Andrea Kotitschke PhD thesis 2009

217 Pages·2009·6.25 MB·English

n

w
o

T

e

p
a
The copyright of this thesis rests with the University of Cape Town. No
C
quotation from it or information der ived from it is to be published
f
o
without full acknowledgement of the source. The thesis is to be used

y
for private study or non-comtmercial research purposes only.
i
s
r

e
v
i
n
U
GENOMIC AND NON-GENOMIC CROSS TALK BETWEEN THE
GONADOTROPIN-RELEASING HORMONE RECEPTOR AND
GLUCOCORTICOID RECEPTOR SIGNALLING PATHWAYS

n
w
o

T
by
e
p
Andrea Kotitschke
a
C

f
o

y
t
i
s
r
Thesis Presented for the Degree of
e
v
i DOCTOR OF PHILOSOPHY
n
U
in the Department of Molecular and Cell Biology
UNIVERSITY OF CAPE TOWN

Supervisor: Professor Janet P. Hapgood

August 2009
ii
DECLARATION

I, the undersigned, hereby declare that the work contained in this thesis is my own
original work and that I have not previously submitted any part of it at any university
for a degree.

n

w
o

T

e
p

a
Signature………………………… C Date…………………………….

f
o

y

t
i
s

r

e

v

i
n
U
iii

ACKNOWLEDGEMENTS

My PhD studies and my thesis have been the most difficult academic challenge I ever had to face thus
far. Many people have each played a significant role during this journey and without the guidance,
support, friendship and patience of the following people; this dissertation would have not been
completed. It is to them that I owe my deepest gratitude.

First and foremost, to my supervisor Prof. Janet P. Hapgood. Your knowledge, conmmitment, wisdom
w
and enthusiasm to the highest standard motivated and inspired me. You have taught me how to think
o
about science in a critical yet excited way, you have helped me to refineT my skills and have shown me

how to approach my work as a scientist and I will always be grateful for this. Thank you for always
e
p
being passionate about science. Thank you for your patience and believing in me. You truly are
a
inspirational. C

f
o
I also gratefully acknowledge my former and current fellow students from the Hapgood laboratory.

y
Especially I would like to say thank you to Kate, Nicky, Donita and Chanel: I have been particularly
t
i
s
privileged to work with every single one of you ladies. From the beginning you all have been with me
r
e
on this journey and we all worked towards the same goal, facing the emotional rollercoaster which
v
brings excitement, fear, firustration and happiness with both work and life. I am exceptionally grateful
n
that we have becomUe friends during this time. You all had an enormous impact on me and my project.
Kate, your enthusiasm, excitement, commitment and hard work is so inspiring and I admire you for
being such a true person. Nicky, you are a gifted scientist, your skills, understanding and personality
made it the most enjoyable work experience. Donita, your strength and passion have been
inspirational throughout. You always reach for the best, expect the most from yourself and the ones
around you. Chanel, you have been a remarkable resource of ideas with your insight. We have
become such a great team, it was a pleasure to work with you. I also would like to thank you for being
a good friend. You always encouraged and motivated me when times were difficult and for this I am
grateful. You all are wonderful examples of graceful and ambitious women.
iv
I also would like to acknowledge the University of Cape Town, especially the Department of Molecular
and Cell Biology and the National Research Foundation who have provided me with scientific
opportunities and financial support, for which I am grateful.

I would like to express my deepest gratitude to my friends and my family here in South Africa as well
as my friends and family in Germany who have walked along side with me.

Einen besonderen Dank möchte ich an dieser Stelle vor allem meinen lieben Eltern, Cordula und
Peter aussprechen. Ich bin Euch unendlich dankbar für Eure Unterstützung, Kraft, Geduld und vor
n
allem Eure Liebe. Jeden Schritt meines Lebens habt ihr liebevoll und umsorgwend begleitet. Ihr gebt
o
mir immer wieder Möglichkeiten, mich weiterzuentwickeln. Nicht zuletzt habt ihr mich unermüdlich und
T
mit Hingabe mit solch Eigenschaften ausgestattet, die mir wesentlich geholfen haben, diese Arbeit zu

e
vollenden. Ich danke Euch dafür, dass Ihr immer an mich geglaubt habt und dass ihr nie an meinen
p
Entscheidungen gezweifelt habt und dass ich immer ich aufa Euch zählen kann.
C

f
My most sincere gratitude goes to Stefanie, youo truly are the best friend one can ask for. Thank you

for always being “on the other end of the liyne” and for being the very beautiful and special person you
t
are. I thank you for your unwavering iemotional and moral support and your endless encouragement
s
r
when it was most required. Your kindness is beyond any doubt. Thank you for all your wise words,
e
v
love and most importantly our unshakeable friendship.
i
n

U
My deepest appreciation to my fiancée who inspired and amazed me every day: Words cannot
describe how grateful I am for all you have done for me. You have been my anchor and my strength.
You kept me focused and encouraged me all the time. You gave me confidence by believing in me,
my work and my abilities. You never gave up, even when I did and you stood by my side so selflessly
and lovingly through all the times of doubts and discouragement. I thank you for your patience, for
your warmth, for understanding, for your sacrifice, your kind and your love. I dedicate this to you.

Thank you to the Lord, Thank you for giving me peace in my heart and guidance in difficult times,
thank you for answering all my prayers and being a comfort and the voice of reason in my head.
v
ABSTRACT

The gonadotropin-releasing hormone receptor (GnRHR), a member of the G protein-coupled receptor
(GPCR) family, is a central regulator of reproductive function in all vertebrates. A central question in
the field is to identify which hormones regulate the GnRHR gene, the signalling pathways that are
activated, the transcription factors involved in mediating responses to various signals, and the
complex interplay between all these factors. This study investigated the mechanisms of regulation of
transcription of the mouse GnRHR gene in the mouse pituitary gonadotrope LßT2 cell line by GnRH
and dexamethasone (dex). Real-time PCR showed that both dex and GnRH increnase transcription of
w
the endogenous mouse GnRHR gene. Chromatin immunoprecipitation (ChIP) assays revealed the
o
presence of the activating protein-1 (AP-1) proteins c-Jun and c-Fos onT the mouse GnRHR promoter

under basal as well dex- and GnRH-stimulated conditions. Furthermore, recruitment of the
e
p
glucocorticoid receptor (GR) to the AP-1 element in response to dex was established by ChIP and
a
interestingly, treatment with GnRH also resulted in recCruitment of the GR in the absence of steroid.
Knockdown of GR levels by small interference RNA decreased the dex- and GnRH-induced
f
o
upregulation of the GnRHR gene revealing a requirement for the GR for both the dex and GnRH

y
response. ChIP and immunofluorescence assays provided evidence that both GnRH and dex
t
i
s
upregulate the GnRHR gene via nuclear translocation and interaction of the GR with the AP-1 region
r
e
on the mouse GnRHR promoter. It was also shown that GnRH activates the unliganded GR by rapid
v
phosphorylation of the GRi at Ser-234 in a GnRHR-dependent fashion to transactivate a GRE-reporter
n
gene in LßT2 and CUOS-1 cells. Using kinase inhibitors a direct link was established between GnRH-
induced protein kinase C and mitogen-activated protein kinase activation, leading to unliganded GR
phosphorylation at Ser-234 and transactivation of the glucocorticoid-response-element (GRE).
Furthermore it was shown that GnRH and dex synergistically activate the endogenous GnRHR
promoter in LßT2 cells, via a mechanism involving SRC-1 recruitment to the GnRHR AP-1 region. The
results suggest a novel mechanism of rapid non-genomic cross talk between the hypothalamic-
pituitary-gonadal and hypothalamic-pituitary-adrenal axes via GnRHR-dependent phosphorylation and
activation of the unliganded GR in response to GnRH.
vi

TABLE OF CONTENTS

LIST OF ABBREVIATIONS xii
THEISIS OUTLINE xv
INTRODUCTION xvii
AIMS AND HYPOTHESIS xix
n
CHAPTER 1
w
o
LITERATURE REVIEW
T
1.1 G-PROTEIN COUPLED RECEPTOR FAMILY 1
e
p
1.1.1 G-protein coupled receptors 1
a
1.1.2 The gonadotropin-releasing hormone receptor C 3
1.1.3 Tissue distribution and physiological function o f the GnRHR 4
f
o
1.1.4 GnRHR signalling pathways 5

y
1.1.5 Mammalian GnRHR gene structure 6
t
i
s
1.1.6 Mouse GnRHR promoter characterization 7
r
e
1.2 TRANSCRIPTIONAL REGULATION OF THE GnRHR GENE 9
v
i
1.2.1 Homologous regulnation of GnRHR gene expression 9
U
1.2.2 Regulation of GnRHR gene expression by gonadal steroid hormones 10
1.2.3 Regulation GnRHR gene expression by glucocorticoids 11
1.3 EFFECTS OF STRESS ON REPRODUCTION 12
1.4 STEROID HORMONE RECEPTORS 13
1.4.1 The glucocorticoid receptor 13
1.4.2 GR mechanism of action 14
1.4.2.1 Control of gene expression by DNA binding of the GR 16
1.4.2.2 Modulation of transcription factor function by the GR 18
1.4.4.3 Modulation of activator protein-1 function by the GR 20
1.4.3 Post-translational modifications of the GR 21
vii
1.5 RAPID STEROID ACTION 23
1.5.1 Rapid non-genomic effects of steroid receptors 23
1.5.2 Rapid GC-mediated effects 25
1.5.2.1 Cross talk of the membrane-bound GR 25
1.5.2.2 Cross talk of the classical GR with intracellular signalling proteins 26
1.5.2.3 Cross talk of the classical GR with GPCRs 27
1.6 GPCR CROSS TALK: FINE-TUNING OF MULTIPLE RECEPTOR
SIGNALLING PATHWAYS 28
n
1.6.1 Microdomains as a functional complex to facilitate signalling cross talk 28
w
1.6.2 Scaffolding proteins as mediators of GPCR cross talk 29
o
1.6.3 Cross talk between GPCRs by heterodimerization T 30

1.6.4 Cross talk of the GnRHR with other receptors e 31
p
CHAPTER 2
a
C
MATERIALS AND METHODS

f
2.1 Compounds and antibodies o 33

2.2 Incubation with test compounds y 33
t
i
2.3 Plasmids s 34
r
2.4 Transformation of bacteerial cells and plasmid preparation 35
v
2.5 Cell culture i 35
n
U
2.6 Transient transfections 36
2.7 RNA interference 36
2.8 Isolation of total RNA 37
2.9 cDNA synthesis 38
2.10 Quantitative real-time PCR 38
2.11 Western blot analysis 39
2.12 Autoradiography and quantification 40
2.13 Immunofluorescence 40
2.14 Chromatin immunoprecipitation assay 41
viii
2.14.1 Chromatin preparation 41
2.14.2 Immmunoprecipitation 42
2.14.3 Reversing formaldehyde crosslinks, DNA purification and
analysis of precipitated DNA 43
2.15 Statistical analysis 44
CHAPTER 3
AN INVESTIGATION INTO HOMOLOGOUS AND GC REGULATION
n
OF THE MOUSE GnRHR GENE IN THE Lb bbb T2 GONADOTROPE CELL LINE
w
3.1 BACKGROUND o 45
T
3.2 AIMS 47
e
3.3 RESULTS p 48
a
3.3.1 Both GnRH and dex increase expression of the enCdogenous mouse GnRHR gene
in LßT2 cells 48
f
o
3.3.2 GnRH but not dex induce a minimal AP-1 reporter construct in LßT2 cells 49

y
3.3.3 GnRH but not dex increases AP-1 protein expression levels 50
t
i
s
3.3.4 Both c-Jun and c-Fos proteins are bound under basal as well as dex- and
r
e
GnRH-stimulated conditions to the AP-1 region of the endogenous GnRHR
v
promoter in intact ceills 53
n
3.3.5 Steroid receptoUr expression in LßT2 cells 55
3.3.6 Both dex and GnRH induce recruitment of the GR to the AP-1 region on the
endogenous GnRHR promoter in intact cells 58
3.3.7 Protein expression levels of the GR remain unchanged in response to dex or GnRH 59
3.3.8 The GR is required for transcriptional regulation of the endogenous GnRHR by GnRH
and dex 61
ix
CHAPTER 4
INVESTIGATION OF LIGAND-INDEPENDENT ACTIVATION OF
THE GLUCOCORTICOID RECEPTOR BY GnRH AND SIGNALLING
PATHWAYS IN THE LßT2 GONADOTROPE CELL LINE
4.1 BACKGROUND 63
4.2 AIMS 65
4.3 RESULTS 66
4.3.1 GnRH mediates nuclear translocation of the unliganded endogenous GR n 66
w
4.3.2 Activation of a GRE by GnRH is GR- and GnRHR-dependent in LßT2 cells 67
o
4.3.3 Ligand-independent activation of a GRE is dependent on both the TGR and the

GnRHR in COS-1 cells 70
e
p
4.3.4 The GnRHR mediates site-specific phosphorylation of the unliganded GR by GnRH
a
in LßT2 cells C 72
4.3.5 Activation of MAPKs by GnRH in LßT2 cells 75
f
o
4.3.5.1 GnRH but not dex rapidly activates c-Jun-N-terminal kinase (JNK) 75

y
4.3.5.2 p38 is rapidly induced by GnRH and not dex in LßT2 cells 77
t
i
s
4.3.5.3 ERK is activated by short-term GnRH treatment while only long-term
r
e
dex stimulation results in ERK activation in LßT2 cells 78
v
4.3.6 Role of Kinases in diex- and GnRH-mediated transactivation and GR phosphorylation 80
n
4.3.6.1 MAPUKs are involved in GnRH- and dex-mediated transactivation of the GR
in LßT2 cells 80
4.3.6.2 MAPK are involved in GnRH-mediated AP-1 transactivation 82
4.3.6.3 GnRH-stimulated phosphorylation of the unliganded GR at Ser-234 is
mediated by a combination of JNK, p38 and ERK 83
4.3.6.4 PKC and c-src are involved in GnRH-mediated transactivation 87
4.3.6.5 PKC and c-src are involved in AP-1 transactivation by GnRH 89
4.3.6.6 PKC is involved in GnRH-mediated GR phosphorylation of Ser-234 89