ANALYSIS OF THE REGULATION OF NFkB BY TPL-2 KINASE
Helen J Coope
A thesis submitted in partial fulfilment of the requirements of the University of London for the degree of Doctor of Philosophy
March 2001
Division of Cellular Immunology National Institute for Medical Research Mill Hill London ProQuest Number: U643313
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NFkB is a ubiquitously expressed transcription factor of particular importance in the immune system. Several signalling pathways regulate NFkB activation, which occurs following the degradation of inhibitory IkB proteins. One pathway of NFkB activation is the signal-induced degradation of NFkB 1 pi 05, when this occurs, associated NFkB proteins transfer to the nucleus. TPL-2 is a proto-oncogene encoding a serine/threonine kinase, which interacts with NFkBI pi 05 and regulates its degradation. The aims of the present study are to investigate the regulation of NFkB 1 pi 05 degradation by TPL-2 and clarify the physiological circumstances in which this is important. Studies of NFkB knockout and transgenic animals reveal roles for NFkB in T cell development and function. TPL-2 is expressed in T cell rich organs. However, in the present study, transgenic mice expressing TPL-2 or dominant negative TPL-2 in the T cell lineage show no defects in thymocyte development, T cell proliferation, IL-2 and TNFa production or NFkB activation. Using stable cell lines, it is shown that TNFa stimulation but not CD3 and CD28 costimulation is a potent stimulus for NFkBI p i05 degradation in T cells. Expression of dominant negative TPL-2 inhibits NFkBI p i05 degradation in these cells, whereas ERK, JNK and P38 activation are unaffected. LPS is a potent inducer of NFkBI p i05 degradation in the monocyte-macrophage cell line
THP-1, which coincides with the activation of the IKK kinases. Expression of dominant negative TPL-2 in these cells inhibits LPS induced NFkBI pi05 degradation, ERK activation and transcription of the TNFa gene. Taken together, these data suggest that
TPL-2 may function in regulation of monocyte or macrophage responses to LPS. Acknowledgements
I would like to thank my supervisor Dr Steve Ley for giving me the opportunity to research this thesis, for his guidance and for providing a stimulating and friendly laboratory in which to work. The Ley lab include: Monica Belich, Soeren Beinke,
Bettina Huhse, Julia Jansen, Valerie Lang, Chien Kuo Lee, Andres Salmeron and
Yasmina Soneji. Thank you all for your good ideas, helpful discussions and for what you have taught me.
I am indebted to many members of the Cellular and Molecular Immunology Divisions at
NIMR for sharing their scientific and technical experience. I would especially like to thank my second supervisor Dimitris Kioussis, for his advice on transgenic analysis,
Monica Belich and Bettina Huhse for willingly sharing their biochemical and molecular biology knowledge, Owen Williams for teaching me flow cytometry, Kathleen Roderick for assistance with genotyping and the brilliant staff of Q building for their services.
I am grateful to friends old and new, inside and outside NIMR, who have helped me to also enjoy my time spent outside the laboratory.
Most importantly, I would like to thank my mother and father, my brother Patrick and my partner Jonathan. Their belief in my ability and unwavering support are an inestimably important part of this thesis. Table of contents
Abstract 2
Acknowledgements 3
Table of contents 4 Table of figures 9 Table of diagrams 10 Table of tables 10
Table of abbreviations 11
CHAPTER 1: Introduction 16
1.1 Regulation of transcription...... 16
1.1.1 NFkB 17
1.1.2 Historical perspectives 17
1.1.3 NFkB activation 18
1.2 The NFkB proteins...... 19
1.2.1 Cloning and functional domains 19
1.2.2 NFkB dimérisation and DNA binding 22
1.3 The IkB proteins...... 25
1.3.1 Cloning and fimctional domains 25
1.3.2 What happens to IkB proteins upon activation? 28
1.3.3 ‘NFkB precursor’ IkB proteins and NFkB activation 31
1.3.4 The IkB kinases 33
4 1.4 Upstream of the IKK complex...... 36
1.4.1 MAP3K Proteins 38
1.4.2 Other kinases 41
1.4.3 IKK autophosphorylation 43
1.4.4 TRAPS: ohgomerisation and ubiquitination in IKK activation 43
1.4.5 Summary of IKK activation. 45
1.4.6 NF kB activation by receptors of the TNF receptor superfamily. 46
1.4.7 NF kB activation by receptors of the IL-1/Toll-like receptor superfamily 48
1.4.8 NFkB activation by T and B cell receptors 49
1.5 Genetic approaches to the analysis of NFkB regulation and function...... 51
1.5.1 Drosophila Melanogaster 51
1.5.2 Mice knockouts / transgenics 54
1.5.3 NFkB in the development of the immune system 54
1.5.4 NFkB in immune responses 56
1.5.5 NFkB and embryonic development 57
1.6 Tumour Progression Locus-2...... 59
1.6.1 TPL-2 knockout mice 64
1.7 The aims of this study ...... 64
CHAPTER 2: Materials and methods 66
2.0 Transgenic mice ...... 66
2.1.1 Genotype 66
2.1.2 Preparation of cells 67
2.1.3 Purification of T lymphocytes 67
5 2.2 Stable cell lines ...... 68
2.3 Lymphocyte analysis ...... 69
2.3.1 Flow cytometry 69
2.3.2 Proliferation Assays 70
2.4 Protein analysis ...... 70
2.4.1 Lysis procedures 70
2.4.2 Immunoprécipitation 71
2.4.3 SDS polyacrylamide gels and Western blotting 71
2.4.4 In Vitro Kinase assays 72
2.4.5 Pulse chase metabolic labelling 73
2.5 Electrophoretic mobility shift assays...... 74
2.5.1 Preparation of cell extracts 74
2.5.2 EMSA 75
2.6 RT-PCR...... 75
2.6.1 RNA extraction 75
2.6.2 RT-PCR 76
CHAPTER 3: Analysis of TPL-2 function using transgenic mice 77
3.1 Results...... 77
3.1.1 Analysis of transgenic TPL-2 expression 78
3.1.2 Analysis of T cell development in transgenic mice 80
3.1.3 Functional analysis of transgenic T cells 84
3.1.4 Analysis of NFkB activation in transgenic T cells 87 3.2 Discussion of Chapter 3...... 90
3.2.1 TPL-2 and NFkB in T cells 90
3.2.2 Measurement of NFkBI pl05 degradation in transgenic T cells 90
3.2.3 Does TPL-2 function in T cells? 94
CHAPTER 4 TPL-2AC mice 96
4.1 Results...... 96
4.1.1 Analysis of thymocytes and T cells from TPL-2AC mice 97
4.2 Discussion of Chapter 4...... 101
4.2.1 What are the possible mechanisms of TPL-2 oncogenesis? 101
4.2.2 The importance of the TPL-2 c-terminal in oncogenesis 103
4.2.3 Co-operation between TPL-2 and other oncogenes 104
CHAPTER 5: Analysis of TPL-2 function in Jurkat cells 106
5.1 Results...... 106
5.1.1 Analysis of signal induced NFkBI pi 05 degradation in E6.1 cells 106
5.1.2 NFkB regulation in TPL-2 or TPL-2KD expressing stable cell lines 109
5.1.3 CD3 and CD28 costimulation of TPL-2KD Jurkat clones 113
5.2 Discussion of Chapter 5...... 116
5.2.1 The role of TPL-2 in CD3 and CD28 signalling 116
5.2.2 Regulation of MAP kinase pathways by TPL-2 117
5.2.3 Are NFkBI pl05 and IkBœ degradation separately regulated? 118
5.2.4 TPL-2KD blocks TNFa induced NFkBI pi05 degradation 119
5.2.5 How might altered NFkBI pi05 degradation affect transcription? 120 CHAPTER 6: Analysis of TPL-2 function in THP-1 cell lines 123
6.1 Results...... 123
6.1.1 Characterisation of NFkB activation in THP-1 cells 123
6.1.2 Analysis of TPL-2KD expressing stable THP-1 cell lines 126
6.1.3 Levels of LPS induced mRNAs in TPL-2KD THP-1 clones 130
6.2 Discussion of Chapter 6...... 133
6.2.1 Regulation of TNFa production 133
6.2.2 TPL-2 in control of septic shock and TNFa production 135
6.2.3 TPL-2 and the TNFa AU Rich Region 140
6.2.4 Are NFkBI pi05 and iKBa degradation separately regulated? 141
6.2.5 Further experiments using THP-1 cells 143
CHAPTER 7: Final discussion 147
7.1 What is the purpose of NFkBI pi 05 degradation? ...... 147
7.2 Specific functions for specific IkB proteins? ...... 148
7.3 Howto define the purpose of NFkBI pl05 degradation ...... 149
7.4 MAP Kinase regulation by TPL-2 ...... 150
7.5 Analysis of NFkB in TPL-2-/- mice ...... 155
7.6 Which molecules co-operate with TPL-2 in endotoxin responses? ...... 153
7.7 Potential clinical significance of TPL-2 ...... 154
REFERENCES 156 Table of figures
Figure 1: Expression of transgenic TPL-2 ...... 78
Figure 2: Association of transgenic TPL-2 withNFxBl pi 05 ...... 79
Figure 3: Thymocyte development in TPL-2 and TPL-2KD transgenic m ice ...... 82
Figure 4: Peripheral T cells inTPL-2 and TPL-2KD transgenic mice ...... 83
Figure 5: Proliferation of TPL-2 and TPL-2KD T cells ...... 85
Figure 6: TNFor and IL-2 production by TPL-2 and TPL-2KD T cells ...... 87
Figure 7: NF^B activation in TPL-2 and TPL-2KD T cells ...... 89
Figure 8: Thymocytes in TPL-2/IC transgenic mice...... 98
Figure 9: Splenocytes in TPL-2/1C transgenic m ice ...... 99
Figure 10: Proliferation of TPL-2/IC T cells...... 100
Figure II: Signal induced NFxfilpl05 degradation in E6.1 Jurkat cells ...... 107
Figure 12: NF/tfilpl05 degradation is inhibited by proteasome inhibitors ...... 108
Figure 13: Expression of TPL-2 and TPL-2KD in stable E6.1 clones ...... 109
Figure 14: TNFor induced NFxfilpl05 degradation in stable E6.1 clones ...... 110
Figure 15: TNFor induced Ixfia degradation in TPL-2 and TPL-2KD E6.1 clones I ll
Figure 16: mRNA induction in E6.1 Jurkat cells treated with CD3 and CD28 or TNForl 13
Figure 17: IL-2 mRNA induction in TPL-2KD expressing E6.1 Jurkat clones ...... 114
Figure 18: MAPK activation in TPL-2KD E6.1 clones ...... 115
Figure 19: JNK activation and IxBa degradation in TPL-2KD E6.1 Jurkat clones 115
Figure 20: Signal induced NFxfil pl05 degradation in THP-1 cells ...... 124
Figure 21: Signal induced IxB a degradation in THP-1 cells ...... 125 Figure 22: Induction of IKK kinase activity by TNFor and LPS in THP-1 cells 126
Figure 23: Stably transfected HA-TPL-2KD associates with endogenous NF/itBl p i05 127
Figure 24: LPS induced N F/tfilpl05 degradation in TPL-2KD THP-1 clones ...... 128
Figure 25: LPS induced I&Bor degradation in HA-TPL-2KD THP-1 clones ...... 129
Figure 26: LPS induced ERK activation in TPL-2KD THP-1 clones ...... 129
Figure 27: TNFor transcription in THP-1 cells stably expressing TPL-2KD ...... 131
Table of diagrams
Diagram 1: Schematic diagram of NFkB activation ...... 18
Diagram 2 The NFkB and IkB proteins ...... 20
Diagram 3: Schematic Overview of mammalian MAPK modules ...... 39
Diagram 4: The TPL-2 containing VA hCD2 transgene cassette ...... 66
Table of tables
Table 1: kB sites from the regulatory regions of several genes...... 23
Table 2: NFkB functions defined by study of knockout and transgenic mice ...... 55
Table 3: Table of antibodies used in FACS analysis ...... 69
Table 4: Table of antibodies used in immunoprécipitation and western blotting ...... 72
Table 5: Primer sequences used in RT-PCR analysis ...... 76
10 Table of abbreviations
AB-IMDM Air buffered Iscoves Modified Dulbeccos Medium
AICD Activation Induced Cell Death
Aly Alymphoplasia
ARE AU Rich Element
ATCC American Type Culture Collection pTRCP P Transducin Repeat containing protein
BCL B Cell Lymphoma e.g. BCL3
BCR B Cell Receptor
CAPS Cyclohexylaminopropane sulfonic acid
CD28RE CD28 Responsive Element
C-EBP CCAAT/Enhancer Binding Protein
CHX Cyclohexamide
CIA Collagen Induced Arthritis
CRP C-Reactive Protein
DAG Diacylglycerol
DD Death Domain
DIF Dorsal Related Immunity Factor
DN Dominant Negative
DNA Deoxyribonucleic acid
DTT Dithiothreitol
EAE Experimental Autoimmune Encephalomyelitis
EBV Epstein Barr Virus
11 ECACC European Collection of Cell Cultures
ECSIT Evolutionarily Conserved Intermediate in Toll pathways
EDTA Ethylamine Diamine Tetraacetic Acid
EMSA Electrophoretic Mobility Shift Assay
ERK Extracellular signal Regulated Kinase
ES Embryonic Stem cell
FADD Fas Associated Death Domain containing protein
FBS Foetal Bovine Serum
FITC Fluoroscein Isothiocyanate
GFP Green Fluorescent Protein
GRR Glycine Rich Region
GSK-3P Glycogen Synthase Kinase 3p
HEPES Hydroxy ethyl piperazine N-2 ethane sulphonic acid
HLH Helix Loop Helix
HAS Heat Stable Antigen
HTLV Human t cell Leukaemia Virus lAP Inhibitor of Apoptosis
ICAD Inhibitor of Caspase Activated DNase
ICAMl Intercellular Adhesion Molecule 1
IkB Inhibitor of NFkB
IKK IkB Kinase
IL Interleukin e.g. IL-2
Ig Immunoglobulin
12 IP Incontinentia Pigmenti
IP Immunoprécipitation
IP3 Inositol 1,4,5 Trisphosphate
IVK In Vitro Kinase assay
JNK c-Jun N-Terminal Kinase
KD Kinase Dead (lacking kinase activity)
LCR Locus Control Region
LPS Lipopolysaccharide
LZ Leucine Zipper
MAPK Mitogen Activated Protein Kinase
MAPKK Mitogen Activated Protein Kinase Kinase (also MAP2K)
MAPKKK Mitogen Activated Protein Kinase Kinase Kinase (also MAP3K)
MAPKKKK Mitogen Activated Protein Kinase Kinase Kinase Kinase
(also MAP4K)
MEF Murine Embryonic Fibroblast
MEK MAPK/ERK Kinase
MEKKl MEK Kinase
MoMuLV Moloney Murine Leukaemia Virus
MS Multiple Sclerosis
MyD88 Myeloid Differentiation marker 88
NAK NF kB activating kinase
NEMO NFkB Essential Modulator (also IKKy)
NFAT Nuclear Factor of Activated T cells
13 NIK NFkB Inducing Kinase
NIMR National Institute for Medical Research
NLS Nuclear Localisation Signal
PAS Protein A Sepharose
PGS Protein G Sepharose
PBS(CMF) Phosphate Buffered Saline (Calcium and Magnesium Free)
PDBu Phorbol 12, 13 Dibutyrate
PE Phycoerythrin
PEST Proline Glutamic acid Serine and Threonine rich region
PGN Peptidoglycan
PEG Phospholipase C
PKC Protein Kinase C
PNPP P Nitrophenyl phosphate
PMA Phorbol Myristate Acetate
PMSF Phenylmethylsulphonyl fluoride
PP2A Protein Phosphatase 2A
PVDF Polyvinylidene
RHD Rel Homology Domain
RIP Receptor Interacting Protein
RNA Ribonucleic Acid
RT-PCR Reverse Transcriptase Polymerase Chain Reaction
SCF Skp-1, Cullin and F Box protein containing complex
SDS Sodium Dodecyl Sulphate
14 SDSPAGE SDS Polyacrylamide Gel Electrophoresis
SODD Silencer of Death Domains
SRR Serine Rich Region
TAK Transforming Growth Factor p Activated Kinase
TCR T Cell receptor
TIR ToMnterleukin 1 receptor domains
TLR Toll-Like Receptor
TNFa Tumour Necrosis Factor a
TNFR Tumour Necrosis Factor Receptor
TPL-2 Tumour Progression Locus 2
TRADD TNF Receptor Associated Death Domain containing protein
TRAP TNF Receptor Associated Factor e.g.TRAF 2
TRIS Tri [hydroxymethyl] amino methane
Ub Ubiquitin
UTR Untranslated Region
Y2H Yeast Two Hybrid technique
15 CHAPTER 1 Introduction
1.1 Regulation of transcription
Complex patterns of gene expression are required during the development of an organism and for cellular responses to external stimuli. The subset of genes a cell expresses determines its cellular phenotype, its growth properties, its differentiation status, its developmental fate and its responses to extracellular events. Gene expression is a tightly controlled process regulated at a number of levels; including differential translation of mRNA, differential stabilisation of mRNA and transcriptional regulation.
Of these, the latter most frequently controls differential rates of protein synthesis.
Eucaryotic cells contain a diverse range of regulatory proteins known as transcription factors, which can positively or negatively influence gene transcription, (reviewed in
Alberts et al., 1994). The formation of transcription factor complexes on the promoter and enhancer elements of genes regulates the recruitment of the RNA polymerase complex to the DNA. Eucaryotic genes are often regulated by a combination of transcription factors, such that a gene is expressed only when an appropriate combination of signals has been received. The activity of these factors can be regulated by a number of distinct mechanisms; control of the induction of the transcription factor
16 Chapter 1 Introduction
gene, its post-translational modification, its interaction with other factors or its subcellular localisation.
1.1.1 NFkB
1.1.2 Historical perspectives
Nuclear Factor kB (NFkB) is a family of transcription factors, members of which are expressed in virtually every eucaryotic cell type, (reviewed in Ghosh et ah, 1998). It was first identified in B cells as a factor bound to the intronic enhancer of the immunoglobulin k light chain gene (Sen and Baltimore, 1986b). Later studies showed that NFkB could be rapidly activated in other cell types by exposure to stimuli such as phorbol esters (Sen and Baltimore, 1986a). This activation does not require de novo protein synthesis; thus identifying NFkB as a ‘primary’ transcription factor. The latency of NFkB in unstimulated cell is due to its cytoplasmic association with an inhibitory protein, IkB, from which it can be dissociated in vitro by detergent treatment (Baeuerle and Baltimore, 1988a). Analysis of the physiological regulation of IkB removal identified the IkB kinases, which are a common step in most pathways of NFkB activation, and target the IkB protein for destruction by the proteasome (DiDonato et al.,
1997; Mercurio et al., 1997; Zandi et al., 1997).
Fourteen years after its discovery, kB elements, the DNA sequences to which NFkB proteins bind have been identified in the promoters or enhancers of more than 150 genes
(Pahl, 1999), regulating processes as diverse as development, proliferation, differentiation and apoptosis. Interestingly many of these genes are implicated in
17 Chapter I Introduction
immunity and inflammation. Consistent with a central role for NFkB in immunity, many of the 150 stimuli which activate NFkB are bacterial products, viral products or inflammatory cytokines (Pahl, 1999).
1.1.3 N F kB activation
1. Receptor activation results in IkB IkBœ phosphorylation by IKK
2. Phosphorylated IkBœ is ubiquitinated by p-TRCP
3. Ubiquitinated IkB is degraded by the proteasome
4. Released NFkB dimers translocate to the nucleus, and regulate transcription
mRNA-4
Diagram 1: Schematic diagram of NFkB activation
An inactive NFkB dimer is tethered in the cytoplasm by its association with a single IkB protein (Baeuerle and Baltimore, 1988b). The mechanisms which regulate the activation of NFkB from such latent cytoplasmic complexes are best known for IkBœ (Diagram 1),
(reviewed in Karin and Ben-Neriah, 2000). In general, when an activating stimulus is delivered to a cell, the IkB kinases become activated and phosphorylate IkBœ (DiDonato et al., 1997; Mercurio et al., 1997; Zandi et al., 1997). Phosphorylated IkBœ can then
18 Chapter 1 Introduction
be recognised by ubiquitinating enzymes (Yaron et aL, 1998), which build polyubiquitin
chains on specific IicBa lysine residues. Ubiquitination then acts as a signal to the 26s
proteasome, which degrades the IkB. The newly released NFkB dimer can then
translocate to the nucleus by binding to nuclear import factors via its nuclear localisation
sequence. In the nucleus the NFkB dimer can bind DNA at decameric consensus
sequences known as kB elements and regulate gene transcription. In the ease of IkBœ
this process is complete within minutes. Once NFkB is activated, an induction of IkBœ
mRNA occurs, due to a KB element within the IkBœ promoter (de Martin et al., 1993).
Newly synthesised IkBœ can enter the nucleus, remove NFkB from its binding sites and
mediate its export to the cytoplasm, thereby switching transcription off (Arenzana-
Seisdedos et al., 1995).
1.2 The NFkB proteins
1.2.1 Cloning and functional domains
In its DNA binding form NFkB is a dimer of proteins from the NFkB/RcI family,
(reviewed in Ghosh et al., 1998). This family includes NFkBI (p50 and its precursor
pl05) NFkB2 (p52 and its precursor p i00), c-Rel, Rel-A (p65) and Rel-B. Mammalian
NFkB proteins have homology with the viral oncogene v-Rel and the Drosophila
melanogaster proteins Dorsal Dif and Relish, see Diagram 2.
The NFkB/RcI family is distinguished by the presence of a Rel homology domain
(RHD); a highly conserved 300 amino acid region containing two immunoglobulin-like
19 Chapter I Introduction
(Ig-like) domains connected by a flexible linker region. The RHD is required for dimérisation of Rel proteins, DNA binding and association with IkB s . It is also the site of a nuclear localisation sequence (NLS).
Rel Homology Domain
p65
c-Rel
LZ RelB
Dorsal
Dif
Relish DD plOO/p52 DD pl05/p50
I k B cx
IkBP
Ik B e
Cactus
Bcl-3 V Ankyrin Repeats
Diagram 2 The NFkB and IkB proteins
Ankyrin repeat domains and Rel Homology domains are shown. Yellow arrows indicate the c- termini of p50 andp52, after processing from NFkBI pI05 and NFkB2 plOO. Death domain (DD), GRR Glycine Rich Region, (SRR) Serine Rich Region and Leucine Zipper (LZ)
20 Chapter 1 Introduction
NFkB proteins can be divided into two groups on the basis of sequences c-terminal to the RHD. Group one comprises NFkBI, NFkB2 and the Drosophila melanogaster protein Relish, which all possess long c-terminal domains containing multiple inhibitory
Ankyrin repeats (see below). Unable to bind DNA in this form they undergo limited proteolysis to yield smaller products NFkBI p50, NFkB2 p52 and Relish p68. A precursor product relationship exists between NFkBI p i05 and p50, in which NFkBI p i05 is processed to p50 by the degradation of its c-terminus (Fan and Maniatis, 1991).
The resulting c-terminal product is not stable in the cell. There is also evidence that p50 is generated co-translationally (Lin et al., 1998). This proposal is not consistent with pulse chase experiments in which NFkBI p i05 labelling occurs during the pulse period and p50 is generated during chase periods (Belich et al., 1999; Orian et al., 2000).
Similarly p52 is generated from NFkB2 p i00 by proteolytic processing. (Heuschet al.,
1999) Both processes are mediated by the proteasome (Heusch et al., 1999; Orian et al.,
1995; Palombella et al., 1994). Thus the generation of p50 and p52 are unusual cases in which the proteasome merely processes its substrate instead of completely destroying it.
Both NFkBI pi 05 and NFkB2 pi 00 contain glycine rich regions (GRR) upstream of the site at which proteasome mediated digestion of the precursor stops. Mutation of these sequences demonstrates that they are required for processing of NFkBI pi05 and
NFkB2 p i00 (Heusch et al., 1999; Lin and Ghosh, 1996; Orian et al., 1999). Lin and
Ghosh propose that the GRR fimctions as a recognition site for an unidentified protease, releasing p50 and permitting the degradation of the p i05 c-terminus. Alternatively the
GRR may form a physical barrier to complete degradation by the proteasome.
Sequences c-terminal to the GRR may also regulate processing; Orian et al
21 Chapter 1 Introduction
demonstrated that lysine residues 441 and 442, which serve as substrates for
ubiquitination are required for processing (Orian et a i, 1999). Some investigators
propose that processing of the NFkB precursors can be increased upon cell stimulation.
Although cell stimulation reduces NFkBI p i05 levels, in many cases the concomitant
increases in p50 are modest and therefore inconsistent with stimulation induced
processing. NFkBI and NFkB2 also contain death domains in their c-termini. These
are protein multimerisation domains involved in connecting ‘death-receptors’ to death
domain containing proteins which regulate the induction of apoptosis and NFkB,
(reviewed in Aravind et al., 1999)
The group two NFkB proteins, mammalian Rel B, c-Rel and Rel-A, and dorsal and dif
from Drosophila lack Ankyrin repeats. Critically however, these group two NFkB
proteins contain transcriptional activation domains c-terminal to the RHD, which are
missing from NFkBI and NFkB2. Thus, most NFkB dimers activate transcription
whereas p50 or p52 homodimers are believed to be repressive in the absence of other
cofactors.
1.2.2 NFkB dimérisation and DNA binding
Of the fifteen possible mammalian NFkB dimer combinations, the most commonly
observed are p50/p65 and p50/p50, some combinations are not observed either in vivo or
in vitro. Furthermore those which do exist are associated with a range of affinities.
These differences have been mapped to primary sequence differences at certain amino
22 Chapter 1 Introduction
acid residues in the dimérisation interface (Sengchanthalangsy et al., 1999). Structures of different NFkB dimers bound to DNA reveal that both immunoglobulin-like domains make contact with DNA, sequence specific recognition largely originates in the n- terminal Ig-like domain whereas the c-terminal domains are also the site of the dimer interface (reviewed in Chen and Ghosh, 1999). Unlike other transcription factors, which use alpha helices to bind DNA, NFkB uses ten flexible loops, which extend from the Ig domains. Primary sequence within these loops is one of the determinants of the affinity of the dimer for the kB site.
Although the k enhancer NFkB binding site was the first to be identified, it is clear that there are many other DNA sequences to which NFkB can bind (kB sites). These sites contain variations on the consensus sequence 5’- GGGRNNYYYCC-3’, see Table 1.
Consensus Sequence G G G R NWTTCC IgK GGGACTTTCC IFNP G GGA AATTCC IL-6 GGGATTTTCC E-selectin GGGG A TTTCC TNFa GG GG CTTTCC IL-2 GG G A TTTCAC GM-CSF GG G A ACTACC IL-2 CD28RE AGAA ATTCC
Table 1: kB sites from the regulatory regions of several genes Adapted from (Chen and Ghosh, 1999).
Different NFkB dimer species have varying affinities for different kB sites. Kunsch et al identified different optimal DNA binding sites for p50, p65 and c-Rel homodimers in
23 Chapter I Introduction
vitro (Kunsch et al., 1992). This specificity reflects what is seen in vivo, in which a
NFkB regulated target gene contains a binding site that preferentially binds a certain dimer combination, e.g. p65 homodimer binding sites in ICAMl and IL4 (Casolaro et al, 1995; Ledebur and Parks, 1995).
The affinity with which NFkB dimers containing transactivating subunits bind to kB sites is not the sole determinant of their transactivation activity. P52/p65 or p52/c-Rel dimers can bind to the kB sites of IgK, HIV-LTR, IL-2R or MHC-1 gene, but can only activate transcription through the IgK and HIV-LTR sites (Perkins et al., 1992). NFkB can bend DNA to which it is bound, and DNA bound NFkB dimers themselves can undergo conformational changes. Both of these events are linked to changes in transactivating activity (Schreck et al., 1990), and may be important in ensuring the productive alignment of the NFkB proteins with the basal transcription machinery.
DNA bound NFkB dimers may be subject to fiirther regulation. Phosphorylation of p50 has been shown to increase the stability of p50 DNA complexes (Li et al., 1994).
Phosphorylation by PKA also regulates the function of p65 containing dimers, by increasing their transcriptional activity. This occurs because phosphorylation unmasks sites on p65 which are required for interaction with the transcriptional co-activator
CBP/p300 (Zhong et al., 1998). A phosphorylation site has been identified on c-Rel which is required for TNFa induced transactivation (Martin and Fresno, 2000).
24 Chapter 1 Introduction
1.3 The IkB proteins
1.3.1 Cloning and functional domains
The Inhibitors of NFkB (IkB) group comprises the ‘small IkBs’; iKBa, IkBP, IkBe,
IkBy and the Drosophila melanogaster protein cactus and the ‘precursor IkBs’, NFkBI pi 05 and NFkB2 pi GO. BCL3 has homology to IkB proteins but does not share their function, see below. All known members of the IkB family contain multiple copies of the Ankyrin repeat, an evolutionarily conserved sequence that mediates the interaction of the IkB with the NFkB RHD. The n-terminus of the IkBœ core is positioned next to the NTS of NFkB (Huxford et a i, 1998; Jacobs and Harrison, 1998), and is thought to obstruct the binding of the NFkB dimer to karyopherins responsible for transporting
NFkB to the nucleus. Other structural features shared by the small IkB proteins are n- terminal regulatory sequences and c-terminal PEST domains. Residues within n- terminal regulatory sequences are phosphorylated in response to stimulation. Mutation of specific residues (Ser32/36 in IkBœ, Ser 19/23 in IkBP and Ser 157/161 in IkBc) blocks the degradation of the IkB (Brown et al., 1995; DiDonato et al., 1996; Whiteside et al., 1997). PEST domains (regions rich in pro line (P), glutamic acid (E), serine (S) and threonine (T)) are found in many proteins with short half-lives (Rogers et al., 1986) and are thought to regulate protein degradation and processing. (Palombella et al.,
1994).
iKBa and IkBP are ubiquitously expressed proteins, which associate with similar combinations of NFkB dimers; both preferentially binding to p50/p65 and p50/c-Rel.
25 Chapter 1 Introduction
They have distinct but overlapping expression patterns, IxBa levels are highest in haemopoietic organs whereas IxBp is distributed equally between haemopoietic and non-haemopoietic organs. Mice in which the IxBa gene is replaced by the IxBp gene expressed under the control of the IxBa promoter are phenotypically normal (Cheng et a i, 1998), confirming that IxBa and IkBP are functionally redundant and differ only in control of their transcription.
Post activation induction of IxBa but not IkBP mRNA is an important part of termination of NFkB activation (Cheng et al., 1998). Once NFkB is activated an induction of IkBœ mRNA occurs, due to multiple kB elements in the IkBœ promoter (de
Martin et al., 1993; Le Bail et al., 1993). Newly synthesised IkBœ enters the nucleus, removes NFkB from its binding sites and mediates its export to the cytoplasm.
(Arenzana-Seisdedos et al., 1995 Arenzana-Seisdedos et al., 1997). Deletion studies have identified both nuclear import and nuclear export tags in IxBa (Arenzana-
Seisdedos et al., 1997; Sachdev et al., 2000; Turpin et al., 1999; Johnson et al., 1999).
Since this rapid resynthesis is limited to IxBa, persistent activation is controlled by
IkBP, whose activation is switched off by the termination of the activating stimulus.
IkBP can associate through its PEST domain with the newly identified KB-Ras proteins
(Fenwick et al., 2000), although the apparent specificity of KB-Ras for IkBP is inconsistent with the IkBœ and IkBP functional redundancy shown by Cheng et al
(Cheng et al., 1998). Transfection of KB-Ras proteins blocks IkBP degradation despite
26 Chapter 1 Introduction
phosphorylation occurring normally (Fenwick et aL, 2000). The mechanisms controlling KB-Ras fonction are not yet known, however, a Drosophila KB-Ras homologue has been identified so this may represent an additional evolutionarily conserved signalling pathway regulating NFkB.
IkB s also contains n-terminal regulatory sequences and a PEST domain, it is degraded in response to a subset of the signals which degrade IkB œ and IkBP but with slower kinetics. It appears to be a specific inhibitor of p65 homodimers and p65/c-Rel heterodimers and is likely to act as a specific inhibitor in the regulation of genes preferentially regulated by such NFkB dimers (Whiteside et aL, 1997).
The class one NFkB proteins, NFkBI pi 05, NFkB2 pi GO and Relish also act as IkB proteins, they contain Ankyrin repeats in their c-termini, which can associate with Rel homology domains. Unlike their n-terminal portions (p52 and p50), NFkB2 pi GO and
NFkBI plG5 are strictly cytoplasmic (Henkel et aL, 1992; Mercurio et aL, 1993). Thus,
NFkBI plG5 or NFkB2 plGG retain associated Rel subunits in the cytoplasm and inhibit
NFkB DNA binding. (Mercurio et aL, 1993; Rice et aL, 1992; Sun et aL, 1994). NFkBI plG5 has been shown to associate with c-Rel, p65 and p5G, whereas plGG has been shown to associate with c-Rel, p65 and RelB (Mercurio et aL, 1993). These precursors most likely retain a single associated Rel subunit in the cytoplasm via interactions involving both the RHD and the Ankyrin repeats of the precursor (Rice et aL, 1992; Sun et aL, 1994). Several groups have demonstrated that the c-terminal Ankyrin repeats in
NFkBI plG5 associate closely with the NTS, thereby blocking nuclear translocation
27 Chapter 1 Introduction
(Henkel et aL, 1992). IicBy corresponds to the c-terminal of NFkBI p i05 and is the product of alternate promoter usage (Inoue et aL, 1992). Its expression is restricted to mature B cell lines from mice and it binds preferentially to NFkBI p50 or NFkB2 p52 homodimers, indicating that it has a highly specialised function.
BCL3 was cloned in 1990 from a chromosomal translocation breakpoint in a B cell chronic lymphocytic leukaemia (Ohno et aL, 1990). Although structurally related to the other IkB proteins, BCL3 is predominantly nuclear and unlike the prototypic IkB proteins, it is not degraded upon activation. BCL3 binds preferentially to NFkBI p50 and NFkB2 p52 homodimers, and can form ternary complexes with p52 or p50 homodimers and activate transcription (Bours et aL, 1993; Fujita et aL, 1993; Pan and
McEver, 1995; Hirano et aL, 1998). The effect of BCL3 on NFkBI pl05 appears to be regulated by BCL-3 phosphorylation status, concentration and interaction with other nuclear cofactors (Bours et aL, 1993; Wulczyn et aL, 1992; Nolan et aL, 1993; Bundy and McKeithan, 1997; Dechend et al, 1999). Thus, in contrast to other IkB proteins,
BCL3 functions as a positive regulator of gene transcription.
1.3.2 What happens to IkB proteins upon activation?
Potent stimuli induce NFkB activation within minutes through IkB phosphorylation, ubiquitination and degradation (Alkalay et aL, 1995; Brown et aL, 1995; DiDonato et aL, 1996). Phosphorylation of conserved serine residues in the n-terminal regulatory sequence of the IkB (Ser 32/36 in IkB œ, Seri 9/23 in IkBP and Ser 157/161 in IkB s) is
28 Chapter 1 Introduction
required for IkB degradation to occur (Brown et aL, 1995; DiDonato et aL, 1996). The phosphorylated serine motif is recognised by an E3 hgase containing p-Transducin repeat containing protein (p-TRCP), which mediates iKBa ubiquitination thereby tagging it for degradation (Yaron et aL, 1998). This sequence DS^’GT^XS’’ , which contains the IkB Kinase (IKK) substrate sites, is common to several proteins that are substrates for p-TRCP, and serves as a consensus recognition site. The most important acceptor residues for ubiquitination are lysine residues 21 and 22 in IkBœ, mutation of these residues inhibits degradation (DiDonato et aL, 1996; Scherer et aL, 1995).
Interestingly, modification of these lysine residues by SUMO blocks ubiquitination and prevents IkBœ degradation (Desterro et aL, 1998). This is a fiirther mechanism for inhibitory regulation of NFkB. Interestingly, the cysteine protease YopJ from Yersinia pestis has recently been shown to regulate sumosylation, and regulate NFkB activation in transfection studies (Orth et aL, 2000). However, the levels at which YopJ interferes with NFkB regulation were not analysed.
In fact ubiquitination requires three protein complexes, (reviewed in Maniatis, 1999):
Ubiquitin is first attached to an El protein, a ubiquitin activating enzyme, activated at a c-terminal glycine residue and transferred to an E2 protein, also known as a ubiquitin conjugating enzyme. The E2 protein functions together with an E3 ligase to covalently attach ubiquitin to a lysine acceptor site in the substrate protein. In most cases E3 ligases function to recruit the target protein to a complex containing the E2, by binding both the E2 and the substrate. The SCF complexes (Skpl, Cullin, and F box protein containing complexes) are one such class of E3 ligases. The F box containing protein,
29 Chapter 1 Introduction
which is responsible for substrate binding is a variable component, in this case P-TRCP is responsible for the recognition of phosphorylated IkB
The first ubiquitin is attached to 8 -NH2 groups of specific internal lysine residues. In successive reactions a polyubiquitin chain is synthesised by addition of activated ubiquitins to lysine residues in the previously conjugated ubiquitin molecule. A typical
Ub-Ub linkage within a polyubiquitin chain recognisable by the proteasome occurs between lysine 48 of one Ubiquitin and the carboxyl terminus of the next one (Chau et a l, 1989).
The 26s proteasome is a large multicatalytic protease, (reviewed in Baumeister et al,
1998). It is composed of two sub complexes; the catalytic 20s particle and the 19s regulatory particle. Electron micrographs show that the catalytic particle is a barrel shaped complex composed of four stacked rings; two outer a rings and two inner p rings in which the catalytic site is found. Either one or both ends of the 20s barrel are capped with a 19s subunit. A ubiquitin binding activity has been identified within the 19s subunit (van Nocker et a l, 1996), implying that the 19s subunit is involved in recognition of polyubiquitinated proteins. Access to the inner compartment of the barrel is restricted to unfolded proteins. It is thought that ATPase complexes within the 19s subunit, which resemble chaperone proteins, are responsible for binding and dissociating complexes and for unfolding substrates (Lupas et a l, 1993). Unfolded substrates are fed into the inner core of the 20s complex where proteolysis occurs. Ubiquitin is released via the actions of isopeptidases.
30 Chapter 1 Introduction
1.3.3 ‘NFkB precursor’ IkB proteins and NFkB activation
Treatment of cells with inducers of NFkB causes the phosphorylation of NFkBI p i05
(Mellits et aL, 1993; Neumann et aL, 1992; Naumann and Scheidereit, 1994). It has been proposed that that this is followed by enhanced processing of NFkBI pl05 to p50,
(Mellits et aL, 1993; Mercurio et aL, 1993; Naumann and Scheidereit, 1994; MacKichan et aL, 1996), allowing a newly released NFkB dimer to translocate to the nucleus. If processing of NFkBI p i05 to p50 was occurring, reduction in p i05 levels should be accompanied by an increase in total p50 levels, however, in most cases the ratio changes observed between p i05 and p50 are small. This gave rise to suggestions that NFkBI p i05 is completely degraded upon stimulation (Belich et aL, 1999; Harhaj et aL, 1996).
Thus, it is possible that two distinct proteolytic mechanisms act upon NFkBI p i05
(Harhaj et aL, 1996); constitutive processing to p50, mediated by the proteasome, and dependant upon the GRR region, and signal induced NFkBI p i05 degradation, in which the entire NFkBI p i05 protein is destroyed by the proteasome, in a process not requiring the GRR region (Belich et aL, 1999; Heissmeyeret aL, 1999).
Although investigators have drawn different conclusions on whether activating signals induce enhanced processing or complete degradation, it is clear that serine phosphorylation in the c-terminal domain of NFkBI pi05 serves to regulate NFkB activation. Deletion analysis demonstrates that PMA induced phosphorylation of NFkBI p i05 occurs largely on the last 6 8 amino acids in NFkBI p i05, and NFkBI p i05 molecules lacking this region cannot undergo signal induced proteolysis (MacKichan et
31 Chapter 1 Introduction
aL, 1996). Mutation of serine residues 894 and 908 blocked degradation of NFkBI pi 05 induced by UV (Fujimoto et aL, 1995). Mutation of serine residues 921, 923 and 932 in
NFkBI pi05 blocked complete degradation of pi05 induced by TNFa (Heissmeyer et aL, 1999) other residues in this region also regulate this process (J Jansen and SC Ley unpublished results).
Although the ubiquitin-proteasome system is firmly implicated in constitutive processing of NFkBI p i05, signal induced ubiquitination has not been clearly demonstrated on NFkBI pi05. However, two motifs within NFkBI p i05 have been identified as potential recognition sites for E3 ligases or ubiquitin conjugation. (Orian et aL, 2000; Orian et aL, 1999). Mutation of residues within the region 446-454 reduces efficiency of in vitro ubiquitin conjugation and processing to p50, but does not block these processes completely. A motif similar to the IKK consensus phosphorylation site and recognition site for p-TRCP is present within residues 918-934 in the NFkBI p i05
C-terminus. p-TRCP can bind to this site in transfection studies and mutation of this site also reduces efficiency of pi 05 processing (Orian et aL, 2000). It is not clear whether these two regulatory sequences co-operate or act independently, perhaps regulating
NFkBI p i05 ubiquitination under different conditions but it is likely that they are recognised by different E3 ligases, and so may have different functions e.g. regulation of constitutive NFkBI p i05 processing and signal induced NFkBI p i05 degradation.
32 Chapter 1 Introduction
1.3.4 The IkB kinases
In 1996 Maniatis’ lab reported the partial purification of a high molecular weight
(TOOkDa) complex from Hela cells which could specifically phosphorylate IxBa on serine 32 and 26 (Chen et aL, 1996) in a ubiquitin dependant manner. Two kinase subunits from this complex were subsequently purified (DiDonato et aL, 1997; Mercurio et aL, 1997; Zandi et aL, 1997) and molecularly cloned (Regnier et aL, 1997; Woronicz et aL, 1997). These kinases are known as IkB kinase (IKK) a (also IKKl) and IKKp
(also IKK2).
IKKa and IKKp have 52% overall amino acid identity, both containing protein kinase domains in their n-termini, a Leucine Zipper (LZ) and a Helix Loop Helix (HLH) motif in their c-terminal portion. Kinase activity of IKKa and IKKp depends upon their dimérisation, which requires intact LZ motifs (Zandi et aL, 1997). Mutations in the IKK helix loop helix motifs also disrupt IKK kinase activity; these are not required for dimérisation but may act to regulate IKK activity by interacting with the kinase domain, as the HLH motif can stimulate IKK activity when co-expressed (Delhase et aL, 1999).
Transfection studies show that IKKa and P phosphorylate IxBa with identical activation kinetics and substrate specificity (Mercurio et aL, 1997; Zandi et aL, 1997).
IKKa/IKKp heterodimers are found in a complex associated with either a dimer or trimer of IKKy, a regulatory subunit (also known as NTkB essential modulator, NEMO)
(Rothwarf et aL, 1998; Yamaoka et aL, 1998). IKK activation depends on the IKKy subunit, as neither NFkB nor IKK activation are detectable following stimulation of
33 Chapter 1 Introduction
IKKy deficient cells with LPS, TNFa, IL-1 or PMA (Rudolph et aL, 2000; Schmidt-
Supprian et aL, 2000; Yamaoka et al., 1998). IKKy has no recognisable catalytic domain, it contains three helical regions and includes a leucine zipper. Deletion analysis shows that the n-terminal portion of IKKy is required for IKKa and IKKp interaction
(Rothwarf et aL, 1998; Yamaoka et aL, 1998; Mercurio et aL, 1999). Interestingly IKKy is a target of the NFkB activating viral protein Tax from the Human T cell leukaemia virus (HTLV) which may activate NFkB by forcing oligomerisation of IKK complexes
{O m etaL, 1999).
Although knockout studies clearly show that IKKa and IKKp are absolutely required for
IxBa degradation (Li et aL, 2000), NFkBI p i05 degradation has not been analysed in these mice. The region 902-936 of NFkBI pi 05 contains two overlapping motifs which are similar to the conserved DS^G'PXS'’ in iKBa, IkBP and IkBc. Transfected IKK can phosphorylate sites within these motifs, causing NFkBI p i05 degradation. Furthermore, mutation of these site blocks TNFa induced NFkBI pi05 degradation (Heissmeyer et aL, 1999). However, genetic studies will be necessary to determine whether IKKa and
IKKp or similar kinases perform this function in vivo.
Are there other IkB kinases? The fact that no active NFkB can be detected in
IKKa/IKKp double mutant cells or embryonic sections (Li et aL, 2000) apparently argues against this. However, the analysis to date on the role of IKKa and IKKp has focussed on a limited number of activating stimuli in a limited number of cell types, so it
34 Chapter 1 Introduction
remains possible that some NFkB activation may proceed independently of IKKa and
IKKp.
Murine IKK-i and its human homologue IKKs have been identified in a novel PMA inducible IKK-like complex which does not contain IKKa or IKKp (Peters et aL, 2000;
Shimada et aL, 1999). The IKKe kinase domain amino acid sequence is 31-33% identical to IKKa and IKKp. Both IKKe and IKKi are most highly expressed in spleen, thymus, peripheral blood, pancreas and placenta. IKKi mRNA is strongly induced in B cells and macrophages in response to Lipopolysaccharide (LPS). Primary sequence analysis shows that like IKKa and IKKp, IKKe has an n-terminal kinase domain and a
HLH motif near the c-terminus. The leucine zipper motif of IKKa and IKKp appears to be lacking fi'om IKKe, but a predicted coiled coil multimerisation domain lies in its place.
Both IKKi and IKKe preferentially phosphorylate serine 36 of IxBa in contrast to IKKa and IKKp, which phosphorylate serine 32 and 36 with similar efficiency (Peters et aL,
2000; Shimada et aL, 1999). In transfection studies, the kinase inactive form of IKKe blocks PMA and CD3 induced but not TNFa induced NFkB activation. This contrasts with the kinase inactive form of IKKp which blocks CD3, PMA, IL-1 and TNFa induced NFkB activation (Peters et aL, 2000). Interestingly, the 350-kDa complex containing IKKe from Jurkat cells has been reported to contain an additional as yet unidentified IKK-like kinase activity, able to phosphorylate IxBa on both serine 32 and
35 Chapter 1 Introduction
36. Taken together these data suggest a role for IKKs in NFkB activation following T cell receptor stimulation and following LPS stimulation in B cells and macrophages.
Three groups have recently described a kinase with more than 60% overall sequence identity to IKKs, termed TBKl, NAK or T2K (Pomerantz and Baltimore, 1999;
Bonnard et aL, 2000; Tojima et aL, 2000). Primary sequence analysis of
NAK/TBK/T2K predicts a similar structure to IKKa and IKKp, which, like IKKs/IKKi lacks one of the activation loop serine residues conserved in IKKa and IKKp. Similar to IKKs/IKKi, baculovirus expressed NAK can phosphorylate IxBa only on serine 36 not serine 32 and IkBP at serine 23, not serine 19. However, NAK is also reported to act as an IKK kinase. Since phosporylation of both iKBa serine residues is required for its degradation, Tojima et al concluded that NAK acts upstream of the IKK complex which it can activate directly through phosphorylation (Tojima et aL, 2000). Since IKKs and
NAK are highly homologous at is unlikely that one functions as an IKK kinase and the other functions as an IkB kinase, further experiments are necessary to resolve this issue.
Indeed, T2K knockout mice exhibit reduced NFkB dependent gene transcription despite normal NFkB DNA binding (Bonnard et aL, 2000), which may indicate that neither prediction is correct and that NAK/TBK/T2K regulates transactivation.
1.4 Upstream of the IKK complex
Eucaryotic cells respond to most NFkB inducing stimuli by activating the IKK complex.
Several important questions regarding the mechanism of IKK activation remain to be answered. How do such diverse stimuli lead to a common event; the activation of IKK?
36 Chapter 1 Introduction
At which step in the signalling cascade are such signals integrated? How is specificity maintained?
NFkB activation following receptor stimulation occurs via different signalling pathways according to the receptors involved. Thus the Toll-like receptors of the innate immune system and IL-1 and IL-18 receptors utilise common signal transduction pathways leading to the activation of IKK. The TNF receptor superfamily utilises a similar system of receptor multimerisation and adapter protein recruitment, but employs different proteins. Genetic studies are now clarifying the mechanism of IKK activation by T and
B cell antigen receptors. Receptor activation events are described below, after examination of the various proposed mechanisms of IKK activation; phosphorylation by upstream kinases, IKK autophosphorylation and polyubiquitination.
Purified IKK complexes are inactivated upon treatment with protein phosphatase 2A
(PP2A). Conversely, treatment of Hela cells with the PP2A inhibitor Okadaic acid stimulates IKK activation (DiDonato et aL, 1997). Taken together these data strongly suggest that IKK activation depends upon phosphorylation. Activation loop sequences necessary for kinase activation are identical in IKKa and IKKp. However, although the
IKKp activation loop sequences are required for TNFa and IL-1 induced NFkB activation, equivalent sequences in IKKa are dispensable (Delhase et aL, 1999). This is supported by apparently normal iKBa degradation in IKKa mice analysed by Hu et al
(Hu et aL, 1999), although, Li et al reported a more significant reduction in NFkB activation in response to TNFa in their IKKa knockout mice (Li et aL, 1999a).
37 Chapter I Introduction
When overexpressed in mammalian cells, numerous protein kinases can activate IKK, or can inhibit IKK in their dominant negative forms. These include some MAPKKK,
Protein Kinase C iso forms and other kinases, see below.
1.4.1 MAP3K Proteins
MAP kinase pathways are signalling cascades present in all eucaryotes, which respond to signals such as mitogens, growth factors and stress,^eviewed in Robinson and Cobb,
1997; Widmann et aL, 1999). They regulate cellular responses such as differentiation, cell cycling and apoptosis by the phosphorylation of proteins containing the core consensus ser/thr-pro. Many of the targets of this class of enzyme are transcription factors e.g. ATF2, or effector proteins e.g. phospholipase A2 iso forms. MAP kinase pathways typically consist of an independently controlled three-kinase module. A MAP kinase (MAPK) is activated upon phosphorylation by a MAPK kinase
(MAPKK/MAP2K) which is regulated by a MAPKK kinase (MAPKKK/MAP3K).
Transmission of signals arises from sequential activation of the components within a module. In mammals at least 5 MAP kinase modules are distinguishable, see Diagram
3. The Extracellular signal Regulated Kinase 1 and 2 (ERK) cascade preferentially regulates growth and differentiation and the c-Jun N-terminal Kinase (INK) and P38 cascades mainly regulate responses to stress e.g. inflammation and apoptosis.
38 Chapter I Introduction
Growth Inflammatory cytokines Stimulus Factors CelluW Stress Serum stress - 4 Ï------MAPKKK Mos/Raf/I PI.-2 IP1-2/MEKK MLK/DLK/TAK/ASK/I PI.-2 ??? ???/rpi,-2 II II MAPKK MEKl/2 MKK4/7 MKK3/6 ??? MKK5 II II I MAPK ERKl/2 INK P38 ERK3 ERK5 1 % ?" # - Response Proliferation Inflammation 999 Proliferation Differentiation Apoptosis Development Development
Diagram 3: Schematic Overview of mammalian MAPK modules Mammalian MAPK modules responsive to growth factors, cellular stress and inflammatory cytokine are shown. Transient transfection studies show that TPL-2(shown in red) can regulate
The MAP3K enzymes are difficult to categorise by sequence alone, however, many
MAP3K share a common feature, the ability to regulate multiple MAPK cascades and
NFkB. Whether this is an experimental artefact or evidence that MAP3K are multifunctional awaits clarification.
Genetic studies have shown that TNPl Receptor Associated Factor (TRAF) proteins play a central role in NFkB activation. The NFkB Inducing Kinase (NIK), which has homology to MAP3K proteins, was identified as a binding partner for TRAF 2 in a yeast two hybrid screen (Malinin et aL, 1997), although this interaction has never been demonstrated occurring between endogenous proteins. NIK is a potent activator of IKK when overexpressed (Ling et aL, 1998), and kinase dead NIK blocks NFkB activation in
39 Chapter 1 Introduction
response to most stimuli. Consequently, NIK is assumed to be directly involved in a
NFkB activation pathway. However, removal of the portion of the TRAF 2 required for
NIK association has no effect upon NFkB activation (Baud et aL, 1999), and the apparent binding preference of NIK for IKKa demonstrated in yeast two hybrid and transfection studies is not consistent with other data suggesting that IKKa is not required for NFkB activation following TNFa stimulation (Ling et aL, 1998; Woronicz et aL, 1997). The naturally occurring Alymphoplasia (aly) mutation in mice is due to a point mutation in NIK, which may prevent its interaction with upstream TRAF proteins
(Shinkura et aL, 1999). TNFa induced NFkB activation is not blocked in Aly embryonic fibroblasts (Shinkura et aL, 1999). However, Aly mice have cell-type specific defects in CD40 induced NFkB activation: dendritic cell activation is normal whereas B cells stimulated through CD40 do not activate NFkB (Garceau et aL, 2000).
Taken together, these data indicate a more restricted role for NIK in IKK activation than previously anticipated.
MEKKl also interacts with TRAF proteins (Baud et aL, 1999) and is involved in regulation of Jun N-terminal Kinase (INK), whose activation often coincides with
NFkB. Dominant negative MEKKl partially inhibits NFkB activation upon overexpression (Nemoto et aL, 1998). However, analysis of MEKKl-/- embryonic stem cells and fibroblasts has shown that although INK activation in response to TNFa, ILl, double stranded RNA and LPS is blocked, NFkB activation is not impaired in response to these stimuli (Xia et aL, 2000; Yujiri et aL, 2000). The possibility that MEKKl regulates NFkB only under certain conditions cannot yet be excluded. Transfection of
40 Chapter 1 Introduction
MEKK2 and MEKK3 can also promote IKK activity and activate NFkB reporter genes
(Zhao and Lee, 1999).
TGPp Activated Kinase 1 (TAKl) is also a MAP3K and has been shown to interact with
IKKa and p. Overexpression of TAKl together with its activator TABl activate NFkB in an IKK dependent manner (Sakurai et aL, 1999). Furthermore, transfection of dominant negative TAKl into cell blocks TNFa induced NFkB activation (Sakurai et a l, 1999).
1.4.2 Other kinases
Lallena et al have shown that several Protein Kinase C (PKC) iso forms regulate NFkB.
PKCa and atypical PKCÇ both activate IKK when overexpressed, furthermore dominant negative PKCÇ severely impairs IKK activation in response to TNFa whereas dominant negative PKCa blocks phorbol ester induced IKK activation (Lallena et aL, 1999). In later studies the same group showed that atypical PKC iso forms are recruited into lL-1 and TNFa signalling through an association with p62. PKC0 is an absolute requirement for NFkB activation in mature T cells, but is dispensable in thymocytes and other cell types, but whether PKC0 acts upon IKK or at another level in NFkB activation is not yet clear (Sun et aL, 2000).
Akt may participate in NFkB regulation by several mechanisms. Akt can phosphorylate
IKKa in response to TNFa and PDGF stimulation (Ozes et aL, 1999; Romashkova and
41 Chapter 1 Introduction
Makarov, 1999). However, these findings are disputed by investigators who believe that
TNFa cannot stimulate Akt activity and that IKKa phosphorylation is not relevant to
IKK activation (Delhase et aL, 2000). It was also reported that Akt plays no part in
IicBa degradation but regulates the transcriptional activity of nuclear NFkB (Madrid et aL, 2000). GSK3p, which regulates NFkB transactivation, is a downstream effector for
Akt and so may mediate these effects (Hoeflich et aL, 2000). These contradictory data may indicate a cell type or stimulus specific function for Akt.
As described above NAK/TBK/T2K is homologous to IKKa and IKKp. However,
NAK cannot directly phosphorylate both of the serine residues required for iKBa degradation and NAK induced iKBa phosphorylation can be blocked by the expression of dominant negative IKKp (Tojima et aL, 2000). Based on this data Tojima et al propose that NAK functions as an IKK kinase.
It seems likely that several physiologically relevant IKK kinases exist, the challenge ahead is identifying the specific conditions in which individual kinases are important.
The involvement of the MAPKKK family of proteins in NFkB stimulation will be best analysed by a thorough genetic approach, which will avoid pitfalls such as non-specific dominant inhibitory effects and overexpression artefacts. This will also allow detailed analysis of cell type or context specific function of these kinases suggested by current data: The involvement of NIK in CD40 signalling in B cells but not dendritic cells
(Garceau et aL, 2000) and the requirement for PKCÔ in NFkB activation in mature T lymphocytes but not thymocytes (Sun et aL, 2000).
42 Chapter 1 Introduction
1.4.3 IKK autophosphorylation
IKK proteins overexpressed in inseet eells are highly aetive in the absence of
stimulation, thus IKK itself may function as an IKK kinase, thereby activating itself,
(reviewed in Karin and Ben-Neriah, 2000). Deng et al used a reconstitution system in
which IKK complexes purified from unstimulated cells were treated with fractions from
TNFa stimulated cell extracts. They found that the only fraction which could stimulate
inactive IKK is the aetive IKK fraction (Deng et aL, 2000). The molecular mass of the
IKK complex increases from 640 kDa to 1.3MDa upon TNFa stimulation (Poyet et ah,
2000) without recruitment of upstream regulators such as TRADD, TRAF 2 or RIP. The
authors propose that the IKK complex is recruited to the activated TNF receptor where
oligomerisation enables IKK autophosphorylation before its release into the cytoplasm.
From a series of experiments in which the recruitment domains of IKKy or RIP are
replaced by inducible oligomerisation modules, Poyet et al showed that oligomerisation
of RIP is sufficient to activate IKKa and IKKp and that this process requires IKKy and
intact IKKa and IKKp activation loop sequences. The Human T cell Leukaemia Virus
(HTLV) protein Tax promotes IKKy oligomerisation, which results in IKK activation.
This mechanism too may rely on IKK autophosphorylation (Chu et aL, 1999). However,
IKK autophosphorylation may not be sufficient for activation, it is possible that
autophosphorylation amplifies IKK kinase activation induced by another kinase.
1.4.4 TRAPS: oligomerisation and ubiquitination in IKK activation
Members of the TRAF (TNF Receptor Associated Factor) group are recruited to
activated IL-1 and TNFa receptor family complexes. There are six known mammalian
43 Chapter 1 Introduction
TRAFs, their importance has been elucidated by generating knockout mice. TRAF 6 knockout mice have an absolute defect in NFkB activation in response to IL-1, LPS,
CD40 and RANK (Lomaga et aL, 1999; Naito et aL, 1999). TRAF 2 deficient mice have a minor defect in NFkB activation following TNFa stimulation but CD40 stimulated NFkB activation is completely blocked (Nguyen et aL, 1999; Yeh et aL,
1997). CD27 and CD40 responses are reduced in TRAF 5 null mice(Nakano et aL,
1999). Furthermore, TRAF 2 or TRAF 6 overexpression are sufficient to activate NFkB
(Rothe et aL, 1995; Cao et aL, 1996).
All TRAFs possess a TRAF domain at their c-terminus, which mediates homo- and heterodimerisation of TRAFs and interactions with receptors and adapters. In a study in which the TRAF domains of TRAF 2 and 6 were replaced with inducible dimérisation domains it was shown that TRAF oligomerisation mimics receptor activation, resulting in activation of NFkB and transcription of physiologically relevant genes (Baud et aL,
1999).
All TRAFs (except TRAF 1) contain an n-terminal RING finger domain. Removal of
TRAF RING finger domains ablates their ability to activate NFkB (Rothe et aL, 1995;
Song et aL, 1997; Takeuchi et aL, 1996). Several RING finger proteins have been shown to fimction as ubiquitin ligases (Lorick et aL, 1999) . Significantly, it has recently been shown that the NFkB inducing role of TRAF 6 is due to its fimction as a ubiquitin E3 ligase. TRAF 6 functions in concert with the E2 ligase Ubcl3/Uevl A, to catalyse the assembly of polyubiquitin chains linked through lysine 63 of Ubiquitin on
44 Chapter 1 Introduction
an unidentified substrate. This process is essential for IKK activation induction by
TRAF 6 . As IKK activation is independent of the proteasome and since the proteasome does not recognise K63 polyubiquitin chains, it seems likely that K63 polyubiquitination regulates the function of an unknown protein upstream of IKK (Deng et aL, 2000)
TRAF 2 can also act in concert with Ubcl3/UevlA to synthesise K63 polyubiquitin chains, perhaps an early indication of a more general effector function for the TRAF proteins (Deng et aL, 2000). It is interesting to note that the enzymes required for synthesis of K63 polyubiquitin chains are conserved from yeast to man (Hofinann and
Pickart, 1999; Spence et aL, 1995), K63 polyubiquitination therefore may be a common mechanism for regulating signalling pathways.
1.4.5 Summary of IKK activation.
There is evidence that activation of the IKK complex may be regulated by at least three mechanisms; phosphorylation by upstream kinases, IKK autophosphorylation and indirectly by K63 polyubiquitination. These mechanisms are clearly not mutually exclusive, and may cooperate to activate the IKK complex in certain circumstances.
Alternatively, IKK may be activated by different mechanisms in different situations or cells. Given that a large number of stimuli activate IKK, this latter possibility seems likely.
45 Chapter 1 Introduction
1.4.6 NF kB activation by receptors of the TNF receptor superfamily.
The TNF receptor family can be categorised according to the presence or absence of a
Death Domain in their cytoplasmic tails, previewed in Wallach et aL, 1999). Death domain containing receptors include TNFRl, TRAIL-Rl and DR3.
Stimulation of TNFRl results in activation of several signalling pathways that lead to
NFkB and JNK activation and the induction of apoptosis (Hsu et aL, 1996). Trimerised death domains of activated receptors serve as a docking site for TRADD (TNFRl associated death domain protein). TRADD functions as a platform to which other signalling proteins are recruited via homotypic death domain interactions. These include
TRAF 2, the kinase RIP (Receptor Interacting Protein) and F ADD (FAS Associated protein with Death Domain) whose functions are described below. These receptor proximal events are regulated by inhibitory SODD (Silencer Of Death Domains) which binds to TNFRl and dissociates upon stimulation (Jiang et aL, 1999). A20 can bind
TRAF 1 and 2 and inhibit NFkB activation when overexpressed (Song et aL, 1996).
Inhibitor of Apoptosis (lAP) proteins are induced by TNFa treatment, bind to TRAF 1 and 2 and inhibit pro-apoptotic signalling (Wang et aL, 1998)
F ADD, which is required for TNFa induced apoptosis recruits procaspases such as caspase 8 or 10 to the TNFR associated multiprotein complex (Yeh et aL, 1998).
Caspases in close proximity can then cleave one another, which results in their activation and release from the receptor. Active cytoplasmic ‘upstream’ caspases can then activate ‘effector’ caspases such as Caspases 3, 6 or 7 which cleave ‘death substrates’ leading to the characteristic cellular changes associated with apoptosis.
46 Chapter 1 Introduction
These include cleavage of ICAD (Inhibitor of Caspase Activated DNase) which leads to the destruction of genomic DNA.
Importantly, although TNFRl can induce apoptosis, it can also signal cell survival, proliferation and differentiation, through NFkB and JNK activation. Genetic studies show that TRAF 2 and MEKKl are required for JNK activation in response to TNFa
(Xia et aL, 2000; Yeh et aL, 1997), presumably stimulating JNK via SEKl activation.
RIP is required for coupling TNFa stimulation to NFkB but is not required for JNK activation (Kelliher et aL, 1998). Interestingly, the RIP kinase domain is not required for NFkB activation (Ting et aL, 1996). Following TNFa treatment RIP can bind through its intermediate domain to IKKy, thereby recruiting the IKK complex to the membrane (Zhang et aL, 2000). The activated IKK complex is subsequently released into the cytoplasm where it has access to its substrate (Zhang et aL, 2000). Although
TNFa induced NFkB activation is only mildly impaired in TRAF 2 null mice, CD40 induced NFkB activation is blocked (Nguyen et aL, 1999). As TRAF 2 participates in
TNFR family induced NFkB activation and is implicated in TNFRl signalling by transfection experiments, another TRAF may substitute for the NFkB inducing function of TRAF 2 in TNFa responses. Genetic analysis of cells lacking multiple TRAFs may clarify this issue.
TNFR family receptors lacking death domains e.g. TNFR2, 0X40 and Lymphotoxin p contain motifs which allow direct binding to TRAF proteins (Ye et aL, 1999). TRAF proteins associate with different affinities for different receptors. Although less is
47 Chapter 1 Introduction
known about the function of TRAFs in signalling by TNFR2-type receptors, it is likely that they will also regulateNF kB and JNK activation in this context.
1.4.7 NF kB activation by receptors of the IL-1/Toll-like receptor
superfamily
A family of receptors recognising conserved motifs from pathogens, including LPS,
peptidoglycan and bacterial DNA, forms the basis of the pathogen recognition strategy
by the innate immune system. These receptors are Toll-like receptors’(TLR), a
reference to Toll, a Drosophila receptor that functions in development and antifungal
immunity (Aderem and Ulevitch, 2000). To date nine mammalian TLR have been
described and the ligands for some of them have been identified, e.g. TLR4 recognises
LPS , TLR9 recognises unmethylated CpG containing DNA (Hemmi et aL, 2000) and
TLR2 appears to be more promiscuous, recognising various fungal. Gram positive and
mycobacterial components Reviewed in Aderem and Ulevitch, 2000). The cytoplasmic
tails of IL-1 R, IL-18R and of the Toll-like receptors contain homologous regions called
TIR domains (Toll/IL-1 receptor domains). All members of the Toll/IL-1 family
function in host defence, and the signal transduction pathways initiated by these
receptors culminate in NFkB and AP-1 activation.
There is accumulating evidence that several components of the IL-1 pathway are also
utilised by the Toll-like receptors. MyD8 8 , which also has a TIR domain, is essential
for both IL-1 and TLR4, TLR2 and TLR9 signalling (Adachi et aL, 1998; Akira, 2000;
48 Chapter 1 Introduction
Hacker et aL, 2000; Kawai et aL, 1999). MyD8 8 associates with the activated IL-1 receptor or TLR4 through a homotypic interaction involving its TIR domain (Muzio et aL, 1998; Muzio et aL, 1997). MyD8 8 can bind to the death domains of IRAKI, 2 or M,
thereby mediating the association of IRAK with the receptor complex (Muzio et aL,
1997; Wesche et aL, 1999; Wesche et aL, 1997). IRAK is phosphorylated, dissociates
from the receptor and associates with TRAF 6 which is essential for IKK and JNK
activation in response to LPS and IL-1 (Cao et aL, 1996; Lomaga et aL, 1999; Naito et
aL, 1999). Upon IL-1 stimulation TRAF 6 associates with p62, which associates with
PKCÇ (Sanz et aL, 2000), which can activate IKKp in transfection studies (Lallena et
aL, 1999). MEKKl is required for JNK activation by TLR4 and IL-1, but is dispensable
for NFkB activation (Xia et aL, 2000). ECSIT (Evolutionarily Conserved Intermediate
in Toll pathways) may regulate the TRAF 6 and MEKKl interaction (Kopp et aL,
1999). As described above TRAF 6 regulates IKK activity by catalysing a
polyubiquitination reaction (Deng et aL, 2000)
1.4.8 NFkB activation by T and B cell receptors
NFkB activation occurring following engagement of the B cell receptor (BCR) (Francis
et aL, 1998; Venkataraman et aL, 1996) regulates BCR induced survival and
proliferation (Grumont et aL, 1998; Kontgen et aL, 1995). It has only recently been
shown that IKK is activated by BCR stimulation (Petro et aL, 2000). Transmission of
signals from the BCR involves multiple protein tyrosine kinases including Syk, Lyn and
BTK. BTK and Syk synergise to activate Phospholipase C y2 (PLCy2), which catalyses
the production of Inositol 1,4,5, trisphosphate (IP3) and Diacylglycerol (DAG), which
49 Chapter I Introduction
can regulate calcium levels and the activity of Protein Kinase C (PKC) isoforms.
Genetic evidence from DT40 knockout B cells and knockout mice indicate that BTK and
PLCy2 are absolutely required for BCR induced NFkB activation (Petro and Khan,
2000; Petro et al., 2000). The mechanism by which IKK becomes activated under these conditions is not yet clear, since PKC0 is required for NFkB activation in T cells, it is possible that another PKC iso form will perform this function in B cells.
TCR-CD3 and CD28 costimulation activate IKK and NFkB in an IKKy dependant manner (Harhaj et al., 2000; Harhaj and Sun, 1998). NFkB is required for the production of cytokines such as IL-2 (Gerondakis et al., 1996) and cell surface receptors such as CD25 (the IL-2 receptor a chain) (Kuang et al., 1993), events which are required for T cell proliferation. Biochemical transmission of signals from the TCR occurs as a result of recruitment of a signalling complex to the site of cell-cell contact with an antigen presenting cell, which initiates the activity of multiple protein tyrosine kinases, (reviewed in Dustin and Chan, 2000). PKC0, a Protein Kinase C isoenzyme is also recruited to this complex. Mature T cells from PKC0 null mice fail to activate
NFkB following CD3 and CD28 co stimulation, indicating that PKC0 is essential in this pathway, although TNFa stimulated NFkB activation is normal (Sun et al., 2000).
Interestingly, NFkB activation in thymocytes following stimulation through CD3 and
CD28 is unaffected indicating developmental differences in the regulation of NFkB activation (Sun et al., 2000). Mice lacking the Rho family GXP exchange factor Vav have partial NFkB activation defects in T lymphocytes (Costello et al., 1999). These mice have defects in IP 3 production, which implies a similar defect in DAG production.
50 Chapter 1 Introduction
Since DAG activates PKC0 it is possible that a Vav regulated PLCy activity lies upstream of PKC0 in T cells. The Rac GTPase has been shown to regulate NFkB in other cell types and receptor systems (Arbibe et al., 2000; Jefferies and O'Neill, 2000), for instance, Racl and PI3K p85 associate with the cytoplasmic tails of TLR in a stimulation dependent manner and regulate transactivation by p65 containing dimers
(Arbibe et al., 2000). IKKs can block CD3 induced NFkB activation, suggesting that
IKKs may also participate in this pathway (Peters et al., 2000), although genetic experiments are required to confirm these data.
1.5 Genetic approaches to the analysis of NFkB regulation and function
Genetic studies on model organisms such as Drosophila and mice have provided critical insights into NFkB regulation and fimction.
1.5.1 Drosophila Melanogaster
Drosophila employ efficient innate host defences against invading micro-organisms
(Imler and Hoffinann, 2000). One facet of this is the production of potent antimicrobial peptides by the fat body, the Drosophila equivalent of the mammalian liver. Seven distinct antimicrobial peptides have been characterised, which are effective against different invading agents (Engstrom, 1999). Drosophila can discriminate between types of invading micro-organism and produce appropriate combinations of antimicrobial peptides. A regulatory scheme is beginning to emerge for the induction of distinct
51 Chapter 1 Introduction
genes, which could provide the framework for these ‘adapted’ responses. Recent studies have indicated distinct but overlapping roles for the three Drosophila NFkB proteins. In flies carrying a deletion of both Dif and Dorsal, induction of the antifungal peptide
Drosomycin was strongly inhibited whereas most antibacterial peptides were unaffected
(Meng et ah, 1999). However, Relish mutant flies have defects in the production of all antimicrobial peptides; production of Diptericin and Cecropin were abolished, levels of other antimicrobial peptides were merely reduced (Hedengren et al., 1999). As in the mammalian system, NFkB proteins form various combinations of hetero- and homodimers, which can differentially regulate the synthesis of the microbial peptides
(Han and Ip, 1999)
To date, two pathways for the activation of the Drosophila immune response have been described,Reviewed in Imler and Hoffmann, 2000). Fungal infection activates the Toll signalling pathway, causing the degradation of cactus, an IkB homologue. Two of the drosophila NFkB proteins, Dif and Dorsal are then able to translocate to the nucleus, leading to the production of Drosomycin. The Toll pathway is triggered through the activation of a serine protease cascade in the haemolymph, which generates the ligand for Toll, Spatzle. The activated receptor signals through the adapter protein Tube,
which is fiinctionally equivalent to MyD 8 8 , and Pelle, a kinase homologous to mammalian IRAK. Similar to IkBœ, cactus degradation requires phosphorylation of its n-terminal domain, however, the kinase(s) responsible for this are as yet unidentified.
The Toll pathway is also critical in early development, where it specifies dorsal-ventral
52 Chapter 1 Introduction
patterning of the embryo see below, however. Dorsal but not Dif is required for this pathway.
The antibacterial immune pathway involves Relish, the other Drosophila NFkB protein,
(reviewed in Imler and Hof&nann, 2000). Relish is homologous to NFkBI p i05 and
NFkB2 p i00 inasmuch as it contains c-terminal Ankyrin repeats, and is believed to act
as an IkB. However, these Ankyrin repeats are structurally dissimilar to all known
NFkB or IkB proteins. Furthermore, unlike NFkBI p i05 and NFkB2 p i00, Relish
contains a transactivation domain. The receptor involved in LPS responsiveness in
Drosophila has not yet been identified, although the Drosophila genome contains at
least eight Toll-like receptor sequences. Activation of Relish occurs by endoproteolytic
cleavage, allowing the n-terminal RHD to translocate to the nucleus. Unlike mammalian
NFkBI p i05, the resulting c-terminal fragment of Relish is stable in the cytoplasm.
Furthermore, the endoproteolytic cleavage event is not mediated by the proteasome
(Stoven et al., 2000). Drosophila homologues of IKKp and IKKy have been identified,
known as dmIKKp and dmIKKy/Kenny respectively (Rutschmann et al., 2000;
Silverman et al., 2000), without which Relish activation and antibacterial defence cannot
occur. Cactus can be degraded in the absence of these proteins suggesting that Relish,
Dif and Dorsal are regulated in different ways.
NFkB signalling also fiinctions during embryogenesis in Drosophila, where it is
involved in the specification of dorsal-ventral polarity, previewed in Govind, 1999).
Ventrally situated maternal cells provide a local factor that stimulates the processing of
53 Chapter 1 Introduction
Spatzle enabling it to bind to Toll receptors on the ventral side of the precellular embryo.
The signal is transduced across the cytoplasm by pelle and tube, causing the degradation
of cactus and thus the nuclear translocation of dorsal. The localised ventral application
of the Spatzle, leads to the establishment of a dorsal-ventral gradient of nuclear levels of
dorsal protein. As dorsal regulates the transcription of genes in a concentration
dependent manner twist and snail are induced in the ventral most regions which gives
rise to mesoderm. Lateral areas (which become neuroectoderm) activate short gastrulation and rhomboid. In both the ventral and lateral regions, nuclear dorsal
represses genes such as twisted gastrulation and decapentaplegic, which are expressed
in the dorsal region of the embryo.
1.5.2 Mice knockouts / transgenics
The generation of mice in which individual members of the NFkB gene family or their
regulators are inactivated has proved an invaluable way to elucidate the physiological
functions of NFkB, and assign roles to individual genes, see summary in Table 2. Some
of these functions are only evident in ‘double knockout’ mice (mice lacking multiple
NFkB proteins) due to functional redundancy between NFkB subunits.
1.5.3 NFkB in the development of the immune system
Most mice with targeted disruption of single NFkB genes survive to birth, however
histological and flow cytometric analyses of lymphoid organs reveal a range of
developmental defects arising from lack of NFkB, summarised in Table 2.
54 Chapter 1 Introduction
Tissue/ NFkB regulated function Genetic evidence from mice Cell type (knockout unless stated otherwise) B cell Development nfkbl/nfkb2 (Franzoso et a l, 1997) B cell Proliferation p65 (Doi et a l, 1997) c-Rel (Grumont et al, 1998) nfkbl (Shae/ûr/., 1995; Snapper et a l, 1996) B cell Survival nfkbl (Grumont et al, 1998) B cell Immunoglobulin heavy chain nflcbl (Snapper et a l, 1996) isotype switching c-Rel (Gerondakis et a l, 1999) p65 (Doi el fl/., 1997) T cell Development/homeostasis iKBa(DN) (Voll et a l, 2000) (Boothby et a l, 1997; Hettmann et a l, 1999) Tcell Proliferation c-Rel (Gerondakis el a/., 1996) p65 (Doi et a l, 1997) nfkblAC(lshikawa e/a/., 1998) Tcell TH2 differentiation p50 (Das et al, 2001) Macrophage Cytokine production c-Rel (Grigoriadis elûf/., 1996) Monocyte Differentiation p65/c-Rel (Grossmann et a l, 1999) Macrophage Protection fi*om apoptosis p65 (Beg and Baltimore, 1996) Dendritic cell Development of CDa DC RelB Wu 1998 Osteoclast Differentiation nfkbl nfkb2 (Franzoso et al, 1997; lotsova et a l, 1997) Liver Protection fi*om apoptosis p65 (Beg and Baltimore, 1996) IKKp (Li et a l, 1999c; Tanaka et a l, 1999) p65/nfkbl (Horwitz el a/., 1997) Erythrocyte Differentiation p65/c-Rel (Grossmann et al, 1999) Neural tube Closure IKKa/lKKP (Li ela/., 2000) Spleen Development of architecture nfkb2 (Caamano ela/., 1998)
Table 2: NFkB functions defined by study of knockout and transgenic mice
Assembly of a pre-TCR is a critical checkpoint in T cell development and coincides with
NFkB activation. Expression of dominant negative IxBa in thymocytes reduces the
55 Chapter 1 Introduction
number of cells successfully undergoing this P selection, clearly defining a role for
NFkB in T cell development (Voll et ah, 2000). IkBs null mice also have defects in thymopoiesis, although other IkB proteins may compensate for IkBs, masking a more severe phenotype (Memet et al., 1999). NFkB is also required for B cell development, and in nfkbl-/- nflcb2-/- double knockout mice and c-Rel-/- p65-/- double knockout mice
B cell development is blocked at the IgM+IgD- stage (Franzoso et al., 1997; Grossmann et al., 1999).
Other immune cell lineages requiring NFkB in their development are monocytes, whose differentiation is inhibited in p65-/- c-Rel-/- mice (Horwitz et al., 1997). Disrupted spleen and lymph node structures in nfkb 2 -/- mice are believed to arise from failure of antigen presentation (Caamano et al., 1998; Franzoso et al., 1998). NFkB2 p52 is the major partner for RelB, which is required for the development of a subset of dendritic cell populations (Burkly et al., 1995; Caamano et al., 1999). Defective or deficient thymic dendritic cells may explain the failure of deletion of autoreaetive thymocytes in
RelB-/- mice (Burkly et al., 1995). Granulocytosis is reported in c-Rel-/- p65-/- mice indicating a role for NFkB in granulocyte homeostasis (Grossmann et al., 1999).
1.5.4 NFkB in immune responses
Requirements for NFkB have been demonstrated in several aspects of the immune response,previewed in Gerondakis et al., 1999). NFkBI p50, p65 and e-Rel regulate lymphocyte activation. LPS or CD40 induced B cell proliferation are inhibited in nflcbl-
56 Chapter 1 Introduction
/- mice, although responses to antigen receptor stimulation are normal (Sha et al., 1995;
Snapper et al., 1996). c-Rel null mice also have proliferative defects in T and B cells and their T cells secrete reduced amounts of IL-2, TNFa, IL-3 and GM-CSF in response to antigen receptor stimulation (Gerondakis et al., 1996; Grigoriadis et al., 1996). When p65-/- bone marrow is reconstituted into SCID mice, T and B cell proliferation is impaired (Doi et al., 1997). B cell heavy chain isotype switching is defective in mice lacking nfkbl, c-Rel or p65 (Sha et al., 1995; Snapper et al., 1996). Furthermore, the lifespan of mature B cells is also regulated by c-Rel, p65 and NFkBI (Grumont et al.,
1998)
Not all defects in immune fimction originate in lymphocytes. Nfkb2-/- T or B cell proliferate normally in culture, and the failure of germinal centre formation and humoral responses in such mice is due to antigen presentation defects in these mice (Caamano et al., 1998; Franzoso et al., 1998). Macrophage activation is impaired in nfkbl-/- and c-
Rel-/- single knockout mice (Grigoriadis et al., 1996; Sha et al., 1995).
1.5.5 NFkB and embryonic development
Early knockout analysis of NFkB proteins revealed few developmental fiinctions for
NFkB, despite the central importance of the NFKB/Dorsal system in Drosophila development. P65-/- mice die at embryonic day 16 due to apoptosis in the foetal fiver
(Beg et al., 1995), however, p65-/- embryos survive when crossed onto a TNFa-/- background (Doi et al., 1999). The purpose of the high levels of TNFa in the foetal
57 Chapter 1 Introduction
liver is unclear. Nfkbl-/-, nfkb2-/- double knockout mice suffer from growth retardation and craniofacial abnormalities (Franzoso et al., 1997; lotsova et al., 1997), associated with osteopetrosis (bone thickening) due to failure of osteoclast differentiation.
Interestingly TRAF 6 mice are also osteopetrotic (Lomaga et al., 1999). Lethally irradiated mice grafted with p65-/- c-Rel-/- bone marrow are anaemic, probably because of their increased number of nucleated erythrocytes, defining a role for NFkB in erythrocyte lineage differentiation (Grossmann et al., 1999).
Ikka-/- mice die soon after birth and exhibit multiple morphological defects (Hu et al.,
1999; Li et al., 1999a; Takeda et al., 1999). Thick shiny skin prevents the emergence of limbs. Skeletal abnormalities reported include fusion of vertebrae, shortened, not fused and incompletely ossified sterna and altered skull shapes. Despite the absence of external limbs, truncated limb bones are present under the skin, however digits are fused
(Li et al., 1999a). Interestingly, defects in the mammalian homologue of twist result in similar skeletal abnormalities (el Ghouzzi et al., 1997), and twist expression is reduced in the limb buds of IKKa null mice (Takeda et al., 1999). These defects are absent from ikkb-/- mice (rescued from embryonic lethality by crossing onto a TNFRl null background) (Li et al., 1999c). Taken together these data suggest that IKKa plays a role in skin and skeletal development, which cannot be compensated by IKKp. The developmental signals responsible for the regulation of IKKa in these conditions are not yet identified.
58 Chapter 1 Introduction
Mice lacking both IKKa and IKKp die at embryonic day 13 from liver apoptosis (Li et al., 2000). However, recovered embryos revealed a failure of neural tube closure associated with increased apoptosis in neuroepithelium. Increased apoptosis was also observed in the spinal cord and dorsal root ganglia. When crossed onto transgenic mice, which carry NFkB dependent LacZ reporter gene, which drives expression of LacZ in tissues in which NFkB is active, no NFkB activation is detectable in the double mutant embryos.
Gene inactivation of the X-linked IKKy gene results in male embryonic lethality (Makris et al., 2000; Rudolph et al., 2000; Schmidt-Supprian et al., 2000), resulting from liver apoptosis. Female heterozygotes develop a skin defect 3-4 days after birth, in which skin becomes hard and inflexible, lacks hair growth and becomes scaly. Concurrently, mice are growth retarded and most females die 6-10 days after birth. Surviving female mice gradually recover, but retain skin patches without hair growth (Makris et al., 2000;
Schmidt-Supprian et al., 2000). Male lethality, and skin defects in females involving keratinocyte hyperproliferation, skin inflammation, hyperkeratosis and increased apoptosis also characterize the human genodermatosis Incontinentia Pigmenti (IP). It has recently been shown that most cases of IP are due to mutations of the IKKy locus
(Smahi et al., 2000).
1.6 Tumour Progression Locus-2
Tumour Progression Locus 2 (TPL-2) is a rat proto-oncogene, which was cloned in 1993 by virtue of the fact that insertion of Moloney Murine Leukaemia Virus (MoMuLY)
59 Chapter 1 Introduction
provirus into the TPL-2 gene confers a growth advantage upon affected cells (Makris et al., 1993; Patriotis et ah, 1993). COT, a human gene encoding a protein that is 90-95% identical to TPL-2, was identified in transformed foci induced in SHOK cells by transfection of DNA from a carcinoma cell line (Miyoshi et ah, 1991).
In TPL-2 and COT, rearrangement of the last coding exon, resulting in a truncated protein, is associated with increased transforming capacity. Overexpressed frill length
TPL-2/C0T has transforming activity in some studies (Aoki et al., 1993; Chiariello et al., 2000), however, other work indicates that truncation of TPL-2/C0T is required for transformation (Ceci et al., 1997). Pro virus insertion into the last intron of TPL-2 has several effects. Transcription of the TPL-2 gene is enhanced, stability of the resulting mRNA is greater which may result from the observed shift in promoter usage (Makris et al., 1993). Under these conditions TPL-2 mRNA terminates in the MoMuLV LTR, a truncation which may lead to increased TPL-2 kinase activity (Ceci et al., 1997). A similar truncation was found in the COT sequence recovered from transformed SHOK cells. Interestingly, a recent publication reported that 40% of human breast tumours tested had increased TPL-2/C0T expression in comparison with adjacent normal tissue
(Sourvinos et al., 1999). Furthermore, expression of truncated but not full length TPL-2 under the control of the proximal Lck promoter in T cell transgenic mice resulted in lymphomas of T cell origin (Ceci et al., 1997).
Both the TPL-2 and COT genes encode two protein species 46kDa and 52kDa, which are generated from alternative sites of initiation of translation (Aoki et al., 1993). TPL-2 is transcribed at low levels in a variety of cell types from the embryo to adulthood, with
60 Chapter 1 Introduction
highest levels in spleen, thymus and lung (Makris et al., 1993; Ohara et al., 1993).
Comparatively high levels of mRNA were also detected in various glandular cells including peptic cells in gastric glands, granular duct cells in salivary glands and goblet cells in colonic glands (Ohara et al., 1995) although the function of TPL-2 in these tissues is not clear.
The TPL-2/COT gene encodes a serine/threonine kinase, with homology to
Saccharomyces cerevisiae STEl 1 and mammalian MEKKl and NIK. The above are all members of the MAPKKK family of proteins, which are involved in the upstream regulation of the Mitogen Activated Protein Kinases (MAPKs). TPL-2/C0T and NIK also share homology with yeast MAPKKKK STE20 (M Belich and SC Ley unpublished observation). Consistent with a MAP3K function, when transfected into cells TPL-2 can activate several MAPKs, including ERK-1, INK, p38y (ERK 6 ) and ERK5 through stimulation of their upstream MAPKKs; MEK, SEK, MKK 6 and MEK5 respectively, highlighted in Diagram 3 (Chiariello et al., 2000; Salmeron et al., 1996). Based upon these data TPL-2/C0T is a highly unusual MAPKKK inasmuch as it can activate all known MAPK cascades.
Following the observation that TPL-2 mRNA is found in T cell rich organs and is induced in mitogen treated spleen cells, much analysis of its function has centred on cells of the T cell lineage. In particular, overexpression of TPL-2/C0T can increases IL-
2 production in the presence or absence of other stimuli (Ballester et al., 1997; Lin et al.,
1999; Tsatsanis et al., 1998a; Tsatsanis et al., 1998b). However, these reports differ on
61 Chapter 1 Introduction
the mechanism of action of TPL-2/C0T, which has been attributed to MAPK mediated activation of the transcription factor Nuclear Factor of Activated T cells (NFAT)
(Tsatsanis et al., 1998a; Tsatsanis et al., 1998b) or IKK mediated NFkB activation (Lin et al., 1999; Tsatsanis et al., 1998b).
In a yeast two hybrid screen using TPL-2 as bait, NFkBI p i05 was identified as a potential binding partner for TPL-2/C0T (Belich et al., 1999). This interaction has been confirmed using endogenous protein from multiple cell types (Belich et ah, 1999 and SC
Ley unpublished results). The interaction occurs through the c-termini of the two proteins, thus NFkBI p50 is unable to bind directly to TPL-2, and similarly TPL-2AC
(which corresponds to the virally truncated oncogenic variant of TPL-2) cannot bind to
NFkBI pi05. Transfection studies showed that TPL-2 can stimulate an NFkB dependent luciferase reporter gene and can increase NFkB DNA binding activity (Belich et al., 1999).
Biochemical analysis demonstrated that TPL-2 mediated NFkB activation occurs through the release ofNF kBI pi05 associated NFkB proteins. Transfection of TPL-2 increases the rate of degradation of cotransfected NFkBI p i05, without significantly changing the rate of production of NFkBI p50. TPL-2 kinase activity is required for this process. Furthermore, NFkBI p i05 lacking the glycine rich region (GRR) required for processing to p50 can be completely degraded by TPL-2 transfection, leading to
NFkB activation.
62 Chapter 1 Introduction
Taken together these data suggest that TPL-2 regulatesNF kB by controlling the degradation of NFkBI pi 05. Signal induced NFkBI p i05 degradation/processing has been widely reported and mechanisms regulating NFkBI p i05 phosphorylation and ubiquitination have been investigated. Despite this, the fimctional importance of the
NFkBI p i05 degradation pathway is unknown.
It has been proposed that the major function of NFkBI pl05 is regulation of NFkBI p50 homodimers (Heissmeyer et al., 1999), however, as NFkBI p i05 also associates with c-
Rel and p65 regulation of p50 cannot be the only purpose of NFkBI pi 05 degradation.
As NFkBI p i05 protein is the cellular source of NFkBI p50, any study wishing to investigate the function of NFkBI pi 05 as an IkB must include measures to ensure that
NFkBI p50 levels remain unchanged. A single study has investigated the inhibitory function of NFkBI pi05 in vivo. Ishikawa et al generated mice in which the NFkBI p50 coding sequence was intact but which lack sequences encoding the c-terminal
Ankyrin repeat containing domain (Ishikawa et al., 1998). Mutant mice exhibit lymphoid hyperplasia, lymphocytic infiltration into lung and liver and increased susceptibility to opportunistic infections. Macrophage and T cell cytokine production was severely impaired, B cell proliferation was increased and T cell proliferation was decreased. As some NFkB regulated genes were upregulated and some were down regulated, NFkBI p i05 positively regulates some genes while negatively regulating others. However, the gene targeting causes an alternative splicing event in NFkBI p50 mRNA, giving rise to a NFkBI p50 iso form which lacks a nuclear localisation sequence
(Ishikawa et al., 1996). Furthermore, overall NFkBI p50 levels are increased in these
63 Chapter 1 Introduction
mice, therefore the observed phenotype may not arise solely from removal of NFkBI p i05 mediated inhibition.
1.6.1 TPL-2 knockout mice
During the writing of this thesis, Dumitru et al published the phenotype of TPL-2 knockout mice (Dumitru et al., 2000). Although several of the in vitro studies described above show that TPL-2 can regulate T cell functions, TPL-2-/- T cells have a normal cell surface phenotype, proliferate normally and show normal responses to T dependent antigens (Dumitru et al., 2000). The most exciting finding of the study is that TPL-2-/- mice are resistant to endotoxic shock. TPL-2-/- macrophages cannot produce TNFa in response to LPS due to a defect in the transport of TNFa mRNA from the nucleus to the cytoplasm. The ERKl and 2 MAP kinase pathways regulate this process, and Dumitru et al demonstrate that ERKl and 2 activation in response to LPS treatment of macrophages are blocked. Thus, TPL-2 has a clear fimction in the regulation of ERKl and 2, however, as discussed below Dumitru et al only perform a preliminary analysis of
NFkB function in TPL-2-/- T cells, hopefully further experiments will clarify the role of
TPL-2 in p i05 and NFkB regulation.
1.7 The aims of this study
As a role for TPL-2 in the regulation of NFkBI pi 05 degradation has been identified in transfection experiments, the aims of this project are to understand more about the biochemical regulation and functional significance of this pathway in vivo. TPL-2
64 Chapter 1 Introduction
proteins including kinase inactive and truncated mutants, which may perturb NFkBI p i05 degradation are expressed. Several experimental systems are used which reflect
TPL-2 expression in vivo, including primary murine T cells, and transformed human T cell and monocyte cell lines. The effects of TPL-2 on different signalling pathways and some of the cellular responses they regulate are analysed.
65 CHAPTER 2 Materials and methods
2.0 Transgenic mice
2.1.1 Genotype
Tpl-2 transgenic mice expressed Tpl-2(AC), Tpl-2 or Tpl-2(R167) under the control of the VAhCD2 promoter and LCR system (Zhumabekov et al., 1995) on a C57B1/10
(BIO) background. TPL-2 mice overexpressed n-terminally myc tagged, full length, wild type TPL-2. TPL-2KD mice overexpressed myc tagged kinase inactive TPL-2, in which a lysine residue (lys 167) in the ATP binding site is substituted with an arginine residue. TPL-2AC mice overexpressed myc tagged TPL-2 which carries the c-terminal truncation equivalent to that caused by pro viral integration arginine 410.
ATG # * STpPpolyAl,A2
5KB human CD2 promoter TPL-2 5.5KB human CD2 LCR BluescriptSK(.) Myc mg
Diagram 4: The TPL-2 containing VA hCD2 transgene cassette The VAHCD2 transgenic vector used by T Ahmad and D Kioussis to generate myc TPL-2,myc TPL-2 AC and myc TPL-2KD transgenic mice is shown, ^indicates the site of TPL-2 c-terminal truncation in TPL-2 AC mice (arginine 410). indicates the site of the lysine to arginine kinase inactivating mutation in the TPL-2KD construct
66 Chapter 2 Methods
All transgenic lines were generated by T Ahmad and D kioussis, NIMR. Genotype analysis was performed by slot and southern blot analysis using protocols described in
(Ausubel F M gral., 1987).
2.1.2 Preparation of cells
Mice were killed by carbon dioxide asphyxiation. Cell suspensions from thymus, spleen and pooled lymph nodes (cervical, inguinal, mesenteric and brachial) were made in cold air buffered Iscoves Modified Dulbeccos Medium (AB-IMDM). The erythrocytes of the spleen were removed by lysis in ACK buffer (0.15M NH 4 CI, ImM KHCO3 , O.IM
NagEDTA, pH to T.2-7.4 with IN HCl) for 5 minutes at room temperature. Cells were then washed twice in AB-IMDM and resuspended in a medium appropriate for the procedure to follow.
2.1.3 Purification of T lymphocytes
T lymphocytes were purified using R&D systems Mouse T cell enrichment columns
(MTCC-500) according to the manufacturer’s instructions. This system allows enrichment of T lymphocytes by the high affinity negative selection of other cell populations. Briefly, the red blood cells from the spleen were removed by ACK lysis prior to purification. Following this, suspensions of spleen and/or lymph node cells were loaded onto columns, incubated at room temperature for 15 minutes and cells which had not been negatively selected (including T cells), were eluted. Purity of the
67 Chapter 2 Methods
resulting cell suspension was tested by flow cytometry, and was routinely 90% CD3 positive.
2.2 Stable cell lines
THP-1 cells (ECACC reference 88081201) and E6.1 Jurkat cells (ECACC reference
88042803) were obtained from ECACC. Both cell lines were maintained in complete
RPMI (Gibco 04191771M) containing 10% Foetal Bovine Serum (FBS).
TPL-2 or TPL-2(A270) containing PMT2 vectors were used to generate stable E6.1
Jurkat cell lines (Kaufman et a l, 1989). PMT2-(TPL-2), PMT2 -(TPL-2A270) or
PMT2-(empty vector) constructs were electroporated together with the selection vector
J6 -hygro. Transfected cells were cloned by limiting dilution, selected for Hygromycin
(Boehringer Mannheim B843555) resistance at 0.5mg/ml and screened for TPL-2 expression by western blotting.
HA-tagged TPL-2(A270) cDNA in the PMX vector (Ingenius MBV 040-10) (Takebe et a l, 1988), which contains an internal neomycin resistance marker, was used to generate
THP-1 stable cells lines. Cells were electroporated, cloned by limiting dilution, screened for Geneticin resistance (G418 Gibco 1181-031), and TPL-2 expression was screened by western blotting
68 Chapter 2 Methods
2.3 Lymphocyte analysis
2.3.1 Flow cytometry
Cell suspension samples for staining were plated into V-bottom 96 well plates at a density of 5x10^ cells per well. Plates were then centrifiiged in a Beckman GS - 6 at
1350rpm for 3 minutes. Incubation of antibody and secondary reagents was performed in FACS medium (Phosphate Buffered Saline without Calcium or Magnesium [PBS-
CMF] plus 0.01% sodium azide and 2% FBS) in a lOOpl volume for 40 minutes on ice.
Antibodies and secondary reagents used were as shown in Table 3:
FACS Antibody Source (hybridoma/manufacturer) TCR ap chain H57 biotin (Pharmingen) Heat Stable Antigen (HS A) YBM5.10 (NIMR) IL-2 receptor a chain (CD25) 7D4 (NIMR) CD44 IM7.8.1 (NIMR) CD69 H1.2F3 (Pharmingen) CD4 H129.19 (Sigma) CDS Clone 2.43 (NIMR) CD3e chain 2C11 (NIMR)
Table 3: Table of antibodies used in FACS analysis
For stains using a biotin conjugated primary, first layer antibodies were removed by two washes in lOOpl of PBS-azide (PBS-CMF with 0.01% azide) before incubation with
Strepdavidin Red 670 (Gibco). On completion of staining, cells were washed twice in
PBS azide and transferred to tubes for data acquisition. This acquisition was performed on a Becton Dickinson FACScan using Cell Quest software for acquisition and analysis.
69 Chapter 2 Methods
2.3.2 Proliferation Assays
Dilutions of anti- CD3 antibody (2C11), in PBS-CMF were coated onto 96 well round bottom plates for 2 hours at 37®C. Unbound antibody was then removed by three washes with PBS-CMF. Purified T cells were adjusted to 2.5x10^ cells/ml in proliferation medium (complete RPMI with 5% FBS and 0.03% 2-Mercaptoethanol) and plated out at a density of 5x10"^ cells/well. All experimental points were triplicates.
After 48 hours of culture at 37°C in 5% CO 2 , 37KBq of thymidine diluted in 25p,l proliferation medium was added to each well. Cells were incubated overnight before harvesting onto filter paper and incorporation quantified by p scintillation counting.
2.4 Protein analysis
2.4.1 Lysis procedures
Washed cells were resuspended in lysis buffer S (25mM Tris-HCl pH7.5, 1% Triton X-
100, 150mM NaCl, 20mM NaF, 90mM Na^PiO?, ImM DTT, ImM EDTA and IM
EGTA plus Antipain, Chymostatin, Leupeptin and Pepstatin A at 5pg/ml each and lOOmM NasVO#) and incubated for 15 minutes at 4°C on a rotor. Lysates were then spun at 1400rpm in a microcentrifuge for 15 minutes after which the supernatant was removed and added to an equal volume of 2X Reducing Sample Buffer (10% SDS, 20% glycerol, 10% 2-ME, 0.0625M Tris and 0.02% Bromophenol blue, adjusted to pH 6 .8 ).
Samples were boiled for 5 minutes and stored at -20®C until use.
70 Chapter 2 Methods
2.4.2 Immunoprécipitation
Antibodies were coupled to Protein A or Protein G Sepharose (PAS/PGS)(Amersham
Pharmacia) for immunopreciptiation according to the protocol described in (Harlow and
Lane, 1988). Briefly, antibodies were rotated with PAS/PGS coupling buffer (0.2M
Na2B4 0 7 , pH 9) at room temperature, after 1 hour beads were washed with coupling buffer and bound antibody was covalently attached by rotation in coupling buffer plus
20mM Dimethyl Pimelidate (Sigma) for 30 minutes. This reaction was terminated by washing and rotation in 0.2M ethanolamine, pH 8 , for 1 hour.
Lysates were precleared with lOpl uncoupled PAS/PGS plus lOpl pre immune serum, for 4 hours to overnight at 4®C. Tubes were then spun down for 2 minutes at ISOOrpm and precleared supernatants were removed. 1 ml volumes of precleared lysate were then added to lOpl packed beads per IP and rotated for 4 hours to overnight at 4°C. Beads were washed five times in cold 1 ml volumes of lysis buffer, then in 1 ml cold dHiO.
Immunoprecipitated proteins were eluted with two incubations in 25pi low pH buffer
(0.2M glycine, 0.05% NP40, pH2.5) for 2 minutes each. Eluates were neutralised with lOpl of IM TRIS pH8 , then added to an equal volume of 2X RSB and heated to 100°C for 5 minutes before loading onto a gel or storage at -20®C.
2.4.3 SDS polyacrylamide gels and Western blotting
SDS PAGE gels and Western transfer were as described (Ausubel F M gr al., 1987).
Proteins were transferred onto Immobilon-P polyvinelidene (PVDF) membranes using a
71 Chapter 2 Methods
tank system containing Cyclohexylaminopropane sulfonic acid (CAPS) at 2.2g/l at
300mA overnight. PVDF membranes were blocked with PBS-CMF with 0.05% tween
20 (PBS-tween) and 5% marvel for 1 hour at room temperature.
Primary and secondary antibodies were incubated in PBS-tween with 5% marval except where specified by the supplier. Multiple wash steps between antibody incubation periods were performed using PBS-tween. Signals were detected using the Amersbam
Pharmacia ECL system.
Target Protein Antibody source Antibody details (order number or epitope) IkB a (mouse and human) Santa Cruz C-21 (SC-371) IKKa (human) NIMR CPLGWEMERLGTGGFGN IKKp (human) NIMR CSRSPGLTTGRGGRWEM IKKy (human) Santa Cruz B-3 (SC-8032) NFkBI p i05 (human) NIMR CKKMPHQYGQEGPLEGKI NFkBI p50 (human) NIMR CMAEDDPYLGRPEQMFHL NFkBI p i05 (mouse) NIMR CNKMPGYGQEGPIEGKI NFkBI p50 (mouse) NIMR MAEDDDPYGTGQMFHLNT Phospho ERK (human) New England Bio labs #9101 Phospho JNK (human) Promega V793B Phospho P38 New England Biolabs #9211 TPL-2 NIMR DLLKHEALNPPREDQPRC Tubulin NIMR Tat-1 hybridoma
Table 4: Table of antibodies used in immunoprécipitation and western blotting
2.4.4 In Vitro Kinase assays
Following stimulation cells were lysed for 15 minutes in IVK buffer (20mM HEPES pH7.6, 240mM NaCL, 1% NP-40, ImM EDTA, 20mM p-glyceropbospbate, 20mM
PNPP, 1 0 0 pM Na 3V0 4 , ImM PMSF, ImM DTT and 5mM Benzamidine) at 5x10^ cells
72 Chapter 2 Methods
per ml, before centrifugation at 1400rpm for 10 minutes. Resulting supernatants were subjected to fiirther centrifugation at 1 0 0 ,0 0 0 xg for 1 0 minutes and precleared overnight with Protein A Sepharose beads and pre-immune rabbit serum. IKK was immunoprecipitated Avith anti-IKKy antibody for three hours, where Protein A sepharose was added for the last hour of the IP. Beads were washed four times with IVK buffer and once with kinase buffer (25mM TRIS pH7.5, 2mM DTT O.lmM Na 3V0 4 , lOmM
MgC12, 20mM PNPP plus Antipain, Chymostatin, Leupeptin and Pepstatin A at 5pg/ml each), before the kinase assay was carried out.
Kinase assays were performed in kinase buffer containing lOpM ATP and O.OlMBq y^^P-ATP in a total volume of 50pl, for 20 minutes at 30®C. GST-lKBa(l-54) was used as a substrate at 2pg per reaction. Reactions were terminated by the addition of 2X RSB and boiling for 5 minutes. Samples were run on 10% SDSPACE gels, transferred to
PVDF and exposed to film. Equal loading of GST-IxBa and the immunoprecipitated
IKKcomplex were verified by western blotting.
2.4.5 Pulse chase metabolic labelling
The protocol for pulse chase metaboHc labelling experiments is based on Current
Protocols in Protein science (Coligan et aL, 1996). THP-1 cells (10^ per experimental point) were washed and resuspended in pulse medium (RPMI without cysteine and methionine [Sigma R7513] containing 5% FBS), at 5x10^ cells/ml and incubated for 15 minutes at 37®C 5% CO 2 in order to deplete methionine and cysteine. Cells were then
73 Chapter 2 Methods
spun down and resuspended at 5xl0^cells/ml in labelling medium (pulse medium containing 0.1 mCi Promix/ml), and incubated for 20 minutes. Promix is an amino acid mixture for in vitro protein labelling containing L [^^S] Methionine and L[^^S]
Cysteine. The pulse period was terminated by removal of the labelling mix and washing with chase medium (complete RPMI containing 5% FBS). Cells were then resuspended at 10^ cells/ml and stimulated. At timepoints shown, cells were lysed in lysis buffer S.
Samples were precleared twice, before immunoprécipitation of NFkBI p i 05. IPs were run on 8 % SDSPAGE gels, which were fixed, bathed in AMPLIFY fluorographic reagent (Amersham Pharmacia) dried and exposed to film. Remaining samples were analysed by western blotting to verify that NFkBI p i 05 immunoprecipitated in different samples were equivalent.
2.5 Electrophoretic mobility shift assays
2.5.1 Preparation of cell extracts
Due to the small number of T lymphocytes available following purification mini extracts were used for bandshifting. These were prepared according to the method described by
Schreiber et al (Schreiber et al., 1989), which entails freeze thawing cell pellets three times in ME buffer containing 20mM HEPES pH 7.8, 450 mM NaCl, 0.4mM EDTA,
5mM DTT, 25% glycerol 0.5mM PMSF plus Antipain, Chymostatin, Leupeptin and
Pepstatin A at 5pg/ml each. Samples were prepared at 1-5x10^ cells per 50pl extract and stored at -70®C until use.
74 Chapter 2 Methods
2.5.2 EMSA
A 7% polyacrylamide gel was prepared in 0.5X TBE and 1% glycerol, and pre-run at
150V in 0.5X TBE and 1% glycerol for 1 hour at 4®C. The binding reaction was performed as described in (Alkalay et al., 1995) in a lOpl volume. Briefly this contained 5pi mini extract sample, lOmM HEPES pH7.9, 60mM KCl, 0.4mM DTT,
10% glycerol, 0.2pg BSA, 1.3pg poly (dldC), and NFkB probe (Promega E3291) ^^P end labeled at 10,000cpm/pl or more. The NFkB probe has been reported to detect a broad spectrum of NFkB dimer combinations (Lenardo and Baltimore, 1989). The reaction mix was incubated on ice for 30 minutes before loading onto the gel. Gels were run for 2 hours at 150V then dried onto paper and exposed to film.
2.6 RT-PCR
2.6.1 RNA extraction
5x10* stimulated THP-1 or Jurkat cells were lysed in 1ml TRIZOL (Life Technologies
15596) for 5 minutes and processed according to the manufacturers instructions.
Briefly, 200pl chloroform was added to each 1ml TRIZOL lysate, samples were shaken vigorously and incubated at room temperature for 5 minutes. Centrifugation of samples at lOOOrpm for 15 minutes at 4°C resulted in phase separation and the RNA containing aqueous phase was removed. RNA was precipitated from the aqueous phase by the addition of 0.5ml isopropanol. Samples were incubated at room temperature for 10 minutes before centrifiigation. RNA pellets were washed with 70% ethanol, resuspended in Rnase free water and adjusted to 1 pg/pl.
75 Chapter 2 Methods
2.6.2 RT-PCR
RT-PCR was performed using the Qiagen One Step RT-PCR system, which enables reverse transcription and PCR to occurs sequentially in one tube. Reaction conditions were 400pM each dNTP, 0.6pM each primer, 5u RNasin (Promega N2511), IX Qiagen
RT-PCR buffer, Ipl enzyme mix and Ipg RNA in a total reaction volume of 25pi and
0.004MBq a^^P dCTP. Thermal cycling conditions varied according to the target RNA and primer pair used.
Samples were analysed by running on a 7% polyacrylamide gel prepared in 0.5X TBE and 1% glycerol, which was dried onto paper and analysed using a STORM 680
Phosphorimager and Molecular Dynamics Image Quant analysis package.
Target Forward Primer Reverse Primer mRNA IL-2 AAA CTC ACC AGG ATG CTC AC TGT TGA GAT GAT GCT TTG AC TNFa CCA AAG TAG ACC TGC CC AGT GAC AAG CCT GTA GCC CA C-IAP-2 GTT GAA GGT TAC ATT TTA GG ATG GGC TGT AAA ATA AGA CC ICAM-1 ACT CCA GAA CGG GTG GAA GGT TCT TGT GTA TAA GCT GG CD25 ACC TGT GAA TGC AAG AGA GG GGC AGG AAG TCT CAC TCT C CD69 TCG TAG CAG AGA CA GCT CT CAC ATT CCA TGC TGC TGA C IL-ip CT GAT GGC CCT AAA CAG ATG AAG TCA AAG ATG AGG GAA AGA AGG P Actin CTC TTT GAT GTC ACG CAC GTG GGG CGC TCT AGG CAC
Table 5: Primer sequences used in RT-PCR analysis
76 CHAPTER 3 Analysis of TPL-2 function using transgenic mice
3.1 Results
Comparatively high levels of TPL-2/C0T transcription in T cell rich organs (Ohara et al., 1993; Patriotis et al., 1993) and several reports showing that TPL-2 can regulate IL-
2 production (Lin et al., 1999; Tsatsanis et al., 1998a; Tsatsanis et al., 1998b) suggest a role for TPL-2 in the T cell lineage. In order to investigate the role of TPL-2 in this cell type, transgenic mice were generated by T Ahmad in this laboratory in collaboration with Dr D Kioussis (NIMR). TPL-2 transgene expression was targeted to the T cell lineage by the use of the VAhCD2 minigene system (Zhumabekov et al., 1995). This vector contains the promoter and Locus Control Region (LCR) from the human CD2 gene and confers integration site independent, copy number dependent transgene expression in T cells from early thymopoiesis.
Three lines of mice were analysed, overexpressing; TPL-2, a kinase inactive mutant
TPL-2 (TPL-2KD) and a c-terminal truncated mutant (TPL-2AC). All transgenic proteins were myc tagged, so that transgenic TPL-2 could be distinguished from endogenous TPL-2. Phosphorimager analysis of slot blot data generated during genotyping of tail DNA suggested that heterozygous TPL-2 mice carry 2-3 copies of the transgene, TPL-2KD mice 4-5 and TPL-2 AC mice carry 5-6 copies (data not shown).
77 Chapter 3 Analysis of TPL-2 function usim transgenic mice
3.1.1 Analysis of transgenic TPL-2 expression
Expression of transgenic TPL-2 in the thymus was confirmed by western blotting
(Figure 1). The epitopes for the anti-TPL-2 antibody are conserved in all the TPL-2 constructs. The lower molecular weight ‘M30’ TPL-2 band, which is translated from a second initiation site (Aoki et al., 1993), was of equal intensity in transgenic and control samples, which may indicate that transgenic TPL-2 is translated primarily from the mye initiation site. Comparison of the intensity of transgenic and non transgenic TPL-2 bands using Image 1 software (Biorad) showed that overexpression of myc-TPL-2 relative to endogenousTPL-2 was 3 fold in TPL-2 mice, 8 fold in TPL-2KD mice and 16 fold in TPL-2AC mice, consistent with predicted transgene copy numbers. Transgenic
TPL-2 was also detected in the spleen of all transgenic lines (data not shown).
mycTPL-2\ 1 2 3 4 A TPL-2__ M30 TPL-2. TPL-2AC/ ^ IB* TPL-2
g NpKBlplOS
Tubulin D
Figure 1: Expression of transgenic TPL-2
The expression of transgenic TPL-2 was determined by western blotting of thymus samples from r/ane 7), 77^L-2 (Aine 2) (Awne j) fTL/O) cofüro/
(lane 4). The same blot was reprobed for NFkBI pl05 (B), NFkBI p50 (C) and tubulin (D). Predicted molecular weights are TPL-2; 55kDa, myc-TPL-2; 56kDa, M30 TPL-2; 46kDa and TPL-2 AC; 43kDa.
78 Chapter 3 Analysis of TPL-2 function usins tramsenic mice
1 2 3 4 A — — NFKBlplOS mycTPL-2v
g T PL -2------► _ TPL-2 M30 TPL-2/Ig ► ##
Figure 2: Association of transgenic TPL-2 with NFkBI pi 05
NFkBI pl05 was immunoprecipitated from whole thymi from TPL-2 (lane 1), TPL-2KD (lane
2), TPL-2 AC (lane 3) and control BIO mice (lane 4). Samples were western blotted for NFkBI pi05 (A) and the association of TPL-2 (B). Different quantities of NFkBI p i 05 immunoprecipitated reflect different sizes o f thymi. Any NFkBI pl05 associated TPL-2AC cannot be visualised due to the presence o f the Ig heavy chain.
In vitro data from several eell lines and yeast two hybrid screens shows that TPL-2 associates with NFkBI pi 05 (Belich el al., 1999). Immunoprécipitation of NFkBI pi 05 from the thymus of a BIO mouse confirmed that endogenous TPL-2 interacts with
NFkBI pi05 in vivo (Figure 2). Furthermore, transgenic (mye) TPL-2 and TPL-2KD also co-immunoprecipitated with NFkBI p i05. Endogenous TPL-2 was present in immunoprecipitates from transgenic thymi indicating that transgenic TPL-2 had not displaced endogenous TPL-2. It was not possible to determine whether mycTPL-2AC or endogenous M30 TPL-2 is associated with NFkBI p i05 from this data due to the presence of the immunoglobulin heavy chain at the same molecular weight. However, previous studies in this laboratory indicate that M30 TPL-2 associates efficiently with
NFkBI p i05 whereas TPL-2AC does not (Belieh et al., 1999).
79 Chapter 3 Analysis o f TPL-2 function usins transsenic mice
3.1.2 Analysis of T cell development in transgenic mice
T cell development takes place in the thymus, where the differentiation of bone marrow derived precursors can be followed by analysis of the expression of cell surface markers including CD4 and CD 8 , (reviewed in Janeway, 1999). Precursor cells first develop to a
CD4-CD8- double negative stage, during which the P chain of the T cell receptor must be rearranged, expressed and functionally selected. Progression of cells that have successfully undergone p selection to the CD4+CD8+ double positive stage is accompanied by rearrangement of the TCRa chain gene. During the thymic selection process, thymocytes bearing receptors with intermediate affinity for thymic ligand survive, whereas those with low affinity fail to be positively selected and die by neglect, and those with high affinity receive a strong TCR mediated signal and die by instruction
(negative selection). Both death by neglect and by instruction occur by apoptosis.
Positive selection of thymocytes is associated with differentiation of CD4+CD8+ double positive cells into CD4+ or CD 8 + single positive thymocytes.
A recent study by Voll et al shows that NFkB is a survival signal for cells undergoing p selection (Voll et al., 2000), since transgenic mice expressing dominant negative IicBa have reduced numbers of p selected double negative thymocytes. Evidence that NFkB regulates the apoptotic death of double positive thymocytes comes from the observation that thymocytes expressing IxBa-DN are resistant to apoptosis induced by anti-CD3 antibodies (Hettmann et ah, 1999). These mice also display a specific reduction in CD 8 single positive thymocytes and splenocytes (Boothby et al., 1997; Hettmann et al..
80 Chapter 3 Analysis o f TPL-2 function usins transsenic mice
1999), which may reflect different roles for NFkB in CD4 and CDS cells in development and survival
In the light of evidence for the importance of NFkB in T cell development, we tested whether thymocyte development was altered in TPL-2 and TPL-2KD mice. As shown in Figure 3 the overall numbers of thymocytes in TPL-2 and TPL-2KD mice and the relative proportions of CD4 and CDS single positive and CD4/CDS double positive were not significantly different (Mann Whitney U Test P >0.05). Furthermore, expression of the T cell receptor, HSA, CD25, CD44 and CD69, which are differentially expressed during thymopoiesis, was very similar to that of control mice (data not shown).
There was no significant change in peripheral T cell numbers in TPL-2 and TPL-2KD mice (Mann Whitney U Test P >0.05, Figure 4), and the peripheral T cells of both transgenic lines showed normal CD4 to CDS cell ratios. Furthermore, expression of the
T cell receptor, and activation markers CD25, CD44 and CD69 was normal on transgenic T cells, (data not shown). Thus, TPL-2 or TPL-2KD transgene expression had no detectable effect on the thymocyte development or mature T cell phenotype of these mice.
81 Chapter 3 Analysis of TPL-2 function usins transsenic mice
Ai Ail
- . -..T. ■■ CD4 ■■ •
- - . . ; .... - ...... 10'* -Jo CD8 Bi Bii
S f- • A/ CD4
■■ ______c
Thymus +/-SEM and %CD4 single %CD8 single % CD4/CD8 double cellularity (N value) positive cells positive cells positive cells (xIO*) ______(+/-SEM)______(+/- SEM) (+/- SEM)______Control mice 2.04 0.1 (n=l3) 10.2 (+/- E3) 4.3 (+/- 0.3) 7&8 (+AE6) TPL-2KD 1.77 0.2 (n=7) 8.9 (+ /-2 .I) 3.3 (+/- 0.4) 81.4 (+/-I.3) TPL-2 2.22 0.4 (n=9) 13 (+/- 1) 4.8 (+/-0.9) 75.8 (+Z-3.2)
Figure 3: Thymocyte development in TPL-2 and TPL-2KD transgenic mice
Thymocytes from TPL-2 (Ai) and littermate (Ail) and TPL-2 KD (Bi) and littermate (Bit) mice were stained with anti-CD8^'^‘^ and anti-CD4^^ antibodies and analysed by flow cytometry. (C)
Total numbers of thymocytes in transgenic and control mice are shown + / - SEM. Mean percentages of cells in CD4 and CDS single positive and CD4/CD8 double positive populations are shown (+/- SEM). . The Mann Whitney U Test was performed using Statview software and indicated that differences in thymocyte numbers and subset proportions were not significant at the 5% level.
82 Chapter 3 Analysis of TPL-2 function usins tramsenic mice
Ai Ail
CD4
► CDS Bi Bii
— :
m
> CD8 c Splenic cellularity +/-SEM and %CD4 single %CD8 single positive (xIO?) (N value) positive cells cells (+/-SEM) (+/- SEM) Control mice 3.3 1.5 (n=13) 17.5 (+/-1.6) 9.58 (+/-0.8) TPL-2KD 4.2 1.2 (n=7) 16.9 (+/-1.09) 7.8 (+/-0.6) TPL-2 3.2 0.3 (n=9) 16.6 (+/-1.68) 8.8 (+/-1.2)
Figure 4: Peripheral T cells inTPL-2 and TPL-2 KD transgenic mice
Splenocytes from TPL-2 (Ai) and littermate (Aii) and TPL-2 KD (Bi) and littermate (Bii) mice were stained with anti-CD^'*‘' and anti-CD4^^ antibodies and analysed by flow cytometry. (C) Total numbers o f splenocytes (after erythrocyte lysis) in transgenic and control mice are shown + / - SEM. Mean percentages of CD4 and CDS single positive cells are shown + / - SEM. The Mann Whitney U Test was performed using Statview software and indicated that these differences were not significant at the 5% level.
83 Chapter 3 Analysis of TPL-2 function usins transsenic mice
3.1.3 Functional analysis of transgenic T cells
T cells are activated upon interaction with the MHC bound peptide antigen for which they are specific (cognate antigen) in conjunction with coreceptor stimulation via CD28,
( reviewed in Dustin and Chan, 2000). Among the first consequences of activation are the secretion of IL-2 (Fraser et al., 1991) and upregulation of CD25, which is a component of the IL-2 receptor, (reviewed in Minami et al., 1993) . These events lead to an autocrine induction of T cell proliferation leading to an increased pool of antigen specific T cells that can then differentiate into effectors.
Stimulation of the IL-2 promoter depends on the activation of several transcription factors, (reviewed in Crabtree and Clipstone, 1994; Jain et al., 1995), including Nuclear
Factor of Activated T cells (NFAT), OCT-1 and NFkB. NFkB regulates not only the kB enhancer element of the IL-2 promoter which lies at -206 to -195 (Hoyos et al., 1989) but also the CD28 responsive element (CD28RE) which lies at -106 to -152 (Fraser et al., 1991). Consistent with this, c-Rel null T cells cannot produce IL-2 and do not proliferate after TCR ligation (Gerondakis et al., 1996). The CD25 (IL-2 receptor a chain) promoter also contains a functional NFkB site (Kuang et al., 1993). Previous data, and transfection studies have demonstrated that TPL-2 can regulate IL-2 production in T cell lines (Tsatsanis et al., 1998a; Tsatsanis et al., 1998b). This prompted us to analyse proliferation and IL-2 production in the TPL-2 and TPL-2KD mice (Figures 5 and 6).
84 Chapter J Analysis of TPL-2 function usins transsenic mice
Ai Aii
o 100 I 100 (I) = 80 control control TPL-2 TPL-2-KD
20: L 20--
0.001 0.01 0.1 1 10 0.001 0.01 0.1 1 10 2C11 (Mg/ml) 2C11 (Mg/ml) Bi Bii I 120 120 Ï100 1 100 o 80 control control TPL-2 T P L -2 -K D
4 0
20 -
0.001 0.01 0.1 1 0.001 0.01 0.1 1 2C11 (pg/ml) 2C11 (Mg/ml)
Figure 5: Proliferation of TPL-2 and TPL-2 KD T cells
(A) Proliferation of TPL-2 (red lines) and littermate control (blue lines) in response to plate bound anti-CDS antibody alone (Ai) or anti-CD3 and anti-CD28 (5pg/ml) antibodies (Ail). (B) Proliferation of TPL-2KD T cells (red lines) and littermate control (blue lines) in response to anti-CD3 (Bi) and anti-CD3 plus anti-CD28 antibodies (Bii). Data is expressed as % of maximum stimulation in that experiment. Experimental points and error bars indicate mean + / - SEM of three mice
Plate bound anti-CD3 antibodies (2C11) were used to mimie TCR stimulation. In highly purified T cell populations, stimulation of the TCR-CD3 complex in the absence of costimulation does not cause activation, (reviewed in Mueller et al., 1989). As the purification method used in these experiments yielded T cells at 90% purity, it is likely that accessory cells remaining in these cultures provided some costimulation. Addition of anti-CD28 antibody to act as a costimulus did increase proliferation further.
85 Chapter 3 Analysis o f TPL-2 function usim transsenic mice
Proliferation of TPL-2 and TPL-2KD transgenic T cells was found to be similar to controls, in response to stimulation with CD3 alone and also CD3/CD28 costimulation
(Figure 5). Consistent with these data, levels of IL-2 production by the transgenic T
cells were also similar to control T cells (Figure 6).
T cells costimulated with anti-CD3 and anti-CD28 antibody also produce TNFa, which
can function in concert with CD3 to stimulate proliferation (Yokota et al., 1988). TNFa
production is regulated at both transcriptional and post-transcriptional levels. Many
response elements have been implicated in TNFa transcriptional regulation, including
NFkB, NFAT, AP-1, AP-2, SP-1 and CRE. The JNK, ERK and P38 MAP kinase
pathways also participate in post-transcriptional regulation of TNFa production, through
the control of mRNA stability, localisation and translation (Dumitru et al., 2000;
Kontoyiannis et al., 1999; Kotlyarov et al., 1999). A transfection study has shown that
TPL-2 can regulate TNFa production in T cell lines, although in this study the
biochemical mechanism underlying the effect was not established (Ballester et al.,
1998). However, in T cells from TPL-2 and TPL-2KD transgenic mice, TNFa
production was found to be similar to controls (Figure 6).
Whilst the absolute quantities of cytokines produced due to inter experimental variation,
the relative amounts of cytokines produced by transgenic and control animals remained
consistent. Thus, TPL-2 does not appear to play a role in TCR induced increases in IL-2
or TNFa production.
86 Chapter 3 Analysis of TPL-2 function usim tramsenic mice
Ai Aii 2500 _ 3 5 0 f 2000 E 300 2 2 5 0 21 5 0 0 a 200 L J 1000 ^ 150 ° TPL-2 500 •“ 100 50 ° Control
0 CO CO CO CN TO Q a Q Q 3 O 13 Ü E Q E w O Q c3 O Bi Bii 5000 300 f 4000 1^250 3 3 0 0 0 0,200 3 2000 f l 5 0 °TPL-2-KD 100 "control 1000 ^ »- 50 a 0 — CO BTO CO Q 3 Q E O N o c Ü 3
Figure 6: TNFa and IL-2 production by TPL-2 and TPL-2 KD T cells
T cells purified from pooled spleen and lymph node cell suspensions were plated onto 24 well plate at 10^ cells/well, and stimulated as labelled with plate bound anti-CD3 antibody (2C11 at
Ipg/ml) + / - anti-CD28 (37.51 at 5jug/ml) antibody. After 24 hours, supernatants were removed and assayed for IL-2 and TNFa by ELISA. Production o f IL-2 (Ai) and TNFa (Aii) by TPL-2 and control cells and IL-2 (Bi) and TNFa (Bii) by TPL-2KD and control cells are shown. Data
shown indicates mean + / - SEM >4 mice per group from representative experiments.
3.1.4 Analysis of NFkB activation in transgenic T cells
Following engagement of the TCR-CD3 complex and coreeeptor stimulation, multiple
biochemical cascades activate many different transcription factors including NFkB, AP-
87 Chapter 3 Analysis of TPL-2 function usins transsenic mice
1 and NFAT, (reviewed in Crabtree and Clipstone, 1994). Nuclear NFkB complexes
induced by T eell activation are predominantly p50/p50 homodimers, p50/p65 heterodimers and p50/c-Rel heterodimers (Hettmann et al., 1999). In electrophoretic
mobility shift assay (EMSA) supershift experiments performed on stimulated T cells
from control (BIO) mice, the predominant complexes observed were p50/p50 and
p50/p65 (Figure 7). In these experiments, no complex which could be reprodueibly
supershifted with an anti-e-Rel antibody was observed.
EMSAs were performed to test whether overall NFkB activation was altered in TPL-2 or
TPL-2KD T cells (Figure 7). Purified T cells were stimulated over a 9 hour timeeourse
because NFkBI p i05 is degraded with a half life of 1-2 hours in other cells types and so
is unlikely to regulate acute NFkB activation (Belieh et al., 1999; Harhaj et al., 1996).
A small increase in DNA binding was seen in all samples following stimulation with
anti CD3 and CD28 antibodies. However, no reproducible defects in NFkB binding
were observed in TPL-2 or TPL-2KD mice compared with control cells (Figure 7).
Acute stimulation of NFkB by CD3 and CD28 eostimulation resulted in a greater
induction of NFkB activation than seen in the long timeeourse experiments. However
no reproducible defects in acute NFkB activation were noted in the TPL-2 and TPL-
2KD T cells (data not shown). Chapter 3 Analysis of TPL-2 function usins transsenic mice
(U IT) VIo Cu CL A a *
p5(Vpi65 * P50/p50
TPL-2KD control B 0 0.5 3 6 9 0 0.5 3 6 9 hours
N FkB
Oct 1
TPL-2 control C 0 0.5 3 6 9 0 0.5 3 6 9 hours
NFkB
* - * Oct 1
Figure 7: NFkB activation in TPL-2 and TPL-2KD T cells
(A) Purified T cells were stimulated and NFkB activation analysed by EMSA using a
radiolabelled kB oligonucleotide probe. The constituents o f stimulated NFkB complexes were tested by supershift analysis in which extracts were preincubated with antibodies: irrelevent serum, anti-p65 antibody, anti-c-Rel antibody and anti-p50 (4). Supershifted complexes are labelled with *. Purified Tpl-2 and control T cells (B) and TPL-2KD and control T cells (C) were costimulated for the times shown with plate bound anti-CDS (2C11 at lOpg/ml) and anti-CD28 (37.51 at
lOpg/ml) antibodies and NFkB activation analysed by EMSA. Oct I DNA binding activity was used to control protein loading.
89 Chapter 3 Analysis of TPL-2 function usins transsenic mice
3.2 Discussion of Chapter 3
3.2.1 TPL-2 and NFkB in T cells
TPL-2 is transcribed in T cell rich organs (Makris et al., 1993), and several in vitro
studies have shown that TPL-2 can regulate T cell functions such as IL-2 and TNFa production (Ballester et al., 1998; Lin et al., 1999; Tsatsanis et al., 1998a; Tsatsanis et al., 1998b). Based on transfection studies this laboratory has shown that TPL-2 can regulateNF kB by promoting the proteolysis ofNF kBI pi 05 (Belich et al., 1999).
Important functions for NFkB have been demonstrated in most cell lineages of the
immune system,(reviewed in Gerondakis et al., 1999). Specifically, during thymic
development, NFkB is involved in the processes of P selection (Voll et al., 2000),
thymocyte apoptosis (Hettmann et al., 1999) and the development or survival of CD8
cells (Boothby et al., 1997; Hettmann et al., 1999; Voll et al., 2000). Genetic evidence
shows that mature T cell functions such as proliferation, IL-2 production and Th2
differentiation are also regulated by NFkB (Das et al., 2001; Gerondakis et ah, 1996).
3.2.2 Measurement of NFkB I pi 05 degradation in transgenic T cells
The experiments shown in Chapter 3 did not pinpoint any phenotypic differences
between TPL-2 and TPL-2KD mice and controls in terms of thymic development, the
cell surface markers of peripheral T cells, IL-2 and TNFa production and T cell
proliferation. Our model for the mechanism of action of TPL-2 predicts that
exogenously expressed TPL-2 may interfere with NFkBI p i05 degradation, therefore
90 Chapter 3 Analysis o f TPL-2 function usim transsenic mice
analysis of NFkBI p i05 degradation in TPL-2 transgenic T cells would be very
informative. At the time these experiments were performed an antibody against mouse
NFkBI p i05 was not available. N- and c- terminal 17mer immunising peptides were
designed and antisera which specifically recognise n- and c-terminal fragments from
mouse NFkBI p i05 have now been generated and were used in Figure 2. Resynthesis
of NFkBI p i05 after stimulation prevents analysis of signal induced NFkBI p i05
degradation by western blotting. This problem can be overcome by the use of protein
synthesis inhibitors or pulse chase metabolic labelling techniques, but both approaches
may be technically difficult in primary T cells or thymocytes. In order to avoid some of
these difficulties, T cells lines were used in further studies, presented in Chapter 6.
If NFkBI p i05 degradation is normal in the transgenic T cells, this could mean that
TPL-2 does not regulate NFkBI pi05 degradation in vivo. However, there are also
several experimental reasons why NFkBI p i05 degradation could be unaffected. TPL-
2KD is expressed in addition to endogenous TPL-2, in order to function as a dominant
negative TPL-2KD must presumably compete with endogenous TPL-2 for access to
substrates and regulators. Although transgenic TPL-2KD associates with NFkBI p i05,
it does not displace endogenous TPL-2 (Figure 2). This endogenous TPL-2 may be
sufficient to phosphorylate target proteins to physiological levels. Generation and
analysis of transgenic mice expressing higher levels of TPL-2KD may reveal whether
this is the case.
91 Chapter 3 Analysis of TPL-2 function usim transsenic mice
Although it was not possible to analyse NFkBI p i05 degradation in the transgenic T cells directly, EMSAs showed that there were no gross alterations in NFkB DNA binding in TPL-2 or TPL-2KD transgenic T cells (Figure 7). IkBœ degradation and
NFkB activation have been reported to occur in response to CD3 and CD28
costimulation (Tong-Starksen et a l, 1987; Verweij et al., 1991), however, when this
work was carried out, it was not known whether CD3 and CD28 eo stimulation stimulate
NFkBI p i05 degradation. Thus, whether the T Cell Receptor utilises the TPL-2/NFkB1
p i05 pathway remains to be investigated. This question is addressed in Chapter 5.
It is not known what contribution NFkBI pi05 degradation makes to total NFkB
activation under endogenous conditions. However, in transiently transfected cells TPL-
2KD expression reduces the NFkB activation detectable by EMSA (Belich et al., 1999).
Furthermore, studies expressing IxBa-DN in transgenic mice and cell lines show that
significant NFkB activation arises from non iKBa associated cytoplasmic NFkB
complexes (Hettmann et al., 1999 Heissmeyeret al., 1999). The nuclear NFkB dimers
which persist when IkBœ degradation is blocked are predominantly p50 homodimers,
which is consistent with the reported low affinity of iKBa and IkBP for p50 homodimers
(Nolan et al., 1993). These data support a model in which NFkBI p i05 degradation
leads to the release of p50 subunits, which dimerise and translocate to the nucleus.
These homodimers may function as transcriptional repressors or in association with
other factors such as BCL3 (Watanabe et al., 1997) or C/EBP (Cha-Molstad et al., 2000)
as transcriptional activators. However, this hypothesis is complicated by the fact that
92 Chapter 3 Analysis o f TPL-2 function usins transsenic mice
NFkB I p i05 also binds to the transactivating Rel subunits c-Rel and p65, which are also regulated by IkB œ, IkBP and IkB s .
Binding of NFkB proteins to kB sequences arises from the degradation not just of
NFkBI p i05, but also degradation of iKBa , IkBP and in some circumstances IkBe.
Thus, if NFkBI pi 05 degradation regulates only a subset of NFkB activation detectable by EMSA, any defect in NFkBI p i05 regulation may not fall within the limits of the resolution of the assay.
Heissmeyer et al developed an EMSA assay for the quantification of BCL3 associated p50 homodimers. Since these complexes arise when IkB degradation is blocked they believe that BCL3 associated homodimers are regulated by NFkBI p i 05 degradation and that the BCL3 IP-EMSA assay is a useful experimental readout for NFkBI p i 05 degradation (Heissmeyer et al., 1999). In order to use this technique to quantify any changes in BCL3 associated p50 homodimers arising from TPL-2 expression, preliminary experiments were performed. None of the commercially available BCL3 antibodies worked in IP-EMSAs or conventional immunoprécipitation and western blotting. An anti-BCL3 antiserum is currently being generated in this laboratory. When this becomes available BCL3 IP-shift assays can be performed and the effects of TPL-2 on BCL3 associated p50 homodimers can be tested.
93 Chapter 3 Analysis o f TPL-2 function usins transsenic mice
3.2.3 Does TPL-2 function in T cells?
During the writing of this thesis, the phenotype of TPL-2 knockout mice was published.
These mice have a major defect in the production of TNFa by macrophages, which is ascribed to defective ERK activation by LPS (Dumitru et al., 2000). However, no T cell phenotype was identified in these TPL-2 knockout mice, despite a detailed analysis of thymocyte and T cell surface markers, T dependent antigen responses, T cell cytotoxic responses, proliferation and TNFa and IL-2 production. This study confirms data in the present report suggesting that TPL-2 does not function, or has a redundant or
(unidentified) highly restricted function in T cells. This raises questions about the interpretation of analysis of TPL-2 tissue distribution (Makris et al., 1993), which shows transcription of TPL-2 in T cell rich organs such as thymus and spleen. Critically,
Makris et al do not identify which cells within these organs transcribe most TPL-2, candidate cell types could include T cells, B cells, macrophages or dendritic cells. The splenocyte and macrophage TNFa production defects of TPL-2 knockout mice show that TPL-2 has a critical function in other immune cells found in these organs (Dumitru et al., 2000). Thus, transgenic mouse lines expressing TPL-2KD in B cells or macrophages may have shown a phenotype. Furthermore, since knockout studies have identified functions for TPL-2 in these cells types, the effectiveness of the TPL-2KD transgene as a dominant negative in vivo could be verified.
It is possible that both this study and Dumitru et al have not stimulated T cells under conditions in which a TPL-2 fonction would be revealed. The induction of TPL-2 mRNA occurring in splenocytes treated with the T cell mitogen Concanavalin A
94 Chapter 3 Analysis o f TPL-2 function usins transsenic mice
suggests that TPL-2 is present in increased quantities after initial T cell activation
(Patriotis et ah, 1993). Therefore TPL-2 may regulate T cell functions occurring after activation, e.g. differentiation, effector function or cell death. Levels of many receptors, such as TNFRl and 4-IBB are similarly induced by T cell activation (Gravestein and
Borst, 1998), thus TPL-2 may participate in signalling in response to these receptors.
Establishing T cell cultures, activating the cells to induce receptors and then restimulating, as Pfeffer et al did when analysing TNF receptor knockout T cells (Pfeffer et ah, 1993), would enable this hypothesis to be tested.
The absence of a clear T cell phenotype in TPL-2KD transgenics (this study) and TPL-2 knockout mice could also be explained by a compensatory mechanism. It is also possible that while TPL-2 performs a non-redundant function in LPS signalling in macrophages, another kinase can compensate for the absence of TPL-2 in T cells. This occurs in the case of the src family kinases Ick and fyn(reviewed in Cheng and Chan,
1997). Lck null mice have an incomplete block in thymic development (Molina et ah,
1992), whereas thymopoiesis in fyn null mice occurs normally (Stein et ah, 1992).
However, inactivation of fyn in lck-/- mice causes more severe defects in thymopoiesis
(Groves et ah, 1996). Analysis of mice with multiple inactivated genes, e.g. TPL-2 and its relative NIK may address whether a similar situation exists in TPL-2-/- cells.
95 CHAPTER 4 TPL-2AC mice
4.1 Results
As described above TPL-2AC transgenic mice have been previously generated in this laboratory. Analysis by T Ahmad showed that these mice develop lymphomas and die
12-16 weeks post-natally, see below. This chapter contains a limited continuation of the analysis of these mice carried out while researching this project. Both TPL-2 and COT were cloned during screens designed to identify genes associated with oncogenesis
(Emy et a l, 1996; Miyoshi et al., 1991; Patriotis et al., 1993). An oncogenic role for
TPL-2 in vivo is supported by reports that 40% of breast cancers tested overexpress
TPL-2 (Sourvinos et al., 1999), and in situ hybridisation studies showing COT overexpression in tumours from gastric and colonic glands (Ohara et al., 1995).
Generation of TPL-2, TPL-2KD and TPL-2AC transgenic mice enabled the Ley laboratory to examine TPL-2 tumourigenicity in vivo. While this work was in progress a similar study was published by another group, using the proximal Lck promoter to drive
TPL-2 or TPL-2AC expression in thymocytes (Ceci et al., 1997). Results generated were essentially similar. TPL-2AC transgenic mice develop metastatic lymphoblastic lymphomas and die 12-16 weeks post-natally (T Ahmad and SC Ley unpublished data and Ceci et al., 1997). Neither group observed tumours in transgenic mice expressing
96 Chapter 4 Analysis of TPL-2 AC transsenic mice
full length TPL-2. Analysis of 12 week old TPL-2AC transgenic mice by T Ahmad revealed grossly enlarged thymi, causing distension of the rib cage and compression of the heart and lungs. Spleen and lymph nodes were enlarged and histological analysis showed widespread infiltration of transformed cells into other tissues, (T Ahmad and SC
Ley unpublished data).
4.1.1 Analysis of thymocytes and T cells from TPL-2AC mice
In the light of the data described above, we chose to investigate whether TPL-2AC had any effects on thymocytes or mature T cells before the development of tumours. Flow cytometric analysis of thymus and spleen from TPL-2AC mice at 4,6 and 8 weeks post- natally showed that a difference between TPL-2AC and control mice is first visible in the thymus 5-6 weeks post-natally. Figure 8, although there is no significant difference in thymic cellularity in mice of this age (Figure 8C). At this time a CD4‘"VCD8”’* population of cells, expressing uniformly low levels of TCR and high levels of CD25 and HSA develops. In week 8 thymi, this cell population predominates relative to CD4 and CD8 single positive cells in all mice examined. Since this ‘extra population’ of cells is not detectable before 6 weeks it is unlikely to represent a block in differentiation arising from the presence of the transgene. This study does not establish whether this
‘extra population’ of cells represents the early stages of tumours seen in older mice, although this could be tested by injection of cell suspensions from this ‘extra population’ and monitoring the recipient mice for the development of tumours. Advanced tumours are phenotypically CD4 or CD8 single positive or CD8/CD4 double positive (T Ahmad and SC Ley unpubhshed data) demonstrating that if the ‘extra population’ observed in
97 Chapter 4 Analysis of TPL-2 AC transsenic mice
TPL-2AC thymi are early tumour eells, further ‘T cell type’ differentiation can still occur. Although tumour development demonstrates that transgenic TPL-2AC is biologically active, thymocyte development occurring before 4 weeks post-natally is normal, in terms of CD4, CD8, CD25, CD44, TCR and HSA expression Figure 8 and data not shown.
A TPL-2AC
i 11 111
^ : .a CIMe
.o' ...... If ...... ,1^ ------► CDS B Age matched littermates
i ii 111
CD4= 0
" ' .:3 • • -► CDS C Thymus Cellularity (xIO ) +/- SEM and (N value) Control mice 2.04 0.1 (n=l3) TPL-2AC mice 2.05 0.09 (n-12)
Figure 8: Thymocytes in TPL-2AC transgenic mice Thymocytes were stained with anti-CDS^"^^ and anti-CD4^ antibodies and analysed by flow cytometry Thymocytes from TPL-2 AC mice at 4 (Ai), 6 (Aii) and 8 weeks (A Hi) are shown and above corresponding littermate thymocytes (BI, Bii and Biii). Representative FACS plots from multiple experiments are shown. Total numbers of thymocytes in transgenic and control mice at
4-7 weeks are shown + / - SEM (C).
98 Chapter 4 Analysis of TPL-2 AC tramsenic mice
A TPL-2AC i 111
CD#
f - f ...... ^ ...... io -► CD8
B Age matched littermates i ii
CD# 4 ; ■ % ■ ...... ^ ■ ‘ ■ , ...... -► CD8 c
Spleen cellularity (x 10^) +/- SEM and (N value) Control mice 3.26 0.5 (n=12) TPL-_2AC mice 3.4 0.3 (n=12)
Figure 9: Splenocytes in TPL-2AC transgenic mice
Splenocytes were stained with anti-CD^”"^ and anti-CD4^ antibodies and analysed by flow cytometry Splenocytes from TPL-2 AC mice at 4 (Ai), 6 (Aii) and 8 weeks (Aiii) are shown above corresponding littermate splenocytes (BI, Bii and Biii). (C) Total numbers of splenocytes after erythrocyte lysis in transgenic and control mice (4-7 weeks) are shown +/- SEM
99 Chapter 4 Analysis ofTPL-2AC transsenic mice
Before 9 weeks post-natally, there is no evidence of transformed cells in the spleen or lymph nodes, (Figure 9), indicating that this occurs later. Peripheral TPL-2AC T cells analysed at this time have a normal phenotype (Figure 9), and proliferate normally after
CD3 ligation (Figure 10). Since tumour development requires the accumulation of multiple mutations, it is likely that in the absence of additional genetics defects, TPL-
2AC cannot regulate proliferation or differentiation of transgenic T cells.
120
100 TPL-2AC c o Control *2 3 2 60 a . (0X E 40 5? 20
0.001 0.01 0.1 1 2C11(ng/ml)
Figure 10: Proliferation of TPL-2AC T cells
Proliferation of TPL-2AC (blue lines) and littermate control (red lines) in response to plate bound anti-CD3 antibody is shown. Data is expressed as % of maximum stimulation in that experiment after subtraction of proliferation in the absence o f exogenous stimuli. Experimental points and error bars indicate mean +/-SEM of three mice
100 Chapter 4 Analysis ofTPL-2AC tramsenic mice
4.2 Discussion of Chapter 4
4.2.1 What are the possible mechanisms of TPL-2 oncogenesis?
Since TPL-2/C0T can participate in several signalling pathways, there are several potential mechanisms for the oncogenic activity of TPL-2/COT. We and others have shown that TPL-2 can activate NFkB, and there is much evidence for deregulated NFkB activity in oncogenesis, (reviewed in Rayet and Gelinas, 1999). Several viruses activate
NFkB as part of their transformation mechanism, e.g. Human T Cell Leukaemia Virus type 1 (HTLV-1), Epstein-Barr Virus (EBV) and the avian Reticuloendotheliosis Virus
(Rev-T) (Gilmore, 1999; Cahir McFarland et al., 1999; Sun and Ballard, 1999).
Chromosomal rearrangement or overexpression of NFkB or IkB genes have been widely reported in haematopoietic and solid tumours, including BCL3, c-Rel, iKBa, NFkBI and
NFkB2, (reviewed in Rayet and Gelinas, 1999). The mechanism of action of NFkB in oncogenesis is likely to be NFkB proteins escaping normal regulatory constraints and entering the nucleus, where they chronically promote expression of genes that enable oncogenesis. NFkB induces expression of several apoptosis inhibitors, such as c-IAPl and 2, TRAFl and BCL-2 homologues Bcl-X and Bfl-l/Al (reviewed in Barkett and
Gilmore, 1999). Additionally, NFkB regulates growth factor production and cell cycle progression, by the regulation of genes such as c-myc and cyclin D1 (Duyao et al., 1990;
Guttridge et al., 1999; La Rosa et al., 1994). NFkB may also regulate later stages of oncogenesis, by the regulation of genes required for invasion and metastasis e.g. VEGF, the matrix metalloproteases and ICAM-1 (Pahl, 1999).
101 Chapter 4 Analysis o f TPL-2 AC transsenic mice
Overexpressed TPL-2 can function as a MAPKKK in several different pathways, resulting in the activation of several MAP Kinases including ERKl, JNKl, P38y and
ERK-5 (Chiariello et aL, 2000; Salmeron et al., 1996). The MAPK pathways transduce signals from a wide variety of stimuli, including growth factors, mitogens, and cellular stress, by regulating transcription, translation and effector protein activity, (reviewed in
Schaeffer and Weber, 1999). Inappropriate activation of MAPK pathways is the proposed mechanism of action of several classes of oncogenes. The v-erbB oncogene from the Avian Erythroblastosis Virus is a mutant EGF receptor, which stimulates
MAPK signalling. The Harvey Sarcoma Virus oncogene V-Ha-ras is closely related to human c-Ha-ras.
Activating mutations in the MAPKK MEKl, which is a substrate for TPL-2, are sufficient for transformation in tissue culture cells (Cowley et aL, 1994; Mansour et aL,
1994). Transformation by the serine threonine kinases Raf and Mos also involves MEK or ERK (Khosravi-Far et aL, 1995) (Kyriakis et aL, 1992; Okazaki and Sagata, 1995).
Several lines of evidence suggest that the INK pathway plays an important role in tumour cells. Ras induced transformation requires c-Jun (Johnson et aL, 1996), as Ras tumorigenicity is suppressed by mutation of the JNK phosphorylation sites in c-Jun
(Behrens et aL, 2000) and expression of the JNK inhibitor JIP-1 inhibits transformation by bcr-abl (Dickens et aL, 1997). Transfection of dominant negative c-Jun inhibits transformation of NIH-3T3 cells induced by COT overexpression (Chiariello et aL,
2000). The authors propose that COT induces c-Jun expression, which is required for transformation, by activating SEK-1, MKK6 and MEK5, which activate JNK-1, P38y
102 Chapter 4 Analysis o f TPL-2 AC tramsenic mice
and ERK5 respectively. However, this proposal is based on transfection assays
(overexpression and dominant negative studies) and does not establish whether COT physiologically (or pathophysiologically) phosphorylates these substrates or after which stimuli this occurs.
Thus, TPL-2 oncogenicity may rely on inappropriate activation of one or more MAPK signalling pathways. It may be possible to test experimentally if these pathways are overactive in TPL-2AC tumours by performing in vitro kinase assays. Although such experiments may implicate a pathway in transformation, a causal link cannot be established directly, as it is possible that altered MAPK signalling may arise as a result of transformation. It may be possible to address this issue using a genetic approach.
Crossing TPL-2AC mice onto transgenic or knock-in mice carrying mutated MAPK or
MAPK targets may identify which pathways are required for transformation. This approach was used to demonstrate that Ras or c-fos induced tumorigenesis requires the n-terminal phosphorylation sites of c-Jun (Behrens et aL, 2000).
4.2.2 The importance of the TPL-2 c-terminal in oncogenesis
Since TPL-2AC mice but not TPL-2 transgenic mice develop tumours (T Ahmad and SC
Ley unpublished results, this report and Ceci et aL, 1997), c-terminal truncation of TPL-
2 appears to be an important event in oncogenesis. Truncation is associated with enhanced expression of TPL-2/C0T, which may arise fi’om increased stability of the
103 Chapter 4 Analysis o f TPL-2 AC tramsenic mice
mRNA or increased efficiency of translation (Ceci et aL, 1997). However, since transgenic mice overexpressing wild type TPL-2 do not develop tumours, overexpression of TPL-2 alone is not sufficient for oncogenesis. In some studies c- terminal truncation of TPL-2 enhances the specific activity of the kinase and this is reversed by co-expression of the TPL-2 c-terminal tail (Ceci et aL, 1997). This led the investigators to propose that TPL-2 is regulated by an intramolecular interaction involving its c-terminal tail. However, the Ley laboratory found that TPL-2 truncation had no detectable effect on its kinase activity, when expressed in COS cells (Salmeron et aL, 1996), and so hypothesised that TPL-2 interacts via its c-terminal with another factor, which regulates TPL-2 localisation and access to its correct substrate. Data presented in this report and other studies carried out in this laboratory show that endogenous TPL-2 associates with endogenous NFkBI p i05, so it is possible that the association of TPL-2 with NFkBI pi 05 prevents activation of the ERK or JNK pathway by TPL-2 (Belich et aL, 1999). According to this model, since TPL-2AC cannot associate efficiently with NFkBI p i05, it is free to activate ERK or JNK which may be the key to its oncogenic capacity. This theory could be tested by analysing ERK or
MEK activation in cell transfected with TPL-2 or TPL-2AC, in the presence and absence ofNFKBl pl05.
4.2.3 Co-operation between TPL-2 and other oncogenes
Deregulation of a proto-oncogene probably means that a rate-limiting step to oncogenesis has been lifted. Since oncogenesis arises from combinations of mutations it
104 Chapter 4 Analysis o f TPL-2 AC transsenic mice
seems likely that synergising oncogenes deregulate different rate limiting steps. The proviral tagging method is a usefiil method for identifying oncogenes and their partners
(Jonkers and Berns, 1996), and indeed was used in the identification of TPL-2 (Patriotis et aL, 1993). In this technique, MoMuLV is injected into neonatal mice and causes T cell lymphomas, because provirus integrates in or near oncogenes, thereby activating them (either by increasing transcription or by mutation). Oncogenes can then be identified by finding the sites of proviral integration. Studies using this technique have shown that certain oncogene combinations are commonly seen but some are rarely found. This lead to the proposal that oncogenes form complementation groups. It may be possible to use this technique to identify which oncogenes co-operate with TPL-2, by injecting MoMuLV into neonatal TPL-2AC transgenic mice, and then identifying sites of proviral integration. This techniques was used to show that pim-1 and c-myc cooperate in oncogenesis (Verbeek et ah, 1991).
105 CHAPTER 5 Analysis of TPL-2 function in Jurkat ceils
5.1 Results
5.1.1 Analysis of signal induced NFkBI p i05 degradation in E6.1 cells
Analysis of TPL-2 and TPL-2KD mice did not identify any functions regulated by TPL-
2 in T cells, but raised the issue that the stimuli which promote NFkB I p i05 degradation are not well characterised. In particular, all experiments in Chapter 3 use CD3 and
CD28 CO stimulation. Although it is well established that T cell activation causes NFkB activation (Tong-Starksen et aL, 1987; Verweij et aL, 1991), it is not known whether
NFkBI pi05 degradation contributes to this process. As only limited biochemical analysis of signalling is possible in the TPL-2 transgenic T cells, we decided to address these questions using the T lymphoblastoid cell line E6.1 Jurkat. TPL-2/COT is expressed Jurkat cells, albeit at low levels (T Ahmad and M Belich unpublished data and
Ballester et aL, 1997). Furthermore it is induced in these cells following stimulation with PDBu and ionophore or PDBu and anti-CD3 antibody (Sanchez-Gongora et aL,
2000).
As shown in Figure 11,when Jurkat cells were treated with the protein synthesis inhibitor cyclohexamide (CHX), NFkBI p i05 levels remained relatively stable during
106 Chapter 5 Analysis of TPL-2 function in Jurkat cells
the 6 hours of the experiment, indicating that endogenous NFkBI p i05 is degraded slowly under unstimulated conditions. Significantly, cells treated with CHX and stimulatory antibodies to CD3 and CD28 degraded IicBa but not NFkBI p i05 over the time course examined. However, when cells were treated with a combination of CHX and TNFa, both NFkBI p i05 and IkBq were degraded. Levels of p50 were stable after
TNFa stimulation, indicating that degradation rather than proteolytic processing of
NFkBI p i05 was occurring. Signal induced NFkBI p i05 degradation occurring in the presence of CHX indicates that the process does not require de novo protein synthesis.
unstimulated TNFa CD3 CD3 & CD28 I ■ - I I------1 I ~ ~ ~ ■ I I------1 02460246 02460246 hours ^ ^ NFkB1p 105
024602460 2 4 6 0 2 4 6 hours B m NFkB lp50
0246024602460246 hours
C ^ iKBa
0246024602460246 hours Tubulin D
Figure 11: Signal induced NFKBlplOS degradation in E6.1 Jurkat cells
E6.1 Jurkat cells were treated for the times shown with cyclohexamide (30/jg/ml) alone, or in the presence of TNFa (20ng/ml) or anti-CD3 antibody (OKT3 at Ipg/ml) in combination with anti- CD28 antibody (9.3 at Ipg/ml). Resulting lysates were analysed by western blotting for levels of NF/cBJpl05 (A), NFfcBlpSO (B), 1/cBa (C) and tubulin (D)
107 Chapter 5 Analysis of TPL-2 function in Jurkat cells
TNFa signals through TNFRl in Jurkat cells, which do not express FNFR2 (Ting et ai,
1996). TNFRl stimulation initiates several pro-apoptotic and anti apoptotic signalling pathways. Proapoptotic pathways result in the activation of caspases, which cleave target proteins whose proteolysis underlies the morphological changes associated with apoptosis,(reviewed in Li and Yuan, 1999). Activation of NFkB under these conditions prevents cell death by inducing expression of anti-apoptotic genes (Beg and Baltimore,
1996; Wang et aL, 1996 Van Antwerp et aL, 1996). Under conditions where the
NFkB mediated protective response is blocked, e.g. when protein or RNA synthesis is inhibited or when iKBa is overexpressed, the apoptotic response predominates.
-MG 132 +MG132 ------1 I------
CHX CHX & TNFa CHX &TNFa CHX A 0246 02 46 0246 0246 hours
B I------1------I------1 I------11------1 02 46 02 46 0246 0246 jubulin
Figure 12; NFkBIpl05 degradation is inhibited by proteasome inhibitors
E6.1 Jurkat cells were pre-treated with cyclohexamide (30pg/ml). The effects of the proteasome inhibitor MG 132 (at 20pM) on basal and TNFa (lOng/ml) induced NFkBI p i 05 degradation were analysed by western blotting for levels ofNFKBlpl05 (A) and tubulin (B)
Clearly the conditions used to analyse NFkBI p i05 degradation in Figure 11 may promote apoptosis. To determine whether this was the case we stimulated Jurkat cells
108 Chapter 5 Analysis of TPL-2 function in Jurkat cells
with TNFa in the presence of the proteasome inhibitor MG 132. TNFa induced NFkBI p i05 degradation was blocked by MG132 (Figure 12), which confirms that the observed degradation was mediated by the proteasome. Furthermore, our laboratory have previously demonstrated by pulse chase metabolic labelling that TNFa stimulation in the absence of CFFX causes NF kBI p i05 degradation (Belich et aL, 1999).
5.1.2 NFkB regulation in TPL-2 or TPL-2KD expressing stable cell lines
Since NFkBI p i05 degradation can be followed directly and a stimulus for this process has been identified Jurkat cells were used for further analysis of TPL-2 function. Clonal cell lines stably overexpressing (untagged) TPL-2 or TPL-2KD were generated.
Expression of TPL-2 was verified by western blotting of whole cell lysates (Figure 13).
Transfected TPL-2 is translated from both the first and the M30 initiation sites.
— (N c(U 0>c — rs| _0 _0
Q_ Û - E CL CL f - H CL H i -
NFk B1p 105
B NFkB1p 50
Figure 13: Expression of TPL-2 and TPL-2KD in stable E6.1 clones
Whole cells lysates from pml2-TPL-2, pmt2-empty vector andpmt2-TPL-2KD transfected stable clones were analysed by western blotting, for (A) NF/cBlpl05, (B) NF/cBlp50 and (C) TPL-2 levels.
109 Chapter 5 Analysis of TPL-2 function in Jurkat cells
As shown in Figure 14, NFkBI p i05 degradation induced by TNFa was blocked in
TPL-2KD stable E6.1 clones. These data confirm pulse chase metabolic labelling experiments performed in these cells (Belich et aL, 1999), which taken together show that TPL-2KD acts as an effective dominant negative in this system. Stable overexpression of kinase active TPL-2 had no effect on NFkBI p i05 degradation
(Figure 14). Therefore, if TPL-2 does participate in the regulation of NFkBI pl05 degradation, other factors may be limiting under these conditions.
empty vector clone TPL-2KD clone
CHX CHX & TNFa CHX CHX & TNFa A I 1 I------1 I------1 I------1 0246 0246 0246 0246 hours NFkB1p 105
Tubulin
empty vector clone______TPL-2 clone______
CHX CHX & TNFa CHX CHX & TNFa ^ 2 4 ? ^ 2 4 6 ' '”0 2 4 6* B 2 4 V hours B NFkB1p 105
Tubulin
Figure 14: TNFa induced NFKBIplOS degradation in TPL-2KD E6.1 clones
TPL-2 KD (A) and TPL-2 (B) expressing cells and empty vector control cells were treated for the times shown with cyclohexamide (SOpg/ml) alone, or in the presence o f TNFa (lOng/ml). Resulting lysates were analysed by western blotting for levels o f NFKBlpl05 (Ai and Bi), and tubulin (Aii and Bii). Representative blots from multiple experiments with similar results are shown.
10 Chapter 5 Analysis of TPL-2 function in Jurkat cells
TNFa induced IkBœ degradation was normal in TPL-2 and TPL-2KD E6.1 cells (Figure
15). Thus, the NFkB regulating effects of TPL-2KD are specific to NFkBI p i05 and
TPL-2 does not regulate IicBa degradation.
empty veetor clone TPL-2KD clone I------1 I------1 0 2 8 15 30 0 2 8 15 30 minutes
A ^ IkBœ
Tubulin empty vector clone TPL-2 clone I------1 0 2 8 15 30 0 2 8 15 30 minutes B _ iKBa
Tubulin
Figure 15: TNFa induced iKBa degradation in TPL-2 and TPL-2KD E6.1 clones
TPL-2 KD (A) orTPL-2 (B) expressing and empty vector control E6.1 Jurkat cells were treated for the times shown with TNFa (20ng/ml). Resulting lysates were analysed by western blotting for levels of IkB a (Ai and Bi) and tubulin (Aii and Bii).
Preliminary experiments showed that TNFa stimulation had no effect on ERK 1/2, P38 or JNKl activation that was detectable by the use of ‘anti-phospho’ antibodies which recognise phosphorylated ‘active’ forms of these MAP kinases. It was therefore not possible to test whether TPL-2KD had any effect on TNFa induced stimulation of these pathways. Flowever, CD3 and CD28 costimulation did activate ERK and P38, see below. Chapter 5 Analysis of TPL-2 function in Jurkat cells
Having shown that TPL-2KD E6.1 clones have a defect in TNFa induced NFkBI p i05 degradation we attempted to identify genes whose transcription may be affected in these cells. As there are no known target genes for regulation by NFkBI p i05 degradation, a panel of NFkB regulated genes that may be induced by TNFRl stimulation of T cells were analysed. E6.1 cells were stimulated for 2-5 hours with TNFa or anti-CD3 and anti-CD28 antibodies before RNA extraction. Induction of candidate mRNAs, including
ICAM-1, C-IAP2, BCL-3, CD25, CD69, IL-2, iKBa and TNFa were analysed by RT-
PCR (Figure 19).
Although CD3/CD28 treatment induced TNFa, CD25 and IL-2 mRNA, none of the candidate mRNAs were induced by TNFa (Figure 19). Identification of further candidate TNFRl induced target genes for analysis was complicated conflicting data on the functions of TNF receptors in T cells. Clearly the risk in using a ‘candidate approach’ to search for genes regulated by a specific signalling pathway carries the risk of failing to identify and analyse the correct target genes. Gene array analysis may be the best way to approach such a question in future studies.
112 Chapter 5 Analysis of TPL-2 function in Jurkat cells
CD3 & CD28 TNFa CD3 & CD28 TNFa 1------1 I------1 1------1 I------1 0 2 4 0 2 4 hours 0 2 4 0 2 4 hours A F CD25 ICAM
B IL-2 G CD69
C H p actin TNFa
D iKBa
P actin
Figure 16: mRNA induction in E6.1 Jurkat cells treated with CD3 and CD28 or TNFa
E6.1 Jurkat cells were stimulated for the times shown with TNFa (20ng/ml) or CD3 (OKT3 at I/ug/ml) and CD2H (9.3 at ljug/ml) and RNA was extracted. ATP RT-PCR was performed, and samples were electrophoresed, the gel dried and analysed by exposure to film or phosphoimager. MRNAs analysed were (A) ICAM-1. (B) IL-2 (C) TNFa (D) IkB a shown above the relevant RNA loading control (F) f3 actin, and (F) CD25, (G) CD69 shown above the relevant loading control (H) J3 actin.
5.1.3 CD3 and CD28 costimulation of TPL-2KD Jurkat clones
As shown in Figure 16, IL-2 mRNA accumulated in Jurkat cells treated with CD3 and
CD28 antibodies. Several reports suggest that TPL-2 can regulate IL-2 production.
Tsatsanis et al propose that TPL-2 regulates NFAT in this process (Tsatsanis et ai,
1998b) Lin et al propose that TPL-2/C0T acts downstream of CD3/CD28 but not
TNFRl in the regulation of the IKK enzymes (Lin el aL, 1999), and regulates IL-2 transeription via a kB element-Iike CD28 responsive element (CD28RE) and an AP-1 site. As shown in Figure 14, TPL-2 regulates NFkBI p i05 degradation in E6.1 Jurkat
113 Chapter 5 Analysis of TPL-2 function in Jurkat cells
cells. In order to test whether l PL-2 also regulates IL-2 production in these eells, RT-
PCR analysis of RNA from CD3 and CD28 costimulated clones was performed. As shown in Figure 17, TPL-2KD and empty vector clones transcribe equivalent quantities of IL-2 mRNA, which is consistent with the normal IL-2 production by TPL-2KD transgenic T cells (Figure 6) and TPL-2-/- T cells (Dumitru et aL, 2000)
Empty vector TPL-2KD#! TPL-2KD#2 r \ t \ r \ 024 024 024 hours
A ^ • - - » IL-2
B «••••• (3 actin
Figure 17: IL-2 induction in TPL-2 KD expressing E6.1 Jurkat clones
Two E6.1 Jurkat cell clones stably transfected with TPL-2KD and an empty vector clone were treated for the times shown with anti-CD3(Ipg/ml) and anti-CD28 (Ipg/ml) antibodies. Induction of IL-2 mRNA was analysed by RT-PCR (A) and equal starting quantities of RNA verified by f actin amplification (B).
I PL-2 activates several MAP Kinase pathways in vitro; ERKl, JNK, P38y and ERK5
(Chiarello et al 2000, Salmeron et al. 1996), and is required for ERK activation in LPS treated macrophages (Dumitru et al, 2000). Flowever, ERK and P38 activation in response to CD3 and CD28 costimulation occurred normally in TPL-2KD Jurkat clones.
Figure 18. JNK activation was not detectable with anti-active JNK antibodies, following
CD3 and CD28 costimulation in these cells, PMA with CD28 antibody however, was a more potent stimulus. TPL-2KD expressing Jurkat cells aetivate JNK normally when treated with PMA and CD28, Figure 19, although it is possible that PMA activates signalling downstream of the function of TPL-2.
114 Chapter 5 Analysis of TPL-2 function in Jurkat cells
empty vector clone TPL-2KD clone 1— ---- 1 0 5 10 30 0 5 10 30 minutes A active ERK 1/2 1 1 0 5 10 30 0 5 10 30 B active P38 1 1 0 5 10 30 0 5 10 30 C mm Tubulin
Figure 18: MAPK activation in TPL-2KD E6.1 clones Empty vector or TPL-2 KD expressing E6.1 Jurkat cells were treated for the times shown with anti-CD3 (0KT3 2pg/ml) and anti-CD28 (9.3 at 2pg/ml) antibodies. Phosphorylation of ERK 1/2 (A) and P38 (B) were analysed using antibodies specific for the pho.sphorylated form of these proteins. Anti-tubulin blots verified equal protein loading (C). A representative experiment from multiple experiments analysing two different TPL-2KD clones is shown.
empty vector clone TPL-2KD clone 1 1 1 "1 0 15 30 60 A 0 15 30 60 A minutes A # e # #
active JNK # # # 1 1 ' 0 15 30 60 a ' ‘ 0 15 30 60 A
B ■ • m m i IkBœ
'o 15 30 60 A* ' 0 15 30 60 A '
C Tubulin
Figure 19: JNK activation andI kB a degradation in TPL-2KD E6.1 Jurkat clones Empty vector or TPL-2 KD expressing E6.1 Jurkat cells were treated for the times shown with PMA (lOng/ml) and anti-CD28 (9.3 at lOpg/ml) antibody. Phosphorylation of JNKl (p46 and p54 isoforms) was analysed using antibody specific for the phosphorylated form of these proteins. Samples in lanes labelled A were treated for 30 minutes with Anisomysin (lOpg/ml) as a positive control for JNKl activation. IkB a degradation under these conditions was analysed (B). Anti-tubulin blots verified equal protein loading (C). A representative experiment from multiple repeats analysing two different TPL-2KD clones is shown.
15 Chapter 5 Analysis of TPL-2 function in Jurkat cells
5.2 Discussion of Chapter 5
5.2.1 The role of TPL-2 in CD3 and CD28 signalling
As shown in Figure 10, when Jurkat cells are stimulated with anti CD3 and anti CD28 antibodies IkBœ but not NFkBI p i05 is degraded. Since the analysis of the TPL-2 transgenic mice described in Chapter 3 focussed on costimulation of T cells via CD3 and
CD28, the fact that these stimuli do not actually cause NFkBI p i05 degradation may be a partial explanation for our failure to identify functional defects in these mice. Taken with the normal phenotype of TPL-2-/- T cells (Dumitru et aL, 2000), these data suggest that TPL-2 may not function in CD3 and CD28 signalling.
Several previous studies have proposed a function for TPL-2 in CD3 and CD28 regulation of downstream signalling pathways. Lin et al showed that TPL-2/C0T regulates IKK activation by CD3 and CD28 but not by TNFa. In a transient transfection study, they demonstrate that TPL-2 regulates an IL-2 enhancer element composed of a kB element-like CD28 responsive element (CD28RE) and an AP-1 site, and that this regulation requires IkB œ degradation (Lin et ah, 1999). However, Jurkat cells stably transfected with TPL-2KD used in our study degrade IkB œ normally when stimulated with TNFa or PMA and CD28 (Figures 15 and 19), furthermore, IL-2 transcription is unchanged in TPL-2KD expressing Jurkat clones (Figure 18). The difference between the present study and that of Lin et al may arise from the lower levels of overexpression achieved by the use of stable transfection techniques, which are less likely to generate non-specific effects. Taken together with normal IL-2 production in TPL-2KD
116 Chapter 5 Analysis of TPL-2 function in Jurkat cells
transgenic T cells and TPL-2-/- knockout T cells (Dumitru et aL, 2000), these data strongly argue against a role for TPL-2 in the regulation of IL-2 transcription.
5.2.2 Regulation of MAP kinase pathways by TPL-2
Transient overexpression of TPL-2 stimulates activates the MAPKKs MEK, SEK and
MKK6, which activate ERK, JNK and P38 respectively (Chiariello et aL, 2000;
Salmeron et aL, 1996). However, as shown in Figures 15 and 16, ERK, JNK and P38 activation in response to CD3 with CD28 or PMA with CD28 stimulation are normal in
TPL-2KD expressing cells. These differences may be a function of the lower level of overexpression achieved using stable transfection compared with transient experiments.
The fact that few of these previous studies examined TPL-2 dominant negative functions in the context of receptor stimulation induced signalling, favouring instead the activation of cells by the overexpression of upstream kinases, may underlie this discrepancy. The failure of dominant negative COT to inhibit T cell activation induced by superantigen presented on antigen presenting cells, is consistent with the present study (Tuosto et aL,
2000). It appears that TPL-2 may only fimction as a dominant negative in MAPK regulation under certain conditions, which complicates elucidation of the physiological role of TPL-2. Data generated in the TPL-2KD Jurkat clones are consistent with the phenotype of the recently published TPL-2 knockout mice. These mice have a very clear defect in ERKl and 2 activation in macrophages stimulated with LPS, which is apparently the basis of reduced TNFa production (Dumitru et aL, 2000). However,
CD3 and CD28 or PDBu and lonomycin induced TNFa production in TPL-2-/- T cells
117 Chapter 5 Analysis of TPL-2 function in Jurkat cells
is normal. Therefore although TPL-2 is vital for ERK activation in response to some receptors, it is dispensable for others.
5.2.3 Are NFkBI p i05 and h cB a degradation separately regulated?
Significantly, the data in Figure 11 identifies a situation where IicBa is degraded but
NFkBI p i05 is not. It has been shown that the IKK enzymes are required for IicBa degradation (Li et aL, 2000), and NFkBI p i05 is degraded when IKKp is overexpressed
(Heissmeyer et aL, 1999). Until genetic experiments show definitively whether IKKa and IKKp are required for NFkBI pi05 degradation, it remains possible that the phosphorylation events required for NFkBI p i05 degradation are mediated by another kinase, perhaps related to IKK. If both IicBa and NFkBI pi 05 degradation require IKK, other control mechanisms may underlie differences in IicBa and NFkBI p i05 responses to stimulation. These may be at the level of IKK regulation; e.g. control of substrate accessibility, or at the level of NFkBI p i05 or IkB e.g. further regulatory phosphorylation events or covalent modifications. Since NFkBI p i05 degradation but not IicBa degradation is blocked in TPL-2KD clones, it is possible that TPL-2 regulates the activity of IKK on NFkBI p i05. A comparison of IKK activation under NFkBI p i05 degrading and non-degrading conditions investigates this question further in
Chapter 6.
118 Chapter 5 Analysis of TPL-2 function in Jurkat cells
5.2.4 TPL-2KD blocks TNFa induced NFkBI pi05 degradation
As shown in Figures 14 and 15, the stable expression of TPL-2KD in E6.1 cells blocks
TNFa induced pi05 degradation, confirming that TPL-2KD can function in the signalling pathway controlling NFkB Ip 105 degradation. Since the are no changes in
ERK, JNK, or P38 activation and IkBœ degradation is normal in these clones, we believe that the effect of TPL-2 on NFkBI p i05 is specific. The CD3 and CD28 responses of TPL-2KD Jurkat cells and TPL-2-/- T cells are similar (Dumitru et aL,
2000). For these reasons we believe that TPL-2KD clones are a good system for analysis of TPL-2 function.
It has been proposed that overexpression of a scaffold protein can dilute the functional components of a signalling pathway and so reduce signalling, which can lead to the misclassification of proteins as inhibitors (Burack and Shaw, 2000). Since NFkBI pi 05 degradation is not blocked in Jurkat clones stably expressing kinase active TPL-2, the scenario described above is unlikely, rather these data suggest that the dominant negative activity of TPL-2KD arises fi*om the inhibition of an endogenous kinase, related to or identical to TPL-2.
Unfortunately, although CD3 and CD28 or Anisomysin elicited detectable P38, ERK and JNK responses in the Jurkat cells, TNFa treatments did not result in a stimulation of these pathways that was detectable by blotting with ‘anti-phospho’ antibodies. This contrasts with reports in other cell types showing that TNFa can activate ERKl/2, JNK and P38 (Beyaert et aL, 1996; Reinhard et aL, 1997; Van Lint et aL, 1992; Vietor et aL,
119 Chapter 5 Analysis o f TPL-2 function in Jurkat cells
1993), although relative activation of the different cascades varies according to the cell type (Wallach et aL, 1999). The use of in vitro kinase assays, which are more sensitive than anti-active MAPK antibodies may detect lower levels of TNFa induced MAPK activation. Since it is now known that TPL-2 knockout mice have ERK activation defects (Dumitru et aL, 2000), and Dumitru et al mention prehminary results showing that TPL-2 functions in TNFa signalling this experiment would be an important part of a continuation of this study.
5.2.5 How might altered NFkBI pl05 degradation affect transcription?
There are multiple possible effects of inhibition of NFkBI pi 05 degradation. Since p50 has higher affinity for NFkBI p i05 than the ‘small’ IkB proteins (a, p and e), it has been proposed that NFkBI p i05 degradation is the primary mechanism regulating p50 homodimers (Heissmeyer et aL, 1999). This is supported by the fact that when dominant negative iKBa is overexpressed, nuclear translocation of p50 is normal
(Heissmeyer et aL, 1999; Hettmann et aL, 1999). As p50 homodimers are repressive
(Ryseck et aL, 1992; Schmid et aL, 1991), one hypothesis is that NFkBI p i05 regulates a slow induction of p50 homodimers which ‘switches off transcription. Consistent with this, LPS treated nfkbl-/- macrophages produce normal levels of TNFa, however, they fail to become LPS tolerant (Bohuslav et aL, 1998). The induction of LPS tolerance coincides with the induction of p50 homodimers onto one of the kB elements in the murine TNFa promoter (Bohuslav et aL, 1998).
120 Chapter 5 Analysis of TPL-2 function in Jurkat cells
BCL3 associated p50 homodimers are transcriptionally active (B o uts et aL, 1993; Fujita et ah, 1993; Pan and McEver, 1995). The genes which are regulated by p50-BCL3 complexes have not yet been identified specifically, however, BCL3 knockout mice have defects in germinal centre formation and antibody responses (Franzoso et aL, 1992;
Schwarz et aL, 1997). C-EBPp is another transcription factor which has been shown to associate with p50 homodimers, regulating that transcription of the acute phase protein
C Reactive Protein (CRP) by hepatocytes (Cha-Molstad et aL, 2000).
The function of NFkBI p i05 is not limited to the regulation of p50 homodimers, as it can also bind to all other members of the NFkB group (Henkel et aL, 1992; Mercurio et al., 1993; Rice et aL, 1992). Thus, NFkBI p i05 and the ‘small’ IkB proteins regulate overlapping populations of NFkB complexes. There is little experimental data to explain the reason why multiple regulatory mechanisms (IkB proteins) regulate a single signalling event (NFkB), but data in this study suggests that the conditions under which the two pathways become activated differ (Figure 10). Furthermore, the slow nature of
NFkBI pi05 degradation implies that this pathway regulates later phases of NFkB activation (Belich et aL, 1999; Harhaj et aL, 1996).
In order to investigate whether there were any changes in TNFa induced gene induction in the TPL-2KD clones RT-PCR analysis was performed. Despite analysing a number of candidate mRNA, it was not possible to identify a gene whose transcription was induced in Jurkat cells by treatment with TNFa (Figure 19). Although some in vitro studies showed that both TNFRl and TNFR2 stimulate T cell proliferation (Gehr et aL,
121 Chapter 5 Analysis of TPL-2 function in Jurkat cells
1992), in vivo studies using TNF receptor knockout mice and receptor blocking antibodies showed that TNF induced T cell proliferation occurs entirely through TNFR2
(Grell et aL, 1998; Tartagliaet aL, 1993; Tartagliaet aL, 1991). During the process of activation induced cell death (AICD), which scales down T cell numbers at the end of the immune response, TNFa is cytotoxic (Zheng et aL, 1995), Despite the well characterised pro-apoptotic functions of TNFRl, most studies indicate that TNFR2 functions in AICD (Alexander-Miller et aL, 1998; Herbe in et aL, 1998; Zheng et aL,
1995). TNFRl is induced on T cells by antigen receptor stimulation (Pfeffer et aL,
1993), and there is some evidence for overlapping functions for TNFRl and TNFR2 in proliferation and apoptosis, there are few data which specifically identify target genes for TNFRl in T cells. The use of gene chip analysis to compare untreated and TNFa stimulated T cells may be the most effective way to address this question.
Cha-Molstad et al have recently shown that the promoter of C-Reactive Protein contains a non-consensus kB site. When incorporated into a reporter gene, this site is responsive to p50 but not p65 transfection (Cha-Molstad et aL, 2000). Since NFkBI p i05 degradation is a major source of p50 homodimers, it is possible that a transgenic reporter construct containing this regulatory region could be used to quantify p50 induction in different mouse tissues. This may identify which tissues and conditions should be used for analysis of p i05 degradation, and enable analysis of p50 homodimer formation in
TPL-2-/- mice. A similar technique was used by Li et al in order to test whether there was any NFkB activation in any of the tissues in mice lacking IKKa and p (Li et aL,
2000).
122 CHAPTER 6 Analysis of TPL-2 function in THP-1 cell lines
6.1 Results
Monocytes are circulating cells of the innate immune system, which differentiate into macrophages upon leaving the bloodstream. Both cell types express pattern recognition receptors, including TLR4 and TLR2, which allow them to recognise microbial pathogens early in infection. Upon exposure to fungal, bacterial or protozoan products, such as LPS, monocytes and macrophages transcribe multiple genes whose products regulate the innate and adaptive immune responses. NFkB is strongly implicated in the regulation of many of these genes, including TNFa, IL-lp, GM-CSF, G-CSF, MCP-1,
NOS and tissue factor (Muller et aL, 1993)
6.1.1 Characterisation of NFkB activation in THP-1 cells
THP-1 is a human leukaemic cell line, which has the phenotypic characteristics of monocytes, including transcription of TNFa, IL-lp and tissue factor in response to LPS.
Published reports of LPS-induced NFkBI p i05 processing or degradation (Donald et aL,
1995; Harhaj et aL, 1996), led us to examine whether THP-1 cells could be used as a
system to analyse the regulation of NFkBI p i05 by TPL-2 and its transcriptional outcomes. Preliminary studies confirmed that LPS stimulates NFkBI p i05 degradation,
123 Chapter 6 Analysis of TPL-2 function in THP-1 cells
Figure 20. Examination of cells by light microscopy did not show any increase in the proportion of cells of apoptotic appearance in cells treated with LPS and CHX. As in
TNFa stimulated Jurkat cells, levels of p50 are constant during NFkBI p i05 degradation, which is consistent with the complete degradation of NFkBI p i05 rather than proteolytic processing to p50. TNFa, which is a potent stimulus for NFkBI p i05 degradation in E6.1 cells (Figure 11), had little detectable effect on NFkBI pl05 degradation in THP-1 cells in the time period examined. Thus, a stimulus may have different effects on NFkBI p i05 regulation depending on which cell type is expressing its receptor.
CHX CHX & LPS CHX & TNFa I------1 I 1 I------1 0 1 3 5 0 0.25 1 3 5 0 0.25 1 3 5 hours A _ . # NFKBI pi05 I ______------1 11------1--I------1 0 1 3 5 0 0.25 I 3 5 0 0.25 1 3 5 hours
B k _ J P NFkBI pl05
0 1 3 5 0 0.25 1 3 5 0 0.25 1 3 5 hours
C 4WW mmrn m tm mrm wamm m m m tm ### ### Tubulin
Figure 20: Signal induced NFkBI pi05 degradation in THP-1 cells THP-1 cells were treated for the times shown with cyclohexamide (10/ug/ml) alone, or in the presence of LPS (lOpg/ml) or TNFa (20ng/ml). Resulting lysates were analysed by western blotting for levels ofNpKBlplOS (A), NF/cBlp50 (B) and tubulin (C)
The expression of a functional TNF receptor on these eells was confirmed in Figure 21 where TNFa stimulation was shown to cause IxBa degradation. As previously reported.
124 Chapter 6 Analysis ofTPL-2 function in THP-1 cells
TNFa induced IicBa degradation occurs more quickly than that triggered by LPS
(Fischer et al., 1999), although both processes require IKKa and IKKp (Li et al., 2000).
no stimulus LPS TNFa I 1 r 0 8 15 3 0 60 120 0 8 15 3 0 60 120 0 8 15 30 60 120 minutes
A IicBa
I 1 I------II------1 0 8 15 30 60 120 0 8 15 3 0 60 120 0 8 15 30 60 120 minutes B ' ## « * m# M# m# mm ## Tubulin
Figure 21: Signal induced iKBa degradation in THP-1 cells THP-1 cells were treated for the times shown with LPS (10/j.g/ml) or TNFa (20ng/ml). Resulting lysates were analysed by western blotting for levels of I/cBa (A) and tubulin (B). Similar degradation kinetics were observed in cells pre-treated with CHX
In order to analyse IKK activity under NFkBI pi 05-degrading and non-NFKBl pi 05 degrading conditions in vitro kinase assays were performed. The IKK complex was immunoprecipitated from cells with anti-IKKy antibody and in vitro kinase assays were performed, using GST-lKBa(l-54) as a substrate. The kinetics of stimulation of IKK kinase activity parallel the kinetics of IicBa degradation (Figures 21 and 22). Figures
20, 21 and 22, show that in TNFa stimulated THP-1 cells, IKK is activated and IxBa is degraded, yet levels ofNF kBI p i05 are stable. Similar data was generated using
NIH3T3 cells, which activate IKK and degrade IicBa in response to TNFa, but do not degrade NFkBI p i05 efficiently under these conditions (data not shown). This provides further support for a model in whichNF kBI p i05 and IicBa degradation are regulated by different signalling pathways. Preliminary experiments were performed to quantify
TPL-2 activation under NFkBI p i05 degrading and non-NFicBl p i05 degrading
125 Chapter 6 Analysis of TPL-2 function in THP-1 cells
conditions. However, technical difficulties with TPL-2in vitro kinase assays prevented the generation o f interpretable results.
TNFa LPS
0 5 15 30 Ih 3h 5h 0 5 15 30 Ih 3h 5h time (mins/hours)
™ GST-lKBa phosphorylation
B “"o 5 15 30 Ih 3h 5h 5 15 30 Ih 3h 5h ' IKKy
Figure 22: Induction of IKK kinase activity by TNFa and LPS in THP-1 cells IKK complexes immunoprecipitated from THP-1 cells stimulated for the times shown with LPS (lOpg/ml) or IN F (20ng/ml) were subjected to in vitrokinase (IVK) assays, using GST-ÏKBa(l- 54) as a substrate (A). Equivalent quantities of IKKywere immunoprecipitated in each sample (B). Similar IKK activation was observed when cells were pre-treated with CHX (lOpg/ml) in addition to LPS or TNFa. No substrate phosphorylation was seen in samples immunoprecipitated with an irrelevent antibody or on GST itself (data not shown).
6.1.2 Analysis of TPL-2KD expressing stable THP-1 cell lines
1 HP-1 cells degrade NFkBI p i05 when stimulated with LPS, they retain in culture the ability to transcribe NFkB regulated genes and express comparatively high levels o f
TPL-2. For these reasons we chose to pursue the dominant negative approach to investigate the function o f TPL-2. In initial experiments THP-1 clones stably transfected with PMT2-TPL-2KD were generated by limiting dilution and TPL-2KD overexpression analysed by western blotting. Levels o f overexpression achieved were low and diminished with time in culture (data not shown). The process was repeated with PMX-HA-TPL-2KD, which uses a different selection marker and has the advantage that transfected protein can be distinguished from endogenous by the presence of an HA-
126 Chapter 6 Analysis of TPL-2 function in THP-1 cells
tag. In this case levels o f overexpression achieved were higher. Densitometric analysis
(using Quantity One software, Biorad) o f immunoprécipitation studies showed that HA-
TPL-2KD was present in NFkBI p i05 containing complexes, at more than 12-fold higher levels than endogenous TPL-2 (Figure 23).
empty vector TPL-2KD clone 2 TPL-2KD clone 20 I------1 I------1 r
A NFkB1p 105
2 3 4 1234 1234 B HA-TPL-2KD- • TPL-2 TPL-2-
Figure 23: Stably transfected HA-TPL-2KD associates with endogenous NFkBI pl05
Precleared cell lysates from empty vector or HA-TPL-2KD expressing clones (2x10' cells) were immunoprecipitated with antibodies to (I) Tpl-2, (2) the HA- tag (3) the n-terminal o f NFkBI pl05 or (4) the c-terminal of NFkBI pl05 and blotted for NFkBI pI05 (A) and TPL-2 (B).
In order to determine whether the overexpression o f TPL-2KD in THP-1 cells altered
NFkB activation, LPS induced degradation o f NFkBI p i05 and IkBœ were analysed.
As in Figure 20, cyclohexamide was used to inhibit resynthesis o f NFkBI pi 05 and LPS induced NFkBI p i05 degradation was followed by western blotting. Preliminary studies showed that NFkBI p i05 was degraded as normal in TPL-2KD clones (data not shown). However, it was noted that HA-TPL-2KD was degraded in a stimulus dependent manner, with similar kinetics to NFkBI p i05, ti/2 approximately 90 minutes.
Due to the poor specificity o f anti-TPL-2 antibodies and low levels of endogenous TPL-
2, it was not possible to determine whether endogenous TPL-2 was also degraded.
Under conditions where protein synthesis is inhibited by treatment with CHX, if TPL-
127 Chapter 6 Analysis of TPL-2 function in THP-1 cells
2KD is degraded and not resynthesised, any effect o f the presence o f TPL-2KD would be lost or reduced. To investigate whether this was the case, LPS inducedNF kBI p i05 degradation in TPL-2KD clones was measured by pulse-ehase metabolic labelling. As shown in Figure 24, an incomplete but reproducible inhibition o fNF kBI p i05 degradation was observed.
Data derived from analysis o f the Jurkat clones predicts that TPL-2 selectively regulates
NFkBI p i05 degradation while having no effect upon other signalling pathways. In order to test whether this is the case in TPL-2KD THP-1 clones, IkBœ degradation in response to LPS was analysed. As shown in Figure 25, IkBœ is degraded in TPL-2KD clones, but with slower kinetics.
unstimulated LPS unstimulated LPS
01240124 01240124 hours
A " ^ ^ NFkBI pl05
B ^ Background band -I L
empty vector clone TPL-2KD clone
Figure 24: LPS induced NFkBI pi 05 degradation in TPL-2 KD TFfP-1 clones
THP-1 cells stably transfected with PMX- empty vector or PMX-HA-TPL-2KD were metabolically labelled with methionine and cysteine for SO minutes before treatment with
LPS (lOpg/ml) or media alone. (A) Immunoprécipitations with NFkBI p i 05 antibodies from lysates made at the times shown were analysed by SDS-PAGE and fluorography. (B) Levels of a non specific band immunoprecipitated with anti-NpKBl p i05 antibody are shown as a control for equal IPs.
128 Chapter 6 Analysis of TPL-2 function in THP-1 cells
TPL-2KD TPL-2KD TPL-2KD EV #2 #5 #20 0 1 2 '' p I 2 * 0 1 7 ^ r i hours
Tubulin
Figure 25: LPS induced IkBœ degradation in HA-TPL-2KD THP-1 clones
THP-l cells stably transfected with PMX empty vector or PMX HA-TPL2KD were treated with cyclohexamide, and stimulated for the times shown with LPS (lOpg/ml). Resulting lysates were analysed by western blotting for levels of I kB a (A) and tubulin (B).
EV TPL-2KD#2 TPL-2KD#20 ‘ Q 15 3 0 6 0 " Q 15 ^ 0 6Q"Q 15 3 0 6rf minutes A ^ ^ Active ERK
Tubulin B
Figure 26: LPS induced ERK activation in TPL-2 KD THP-1 clones
Empty vector or TPL-2KD expressing THP-1 cells were stimulated for the times shown with LPS (lOpg.ml) and resulting lysates analysed by western blotting for (A) ERK activation and (B) tubulin loading control.
1 PL-2 regulates several different signalling pathways when transiently expressed, these
include ERK, JNK and P38 MAP kinase pathways (Chiariello et al., 2000; Salmeron el al., 1996). Preliminary studies were undertaken to test whether ERK, JNK or P38
activation in response to LPS were altered in TPL-2KD expressing THP-1 clones. LPS
stimulates ERKl (p42) but not ERK2 (p44) in these cells (data not shown), although
LPS activates ERKl and 2 in primary murine macrophages (Dumitru et ah, 2000).
129 Chapter 6 Analysis o f TPL-2 function in THP-1 cells
Interestingly, TPL-2KD clones failed to activate ERK in response to LPS (Figure 26), suggesting that TPL-2 regulates ERK activation in macrophage responses to LPS.
Using ‘anti-phospho’ antibodies to detect active MAPK it was not possible to detect any activation of JNK or P38 in response to LPS. Given time, performing in vitro kinase assays, which are more sensitive, would determine whether the defects of TPL-2KD in
LPS signalling are limited to regulation of NFkB and ERK.
6.1.3 Levels of LPS induced mRNAs in TPL-2KD THP-1 clones
TPL-2KD expressing THP-1 clones show diminished ERK activation and NFkBI p i05 degradation in response to LPS stimulation. Does this translate into altered gene expression? LPS treatment of macrophages results in the activation of a broad array of genes, several of which are regulated by NFkB (Muller et al., 1993; Pahl, 1999). These include TNFa, lL-1 and MCP-1. TNFa regulates many processes, (reviewed in
Aggarwal and Natarajan, 1996; Vassalli, 1992), including the induction of acute phase proteins and activation of the vascular endothelium. lL-1 also regulates many processes, which include lymphocyte activation and chemokine production, (reviewed in ONeill and Dinarello, 2000). MCP-1 recruits immune cells to infection sites, (reviewed in
Ward et ah, 1998).
Preliminary studies were undertaken to identify whether there were any defects in transcription in LPS treated HA-TPL-2KD clones. RT-PCR analysis showed that TNFa
130 Chapter 6 Analysis of TPL-2 function in THP-1 cells
expression was inhibited in multiple high-expressing clones (Figure 27). Technical difficulties with IL-1|3 and MCP-1 PGR reactions prevented analysis of IL-lp and MCP-
1 induction and there was insufficient time to overcome this.
Empty vector TPL-2KD#5 TPL-2KD#20 ------1 1 I------1 I------1 0 2 4024024 hours
A . . . mRNA
B ########## ## ###### P actin mRNA
Figure 27: TNFa transcription in THP-1 cells stably expressing TPL-2 KD
TIIP-l cells stably transfected with PMX empty vector or PMX-HA-TPL-2KD were stimulated for the times shown with LPS (lOpg/ml). RNA was extracted and analysed by RT-PCR for levels of TNFa (A) and fi actin mRNA (B).
PCR conditions used for TNFa amplification were not saturated and the major product is the correct size. It was necessary to use ‘Touchdown PCR’ to ensure specific amplification of TNFa mRNA due to its low abundance in the cell. As amplification cycle conditions vary during the PCR reaction this is not a suitable technique for
quantitative analysis; northern blotting should be used to confirm these results. To
confirm the RT-PCR data, levels of TNFa in supernatants from stimulated THP-1 cells
were quantified by ELISA. In order to ensure that TNFa was shed from the cell surface
into the supernatant, cells were costimulated with PMA, however, under these
conditions TNFa was not detectable. Subsequent experiments by collaborators using a
more sensitive method showed that the ECACC THP-1 cells used in this study do
produce low levels of TNFa, however under identical conditions, THP-1 cells from
131 Chapter 6 Analysis o f TPL-2 function in THP-1 cells
ATCC produce 10-100 times more TNFa (H Allen and SC Ley unpublished data).
Given more time it may be possible to modify the experimental conditions used and test whether the reduced levels of TNFa mRNA seen in LPS stimulated clones are reflected in reduced levels of TNFa protein.
132 Chapter 6 Analysis of TPL-2 function in THP-1 cells
6.2 Discussion of Chapter 6
6.2.1 Regulation of TNFa production
TNFa production is a tightly controlled process, which is regulated at both transcriptional and post-transcriptional levels (Raabe et al., 1998). Numerous lines of
evidence implicate NFkB in TNFa gene regulation. Several kB like binding sequences
are found in the TNFa promoter (Shakhov et al., 1990), c-Rel-/- mice do not transcribe
TNFa in response to LPS (Grigoriadis et al., 1996) and primary macrophages infected
with IicBa have an 80% reduction in TNFa production (Foxwell et al., 1998). Although
LPS induction of TNFa is normal in nfkbl/nfkb2 double knockout mice (Franzoso et
al., 1997), in nfkbl^®^ mice, (which lack the NFkBI p i05 c-terminus) thymic TNFa
mRNA levels are reduced whereas, LPS stimulated peritoneal macrophages make more
TNFa than control cells (Ishikawa et al., 1998). The relative importance of the different
kB sequences is not clear due to conflicting data from different experimental systems.
Species variation has contributed to the confusion about the role of NFkB in TNFa gene
regulation, as high affinity kB sequences in the murine promoter are absent from the
human promoter (Kuprash et al., 1999).
There are several potential binding sites for other transcription factors in the TNFa
promoter, including CRE, SPl Egr and NFAT. Following LPS stimulation, ERK
substrates Elk-1 and Ets bind to NFAT sites, while JNK and P38 targets ATF-2 and c-
Jun bind to a CRE site (Tsai et al., 2000). The importance of the ERK, JNK and P38
133 Chapter 6 Analysis o f TPL-2 function in THP-1 cells
pathways in TNFa transcription is underscored by the observation that MEK and P38
inhibitors inhibit LPS induced TNFa mRNA levels, and ATF-2 knockout macrophages make reduced levels of TNFa mRNA (Rutault et al., 2000; Tsai et al., 2000). Different
TNFa inducing stimuli induce different transcription factor complexes on the TNFa promoter (Tsai et al., 1996; Zagariya et al., 1998), which may explain some of the
conflicting data on the relative importance of different signalling pathways and
transcription factors in this regulation.
Recent studies have identified the ERK, JNK and P38 MAP kinase pathways as players
in post-transcriptional regulation of TNFa mRNA. Treatment of THP-1 cells with
SB203580, which specifically blocks P38, inhibits LPS induced TNFa mRNA
accumulation, by destabilising the mRNA (Rutault et al., 2000). Gene inactivation of
the P38 target MAPKAP kinase 2, prevents TNFa synthesis despite normal TNFa
mRNA levels (Kotlyarov et al., 1999). The evidence for JNK involvement in TNFa
gene regulation is less clear, since it is based upon the use of dexamethasone as a JNK
inhibitor. However, in cells treated with dexamethasone, JNK is inhibited and TNFa
translation is blocked (Swantek et al., 1997). Murine macrophages treated with the
MEK inhibitor PD98059 respond to LPS by inducing TNFa mRNA but no TNFa
protein is produced (Dumitru et al., 2000).
The AU Rich Region (ARE) in the 3’ UTR of the TNFa transcript regulates TNFa
mRNA stability, localisation and translation (Kontoyiannis et al., 1999). Mice lacking
these sequences (T N F a^^/- mice) have increased levels of constitutive and inducible 134 Chapter 6 Analysis o f TPL-2 function in THP-1 cells
TNFa production, and are sensitised to the lethal effects of LPS (Kontoyiannis et al.,
1999). Treatment of these mice with dexamethasone, SB203580 or PD98059 has shown
that the ARE can be regulated by the ERK, P38 and JNK pathways (Dumitru et al.,
2000; Kontoyiannis et al., 1999). The ARE has been previously shown to perform
multiple functions in the post-transcriptional regulation of TNFa (Kontoyiannis et al.,
1999). These include causing mRNA destabilisation in unstimulated conditions,
regulation of permanent translational silencing and increased mRNA stability and
translational derepression under stimulated conditions. Dumitru et al have shown that
the TNFa ARE also regulates nucleus to cytoplasm transport of TNFa mRNA (Dumitru
et al., 2000).
6.2.2 TPL-2 in control of septic shock and TNFa production
TNFa regulates many physiological processes, among these is activation of the vascular
endothelium. This enables increased access for antibodies and immune cells to sites of
infection, increased drainage of fluid into lymph nodes and clotting in small blood
vessels. However, this process becomes harmful in septic shock which is characterised
by fever, hypotension and intravascular coagulation, leading to multiple organ failure,
(reviewed in Aggarwal and Natarajan, 1996; Kollias et al., 1999; Vassalli, 1992).
TNFRl knockout mice are resistant to septic shock, demonstrating the importance of
TNFa in this process (Pfeffer et al., 1993). Similarly, TPL-2 knockout mice are
resistant to LPS and galactosamine induced septic shock, which is due to a failure of
135 Chapter 6 Analysis of TPL-2 function in THP-1 cells
TNFa production (Dumitru et al., 2000). TNFa signalling is not effected as TPL-2-/- mice are sensitive to septic shock induced by the injection of TNFa and galactosamine
(Dumitru et al, 2000). TPL-2 is not required for TNFa production in response to all
stimuli: TPL-2-/- splenocytes and TPL-2KD transgenic T cells produce normal levels of
TNFa after CD3 and CD28 co stimulation (Figure 6) and Dumitru et al., 2000).
Both TPL-2KD expressing THP-1 clones and TPL-2 knockout macrophages have
defects in the regulation of TNFa production in response to LPS (Figure 27 and Dumitru
et al., 2000). In the TPL-2KD THP-1 clones, LPS fails to induce TNFa mRNA,
whereas normal levels of TNFa are transcribed in TPL-2-/- macrophages. In the latter,
failure of TNFa production arises from a post-transcriptional effect, in which transport
of TNFa mRNA between the nucleus and cytoplasm is blocked (Dumitru et al., 2000).
Differences between the experimental systems used that may account for these
contrasting results are described below.
In TPL-2 null macrophages, TPL-2 is vital for LPS induced ERKl and 2 activation.
Normal levels of LPS stimulated TNFa mRNA induction in TPL-2-/- macrophages
indicate that TPL-2 does not function in regulation of TNFa transcription or has a
redundant function in this process (Dumitru et al., 2000). TPL-2 is required however for
the transport of TNFa mRNA from the nucleus to the cytoplasm, using an ARE
dependent process.
136 Chapter 6 Analysis of TPL-2 function in THP-I cells
In (human) THP-1 cells and the murine monocytic cell line J774, treatment with the
MEK inhibitor PD98059 inhibits induction of LPS mRNA following TNFa stimulation
(Rutault et al., 2000; Tsai et al., 2000). Although ERKl and 2 activation is completely blocked in TPL-2-/- mice, TNFa is still transcribed (Dumitru et al., 2000). This may signify differences in the role of ERK in TNFa transcription between primary murine and cultured cells.
In THP-1 clones, the block in TNFa transcription may arise from TPL-2KD possessing non-specific dominant negative activity, e.g. blockade of other kinases which regulate
TNFa mRNA. Alternatively, TPL-2KD may block a physiological TPL-2 function in the regulation of TNFa transcription, which is compensated in TPL-2-/- mice.
The dominant negative approach gives rise to more severe functional defects than gene inactivation in studies of the fimction of src family kinases Ick and fyn, reviewed in
(Cheng and Chan, 1997). Thymocyte development is normal in fyn-/- mice (Appleby et al., 1992; Stein et al., 1992). However, Ick-/- mice exhibit thymic atrophy and have 10-
20 fold fewer double positive thymocytes than normal (Molina et al., 1992). This incomplete inhibition suggests that another signalling molecule may compensate for the absence of Ick in the transduction of pre-TCR signals. The observation that double positive thymocytes are completely absent in fyn/lck double knockout mice supports this idea (Groves et al., 1996; van Oers et al., 1996; van Oers et al., 1995). Critically, in mice expressing a dominant negative Ick transgene the developmental block is also complete (Anderson et al., 1993; Hashimoto et al., 1996; Levin et al., 1993).
137 Chapter 6 Analysis o f TPL-2 function in THP-1 cells
Presumably, the presence of dominant negative Ick prevents signalling either by either
Ick, fyn or another compensating kinase.
If a similar situation occurs in TPL-2 signalling, this would predict that TPL-2 regulates both TNFa transcription and mRNA localisation. The TPL-2 function in TNFa transcription may be compensated in TPL-2 knockout cells, but the TPL-2 function in mRNA regulation cannot.
The biochemical events that underlie TNFa production defects in TPL-2KD THP-1 clones and TPL-2-/- macrophages are not yet fully characterised. This study has shown that overexpression of TPL-2KD in THP-1 cells results in a blockade in LPS stimulated
ERK activation and NFkBI p i05 degradation (Figure 24 and 26). Since both NFkB and
ERK are implicated in TNFa transcription (Collart et aL, 1990; Tsai et al., 2000), either or both of these disrupted processes may be the reason for why LPS does not stimulate
TNFa mRNA accumulation in these clones.
Dumitru et al address the function of NFkB in the TPL-2 knockout phenotype by examining the susceptibility of nfkbl-/- mice to septic shock and by performing a single timepoint NFkB EMSA using TPL-2-/- samples (Dumitru et aL, 2000). Since nfkbl mice are susceptible to septic shock they conclude that NFkB ‘cannot be an important determinant of the TPL-2 knockout phenotype’. This experiment does not fully address the question of TPL-2 regulation of NFkBI p i05. Since we propose that
TPL-2 promotes the proteolytic removal of NFkBI pl05, removal of NFkBI pl05 by 138 Chapter 6 Analysis o f TPL-2 function in THP-1 cells
gene inactivation may mimic the effects of TPL-2 activation, rather than inactivation.
Furthermore, although the EMSA is a good assay for total NFkB activation, as discussed above it is not a sensitive system for the measurement of NFkBI p i05 degradation.
Interestingly, although EMSAs performed by Dumitru et al show that there is no absolute defect in NFkB activation in TPL-2-/- cells, the lower band, usually composed
of p50 homodimers appears to be reduced in the single timepoint shown (Dumitru et aL,
2000).
It would be interesting to conduct a more thorough analysis of NFkB function in the
absence of TPL-2 using these mice. Thereby addressing whether TPL-2 regulates ERK
alone, or ERK and NFkB, and whether there is crosstalk between these processes. In
particular, it would be interesting to test our hypothesis that TPL-2 regulates NFkBI
p i05 degradation, which regulates NFkB activation. Murine embryonic fibroblasts
(MEFs) can be used for direct analysis of LPS, TNFa or IL-1 induced NFkBI p i05
degradation, using pulse chase metabolic labelling (A Salmeron and SC Ley,
unpublished results). Dumitru et al have shown that there is no overt change in NFkB
activation measurable by EMSA in LPS stimulated TPL-2-/- macrophages (Dumitru et
aL, 2000). However, a study of LPS induced NFkB activation (by EMSA) over a 6 hour
timecourse would be more likely to include timepoints at which NFkBI p i05
degradation is occurring. If different NFkB DNA binding complexes could be resolved
this would allow the effects of TPL-2 on p50 homodimer formation to be monitored.
139 Chapter 6 Analysis o f TPL-2 function in THP-1 cells
6.2.3 TPL-2 and the TNFa AU Rich Region
TPL-2-/- mice are defective in TNFa production by LPS stimulated macrophages, despite equivalent induction of TNFa mRNA in TPL-2-/- and +/+ macrophages
(Dumitru et aL, 2000). Dumitru et al ascribe the effect of TPL-2 on TNFa production to regulation of the ARE in the 3’ untranslated region (UTR) of TNFa mRNA. They show that while TNFa production is blocked in TPL-2-/- and TPL-2-/-TNF-/- mice, TPL-2-/-
TNpAAR^/- mice (in which a single copy of the TNFa gene is present with the ARE
sequence removed) produce the same elevated levels of TNFa as TNF^'^^/- mice
(Dumitru et aL, 2000). They conclude from this data that TPL-2 functions in a pathway
that targets the ARE of TNFa mRNA. While this may be correct, it is not the only
conclusion that can be drawn. Since removal of the TNFa ARE results in increased
levels of TNFa mRNA, the ARE can be described as an inhibitory sequence
(Kontoyiannis et aL, 1999). The increased levels of TNFa seen in TNF^*^^/- mice
occur in the presence or absence of TPL-2 (Dumitru et aL, 2000), thus it is possible that
TPL-2 and ARE regulate TNFa mRNA via parallel pathways. Interestingly however, an
examination of the localisation of LPS induced TNFa mRNA showed an inhibition of
nucleus to cytoplasm transport in TPL-2-/- macrophages (Dumitru et aL, 2000). This
phenomenon was also seen in cells treated with the MEK inhibitor PD98059 suggesting
that TPL-2 is acting through the ERK pathway in this fimction.
140 Chapter 6 Analysis of TPL-2 function in THP-1 cells
6.2.4 Are NFkBI p i05 and iKBa degradation separately regulated?
Figures 20, 21 and 22 show that in THP-1 cells stimulated with TNFa, IicBa is degraded and IKK is activated but NFkBI p i05 is not degraded. In Jurkat cells stimulated with
CD3 and CD28, IicBa is degraded but NFkBI p i05 levels remain constant (Figure 11).
Similarly, in NIH3T3 cells stimulated with TNFa, IKK is activated, IicBa is degraded
but NFkBI p i05 is not (data not shown). Since NFkBI p i05 and IicBa degradation do
not always occur in parallel, there must be some element of separate regulation of these
two processes. The observation that in Jurkat cells stably expressed TPL-2KD blocks
NFkBI p i05 degradation but not IicBa degradation (Figure 14 and 15) supports this
idea. As distinct functions for the different IkB proteins are not well defined, it is not
easy to predict how the degradation of the different of IkB proteins may be regulated.
Although IKKa and IKKp are required for IicBa degradation (Li et aL, 2000), no
published data directly addresses whether IKK is required for NFkBI p i05 degradation.
However, NFkB activation is not detectable in IKKa/p null mice, either by in vitro
stimulation of MEFs or by analysis of icB-lacZ reporter transgene expression on
embryonic sections (Li et aL, 2000). Since it is thought that NFkBI p i05 degradation
leads to NFkB DNA binding, it seems likely that IKKa and p are required for NFkBI
p i05 degradation, either directly or by regulating the expression of another required
factor.
141 Chapter 6 Analysis o f TPL-2 function in THP-1 cells
In this model, control mechanisms must be in place to regulate the differential activity of
IKK towards IkBœ and NFkBI p i05. As discussed above, these control mechanisms could take several forms, e.g. a requirement for a separate signalling pathway converging on NFkBI pi 05. Alternatively, although IKK is activated by these stimuli, it
is possible that IKK substrate access is controlled, thus a permissive mechanism may be
in place to control whether or not IKK ‘sees’ NFkBI p i05 (or the intermediate that phosphorylates NFkBI p i05). A similar situation is seen in MAP kinase signalling in yeast, although Ste 11 participates in two MAP kinase cascades, no crosstalk is seen between the two pathways, (reviewed in Burack and Shaw, 2000). Stel 1 functioning in the mating pathway is held in place by association with Ste5, and Stel 1 functioning in the osmosensing pathway is held in place by association with pbs2. It is likely that the
IKK kinase assays performed in this study are analysing total IKK activation, rather than
IKK activity towards a specific substrate.
As TPL-2 is tightly associated with NFkBI pi05 under physiological conditions
(Figures 2 and 23), and TPL-2KD blocks TNFa and LPS induced NFkBI p i05
degradation (Figures 14 and 24), we hypothesised that TPL-2 may participate in a process that regulates whether NFkBI pi05 degradation occurs in addition to IkBœ
degradation. In order to analyse TPL-2 activity under NFkB activating conditions in
which NFkBI p i05 is or is not being degraded, in vitro kinase assays were performed.
However, due to technical difficulties with the kinase assay, the results generated were
inconclusive.
142 Chapter 6 Analysis o f TPL-2 function in THP-1 cells
6.2.5 Further experiments using THP-1 cells
Given time, many further questions regarding the fimction of TPL-2 could be addressed using the THP-1 system. This study has shown that TPL-2KD can block LPS induced
TNFa mRNA induction. Dumitru et al have shown that TNFa and to a lesser extent IL-
1 production are inhibited in TPL-2 knockout mice (Dumitru et aL, 2000). A"gene chip’ approach has recently been used to analyse which mRNA are induced when THP-1 cells and primary monocytes are stimulated with LPS, peptidoglycan (PGN) or Gentamicin killed Staphylococcus Aureus. The study confirms that THP-1 cells and monocytes induce similar patterns of gene expression upon stimulation. Although many studies analyse induction of TNFa and IL-ip in response to LPS, the chemokines MCP-1, MIP-
la, MIP-lp and MIP-2a showed greatest induction in these conditions (Wang et aL,
2000). It would be interesting to investigate which of these mRNA were effected in
TPL-2KD expressing THP-1 clones, e.g. using RNase protection assays. Furthermore,
now that convincing evidence is available on the cell type in which TPL-2 operates, it is
possible to examine other receptor signalling pathways which operate in monocytes and
macrophages. The IL-1 receptor and TLR4 (through which the LPS signal is
transduced) utilise similar signalling pathways to activate NFkB and JNK (Adaehi et aL,
1998; Lomagaet aL, 1999; Thomas et aL, 1999), therefore analysis of IL-1 receptor
signalling in TPL-2KD THP-1 cells or TPL-2-/- macrophages may be informative.
Although IL-1 receptors are not constitutively expressed on THP-1 cells, they can be
induced by 24 hour culture in the presence of PMA.
143 Chapter 6 Analysis o f TPL-2 function in THP-1 cells
TPL-2-/- mice are susceptible to septic shock induced by TNFa and D galactosamine injection (Dumitru et ah, 2000). Moreover, in the data shown by Dumitru et al, these mice appear to be more susceptible to these stimuli than control mice, which would be consistent with defective NFkB activation. The authors also claim that they have preliminary evidence of a role for TPL-2 in TNFa signal transduction. Therefore, analysis of TNFa signalling in TPL-2KD THP-1 cells may be a useful way to address these issues.
A fundamental difference between this study and that of Dumitru et al is the use of gene
inactivation compared with dominant negative kinase expression. The use of antisense techniques can remove TPL-2 from cells, allowing analysis of signalling function and
transcriptional regulation in the complete absence of TPL-2. This approach has been
successfully used to interpret the function of the aPKC interacting protein p62 (Sanz et
ah, 2000). The outcome of ‘knocking out’ TPL-2 from THP-1 cells using antisense
techniques would address whether the differences between TPL-2-/- macrophages and
TPL-2KD THP-1 cells arise from differences between transformed (human) THP-1 cell
and primary murine cells or from the different signalling disruption caused by dominant
negative and knockout approaches.
It is interesting to note that the two stimuli identified by this study which cause NFkBI
p i05 degradation, TNFa and LPS, are the two stimuli identified by Dumitru et al whose
responses are disrupted in TPL-2-/- mice, although they believe that TPL-2 fimctions as
a MAPK regulator in these pathways (Dumitru et ah, 2000). Since NFkBI p i05 and
144 Chapter 6 Analysis of TPL-2 function in THP-1 cells
TPL-2 are closely associated physiologically (Figure 2 and 23), it may be interesting to analyse what effect NFkBI p i05 association or degradation has on ERK activation.
Mutations in NFkBI p i05 which may prevent TPL-2 binding have been identified in this laboratory (S Beinke and SC Ley unpublished results), if LPS induced ERK activation were reduced in THP-1 clones stably expressing this mutant NFkBI p i05, this would suggest that NFkBI p i05 is required for LPS induced ERK activation. A different approach may address whether the process of NFkBI p i05 degradation is important for ERK activation. Heissmeyer et al describe a mutation in NFkBI p i05 which renders it resistant to signal induced degradation (Heissmeyer et aL, 1999). It is possible that this type of construct could be used to block the effects of NFkBI p i05 degradation as has been shown for IkB (Boothby et aL, 1997; Brown et aL, 1995). LPS
induced ERK activation could be tested in THP-1 cell lines stably expressing non-
degradable NFkBI p i05 in order to rule out any connection between NFkBI p i05
degradation and ERK activation.
Non-degradable NFkBI p i05 mutants could also be used to generate knock-in mice,
which express only non-degradable NFkBI p i05. NFkBI p i05 has at least two
functions; acting as the source of p50 and as an IkB. If as predicted p50 generation was
unaffected in such mice, then the mutation would interfere only with the postulated IkB
function for NFkBI p i05, and would allow this function to be analysed separately.
Several groups generated IkBœDN transgenic mice, in which the IKK phosphorylation
site were mutated, and reported similar phenotypes (Boothby et aL, 1997; Hettmann et
145 Chapter 6 Analysis o f TPL-2 function in THP-1 cells
aL, 1999). A more effective dominant negative fimction was seen in transgenic mice expressing IkBœ with multiple mutations, including the IKK phosphorylation sites ser
32 and 36, the ubiquitin acceptor sites lys 21 and 22 and the tyrosine phosphorylation site at tyr 42 (Voll et aL, 2000). Thus, if further data becomes available on the relative importance of the ubiquitination sites in NFkBI p i05 degradation, or other phosphorylation sites, it is possible that a NFkBI p i05 knock-in construct containing multiple mutations may be the most effective approach.
Phenotypic and signalling analysis of knock-in mice or cell lines expressing this protein may illuminate the physiological functions of NFkBI pi 05 degradation. This approach will not specifically address the function of TPL-2 in these processes, however, phenotypic comparison of cells or mice lacking TPL-2 with those in which NFkBI pi 05 cannot be degraded may help to address whether TPL-2 is absolutely required for
NFkBI p i05 degradation and whether TPL-2 function is limited to regulation of ERK.
146 CHAPTER 7 Final discussion
Based on in vitro data suggesting that TPL-2 can regulate NFkB via the control of
NFkBI pi05 proteolysis (Belich et aL, 1999), this study aimed to analyse this regulation of this pathway, the conditions under which it operates and its effects. Data generated has shown that overexpressed TPL-2KD can disrupt NFkBI p i05 degradation stimulated by TNFa treatment of Jurkat cells or LPS treatment of THP-1 cells. It has also been shown that expression of TPL-2KD in THP-1 cells blocks LPS induced TNFa mRNA induction, although the specific signalling event underlying this is not necessarily NFkB.
7.1 What is the purpose of NFkBI pi 05 degradation?
It was known as long ago as 1991 that certain stimuli promote the proteolytic destruction
of NFkBI p i05 (Riviere et aL, 1991), although only recent studies have shown that
signal induced NFkBI p i05 destruction is more consistent with complete degradation of
NFkBI p i05 than proteolytic processing to p50 (Belich et aL, 1999; Harhaj et aL, 1996;
Heissmeyer et aL, 1999). The function of NFkBI p i05 is not limited to the iKB-like
function of inhibiting the nuclear translocation of NFkB proteins, NFkBI p i05 is also
the precursor to the NFkB subunit p50. Thus, it is difficult to determine from the
147 Chapter 7 Final discussion
ubiquitous cellular distribution of NFkBI p i05 whether p i05 degradation is important in all cell types or in certain specific contexts. In contrast TPL-2 is expressed at highest levels in thymus, spleen, liver and certain glandular tissues (Makris et aL, 1993; Ohara et aL, 1993). It is therefore possible that either NFkBI p i05 degradation is only regulated in some tissues or that other kinases regulate this process in tissues which express no TPL-2. Since TPL-2 mRNA is induced in splenocytes treated with the T cell mitogen Concanavalin A, TPL-2 function may also be controlled at the level of gene induction (Patriotis et aL, 1993).
7.2 Specific functions for specific IkB proteins?
The mammalian IkB protein group includes lKBa,p,8,y and the precursor proteins
NFkB2 p i00 and NFkBI p i05. Mice in which the IkBœ coding sequence is replaced with the IkBP coding sequence remaining under the control of the IkBœ promoter are phenotypically normal (Cheng et aL, 1998). This lead to the proposal that although these two IkB proteins appear biochemically identical, differential control of their transcription, especially in post-activation inhibition means that they perform distinct functions. Similarly, IkB8 has localised tissue distribution and associates preferentially with c-Rel containing NFkB dimers (Whiteside et aL, 1997). Presumably, in this model the unique function of NFkBI pi05 is its relatively high affinity for p50 and/or the relatively slow nature of its degradation. Other signalling proteins have temporally restricted functions; e.g. Ras mediates early ERK activation in PCI2 cells, however,
Rap-1 mediates the persistent ERK activation required for neuronal differentiation of these cells (York et aL, 1998).
148 Chapter 7 Final discussion
7.3 How to define the purpose of NFkBI pi 05 degradation
In the TPL-2KD transgenic mouse analysis it was hoped that the identification of a phenotype would identify the physiological processes in which TPL-2 participates. The role of NFkBI pi 05 degradation in such a process may then be analysed. This reflects a general obstacle in the study of NFkBI pi 05 degradation; how to identify circumstances under which NFkBI pi05 degradation is important. Ishikawa et al addressed this question by the generation of nfkbl ACT mice, in which p50 is expressed but the NFkBI p i05 c-terminal is removed by gene inactivation (Ishikawa et aL, 1998). These mice show chronic inflammation and increased susceptibility to opportunistic infections, which underlines the importance of the NFkBI pi 05 c-terminal. Interestingly, levels of some NFkB regulated mRNAs were increased whereas other were decreased, which may indicate that p50 homodimer fimction is context dependent. However, interpretation of this study in terms of the IkB function of NFkB I pi 05 is limited by the fact that the gene inactivation has not simply removed the NFkBI p i05 IkB fimction but has also generated more p50.
Assuming that regulation of nuclear p50 is a primary role of NFkBI pl05 degradation, a
‘reporter transgene’ system could be used to analyse NFkBI p i05 activity in different tissues. Expression of a reporter gene such as luciferase, green fluorescent protein
(GFP) or LacZ under the control of a p50 homodimer responsive kB element could be monitored in mice by histology or FACS analysis. The expression pattern of the reporter protein could then be used to indicate which tissues were generating p50 homodimers. It may then be possible to analyse NFkBI p i05 degradation in such
149 Chapter 7 Final discussion
tissues. P50 reporter miee could then be crossed with mice in which a candidate NFkBI p i05 regulator was altered by transgenesis or gene inactivation.
An alternative approach to the question of the importance of NFkBI p i05 degradation would be the use of non- degradable NFkBI pi 05 mutants (Heissmeyer et al., 1999). If p50 generation from such mutant was unaffected then analysis would specifically address the IkB function of NFkBI p i05 (not its precursor function). This construct could then be used for the generation of transgenic or knock-in miee. Any phenotype seen in these mice may have arisen from the failure of signal induced NFkBI p i05 degradation and would then identify tissues or processes in which NFkBI p i05 degradation is important. Similar approaches were taken by several groups to address the role of NFkB in T cells (Boothby et al., 1997; Hettmann et al., 1999; Voll et al.,
2000).
7.4 MAP Kinase regulation by TPL-2
The recently published TPL-2 knockout mouse has confirmed in vitro data suggesting that TPL-2 has a function in MAP kinase regulation (Dumitru et al., 2000).
Interestingly, it is not clear from the paper exactly where TPL-2 is acting in this pathway. Since both TPL-2 and NIK have homology with the MAPKKK Kinase Ste20 in addition to MAPKKK homologies, it is possible that TPL-2 could be functioning in either of these roles. Since treatment of mice with the MEK inhibitor PD98059 also causes defects in ERK activation and TNFa production, and MEK phosphorylation is reduced in TPL-2-/- macrophages, it is likely that TPL-2 is acting upstream of MEK.
150 Chapter 7 Final discussion
So does a proven MAPK regulation function for TPL-2 exclude a role for TPL-2 in
NFkBI pi 05 regulation? These are many examples of kinases that function in multiple pathways, e.g. Stell. Stell is a MAP3K from Saccharomyces cerevisiae which functions in multiple MAPK cassettes, including the mating and osmosensing HOG pathway, however, no ‘cross talk’ between pathways has been observed. The components of the mating response, Stel 1 (MAP3K), Ste7 (MAP2K) and Fusl (MAPK) are held together by association with the scaffold protein Ste5 (Choi et al., 1994). The components of the HOG pathway S tell, Pbs2 and Hogl are all associated via the
MAP2K Pbs2 (Posas and Saito, 1997). In mammalian cells, mutations in the MAP3K
Rafl which prevent its binding to its MAP2K MEK 1/2, impairs only a subset of the cellular responses to Raf activation (Pearson et at., 2000). Interestingly, Rafl induced,
NFkB mediated neurite outgrowth was normal in such cells.
Based on the example of Stell or Rafl, is it possible that TPL-2 has more than one function? e.g ERK and NFkBI pi05 regulation? Is the pathway that TPL-2 regulates controlled by its localisation? In thorough Yeast Two Hybrid (Y2H) screening carried out in this laboratory using the conventional Y2H (Fromont-Racine et al., 1997) and the
Ras Recruitment system (Broder et al., 1998), NFkBI p i05 was the only TPL-2 interacting protein identified (M Belich unpublished results). It is well known that some interactions are more readily detectable by Y2H screening than others however, which may be the reason why no other proteins were found. MEK was never identified in these screens however, it is possible that the ‘kinase substrate’ complex formed is so transient that is not readily detected by Y2H, which is most useful in identification of
151 Chapter 7 Final discussion
more stable interactions, e.g. the molecular complex assembled upon TNF receptor activation. Immunodepletion studies have shown that in HELA cells 100% of TPL-2 is associated with NFkBI p i05, which would argue against two pools of TPL-2 regulating two different signalling pathways. However, whether all TPL-2 is associated with
NFkBI p i05 in other cell types has not yet been addressed.
Is it possible that TPL-2 activity towards the ERK pathway is regulated by its interaction with NFkBI pi 05? Since TPL-2 AC cannot interact with NFkBI pi 05 yet is oncogenic it is tempting to speculate that NFkBI p i05 association does in some way control TPL-2 activity. We have not identified any conditions under which TPL-2 disassociates from
NFkBI p i05, although it has been noted that TPL-2 and NFkBI p i05 are degraded with similar kinetics. It may be possible to test the theory that the interaction between
NFkBI p i05 and TPL-2 regulates TPL-2 activity towards the ERK pathway by analysing ERK activation in cells expressing mutant NFkBI p i05 which cannot interact with TPL-2 or cannot be degraded.
This study has concentrated on using a dominant negative approach in order to dissect the function of TPL-2 in NFkB regulation. Similar approaches identified functions for
NIK and MEKKl in NFkB regulation by IKK activation (Lee et al., 1997; Malinin et al., 1997; Regnier et al., 1997), both of which, like TPL-2 have been shown to function in MAP Kinase regulation. However, when MEKKl knockout mice and ES cells were analysed, although JNK activation was compromised, MEKKl was not found to be critical for NFkB activation (Xia et al., 2000; Yujiri et al., 2000). However, this was
152 Chapter 7 Final discussion
only tested in ES cell, fibroblasts and macrophages. NIK was shown to have a cell-type specific function in NFkB activation (Garceau et al., 2000). The aly mouse carries mutant NIK genes, which results in defects in NFkB activation in response to CD40 in dendritic cells but not B cells. However, NIK is only required for NFkB activation in FI------^ response to a subset of dendritic cell NFkB stimuli, as EPS responses are unaffected.
Interestingly, it is not clear what the aly mutation actually does to NIK, although it has been proposed to uncouple NIK from upstream regulation by the TRAF proteins.
Thus, at least one MAPKKK (NIK) regulates NFkB activation. However the receptor specific and cell type specific defects seen in aly mice suggest certain themes in NFkB regulation by MAPKKK. It is possible that the since NFkB defects are limited to
B cells in aly mice that NIK has a specific function in this cell type, and so by implication, other kinases perform this function in other cell types. A similar situation is seen in ERK regulation, in which A-Raf, B-Raf and TPL-2 function as MAP3K under different conditions (Bogoyevitch et al., 1995; Dumitru et al., 2000; Yamamori et al.,
1995). This scenario would explain why so many kinases have been implicated as IKK kinases, and why no single candidate IKK kinase is absolutely required for NFkB activation.
7.5 Which molecules co-operate with TPL-2 in endotoxin responses?
Dominant negative and knockout studies have partially defined the signal transduction proteins involved in EPS responses, (reviewed in O'Neill and Dinarello, 2000), these include MyD88, IRAKI,2 and M, and TRAF 6 (Kawai et al., 1999; Swantek et al.,
153 Chapter 7 Final discussion
2000) (Li et a l, 1999b; Lomagaet ah, 1999; Wesche et ah, 1999). Of these IRAK-1 and MyD88 are required for LPS induced endotoxic shock. Data on the role of IRAK-1 and MyD88 in ERK stimulation is less clear. MyD88 gene inactivation does not prevent but delays ERK, JNK, P38 and NFkB activation in response to LPS (Kawai et al.,
1999). This suggests that MyD88 transduces signals upstream of TPL-2 and other pathways early in activation, but that this function can be performed by other proteins late in activation. IRAK I null mice have defects in LPS induced JNK and P38
(Swantek et al., 2000), however, the ERK pathway is only partially compromised. It is possible that IRAK-2 or IRAK M can compensate for IRAK-1 in this function. Thus, the emerging data suggests that TPL-2 may act downstream of MyD88 and IRAK-1, and upstream of MEK in macrophage LPS signalling.
7.6 Analysis of NFkB in TPL-2-/- mice
As discussed above, a function for TPL-2 in MAPK regulation does not necessarily preclude a function in NFkBI p i05 and NFkB regulation. Further analysis of NFkBI p i05 function in TPL-2-/- cells may clarify the significance of the results of this study showing that TPL-2 regulates NFkBI p i05 degradation. In particular, as macrophage and splenocyte (probably B cell or macrophage) function is specifically compromised in
TPL-2-/- mice, attention should focus on these cell types. Examination of NFkB activation should include analysis at the late timepoints during which NFkBI p i05 degradation is occurring. In particular, formation of p50 homodimers could be examined by BCL-3 IP-EMSA or by conventional EMSA. Crossing TPL-2-/- mice with
154 Chapter 7 Final discussion
NFkB reporter mice may also address whether NFkB activation is compromised in any other tissues, either by FACS analysis, or by histological analysis.
7.7 Potential clinical significance of TPL-2
The resistance of TPL-2 knockout mice to septic shock induced by LPS and
Galactosamine appears to arise from a defect in TNFa synthesis (Dumitru et al., 2000).
This data implies that TPL-2 may be a suitable clinical target for the treatment of septic shock and other conditions in which TNFa has a pathological function, such as
Inflammatory Bowel Disease, Arthritis or Multiple Sclerosis (MS) (Keffer et al., 1991;
Kontoyiannis et al., 1999; Ruddle et al., 1990; Targan et al., 1997). These propositions could be tested by subjecting TPL-2-/- mice to experimental models of disease. For example: Experimental Autoimmune Encephalomyelitis (EAE) an autoimmune model for human MS or Collagen Induced Arthritis (CIA), (reviewed in Kollias et al., 1999).
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