Generation of sphingosine-1-phosphate by apoptotic cells and its impact on macrophage polarization in cancer development [Elektronische Ressource] / vorgelegt von Andreas Weigert

Andreas Weigert

Sujets

• Gesundheit

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2008
• Nombre de lectures: 6
• Langue: English
• Poids de l'ouvrage: 14 Mo

Extrait

Aus dem Fachbereich Medizin
der Johann Wolfgang Goethe-Universität
Frankfurt am Main

Institut für Biochemie I – Pathobiochemie

und dem Fachbereich Biologie
der Technischen Universität Kaiserslautern

Institut für Zellbiologie


Generation of sphingosine-1-phosphate by apoptotic cells and its
impact on macrophage polarization in cancer development



Dissertation
zur Erlangung des Doktorgrades der theoretischen Medizin
des Fachbereiches Medizin der Johann Wolfgang Goethe-Universität
Frankfurt am Main



vorgelegt von
Andreas Weigert
Worms





Frankfurt am Main 2007
























Dekan: Prof. Dr. med. J. Pfeilschifter
Referent: Prof. Dr. med. T. Deller
Koreferent: Prof. Dr. med. S. Harder

Tag der mündlichen Prüfung: 27. März 2008














Was man in der Natur Geheimnisvolles pries,
das wagen wir verständig zu probieren,
und was sie sonst organisieren ließ,
das lassen wir kristallisieren.

Johann Wolfgang von Goethe, Faust II



Index I
Index
1 SUMMARY 1
2 ZUSAMMENFASSUNG 3
3 INTRODUCTION 5
3.1 Cell death 5
3.1.1 Roads to apoptosis 6
3.1.2 Death in disease 8
3.2 Phagocytosis of apoptotic cells 10
3.2.1 Attraction 10
3.2.2 Recognition 11
3.2.3 Removal 13
3.3 Macrophage polarization 15
3.3.1 Macrophage phenotypes 15
3.3.2 Macrophage polarization by apoptotic cells 17
3.3.3 Macrophages in cancer development 21
3.4 Sphingosine-1-phosphate 23
3.4.1 Sphingosine kinases 25
3.4.2 Sphingosine-1-phosphate and cancer 28
3.5 Aims of this study 30
4 MATERIALS AND METHODS 32
4.1 Materials 32
4.1.1 Chemicals and Reagents 32
4.1.2 Buffers and Solutions 34
4.1.3 Kits 40
4.1.4 Antibodies 40
4.1.5 Media and reagents for cell culture 41
4.1.6 Stimulants and Inhibitors 42
4.1.7 Oligonucleotides 42
4.1.8 Expression plasmids 44
4.1.9 Cell lines and bacteria 44
4.1.10 Mice 46 Index II
4.1.11 Instruments 46
4.1.12 Software 47
4.2 Methods 48
4.2.1 Cell culture 48
4.2.2 Human monocyte isolation and culture 48
4.2.3 Induction of apoptosis and necrosis 49
4.2.4 Production and characterization of conditioned media 49
4.2.5 Co-culture experiments 50
4.2.6 Quantification of cell death 51
4.2.7 Cell death quantification in co-cultures 52
4.2.8 Caspase activity assays 52
4.2.9 Protein determination (Lowry method) 52
4.2.10 SDS-PAGE and Western blot analysis 53
4.2.11 Cellular fractionation 53
4.2.12 Electrophoretic mobility shift assays (EMSA) 54
4.2.13 Quantification of cytokine release from co-cultures 54
4.2.14 Down-regulation of S1PR1 55
4.2.15 S1P quantification in cell culture supernatants 55
4.2.16 Sphingosine kinase activity assay 56
4.2.17 Transformation of bacteria by the heat shock protocol 56
4.2.18 Bacterial culture and plasmid preparation 57
4.2.19 Site-directed mutagenesis 57
4.2.20 Transfection of eukaryotic cells 58
4.2.21 Stable SphK2 knock-down 59
4.2.22 Immunofluorescence staining 59
4.2.23 Tumor growth in nude mice 60
4.2.24 Statistical analysis 61
5 RESULTS 62
5.1 Macrophages cheat death after interaction with apoptotic cells 62
5.1.1 Macrophage protection against apoptosis by apoptotic cells 63
5.1.2 Survival is transmitted by a soluble factor 64
5.1.3 Survival is irrespective of AC cell line and pro-apoptotic stimulus 65
5.1.4 Characterization of the protective factor 66
5.1.5 Characterization of the protective principle 67
5.1.6 Contribution of sphingosine-1-phosphate towards protection 69
5.1.7 S1P production during apoptosis is performed by SphK2 73
5.2 The mechanism of S1P production by apoptotic cells 74 Index III
5.2.1 A truncated sphingosine kinase 2 is released during apoptosis 74
5.2.2 Caspase-1 cleavage sites in SphK2 75
5.2.3 Caspase-1 inhibition abrogates SphK2 release 76
5.2.4 Mutational analysis of caspase-1 cleavage sites in SphK2 78
5.2.5 Caspase-1 and SphK2 co-localize at the plasma membrane 80
5.2.6 Mutation of the PS binding site in SphK2 prevents its cleavage 82
5.2.7 Lowering PS exposure attenuates SphK2 release 83
5.3 Tumor cell apoptosis polarizes macrophages via S1P 85
5.3.1 Co-cultured MCF-7 cells alter the cytokine profile of macrophages 85
5.3.2 Viability changes of cancer cells in co-cultures with macrophages 89
5.3.3 Alternative macrophage activation demands tumor cell apoptosis 91
5.3.4 MCF-7 cell-derived S1P accounts for macrophage polarization 94
5.3.5 MCF-7 cells or authentic S1P impair NF-κB activation in macrophages 97
5.4 SphK2 is important for tumor growth in vivo 99
5.4.1 Knock-down of SphK2 in MCF-7 cells 99
5.4.2 Growth of MCF-7-siSphK2 tumors is impaired in nude mice 100
6 DISCUSSION 102
6.1 Macrophage survival induced by apoptotic cells 103
6.2 The mechanism of S1P release from apoptotic cells 107
6.3 Macrophage polarization by S1P 110
6.4 In vivo evidence for SphK2 implication in cancer development 115
6.5 Concluding remarks 117
7 REFERENCES 118
8 PUBLICATIONS 127
9 ACKNOWLEDGEMENTS 128

10 CURRICULUM VITAE 129 List of Figures IV
List of Figures

Figure 1: Signaling towards apoptosis........................................................................................8
Figure 2: Phagocytosis of AC. ..................................................................................................12
Figure 3: The M-Phenotypes. ...................................................................................................16
Figure 4: Macrophage polarization by AC. ...............................................................................20
Figure 5: Production and actions of sphingosine-1-phosphate. ...............................................24
Figure 6: Sphingosine kinases and their different functions in apoptosis. ...............................27
Figure 7: AC attenuate caspase-3 activation in murine and human macrophages. ................62
Figure 8: AC attenuate macrophage apoptosis........................................................................63
Figure 9: Macrophage protection caused by an apoptotic cell-derived soluble factor. ............64
Figure 10: Protection of primary human macrophages by ACM is PI3K-, Ca2+- and
ERK1/2-dependent. .................................................................................................68
Figure 11: Sphingosine-1-phosphate conveys protection. .......................................................71
Figure 12: Measurement of S1P-secretion by AC....................................................................72
Figure 13: SphK2 expression in AC contributes to the release of S1P....................................73
Figure 14: A truncated active SphK2 (t-SphK2) is released into the supernatant of
apoptotic Jurkat cells. ..............................................................................................75
Figure 15: Caspase-1 cleavage sites in SphK2 are conserved in mammals...........................76
Figure 16: Release of SphK2 demands active caspase-1. ......................................................77
Figure 17: Overexpression of SphK2 in HEK293 and NIH 3T3 cells. ......................................79
Figure 18: Mutation of the N-terminal caspase-1 cleavage site or the phosphatidylserine
binding site in SphK2 inhibits cleavage and release of SphK2 during apoptosis....81
Figure 19: PS binding site is highly conserved between human SphK1 and SphK2. ..............83
Figure 20: SphK2 release during apoptosis is coupled to phosphatidylserine exposure.........84
Figure 21: Co-culture of human macrophages with MCF-7 cells induced a cytokine shift. .....86
Figure 22: Co-culture with MCF-7 cells induced an alternative activation profile in human
macrophages. ..........................................................................................................88
Figure 23: Impact of human primary macrophages on the viability of different human
cancer cell lines. ......................................................................................................90
Figure 24: Activation profile of human macrophages after co-culture with different human
tumor cell lines. .....................................................