Uncoupling protein-2 accumulates rapidly in the inner mitochondrial membrane during

216 Pages·2012·2.47 MB·English

Uncoupling protein-2 accumulates rapidly in the
inner mitochondrial membrane during mitochondrial
reactive oxygen stress in macrophages

Tindaro Matthew Giardina

This thesis is presented for the degree of Doctor of Philosophy of
The University of Western Australia,
School of Medicine and Pharmacology

December 2011
Uncoupling protein-2 accumulates rapidly in the inner mitochondrial membrane
during reactive oxygen stress in macrophages
ABSTRACT

UCP2 (Uncoupling protein 2) belongs to the family of mitochondrial carrier proteins.
It is expressed in the inner mitochondrial membrane (IMM) and is a known regulator
of mitochondrial function. UCP2 is best characterized as a regulator of reactive
oxygen species (ROS) during electron transport in the IMM.

Macrophages exist in various tissue environments and represent many cell types
including tissue macrophages, inflammatory macrophages, dendritic cells and
osteoclasts (Gordon et al., 2005). These various cell types have specialisation that
reflects the tissue environment that they populate or are recruited to. Recruitment of
macrophages to inflammatory tissue sites for example is associated with the
production of intracellular ROS as an important host defense mechanism against
pathogenic organisms that can find sanctuary intracellulary (Arsenijevic et al., 2000).
Production of ROS may have more defined roles in cell signaling, regulation and
recruitment (Jackson, 2002). There is limited information of how ROS and in
particular the type of ROS species affect the various points of UCP2 regulation
(including protein synthesis, gene transcription, mRNA abundance and protein
degradation rate) in macrophages. Understanding the points of regulation of UCP2 in
macrophages is important clinically in disease states including atherosclerosis and
ischemic insult/ re-perfusion injury where ROS elevation plays a role in progression
of these disease states. The presence of UCP2 mRNA, but not protein, in many non-
macrophage tissues then suggests that similar regulation may be available beyond
macrophage-lineage cells, allowing UCP2 protein expression, under conditions of
mitochondrial oxidative stress, and perhaps other environmental stresses.

This project found in a primary and cell line (RAW ) macrophage model that
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UCP2 protein abundance was increased specifically by mitochondrial generated
superoxide (O •-) and not by extra-mitochondrial cellular generated O •- or hydrogen
2 2
peroxide (H O ) (Chapter 2). The nitric oxide (NO) generator SNAP similarly
2 2
increases UCP2 protein expression (Chapter 2).

The induction of UCP2 protein is not due to increased UCP2 mRNA levels, nor
i
Uncoupling protein-2 accumulates rapidly in the inner mitochondrial membrane
during reactive oxygen stress in macrophages
increased transcription of the Ucp2 promoter (Chapter 3). In contrast, when protein
synthesis was halted with cycloheximide, UCP2 protein decayed at the same rate in
unstressed cells as cells experiencing mitochondrial oxidative stress (after exposure to
diethyldithiocarbamate (DETC), an inhibitor of mitochondrial superoxide
dismutatases, antimycin A (AA) or rotenone (Chapter 3), indicating that UCP2
induction is not a consequence of protein stabilization. UCP2 accumulated under
mitochondrial oxidative stress, suggesting that expression is regulated at translational
or post-translational steps induced by i) DETC and ii) AA (Chapter 3). The
availability of glutamine is described in the literature to permit UCP2 translation.
However, results presented here from in vitro studies in macrophages show that UCP2
translation was initiated with glutamine levels higher than that found in ischaemic
tissues (Chapter 5).

Experiments investigating the turnover of UCP2 protein show that inhibition of the
cytosolic 26S proteasome did not increase UCP2 protein abundance (Chapter 3), so in
this model it is not a substrate for the extramitochondrial proteolytic pathway.
Therefore, UCP2 protein degradation is probably achieved through other, as yet
unidentified pathways.

Ectopically expressed UCP2 has well-described roles in the regulation of
mitochondrial ROS and function. UCP2 transiently expressed in HEK 293 cells
localizes to mitochondria, and reduces ROS levels and mitochondrial membrane
potential ( ) and is consistent with increased UCP2 protein abundance in response
m
to mitochondrial O •- production, as in macrophages (Chapter 4). Further, UCP2
2
upregulation through exogenous application of glutamine also reduces ROS
production (Chapter 4). Similarly the antioxidant butylated hydroxy anisole (BHA)
lowers ROS production and negatively regulates UCP2 protein expression (Chapter
4). Acrolein, a ROS-derived product of lipid peroxidation that has been linked to
activation of UCP2 function elsewhere, can similarly reduce ROS levels and  in
m
RAW macrophages, without altering UCP2 abundance (Chapter 4). These data
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highlights the relationship between ROS levels and UCP2 protein expression.

Other physiological processes of increasing endogenous ROS in macrophages
ii
Uncoupling protein-2 accumulates rapidly in the inner mitochondrial membrane
during reactive oxygen stress in macrophages
including hypoxia likewise increase UCP2 protein expression (Chapter 4 and Chapter
5). Regulation by hypoxia is similar to that of mitochondrial O •-, that is, it occurs at
2
the level of protein synthesis, without changes in gene transcription or mRNA
abundance (Chapter 4).

The overall data therefore supports a novel model in which UCP2 is regulated
adaptively in macrophages, to assist in maintaining function in adverse tissue
environments including hypoxia that lead to increased ROS production. Regulation by
mitochondrial ROS is at the level of protein synthesis, which is intrinsically involved
in a feedback loop that may involve the degradation pathway. The presence of UCP2
mRNA, but not protein, in many non-macrophage tissues suggests that similar
regulation may be available beyond macrophage-lineage cells, allowing UCP2 protein
expression.

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Table of Contents
TABLE OF CONTENTS

Abstract...............................................................................................................................i
Table of Contents.............................................................................................................iv
List of Abbreviations.......................................................................................................xi
Publications and Conference Proceedings...................................................................xiv
Acknowledgements.........................................................................................................xv
Declaration......................................................................................................................xvi

CHAPTER 1: MACROPHAGES, MITOCHONDRIA, REACTIVE OXYGEN
SPECIES AND UNCOUPLING PROTEINS
1. INTRODUCTION.....................................................................................................1
1.1 Biology of macrophage lineage cells..........................................................................2
1.1.1 The origin of hematopoietic lineages.....................................................................2
1.1.2 Granulocyte/macrophage lineage differentiation..................................................3
1.1.3 Phenotypes and functions of macrophage lineage cells........................................4
1.1.3.1 Functions of macrophages..............................................................................5
1.1.3.2 Activation of macrophages.............................................................................6
1.1.4 Energy metabolism and energy production in macrophages................................7
1.1.4.1 Substrates for energy production....................................................................7
1.1.4.2 Substrate metabolism for energy production..................................................7
1.2 The function of mitochondrial oxidative phosphorylation......................................9
1.2.1 Complex I.............................................................................................................10
1.2.2 Complex II............................................................................................................11
1.2.3 Complex III..........................................................................................................11
1.2.4 Complex IV and V- the ATP generator................................................................12
1.2.5 Energy production by the ETC and ROS production...........................................13
1.3 Reactive oxygen species............................................................................................13
1.3.1 Sites of ROS generation.......................................................................................14
1.3.1.1 Extramitochondrial sources of ROS.............................................................14
1.3.1.2 Mitochondrial source of ROS.......................................................................15
1.3.2 Regulation of ROS production.............................................................................18
1.3.2.1 Cellular defenses against ROS......................................................................19
1.4 Uncoupling proteins are mitochondrial regulators of ROS..................................20
1.4.1 Mitochondrial uncoupling...................................................................................20
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1.4.2 Phylogenesis of uncoupling proteins...................................................................21
1.4.2.1 Gene structure of uncoupling proteins..........................................................22
1.4.2.2 Protein structures and features of uncoupling proteins.................................24
1.4.2.3 Mechanism of uncoupling.............................................................................26
1.4.2.4 Tissue distribution and protein expression...................................................27
1.4.3 Role and regulation of uncoupling proteins........................................................28
1.4.3.1 UCP1.............................................................................................................28
1.4.3.2 UCP3.............................................................................................................29
1.4.3.3 UCP2.............................................................................................................32
1.4.3.3.1 Activation of UCP2................................................................................32
1.4.3.3.2 Regulation of UCP2...............................................................................33
1.4.3.4 Involvement of UCP2 in disease...................................................................34
1.4.3.4.1 The metabolic syndrome........................................................................34
1.4.3.4.2 Atherosclerosis.......................................................................................35
1.4.3.4.3 Neuroprotection.....................................................................................36
1.5 Mitochondrial protein uptake and turnover..........................................................37
1.5.1 Import of mitochondrial proteins.........................................................................37
1.5.2 Turnover of mitochondrial proteins.....................................................................38
1.5.2.1 UCP2 turnover of uncoupling proteins.........................................................40
1.6 Project Summary and Aims.....................................................................................41
1.6.1 Significance..........................................................................................................43
-
CHAPTER 2: MITOCHONDRIAL ELEVATION OF O • SPECIFICALLY
2
INCREASES UCP2 PROTEIN
2. INTRODUCTION.......................................................................................................44
2.2 EXPERIMENTAL PROCEDURES.......................................................................44
2.2.1 Reagents...............................................................................................................44
2.2.2 Cell culture...........................................................................................................44
2.2.2.1 Cell lines.......................................................................................................44
2.2.2.2 Isolation and culture of bone marrow-derived macrophages........................45
2.2.2.3 Cryopreservation of cell lines.......................................................................45
2.2.3 Measurement of reactive oxygen species.............................................................46
2.2.3.1 Measurement of superoxide anions..............................................................46
2.2.3.2 Measurement of hydrogen peroxide.............................................................46
2.2.4 UCP2 Extractions................................................................................................47
2.2.4.1 Whole cell preparations................................................................................47
2.2.5 UCP2 protein detection by Western blotting.......................................................47
2.2.5.1 SDS-PAGE...................................................................................................47
2.2.5.2 Protein transfer..............................................................................................48
2.2.5.3 Protein detection- Enhanced chemiluminescence (ECL).............................48
2.2.5.4 Determination of the linear range for detection of UCP2 and -actin on
X-ray film..................................................................................................................49
2.2.6 Development of a UCP2 overexpression system.................................................50
2.2.6.1 Details of the UCP2 overexpression vectors................................................50
2.2.6.2 Sequence amplifcation..................................................................................51
2.2.6.3 Purification of the amplified product............................................................52
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2.2.6.4 Cloning of the amplified UCP2 insert into pGEM-Teasy carrier vector......53
2.2.6.4.1 Ligation..................................................................................................53
2.2.6.4.2 Transformation of E.coli with plasmid DNA........................................53
2.2.6.4.3 Isolation and purification of plasmid DNA...........................................53
2.2.6.4.4 Restriction enzyme digest of UCP2-pGEM-Teasy................................54
2.2.6.5 Cloning of the UCP2 insert into the pCDNA 3.1 expression vector............54
2.2.6.6 Plasmid DNA sequencing and analysis........................................................54
2.2.6.6.1 Restriction digest confirming size of insert and target vector...............54
2.2.6.6.2 Plasmid DNA sequencing......................................................................55
2.2.7 Development of a UCP2 siRNA knockdown system............................................56
2.2.7.1 Construction of the siRNA vector.................................................................56
2.2.7.2 Digestion and ligation of the UCP2 mRNA insert and siRNA target
vector.........................................................................................................................57
2.2.7.3 Restriction digest to confirm size of insert and target vector.......................58
2.2.7.4 Virus incubation and collection of virus particles........................................59
2.2.7.4.1 Lipofectamine transfection....................................................................59
2.2.7.4.2 Virus collection......................................................................................60
2.2.7.5 Determining virus infection titers.................................................................60
2.2.7.6 Viral infection of HEK 293 cells..................................................................60
2.2.8 Validation of UCP2 overexpression system and antibody specificity.................61
2.2.8.1 UCP2 overexpression....................................................................................61
2.2.8.2 UCP2 protein knockdown.............................................................................61
2.2.9 Real-Time Polymerase Chain Reaction Amplification........................................61
2.2.9.1 General handling procedure..........................................................................61
2.2.9.2 Extraction of total RNA................................................................................62
2.2.9.2.1 Quantitation of RNA..............................................................................62
2.2.9.3 cDNA synthesis............................................................................................62
2.2.9.4 Real time PCR sample preparation...............................................................63
2.2.10 Creation of UCP2 transcriptional promoter plasmid........................................64
2.2.10.1 Amplification of the -2746 to +100 muring UCP2 promoter region..........64
2.2.10.2 Purification of the amplified -2746 to +100 UCP2 promoter PCR
product......................................................................................................................65
2.2.10.3 Restriction enzyme digest of -2746 to +100 UCP2 promoter and pGL3-
basic vector...............................................................................................................65
2.2.10.4 Preparation of the -2746 to +100 UCP3 promoter insert and pGL3-basic
vector for cloning......................................................................................................66
2.2.10.4.1 Ligation................................................................................................66
2.2.10.4.2 Transformation of E.coli with plasmid DNA......................................66
2.2.10.4.3 Isolation and purification of plasmid DNA.........................................67
2.2.10.5 Plasmid DNA sequencing and analysis......................................................67
2.2.10.5.1 Restriction digest confirming size of insert and target vector.............67
2.2.10.5.2 Plasmid DNA sequencing....................................................................68
2.2.10.6 Transient transfection of RAW macrophages with the -2746 to +100
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UCP2 promoter luciferase vector..............................................................................69
2.2.10.6.1 Luciferase assay...................................................................................69
2.2.11 ATP cell viability................................................................................................70
2.2.12 Statistical Analysis.............................................................................................70

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2.3 RESULTS..................................................................................................................71
2.3.1 Inhibitors of mitochondrial complexes I and III and the superoxide dismutase
inhibitor increase reactive oxygen species in macrophages.........................................71
2.3.2 Agents that increase O •- and H O production from non-mitochondrial
2 2 2
sources..........................................................................................................................74
2.3.3 Increased mitochondrial production of O •-, but not increased non-
2
mitochondrial O •- or isolated increased H O , is associated with increased UCP2
2 2 2
protein expression.........................................................................................................77
2.3.3.1 Confirming UCP2 antibody specificity........................................................77
2.3.3.2 Increased mitochondrial production of O •- leads to increased UCP2
2
abundance.................................................................................................................78
2.3.3.3 Increased non-mitochondrial O •- or H O fail to alter UCP2 abundance...82
2 2 2
2.3.3.4 Downstream products of O •- generation regulate UCP2 protein
2
expression.................................................................................................................84
2.3.3.5 UCP2 protein induction is not accompanied by an increase in ANT...........85
2.3.4 Increased UCP2 protein expression from O •- exposure is not through an
2
increase in transcription...............................................................................................86
2.3.4.1 Transcription is not required for the increase in UCP2 protein abundance
that is associated with elevated mitochondrial O •-..................................................86
2
2.3.4.2 UCP2 promoter reporter activity does not correlate with UCP2 protein
content in maacrophages exposed to mitochondrial O •-..........................................87
2
2.3.4.3 UCP2 mRNA does not increase in macrophages exposed to
mitochondrial O •-.....................................................................................................89
2
2.3.5 There is no evidence that reductions in ucp2 promoter activity or ucp2 mRNA
abundance by mitochondrial toxins is due to a decline in ATP levels..........................91
2.4 DISCUSSION............................................................................................................93
2.5 CONCLUSION.........................................................................................................95
CHAPTER 3: MITOCHONDRIAL ACCUMULATION OF UCP2 PROTEIN
-
FROM EXPOSURE TO MITOCHONDRIAL DERIVED O • IS A
2
CONSEQUENCE OF INCREASED PROTEIN TRANSLATION AND POSSIBLY
PROTEIN STABILITY
3. INTRODUCTION.......................................................................................................96
3.2 EXPERIMENTAL PROCEDURES.......................................................................96
3.2.1 Extraction and collection of protein extracts.......................................................96
3.2.1.1 Whole cell preparations................................................................................96
3.2.1.2 Mitochondrial isolation.................................................................................97
3.2.1.3 Mitoplast isolation........................................................................................97
3.2.1.4 IMS isolation.................................................................................................98
3.2.3 Western blot.........................................................................................................98
3.2.3.1 Determination of UCP2 protein stability......................................................98
3.2.4 Mitochondrial membrane potential.....................................................................98
3.2.5 Statistical analysis...............................................................................................99
3.3 RESULTS................................................................................................................100
3.3.1 Translational regulation....................................................................................100
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3.3.1.1 Induction of UCP2 following mitochondrial O •- generation requires de-
2
novo protein synthesis.............................................................................................100
3.3.2 Accumulation of UCP2 protein during increased mitochondrial O •-
2
generation occurs within the mitochondrial inner membrane....................................102
3.3.3 UCP2 protein accumulation is not through action of mitochondrial O •- on
2
proteosomal degradation............................................................................................103
3.3.4 UCP2 accumulation is not dependent on mitochondrial membrane potential..104
3.4 DISCUSSION..........................................................................................................106
3.5 CONCLUSION.......................................................................................................109
CHAPTER 4: STRATEGIES THAT MODULATE UCP2 ABUNDANCE
REGULATE CHANGES IN PUTATIVE UCP2 FUNCTION
4.1 INTRODUCTION...................................................................................................111
4.1.1 In vivo activators of UCP2................................................................................111
4.1.2 Cellular effects of UCP2....................................................................................112
4.1.2.1 Over-expression studies..............................................................................112
4.1.2.2 UCP2 knockdown studies...........................................................................113
4.1.2.3 Antioxidants regulate UCP2 putative function and protein abundance......114
4.2 EXPERIMENTAL PROCEDURES.....................................................................115
4.2.1 Creation of the pEGFP-UCP2 fusion construct................................................115
4.2.1.1 Amplification of the UCP2 coding region..................................................115
4.2.1.2 Purification of the amplified UCP2 PCR product.......................................115
4.2.1.3 Restriction enzyme digest of the rat UCP2 cDNA and pEGFR-N1 fusion
vector.......................................................................................................................115
4.2.1.4 Preparation of rat UCP2 cDNA and pEGFP-N1 fusion vector for cloning 116
4.2.1.4.1 Ligation................................................................................................116
4.2.1.4.2 Transformation of E.coli plasmid DNA...............................................117
4.2.1.4.3 Isolation and purification of plasmid DNA.........................................117
4.2.1.5 Plasmid DNA sequencing and analysis......................................................117
4.2.1.5.1 Restriction digest confirming size of insert and target vector.............117
4.2.1.5.2 Plasmid DNA sequencing....................................................................118
4.2.2 Transient transfection of pCDNA-UCP2 and pEGFP-UCP2 fusion vectors....119
4.2.3 Generation of mitochondrially deficient (p0) cell lines.....................................119
4.2.4 Western blotting.................................................................................................120
4.2.4.1 Measurement of UCP2 protein levels.........................................................120
4.2.5 Calcein cell number estimates...........................................................................120
4.2.6 Measurement of m.........................................................................................121
4.2.7 Measurement of O •- and H O species............................................................121
2 2 2
4.2.7.1 RAW macrophage cell line..................................................................121
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4.2.7.2 Transiently-transfected UCP2-HEK 293 cells............................................121
4.2.8 Confocal Microscopy.........................................................................................122
4.2.8.1 Detecting O •- by DHE...............................................................................122
2
4.2.8.2 Mitochondrial localisation of the UCP2-EGFP fusion protein...................122
4.2.9 Statistical Analysis.............................................................................................122

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4.3 RESULTS................................................................................................................123
4.3.1 UCP2 transiently expressed in HEK 293 cells is localised in mitochondria
and replicates m and O •- production regulation observed in RAW cells that
2 264.7
contain native UCP2 protein expression....................................................................123
4.3.1.1 UCP2 is localised to the mitochondria of HEK 293 cells...........................123
4.3.1.2 Transient expression of UCP2 in HEK 293 cells lowers mitochondrial
membrane potential.................................................................................................125
4.3.1.3 Transient expression of UCP2 in HEK 293 cells lowers mitochondrial
O •-..........................................................................................................................126
2
4.3.2 Agents that modulate UCP2 protein abundance or activity regulate function..128
4.3.2.1 Glutamine enhances UCP2 protein abundance by increasing translation of
the ucp2 gene..........................................................................................................128
4.3.2.2 Glutamine exposure increases UCP2 protein expression...........................129
4.3.2.3 Glutamine lowers  m and ROS production.............................................131

4.3.2.4 BHA lowers UCP2 abundance and associates with reduced reactive
oxygen production and Δψm..................................................................................132
4.3.2.5 The reactive aldehyde acrolein associates with increased UCP2 function
to lower  m and O •- production, despite lowering UCP2 protein abundance....135
 2
4.3.2.6 Mitochondria-deficient macrophages maintain lower Δ m, produce less
ψ
mitochondrial O •- and fail to increase UCP2 when exposed to inhibitors of
2
mitochondrial complex I.........................................................................................137
4.4 DISCUSSION..........................................................................................................140
4.5 CONCLUSION.......................................................................................................142
CHAPTER 5: HYPOXIA, BUT NOT ANOXIA, INDUCES EXPRESSION OF
UCP2 IN A MACROPHAGE CELL LINE
5.1 INTRODUCTION...................................................................................................143
5.1.1 Ischemia and UCP2 regulation.........................................................................143
5.2 EXPERIMENTAL PROCEDURES.....................................................................145
5.2.1 In vitro anoxic and hypoxic cell models............................................................145
5.2.1.1 In vitro anoxic model..................................................................................145
5.2.1.2 In vitro hypoxic model................................................................................146
5.2.1.3 Protein analyses..........................................................................................146
5.2.2 Transcription studies.........................................................................................146
5.2.3 UCP2 mRNA analysis........................................................................................147
5.2.4 Reactive oxygen species measurement...............................................................147
5.2.5 Mitochondrial membrane potential...................................................................147
5.2.6 ATP cell viability................................................................................................148
5.2.6.1 In vitro hypoxic model................................................................................148
5.2.6.2 In vitro anoxia model..................................................................................148
5.2.7 Statistical Analysis.............................................................................................148
5.3 RESULTS................................................................................................................149
5.3.1 Anoxia prevents UCP2 induced expression.......................................................149
5.3.2 Hypoxia induces UCP2 protein in macrophages...............................................151
5.3.3 Hypoxia does not alter ucp2 transcription........................................................153
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