B2
Modelling of the NF-kappaB dependent Apoptosis/Regeneration
switch in Hepatocytes
Projectleaders:
Experimental:

Prof. Irmgard Merfort, Dept. of Pharmaceutical Biology and Biotechnology,
University Freiburg, +49 761 203 8373; merfort@pharmazie.uni-freiburg.de

PD Dr. Johannes Bode & PD Dr. Freimut Schliess, University Hospital
Düsseldorf, +49 211 81 18941, schliess@med.uni-duesseldorf.de

Dr. R. Nitschke, Life Imaging Center, University Freiburg,
+49 761 203 2934, roland.nitschke@biologie.uni-freiburg.de
Modeling:

Dr. H. P.Fischer, Genedata AG, München
+41 61 69 76767 Hans-Peter.Fischer@genedata.com,

Prof. J. Timmer, Center for Data Analysis and Modeling, University of
Freiburg, +49 761 203 5829, jeti@fdm.uni-freiburg.de
Summary:
Regeneration of hepatocytes is a complex growth process being regulated by a
coordinated network of different signal transduction pathways. In the initial phase of
regeneration the cytokine TNF-alpha plays a key role activating the transcription
factor NF-kappaB besides induction of other signalling cascades. Additionally,
activation of NF-kappaB in hepatocytes further occurs in response to IL-1beta.
Whereas TNF-alpha and other cytokines, such as IL-6 are important for the initiation
and propagation of liver regeneration, IL-1beta is a potent inhibitor of liver
regeneration and hepatocyte proliferation. NF-kappaB activation represents a key
event in IL-1beta signalling which does not only influence NF-kappaB target genes
but also antagonizes signalling via the Jak/STAT cascade important for the signaltransduction of cytokines and growth factors such as IL-6. We therefore attempt to
model the time and concentration dependent activation of NF-kappaB and its target
genes in response to IL-1beta and its impact on STAT3 signalling and hepatocyte
proliferation.
NF-kappaB was shown to be crucially involved in the conversion of hepatocytes from
proliferation into apoptosis after TNF-alpha stimulation, suggesting that NF-kappaB
plays a pivotal role in the regulation of this switch. Therefore, our project aims to
model TNF-alpha-induced NF-kappaB signal transduction in regenerating primary
hepatocytes from B6 mice under SOP conditions (Klingmüller et al., 2006) with a
special focus on the NF-kappaB dependent switch between survival, apoptosis and
regeneration. To investigate this tempting subject, it is proposed to analyse the target
genes of the NF-kappaB transcription factor in different cellular stages (blocked in
survival, apoptotic and proliferating) with means of microarray analysis and single cell
based expression measurement. Extensive data analysis on our own as well as
literature derived array data will be accomplished to distinguish direct NF-kappaB
target genes from secondary influenced cascade genes. Genome- wide promoteranalysis of target genes is expected to identify signatures for regeneration specific
promoter sequences including other transcription factors binding sites.
Work Plan:
Year 1:
(a)
(b)
(c)
(d)
(e)
Merfort: Whereas in the initial funding period, our work has been focussed on
time resolved analysis of NF-kappaB target genes on quiescent primary
hepatocytes we now extend the study to cultural conditions for the induction of
apoptosis and proliferation in primary hepatocytes and contribute to the
development of SOPs by analysing proliferation observed in response to
mitogenic stimulation (in cooperation with Project A1, B1, B3, B5, C1).
Merfort: Bode, GeneData, Timmer: We will focus on TNF-alpha, together with
IL-6 (cooperation Project B1) a key factor that synergize in promoting the
induction of DNA synthesis during the priming phase of hepatocyte regeneration.
Studies will be performed with TNF-alpha stimulation at apoptotic as well as
proliferating conditions using qPCR and protein techniques to systematically
identify correct time points.
Bode, Schliess, Merfort, Nitschke, Timmer: We will quantitatively study the
effect of IL-1ß on proliferating hepatocytes and the role of NF-kappaB for this
effect emphasizing on the inhibitory effects of IL-1beta on liver regeneration (in
cooperation with Project B1, C1). Methods such as EMSA and western blots will
be used. A model of IL-1beta derived modulation of NF-kappaB signalling
properties will be established.
Merfort, GeneData: Gene expression data collected in the first funding period
revealed NF-kappaB target genes with the annotation “survival” to be upregulated
in cultured hepatocytes compared to a regeneration model of partial hepatectomy
in vivo (data from Project B9). Following more extensive data analysis using the
tools from the established data management software Phylosopher® and
Expressionist®, we will identify common sequence-signatures that can be
attributed to the survival gene subgroup. In a similar approach the effect of costimulation with TNF-alpha and FasL on gene expression will be investigated
(cooperation Project C1).
Bode, Merfort: Expression dynamics of a certain set of target genes either
induced or suppressed by NF-kappaB important for cell cycle control and acute
phase response (serum amyloid A, -fibrinogen, p21WAF, cyclin D, c-Myc) will be
predicted and measured using real time PCR and Western blot analysis.
Year 2
(a)
Merfort, Bode, GeneData, Timmer: Following real time PCR- analysis of TNFalpha cytokine response on apoptotic and proliferative hepatocytes, genomewide gene profiling using established microarray technology will follow to identify
NF-kappaB related genes differently expressed at apoptotic and proliferating
conditions. Based on results from data-analysis on the data achieved, a time
resolved model for the different hepatocyte outcome will be developed.
(b)
Merfort: Based on the results of year 1, natural compounds, such as
sesquiterpene lactones and the phenolics curcumin and silybin, will be studied in
which way they influence the switch regeneration/apoptosis on the gene level
using qPCR and microarray technology. Previous results have proven
sesquiterpene lactones as suitable NF-kappaB inhibitors (Wagner et al., 2006).
Moreover, our recent review on NF-kappaB inhibitors reveal more candidates
(Merfort, 2006 in press).
(c)
Bode, Merfort: Data retrieved from target gene analysis for cell cycle and acute
phase control will be correlated with data from ChIP-experiments (SOPs for ChIP
technology will be developed in cooperation with Project B8 and B1) in order to
model promoter binding of NF-kappaB and for the SAA and -fibrinogen gene
also STAT3. Polymerase binding will be determined as an indicator of
transcription-start.
(d)
Merfort, Nitschke, GeneData: Experimental promoter analysis such as DNA-
protein binding analysis will be done by chromatin immuno precipitation analysis
(ChIP) followed by real time PCR or microarray analysis. In order to quantitatively
analyse this gene expression model, expression strength of this type of
promoters will be analyzed by means of a quantitative fluorescent gene reporter
construct driven by selected gene promoters and quantified on single cell level.
(e)
Merfort, GeneData: Genome wide gene expression analysis is suitable for the
identification of new target genes. Based on initial experiments, we propose to
use qPCR or the recently developed liquid chip technology (Qiagen/Luminex) to
analyse time resolved a restriced number of target genes (up to 100) following a
multitude of different cytokine stimuli, with the aim to analyse cross-talks to
related signalling pathways, such as the STAT3 pathway, stimulated by IL-6
(cooperation Project B1) or the Wnt pathway (cooperation Project B3).
Year 3
(a)
Merfort, Bode, GeneData, Timmer: Based on qPCR and microarray Data
analysis, selective quantitative protein technologies will be applied to follow
relevant protein modifications and expression variations extracted from the
modelling approach. If applicable, higher resolution target gene analysis will be
performed based on the techniques described above.
(b)
Bode: Data derived from IL-1beta dependent ChIP- assays will be analyzed to
identify NF-kappaB dependent target gene subsets regulated by IL-1beta.
Further, the input of IL-1beta dependent NF-kappaB activation on STAT3 activity
will be determined and modulated (in cooperation Project B1).
(c)
Merfort, GeneData: NF-kappaB activation leads to a different outcome,
depending on the stimulus received and the cellular response. This information is
preserved along the pathway resulting in activation of different NF-kappaB target
gene subsets. In a top-down approach, we will apply extensive data analysis
towards own and literature derived expression profiles of NF-kappaB induction in
hepatocytes to identify co-regulated or in other perspectives correlated target
gene subsets. The analysis will be focus towards establishing a model of distinct
target gene subsets including identification of typical “lead genes” representing
the dynamic and temporal behaviour of the cluster.
(d)
Merfort, Nitschke: Signatures identified from quantitative fluorescent gene
reporter assay approach with potential for pharmaceutical screening shall be
further developed towards pharmaceutical application (e.g. in collaboration with
Evotec AG).
(e)
Timmer: To elucidate mechanism regulating apoptosis or proliferation and
thereby the onset of hepatocyte regeneration by a systems biology approach, we
will initially complete the submodules of signaling pathways activated during the
priming, proliferation and termination phase of regeneration. For NF-kappaB
signaling, the effects of different stimuli (especially TNF-alpha and IL-1beta) will
be employed to establish a generalized model for this signalling pathway.
Milestones:
Establishing cultural conditions for triggering apoptosis and proliferation in
primary hepatocytes.
Establishing a FACS based system to differentiate proliferating hepatocytes
from non parenchymal cell contamination.
Determination of the time and concentration dependent impact of IL-1beta on
the proliferation of primary hepatocytes.
Characterization of a non-TNF-alpha influenced promoter for reference
expression in a hepatocyte cell line. Quality control analysis of the normalizer.
Establishing an SOP development for ChIP-analysis for polymerase binding to
-
the promoter of selected target genes in primary murine hepatocytes.
ChIP-analysis and modulation of NF-kappaB DNA binding in response to IL1beta in the presence or absence of IL-6 and correlation to the data for
polymerase binding and its influence on STAT3 DNA binding.
Experimental analysis of different cytokine stimuli on lead gene regulation.
Influence of NF-kappaB inactivation by retroviral transfection of a non
degradable mutant of IkappaB-alpha or specific peptide inhibitors of NF-kappaBactivation on the impact of IL-1beta on STAT3-activity.
Validation of the luminex- systems with 10 target genes and comparison to
qPCR results.
Budget Merfort
Personal:
Postdoc- Position (continuation Titus Sparna), BATIIa
Ph.D.- Position (continuation of Claudia Kern), BATIIa/2
171.300
85.650
256.950
Consumables:
Materials for EMSA and western blots analysis (glasware, isotopes,
membranes, antibodies, recombinant proteins, enzymes, materials for
cell culture)
qRT-PCR, Taqman® (primer, RNA polymerases, kits for RNA
isolation, DNA synthesis, kits for bioanalyzer)
external microarray analysis, kits for RNA isolation, kits for bioanalyzer
50 000
40 000
100 000
190.000
Budget Bode & Schliess
Personal:
Ph.D.- Position (continuation of Claudia Kern), BATIIa/2
Technician, BATVb
85.650
123 300
208.950
Consumables:
Materials for ChIP analysis (primary and secondary antibodies for NFkappaB subunits, secondary antibodies, membranes)
External luminex bead application
Materials for quantitative fluorescent gene reporter constructs
50.000
30.000
20.000
90.000
Budget Nitschke
Personal
Technician, BATVb/2
61 650
Consumables
Begründung????
10 000
Budget Travel:
Trips to Heidelberg, Düsseldorf to Freiburg and Freiburg-Düsseldorf for
scientific experience exchange, participation in one European
conference in the first two years and 1 international conference in the
10.000
third year
References:
Klingmüller, U. et al. Primary mouse hepatocytes for systems biology approaches: a
standardized in vitro system for modelling of signal transduction pathways. IEE
Proc Systems Biology in press
Merfort I., Patented inhibitors (2002-2005) of the transcription factor NF-kappaB,
Expert Opinion (2006) in press
Wagner, S., A. Hofmann, B. Siedle, L. Terfloth, I. Merfort, J. Gasteiger Development
of a structural model for NFkappaB inhibition of sesquiterpene lactones using selforganizing neural networks. J Med Chem (2006) 49, 2241-2252