The rainbow trout glucocorticoid receptors [Elektronische Ressource] : a mechanistic and toxicological study / von Heidi Becker

Heidi Becker

Sujets

• Biologie

Informations

• Publié par: martin-luther-universitat_halle-wittenberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 28
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

The rainbow trout glucocorticoid receptors -
a mechanistic and toxicological study
Dissertation
zur Erlangung des akademischen Grades
doctor rerum naturalium (Dr.rer.nat.)
vorgelegt der
Mathematisch-Naturwissenschaftlich-Technischen Fakult at
(mathematisch-naturwissenschaftlicher Bereich) der
Martin-Luther-Universitat Halle-Wittenberg
von Frau Heidi Becker
geb. am 16. M arz 1979 in Schkeuditz
Gutachterin bzw. Gutachter:
1. Prof. Dr. H.-J. Ferenz, Universit at Halle
2. PD Dr. habil. Kristin Schirmer, Helmholtz-Zentrum fur Umweltforschung
3. Prof. Dr. Christer Hogstrand, King’s College London
Halle(Saale),November2007
Verteidigt am 16. Juni 2008
urn:nbn:de:gbv:3-000014392
[http://nbn-resolving.de/urn/resolver.pl?urn=nbn%3Ade%3Agbv%3A3-000014392]Anfertigung der Doktorarbeit am
Helmholtz-Zentrum fur Umweltforschung GmbH - UFZ
Department Zelltoxikologie
unter Betreuung von Dr. Kristin Schirmer
sowie am
King’s College London, UK
Nutritional Sciences
unter Betreuung von Dr. Nic Bury
und Betreung von
Prof. Dr. H.-J. Ferenz
Institut fur Zoologie
Universitat Halle-WittenbergContents
Summary ix
Zusammenfassung xi
1 Introduction 1
1.1 Overview of the corticosteroid receptor system in sh . . . . . . . . . . 1
1.2 Mechanistic properties of mammalian glucocorticoid receptors . . . . . 3
1.2.1 Structure of the glucocorticoid receptor . . . . . . . . . . . . . . 3
1.2.2 Glucocorticoid receptor functioning . . . . . . . . . . . . . . . . 5
1.2.3 Nuclear localisation, tra cking and shuttling of GR . . . . . . . 5
1.2.4 Activation and repression of genes by the GR . . . . . . . . . . 7
1.3 Mechanistic properties of sh glucocorticoid receptors . . . . . . . . . . 8
1.3.1 Molecular characterization of glucocorticoid receptors in sh . . 8
1.3.2 The DNA binding region in sh glucocorticoid . . . . 9
1.3.3 Transactivational capacities of rainbow trout glucocorticoid re-
ceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.4 Toxicological analysis of sh glucocorticoid receptors . . . . . . . . . . 10
1.4.1 Direct interaction of contaminants with GR . . . . . . . . . . . 11
1.4.2 Indirect impact of contaminants on GR . . . . . . . . . . . . . . 11
1.5 Speci c objectives of this thesis . . . . . . . . . . . . . . . . . . . . . . 12
iContents ii
2 Material and Methods 13
2.1 Screening for a sh cell model and method development . . . . . . . . . 13
2.1.1 Routine growth of cell lines . . . . . . . . . . . . . . . . . . . . 13
2.1.2 RT-PCR of rtGR1 and rtGR2 mRNA . . . . . . . . . . . . . . . 15
2.1.3 Method development for the transfection of RTG-2 . . . . . . . 16
2.2 Mechanistic studies of sh glucocorticoid receptors . . . . . . . . . . . . 18
2.2.1 Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.2.2 Nuclear import studies using COS-7 and RTG-2 . . . . . . . . . 21
2.3 Toxicological analysis of sh glucocorticoid receptors . . . . . . . . . . 22
2.3.1 Transactivation assay using COS-7 . . . . . . . . . . . . . . . . 22
2.3.2 Exposure of COS-7 and RTG-2 cells with pollutants . . . . . . . 23
2.3.3 Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3 Results 27
3.1 Screening for a sh cell model and method development . . . . . . . . . 27
3.1.1 Screening of sh cell lines . . . . . . . . . . . . . . . . . . . . . 27
3.1.2 Development of a transfection technique for RTG-2 and COS-7 . 28
3.2 Mechanistic studies of sh glucocorticoid receptors . . . . . . . . . . . . 29
3.2.1 Cellular localisation of GFP-rtGR1 and GFP-rtGR2 . . . . . . . 29
3.2.2 Time and dose-dependent pattern of GFP-rtGR1 and GFP-rtGR2
nuclear transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.2.3 Ligand speci city of GFP-rtGR1 and GFP-rtGR2 . . . . . . . . 34
3.2.4 Domain swap mutants of sh GR and rat GR . . . . . . . . . . 37
3.3 Toxicological analysis of sh glucocorticoid receptors . . . . . . . . . . 41
3.3.1 Screening for environmental contaminants impairing rtGR1 and
rtGR2 action . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.3.2 Alteration of subcellular localisation of GFP-rtGR1 and GFP-
rtGR2 by selected environmental contaminants in the absence of
cortisol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
3.3.3 Kinetics of GFP-rtGR1 and GFP-rtGR2 nuclear transfer in the
presence by selected environmental contaminants . . . . . . . . 44
3.3.4 In uence of selected ental contaminants on transactiva-
tional activity of rtGR1 and rtGR2 . . . . . . . . . . . . . . . . 47Contents iii
4 Discussion 51
4.1 Mechanistic studies of sh glucocortidoid receptors . . . . . . . . . . . 51
4.1.1 Identi cation of a novel NLS in the A/B-domain of rtGR1 and
rtGR2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
4.1.2 Determinants of nuclear translocation of GFP-tagged rtGR1 and
rtGR2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.2 Toxicological analysis of sh glucocorticoid receptors . . . . . . . . . . 56
4.2.1 Impact of Nonylphenol on action of rtGR1 and rtGR2 . . . . . 56
4.2.2 Impact of Diclofenac on action of rtGR1 and rtGR2 . . . . . . . 57
4.2.3 Impact of Cadmium on action of rtGR1 and rtGR2 . . . . . . . 58
4.2.4 Impact of Copper on action of rtGR1 and rtGR2 . . . . . . . . 58
4.2.5 Impact of H O on action of rtGR1 and rtGR2 . . . . . . . . . 582 2
4.2.6 Additional pollutants tested for their impact on trout GRs . . . 59
5 Concluding Remarks and Future Directions 60
6 References 62
A Appendix xiii
A.1 Desteroidized FBS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
A.2 Reverse transcription of RNA to rst strand cDNA . . . . . . . . . . . xiv
A.3 Polymerase chain reaction . . . . . . . . . . . . . . . . . . . . . . . . . xiv
A.4 Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv
A.5 Maps of expression vectors . . . . . . . . . . . . . . . . . . . . . . . . . xvi
A.6 Competent cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xviii
A.7 Transactivation assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . xviii
B Acknowledgement xx
C Versicherung an Eides statt xxii
D Curriculum Vitae xxiiiList of Tables
2.1 Cell lines used for studying the glucocorticoid receptors . . . . . . . . . 14
2.2 Primer sequences used for RT-PCR . . . . . . . . . . . . . . . . . . . . 16
2.3 Strategies for cloning rainbow trout GRs into pEGFP-C1 . . . . . . . . 19
2.4 Primers used for cloning . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.5 Environmental contaminants tested for impact on rtGR1 and rtGR2
functioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.5 Environmental contaminants tested for impact on rtGR1 and rtGR2
functioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.1 Presence of rtGR1 and rtGR2 mRNA in diverse sh cell lines . . . . . 28
3.2 Impact of xenobiotics on rtGR1 and rtGR2 action . . . . . . . . . . . . 41
ivList of Figures
1.1 Structural and functional features of mammalian GR . . . . . . . . . . 4
1.2 Transactivation activity of rtGR1 and rtGR2 . . . . . . . . . . . . . . . 10
3.1 Characterisation of expression levels and function of the GFP-tagged
rtGR1 and rtGR2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.2 Subcellular localisation of GFP-tagged rtGR1, rtGR2, and rat GR 30C656G
3.3 Kinetics of GFP-rtGR1 and GFP-rtGR2 nuclear transfer transiently ex-
pressed in COS-7 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.4 Kinetics of GFP-rtGR1 and GFP-rtGR2 nuclear transfer transiently ex-
pressed in RTG-2 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.5 Ligand speci city of GFP-rtGR1 and GFP-rtGR2 transiently expressed
in COS-7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.6 Ligand speci ty of GFP-rtGR1 and GFP-rtGR2 transiently expressed
in RTG-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.7 Transactivation activity of rtGR1 and rtGR2 in the presence of proges-
terone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.8 Nuclear localisation of na ve receptors can be linked to certain regions
of the trout GRs. Chimers expressed in COS-7 cells . . . . . . . . . . . 39
3.9 Nuclear localisation of na ve receptors can be linked to certain regions
of the trout GRs. Chimers expressed in RTG-2 cells . . . . . . . . . . . 40
3.10 In uence of environmental contaminants on GFP-rtGR1 subcellular lo-
calisation in the absence of cortisol . . . . . . . . . . . . . . . . . . . . 43
3.11 In uence of environmental contaminants on GFP-rtGR2 subcellular lo-
calisation in the absence of cortisol . . . . . . . . . . . . . . . . . . . . 44
vList of Figures vi
3.12 Kinetics of GFP-rtGR1 nuclear transfer is altered in the presence of
contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.13 Kinetics of GFP-rtGR2 nuclear transfer is altered in the presence of
contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3.14 Kinetics of GFP-rtGR1 and GFP-rtGR2 nuclear transfer is altered after
2 h expsoure to hydrogen peroxid . . . . . . . . . . . . . . . . . . . . . 47
3.15 Transactivation activity of rtGR1