Nuclear {factor-_k63b [factor-kappa-B] signaling pathway controls a pro-inflammatory and pro-atherogenic gene expression program in endothelial cells [Elektronische Ressource] / vorgelegt von Sybille Sophie Kempe González

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: universitat_ulm
• Publié le: 01 janvier 2008
• Nombre de lectures: 35
• Langue: English
• Poids de l'ouvrage: 70 Mo

Extrait

FAKULTÄT FÜR NATURWISSENSCHAFTEN
UNIVERSITÄT ULM


Nuclear FactorB signaling pathway controls a pro-
inflammatory and pro-atherogenic gene expression
program in endothelial cells.

Dissertation
zur Erlangung des Doktorgrades (Dr. Rer. Nat)
an der Fakultät für Naturwissenschaften
der Universität Ulm

vorgelegt von
Sybille Sophie Kempe González

aus
Concepción, Chile
2008


Erstgutachter:
Prof. Dr. Thomas Wirth, Institut für Physiologische Chemie, Universität Ulm.


Zweitgutachter:
Prof. Dr Klaus-Dieter Spindler, Abt. Allgemeine Zoologie und Endokrinologie,
Universität Ulm.

Tag der Promotion: 19.12.2008



Die Arbeiten in Rahmen der vorliegenden Dissertation wurden in der Institut für
Physiologische Chemie der Universität Ulm durchgeführt und von Herrn Prof.
Dr. Thomas Wirth betreut.


2
Acknowledgements

I thank Prof. Thomas Wirth for the opportunity to work in his lab, for all the support,
experience and advice he gave me during these years.

I thank Prof. Klaus-Dieter Spindler, from the Dept. of General Zoology and
Endocrinology at the University of Ulm, who agreed to revise this thesis.

I thank Dr. Bernd Baumann for correcting part of this thesis and Kay Klapproth for
translating to german the summary.

I wish to thank each of my collegues and friends of Physiologische Chemie for the
nice time we spent together. A very special thank to the former members who received me in
the lab and introduced me in Germany, in particular Dr. Andrea Lasar and Dr. Ninel Azotei.
Also to Dr. Alexei Ushmorov who shared with me all his technical and scientific experience,
Dr. Ralf Marienfeld for all the NF B advice and Dr. Cornelia Brunner for helpful promoter
discussions. I thank my friend and colleague Eniko Kokai with whom I shared a great time
doing research and became a good team.

I thank my mother and sister for their constant support, my mother-in-law for her
invaluable help, and all my professors and teachers who build up my way to this stage.

Finally, I thank my life friend and husband, Joaquin, for his complete support and
comprehension during this time.

I dedicate this thesis to our son Sebastian.
3
Data presented in this thesis are published in the following
papers:


1) NF  B CONTROLS THE GLOBAL PRO-INFLAMMATORY RESPONSE IN
ENDOTHELIAL CELLS: EVIDENCE FOR THE REGULATION OF A PRO-
1 2 1ATHEROGENIC PROGRAM. Sybille Kempe , Hans Kestler , Andrea Lasar and
1Thomas Wirth . Nucleic Acids Res., 2005 Sep 21; 33(16):5308-19

2) EPSTEINBARR VIRUS INDUCED GENE-3 IS EXPRESSED IN HUMAN
1 2 1ATHEROMA PLAQUES. Sybille Kempe , Philipp Heinz , Enikö Kokai , Odile
2 1Devergne, Nikolaus Marx and Thomas Wirth , 2008. (manuscript submitted).



4
Contents

Abbreviations ……… …………… …………………………………………………. ………8
Summary……………………………………..……………………………………...….......13
Zusammenfassung………………………………………………………………..………15
I) Introduction……..……………………………………………………...…………….18
1) Nuclear factor kappa B (NFB) transcription factors: master regulators of the
inflammatory response....................................................................................................18
1.1) Canonical and non-canonical pathways………………………………............21
1.2) Activation and regulation of IKK complex.……………………….................23
1.2.1) TRAFS are key intermediates in most NF B pathways…………….24
1.2.2) RIPs are adapters in the canonical NF B pathway……………….....25
1.2.3) IKK activation in the canonical NFB pathway…………………….26
1.2.4) IKK activation in the non- canonical NFB pathway……………....28
1.3) Degradation of I B proteins.………………………………………….….…...28
1.4) Activation and processing of NFB subunits………………………….........29
1.5) Regulation of nuclear NF B function.………………..…………….…….....30
1.6) Termination of NFB response………………..………………….…………31
2) Role of NFB in inflammation………...……..………………………………….…....33
3) TNF  is a potent pro-inflammatory cytokine…………………………………………..35
3.1) TNF signaling to NFB……………………………………………………..35
3.2) TNF signaling to JNK and p38-MAPK……………………………..………..36
4) Role of the vascular endothelium in inflammation……..…………………………...….37
5
4.1) The endothelium in acute inflammation………………………………...…….38
4.1.1) Type-I activation of endothelial cells……...……….38
4.1.2) Type-II activation of endothelial cells……………………….………..39
4.2) Endothelium in chronic inflammation……………………….………………..41
5) Atehrosclerosis a chronic inflammatory disease of the vessels wall…..……………….42
II) Aim of the study…………………………………………………………..................48
III) Results and Discussion………………………………….………………………...49
1) Determination of IKK/NF B gene expression profile in endothelial cells………...…49
1.1) Genetic manipulation of NF- B activity in HMEC-1 cells………………...…49
1.2) Identification of TNF- regulated genes in HMEC-1 cells and dependence on
the IKK/NF- B pathway……………………………………………………...50
1.2.1) Identification of up-regulated genes………………..…………..……..51
1.2.2) Identification of down-regulated genes………….……………………58
1.3) Validation of microarray data by RT-PCR analyses…………………….........61
1.4) Analysis of NFB transcriptional regulation of human endothelial lipase
promoter……………………………………………..………………..………63
2) Development of endothelial lipase conditional transgenic mice……………………….65
2.1) Construction and functional validation of tetO:LIPG transgene……………..67
2.2) Luciferase activity in VE/EL transgenic mice………………...…………..…..69
2.3) Lipid profile analysis of VE/EL transgenic mice……………………………..69
3) Analysis of Ebi3 expression in human atheroma plaques………………………….......73
3.1) Immunohystochemistry analysis of Ebi3 expression in human atheroma
plaques……………………………………………………………………...…73
3.2) Analysis of Ebi3 expression in activated smooth muscle cells……………….74
6
3.3) Activation of the nuclear transcription factor PPAR impairs cytokine-induced
Ebi3 expression in AoSMCs…………...……………………….…………….75
IV) Conclusion……………………………………………………………….………..…77
V) References……………………………………………………………..……………..78
VI) Papers….….………………………………………..……........96
VII) Appendix. Gene expression profile tables ………………………….…………138
Erklärung ……………………………………............…………………………….………148
Curriculum vitae ……………………………….. .. .. .. .. . ………………… ………..……..149


7
Abbreviations

ACS Acute coronary syndrome
AoSMCs Aortic smooth muscle cells
AP-1 Activator protein 1
ApoE Apolipoprotein E
ATF2 Activating transcription factor 2
ASK Apoptosis signal-regulating kinase 1
Bcl- B-cell leukaemia
CA IKK2 Constitutive active IKK2/ beta
cAMP Cyclic adenosine monophosphate
CARD Caspase activation and recruitment domain
CASP Caspase
CBP/p300 CREB binding protein
CD40 CD40 molecule, TNF receptor superfamily member 5
CFLAR Caspase 8 and Fas-associated death domain-like apoptosis regulator
cFLIP CFLAR
CHD Coronary heart disease
ChIP Chromatin immunoprecipitation assay
CHOP CCAAT/enhancer binding protein (C/EBP) homologous protein
cIAPs inhibitor of apoptosis proteins
CK2 Casein kinase-2
CCL chemokine (C-C motif) ligand
COX Cyclooxygenase
CpG Cytosine-Guanine-rich regions
CSF3 Colony stimulating factor-3
CTSS Cathepsin S
CREB Cyclic AMP-Response Element Binding Protein
CXCL chemokine (C-X-C motif) ligand
8
CXCLR chemokine (C-X-C motif) ligand receptor
CYLD cylindromatosis (turban tumor syndrome)
DCs Dendritic cells
DNA Desoxyribonucleic acid
DUB Ubiquitin D
Ebi3 Epstein-Barr induced gene 3
ECs Endothelial cells
ECMV Encephalomyocarditis virus
EL Endothelial lipase
ELK Ets-like protein
EMSA Electromobility shift assay
eNOS Endothelial nitric oxide synthase
ERK Extracellular signal regulated kinase
Foxo Forkhead transcription factor O
Gal Galectin
GBP Guanylate binding protein
GPCRs G-protein coupled receptors
HDAC Histone deacetylase
HDL High-density lipoprotein
HDMEC Human dermis microvascular endothelial cells
HMEC-1 Human microvascular endothelial cell line-1
HSVEC Human safena vein endothelial cells
HUVEC Human umbilical vein endothelial cells
ICAM-1 Intracellular adhesion molecule-1
IFN Interferon
I B Inhibitory kappa B
IKK IkB kinase
IL Interleukin
IL-27R Interleukin 27 receptor
IRES Internal ribosome entry site
9
JNK c-Jun N-terminal kinase
B kappa light chain enhancer
kDa kilo Dalton
KLF2 Kruppel-like factor 2
LDL Low-density lipoprotein
LDLR Low-density lipoprotein receptor
LIPG Endothelial lipase gene
LPS Lipopolysaccharide
LT R Lymphotoxin beta receptor
MALT Mucosa associated lymphoid tissue lymphoma translocation gene
MAPK Mitogen activated protein kinase
MCP Monocyte chemoattractant protein
MEF myeloid elf-1-like factor
MEKK Mitogen-activated protein kinas