by
University of Toronto
University of Toronto
Chemokines are responsible for directing leukocyte migration and triggering firm
critical biological roles beyond chemo-attraction. Throughout this thesis, I describe the
importance of the CCL5/CCR5 axis in the context of the immune response and cancer
mediate CD4+ T cell migration that is partially dependent on mTOR activation. CCL5
provide evidence that CCL5 initiates rapid translation of cyclin D1 and MMP-9, known
related proteins may “prime” T cells for efficient migration. During an immune response,
recently recruited T cells are exposed to high CCL5 concentrations. The propensity of
investigate their effects on T cell function. I show that at these high doses, CCL5 induces
ii
phosphorylation signalling events are important in CCL5-mediated apoptosis. Our data
suggest that CCL5-induced cell death, in addition to Fas/FasL mediated events, may
abolished by anti-CCR5 antibody and rapamycin. CCL5 induces the formation of the
eIF4F translation initiation complex, and mediates a rapid up-regulation of cyclin D1, c-
Myc and Dad-1 protein expression. Thus, our data demonstrate the potential for breast
proliferative and survival advantage. Taken altogether, each of these studies reinforces
the notion that chemokines are not only potent chemotactic mediators, but are key
iii
ACKNOWLEDGEMENTS
Thank you, Eleanor, for being so supportive of me through the years. You always guided
me in the right direction, and gave me words of encouragement through the lean times.
Thank you for always being accessible, and taking the time to discuss my research plans.
I am especially thankful for taking me to multiple international meetings, more than any
laboratory in the department. You encouraged me to give oral presentations, and by
doing so, I now have the confidence and experience to speak in front of an audience.
Your continued commitment to your family and the scientific community is contagious,
and look forward to working with you in the near future.
To all past and present Fish Pond members, thank you for all your scientific and
emotional support through the years. Beata, you were there for me from the very
beginning, and took this skinny (well, skinnier) and bewildered student from Vancouver
under your wing. Thank you for teaching me everything I know and for being such a
great friend. Jiabing, thank you for teaching me the art of molecular biology, and for
being such a calm influence in the lab. Jyothi, Raj, Anna, Joanna, Celeste and Melissa, it
was such a privilege to work with all of you, and I enjoyed being the only guy in the lab
(at the time). Thanks for making me feel like a part of the team and giving me a crash
course on female psychology (I listened attentively but forgot most of it). Sham, I
enjoyed working with you and I wish you luck with your medical career. Ramtin, I’m
glad you decided to join the Fish lab, and I knew we would get along from the moment
we shook hands. Friends usually encourage you, but only true friends challenge you and
point out your flaws, and that’s exactly what you did. I wouldn’t have been as successful
without your support, and I leave here, but never leave behind, my true friend, confidant,
and scientific partner. Carole, I’m going to miss all your unanswerable questions, but
I’m always a phone call away! Thank you for being such a great mentor and friend, and
will definitely miss Kaycee and Kip. Behnam, I enjoyed working with you, but more so
our time outside the lab. I wish you all the luck with your studies and your backhand.
Danlin, thank you for all your input and help throughout my studies, and I look forward
to working with you in the future. Just make sure you don’t develop a drinking habit.
Daniel, I really enjoyed working with you also, and our many discussions on protein
translation. I think you’re well on your way towards obtaining your Ph.D., as long as
Tim Horton’s doesn’t file for Chapter 11. Cole, thank you for all your help in the lab, but
telling me you’re CCR5Δ32 homozygous AFTER leaving the lab didn’t help. Erin, it’s
been a short time, but a blast! I hear the CBS ghosts don’t bother you if you keep smiling.
Joanne, you’re like a rainbow on a rainy day, and I wish you luck with your future studies.
Olivia, I enjoyed our short time working together, and am confident that you will take
this project to new heights.
iv
To my mom, dad and Arnold, thank you for all your emotional and financial support over
the years. Knowing that I could fly back to Vancouver and get some TLC (tender love
and care) from my family was all the motivation I needed during my studies. Mom, you
are an incredibly courageous woman, and even with all the pain you continue to suffer,
you always manage to give me encouragement and comfort. Dad, you always put a smile
on my face and I thank you for all your support. Your stock tips, however, is at best
50/50, equivalent to a coin toss. Arnold, thank you for taking care of the family back
home, and I see tremendous growth in you while I was away. Yes, I will practice my
golf game more, but I rather cheat to beat you.
Ada, you held my hand through the tougher stretches of my studies, and for that I will be
eternally grateful. Knowing that I can count on your at any time means the world to me,
and we have a lifetime of pillow talks to look forward to. I do hope your carpal tunnel on
your left wrist can handle some more extra weight on your finger. Mr. and Mrs. Man, I
am truly grateful for feeding me and supporting me over the course of my studies. I look
forward for more discussions and meals with you, but I’ll treat this time.
I want to thank the “1002” boys, Jeff and Cliff for their friendship during our Toronto
days, and I wish you two nothing but the best. See you guys at the top! I also thank
“turtle” and my late cat, Tama, for many good times.
Finally, I would like to thank the chair, all faculty members, graduate coordinators and
fellow students in the Immunology department for all their support and friendship, and
look forward to working with all of you again in the future. Playing left field for the
Immunology softball team was truly a blast!
v
TABLE OF CONTENTS
Title Page ……………………………………………………………………………...…..i
Abstract …………………………………………………………………………………..ii
Acknowledgements …………………………………………………………...………….iv
Table of Contents …………………………………………………………………….….vi
List of Figures ……………………………………………………………………...…….x
List of Tables ……………………………………………………………………..……..xii
List of Abbreviations ………………………………………………………….……...xiii
vi
1.4.3. Human Immunodeficiency Virus (HIV) Infection …………..….66
2.4. Results
2.4.1. µM concentrations of CCL5 induce apoptosis in CCR5 expressing T
cells ………………………………………………………………80
2.4.2. CCL5 induced cell death is mediated by the mitochondrial and
apoptosome pathway ………………………………………...…..80
2.4.3. µM concentrations of CCL5 induce apoptosis in CCR5 expressing
primary T cells ……………………………………………..……92
2.4.4. Expression of intact CCR5, but not CCR5Y339F, renders PM1 cells
susceptible to CCL5-inducible apoptosis …………………..……92
2.4.5. CCL5-induced cell death is dependent on GAG interactions ….…95
2.4.6. Aggregation of CCL5 is required for CCL5-induced cell death .104
vii
3.3.4. Chemotaxis Assay …………………………………………...…121
3.3.5. Semi-quantitative RT-PCR ………………………………….…122
3.3.6. Polysome gradients ………………………………………….…122
3.3.7. Statistical Analysis …………………………………………..…123
3.4. Results
3.4.1. CCL5-mediated chemotaxis of activated CD4+ T cells is mTOR
dependent ……………………………………………………….124
3.4.2. CCL5 induces phosphorylation of mTOR, p70 S6 kinase and S6
ribosomal protein …………………………………………….…129
3.4.3. CCL5-mediated 4E-BP1 phosphorylation is PI-3’K-, PLD- and
mTOR- dependent …………………………………………...…134
3.4.4. CCL5 initiates protein translation through formation of the eIF4F
complex ……………………………………………………...…134
3.4.5. CCL5-inducible protein translation of cyclin D1 and MMP-9 is
mTOR-dependent ……………………………………………....144
4.4. Results
4.4.1. CCL5-CCR5 inducible MCF-7 proliferation is dependent on
mTOR ……………………………………………………….…165
4.4.2. CCL5 activation of CCR5 leads to the formation of the eIF4F
complex through mTOR …………………………………….…168
4.4.3. CCL5 induces protein translation of proliferation and survival
proteins …………………………………………………………171
4.4.4. CCL5 facilitates recruitment of a subset of mRNAs to
polysomes .………………………………………………..……174
viii
CHAPTER 5: Discussion and Future Directions …………………………...…182-202
ix
LIST OF FIGURES
CHAPTER 1
CHAPTER 2
CHAPTER 3
x
Figure 3.9. CCL5-inducible upregulation of cyclin D1 and MMP-9 protein levels
is dependent on mTOR-mediated mRNA translation …………..…145
Figure 3.10. Possible model for CCL5-mediated mRNA translation in CD4+
Tcells ……………………………………………………………....152
CHAPTER 4
CHAPTER 5
xi
LIST OF TABLES
CHAPTER 1
xii
LIST OF ABBREVIATIONS
Ab Antibody
ADP Adenosine diphosphate
AICD Activation induced cell death
AOP-CCL5 Aminooxypentane-CC chemokine ligand 5
APC Antigen presenting cell
Arp2/3 Actin-related proteins 2/3
Bcl-2 B cell lymphoma-2
bp Base pair
BRET Bioluminescence resonance energy transfer
CCL5 CC chemokine ligand 5
CCR5 CC chemokine receptor 5
CCX-CKR ChemoCentryx chemokine receptor
Cdc42 Cell division cycle 42
c-Myc Cellular-myelocytomatosis virus oncogene
CS Chondroitin sulphate
C-terminus Carboxy-terminus
CTL Cytotoxic T lymphocyte
CXCL CXC chemokine ligand
CXCR CXC chemokine receptor
CX3CL CX3C chemokine ligand
CX3CR CX3C chemokine receptor
DAD Defender against cell death
DAG Diaceylglyerol
DARC Duffy antigen receptor for chemokines
DC Dendritic cell
DNA Deoxyribonucleic acid
DPG Diphosphoglycerate
DRY Aspartate-Arginine-Tyrosine
DS Dermatan sulphate
DTT Dithiothreitol
ECL Extra-cellular loop
EDTA Ethylenediamine tetra-acetic acid
EGTA Ethylene glycol-bis (2-aminoethylether)-N’N’N’N’-tetra-acetic
acid
EGF Epidermal growth factor
eIF Eukaryotic translation initiation factor
ELR Glutamate-Leucine-Arginine
ERK Extracellular signal-related kinase
F-actin Filamentous actin
FACS Flourescence activated cell sorter
FAK Focal adhesion kinase
FasL Fas antigen ligand
FCS Fetal calf serum
xiii
FITC Flourescein isothiocynate
FKBP12 FK506-binding protein of 12kDa
FLF Fulminant liver failure
FRAP/mTOR FKBP 12-rapamycin-associated protein/mammalian target of
rapamycin
FRET Fluorescence resonance energy transfer
GABA γ-aminobutyric acid
GAG Glycosaminoglycan
GAP GTPase activating protein
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
GDP Guanosine diphosphate
GEF Guanidine nucleotide exchange factor
GFP Green fluorescent protein
GM-CSF Granulocyte-macrophage colony-stimulating factor
gp120 Glycoprotein of 120kDa
GPCR G-protein coupled receptor
GRK G-protein receptor kinase
GTP Guanosine triphosphate
HA Hyaluronic acid
HEK Human embryonic kidney
HEV High endothelial venule
HIV Human immunodeficiency virus
HLA Human leukocyte antigen
HRP Horseradish peroxidase
HS Heparin sulphate
IP3 Inositol 1,4,5-phosphate
Jnk c-Jun N-terminal kinase
kDa Kilodalton
KS Keratin sulphate
KSHV Karposi’s sarcoma-associated herpes virus
ICAM Intracellular adhesion molecule
IFN Interferon
IL Interleukin
IP Immunoprecipitation
IRES Internal ribosomal entry segment
Jak Janus kinase
LFA Lymphocyte function-associated antigen
LPS Lipopolysaccharide
MAPK Mitogen-activated protein kinase
MCP Macrophage chemo-attractant protein
MEF Murine embryonic fibroblast
Met-CCL5 Methionine-CC chemokine ligand 5
Met-tRNA Methionine-transfer ribonucleic acid
MHC Major histocompatibility complex
MIP Macrophage inflammatory protein
MLCK Myosin light chain kinase
xiv
µM Micromolar
MMP Matrix metalloproteinase
mRNA Messenger RNA
mTOR Mammalian target of rapamycin
mTORC1 Mammalian target of rapamycin complex 1
NF-κB Nuclear factor-kappa B
NK Natural killer
NMR Nuclear magnetic resonance
nM Nanomolar
NP-40 Nonidet-40
N-terminus Amino-terminus
OD Optical density
OX-PHOS Oxidative phosphorylation
p38 38kDa stress-activated kinase
PA Phosphatidic acid
PARP Poly ADP ribose polymerase
PBS Phosphate buffered saline
PCR Polymerase chain reaction
PDK Phosphoinositide-dependent kinase
PGE2 Prostaglandin E2
PH Pleckstrin homology
PHA Phytohaemagglutinin
PHAS Phosphorylated heat and acid soluble protein
PI Propidium iodide
PI-3’K Phosphatidylinositol 3-kinase
PIKK Phosphoinositide kinase-related kinase
PIP3 Phosphatidylinositol 3,4,5-phosphate
PKB Protein kinase B
PKC Protein kinase C
PKR Protein kinase R
PLCβ Phospholipase Cβ
PLD Phospholipase D
PMA Phorbol-12-miristate-13-acetate
PMSF Phenylmethylsulfonylflouride
PRR Pattern-recognition receptors
PTEN Phosphatase and tensin homolog deleted in chromosome ten
pTx Pertussis toxin
RA Rheumatoid arthritis
Rac Ras-related C3 botulinum toxin substrate
RANTES Regulated on activation normal T cell expressed and secreted
Raptor Regulatory associated protein of mTOR
Rheb Ras-homolog enriched in brain
Rho Ras homolog gene family
Rictor Rapamycin-insensitive companion of mammalian target of
rapamycin
RNA Ribonucleic acid
xv
ROCK Rho kinase
ROS Reactive oxygen species
rpS6 Ribosomal protein S6
RT-PCR Reverse transcription-polymerase chain reaction
SDF Stromal derived factor
SDS Sodium dodecyl sulphate
SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
SH2 Src-homology 2
SHIP Src-homology 2 domain-containing inositol phosphatase
S6K S6 kinase
Stat Signal transducer and activator of transcription
TAM Tumor associated macrophages
TBS Tris buffered saline
TCR T cell receptor
Th T helper
TIL Tumor infiltrating T lymphocytes
TLR Toll-like receptor
TM Trans-membrane
TNFα Tumor necrosis factor α
TNFR Tumor necrosis factor receptor α
TOP Tract of oligopyrimidines
TRAIL TNF-related apoptosis-inducing ligand
TSC Tuberous sclerosis complex
TXP Threonine-X-Proline
UTR Untranslated region
VCAM Vascular cell adhesion molecule
VEGF Vascular endothelial growth factor
VLA Very late antigen
WASp Wiskott-Aldrich syndrome protein
WAVE/Scar Wiskott-Aldrich syndrome protein family verprolin-homologous
protein/suppressor of cyclic adenosine monophosphate receptor
XCL XC chemokine ligand
XCR XC chemokine receptor
ZAP-70 Zeta-associated protein tyrosine kinase of 70kDa
4E-BP 4E-binding protein
7-AAD 7-amino actinomycin D
xvi
Chapter 1
Introduction
Murooka, T.T., Ward, S.E., and Fish, E.N. (2005). Chemokines and cancer. Cancer
Treat Res 126, 15-44.
Galligan C.L., Murooka, T.T., Rahbar, R., Baig, E., Majchrzak-Kita, B., and Fish, E.N.
(2006). Interferons and viruses: signalling for supremacy. Immunol Res 35, 27-40.
1.1. Chemokine Superfamily
1.1.1. Classification
The chemokines are soluble, small molecular weight (8-14 kDa) and basic
cytokines that bind to their cognate seven trans-membrane G-protein coupled receptors
(GPCRs) to elicit directed cell migration. Since their initial discovery almost 30 years
ago, approximately 47 human chemokines have been identified to date (Table 1.1). They
are separated into four sub-families based on the relative positioning and presence of the
first two cysteine residues at the N-terminus (Zlotnik and Yoshie, 2000). The cysteine
residues in CXC chemokines are separated by one non-conserved amino acid, whereas in
CC chemokines, the first two cysteine residues are adjacent. The XC chemokines lack
the first consensus cysteine, whereas the CX3C chemokine CX3CL1 is characterized by
three non-conserved amino acids between the first two cysteine residues. In 2000, a
system of nomenclature was introduced in which each ligand and receptor is identified by
its sub-family and given an identifying number (Bacon et al., 2002; Murphy et al., 2000).
For example, the CXC chemokine SDF-1α (stromal-derived factor 1α) is now known as
CXCL12 for CXC chemokine ligand 12, and the CC chemokine RANTES (regulated on
activation normal T cell expressed and secreted) is now known as CCL5 for CC
chemokine ligand 5. Throughout this thesis, chemokine ligands and receptors will be
referred to by the new nomenclature, with their corresponding original names found in
Table 1.1. This thesis will review our general understanding of chemokine/chemokine
receptor structure and function, with a major emphasis on the CC chemokine CCL5 and
2
Table 1.1. The Chemokine Superfamily and Nomenclature
CXC Chemokines
CXCL1 Groα/MGSAα Gro/KC CXCR2, CXCR1
CXCL2 Groβ/MGSAβ MIP-2 CXCR2
CXCL3 Groγ Dcip CXCR2
CXCL4 PF4 PF4 CXCR3b
CXCL5 ENA-78 LIX CXCR2
CXCL6 GCP-2 CXCR1, CXCR2
CXCL7 NAP-2 Ppbp CXCR2
CXCL8 IL-8 CXCR1, CXCR2
CXCL9 MIG MIG CXCR3, CXCR3b
CXCL10 IP-10 IP-10 CXCR3, CXCR3b
CXCL11 I-TAC I-TAC CXCR3, CXCR3b, CXCR7
CXCL12 SDF-1α/β SDF-1α/β CXCR4, CXCR7
CXCL13 BLC, BCA-1 BLC, BCA-1 CXCR5
CXCL14 BRAK, Bolekine BRAK, Boleine Unknown
CXCL15 none Lungkine Unknown
CXCL16 none CXCL16 CXCR6
CXCL17 DMC DMC Unknown
CC Chemokines
CCL1 I-309 TCA-3 CCR8
CCL2 MCP-1 JE CCR2
CCL3 MIP-1α/LD78α MIP-1α CCR1, CCR5
CCL4 MIP-1β MIP-1β CCR5
CCL5 RANTES RANTES CCR1, CCR3, CCR5
CCL7 MCP-3 MARC CCR1, CCR2, CCR3
CCL8 MCP-2 MCP-2, MCP-5 CCR1, CCR2, CCR3, CCR5
CCL11 Eotaxin Eotaxin CCR3
CCL13 MCP-4 CCR1, CCR2, CCR3
CCL14 HCC-1 CCR1
CCL15 HCC-2/LKN1/MIP-1γ CCL9, MIP-1γ CCR1, CCR3
CCL16 HCC-4/LEC/LCC-1 CCR1, CCR2, CCR5
CCL17 TARC TARC CCR4
CCL18 DC-CK1/PARC/AMAC-1 Unknown
CCL19 MIP-3β/ELC MIP-13β CCR7
CCL20 MIP-3β/LARC MIP-α/LARC CCR6
CCL21 SLC/6Ckinase CCL21a, b, c/SLC CCR7
CCL22 MDC/STCP-1 ABCD-1 CCR4
CCL23 MPIF/CKβ8 CCL6/C10 CCR1
CCL24 Eotaxin-2/MPIF-2 Eotaxin-2 CCR3
CCL25 TECK TECK CCR9
CCL26 Eotaxin-3 CCL26l CCR3
CCL27 CTACK/ILC CTACK/ILC CCR10
CCL28 MEC MEC CCR3, CCR10
C Chemokines
XCL1 Lymphotactin/SCM-1α Lymphotactin XCR1
XCL2 SCM-1β XCR1
CX3C Chemokine
CX3CL1 Fractalkine Fractalkine CX3CR1
3
Figure 1.1 Chemokines share similar structural elements
Overlayed monomeric minimized mean structure of CCL2 (yellow), CCL5 (blue) and
CCL11 (red) shows similar structural elements despite a low level of sequence homology.
4
C-terminal α-helix
30s loop
β1
N-loop
N-terminus
β2
β3
310 helix
40s loop
5
Chemokines are also functionally classified as homeostasis or inflammation.
and the migration of cells to and within secondary lymphoid organs (Moser et al., 2004).
Most chemokines are secreted from the cell, with the exception of CX3CL1 and CXCL16,
which are tethered to the extracellular surface through a trans-membrane stalk (Zlotnik
and Yoshie, 2000). These chemokines can also be released in soluble form after
suggesting that other chemokine receptors may compensate for the lack of CCR5 (Zhou
et al., 1998).
The CXC chemokines can be further subdivided into ELR+ and ELR-
motif preceding the CXC sequence. ELR+ chemokines are potent promoters of
CXCL3, CXCL5, CXCL6, CXCL7 and CXCL8 are all ELR+ chemokines, with CXCL12
the only ELR- chemokine with angiogenic properties (Luker and Luker, 2006; Moser et
al., 2004; Orimo et al., 2005). The role of chemokines in angiogenesis is discussed in
6
1.1.2. Chemokine Structure
resonance (NMR) and/or X-ray crystallography. Studies have revealed that the three
dimensional structure of CCL5 is similar to that of CCL2, CCL3, CCL4 and CXCL8,
despite a relatively low level of sequence homology (Baldwin et al., 1991; Czaplewski et
al., 1999; Handel and Domaille, 1996; Lodi et al., 1994) (Figure 1.1). This “chemokine
fold” structure consists of three anti-parallel β-strands (β(1), β(2) and β(3)) overlaid by a C-
terminal α-helix. Upstream of the β-sheets is the flexible N-terminal region, followed by
a long N-loop and a short 310 helix. Two characteristic disulphide bridges between the
first and third, and the second and fourth cysteine residues stabilize the three dimensional
activation, since modifications in this region have been shown to affect function (Gong
and Clark-Lewis, 1995; Jarnagin et al., 1999; Mizoue et al., 2001). In some instances, N-
(Met-CCL5) and CCL2 (Met-CCL2) both produced antagonists for CCR5 and CCR2,
et al., 2000; Simmons et al., 1997). In addition to the N-terminus, the N-loop between
the first two cysteines and the 310 helix contains residues involved in receptor binding
(Crump et al., 1997; Pakianathan et al., 1997). Taken together, in a hypothesized two-site
model of chemokine receptor activation, the core domain of chemokines (which differs
7
for each chemokine) binds to the extracellular loops of the receptor to help position the
N-terminal signalling domain of the ligand within the helical bundle of the receptor.
chemokines with N-terminal modifications have been identified (Proost et al., 2006). The
bioactivity. For example, the serine protease CD26 (also known as dipeptidyl peptidase
IV) is capable of mediating N-teriminal CCL5 cleavage, resulting in a CCL5 variant (3-
68) that exhibited reduced chemotactic and intracellular calcium mobilization ability
(Proost et al., 1998; Struyf et al., 1998). Thus, by altering the N-terminus, proteases can
alter chemokine function by directly affecting receptor binding. The data demonstrate the
It has been known for some time that chemokines form oligomers in solution, but
through residues near their N-terminus surrounding the first two cysteine residues, while
CXC chemokines predominantly dimerize through residues in the first strand of β(1)
(Proudfoot, 2006). Intriguingly, CCL5 not only forms dimers, but has a tendency to
Extensive mutational studies have produced mutant CCL5 molecules that display unique
8
nitrogen ([Nme-7T]-CCL5), is monomeric and does not oligomerize on immobilized
(Proudfoot et al., 2003). However, when tested in vivo using a peritoneal recruitment
assay, [Nme-7T]-CCL5 failed to recruit cells. In the same study, the dimeric [E66S]-
CCL5 mutant, but not the tetrameric [E26A]-CCL5 mutant, failed to recruit cells in vivo,
although both retained chemotactic ability in vitro. The data suggest that not only is
CCL5 aggregation required for biological activity in vivo, but a minimal quaternary
chemotaxis with wildtype potency and efficacy in vitro, while failing to do so in vivo
immobilize and concentrate chemokines at tissue sites. GAGs are normally attached to
proteins on the cell surface and/or the extracellular matrix to form proteoglycans
(Proudfoot, 2006). GAGs are widely diverse, and consist of repeating disaccharide units
sulphation patterns. A common feature of GAGs is their overall negative charge due to
the density of sulphate and carboxylate groups on the GAG chains. This suggests an
electrostatic interaction with the basic, positively charged chemokines (Kuschert et al.,
1999). There are several classes of GAGs, the most ubiquitous being heparin sulphate
9
(HS), a polysaccharide that is expressed on virtually every cell in the body. Others
include heparin, produced almost exclusively by mast cells; chondroitin sulphate (CS)
and dermatan sulphate (DS), found on cell surfaces and the extracellular matrix; keratin
sulphate (KS), found as part of the cornea and cartilage; and hyaluronic acid (HA).
Interestingly, chemokines have been shown to have a hierarchical preference for GAGs.
For example, CCL5 binding affinity for different GAGs was determined as heparin > DS
interactions may have important implications in vivo (Kuschert et al., 1999). The GAG
binding residues on various chemokines have been identified, described as XBBXBX and
XBBBXXBX (where B is a basic amino acid and X is any amino acid). In some cases,
the GAG binding epitopes can overlap with the receptor binding domains (Hileman et al.,
1998). Specific residues critical for GAG binding of chemokines CCL2, CCL3 and
CCL4 have now been identified (Chakravarty et al., 1998; Koopmann et al., 1999;
Koopmann and Krangel, 1997; Lau et al., 2004; Laurence et al., 2001; Martin et al.,
2001; Sadir et al., 2001; Vita et al., 2002). Proudfoot and colleagues identified the
heparin-binding BBXB motif found within the 40s loop for CCL5. An alanine mutant,
recruitment activity in vivo, although in vitro activity was retained (Proudfoot et al.,
2003; Shaw et al., 2004). Intriguingly, mixing both [44AANA47]-CCL5 and intact CCL5
resulted in heterodimers that were unable to recruit cells into the peritoneal cavity in vivo
10
chemokine aggregation, local retention and the establishment of a chemokine
(Amara et al., 1999; Cinamon et al., 2001; Kuschert et al., 1999; Netelenbos et al., 2002;
Pablos et al., 2003; Proudfoot et al., 2003). These immobilized chemokines allow
chemokine receptors (GPCRs). In most cases, ligand binding causes the dissociation of
Gαi from the Gβγ subunit of the heterotrimeric G-proteins, leading to the activation of a
Clayman, 2001; Richmond, 2002; Rossi and Zlotnik, 2000). Specifically, PLCβ
by pertussis toxin (PTx), a bacterial toxin that catalyzes the ADP-ribosylation of the Gαi
have been reported to associate with other PTx-insensitive G-proteins, including Gq/11 or
G16, (Mellado et al., 2001b). Furthermore, CCR2 and CCR5 have been demonstrated to
11
1.1.4.1. Jak-Stat Pathway
The Jak-Stat pathway is the principle signalling mechanism for many cytokines
and growth factors. It is clear through numerous studies that chemokines can activate the
pathway (Mellado et al., 1998; Rodriguez-Frade et al., 1999; Shahrara et al., 2003; Vila-
Coro et al., 1999a; Wong and Fish, 1998; Wong et al., 2001). Generally, activation of
Jaks occurs upon ligand-mediated receptor dimerization, when two Jaks are brought into
then directly phosphorylate a single tyrosine residue within the carboxy terminus of Stats
(Fu, 1992). Phosphorylated Stats then dimerize through their SH2 domains, translocate
to the nucleus and bind specific DNA sequences to regulate gene transcription (Darnell,
1998). CCL5 induced rapid tyrosine phosphorylation of CCR5, Jak2 and Jak3 in a PTx-
insensitive manner in PM1 T cells, suggesting that these events were independent of G-
protein signalling (Wong et al., 2001). Subsequent studies have shown that both CCL3
and CCL5 mediated Stat1:Stat1 and Stat1:Stat3 homo- and hetero-dimer formation in
Molt-4 and Jurkat T cells (Wong and Fish, 1998). Other studies have demonstrated that
293 cells (Mellado et al., 2001b). Similarly, CXCL12 has been shown to induce Jak2 and
Jak3 activation in T cells, although subsequent studies have not been able to reproduce
these finding (Moriguchi et al., 2005; Soriano et al., 2003; Vila-Coro et al., 1999b).
Jak2 phosphorylation and its association with PI-3’K to possibly modulate cell migration
12
(Zhang et al., 2001). Taken altogether, chemokines activate the Jak-Stat pathway to
invoke various biological responses, where specific usage of various Jak and Stat
molecules seems to be largely ligand and cell type specific (Wong and Fish, 2003).
13
1.2. Chemokine receptors
1.2.1. Classification
ligands they are receptors for: CC chemokines bind to CC chemokine receptors (CCRs),
XC chemokine receptors (XCRs) , and CX3CL1 is the ligand for the CX3CR1 receptor
(Bacon et al., 2002; Murphy et al., 2000). The CC chemokine receptor 5, CCR5, contains
352 amino acids and has a calculated molecular mass of 40.6 kDa. CCR5 shares 71%
sequence identitiy with CCR2, and is the receptor for CCL3, CCL4 and CCL5 (Figure
1.2) (Combadiere et al., 1996; Raport et al., 1996; Samson et al., 1996). A number of
non-functional CCR5 variants have been identified, the most important being the
truncated CCR5Δ32 variant that is non-functional and not expressed on the cell surface
KHSV-GPCR. This receptor shares a high degree of homology with human CXCR2
14
Figure 1.2 Two-dimensional diagram of CCR5 depicting residues critical for ligand
binding, receptor integrity, internalization and signal transduction
15
Tyrosine sulfation sites
Extracellular
Domain
Palmitoylatio
n sites
Trans-membrane
Domain
Y12
G-protein Intracellular
binding Y307 Domain
Y339
Serine phosphorylation
16
Once expressed in endothelial cells, KSHV-GPCR can trigger a constitutive signal
sufficient to induce Kaposi-like sarcomas in mice (Bais et al., 1998; Sodhi et al., 2006).
Altered chemokine expression has also been reported in Kaposi’s sarcoma herpes virus-
infected cells. The virus has acquired genes encoding three chemokines, viral
macrophage inflammatory proteins (vMIP)-I, -II and –III (Nakano et al., 2003).
Recombinant vMIP-I and –II induced calcium mobilization and are chemotactic for
receptors) have been described, namely DARC (Duffy Antigen Receptor for
DRY (Asp-Arg-Tyr) motif in the second intracellular loop and therefore cannot couple
chemokines (Pogo and Chaudhuri, 2000). The four extracellular domains of DARC are
17
essential for chemokine binding, but how they are able to bind multiple chemokines is
unclear (de Brevern et al., 2005). The role of DARC during an immune response differs
chemokine sink, both neutralizing excess chemokine in the bloodstream and preventing
chemokine diffusion into distant tissues or organs. This was demonstrated in DARC-
wildtype mice (Dawson et al., 2000). The data suggest that in the absence of DARC,
excess inflammatory chemokines are allowed to reach distal sites. In contrast, DARC
transcytosis from the basolateral to the apical side of endothelial cells, as well as their
DARC seems to have two distinct functions in vivo: (1) DARC expressed on erythrocytes
acts as a chemokine sink to limit chemokine circulation to distant tissues and (2) DARC
expression on endothelial cells aid in the transcytosis and presentation of chemokines for
CCL3L1, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL17 and
CCL22), yet does not mediate chemotaxis or signalling (Hansell et al., 2006). Once
18
bound, the ligand-D6 complex is rapidly internalized and targeted for degradation. Like
other signalling chemokine receptors, D6 is recycled back to the cell surface for
chemokines (Jamieson et al., 2005). How D6 is able to internalize bound ligand without
phosphorylated on its C-terminal serine residues, but does not require β-arrestin 2
recruitment for internalization and degradation of CCL3 (Weber et al., 2004). Thus, D6
The recently described CCX-CKR binds CCL19, CCL21 and CCL25, yet
mediates neither chemotaxis nor signal transduction (Comerford et al., 2006). CCX-CKR
CCL19, CCL21 and CCL25 are critical mediators of lymph node organogenesis,
cells, naïve T cells and some memory T cell subsets into T-cell compartments within
secondary lymphoid organs (Campbell et al., 2003; Cyster, 2005; Misslitz et al., 2004;
Muller et al., 2003; Uehara et al., 2002; Ueno et al., 2004). CCX-CKR may actively
19
regulate migratory events within secondary lymphoid tissues to modulate immune
responses.
bovine rhodopsin as the only experimental 3D structure available for any GPCRs
(Palczewski et al., 2000). All chemokine receptors are seven trans-membrane receptors,
with their N-terminus outside the cell, three extracellular and intracellular loops and a C-
Chemokine receptors have disulphide bridges in their extracellular domains that provide
structure to the overall receptor. Generally, one disulfide bridge connects the N-terminus
to the third extracellular loop (ECL), while the second links the first and second ECL.
function. For example, CCR5 is palmitoylated in its C-terminal domain on three cysteine
residues which are critical for intracellular trafficking. CCR5 mutants lacking these
palmitoylation residues are not expressed on the cell surface and remain sequestered in
receptor affinity for the ligand, as well as enhancing the usage of CCR5 by HIV-1 virus
as a cofactor for viral infection. With the exception of decoy receptors, most chemokine
receptors are coupled to the heterotrimeric G-proteins through the conserved DRY motif
20
1.2.4. Chemokine Ligand Binding Domains
The ligand binding regions of chemokine receptors have been defined through
various mutagenesis studies. The N-terminal domain of several receptors, namely CCR2,
CCR3, CCR5 and CXCR1 is crucial for ligand binding. CCR5 mutants with N-terminal
residues 2-13 exhibited weak responses to CCL4 and CCL5. Charged and aromatic
residues in this region, namely Asp-2, Tyr-3, Tyr-10, Asp-11, and Glu-18, are critical for
mutagenesis studies by Blanpain and colleagues have identified the extracellular loop
bonds in the extracellular domains maintain the structure of the receptor helical bundle.
In CCR5, alanine substitution of any of the four extracellular domain cysteine residues,
namely Cys-20, Cys-101, Cys-178 and Cys-269, dramatically reduced receptor cell
surface expression and resulted in mutant receptors unable to bind CCL4 (Blanpain et al.,
1999b). Mutations to Cys-101 or Cys-178, predicted to link ECL1 and ECL2 of CCR5,
abolished recognition by anti-CCR5 antibodies. The epitope for the monoclonal antibody
2D7 that completely blocks CCR5 ligand binding and chemotaxis was mapped to the
second ECL of CCR5 (Wu et al., 1997). Furthermore, ECL2 specific monoclonal
antibodies are more efficient than antibodies against the N-terminus in blocking CCL4
and CCL5 binding (Lee et al., 1999). Taken altogether, disulfide bonds linking the ECLs
are required for maintaining structural integrity necessary for ligand binding and receptor
activation. Thus, two hypothetical interactions are believed to play a role in CCR5
21
activation: the globular body of the chemokine ligand contacts the N-terminus and the
extracellular loops of the receptor to orient the ligand N-terminus among the trans-
membrane helices. Indeed, the core domains of CCL3 and CCL5 bind distinct residues in
The trans-membrane region of CCR5 has also been shown to be important for
ligand binding and/or receptor activation. Mutagenesis of the Thr-X-Pro (TXP) motif in
chemokine binding and functional responses (Govaerts et al., 2001). More recently, an
interaction between the arginine of the DRY motif and the cytosolic ends of TM6 was
shown to play a role in the transition from an inactive to active state (Springael et al.,
2007). The data reinforce the notion that trans-membrane regions contain important
structural elements for proper CCR5 ligand binding and subsequent receptor activation.
after serine phosphorylation by PKC and G-protein receptor kinases (GRKs) of their C-
337, Ser-342 and Ser-349 (Oppermann et al., 1999). Specifically, Ser-337 is exclusively
(Pollok-Kopp et al., 2003). Mutation to any two serine residues abrogated ligand induced
22
GRK2+/- mice displayed enhanced CCR5-mediated calcium mobilization and chemotaxis,
arrestins, which are large, multi-functional proteins that block further G-protein coupling
and attenuate additional signalling (Oppermann et al., 1999; Shenoy and Lefkowitz,
heavy chain and the β2-adaptin subunit of the heterotrimeric AP-2 adaptor complex
endosomes and are recycled back to the cell surface in their dephosphorylated form
(Blanpain et al., 1999c; Mueller and Strange, 2004; Pollok-Kopp et al., 2003).
β-arrestin 1/2, demonstrating that these molecules are critical for receptor internalization
dispensible for CCR5 internalization, as the CCR5 mutant R126N (where Arg-126 of the
DRY motif is replaced by Asn) abolished G-protein activation but there was no effect on
antibodies bound efficiently to CCR5 but did not induce internalization, suggesting that
CCR5 must exist, at a minimum, as a dimer for the internalization process to occur
arrestins not only function to prevent further G-protein signalling, but also recruit and
initiate new signals themselves, such as Erk1/2 (Perry and Lefkowitz, 2002).
Additionally, β-arrestin ½ act as scaffolds that connect activated GPCRs with tyrosine
kinases c-Src, PI-3’K and NF-κB pathways (Lefkowitz and Shenoy, 2005).
23
1.2.6. Receptor Homo- and Hetero-Dimerization
chemokine receptors form functional dimers or even higher order oligomers (Hereld and
Energy Transfer) have allowed for the monitoring of chemokine receptor interactions in
live cells. These techniques are based on the non-radiative transfer of energy between an
energy donor and an energy acceptor that occurs only when the two are in close
proximity, typically within 100Å (Kroeger and Eidne, 2004). Numerous studies have
demonstrated that chemokine receptors CXCR2, CXCR4, CCR2 and CCR5 homo-
dimerize on the cell surface. CCR5 has been shown to homo-dimerize shortly after
synthesis in the endoplasmic reticulum (Issafras et al., 2002). Consistant with this, CCR5
dimers on the cell surface were observed in the absence of ligand, suggesting that ligand
binding is not a pre-requisite for CCR5 dimerization (El-Asmar et al., 2005; Issafras et al.,
CCR2b, where Tyr-139 in the DRY motif was mutated to phenylalanine (CCR2bY139F),
1998). The data suggest that CCR2 dimerization is a pre-requisite for its function, and
that CCR2bY139F may act as a dominant negative by associating with intact CCR2 to
24
Chemokine receptors also form hetero-dimers with other chemokine receptors.
FRET analysis showed that CCR2b and CCR5 were able to form functional hetero-
dimers when co-expressed in cells (El-Asmar et al., 2005; Hernanz-Falcon et al., 2004;
Issafras et al., 2002; Mellado et al., 2001c). Such hetero-dimers are as abundant as
homo-dimers, and are only able to bind a single chemokine ligand of either cognate
receptor at any one time (El-Asmar et al., 2005). In fact, CCL5 efficiently inhibits CCL2
binding only when both CCR5 and CCR2 are co-expressed, again suggesting that the
dimerize with CCR2, but not CCR5 when co-expressed in cells (Babcock et al., 2003;
metabotropic receptor GABAB1 with GABAB2 is absolutely required for their cell surface
expression and proper function (Pin et al., 2003). The functional consequence of
that CCR2b and CCR5 homo- and hetero-dimers activate distinct signal transduction
pathways. Specifically, they showed that both CCR2b and CCR5 homo-dimers triggered
respective ligands. In the presence of both CCL2 and CCL5, they had a synergistic affect
on the CCR2b/CCR5 hetero-dimers, activating PI-3’K through Gq/11 and lowering the
threshold for calcium mobilization. However, subsequent studies have not been able to
reproduce these findings (El-Asmar et al., 2005; Springael et al., 2005). Additionally,
these results are incompatible with more current data showing that hetero-dimers respond
to only one ligand. Taken altogether, initial excitement over the possibility that different
25
combinations of chemokine receptor hetero-dimers may lead to distinct biological
More recently, chemokine receptors have been reported to form hetero-dimers with
receptors belonging to other families. For example, CCR5 and CXCR4 were reported to
interact with opioid receptors, although the physiological relevance remains unclear
(Chen et al., 2004; Pello et al., 2008; Suzuki et al., 2002). Recent studies have
signalling in response to ligands for both receptor (Pello et al., 2008). It is intriguing to
26
1.3. Chemokine/Chemokine Receptor Function and the
Immune Response
1.3.1. Chemotaxis
numerous biological processes including proper tissue development, wound healing and
protection against invading pathogens. Chemotaxis requires the activation and re-
surface. Numerous external stimuli that engage various cell surface receptors and
membrane receptors for a chemo-attractant bind their cognate ligand(s) and cluster at the
leading edge of the cell, known as the lamellipodium. This leads to the accumulation of
intracellular signalling and lipid molecules at this leading edge, causing the cell to
polarize. Second, there is formation of adhesions that attach the protrusion to the
substratum on which the cell is rolling. These act as traction points for migration,
complete the cycle, adhesion molecules detach at the back of the cell (termed the
uropodium) coupled with contractions to move the cell body forward (Giannone and
Sheetz, 2006; Hynes, 2002; Nelson and Nusse, 2004). F-actin polymerization is localized
at the lamellipodium, critical for the assembly of cellular protrusions (Cory et al., 2003;
27
Pollard and Borisy, 2003). Not surprisingly, lamellipodia contain numerous actin-
modifying enzymes, namely the Arp2/3 complex, WAVE/Scar and WASp (Myers et al.,
assembled at the uropodium and lateral sides of the cell, where it provides rigidity to the
filaments at the uropodium provides the mechanical force needed to move the cell
(PI(3,4,5)P3) have been widely implicated in controlling cell migration and polarity. The
PI-3’K family of proteins are defined as lipid kinases that phosphorylate the 3’-OH
al., 2001). Members of the family are grouped into four classes (IA, IB, II and III) on the
basis of their structure and substrate specificity. Class IA and IB PI-3’K members are the
best characterized and are primarily responsible for the production of PI(3,4,5)P3 in
receptor tyrosine kinases and exist as a stable hetero-dimer, consisting of one of three
catalytic isoforms (p110α, p110β or p110δ) that associate with any one of the five
regulatory isoforms (p85α, p55β, p50α, p85β or p55γ). Class IB PI-3’K is activated by
the G protein βγ subunit, and consists of a p101/p87 regulatory subunit and a p110γ
catalytic subunit. Class II PI-3’K poorly phosphorylates PI(4,5)P2 and its biological
28
function is not well understood (Falasca and Maffucci, 2007). Class III PI-3K is
homologous to the yeast protein Vps34p and regulates intracellular vesicle trafficking
responsible for the generation and accumulation of PI(3,4,5)P3 at the leading edge of the
is the PH domain containing Protein Kinase B (PKB, also known as Akt), which is
Ser-473 within the hydrophobic motif, either by mTORC2 or DNA-PKCS (Feng et al.,
2004; Manning and Cantley, 2007). PKB is largely responsible for activation of a wide
range of signalling cascades, many intimately involved in cell cycle progression, cell
et al., 2000). Another important phosphatase, the Phosphatase and Tensin Homolog
al., 1998). These phosphatases are critical suppressors of constitutive PI-3’K activity,
29
also associated with maintaining localized PI-3’K activation at the leading edge of the
chemotaxis and polarization were dependent on PI-3’K activation (Turner et al., 1995b).
Subsequent studies have shown that other chemokines, namely CCL2 and CXCL12,
Turner et al., 1998). It is now clear that localized PI-3’K activation at the lamellipodium
GFP-tagged PH domains that selectively bind PI(3,4,5)P3 accumulate at the leading edge
of polarized cells undergoing chemotaxis (Rickert et al., 2000; Servant et al., 2000).
Coincidently, studies have shown that PTEN is largely excluded from the leading edge of
the migrating cell and accumulates at the trailing edge. The net effect is a transient
increase in the level of PIP3 at the lamellipodium. The crucial role of PTEN is
reduce or enhance leukocyte motility, respectively (Fox et al., 2002). Presumably, the
localized.
In recent years, much of the focus has been on elucidating the role of different PI-
30
pharmacological inhibitors. The PI-3’Kγ isoform is undoubtedly a key regulator of
This seems to be the case for neutrophils and macrophages, where p110γ-deficiency leads
to defective chemotaxis towards several chemokines (Hirsch et al., 2000; Li et al., 2000;
Sasaki et al., 2000). However, B cells do not utilize p110γ, but rather use p110δ for
CCL19 and CCL21 was not completely abrogated, suggesting that other PI-3’K isoforms
and/or PI-3’K-independent events are required for efficient migration (Reif et al., 2004).
Certainly, studies have shown that the Class IA p85/p110 hetero-dimer contributes to the
signals that determine optimal chemotactic migration towards CCL5 and CXCL12 in T
cells (Curnock et al., 2003; Turner et al., 1995b). In fact, the regulatory subunit p85 co-
association with CCR5 has not been shown (Vicente-Manzanares et al., 1999). The
of effector molecules, including p56 lck, focal adhesion kinase (FAK) and zeta-associated
protein (ZAP-70) (Bacon et al., 1996; Vanhaesebroeck et al., 2001; Wong et al., 2001).
Although speculative, these proteins may be able to couple the p85/p110 hetero-dimer
31
The Rho family of small GTPases are key regulators of the actin/myosin
cytoskeleton during chemotaxis, the most well-known members being Rho, Rac and
Cdc42 (Raftopoulou and Hall, 2004). They act as molecular switches by cycling between
GDP-bound, inactive and GTP-bound, active forms. Rho GTPases are intimately
regulated by guanidine nucleotide exchange factors (GEFs) that catalyze the exchange of
GDP for GTP. Many RhoGEFs contain a PH domain, allowing them to accumulate at the
leading edge of the migrating cell in response to phospholipids. Indeed, GFP reporter
studies have demonstrated that both Rac1 and Cdc42 are exclusively recruited to and
activated at the lamellipodium (Itoh et al., 2002; Kraynov et al., 2000; Srinivasan et al.,
2003). Interestingly, Rac1 can stimulate PI-3’K activity, possibly establishing a positive
feedback loop for sustained asymmetrical accumulation of PI(3,4,5)P3 at the leading edge
(Wang et al., 2002). It is now clear that Rac1 and Cdc42 are crucial regulators of F-actin
(Raftopoulou and Hall, 2004). A family of WAVE/Scar and WASp proteins bridge Rac1
and Cdc42 to the Arp2/3 complex, that functions to nucleate actin polymerization and
facilitate branching of actin filaments (Pollard and Borisy, 2003). Specifically, Rac1,
through its binding to IRSp53, regulates WAVE dependent Arp2/3 complex activation
(Miki et al., 2000). Cdc42 directly binds to N-WASP, exposing the domains that activate
the Arp2/3 complex (Suetsugu et al., 1998). These dynamic actin structures at the
leading edge enable cells to form protrusion on the substratum in preparation for
migration. Migrational studies with Rac1 and Rac2 double-deficient hematopoietic cells
and neutrophils revealed that the cells were unable to respond to chemokines because of
defective F-actin polymerization (Gu et al., 2003). In contrast to Rac and Cdc42, Rho
32
seems to accumulate at the rear of the cell, where it regulates the assembly of contractile,
actin:myosin filaments through its effectors Rho kinase (ROCK) and myosin light chain
kinase (MLCK) (Amano et al., 1997; Amano et al., 1996; Ohashi et al., 2000; Sumi et al.,
the migrating cell. Notably, CCL5 was shown to induce RhoA activation in Jurkat T
cells, although its role in chemotaxis was not investigated (Bacon et al., 1998). A
endothelial cells (Honing et al., 2004). There is also evidence that RhoA, acting through
mDia, has a direct positive effect on microtubule stability at the leading edge (Palazzo et
al., 2001). Recent studies have shown that mDia1-deficient T cells exhibit reduced
The Mitogen-Activated Protein Kinase (MAPK) pathways that activate Erk, Jnk
and p38 kinases elicit wide-ranging cellular outcomes, including regulating gene
expression, cell proliferation and cell motility (Pullikuth and Catling, 2007). MAPK
signalling cascades comprise a core hierarchy of three kinases, each of which is activated
through phosphorylation by the kinase positioned upstream of it. Thus, the MAPKs are
phosphorylated and activated by the MAPK kinases (MAPKKs), which are themselves
activated by the MAPKK kinases (MAPKKK) (Figure 1.3). Numerous growth factors
and cytokines signal through MAPKs to induce cellular proliferation and the
transcriptional activation of cytokine genes (Pullikuth and Catling, 2007). Given that
33
Figure 1.3 The MAPK Signalling Cascade
34
Mekk1-4, Tak1-3, Tao1-3,
MAPKKK Raf Ask1-2, Tpl2, Mlk3
Mekk1 Tak
Biological Response
35
chemokines are potent inducers of cytokines and proliferation, it is not surprising that
chemokines can activate multiple MAPK signalling cascades. For example, ligands for
CCR5 have been demonstrated to activate Erk, Jnk and p38 signalling pathways (Brill et
al., 2001; Ganju et al., 1998; Kraft et al., 2001; Misse et al., 2001; Wong et al., 2001)
increased astrocyte proliferation (Bajetto et al., 2001). Several studies have demonstrated
of focal adhesions. Active Erk localizes to adhesions at the uropodium and facilitates
their disassembly to promote motility (Suetsugu et al., 2006; Webb et al., 2004).
Although a specific mechanism has not been described, sustained Erk phosphorylation
et al., 2001). Disassembly of adhesions by MAPKs at the rear of the cell allows for the
migrating cell to push forward. Thus, MAPKs may play an unexpected role in
Accumulating evidence has shown that chemokines invoke both apoptotic and
anti-apoptotic events in a wide range of cell types. Whether a chemokine protects from
or induces cell death depends on the chemokine, its concentration and/or the target cell.
36
(CD95/CD95L) interactions have been described as important inducers of AICD in T
cells, although different effectors, including c-Myc and TRAIL, have also been described
(Green et al., 2003; Ju et al., 1995). Several reports have demonstrated that chemokines
can potentiate T cell death. CXCL12 induces apoptosis of Jurkat T cells through a
Similarly, XCL1 can co-stimulate the apoptosis of CD4+ T cells triggered through the
caspase-7 and PARP cleavage (Cerdan et al., 2001). These studies indicate that
AICD. Mellado and colleagues reported that melanoma tumour cell-derived CCL5
2001a). CCL5-CCR5 mediated caspase-3 activation and cell death were also reported in
neuroblastoma cells, and there is also evidence that the HIV-1 envelope-mediated
liver failure (FLF), by preventing hepatic NKT cell apoptosis (Ajuebor et al., 2005).
Work from our laboratory has demonstrated that CCL5-CCR5 interactions induce T cell
death (Murooka et al., 2006) (Chapter 2). Specifically, we showed that CCL5
37
subjected to µM concentration of CCL5, cells undergo apoptosis through cytosolic
followed by poly ADP ribose polymerase (PARP) cleavage. We showed that CCL5-
kinases initiated through the Tyr-339 residue found on the C-teriminus of CCR5. Finally,
we showed that CCL5-GAG interactions and CCL5 oligomerization are important pre-
requisites to initiate a cascade of events resulting in T cell death. Taken together, our
data suggest that CCL5-induced cell death, in addition to CD95/CD95L mediated events,
CCL3, CCL4 and CCL5, either individually or in combination, will reduce anti-CD3-
induced apoptosis of T cell blasts. These chemokines do not affect CD3 or Fas cell
surface expression levels, suggesting that they reduce AICD downstream of Fas (Pinto et
al., 2000). Interestingly, Tyner and colleagues have reported that virus-inducible CCL5 is
protective effects of CCL5 are dependent on CCR5 and activation of the PI-3’K/Akt and
Mek/Erk signalling pathways (Tyner et al., 2005). Although apparently contradicting our
data (Murooka et al, 2006), the cell lineage studied (macrophages vs T cells) and the
lower dose of CCL5 employed may explain these different observations. CCL1 activation
Spinetti et al., 2003). Viewed altogether, conflicting data in regard to the pro- or anti-
38
apoptotic properties of several chemokines reflect the need for further studies. The
factors, such as the nature of the chemokine, whether it exhibits aggregation and GAG-
binding, the chemokine dose effect, the nature of the specific cognate receptor, and the
lineage of the target cell. These factors are particularly important when considering
shown to co-stimulate T cell activation. For example, CXCL12 can co-stimulate anti-
CD3 stimulation of CD4+ T cells in the context of proliferation and IL-2, IFNγ, IL-4 and
IL-10 production. CXCL12 treatment alone did not have the same effect, suggesting that
the chemokine functions as a co-stimulator for T cells (Nanki and Lipsky, 2000). Such
co-stimulation was PTx-sensitive, but not altered by anti-CD25 antibodies, indicating the
dependence on G-protein, but not IL-2, mediated signalling (Nanki and Lipsky, 2001).
Furthermore, CXCL12 stimulated the physical association between CXCR4 and the TCR
to initiate signalling through ZAP-70 (Kumar et al., 2006). CCL5 is also a T cell co-
stimulatory molecule in the context of CD3 stimulation (Makino et al., 2002; Taub et al.,
1996). Studies in CCL5 deficient mice showed impaired T cell proliferation and cytokine
proliferation and cytokine production, dependent on IL-2 and extracellular calcium (Taub
39
et al., 1996). In the same study, CCL3, CCL4 and CCL5 all induced expression of B7.1
cell activation. In Jurkat T cells, Dairaghi and colleagues showed that T cell responses to
CCL5 are dependent on the level of CD3 cell surface expression (Dairaghi et al., 1998).
Interestingly, CCR5 constitutively co-localizes with CD4 on the cell surface (Xiao et al.,
cytokine production. This unexpected property of CCL5 demonstrated that high doses of
CCL5 can bypass T cell receptor recognition of antigen to activate T cells (Bacon et al.,
1995; Dairaghi et al., 1998). Since these initial observations, it is now apparent that at
these µM concentrations, CCL5 forms large oligomers with a mass greater than 100 kDa
(Appay et al., 1999; Appay et al., 2000). CCL5 variants with a Glu-26 to alanine
form higher order aggregates at µM concentrations (Appay et al., 1999; Czaplewski et al.,
1999). These mutants are unable to activate T cells, demonstrating that the aggregating
properties of CCL5 are important for T cell activation (Appay et al., 1999; Appay et al.,
2000). Notably, the non-aggregating mutants retain their ability to signal via classical G-
protein dependent pathways in vitro. Whether high CCL5 concentrations are attainable in
vivo is unclear. Certainly, unusually high CCL5 concentrations may be realizable at site
and/or extracellular matrix GAGs. In addition, the unique ability of CCL5 to form higher
order aggregates, facilitated through GAG-binding, may also lead to an increase in local
CCL5 concentration (Appay et al., 1999; Appay et al., 2000; Czaplewski et al., 1999;
40
Hoogewerf et al., 1997; Kuschert et al., 1999; Martin et al., 2001; Proudfoot et al., 2001;
differentiation, cell cycle progression, cell growth and apoptosis. Not surprisingly,
highly regulated process is mRNA translation, (Proud, 2007). Once mRNAs are
transcribed, processed and exported into the cytoplasm, they are available for translation
through two principle pathways. The first involves the binding of translation initiation
factors (eIFs) to the 7-methyl guanosine residue (m7GpppN, where m is a methyl group
and N is any nucleotide) that caps the 5’ end of all nuclear-encoded eukaryotic mRNAs,
termed cap-dependent translation. Specifically, the interaction of the cap structure with
the 5’end of the mRNA (Richter and Sonenberg, 2005). A second pathway uses complex
secondary structure elements in the mRNA, called Internal Ribosomal Entry Segments
(IRES), to recruit small ribosomal subunits, independently of the cap structure, referred to
mRNAs are translated in a cap-dependent manner, eIF4E represents the rate-limiting step
mTOR during development (Gangloff et al., 2004; Martin and Sutherland, 2001). mTOR
41
possesses a carboxy-terminal region sharing significant homology with lipid kinases,
especially with PI-3’K, and has been assigned to a larger protein family termed the
proliferative properties. In the early 1990s, rapamycin was shown to bind to a small
protein receptor called FKBP12 (FK506-binding protein 12kDa), and the complex
specifically interacted with mTOR to inhibit its function (Sabatini et al., 1994; Sabers et
al., 1995). However, there is controversy whether rapamycin directly inhibits the intrinsic
from interacting with its substrates (Edinger et al., 2003b; Peterson et al., 2000). mTOR
and phosphorylates p70 S6K1 and initiation factor 4E binding proteins (4E-BPs), and
(Dann et al., 2007; Gingras et al., 1998; Hay and Sonenberg, 2004). mTORC1 is a
Companion of mTOR), Sin1 and mLST8 (Jacinto et al., 2004; Kim et al., 2002). Given
the importance of mTOR in development and protein translation, its activation is under
exquisite control by several molecules. The major upstream positive regulator is the
small GTPase, Rheb (Ras-Homolog Enriched in Brain) (Saucedo et al., 2003). Similar to
other GTPases, GTP-bound Rheb, but not GDP-bound, is active and stimulates mTOR
kinase activity (Long et al., 2005). Rheb activity is negatively regulated by the
mammalian TSC1/2 (Tuberous Sclerosis Complex 1/2), by increasing the intrinsic GTP
42
hydrolysis of Rheb (Inoki et al., 2003). Thus, TSC1/2 is a potent negative regulator of
mTOR by inactivating Rheb activity. It is now clear that TSC1/2 represent tumour
suppressor proteins, where mutation to either one is sufficient to cause TSC tumor
formation in a number of target organs (Yang and Guan, 2007). TSC1 and TSC2 form a
physical and functional complex, where TSC1 stabilizes the complex and TSC2 exerts
GTPase activating protein (GAP) activity. Thus, mutations in the TSC1/2 complex lead
Upstream of TSC1/2 is PKB, an important survival kinase with a wide array of effector
molecules. PKB has been shown to phosphorylate TSC2 directly on multiple sites to
inhibit its function (Inoki et al., 2002). Given that PI-3’K is largely responsible for the
recruitment and activation of PKB, as described earlier, it is now well established that PI-
3’K is responsible for indirectly activating mTOR activity (Figure 1.4). Another
of phosphatidic acid (PA). Several studies reported that PA was required for mTOR-
dependent S6K activation and 4E-BP1 phosphorylation in several cell types (Fang et al.,
2001; Foster, 2007; Hornberger et al., 2006). Interestingly, PA seems to compete for
A wide range of factors, including hormones, growth factors, mitogens and amino
acids, can initiate protein translation. eIF4E availability represents the rate-limiting step
for cap-dependent translation and thus act as the node of convergence for a number of
upstream signalling events. Three eIF4E inhibitory proteins, the 4E-BPs (4E-BP1-3, also
43
Figure 1.4 Regulation of cap-dependent mRNA translation
44
Growth factors, hormones,
mitogens, cytokines, chemokines
PI-3’K
Energy
PIP2 PIP3 starvation
Ras PTEN
PDK1
Raf AMPK
PKB
Mek
TSC1/2
*
Erk
Rheb-GTP
4E-BP1 S6K1
eIF4E eIF4B
Cap-dependent translation
(e.g., cyclin D1, VEGF, c-Myc)
45
known as PHAS-1-3 for Phosphorylated Heat and Acid Soluble protein stimulated by
proteins that compete with eIF4G for an overlapping binding site on eIF4E. Indeed,
through X-ray crystallographic analysis, peptides derived from the regions of eIF4G and
4E-BP1 form nearly identical α-helical structures that lie along the same convex region of
eIF4E (Marcotrigiano et al., 1997; Matsuo et al., 1997). Notably, the eIF4G and 4E-BP1
binding sites on eIF4E do not overlap the cap binding sites. By sequestering eIF4E, 4E-
BPs are negative regulators of mRNA translation that requires high levels of available
The number of phosphorylation sites is controversial, but the most critical sites for eIF4E
release are located on Thr-37, Thr-46, Ser-65, and Thr-70 (Hay and Sonenberg, 2004). In
represent the priming sites, and are phosphorylated by mTOR in vitro (Brunn et al., 1997;
Burnett et al., 1998). Phosphorylation at these priming sites is critical for subsequent
BP1 from eIF4E (Hay and Sonenberg, 2004). X-ray crystallography studies have
revealed that these residues on 4E-BP1 are all in close proximity to acidic amino acid
phosphate groups would likely lead to electrostatic repulsion of the acidic residues, to
mediate release of 4E-BP1 from eIF4E (Gross et al., 2003). Additionally, mTOR
controls the translation of 5’-TOP (tract of oligopyrimidines) mRNAs which often encode
46
for cytoplasmic ribosomal proteins (Meyuhas, 2000; Ruvinsky and Meyuhas, 2006).
is unclear and studies have shown that S6K1 and its effector molecule rpS6 are
dispensable for their translation (Ruvinsky et al., 2005). Taken together, mTOR is a
critical regulator of the translational machinery by: (1) directly influencing eIF4E
availability for 5’-capped mRNA translation initiation and (2) up-regulating ribosomal
complex comprised of a large, scaffold protein eIF4G, the RNA helicase eIF4A and the
cap-binding eIF4E (Figure 1.5). eIF4G also associates with eIF3, a multi-subunit,
ribosome-associated initiation factor, to bridge the mRNA to the 40S ribosomal subunit.
The 40S ribosomal subunit is bound to eIF2, GTP and the initiator methionine-transfer
RNA (Met-tRNA), and the entire complex is termed the 43S pre-initiation complex
(Proud, 2007). In addition, the N-terminus of eIF4G binds the poly(A) binding protein
(PABP), leading to the circularization of the mRNA via the cap-eIF4F-poly(A) tail bridge,
which enhances mRNA translation. Optimal binding and passage along the 5’-UTR
towards the initiation codon by the translation initiation complex is often hindered by
long, complex secondary structures that are found in the 5’-UTR of some mRNAs
(Richter and Sonenberg, 2005). The helicase function of eIF4A is enhanced by eIF4B,
and is critical for unwinding the inhibitory secondary structures present in the 5’UTR.
47
Figure 1.5 eIF4F formation and ribosome recruitment
48
eIF4A
eIF4G
A B
eIF4E
cap
AUG
4E-BP1 mRNA
eIF4F
complex
ATP Pi
ADP
P cap
AUG C
eIF4B
AUG
ATP
mRNA unwinding
ADP + Pi
AUG D
eIF2 eIF1A
+
GTP
+
+ 40S
Met-tRNA eIF3
49
Once the 43S pre-initiation complex is bound to mRNA, it is thought to scan the mRNA
in the 5’ to 3’ direction (Proud, 2007). When it encounters an AUG start codon in the
proper sequence context, other factors, including the 60S ribosomal subunit, are recruited
Two mRNA transcripts may be translated at very different rates, depending on the
length and structure of their 5’-UTRs. As mentioned earlier, the helicase activity of the
eIF4F translation initiation complex is crucial for unwinding inhibitory structures within
the 5’-UTR. Those mRNAs that are well translated when eIF4E availability is low are
termed “strong” mRNAs, and have relatively short, unstructured 5’-UTRs (e.g. β-actin,
levels, and typically encode for proliferative and survival proteins (e.g. c-Myc, vascular
endothelial growth factor (VEGF), bcl-2) (Armengol et al., 2007; Graff and Zimmer,
2003). This ensures that proliferation and survival proteins are preferentially synthesized
Several published reports suggest a role for both mTOR and p70 S6K1 in cellular
arterial E47 smooth muscle cells is sensitive to rapamycin (Sakakibara et al., 2005).
Several chemokines have been reported to activate S6K1, but this activation was studied
in the context of cell survival and proliferation, not migration (Hwang et al., 2003; Joo et
50
al., 2004; Lee et al., 2002; Loberg et al., 2006). As mentioned previously, a G protein-
However, the specific role for mTOR-dependent protein translation in T cell chemotaxis
mediated T cell chemotaxis in vitro (Murooka et al., 2008). CCL5 induced rapid
its dissociation from eIF4E. Subsequently, eIF4E associated with the scaffold protein
eIF4G, forming the eIF4F translation initiation complex. Indeed, CCL5 initiated active
induced protein translation of cyclin D1 and MMP-9, known mediators of migration. Our
51
1.4. Chemokine/Chemokine Receptors and Disease
inflammatory disease that affects synovial tissue in multiple joints. Such chronic
inflammation leads to severe morbidity and progressive structural damage to the joints.
While genetic associations between RA and variants of the human leukocyte antigens
(HLA) are well established, other genes also play important roles in RA susceptibility,
by extensive infiltration of activated T cells, B cells and macrophages into affected joints,
leading to the expansion of the synovial tissue. Inflammatory chemokines are critical for
actively recruiting leukocytes into inflamed synovial joints. Indeed, analysis of synovial
tissue and synovial fluid from patients with RA, revealed abundant expression of a wide
range of inflammatory chemokines and their receptors (Haringman et al., 2004; Hosaka et
al., 1994; Wong et al., 2003). RA synovial fibroblasts produce chemokines CCL2, CCL3,
CCL4 and CCL5 in response to TNFα, IL-1α and IL1β (Hosaka et al., 1994; Luster,
joints and stimulate cells to release additional inflammatory mediators. For example,
CXCL12 resulted in enhanced IL-6 and IL-8 production (Nanki and Lipsky, 2001). Thus,
52
both through recruitment of activated leukocyte and direct modulation of cytokine
Animal models for RA have been used extensively to examine the role of
expression profiles in affected tissues are comparable between human and rodent models
of RA. High levels of CCL3 and CCL5 in synovial fluid are present early and in later
stages of disease in both human patients and murine collagen-induced arthritic mice
in the synovial joints (Suzuki et al., 1999). Indeed, upregulation of CCR1, CCR2 and
CCR5 mRNA levels coincide with peak inflammation in the joints of rat adjuvant-
induced arthritic mice (Shahrara et al., 2003). The data suggest that CCR5 is one of
implications are that individuals with altered/reduced CCR5 expression exhibit less
severe and/or slower progression of RA. To this end, several studies have focused on
cohorts of RA patients that are homozygous for the CCR5Δ32 allele, a non-functional
confirmed the negative association between CCR5Δ32 and RA, indicating that CCR5Δ32
showed that CCR5Δ32 homozygosity conferred a much greater protective effect than
53
this analysis took into account published studies conducted in populations of European
ancestry, where the CCR5Δ32 allelic frequency is approximately 5-10% (Cooke et al.,
1998; Garred et al., 1998; Gomez-Reino et al., 1999; Pokorny et al., 2005; Samson et al.,
1996; Zapico et al., 2000). Whether these results are relevant for different ethnic groups
is unclear (John et al., 2003; Zuniga et al., 2003). Nevertheless, the data suggest the
possibility that CCR5 blockade may have therapeutic potential in selected cohorts of RA
patients.
new, yet there are no clinical applications of this approach approved or in the clinic to
date. There is accumulating evidence in rodents that targeting the chemokine system can
dampen arthritic inflammation. Notably, targeting CCL2 or its receptor CCR2 in mice
1997; Ogata et al., 1997). In a rat adjuvant model of arthritis, administration of CCL5
and subsequent reductions in joint damage (Barnes et al., 1998). Similarly, a non-peptide
CCR5 antagonist TAK-779 significantly reduced both incidence and severity of collagen-
induced arthritis in mice by reducing T cell migration (Yang et al., 2002). The
chemotaxis of monocytes towards patient synovial fluid was significantly reduced with
(Barsante et al., 2008). Taken altogether, antagonists for various chemokine receptors,
that reduce leukocyte migration to affected tissues and dampen cytokine production in the
54
joints, may prove to be effective in RA. Currently, a fully humanized monoclonal
Inc.
1.4.2. Cancer
initiating angiogenesis. To date, chemokines and their receptors have been implicated in
all steps of tumorigenesis, including the control of leukocyte infiltration into tumors,
initiation of primary tumor growth and survival, regulation of angiogenesis, and the
control of tumor cell adhesion, invasion and migration (Figure 1.6) (Murooka et al.,
2005). Understanding the complex role chemokines play at each stage of tumorigenesis
monocytes/macrophages, T cells, dendritic cells, and mast cells. The influx of immune
cells into solid tumors was initially believed to reflect an anti-tumor immune response.
55
Figure 1.6 Chemokines and Cancer
The roles of chemokines and their receptors in various steps of tumorgenesis, namely
leukocyte infiltration into tumors, initiation of primary tumor growth and survival,
regulation of angiogenesis, and the control of tumor cell adhesion, invasion and migration,
are shown.
56
1 – Neoplastic Transformation 4 - Angiogenesis
Abnormal chemokine Tumor-produced
and chemokine receptor chemokines stimulate
expression in angiogenesis, causing
transformed cells up- neighbouring blood
regulate growth and vessels to grow into the
survival factor tumor
Murooka, TT, Ward, SE and Fish, EN. Cancer Treat Res 126 (2005)
57
metastasis. Tumor associated macrophages (TAMs) have pro-tumor functions by virtue
of their release of growth factors, such as epidermal growth factor (EGF), and their
production of angiogenic mediators, including VEGF and basic fibroblast growth factor
(bFGF) (Mantovani et al., 1992). TAMs are also a source of IL-10 and prostaglandin E2
Over two decades ago, Bottazzi and colleagues showed that CCL2 is expressed
and secreted by most tumor cell lines (Bottazzi et al., 1990; Bottazzi et al., 1983).
by tumors and stromal cells, and is implicated in breast, ovarian, bladder, and lung cancer
(Bottazzi et al., 1990; Bottazzi et al., 1983; Frederick and Clayman, 2001; Silzle et al.,
the existence of an amplification loop for their recruitment. Correlative studies in breast
cancer patients showed that CCL2 expression levels are directly proportional to TAM
accumulation (Saji et al., 2001; Ueno et al., 2000). These studies also identified a
significant correlation between CCL2 levels and several potent angiogenic factors,
namely VEGF, thymidine phosphorylase (TP) and CXCL-8. Other studies have
demonstrated a pivotal role for tumor-derived CCL5 in leukocyte infiltration. CCL5 was
highly expressed in high grade breast tumors, while breast tumor cell lines express
functional CCL5 in culture that induces monocyte migration in vitro (Adler et al., 2003;
Azenshtein et al., 2002; Luboshits et al., 1999; Robinson et al., 2003; Saji et al., 2001).
58
CCL5 using a murine model of breast cancer. Administration of the CCR1/CCR5
tumors, which correlated with reduced tumor burden (Robinson et al., 2003). Similar
conclusions can be drawn from studies where mammary carcinoma cells expressing
lower levels of CCL5 exhibit a decrease in tumor growth in vivo (Adler et al., 2003).
Following the recruitment of TAMs, both CCL2 and CCL5 also stimulate the release of
tumor-promoting factors by TAMs, namely MMP-9 and TNF-α (Azenshtein et al., 2002;
Robinson et al., 2002; Saji et al., 2001). Viewed altogether, chemokines are important
for the recruitment of tumor-promoting inflammatory cells into the tumor site, considered
Chemokines act as growth and survival factors for various tumors, generally in an
signalling axis that has direct pro-tumor growth effects on tumor cells. Upregulation of
cancer, glioblastoma, leukemia, multiple myeloma, prostate cancer, oral squamous cell
carcinoma and pancreatic cancer (Chan et al., 2003; Floridi et al., 2003; Koshiba et al.,
2000; Moller et al., 2003; Sehgal et al., 1998a; Sehgal et al., 1998b; Sun et al., 2003;
Uchida et al., 2003; Zeelenberg et al., 2003). CXCL12 increases DNA synthesis and
dependent manner (Barbieri et al., 2006; Florio et al., 2006; Mowafi et al., 2008).
59
and neuroepithelioma cell lines correlated with Erk1/2 and PKB activation (Barbero et al.,
loss of cell-to-cell contact (Ueda et al., 2006). The contributions of CCL5 and CCR5 in
the pathogenesis of breast cancer have been investigated by several groups. CCL5 is
reported to be highly expressed in high grade breast tumors (Azenshtein et al., 2002;
Luboshits et al., 1999; Niwa et al., 2001; Yaal-Hahoshen et al., 2006). Serum CCL5
levels were elevated in patients with high grade tumors compared to low grade tumors
(Niwa et al., 2001). Indeed, several breast cancer cell lines migrate towards CCL5
(Azenshtein et al., 2002; Luboshits et al., 1999; Robinson et al., 2003; Youngs et al.,
1997). This suggests the possibility that local production of CCL5 by tumor cells, or
other cells within the tumor microenvironment, results in CCL5 exerting effects directly
on breast tumor cells. Certainly, there is conflicting reports for the direct pro-growth
effects of CCL5 in breast cancer (Adler et al., 2003; Jayasinghe et al., 2008). Our data
support a pro-proliferative role for CCL5 in breast cancer. Specifically, CCL5 actively
promoted translation of proliferative and survival proteins, namely cyclin D1, c-Myc and
rapamycin-dependent manner (Murooka et. al., unpublished data). Thus, our data
demonstrate the potential for breast cancer cells to exploit downstream chemokine
signalling pathways for their proliferative and survival advantage through expression of
derived CCL5 did not contribute to breast tumor formation in vivo (Jayasinghe et al.,
2008). One explanation for these contradictory results is the concentration differences in
60
CCL5 in these two studies. While we observed significant CCL5-mediated proliferative
effects at 10 nM, CCL5 expression levels by 4T1 breast cancer cells reported by
Jayasinghe and colleagues was approximately 100 fold less (Jayasinghe et al., 2008).
The data suggest that a threshold level of CCL5 is required in order for CCL5 to invoke a
studies showing that CCL5 content within tumor lesions is markedly higher in more
aggressive forms of breast cancer (Bieche et al., 2004; Niwa et al., 2001). Such a
threshold level may be attainable through the propensity of CCL5 to bind, oligomerize
and accumulate on GAGs at their secretion site (Proudfoot et al., 2003). Others have
and migratory potential of gastric carcinoma cells (Hashimoto et al., 2008). CXCL8 has
been shown to up-regulate cyclin D1 at the level of translation in prostate cancer cells
(MacManus et al., 2007). Sodhi and colleagues show that endothelial-specific expression
(Sugasawa et al., 2008). Notably, TILs rather than tumor cells, were the source of CCL5.
Angiogenesis involves the formation of new vessels from pre-existing ones and is
61
accumulating evidence that CXC chemokines regulate angiogenesis thereby promoting
containing the ELR motif at their NH2 terminus (ELR+) are potent promoters of
angiogenesis. These chemokines were directly chemotactic for endothelial cells and
Strieter et al., 1992). In contrast, CXC chemokines lacking this motif (ELR-) were potent
angiostatic factors (Strieter et al., 1995). These molecules were able to inhibit new vessel
et al., 1995; Belperio et al., 2000; Sgadari et al., 1996). ELR+ chemokines that promote
angiogenesis include CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8.
Generally, the ELR- chemokines are IFN-γ inducible and inhibit angiogenesis (Belperio
et al., 2000). CXCL4, CXCL9, and CXCL10 are ELR- chemokines that inhibit
CXCL8 was the first chemokine to display potent angiogenic activity when
implanted into rat cornea and to induce proliferation and chemotaxis of human umbilical
bronchogenic carcinoma (Arenberg et al., 1996; Smith et al., 1994). Increased levels of
62
CXCL8 were detected in tumor tissue compared with normal lung tissue, and CXCL8
antibodies reduced human prostate tumor growth and tumor-related angiogenesis in SCID
mice (Moore et al., 1999). The angiogenic effects of CCL2 and CCL5 are less well
pro-angiogenic TAMs. Several correlative studies have shown that CCL2 is co-
expressed with known angiogenic factors VEGF and CXCL8 (Saji et al., 2001; Ueno et
al., 2000). Additionally, CCL5 has been shown to directly up-regulate MMP-9
expression in breast cancer cells (Azenshtein et al., 2002). Given the importance of
increased tumor vascularity to support tumor growth and spread, the angiogenic
metastasis in breast cancer (Muller et al., 2001). Breast cancer typically metastasizes to
regional lymph nodes, bone marrow, lung, and liver. Comparing the expression levels of
17 chemokine receptors in seven human breast cancer cell lines and normal primary
mammary epithelial cells, their data revealed that the breast cancer cells exhibited
specific patterns of receptor expression. Specifically, CXCR4 and CCR7 are highly
expressed in breast cancer cells, malignant breast tumors, and metastatic cells.
63
Subsequent studies examined patterns of expression for the ligands CXCL12, CCL19,
and CCL21, in different tissues. The highest levels of expression of CXCL12 were
detected in lymph nodes, lung, liver, and bone marrow, corresponding to the typical sites
of breast cancer metastasis. Low levels of CXCL12 were found in tissues that are not
typically associated with breast cancer metastases, such as the skin, brain, and kidneys.
CCL19 and CCL21 expression levels were highest in lymph nodes, although CCL21 was
expressed at higher levels, suggesting that this chemokine played a more prominent role
in the homing of breast cancer cells to the lymph nodes via its interaction with CCR7. To
determine whether the pattern of chemokine receptor expression observed was unique to
breast cancer, Muller and colleagues then examined chemokine receptor expression
breast cancer, but also metastasizes within the skin. Interestingly, the authors showed
that melanoma cells expressed CXCR4 and CCR7, similar to breast cancer cells, but also
expressed higher than normal levels of CCR10, which interacts with the skin-specific
homeostatic chemokine, CCL27. Expression of CXCR4 in breast cancer cells has since
promoter of breast cancer metastasis in vivo. These studies addressed the complicated
interplay between breast tumor cells and the tumor-associated stroma (Karnoub et al.,
2007). The de novo production of CCL5 from mesenchymal stem cells acted directly on
cancer cells to enhance their motility, invasion and metastasis. Such tumor cell-
64
mesenchymal stem cell interactions were largely dependent on CCL5-CCR5.
Interestingly, the metastatic potential of tumor cells was reversible, suggesting that
was dependent on CCR5 trans-activation via CCL5 expression (Mira et al., 2001). In an
significantly fewer lung metastases than their wildtype counterparts (van Deventer et al.,
2005). Subsequent studies by the same group showed that CCR5 expression of
pulmonary mesenchymal cells was responsible for lung metastases through MMP-9
expression (van Deventer et al., 2008). These data lead us to hypothesize that CCL5-
CCR5 signalling in both stromal and breast cancer cells leads to phenotypic changes that
favour increased motility and invasiveness. Our data suggest that mTOR-dependent
CXCR1 and CXCR2 are expressed at higher levels in highly metastatic human melanoma
cell lines than in non-metastatic melanoma cells (Varney et al., 2003). In the same study,
neutralizing antibodies directed against these receptors were shown to inhibit both the
proliferation and invasive potential of melanoma cells, regardless of whether or not the
cells had been stimulated by CXCL8. In addition to its role in breast cancer metastasis,
CCR7 is associated with lymph node metastasis of esophageal squamous cell carcinoma,
with high levels of CCR7 expression correlating with lymphatic permeation, lymph node
65
metastasis, and poor survival (Ding et al., 2003). Interesting new studies showed that
bone loss (Kinder et al., 2008; Zhu et al., 2007). Thus, localized expression of CCL2 was
responsible for breast cancer metastasis to the bone, and their subsequent erosion.
The relationship between the chemokine system and invading pathogens is highly
complex. While chemokines are highly expressed and are essential to coordinate the host
immune response, some are exploited by viruses for their pathogenicity. Furthermore,
viruses have acquired the ability to interfere with host chemokines to disrupt the immune
response. Different viruses encode for proteins that exhibit high homology with
proteins bind to host chemokines and interfere with their binding to their cognate
receptors. Viruses use these molecules to evade the protective mechanisms of the host
for their own survival advantage (Finlay and McFadden, 2006; Murphy, 2001; Seet et al.,
2003).
Over 10 years ago several groups demonstrated that the chemokine receptors
CCR5 and CXCR4 were essential co-receptors for HIV entry into host cells (Choe et al.,
1996; Dean et al., 1996; Doranz et al., 1996; Liu et al., 1996). Initial observations
revealed that the CC chemokines CCL3, CCL4 and CCL5 exerted HIV suppressive
activity (Cocchi et al., 1995). The HIV-1 envelope protein gp120 forms a tri-molecular
complex with host cell CD4 and either CCR5 or CXCR4. This results in the exposure of
66
a cryptic fusogenic peptide of gp41 from the HIV-1 envelope protein, which mediates
fusion between the viral envelope and host cell membranes (Berger et al., 1999; Wyatt
and Sodroski, 1998). It is now understood that CCR5 is utilized by macrophage tropic
HIV strains to infect mononuclear phagocytes, primary T cells and DCs, while CXCR4 is
used by HIV strains that infect CD4+ T lymphocytes. By infecting T cells and
that orchestrate antiviral immunity (Gerard and Rollins, 2001; Horuk, 1999). Several
prominent mutations within the coding regions of CCR5 alter susceptibility of the host to
HIV-1 infection. As described earlier, individuals who are homozygous for the defective
allele CCR5Δ32 are largely resistant to HIV infection (Liu et al., 1996; Samson et al.,
1996). In fact, individuals who were heterozygous for the mutant CCR5 allele progress
slower towards AIDS (Dean et al., 1996). The utilization of chemokine receptors by HIV
is not restricted to CCR5 and CXCR4, as CCR3, CCR2b and CCR8 are capable of
mediating infection (Choe et al., 1996; Doranz et al., 1996; Horuk et al., 1998). Another
Since the initial reports of the HIV suppressive properties of CCL3, CCL4 and
CCL5, subsequent studies have demonstrated their protective roles in vivo. In one study,
high levels of CCL5 were found in both HIV-exposed humans and vaccinated monkeys
who were resistant to HIV or SIV infection, respectively (Furci et al., 1997; Wang et al.,
1998; Zagury et al., 1998). Another study showed that the number of CCL3 gene
duplications inversely predicted the risk of HIV infection and rate of disease progression
67
(Gonzalez et al., 2005). It is conceivable that these individuals with higher CCL3
expression limit HIV infection by CCL3 binding to and internalizing CCR5. The CCR5
internalize and prevent receptor recycling to the cell surface compared to wildtype CCL5
(Mack et al., 1998; Signoret et al., 2000). Thus, cell surface CCR5 availability is a
from HIV infection (Signoret et al., 1997). Viewed altogether, chemokines inhibit the
initial stages of HIV entry by either blocking the binding of the viral envelope protein
(Appay and Rowland-Jones, 2001; Ward et al., 1998). Small molecule inhibitors that
target these chemokine receptors are currently in advanced-stage clinical trials in HIV,
with the CCR5 inhibitor Maraviroc recently being approved for clinical use in the US
(MacArthur and Novak, 2008). It is important to note that high concentrations of CCL5
unexpectedly enhanced HIV infection in vitro (Gordon et al., 1999; Trkola et al., 1999).
This is due to the propensity of CCL5 to aggregate and oligomerize at these high
68
1.5. Hypothesis and Objectives
Hypothesis:
Objectives:
T cell chemotaxis.
69
Chapter 2
1
Division of Cellular and Molecular Biology, Toronto General Research Institute
University Health Network & Department of Immunology, University of Toronto
2
Serono Pharmaceutical Research Institute, Geneva, Switzerland
Murooka, T.T., Wong, M.M., Rahbar, R., Majchrzak-Kita, B., Proudfoot, A.E., and Fish,
E.N. (2006). CCL5-CCR5-mediated Apoptosis in T cells: Requirement for
Glycosaminoglycan Binding and CCL5 Aggregation. J Biol Chem 281, 25184-25194.
T.T.M. performed experiments in Fig. 2.1A, 2.1E, 2.3, 2.4A, C, D, E, 2.5, 2.6, 2.8, 2.9,
analyzed the data and drafted the manuscript.
M.W. performed experiments in Fig 2.1B, C, D and drafted the manuscript.
R.R. performed experiments in Fig. 2.7 and analyzed the data.
B.M-K. performed experiments in Fig. 2.2, 2.4B
A.E.I.P. provided valueable reagents.
E.N.F. designed research, analyzed the data and drafted the manuscript.
70
2.1. Abstract
CCL5 (RANTES) and its cognate receptor, CCR5, have been implicated in T cell
directional signal. In two CCR5 expressing human T cell lines, PM1.CCR5 and
CCL5 induce apoptosis. CCL5-induced cell death involves the cytosolic release of
cytochrome c, the activation of caspase-9 and caspase-3 and poly ADP ribose polymerase
MOLT4 cells lacking CCR5 expression are resistant to CCL5-induced cell death.
surface GAG binding. The addition of exogenous heparin and chondroitin sulfate, and
GAG digestion from the cell surface protects cells from apoptosis. Moreover, the non-
GAG binding variant, [44AANA47]-CCL5, fails to induce apoptosis. To address the role
forms dimers and E26A, that form tetramers at µM concentrations, were utilized. Unlike
native CCL5, the E66S mutant fails to induce apoptosis, suggesting that tetramers are the
minimal higher ordered CCL5 aggregates required for CCL5-induced apoptosis. Viewed
altogether, these data suggest that CCL5-GAG binding and CCL5 aggregation are
apoptosis.
71
2.2. Introduction
pro-adhesive effects. They are responsible for directing leukocyte migration by forming
chemokine gradients and triggering firm arrest by activating integrins on the leukocyte
cell surface. It is now apparent that chemokines exhibit critical functions in many diverse
developmental and immunological operations (Aliberti et al., 2000; Ansel et al., 2000;
Karpus et al., 1997; Makino et al., 2002; Nagasawa et al., 1996; Szekanecz and Koch,
for T cells of the Th1 and memory phenotype (Schall et al., 1990; Siveke and Hamann,
1998). This may be due to CCL5 binding to CCR5, which is predominantly expressed on
memory Th1 T cells (Kawai et al., 1999; Rabin et al., 1999). Given the prevalence of
memory Th1 T cells in inflammatory diseases and the coincident increased expression of
CCL5 and CCR5, CCL5-CCR5 mediated events in T cells may be critical in disease
(Makino et al., 2002; Taub et al., 1996). Mice deficient in CCL5 demonstrate impaired T
(Makino et al., 2002). Anti-CD3 stimulation of T cells together with nM CCL5 treatment
72
that high doses of CCL5 can bypass T cell receptor (TCR) recognition of antigen to
activate T cells (Bacon et al., 1995; Dairaghi et al., 1998). At these µM concentrations,
CCL5 forms large oligomers with a mass greater than 100 kDa (Appay et al., 1999;
Appay et al., 2000). Mutation of the acidic amino acid residues glutamate 26 to alanine
(E26A), or glutamate 66 to serine (E66S), in CCL5, results in CCL5 variants that are
Czaplewski et al., 1999). These mutants are unable to activate T cells, implying that the
aggregating properties of CCL5 are important for T cell activation (Appay et al., 1999;
Appay et al., 2000). Notably, the non-aggregating mutants retain their ability to signal
via classical G-protein dependent pathways in vitro. CCL5, as well as other chemokines,
can bind to glycosaminoglycans (GAGs) on the cell surface or the extracellular matrix
(ECM) to increase relative chemokine concentrations (Ali et al., 2000; Hoogewerf et al.,
1997). The predominant GAG binding site for CCL5 has been shown to be the BBXB
motif in the 40s loop (Martin et al., 2001; Proudfoot et al., 2001) and GAG binding in
vivo has been shown to be critical for CCL5 function (Proudfoot et al., 2003). Residues
critical for GAG binding of other chemokines including CCL3, CCL4, and MCP-1 have
now been identified (Chakravarty et al., 1998; Koopmann et al., 1999; Koopmann and
Krangel, 1997; Lau et al., 2004; Laurence et al., 2001; Martin et al., 2001; Sadir et al.,
2001; Vita et al., 2002). Whether the interaction of CCL5 with GAGs induces cellular
activation through a novel signaling mechanism is not clear. However, CCL5 and its
receptor presentation.
73
In this study we examined CCL5 activity in T cells in the context of GAG-binding,
aggregation and apoptosis. We present evidence that CCL5 aggregates form at high
ligand concentrations and that these may induce apoptosis in T cell lines and in primary
involves the cytosolic release of the mitochondrial pro-apoptotic factor cytochrome c, the
activation of caspases -9 and -3 and poly ADP ribose polymerase (PARP) cleavage.
Additionally, we provide evidence for the critical role of intracellular Tyrosine (Y)
residue 339 of CCR5 in mediating cell death that is independent of G-protein mediated
events. Finally, we show that CCL5-GAG interactions and CCL5 oligomerization are
together, our data suggests a potential novel role for CCL5 in determining T cell fate
74
2.3. Materials and Methods
the anti-CCR5 monoclonal antibody (2D7) were obtained from the National Institutes of
Health AIDS Research and Reference Reagent Program. All cells were maintained in
culture in RPMI 1640 (Gibco-BRL) supplemented with 10% fetal calf serum (Gibco-
BRL), 100 units/ml penicillin, 100 mg/ml streptomycin (Gibco-BRL) and 25 µg/ml
(1:1000) were purchased from Cell Signaling, anti-cytochrome c antibody (1:1000) was
purchased from Santa Cruz, and anti-PARP antibody (1:2000) was purchased from BD
(1:2000) were purchased from R & D Systems. Heparin sodium salt, chondroitin sulfate
A and chondroitinase ABC were from Sigma-Aldrich. JC-1 was purchased from
Molecular Probes. WT CCL5, CCL5 aggregation mutants E26A and E66S and
experiments.
Human peripheral blood derived T cells were isolated from consenting healthy
donors, as approved by the UHN research ethics committee. For activation, 106 resting T
cells/ml were cultured with 1 µg/ml PHA and 2 ng/mL IL-12 for 2 days, then cultured for
an additional 3 days in the presence of 100 U/ml hrIL-2. T cells were then stained with
75
anti human CCR5 antibody (2D7) and sorted for CCR5- and CCR5+ T cells. Sorted cells
Actively growing PM1.CCR5 cells were incubated with 10 µg/ml CCL5 for 24h.
In experiments where cellular surface GAGs were enzymatically digested, cells were first
chondroitinase ABC (1 U/ml) for 1 h at 370C and 5% CO2. Cells were then washed three
times and incubated with 10 µg/ml CCL5 for 24h and analyzed by Annexin V/7-AAD
analysis. For experiments where cell surface CCL5 was measured, PM1.CCR5 cells
either untreated or pretreated with chondroitinase ABC were incubated with 10 µg/ml
CCL5 for 1 h on ice. Cells were collected, washed three times with ice cold PBS and
anti-mouse IgG antibody. As isotype controls, cells were incubated with FITC labeled
The MTT assay was performed as previously described (Uddin et al., 1997).
Annexin V-FITC and 7-AAD staining were carried out according to the manufacturer’s
protocol (BD Pharmingen). Briefly, native PM1, PM1.CCR5, native MOLT-4 and
MOLT-4.CCR5 cells were incubated with 10 µg/ml CCL5 for 24h. Cells were then
collected, and 1 x 105 cells were incubated in 100 µl of binding buffer together with
Annexin V-FITC and 7-AAD for 15 min. Samples were analyzed immediately by flow
76
cytometry (FACSCalibur, BD). DNA fragmentation was analyzed using an apoptotic
DNA ladder kit (Roche Diagnostics, Germany) according to the manufacturer’s protocol.
DNA isolated from cells was resolved in a 2% agarose gel containing ethidium bromide
PM1.CCR5 cells were incubated with 10 µg/ml CCL5 for the times indicated,
1 x 106 cells/ml. JC-1 was added at a final concentration of 2µM and incubated at 370C
and 5% CO2 for 30 min. Cells were washed two times in PBS and resuspended in 1mL
Motif #40015) according to the manufacturer’s protocol. Cell lysates were resolved by
Cells were incubated with 10 µg/ml CCL5 for the times indicated, pelleted by
centrifugation, washed with ice-cold PBS and lysed in 100 μL of lysis buffer (1% Triton
EGTA, 0.2 mM PMSF). Protein concentration in lysates was determined using Bio-Rad
DC protein assay kit (BioRad laboratories). 40 μg of protein lysate per sample was
77
denatured in 5X sample buffer and resolved by SDS-PAGE gel electrophoresis. The
with 5% BSA (w/v) in TBS for 1hr at room temperature. Membranes were probed with
the specified antibodies. Proteins were visualized using the ECL detection system
(Pierce).
conjugated anti-mouse IgG antibody. As isotype controls, cells were incubated with
FITC labeled isotype control IgG antibody (eBioscience) and analyzed using the
FACSCalibur and CellQuest software. Cells were gated based on forward and side
scatter. For intracellular caspase-3 activity analysis, 1 x 106 cells were treated with CCL5
for the indicated times, fixed and permeabilized with 0.5% saponin on ice. Cells were
then incubated with FITC labeled anti-active caspase-3 (Transduction Laboratories) and
The pEF-BOS-CCR5 carrying the human CCR5 gene was obtained from Dr.
using the QuickChange Site-Directed Mutagenesis Kit (Stratagene) using the following
78
cgctcgttcgagtcaaaagtgggctaggtgacccc 3’ (reverse). Mutation was confirmed by
sequencing (ACGT Corporation, Toronto). Intact CCR5 and CCR5Y339F genes were
then subcloned into the pUMFG retroviral vector (a gift from Dr. Jeffery Medin, Division
method. At 48 h post-transfection, the viral supernatant was collected and used for PM1
transfection, as described (Kinsella and Nolan, 1996). Positive transfectants were FACS
sorted using anti-human CCR5 antibody and used for subsequent experiments.
between groups.
79
2.4. Results
(Boehme et al., 2000) and death (Colamussi et al., 2001; Jinquan et al., 2003; Kaul and
Lipton, 1999; Zhang et al., 2005) of various cell types. To investigate the biological
were treated with different doses of CCL5 and the viability of cells assessed by the
apoptosis marker annexin V and the permeability indicator 7-amino actinomycin D (7-
AAD). At 10 ng/ml – 1 µg/ml (nM) doses, CCL5 treatment did not affect viability, but at
10 µg/ml (µM) doses CCL5 induced apoptosis (Figure 2.1.A). Classical apoptotic cell
death may be distinguished by DNA fragmentation, revealed when cells were treated with
cell line, MOLT-4.CCR5 (Figure 2.1.C). By contrast, native PM1 and MOLT-4 cells
lacking CCR5 expression were not susceptible to CCL5-inducible apoptotic cell death
(Figure 2.1.D, E). Notably, the PM1 and MOLT-4 cell lines do not express CCR1, an
costimulation through CD28 in the context of CD3 protects cells from AICD (Collette et
al., 1998; Noel et al., 1996). Perhaps, at µM doses CCL5 bypasses the T cell receptor to
80
Figure 2.1. µM concentrations of CCL5 induce apoptosis in PM1.CCR5 T cells.
(A) 2 x 105 PM1.CCR5 cells/ml were treated with varying doses of CCL5 for 24 h and
percent apoptotic cells were detected by staining with Annexin V-FITC and 7-AAD.
Data are representative of three independent experiments (mean ± S.D.) **p<0.01. (B) 2
x 105 PM1.CCR5 cells/ml were either left untreated (control), treated with CCL5 (10
µg/ml) for either 24h or 48h. DNA fragmentation assay was performed as described in
Experimental Procedures. Cells treated with PMA and ionomycin (P+I) for 24 h served
as a positive control. Data are representative of two independent experiments. (C) 2 x
105 PM1.CCR5 and MOLT-4.CCR5 T cells/ml were either left untreated or treated with
10 µg/ml CCL5 for 24 h. Apoptotic cells were detected by staining with Annexin V-
FITC and 7-AAD. The percentage of the cell population in each quadrant is indicated to
the right of each FACS blot. Data are representative of five independent experiments.
(D) Cell surface CCR5 expression was determined for native PM1, PM1.CCR5, native
MOLT-4, and MOLT-4.CCR5 cells by FACS. The dotted line represents the
fluorescence intensity using a FITC labeled isotype control IgG antibody. The bold solid
line and the grey solid line represents the fluorescence intensity using primary anti-CCR5
antibody in conjunction with the secondary anti-mouse FITC. Data are representative of
three independent experiments. (E) 2 x 105 native PM1 and native MOLT-4 cells/ml
were either left untreated or treated with 10 µg/ml CCL5 for 24 h. Apoptotic cells were
detected by staining with Annexin V-FITC and 7-AAD. The percentage of the cell
population in each quadrant is indicated to the right of each FACS blot. Data are
representative of three independent experiments.
81
Figure 2.1.
A B
70
**
60
% Annexin V positive
50
40
30
20
10
0
Control 10ng/mL 100ng/mL 1µg/mL 10µg/mL
CCL5 concentration
C
Untreated CCL5 treated
0 7 15 53
92 0 29 3
7-AAD
7-AAD
PM1.CCR5
0 4 0 15
95 1 35 50
7-AAD
7-AAD
MOLT-4.CCR5
82
D
Native PM1 Native MOLT-4
PM1.CCR5 MOLT-4.CCR5
CCR5-FITC CCR5-FITC
E
Untreated CCL5 treated
1 8 0.5 9
91 0 90 0.5
7-AAD
7-AAD
PM1
0 2 0 8
97 1 92 0
7-AAD
7-AAD
MOLT-4
83
induce apoptosis. The Fas/FasL apoptotic pathway has been studied extensively in CD4+
T cells. We did not observe any change in Fas or FasL expression following CCL5
treatment for 24 hours (Figure 2.2.). Moreover, pretreatment of cultures with the anti-
FasL monoclonal antibody, NOK1, did not prevent CCL5-mediated apoptosis (Figure
reduction in ΔΨm (Figure 2.4.A). Indeed, the results in Figure 2.4.B reveal a CCL5
fragment) and caspase-3 (17 kDa and 19 kDa fragments) at 8 h that is sustained for 24 h
(Figure 2.4.C). The activation of caspase-3 was further confirmed by intracellular FACS
caspase-3 was detected (Figure 2.4.D). Finally, we examined the cleavage of the
endogenous caspase-3 substrate PARP, in similar time course studies. The 85 kDa
cleavage fragment of PARP was detected at 8 hours post-CCL5 treatment and to a greater
84
Figure 2.2 CCL5 does not affect Fas/FasL expression in T cells.
85
Figure 2.2.
PM1.CCR5
IgG control
Anti-FasL
Anti-Fas
MOLT-4.CCR5
86
Figure 2.3. FasL neutralizing monoclonal antibody NOK1 does not block CCL5-
mediated apoptosis in PM1.CCR5 cells.
PM1.CCR5 cells were pretreated with either IgG control or NOK1 antibody (10 µg/ml)
for 30 minutes and either left untreated or treated with 10 µg/ml CCL5 for 24 h.
Apoptotic cells were detected by staining with AnnexinV-FITC and 7-AAD. The
percentage of the cell population in each quadrant is indicated to the right of each FACS
blot. Data are representative of two independent experiments.
87
Figure 2.3.
IgG
0 13 2 50
85 2 42 7
NOK1
7-AAD
Annexin V-FITC
88
Figure 2.4. µM concentrations of CCL5 induce cytochrome c release, caspase-9 and
caspase-3 activation and PARP cleavage.
(A) 1 x 106 PM1.CCR5 cells were treated with 10 µg/ml CCL5 for the indicated times.
Cells were collected and stained with 2 µM JC-1, and analyzed by FACS. Mitochondrial
depolarization was measured by decrease of JC-1 accumulation in the mitochondria (thus
an increase in JC-1 monomers) due to loss of membrane potential. CCCP was used as
positive control and gating correction (data not shown). Data are representative of two
independent experiments. (mean ± S.D.) *p<0.05, **p<0.01 (B) PM1.CCR5 cells were
either left untreated or treated with 10 µg/ml CCL5 for the indicated times. Cells were
harvested and the cytosolic fraction isolated. The resulting lysates were resolved by
SDS-PAGE and immunoblotted with anti-cytochrome c antibody. Membranes were
stripped and reprobed for tubulin as loading control. The relative fold increase of
cytochrome c levels is shown as signal intensity over loading control. Data are
representative of two independent experiments. (C) PM1.CCR5 cells were either left
untreated or treated with 10 µg/mL CCL5 for the indicated times. Cells were harvested
and lysates were resolved by SDS-PAGE and immunoblotted with anti-caspase-9 or anti-
cleaved caspase-3 antibody. Membranes were stripped and reprobed for tubulin as a
loading control. The relative fold increase of protein level is shown as signal intensity
over loading control. Data are representative of two independent experiments. (D) 1 x
106 PM1.CCR5 cells were either left untreated or treated with 10 µg/ml CCL5 for the
times indicated, fixed with 2% paraformaldehyde and permeabilized with 0.5% saponin.
Cells were then stained with an anti-active caspase-3-FITC antibody. Data are
representative of three independent experiments. (E) PM1.CCR5 cells were either left
untreated or treated with 10 µg/mL CCL5 for the indicated times and immunoblotted
with anti-PARP antibody. The relative fold increase of protein level is shown as signal
intensity over loading control.
89
Figure 2.4.
A 100
90
80
70 *
* **
% ΔΨm
60
50
40
30
20
10
0
0 2 4 8 16 24
B cytochrome c
3.5
0 2 4 12 24 Signal Intensity
2.5
2
WB: cytochrome c
1. 5
WB: tubulin 1
0.5
0
0 2 4 12 24
C
CCL5 treated (time [hrs])
0 2 4 8 16 24
37kDa Cleaved Caspase-9
WB: tubulin
caspase-9 caspase-3
4.5 6
4 17k D a
5
3.5 19k D a
Signal Intensity
Signal Intensity
3 4
2.5
3
2
1.5 2
1
1
0.5
0 0
0 2 4 8 16 24 0 2 4 8 16 24
90
D
Untreated
10h
Counts
24h
48h
Active caspase-3-FITC
E
CCL5 treated (time [hrs])
0 2 4 8 16
WB: Cleaved PARP
WB: tubulin
2.5
2
Signal Intensity
1.5
0.5
0
0 2 4 8 16
91
2.4.3. µM concentrations of CCL5 induce apoptosis in CCR5 expressing primary T
cells
Human primary T cells were isolated from peripheral blood from healthy donors
based on cell surface CCR5 expression, and were >95% CD3 positive (Figure 2.5.A). To
cells, CCR5+ and CCR5- primary T cells were treated with CCL5 for 24 h. Consistent
with our data for PM1.CCR5 cultures, CCL5 inducible apoptosis was dependent on
CCR5 expression, did not occur at 100 ng/ml – 1 µg/ml (nM) CCL5 doses, but required
10 µg/ml (µM) doses (Figure 2.5.B). Figure 2.5.C reveals a CCL5 inducible and time-
confirm that CCL5 induces apoptosis in T cells in a CCR5- and mitochondrial pathway-
dependent manner.
2.4.4. Expression of intact CCR5, but not CCR5Y339F, renders PM1 cells susceptible
to CCL5-inducible apoptosis
CCL5 mediated CCR5 activation results in the exchange of GTP for GDP by the
Gα subunit, the dissociation of heterotrimeric G-proteins into Gα and Gβγ subunits and
subsequent signal transduction. Additionally, we and others have provided evidence for
intracellular residue Y339 was mutagenized to phenylalanine (F). Vectors for intact
92
Figure 2.5. µM concentrations of CCL5 induce apoptosis in human primary T cells.
93
Figure 2.5.
A Pre-sort Post-sort
B 15
CCR5+ T cells
*
% Annexin V positive
CCR5− T cells
10
0
Untreated 100ng/mL 1ug/mL 10ug/mL
CCL5 concentration
C
CCL5 treated (time [hrs])
0 2 4 8 16 24
WB: Cleaved Caspase-9
WB: tubulin
4
Signal Intensity
3.5
2.5
1.5
0.5
0
0 2 4 8 16 24
94
CCR5 and CCR5Y339F cDNA were constructed (as described in Materials and
Methods) and introduced into native PM1 cells. Each transfectant was analyzed for cell
surface CCR5 expression using an anti-human CCR5 antibody (Figure 2.7.A), which
does not distinguish among the intact or mutant receptors, and clones exhibiting similar
ectopic expression levels were selected for use. CCL5 binding and receptor
doses of CCL5 would induce apoptosis in PM1 cells expressing Tyrosine-339 deficient
mutant CCR5. The data in Figure 2.7.B. show that CCL5 induced apoptosis in PM1 cells
components of the cell surface and extracellular matrix, thereby creating a concentration
gradient for cells to migrate along via a haptotactic mechanism (Amara et al., 1999;
Baltus et al., 2003; Cinamon et al., 2001; Kuschert et al., 1999; Netelenbos et al., 2002;
Pablos et al., 2003). Different studies suggest that chemokine-GAG interactions enhance
the cell surface (Ali et al., 2000; Burns et al., 1998; Hoogewerf et al., 1997). Binding
studies with immobilized heparin and HUVECs revealed that the affinity interaction of
CCL5 to GAGs can be ablated by the addition of heparin, heparin sulfate, chondroitin
sulfate and dermatan sulfate (Kuschert et al., 1999). Cell surface CCL5 binding was
95
Figure 2.6. CCL5 binding and receptor internalization of PM1.CCR5 and
PM1.CCR5Y339F cells.
(A) PM1.CCR5 and PM1.CCR5Y339F cells were either left untreated or treated with
250 nM CCL5 at 37˚C for the times indicated. Cells were collected on ice, washed, and
stained for cell surface CCR5 expression. % CCR5 internalization was calculated as the
MFI of treated cells / MFI of untreated cells x 100% (± S.D.) (B) PM1.CCR5 and
PM1.CCR5Y339F cells were either left untreated or treated with 250 nM CCL5 for 1h on
ice. Cells were collected, washed, and stained for CCR5 and CCL5. The data are shown
as the ratio of CCL5 MFI and CCR5 MFI (± S.D.)
96
Figure 2.6.
A
120
PM1.CCR5
100 PM1.CCR5Y339F
% Internalization
80
60
40
20
0
0 5 15 30 60
Time (min)
B
100
CCL5 MFI / CCR5 MFI Ratio
80
60
40
20
0
PM1.CCR5 PM1.CCR5Y339F
97
Figure 2.7. Introduction of CCR5 but not CCR5Y339F into PM1 T cells renders
them susceptible to CCL5-inducible apoptosis.
(A) cDNA for intact CCR5, CCR5Y339F or vector alone was introduced by retroviral
transduction into native PM1 cells. Cell surface CCR5 expression of all transfectants was
examined by FACS. The dotted line represents the fluorescence intensity using FITC
labeled isotype control IgG antibody. The bold solid line represents the fluorescence
intensity using primary anti-CCR5 antibody in conjunction with the secondary anti-
mouse FITC. (B) 2 x 105 PM1.vector, PM1.CCR5 and PM1.CCR5Y339F cells/ml were
either left untreated or treated with 10 µg/ml CCL5 for 24 h. % Apoptotic cells were
detected by staining with Annexin V-FITC and 7-AAD. Data are representative
experiment of five independent experiments. (mean ± S.D.) **p<0.01
98
Figure 2.7.
A
PM1.vector PM1.CCR5 PM1.CCR5Y339F
B
60
**
50
% Annexin V positive
40
30
20
10
99
observed in both native PM1 and PM1.CCR5 cells, although consistently higher CCL5
receptor (Figure 2.8.A). The data suggest that PM1 T cells have GAGs on the cell
surface that are able to bind and sequester CCL5. To address the role of GAG
interactions in CCL5-induced cell death, PM1.CCR5 cells were treated with CCL5 and
sulfate rescued PM1.CCR5 cells from CCL5- induced cell death: 10 µg/ml of heparin or
PM1.CCR5 cells were treated with chondroitinase ABC to enzymatically remove the
GAGs from the cell surface. Chondroitinase treatment significantly reduced the ability of
CCL5 to bind to the cell surface (Figure 2.8.C) without altering CCR5 expression itself
PM1.CCR5 cells from CCL5-induced death. Similarly, when PM1.CCR5 cells were
(Proudfoot et al., 2003) we did not observe apoptosis (Figure 2.8.F). Moreover, when
CCL5 and intact CCL5, which had been pre-mixed for 4 h at room temperature, the
resulting heterodimer did not induce apoptosis (Figure 2.8.G). The data suggest that in
100
Figure 2.8. CCL5-GAG interactions are important for apoptosis.
(A) Native PM1 and PM1.CCR5 cells were either left untreated or treated with 10 µg/ml
CCL5 for 1 hr on ice. CCL5 binding to the cell surface was determined by FACS
analysis. The solid line represents staining with the FITC-labeled anti-CCL5 antibody
and the dotted line staining with the FITC-labeled isotype control antibody. Mean
fluorescence intensity is indicated in each FACS histogram. Data are representative of
two independent experiments. (B) 2 x 105 PM1.CCR5 cells/ml were either left untreated
or treated with 10 µg/ml CCL5, in the presence or absence of increasing doses of heparin
or chondroitin sulfate A, for 24 h. Cell viability was determined using the MTT assay.
Data are representative of three independent experiments. (mean ± S.D.) **p<0.01 (C)
PM1.CCR5 cells were either pretreated with PBS or chondroitinase ABC prior to CCL5
treatment. CCL5 binding to the cell surface was determined by FACS analysis. Data are
representative of three independent experiments. (D) PM1.CCR5 cells were either
pretreated with PBS or chondroitinase ABC and cell surface CCR5 expression
determined by FACS analysis. (E) PM1.CCR5 cells were either pretreated with PBS or
chondroitinase ABC prior to 24 h CCL5 treatment. Apoptotic cells were detected by
staining with Annexin V-FITC and 7-AAD. Data are representative of three independent
experiments. (mean ± S.D.) **p<0.01 (F) PM1.CCR5 cells were either left untreated or
treated with 10 µg/mL CCL5 or [44AANA47]-CCL5 for 24 h. Additionally, equal
concentrations of [44AANA47]-CCL5 and wildtype CCL5 were preincubated for 4 h at
room temperature, then PM1.CCR5 cells stimulated for 24 hours (1:1 [44AANA47]-
CCL5:CCL5)~ heterodimer. Apoptotic cells were detected by staining with Annexin V-
FITC and 7-AAD. Data are representative of three independent experiments. (mean ±
S.D.) **p<0.01
101
Figure 2.8.
A
Untreated CCL5 treated
Mean: 6.4 Mean: 63.3
PM1
PM1.CCR5
B
% Viability
% Viability
CCL5 + + + + CCL5 + + + +
Heparin - + + + Chondroitin
µg/mL 0 1 10 100 Sulfate - + + +
µg/mL 0 1 10 100
102
C D
Chondroitinase
PBS alone PBS +CCL5 +CCL5
PBS alone
Chondroitinase
treated
E F **
** 60
50
50
40
% Annexin V positive
% Annexin V positive
40
30
30
20
20
10
10
0 0
[44AANA47]-
Chondroitinase
Chondroitinase
Heterodimer
PBS alone
PBS + CCL5
Untreated
CCL5
+ CCL5
CCL5
103
2.4.6. Aggregation of CCL5 is required for CCL5-induced cell death
al., 1999; Czaplewski et al., 1999). Certainly, CCL5 oligomerization is necessary for
CCL5-induced PM1.CCR5 cell death, experiments were conducted using the E26A and
where native CCL5 forms large oligomers, E26A and E66S form tetramers and dimers,
respectively (Czaplewski et al., 1999). The results in Figure 2.9. show that the E66S
mutant did not induce cell death even at 100 µg/ml doses, in contrast to both the intact
CCL5 and the mutant E26A. The data suggest that CCL5 tetramers are the minimal
104
Figure 2.9. The CCL5 aggregation mutant E66S does not induce PM1.CCR5 cell
death.
2 x 105 PM1.CCR5 cells/ml were either left untreated or treated with 10 µg/ml of CCL5,
E26A, E66S or 100 µg/mL E66S. Apoptotic cells were detected by staining with
Annexin V-FITC and 7-AAD. Data are representative of three independent experiments.
(mean ± S.D.) **p<0.01
105
Figure 2.9.
70
**
60 **
% Annexin V positive
50
40
30
20
10
0
Untreated CCL5 E26A E66S 100µg/ml
E66S
E66S
10µg/ml
106
2.5. Discussion
to their roles in the recruitment of T cells to sites of inflammation and in triggering their
invoke many different signaling cascades that determine the functional outcome of the
target cell. Herein we report on CCL5 activity in T cells in the context of GAG-binding,
associated with CCR5 mediated cytochrome c release, and caspase-9 and -3 activation
(Mellado et al., 2001a). CCL5-CCR5 mediated caspase-3 activation and cell death have
been reported in neuroblastoma cells, and there is also evidence that HIV-1 virion -
mediated apoptosis of bystander uninfected CD4+ T cells, which leads to T cell depletion
The cell suicide machinery can be induced by several factors, which then
converge to activate caspases via two pathways: one involving caspase-8 recruitment to
death receptors (TNF or CD95) and the other involving the mitochondrial/apoptosome
pathway [reviewed in (Creagh et al., 2003)]. Our studies show that CCL5 induced
107
the data indicated that CCL5-inducible apoptosis in CCR5-expressing T cells is mediated
residues, at position 127, 307 and 339. Y127 lies in the second intracellular loop of the
receptor in the DRY motif, highly conserved among CC chemokine receptors and
motif in CCR5 results in a non-functional receptor with reduced surface expression and
al., 2001). The other two intracellular tyrosine residues of CCR5, Y307 and Y339, reside
in the C-terminal tail of the receptor. While Y307 is conserved among CC chemokine
receptors, Y339 is unique to CCR5 and CCR4. In other studies, we have evidence that
mediated by Y339 and not Y307 (Rahbar et al., 2006). Accordingly, we focused on
effectors in mediating cell death. Dong et al. reported that whether HEK293 cells
express intact CCR5 or the tyrosine mutant variant, CCR5Y339F, CCL5-receptor binding
is unaffected (Dong et al., 2005). In agreement, we do not observe a defect in the kinetics
CCR5, but not in those expressing CCR5Y339F. The data suggest that Y339 may be a
108
critical target for effector recruitment after CCR5 dimerization, an obligatory step to
the DRY motif of CCR2 has been identified as the primary target for Jak2 mediated
that heparin and chondroitin sulfate compete for CCL5-cell surface GAG interactions,
thereby effectively blocking cell death. Apparently, heparin is more potent than
chondroitin sulfate in protecting PM1.CCR5 cells from CCL5-induced cell death. This
result is consistent with CCL5 exhibiting a greater affinity for heparin than chondroitin
sulfate (Kuschert et al., 1999). The amino acid residues R45, K46 and R47 in CCL5
comprise a BBXB motif that is important for heparin binding (Martin et al., 2001;
Proudfoot et al., 2001). Other chemokines including CCL3, CCL4 and CXCL12 have a
similar motif (Chakravarty et al., 1998; Koopmann et al., 1999; Koopmann and Krangel,
1997; Laurence et al., 2001; Martin et al., 2001; Sadir et al., 2001; Vita et al., 2002). We
found that enzymatic digestion of cell surface chondroitin sulfate by chondroitinase ABC
treatment protected cells from CCL5-induced death. This was consistent with a
significant decrease in CCL5 binding to the cell surface after chondroitinase ABC
treatment, despite no effect on CCR5 cell surface expression (Fig 5C,D), in further
109
support of a role for GAGs in sequestering chemokines and facilitating chemokine
receptor binding. Proudfoot et al. have demonstrated that CCL5 GAG mutants
and no recruitment activity in vivo, although in vitro activity is retained (Proudfoot et al.,
intact CCL5 results in heterodimers that are unable to recruit cells into the peritoneal
cavity in vivo (Johnson et al., 2004). We observe that this heterodimeric mixture will not
disrupt CCL5 oligomerization on GAGs. Taken together, our data suggest that in the
mutants variably affected cell death. Specifically, the E66S mutant did not induce cell
death, even at concentrations reaching 100 µg/mL, in contrast to both the native
aggregating CCL5 and the mutant E26A. The data suggest that CCL5 tetramers are the
minimal higher order aggregates required for inducing T cell death, in agreement with
evidence that tetramers are the minimal order aggregates required to recruit cells in vivo
110
The ability of CCL5 to induce at least two distinct biological outcomes –
et al., 1995). The effects of µM CCL5 in T cells have been well documented, ranging
infection (Appay et al., 1999; Appay et al., 2000; Bacon et al., 1995; Bacon et al., 1996;
Chang et al., 2002; Dairaghi et al., 1998; Szabo et al., 1997; Turner et al., 1996). As an
extension of these, the present study describes a potential novel mechanism by which
to invoke this outcome, the important question is whether these concentrations of CCL5
are achievable or likely in vivo. Certainly, unusually high CCL5 concentrations may be
by cell surface and/or extracellular matrix GAGs. In addition, the unique ability of CCL5
local CCL5 concentration (Appay et al., 1999; Appay et al., 2000; Czaplewski et al.,
1999; Hoogewerf et al., 1997; Kuschert et al., 1999; Martin et al., 2001; Proudfoot et al.,
2001; Proudfoot et al., 2003). We, therefore, infer that the CCL5-CCR5 induced
apoptosis of T cells we observe is not likely an in vitro artifact, but is attainable in vivo.
111
We argue against the possibility of CCL5 aggregates blocking the interaction of growth
factors with their receptors and indirectly inducing apoptosis, as the viability of native
PM1 and MOLT-4 cells lacking CCR5 expression, yet able to sequester CCL5 aggregates
cells, although different effectors, including c-Myc and TRAIL, have also been identified.
contradicting our findings, the lineage of the cell type studied and the lower dose of
CCL5 employed, may explain these different observations. In the present study, we
112
Chapter 3
Murooka, T.T., Rahbar, R., Platanias, L.C., and Fish, E.N. (2008). CCL5-mediated T-
cell chemotaxis involves the initiation of mRNA translation through mTOR/4E-BP1.
Blood 111, 4892-4901.
T.T.M. performed all experiments, analyzed the data and drafted the manuscript.
R.R. analyzed the data and edited the manuscript.
L.C.P. designed research.
E.N.F. designed research, analyzed the data and drafted the manuscript.
113
3.1. Abstract
and their chemokine receptors, to invoke signaling events to direct cell migration. Here,
we examined the role for CCL5-mediated initiation of mRNA translation in CD4+ T cell
chemotaxis. Using rapamycin, an inhibitor of mTOR, our data show the importance of
which resulted in its dissociation from the eukaryotic initiation factor-4E. Subsequently,
eIF4E associated with scaffold protein eIF4G, forming the eIF4F translation initiation
complex. Indeed, CCL5 initiated active translation of mRNA, shown by the increased
rapamycin treatment. Notably, CCL5 induced protein translation of cyclin D1 and MMP-
114
3.2. Introduction
biological processes including proper tissue development, wound healing and protection
molecules that play a vital role in many of these biological processes. The chemokines
are a large family of mainly secreted, 8-10 kDa proteins subdivided into 4 families based
on the relative positioning of the first two cysteine residues near the N-terminus (Luster,
1998; Stein and Nombela-Arrieta, 2005; Zlotnik et al., 1999). Chemokine ligands
immune cells to migrate via a haptotactic mechanism (Amara et al., 1999; Cinamon et al.,
2001; Kuschert et al., 1999; Netelenbos et al., 2002; Pablos et al., 2003; Proudfoot et al.,
number of signaling, adhesion and cytoskeletal molecules at the cell surface (Raftopoulou
chemokines and is chemotactic for Th1 T cells, macrophages, dendritic cells and NK
cells through the expression of CCR1 and/or CCR5 (Kawai et al., 1999; Lederman et al.,
2006; Rabin et al., 1999; Schall et al., 1990; Siveke and Hamann, 1998). It is now clear
115
that signaling through CCR5 controls a multitude of cellular functions, including
1995; Bacon et al., 1998; Bacon et al., 1996; Dairaghi et al., 1998; Ganju et al., 2000;
Ganju et al., 1998; Murooka et al., 2006; Rahbar et al., 2006). Through studies with PI-
activation is critical for chemotaxis (Turner et al., 1995a; Ward, 2004; Wymann and
Marone, 2005). Chemokines activate the PI-3’Kγ isoform by the βγ subunits of trimeric
G proteins at the cell membrane, although contributions from other isoforms cannot be
discounted (Curnock et al., 2003; Curnock and Ward, 2003; Sasaki et al., 2000). Studies
with the mTOR inhibitor, rapamycin, have underscored the role for mTOR in fibronectin
and GM-CSF induced cellular migration downstream of PI-3’K (Daniel et al., 2004;
Gomez-Cambronero, 2003; Sakakibara et al., 2005; Sun et al., 2001). mTOR possesses a
carboxy-terminal region sharing significant homology with lipid kinases, especially with
PI-3’K, and has been assigned to a larger protein family termed the PIKKs
S6K1 and initiation factor 4E binding proteins (4E-BPs), and mTOR Complex2, which is
rapamycin-resistant and phosphorylates PKB (Dann et al., 2007; Gingras et al., 1998;
Hay and Sonenberg, 2004). mTOR Complex1 is responsible for the phosphorylation of
S6K1 on Threonine-389 (Hay and Sonenberg, 2004; Loewith et al., 2002; Um et al.,
sites Serine-65 and Threonine-70 are LY294002 and rapamycin sensitive (albeit in
varying degrees), indicating that 4E-BP1 is regulated by both mTOR and PI-3’K (Hay
116
and Sonenberg, 2004; Proud, 2007). Another important modulator of mTOR activity is
phospholipase D (PLD), for which the primary alcohol, 1-butanol, but not tert-butanol,
types(Fang et al., 2001; Foster, 2007; Hornberger et al., 2006). Indeed, CCL5 has been
shown to stimulate PLD activity in Jurkat T cells, but its role in chemotaxis has not been
studied (Bacon et al., 1998). mTOR-dependent modulation of S6K1 and 4E-BP1 has
translation and cell growth (Gingras et al., 2004; Wullschleger et al., 2006). Here, we
examine for the first time the effects of CCL5 on the translation initiation of rapamycin-
multiple levels in mammalian cells. Changes in translation rates often correlate with
changes in the level of eIF4E, and thus its availability is under tight control. Three eIF4E
comprised of an eIF4G backbone, the cap-binding eIF4E and the RNA helicase, eIF4A.
This complex facilitates ribosome binding and passage along the 5’-UTR (untranslated
region) towards the initiation codon (Richter and Sonenberg, 2005; von der Haar et al.,
mRNAs which often encodes for cytoplasmic ribosomal proteins (Meyuhas, 2000;
117
Ruvinsky and Meyuhas, 2006). Although 5’-TOP mRNA translation is sensitive to
rapamycin, the exact mechanism is unclear and recent studies have shown that S6K1 and
its effector molecule rpS6 are dispensable for their translation (Ruvinsky et al., 2005).
Taken together, mTOR is a crucial regulator of the translational machinery by: (1)
directly affecting eIF4F availability for 5’-capped mRNA translation initiation and (2)
expression. Translational control allows for the rapid production of proteins without the
need for mRNA transcription, processing and export into the cytoplasm. In the present
study, we examined the role for CCL5-mediated initiation of mRNA translation in the
“prime” cells for directed cellular migration, thus allowing for a more rapid and effective
immune response.
118
3.3. Materials and Methods
previously described (Murooka et al., 2006). Cells were maintained in RPMI 1640
supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 mg/ml streptomycin
and 2 mM L-glutamine (Gibco-BRL). Briefly, CD4+ T cells were purified using the
(eBiosciences) with soluble 5 ng/ml hrIL-12 (Bioshop, Canada) and 2.5 µg/ml anti-IL-4
antibody (eBiosciences) for 2 days, and further expanded in culture supplemented with
100 U/ml hrIL-2 (Bioshop, Canada) for 3 days. T cell purity and CCR5 expression were
confirmed at day 5 by FACS analysis using anti-human CCR5 antibody (2D7) and anti-
human CD3 antibody (BD Biosciences). Antibodies for phospho-eIF4E (Ser-209), eIF4E,
mTOR were purchased from Cell Signaling Technology. Antibody for human cyclin D1
(DCS-6), eIF4E (P-2) and eIF4G (H-300) were purchased from Santa Cruz
Systems. Polyclonal rabbit antibody against human MMP-9 was purchased from
LY294002 were all obtained from Calbiochem. 1-butanol and tert-butanol were
119
Chemical Company. 2,3-diphosphoglycerate (DPG) has been shown to inhibit PLD and
was purchased from Sigma-Aldrich (Canada).(Kanaho et al., 1993; Kusner et al., 1996)
CCL3 (LD78β) was purchased from Peptrotech (USA). CCL5 was a generous gift from
T cells were serum starved in RPMI 1640/0.5% BSA overnight to reduce the
effects of the various growth factors found in fetal calf serum (FCS) on mTOR and
protein translation. Cells were incubated with 10 nM CCL5 for the times indicated,
collected, washed two times with ice-cold PBS and lysed in 100 μl lysis buffer (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, cells were pretreated for 1 hour with the amount of
inhibitor indicated prior to CCL5 treatment. Protein concentration was determined using
the Bio-Rad DC protein assay kit (BioRad laboratories). 30 μg of protein lysate was
blocking with 5% BSA (w/v) in TBS for 1 hour at room temperature. Membranes were
probed with the specified antibodies overnight in 5% BSA (w/v) in TBST (0.1% Tween-
20) at 4°C and the respective proteins visualized using the ECL detection system (Pierce).
rabbit anti-eIF4G polyclonal antibody (H-300) were added to 500 µg of protein lysates. 2
120
were used as controls. Antibodies were pulled down with protein A/G-sepharose beads
(Santa Cruz Biotechnology) and washed six times with lysis buffer. Beads were then
1 x 106 cells were incubated with mouse anti-human CCR5 antibody for 45
minutes on ice and washed three times with ice-cold FACS buffer (PBS/2% FCS). Cells
control, cells were incubated with FITC-conjugated isotype control IgG antibody
(eBioscience). Cells were analyzed using the FACSCalibur and CellQuest software (BD
Biosciences).
T cell chemotaxis was assayed using 24-well Transwell chambers with 5µm pores
(Corning). A total of 1 x 105 cells in 100 µl chemotaxis buffer (RPMI 1640/0.5% BSA)
were placed in the upper chambers. CCL5, diluted in 600 µl chemotaxis buffer, was
placed in the lower wells and the chambers incubated for 2 hours at 37ºC. Migrated cells
located in the bottom wells were collected, washed once with PBS and counted by FACS.
were pretreated for 1 h at the indicated inhibitor concentrations and placed in the upper
chambers. Cell viability, as measured by PI staining (Figure 3.3), was not affected at any
121
3.3.5. Semi-quantitative RT-PCR
T cells (1 x 107) were serum starved in RPMI 1640/0.5% BSA overnight and
incubated at 37°C with 10 nM CCL5 for the times indicated. Cells were collected,
washed twice with ice-cold PBS and lysed with the RLT buffer (Qiagen). The resulting
lysates were homogenized with a QIA shredder column and total RNA extracted using
the RNeasy Mini kit (Qiagen). 2 µg of RNA was reverse transcribed using M-MLV
reverse transcriptase (Invitrogen). cDNA was then serially diluted in dH20 as indicated
and amplified for human cyclin D1, MMP-9 and GAPDH using the following primers
Activated CD4+ T cells were serum-starved and treated with 10 nM CCL5 for 1
hour before lysis in ice-cold Nonidet P-40 lysis buffer (10 mM Tris-HCl (pH 8.0), 140
mM NaCl, 1.5 mM MgCl2, and 0.5% Nonidet P-40) supplemented with RNaseOut RNase
centrifugation at 3,000 x g for 2 minutes at 4 ºC. The supernatant was supplemented with
665 µg/ml heparin, 150 µg/ml cycloheximide, 20 mM DTT and 1 mM PMSF and
supernatant was then layered onto a 30 ml linear sucrose gradient (15-40% sucrose (w/v)
122
supplemented with 10 mM Tris-HCl (pH 7.5), 140 mM NaCl, 1.5 mM MgCl2, 10 mM
DTT, 100 µg/ml cycloheximide, and 0.5 mg/ml heparin) and centrifuged in a SW32
swing-out rotor (Beckman) at 32,000 rpm for 2 hours at 4 ºC without a brake. Fractions
(750 µl) were carefully collected from the center of the column using a pipette and
precipitation and dissolved in RNase free water before being analyzed by electrophoresis
each fraction was quantified at optical density (OD) of 254 nm. OD readings for each
fraction were plotted as a percentage of the total RNA of all fractions to facilitate visual
between groups.
123
3.4. Results
with cytokines induced CCR5 expression (Figure 3.1.A, left panel). Notably, we observe
no expression of CCR1 in our activated CD4+ T cells. T cell populations used for
subsequent experiments were consistently >95% CD3 and CD4 positive. CCR5
pretreatment with anti-CCR5 antibody (5 µg/ml, 2D7) (Figure 3.1.A, right panel).
Subsequent experiments examined the effects of the PI-3’K inhibitor LY294002 or the
cell chemotaxis in a dose-dependent manner. The data suggest that both PI-3’K and
experiments with CCL3/MIP1α, another agonist ligand of CCR5, revealed that CCL3-
(Figure 3.2.). Notably, we find that both CCL3/MIP1α and CCL4/MIP1β are poor
effectors of CCR5-mediated T cell chemotaxis when compared with CCL5, with CCL3
exhibits a migration index approximately 5 fold over control (Figure 3.1.A), the
124
Figure 3.1. CCL5-mediated chemotaxis of activated CD4+ T cells is dependent on
PI-3’K and mTOR.
(A) Activated peripheral blood (PB) T cells were stained with anti-CCR5 and anti-CD3
antibodies (solid line) or isotype controls (dotted line) and analyzed by FACS. CCL5-
mediated chemotaxis is presented as migrated cells per well. (B) Activated PB T cells
were pretreated with either DMSO (carrier) or the specified inhibitors for 1hr at the
concentrations indicated, and CCL5-mediated chemotaxis measured using 10 nM CCL5.
The data are presented as % migration, with the number of migrated cells at 10 nM CCL5
taken as 100%. Representatives of three independent experiments are shown (± S.D.) *
p<0.05 (C) Activated PB T cells were pretreated with either ethanol (carrier) or AS-
252424 for 1 hr at the concentration indicated, and CCL5-mediated chemotaxis measured
using 10 nM CCL5. Data are representative of two independent experiments (± S.D.) *
p<0.05 (D) Activated PB T cells pretreated with either DMSO (carrier), cycloheximide
or actinomycin D for 30 min at the concentrations indicated. The data represent means ±
S.D. of 3 independent experiments. * p<0.05
125
Figure 3.1.
A 30
CCR5 CD3
% input cells
97% 20
10
0
0 1 10 100 anti-
CCR5
CCL5 (nM)
B * * C
120 * 120 * 120 *
100 100 100
% Migration
% Migration
% Migration
80 80 80
60 60 60
40 40 40
20 20 20
0 0 0
0 5 10 20 0 10 20 50 0 1 2.5
LY294002 (µM) Rapamycin (nM) AS-252424 (µM)
D
*
120 120 *
100 100
% Migration
% Migration
80 80
60 60
40 40
20 20
0 0
0 1 5 0 0.1 1 10
126
Figure 3.2. CCL3/MIP1α-dependent T cell chemotaxis is not dependent on mTOR.
(A) Activated PB T cells were pretreated with either DMSO (carrier) or rapamycin for 1
hr at the concentrations indicated, and CCL3-mediated chemotaxis measured using 10
nM CCL3 (LD78β). The data are presented as % migration, with the number of migrated
cells at 10 nM CCL3 taken as 100%. Representatives of two independent experiments
are shown (± S.D.). * p<0.05 (B) Activated PB T cells were either left untreated or
treated with 10 nM CCL3 for the indicated times. Cells were harvested and lysates
resolved by SDS-PAGE and immunoblotted with anti-phospho-4E-BP1 (Thr-37/46)
antibody. Membranes were stripped and re-probed for 4E-BP1 as a loading control. The
relative fold increase of 4E-BP1 phosphorylation is shown as signal intensity over
loading control to the right of each blot. Representatives of two independent experiments
are shown (± S.D.)
127
Figure 3.2.
A 120
100
% Migration
80
60
40
20
0
0 20 50 100
Rapamycin (nM)
B
+ CCL3 (min)
0 5 15 30
p-4E-BP1(thr 37/46)
4E-BP1
1.2
1
Fold Induction
0.8
0.6
0.4
0.2
0
0 5 15 30
Time (min)
128
transwell experiments. To further investigate the involvement of mRNA translation on
mediated T cell chemotaxis by inhibitors at the doses employed is not due their toxicity
protein
Threonine-389 in vitro (Burnett et al., 1998). In time course studies we show that T cells
Serine-2448 and S6K1 on Threonine-389, within 5 minutes (Figure 3.4.A, B). We also
downstream effector of S6K1, on Serine-235/236 (Figure 3.4.C). Although rpS6 does not
for S6K1 activity. Taken together, the data suggest that CCL5 is able to activate the
129
Figure 3.3. Effect of various inhibitors on T cell viability and adhesion.
(A) Activated T cells were treated with either DMSO (carrier), ethanol (carrier) or the
specified inhibitors for 3 hrs at the concentrations indicated, stained with propidium
iodide and analyzed by FACS. Cells negative for PI stain were considered viable. The
data represent means ± S.D. of at least 2 independent experiments. (B) 2 x 105 T cells
were either left untreated, treated with DMSO (0.1% v/v) or ethanol (0.1% v/v) for 3 hrs,
plated onto fibronectin-coated wells and incubated for 2 hrs at 37˚C. Cells were washed,
fixed in 95% ethanol and stained with crystal violet (2% w/v). 100 µl of solubilization
buffer was added and the absorbance read at 570 nm. Data are representative of two
independent experiments performed in triplicate. The data show that the presence of
DMSO or ethanol as a carrier did not affect T cell adhesion to fibronectin.
130
Figure 3.3.
A
100 100 100
% Viability
% Viability
% Viability
80 80 80
60 60 60
40 40 40
20 20 20
0 0 0
0 20 50 0 10 20 0 1 2.5
Rapamycin (nM) LY294002 (µM) AS-252424 (µM)
% Viability
% Viability
80 80 80
60 60 60
40 40 40
20 20 20
0 0 0
0 0.1 1 10 0 1 5 0 250 500
Cycloheximide (µg/ml) Actinomycin D (µg/ml) 2,3-DPG (µM)
100
80
B
% Viability
60 0.08
Arbitrary units (A570 )
40 0.06
20
0.04
0
0.02
0
0.1% tert-butanol
0.1% 1-butanol
0
Untreated DMSO ethanol
131
Figure 3.4. CCL5-dependent phosphorlyation of mTOR, p70 S6K1 and ribosomal
protein S6 in T cells.
Activated PB T cells were either left untreated or treated with 10 nM CCL5 for the
indicated times. Cells were harvested and lysates resolved by SDS-PAGE and
immunoblotted with (A) anti-phospho-mTOR (Ser-2448) antibody, anti-phospho-p70 S6
kinase (Thr-389) antibody, or anti-phospho-rpS6 (Ser-235/236) antibody. Membranes
were stripped and re-probed for the appropriate loading controls. (B) The relative fold
increase in phosphorylation is shown as signal intensity over loading control. Data are
representative of two independent experiments.
132
Figure 3.4.
A + CCL5 (min)
0 5 15 30
p-mTOR
mTOR
p-p70S6K1
p70S6K1
p-rpS6
rpS6
B
p-mTOR p-p70S6K1 p-rpS6
1.5 2.5 3
2.5
2
Fold Induction
Fold Induction
Fold Induction
1 2
1.5
1.5
1
0.5 1
0.5 0.5
0 0 0
0 5 15 30 0 5 15 30 0 5 15 30
Time (min) Time (min) Time (min)
133
3.4.3. CCL5-mediated 4E-BP1 phosphorylation is PI-3’K-, PLD- and mTOR-dependent
(Hay and Sonenberg, 2004). In PB T cells, CCL5 induced a rapid phosphorylation of 4E-
BP1 on both Threonine-37/46 and Threonine-70 sites (Figure 3.5.A). The roles of PI-3’K,
Consistent with their inhibitory effects on 4E-BP1 phosphorylation, both PLD inhibitors
dependent manner (Figure 3.6.A). Notably, CCL3 did not induce phosphorylation of 4E-
BP1 on Threonine-37/46, consistent with our findings that rapamycin also does not affect
3.4.4. CCL5 initiates protein translation through formation of the eIF4F complex
The preceding suggests that CCL5 may regulate eIF4E availability through
the formation of the eIF4F complex, which also includes eIF4G, a multi-domain scaffold
protein, and eIF4A, a RNA helicase that is required to unwind regions of the secondary
structure in the 5’-UTRs of mRNAs (Richter and Sonenberg, 2005; von der Haar et al.,
2004). To determine whether CCL5 mediates the formation of the eIF4F complex, cells
were treated with CCL5 for 30 minutes and cell lysates immunoprecipiated for eIF4E and
134
Figure 3.5. CCL5 phosphorylates the 4E-BP1 repressor of mRNA translation
through PI-3’ kinase and mTOR.
(A) Activated PB T cells were either left untreated or treated with 10 nM CCL5 for the
indicated times. Cells were harvested and lysates resolved by SDS-PAGE and
immunoblotted with anti-phospho-4E-BP1 (Thr-37/46) antibody or anti-phospho-4E-BP1
(Thr-70) antibody. Membranes were stripped and re-probed for 4E-BP1 as a loading
control. The relative fold increase of 4E-BP1 phosphorylation is shown as signal
intensity over loading control to the right of each blot. (B) Activated PB T cells were
pretreated with either DMSO (carrier), 20 µM LY294002 or 50 nM rapamycin for 1 hr
prior to 15 min treatment with 10 nM CCL5. Cells were harvested and lysates resolved
by SDS-PAGE and immunoblotted with anti-phospho-4E-BP1 (Thr-37/46) antibody.
Membranes were stripped and re-probed for 4E-BP1 as a loading control. The relative
fold increase of 4E-BP1 phosphorylation is shown as signal intensity over loading control.
Data are representative of two independent experiments.
135
Figure 3.5.
Thr 37/46
3
A 2.5
Fold Induction
2
+ CCL5 (min) 1.5
1
0 5 15 30 0.5
p4E-BP1 (Thr 37/46) 0
0 5 15 30
4E-BP1
Time (min)
Fold Induction
2
1
B 0
0 5 15 30
LY294002 – – – + Time (min)
Rapamycin – – + –
CCL5 – + + +
p-4E-BP1 (Thr 37/46)
4E-BP1
3.5
3
Fold Induction
2.5
2
1.5
1
0.5
0
DMSO + CCL5
DMSO (carrier)
Rapamycin
LY294002
136
Figure 3.6. CCL5-mediated PLD activation regulates T cell migration.
(A) Activated PB T cells were pretreated with either ethanol (carrier) or the specified
inhibitors for 1hr at the concentrations indicated, and CCL5-mediated chemotaxis
measured using 10 nM CCL5. The data are presented as % migration, with the number
of migrated cells at 10 nM CCL5 taken as 100%. Data representative of three
independent experiments are shown (± S.D.) * p<0.05 (B) T cells were pretreated with
either ethanol (carrier), 500 µM 2,3-DPG or 0.1% 1-butanol for 1 hr prior to 15 min
treatment with 10 nM CCL5. Cells were harvested and lysates resolved by SDS-PAGE
and immunoblotted with anti-phospho-4E-BP1 (Thr-37/46) antibody. Blots were
stripped and reprobed with anti-4E-BP1 antibody as a loading control. The relative fold
increase of 4E-BP1 phosphorylation is shown as signal intensity over loading control.
Data are representative of two independent experiments.
137
Figure 3.6.
A
* *
120
120
100
100
% Migration
% Migration
80 80
60 60
40 40
20 20
0 0
0.1% 1-butanol
0.1% tert-butanol
0
0 250 350 500
2,3-DPG (µM)
B
1-butanol – – – +
2,3-DPG – – + –
CCL5 – + + +
p-4E-BP1 (Thr 37/46)
4E-BP1
2.5
Fold Induction
2
1.5
1
0.5
0
2,3-DPG
1-butanol
ethanol (carrier)
ethanol + CCL5
138
eIF4G. As shown in Figure 3.7.A, CCL5 induced an association between eIF4E and
eIF4G. To examine the phosphorylation status of eIF4E, cells were treated with 10 nM
CCL5 and the cell lysates resolved by SDS-PAGE gel electrophoresis. As shown in
was performed. Cells were treated with CCL5 for 1 hour and RNAs from each fraction
to examine polysome integrity. The distribution of 28S, 18S and 5S rRNA in untreated
cells were visualized by ethidium bromide staining (Figure 3.8, upper panel). CCL5
molecular-weight polysomes deep in the sucrose gradient (fractions 16-20) (Figure 3.8,
lower panel). Pretreatment with rapamycin inhibited the formation of heavy polysomes.
Viewed altogether, these data show that CCL5 promotes an mTOR-dependent active
139
Figure 3.7. CCL5 induces formation of the eIF4F initiation complex.
(A) Activated PB T cells were either left untreated or treated with 10 nM CCL5 for 30
min, lysed and immunoprecipitated with either anti-eIF4E or anti-eIF4G antibodies.
Samples were resolved by SDS-PAGE and immunoblotted with anti-eIF4E and anti-
eIF4G antibody. Whole molecule mouse or rabbit IgG was used as control. (B) Cells
were either left untreated or treated with 10 nM CCL5 for the indicated times, then
lysates resolved by SDS-PAGE and immunoblotted with anti-phospho-eIF4E (Ser-209)
antibody. Membranes were stripped and reprobed for eIF4E as a loading control. Data
are representative of two independent experiments. Values denoting the extent of
phosphorylation are shown in the right hand panel.
140
Figure 3.7.
A
+ CCL5 (min) + CCL5 (min)
IgG (HC)
B p-eIF4E
5
+ CCL5 (min)
Fold Induction
4
0 5 15 30 3
p-eIF4E
2
eIF4E 1
0
0 5 15 30
Time (min)
141
Figure 3.8. CCL5-inducible protein translation enhances mRNA association with
polyribosomes.
PB T cells were pretreated with either DMSO (carrier) or 50 nM rapamycin for 1 hr,
followed by 10 nM CCL5 for 1 hr. Cells were harvested, lysed and lysates layered onto a
sucrose gradient. Fractions were collected after centrifugation, RNAs extracted and
quantified at optical density (OD) 254 nm. A representative gel profile of fractions from
untreated cells is shown to visualize the distribution of 5S, 18S and 28S rRNAs as an
indicator of the polyribosome integrity (upper panel). OD readings for each fraction were
plotted as a percentage of the total RNA of all fractions and are shown as a function of
gradient depth (lower panel). Actively translated mRNA is associated with high-
molecular-weight polysomes deep in the gradient (shaded region). Data are
representative of two independent experiments.
142
Figure 3.8.
28S
18S
5S
14 Untreated
12
CCL5
CCL5 + Rapamycin
10
% Total RNA
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Fraction number
143
3.4.5. CCL5-inducible protein translation of cyclin D1 and MMP-9 is mTOR-
dependent
substantial secondary structures in the 5’-UTR (De Benedetti and Graff, 2004). Among
these, both MMP-9 and cyclin D1 have recently been shown to promote cellular motility
(Hu and Ivashkiv, 2006; Khandoga et al., 2006; Li et al., 2006a; Li et al., 2006b;
Neumeister et al., 2003; Xia et al., 1996). Accordingly, we conducted studies to examine
whether CCL5 initiated the translation of cyclin D1 and MMP-9 levels. Serum starved T
cells were pretreated with either DMSO or rapamycin for 1 hour and treated with 10 nM
CCL5 in time course experiments. CCL5 induced upregulation of both cyclin D1 and
MMP-9 protein levels within 60 minutes, whereas rapamycin completely abolished this
induction (Figure 3.9.A). The observed increases in cyclin D1 and MMP-9 protein levels
were not due to increased mRNA transcription, as their mRNA levels remained
144
Figure 3.9. CCL5-inducible upregulation of cyclin D1 and MMP-9 protein levels is
dependent on mTOR-mediated mRNA translation.
145
Figure 3.9.
A 2.5
Cyclin D1
Fold Induction
0 30 60 90 0 30 60 90 1.5
cyclin D1
1
β-tubulin
0.5
0
0 30 60 90
Time (min)
Untreated
Active MMP-9 Rapamycin
3.5
UT + CCL5 (min) Rapamycin + CCL5 (min) 3
Fold Induction
0 30 60 90 0 30 60 90 2.5
pro 2
active MMP-9
1.5
β-tubulin
1
0.5
0
0 30 60 90
Time (min)
B CCL5 (min)
0 60
GAPDH
146
3.5. Discussion
towards understanding the complex signaling cascades coordinating cell migration, which
include the activation of many distinct tyrosine kinases, lipid kinases, and MAPKs.
initiation and up-regulates ribosomal protein levels through 5’-TOP mRNA translation.
Published reports suggest a role for both mTOR and p70 S6K1 in cellular migration of
of human arterial E47 smooth muscle cells is sensitive to rapamycin (Sakakibara et al.,
2005). Several chemokines have been reported to activate S6K1, but this activation was
studied in the context of cell survival and proliferation, not migration (Hwang et al.,
2003; Joo et al., 2004; Lee et al., 2002; Loberg et al., 2006). Interestingly, a G protein-
coupled receptor (vGPCR), which belongs to the CXC chemokine receptor family, is
147
encoded by the Kaposi’s sarcoma-associated herpesvirus (KSHV or HHV8) and exhibits
growth (Montaner, 2007; Sodhi et al., 2006). Here, we show the importance of mTOR in
have been identified including rpS6, eIF4B and eEF2 (Proud, 2007; Ruvinsky and
(Raught et al., 2004). The role for rpS6 is less well understood, previously believed to be
associated with 5’ TOP mRNA translation. However, studies with knock-in mice in
which all five regulated sites of S6 phosphorylation were altered to alanines (S6[5A])
demonstrated that rpS6 is not required for 5’TOP mRNA translation, but rather for
sequential manner (Brunn et al., 1997; Burnett et al., 1998). mTOR phosphorylates
leading to its release from eIF4E. Here we show CCL5 mediates a rapid phosphorylation
Threonine-37/46 is dependent on PI-3’K, PLD and mTOR. Therefore, the data indicate
that CCL5-mediated activation of the PI-3’K and PLD signaling pathways may converge
148
at the level of mTOR to modulate downstream 4E-BP1 phosphorylation. 4E-BP1 hyper-
phosphorylation releases eIF4E to allow for association with the scaffold protein eIF4G,
which along with the RNA helicase eIF4A, forms the eIF4F hetero-trimeric initiation
complex (Richter and Sonenberg, 2005; von der Haar et al., 2004). By binding to the 5’-
cap structure of mRNA through eIF4E, the eIF4F initiation complex facilitates ribosome
binding and its passage along the 5’-UTR towards the initiation codon. eIF4E is also
is still unclear. Several reports demonstrated that phosphorylated eIF4E actually had
reduced m7G cap-binding ability (McKendrick et al., 2001; Scheper et al., 2002). Proud
and colleagues suggested that phosphorylation of eIF4E may allow the eIF4F complex to
detach from the 5’-cap during scanning to either accelerate translation or to allow a
second initiation complex to bind the mRNA (Proud, 2007). Our data support this model,
as phosphorylation of eIF4E was not evident until 15 minutes post CCL5 treatment
(Figure 3.7.B). This allows time for eIF4E to bind the cap structure and recruit
eIF4G/eIF4A and other associated factors such as eIF3 and the 40S subunit before the
complex is released for scanning. Consistent with this, CCL5 initiated active translation
the sucrose gradient we analyzed (fractions 16-20). The presence of polysomes was
significantly reduced in the presence of rapamycin, further supporting the role for mTOR
in translation initiation.
It is well known that cap structures at the 5' end of mRNA are required for
efficient translation, nuclear export and protection from 5' exonucleases. Once bound by
149
eIF4F, ribosome binding and scanning can commence along the 5’-UTR towards the
initiation codon. Unlike mRNAs with short 5’UTRs (e.g. β-actin), a subset of mRNAs
with lengthy, highly structured 5’UTRs are poorly translated when eIF4F levels are low
(De Benedetti and Graff, 2004; Graff and Zimmer, 2003). Among these, cyclin D1 and
MMP-9 have been implicated in cellular migration of a number of cell types (Hu and
Ivashkiv, 2006; Khandoga et al., 2006; Li et al., 2006a; Li et al., 2006b; Neumeister et al.,
2003; Sun et al., 2001). Li and colleagues showed that cyclin D1-deficient mouse
compared to wildtype MEFs (Li et al., 2006b). Migratory defects in cyclin D1-deficient
MEFs were not a direct consequence of reduced DNA synthesis, but rather through de-
repression of ROCKII and TSP-1 expression. Use of PI-3’K and mTOR inhibitors in
cancer cell lines decreased cyclin D1 levels, where eIF4E over-expression led to its
increased production (Gao et al., 2004; Pene et al., 2002; Rosenwald et al., 1995;
Rosenwald et al., 1993). IL-8 has been shown to up-regulate cyclin D1 at the level of
translation in prostate cancer cell lines (MacManus et al., 2007). Similarly, in MMP-9
knockout mice, bone marrow-derived dendritic cell (BM-DC) migration to CCL19 was
(Hu and Ivashkiv, 2006). Therefore, we investigated the ability of CCL5 to initiate
of cyclin D1 and MMP-9 protein levels occurred within 1 hour of CCL5 treatment. Up-
regulation of protein levels was not due to increased transcription since we did not
observe significant mRNA synthesis of these genes within this time frame. Interestingly,
others have shown that CCL5-mediated increases in MMP-9 protein levels are detectable
150
early without significant upregulation of mRNA, indicating that the early effect of CCL5
on MMP-9 secretion was independent of mRNA synthesis (Chabot et al., 2006). Since T
degradation of matrix proteins by MMP-9, rapid production of the protease during the
forming a leading edge and directional migration towards the source of chemokines
(Gomez-Mouton et al., 2004). In a typical chemotaxis assay, migrated cells are collected
cells likely do not take 2 hours to migrate through the membrane pores. We have
unpublished data demonstrating that rapamycin does not affect CCL5-mediated actin
polymerization, indicating that mTOR plays no role in the initial stages of migration.
3.10.). CCR5 is the receptor for several chemokines, specifically CCL3, CCL4, and
CCL5. We observe that CCL3 and CCL5 differentially activate mTOR signaling. mTOR
The identification of additional proteins that are regulated by CCL5 at the level of
151
Figure 3.10. Possible model for CCL5-mediated mRNA translation in CD4+ Tcells.
CCL5 activates the mTOR pathway and subsequent phosphorylation of p70 S6K1 and
4E-BP1. Hyper-phosphorylation of 4E-BP1 leads to its release from eIF4E where it
binds to eIF4G to form the eIF4F initiation complex. Through eIF4E, eIF4F binds to the
mRNA 5’-cap structure and facilitates ribosome binding and unwinding secondary
structure in the 5’-UTR. Translation initiation leads to a rapid upregulation of cyclin D1
and MMP-9 protein levels to “prime” T cells for directed cell migration. S6K1 has been
shown to phosphorylate eIF4B (RNA-binding protein that enhances activity of the eIF4A
helicase) in response to insulin (dotted line).
152
Figure 3.10.
CCL5
CCR5
PLD PI-3’K
mTOR
S6K 4E-BP1
eIF4E
rpS6 Cyclin D1
eIF4E MMP-9
AUG
? eIF4G eIF4A
eIF4B
Cell Migration
153
translation is currently under investigation. Translational control generates a rapid
production of proteins without the need for mRNA transcription, processing and export
into the cytoplasm. As migratory responses must be both initiated and resolved with
speed and precision, it is beneficial that chemokines can effect translation and rapidly
influence the protein pool within the migrating cell. Our data describe a novel
mechanism by which the chemokine CCL5 may regulate translation of mRNAs that
154
Chapter 4
Murooka, T.T., Rahbar, R. and Fish, E.N. CCL5 promotes breast cancer proliferation
through mTOR/4E-BP1 dependent mRNA translation.
T.T.M. performed all experiments, analyzed the data and drafted the manuscript.
R.R. analyzed the data and edited the manuscript.
E.N.F. designed research, analyzed the data and drafted the manuscript.
155
4.1. Abstract
evidence identifies a role for chemokines and their cognate receptors in cancer
chemokine receptor, CCR5, when expressed in the breast cancer cell line, MCF-7. At
induced the formation of the eIF4F translation initiation complex through an mTOR-
up-regulation of protein expression for cyclin D1, c-Myc and Dad-1, without affecting
their mRNA levels. CCL5 increased the recruitment of cyclin D1 and Dad-1 mRNAs to
polysomes, indicating that their protein expression was regulated at the level of
156
4.2. Introduction
for neoplastic growth (O'Hayre et al., 2008). Given that chemokines are important
including the control of leukocyte infiltration into tumors, initiation of primary tumor
growth, survival, invasion and organ-specific metastasis (Muller et al., 2001; O'Hayre et
components of the cell surface and extracellular matrix, and interact with seven
the β-chemokines and is chemotactic for T cells, macrophages, NK cells and eosinophils
through CCR1 and/or CCR5 (Kameyoshi et al., 1992; Schall et al., 1990; Taub et al.,
production, survival and apoptosis (Bacon et al., 1995; Bacon et al., 1998; Bacon et al.,
1996; Dairaghi et al., 1998; Ganju et al., 2000; Ganju et al., 1998; Murooka et al., 2006;
Rahbar et al., 2006; Tyner et al., 2005). Several studies have demonstrated a pivotal role
157
for the CCL5/CCR5 axis in breast cancer progression. CCL5 was reported to be highly
expressed in high grade tumors and was a predictor of rapid disease progression in stage
II breast cancer patients (Azenshtein et al., 2002; Luboshits et al., 1999; Niwa et al.,
2001; Yaal-Hahoshen et al., 2006). Additionally, serum CCL5 levels were elevated in
patients with high grade tumors compared to low grade tumors (Niwa et al., 2001).
Breast cancer cell lines have been shown to respond to and migrate towards CCL5, as
Luboshits et al., 1999; Robinson et al., 2003; Youngs et al., 1997). Finally, the CCR5
tumors, resulting in a reduction in tumor mass in mice (Robinson et al., 2003). Viewed
altogether, these studies demonstrate that CCL5 can influence breast cancer progression
metabolism, nutrient sensing, translation and cell growth (Gingras et al., 2004;
Wullschleger et al., 2006). We have previously shown that CCL5 initiates mRNA
availability of eIF4E through 4E-BP1 phosphorylation (Beretta et al., 1996). The eIF4F
complex, which is comprised of an eIF4G backbone, the cap-binding eIF4E and the RNA
helicase eIF4A, binds the mRNA 5’-cap structure (m7GpppN). eIF4F unwinds the
158
secondary structure in the 5'-untranslated region (UTR) of mRNA and facilitates binding
of the mRNA to the 40S ribosomal subunit (Richter and Sonenberg, 2005; von der Haar
(Meyuhas, 2000). Taken together, mTOR regulates the translation of a subset of mRNAs
with lengthy, highly structured 5’-UTRs, which typically encode growth and survival
Here, we demonstrate that the CCL5/CCR5 signaling axis can directly stimulate
ectopic expression of CCR5 provides MCF-7 cells with a proliferative advantage when
cultured in the presence of exogenous CCL5. Through the formation of the eIF4F
cyclin D1, c-Myc and defender against cell death-1 (Dad-1). The data illustrate the
potential for breast cancer cells to exploit downstream chemokine signaling pathways for
receptors.
159
4.3. Materials and Methods
MCF-7 breast cancer cells were a generous gift from Dr. Jeffery Medin (Division
maintained in DMEM supplemented with 10% fetal calf serum, 100 units/ml penicillin,
100 mg/ml streptomycin and 2 mM L-glutamine (Gibco-BRL). Antibodies for eIF4E and
4E-BP1 were purchased from Cell Signaling Technology. Antibody for human cyclin D1
(DCS-6), eIF4G (H-300), phospho-Erk (E-4) and Erk1 (K-23) were purchased from Santa
Cruz Biotechnology (Santa Cruz, USA). Murine monoclonal anti-β-actin antibody was
Myc antibody was a generous gift from Dr. Linda Penn (Ontario Cancer Institute,
Toronto, Canada). CCL5 was a generous gift from Dr. Amanda Proudfoot (Geneva
purchased from Amersham Biosciences. Rapamycin was obtained from Calbiochem and
resuspended in DMSO.
Full-length human CCR5 cDNA was generated by PCR using the pEF.BOS-
CCR5 vector, as previously described (Rahbar et al., 2006). Specific human CCR5
forward and reverse primers containing the BamH1 and NotI restriction sites,
respectively, and the FLAG epitope DYKDDDDK on the N-terminus, were used: FP 5’
ggatccatggactacaaggacgatgatgac gccgattatcaagtgtcaagtcca 3’ RP 5’
160
tgcggccgctcacaagcccacagatatttc 3’ (95°C 1 min, 64°C 30 sec, 72°C 75 sec, 30 cycles).
Human CCR5 was subcloned into pcDNA3.1+/Zeo+ vector (Invitrogen) and the
orientation and integrity of the insert confirmed by DNA sequencing (ACGT Corp.,
Toronto, Canada). To establish the MCF-7.CCR5 cell line, subconfluent MCF-7 cells in
manufacturer’s protocol (Roche). Cells were selected in 250 µg/ml zeocin for 4 weeks
and FACS sorted for CCR5-positive clones. Stable CCR5 transfectant cell lines were
designated MCF-7.CCR5, whereas cells transfected with vector were designated MCF-
7.vector.
MCF-7.vector and MCF-7.CCR5 cells (5 x 103) were seeded into 24-well plates in
DMEM/2% fetal calf serum (FCS). Cells were incubated with either 1 or 10 nM CCL5
for the days indicated, collected and counted with a hemocytometer. Cells were fed with
fresh media and CCL5 every other day. In CCR5 blocking studies, cells were pretreated
with the anti-CCR5 antibody (5 µg/mL) for 1 hour prior to CCL5 stimulation. To
determine the role of mTOR, cells were pretreated with rapamycin at the indicated doses
for 1 hour prior to CCL5 stimulation. Cells were subsequently fed with fresh media
161
MCF-7.CCR5 cells (4 x 105) were serum starved in DMEM/0.5% BSA + 0.5%
fetal calf serum (FCS) to reduce the effects of the various growth factors found in fetal
calf serum on mTOR and protein translation. Cells were incubated with 10 nM CCL5 for
the times indicated, collected, washed with ice-cold PBS and lysed in 200 μl lysis buffer
(1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA,
A). In experiments where rapamycin was used, MCF-7.CCR5 cells were pretreated for 1
hour prior to CCL5 treatment. Protein concentration was determined using the Bio-Rad
sample reducing buffer and resolved by SDS-PAGE gel electrophoresis. The separated
BSA (w/v) in TBS for 1 hour at room temperature. Membranes were probed with the
specified antibodies overnight in 5% BSA (w/v) in TBST (0.1% Tween-20) at 4°C and
the respective proteins visualized using the ECL detection system (Pierce). For
negative control. Beads were washed three times with lysis buffer, denatured in 5X
1 x 106 cells were incubated with mouse anti-human CCR5 antibody for 45
minutes on ice and washed three times with ice-cold FACS buffer (PBS/2% FCS). Cells
162
Cells incubated with FITC-conjugated anti-mouse IgG antibody alone was used as
control. Cells were analyzed using the FACSCalibur and CellQuest software (BD
Biosciences).
MCF-7.CCR5 cells were serum-starved and treated with 10 nM CCL5 for 1 hour
before lysis in ice-cold Nonidet P-40 lysis buffer (10 mM Tris-HCl (pH 8.0), 140 mM
NaCl, 1.5 mM MgCl2, and 0.5% Nonidet P-40) supplemented with RNaseOut RNase
centrifugation at 3,000 x g for 2 minutes at 4 ºC. The supernatant was supplemented with
for 5 minutes at 4 ºC to eliminate mitochondria. The supernatant was then layered onto a
(pH 7.5), 140 mM NaCl, 1.5 mM MgCl2, 10 mM DTT, 100 µg/ml cycloheximide) and
without a brake. Fractions (1 mL) were carefully collected from the center of the column
using a pipette and digested with 100 µg of proteinase K in 1% SDS and 10 mM EDTA
followed by ethanol precipitation and dissolved in 20 µl RNase free water before being
from each fraction was quantified at optical density (OD) of 254 nm. OD readings for
each fraction were plotted as a percentage of the total RNA of all fractions to facilitate
163
4.3.7. RT-PCR
Total RNA was isolated using the RNease Mini-Kit (Qiagen). For the reverse
For semi-quantitative PCR of total RNA, cDNAs were diluted 1:3 and 1:9 in water and
used for subsequent amplification of human cyclin D1, c-Myc, Dad-1, PKR and β-actin
Myc, FP 5’ cccggaattcgcccctcaacgttagcttc 3’ RP 5’
cycles); Dad-1, FP 5' agttcggttactgtctcctcg 3' RP 5' tgtgtccataagctgccatc 3' (95°C 1 min,
1 min, 58°C 40 sec, 72°C 1 min, 23 cycles). For polysomal PCR, 1 µl of cDNA from
each fraction was used. Aliquots were loaded onto 1-1.2% agarose gels and visualized
between groups.
164
4.4. Results
towards CCL5 in a G-protein dependent manner (Youngs et al., 1997). However, the
MCF-7 cells provided to us did not express cell surface CCR1 or CCR5, as
heterogeneity of different MCF-7 cell lines (Prest et al., 1999). Thus, the stable sub-cell
Methods, to examine the potential proto-oncogenic role of CCR5. Cell surface CCR5
expression was confirmed in the transfected cells by FACS analysis (Figure 4.1.A). To
examine their proliferative capacity, MCF-7.vector and MCF-7.CCR5 cells were cultured
CCL5, which was not observed in MCF-7.vector cells (Figure 4.1.B). The presence of
anti-CCR5 antibody abrogated this CCL5-induced growth effect (Figure 4.1.B, right
panel). Subsequent experiments examined the role of mTOR and mRNA translational
4.1.C), underscoring the role mTOR plays in MCF-7 breast tumor growth (Noh et al.,
165
Figure 4.1. CCL5-mediated MCF-7 proliferation is dependent on mTOR.
(A) MCF-7 cells were transfected with either pcDNA3 vector or pcDNA.CCR5 plasmid
and selected for 4 weeks. Stable sub-cell lines were stained with anti-CCR5 (solid line)
or isotype controls (dotted line) and analyzed by FACS. (B) 5 x 103 MCF-7.vector or
MCF-7.CCR5 cells were seeded into 24 well plates and stimulated with CCL5. Cells
were fed with fresh media containing the indicated doses of CCL5 every other day.
MCF-7.CCR5 cells were pretreated with 5 µg/ml anti-CCR5 mAb (2D7) for 1 hr prior to
CCL5 stimulation. Cells were trypsinized and counted with a hemocytometer. * p<0.05
(C) MCF-7.CCR5 cells were pretreated with either DMSO (carrier) or rapamycin at the
indicated doses for 1 hr prior to CCL5 stimulation. Cells were fed with fresh media
containing the indicated doses of rapamycin and CCL5 every other day. The data
represent means ± S.D. of 3 independent experiments. * p<0.05
166
Figure 4.1.
A
MCF-7.vector MCF-7.CCR5
CCR5 CCR5
B MCF-7.vector MCF-7.CCR5
* *
140000 140000
120000 120000
Cell number
Untreated
Cell number
20000 20000
0 0
0 4 5 0 4 5
Time (Days) Time (Days)
C
MCF-7.CCR5
140000
* *
120000
Untreated
Cell number
20000
0
0 5
Time (Days)
167
4.4.2. CCL5 activation of CCR5 leads to the formation of the eIF4F complex through
mTOR.
formation of the eIF4F complex, MCF-7.CCR5 cells were treated with CCL5 for up to 60
sepharose beads that mimic the 5’ cap, was used to affinity pull-down eIF4E cell lysates
(Haller and Sarnow, 1997). As shown in Figure 4.2.A, treatment with 10 nM CCL5 led
to the dissociation of eIF4E and 4E-BP1, which was sensitive to rapamycin treatment.
The consequent CCL5-induced association of eIF4E with eIF4G was likewise blocked by
treatment with rapamycin. These findings suggest that CCL5-CCR5 interactions result in
7.CCR5 cells were treated with 10 nM CCL5 for 1 hour, then cell extracts subjected to
sucrose gradient centrifugation and serial fractions collected. RNA from each fraction
extracted was analyzed by agarose gel electrophoresis to ensure polysome integrity. The
distribution of 18S and 28S rRNA in fractions derived from cells either treated with
CCL5 or left untreated was visualized by ethidium bromide staining (Figure 4.2.B, upper
panel). CCL5 initiated active translation of mRNA, as shown by the increased presence
168
Figure 4.2. CCL5 induces formation of the eIF4F initiation complex and enhances
mRNA association with polyribosomes.
(A) 1 x 106 MCF-7.CCR5 cells were pretreated with either DMSO (carrier) or 50 nM
rapamycin for 1 hr prior to 10 nM CCL5 treatment for the indicated times. Cells were
lysed and immunoprecipitated with 7-methyl GTP sepharose beads overnight. Beads
were washed, resolved by SDS-PAGE and immunoblotted with anti-eIF4E, anti-eIF4G or
anti-4E-BP1 antibodies. Unconjugated sepharose beads were used as negative control
(neg). (B) MCF-7.CCR5 cells were pretreated with either DMSO (carrier) or 50 nM
rapamycin for 1 hr, followed by 10 nM CCL5 for 1 hr. Cells were harvested, lysed and
lysates layered onto a sucrose gradient. Fractions were collected after centrifugation,
RNAs extracted and quantified at optical density (OD) 254 nm. Representative gel
profile of fractions from untreated and CCL5-treated cells are shown to visualize the
distribution of 5S, 18S and 28S rRNAs as an indicator of the polyribosome integrity
(upper panel). OD readings for each fraction were plotted as a percentage of the total
RNA of all fractions and are shown as a function of gradient depth (lower panel).
Actively translated mRNA is associated with high-molecular-weight polysomes deep in
the gradient (shaded region). Data are representative of two independent experiments.
Figure 4.2.
169
A 7-methyl GTP Sepharose neg
CCL5 (min) 0 30 60 0 30 60
Rapamycin - - - + + +
eIF4E
4E-BP1
CCL5 (min) 0 30 60 0 30 60
Rapamycin - - - + + +
eIF4E
eIF4G
B
15% Sucrose 40%
Untreated
CCL5
Fraction # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
14
12
Untreated
10 CCL5
% Total RNA
CCL5 + Rapamycin
8
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Fraction numbe r
170
(Figure 4.2.B, lower panel). Pretreatment with rapamycin inhibited the formation of
heavy polysomes. Viewed together, these data suggest that CCL5 may exert its
proliferative effect by actively translating mRNAs involved in cell growth and survival.
with substantial secondary structures in their 5’-UTR. A large number of these mRNAs
encode for proliferation and survival proteins (Graff and Zimmer, 2003; Mamane et al.,
translation of cyclin D1, c-Myc and Dad-1, because of their well-studied roles in cell
cycle progression and survival. In time course studies, MCF-7.CCR5 cells were
pretreated with either DMSO (carrier) or rapamycin for 1 hour prior to treatment with 10
nM CCL5. CCL5 treatment rapidly up-regulated cyclin D1, c-Myc and Dad-1 protein
levels in a time dependent manner, whereas rapamycin treatment reduced their induction
(Figure 4.3.A). Notably, rapamycin treatment did not affect CCL5-mediated Erk1/2
phosphorylation, consistent with data that mTOR is not placed upstream of Erk1/2
(Steelman et al., 2008). We provide evidence that the increases in cyclin D1, c-Myc and
Dad-1 protein levels were not due to increased gene transcription, as their mRNA levels
171
Figure 4.3. CCL5 mediates upregulation of proliferative and survival proteins
through a mTOR dependent mechanism.
(A) MCF-7.CCR5 cells were either pretreated with DMSO (carrier) or 50 nM rapamycin
for 1 hr prior to treatment with 10 nM CCL5 for the indicated times. Cells were
harvested and lysates resolved by SDS-PAGE and immunoblotted with anti-cyclin D1,
anti-Dad-1, anti-c-Myc, anti-phospho-Erk1/2, anti-Erk1/2 or β-actin. Data are
representative of two independent experiments. (B) MCF-7.CCR5 cells were either
pretreated with DMSO or 50 nM rapamycin for 1 hr prior to treatment with 10 nM CCL5
for 1hr and total mRNAs extracted. RT-PCR (undiluted, 1:3, 1:9) was performed using
primer sets specific for cyclin D1, Dad-1, β-actin, c-Myc and PKR, as described in
Materials and Methods.
172
Figure 4.3.
A
CCL5 Rapamycin + CCL5
hrs: 0 0.5 1 2 0 0.5 1 2
c-Myc
cyclin D1
β-actin
Dad-1
p-Erk1/2
Erk1/2
β-actin
B Rapamycin
UT CCL5
+ CCL5
Dad-1
cyclin D1
c-Myc
β-actin
PKR
173
4.4.4. CCL5 facilitates recruitment of a subset of mRNAs to polysomes.
was due to effects on protein stability, the distribution of cyclin D1 and Dad-1 mRNA
along the sucrose density gradient was examined. MCF-7.CCR5 cells were pretreated
with either DMSO (carrier) or 50 nM rapamycin, then treated with 10 nM CCL5 for 1
hour. Cell extracts were subjected to sucrose density centrifugation, fractions collected
and RNA prepared. RT-PCR was performed on each fraction, the cDNAs analyzed by
agarose gel electrophoresis, and each amplified band was quantified by densitometry.
Total RNA was designated as the sum of the band density values of all fractions. As
shown in Figure 4.4., CCL5 induced the shifting of Dad-1 and cyclin D1 mRNAs to
heavier polysome fractions, which was inhibited by rapamycin. CCL5 did not induce the
for PKR, a protein not known to be regulated by CCL5. Both ß-actin and PKR mRNA
profiles in the sucrose gradient were largely unaffected by rapamycin. The data suggest
174
Figure 4.4. CCL5 faciliates recruitment of a subset of mRNAs to polysomes.
(A) MCF-7.CCR5 cells were either pretreated with DMSO or 50 nM rapamycin for 1 hr
prior to treatment with CCL5 for 1 hr. RNA from 20 fractions was extracted and reverse
transcribed into cDNA. RT-PCR was performed to assess mRNA levels of cyclin D1,
Dad-1, β-actin and PKR within each fraction. Aliquots from each reaction was loaded
onto an agarose gel and visualized by ethidium bromide. Amplified PCR bands from
fractions were quantified by densitometry and plotted as a % of total RNA to the right of
each gel. Polysomes are found in fractions 17-20 (shaded region). Data are
representative of two independent experiments.
175
Figure 4.4.
12
UT 10
% total RNA
Dad-1 CCL5 8
Rapamycin
+CCL5
6
Fraction #: 1 3 5 7 9 11 12 13 14 15 16 17 18 19 20 4
polysome
2
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
14
UT
% total RNA
cyclin D1 CCL5 9
Rapamycin
+CCL5
Fraction #: 1 3 5 7 9 11 12 13 14 15 16 17 18 19 20 4
polysome
-1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
12
UT 10
% total RNA
CCL5
β-actin 8
Rapamycin
+CCL5 6
Fraction #: 1 3 5 7 9 11 12 13 14 15 16 17 18 19 20 4
polysome 2
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
14
12
UT
% total RNA
10
PKR CCL5
Rapamycin 8
+CCL5 6
Fraction #: 1 3 5 7 9 11 12 13 14 15 16 17 18 19 20 4
polysome 2
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fraction #
Untreated
CCL5
CCL5 + Rapamycin
176
4.5. Discussion
and angiogenic factors. Many of these biological response modifiers activate signaling
has been shown to correlate with poor breast cancer prognosis (Armengol et al., 2007).
Similarly, eIF4E over-expression has been associated with the malignant progression of
different cancers including breast, colon, lung and prostate (De Benedetti and Graff,
2004; Graff et al., 2008; Zhou et al., 2006). We have previously shown that CCL5
cascade, thereby modulating CD4+ T cell chemotaxis (Murooka et al., 2008). In the
present study, we provide evidence that CCL5 activation of CCR5 results in signaling
Accumulating evidence indicates that eIF4E may act as the node of convergence
(Noh et al., 2004; Sabatini, 2006). The two major substrates of mTOR are the
serine/threonine kinase p70 S6K and the eIF4E-binding protein 4E-BP1, both shown to
phosphorylation releases eIF4E, allowing it to associate with the scaffold protein eIF4G,
which, along with the RNA helicase eIF4A, forms the eIF4F heterotrimeric initiation
177
complex. By binding to the 5’-cap structure of mRNAs through eIF4E, the eIF4F
complex facilitates ribosome binding and its passage along the 5’-UTR towards the
initiation codon. Although increased availability of eIF4F initiates the translation of all
cap-dependent mRNAs, a subset of mRNAs that contain lengthy, highly structured 5’-
UTRs are the most sensitive. These mRNAs typically encode for growth and survival
proteins (e.g. cyclin D1, VEGF, bcl-2), and are poorly translated when eIF4F availability
is limited (De Benedetti and Graff, 2004; Graff et al., 2008). Once eIF4F complex levels
are high, these mRNAs are preferentially translated and play critical roles in cell growth,
NIH 3T3 cell line. These mRNAs encoded for a number of ribosomal proteins, anti-
apoptotic proteins and cell growth-related factors (Averous et al., 2008; De Benedetti and
Graff, 2004; Mamane et al., 2007). We have extended these findings to investigate the
potential for CCL5 to regulate translation of the mRNAs for cyclin D1, c-Myc and Dad-1.
The oncogenic properties of cyclin D1 during mitosis have been well characterized, and
its over-expression is common in many human cancers (Knudsen et al., 2006). Similarly,
the proto-oncogene c-Myc is over-expressed in many cancers, and high expression levels
correlate with advanced disease stage (Pelengaris et al., 2002; Vogelstein and Kinzler,
2004). Notably, eIF4E and c-Myc synergistically have anti-apoptotic effects on cells,
c-Myc decreased MCF-7 growth rate both in vitro and in vivo (Wang et al., 2005). Dad-
178
1-deficiency results in embryonic lethality in mice, associated with an increased
frequency of apoptosis observed in selective tissues (Hong et al., 2000). Herein we show
that CCL5 rapidly up-regulates cyclin D1, c-Myc and Dad-1 protein levels without
these translational events is reinforced by our observation that mRNAs for β-actin and
PKR did not redistribute along the sucrose gradient following CCL5 treatment of cells.
Increased eIF4E availability does not affect all cap-dependent mRNA translation, but
may allow breast cancer cells to take advantage of CCL5 which accumulates within the
proliferation.
Previous studies have described the proto-oncogenic roles of both CCL5 and
CCR5 in several cancer types (Aldinucci et al., 2008; Azenshtein et al., 2002; Luboshits
et al., 1999; Robinson et al., 2003; Sugasawa et al., 2008; Vaday et al., 2006; Youngs et
al., 1997). However, there are conflicting reports regarding the direct role of CCL5 in
breast tumor cell growth (Adler et al., 2003; Jayasinghe et al., 2008). Our data support
the proliferative role of CCL5 in breast cancer. This is in contrast to studies showing that
tumor-derived CCL5 did not contribute to breast tumor formation in vivo (Jayasinghe et
al., 2008). One explanation for these discrepant results is the concentration of CCL5 in
nM, CCL5 produced by 4T1 breast cancer cells reported by Jayasinghe and colleagues
179
was approximately 100 fold less (Jayasinghe et al., 2008). We infer that a threshold level
of CCL5 is required in order for CCL5 to invoke a proliferative response in breast cancer
cells. The hypothesis is supported by several studies showing that CCL5 content within
tumor lesions is markedly higher in more aggressive forms of breast cancer (Bieche et al.,
2004; Niwa et al., 2001). Such a threshold may be attainable through the propensity of
CCL5 to bind, oligomerize and accumulate on GAGs at their secretion site (Proudfoot et
al., 2003).
al., 2008). CXCL8 has been shown to up-regulate cyclin D1 at the level of translation in
prostate cancer cells (MacManus et al., 2007). Sodhi and colleagues show that
CCL5 was implicated in mediating pro-growth and anti-apoptotic effects of gastric cancer
this signaling pathway may have therapeutic potential as anti-cancer drugs. Certainly,
rapamycin and its derivatives are currently being evaluated in multiple cancer clinical
180
trials (Guertin and Sabatini, 2007). In addition, Graff and colleagues have successfully
mice (Graff et al., 2007). Small molecule inhibitors of eIF4E-eIF4G interaction were
also reported to reduce proliferation in several cancer cell lines (Moerke et al., 2007).
These initiatives have proven successful thus far, and warrant clinical investigations to
181
Chapter 5
Murooka, T.T., Ward, S.E., and Fish, E.N. (2005). Chemokines and cancer. Cancer
Treat Res 126, 15-44.
Galligan C.L., Murooka, T.T., Rahbar, R., Baig, E., Majchrzak-Kita, B., and Fish, E.N.
(2006). Interferons and viruses: signalling for supremacy. Immunol Res 35, 27-40.
182
Chemokines and the Immune Response
Chemokine and chemokine receptors are largely responsible for orchestrating leukocyte
trafficking between infected tissues and the secondary lymphoid organs during an
Activation through chemokine receptors facilitates the transition of leukocytes from fast
to slow rolling and finally, to firm adhesion. Chemokine gradients found within the
tissues determine where the leukocytes ultimately localize to. Importantly, some
cytokine expression, mediate co-stimulation of T cells, and determine T cell fate. Thus,
the chemokine system plays critical roles in all facets of both the innate and adaptive
immune response.
During immunological insult, the innate immune response is the first line of
receptors (TLRs) function as PRRs and recognize conserved molecular patterns shared by
pathogens (Akira et al., 2001). Resident tissue macrophages and immature dendritic cells
express multiple TLRs and are the primary activators of innate immunity through the
183
Figure 5.1 Chemokines mediate leukocyte migration from blood to extravascular
tissue
184
Blood vessel flow
Activation
Adherance
Rolling
Extravasation
Endothelium
Inflamed tissue
Glycosaminoglycan
Inflammatory chemokine
Integrin interaction
185
These chemokines, namely CXCL8, CCL3, CCL4, CCL5 and CXCL10, are largely
responsible for the recruitment of additional immature dendritic cells, neutrophils and NK
cells into infected tissues. They function to engulf or specifically kill infected cells to
clear invading microbes and contain larger parasites (Akira et al., 2001). Of particular
dinucleotides (Muzio et al., 2000). Immature DCs express chemokine receptors CCR1,
CCR5 and CCR6 which keep them within tissues (Sozzani et al., 2000). However, upon
chemokine receptors and up-regulate CCR7 expression (Dieu et al., 1998). The switch in
chemokine receptor expression results in the net migration of maturing DCs from
peripheral tissues to the afferent lymphatics, which express ligands for CCR7, CCL19
and CCL21 (Martin-Fontecha et al., 2003). Once in lymph nodes, CCR7 also allows
mature DCs to enter the T cell areas in the deep cortex (Gunn et al., 1999). Thus, the
change in the DC migratory pattern upon antigen uptake is vital for the induction of the
Naïve T cells continuously circulate the periphery, entering LNs via High
Endothelial Venules (HEVs). They express the adhesion molecule CD62L (L-selectin),
LFA-1 and α4β7, and the chemokine receptor CCR7. CD62L mediates tethering and
rolling of naïve T cells on the endothelium of HEVs (Mora and von Andrian, 2006). This
allows naïve T cells to home into and be retained in lymphoid tissues via their ability to
respond to CCL21 synthesized in HEVs and by lymphatic endothelial cells (Gunn et al.,
186
1999). Once in the T cell zones, T cells and mature DCs continuously interact with one
another until a “match” is found. The resulting activation of T cells involves alterations
function they acquire. CCR5 and CXCR3 pre-dominate on primarily cytotoxic, IFNγ-
driven Th1 cells, while CCR4 and CCR8 are preferentially expressed on humoral, IL-4-
dependent Th2 cells (Luther and Cyster, 2001). Some activated CD4+ T cells up-
regulate CXCR5, allowing them to migrate towards the edges of B follicles to provide
help to B cells (Schaerli et al., 2000). Recently activated T cells down-modulate CCR7
expression and eventually re-express the S1P receptor (also known as endothelial
Thus, activated T cell egress is also an active process, responding to the S1P
concentration gradient that is present between the interior of the lymphoid tissue and the
adjacent blood or lymph (Cyster, 2005). The S1P receptor agonist, FTY720, displays
and preventing lymphocyte release from lymphoid organs (Matloubian et al., 2004).
Taken together, chemokine receptor switching ensures that only activated T cells are
Once in the circulation, activated T cell recruitment involves their rolling on the
endothelial surface. This process is primarily mediated by the selectin family as well as
the adhesion molecule VLA-4 (Alon et al., 1995). The inflammatory chemokine CCL5 is
187
endothelial cells via GAGs. Rolling of T cells is gradually replaced by more firm
(Johnston and Butcher, 2002). CCR5 activation on T cells leads to their firm adhesion
through ICAM-1 and VCAM-1 on endothelial cells. After undergoing diapedesis, T cells
ultimately localize to the focus of infection via a CCL5 concentration gradient found
immune responses. Thus, there is much interest in elucidating the molecular mechanisms
and signalling pathways that control lymphocyte trafficking. As discussed earlier, naïve
T cells express a unique array of molecules, namely CD62L, CCR7 and CXCR4, to help
maintain their retention within lymphoid organs. Recent studies by Sinclair and
treated CTLs led to their increased retention in both the lymph node and spleen compared
factor for both CD62L and CCR7 (Bai et al., 2007; Carlson et al., 2006). Thus, both PI-
3’K and mTOR are responsible for regulating T cell egress in vivo by directly regulating
188
the expression of chemokine receptors and adhesion molecules. The data in Chapter 3
show that mTOR plays an important role in CCL5-mediated CD4+ T cell chemotaxis in
protein translation, specifically cyclin D1 and MMP-9 (Figure 3.1, 3,8). The data
mRNAs during T cell migration (Murooka et al., 2008). When considering the data from
these two studies (Murooka et al., 2008; Sinclair et al., 2008), an intriguing story
cytokine production, including IL-2. By up-regulating CD25, the α-subunit of the IL-2
receptor, T cells display increased IL-2/IL-2R signal transduction through PI-3’Kδ and
KLF2 activity, causing down-regulation in CD62L, CCR7 and S1P1 mRNA expression.
further promoting T cell egress. Once out in the periphery, mTOR plays a positive role in
effector T cell migration towards inflamed peripheral tissue. T cells respond to a CCL5
possibly “prime” T cells for efficient migration. Once localized within inflammatory
sites, effector T cells exert their specialized functions to control and clear pathogens. The
implications are that rapamycin may exert its potent immuno-suppressive properties by
189
limiting activated effector T cell migration into inflamed tissue and simultaneously
preventing their egress from secondary lymphoid organs. Whether mTOR plays a role in
190
Figure 5.2 Illustration of the role of mTOR activity in T cell migration in vivo.
Naïve T cells have high expression of KLF2. KLF2 up-regulates the expression of cell
surface CD62L, CCR7 and S1P1 to ensure the normal recirculation of T cells into and out
of secondary lymphoid organs. After a productive encounter with antigen-presenting
cells, the IL-2/IL-2R signalling pathway suppresses KLF2 activity through PI-3’K/mTOR.
Suppression of KLF2 leads to down-regulation of CD62L and CCR7 expression,
promoting T cell egress from lymphoid tissue. Expression of S1P1 is similarly
suppressed, although it is eventually re-expressed in activated T cells to allow their egress
mediated by an alternative mechanism. Activated T cells up-regulate CCR5 and respond
to the CCL5 concentration gradient in the periphery. CCL5-mediated CD4+ T cell
chemotaxis is dependent on mTOR activity. Through mTOR, rapid translation of
chemotaxis-related mRNAs “prime” T cells for efficient chemotaxis towards the site of
inflammation.
191
Lymph node
Naïve T cells
Naïve T cells
HEV
KLF2
CD62L CCR7
S1P1
LN homing and
recirculation
Activated T cells
IL-2/IL-2R
complex
CCL5 mTOR
concentration
gradient
KLF2
Decreased LN homing
4E-BP1
eIF4E
Increased translation of
chemotaxis-related mRNAs
192
cell migration mediated by other chemokines, namely CXCL12, CCL19 and CCL21, in T
cells as well as other cell types, namely B cells and macrophages, have not been studied.
The intriguing aspect of these studies is the possible cross-talk between the
that energy demands for the highly energy-taxing process of cell migration are met? Can
chemokines play a role in regulating cellular metabolism and nutrient uptake during
migration? Several studies have shown that stimulation through the TCR and co-
demands of increased cell growth, proliferation and gene transcription. In fact, T cell
high throughput glycolysis, termed aerobic glycolysis (Fox et al., 2005; Krauss et al.,
2001). Such a switch in metabolism is important for both energy production and
metabolic intermediates required for nucleotide, protein and lipid biosynthesis. Sustained
T cell activation leads to Ca2+-dependent increases in reactive oxygen species (ROS) and
has implications in shaping the T cell response (Jones et al., 2007). Additionally,
transporter, Glut1, through PKB (Frauwirth et al., 2002; Rathmell et al., 2003).
Altogether, the data illustrate that recently activated T cells are metabolically equipped to
sustain rapid cell growth and proliferation. Once effector T cells leave lymphoid organs,
do they require sustained signalling in order to maintain their anabolic metabolism and
nutrient uptake? If so, can inflammatory chemokines deliver that signal, possibly through
193
mTOR activation? Since we have shown that CCL5-mediated mTOR activation leads to
mRNA translation, it will be interesting to investigate whether CCL5 has other mTOR-
mediated effects on T cells. Certainly, several studies demonstrated that mTOR kinase
associated 4F2 heavy chain redistribution to the plasma membrane (Edinger et al., 2003b;
Edinger and Thompson, 2002). In fact, maintenance of nutrient transporters on the cell
surface depends on ongoing signal transduction (Edinger et al., 2003a). When cells are
deprived of IL-3, the turnover of nutrient transporters Glut1 and 4F2hc rapidly decreased
rapamycin treatment decreased glycolytic rates in FL5.12 cells (Edinger et al., 2003b).
Microarray analysis of yeast and mammalian cells treated with rapamycin showed
1999; Peng et al., 2002). mTOR-dependent uptake of nutrients and glycolytic metabolism
may be important to support increased protein translation and expansion in cell size, also
regulated through mTOR. Further studies are required to determine whether CCL5-
mediated mTOR activation affects cellular metabolism and nutrient uptake in T cells.
proteins, such as the 4F2 heavy chain and the glucose transporter Glut1, has not been
studied. Flow cytometric studies using PM1.CCR5 T cells and primary activated CD4+
T cells to determine whether CCL5 can up-regulate or sustain Glut1 and 4F2 expression
and/or MAPK signalling pathways on Glut1 and 4F2 expression can be addressed using
the appropriate pharmacological inhibitors. If indeed Glut1 protein and cell surface
194
expression is up-regulated or maintained by CCL5, glucose uptake and metabolism can
be directly measured within cells. Glycolytic rates can be calculated by measuring the
experiments of these nutrient receptors assessing the impact on cellular migration can
experiments, T cells are treated with CCL5 in the presence or absence of rapamycin and
are isolated, purified and subjected to microarray analysis, to identify a subset of mRNAs
that are regulated by CCL5 at the level of translation. Specifically, proteins involved in
intriguing to speculate that besides providing migrational cues, CCL5 may regulate
195
5.2. CCL5 determines T cell Fate through AICD
concentrated within tissue sites. Accordingly, recently activated T cells recruited from
the lymphoid organs to a site of infection, are exposed to high CCL5 concentrations. The
prompted studies to investigate their effects on T cell function. It is now apparent that at
these concentrations, CCL5 forms large oligomers with a mass greater than 100 kDa
(Appay et al., 1999; Appay et al., 2000). Previous studies showed that CCL5 stimulated
expression and cytokine production, only at these high concentrations (Bacon et al., 1995;
Dairaghi et al., 1998). This unexpected property of CCL5 demonstrated that high doses
activation may play a role in Activation-Induced Cell Death (AICD). AICD mediates the
removal of the activated and expanded T cells after an immune response (Krammer et al.,
2007). Typically, TCR re-stimulation of already expanded T cells in the absence of co-
stimulation leads to the efficient induction of cell death, in most cases through CD95, but
other mechanisms have also been described, namely TNFR1 and granzyme B (Devadas et
Weber and Krammer, 2003). Our data in Chapter 2 show that high, µM CCL5
concentrations induce T cell death (Murooka et al., 2006). Specifically, we show that
196
CCL5 aggregation at high ligand concentrations induces apoptosis in PM1, MOLT-4 and
activated peripheral blood T cells in a CCR5-dependent manner (Figure 2.1, 2.5). When
caspase-3, followed by poly ADP ribose polymerase (PARP) cleavage (Figure 2.4). In
approximately 60% of the cells after 24 hours, whereas ex vivo activated T cells exhibited
approximately 9% apoptotic death. The data suggest that the sensitivity to CCL5-
mediated apoptosis is higher in the two T cell lines. It is also possible that 24 hours is not
apoptosis of Jurkat T cells was not observed until after 3 days in culture. The prolonged
lag period observed may reflect changes in gene expression of the death receptors
CD95/CD95L (Colamussi et al., 2001). Thus, additional time course studies with ex vivo
T cells are necessary to determine whether a similar lag period also exists in CCL5-
mediate apoptosis. The result from such studies may reveal that CCL5-mediated AICD
of T cells does not occur immediately, but rather is achieved over several days. This
would be in agreement with the overall kinetics of the T cell immune response, where T
cell function can be gradually “turned off” by prolonged exposure to high CCL5 doses.
Taken altogether, our data suggest that CCL5-induced cell death, in addition to
immunological response.
197
Because µM concentrations of CCL5 are required to invoke this outcome, the
vivo. Certainly, unusually high CCL5 concentrations may be realizable at sites of acute
extracellular matrix GAGs. In addition, the unique ability of CCL5 to form aggregates,
concentration (Appay et al., 1999; Appay et al., 2000; Czaplewski et al., 1999;
Hoogewerf et al., 1997; Kuschert et al., 1999; Martin et al., 2001; Proudfoot et al., 2001;
Proudfoot et al., 2003). We, therefore, infer that the CCL5-CCR5 induced apoptosis of T
cells we observe is not likely an in vitro artifact, but is attainable in vivo. However, this
hypothesis remains an assumption, as CCL5 levels at inflammatory sites have never been
inflammatory site. Such studies are experimentally challenging, because of the ability of
CCL5 to bind GAGs, either expressed on the extracellular matrix or cell surfaces, and the
198
5.3. CCL5 promotes breast cancer proliferation
act directly on tumor cells to regulate proliferation and survival through an autocrine loop,
release of growth factors. There is accumulating evidence for the pathogenic role of both
CCL5 and CCR5 in breast cancer. The CCL5/CCR5 axis has been associated with active
recruitment of TAMs, as well as their direct proliferative role in breast cancer cells.
Robinson and colleagues showed that administration of the CCR1/CCR5 antagonist, Met-
CCL5, significantly reduced the extent of macrophage infiltration within tumors, which
correlated with reduced tumor burden (Robinson et al., 2003). Breast tumor cells
expressing lower levels of CCL5 exhibited decreased growth in vivo (Adler et al., 2003).
In vitro studies have shown that both CCL2 and CCL5 stimulate the release of tumor-
promoting factors by macrophages, namely MMP-9 and TNFα (Azenshtein et al., 2002;
Robinson et al., 2003; Saji et al., 2001). The data indicate that inflammatory chemokines
can actively recruit tumor-promoting leukocytes into the tumor microenvironment, thus
We investigated the possibility that CCL5 has direct proliferative and survival
effects on breast cancer cells mediated by mTOR. The data in Chapter 4 show that
exogenous CCL5 induced MCF-7 breast cancer cell proliferation (Figure 4.1.).
namely cyclin D1, c-Myc and defender against cell death-1 (Dad-1) in a rapamycin-
199
dependent manner (Figure 4.3, 4.4.). The implications are that breast cancer cells can
exploit downstream chemokine signalling pathways for their proliferative and survival
studies showing that tumor-derived CCL5 did not contribute to breast tumor formation in
vivo (Jayasinghe et al., 2008). One explanation for these conflicting results is the
mediated proliferative effects at 10 nM, CCL5 produced by 4T1 breast cancer cells,
reported by Jayasinghe and colleagues, was approximately 100 fold less (Jayasinghe et al.,
2008). The data suggest that a threshold level of CCL5 is required to invoke a
studies showing that CCL5 content within tumor lesions is markedly higher in the more
aggressive forms of breast cancer (Bieche et al., 2004; Niwa et al., 2001). This threshold
bind, oligomerize and accumulate on GAGs at their secretion site (Proudfoot et al., 2003).
that tumor cells switch from oxidative phosphorylation to aerobic glycolysis, even when
oxygen is non-limiting (Bauer et al., 2004; Elstrom et al., 2004). Glycolysis yields much
less ATP per glucose molecule utilized compared to oxidative phosphorylation, but
provides cells with metabolic intermediates critical for cell growth. For example, the
200
Christofk and colleagues demonstrated that a switch in a splice isoform of the glycolytic
enzyme pyruvate kinase is necessary for the metabolic switch to aerobic glycolysis.
RNA knockdown of the M2, but not the M1 isoform, reduced lactate production and
reduced tumor formation in vivo (Christofk et al., 2008a). Furthermore, the M2 isoform
2008b). The implications are that tyrosine phosphorylation signalling effectors can
potentially regulate glycolysis through the glycolytic enzyme pyruvate kinase. This is
consistent with studies showing that mammalian cells require exogenous signals to alter
their cellular metabolism. For example, hyperglycemia associated with Type I diabetes
glucose (Saltiel and Kahn, 2001). Further studies are needed to investigate the effects of
CCL5 on cellular metabolism and nutrient uptake, possibly mediated by mTOR, in breast
will be of interest. The impact of CCL5 on the expression of the glucose transporter
Glut1 and the amino acid transporter-associated protein, 4F2, can be assessed by flow
cells, and the role of PI-3`K and mTOR can be assessed using the appropriate
of these nutrient receptors to address their contributions to cell size, proliferation and
survival would be of interest. Results from these studies would provide insights into the
201
metabolism, amino acid uptake and increased translation of proliferation and survival
proteins.
202
5.4. Conclusions
pro-adhesive effects. They are responsible for directing leukocyte migration by forming
chemokine gradients and triggering firm arrest by activating integrins on the leukocyte
cell surface. Throughout this thesis, I have described the importance of the CCL5/CCR5
axis in the context of the immune response and cancer biology. Firstly, I showed that
“prime” T cells for efficient migration. Secondly, I show that high concentrations of
CCL5 at the inflammatory sites can instruct effector T cells to undergo apoptosis. The
data suggest that CCL5-induced cell death, in addition to CD95/CD95L mediated events,
cancer. CCL5 can directly induce proliferation of MCF-7 breast cancer cells through
increased translation of proliferation and survival proteins. These studies reinforce the
notion that chemokines are not only potent chemotactic mediators, but are key effectors
203
Chapter 6
204
Chapter 2 was published as:
Murooka, T.T., Wong, M.M., Rahbar, R., Majchrzak-Kita, B., Proudfoot, A.E., and Fish,
E.N. (2006). CCL5-CCR5-mediated Apoptosis in T cells: Requirement for
Glycosaminoglycan Binding and CCL5 Aggregation. J Biol Chem 281, 25184-25194.
Murooka, T.T., Rahbar, R., Platanias, L.C., and Fish, E.N. (2008). CCL5-mediated T-
cell chemotaxis involves the initiation of mRNA translation through mTOR/4E-BP1.
Blood 111, 4892-4901.
Murooka, T.T., Rahbar, R., Platanias, L.C., and Fish, E.N. CCL5 promotes breast cancer
proliferation through mTOR/4E-BP1 dependent mRNA translation.
Murooka, T.T., Ward, S.E., and Fish, E.N. (2005). Chemokines and cancer. Cancer
Treat Res 126, 15-44.
Galligan C.L., Murooka, T.T., Rahbar, R., Baig, E., Majchrzak-Kita, B., and Fish, E.N.
(2006). Interferons and viruses: signalling for supremacy. Immunol Res 35, 27-40.
205
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