Post on 03-Mar-2020
The Role of CCL5/RANTES in Regulating
Cellular Metabolism in Activated T cells
by
Olivia Chan
A thesis submitted in conformity with the requirements for the degree of
Master of Science
Graduate Department of Immunology
University of Toronto
© Copyright by Olivia Chan, 2011
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The Role of CCL5/RANTES in Regulating Cellular Metabolism in Activated T cells
Olivia Chan
Master of Science
Graduate Department of Immunology
University of Toronto, 2011
Recruitment of effector T cells to sites of infection is essential for an effective
adaptive immune response. The inflammatory chemokine CCL5/RANTES activates its
cognate receptor, CCR5, to initiate cellular functions including chemotaxis. This thesis
describes the signaling events invoked by CCL5 and its ability to regulate the energy
status of activated T cells. CCL5 treatment in ex vivo activated human T cells induced the
activation of AMPK and downstream substrates ACC1, PFKFB2 and GSK-3. Evidence
is provided that CCL5 treatment is able to induce glucose uptake in an mTOR-dependent
manner. Using 2-deoxy-D-glucose, an inhibitor of glucose uptake, and Compound C, an
inhibitor of AMPK, evidence is provided that demonstrate that CCL5-mediated
chemotaxis is dependent on metabolic events, since these inhibitors perturb chemotaxis in
a dose-dependent manner. Collectively, these studies suggest that CCL5 may also
influence the metabolic status of activated T cells by simultaneously activating the
AMPK and mTOR pathways.
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ACKNOWLEDGEMENTS
I would like to dedicate this thesis to everyone who supported me throughout my
graduate studies, and would like to express my gratitude to the individuals whom,
without which, none of this could have been possible.
Eleanor, your guidance, without fail, has always pointed me in the right direction.
Whenever things looked grim, your encouragement and advice would always put me
back on the right path. Thank you for your patience and mentorship throughout my
studies – you have been an incredible driving-force that has helped me grow both as a
scientist and as an individual. I am especially thankful for the opportunity to have gone to
multiple international conferences, and the chance to present my work around the world.
I look forward to working with you in the future, as we further embark on the „CCL5-era‟
in the lab!
To my supervisory committee members, Dr. Pam Ohashi and Dr. Juan Carlos
Zuniga-Pflucker, I am grateful for your helpful guidance and input into my work. Also,
thank you Dr. Shannon Dun for taking the time to discuss my research plans and long
term goals.
To past and present Fishies, I offer my sincerest thanks for all your emotional and
scientific support (especially your PBMCs). Beata, you‟ve given multi-tasking a whole
new meaning, and I look to you as a constant source of inspiration and motivation.
You‟ve been the shoulders I could always depend on and a friend I could always turn to.
Thank you for holding my hand through some of the toughest times during my studies.
Daniel, without a doubt, you‟ve been an invaluable „metabolism-and-protein-translation-
buddy‟. Thank you for our many scientific discussions – which have always made the
realm of metabolism a little less daunting. Thank you for all your encouragement and
friendship throughout the years, and keep working on your chopstick skills! Carole, I‟ve
always been determined to answer at least one of your unanswerable questions! Thank
you for taking the time to guide and mentor me throughout my project and for letting me
pick-your-brain about experimental design. I wish you, Kip and Kaycee all the very best.
Leesa, I can‟t help but chuckle whenever I come across or hear about PKR (I think that
video is still on my desktop…). Thank you for sharing your incredible energy with the
lab, and although there is much work to be done to improve your Chinese accent, there is
no doubt that you are well on your way towards obtaining your Ph.D. Ben, thank you for
bringing Fail Blog into our daily lunch routine (or was that Craig?). I wish you all the
best in your Ph.D. journey, and remember to take advantage of the lab hammock when
you truly need it! Craig, I look forward to the day when you and Ben revolutionize IGSA
(with karaoke nights!). Thank you for bringing your calming-influence to the lab and I
wish you best of luck in your Ph.D. studies. Thomas, Danlin and Ramtin, thank you for
your continuous support throughout the years and for always being an email away!
You‟ve been brilliant mentors and role models, and I wish you all happiness in your new
lives.
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To my Mom, Dad and Oscar, thank you for your constant vote of confidence.
Mom and Dad, you give me strength and hope during my rough patches – thank you for
believing, before I did, in my ability to achieve something substantial. Oscar, you never
fail to cheer me up following rather disappointing experiments. Thank you for your
tremendous support and encouragement, and I can‟t wait to see what life has in store for
you!
Thanks to all my friends and labmates who have donated blood to „fuel‟ the
studies undertaken in my project. None of this would have been possible without your
generous donations; and I may owe royalties to many of you.
Finally, a heartfelt thank you goes to my fellow students of the Immunology
Department for their support and friendship. Nothing beats sharing a pint of beer together
when experiments go awry, and I look forward to working with all of you again in the
future.
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Table of Contents
Title Page………………………………………………………………………………….. i
Abstract…………………………………………………………………………………ii-iii
Acknowledgments……………………………………………………………. ………..iv-v
Table of Contents……………………………………………………………………….v-vi
List of Figures………………………………………………………………………….... vii
List of Tables…………………………………………………………………………….viii
List of Abbreviations………………………………………………………………….. ix-xi
CHAPTER 1: INTRODUCTION…………………………………………………... 1-52
1.1 Chemokine Superfamily……………………………………………………………….2
1.1.1. Classification……………………………………………………………………2
1.1.2. Chemokine Structure……………………………………………………………4
1.1.3. Chemokine-mediated Signaling………………………………………………... 7
1.1.3.1. Jak-Stat Pathway…………………………………………………………11
1.1.3.2. MAPK Signaling Cascade……………………………………………….12
1.2 Chemokine Receptors……………………………………………………………….. 15
1.2.1. Classification…………………………………………………………………. 15
1.2.2. Chemokine Receptor Structure and Ligand Binding…………………………. 18
1.2.3. Receptor Dimerization and Internalization…………………………………… 19
1.3. Chemokine/ Chemokine Receptor Functions………………………………………. 20
1.3.1. Chemotaxis…………………………………………………………………… 20
1.3.1.1. Cellular Polarization……………………………………………………. 20
1.3.1.2. The Rho Family GTPases in Cytoskeletal Rearrangement…………….. 21
1.3.1.3. Activation of the PI-3‟K Pathway……………………………………… 22
1.3.1.4. The mTOR/4E-BP1 Pathway and Chemotaxis………………………… 24
1.3.2. Role in determining Cellular Fate…………………………………………….. 32
1.3.2.1. T cell Differentiation and Activation…………………………………… 33
1.3.2.2. Role in Cell Death………………………………………………………. 35
1.4. mTOR Signaling and Metabolic Regulation……………………………………....... 36
1.4.1. Growth Factor and Nutrient-Sensing by mTOR……………………………… 36
1.4.1.1. AMPK-regulation of mTOR……………………………………………. 39
1.4.2. mTOR Signaling in Lymphocyte Trafficking………………………………… 40
1.4.3. mTOR-mediated Proliferation………………………………………………... 41
1.5. Energy Metabolism and the T cell Response……………………………………….. 43
1.5.1. Regulation of T lymphocyte Metabolism…………………………………….. 43
1.5.2. Quiescent Cells and Oxidative Phosphorylation……………………………… 44
1.5.3. Proliferating Lymphocytes and Glycolysis…………………………………... 45
1.5.3.1. PI-3‟K Signaling in Aerobic Glycolysis………………………………... 46
1.5.4. Energy Regulation during an Immune Response…………………………….. 48
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1.6. Thesis Hypothesis and Objectives………………………………………………….. 52
CHAPTER 2: MATERIALS AND METHODS…………………………………. 53-57
2.1. Cells and Reagents ………………………………………………………………….54
2.2. Immunoblotting …………………………………………………………………….55
2.3. Flow Cytometric Analysis ………………………………………………………….55
2.4. Chemotaxis Assay …………………………………………………………………..56
2.5. Glucose Uptake Assay ………………………………………………………………56
2.6. AMPK Antibody Signaling Array ………………………………………………….57
2.7 Statistical Analysis……………………………………………………………………57
CHAPTER 3: RESULTS…………………………………………………………... 58-87
3.1. CCL5 induces phosphorylation of proteins in the AMPK signaling pathway……... 59
3.2. CCL5-mediated glucose uptake is mTOR-dependent……………………………… 69
3.3. CCL5-mediated glucose uptake is not accompanied by changes in the surface
expression of nutrient receptors ………………………………….....................................74
3.4. Glucose uptake and AMPK signaling are required for efficient CCL5-mediated
chemotaxis ………………………………………………………………………………80
3.5. CCL5-induced AMPK signaling phosphorylates the 4E-BP1 repressor of mRNA
translation………………………………………………………………………………... 80
CHAPTER 4: DISCUSSION………………………………………………………. 88-97
CHAPTER 5: FUTURE DIRECTIONS………………………………..…………98-101
CHAPTER 6: REFERENCES…………………………………………………...102-118
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LIST OF FIGURES
CHAPTER 1
Figure 1.1. Chemokines share similar structure………………………………………….. 5
Figure 1.2. Chemokine-induced signaling pathways……………………………………... 9
Figure 1.3. The MAPK signaling cascade………………………………………………. 13
Figure 1.4. Two-dimension depiction of CCR5 and residues critical for ligand binding
and signaling transduction………………………………………………………………. 16
Figure 1.5. mTOR signaling complexes………………………………………………….26
Figure 1.6. Regulation of cap-dependent mRNA translation ……………………………30
CHAPTER 3
Figure 3.1. CCR5 surface expression is induced upon T cell activation in the presence of
cytokines………………………………………………………………………………….60
Figure 3.2. CCL5 induces phosphorylation of proteins in the AMPK signaling pathway
…………………………………………………………………………………………… 64
Figure 3.3. CCL5 activates the energy-sensing kinase AMPK and downstream substrate
GSK-3β………………………………………………………………………………... ...67
Figure 3.4. CCL5-mediated glucose uptake is mTOR-dependent……………………….71
Figure 3.5. CCL5 increases cell surface expression of GLUT-1 and CD98 …………….76
Figure 3.6. Glucose uptake and AMPK signaling are required for efficient CCL5-
mediated chemotaxis……………………………………………………………………...81
Figure 3.7. Effects of 2-DG and Compound C on T cell viability ……………………….83
Figure 3.8. CCL5-induced AMPK signaling phosphorylates the 4E-BP1 repressor of
mRNA translation…………………………………………………………………………86
CHAPTER 4
Figure 4.1. Illustration of the AMPK and mTOR signaling cascades…………………... 92
viii
LIST OF TABLES
CHAPTER 1
Table 1.1. The Chemokine Superfamily and Classification………………………….. 3
Table 3.1. List of AMPK Signaling Phospho-Specific Antibodies..…………………62
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LIST OF ABBREVIATIONS
ACC Acetyl CoA carboxylase
AICAR 5-aminoomidazole-4-carboxyamide
AICD Activation induced cell death
AMPK Adenosine monophosphate-activated protein kinase
AOP-CCL5 Aminooxypentane-CC chemokine ligand 5
ATP Adenosine-5‟-triphosphate
APC Antigen presenting cell
Bcl-2 B cell lymphoma 2
C-terminal Carboxy-terminal
CCL5 CC chemokine ligand 5
CCR5 CC chemokine receptor 5
CCX-CKR ChemoCentryx chemokine receptor
CXCL CXC chemokine ligand
CXCR CXC chemokine receptor
CX3CL CX3C chemokine ligand
CX3CR CX3C chemokine receptor
DAD Defender against cell death
DAG Diacylglycerol
DARC Duffy antigen receptor for chemokines
DC Dendritic cell
DN Double negative
DNA Deoxyribonucleic acid
DP Double positive
EDTA Ethylenediamine tetra-acetic acid
EGTA Ethylene glycol-bis (2-aminoethylether)-N‟N‟N‟N‟-tetra-acetic
eIF Eukaryotic translation initiation factor
ERK Extracellular signal-related kinase
F1,6BP Fructose-1,6-biphosphate
F2,6BP Fructose-2,6-biphosphate
F6P Fructose-6-phosphate
FKBP12 FK506-binding protein 12kDa
FOXO Forkhead box class O
GAG Glycosaminoglycan
GDP Guanosine diphosphate
GTP Guanosine triphosphate
GLUT Glucose transporter
gp120 Glycoprotein of 120 kDa
GPCR G-protein coupled receptor
GPK G-protein receptor kinase
GSK Glycogen synthase kinase
HEK Human embryonic kidney
HEV High endothelial venules
HXK Hexokinase
HIV Human immunodeficiency virus
x
IFN Interferon
IGF Insulin-like growth factor
IL Interleukin
IP3 Inositol 1,4,5- phosphate
IRS Insulin receptor substrate 1
Jak Janus kinase
JNK Jun NH2-terminal protein kinase
kDa Kilodalton
KLF Kruppel-like factor
KRH Krebs-Ringer-HEPES
LKB1 Liver kinase B1
LKLF Lung Kruppel-like factor
m7GpppN 7-methyl guanosine residue
MAPK Mitogen-activated protein kinase
MAPKK MAPK kinases
MAPKKK MAPKK kinases
MAPKAP MAP kinase-activation protein
Met-CCL5 Methionine-CC chemokine ligand 5
MDCK Madin-Darby canine kidney
μM Micromolar
mTOR Mammalian target of rapamycin
mTORC Mammalian target of rapamycin complex
N-terminal Amino-terminal
nM Nanomolar
NMR Nuclear magnetic resonance
NP-40 Nonidet-40
PAMPs Pathogen-associated molecular patterns
PB Peripheral blood
PBS Phosphate buffered saline
PDK Phosphoinositide-dependent protein kinase
PFA Paraformaldehyde
PFK Phosphofructokinase
PFKFB2 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; or PFK-2
PH Pleckstrin homology
PI-3‟K Phosphatidylinositol 3-OH kinase
PIKK Phosphatidylinositol kinase-related kinase
PIP3 Phosphatidylinositol (3,4,5) triphosphate
PIP2 Phosphatidylinositol (3,4) biphosphate
PKB Protein kinase B
PKC Protein kinase C
PLC Phospholipase C
PMSF Phenylmethylsulfonylflouride
PRR Pattern recognition receptor
PTEN Phosphatase and tensin homolog deleted in chromosome ten
PTx Pertussis toxin
RAG Recombinase-activating gene
Raptor Regulatory associated protein of mTOR
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Rheb Ras-homolog enriched in brain
Rictor Rapamycin insensitive companion of mTOR
rpS6 Ribosomal protein S6
S1P Sphingosine 1-phosphate
S6K S6 kinase
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SH2 Src homology 2
Stat Signal transducers and activators of transcription
Th T helper
TBS Tris buffer saline
TCR T cell receptor
TNF Tumor necrosis factor
TRAF TNF-receptor-associated factor
TSC Tuberous sclerosis complex
TOP 5‟ tract of oligopyrimidine
VLA Very late antigen
Vps34 Vacuolar protein sorting 34
XCL XC chemokine ligand
XCR XC chemokine receptor
2-DG 2-deoxy-D-glucose
4E-BP 4E-binding protein
4F2HC 4F2 heavy chain
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CHAPTER 1
INTRODUCTION
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1.1. Chemokine Superfamily
1.1.1. Classification
Chemokines are soluble, low molecular weight (8-14 kDa) chemotactic cytokines
that bind to their cognate seven trans-membrane G-protein coupled receptors (GPCRs) to
mediate cellular migration. To date, there are over 40 characterized human chemokines,
all of which have two to four highly conserved cysteine residues (Table 1.1.) as well as a
number of virally encoded chemokine-like proteins (Alcami, 2003). The chemokine
superfamily can be separated into four sub-families based on the presence and relative
positioning of the first two cysteine residues at the N-terminus. The cysteine residues in
the CXC (or α) family are separated by a non-conserved amino acid, while in the CC (or
β) family, these cysteine residues are adjacent to each other. The XC (or γ) chemokines
have only a single cysteine residue, while the CX3C (or δ) chemokine, CX3CL1, has three
non-conserved amino acids between the first two cysteine residues.
Chemokines are also functionally categorized depending on whether they are
constitutively produced or are inducible. Constitutive, or homeostatic, chemokines are
involved in basal leukocyte migration and development, whereas inducible, or
inflammatory, chemokines control the recruitment of effector leukocytes during an
immunological insult (Proudfoot, 2002). It is important to note that this division is not
absolute, given that several chemokines cannot be assigned unambiguously to either one
of the two functional categories. CXCL9, for instance, is a “dual-function” chemokine
that is up-regulated under inflammatory conditions, but also participates in T cell
lymphopoiesis (Moser et al., 2004). Most chemokines are secreted from the cell, with the
3
Table 1.1. The Chemokine Superfamily and Classification
Systematic Names Alternate Names Receptor(s) Expression Profile
CXC Chemokines CXCL1 Groα/MGSAα CXCR2, CXCR1 Inducible
CXCL2 Groβ/MGSAβ CXCR2 Inducible
CXCL3 Groγ CXCR2 Inducible
CXCL4 PF4 CXCR3b Inducible
CXCL5 ENA-78 CXCR2 Inducible
CXCL6 GCP-2 CXCR1, CXCR2 Inducible
CXCL7 NAP-2 CXCR2 Inducible
CXCL8 IL-8 CXCR1, CXCR2 Inducible
CXCL9 MIG CXCR3, CXCR3b Dual-function
CXCL10 IP-10 CXCR3, CXCR3b Dual-function
CXCL11 I-TAC CXCR3, CXCR3b, CXCR7 Dual-function
CXCL12 SDF-1α/β CXCR4, CXCR7 Constitutive
CXCL13 BLC, BCA-1 CXCR5 Constitutive
CXCL14 BRAK, Bolekine Unknown Constitutive
CXCL15 none Unknown Constitutive
CXCL16 none CXCR6 Dual-function
CXCL17 DMC Unknown Unknown
CC Chemokines CCL1 I-309 CCR8 Dual-function
CCL2 MCP-1 CCR2 Inducible
CCL3 MIP-1α/LD78α CCR1, CCR5 Inducible
CCL4 MIP-1β CCR5 Inducible
CCL5 RANTES CCR1, CCR3, CCR5 Inducible
CCL7 MCP-3 CCR1, CCR2, CCR3 Inducible
CCL8 MCP-2 CCR1, CCR2, CCR3, CCR5 Inducible
CCL11 Eotaxin CCR3 Inducible
CCL13 MCP-4 CCR1, CCR2, CCR3 Inducible
CCL14 HCC-1 CCR1 Inducible
CCL15 HCC-2/LKN1/MIP-1γ CCR1, CCR3 Inducible
CCL16 HCC-4/LEC/LCC-1 CCR1, CCR3 Dual-function
CCL17 TARC CCR4 Dual-function
CCL18 DC-CK1/PARC/AMAC-1 Unknown Constitutive
CCL19 MIP-3β/ELC CCR7 Constitutive
CCL20 MIP-3β/LARC CCR6 Dual-function
CCL21 SLC/6Ckinase CCR7 Constitutive
CCL22 MDC/STCP-1 CCR4 Dual-function
CCL23 MPIF/CKβ8 CCR1 Constitutive
CCL24 Eotaxin-2/ MPIF-2 CCR3 Inducible
CCL25 TECK CCR9 Dual-function
CCL26 Eotaxin-3 CCR3 Inducible
CCL27 CTACK/ILC CCR10 Inducible
CCL28 MEC CCR3, CCR10 Inducible
C Chemokines XCL1 Lymphotactin/ SCM-1α XCR1 Inducible
XCL2 SCM-1β XCR1 Inducible
CX3C Chemokines CX3CL1 Fractalkine CX3CR1 Inducible
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exception of CXCL16 and CX3CL1, which are membrane-bound proteins (Bazan et al.,
1997). These proteins can also exist as soluble glycoproteins upon protease cleavage of
their trans-membrane stalks. Thus far, 47 human chemokines have been described, many
of which bind to several of the 18 described human chemokine receptors. Although there
is considerable redundancy in the chemokine system, chemokine ligand-receptor binding
does not usually cross the CC versus CXC chemokine boundaries. This redundancy also
ensures sufficient levels of robustness within the system such that essential processes are
not compromised by chance mutations.
This thesis will review our general understanding of chemokine-chemokine
receptor function and signaling, with an emphasis on the CC chemokine CCL5 and its
receptor, CCR5 in activated T cells.
1.1.2. Chemokine Structure
Although chemokines have relatively low sequence identity, they share
considerable amount of structural homology. The three-dimensional structure of CCL5,
for example, is similar to that of CCL2, CCL3, CCL4 and CXCL8, wherein they all have
the same monomeric fold. This “chemokine fold” consists of three anti-parallel β-sheets,
a carboxy (C) terminal helix and a flexible amino (N) terminal region (Figure 1.1.). Two
disulfide bonds exists between the first and third, and the second and fourth cysteine
residues to stabilize the conformation. The N-terminal segment of many chemokines,
including CCL5, is important for receptor binding. Chemical modifications to the N-
terminus of CCL5, such as the addition of amino-oxypentane (AOP-CCL5), methioine
(Met-CCL5), or pharmacophore grafting CCL5 (PSC-CCL5), have been shown to result
5
Figure 1.1. Chemokines share similar structures
Superimposed structures of CCL2 (yellow), CCL5 (blue) and CCL11 (red) revealing
similar structural elements despite low sequence homology.
6
Adapted from M. Crump et al., J. Biol. Chem 273 (1998)
7
in antagonists for CCR5 (Proudfoot et al., 1996; Simmons et al., 1997; Gaertner et al.,
2008). AOP-CCL5 and Met-CCL5 act as competitive inhibitors of CCL5 and CCL3 by
binding to CCR5 with high affinity without inducing signaling.
Chemokine oligomers have been the topic of interest in recent years, as
accumulating evidence point out distinct biological functions of chemokine monomers
and higher order multimers. While chemokine monomers are thought to be the receptor-
binding unit, it has been postulated that chemokine dimers bind to the
glycosaminoglycans (GAGs) on cell surfaces to generate a chemical gradient for
chemotaxis. CCL5 has the ability to self-aggregate, and more intriguingly, is able to form
multimers at high concentrations (Appay et al., 2000). The amino acids involved in this
self-aggregation are the negatively charged glutamine residues Glu66 and Glu26. Several
positively charged residues are found on the surface of CCL5, making it likely for ionic
bonding to occur for the formation of multimers (Appay and Rowland-Jones, 2001). At
high concentrations, CCL5 does not behave as a typical chemokine. Not only is it able to
induce the activation of T cells independent of antigen (Appay et al., 2000), micromolar
concentrations of CCL5 initiate signaling cascades that are distinct from typical
monomeric chemokines to induce T cell apoptosis (Section 1.1.3. and Section 1.3.2.2.).
The biological significance of aggregated CCL5 will be further discussed in Section
1.3.2.2.
1.1.3. Chemokine-mediated Signaling
Chemokine-induced signaling is mediated by the binding and activation of a G-
protein coupled receptor (GPCR). This binding results in the dissociation of
heterotrimeric G-protein into Gi and G subunits and leads to the activation of a
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number of signaling cascades (Figure 1.2.). For instance, free G can then activate the
phosphoinositide-3-OH kinase (PI-3‟K) pathway and/or the pathway that involves
phospholipase C (PLC), among others. PLC activation leads to the generation of two
messenger molecules, inositol-1,4,5- trisphosphate (IP3) and diacylglycerol (DAG). The
first mediates the transient increase in cytosolic calcium, while the second activates
protein kinase C (PKC) and phosphorylates a number of serine residues at the C-terminus
of CCR5, namely Ser336, Ser337, Ser342 and Ser349 (Mellado et al., 2001). The
majority of chemokine-mediated signaling responses are inhibited by pertussis toxin
(PTx), a bacterial toxin that prevents the Gi subunit from interacting with GPRCs. In
some studies, however, chemokine receptors have been reported to couple to PTx-
insensitive G-proteins, such as Gq or G16, where the receptor/G protein pairings may be
cell-specific (Al-Aoukaty et al., 1996; Arai and Charo, 1996).
9
Figure 1.2. Chemokine-induced signaling pathways
Schematic model depicting the signaling pathways activated following chemokine
binding to its receptor. Some of the molecules involved in these pathways and the
effects they promote are shown. A detailed description of these signaling pathways is
presented throughout this Thesis.
10
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1.1.3.1. Jak-Stat Pathway
Many cytokines and growth factors signal through the Jak-Stat pathway to
mediate their biological effects. Upon ligand binding, receptor dimerization occurs and
brings two Janus kinases (Jaks) into close proximity to each other. This allows for trans-
phosphorylation of the Jaks to occur, as well as the tyrosine phosphorylation of the
receptor (Rodriguez-Frade et al., 2001). Active Jaks induce the phosphorylation of signal
transducer and activator of transcription (Stat) proteins, subsequently allowing them to
dimerize via their SH2-domains, and translocate into the nucleus where they regulate
gene transcription. Several studies have established that chemokines can also invoke Jak-
Stat signaling (Rodriguez-Fade et al., 1999; Wong and Fish, 1998; Wong et al., 2001).
Nuclear extracts from Molt-4 and Jurkat T cells treated with CCL3 or CCL5 contained
phosphorylated Stat1:Stat1 and Stat1:Stat3 dimers that were bound to the DNA
complexes of a Stat-inducible gene, c-fos (Wong and Fish, 1998). Furthermore, CCL5
treatment of PM1 T cells resulted in the rapid phosphorylation/ activation of CCR5, Jak2
and Jak3 in a PTx-insensitive manner. These data are suggestive that these CCL5-
mediated phosphorylation events are not dependent on Gi -protein signaling (Wong et
al., 2001). Other studies in HEK-293 cells have shown that Jak1, but not Jak2 or Jak3,
associate with the CCR5 receptor upon CCL5 treatment, and this association promotes
the activation of STAT5b (Rodriguez-Frade et al., 1999). These data indicate that the
recruitment of Jaks and Stats to CCR5 upon CCL5 treatment appear to be cell type
specific, however, it is clear that CCL5 engages this signaling pathway to activate various
biological processes (Wong and Fish, 2003).
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1.1.3.2. MAP Kinase Cascade
Signaling through the mitogen-activated protein kinase (MAPK) pathways
regulate several different cellular responses including oncogenesis, cell proliferation and
inflammation (Johnson and Lapadat, 2002). The three major MAPK signaling pathways
include the extracellular signal-regulated protein kinase (ERK 1/2), p38, and c-JUN NH2-
terminal protein kinases (JNKs). All MAPKs are activated through a kinase cascade
where MAPKK kinases (MAPKKKs) activate MAPK kinases (MAPKKs), which then
active MAPKs by phosphorylating the threonine and tyrosine residues within the
activation loop. This hierarchy of kinase signaling is shown in Figure 1.3. Chemokine
signaling has been reported to active MAPK signaling, and evidence has accumulated for
MAPKs contributing to chemokine-mediated cell migration. CCL5-CCR5 interactions in
PM1 T cells, for example, activate p38 and its downstream substrate MAP kinase-
activation protein (MAPKAP) kinase-2 (Wong et al. 2001). Indeed, additional studies
have shown that CCR5 activation can also activate ERK to promote cytoskeletal
13
Figure 1.3. The MAPK signaling cascade
The MAPK pathways activate ERK, JNK and p38 kinases to elicit a wide-range of
biological outcomes, including gene expression, proliferation and cellular motility.
14
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rearrangement and integrin activity to promote T cell chemotaxis (Brill et al., 2001;
Ganju et al., 1998). The emerging roles of MAPKs in cytoskeletal dynamics will be
further discussed in Section 1.3.1.4.
1.2. Chemokine Receptors
1.2.1. Classification
18 human chemokine receptors have been described to date (Table 1.1).
Chemokines exert their biological effects by binding to and activating their cognate
GPCR on target cells. Similar to their chemokine ligands, chemokine receptors can also
be sub-divided into four major families, CR, CCR, CXCR and CX3CR. This
classification is based on the sub-family of chemokine ligands they are receptors for;
therefore CC chemokines bind to CC chemokine receptors (CCRs), CXC chemokines
bind to CXC chemokine receptors (CXCR), XC chemokines bind to XC chemokine
receptors (XCRs) and CX3CL1 binds to the CX3CR1 receptor (Mellado et al., 2001). The
human CC chemokine receptor 5, CCR5, comprises 352 amino acids with a molecular
mass of 40.6 kDa, and is the receptor for CCL3, CCL4, and CCL5 (Figure 1.4.). It shares
83% sequence identity with the mouse CCR5 and 71% sequence identity with CCR2
(Appay and Rowland-Jones, 2001; Raport et al., 1996; Samson et al., 1996). Non-
functional CCR5 variants exist in the human population, the best-studied being the
truncated CCR5Δ32 variant that is not expressed on the cell surface (Smith et al., 1997;
Samson et al., 1996).
Three atypical chemokine receptors which function as interceptors (internalizing
receptors) have been described. These receptors are DARC (Duffy Antigen Receptor for
16
Figure 1.4. Two-dimension depiction of CCR5 and residues critical for ligand
binding and signaling transduction
17
Adapted from M. Oppermann Cellular Signaling 16 (2004)
Disulfide bonds
G-protein binding
Tyrosine sulfation sites
Serine phosphorylation sites
Extracellular domain
7-Trans-membrane domains
Intracellular domain
18
Chemokines), CCX-CKR (ChemoCentryx Chemokine Receptor) and D6. Despite
structural similarities and high-affinity ligand binding, these receptors fail to invoke
signaling cascades observed for other chemokine receptors (Weber et al., 2004). Also,
given their predominant expression on non-leukocytic cells, these receptors play a
minimal role in directing leukocyte migration. Instead, decoy receptor-like functions have
been reported. D6 is able to internalize inflammatory chemokines in a non-specific
manner, and target them for degradation (Fra et al., 2003; Graham, 2009). As a scavenger
receptor, D6 has an essential role in the regulation of inflammatory responses and limits
chemokine circulation following an immune response.
1.2.2. Chemokine Receptor Structure and Ligand Binding
Chemokine receptors are seven trans-membrane receptors with an N-terminal
segment, seven hydrophobic trans-membrane domains, and a cytoplasmic C-terminal tail
containing multiple serine/threonine and tyrosine phosphorylation residues (Figure 1.4.).
Chemokine receptors have disulfide bridges between cysteine residues in the extracellular
loop to stabilize the receptor conformation important for ligand binding (Oppermann
2004). The N-terminal tail of several receptors, including CCR2, CCR3, CCR5 and
CXCR1, is essential for ligand binding. NMR spectroscopy studies have identified that
the first 25 amino acid residues of CCR5 are essential for CCL5 binding. In addition,
several site-directed mutagenesis studies reveal that these residues are crucial for the viral
entry and interactions with the HIV-1 gp120 protein (Duma et al., 2006; Farzan et al.,
1998). CCR5 is also post-translationally modified by O-linked glycosylations and
tyrosine sulfations of the N-terminus. These modifications have been reported to be
necessary for CCL3 and CCL4 binding, as well as enhancing the usage of CCR5 by HIV-
19
1 as a co-receptor for viral entry (Farzan et al., 1999; Dragic, 2001; Oppermann, 2004).
Upon ligand binding, CCR5 is phosphorylated on conserved serine residues Ser-336, Ser-
337, Ser-342 and Ser-349 by PKC and G-protein receptor kinases (GPK) (Oppermann et
al., 1999). With the exception of decoy/scavenger receptors, most chemokine receptors
have a conserved DRYLAIV motif in the second intracellular loop that is coupled to the
heterotrimeric G-proteins for intracellular signaling (Oppermann, 2004).
1.2.3. Receptor Dimerization and Internalization
Several chemokine receptors, namely CXCR2, CXCR4, CCR2 and CCR5, homo-
or hetero-dimerize on the cell surface (Angers et al., 2002). CCR5 dimerization was
originally thought to occur only upon ligand binding, as dimerization was postulated to
be crucial for the initiation of chemokine signaling (Rodriguez-Frade, 2001). Subsequent
studies have shown that CCR5 can homo-dimerize shortly after synthesis in the
endoplasmic reticulum, and can form dimers on the cell surface in the absence of ligand
(Issafras et al., 2002; El-Asmar et al., 2005). Interestingly, the simultaneous presence of
CCL2 and CCL5 can induce the formation of a functional CCR2-CCR5 hetero-dimer
(Mellado et al., 2001). In contrast to CCR2 and CCR5 homo-dimers, this complex
promoted the recruitment of PTx-insensitive Gq/11, and showed distinct phosphoinositide-
3-OH kinase (PI-3‟K) activation kinetics. These hetero-dimers are as abundant as homo-
dimers, and are only able to bind a single chemokine ligand of either cognate receptor at
one time (El-Asmar et al., 2005).
Ligand binding to chemokine receptors induces rapid receptor phosphorylation as
well as the clathrin-dependent endocytosis of receptors into cellular endosomes. Many
chemokine receptors are internalized through the -arrestin/clathrin-mediated pathway,
20
namely CXCR1, CXCR2, CXCR4, CCR5, CCR7 and the decoy receptor D6 (Barlic et al.,
1999; Yang et al., 1999; Signoret et al., 1997; Signoret et al., 2005; Otero et al., 2006;
Bonecchi et al., 2008). Electron microscopy and immunofluorescent studies demonstrated
that upon CCL5 binding, -arrestins are recruited to the plasma membrane where they act
as scaffold proteins that link the phosphorylated CCR5 together with clathrin-coated pits
and target receptors for recycling (Signoret et al., 2005). CCR5 accumulates in peri-
nuclear recycling endosomes and soon returns back to the cell surface in a
dephosphorylated form. CXCR4, on the other hand, undergoes similar clathrin-dependent
endocytosis but is targeted to late endosomal and lysosomal compartments where it is
ubiquitinated and degraded (Marchese and Benovic, 2001).
1.3. Chemokine/ Chemokine Receptor Functions
1.3.1. Chemotaxis
Directed cell migration, or chemotaxis, is a highly coordinated process that is
crucial for a wide spectrum of biological processes including leukocyte effector functions
during an immune response, development and wound healing. Chemotaxis involves the
sensing of a chemokine gradient for the generation of a „leading edge‟, rearrangement in
the cytoskeleton, and the initiation of the leukocyte adhesion cascade in order for
leukocyte trafficking to occur.
1.3.1.1. Cellular Polarization
When cells encounter a migration-promoting agent they quickly become polarized
and extend protrusions in the direction of migration. First, filamentous F-actin within the
21
cell becomes concentrated at the lamellipodium, or the leading edge, and a dynamic
pseudopod is extended. The pseudopod is an extension of the cell membrane which
adheres to the extracellular matrix and acts as a traction site for migration as the cell
moves forward. These adhesions disassemble as the cell detaches at the rear (termed the
uropod), and this cyclic process begins again with the protrusions at the leading edge
moving forward and re-adhering (Ridley et al., 2003). While the front of the cell
contains actin filaments which aid in the formation of new pseudopods, the back of the
cell is rich in myosin filaments that anchor the cell to the extracellular matrix during
migration (Haastert and Devreotes, 2004).
1.3.1.2. The Rho Family GTPases in Cytoskeletal Rearrangement
The ubiquitously expressed Rho family of GTPases are key regulators of
cytoskeleton rearrangement, cell polarity, gene expression, microtubule dynamics and
vesicular trafficking. While 20 members have been characterized in mammals, the most
well known members of the family are Rho, Rac and Cdc42 (Raftopoulou and Hall,
2004). These regulatory proteins act as molecular switches by cycling between GDP-
bound, inactive and GTP-bound, active forms to control signal transduction. Once
activated, Rac and Cdc42 localize to the front of migrating cells to modulate the
polymerization of actin to form pseudopods at the leading edge. In contrast, Rho
localizes at the rear of the cell where it regulates the contraction and retraction of the cell
(Raftopoulou and Hall, 2004). It comes as no surprise that the Rho family GTPases are
essential for chemokine-triggered migration. In macrophages, CCL5 activates Rac and its
downstream substrate PAK2 to induce macrophage polarization and directional migration
(Weiss-Haljiti et al., 2004). Subsequent studies extended these findings and showed that
22
other β-chemokines, CCL3 and CCL4, activate Rac for lamellipodia formation to occur
in macrophages (Di Marzio et al., 2005). These studies provide evidence for Rac in
mediating cytoskeletal rearrangement and protrusions at the leading edge downstream of
CCR1 and CCR5 ligation. Specifically, chemokine activation of GPCRs and subsequent
activation of the PI-3‟K pathway were prerequisites for Rac-induced cytoskeletal
modifications. The PI-3‟K signaling pathway is discussed below.
1.3.1.3. Activation of the PI-3’K Pathway
One signaling pathway critical for the regulation of cellular migration is initiated
by PI-3‟K and its lipid product phosphatidylinositol (3,4,5) triphosphate (PIP3). The
family of PI-3‟Ks have been divided into four classes (class IA, IB, II and III) based on
their structure and substrate specificity. Class IA and IB PI-3‟Ks are heterodimeric
enzymes that are primarily responsible for the generation of PIP3 from
phosphatidylinositol (3,4) biphosphate (PIP2) upon GPCR-signaling. Class IA PI-3‟Ks
are comprised of a 110 kDa catalytic subunit and an adaptor regulator subunit. Four
catalytic isoforms (α, β, γ, δ) and five regulatory subunits (p85α, p85β, p55α, p55γ and
p50α) exist. On the other hand, class IB PI-3‟Ks have a p110γ catalytic subunit that binds
one of two regulatory subunits, p101 or p84. The class II PI-3‟Ks, which include PI3K-
C2α, PI3K-C2β and PIK3-C2γ isoforms, poorly phosphorylates PIP2, and their
importance in cell signaling, is less clear (Kok et al., 2009). Vacuolar protein sorting 34
(vps34) is the sole class III PI-3‟K, and has been implicated in endocytosis, autophagy
and nutrient sensing (Backer, 2008).
During chemotaxis, class I PI-3‟Ks translocate to the leading edge of the cell
where they phosphorylate the 3‟-OH position of the inositol ring of PIP2 to generate PIP3.
23
PIP3 is then able to bind to the pleckstrin homology (PH) domains of signaling proteins
necessary for chemotaxis and localize them to lamellipodia. Important PIP3–binding
proteins include the serine/threonine kinase protein kinase B (PKB, or Akt) and
phosphoinositide-dependent protein kinase 1 (PDK1). Co-localizing PDK1 and AKT
promote PDK1-dependent phosphorylation/activation of AKT at Thr-308. Once activated,
Akt phosphorylates a number of critical signaling molecules and transcription factors that
are involved in cell motility, metabolism, protein translation, and cell survival (Finlay
and Cantrell, 2010). The lipid phosphatase PTEN (Phosphatase and Tensin Homolog
Deleted in Chromosome Ten) is a critical negative regulator of PIP3 levels. PTEN
primarily localizes at the back of the cell where it dephosphorylates PIP3 to PIP2, ensuring
the polarization of PIP3 at the leading edge. Cells that lack PTEN extend multiple
pseudopodia simultaneously, which impairs progress towards the chemoattractant and
invokes a lack of directionality (Lijima and Devreotes, 2002).
PI-3‟K activity is rapidly stimulated by chemoattractants including CXCL12,
CCL5, CCL19, and CCL21, among others (Ward, 2004). CCL5-induced chemotaxis and
polarization of human T lymphocytes were reported to correlate with an increase in PI-
3‟K activity and PIP3 accumulation at the leading edge. Moreover, the chemotactic
effects of CCL5 were inhibited by wortmannin, the fungal metabolite which potently
inhibits PI-3‟K activity (Turner et al., 1995). The differential roles for PI-3‟K isoforms in
mediating chemotaxis have been investigated in studies using gene-specific knockout
mice, and it has become clear that PI-3‟Kγ is a key regulator of chemotaxis. PI-3‟Kγ (or
p110γ) is activated by G-protein βγ subunits from activated chemokine GPCRs.
Neutrophils and macrophages purified from PI-3‟Kγ-null mice display impaired motility
and reduced chemoattractant-mediated migration. Moreover, these neutrophils are unable
24
to generate PIP3 at the leading edge and are unable to activate downstream effector Akt in
response to the chemoattractant N-formyl-met-leu-phe (Hannigan et al., 2002). While
numerous studies have underscored the importance of p110γ in mediating chemotaxis,
evidence has accumulated which indicates other PI-3‟K isoforms are crucial for
chemotaxis. Studies conducted by Reif and colleagues demonstrated that B cell homing
to Peyer‟s patches and splenic white pulp was impaired only in B cells deficient in p110
and not p110γ. Furthermore, the chemotactic ability of PI-3‟Kγ-deficient macrophages
and T cells in response to chemokines is not completely abrogated (Hirsch et al., 2000;
Reif et al., 2004). This is suggestive that other PI-3‟K isoforms and/or additional PI-3‟K-
independent pathways may be important for directional migration.
While PI-3‟Ks play a critical role in mediating chemotaxis, the most intriguing
aspect of these kinases is their ability to affect an extraordinarily diverse number of
cellular functions including cell survival, receptor trafficking, metabolism and
differentiation. Their roles in regulating metabolism will be discussed in Section 1.5.2.1.
1.3.1.4.The mTOR/4E-BP1 Pathway and Chemotaxis
The evolutionarily conserved mammalian target of rapamycin (mTOR) is a
serine/threonine kinase that is downstream of PI-3‟K activation. Much like PI-3‟K,
mTOR senses and integrates a plethora of extrinsic signals to regulate cellular growth,
proliferation, differentiation, metabolism and migration (Murooka et al., 2008; Sinclair et
al., 2008; Peter et al., 2010). As will be further discussed in Section 1.4., mTOR serves as
a central regulator of nutrient-sensing and metabolism which have important
ramifications on energy-consuming processes like chemotaxis and protein translation
(discussed below).
25
Protein translation is a highly regulated process that affects development, cell
cycle progression and apoptosis. Following transcription in the nucleus, mRNAs are
processed and exported to the cytoplasm where they are translated. Ribosomes are
recruited to the 5‟ end of an mRNA and begin translation where a start codon sequence
(RNA sequence with AUG) is located. The binding of ribosomes to mRNAs is facilitated
in one of two ways. First, eukaryotic translation initiation factors (eIFs) recognize and
bind to the 7-methyl guanosine residue (m7GpppN; where „m‟ is a methyl group and „N‟
is any nucleotide) that cap the 5‟ end of all nuclear-transcribed mRNAs – this is termed
cap-dependent translation. eIF4E binds the 5‟ cap together with other initiation factors,
eIF4G, eIF4A and eIF4B to form the eIF4F-complex that is responsible for unwinding
mRNA and allowing for ribosomal binding to occur (Hay and Sonenberg, 2004).
Ribosomes can also be recruited to complex RNA structural elements termed an internal
ribosomal entry segment (IRES), and initiate cap-independent translation (Stoneley and
Willis, 2004). Notably, numerous components of cap-dependent translation are regulated
by mTOR, namely eIF4B, eIF4G and eIF4E.
mTOR exists in two complexes: mTOR complex 1 (mTORC1), which is sensitive
to rapamycin, an anti-fungal macrolide, and mTOR complex 2 (mTORC2), which is
rapamycin-insensitive (Figure 1.5) (Hay and Sonenberg, 2004). mTORC1 consists of
catalytic mTOR, Raptor (regulatory associated protein of mTOR), mLST8, PRAS40 and
Deptor, while mTORC2 is made up of mTOR, Rictor (rapamycin-insensitive companion
of mTOR), mSIN1, Protor-1, mLST8 and Deptor (Laplante and Sabatini, 2009).
Although mTORC1 and mTORC2 share several accessory proteins, they regulate distinct
signaling pathways and relatively little is known about mTORC2 biology.
26
Figure 1.5. mTOR signaling complexes
The serine/threonine protein kinase mTOR exist as two complexes (mTORC1 and
mTORC2) that are structurally and functionally distinct.
27
28
mTORC1 promotes mammalian protein translation via activation of the p70
ribosomal S6 kinase 1 (S6K1) and via inhibition of the eukaryotic initiation factor 4E
(eIF4E)-binding protein 1 (4E-BP1). mTORC1 phosphorylation and activation of S6K1
at Thr389 leads to the subsequent phosphorylation of rpS6 (40S ribosomal protein S6).
These phosphorylation events promote the translation of a subset of mRNAs that contain
a 5‟ tract of oligopyrimidine (TOP). These mRNAs encode components of the translation
machinery, including ribosomal proteins and elongation factors, wherein translation of
these mRNAs up-regulate global translation capacity (Wullschleger et al., 2006). The
eIF4E-binding proteins are a family of translational repressor proteins, consisting of three
members, 4E-BP1, 4E-BP-2 and 4E-BP3. These compete with eIF4G for the overlapping
binding site on eIF4E. The sequestering of eIF4E prevents the formation of a functional
initiation complex and mRNA translation is inhibited. Binding of the 4E-BPs to eIF4E is
regulated by phosphorylation events: hypo-phosphorylated 4E-BP binds with high
affinity to eIF4E, whereas the hyper-phosphorylation of specific serine and threonine
residues prevent this interaction. Several phosphorylation sites have been reported on 4E-
BP1, but the most important for eIF4E release are Thr37, Thr46, Ser65 and Thr70 which
occur in an ordered, hierarchical manner (Hay and Sonenberg, 2004). Phosphorylation of
two priming sites, Thr37/46, by mTORC1 is required for the subsequent phosphorylation
of Thr70, Ser65, and finally the release of 4E-BP1 from eIF4E and enabling cap-
dependent translation (Hay and Sonenberg, 2004). Taken together, the association of
mTORC1 with S6K1 and 4E-BP1 co-ordinate mRNA translation by (1) up-regulating
translational machinery through 5‟TOP mRNA translation and (2) directly enabling
29
eIF4E availability for 5‟capped mRNA translation initiation. mTOR-mediated translation
is depicted in Figure 1.6.
Many facets of an immune response are influenced by mTOR/4E-BP1-mediated
protein translation, including lymphocyte proliferation (Section 1.4.2.), growth and
cellular migration. The expression and activity of cytoskeletal regulators, Rho, Rac and
Cdc42, are regulated by mTORC1-modulation of 4E-BP1 and S6K1 activity (Liu et al.,
2010). Inhibition of mTORC1 with rapamycin inhibits F-actin reorganization and cell
motility induced by IGF-1 (type I insulin-like growth factor) in a number of tumor cell
lines. Specifically, rapamycin inhibits mTORC1-mediated protein synthesis and activity
of small GTPases, Rho, Rac and Cdc42, leading to decreased F-actin polymerization,
reduced lamellipodium formation, and decreased cell motility. Furthermore, Liu and
colleagues reported that mTORC1-mediated 4E-BP1 and S6K1 signaling pathways are
involved in rapamycin inhibition of Rho, Rac and Cdc42 expression. Cells transfected
with a constitutively hypo-phosphorylated 4E-BP1 bind with high affinity to eIF4E and
inhibited its ability to initiated cap-dependent translation. In response to IGF-1
stimulation, these cells also displayed a reduced expression of Rho, Rac and Cdc42,
mimicking the effects of rapamycin. Down-regulating S6K1 also impaired the expression
of GTPases in different tumor cells lines. Collectively, both 4E-BP1 and S6K1 pathways
are essential for mTORC1-regulation of GTPase expression and cell motility.
CCL5-mediated chemotaxis is also influenced by mTOR/4E-BP1-mediated
protein translation (Murooka et al., 2008). CCL5 induces rapid phosphorylation of mTOR
as well as its downstream substrates S6K1, rpS6 and 4E-BP1 in human peripheral blood
(PB) CD4 T cells (Murooka et al., 2008). This CCL5-induced phosphorylation/ de-
activation of 4E-BP1 lead to the dissociation of eIF4E and subsequent eIF4F-complex
30
Figure 1.6. Regulation of cap-dependent mRNA translation
4E-BP1 binds to eIF4E and prevents its interaction with eIF4G and other initiation
factors, thereby preventing protein translation. mTOR directly phosphorylates 4E-BP1
and results in its release from eIF4E. eIF4E, as part of the eIF4F complex, binds the 5‟
cap of an mRNA and recruits eIF3, 40S ribosomal subunit along with associated
complexes necessary for translation.
31
Adapted from Hay and Sonenberg, Genes Dev.18 (2004)
32
formation. Indeed, CCL5 initiates active mRNA translation, evidenced by the increased
presence of high-molecular-weight polysomes which were reduced by rapamycin
inhibition. Importantly, CCL5 initiates the translation of chemotaxis-promoting proteins
cyclin D1 and MMP-9 (matrix metalloproteinase-9) in an mTOR-dependent manner. The
expression of cyclin D1 prevents cell-cell and cell-matrix adhesion and ensures motility
during chemotaxis, whereas MMP-9 degrades proteins of the extracellular matrix (Xia et
al., 1996; Li et al., 2006). Taken together, CCL5 is able to regulate eIF4E availability and
mRNA translation machinery by phosphorylating and inhibiting 4E-BP1 in an mTOR-
dependent manner. This enables CCL5-mediated expression of proteins that contribute to
efficient chemotaxis.
Eukaryotic protein synthesis can be regulated by transcriptional and translational
processes that contribute to a cell‟s decision to grow, proliferate or undergo apoptosis.
The activation of mTORC1 and subsequent signaling of S6K1 and 4E-BP1 promotes the
translation of 5‟TOP mRNAs and influences eIF4E availability for 5‟-capped mRNA
translation. These events regulate the effectiveness of an immune response by promoting
the expression of chemotaxis-related proteins as well as positively controlling
proliferation (Section 1.4.2.). Upstream regulators of mTOR activity will be further
presented in Section 1.4.1.
1.3.2. Role in determining Cellular Fate
Emerging evidence suggests that chemokines can influence cellular fate during an
immune response by affecting cell differentiation, activation and apoptosis (Luther and
Cyster, 2001; Vlahakis et al., 2002). Their ability to induce these changes is critical to the
outcome of an infection.
33
1.3.2.1. T cell Differentiation and Activation
Upon activation by APCs, CD4 T cells alter their cytokine production, increase
cellular proliferation and acquire different effector functions necessary for an effective
adaptive immune response. Interferon γ (IFN-γ)-producing T helper (Th) 1 cells are
important for the clearance of intracellular pathogens, while interleukin (IL)-4, IL-5 and
IL-10 producing Th2 cells initiate antibody production by B cells to eliminate
extracellular pathogens. Depending on the effector functions acquired, different
chemokine receptors are expressed: CCR5 and CXCR3 predominate on Th1 cells,
whereas CCR4 and CCR8 are preferentially expressed on Th2 cells (Luther and Cyster,
2001). The polarization of an immune response towards a Th1 or Th2 response is
dependent on a combination of host genetic factors, the type and amount of antigen
encountered and the cytokines elicited by infectious agents or any other insult
(Romagnani, 1997). Moreover, chemokine receptors and the chemokine milieu can
influence T cell fate and determine T helper cell polarization.
For instance, microbial challenge with Toxoplasma gondii is able to induce the
production of CCL3, CCL4 and CCL5. These chemokines activate CCR5 and signal the
production of IL-12 by CD8α dendritic cells to initiate a Th1 response for clearance of
the parasite (Aliberti et al., 2000). Mice deficient in CCR5 and CCR2 have also
elucidated the role of chemokine receptors in directing differentiation. When challenged
with a colitis-inducing agent or an opportunistic pathogen, CCR5-/-
and CCR2-/-
mice
displayed a Th2-skewed profile compared to their wild-type counterparts. These data
indicate the importance of these receptors in mediating a Th1 response (Andres et al.,
2000; Trayner et al., 2002). Taken together, chemokines are able to promote Th1 and Th2
34
differentiation and direct the polarization of an immune response. Distinct chemokine
receptors also serve as Th1 versus Th2 markers and ensure that only the appropriate
effector T cells are recruited to the sites of inflammation. For instance, rheumatoid
arthritis is a Th1 disease characterized by inflammation of the synovial tissue of multiple
joints. An abundance of inflammatory chemokines including CCL2, CCL3, CCL4 and
CCL5 are produced, which in turn promote the recruitment of T cells and macrophages
that express receptors for these chemokines (CCR1, CCL2 and CCR5) (Luster, 1998) In
fact, most of the T cells infiltrating affected rheumatic joints express CCR5 (Suzuki et al.,
1999).
A number of chemokines affect T cell fate by acting as co-stimulatory molecules
to promote lymphocyte activation. T cells derived from mice deficient in CCL5 have a
reduced capacity to proliferate and secrete inflammatory cytokines IL-2 and IFN-γ in
response to antigen or CD3 ligation (Makino et al., 2002). In the context of CD3 T cell
stimulation, nanomolar (nM) concentrations of CCL5 results in T cell proliferation and
cytokine production. At higher micromolar (M) concentrations, CCL5 induces antigen-
independent activation of T cells measurable by increased proliferation, IL-2 receptor
expression, and release of IL-2, IL-5, IFN-γ and CCL3 (Bacon et al., 1995; Appay and
Rowland-Jones, 2001). The ability of CCL5 to bypass antigen recognition to activate T
cells is also seen in other chemokines including CCL3, CCL4 and CCL2 (Taub et al.,
1996). Their ability to co-stimulate purified human T cells and different T cell clones
may be due to their capacity to induce proliferation, IL-2 production, calcium flux, and
up-regulation of B7.1 (CD80) on antigen presenting cells (APC). CCL5-induced
activation is not restricted to T cells, since activation in monocytes and neutrophils have
also been reported (Appay et al., 1999).
35
This mitogen-like property of CCL5 has been attributed to its ability to form
multimers at high concentrations. The amino acids involved in CCL5-oligomerization are
the negatively charged Glu66 and Glu26. Importantly, CCL5 variants with a Glu26 to
alanine mutation (E26A-CCL5), or a Glu66 to serine mutation (E66S-CCL5) were unable
to form aggregates at M concentrations and were unable to activate T cells (Appay et al.,
1999). Furthermore, CCL-5 aggregates initiate signaling programs that are distinct from
non-aggregated CCL5 to induce T cell activation as well as cellular apoptosis (Section
1.1.3. and Section 1.3.2.2).
1.3.2.2. Role in Cell Death
Different chemokines have been reported to induce pro- and/or anti-apoptotic
events when engaging their chemokine receptors. Their ability to protect or enhance cell
death depends on the chemokine in question, their concentration and the targeted cell
type. CXCL12, for example, is a chemoattractant for T cells and monocytes, but triggers
cell death in neurons (Berndt et al., 1998). Subsequent studies have investigated the
dichotomy in CXCL12 signaling to address why CXCL12 stimulation of T cells does not
result in cell death while in other cell types it does. Vlahakis and colleagues proposed that
CXCL12 is able to simultaneously activate pro-survival and pro-death signals in CD4 T
cells, with a net result of cell survival. They provide evidence that CXCL12-CXCR4
signaling results in the activation of ERK 1/2 and PKB/Akt for cell survival, as well as
activation of p38 for apoptosis. This model suggests that the default cell fate is apoptosis
and CXCL12-induced Akt signaling is able to „override‟ pro-apoptotic events for cell
survival. It is inferred that there is a lack of Akt signaling downstream of CCR4 ligation
in neurons, which results in cell death.
36
CCL5-CCR5 interactions have also been shown to induce cell death (Murooka et
al., 2006). Micromolar (M) concentrations of CCL5 are able to induce apoptosis in PM1,
Molt-4, and activated human PB-T cells in a CCR5-dependent manner. At these high
concentrations, CCL5 aggregates and forms multimers to induce cell death through the
release of cytochrome c, caspase-9 and caspase-3. Interestingly, CCL5-mediated
apoptosis is independent of GPCR-signaling, but rather dependent on tyrosine activity at
Tyr-339 found in the C-terminus of CCR5 (Murooka et al., 2006). Thus, CCL5 is able to
induce two distinct signaling pathways in T cells. At nM concentrations, CCL5 acts as a
typical chemokine to induce PTx-sensitive GPCR-mediated signaling that is associated
with a transient calcium influx resulting in chemotaxis and cell polarization. However, at
M concentrations, CCL5 triggers a tyrosine phosphorylation pathway that leads to
prolonged calcium influx and T cell death (Bacon et al., 1995; Appay and Rowland-Jones,
2001; Murooka et al., 2006).
The ability for CXCL12 and CCL5 to induce two distinct signaling outcomes,
chemotaxis and apoptosis, may be important for the resolution of an immune response.
At an inflammatory site where high concentrations of CCL5 may be attainable,
chemokine-mediated apoptosis may play a regulatory role in resolving an immune
response by inducing clonal deletion similar to activation induced cell death (AICD).
1.4. mTOR Signaling and Metabolic Regulation
1.4.1. Growth Factor and Nutrient-sensing by mTOR
The mammalian target of rapamycin (mTOR) is an evolutionarily conserved
regulator of cell metabolism, growth, proliferation and survival (Figure 1.5.). Eukaryotic
37
TOR proteins possess a C-terminal serine/threonine kinase domain that resembles the
catalytic domain of PI-3‟K, and belong to a group of kinases known as the
phosphatidylinositol kinase-related kinase (PIKK) family (Wullschleger et al., 2006).
Rapamycin is a potent immuno-suppressive agent that forms a complex with intracellular
cofactor FKBP12 (FK506-binding protein 12kDa) and inhibits mTORC1 activity by
binding to its N-terminus. mTOR positively controls cell growth by integrating signals
from growth factors, nutrients and energy status in order to modulate cell metabolism,
cell fate (Section 1.4.2.) and different aspects of an immune response, including
chemotaxis and protein translation (Section 1.3.1.4.).
Many growth-factor signals converge on the PI-3‟K/Akt pathway to subsequently
activate mTOR (Figure 1.2.). The binding of insulin or insulin-like growth factor (IGF)
to their receptors leads to the recruitment of IRS1 (insulin receptor substrate 1) together
with PI-3‟K and Akt to the plasma membrane. PI-3‟K-mediated activation of Akt
subsequently phosphorylates and inactivates the tuberous sclerosis complex (TSC 1/2),
an indirect inhibitor of mTOR. It is clear that TSC 1/2 are a complex of tumor suppressor
proteins, where mutations in either TSC1 or TSC2 lead to a hyper-active mTOR. These
mutations manifest as tumor formations in a number of target organs (Garami et al.,
2003). TSC 1/2 function as a GTPase-activating protein for the small GTPase, Rheb
(Ras-homolog enriched in brain) which is an upstream positive regulator of mTOR. TSC
1/2 negatively regulates Rheb activity by promoting GTP hydrolysis and causing Rheb to
remain in its inactive, GDP-bound state (Inoki et al., 2003). In its active, GTP-bound
form, Rheb directly interacts with mTOR and stimulates its activity (Long et al., 2005).
Active mTOR can subsequently regulate growth and survival by biosynthesis of proteins,
lipids and organelles through S6K1 and 4E-BP1 signaling (Laplante and Sabatini, 2009).
38
Nutrient signals, especially those from amino acids, regulate mTOR activity by
inhibiting autophagy and inducing S6K1 activation (Wullschleger et al., 2006).
Withdrawal of the essential amino acid, leucine, results in the rapid dephosphorylation of
mTOR effectors S6K and 4E-BP1 (Hay and Sonenberg, 2004). The activation of mTOR
by amino acids is TSC1/2-independent, given that S6K1 remained sensitive to amino acid
withdrawal in cells lacking TSC1 or TSC2 (Nobukuni et al., 2005). Moreover, levels of
GTP-bound Rheb remain unchanged in cells that were starved of amino acids despite
dephosphorylation of mTOR – suggesting that amino acids may invoke signaling in a
pathway distinct from the insulin-mediated TSC1/2-Rheb axis (Zhang et al., 2003).
Indeed, studies suggest that the class III PI-3‟K, vps34, signals amino acid availability to
mTOR, independent of TSC1-TSC2/Rheb (Nobukuni et al., 2005). Removal of amino
acids causes a decrease in vps34 kinase activity and S6K1 phosphorylation levels.
However, the exact role of vps34 in nutrient sensing and how this information is relayed
to mTOR remains to be established. mTOR signaling can subsequently up-regulate the
surface expression of a number of nutrient receptors. These, among others, include
glucose transporters, GLUT-1 and GLUT-4, iron/transferrin receptors, and 4F2HC (4F2
heavy chain; CD98), the invariant heavy chain that associates with different light chains
to form the heterodimeric amino acid transporter family (Edinger, 2007). Collectively,
nutrient signals are able to stimulate cell growth and proliferation via the mTOR pathway,
in part by regulating nutrient transporter expression in an mTOR-dependent manner.
Given that cell growth depends on a high rate of protein synthesis mediated by
mTOR signaling, a high level of cellular energy is required to fuel mTOR-mediated
processes. The energy status of the cell is signaled to mTORC1 through the AMP-
39
activated protein kinase (AMPK), a master sensor of intracellular energy status and
inhibitor of mTORC1 (Wullschleger et al., 2006; Gwinn et al., 2008).
1.4.1.1. AMPK-regulation of mTOR
During nutrient deprivation or environmental stress – such as glucose withdrawal
or hypoxia – mammalian cells are able to sense declining intracellular energy levels and
attempt to restore homeostasis through AMPK signaling. AMPK is a highly conserved
heterotrimeric kinase complex composed of a catalytic α-subunit and two regulatory, β
and γ subunits. Under conditions of nutrient deprivation, intracellular ATP levels decline
while AMP levels rise and AMPK becomes activated (Shaw, 2009). AMP directly binds
to the γ-subunit of AMPK and allosterically activates AMPK. AMPK can also be
activated by Thr172 phosphorylation of its activation loop located on the α-subunit. The
major upstream regulator of AMPK is the serine/threonine kinase LKB1 (liver kinase B1)
which phosphorylates AMPK at its activation loop under low ATP conditions. Once
activated, AMPK acts as a metabolic checkpoint – halting cell growth and suppressing
ATP-consuming processes such as protein synthesis while activating ATP-generating
processes like fatty acid oxidation.
The inhibitory effects of AMPK ensure that cells do not continue to grow under
unfavorable conditions when nutrients are lacking. AMPK activation is able to down-
regulate mTORC1-mediated processes, like growth and protein translation, both of which
require high levels of cellular energy. Cells deprived of nutrients or treated with the
AMPK activator, AICAR (5-aminoomidazole-4-carboxyamide), exhibit a decrease in
mTOR activity where the phosphorylation of S6K1 and 4E-BP1 are reduced (Kimura et
al., 2003). Active AMPK can directly phosphorylate TSC2, which contributes to
40
mTORC1 suppression by limiting Rheb activity (Inoki et al., 2003). In addition, AMPK
can directly inhibit mTORC1 by phosphorylating the Raptor subunit which induces 14-3-
3-binding of Raptor thereby affecting its conformational state (Gwinn et al., 2008).
Activation of AMPK leads to a metabolic checkpoint where cells will undergo cell-cycle
arrest. However, cells that lack components of the AMPK pathway, including upstream
activator LKB or downstream effector TSC2, continue cycling and subsequently undergo
apoptosis (Inoki et al., 2003; Gwinn et al., 2008). Specifically, the failure to down-
regulate mTORC1 under conditions of energy deprivation induces cell death. This
metabolic checkpoint function of AMPK has been further emphasized in studies using
various cell types under conditions of low glucose, hypoxia and treatment with glycolytic
inhibitors (Shaw et al., 2004; Buzzai et al., 2007). These studies underscore the
importance for AMPK-mediated inhibition of mTORC1 under conditions of energy stress
to halt cell-cycling and prevent cell death.
1.4.2. mTOR Signaling in Lymphocyte Trafficking
Naïve T lymphocytes constantly circulate the body through the blood, lymphatics
and secondary lymphoid organs by trans-endothelial migration via high endothelial
venules (HEVs). Lymphocyte entry depends on a unique set of molecules that are
constitutively expressed on naïve T cells, including CCR7, CD62L (L-selectin), and
CXCR4 (Sinclair et al., 2008; Finlay and Cantrell, 2009). Lymphocytes move into
secondary lymphoid organs by responding to a gradient of CCR7 ligands, CCL19 and
CCL21, and CD62L that mediate the capture and rolling of naïve lymphocytes on the
HEVs. These molecules ensure the retention of lymphocytes within lymphoid tissues
where they interact with APCs until a “match” is found. Upon activation, T cells alter
41
their expression of chemokine receptors and adhesion molecules to facilitate changes in
their migratory pattern. Instead of residing in lymphoid tissues, effector T cells migrate to
nonlymphoid tissues and sites of inflammation. Activated T cells down-regulate CCR7
and CD62L and up-regulate receptors that aid in the homing to sites in the periphery such
as VLA-4 (very late antigen 4), P- and E-selectin ligands, sphingosine 1-phosphate
receptor type 1 (S1P1) and inflammatory chemokines CCR5 and CXCR3 (Cyster, 2005;
Mora and von Andrian, 2006). The down-regulation of CCR7 and CD62L is mediated by
signaling through PI-3‟Kδ/mTOR, suppressed by LY294002 and rapamycin (Sinclair et
al., 2008). Together, the p110δ subunit of PI-3‟K promotes CD62L down-regulation by
proteolysis and mTOR regulates the expression of KLF2, a key transcription factor of
CCR7 and CD62L. The most intriguing aspect is the potential link between the PI-
3‟K/mTOR nutrient-sensing pathway, and its ability to affect chemotaxis by regulating
chemokine receptor expression. Given that mTOR is a nutrient sensor that promotes cell
growth, these data, together with those discussed in Section 1.3.1.4., suggest that mTOR
may potentially be able to integrate cellular energy levels and promote trafficking by
controlling homing receptor expression together with promoting mRNA translation of
proteins important for chemotaxis.
1.4.3. mTOR-mediated Proliferation
As a critical player in metabolic regulation, deregulation of mTOR can manifest
as different cancers and metabolic diseases, including diabetes and obesity (Sabatini,
2006; Dann et al., 2007).
Several upstream and downstream components of the mTOR pathway are altered
in cancer. Up-regulation and/or mutations of PI-3‟K, Akt, loss of PTEN, mutations of
42
TSC1 and TSC2, among others, have all been identified in different types of cancers
(Cully et al., 2006; Dann et al., 2007). Intriguingly, aberrantly high mTORC1 activity
appears to be an underlying cause of different cancers and hamartoma syndromes, which
are benign tumors that contain architecturally disorganized cells (Inoki et al., 2005; Tee
and Blenis, 2005; Wullscleger et al., 2006). These observations have influenced clinical
trials using rapamycin and its derivatives as anti-cancer agents to inhibit growth. Results
from clinical trials show that mTOR inhibitors are generally well tolerated and induce
tumor regression in a subset of patients (Dancey, 2005; Vignot et al., 2005). Rapamycin
derivatives have also been used as immunosuppressive agents in organ transplants
(Wullscleger et al., 2006).
Many cancers are characterized by abnormal chemokine production and
chemokine receptor signaling. This allows chemokines to enhance tumor growth,
metastasis and survival by initiating angiogenesis or promoting a pro-inflammatory
microenvironment (Murooka et al., 2005; Viola and Luster, 2008). For example,
CCL5:CCR5 signaling has been demonstrated to enhance breast cancer progression and
proliferation (Murooka et al., 2009). Breast tumor cells expressing lower levels of CCL5
exhibited a decreased growth rate in vitro (Alder et al., 2003). In contrast, CCL5 is highly
expressed in high grade tumors and was a predictor of rapid disease progression in breast
cancer patients (Yaal-Hahosheney al., 2006). The ability for CCL5 to promote growth
and proliferation is, in part, due to its ability to invoke the mTOR/4E-BP1 pathway and
up-regulate protein translation (Murooka et al., 2009). Studies in the breast cancer cell
line, MCF.7, have demonstrated that CCL5:CCR5 signaling directly promotes
proliferation and survival through mTOR signaling. Specifically, CCL5 engages the
mTOR/4E-BP1 pathway and promotes the translation of proliferative and survival
43
proteins, namely cyclin D1, c-Myc and defender against cell death-1 (DAD-1) in a
rapamycin-sensitive manner (Murooka et al., 2009). These findings suggest that breast
cancer cells can exploit chemokine-induced signaling and mTOR-mediated effects to
promote proliferation and survival.
1.5. Energy Metabolism and the T cell Response
1.5.1. Regulation of T lymphocyte Metabolism
Upon antigen presentation and activation by APCs, lymphocytes undergo rapid
and extensive expansion. Resting lymphocytes shift from a quiescent phenotype to a
highly proliferative and secretory state which requires a considerable amount of energy
and cellular resources. In order to match the energetic demands of the transcriptional and
translational programs that promote growth and effector functions, activated T cells must
alter their metabolism to support these activities (Frauwirth and Thompson, 2004; Fox et
al., 2005; Pearce, 2010). Interestingly, T cells lack the cell-autonomous ability to control
their uptake of metabolites necessary for adenosine-5‟-triphosphate (ATP) generation
(Fox et al., 2005). T cells must therefore rely on external, „instructional‟ signals to access
nutrient molecules such as glucose, amino acids and fatty acids from the environment.
Specifically, T cells receive these instructional signals via cytokine, antigen and co-
stimulatory receptor signaling. At a fundamental level, it is cellular metabolism which
governs T cell function and differentiation, which in turn ultimately influences the
outcome of an adaptive immune response.
At different stages of T cell development, different growth-factor signals are
required to maintain nutrient uptake. IL-7, for instance, is a survival factor for early
44
thymocytes, resting naïve T cells as well as memory T cells (Ma et al., 2006; Surh and
Sprent, 2008). T cell activation results in the down-regulation of the α-chain of the IL-7
receptor, and effector T cells become more dependent on IL-2. Withdrawal of growth-
factors, such as IL-2 or IL-7, results in the decline of cellular metabolism that is
characterized by decreased surface expression of nutrient receptors, decreased rate of
nutrient uptake and changes in mitochondrial integrity. These changes lead to the release
of apoptotic factors into the cytosol and the commitment to cell death by apoptosis (Cory
and Adams, 2002). Cytokines, such as IL-4, IL-2 and IL-7 modify the expression and
activity of the pro-survival B-cell lymphoma 2 (Bcl-2) proteins and prevent apoptotic
events from occurring. It is clear that the decision for T cells to live or die is regulated, in
part, by cytokine-dependent effects on T cell metabolism.
1.5.2. Quiescent Cells and Oxidative Phosphorylation
Naïve and memory T cells exist as relatively quiescent cells that consume glucose
and other essential nutrients at a low rate to supply energy to maintain normal
homeostatic functions (Fox et al., 2005). Their intracellular stores of ATP are largely
generated by the combined breakdown (or catabolism) of glucose, amino acids and lipids
through oxidative phosphorylation that take place within mitochondria. Oxidative
phosphorylation generates large amounts of ATP (36 ATP molecules per glucose
molecule) using the energy liberated by the stepwise transfer of free electrons from
nutrient intermediates to oxygen.
IL-4 and IL-7 are essential for the survival of naïve T cells (Vella et al., 1997; Fox
et al., 2005). Freshly isolated naïve T cells cultured without IL-4 or IL-7 die, even in the
presence of high levels of extracellular nutrients. The anti-apoptotic effects of IL-4 and
45
IL-7 have been associated with their ability to maintain levels of survival-promoting Bcl-
2 and Bcl-X1 in immature T cells. Quiescent cells can also rely on autophagy, the break
down of intracellular components, to fuel oxidative phosphorylation (Lum et al., 2005;
Lum et al., 2005b). Indeed, in the absence of growth factors, cells will rely on the
breakdown of cellular components to maintain survival. It is becoming clear that
quiescence is a state that is actively maintained by the expression of transcription factors
such as FOXO (Forkhead box class O) and LKLF (lung Kruppel-like factor). IL-7 can
induce the expression of these transcriptional factors to regulate genes that inhibit cellular
activation, cell-cycle progression and modulate metabolic pathways to maintain the
quiescent phenotype (Yusuf and Fruman, 2003; Sinclair et al., 2008). Indeed, quiescence
in lymphocytes is an actively maintained state that is under tight transcriptional control
rather than a default fate that is determined by a lack of mitogenic signals.
1.5.3. Proliferating Lymphocytes and Glycolysis
Antigenic stimulation of T cells is followed by a metabolic switch from oxidative
phosphorylation to glycolysis which is required to support growth, proliferation and
effector functions in activated lymphocytes (Frauwirth and Thompson, 2004; Pearce
2010). In mature T cells, ligation of the T cell receptor together with CD28 promotes
metabolic pathways that shunt lipids and amino acids into the production (or anabolism)
of macromolecules. As a consequence, ATP production becomes dependent on the
degradation of glucose by glycolysis. Strikingly, even in the presence of sufficient
oxygen to support oxidative phosphorylation, expanding T cells prefer to ferment glucose
to meet their energy demands. This phenomenon, known as aerobic glycolysis or the
Warburg effect, is hallmark of many transformed cells (Warburg, 1956; Elstrom et al.,
46
2004). In addition, limiting glucose availability to rapidly proliferating lymphocytes will
cause cell death, despite the availability of alternative fuels such as fatty acids or amino
acids. Taken together, effector T cells rely exclusively on glucose metabolism for ATP
production and survival. Although oxidative phosphorylation is the more efficient way of
generating ATP compared to aerobic glycolysis (2 ATP molecules per glucose molecule),
it remains unclear why proliferating T cells favor this form of metabolism. One
explanation is that glycolysis is a process which leaves many cellular metabolites
untouched; building blocks like amino acids and fatty acids can then be incorporated into
macromolecules to help activated T cells expand and proliferate (Maciver et al., 2008;
Vander Heiden et al., 2009; Pearce, 2010). At the expense of ATP production, effector T
cells rely on the hyper-induction of glycolysis initiated by PI-3‟K signaling to support
their increased metabolic needs (Frauwirth et al., 2002; Seder et al., 1994).
1.5.3.1. PI-3’K Signaling in Aerobic Glycolysis
Many growth-factors that control aerobic glycolysis bind to their surface receptors
and initiate signaling of the PI-3‟K pathway (Frauwirth et al., 2002; Fung et al., 2002;
Wofford et al., 2008). Activated PI-3‟K generates the lipid messenger PIP3 which is
required for the recruitment and activation of downstream effectors, including the
serine/threonine kinases PKB/Akt and mTOR. Growth-factor-mediated PI-
3‟K/Akt/mTOR activation enhances a number of metabolic activities that support the
energy demands of activated T cells.
First, cell surface expression of nutrient receptors can be enhanced to increase
nutrient uptake. CD28 co-stimulation together with CD3 ligation induces sustained Akt
activation along with an increase in glucose transporter, GLUT-1, surface expression in
47
human peripheral blood T cells (Frauwirth et al., 2002). In contrast, resting T cells
express low levels of GLUT-1, the primary glucose transporter of hematopoietic cells. In
fact, activation by anti-CD3 or anti-CD28 alone does not change GLUT-1 expression and
is unable to sustain Akt phosphorylation. The synergistic/co-operative effects of anti-
CD3/anti-CD28 stimulation of Akt and GLUT-1 are sensitive to PI-3‟K inhibition by
LY294002. Frauwirth and colleagues further demonstrated that CD3/CD28-stimulated T
cells exhibit increased glucose uptake that is predominantly converted and secreted as
lactate. As much as 90% of the glucose consumed is converted to lactate instead of being
processed into macromolecules – indicative of limited oxidative phosphorylation in
activated T cells. Subsequent studies using growth-factor-dependent cell lines and ex vivo
activated lymphocytes have shown similar results. Specifically, PI-3‟K/Akt signaling is
necessary for GLUT-1 translocation from intracellular stores to the cell surface, efficient
glucose import and lactate secretion to occur (Bentley et al., 2003; Wieman et al., 2007;
Wofford et al., 2008). Interestingly, the Wieman group noted that mTOR signaling was
not required for IL-3-mediated GLUT-1 expression in FL5.12 cells, however, mTOR
inhibition with rapamycin greatly diminished glucose uptake. It can be speculated that
mTOR signaling may promote GLUT-1 activity rather than receptor levels.
Changes in gene expression and enzymatic activity induced by Akt can also
increase the rate of glycolysis. For example, cells with constitutively active Akt have
increased total cellular hexokinase (HXK) activity, the enzyme responsible for
phosphorylating and trapping glucose in the cell and ensuring its entry into the glycolytic
pathway (Rathmell et al., 2003). Moreover, constitutively active Akt is able to induce
glucose uptake, maintain HXK activity and prevent cellular apoptosis in the absence of
extrinsic factors. Phosphofructokinase (PFK) is the enzyme that catalyzes the rate-
48
limiting step in glycolysis by phosphorylating fructose 6-phosphate to fructose 1,6-
biphosphate. This step fully commits glucose to glycolysis. Studies have shown that
insulin-activated-Akt can stimulate PFK activity in the heart to up-regulate glycolysis
(Deprez et al., 1997). Taken together, PI-3‟K signaling acts to coordinate energy
metabolism by regulating glucose transport and key glycolytic enzymes.
1.5.4. Energy Regulation in an Immune Response
The ability for lymphocytes to alter their metabolism and co-ordinate glycolysis
not only affects their decision to proliferate (Section 1.5.3.) or quiesce (Section 1.5.2.), it
can also determine lymphocyte function in an immune response. At each stage of
development, lymphocyte metabolism is regulated to eventually fuel an effective immune
response (Fox et al., 2005).
The development of αβ cells in the thymus is a highly regulated process. The
earliest thymocyte precursors lack the expression of CD4 and CD8 co-receptors and are
referred to as double-negative (DN) cells. DN precursors can be divided into four
populations based on their expression of CD44 and CD25: DN1 (CD44+CD25
-), DN2
(CD44+CD25
+), DN3 (CD44
-CD25
+), and DN4 (CD44
-CD25
-) (Godfrey et al., 1993).
Rearrangement of the T cell receptor (TCR) β locus is catalyzed by recombinase-
activating gene 1 (RAG-1) and RAG-2 during the transition of cells from DN2 to DN3.
The β chain of DN3 cells must pair with a surrogate pre-TCR α chain and CD3 molecules
in order to form the pre-TCR. Only DN3 cells that generate a functional TCR β chain will
further differentiate and enter the first check point of thymopoiesis, β-selection. Both
Notch and IL-7 signaling are important during β-selection for the maintenance of glucose
metabolism and survival in thymocytes (Ciofani and Zuniga-Pflucker, 2005; Wofford et
49
al., 2008). Notch signaling not only directs T cell lineage commitment (versus B cell
lineage), its presence is also required throughout DN stages for continued differentiation
of αβ T cells towards a double positive (DP) CD4+CD8
+ T cell (Wolfer et al., 2002). The
signaling cascades invoked by Notch signaling during β-selection were studied using DN
thymocytes from RAG-2 deficient mice, which fail to produce an endogenous TCR-β
chain. Only in DN cells that were retrovirally transduced with TCR-β and cultured on
stromal cells in the presence of Notch ligand, Delta-like 1, did cells survive and further
differentiate into CD4+CD8
+ double positive T cells (Ciofani and Zuniga-Pflucker, 2005).
In the absence of Notch ligand, thymocytes underwent apoptosis with increased caspase 3
activity and a loss of mitochondrial membrane potential. In addition, Notch signaling was
required to maintain GLUT-1 expression and glucose metabolism in a PI-3‟K/Akt-
dependent manner to promote survival in these pre-T cells. Constitutively active Akt was
also sufficient to maintain thymocyte glucose metabolism for β-selection in the absence
of Notch. Collectively, these data provide evidence that glucose metabolism is a means
for Notch to promote survival during β-selection. By controlling thymocyte metabolism,
Notch regulates the development and selection of T lymphocytes.
Another important response regulated by lymphocyte metabolism is the
generation of memory CD8 T cells. In response to an infection CD8 T cells undergo
expansion to become antigen-specific effector cells – a process that is accompanied by a
metabolic conversion from oxidative phosphorylation to rapid glycolysis (Section 1.5.3.).
Long-lived memory CD8 T cells arise when effector T cells contract and once again
become quiescent cells that rely on oxidative phosphorylation after an infection. The
exact mechanism which regulates the transition to the memory phenotype remains
unclear; however a successful metabolic transition may be required for the differentiation
50
to CD8 memory T cells after an infection (Pearce et al., 2009; Araki et al., 2009). To
investigate the mechanisms underlying memory T cell development, Pearce and
colleagues studied mice with a T cell-specific deletion of the tumor necrosis factor (TNF)
receptor-associated factor 6 (TRAF6). TRAF6 is an adaptor protein in the TNF-receptor
and interleukin-1R/Toll-like receptor superfamily and is a known negative regulator of T
cell activation. Following bacterial infection, TRAF6-deficient T cells mounted normal
antigen-specific effector CD8 T cell responses, but fewer memory CD8 T cells were
detected 60 days after infection. In addition, these mice failed to respond robustly to re-
infection, indicative that a lack of memory T cells was generated in the T-cell specific
TRAF6-deficient mice. Most interestingly, microarray analyses that compared gene
expression between wild-type and TRAF6-deficient CD8 T cells indicated that TRAF-
deficient cells displayed defects in the expression of genes in several metabolic pathways,
including fatty acid metabolism (Pearce et al., 2009). The oxidation of fatty acids, similar
to autophagy, can occur when growth-factor signals are withdrawn and glycolysis
becomes compromised (Rathmell et al., 2005). Although TRAF6-deficient CD8 T cells
were able to initiate glycolysis during activation, they had a reduced capacity to oxidize
fatty acids when IL-2 was withdrawn. Taken together, TRAF6-deficient CD8 T cells fail
to persist as long-lived memory cells due to their inability to engage fatty acid oxidation
pathways. This switch may be imperative for their survival when growth factors such as
IL-2 become limiting following the peak of an immune response. The conversion in
metabolism during the contraction phase may be required in memory T cell development
for a successful and accelerated secondary response.
It is becoming clear that changes in T cell metabolism can determine T cell fate
and further influence the outcome of an immune response. The ability for growing
51
thymocytes to acquire nutrients will determine their survival and selection in the thymus.
Similarly, the conversion between differing metabolic states is required for effective
generation of a given T cell fate.
52
1.6. Hypothesis and Objective
Hypothesis: CCL5-mediated mTOR activation modulates cellular metabolism by
directly regulating nutrient uptake and glucose metabolism in activated T cells.
Given that mTOR is a central regulator of nutrient sensing and processes that are
induced by CCL5, including mRNA translation and chemotaxis, that are energy-taxing,
CCL5/mTOR signaling may influence cellular metabolism to match energy demands of
activated T cells.
Objective: Examine the role of CCL5-CCR5 mediated signaling in regulating T cell
metabolism in the context of chemotaxis.
53
CHAPTER 2
MATERIALS AND METHODS
54
2.1. Cells and Reagents
Human peripheral blood (PB)-derived T cells were isolated from consenting
healthy donors, approved by the UHN REB. Cells were maintained in RPMI 1640
supplemented 10% dialyzed fetal calf serum (Sigma), 100 units/ml penicillin, 100 mg/ml
streptomycin and 2mM L-glutamine (Invitrogen). CD3+ T cells were purified using the
StemSep T cell enrichment cocktail, according to the manufacturer‟s specifications
(StemCell Technologies). T cells were subsequently activated in the presence of 10
µg/ml anti-CD3 antibody (eBiosciences), 5 µg/ml anti-CD28 antibody (eBiosciences),
and 5ng/ml hrIL-12 (Bioshop, Canada) for 2 days, and further expanded in culture
supplemented with 100U/ml (10ng/ml) hrIL-2 (Bioshop, Canada) for 3 days. T cell
purity and CCR5 expression were confirmed at day 6 by flow cytometric analysis using
anti-human CCR5 antibody (2D7; BD Pharmingen), anti-human CD3, anti-human CD4
and anti-human CD8 antibodies (eBiosciences). Antibodies for phospho-AMPKα (Thr-
172), AMPKα, phospho-GSK-3β (Ser-9), phospho-4E-BP1 (Thr-37/46) and 4E-BP1,
were purchased from Cell Signaling Technology. Mouse monoclonal anti-tubulin
antibody was purchased from R & D Systems. Purified mouse anti-human CD98
(4F2HC) and GLUT-1 antibodies were obtained from Santa Cruz Biotechnology Inc. and
R & D Systems, respectively. Rapamycin, Compound C and AICAR were obtained from
Calbiochem. 2-deoxy-D-glucose was purchased from Sigma. Toxicity studies were
performed on splenocytes derived from C57Cl/6 mice purchased from Jackson
Laboratory (Bar Harbor, ME). CCL5/RANTES was a generous gift from Dr. Amanda
Proudfoot (Geneva Research Centre, Merck Serono International). The CCR5 antagonist,
TAK-779, was kindly provided by Dr. Clifford Lingwood (University of Toronto,
Sickkids Hospital).
55
2.2. Immunoblotting
Cells were incubated with 10 nM CCL5 for the times indicated, collected, washed
twice with ice-cold PBS and lysed in 100 l lysis buffer (1% Triton X-100, 0.5% NP-40,
150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM PMSF, 10
µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin A). For all experiments using
inhibitors or activators, cells were pretreated for 1 hr with the amount indicated prior to
CCL5 treatment. Protein concentration was determined using the Bio-Rad DC protein
assay kit (BioRad laboratories). 50 g of protein lysate were denatured in 5x sample
reducing buffer and proteins resolved by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). The separated proteins were transferred to a nitrocellulose
membrane followed by blocking with 5% BSA (w/v) in 1x TBST (0.1% Tween-20) for
1hr at room temperature. Membranes were probed with the specified antibodies overnight
in 5% BSA (w/v) in TBST at 4°C and the respective proteins visualized using the ECL
detection system (Pierce).
2.3 Flow Cytometric Analysis
1 x 106 cells were incubated with mouse anti-human CCR5 antibody for 30
minutes on ice and washed twice with ice-cold FACS buffer (PBS/2% FCS). Cells were
then incubated with AlexaFlour 488-conjugated anti-mouse IgG antibody (eBiosciences).
As control, cells were incubated with AlexaFlour 488-conjugated antibody alone. T cell
purity was determined by incubating cells with a PE-conjugated anti-human CD3
antibody. As isotype control, cells were incubated with PE-labeled isotype control IgG
56
antibody (eBiosciences). For GLUT-1 and CD98 (4F2HC) surface expression, cells were
collected and washed twice with ice-cold FACS buffer and fixed with 2% PFA at room
temperature for 20 minutes. Cells were then washed twice with FACS buffer and
incubated with mouse anti-human GLUT-1 or mouse anti-human CD98 for 30 minutes
on ice. Cells were then washed twice and incubated with AlexaFlour 488-conjugated
anti-mouse IgG antibody. Cells were analyzed using the FACSCalibur and FlowJo
software (BD Biosciences).
2.4 Chemotaxis Assay
T cell chemotaxis was assayed using 24-well Transwell chambers with 5 µm
pores (Corning). A total of 1 x 105 cells in 100µl chemotaxis buffer (RPMI 1640/0.5%
BSA) were placed in the upper chambers. CCL5, diluted in 600µl chemotaxis buffer,
was placed in the lower wells and the chambers incubated for 2 hrs at 37ºC. Migrated
cells located in the bottom wells were collected, and counted with a hemocytometer. All
experiments were conducted in triplicate. In experiments involving inhibitors, cells were
pretreated for 1 hr at the indicated inhibitor concentrations and placed in the upper
chambers. Cell viability, as measured by PI staining, was not affected by any of the
doses of inhibitors used in this study.
2.5 Glucose Uptake Assay
3-5 x 106 cells were washed with PBS and resuspended in 500µl of Krebs-Ringer-
HEPES (KRH) (at pH 7.4, 136 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl2, 1.25mM
MgSO4, and 10 mM HEPES). 2-Deoxy-D-[H3] glucose (2Ci/ reaction; Perkin Elmer)
was added in the presence of CCL5 and the reaction mixture incubated at 37C.
57
Reactions were quenched by the addition of ice-cold 200 M phloretin (Calbiochem)
followed by immediate centrifugation through an oil layer (1:1 phthalic acid and
dibutlylpthalate from Sigma-Aldrich). Cell pellets were washed and solubilized in 1M
NaOH for 1 hr, and radioactivity was measured using a scintillation counter. In
experiments involving inhibitors, cells were pretreated for 1h before the addition of 2-
Deoxy-D-[H3] glucose and CCL5.
2.6 AMPK Signaling Antibody Array
Phosphorylation events in the AMPK signaling pathway were examined using the
Full Moon BioSystems Antibody Microarray, according to the manufacturer‟s
specifications (Full Moon BioSystems, Inc.). Briefly, 5 x 106 cells were stimulated with
CCL5 for 10 minutes, washed with ice-cold PBS, and lyzed with 200 µl of Extraction
Buffer. Protein samples were biotinylated and were added to a microscope slide
chambers that had specific antibodies bound to its surface. Cy3-streptavidin was added,
and fluorescence was detected using the Axon GenePix 400A Microarray Scanner at
PMT voltages between 300-400 (Molecular Devices).
2.7 Statistical Analysis
Statistical significance was analyzed with repeated-measures analysis of variance
(ANOVA). A level of p<0.05 was chosen to identify significant differences. All data are
expressed as mean ± S.E.M. (standard error of means).
58
CHAPTER 3
RESULTS
O.C. performed all experiments and analyzed the data
Dr. E.N.F. designed research and analyzed the data
59
3.1. CCL5 induces phosphorylation of proteins in the AMPK signaling pathway
To investigate potential metabolic changes induced by CCL5, studies were
conducted to examine phosphorylation events in the energy-sensing, AMPK signaling
pathway. The phosphorylation/activation of AMPK is an indication of a decrease in
intracellular ATP levels – which may initiate processes that attempt to restore
homeostasis by up-regulating ATP-generating processes, such as glycolysis, and
suppressing ATP-consuming processes, such as cell cycling (Shaw et al., 2004).
Ex vivo cytokine activation of peripheral blood (PB) CD3+ T cells (as described
in Materials and Methods) induced the surface expression of CCR5 (Figure 3.1.A). T cell
populations used in all experiments were consistently 70% - 75% CCR5 positive, >90%
CD3 positive, and were a heterogeneous population of CD4+ and CD8+ T cells (Figure
3.1.B and Figure 3.1.C). To avoid confounding data attributed to IL-2 effects PB T cell
cultures that were used for CCL5 treatment experiments were only stimulated with IL-2
on days 2 and 4, and then CCL5 treated on day 6. IL-2 treated cultures that served as
positive controls were stimulated with IL-2 on days 2, 4 and 6.
At the onset, a global screening approach was undertaken to examine signaling
events in the AMPK pathway. The Antibody Microarray platform from Full Moon
BioSystems measures the phosphorylation of upstream and downstream substrates of
AMPK. The different phospho-specific antibodies in each array are listed in Table 3.1.
Activated PB T cells either left untreated (control) or treated with 10 nM CCL5 for 10
minutes, were lysed and proteins biotinylated. Biotinylated proteins were then introduced
on to the microarray slide chambers which were conjugated with antibodies specific for
the AMPK signaling cascade. T cell bound proteins were identified using a Cy3-
streptavidin detection system.
60
Figure 3.1. CCR5 surface expression is induced upon T cell activation in the
presence of cytokines
(A) PB T cells were activated in the presence of IL-12 and IL-2 to induce the surface
expression of CCR5. (B) CD3 expression was measured with anti-CD3 antibody staining
(black), using the appropriate isotype control (grey). Representatives of three independent
experiments are shown. (C) T cell populations were a heterogeneous mix of CD4+ T
cells and CD8+ T cells; majority of which were CD4+.
61
Figure 3.1
62
Table 3.1. List of AMPK Signaling Phospho-Specific Antibodies
Phospho-antibody Phosphorylation
Sites
Phospho-antibody Phosphorylation
Sites
4E-BP1 Ser65 Thr45
mTOR
Ser2448
Thr36 Thr70
Ser2481
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2)
Ser483
p21Cip1
Thr2446
ACC1 Ser79
Thr145
AKT Ser80 Thr308
p53
Ser15 Ser392
Ser473 Tyr326
Ser20 Ser46
AKT1
Ser124 Thr72
Ser315 Ser6
Ser246 Tyr474
Ser33 Ser9
Thr450
Ser366 Thr18
AKT1S1 Thr246
Ser37 Thr81
AKT2 Ser474
Ser378 AMPK1 Thr174
p70 S6 Kinase
Ser411
AMPK1/AMPK2 Ser485/491
Ser424
AMPK/beta 1 Ser182
p70S6K
Ser371 Thr389
CaMK1-alpha Thr177
Ser418 Thr421
CaMK2 Thr305
Thr229 CaMK2-beta/gamma/delta Thr287
p70S6k beta Ser423
CaMK4 Thr196/200
PI3-kinase p85-alpha Tyr607
CaMKII Thr286
PI3-kinase p85-subunit alpha/gamma Tyr467/199
Cyclin B1 Ser126
PKA CAT Thr197
Ser147
PKA-R2B Ser113
EEF2 Thr56
PLC-beta Ser1105
eEF2K Ser366
PLC-beta3 Ser537
eNOS
Ser1177
RapGEF1 Tyr504
Ser615
SREBP-1 Ser439
Thr495
Tuberin/TSC2
Ser939
HNF4 alpha Ser304
Thr1462
HSF1 Ser303
HSL
Ser552/563
Ser554
LKB1 Ser428
MEF2A
Thr189 Thr312
Ser408 Thr319
MEF2C Ser396
MEF2D Ser444
63
The microarray slide images generated are shown in Figure 3.2.A. and phosphorylation
events were quantitated and represented as fold induction in Figure 3.2.B. PB T cells
treated with 10 nM CCL5 for 10 minutes induced the rapid phosphorylation of a number
of signaling effectors in the AMPK signaling pathway, as well as effectors in the PI-
3‟K/Akt and mTOR/4E-BP1 cascades. Notable proteins phosphorylated by are indicated
with red arrows. These included phosphorylation of PFKFB-2 (6-phosphofructo-2-
kinase/fructose-2,6-biphosphatase 2 or PFK-2), a positive regulator of glycolysis, ACC-1
(acetyl-CoA carboxylase 1), an enzyme important for fatty acid synthesis and inhibitor of
fatty acid oxidation, and the master regulators of energy status LKB1, AMPK1/AMPK2
and mTOR.
64
Figure 3.2. CCL5 induces phosphorylation of proteins in the AMPK signaling
pathway
(A) The AMPK Signaling Phospho-Specific Antibody Array includes six replicates
(vertical columns) of phospho-specific antibodies and their non-phospho pairs, targeted
against proteins in the AMPK signaling pathway. These antibodies are covalently
immobilized on microscope slides. Biotinylated protein lysates were added to microscope
slide chambers and fluorescence from Cy3-streptavidin was measured with the Axon
GenePix 400A Microarray Scanner (B) The extent of protein phosphorylation (mean
fluorescence intensity, MFI) was normalized within each slide and compared between
untreated control and CCL5 treated cells. The data are represented as fold CCL5-
induction relative to untreated controls. Important proteins phosphorylated by CCL5 are
indicated (red arrows).
65
Figure 3.2
A
Control 10 nM CCL5
B
66
To validate the antibody array findings for AMPK phosphorylation, Western
immunoblot time-course studies (0-60 minutes) were performed. PB T cells treated with
10 nM CCL5 induced the maximal phosphorylation and activation of AMPK-α on
Thr172 by 10 minutes. Phosphorylation at this activation loop threonine residue is
absolutely required for AMPK activation (Zhou et al., 2001; Gwinn et al., 2008). CCL5
also induced the phosphorylation of GSK-3β (glycogen synthase kinase 3 β), a
downstream substrate of AMPK, on Ser9, with peak phosphorylation detected at 10
minutes post-CCL5 treatment (Figure 3.3.). GSK-3β is a constitutively active
serine/threonine kinase that regulates glycogen synthesis, gene transcription, protein
translation and cell proliferation (Doble and Woodgett, 2003; Jope and Johnson, 2004). It
is in its phosphorylated/ inactive form that GSK-3β is able to de-repress downstream
signaling mediated by glycogen synthase, eIF2B, NF-κB, and other downstream
substrates. Thus, the inhibitory effect of CCL5 treatment on GSK-3β may not only affect
glycogen metabolism, but may also promote energy storage in activated T cells.
67
Figure 3.3. CCL5 activates the energy-sensing kinase AMPK and downstream
substrate GSK-3β
Activated PB T cells were either left untreated, or treated with 10 nM CCL5 for the
indicated times. Cells were harvested and protein lysates resolved by SDS-PAGE and
immunoblotted with anti-phospho-AMPKα (Thr 172) or anti-phospho-GSK-3β (Ser 9)
antibodies. Membranes were stripped and re-probed for loading. Relative
phosphorylation is shown as signal intensity over loading control. Data are representative
of two independent experiments. *p<0.01
68
Figure 3.3
69
3.2. CCL5-mediated glucose uptake is mTOR-dependent
The preceding data suggest that CCL5 may regulate the metabolic state of T cells
through AMPKα activation. AMPK acts as a “fuel gauge” to initiate pathways for ATP
regeneration and is able to induce glucose uptake in a variety of cell types during energy
stress (Fujii et al., 2006; Gwinn et al., 2008). The nutrient-sensitive mTOR is also able to
respond to extracellular nutrient signals to regulate growth and glycolysis (Wullschleger
et al., 2006). Glucose uptake was therefore examined to determine if the signaling events
invoked by CCL5 could lead to active glucose uptake in activated T cells. For these
experiments, cells were resuspended in Kreb‟s Ringer HEPES (KRH) buffer, a salt
solution that maintains pH and osmotic balance and provides cells with water and
essential inorganic ions. This buffer is free of glucose. As shown in Figure 3.4.A, CCL5
treatment was able to stimulate glucose uptake in a dose-dependent manner, with
maximal uptake using 10 nM CCL5 (1.2-1.4 fold increase). This effect was abrogated by
pretreatment with the glucose analogue and anti-cancer agent, 2-deoxy-D-glucose (2-DG).
2-DG is structurally similar to glucose, differing at the second carbon by a substitution of
hydrogen for a hydroxyl group (Aft et al., 2002). 2-DG is transported into cells through
glucose transporters and is phosphorylated by hexokinase (HXK), but is not metabolized
any further (Figure 3.4.C.). The accumulation of 2-DG in the cell interferes with
glycolysis and subsequent glucose uptake by inhibiting the activity of glycolytic enzymes
(Ralser et al., 2008). In agreement with previous studies, IL-2 treatment results in a 1.5-
1.9 fold increase in glucose uptake (Wofford et al., 2008).
Next, the specific contribution of mTOR signaling to the CCL5-mediated increase
in glucose uptake was examined using rapamycin. Indeed, inhibiting mTOR effectively
reduced CCL5 mediated glucose uptake by 1.5 fold (Figure 3.4.B.), supporting the
70
hypothesis that CCL5-mediated mTOR activation promotes the up-regulation of nutrient
uptake and glucose metabolism in activated T cells.
Finally, to confirm that CCL5 specifically induces glucose uptake through CCR5
activation, the CCR5 antagonist TAK 779 was employed. TAK 779 is a small chemical
molecule that binds to CCR5 trans-membrane helices 1, 2, 3, and 7 to induce
conformational changes in CCR5. These conformational changes disrupt ligand binding
and inhibit ligand-mediated signaling (Baba et al., 1999; Dragic et al., 2000). A marked
reduction in glucose uptake was observed in T cells pretreated with TAK 779 (Figure
3.4.C.). These data indicate that CCL5 binding to CCR5, and not CCR1 or CCR3, is
required for glucose uptake.
71
Figure 3.4. CCL5-mediated glucose uptake is mTOR-dependent
(A) Activated PB T cells were suspended in KRH buffer and were either left untreated,
treated with 20 ng/ml IL-2, or different doses of CCL5 for 2hr. In parallel, cells were pre-
treated with 10 mM of 2-DG for 1hr prior to treatment with 10 nM CCL5. At time 0, 2
μCi/rxn of 2-deoxy-D-[3H] glucose was added to the cultures. Reactions were quenched
with ice-cold phloretin, cells solubilized with NaOH and radioactivity measured with a
scintillation counter. Data are representative of three independent studies. (B) Cells were
pretreated with either DMSO (carrier) or 50 nM of rapamycin for 1hr prior to treatment
with 10 nM CCL5. Tritiated-glucose uptake was measured as in (A). Data are
representative of two independent studies. (C) Cells were pretreated with CCR5
antagonist, TAK-779 for 1hr prior to treatment with 10 nM CCL5. Tritiated-glucose
uptake was measured as in (A). Data are representative of two independent studies. (D)
Schematic model depicting the pathway in which glucose is imported through glucose
transporters and metabolized by enzymes hexokinase (HXK) and phosphofructokinase-1
(PFK-1). 2-DG is taken up into cells through glucose transporters and trapped within
cells once phosphorylated by HXK.
72
Figure 3.4
73
D
74
3.3. CCL5-mediated glucose uptake is not accompanied by changes in the surface
expression of nutrient receptors
The ability of CCL5 to stimulate glucose uptake may be facilitated through
enhanced surface expression of nutrient receptors. Glucose uptake is mediated by a
family of facilitative integral membrane glucose transporters (GLUTs) that are expressed
on the cell surface. In lymphocytes, facilitated diffusion is primarily controlled by
GLUT-1, a ubiquitously expressed glucose transporter that is highly up-regulated upon
CD3/CD28 ligation (Frauwirth and Thompson, 2004; Maciver et al., 2008). Activated
lymphocytes also increase expression of the insulin-sensitive GLUT-3 and GLUT-4
receptors. Numerous growth signals mediate cell-surface trafficking and expression of
GLUT-1 through the PI-3‟K/Akt pathway, thereby increasing glucose uptake and
glycolytic flux (Frauwirth et al., 2002; Bentley et al., 2003; Wofford et al., 2008).
Another key nutrient receptor that is regulated by this pathway is CD98, a critical
component of the amino acid-transporter complex. Accordingly, we undertook studies to
examine the ability of CCL5 to regulate the surface expression of GLUT-1 and CD98.
Whereas naïve T cells express low levels of GLUT-1 and CD98 (Figure 3.5.A.
and Figure 3.5.B.), their cell surface expression is strongly induced upon T cell
activation. In time course studies, CCL5 treatment did not further increase GLUT-1
expression at 2, 4, 6 and 8 hours post CCL5 treatment (Figure 3.5.C.), with evidence of
enhanced expression only by 24 hours post-treatment. Notably, neither IL-2 nor CCL5
led to increased GLUT-1 expression at earlier time points that corresponded with
enhanced glucose uptake. Given that GLUT-1 surface expression is strongly induced
upon T cell activation the effects of CCL5 on glucose uptake in the absence of a
concomitant increase in GLUT-1 expression might be anticipated. Indeed, published
75
studies using different diabetes models have demonstrated that alterations in glucose
uptake are not necessarily accompanied by changes in GLUT-1 or GLUT-4 expression
(Pedersen et al., 1990; Kahn et al., 1991). Thus, CCL5-mediated mTOR signaling may
increase the functionality or intrinsic activity of GLUT-1 to induce glucose uptake
without altering surface expression. The increase in GLUT-1 expression seen at 24 hours
may also reflect changes in transcriptional activation induced by CCL5 treatment.
Certainly, insulin is able to promote GLUT-1 protein expression by increasing GLUT-1
mRNA levels and GLUT1 gene transcription through Akt activation (Barthel et al., 1999).
Similar to the late induction of GLUT-1, CCL5 likewise resulted in enhanced
CD98 expression at 8 hour and 24 hour post-treatment, but not at earlier time points
(Figure 3.5.D.). Amino acid transport is mediated by CD98 when it forms disulfide-
linked hetero-dimers with other membrane-spanning light chains on the cell surface
(Edinger, 2007). CD98 is a ubiquitously expressed membrane protein that is strongly
induced following T cell activation, and also following IL-2, IL-15 or insulin treatment
(Deves and Boyd, 2000; Cornish et al., 2006; Edinger, 2007). In agreement with these
studies, CCL5 is also able to up-regulate the surface expression of CD98, albeit not to the
same extent as IL-2.
76
Figure 3.5. CCL5 increases cell surface expression of GLUT-1 and CD98
(A) Surface GLUT-1 levels and (B) CD98 levels were determined by FACs on freshly
isolated T cells or T cells activated with α-CD3 and α-CD28 for 2 days. (C) Activated
PB T cells were treated with 10 nM CCL5 or 20 ng/mL IL-2 for the indicated times.
Cells were fixed with 2% PFA and stained for cell surface GLUT-1 or (D) CD98
expression and analyzed by FACS. Data are representative of three independent studies.
77
Figure 3.5.
A B
78
C
GLUT-1
79
D
CD98
80
3.4. Glucose uptake and AMPK signaling are required for efficient CCL5-mediated
chemotaxis
To investigate whether CCL5-mediated glucose uptake is important for mediating
T cell chemotaxis, inhibition studies were performed using the glucose uptake inhibitor,
2-DG. As shown in Figure 3.6.A., pre-treatment with 2-DG reduced CCL5-mediated T
cell chemotaxis. These data suggest that efficient chemotaxis requires a steady supply of
glucose, and that glucose metabolism may contribute to CCL5-mediated cellular
migration of T cells. Next, the role of AMPK signaling was evaluated in chemokine-
induced chemotaxis. We examined the effects of the AMPK inhibitor, Compound C, on
CCL5-mediated T cell migration. The data reveal that AMPK inhibition reduced CCL5-
inducible T cell chemotaxis (Figure 3.6.B). The reduction in CCL5-mediated chemotaxis
by the inhibitors, 2-DG and Compound C, at the doses employed, was not due to any
cytotoxic effects (Figure 3.7.)
3.5. CCL5-induced AMPK signaling phosphorylates the 4E-BP1 repressor of mRNA
translation
The preceding data have suggested that CCL5 may activate both AMPK and
mTOR signaling simultaneously, despite AMPK being regarded as an inhibitor of mTOR
activity. Thus, the role of AMPK signaling in CCL5/mTOR-mediated phosphorylation of
4E-BP1 was examined. mTOR-mediated phosphorylation of 4E-BP1 is required for the
release of the initiation factor, eIF4E, and the de-repression of mRNA translation (Hay
and Sonenberg, 2004). In PB T cells, CCL5 induces the phosphorylation of 4E-BP1 in a
PI-3‟K/mTOR-dependent manner (Murooka et al., 2008). The role of AMPK signaling in
CCL5-dependent 4E-BP1 phosphorylation was determined using AICAR, an AMPK
81
Figure 3.6. Glucose uptake and AMPK signaling are required for efficient CCL5-
mediated chemotaxis
(A) Activated PB T cells were either left untreated or pretreated with 2-DG at the doses
indicated for 1 hr. A total of 1 x 105 cells in 100 μl chemotaxis buffer were then placed in
the upper chamber of Transwell chambers. CCL5-mediated chemotaxis was measured
using 10 nM CCL5. Data are presented as % migration, with the number of migrated
cells at 10 nM CCL5 taken as 100%. Data are representative of three independent
experiments. (B) Activated PB T cells were pretreated with either DMSO (carrier) or
different doses of Compound C for 1 hr. CCL5-mediated chemotaxis was measured as in
(A). Data are representative of three independent experiments. *p<0.01
82
Figure 3.6
83
Figure 3.7. Effects of 2-DG and Compound C on T cell viability
Activated T cells were either left untreated, treated with DMSO (carrier), 2-DG (A) or
Compound C (B) for the indicated times. Cell viability was determined by propidium
iodide staining analyzed by FACS. Cells negative for PI stain were considered viable.
84
Figure 3.7.
A
B
85
activator, and Compound C, an inhibitor of AMPK. As shown in Figure 3.8.,
pretreatment of PB T cells with Compound C reduced 4E-BP1 phosphorylation of
Thr37/46, which indicates AMPK signaling to be required for CCL5-mediated 4E-BP1
deactivation. Given 4E-BP1 to be an important downstream substrate of mTOR, these
data suggest that AMPK activation does not strictly inhibit mTOR activity and that
biological events invoked by CCL5, such as protein translation and migration, may
require both AMPK and mTOR to be active simultaneously.
86
Figure 3.8. CCL5-induced AMPK signaling phosphorylates the 4E-BP1 repressor
of mRNA translation
Activated PB T cells were pre-treated with either DMSO (carrier), 1 mM AICAR or 10
μM Compound C for an hour prior to 10 min treatment with 10 nM CCL5. Cells were
harvested and protein lysates resolved by SDS-PAGE and immunoblotted with anti-
phospho-4E-BP1 (Thr 37/46) antibodies. Membranes were stripped and re-probed for
4E-BP1 as loading control. Relative phosphorylation is shown as signal intensity over
loading control. Data are representative of two independent experiments. *p<0.05
87
Figure 3.8.
88
CHAPTER 4
DISCUSSION
89
Recruitment of immune cells to a site of infection is imperative for an effective
immune response. The infiltration of antigen-specific effector T cells is a highly
organized process that is co-ordinated by chemokines, and contributes to the clearance of
foreign pathogens. Activated T cells are able to navigate to sites of infection with the help
of inflammatory chemokines such as CCL5 that are deposited on the GAGs of
endothelial cells. T cell migration along a CCL5 gradient establishes cell polarization and
promotes directional migration through cytoskeletal rearrangements when CCL5
activates its cognate receptor, CCR5. In addition to promoting lymphocyte trafficking,
CCL5 also regulates a number of cellular processes including cell proliferation, protein
translation and T cell fate.
Previous studies have shown that CCL5/CCR5 signaling in primary T cells
activate the mTOR/4E-BP1 pathway to directly modulate mRNA translation (Murooka et
al., 2008). The ability of CCL5 to regulate protein translation is mediated by the
inhibition of 4E-BP1, an inhibitor of translation, and de-repression of the initiation factor,
eIF4E. Moreover, CCL5-mediated mTOR activation influences T cell chemotaxis by
initiating the translation of chemotaxis-related proteins, including MMP-9 and cyclin D1.
Taken together, CCL5 up-regulation of chemotaxis-related proteins may “prime” T cells
for efficient migration.
CCL5-mediated chemotaxis and mRNA translation, two processes that are
affected by mTOR, consume high levels of cellular energy (Hay and Sonenberg, 2004).
Indeed, mTOR is a central regulator of nutrient sensing, cell size and glycolysis. This
raised the possibility that CCL5 may be linked to cellular metabolism by activating
90
mTOR and directly regulating nutrient uptake. Its ability to do so may be required to
meet energy demands of migration and protein translation in activated T cells.
Initial studies investigated the activation of AMPK, the energy-sensing kinase that
is activated under conditions of energy stress. CCL5 induced the rapid
phosphorylation/activation of AMPK at its activation loop, in addition to the
phosphorylation of a number of downstream substrates including ACC1, PFKFB2 and
GSK-3β. The ability of CCL5 to induce AMPK activation suggests that intracellular
levels of ATP may be declining due to energy-taxing processes invoked by CCL5. As a
result, CCL5 may simultaneously initiate processes that attempt to increase intracellular
nutrient and energy levels, while suppressing cell growth and biosynthetic processes via
AMPK activation. Previous studies in various tissues have demonstrated that AMPK
acutely inhibits fatty acid and cholesterol synthesis by phosphorylating and inactivating
metabolic enzymes ACC1, SREBP1 and HMG-CoA reductase (Marsin et al., 2000; Li et
al., 2011). ACC1 is an enzyme that catalyzes the formation of essential substrates
necessary for fatty acid synthesis and is also a potent inhibitor of lipid oxidation
(Brownsey et al., 2006). AMPK-mediated phosphorylation of ACC1 allosterically
inactivates it and prevents the biosynthesis of fatty acids. Fat and liver cells treated with
adrenaline or glucagon – hormones released into the bloodstream during exercise, stress
and starvation – leads to the rapid phosphorylation and inactivation of ACC1 which is
predominately mediated by AMPK. Several RNA interference studies have also
demonstrated the importance of ACC1-mediated lipogenesis for the growth of various
tumor cell lines (Brusselmans et al., 2005; Chajes et al., 2006). Here, CCL5-mediated
phosphorylation of ACC1 may prevent lipid biosynthesis as a means to conserve energy
and limit cellular growth.
91
AMPK activity is also able to stimulate glycolysis through glucose transporter
translocation and phosphorylation/activation of the bi-functional enzyme, PFKFB2
(Marsin et al., 2000; Fujii et al., 2006). The first irreversible and commitment step in
glycolysis is the conversion of fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate
(F1,6BP) which is catalyzed by 6-phosphofructo-1-kinase (PFK-1). A potent stimulator
of PFK-1 is fructose-2,6-bisphosphate (F2,6BP), the concentration of which is controlled
by PFKFB2. PFKFB2 is a bi-functional enzyme that can act as a kinase and
phosphorylate F6P to F2,6BP or act as a phosphatase and dephosphorylate F2,6BP to F6P.
This kinase:phosphatase activity is regulated, in part, by the phosphorylation of Ser466
and Ser483 on PFKFB2 (Bertrand et al., 1999; Marsin et al., 2000; Carlet et al., 2010).
Specifically, phosphorylation of these sites synergistically promotes PFKFB2 affinity for
F6P and catalyzes F6P conversation to F2,6BP, whereas dephosphorylation of the same
residues increases the phosphatase activity. In both skeletal and cardiac muscles, insulin,
epinephrine, and ischemia are able to increase glycolysis and F2,6BP concentrations by
activating PFKFB2. CCL5-induced activation of PFKFB2 may contribute to ATP
production by increasing F2,6BP levels and, as a consequence, PFK-1 activity and thus
glycolysis.
CCL5 was also able to promote glucose uptake in an mTOR-dependent manner,
although this increase in nutrient uptake is not accompanied by changes in GLUT-1 nor
CD98 surface expression. Glucose transport across the plasma membrane of lymphocytes
is mediated by specific GLUT proteins: GLUT-1 is responsible for basal glucose
transport, while GLUT-3 and GLUT-4 mediate glucose uptake in response to insulin
stimulation (Calder et al., 2007). Upon activation during an immune response,
lymphocytes increase glucose utilization and rely heavily on glycolysis over oxidative
92
phosphorylation for the generation of ATP (Frauwirth and Thompson, 2004; Fox et al.,
2005). CD3/CD28 ligation is able to stimulate glucose transport, increase GLUT-1
surface expression and promote glycolysis via PI-3‟K/Akt signaling (Frauwirth et al.,
2002; Frauwirth and Thompson, 2004). Intriguingly, increased glucose transport can be
detected well before increased GLUT-1 expression, the major GLUT isoform expressed
on lymphocytes, suggestive that enhanced nutrient uptake is not necessarily accompanied
by increased transporter expression. Several studies in muscle cells, adipose tissues and
diabetic models have also demonstrated that hormone-induced changes in glucose uptake
can occur without affecting glucose transporter expression and translocation (Pedersen et
al., 1990; Kahn et al., 1991; Sweeny et al., 2001; Somwar et al., 2002). The CCL5-
stimulated glucose uptake in the absence of enhanced GLUT-1 expression that we
observe suggests that CCL5 may promote GLUT-1 intrinsic activity in order to promote
glucose uptake. CCL5 stimulation of activated PB T cells induced an up-regulation in the
surface expression of CD98, the heavy chain of the hetero-dimeric amino acid transporter
complex. Despite only a modest increase induced by CCL5, up-regulation of CD98
expression could subsequently modulate amino acid uptake and amino acid incorporation
into proteins. Given its ability to promote mTOR-dependent mRNA translation, CCL5
may also modulate the pool of amino acid „building blocks‟ required to fuel protein
synthesis by regulating CD98 expression.
As mentioned, mTORC1 integrates numerous intracellular and extracellular
signals and is a central regulator of metabolism, growth, cellular migration and protein
synthesis. Studies using the specific mTOR inhibitor, rapamycin, have underscored the
importance of mTORC1 signaling for glucose uptake (Wieman et al., 2007; Buller et al.,
2008). The Wieman group demonstrated that IL-3 dependent hematopoietic FL5.12 cells
93
activate the PI-3‟K/Akt/mTOR pathway following IL-3 treatment, to stimulate glucose
uptake and GLUT-1 trafficking. Interestingly, mTORC1 activity was not required to
maintain surface expression of GLUT-1, although inhibition of mTORC1 by rapamycin
greatly diminished IL-3 mediated glucose uptake. These data suggest that mTOR
signaling may only be required to promote GLUT-1 intrinsic activity to enhance glucose
uptake. We provide evidence that CCL5 is also able to induce glucose uptake in an
mTOR-dependent manner. Although rapamycin reduced CCL5-mediate glucose uptake,
this reduction in glucose uptake was less than that observed for 2-DG. It may be that
other mTORC1-independent mechanisms are also responsible for regulating metabolism
in activated T cells, including the MAPKs p38 (Somwar et al., 2002), ERK1/2 (Carr et al.,
2010) and other AMPK-signaling effector molecules (Finlay and Cantrell, 2011).
To investigate whether glucose uptake contributed to efficient CCL5-mediate
chemotaxis, the non-metabolized glucose analog, 2-DG, was employed. 2-DG is a potent
inhibitor of glucose metabolism and ATP production, and has been examined as a
chemotherapeutic agent, given that cancerous cells exhibit an increased rate of glucose
uptake (Aft et al., 2002). Prolonged 2-DG treatment in various cancer cell lines interferes
with glycolysis, contributing to decreased cell growth, decreased clonogenictiy and
enhanced apoptosis through caspase-3 release. Notably, our chemotactic studies using 2-
DG avoided prolonged drug exposure to avoid toxicity and cell death. Glucose uptake
inhibition by 2-DG pre-treatment reduced the ability of T cells to migrate towards a
CCL5 gradient, in a dose-dependent manner. Proliferating lymphocytes depend on
growth factor signals to promote glucose uptake to maintain survival (Fox et al., 2005).
Even in the presence of alternative energy sources, such as glutamine, T cells maintained
in glucose-free medium fail to proliferate, underscoring the essential and non-redundant
94
role of glucose in supporting T cell viability. In the present study, the inability of effector
T cells to take up glucose had an impact on migration. Previous studies have
demonstrated that the ability of tumor cells to metastasize to secondary sites in response
to a chemoattractant is also dependent on active glycolysis (Beckner et al., 1990;
Kroemer and Pouyssegur, 2008).
For optimal T cell migration orchestrated by CCL5, we hypothesized that AMPK
stimulation of ATP-generating processes may be necessary. CCL5-mediated T cell
chemotaxis was examined following AMPK inhibition by Compound C. Indeed,
Compound C pre-treatment reduced CCL5-mediated chemotaxis in a dose-dependent
manner, suggesting that T cell migration in response to CCL5 is partially dependent on
AMPK signaling. Importantly, while AMPK is most well known for its role as an energy
sensor, AMPK signaling also regulates a number of non-metabolic processes, including
cell division, cell polarity and directional cell migration (Williams and Brenman, 2008;
Nakano et al., 2010). Recent studies have demonstrated that AMPK activation and
signaling to downstream substrates are able to regulate microtubule dynamics and cell
polarization to promote migration in the 293 T cell line (Nakano et al., 2010). Moreover,
F-actin polymerization and Rho GTPase activation were reported following AMPK
activation in epithelial MDCK cells (Miranda et al., 2010). AMPK inhibition by
Compound C may prevent processes that directly promote CCL5-mediated chemotaxis or
indirectly affect ATP-generation. Collectively, these data suggest that both glucose
metabolism and AMPK signaling have roles in efficient T cell migration.
Studies undertaken in this thesis have identified AMPK as a novel downstream
substrate of CCL5 signaling in activated T cells. In addition, the present studies have
identified a role for CCL5-mediated mTOR signaling in modulating glucose metabolism
95
by enhancing glucose uptake. Taken together, CCL5 may simultaneously induce
signaling events in both the mTORC1 and AMPK pathways. Intriguingly, whereas
AMPK is active under nutrient-poor conditions, mTOR is active during energy-rich
coniditions. More importantly, the current literature indicates that AMPK activation
during energy deprivation indirectly suppresses mTOR activity by
phosphorylating/activating TSC2 (Inoki et al., 2003), or directly inactivates mTOR by
targeting its Raptor subunit (Gwinn et al., 2008). Data generated herein suggest that
CCL5 is able to activate both pathways simultaneously in order to maintain homeostasis:
mTOR-dependent processes such as protein translation and chemotaxis are energy taxing
(Hay and Sonenberg, 2004), which may require AMPK signaling to initiate ATP-
generating processes. AMPK-mediated inhibition of fatty acid biosynthesis together with
its ability to stimulate glycolysis may generate the energy needed to fuel CCL5-mediated
processes during an immune response.
Chemokines are critical for the successful recruitment of leukocytes to sites of
inflammation during an immune response. Certainly, the mTOR signaling cascade
orchestrates aspects of cellular migration including mRNA translation of chemotaxis-
related proteins (Murooka et al., 2008) and the expression of lymph node homing
receptors, CD62L, CCR7 and CXCR4 (Sinclair et al., 2008). The present studies
addressed the cross-talk between lymphocyte migration and cellular metabolism and have
shown that the inflammatory chemokine, CCL5, exhibits functions beyond that of a
chemotactic cytokine. In addition to its ability to direct PB T cell migration, CCL5-
mediated mTOR activation is also able to modulate cellular metabolism by directly
regulating glucose uptake in order to match the energy demands of chemotaxis.
96
Figure 4.1. Illustration of the AMPK and mTOR signaling cascades
AMPK activation upon energy stress leads to the activation of ATP-generating processes
such as glycolysis, and the inhibition of ATP-consuming processes such as fatty acid
synthesis. CCL5-mediated PI‟3-K/Akt/mTOR signaling results in the phosphorylation of
p70 S6K1 and 4E-BP1. Hyper-phosphorylation of 4E-BP1 leads to the release of eIF4E
and the formation of the initiation complex for protein translation. A possible model for
CCL5-mediated metabolic changes in activated T cells may be through the simultaneous
activation of both the AMPK and mTOR signaling pathway.
97
Figure 4.1.
98
CHAPTER 5
FUTURE DIRECTIONS
99
A complex network of chemokines and chemokine receptors influence the growth
and progression of many cancers. Tumor-associated chemokines are able to promote
tumor growth directly by stimulating proliferation and/or survival, or indirectly by
initiating angiogenesis (Vicari and Caux, 2002; Balkwill, 2004; Murooka et al., 2009).
CCL5 is highly expressed in the microenvironment of breast and prostate cancers. Plasma
levels of CCL5 are correlated with breast cancer disease severity, where patients with a
more advanced disease express increased levels of CCL5 compared to patients who are in
clinical remission (Adler et al., 2003; Yaal-Hahoshen et al., 2006). Various aspects of
tumorigenesis are regulated by CCL5, including leukocyte infiltration into primary
tumors, metastasis, tumor growth and survival (Niwa et al., 2001; Azenshtein et al., 2002;
Murooka et al., 2009). The ability of CCL5/CCR5 signaling to modulate breast cancer
cell metabolism has not been addressed. Previous studies have demonstrated that CCR5
expression on the breast cancer cell line, MCF-7, is able to enhance mTOR-dependent
proliferation of cells when cultured in the presence of CCL5 (Murooka et al., 2009).
Moreover, CCL5-mediated mTOR signaling also enhanced mRNA translation of pro-
survival proteins cyclin D1, c-Myc and defender against cell death-1 (DAD-1).
Collectively, the proto-oncogenic role of CCL5 can be attributed, in part, to its ability to
promote mTOR-dependent mRNA translation in breast cancer cells. Further studies are
required to determine whether CCL5-mediated mTOR signaling can affect cancer cell
metabolism and nutrient uptake.
To elucidate CCL5-mediated metabolic changes in breast cancer cells, CCR5
expressing MCF-7 cells will be employed to measure changes in glucose uptake invoked
by CCL5. We hypothesize that CCL5 may provide a proliferative advantage in cancer
100
cells by stimulating nutrient uptake and promoting glucose metabolism to fuel the high
rate of proliferation. The role of CCL5-CCR5 mediated PI-3‟K/Akt/mTOR and AMPK
signaling in glucose uptake will be addressed using the appropriate pharmacological
inhibitors. If, indeed, CCL5 can promote enhanced glucose uptake in breast cancer cells,
it would be intriguing to determine if this can further support breast cancer metastasis and
invasion. Cell invasion studies using Matrigel-coated Transwell chambers, together with
glucose uptake inhibitors and signaling inhibitors will address if CCL5-mediated glucose
uptake is necessary for MCF-7 invasion.
We report herein the ability of CCL5 to up-regulate glucose uptake in PB CD3+ T
cells and its importance in mediating chemotaxis. Further studies are required to
determine whether CCL5 can regulate the catalytic activity of key glycolytic enzymes in
order to promote glucose metabolism and ATP generation for effector T cell functions.
Specifically, enzymatic activity of hexokinase (HXK) and phosphofructokinase-1 (PFK-
1) will be measured to access if CCL5 can enhance glycolysis in addition to glucose
uptake. Furthermore, the rates of oxygen consumption and glycolysis can be measured
using the Seahorse Bioscience platform. Seahorse Bioscience provides a means of
measuring cellular metabolism and metabolic activity in real-time. This platform is a
non-invasive and non-destructive way of determining the rate of oxygen consumption,
which is a measure of oxidative phosphorylation, and the rate of acidification or lactate
production, which is a measure of glycolysis. The ability of CCL5 to modulate cellular
metabolism of PB CD3+ T cells and MCF-7 can be determined, in real-time, with the XF
Analyser.
Finally, we have demonstrated the ability of CCL5 to simultaneously activate
mTOR and AMPK signaling pathways in PB CD3+ T cells, even though the current
101
literature indicates that AMPK activation is able to suppress mTOR signaling (Inoki et al.,
2003; Gwinn et al., 2008). We propose that homeostasis is maintained when both mTOR
and AMPK are activated in conjunction. However, these homeostatic signaling cascades
may be disrupted in the context of breast cancer cells that have a proliferative advantage
in response to CCL5. The activation/signaling profile of mTOR and AMPK in CCL5-
stimulated breast cancer cells can be addressed using Western immunoblotting studies.
These studies will address if CCL5, a chemokine prevalent in the microenvironment of
breast cancer tumors, can enhance proliferation by altering signaling pathways that
regulate nutrient uptake and growth.
102
CHAPTER 6
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