Regulation of arginase II expression in macrophages by supernatants of apoptotic cells [Elektronische Ressource] / vorgelegt von Vera Diana Barra

Vera Mace

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2010
• Nombre de lectures: 44
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

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


Gustav-Embden Zentrum für Biologische Chemie
Institut für Biochemie I – Pathobiochemie
Direktor: Prof. Dr. Bernhard Brüne


Regulation of arginase II expression in macrophages by
supernatants of apoptotic cells



Dissertation

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



vorgelegt von
Vera Diana Barra
aus Coimbra (Portugal)





Frankfurt am Main, 2010


























Dekan: Prof. Dr. med. J. Pfeilschifter
Referent: Prof. Dr. rer. nat. B. Brüne
Koreferentin: Prof. Dr. med. D. Meyer zu Heringdorf


Tag der mündlichen Prüfung: 11.03.2011











Das tiefste und erhabenste Gefühl, dessen wir fähig sind,
ist das Erlebnis des Mystischen.
Aus ihm allein keimt wahre Wissenschaft.
Wem dieses Gefühl fremd ist,
wer sich nicht mehr wundern und in Ehrfurcht verlieren kann,
der ist seelisch bereits tot.



Albert Einstein

deutsch-amerikanischer Physiker (1879 - 1955)








Index I
Index

1 SUMMARY 1
2 ZUSAMMENFASSUNG 3
3 INTRODUCTION 5
3.1 Homeostasis and apoptotic cell death 5
3.1.1 Tissue homeostasis 5
3.1.2 Apoptosis 5
3.2 Phagocytosis of apoptotic cells 7
3.2.1 Phagocyte attraction 7
3.2.2 Recognition and removal of apoptotic cells 8
3.2.2.1 Innate recognition of ‘nonself’ molecules by phagocytes 8
3.2.2.2 Recognition of modified ‘self’ molecules by phagocytes 9
3.2.2.3 Recognition of non-detaching ‘self’ molecules by phagocytes 12
3.2.2.4 Removal of apoptotic cells 12
3.3 Macrophage populations 13
3.3.1 M1 phenotype: classically activated or host defense macrophages 13
3.3.2 M2 phenotype: alternatively activated macrophages 14
3.3.2.1 M2a phenotype: wound-healing macrophages 15
3.3.2.2 M2b phenotype: hybrid macrophages 16
3.3.2.3 M2c phenotype: regulatory macrophages 16
3.3.2.4 Role of extracellular signal-regulated kinase in regulatory macrophage phenotype 18
3.3.2.5 Role of cyclic adenosine monophosphate in regulatory macrophage phenotype 18
3.3.3 M2 macrophages in cancer 19
3.4 Macrophage polarization by apoptotic cells 20
3.4.1 Attenuated pro- and enhanced anti-inflammatory response of macrophages
towards apoptotic cells 21
3.4.2 Attenuated nitric oxide and reactive oxygen species formation in macrophages
in response to apoptotic cells 23
3.4.3 Impact of soluble factors released from apoptotic cells on macrophages 25
3.4.3.1 Effects of IL-4, IL-10 and TGF-β on macrophages 25
3.4.3.2 Effect of S1P on macrophages 27
3.5 Cyclic adenosine monophosphate signaling pathways 30
3.5.1 Properties of A kinase-anchoring proteins 31
3.6 Regulation of arginase II expression 33 Index II
3.7 Aims of the studies 35
4 MATERIALS AND METHODS 36
4.1 Materials 36
4.1.1 Cells 36
4.1.2 Bacteria 36
4.1.3 Chemicals and reagents 37
4.1.4 Antibodies 39
4.1.5 Plasmids 39
4.1.6 Oligonucleotides 40
4.1.7 Instruments and Software 41
4.2 Methods 43
4.2.1 Cell biology 43
4.2.1.1 Cell culture 43
4.2.1.2 Isolation and culture of murine peritoneal macrophages 43
4.2.1.3 Generation of apoptotic Jurkat and MCF-7 cells 43
4.2.1.4 Generation of modified conditioned media 44
4.2.1.5 Generation of a stable sphingosine kinase 2 knock-down in MCF-7 cells 44
4.2.1.6 Transient transfection 44
4.2.1.7 CREB decoy experiments 45
4.2.2 Biochemistry 46
4.2.2.1 Protein determination (Lowry) 46
4.2.2.2 Nuclear protein extraction 46
4.2.2.3 SDS-PAGE/Western analysis 46
4.2.3 Molecular biology 47
4.2.3.1 Reporter assay 47
4.2.3.2 Electrophoretic mobility shift assay (EMSA) 47
4.2.3.3 Enzyme immunoassay (EIA) 48
4.2.3.4 Construction of the pGL3-mpARG II deletion constructs 48
4.2.3.5 Site directed mutagenesis for generation of point mutations 49
4.2.4 Microbiology 50
4.2.4.1 Transformation of bacteria by heat-shock 50
4.2.4.2 Bacterial culture and plasmid preparation 51
4.2.5 Statistical analysis 51
5 RESULTS 52
5.1 Sphingosine-1-phosphate (S1P) from apoptotic cells (ACs) activates S1P
receptor 2 (S1P ) to up-regulate arginase II (ARG II) in macrophages (MΦs) 52 2
5.1.1 ACs secrete soluble lipid factors that up-regulate ARG II expression 52
5.1.2 Lipid factors in apoptotic cell-conditioned medium (CM) up-regulate ARG II 53
5.1.3 S1P mediates ARG II up-regulation via S1P 56 2Index III
5.2 Extracellular signal-regulated kinase 5 (ERK5) contributes to CM-mediated
ARG II up-regulation in MΦs 58
5.2.1 ERKs contribute to ARG II up-regulation 58
5.2.2 ERK5 signaling induces ARG II expression 59
5.3 Cyclic adenosine monophosphate (cAMP) responsive element binding protein
(CREB) contributes to ARG II up-regulation in MΦs 60
5.3.1 Murine ARG II promoter (mpARG II) analysis 61
5.3.2 CREB contributes to CM-mediated ARG II up-regulation 61
5.4 S1P and ERK5 are required for CREB activation and subsequent ARG II up-2
regulation in MΦs 64
5.4.1 S1P mediates ERK5 phosphorylation 64 2
5.4.2 S1P and ERK5 induce CREB binding to the target sequence in mpARG II 67 2
5.5 Mechanism of ERK5-dependent CREB activation in MΦs 68
5.5.1 cAMP signaling contributes to CM-mediated ARG II induction 68
5.5.2 Combined cAMP elevation and phosphodiesterase 4 (PDE4) inhibition mimic
the CM effect on ARG II expression 69
5.5.3 CM induces cAMP accumulation in RAW264.7 MΦs 71
6 DISCUSSION 73
6.1 Mechanism mediating ARG II up-regulation in MΦs in response to CM 75
6.1.1 AC-derived S1P contributes to ARG II up-regulation 75
6.1.2 S1P contributes to ARG II expression by activating S1P 76 2
6.1.3 ERK5 signaling accounts for CM-mediated ARG II induction 77
6.1.4 CREB DNA-binding and transactivation is necessary for CM-dependent
ARG II up-regulation 78
6.1.5 S1P triggers ERK5 signaling and subsequent CREB activation resulting in 2
ARG II induction 79
6.1.6 Mechanism of CREB activation by ERK5 80
6.1.6.1 Adenylyl cyclase, PKA and PDE4 regulate ARG II expression 82
6.1.6.2 Postulation of an AKAP complex involved in ARG II up-regulation 82
6.2 Concluding remarks 83
7 REFERENCES 86
8 APPENDICES 101
8.1 Buffers and solutions 101
8.1.1 Buffer for cell biology 101 Index IV
8.1.2 Buffers and solutions for protein analysis 101
8.1.3 Buffers for molecular biology and microbiology 103
8.1.4 Buffers for luciferase assay 104
9 PUBLICATIONS 105
10 DANKSAGUNG 106
11 CURRICULUM VITAE 107
12 SCHRIFTLICHE ERKLÄRUNG 108

List of Figures V
List of Figures

FIGURE 3.1: Scheme of apoptosis vs. necrosis. 6
FIGURE 3.2: Mechanisms of apoptotic cell-recognition by macrophages. 11
FIGURE 3.3: Macrophage populations. 17
FIGURE 3.4: Scheme of the reaction catalyzed by nitric oxide synthase (NOS). 23
FIGURE 3.5: Scheme of the reaction catalyzed by arginase (ARG). 24
FIGURE 3.6: Apoptotic cell-mediated polarization of macrophages. 26
FIGURE 3.7: Sphingolipid metabolism. 28
FIGURE 3.8: Sphingosine-1-phosphate receptors and effectors. 29
FIGURE 3.9: Cyclic adenosine monophosphate signaling pathways. 31
FIGURE 3.10: Constitution of muscle A kinase-anchoring protein. 32
FIGURE 4.1: Restriction enzymes used to generate mpARG II promoter constructs. 49
FIGURE 5.1: CM-M up-regulates ARG II in MΦs in a protein-independent way. 53
FIGURE 5.2: Contribution of glycerophospholipids to ARG II induction in MΦs. 54
FIGURE 5.3: Contribution of sphingolipids to ARG II induction in MΦs. 55
FIGURE 5.4: S1P contributes to ARG II up-regulation. 56
FIGURE 5.5: Sub-optimal concentrations of CM-J cannot be rescued by S1P to
restore ARG II up-regulation. 57
FIGURE 5.6: S1P mediates ARG II up-regulation via S1P . 58 2
FIGURE 5.7: ERKs contribute to ARG II up-regulation. 59
FIGURE 5.8: ERK1/2 is not involved in ARG II up-regulation. 59
FIGURE 5.9: ERK5 signaling induces ARG II. 60
FIGURE 5.10: mpARG II analysis. 61
FIGURE 5.11: 262 bp fragment of mpARG II containing a CREB binding site. 62
FIGURE 5.12: Enhanced CREB binding to mpARG II oligonucleotides. 63
FIGURE 5.13: CREB decoy in RAW264.7 MΦs prevents ARG II induction. 64
FIGURE 5.14: CM-M induces ERK5 phosphorylation in RAW264.7 MΦs. 65
FIGURE 5.15: S1P mediates ERK5N phosphorylation. 66 2
FIGURE 5.16: Authentic S1P provokes ERK5N phosphorylation. 66
FIGURE 5.17: S1P and MEK5 provoke CREB binding to target sequence. 67 2
FIGURE 5.18: Physiological relevance of S1P and ERK5 signaling. 68 2
FIGURE 5.19: Adenylyl cyclase contributes to ARG II up-regulation. 69
FIGURE 5.20: PKA contributes to ARG II up-regulation. 69
FIGURE 5.21: Elevation of cAMP combined w