Detection of NADPH oxidase subunits NOX1 and NOX4 in lung adenocarcinoma A549 cells, and impact of NOX1 on Nrf2- and HIF-1-dependent gene regulation [Elektronische Ressource] / by Malec, Viktor
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Detection of NADPH oxidase subunits NOX1 and NOX4 in lung adenocarcinoma A549 cells, and impact of NOX1 on Nrf2- and HIF-1-dependent gene regulation [Elektronische Ressource] / by Malec, Viktor

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139 Pages
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Detection of NADPH oxidase subunits NOX1 and NOX4 in lung adenocarcinoma A549 cells, and impact of NOX1 on Nrf2- and HIF-1- dependent gene regulation Inaugural Dissertation submitted to the Faculty of Medicine in partial fulfillment of the requirements for the PhD-Degree of the Faculties of Veterinary Medicine and Medicine of the Justus Liebig University Giessen by Malec, Viktor born in Liberec, Czech Republic Giessen 2010 From the Department of Internal Medicine II Director: Prof. Dr. Werner Seeger of the Faculty of Medicine of the Justus Liebig University Giessen First Supervisor and Committee Member: PD Dr. Jörg Hänze Second Supervisor and Committee Member: Prof. Dr. Thomas Kietzmann Committee Members: Prof. Dr. Ernst Petzinger Prof. Dr. Andre Menke thDate of Doctoral Defense: 30 March 2010 Table of content I. Table of content……………………………………………………………….1 II. List of figures…………………………………………………….……………4 III. Table of abbreviations…………………………………………………………6 I. Table of content 1. Introduction................................................................................................................. 9 1.1 NADPH oxidase........................................................................................................ 9 1.1.

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Detection of NADPH oxidase subunits NOX1 and NOX4 in lung
adenocarcinoma A549 cells, and impact of NOX1 on Nrf2- and HIF-1-
dependent gene regulation





Inaugural Dissertation
submitted to the
Faculty of Medicine
in partial fulfillment of the requirements
for the PhD-Degree
of the Faculties of Veterinary Medicine and Medicine
of the Justus Liebig University Giessen


by
Malec, Viktor
born in
Liberec, Czech Republic




Giessen 2010







From the Department of Internal Medicine II
Director: Prof. Dr. Werner Seeger
of the Faculty of Medicine of the Justus Liebig University Giessen










First Supervisor and Committee Member: PD Dr. Jörg Hänze
Second Supervisor and Committee Member: Prof. Dr. Thomas Kietzmann
Committee Members: Prof. Dr. Ernst Petzinger
Prof. Dr. Andre Menke






thDate of Doctoral Defense: 30 March 2010
Table of content
I. Table of content……………………………………………………………….1
II. List of figures…………………………………………………….……………4
III. Table of abbreviations…………………………………………………………6

I. Table of content
1. Introduction................................................................................................................. 9
1.1 NADPH oxidase........................................................................................................ 9
1.1.1 Functions of NADPH oxidases and NOX subunits ........................................... 9
1.1.2 Subunits of NADPH oxidases.......................................................................... 11
1.1.2.1 NOX1 and NOX4 ..................................................................................... 11
NOX1................................................................................................................ 11
NOX4................................................................................................................ 13
1.1.2.2 p22phox, NOXO, NOXA, p40phox, Rac ................................................. 14
p22phox............................................................................................................. 14
NOXO............................................................................................................... 14
NOXA............................................................................................................... 14
p40phox............................................................................................................. 15
Rac GTPase....................................................................................................... 15
1.1.2.3 Assembling and activation of NADPH oxidase subunits ......................... 15
1.2 Reactive oxygen species ......................................................................................... 18
1.2.1 Types of ROS, oxidative stress and antioxidants............................................. 18
1.2.2 Cellular sources of ROS................................................................................... 19
1.2.3 Mechanisms of ROS impact on cell signaling................................................. 19
1.2.4 ROS, oxygen sensing and hypoxia inducible factor 1 ..................................... 20
1.2.4.1 HIF-1 is a target of oxygen sensors .......................................................... 20
1.2.4.2 Regulation of HIF by the oxygen sensors prolyl 4-hydroxylase and
asparaginyl hydroxylase........................................................................................ 21
1.2.4.3 Impact of reactive oxygen species on HIF regulation .............................. 22
1.3 Nuclear factor (erythroid-derived 2)-like 2............................................................. 24
1.3.1 Regulation of Nrf2 stability and activity ......................................................... 24
1.3.2 Nrf2 target genes.............................................................................................. 25
1.4 Thioredoxin system................................................................................................. 27
1.4.1 Thioredoxin...................................................................................................... 27
1.4.2 Thioredoxin reductase...................................................................................... 28
1.4.3 Function of the thioredoxin system.................................................................. 29
2. Aims of the work ..................................................................................................... 30
Part I.............................................................................................................................. 30
Part II ............................................................................................................................ 30
3. Materials and Methods........................................................................................... 32
3.1 Materials ................................................................................................................. 32
3.1.1 Materials and instruments................................................................................ 32
3.1.2 Chemical agents............................................................................................... 32
3.1.3 Buffers.............................................................................................................. 33
3.1.4 Antibodies........................................................................................................ 34
3.1.5 Oligonucleotide sequences............................................................................... 34
1
3.1.6 siRNAs............................................................................................................. 35
3.1.7 Plasmids ........................................................................................................... 35
3.1.8 Cell culturing ................................................................................................... 36
3.1.8.1 Bacteria, mediums for bacteria culturing and transformation .................. 36
3.1.8.2 Mammalian cells and cell culturing mediums .......................................... 36
3.2 Methods................................................................................................................... 37
3.2.1 Biochemical methods....................................................................................... 37
3.2.1.1 Subcellular fractionation........................................................................... 37
A) Isolation of cytosol, nuclei, membranes and cytoskeleton .......................... 37
B) Separation of Tx-100 soluble and insoluble fraction................................... 38
C) Isolation of plasma membrane..................................................................... 38
D) Isolation of nuclei ........................................................................................ 38
E) Subcellular fraction of samples for HPLC................................................... 38
3.2.1.2 Protein concentration measurement.......................................................... 39
3.2.1.3 Western blot.............................................................................................. 39
3.2.1.4 HPCL protein purification ........................................................................ 40
A) Anion exchange chromatography................................................................ 40
B) Reverse phase chromatography ................................................................... 41
3.2.1.5 2D gel electrophoresis............................................................................... 41
3.2.1.6 MALDI-TOF MS...................................................................................... 42
3.2.1.7 Reactive oxygen species measurement..................................................... 43
3.2.1.8 Luciferase activity..................................................................................... 44
3.2.2 Molecular biological methods.......................................................................... 44
3.2.2.1 Transformation of bacteria and isolation of plasmid ................................ 44
3.2.2.2 RNA isolation, reverse transcription and real-time PCR.......................... 44
3.2.2.3 DNA agarose gel electrophoresis.............................................................. 45
3.2.2.4 RNA interference by synthetic siRNA ..................................................... 46
3.2.2.5 Plasmids .................................................................................................... 46
3.2.3 Cell biological methods ................................................................................... 46
3.2.3.1 Cell culturing under different oxygenation conditions ............................. 46
3.2.3.2 Cell transfection by siRNA and plasmid .................................................. 47
4. Results ........................................................................................................................ 48
4.1 Detection of NOX1 and NOX4 proteins, and analysis of NOX1- and NOX4-
dependent ROS generation ........................................................................................... 48
4.1.1 Expression of NOX1 and NOX4 mRNA in A549 cells .................................. 48
4.1.2 Schema of binding sites of different NOX1 and NOX4 antibodies to NOX1
and NOX4 proteins ................................................................................................... 49
4.1.3 Detection of putative NOX1 and NOX4 proteins by different antibodies in
total cell lysates......................................................................................................... 50
4.1.3.1 NOX1 antibodies (A549 versus CaCo2)................................................... 50
4.1.3.2 NOX4 antibodies (A549 versus HUVEC)................................................ 51
4.1.4 Detection of putative NOX1 and NOX4 proteins, p22phox and NOXO1 in cell
compartments............................................................................................................ 52
4.1.4.1 NOX1 in cytosol, membrane, nuclei and cytoskeleton............................ 52
4.1.4.2 NOX4 in cytosol, membrane, nuclei oskeleton............................ 53
4.1.4.3 p22phox and NOXO1 in cytosol, membrane, nuclei and cytoskeleton... 53
2
4.1.5 Detection of putative NOX1 and NOX4 proteins, p22phox and NOXO1 in Tx-
100 soluble and insoluble fraction ............................................................................ 55
4.1.5.1 NOX1 in Tx-100 soluble and insoluble fraction...................................... 55
4.1.5.2 NOX4 in Tx-100 sond insoluble fraction...................................... 56
4.1.5.3 p22phox and NOXO1 in Tx-100 soluble and insoluble fraction.............. 56
4.1.6 Detection of putative NOX1, NOX4 proteins and p22phox in the plasma
membrane of A549 cells ........................................................................................... 58
4.1.7 Knock-down of NOX1 and NOX4 in A549 cells by siRNA........................... 59
4.1.8 Interaction of NOX1wch and NOX4jh antibodies with recombinant NOX1 and
NOX4 proteins.......................................................................................................... 61
4.1.9 Localization of NOX1, NOX4, p22phox and NOXO1 in cell nuclei.............. 62
4.1.10 Regulation of NOX1 and NOX4 stability by p22phox.................................. 64
4.1.11 Purification of putative NOX1 and NOX4 proteins ...................................... 66
4.1.11.1 Strategy of purification ........................................................................... 66
4.1.11.2 Homogenization and fractionation.......................................................... 67
4.1.11.3 Chromatographic or 2D gel electrophoretic protein purification for
MALDI-TOF MS.................................................................................................. 68
4.1.12 Effect of NOX1 and NOX4 knock-down on ROS generation in A549 cells 76
4.1.13 Expression of NOX1 and NOX4 under normoxic and hypoxic conditions... 77
4.1.14 NOX1-derived ROS generation under normoxic and hypoxic conditions .... 78
4.2 Cross-talk between NOX1 and redox state-dependent transcription factors Nrf2 and
HIF-1 in intermittent hypoxia ....................................................................................... 79
4.2.1 Regulation of HIF-1α and Nrf2 under continuous long term and intermittent
hypoxia...................................................................................................................... 80
4.2.2 Regulation of HIF-1α, Nrf2, Trx1 and NOX1 expression in A549 cells treated
by different alternating hypoxic and normoxic intervals.......................................... 81
4.2.3 Reactive oxygen species in A549 cells treated by different alternating hypoxic
and normoxic intervals.............................................................................................. 83
4.2.4 Regulation of Nrf2 by NOX1 .......................................................................... 84
4.2.5 Regulation of Trx1 expression by NOX1 ........................................................ 86
4.2.6 Regulation of Nrf2 and Trx1 expression by NOX1 under conditions of
hypoxia-reoxygenation and intermittent hypoxia ..................................................... 87
4.2.7 Regulation of HIF-1α by Nrf2, Trx1 and NOX1 under conditions of
continuous and intermittent hypoxia......................................................................... 88
4.2.8 Signaling pathway of NOX1 and Trx1 causing induction of HIF-1α in
intermittent hypoxia.................................................................................................. 90
5. Discussion ................................................................................................................. 92
5.1 Detection of NOX1 and NOX4 proteins and analysis of NOX1- and NOX4-
dependent ROS generation ........................................................................................... 92
5.1.1 Detection and analysis of cellular localization of NOX1 and NOX4 proteins
using different antibodies.......................................................................................... 92
5.1.1.2 NOX1........................................................................................................ 92
NOX1wch ......................................................................................................... 93
NOX1ab55831 .................................................................................................. 96
MOX1 H-15...................................................................................................... 96 75...................................................................................................... 96
3
NOX1jh............................................................................................................. 97
mAb54.1............................................................................................................ 97
5.1.1.3 NOX4........................................................................................................ 98
NOX4jh............................................................................................................. 98
NOX4 N-15 & NOX4 H-300............................................................................ 99
5.1.2 Detection of p22phox and NOXO1 proteins in different cell compartments 100
5.1.3 Effect of p22phox knock-down on NOX1 and NOX4 stability .................... 101
5.1.4 Purification of putative NOX1 and NOX4 proteins ...................................... 101
5.1.5 Contribution of NOX1 and NOX4 to ROS generation in A549 cells............ 104
5.2 Regulation and cross-talk of NOX1, Nrf2, Trx1 and HIF-1α under different
oxygenation conditions ............................................................................................... 105
5.2.1 NOX1, NOX4, HIF-1α and Nrf2 expression, and NOX1-dependent ROS
generation under different hypoxic conditions ....................................................... 106
5.2.2 Intermittent hypoxia and its relevance for physiological and
pathophysiological conditions ................................................................................ 106
5.2.2.1 Regulation of HIF-1α, Nrf2, Trx1 and NOX1 expression, and NOX1-
dependent ROS generation in A549 cells treated by different alternating hypoxic
and normoxic intervals........................................................................................ 107
5.2.2.2 Regulation Nrf2 and Trx1 expression by NOX1 .................................... 109
5.2.2.3 Trx1 mediated regulation of HIF-1α by Nrf2 in NOX1-dependent manner
............................................................................................................................. 110
6. Summary.................................................................................................................. 113
7. Zusammenfassung................................................................................................. 115
8. References ............................................................................................................... 118
9. Appendix ................................................................................................................. 136
9.1 Acknowledgments................................................................................................. 136
9.2 Curiculum Vitae.................................................................................................... 137

II. List of figures
Figure 1: Activation and organization of NADPH oxidase subtypes............................... 10
Figure 2: Proposed organization of NADPH oxidase subunits ........................................ 16
Figure 3: Activation of NADPH oxidase 2....................................................................... 17
Figure 4: Regulation of HIF-1α in hypoxia...................................................................... 22
Figure 5: ROS in oxygen sensing ..................................................................................... 24
Figure 6: Mechanisms of Nrf2 activation ......................................................................... 26
Figure 7: Dual role of Nrf2 in cancerogenesis.................................................................. 28
4
Figure 8: Expression of NOX1 and NOX4 mRNA in A549 cells.................................... 49
Figure 9: Binding sites of NOX1 and NOX4 antibodies .................................................. 50
Figure 10: Detection of putative NOX1 and NOX4 proteins in total cell lysates
employing different NOX1 and NOX4 antibodies ................................................... 51
Figure 11: Detection of putative NOX1 and NOX4 proteins, p22phox and NOXO1 in
different cell compartments ...................................................................................... 55
Figure 12: Detection of putative NOX1 and NOX4 proteins, p22phox and NOXO1 in Tx-
100 soluble and insoluble fractions........................................................................... 58
Figure 13: Detection of putative NOX1, NOX4 proteins and p22phox in plasma
membrane of A549 cells ........................................................................................... 59
Figure 14: Knock-down of NOX1 and NOX4 in A549 cells by siRNA .......................... 60
Figure 15: Interaction of NOX1wch and NOX4jh antibodies with recombinant NOX1
and NOX4 proteins ................................................................................................... 62
Figure 16: Localization of NOX1, NOX4, p22phox and NOXO1 in cell nuclei ............. 64
Figure 17: Regulation of NOX1 and NOX4 stability by p22phox ................................... 65
Figure 18: Schema of purification strategy for the identification of putative NOX1 and
NOX4 proteins.......................................................................................................... 67
Figure 19: Homogenization and fractionation of A549 cells and HUVEC...................... 68
Figure 20A-H: Purification of proteins by chromatography or 2D gel electrophoresis for
further analysis by MALDI-TOF MS....................................................................... 69
Figure 21: Effect of NOX1 and NOX4 knock-down by siRNAs on the ROS generation in
A549 cells ................................................................................................................. 77
Figure 22: Expression of NOX1 and NOX4 under normoxic and hypoxic conditions.... 78
Figure 23: Total ROS and NOX1-derived ROS levels under normoxic and hypoxic
conditions.................................................................................................................. 79
Figure 24: Regulation of HIF-1α and Nrf2 under continuous long term and intermittent
hypoxias .................................................................................................................... 81
Figure 25: Regulation of HIF-1α, Nrf2, Trx1 and NOX1 expression in A549 cells treated
by different alternating hypoxic and normoxic intervals.......................................... 83
Figure 26: Total ROS and NOX1-derived ROS levels in A549 cells treated by different
alternating hypoxic and normoxic intervals.............................................................. 84
5
Figure 27: Regulation of Nrf2 by NOX1 in A549 cells.................................................... 86
Figure 28: Regulation of Trx1 expression by NOX1 in A549 cells ................................. 87
Figure 29: Regulation of Nrf2 and Trx1 expression by NOX1 under conditions of
hypoxia-reoxygenation and intermittent hypoxia ..................................................... 88
Figure 30: Regulation of HIF-1α by Nrf2, Trx1 and NOX1 under conditions of
continuous and intermittent hypoxia......................................................................... 89
Figure 31: Signaling pathway of NOX1 and Trx1 causing the induction of HIF-1α after
intermittent hypoxia exposure................................................................................... 91
Figure 32: Schematic diagram of the regulation of HIF-1α by NOX1, Nrf2 and Trx1
during intermittent hypoxia exposure of A549 cells................................................. 91

II. Table of abbreviations
AA amino acid DGR double glycine repeat
ACN acetonitrile DMSO dimethylsulfoxide
AEC anion exchange chromatography dNTP deoxyribose nucleotide triphosphate
AIR autoinhibitory region ddNTP di-deoxyribose nucleotide triphosphate
Akt corresponds to PKB (protein kinase B) DPI diphenyleneiodonium chloride
(homologue of v-Akt)
Ang II angiotensin 2 dsDNA double stranded DNA
AP-1 activator protein-1 DTT dithiothreitol
ARE antioxidant response element Duox dual oxidase
ARNT aryl hydro-carbon nuclear translocator ECL enhanced chemiluminescence
ASK1 apoptosis signal-regulating kinase 1 EDTA ethylendinitrilo-N,N,N’,N’,-tetra-acetate
ATCC American Type Culture Collection EFP1 EF-hand binding protein 1
ATF-1 activating transcription factor 1 EGF epidermal growth factor
bHLH basic helix-loop-helix ER endoplasmic reticulum
BSA bovine serum albumin ERK extracellular signal-regulated kinase
bp base pair ETC electron transport chain
C cytosol FAD flavin adenine dinucleotide
CaCo2 colorectal carcinoma (cells) FCS fetal calf serum
CBB Coomassie Brilliant Blue FIH factor-inhibiting HIF-1
CBP CREB-binding protein FT flow through
cDNA complementary deoxyribonucleic acid GATA6 GATA (nucleotide sequence), (GATA
transcription factor 6)
Csk cytoskeleton GDP guanosine-5'-diphosphate
C-TAD C-terminal transactivation domain gp91phox glycoprotein-91kDa-phagocytic-oxidase
Cul cullin Gth glutathione
6
GTP guanosine-5'-triphosphate NADPH nicotinamide adenine dinucleotide
phosphate
H2 2 h of hypoxia NF- κB nuclear factor kappa-light-chain-enhancer
of activated B cells
H6 6 h of hypoxia NIH3T3 3-day transfer, inoculum 3 x 105 cells
H2R H2 followed by 2 h of reoxygenation NOX NADPH oxidase
HA hemagglutinin NOX1v NOX1variant
HIF-1 hypoxia inducible factor 1 NOXA NOX activator protein
HPLC high performace liquid chromatography NOXO NOX organizer protein
HRE hypoxia response element NQO1 NADPH quinone-oxido-reductase
HRP horseradish peroxidase Nrf2 nuclear factor (erythroid-derived 2)-like 2
HUVEC human umbilical vein endothelial cells N-TAD N-terminal transactivation domain
IEF isoeletric focusing Nu nuclei
IH4 IH (2x2 h of hypoxia interupted with NuInslear insoluble
2 h of rexygenation)
IH6 IH (3x2 h of hypoxia interupted with NuSol nuclear soluble
2x2 h of rexygenation)
IH4R IH (IH4 followed by 2 h of rexygenation) OD optical density
IKK I κB kinase (I κB is inhibitor of kappa B) ODDD oxygen-dependent degradation domain
IP3 inosiotol triphosphate p pellet
IPG immobilized pH gradient PAGE polyacryl-amid gel electrophoresis
JNK c-jun N-terminal kinase PAS Per, ARNT, SIM
kb kilo base PB1 phox-Bem1
kDa kilo Dalton PBS phosphate buffer saline
Keap1 Kelch-like ECH-associated protein 1 PCR polymerase chain reaction
Km Michaelis constant PDGF platelet-derived growth factor
LB Luria Bertani PGF2 α prostaglandin F2 α
Luc luciferase PGK phosphoglycerate kinase
mAb monoclonal antibody Phox phagocyte oxidase
MAF avian musculoaponeurotic fibrosarcoma PHD prolyl hydroxylase
(protooncogene)
MAPK mitogen-activated protein kinase PI3K phosphatidylinositol 3-kinase
MALDI- matrix assisted laser desorption/ionisation- PKC protein kinase C
TOF time of flight
Me membrane Pl.Me. plasma membrane
MEK MAPK/ERK kinase p"x"phox protein "x kDa" phox (e.g.p67phox,
p47phox)
mRNA messenger RNA PtdInsP phosphatidylintositol phosphate
MS mass spectrometry PVDF polyvinylidene fluoride
mTOR mammalian target of rapamycin pVHL von Hippel-Lindau protein
M-MuLV moloney murine leukemia virus PX phox homology
Mw molecular weight Ref-1 redox factor 1
N normoxia RhoA Ras homolog gene family, member A


7
RhoGDI RhoGDP-dissociation inhibitor
RNA ribonucleic acid
RNase ribonuclease
ROS reactive oxygen species
Rpm rotations per minute
RPC reverse phase chromatography
RT room temperature
RT- reverse transcription-PCR
PCR
s supernatant
SDS sodium dodecyl sulfate
SEM standard error of the mean
SH3 Src homology 3
siRNA small interfering RNA
SOC super optimal broth with catabolite repression
STAT signal transducers and activators of
transcription
TAD transactivation domain
TEMED N’,N’,N’,N’-Tetra methyl diamine
TFA trifluoroacetic acid
T.Me. total membrane
TPR tetratricopeptide repeat
Trx thioredoxin
TrxR thioredoxin reductase
TSR template suppression reagent
Tx-100 Triton X-100
UV ultra violet
VEGF vascular endothelial growth factor
VHL von Hippel Lindau
VSMC vascular smooth muscle cells
2D-GE two dimensional gel electrophoresis










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