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1. General description of the project.
As explained in the Form C, the research proposal will be articulated around two main objectives: a
better understanding of the signal transduction leading to (i) NF-B activation and (ii) cell death both in
vitro and in vivo.
Aim:
Increase our understanding of the molecular and cellular events that (i) determine the sensitivity for
and onset of inflammation, (ii) participate in and control either positively or negatively the propagation
of the inflammatory cascade, (iii) participate in the resolution of inflammation, and (iv) upon their
dysfunction contribute to acute and chronic inflammatory disease.
Overall strategy:
An integrated and multi-level approach based on three interacting research axes will be followed.
Constituting a first axis, cellular models of signalling molecules involved in NF-B activation and NFB target gene expression and their modulation by intrinsic and microbial-induced signalling pathways
will be studied. This will be extended into the exploration/validation of the role of presumed key
molecules in selected mouse models of inflammatory disease. Inversely, starting from animal models
for human inflammatory diseases, mechanisms implicated in various stages of inflammatory diseases
or exerting control over the inflammatory process will be studied and translated to the molecular level
in order to elucidate the underlying (anti-)inflammatory signalling pathways. Interconnected with this
bidirectional approach, molecular mechanisms contributing to or activated by cell death processes will
be dissected in cellular models for their implications for inflammatory pathways and analysed in the
living organism, thus constituting research axes two and three.
Part 1. Signal transduction leading to NF-B activation.
NF-B is a dimeric transcription factor composed of five proteins (RelA, p50, c-Rel, p52 and RelB)
maintained inactive in the cytoplasm by inhibitory proteins (IBs). Upon activation, NF-B is released
from the inhibitory proteins and translocate to the cell nucleus where it transactivates target genes.
There are two distinct NF-B-activation pathways: the classical and the alternative. The classical
pathway was the first being characterized and is triggered by pro-inflammatory signals such as
cytokines, bacterial and viral infections, all of which activate the IKK complex.
This complex is
composed of two catalytic subunits, IKK and IKK and a regulatory subunit IKK (also known as
NEMO). This complex phosphorylates NF-B bound IBs, thereby targeting them for proteasomal
degradation and liberating NF-B dimers to enter the nucleus and mediate transcription of target
genes. This reaction mostly depends on the catalytic subunit IKK which carries out IB
phosphorylation. The alternative NF-B activation pathway which is triggered by some ligands from
the TNF family (such as BAFF, LT, CD40L,….) involves the upstream kinase NF-B-inducing kinase
(NIK) activating IKK homodimers, independently of either IKK or IKK.
phosphorylation and processing of p100.
This leads to the
The two pathways switch on different gene sets and
therefore mediate different immune functions. The contribution of the classical pathway to acute
inflammation and cell survival mechanisms is well accepted, and sustained NF-B in various
malignancies has been described. Owing to the variety of target genes activated by the classical
pathway, which include those encoding cytokines, chemokines, proteases and inhibitors of apoptosis,
it has been proposed that the classical NF-B activation pathway might also link inflammation to
tumour promotion and progression. Therefore, an emerging link between chronic inflammation and
cancer is being established and some cancers can also be considered as chronic inflammatory
diseases. In this part of the proposal, we will better characterize and compare the two signal
transduction pathways leading to NF-B activation, as well as their crosstalk with some other
signalling pathways that contribute to proinflammatory gene expression. In addition, we will address
several molecular mechanisms of signal attenuation that determine the kinetics and strength of NF-B
activation. Regulation of NF-B-dependent gene expression will not only be studied at the level of the
initiating and propagating signalling events in the cytoplasm, but also at the level of the gene in the
nucleus.
WP1: Study of the classical and alternative pathways of NF-B activation in cellular models.
Although the knowledge of NF-B activation in response to various receptors has increased
considerably and has uncovered a crucial role for protein-protein interactions, multiple aspects remain
unclear. We will analyse the potential role of specific signalling molecules at the cross-road of NF-B
and other signalling pathways (e.g the IRF pathway) that determine a proper immune response.
Special attention will also be given to the molecular mechanisms that regulate the multiple proteinprotein interactions in NF-B signalling in response to various stimuli (e.g. TNF, TLRs and TCR). In
this context, we will study the role of protein ubiquitination and acetylation.
WP1.1 Characterization of signalling molecules at the cross-road of NF-B and other signalling
pathways
Molecular mechanisms of IRF and NF-B activation through TANK
ULG2 studies the scaffold protein TANK/I-TRAF, which is a TRAF2 and IKK-interacting protein that is
required for the TNF-induced and NF-B-dependent expression of a selection of target genes (Bonif et
al., 2006). The fact that TANK also binds TBK1 and IKK kinases, which are essential components of
the IRF signalling pathway (Fitzgerald et al., 2003), suggests also a role for TANK in IRF activation.
This will be further addressed by deciphering the phase of NF-B and IRF activation in TANK-deficient
macrophages treated with various stimuli, by identifying TANK-dependent genes in LPS-treated
macrophages through micro-array analyses, and by functionally characterizing the role of novel TANKinteracting proteins (e.g. IRF7) that were recently identified by yeast two-hybrid screening (ULG2,
unpublished data). Moreover the role of specific posttranslational modifications of TANK
(phosphorylation, ubiquitination) will also be studied by ULG2 and UG2.
Caspase-mediated activation of NF-B
The proteolytic activity of caspases mainly connotes their central role in apoptosis and inflammation.
UG2 and others recently discovered that caspase-1, -2 and -8 can also activate NF-B through the
recruitment of specific NF-B-signalling molecules in different caspase containing protein complexes:
the inflammasome (caspase-1 and caspase-8) or the stressosome (caspase-2) (reviewed in Lamkanfi
et. al., 2006). In this project, the formation of the stressosome under different conditions of cellular
stress (DNA damage, ATP release, ER stress, mitochondrial ROS, heat, TLR) will be studied by UG2
in cells that are deficient in different signalling molecules, using gel filtration, blue native
electrophoresis, confocal microscopy, immunoprecipitation and mass spectrometry analysis (see also
WP6.2). Structure-function analysis of the CARD domains of caspase-1 and caspase-2 will reveal the
potential of CARD-only proteins to interfere with NF-B activation in macrophages (UG2 and ULG1).
Modulation of NOD2-dependent NF-B signalling by the actin cytoskeleton
The cytosolic Nod2 protein plays a key role in sensing bacteria and generating an inflammatory
immune response that is mediated by the activation of NF-B (reviewed by Strober et al., 2006).
Specific mutations in the Nod2 gene have been associated with Crohn disease (CD). To date, little
information exists about the regulation of the Nod2-signalling pathway. ULG1 recently showed that
Nod2 is recruited in membrane ruffles through Rac1 and that actin disruption significantly increased
Nod2-mediated NF-B activation (unpublished data). The recruitment of Nod2 in dynamic cytoskeletal
structures could be a strategy to both repress Nod2-dependent NF-B signalling in unstimulated cells
and rapidly mobilize Nod2 during infection with invasive pathogens that interfere with the cytoskeleton
machinery. ULG1 will investigate this further in a model of apical invasion of Nod2-transfected
polarized intestinal epithelial cells by Salmonella. Nod2 recruitment to Salmonella invasion sites and
interactions of Nod2 with proteins involved in actin reorganization (e.g. Rac1) will be examined by
colocalization (confocal microscopy) and coimmunoprecipitation assays. The role of Salmonellainduced reorganisation of the cytoskeleton machinery will be analysed by ULG1 and UG2 using
specific Salmonella type III secretion mutants or cells expressing specific Rac1 mutants. ULG1 and
UG2 will also try to determine the domains and/or residues of Nod2 involved in its recruitment to
invasion sites as well as the behaviour of CD-associated Nod2 mutants.
Role of Protein kinase D in NF-B signalling pathways
KUL in collaboration with UG2 aims to investigate the contribution of Protein Kinase D (PKD) in
signalling towards NF-B in response to Reactive Oxygen Species (ROS), TLR or TCR stimulation.
ROS leads to Abl-mediated tyrosine phosphorylation of PKD, which is both necessary and sufficient to
activate NF-B (Storz and Toker, 2003). KUL recently found that tyrosine phosphorylated PKD induces
an invasive phenotype in normal and malignant cells (unpublished data) and will investigate in
collaboration with UG2
the underlying molecular mechanisms of NF-B activation by tyrosine
phosphorylated PKD, as well as the role of NF-B activation in the invasion promoting effect of PKD.
Furthermore, KUL and UG2 will investigate whether these effects can be regulated by ABINs and
whether other tyrosine kinases (besides Abl) can phosphorylate PKD.
PKD is a signalling target of PKC and the latter plays a crucial role in TLR4-induced NF-B activation
(Castrillo et al., 2001). KUL and UG2 will therefore investigate whether TLR4 activates PKD, whether
this activation occurs through PKC and whether PKD is required for TLR4-induced NF-B activation.
Since PKD has been shown to associate with the kinase Btk and since Btk plays a crucial role in TLRinduced NF-B activation (Jefferies et al., 2003), KUL and UG2 will also investigate the potential role
for a PKD-Btk complex in TLR-induced NF-B activation. A role for PKD-mediated activation and
phosphorylation of HPK1 in TCR-induced NF-B activation has recently been demonstrated (Arnold et
al., 2005). Since PKD is a downstream target of PKC-, KUL and UG2 want to investigate whether
PKD is part of the PKC-/CARMA/Bcl10/MALT induced NF-B activation pathway in response to TCR.
Molecular crosstalk between oxysteroid nuclear receptors and NF-B in foamy macrophages
M. tuberculosis infects and survives within macrophages, the cells that are out to provide an effective
initial barrier to contain infection. Mycolic acid (MA), a characteristic biolipid of the cell wall of M.
tuberculosis was recently discovered by UG1 to mimic pathogen-associated host innate immune
response (Korf et al., 2005). A major feature of the MA-induced macrophage response is a disruption
of lipid homeostasis, resulting in excessive cholesterol accumulation and the development of foamy
macrophages. This imbalance between cholesterol uptake and export was reflected also at the
molecular level as apparent from the increased mRNA levels of CD36, involved in the uptake of
oxidized LDL, and the decreased levels of the cholesterol transporter, ABCA1, and its extracellular
acceptor, APOE. Finally, a striking induction of the cholesterol-sensing oxysteroid nuclear receptor,
LXR, was observed. LXR and other lipid-activated transcription factors such as PPAR, functioning
as lipid sensors and guarding the lipid homeostasis within the cell, have also been implicated in the
control of innate immune response genes (Joseph et al., 2004). In agreement herewith, MA-treated
macrophages exhibit a markedly altered responsiveness to LPS, increased expression of TNF and
IDO, decreased expression of IL-10, and novel expression of IFN- and MPO. Within the present
proposal, UG1 intends to define in more details the molecular pathways through which accumulation
of cholesterol modifies innate immune functions of MA-induced foamy macrophages. TLR4 signalling
pathways leading to NF-B and IRF3/STAT1 activation will be verified in macrophages treated in
culture with MA under conditions of normal and low oxidized LDL, thus allowing verifying the
interference of cholesterol accumulation. The cross-talk between oxysteroid receptors and NF-B will
be verified by using macrophages isolated from mouse strains deficient for the A and b LXR
paralogues and/or treated with LXR and PPRA agonists. Also the response pattern of macrophages
deficient for TANK, a scaffold protein required for the NF-B dependent expression of a subset of
target genes will be investigated by UG1 and ULG2.
Regulation of IKK complex activity through protein acetylation.
In addition to protein ubiquitination (Chen, 2005), protein acetylation has emerged as a key regulatory
element in the field of NF-B-dependent gene expression (Quivy and Van Lint, 2004). ULG1 has
demonstrated a strong synergistic transcriptional activation of NF-B-dependent promoters (IL-6, HIV-
1, ICAM-1, IL-8) by NF-B inducers of the classical pathway (TNF, IL-1, PMA and pervanadate) and
deacetylase (HDAC) inhibitors. Mechanistically, HDAC inhibitors prolonged the activated IKK complex
activity. This prolongation could explain the persistent proteasome-mediated degradation of the
inhibitory protein IB and the resulting prolonged presence and DNA-binding of NF-B in the nucleus
(Quivy et al., 2002; Adam et al., 2003; Quivy and Van Lint, 2004). These results suggest that HDAC(s)
control(s) directly or indirectly the activity of the IKK complex. In this regard, we have shown that
several HDACs interact with the IKK complex probably via IKK. Within the current proposal, ULG1
will compare, in normal versus HDAC knock down cell lines, the activation kinetics of the NF-B
pathway and their functional consequences. Interestingly with pervanadate, in contrast to the other
NF-B inducers, ULG1 observed no prolongation of IKK activation by HDAC inhibitors, but a
downregulation of the IB
mRNA transcription, and UG1, ULG1 will examine the molecular basis
for this difference.
WP1.2. Study of the alternative pathway of NF-B activation in cellular models.
In our studies on the alternative NF-B activation pathway, ULG1 will mainly focus on NF-B
activation in response to the Lymphotoxin- Receptor (LTR) (Dejardin et al., 2002). Biochemical and
genetic studies have demonstrated a role for this receptor in the development, as well as in the
maintenance of the architecture, of secondary lymphoid organs. Abnormal spatio-temporal expression
of the LTR is associated with the formation of ectopic lymphoid structures, which is a hallmark of
most chronic inflammatory diseases. So far, the only kinases known to play a role in the activation of
the alternative pathway are NIK and IKK but the mechanisms controlling their kinase activity is not
well defined (Pomerantz and Baltimore, 2002). Phosphorylation and dephosphorylation of signalling
proteins allow to turn on and/or off most signalling pathways in eukaryotic cells and have been studied
for decades. Although the phosphoacceptor sites within the kinase domain of NIK and IKK that
modulate their kinase activity have been identified, it is still not known whether they are the only
requirement for the full activation of the enzymatic activities of NIK and IKK mediating the processing
of p100. There is a growing body of evidence that ubiquitination and de-ubiquitination activities tightly
control a wide range of signalling pathways, TNF signalling being one striking example (reviewed by
Chen, 2002). Whereas the mechanism of A20-mediated de-ubiquitination of polyubiquitinated RIP via
K63-linkage followed by the polyubiquitination of RIP via K48-linkage is well defined for the negative
feedback regulation of TNF-induced NF-B signalling (reviewed by Heyninck and Beyaert, 2005), up
to now no such mechanism has been discovered for the alternative NF-B pathway. ULG1, EU1 &
UG2 will investigate if and how monoubiquitination and/or the various ubiquitin linkages of
polyubiquitination reactions control positively and/or negatively the activation of the alternative
pathway.
Another goal is to understand how TRAF proteins control the induction of the alternative pathway.
Indeed, preliminary data indicate that TRAF2 and TRAF3 have an inhibitory function on the alternative
NF-B pathway as opposed to their positive role in the classical NF-B pathway. How the inhibitory
function of TRAF proteins is alleviated upon ligation of the LTR is still an unanswered question. First
of all, post-translational modifications of TRAF proteins that occur following the engagement of the
LTR will be investigated by ULG1 and UG2. In this regard, the putative role of the protein TANK/ITRAF will be studied. Indeed, it has been shown that TANK exclusively binds TRAF2 and TRAF3. The
putative role of c-IAP1 for the steady state of TRAF2 and TRAF3 will also be investigated by ULG1
and ULG2.
Finally, a forward genetic approach based on the use of a chemical mutagenic compound to identify
new proteins specific to the LTR-mediated alternative NF-B pathway will be conducted by ULG1,
ULG2, and EU1. A particular attention will be given to new IKK-interacting partners (EU1) via an in
vivo biotinylation approach (De Boer et al., 2003). Potential new candidates will be targeted with
siRNA or lentivirus-expressing shRNA to downregulate their level of expression both in vitro system
and in vivo. The effect of the RNA interference will be evaluated by ULG1 in vivo in an adjuvant
arthritis model in rats for which it would be feasible to inject the siRNA or lentirus-expressing shRNA
directly into the joints.
WP2. Signal modulation of NF-B activation
An effective immune system requires rapid and appropriate activation of an inflammatory response
upon infection, in order to terminate the spread of the infection as quickly as possible. However, as the
inflammatory response evolved at the cost of self-tissue damage, failure of resolution can have
detrimental effects to the organism and can result in the development of chronic inflammatory
disorders. Because of this inherent danger of inflammation, the crucial activation steps of this process
are tightly regulated. Hence, many cellular proteins control the NF-B-signalling pathways induced by
pathogens and pro-inflammatory cytokines. Moreover, also microbes themselves have developed
several strategies to modulate NF-B and IRF mediated signalling and evade the immune system.
These checkpoints act at different levels in NF-B signalling and apply various strategies to inhibit NFB activation, such as interfering with protein-protein interactions, ubiquitination and acetylation.
Understanding these negative regulatory mechanisms of NF-B activation is of great value for the
development of new approaches to treat NF-B-mediated inflammatory diseases.
WP2.1. Mechanisms of signal attenuation of NF-B activation in the cytoplasm.
UG2 aims to study the negative feedback regulation of NF-B activation by A20 and ABINs
(respectively ABIN-1, -2 and –3). They previously showed that ABINs can inhibit NF-B activation
upstream of IKK in response to various stimuli in vitro as well as in murine models of hepatitis and
asthma (e.g. Wullaert et al., 2005; El Bakkouri et al., 2005). To further elucidate their mechanism of
action as well as the mechanisms that regulate their activity, A20 and ABINs will be subjected to
detailed protein-protein interaction studies. Yeast two-hybrid screening already revealed some
interesting candidates that are currently analysed (UG2 & ULG2). UG2 will also identify novel A20and ABIN-interacting proteins via ‘Mammalian Protein-Protein Interaction Trapping’ (MAPPIT)
(Eyckerman et al., 2001), as well as via TAP-tagged affinity purification of an ‘in vivo’ expressed TAPA20 transgene (see also WP5). The role of A20, ABINs and their interacting proteins will be
functionally analysed using RNAi as well as cells derived from knockout mice. Conditional ABIN-1 and
–3 knockout mice will therefore be generated by UG2 during this project (see also WP5). Interesting
perspectives are also gained from the recent demonstration that A20 has dual ubiquitin ligase and
deubiquitinase activities (reviewed by Heyninck and Beyaert, 2005). Using complementation studies of
A20-deficient MEF cells with specific A20 mutants, UG2 will investigate the specific role of this dual
ubiquitin-editing activity of A20 in the regulation of specific ubiquitin-dependent protein-protein
interactions that are involved in NF-B and IRF activation. In addition, UG2 will identify and
characterize several known and novel A20-binding proteins as potential substrates or regulators of
A20 activity.
UG2 studies the regulation of NF-B activation by MyD88s, which they previously identified as an
alternative splice variant of the IL-1R and TLR-adaptor protein MyD88 that is produced in LPS-treated
monocytes (Janssens et al., 2002). MyD88s prevents IL-1 and TLR induced NF-B activation by
inhibiting the recruitment of IRAK4 to the receptor complex. The effect of MyD88s will now also be
analysed on other MyD88-dependent and MyD88-independent signalling pathways, including the IRF
pathway (in collaboration with ULG2).
UG2 has clear evidence that TNF leads to a serious and irreversible inhibition of ligand binding of the
glucocorticoid receptor (GR) and that this forms the basis of excessive NF-B activity (unpublished
data). In the context of this project, UG2 will try to identify the mechanism of GR inhibition as well as
its protection by HSP70 (Van Molle et al., 2002). Therefore, they will analyse GR modifications in
response to TNF, thereby focusing on the role of IKKs, MAP kinases and MKPs (UG1 and UG2).
Therefore, UG1 and UG2 will analyse GR modifications in response to TNF, thereby focusing on the
role of IKKs, MAP kinases and MKPs.
WP2.2. Interference of pathogens with NF-B and IRF signalling pathways.
ULG1 showed that Varicella-zoster virus (VZV) can activate NF-B and IRF3 via TLR-dependent and independent signalling, but that infected cells become resistant to TNF and to IFN- (unpublished
data). In this project ULG1 will try to identify the viral components and the intracellular signalling
components involved in the activation of NF-B and IRF3 through TLR2 and TLR3, and investigate the
potential role of intracellular receptors such as RIG1 or PKR. In addition, we will study the putative role
of two viral kinases in NF-B and IRF3 activation, as well as the molecular basis for the inducible
resistance to TNF and IFN-. The latter will be addressed by detailed analysis of the signalling
pathways in infected cells and by microarray analysis of the genes modulated by VZV infection (in
collaboration with UG2 and ULG2).
WP3: Nuclear mechanisms in NF-B signalling.
Once NF-B has migrated into the nucleus, it transactivates a large variety of target promoters with a
gene specificity and an activation strength and duration, which is ensured by post-translational
modifications (including phosphorylation and acetylation), by the availability of co-acting transcription
factors and non-DNA-bound cofactors, and by the recruitment of chromatin-modifying enzymatic
complexes.
WP3.1. Characterization of cofactor complexes involved in NF-B-driven gene expression
UG1 and others have demonstrated crucial involvement of multiple kinases (i.e. PKAc, RSK1/2,
MSK1/2, and IKK) to fine-tune NF-B-dependent gene expression at the transcription factor, cofactor
(SMRT, CBP) and chromatin (histone H3) level (Vermeulen et al., 2003). However, the interplay with
other cofactor families [histone acetyltransferases (HATs), histone deacetylases (HDACs), histone
methyltransferases (HMTs)] is less well understood. UG1, EU1 and EU2 will examine time-dependent
cofactor associations, localization and nucleocytoplasmic shuttling dynamics during gene activation
(via classical or alternative pathways) or gene repression by glucocorticoids and/or PPAR nuclear
receptor agonists by means of immunoaffinity and/or immunofluorescence approaches. Functional
involvement of individual or tandem cofactor complexes in gene expression/repression of IL-6 gene
will be further evaluated by siRNA tools or KO cells. Co-immunoprecipitation assays of tagged cofactors in the presence of absence of kinase inhibitors, deacetylase inhibitors (HDACs), or
(de)methylase inhibitors should allow dissecting relationships in co-factor complex formation and/or
activities in a gene-specific way.
WP3.2. Role of epigenetic modifications in IL-6 transcriptional regulation.
IL-6 is a tightly controlled inflammatory cytokine which preserves immune homeostasis, whereas
excessive IL-6 amounts causes chronic inflammatory disorders and tumorigenesis. Various stimuli,
like oxidative stress, viruses (HIV, HTLV), DNA damage, classical (TNFR, TLR, IL1R) and alternative
(CD40, BAFF, LT) NF-B signalling pathways, can trigger IL-6 gene expression. Previous studies
have focused on IL-6 promoter chromatin regulation (nucleosome mapping, chromatin accessibility
assays) in benign and metastatic breast cancer cells, which differentially express the IL-6 gene
(Ndlovu et al., 2006). Within the current proposal, UG1 will further analyse IL-6 promoter DNA
methylation patterns in presence or absence of DNA methylation inhibitors (5’-azacytidine) or PARP
inhibitors (i.e. aminobenzamide, which has been described to elicit DNA methylation) in relation to
chromatin accessibility in breast cancer cells (MDA-MB231 vs. MCF7), epithelial cells (A549, TC10)
and immune cells (primary B cells, dendritic cells) in response to different stimuli.
Toll-like receptors (TLRs), which activate innate and adaptive immune responses, are thought to be
restricted to immune cells. However, TLRs are also (over)expressed on (metastatic) breast tumor
cells, suggesting that TLR activation may be an important event in tumor cell immune evasion. UG1
will further analyse in breast cancer cells the effect of epigenetic cancer drugs (5’-azacytidine, HDAC
inhibitors, PARP inhibitors) on the “inflammatory” epigenetic network (DNA methylation, histone
modifications, nucleosome remodelling) and IL-6 gene expression modifications in response to
endogenously generated (HSPs, HMBG1) or exogenous (LPS) stimuli and/or TLR antagonists (siRNA,
E5564, peptides)
WP3.3. Combinatorial control: NF-B activation and other activation cascades.
Bacterial toxins were found to interfere with pathogen responses via MAPK and NF-B signalling
pathways. This allowed the identification of a subclass of MAPK-sensitive NF-B target genes with
combinatorial promoter regulation by CREB and NF-B. UG1 will focus on the role of MSK/RSK
kinases in mediating gene-specific crosstalk between CREB and NF-B (dimerization, localization,
modifications, chromatin accessibility) at the IL-6 gene promoter. Another example of combinatorial
control concerns differential gene repression efficacy of glucocorticoids on NF-B (De Bosscher et al.,
2003) versus NF-B-IRF3 target genes in response to TLR signalling. UG1 will focus on promoterspecific GR-NF-B versus GR-NF-B/IRF3 transrepression mechanisms during inflammatory gene
responses. As PPAR negatively interferes with inflammatory gene expression through interference
with NF-B (Delerive et al., 1999), UG1 and EU2 will additionally explore promoter-specific PPAR-NFB versus PPAR-NF-B/IRF3 transrepression mechanisms and interference between PPAR and GR.
WP3.4. Implication of Sir2/Sirt1 in NF-kB-driven gene expression associated with aging.
As aging is associated with accumulation of DNA damage and with increased IL-6 expression levels,
various strategies are aimed to protect against inflammation syndrome-related aging discomforts.
Interestingly, caloric restriction or caloric mimetics (polyphenolic compounds) protect against aging via
regulation of Sir2/Sirt1 (a class III HDAC). Important targets of Sirt1 are p53, NF-B and Ku70. UG1
and ULG1 will investigate IL-6 gene regulation by p53, NF-B and Ku70 in response to DNA damage
pathways (ATM/PIDD) in wild-type and Sir2 KO cells. Furthermore, UG1 and ULG1 will investigate
concomitant acetylation of selected factors which may affect cofactor interaction and/or subcellular
localization.
Part 2. Inflammation regulation in animal disease models
The real biological significance of molecules involved in the regulation of NF-B and members of the
IRF transcription factors, but also of genes induced or repressed by these transcription factors, can
only become clear by studying them in in vivo models of pathophysiological relevance. This constitutes
the major focus of part 2 of the project. Specifically, the NF-B signalling pathways and the
involvement of agonists of NF-B (e.g. cytokines, pathogen-associated molecular patterns leading to
TLR-activation, cellular stress), NF-B itself and its effector genes will be studied ex vivo and in vivo.
Tissues or enriched cell populations isolated from mice exposed to various types of inflammatory
insults will be analysed to complement the data obtained in parts 1 and 3 of the research proposal. In
addition, in animal models representing various types of inflammation, we will pursue proof of concept
for the involvement of key molecules (agonists and antagonists of NF-B, or NF-B-regulatory
proteins) in inflammation using specific knockout and knockin mice. From this analysis we hope on the
one hand to gain new insights into the biological role of several genes and molecules studied in parts
1 and 3, but, on the other hand, we also hope to gain more insight into the mechanism of a selected
group of diseases such that in vitro data become easier to interpret.
WP4: Study of inflammatory processes in animal disease models.
Focusing on models of allergic, infectious and septic inflammation, this workpackage addresses
regulatory processes involved in the inflammatory cascade, the underlying molecular and cellular
actors and their regulation by NF-B signalling pathways.
WP4.1. Inflammatory responses upon Pseudomonas aeruginosa lung infection
Pseudomonas aeruginosa is an important nosocomial pathogen in patients with significant underlying
diseases, and colonization is frequently selected by broad-spectrum antimicrobial usage. In patients
with damaged airways from mechanical ventilation, trauma, or antecedent viral infection, P.
aeruginosa colonization of the respiratory tract is often followed by acute pneumonia, sepsis, and
death. In addition, patients with cystic fibrosis typically die of recurrent lung infections with P.
aeruginosa, which lead to chronic lung disease and eventual respiratory failure by their mid-30s. UG2
is studying the role of the bacterial type III secretion system (TTSS) of P. aeruginosa during lung
infection, using several acute and chronic mouse lung infection models. More specifically, UG2
focuses on the production of IL-1 and cell death in alveolar macrophages and lung epithelial cells,
and could recently demonstrate an important role for specific TTSS effector proteins (e.g. ExoS;
unpublished data). Using infection of mice with different bacterial mutants and complementation
studies one will now further elucidate the underlying mechanisms. This will also involve the
identification and functional analysis of host cell proteins that interact with TTSS proteins via yeast
two-hybrid screening. Interestingly, UG2 also found that cell death responses to P. aeruginosa differ
depending on the cell type and cell source, which will be further characterized at the molecular level.
The physiological relevance of the findings will be validated in acute and chronic lung infection models
in mice. The role of caspase-1 in all these events will be investigated by comparing the inflammatory
response upon infection of wild type mice versus caspase-1 deficient mice. Because of the well known
role of purinergic receptors in caspase-1 activation, UG2 in collaboration with ULG2 will also analyse
inflammatory responses of P2X1-deficient mice to P. aeruginosa lung infection. UG2 is studying the
role of type I and type III IFNs in endotoxemia and sepsis, using receptor knockout animals. Several
models of Gram-positive and Gram-negative sepsis are used, such as Klebsiella pneumoniae,
Salmonella typhimurium, Pseudomonas aeruginosa, and Listeria monocytogenes. Furthermore, a link
between expression of MMPs and IFN- was found, illustrating that several MMPs may be involved in
regulating the response to LPS and other inflammatory stimuli.
WP4.2. Inhibition of chronic inflammation by a natural IKK antagonist
UG1 has an experience in the analysis of the molecular mechanisms responsible for the induction
and/or the repression of inflammatory genes. For these studies, UG1 has used chemical compounds
and biological macromolecules, as well as a number of natural, plant-derived compounds. One such
compound, derived from an indigenous plant in Palestine, has shown a very effective antiinflammatory activity, both in vitro (cell cultures) and in vivo (acute inflammation model). The chemical
structure of this compound is described as a steroidal lactone, and it shows an entirely novel, but
highly specific inhibitory activity towards IKK. Since the natural compound is a specific NF-B
inhibitor, it dissociates NF-B- from AP-1-mediated effects, and might thus have a more selective
activity (as compared to e.g. glucocorticoids). In view of these properties, UG1 and UG2 will be
interesting to test this compound in mouse models of chronic inflammation, i.e. in chronic relapsing
remitting EAE (CREAE), in endotoxemia models and in DSS-induced colitis.
WP4.3. Study of inflammatory pathways in experimental asthma and other types of bronchial
inflammation.
In allergic responses, the combined action of humoral factors, especially IgE, and topically produced
mast cell inflammatory mediators and Th2 cytokines culminates into a local inflammatory response
featuring the recruitment of eosinophils, mast cells and macrophages, and is reminiscent of the
pathological features observed in asthma patients. Focusing on alveolar and interstitial lung
macrophages, a comparative multi-model transcriptome analysis of highly enriched macrophages by
UG1 revealed a striking induction of MMP-12 during experimental asthma but also during other types
of bronchial inflammation (experimental COPD, silicosis and LPS-induced acute alveolitis). The value
of MMP-12 as a universal marker for elicited inflammatory macrophages will be verified and its
expression regulation determined using mice genetically engineered for modified NF-B signalling. In
agreement with literature (Pouladi et al., 2004), UG1 found that MMP-12-deficiency strongly affected
eosinophilic airway inflammation in response to allergen but, strikingly, not LPS-induced neutrophilic
inflammation (acute alveolitis) nor models of extrinsic allergic alveolitis (Th1 mediated). From this
perspective, the differential role of MMP-12 in these opposite inflammatory models will be further
investigated by UG1 and UG2 with special emphasis on the potential role of MMP-12 in promoting the
processing of selected chemokines or their release from the extracellular matrix. Also the involvement
of MMP-12 in promoting macrophage-mediated allergen transport from the bronchoalveolar lumen into
the lung and the periphery will be determined.
Transcriptome profiling of resident alveolar and peritoneal macrophages isolated under noninflammatory conditions revealed striking differences in basal expression levels of genes involved in
arachidonic acid metabolism (COX1, LTC4 synthase, Ptgi synthase, 15-LOX). Topical application of
specific chemical inhibitors indicates a role of this differential eicosanoid production potential in
regulating the sensitivity for developing allergic airway inflammation. Besides chemical inhibitors also
lentiviral transfer of GPx4, a ROOH-scavenging antioxidant enzyme with inhibitory activity on COXs
and LOXs, will be studied (Heirman et al., 2005; see WP 6.3). The contribution in this differential
eicosanoid profile of constitutive NF-B signalling, a possible consequence of the continuous exposure
of pulmonary mucosal surfaces to airborne environmental constituents, will be determined in alveolar
macrophages but also in primary alveolar type II cells by biochemical analysis and by expression
analysis of additional NF-B target genes in wild-type mice and mice genetically engineered for
modified NF-B signalling (with ULG1).
Although asthma and its experimental murine counterpart represent a Th2-driven eosinophilic type of
inflammation, TLR agonists such as LPS but also other PAMPs play an important role in allergic
sensitization and its progression to asthma by triggering danger signalling necessary for immune
reactivity (Eisenbarth et al., 2002). In this context, UG1 will focus on LPS and mycolic acid (MA) from
Mycobacterium tuberculosis. Whereas acute exposure to LPS promotes allergic sensitization, the
effects of chronic LPS exposure regimens leading to macrophage/monocyte tolerance for endotoxin
will be determined and its consequences for the development of allergic sensitization and inflammation
verified. Similarly, the Treg-mediated tolerogenic mechanism induced by intratracheal instillation of the
M. tuberculosis cell wall component, MA, will be verified (Korf et al., 2006). The molecular crosstalk
between oxysteroid nuclear receptors and NF-B in MA-treated macrophages will be determined (see
WP 2.2) and its relation to the primary candidate tolerogenic function, expression of indoleamine 2,3dioxygenase (IDO) by the treated macrophages, verified.
WP4.4. Sepsis and endotoxemia
NF-B regulation is a central and essential feature in acute inflammatory shock as occurs in
endotoxemia and sepsis. To dissect the importance of several molecules in acute inflammation, UG2
will be focusing on TLR4- and TLR3-induced pathologies and focus on the cytokines TNF and IFN-,
the proteases MMP-7 and MMP-8 and the model organism Mus spretus. TNF-induced lethal
inflammatory shock is important in view of the potential application of TNF as an anticancer weapon.
UG2 will study the genetics of new TNF-resistant mouse strains (generated in house by an ENU
approach), the non-linear gene dosage effects of the P55-TNF receptor in relation to ERK kinase and
lipid rafts, the mechanism of glucocorticoid receptor (GR) inactivation and the protection of
MMP2/MMP9 double knockout mice. IFN- is another important gene product resulting from NF-B
and IRF-3/7 activation. Using knockout mice, UG2 will evaluate the real importance of IFN- in
endotoxemia and in mouse models of gram-negative sepsis. Finally, inflammatory cells have to move
during endotoxemia and sepsis. Proteases play a role in the degradation of extracellular matrices and
activation of cytokines and chemokines. Several members of the MMP family are induced by NF-B
and seem to play an essential mediating role in endotoxemia (Wielockx et al., 2001; Van Lint et al.,
2005). UG2 will focus on MMP-7 and MMP-8. UG2 will try to find the molecular basis of the protection
against LPS as seen in MMP7- and MMP8-knockout mice. In case of MMP-8, an interaction with IFN-
is possible. Finally, in the LPS resistant mouse strain (SPRET) derived from Mus spretus, the very
robust resistance is likely to be mediated by regulation of and interactions between NF-B and
members of the IRF family (Mahieu et al., 2006). This will be studied using gene expression profiles
on bone marrow derived macrophages and by comparing the results with those obtained in LPS
tolerized mice. A selected group of NF-B regulators will be studied in macrophages and ES cells
derived from the SPRET mice.
WP5. Study of selected signalling molecules using mouse transgenesis.
WP5.1. Deciphering the molecular mechanisms underlying IRF and NF-B activation pathways in vivo.
Despite substantial progress was made during the last years in understanding the NF-B and IRF
signalling pathways and the regulatory role of intermediate signalling molecules (Moynagh, 2005;
Kawai and Akira, 2006), little is still known about the physiological function of these signalling proteins
during normal tissue homeostasis and disease. It is our aim to understand the function, activation and
regulation of NF-B and IRF activation by TANK/I-TRAF, TRAF6, FADD, MyD88, A20, and ABINs
using conditional gene targeting (knock-outs or knock-ins) in the mouse.
For TANK/I-TRAF, ULG2 will first determine the physiological relevance of TANK/I-TRAF as a
signalling molecule in the TLR-dependent pathways and second decipher the role of the LPSmediated TANK/I-TRAF post-translational modifications (phosphorylations and ubiquitinations) in vivo.
A mouse model where these targeted residues are mutated through homologous recombination
(knock-in mice) will be generated by ULG2. The phenotype of such mice will be evaluated, especially
upon microbial challenges (LPS, viral infections, etc…) first at the molecular level by monitoring the
phase of IRF and NF-B activations and the induction of their target genes and secondly, at the
cellular level by following the apoptotic/necrotic status of the stimulated macrophages. In the second
part of the project, several models of acute and chronic inflammation (such as TNF-induced lethal
hepatitis, LPS-induced lethal shock, DSS-induced colitis and extrinsic allergic alveolitis will be studied
in the TANK/I-TRAF transgenic mice by ULG2, UG1 and UG2.
A20-deficient mice have previously been described and were shown to develop severe inflammation
and cachexia, to be hypersensitive to both lipopolysaccharide and TNF, and to die prematurely (Lee et
al., 2000). To investigate the physiological role of A20 and the A20-interacting proteins ABIN-1 and
ABIN-3 in several disease models, UG2 would like to generate mice with conditional alleles for the
A20, ABIN-1 or ABIN-3 gene. This will allow deletion of the gene in specific cell types and tissues (e.g.
macrophages, hepatocytes, lung or intestinal epithelial cells, T cells, keratinocytes) by crossing these
mice to cell-specific Cre transgenic lines that are already available in the UG2 laboratory. Moreover,
UG2 will generate mice which are mutated in respectively the ubiquitin ligase or the de-ubiquitinase
domains of A20, allowing to determine the relative contribution of both activities in the NF-B inhibitory
function of A20. UG2 will also generate a knockin mouse in which the endogenous A20 gene is
replaced by A20 fused to a short linker (TAP tag) that allows rapid and specific tandem affinity
purification (TAP) of protein complexes from selected tissues prepared from mice treated with specific
stimuli (e.g. TNF) or from disease developing mice (e.g. asthma or Crohn’s disease).
UG2 will also study the specific role of TRAF6 and FADD expression in epithelial cells versus
macrophages during the pathogenesis of asthma and bacterial (Pseudomonas) or viral (Influenza)
lung infection. Therefore, they will be crossing mice with conditional alleles for TRAF6 and FADD
(available through collaboration with Dr. M. Pasparakis, University of Koln, Germany) to specific Cre
transgenic lines and test the resulting mice in mouse models for asthma and lung infection.
Finally, UG2 will generate mice which are no longer able to produce the alternative splice variant of
MyD88 by knocking in a MyD88 gene that lacks intron 2. Such mice will allow to analyse the specific
role of MyD88s in immune responses to infection (e.g. Pseudomonas, Mycobacterium).
WP5.2. Signalling of inflammation by purine-receptors in vivo
In inflammatory arthridities such as rheumatoid arthritis, cognate lymphocytes have long been
considered instigators of autoimmunity, but accumulating evidence indicates that innate immune cells
such as neutrophils and mast cells are responsible for a vast majority of acute and ongoing
inflammation (Johnsson et al., 2005). However, the molecular mechanisms that govern them remain
largely unknown. Recent work has shown that the forkhead transcription factor Foxo3a, known to
inhibit NF-B in T cells (Lin et al., 2004), contributes to inflammation by ensuring neutrophils survival in
models of murine arthritis and peritonitis (Johnson et al., 2005).
ULG2 will investigate the role of purine P2X 1 receptors in animal models of neutrophilic inflammation.
P2X receptors are membrane ion channels that open in response to binding of extracellular ATP
(North, 2002). Neutrophils express at the least three P2X subtypes P2X 1, P2X5 and P2X7. P2X1deficient mice (Mulryan et al., 2000), available in the ULG2 laboratory, will be compared to wild-type
animals in dextran sulfate sodium (DSS)-induced acute and chronic colitis models for IBD (Pizarro et
al., 2003). Recent unpublished data from ULG2 suggest a protective role for P2X1 in DSS-induced
acute colitis. Similary, inflammatory responses of P2X 1-deficient mice will be tested in acute and
chronic models for Pseudomonas lung infection (in collaboration with UG2). In the latter case, special
attention will be given to the role of P2X1 and P2X7 in the activation of caspase-1 mediated IL-1
production and cell death, because of the well known role of purinergic receptors in caspase-1
activation (Mariathasan et al., 2006) (see also WP6.1).
Using P2X1-deficient neutrophils as well as pharmacological activation of human neutrophils with
selective P2X agonists, ULG2 will further investigate in vitro the contribution of P2X1 to neutrophil
functions (chemotaxis, phagocytosis, degranulation, ROS production, cytokine secretion, adhesion,
apoptosis). P2X receptor-dependent signalling that are important in these cellular processes will be
examined, with special emphasis on the contribution of PI-3K-, MAPK-, NF-B- and Foxo3adependent pathways, the activation of NALP-containing inflammasomes, and the involvement of the
cytoskeletal proteins Rac1 and Rac2. The Rac proteins are indeed required for normal neutrophil
chemotaxis and bacteria killing. The molecular mechanisms of P2X-mediated neutrophil apoptosis will
also be studied. The identification of the signalling molecules activated downstream of neutrophilic
P2X1 will then enable us to further address their role in vivo in murine models of inflammatory
diseases.
Part 3. Signal transduction leading to cell death.
WP6: Cell death pathways: linking cell death to inflammation and therapy
Current knowledge indicates that most of the molecular signalling complexes leading to the activation
of NF-kB and inflammation are also involved in the initiation of apoptotic and necrotic cell death
processes. Moreover, in certain conditions cell death, may even be considered as an important
inflammation-modulating process. In models of ischemia-reperfusion, prevention of cell death
contributes to reduced inflammation suggesting that cell death may precede or amplify the
inflammation process (Daemen et al., 1999). Similarly,
ongoing inflammation during rheumatoid
arthritis has been attributed to necrotic cell death processes, for example by the release of HMGB1
from the dying cells (Andersson et al., 2004). Therefore, a better understanding of the molecular
mechanisms governing the impact of cell death on the inflammatory process could provide new
molecular basis for therapeutic intervention. The general aim of this workpackage is to integrate
different levels of cell death research: morphology of cell death, intracellular signal transduction,
intercellular communication (phagocytosis, inflammation, immunomodulation), involvement of cell
death in pathologies and use of cell death related molecules as therapeutic targets to influence
inflammation. Since the core signalling pathways paradigms have been worked out for apoptosis, the
challenge today is to determine if and how the process is manipulated by intracellular pathogens to
their advantage,
to discover
alternative cell death pathways underlining the various cell death
processes and the molecular links and crosstalks with inflammatory signalling.
WP6.1. Molecular signalling in cell death
This work package combines the efforts of different groups in the program (ULG1, ULG2, UG1, UG2,
KUL) to integrate the knowledge and share the tools and methods, to study the induction or modulation
of the cell death processes elicited by different stimuli such as bacterial and viral infection,
photodynamic therapy (PDT), endoplasmatic reticulum (ER) stress, TLR ligands and TNF ligation. We
believe sharing data in an early stage on cell death pathways elicited by different stimuli will be of
synergistic value to the whole consortium in identifying points of convergence or crosstalks.
The following items will be addressed:
RIP1–mediated programmed necrosis. RIP1 is a serine/threonine kinase containing a dead domain
(DD). It plays a central role in NF-B activation and necrotic cell death induced by TNF, dsRNA and
hypoxia. In TLR3 response to viral infection or dsRNA, RIP1 is required in addition for signalling to
apoptosis (Kalai et al. 2002, Kaiser and Offermann (2005). Therefore, UG2 will study the involvement
of RIP1 in necrotic cell death induced by photochemically generated oxidative stress with KUL.
Structure-function analysis of RIP1 carried out by UG2 suggests that the NF-B activation and necrotic
cell death promoting functions of the protein can be separated. The UG2 group is developing
conditional transgenic mice overexpressing the RIP1 mutants lacking necrotic signalling and
conditional knock-in mice. When the mice are available, they will be also analysed in the various
disease models and infection models available in the consortium including viral and bacterial infection
models.
ER stress mediated cell death. Cell death signalling pathways initiated by ER stress are still poorly
understood. The ER is especially developed in B cell lymphoma and myeloma cells. Therefore, with
the hope that a better understanding of these pathways may lead to development of new treatments
for these types of cancer, UG2 and KUL will study the involvement of PKR, PERK, IRE-1, ATF-6,
caspases, p53 and BCL2 family members, in cell death induced by ER-stress agents such as
thapsigargin, tunicamycin, brefeldin A and hypericin-PDT in B cell lymphoma and myeloma cells.
The KUL group has recently reported that ER-Ca2+ depletion caused by hypericin-PDT, a paradigm of
an anticancer treatment utilizing reactive oxygen species (ROS) to kill the cancer cells or endothelial
cells (Dolmans et al., 2003), engages an autophagic pathway which causes cell death in apoptoticdeficient cells (Buytaert et al., 2006). Given the still uncertain role of autophagy as a tumor suppression
mechanism and as a death pathway in response to anticancer therapy KUL will focus on the molecular
mechanisms and role of autophagy as principal backup lethal program activated by PDT. The crucial
question is how and when autophagy, which is in essence a survival mechanism following cellular
stress, turns into a veritable cell death mechanism and whether it uses caspase-dependent or
caspase-independent cell death pathways. Thus, the KUL group will undertake a thorough comparison
of the effects of PDT in MEFs or human cancer cell lines with RNAi silenced cells, or in apoptosisdeficient cells (with UG2). Analysis of the crosstalk between the apoptotic and autophagic machinery
will clarify whether these pathways actively suppress/influence each other or are promoted
independently. The role of calpains and of the death kinases RIP1 and DAPK as mediators of
autophagic cell death, and the interaction between Bcl-2 proteins with Beclin-1 will be investigated. The
fact that RIP1, a pronecrotic kinase, also plays a role in autophagic cell death (Yu et al., 2004), and
that caspase-inhibition apparently promotes autophagy, suggests an intricate relationship between
these three cell death pathways. The UG2 group will also evaluate the RIP1 mutants for this function
and the effects of caspases in the regulation of Beclin-1following growth factor depletion or cellular
stress (PDT with KUL).
Death pathways in T-lymphocytes and neutrophils. During the inflammatory reaction, activated tissueresident macrophages and mast cells release various mediators (histamine, leukotrienes, chemokines,
TNF,..) that are perceived by neutrophils, which in turn respond by a ROS production and
degranulation. ROS strongly influence lymphocytes and other cells that are present in the tissue or in
the close vicinity. Recently, the ULG1 laboratory has shown that T lymphocytes exposure to ROS
leads to apoptosis by a still undefined pathway. This ROS mediated pathway can be suppressed by
the expression of the lipid phosphatase SHIP-1, which also counteracts Fas ligand induced T cell
apoptosis (Gloire et al., 2006). Hence, ULG1 will be involved in the molecular characterization of (i)
the apoptosis pathways in T lymphocytes subjected to an oxidative stress with a particular attention to
the role of the SHIP-1 phosphatase (ii) the domain of SHIP-1 that are required for acting as protector of
apoptosis, and (iii) the signalling pathways influenced by SHIP-1. In addition, ULG1 will determine
whether or not SHIP-1 is also protecting T lymphocytes from apoptosis induced by a series of
compounds known to trigger apoptosis at various cellular sites (mitochondria, ER, nucleus...) (in
collaboration with KUL).
Preliminary in vitro results from ULG2 reveal that the engagement of P2X1 receptors contributes to the
activation and cell death of human blood neutrophils. Within the framework of the WP6.1, ULG2 will
characterize the cell death modalities, apoptotic, necrotic or autophagic, of wild-type and P2X1-deficient
neutrophils ex vivo (see also WP4.6). The intracellular signalling pathways activated upon stimulation
of murine and human neutrophils with ATP or stable P2X-subtype selective analogs, used alone and in
combination with relevant pro-inflammatory molecules or Toll-like receptor agonists, will be studied. In
particular, the role of caspase-1 activation by the NALP-containing inflammasomes (caspase-1dependent production of IL-1 and IL-18) and caspase-11 up-regulation, and signalling through
caspase-8 and caspase-3 will be analysed (with UG2).
Caspase-1 in inflammation and cell death (pyroptosis). Caspase-1 is a crucial mediator in inflammation
due to the proteolytic activation of pro-IL1. In humans several caspase-1 regulatory gene products
have been reported that originated recently by gene duplication of the caspase-1 gene and that
modulates caspase-1 activation. These gene products consist of the prodomain of caspase-1 and are
therefore called caspase-1 CARD-only proteins and include COP, INCA and ICEBERG (Lamkanfi et
al., 2004a). UG2 has also demonstrated that the prodomain of caspase-1 is able to recruit RIP2 and
TRAF6 leading to activation of NF-B, a feature shared by COP, but not by the more distantly related
INCA and ICEBERG (Lamkanfi et al. 2004a, 2006). Recently caspase-1 has also been implicated in
pyroptosis of Salmonella-infected macrophages (Franchi et al. 2006). In the UG2 group, the molecular
mechanism of this bacteria-induced caspase-1 activation is unraveled and the type of cell death is
analysed by time laps. Moreover, CARD-only mutants of caspase-1 are developed that interfere both
with caspase-1 activation and caspase-1-mediated NF-B activation, and which apparently block
induction of pyroptosis. The development of transgenic mice of this CARD-only mutant will unable UG2
to evaluate the modulatory effects on inflammation and pyroptosis, and to test various disease and
infection models available in the consortium.
Terminal differentiation of the keratinocytes. Terminal differentiation of the keratinocytes can be
considered as a special type of programmed cell death in which the dead body corpses (corneocytes)
are not removed by phagocytosis but remain and function as a barrier against water loss and infection
(Lippens et al. 2005). UG2 group has identified caspase-14 as being specifically expressed and
proteolytically activated during terminal differentiation of keratinocytes. UG2 has developed a caspase14 knockout mice, which has a severe barrier phenotype. This knockout mouse will be analysed in
inflammation
models
(psoriasis
induced
by
ubiquimod
a
TLRx
ligand),
infection
models
(Pseudomonas), damage models (UVB, PDT in collaboration with KUL) and skin cancer development
(melanomas, papillomas, squamous and basal cell carcinomas) whether or not in p53 deficient
background.
WP6.2 Identification of protein complexes and key executioner molecules in cell death and
inflammation
Cell death and inflammation processes are often initiated by molecular complexes that consist of a
platform/sensor molecule (TNF receptor, Apaf-1, NALPs), adaptor molecules combining two
recognition motifs (TRADD, FADD, RAIDD, PYCARD, CARDINAL) and effector proteins (caspase or
RIP kinases). Cellular stress, infection, death domain ligands, organelle perturbation lead to the
activation of a platform, often an ATP-dependent process, the recruitment of appropriate adaptors, and
the recruitment and activation of the effector proteins. This initiates cellular responses such as NF-B
activation, necrosis or apoptosis. In this WP6.2 the protein complexes initiating necrotic cell death
(necrosome) or cellular stress (stressosome) following DNA damage, heat, ischemia-reperfusion,
oxidative stress, hypoxia, will be studied.
The necrosome and stressosome. In preliminary experiments UG2 has already described a complex
containing PIDD, RAIDD, caspase-2, IKK, IKK, NEMO, Hsp90, RIP1. This complex, called
stressosome, has been demonstrated by gel filtration and coimmunoprecipitation experiments, and is
formed in conditions of heat treatment, hydrogen peroxide stress, hypoxia. This stressosome complex
has in theory many different outcomes in inflammation (NF-B), apoptosis (caspase-2) and necrosis
(RIP1). UG2 is now exploring this complex formation in cell deficient for certain of these proteins (by
RNAi) and evaluate the effect on the biological outcome (NF-B activation, apoptosis, necrosis). In
collaboration with KUL, UG2 will evaluate whether the stressosome is formed in UVB exposed normal
or transformed keratinocytes, its contribution to sunburn cell formation and its crosstalk with the
proapoptotic p38 MAPK (Van Laethem et al., 2004) and p53 pathways. In order to identify the crucial
signal transduction proteins and substrates in necrotic cell death, the UG2 group is now setting up a
RNAi library screening. In parallel UG2 is also following a biochemical approach to identify a possible
necrosome complex containing RIP1 using gel filtration, coimmunoprecipitation and two dimensional
blue native gel electrophroresis/PAGE and mass spectrometry (see also WP1.1)
WP6.3 Intercellular crosstalk between cell death and inflammation
Several factors that modulate the innate immune system have been identified in dying cells. These
include intracellular factors HMGB1, Hsp70 and modified phospholipids. Cell death coupled with
phagocytosis of the dying cell is an important alternative mechanism for clearance when an infected
cell fails to eliminate an intracellular pathogen. Understanding better how cell death and phagocytosis
of dying cells promote clearance of pathogens and development of immunity and how these pathways
are deregulated during infection, may help in designing new and better vaccination strategies and
antitumor approaches.
Signals from dying cells. Within UG1 and UG2 a microarray analysis has been performed on
macrophages (naïve macrophages, M1 “killer” macrophages, M2 “healer” macrophages) (Mantovani et
al., 2005) that have been exposed to living, apoptotic or necrotic dying cells. The preliminary results
show that apoptotic dying cells strongly activate genes involved in tissue repair (VEGF, MMPs, etc.).
UG1 and UG2 want to validate these results and expand them to other relevant cellular coincubation
systems (neutrophils, endothelial cells, dendritic cells).
KUL will explore the way by which the robust expression of HSP70 in response to PDT affects cell
death (intracellular) and modulate the immune response (intercellular). KUL will explore how surface
expression/release of HSP70 in PDT-stressed living cells or dying cells (and the dependence on the
cell death modalities) modulates their recognition by macrophages and release of pro-inflammatory
cytokines. The use of hsp70-/- MEFs (with UG2), stably transfected siRNA-hsp70 cancer cell lines and
apoptosis-deficient cells, will be very instrumental for this analysis. Furthermore, KUL will disclose
whether the cytoprotective function of HSP70 in PDT relays on its powerful chaperone activity, the
inhibition of key modulators of the apoptotic machinery and/or the protection from lysosomal damage
(with UG1 & UG2).
Tumor-derived eicosanoids and MMPs. Tumor-derived eicosanoids and matrix metalloproteinases
(MMPs) are crucial regulators of inflammation, modulate sensitivity of tumor cells to death-inducing
insults and are deeply implicated at the tumor invasion/metastatic front. These molecules are
promising targets in many disease pathways and are explored at the molecular and in vivo levels by
several laboratories of this consortium.
UG1 has shown that overexpression of the antioxidant enzyme glutathione peroxidase-4 (GPx4) result
in pronounced suppression of solid tumor growth in weakly malignant tumors, whereas no effect is
observed in modestly and strongly malignant tumors (Heirman et al. 2006). This differential effect of
GPx4 on solid tumor growth correlated with the expression levels of cyclooxygenase-2 (COX-2) and
VEGF in the tumor models (e.g. COX-2high/VEGFlow in weakly malignant tumor, COX-2low/VEGFhigh in
modestly/strongly malignant tumors). Furthermore, tumor growth-inhibiting effect of GPx4 in the weakly
malignant tumor model was due to the inhibition of COX-2 expression and COX-2 activity in the tumor
cells. UG2 will now concentrate on the clarification of the mechanisms and signalling pathways that
define the malignant potential of tumors. As COX-2 expression is mainly dependent on p38 MAPK
signalling pathways and NF-B activity, the constitutive activity of these signalling molecules in low
malignancy tumors will be verified and compared to the activity levels in high malignancy tumors. As
VEGF expression is controlled by the transcription factor HIF-1, also HIF-1 basal expression levels
will be verified in the respective tumor models. As GPx4 inhibits basal COX-2 expression and COX-2
induction by TNF treatment, the interference of the phospholipid hydroperoxide scavenger with p38
and NF-B pathways will be verified in order to look for regulatory feedback loops between COX–
derived prostanoids and p38/NF-B pathways (with KUL & UG2). To establish the relevance for human
malignancies, the tumor-sensitizing effect of GPx4 on angiostatic therapy applying drugs presently in
clinical use (thalidomide, others), will be evaluated. Also, the malignancy predictive value of the
polarized COX-2/VEGF expression ratio will be verified by in silico analysis of public databases along
with expression analysis on cancer biopsies.
KUL and ULG1 will focus on the molecular pathways modulating the expression/activity of COX-2
(Hendrickx et al. 2003; 2005; Volanti et al. 2005) and MMPs in PDT treated cells using
photosensitizers targeted to different subcellular localization (with UG2). The significance of MMP-1,
MMP-10 and MMP-13, as major MMP members induced by hypericin-PDT in promoting motility,
invasion and metastasis of the surviving cancer cells will be challenged in specific MMP knockout
genetic models (with UG2). As recent transcriptomic analysis of bladder cancer cells treated with
hypericin-PDT either alone or in combination with a p38 MAPK antagonist discloses that the
expression of key pro-inflammatory, proangiogenic and metastastic genes is dependent on the p38
MAPK pathway, KUL and ULG1 will analyse the underlying mechanisms (e.g. transcriptional, mRNA
stability) leading to their up-regulation and validate these targets at the functional level. A transgenic
zebrafish (fli1:EGFP) model will be used to visualize and validate the effect of the inhibition
(pharmacological or morpholino knockdown) of proangiogenic signalling pathways in PDT at the
organism level.
WP6.4 Cell death signalling molecules as a therapeutic agent
Defects in apoptosis programs may contribute to tumor progression and treatment resistance and may
be caused by deregulated expression and/or function of anti-apoptotic or pro-apoptotic molecules. The
recent success using therapeutics that specifically target deregulated signal transduction pathways in
cancer cells, underscores the importance of identifying deranged pathways in cancer therapy and
evidences that the most adroit strategy to increase therapeutic potential of conventional anticancer
therapies relies in the simultaneous targeting of key elements of (pathogenic) signal transduction
cascades.
Targeted genes with strong immunomodulatory effects include the caspase-1 derived COP, INCA and
ICEBERG, which lead to CARD-only proteins that modulate the activation of caspase-1 by interfering
with the recruitment of caspase-1 in inflammasome complexes and/or preventing the recruitment of
other immunomodulatory pathways (RIP2, TRAF6). Moreover, in human also caspase-12 has also
evolved to a shortened and catalytically inactive version of caspase-12 that would modulate immune
responses during sepsis (Saleh et al., 2004). In view of these strong modulatory effects of CARD-only
proteins UG2 is identifying mutant forms of caspase-1 and caspase-2 that have very potent antiinflammatory properties and which are able to interfere with inflammasome and stressosome
formation. On the other hand, certain CARD-only proteins derived from caspase-1 and caspase-2, are
also strong activators of NF-B through the recruitment of RIP2/TRAF6 (Lamkanfi et al. 2004b) and
RIP1/TRAF2 (Lamkanfi et al. 2005), respectively.
Signal transduction-based therapy in cancer. Inhibitor of apoptosis proteins (IAPs) are expressed at
high levels in many tumors and have been associated with refractory disease and poor prognosis.
Since IAPs block apoptosis at the effector phase strategies targeting IAPs may prove to be especially
effective to overcome resistance. To target IAP expression in cancers, EU3 has developed cellpermeable Smac peptides, which strongly sensitized even resistant tumor cells for apoptosis induced
by death receptor ligation, chemotherapy or -irradiation in vitro and also in vivo in an intracranial
mouse model of malignant glioma (Fulda et al., 2002). Moreover, ectopic expression of mitochondrial
or cytosolic Smac sensitize various cancer types for -irradiation-induced apoptosis (Giagkousiklidis et
al., 2005) and exert an inhibitory effect on cell proliferation under conditions where cell-cell contact is
lost (Vogler et al., 2005). With the primary aim of evaluating IAP antagonists as novel cancer
therapeutics to potentiate the efficacy of cytotoxic therapies selectively in cancer cells, the following
questions will be addressed: what is the impact of different IAP antagonists (Smac peptides, full-length
and cytosolic Smac, small molecule IAP antagonists) on cancer cell sensitivity towards cytotoxic
therapies (chemotherapy, cytotoxic ligands,-irradiation)? Do IAP antagonists selectively sensitize
malignant cells compared to nonmalignant cells for apoptosis? Which signalling pathways, e.g. NF-B,
are modulated by IAP antagonists? Finally, we will evaluate the therapeutic potential of IAP
antagonists in combination with cytotoxic therapies in an orthotopic mouse model of human malignant
glioma in vivo.
KUL is investigating the therapeutic efficacy of hypericin-based PDT for superficial bladder tumors
(Kamuhabwa et al. 2004). In this WP6.4 an orthotopic rat bladder tumor model, which is currently used
as a preclinical in vivo model in our laboratory, will be used to validate the knowledge obtained from in
vitro studies. The small intravesical volume (300 µl) of the rat bladder and the readily accessibility of
the urothelium tumor lesions, makes it possible to instillate hypericin in the presence of cell permeable
pharmacological inhibitors of known (e.g. p38 MAPK/COX-2, MMPs) or newly identified molecular
targets revealed by the approaches described in WP6.1-3, prior to light delivery. IAP antagonists (e.g.
cell permeable Smac peptides) will be also explored in combination with hypericin-PDT (with EU3).
This preclinical model will reveal the in vivo significance of the signalling pathways associated with
hypericin-based PDT and whether their inhibition is of therapeutic benefit and leads to an improvement
of the tumoricidial effects of PDT. In addition the potential participation of autophagy as in vivo
response to PDT will be assessed (UG1, UG2, ULG1).
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