The role of Men1 in pituitary gland tumourigenesis [Elektronische Ressource] / presented by Lars Gredsted

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Dissertationsubmitted to theCombined Faculties for the Natural Sciences and for Mathematicsof the Ruperto-Carola University of Heidelberg, Germanyfor the degree ofDoctor of Natural Sciencespresented byDiplom-Biochemist Lars GredstedBorn in Copenhagen, DenmarkOral examination:The Role of Men1 in Pituitary Gland TumourigenesisReferees: Priv. Doz. Jochen WittbrodtProf. Dr. Günther SchützAcknowledgementsAcknowledgementsThe work presented in this thesis would not have been possible without the help andassistance from many people both inside and outside the lab.First of all I would like to thank Mathias Treier for his advise and support throughout my fouryears in his lab. I have really learned a lot during my time at EMBL.I would also like to thank the other members of my TAC committee, Jochen Wittbrodt andWalter Wittke and my second “Gutachter” from the university of Heidelberg Günther Schützfor constructive discussions of my project.I would like to thank all past and present members of the Treier lab; Vitor, Catherine, Katrin,Anna Corinna, Uli, Eve, Sandra, Dirk, Maria, Henry and Thomas for making many of thelong working days entertaining through discussions about most things imaginable includingscience. A special thanks to Katrin for helping with the ES cell tissue culture, Sandra forhelping with immunohistochemistry and Henry for helping with the Genespring analysis ofthe microarray data.

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Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences
presented by
Diplom-Biochemist Lars Gredsted
Born in Copenhagen, Denmark
Oral examination:The Role of Men1 in Pituitary Gland Tumourigenesis
Referees: Priv. Doz. Jochen Wittbrodt
Prof. Dr. Günther SchützAcknowledgements
Acknowledgements
The work presented in this thesis would not have been possible without the help and
assistance from many people both inside and outside the lab.
First of all I would like to thank Mathias Treier for his advise and support throughout my four
years in his lab. I have really learned a lot during my time at EMBL.
I would also like to thank the other members of my TAC committee, Jochen Wittbrodt and
Walter Wittke and my second “Gutachter” from the university of Heidelberg Günther Schütz
for constructive discussions of my project.
I would like to thank all past and present members of the Treier lab; Vitor, Catherine, Katrin,
Anna Corinna, Uli, Eve, Sandra, Dirk, Maria, Henry and Thomas for making many of the
long working days entertaining through discussions about most things imaginable including
science. A special thanks to Katrin for helping with the ES cell tissue culture, Sandra for
helping with immunohistochemistry and Henry for helping with the Genespring analysis of
the microarray data.
A big thanks must go to everybody at the animal house for taking care of our mice and for
always being helpful even when I was late with requests. I would also like to thanks the
people past and present in the transgenic facility, without whom my mouse would still be a
model only.
Thanks to all the friends I have made at EMBL that have made these years some of the best of
my life. EMBL is a truly international place and I feel very fortunate to have been given the
chance to make friends from so many countries of the world.
I would also like to thank my parents who have always supported me whatever decision I
have made right or wrong.
Finally the biggest thanks must go to Rebecca, for always being there to support me and cheer
me up. I dedicate this these to youIndex
SUMMARY 1
1 INTRODUCTION 2
1.1 Growth control in the pituitary gland 2
1.1.1 The pituitary gland 2
1.1.2 Development of the pituitary gland 3
1.1.3 Control of pituitary gland growth 5
1.1.4 Pituitary gland tumours 6
1.2 Multiple endocrine neoplasia 9
1.2.1 Multiple endocrine neoplasias 9
1.2.2 Multiple Endocrine Neoplasia type 1 (MEN1) 11
1.2.3 MEN1 12
1.2.4 MENIN 13
1.3 Menin as a regulator of transcription 14
1.3.1 Menin interacting proteins 14
1.3.2 Menin and JunD 15
1.3.3 mSin3A and Menin 16
1.3.4 Menin and NF-κB 17
1.3.5 Smad transcription factors and Menin 19
1.3.6 TGF-β signalling and growth control 21
1.3.7 MLL2 Histone methylation complex and Menin 22
1.3.8 Menin regulation of telomerase activity 23
1.3.9 Menin regulated transcription and MEN1 24
1.4 AIMS OF STUDY. 26
2 RESULTS 28
2.1 Generation of Men1 deficient pituitary glands 28
2.1.1 Construction of the Men1 conditional knockout vector 28
LoxP-Neo2.1.2 Establishment of Men1 allele 30
2.1.3 FlpE and Cre mediated recombination 30
LoxP-Frt/Frt LoxP-Null/Null 2.1.4 Viability of Men1 mice and absence of Menin in Men1 embryos 34
2.1.5 Pituitary gland specific cre mouse lines 36
2.1.6 Pituitary gland specific deletion of Men1 38
2.1.7 Pituitary gland phenotype 38
2.1.8 Pituitary gland histology 41
2.2 Molecular characterisation of Men1 deficient pituitary glands 45
2.2.1 Analysis of Menin expression in Men1 deficient pituitary glands 45
2.2.2 Analysis of growth and apoptosis in Men1 47
2.2.3 Microarray analysis of expression pattern in Men1 deficient pituitary glands 50
2.2.4 Genes deregulated in Men1 deficient pituitary glands 54
2.2.7 VIP, Cdc2/cyclin B and IGF1 57
2.2.8 Analysis by in situ hybridisation of deregulated genes 62
2.2.9 Analysis of VIP expression by immunohistochemistry 65
2.3 Screening for novel Menin interaction partners 67
2.3.1 Yeast two hybrid screening for novel Menin interactors 67
2.3.2 Previously characterised genes as novel Menin interactors 69
Trip11/GMAP210/Trip230 69
Spectrin β2/ELF 69
Ldb1a 70Index
2.3.3 Novel proteins interacting with Menin 70
2.3.4 Expression pattern of Gad67 and cloning of cDNA 71
3 DISCUSSION 74
3.1 Men1 loss leads to pituitary gland hyperplasia and adenoma formation. 74
3.2 Gene expression profiling suggest novel mechanism of MEN1 tumourigenesis 77
3.3 VIP, IGF1 and Cdc2 are upregulated in Men1 deficient pituitary glands 78
3.4 VIP and pituitary gland proliferation 81
3.5 IGF and pituitary gland proliferation 82
3.6 VIP and IGF1 in Cancer 83
3.7 Regulation of VIP and IGF expression 83
3.8 Menin interacting proteins 84
3.9 VIP and IGF1 as mediators of MEN1 tumourigenesis 86
LIST OF ABBREVIATIONS 89
4 MATERIALS AND METHODS 91
4.1 Materials 91
4.1.1 Chemicals 91
4.1.2 Equipment, plastic ware and other materials 92
4.1.3 Enzymes 94
4.1.4 Molecular weight markers 94
4.1.5 Oligonucleotides 94
Targeting vector synthesis 94
Cloning of pGad67 95
Mouse genotyping: 95
In situ probes 95
4.1.6 Antibodies 96
Primary antibodies: 96
Secondary antibodies: 96
4.1.7 Plasmid vectors 96
4.1.8 Commercial kits 97
4.1.9 Generally used solutions 97
4.1.10 Generally used media for bacteria and yeast 98
4.1.11 Cells 98
Bacterial strains 98
Yeast strains 98
ES cells 99
4.2 Methods 99
4.2.1 DNA - Plasmids 99
Preparation of plasmid DNA from bacteria 99
Purification of supercoiled DNA by CsCl gradient centrifugation 99
Plasmid extraction from yeast 100
Spectrophotometric determination of DNA and RNA concentration 100
DNA restriction and Klenow treatment 100
Electrophoresis of DNA 100
Isolation and purification of DNA from preparative agarose gels 101
DNA ligation 101
Preparation of chemocompetent Escherichia coli XL-10 cells 101
Transformation of chemocompetent Escherichia coli XL-10 cells 101
Preparation and transformation of electrocompetent E.coli XL-10 cells 102
Transformation of yeast 102
4.2.2 DNA - λ phage 103
Culture and preparation of bacteria for infection with λ-phage 103
Infection with and plating of λ-phage 103Index
Detection of specific λ-phage plaques by southern blot 103
Picking λ-phage plaques 104
Extraction of λ-phage DNA 104
4.2.3 DNA - Genomic 104
Preparation of genomic DNA 104
Polymerase Chain Reaction (PCR) 105
Southern blot analysis 105
Radiolabelling of DNA probes for southern blot analysis 106
4.2.4 RNA 106
Microarray analysis of pituitary gland expression pattern 106
4.2.5 DNA constructs 107
Construction of the Men1 targeting vector 107
Cloning of full length Gad67 107
Generation of Pit-1-Cre transgene 108
DNA constructs for in situ probes 108
4.2.6 Cell culture methods 109
Culture conditions 109
Trypsinisation of cells 109
Mitomycin C treatment of Mouse Embryo Fibroblasts 109
Freezing and thawing cells 109
Electroporation of ES cells 110
Isolation of individual ES cell colonies 110
ES cell injection into blastocysts and chimera production 111
Establishment of MEFs 111
4.2.7 Tissue sectioning 111
Tissue preparation and fixation 111
Cryosectioning 112
Paraffin embedding and mounting 112
Vibrotome sectioning 112
4.2.8 Histochemistry and Immunohistochemistry 112
Hematoxylene and Eosin staining 112
Immunofluorescence 113
Immunohistochemistry 113
4.2.9 In situ hybridisation 115
Generation of in situ probes by in vitro transcription 115
Hybridisation 115
4.2.10 Mouse methods 116
4.2.11 Proteins 117
Protein concentration meassurements 116
SDS-PAGE 116
Western blotting 117
Purification of GST-Menin 117
REFERENCES 118Summary
Summary
The pituitary gland is a key regulator of growth, metabolism and sexual development. The
pituitary gland integrates signals from the hypothalamus and from peripheral endocrine glands
and responds to changing physiological needs by secreting a series of hormones that regulate
the activity of various endocrine glands as well as acting directly on many tissues. Tumours of
the pituitary gland are relatively frequent possibly due to the plasticity of the gland. Pituitary
gland tumours occur both sporadically and as part of inherited multiple endocrine neoplasia
(MEN) syndromes. MEN1 is one of these inherited syndromes. People suffering from MEN1
develop tumours of the pituitary gland, the parathyroid glands, the pancreatic islets and the
adrenal glands. MEN1 is caused by a loss of function mutation in the tumour suppressor gene
MEN1. Men1 expression is found in all tissues in the mouse and not only in the endocrine
system. Menin the protein encoded by Men1, shares no homology with any known proteins
and contains no recognisable protein domains. Menin is believed to function as a regulator of
transcription through binding to several specific transcription factors. These include Smad
transcription factors, JunD and members of the NF-kB family of proteins.
To investigate the phenotype of Men1 deficiency and to elucidate the mechanism of MEN1
tumourigenesis I have generated a conditional Men1 mouse model that enables me to
specifically delete Men1 in the pituitary gland. Men1 deficient pituitary glands are
hyperplastic as early as 7 weeks of age. The hyperplasia often develops into massive
adenomas by 40 weeks of age. Both hyperplasia and adenoma formation shows a gender
difference and is more pronounced in female mice. Analysis of the Men1 deficient pituitary
glands revealed pituitary gland overproliferation by 12 weeks of age before the development
of adenomas. Microarray analysis of the Men1 deficient pituitaries identified two growth
factors that were significantly overexpressed in Men1 deficient pituitary glands. Both of these
factors also showed a clear gender difference in their expression levels. The overexpression of
these growth factors in the Men1 deficient pituitaries was confirmed by in situ hybridisation.
1Introduction
1 Introduction
1.1 Growth control in the pituitary gland
1.1.1 The pituitary gland
The pituitary gland is the master gland of the endocrine system. It integrates signals from the
hypothalamus, to which it is connected to by the pituitary stalk and regulates the activity of
the thyroid gland, the adrenal gland and the gonads.
The pituitary gland has three anatomical and functional parts, the posterior pituitary gland,
which is of neuroectodermal origin, and the intermediate and anterior pituitary gland, which
derives from the oral ectoderm. The posterior pituitary gland contains axons from neurons in
the hypothalamus that secrete Vasopressin and Oxytoxin. The intermediate gland (which is
not present in humans) secretes Melanocyte Stimulating Hormone (MSH). The anterior
pituitary gland consists of five major cell types, the lactotropes (10-25 percent), corticotropes
(15-20 percent), thyrotropes (3-5 percent), gonadotropes (10-15 percent) and somatotropes
(40-50 percent). In response to specific releasing hormones secreted from the hypothalamus
each cell type secretes their distinct hormone. Thyrotropin Releasing Hormone (TRH)
stimulates Thyroid Stimulating Hormone (TSH) release from the thyrotropes. Gonadotropin
Releasing Hormone (GnRH) stimulates release of Luteinising Hormone (LH) and Follicle-
Stimulating (FSH) from gonadotropes. Growth hormone releasing hormone
(GHRH) stimulates Growth Hormone (GH) release from the somatotropes and finally
Corticotropin Releasing Hormone (CRH) stimulates Adrenocorticotropin (ACTH) release
from the corticotropes. A hypothalamic Prolactin Releasing Hormone (PRRH) has been
characterised that binds to the G-protein coupled receptor hGR3 (Hinuma et al., 1998),
although subsequent studies have suggested that PRRH is only functional in females and only
at high concentrations (Samson et al., 1998) and that much of its function may be mediated by
the hypothalamus (Seal et al., 2000).
TSH, LH, FSH and ACTH exert their effects through peripheral endocrine organs whereas
Prolactin and GH act directly on their target organs to regulate reproduction and growth
respectively.
During adult life, the activity and size of the pituitary gland changes with age and in response
to certain physiological events such as pregnancy. Accordingly the pituitary gland is subjected
to a very complex regulation consisting of hormonal regulation from the hypothalamus, as
2Introduction
well as feedback control from the peripheral target glands and other endocrine glands that
regulate the activity and growth of the pituitary gland directly and indirectly through the
hypothalamus. This plasticity of the pituitary gland, which is a consequence of the necessity
to adapt to changing physiological needs, may be the reason why pituitary gland neoplasias
are more frequent than tumours of other tissues. To elucidate the mechanism of pituitary gland
tumourigenesis in both sporadic cases and in the context of inherited tumour syndromes like
MEN1, I will in the following sections discuss the development of the pituitary gland and the
factors that control its growth as well as what is known about factors contributing to
tumourigenesis of the pituitary gland and other endocrine glands.
1.1.2 Development of the pituitary gland
A schematic representation of the development of the pituitary gland is shown on Figure 1.1.
The pituitary gland and the hypothalamus develop from a close association between two
embryonic tissues, the neural ectoderm and the oral roof ectoderm. At embryonic day E8.5 of
mouse development, the oral comes into contact with the neural ectoderm from
which it receives inductive signals, mainly BMP4. These signals induce the onset of pituitary
gland organogenesis (Treier et al., 1996)(Dasen et al., 1999). Shh and Hnf3ß, a winged helix
transcription factor, which were previously uniformly expressed in the oral ectoderm, are now
excluded from the contact region with the ventral diencephalon. This creates a molecular
compartment within the oral ectoderm, which later becomes the anterior pituitary gland. At
the same time, Lhx3, a member of the LIM homeodomain family of transcription factors, is
expressed in this molecular compartment and commits the oral ectoderm to the pituitary gland
fate. Subsequently at E9.0, the oral ectoderm invaginates to form a structure called Rathke's
pouch which is the pituitary gland primordium (Figure 1.1 A). The formation of Rathke’s
pouch is followed by proliferation of cells from the ventral part of the pituitary gland. This
proliferation is blocked in the absence of Lhx3 or FGF8 from the ventral diencephalon. In the
definitive Rathke’s pouch, BMP2 is induced at the boundary between Rathke’s pouch and the
oral ectoderm and a ventral to dorsal BMP2 activity gradient is created. Concomitant with the
invagination of the oral ectoderm, a portion of the ventral diencephalon (the infundibulum)
evaginates and contacts the dorsal portion of Rathke's pouch directly. FGF8 originating from
the infundibulum establishes a dorsal to ventral activity gradient that functions
antagonistically to the opposing ventral to dorsal BMP2 gradient (Figure 1.1 B) (Treier et al.,
1998). These two transient gradients establish patterns of overlapping expression of several
3Introduction
transcription factors in Rathke’s pouch. Many of these transcription factors exhibit temporally
and spatially restricted domains of expression within the developing pituitary gland resulting
in the appearance of the different hormone secreting cell types (Figure 1.1 C) (Treier et al.,
1998).
A series of mice with pituitary gland phenotypes have contributed to the identification of
factors involved in specifying the different pituitary gland cell types. The Ames dwarf mouse
has a hypoplastic pituitary gland. In the Ames mouse, somatotropes, lactotropes and
thyrotropes are specified but fail to proliferate. Accordingly the number of these cell types is
less than 1 percent of wildtype numbers (Sornson et al., 1996). The gene responsible for this
phenotype was cloned by positional cloning and named Prop-1. Prop-1 is a homeodomain
transcription factor that is exclusively expressed in the pituitary. Its expression can be
4