, through a TOS (TOR signaling) motif [123], and then either raptor or rictor which are rapamycin sensitive and insensitive, respectively [124-126]. mTOR is regulated by a number of extracellular cues, including growth factors such as insulin and amino acids, mediating cell growth, metabolism and proliferation [127]. The role of mTOR in the cell, when proper nutrients are available, is to maintain ribosomal biogenesis, translation initiation and nutrient import [121]. Specifically, mTOR activity is regulated by the availability of branch chain amino acids, where the lack of, for example leucine, leads to inhibition of mTOR-dependent downstream events. The primary downstream effectors of mTOR are 4EBP1 and P70 S6K. The role of 4EBP1 is to regulate cap dependent translation of mRNA. When 4EBP1 is phosphorylated by mTOR, it causes dissociation from the cap binding protein eIF4E, allowing for its binding to mRNA and increasing eIF4E dependent translation. P70 S6K functions to phosphorylate S6, a major ribosomal protein [128]. Phosphorylation of the S6 ribosomal protein functions to selectively increase the translation of a subset of mRNA that contains a tract of pyrimidines [129]. These mRNAs account for approximately 30% of the total mRNA in the cell and encode for ribosomal proteins, as well as, translation initiation and elongation factors [130]. Through the regulation of the translational components 4EBP1 and p70 S6K mTOR regulates cell growth and proliferation, mediated by the availability of growth factors and nutrients.

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63 Materials and Methods Isolation of Total RNA RNA isolation was performed as described in Chapter 2. Protein Isolation and Immunoblot Analysis Protein isolation was performed as described in Chapter 2 with the following modifications for the indicated antibody which were obtained from Cell signaling, Danvers, MA: phospho-p70S6 kinase (Thr389) (#9205), phospho-4E-BP1 (Thr37/46) (#9459); phospho-p70S6 kinase (Th421/Ser424) (#9204). Samples immunoblotted for p70S6 kinase (Thr389) and p70S6 kinase (Th421/Ser424) were run on a 7.5% gel. Samples immunoblotted for phospho-4E-BP1 (Thr37/46) were run on a 12.5% gel. The gels were then transferred to a nitrocellulose membrane and blocked for 1 h at room temperature in 5% non-fat milk. The primary antibodies were diluted 1:1000 in 5% BSA in TBST incubated with the membrane overnight at 4 o C. Anti-rabbit secondary antibody was used at 1:2000 diluted in 5% non-fat milk for 1 h at room temperature. Reagents Used From Calbiochem, San Diego, CA: SB202190 (#559388); SB203580 (#559389); PD98059 (#513000); JNK (c-Jun N-terminal kinase) inhibitor II (#SP600125); U0126 (#662005). Rapamycin was obtained from LC laboratories, Woburn, MA #ASW-104. Results Requirement of ERK1/2 Signaling To investigate the signaling mechanisms involved in the regulation of MnSOD in response to amino acid deprivation, two regulatory pathways known to be regulated by amino acid availability were evaluated, MAP kinase signaling and the protein kinase

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64 mTOR. Previous data has linked the availability of amino acids to the phosphorylation of the MAP kinase ERK1/2 [105,106]. However, with the unique nature of the MnSOD induction, relative to other amino acid regulated genes, other members of the MAPK and JNK pathways were also tested. In order to study the potential signal transduction pathways involved in the regulation of MnSOD, inhibitors of the MAP kinase pathways were used. Specifically, SB203580 (10 M) and SB202190 (10 M) were employed to inhibit p38 kinase and SP600125 (20 M) as a JNK inhibitor. Two different inhibitors of the ERK1/2 kinase, PD98059 (50 M) and U0126 (50 M), were also utilized. PD98059 and U0126 are both selective and cell-permeable inhibitors of MEK kinase. However, PD98059 inhibits the active form of MEK whereas UO126 inhibits both the active and inactive MEK [131,132]. The concentrations utilized for these inhibitors have been demonstrated previously to inhibit the activation of these kinases in HepG2 cells [99,133,134]. Inhibitors for p38 MAPK and JNK had no effect on the induction by HIS or EBSS +GLN (Figure 4-1). Inhibitors targeting ERK1/2 activity, PD98059 and U0126, blocked both the HIS and EBSS +GLN inductions (Figure 4-2). FED-HISEBSS+GLNFED-HISEBSS+GLNFED-HISEBSS+GLNNo TreatmentSB 202SB 203FED-HISEBSS+GLNJNK inhibitorMnSODL7a FED-HISEBSS+GLNFED-HISEBSS+GLNFED-HISEBSS+GLNNo TreatmentSB 202SB 203FED-HISEBSS+GLNJNK inhibitorMnSODL7a Figure 4-1. Northern analysis of HepG2 cells treated for 12 h in FED, -HIS, EBSS or EBSS +GLN with the indicated inhibitor; SB202 (10 M), SB203 (10 M), or a JNK inhibitor (20 M). Gene specific probes for MnSOD and L7a (loading control) were used to determine the relative amounts of mRNA.

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65 MnSODL7a FED-HISEBSS+GLNNo TreatmentPD 98059U0126 FED-HISEBSS+GLNFED-HISEBSS+GLN MnSODL7a FED-HISEBSS+GLNNo TreatmentPD 98059U0126 FED-HISEBSS+GLNFED-HISEBSS+GLN Fold Induction*** FED -HIS EBSS +GLNNo TreatmentPD 98059U012601234Fold Induction*** FED -HIS EBSS +GLNNo TreatmentPD 98059U012601234AB MnSODL7a FED-HISEBSS+GLNNo TreatmentPD 98059U0126 FED-HISEBSS+GLNFED-HISEBSS+GLN MnSODL7a FED-HISEBSS+GLNNo TreatmentPD 98059U0126 FED-HISEBSS+GLNFED-HISEBSS+GLN Fold Induction*** FED -HIS EBSS +GLNNo TreatmentPD 98059U012601234Fold Induction*** FED -HIS EBSS +GLNNo TreatmentPD 98059U012601234AB Figure 4-2. Evaluation of HepG2 cells treated for 12 h in the indicated medium and the indicated inhibitor. A) Representative northern blot of HepG2 cells treated for 12 h in FED, -HIS, EBSS or EBSS +GLN with the indicated inhibitor; PD98059 (50 M) or U0126 (50 M). B) The corresponding densitometry. Gene specific probes for MnSOD and L7a (loading control) were used to determine the relative amounts of mRNA. Densitometry was quantitated from multiple experiments using a Microtek scan maker 9600XL and analyzed with the UN-SCAN-IT program. Data points are represented as relative fold induction, as compared to the FED or EBSS condition; all samples were normalized to the internal control, L7a. Data points are the means +/SEM (n 3). An asterisk (*) denotes significance as determined by a Students t-test to a value of p 0.05.

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66 Amino Acid Deprivation and mTOR Signaling mTOR is a key regulator of cell growth, predominantly through the regulation of translation events. The availability of nutrients, such as amino acids and other growth factors, are required for mTOR to remain active. Unlike GCN2, which senses uncharged tRNA, mTOR senses the levels of intracellular pools of amino acids (although the mechanism by which this occurs is still unknown) [111,135]. The inhibition of mTOR, through nutrient withdrawal or the use of rapamycin, causes a global decrease in general protein synthesis due to a loss of phosphorylation of key translational components [121]. With mTOR at the center of regulation by growth factors and amino acids, this pathway was evaluated by use of the potent mTOR inhibitor rapamycin. To determine the involvement of mTOR in the regulation of MnSOD, HepG2 cells were treated in the FED condition with increasing concentrations of rapamycin, with the hypothesis that this would mimic amino acid deprivation and cause an induction of MnSOD mRNA levels. Interestingly, rapamycin treatment in FED medium did not induce MnSOD mRNA levels (Figure 4-3). However, rapamycin treatment did cause a concentration dependent inhibition of the MnSOD induction in HIS conditions (Figure 4-3). Furthermore, rapamycin had no effect on the induction of MnSOD mRNA levels when cells were incubated in EBSS +GLN conditions (Figure 4-3). A quantitative summary of this data is also shown (Figure 4-4). The ability of rapamycin to distinguish between the effects of -HIS and EBSS +GLN conditions is in contrast to the ERK1/2 inhibitors ability to block both modes of MnSOD induction. Furthermore, the ability of rapamycin to distinguish between the two conditions strongly suggests that, although necessary for the induction by HIS, the addition of GLN to EBSS may constitute a distinct but interdependent pathway for induction of MnSOD.

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67 FED-HISEBSSEBSS + GLN L7aMnSODRapamycin(M)( 00100101501001015 FED-HISEBSSEBSS + GLN L7aMnSODRapamycin(M)(Rapamycin(M)( 00100101501001015 Figure 4-3. Northern analysis of HepG2 cells treated for 12 h in FED, -HIS, EBSS or EBSS +GLN with increasing concentrations of rapamycin. Gene specific probes for MnSOD and L7a (loading control) were used to determine the relative amounts of mRNA. Rapamycin (M)-HIS*** FED01510Fold induction01234 Rapamycin (M)-HIS*** FED01510Fold induction01234 Rapamycin (M)EBSS+GLN Fold induction01234EBSS01510Rapamycin (M)EBSS+GLN Fold induction01234EBSS01510Rapamycin (M)-HIS*** FED01510Fold induction01234 Rapamycin (M)-HIS*** FED01510Fold induction01234 Rapamycin (M)EBSS+GLN Fold induction01234EBSS01510Rapamycin (M)EBSS+GLN Fold induction01234EBSS01510 Figure 4-4. Densitometry of Northern analysis of HepG2 cells treated for 12 h in FED, -HIS, EBSS or EBSS +GLN with increasing concentrations of rapamycin (as in Figure 4-3). Data points are represented as relative fold induction, as compared to the FED or EBSS condition; all samples were normalized to the internal control, L7a. Data points are the means +/SEM (n 3). An asterisk (*) denotes significance as determined by a Students t-test to a value of p 0.05. Given the unexpected results obtained with rapamycin treatment, additional experiments to fully understand the mechanism underlying this phenomenon was employed. To further define mTORs role in the amino acid dependent gene regulation of MnSOD, two downstream effectors of mTOR, p70S6K and 4EBP-1 [136,137] were

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68 evaluated. As previously discussed, both mTOR and ERK1/2 lead to the phosphorylation of p70S6K. mTOR phosphorylates p70S6K at threonine 389, an event that is rapamycin sensitive [130,138]. A p70S6K immunoblot for cells in a variety of growth conditions in conjunction with exposure to the inhibitors rapamycin, PD98059 or U0126. The FED condition demonstrates mTOR-dependent phosphorylation of threonine 389 (T389) (Figure 4-5), a relevant mTOR target [138]. The antibody for p70S6K also recognizes an 85kD isoform of p70S6K which is marked by an asterisk (*) [139]. Interestingly, deprivation of a single essential amino acid (-HIS) did not block this phosphorylation at position T389, but, as expected, total amino acid deprivation or rapamycin treatment blocked this phosphorylation. GLN add back to EBSS did not restore phosphorylation at T389, further implicating two distinct but interconnected pathways for MnSOD induction. Inhibition of ERK1/2 caused an ~50% reduction in the phosphorylation of T389 as compared to untreated FED or HIS conditions. FED-HISEBSS+GLNFED-HISEBSS+GLNFED-HISEBSS+GLNPD98059U0126 4EBP1-pP70 S6K(421/424)FED-HISEBSS+GLNFED-HISEBSS+GLNRapamycinP70 S6K(389) ****FED-HISEBSS+GLNFED-HISEBSS+GLNFED-HISEBSS+GLNPD98059U0126 4EBP1-pP70 S6K(421/424)FED-HISEBSS+GLNFED-HISEBSS+GLNRapamycinP70 S6K(389) FED-HISEBSS+GLNFED-HISEBSS+GLNFED-HISEBSS+GLNPD98059U0126 4EBP1-pP70 S6K(421/424)FED-HISEBSS+GLNFED-HISEBSS+GLNRapamycinP70 S6K(389) **** Figure 4-5. Immunoblot analyses of HepG2 cells incubated for 12 h in FED, -HIS, EBSS or EBSS +GLN medium with the indicated inhibitor and immunoblotted for the respective proteins. An asterisk (*) marks the 85kD isoform of the p70S6 kinase protein. The phosphorylation at sites T421 and S424 in the autoinhibitory domain of p70S6K, which are believed to be important as the first sites leading to kinase activation as well as potential sites for phosphorylation by ERK1/2 [140,141] were also evaluated.

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69 Conditions for both HIS and EBSS +GLN increased the phosphorylation at these sites and inhibition of either mTOR or ERK1/2 activity by rapamycin or PD98059/U0126 [142], respectively, blocks the p70S6K phosphorylation at T421 and S424 (Figure 4-5). This correlates with the importance of mTOR and ERK1/2 in the amino acid dependent induction of MnSOD. Another translational regulatory protein phosphorylated by mTOR, eukaryotic initiation factor 4E-binding protein (4EBP1), was also evaluated. When mTOR is active, 4EBP1 is phosphorylated at four sites, threonines 37 and 46, serine 65 and threonine 70 [143]. The phosphorylation of threonines 37 and 46 is required for the subsequent phosphorylation of the other two sites [144]. Furthermore, in vitro studies have demonstrated that mTOR can directly phosphorylate threonines 37 and 46 [145,146] A phospho-peptide antibody for positions T37/S46 of 4EBP-1 shows that the phosphorylation of these sites is unchanged in FED versus HIS, whereas EBSS significantly reduces the phosphorylation at these sites (Figure 4-5). The addition of GLN to EBSS stimulates the partial re-phosphorylation at these sites, with no effect in any condition when rapamycin or PD98059/U0126 is included. This observation argues for a glutamine dependent phosphorylation of 4EBP-1 that is independent of both mTOR/raptor and ERK1/2. However, the +GLN response could be explained by regulation through a known alternative rapamycin insensitive pathway, mTOR/rictor [126,147-149]. Discussion While organisms have adapted metabolic strategies to accommodate changes in the availability of critical nutrients, how these changes are sensed by the cell still remains to be fully understood. However, two pathways known to mediate downstream effects in

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70 response to amino acid availability are through ERK1/2 signaling and the protein kinase mTOR. The data presented here, and summarized (Figure 4-6), implicates the importance of MEK/ERK and mTOR/raptor signaling pathways in the regulation of MnSOD mRNA in response to amino acid deprivation. Erk1/2 P70 S6K PThr421/Ser 424 PThr389 mTOR TSC2 GAP TSC1 Rheb-GDP TSC2 GAP TSC1 P Rheb-GTP PTranslation ON MnSOD gene ? S6 P P P P P eIF4E 4EBP-1 P P P P PThr37/Ser 46 eIF4E 4EBP-1Translation OFF(inactive)(active) Rapamycin PD98059/U0126 Cytoplasm MEK 1/2 Erk1/2 P70 S6K PThr421/Ser 424 PThr389 mTOR TSC2 GAP TSC2 GAP TSC1 Rheb-GDP TSC2 GAP TSC2 GAP TSC1 P Rheb-GTP P PTranslation ON MnSOD gene ? S6 P P P P P eIF4E 4EBP-1 P P P P PThr37/Ser 46 eIF4E 4EBP-1Translation OFF(inactive)(active) Rapamycin PD98059/U0126 Cytoplasm MEK 1/2 Figure 4-6. Relevant signal transduction pathways involved in the induction of MnSOD in response to amino acid deprivation. MAP kinase signaling is a signal transduction pathway orchestrating a large number of extracellular cues to mediate a number of intracellular responses. In particular, it has been demonstrated that ERK1/2 activation occurs in response to amino acid deprivation [105,106]. To evaluate the contribution of MAP kinase signaling in the regulation of MnSOD, several inhibitors were used. As determined by the MAP kinase inhibitors tested, only the MEK/ERK1/2 pathway was required for MnSOD mRNA induction by amino acid deprivation. As discussed, crosstalk between the MAP kinase pathways can occur. However, at this time, ERK1/2 is the only known target for

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71 MEK1/2. Utilization of inhibitors specific for MEK1/2 blocked MnSOD, induction, supporting the importance of ERK1/2 activity. Furthermore, evaluation of the downstream effectors of ERK1/2 signaling, by immunoblot analysis, confirmed the activity of this enzyme. The phosphorylation of T421 and S424 of p70S6K demonstrates that, through both means of MnSOD induction (HIS and EBSS +GLN), ERK1/2 activity is increased. Additionally, phosphorylation at these sites was also blocked by both of the MEK1/2 inhibitors, as well as rapamycin. This strongly implicates a role for ERK1/2 signaling in the regulation of MnSOD in response to amino acid deprivation. mTOR, a central mediator of growth, metabolism and proliferation [127], was also evaluated as a potential contributor to the induction of MnSOD. mTOR regulation is mediated by amino acid availability and is active when amino acid levels are sufficient. Initial studies to determine the involvement of mTOR in the regulation of MnSOD utilized the inhibitor rapamycin, which should mimic amino acid deprivation. However, inhibition of mTOR alone did not induce MnSOD mRNA levels. Furthermore, the induction by histidine deprivation was blocked by rapamycin treatment, whereas the EBSS +GLN induction was not affected, indicating an interdependent regulation for this pathway. These data led to the hypothesis that mTOR activity and signaling may be required for MnSOD induction, but not involved in the activation of signaling pathways regulating this induction. To further understand the role of mTOR in the induction of MnSOD, the downstream effectors p70S6K and 4EBP-1 were evaluated to determine the signaling status of mTOR. A major rapamycin sensitive target of mTOR is p70S6K T389. Immunoblot analysis with a phospho-specific antibody to this protein was used to determine the status

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72 of mTOR activity. As expected, T389 phosphorylation did occur when HepG2 cells were incubated in FED medium. Additionally, as hypothesized, histidine deprivation still resulted in the phosphorylation of T389, indicating that mTOR signaling was occurring. Equally important, inhibition of mTOR with rapamycin blocks both the phosphorylation of T389 and mRNA levels of MnSOD in response to histidine deprivation. On the other hand, EBSS or EBSS +GLN medium did not result in the phosphorylation of T389, nor did rapamycin treatment block the induction of MnSOD mRNA in EBSS +GLN medium. These data suggest a requirement of mTOR activity for the induction of MnSOD by histidine deprivation and a separate rapamycin insensitive pathway for the induction occurring in response to EBSS +GLN. Recent reports have associated ERK1/2 signaling and the regulation of mTOR, through the regulation of TSC1 (tuberin) and TSC2 (hamartin) [150-153]. TSC1 and TSC2 are tumor suppressor genes that normally participate in cell growth and proliferation. However, mutations in these genes cause an autosomal dominant genetic disease, tuberous sclerosis complex (TSC), characterized by benign tumors (hamartomas and hamartias) affecting many organs. TSC1 and TSC2 form a heterodimer and, through the GTPase activity of TSC2, function to inhibit Rheb (Ras homologue enriched in brain), whose activity is required for mTOR signaling to occur [151]. Ma et al. [150] recently published a paper demonstrating the phosphorylation of TSC2, through treatment with phorbol 12-myristate 13-acetate (PMA), which was blocked by the ERK1/2 inhibitor U0126. Activation of ERK1/2 leads to the phosphorylation of TSC2 and inhibition of its ability to deactivate Rheb and thus mTOR activity is maintained (Figure 4-6) [150]. Therefore, inhibition of ERK1/2 signaling would also block mTOR

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73 activity and ultimately the induction of MnSOD by amino acid deprivation. The connection of ERK1/2 signaling to mTOR fits well with the data presented here and establishes an amino acid dependent regulation pathway for MnSOD that is completely novel when compared to the cellular mechanisms currently proposed for the induction of gene expression via amino acid deprivation.

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CHAPTER 5 TRANSCRIPTIONAL REGULATION OF MANGANESE SUPEROXIDE DISMUTASE BY FORKHEAD BINDING PROTEINS IN RESPONSE TO AMINO ACID DEPRIVATION Introduction The transcriptional regulation of MnSOD gene has been extensively studied in the human, mouse and rat species in response to a number of stimuli [18,23,24,39,43,154-158]. Given that the characterization of MnSOD induction in response to amino acid deprivation has been limited to the human species, the transcriptional regulation of the human MnSOD will be discussed, although many comparisons can be made between species. The human MnSOD gene has a typical housekeeping basal promoter that it is GC rich and contains no TATA or CATT box [159]. In vivo footprinting with purified SP1 and AP2 proteins identified eight SP1 sites and nine AP2 sites within an 250 bp proximal promoter [159]. Further studies demonstrated a requirement of SP1 binding for the activation of MnSOD gene transcription to occur with AP2 functioning to block SP1 binding [39,159]. As previously discussed, MnSOD is inducible in response to several pro-inflammatory stimuli including lipopolysaccharide (LPS), interleukins 1 and 6 (IL-1 and IL-6), tumor necrosis factor alpha (TNF-) and interferon[15,20,21,154]. The 5 region of the MnSOD promoter does contain several NF-B and AP1 elements, however, this region alone is not inducible with these stimuli, requiring an enhancer element located within intron two [160]. The significant induction of MnSOD gene expression by pro-inflammatory cytokines [15,20,21] has been proposed to contribute to cell survival by reducing the 74

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75 increased levels of reactive oxygen species (ROS) associated with the inflammatory response [4,5,15,16]. ROS are highly reactive and cause extensive damage to cellular components including lipids, DNA and proteins, contributing to the pathogenesis of disease [6-8] and aging [87]. A critical consequence of nutrient availability and subsequent metabolism is the generation of ROS with previous estimates indicating 1-3% of consumed oxygen in the basal state is released as superoxide radicals from mitochondrial electron transport [3]. The connection between nutrient levels and the generation of ROS is also thought to help delay the aging process through caloric restriction [85], which may be explained by a reduction in metabolic flux and a concomitant decline in ROS production [87]. This observation is also consistent with the mitochondrial theory of aging which implicates continuous generation of ROS as a critical factor for damage to mitochondrial DNA as well as oxidative reactions with components of the cytosol and nucleus [86,161]. In C. elegans, a model organism for aging studies due to ease of genetic manipulation and short life span (19 days), two genes have been identified in the regulation of aging, age-1, a homologue of the mammalian phosphatidylinositol-3-OH (PI3) kinase, and daf-2, a homologue of the insulin or insulin-like growth factor receptor family [162]. The age-1 mutation was identified in a screen for long lived mutants and conferred an approximately 65% increase in life span [163]. Mutations affecting dauer formation, a developmentally arrested stage occurring in C. elegans when there is a limited amount of food, led to the identification of the daf-2 gene [164]. Mutations in the daf-2 gene caused an increase in dauer formation marked by long life span, decreased growth, increased resistance to starvation and reproductive immaturity causing an

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76 increase in life span twice that of the wild type counterpart [164,165]. Subsequently, Honda et al. [166] demonstrated that mutations in the age-1 and daf-2 genes caused a life span extension phenotype by conferring an increase resistance to oxidative stress corresponding with an increase in MnSOD levels. The mutant phenotypes of age-1 and daf-2 were demonstrated to require the activity of the downstream target daf-16 [165,167] and, soon after, daf-16 were established to directly regulate the transcription of genes involved in metabolism and development leading to the dauer formation and increased lifespan [168]. Work by Furuyama et al. [169] determined the canonical binding site (TTGTTTAC) for daf-16 and, through computer analysis, demonstrated that the C. elegans sod3 promoter contained a daf-16 binding element (DBE). Subsequently, Kops et al. demonstrated that overexpression of the daf-16 human homologue, FOXO3a, could protect human colon carcinoma cells from oxidative stress through increases in MnSOD mRNA and protein levels [170]. Furthermore, overexpression of FOXO3a caused an increase in activity of a luciferase reporter construct, and mutational analysis led to the identification of one functional inverse DBE at position -1249 of the MnSOD gene [170]. FOXO proteins belong to the FOX ( f orkhead b ox ) family of winged helix/forkhead transcription factors consisting of over 100 family members ranging from FOXA to FOXS [171-173]. Classification of these transcription factors is based on the conserve DNA binding domain which binds as a monomer forming three alpha-helices and two characteristic large loops or wings[171,174]. The forkhead member of the class O (other), commonly referred to as FOXO, comprises a group of the following functionally related proteins: FOXO1(forkhead in rhabdosarcoma or FKHR), FOXO3a (FKHR-like

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77 1or FKHR-L1), FOXO4 (acute lymphocytic leukemia-1 fused gene from chromosome X or AFX) and FOXO6 [172,173]. The FOXO class of proteins are implicated in the regulation of a variety of cellular processes, including the cell cycle, apoptosis, DNA repair, stress resistance, and metabolism [170,173,175-178]. Although FOXO proteins share a high level of homology, knock out studies in mice have demonstrated different functions for each isoform [179]. Foxo1 homozygous null mutants die before birth [179], whereas haplo-insufficiency rescues the diabetic phenotype in insulin resistant mice (Irs2 -/-) by reducing hepatic expression of glucogenetic genes and increasing -cell proliferation [179-181]. Foxo3a-/female mice have age dependent abnormal ovarian follicular development, mild anemia and decreased glucose uptake in glucose-tolerance tests [179,182]. Foxo4 -/mice have no phenotype and are indistinguishable from their littermates [179]. FOXO binding proteins are negatively regulated by the presence of growth/survival signals through protein kinase B (PKB) [175]. When nutrients are available, PKB is in its active state and phosphorylates FOXO proteins, facilitating the binding of the chaperonin 14-3-3, which can then be exported out of the nucleus and thus causing retention of the FOXO proteins in the cytostol [183-185]. On the other hand, removal of growth factors impairs PKB signaling and the phosphorylation of the FOXO protein, resulting in its localization to the nucleus and activation of its target genes [183-185]. Materials and Methods Growth Hormone Reporter Constructs Regions of the MnSOD gene were cloned into a human growth hormone reporter plasmid [186]. A PAC (phage artificial chromosome) clone, obtained from the Sanger center (RP1-56L9), containing the entire human MnSOD gene was used to clone regions

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78 of interest into the growth hormone vector. A 3.6 kb BamHI fragment of the human promoter was digested from the PAC. This fragment was then run on a 0.7% agarose gel, gel purified and cloned into the BamHI site of a promoterless growth hormone reporter plasmid pGH. This construct was sequenced and analyzed by restriction digest for confirmation of the correct sequence and orientation. The unique restriction enzyme sites were used to generate a 1.4, 1.3, 1.1 and 0.83 kb promoter fragments. Briefly, the constructs were generated using an XbaI site at the 5 end of the promoter fragment along with the following 3 restriction enzymes NruI (1.4), SexAI (1.3), XmnI (1.1) and SpeI (0.83). To generate the promoter constructs in conjunction with the human enhancer a 488 bp fragment was digested from a construct previously generated in the lab and described in [43]. The 488 bp human enhancer fragment was digested from the HindIII site of the pGH contruct and religated into the HindIII site of the GH constructs containing the indicated human MnSOD promoter. The TKGH with the human enhancer construct was already generated and is described in [43]. Overexpression Plasmids The FOXO3a and FOXO1 plasmids were generously provided. The FOXO3a plasmid was from Dr. Burrenger and is described in [170]. The FOXO1 plasmid was from Dr. Tang and described in [185]. Briefly, the FOXO3a plasmid is an estrogen receptor (ER) fusion protein that remains inhibited (localized to the cytosol) until treated with 4-Hydroxytamoxifen (4-OHT), a modified ligand for the ER receptor. The protein is constitutively active due to the mutations of the PKB sites to alanines, leaving this protein unable to be phosphorylated. The FOXO1 plasmid is not an ER fusion protein but it is constitutively active due to similar mutations.

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79 Quick Change PCR To delete the FOXO site from the growth hormone promoter constructs the Stratagene QuickChange Site-Directed Mutagenesis Kit #200518 was used. Briefly, 25 ng of either the human MnSOD 3.4 promoter GH construct or the human MnSOD 3.4 promoter + enhancer fragment GH construct used as the template. 125 ng of each of the following primers Forward: ATT CTT CTG ACG TCT GCC CAG CCC TTC CTG Reverse: CAG GAA GGG CTG GGC AGA CGT CAG AAG AAT. Also added to the reaction was a 1X final concentration of reaction buffer, 8 mM DNTP mixture, water and PFU enzyme. A PTC 100 peltier thermal cycler was use with the following parameters: Cycle 1 (95 o C for 30 seconds) X 1 cycle. Cycle 2 (95 o C for 30 seconds, 55 o C for 1 minute, 68 o C for 14 minutes) X 18 cycles. The reaction was then incubated on ice for two minutes and 1 L of DPNI was added to remove the parental strand, leaving only the mutated desired product which was subsequently transformed into XL-10 gold competent cells and incubated on a plate with ampicilin. From the resulting colonies, the plasmids were isolated and sequenced for verification. Transient Transfection of Reporter Constructs HepG2 cells were cultured as described previously and transfected at approximately 50% confluency in a 10 cm dish. The reporter plasmid containing different regions of the MnSOD gene were transiently transfected using a FUGENE 6 transfection reagent (Roche #11 814 443 001). For each 10 cm dish, 15 L of the FUGENE 6 transfection reagent was diluted to a final volume of 600 L in serum free MEM. 5 g of reporter plasmid was then added and the reaction was incubated at room temperature for 30 minutes and then transferred to HepG2 cells. After 24 h, the cells were split 1:10 into 35 mm dishes and incubated for another 12 h after which the cells

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80 were then incubated in either FED or -HIS media. The promoterless hGH was also transfected to ensure that the transfection itself or the hGH plasmid does not have an effect on the MnSOD message. Northern analysis was used evaluate the effect of histidine starvation on the hGH message. A fragment from the hGH cDNA was used to create a probe for northern analysis. Transfection of FOXO Expression Constructs HepG2 cells were grown to 60 % confluency in 35 mm dishes and transfected with 0.5 g of growth hormone construct containing the 1.3 kb promoter fragment and increasing concentrations of either the FOXO1 or FOXO3a plasmid (0, 0.5, 1 or 5 g) using FUGENE6 transfection reagent. 24 h post transfection, 500 nM of 4-OHT was added to the FOXO3a series of transfections, including the zero g of plasmid to control for effects caused by this treatment. 48 h post transfection, cells were collected and total RNA was isolated as described in Chapter two. Cell Culture and Transfection of siRNAs Human hepatoma (HepG2) cells were maintained in MEM supplemented with 25 mM NaHCO 3 2 mM glutamine, antibiotic/antimycotic (ABAM) and 10% FBS at 37C with 5% CO2. The cells were grown to 70-85% confluency on 10cm dishes and then split 1:12 into 35 mm dishes and grown to 40% confluency. The cells were then transfected with a final concentration of 100 nM SMARTpool FOXO3a siRNA (Dharmacon) using DharmaFECT 4 siRNA Transfection Reagent (Dharmacon). To control for off target effects of siRNA, a cyclophilin siRNA (Dharmacon) was also transfected. After 60 h, cells were washed three times with PBS to ensure all histidine containing medium is removed. Cells were then incubated in MEM (FED), MEM-HIS, EBSS or EBSS+GLN medium. To ensure the amino acid levels in general are not depleted, the cells received

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81 fresh medium 12 h before the start of all experiments (48 h post transfection). After 12 h of amino acid deprivation, cells were collected and MnSOD expression was determined by northern blot, immunoblot or real time. Generation of cDNA To generate cDNA for real-time PCR analysis SuperScript first strand sythesis kit from invitrogen #12371-019 was used. 1 g of total RNA isolated as described in Chapter two was used as the template to which the following components were added: 1 L of a 10 mM dNTP mix, 0.5 g of Oligo(dT), and water. This reaction was incubated at 65 o C for 5 minutes then placed on ice for 2 minutes. To this reaction the following was added, 2 L of 10 RT buffer, 25 mM MgCl 2 2 L of 0.1 M DTT, and 1 L of RNAseOUT recombinant RNAase inhibitor. The reaction was then incubated for 2 min. at 42 o C, 50 units of SuperScript II RT was added to each reaction and then incubated for an additional 50 min. at 42 o C. The reaction was then terminated by incubation at 70 o C for 15 min. and then incubated on ice for for at least 5 min.. 2 units of Rnase H was then added and incubated at 37 o C for 20 min.. The sample was then diluted with 79 L of water and stored at -20 o C. Real-Time PCR We used 2 L of cDNA generated from first strand synthesis (as described above) was used as the template for real time PCR. To this, 0.3 M of each primer was added, 12.5 L of iTaq SYBER Green Supermix with ROX (Bio Rad, Hercules, CA #170-8851 and water to a final volume of 25 L. The Applied Biosystems, Foster City, CA 7000 sequence detection system was used with the following parameters: Cycle 1 (95 o C for 10 minutes) X 1, Cycle 2 (95 o C for 15 seconds, 60 o C for 1 minute) X 40 cycles. The

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82 CT method was used to determine the relative fold changes, normalized to the cyclophilin A gene, and is described in [187] Real time primers: FOXO3A: F: (5-TGG ATG CTG ATG GGT TGG A-3) R: (5-ATG GCG TGG GAT TCA CAAAG-3). FOXO4: F: (5-AGC GAC TGA CAC TTG CCC A -3) R: 5-GCC TCG TTG TGA ACC TTG ATG-3). FOXO1: (F: 5-TGG TCA AGA GCG TGC CCT AC-3) R: (5-GCT CGG CTT CGG CTC TTA G-3). Cyclophilin A: F: (5-CAT CCT AAA GCA TAC GGG TCC-3) R (5-GCT GGT CTT GCC ATT CCT G). Protein Isolation and Immunoblot Analysis Protein isolation and immunoblot analysis was performed as in Chapter two with modifications for the indicated antibody. Samples immunoblotted for FOXO1 were run on a 7.5% gel and transferred to a nitrocellulose membrane and blocked for 1 h at room temperature in 7% non-fat milk. 1.25 g/mL of Anti-FOXO1 (Chemicon #Ab3130) was diluted in 5% BSA in TBST and the membrane was incubated overnight at 4 o C. Anti-rabbit secondary antibody was used at 1:5000 diluted in 5% non-fat milk for one h at room temperature. Samples immunoblotted for FOXO3a (Upstate 07-702) were run on a 7.5% gel and transferred to a nitrocellulose membrane and blocked for 1 h at room temperature in 7% non-fat milk. 1.25 g/mL of Anti-FOXO3a was diluted in 5% BSA in TBST and the membrane was incubated overnight at 4 o C. Anti-rabbit secondary antibody was used at 1:5000 diluted in 5% non-fat milk for one h at room temperature. Isolation of Nuclei HepG2 cells were cultured as described in Chapter two. Following incubation for 6 h in the indicated medium, cells from two 150 mm dishes (per condition) were placed on ice and rinsed two times with ice cold PBS. 5 mL of PBS was then added to each plate

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83 and cells were scraped into a 15 mL tube. The cells were then centrifuged at 514g for 10 minutes. The PBS was aspirated and the pellet was transferred to a 1.5 mL eppendorph tube and resuspended by pipetting up and down until there were no clumps in 900 L of lysis buffer containing 20 mM HEPES (pH 7.6), 10 mM NaCl, 1.5 mM MgCl 2 0.2 mM EDTA, 20% glycerol, and added just prior to use, 0.1 M DTT and 1X protease inhibitor tablet (from complete mini pellet dissolved in 1 mL water) and incubated on ice for 15 min.. 100 L of a 10% Triton X-100 solution (final concentration is 0.1%) was added, vortexed briefly and the cells were centrifuged at 5200 rpm for 10 minutes at 4 o C. The supernatant containing the cytosolic fraction was removed and stored at -80 o C for later use. To each sample 500 L of nuclear extraction buffer containing 20 mM HEPES (pH 7.6), 400 mM NaCl, 1.5 mM MgCl 2 0.2 mM EDTA, 20% glycerol, and added just prior to use, 0.1 M DTT and 1X protease inhibitor tablet (from complete mini pellet dissolved in 1 mL water). The samples were then gently rocked for two h at 4 o C and then centrifuged for 10 min. at 14,000 rpm. The supernatant containing the nuclear extract was collected and stored for later use. Reagents Used 4-Hydroxytamoxifen (4-OHT) (#H7904) and insulin (#I-9278) were obtained from Sigma. LY294002 was obtained from Calbiochem (#440204). Results Endogenous Regulation of MnSOD by FOXO Transcription Factors Extensive studies in C. elegans have demonstrated that limitation of food supply causes an increase in life span which is dependent on the transcription factor daf-16, conferring increased resistance to oxidative stress and an increase in MnSOD levels [162,165-168,188]. Studies in humans demonstrated that MnSOD levels can be

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84 increased by overexpression of the daf-16, homologue FOXO [170,189]. Given that in C. elegans the FOXO homologue daf-16 is regulated by the limitation of food, the regulation of this protein in response to amino acid deprivation was evaluated. To determine if FOXO proteins may play a part in the regulation of MnSOD, modulators of the FOXO intracellular signaling pathways were used in combination with histidine deprivation. Protein kinase B (PKB) regulates the cytoplasmic localization of the FOXO proteins by phosphorylation. Insulin is a known activator of the PKB pathway and thus proposed to inhibit of the transcriptional activity of the FOXO proteins [175,176]. To evaluate this possibility, HepG2 cells were incubated in FED or HIS medium and treated with increasing amounts of insulin. Insulin does decrease the induction of MnSOD by amino acid deprivation indicating that the FOXO proteins may have a role in this regulation (Figure 5-1). Activation of PKB with insulin causes localization of the FOXO proteins to the cytoplasm, while, on the other hand, blocking PKB activity inhibits the phosphorylation of the FOXO proteins and causes translocation of the FOXO proteins to the nucleus to activate transcription of target genes. LY294002, an inhibitor of PKB [190], was tested on HepG2 cells in FED, -HIS, EBSS and +GLN conditions. The inhibitor LY294002, in all medium conditions tested, caused an increase in MnSOD mRNA levels, further implicating the role FOXO proteins may play in the regulation of MnSOD (Figure 5-2). Transcriptional Regulation of MnSOD in Response to Amino Acid Deprivation To determine DNA regulatory elements involved in MnSOD gene induction in response to histidine, a human growth hormone (hGH) reporter plasmid was used. An advantage to using the hGH plasmid is that it is an intact eukaryotic gene, complete with introns and exons. Thus, the expressed message will undergo splicing and maturation in

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85 00.010.050.10.250.51InsulinM00.010.050.10.250.51 FED-HISMnSOD 00.010.050.10.250.51InsulinM00.010.050.10.250.51 FED-HISMnSOD Figure 5-1. Northern blot of HepG2 cells incubated in FED or HIS medium, with increasing concentrations of insulin. After 12 h, cells were collected and total RNA was isolated. A gene specific probe for MnSOD was used to determine the relative amounts of mRNA. -+-+-+-+ EBSSFED-HIS+GLNLY294002 MnSOD -+-+-+-+ EBSSFED-HIS+GLNLY294002 MnSOD Figure 5-2. Northern blot of HepG2 cells incubated in EBSS, FED, -HIS or +GLN medium with or without the PKB inhibitor, LY294002. After 12 h, cells were collected and total RNA was isolated. A gene specific probe for MnSOD was used to determine the relative amounts of mRNA.

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86 a manner identical to endogenous mRNAs. Another advantage is that growth hormone expression is usually confined to the pituitary gland, eliminating any background in the HepG2 cells. In order to confirm this, cells which were not transfected with hGH plasmid were also probed with a hGH cDNA probe confirming that there was no endogenous growth hormone mRNA in any cell lines used. By placing MnSOD gene elements in front of the growth hormone message, in conjunction with the MnSOD promoter, the contributions of these regions to the induction seen by histidine starvation were evaluated. As previously demonstrated, the MnSOD promoter contains a FOXO binding site at 1249 bp upstream of the transcriptional start site [170]. To determine if histidine deprivation caused an increase in the MnSOD promoter activity, a 3.4 kb promoter fragment was cloned into the BamHI site of the hGH reporter plasmid. To further define the promoter, this plasmid was then used to create two more constructs using convenient restriction sites within the MnSOD promoter, creating 1.4kb and 0.83kb fragments. These constructs were then transfected into HepG2 cells as described in the materials and methods section. To ensure that there was little to no amino acid deprivation before the start of the experiment, fresh MEM was given to the cells 12 h before the start of the experiment. There is a small, but reproducible, induction with the 3.4 and 1.4kb constructs and no effect with the 0.83kb promoter fragment in response to histidine deprivation (Figure 5-3). A summary of the constructs generated and the corresponding results from the promoter deletion analysis are also shown (Figure 5-4). Previous studies have demonstrated that, in the rat, the MnSOD promoter has an approximately 2 fold induction by IL-1. Initially, this was included as a positive control,

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87 3.4 kb1.4 kb0.83 kbFED-HIS+IL-1FED-HIS+IL-1FED-HIS+IL-1 GH 3.4 kb1.4 kb0.83 kbFED-HIS+IL-1FED-HIS+IL-1FED-HIS+IL-1 GH Figure 5-3. Representative northern blot of HepG2 cells transfected with a growth hormone construct containing the indicated promoter fragments. 36 h post transfection, cells were cultured in FED, HIS medium or treated with IL-1 for 12 h. A gene specific probe for human growth hormone message (GH) was used to determine the relative amounts of messenger RNA (mRNA). -3.4-1.4-1.3-1.1-0.83 +1NruISexAIXmnISpeI -His +++-Human MnSOD promoter Human growth hormone -3.4-1.4-1.3-1.1-0.83 +1NruISexAIXmnISpeI -His +++-Human MnSOD promoter Human growth hormone Figure 5-4. Human growth hormone constructs used to evaluate the human MnSOD promoter deletions. The white boxes represent the growth hormone exons, and the striped boxes represent the untranslated message of growth hormone. Below the reporter constructs is a summary of results of the corresponding deletions of the human MnSOD promoter following histidine deprivation. treatment. Given that the enhancer element is known to regulate MnSOD induction by cytokines, this region in conjunction with each of the previously described promoter constructs, were also evaluated following histidine deprivation. The addition of the enhancer element does not seem to add the relative fold induction through amino acid deprivation (Figure 5-5). However, the addition of the enhancer element did, as expected, induce as a result of IL-1 treatment.

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88 3.4 kb + enhancer1.4 kb + enhancer0.83 kb+ enhancer GHFED-HIS+IL-1FED-HIS+IL-1FED-HIS+IL-1 3.4 kb + enhancer1.4 kb + enhancer0.83 kb+ enhancer GHFED-HIS+IL-1FED-HIS+IL-1FED-HIS+IL-1 Figure 5-5. Representative northern blot of HepG2 cells transfected with a growth hormone construct containing the respective promoter fragments and the human MnSOD enhancer. 36 h post transfection, cells were cultured in FED, HIS medium or treated with IL-1 for 12 h. A gene specific probe for human growth hormone message (GH) was used to determine the relative amounts of messenger RNA (mRNA). Although the enhancer does seem to increase the overall expression levels of the promoter constructs, the relative fold induction does not change. To determine the contribution of the enhancer fragment to the induction of MnSOD by amino acid deprivation, or just the overall transcription levels of the reporter plasmid, the enhancer in front of a heterologous promoter TK (thymidine kinase) was evaluated. The heterologous promoter has not been established to be inducible by amino acid deprivation. The enhancer does not respond to HIS conditions when the human MnSOD promoter is not present (Figure 5-6). As expected, the human enhancer alone in conjunction with a heterologous promoter does respond to IL-1 (Figure 5-6). Given that the induction is lost when you delete the promoter from 1.4kb to 0.83kb further deletions of the promoter constructs were generated from unique restriction sites within this region (Figure 5-7). These experiments further defined a region between 1.3 and 1.1kb. Kops et al.[170] gave insight to a potential mechanism when they published data reporting control of the MnSOD human promoter through the overexpression of the transcription factor FOXO3a. This element was in the region between the 1.8kb

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89 TK promoter+ enhancerFED-HIS+IL-1 GH TK promoter+ enhancerFED-HIS+IL-1 GH Figure 5-6. Northern blot of HepG2 cells transfected with a growth hormone construct containing the TK promoter and the human MnSOD enhancer. 36 h post transfection, cells were cultured in FED, HIS medium or treated with IL-1 for 12 h. A gene specific probe for human growth hormone message (GH) was used to determine the relative amounts of messenger RNA (mRNA). fragment and the 0.83kb fragment. To test the role of FOXO3a in the involvement of MnSOD regulation through amino acid deprivation, constructs were generated from the 1.3kb promoter construct that lacked the canonical FOXO DNA binding site; a summary of the constructs tested and the results are shown (Figure 5-7). Additionally, these constructs were also tested in conjunction with the enhancer. Representative northern blots (Figure 5-8) with the corresponding densitometry (Figure 5-9) are also shown. The importance of the FOXO site was determined by deletion of the site and the corresponding reduction of the reporter plasmid response to histidine deprivation. In order to further evaluate the role of the FOXO proteins and the regulation of the MnSOD promoter, the FOXO1 and FOXO3a proteins were overexpressed with the growth hormone reporter plasmid. These results show that FOXO1 and FOXO3a can induce the promoter construct, further implicating a role for the FOXO proteins in the regulation of MnSOD (Figure 5-10).

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90 -3.4-1.4-1.3-1.1-0.83 +1NruISexAIXmnISpeI Foxo3a -1.3Foxo3a -His +-++--3.4-1.4-1.3-1.1-0.83 +1NruISexAIXmnISpeI Foxo3a -1.3Foxo3a -His +-++-Figure 5-7. Human MnSOD promoter constructs generated and tested by histidine deprivation. The + and indicate responsive to histidine deprivation. FED-HIS FED-HIS 1.3 kb1.3 kb+ enhancer GH 1.3 kb Foxo3a FED-HIS FED-HIS 1.3 kb Foxo3a+ enhancer GH FED-HIS FED-HIS 1.3 kb1.3 kb+ enhancer GH FED-HIS FED-HIS 1.3 kb1.3 kb+ enhancer GH 1.3 kb Foxo3a FED-HIS FED-HIS 1.3 kb Foxo3a+ enhancer GH 1.3 kb Foxo3a FED-HIS FED-HIS 1.3 kb Foxo3a+ enhancer GH Figure 5-8. Northern analysis of growth hormone constructs evaluating the contribution of the FOXO binding site to MnSOD induction. The top panel is a representative northern blot of HepG2 cells transfected with a growth hormone construct containing the 1.3 kb promoter fragment alone or with the MnSOD enhancer fragment. The bottom panel is a representative northern blot of HepG2 cells transfected with a growth hormone construct containing the 1.3 kb promoter fragment with the FOXO site deleted alone or with the MnSOD enhancer fragment. In both panels, 36 h post transfection, cells were cultured in FED or HIS medium for 12 h. A gene specific probe for human growth hormone message (GH) was used to determine the relative amounts of messenger RNA (mRNA).

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91 00.20.40.60.811.21.41.61.821.3 kb 1.3 kb Foxo0.83 kbRelative fold induction FED levels** 00.20.40.60.811.21.41.61.821.3 kb 1.3 kb Foxo0.83 kbRelative fold induction FED levels** Figure 5-9. Densitometric analysis of northern blots as shown in Figure 5-9. An asterisk (*) marks significance as determined by a students t-test to a value of p 0.05. FOXO1FOXO3a GH FOXO1FOXO3a GH Figure 5-10. Overexpression of FOXO proteins with MnSOD growth hormone reporter plasmid. Northern blot analysis of HepG2 cells transfected as described in the materials and methods section with 0.5 g of growth hormone construct containing the 1.3 kb promoter fragment with increasing concentrations of either the FOXO1 or FOXO3a plasmid (0, 0.5, 1 or 5 g). Cells were collected and total RNA was isolated. A gene specific probe for human growth hormone message (GH) was used to determine the relative amounts of messenger RNA (mRNA). FOXO Messenger RNA Levels in Response to Amino Acid Deprivation Given that MnSOD is induced by amino acid deprivation, potentially through the FOXO family of proteins mediated by a conserved DBE, the gene structure of the FOXOs were also evaluated. Three of the FOXO genes, FOXO1, FOXO3a and FOXO4, all had at least one DBE in their promoter region. Real time analysis of the three different mRNAs (Figure 5-11) were evaluated demonstrating that FOXO1 mRNA is

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92 induced only in the +GLN condition. However, FOXO3a mRNA levels are induced in response to all three conditions tested. The FOXO4 mRNA levels seem to be repressed in response to changes in nutrient levels. In view of the fact that only FOXO3a was induced by histidine deprivation, further analysis of the mRNA (Figure 5-12) and protein (Figure 5-13) was performed, demonstrating a time dependent induction in both cases. Although the reporter deletion, insulin and LY294002 data implicate FOXO proteins in the regulation of MnSOD in response to amino acid deprivation, they do not provide a direct link between MnSOD and a specific FOXO protein. Therefore, to obtain direct evidence, an siRNA to FOXO3a was used. In order to verify that FOXO3a expression levels were reduced, real time PCR and western blot analysis was used. Real time analysis demonstrated that FOXO3a mRNA levels were reduced by 50%, with the corresponding reduction of protein levels shown in the immunoblot (Figure 5-14). Also shown is that reducing FOXO3a levels by siRNA does not block the induction of MnSOD at the mRNA level (Figure 5-15). 00.511.522.533.544.55FED-HISEBSS+GLNRelative fold induction FOXO1 FOXO3 FOXO4 Fold Induction (CT) 00.511.522.533.544.55FED-HISEBSS+GLNRelative fold induction FOXO1 FOXO3 FOXO4 Fold Induction (CT) Figure 5-11. Real time PCR analysis from total mRNA isolated from HepG2 cells incubated for 12 h in the indicated medium. Fold induction is compared to HepG2 mRNA isolated at the start of the experiment, zero. Data points represent the relative fold induction as determined by CT.

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93 012345626912Hours FED -HISFold Induction (CT) 012345626912Hours FED -HISFold Induction (CT) Figure 5-12. Real time PCR analysis from total mRNA isolated from HepG2 cells incubated for 12 h in of FOXO3a mRNA levels in FED or HIS medium. Fold induction is compared to HepG2 mRNA isolated at the start of the experiment, zero. Data points were determined by CT and are the means +/SEM (n =3). +12247212247248 Hrs-Foxo3 FED-HIS11892 +12247212247248 Hrs-Foxo3 FED-HIS11892 Figure 5-13. Immunoblot analysis of FOXO3a protein in FED or HIS medium for the indicate amount of time. -FOXO3a No TransfectionDharmafectaloneCyclophilinsiRNAFOXO3a siRNA -FOXO3a No TransfectionDharmafectaloneCyclophilinsiRNAFOXO3a siRNA Figure 5-14. Immunoblot analysis of FOXO3a protein from total protein isolated from HepG2 cells transfected with the indicated siRNA.

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94 No TransfectionDharmafectaloneCyclophilinsiRNAFOXO3a siRNANo TransfectionDharmafectaloneCyclophilinsiRNAFOXO3a siRNA FED-HISMnSOD No TransfectionDharmafectaloneCyclophilinsiRNAFOXO3a siRNANo TransfectionDharmafectaloneCyclophilinsiRNAFOXO3a siRNA FED-HISMnSOD Figure 5-15. Northern blot analysis of HepG2 cells in FED or HIS MEM, transfected with the indicated siRNA. A gene specific probe for MnSOD was used to determine the relative amounts of mRNA. Localization of FOXO Proteins Despite 50% reduction in FOXO3a levels, there was no reduction of MnSOD mRNA levels. In order to determine any roles that the FOXO proteins may be having in the regulation of MnSOD, the localization of the FOXO1 and FOXO3a proteins was determined. HepG2 cells were incubated in FED, -HIS, EBSS or +GLN medium conditions and the cytoplasmic or nuclear fractions were isolated and immunoblotted for the indicated protein. The FOXO3a protein is not localized in the nucleus in any of the given medium conditions (Figure 5-16). FOXO1 is localized in the nucleus; however,

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95 this protein is not inducible. Furthermore, the data on FOXO1 is also in contradiction to the model currently proposed to regulate FOXO1 [175,183-185]. FOXO3a FOXO1FED-HISEBSS+GLNFED-HISEBSS+GLN CytosolicNuclear FOXO3a FOXO1FED-HISEBSS+GLNFED-HISEBSS+GLN CytosolicNuclear Figure 5-16. Immunoblot of cytoplasmic and nuclear fractions isolated from HepG2 cells incubated for 6 h in the indicated medium. Discussion The transcriptional regulation of MnSOD in response to cytokine induction has been well studied. The data presented here establish a mechanism of transcriptional regulation by amino acid deprivation through forkhead binding proteins. Previous data has suggested the role of forkhead binding proteins and MnSOD through overexpression studies [170,189]. Analysis of the human MnSOD promoter with a growth hormone reporter plasmid resulted in delineating a 1.4 kb responsive promoter fragment, although deletion of this promoter fragment to 1.3 kb resulted in a loss of the responsiveness to histidine

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96 deprivation. Further analysis of this 100 base pair region revealed a functional forkhead binding site that was responsive to histidine deprivation, and deletion of this site from the reporter construct blocked the induction seen with histidine deprivation. Addition of the enhancer region did increase the overall expression level of the growth hormone message, however did not change the relative fold induction. Data to support that the enhancer is not involved in this regulation was further demonstrated by use of the heterologous TK promoter. However, deletion of the forkhead binding site blocked the induction seen with histidine deprivation in both constructs, demonstrating the importance of this DNA binding site to the induction of MnSOD by amino acid deprivation. Overexpression of either FOXO3a or FOXO1 along with the 1.3 kb promoter construct resulted in increased expression levels of the reporter plasmid, further implicating a role for these transcription factors in the regulation of MnSOD. However, overexpression studies do not link a stimulus to the regulation of these transcription factors. To help establish a connection between amino acid deprivation and the regulation of these forkhead binding proteins, studies evaluating the forkhead signaling pathway were employed. Under normal cellular conditions, FOXO proteins are thought to be localized in the cytoplasm, and this regulation is orchestrated through the protein kinase B (PKB). Under conditions of nutrient withdrawal, PKB is inactive and FOXO proteins translocate to the nucleus regulating the transcription of target genes. To evaluate the role of FOXO proteins in the regulation of MnSOD, two different conditions regulating the activity of PKB were used. Co-treatment of histidine deprived cells with insulin, a stimulus known

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97 to activate PKB and therefore retain FOXO proteins in the cytoplasm, caused a decrease in MnSOD mRNA levels [191]. Similarly, treatment with LY294002, a compound known to block PKB activity, caused an induction of MnSOD mRNA levels [175,176]. These data provide a link between amino acid deprivation, the regulation of FOXO proteins and the regulation of MnSOD gene expression. Furthermore, the mRNA levels for FOXO3a were elevated in response to histidine deprivation, implicating a level of transcriptional regulation previously not established. siRNA to FOXO3a was the used to further evaluate its role in the regulation of MnSOD in response to histidine derivation. The results from these experiments demonstrated that FOXO3a may not be the FOXO transcription factor responsible for the induction of MnSOD. Furthermore, a comparison of the localization of the FOXO proteins provides data supporting a role for FOXO1 in the regulation of MnSOD.

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CHAPTER 6 DISCUSSION AND FUTURE DIRECTIONS Discussion Amino acid dependent regulation of gene expression plays a significant role in cellular growth and metabolism. Amino acid deprivation causes a specific increase in the regulation of protective genes while decreasing global protein synthesis in an effort to maintain proper amino acid levels. The data presented here provide evidence of an amino acid dependent control of the cytoprotective, mitochondrial localized, free radical scavenger, MnSOD. Initial studies were aimed at identifying a potential role for MnSOD in response to amino acid deprivation. These early studies revealed that MnSOD mRNA and protein levels were elevated by amino acid deprivation, specifically in response to essential amino acids, with the exception of tryptophan. Interestingly, analysis of other nutrients, such as glucose, serum, IGF, EGF and vitamins, demonstrated that they had no effect on MnSOD levels. This was unexpected because other genes known to be regulated by amino acids can also be regulated by changes in other nutrients levels. Another unanticipated result was that total amino acid deprivation did not lead to the induction of MnSOD mRNA levels. This is contrary to effects on other amino acid regulated genes, such as ASNS [1,103], which are regulated by depletion of a single or total amino acid depletion as well as glucose deprivation. These data led to further analysis of the components required for the induction of MnSOD, revealing glutamine as a necessary component in this induction. 98

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99 ASNS has been well characterized in the liver, and, as such, liver-derived cell types initially examined. Two liver cell lines, HepG2 and HUH7, were tested and responded similarly in response to amino acid deprivation. Several other cell lines were also tested, but, in these cases, MnSOD mRNA levels were not elevated in response to amino acid deprivation. Given the requirement of glutamine for the induction of MnSOD and the localization to the mitochondria of this enzyme, the contribution of the TCA cycle to the gene regulation of MnSOD was evaluated. To test this, several inhibitors blocking various steps of the TCA cycle were used and the mRNA levels of MnSOD were evaluated. This revealed a requirement for the signaling through the TCA cycle. These results support the requirement of glutamine in the induction of MnSOD by histidine deprivation. A direct effect of the TCA cycle is the production of reducing units used to drive the electron transport chain, ultimately leading to the production of cellular energy in the form of ATP. Therefore, the contribution of these products to the induction of MnSOD by amino acid deprivation was evaluated, revealing a requirement of the electron transport chain and a functional mitochondrial membrane potential but no dependency on ATP levels. To further characterize the regulation of MnSOD, potential signaling pathways were also evaluated. Given that MnSOD is not induced in response to total amino acid deprivation, the classic GCN2 pathway does not seem to be responsible for the transcriptional activation of MnSOD. Therefore, other relevant signaling pathways were evaluated. As previously discussed, a major stress signaling pathway is the mitogen activated protein kinase (MAP) pathway. Several inhibitors of this pathway were utilized

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100 and revealed a requirement for ERK1/2 signaling in the induction of MnSOD. Furthermore, this also revealed a requirement for the mTOR signaling pathway. Further analysis of the mTOR pathway, utilizing the inhibitor rapamycin, demonstrated a separation of the histidine deprivation and the requirement of glutamine in the induction of MnSOD. As previously discussed, amino acid deprivation has been demonstrated to regulate a number genes through different mechanisms including transcription, translation and mRNA stability [1]. Analysis of the human MnSOD promoter with a growth hormone reporter construct provided data demonstrating a 1.4kb responsive promoter fragment, indicating a transcriptionally based mechanism for the induction of MnSOD by amino acid deprivation. Analysis of the amino acid responsive human promoter led to the identification of a forkhead binding site which, when deleted, resulted in loss of the responsiveness to histidine deprivation. The forkhead binding site is a consensus sequence for the FOXO family of transcription factors. Overexpression of either FOXO3a or FOXO1 with the human MnSOD promoter resulted in increased expression levels, implicating a transcriptional role for these transcription factors. Studies evaluating the forkhead signaling pathway supported a role for the FOXO proteins in the regulation of MnSOD. To identify which FOXO transcription factor is responsible for the induction, an siRNA to FOXO3a was used. The results from these experiments did not block the induction of MnSOD in response to amino acid deprivation. However, data demonstrating the localization of the FOXO proteins support a role for FOXO1 in the regulation of MnSOD.

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101 In summary, amino acids are important to protein synthesis, gene regulation and overall homeostasis of the body. The results presented here provide data supporting a novel regulatory mechanism of MnSOD through the metabolic control of amino acids. The relevant amino acid signal transduction pathways and the involvement of potential transcription factors in the regulation of MnSOD are also characterized. Future Directions The data presented here demonstrate that MnSOD is induced in response to essential amino acid deprivation, with a requirement for the non-essential amino acid glutamine. The characterization of MnSOD gene induction in response to amino acid deprivation provides a solid foundation for the continuation of this project. One avenue of pursuit is to evaluate the protective nature of MnSOD in relation to amino acid deprivation and cell survival. Previous studies have demonstrated the importance of this enzyme by overexpression studies conferring a level of protection in response to IL-1, TNF, chemotherapeutic drugs, hyperoxia, ionizing radiation and injury [25-29]. Furthermore, data have also implicated MnSOD in aging, where loss of this enzymes activity leads to the premature accumulation of age related diseases and also mitochondrial dysfunction. Amino acid deprivation, through caloric restriction, has also been associated with an increase in life span [85]. Due to the protective properties of MnSOD, correlative data have supported a role for MnSOD in conferring increase in life span [86,161]. One example is the increase of MnSOD mRNA and protein levels in C. elegans and the associated increase of life span by as much as twice that with low levels of this enzyme [166].

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102 Determining the beneficial effects of MnSOD induction through amino acid deprivation is a very interesting area of research. Linking amino acid deprivation directly to cell survival through MnSOD may help to provide evidence for the mechanisms underlying increased life span in response to caloric restriction. To evaluate the contribution of MnSOD to cell survival, experiments must be designed to reduce the expression of MnSOD and evaluating cell survival by utilizing pro-apoptotic markers, growth curves and production of ROS. One useful approach is through the use of siRNA against MnSOD to reduce its expression levels, incubating cells in the various medium conditions and then comparing the survival of these cells to those not transfected with the siRNA. Another area of interest is the contribution of glutamine to the signaling of MnSOD. The data presented here demonstrated the importance of glutamine to the induction of MnSOD and established a correlative link between the TCA cycle and a functional mitochondrial membrane potential. It would be interesting to determine if the media conditions established for the induction of MnSOD would cause a similar induction to other mitochondrial associated or cell survival proteins such as glutathione peroxidase or BCL2. Metabolic signals from the mitochondria, through retrograde signaling, regulate the transcription of several nuclear encoded genes essential to the mitochondria [192,193]. It would be interesting to determine if amino acid deprivation, through a similar mechanism used for MnSOD, would have an effect on the gene regulation of the TCA cycle enzymes. Research in yeast has demonstrated that the regulation of several of the genes encoding for the components of the TCA cycle are altered when the enzymes comprising

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103 this cycle are blocked through the use of mutants [194]. Specifically, these researchers noted that increased mitochondrial dysfunction resulted in a decrease in the production of genes encoding for oxidative respiration. This correlates well with the data presented in Chapter three, indicating that the signals required for MnSOD induction include those from the TCA cycle and electron transport chain mediated through an intact mitochondrial membrane potential. Another area of future studies lies in further understanding the regulation of FOXO proteins by amino acid deprivation. The data presented here demonstrate that FOXO1 and FOXO3a proteins can induce the MnSOD promoter when overexpressed. However, the data also demonstrate that FOXO3a is located in the cytosol whereas FOXO1 is sequestered in the nucleus. Although overexpression studies are valuable, the regulation of the endogenous genes needs to be explored. Chromatin immunoprecipitation analysis would be valuable to understanding the regulation of this protein in context with MnSOD. Furthermore siRNA to FOXO1 would help to demonstrate if this protein is involved in the regulation of MnSOD. Understanding the role of endogenously regulated FOXO proteins may have its limitations. To overcome this, overexpression of the unmodified protein may add insight to their regulation. By overexpressing these proteins and incubating in the various media, localization, transcriptional activation and possibly ChIP may be able to elucidate the mechanisms regulating these proteins. Furthermore, recent studies have implicated a role for the acetylation modulating the activity of the FOXO proteins [195-197]. Brunet et al.[195] have recently demonstrated that that, in response to oxidative stress, SIRT1 caused the deacetylation of the FOXO transcription factors. The deacetylation of this protein increased the cells

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104 ability to resist cell death. It would be interesting to determine if amino acid deprivation would have an effect on the acetylation status of these proteins and their affect on MnSOD gene expression. Additionally, determining if there is a coordination of gene regulation by amino acid deprivation mediated through the ERK1/2 MAP kinase pathway, and possibly mTOR, to the regulation of FOXO proteins may help to further our understanding of the underlying mechanisms of gene regulation of MnSOD.

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BIOGRAPHICAL SKETCH Kimberly Jean Aiken was born on February 27, 1977, to Robert and Jane Aiken in St. Albans Vermont. In 1988, her family moved to Boca Raton, Florida where she eventually attended Spanish River High School. In 1995, she began her undergraduate studies at the University of Florida in microbiology. While at the University of Florida, she gained an interest in research and began volunteering in Dr. Laurence Morels laboratory. This volunteer position led to working in the laboratory earning research credits toward her degree. In December of 1999, she graduated with a Bachelor of Science degree from the College of Liberal Arts and Sciences at the University of Florida. After graduation, she continued working in Dr. Morels laboratory as a full time laboratory technician and decided to continue her education by joining the Interdisciplinary Doctoral Program (IDP) for her graduate studies at the University of Florida. In 2001, she entered into the doctoral program and in 2002, joined the laboratory of Dr. Harry Nick. 121