Functional Role of a Constitutively Active Dioxin/Ah Receptor in a Transgenic Mouse Model

68 Pages·2003·1.22 MB·English

From Institute of Environmental Medicine and
Institution for Cell and Molecular Biology
Karolinska Institutet, Stockholm, Sweden

Functional Role of a
Constitutively Active
Dioxin/Ah Receptor in a
Transgenic Mouse Model
Patrik Andersson

Stockholm 2003

i
All previously published papers were reproduced with permission from the publisher.

Published and printed by Karolinska University Press
Box 200, SE-171 77 Stockholm, Sweden
' Patrik Andersson, 2003
ISBN 91-7349-469-0
ii
To Charlotte and Linus
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Abstract
The dioxin/ Aryl hydrocarbon receptor is a ligand-activated transcription factor that mediates
most (if not all) of the toxic effects of the group of highly potent environmental pollutants
collectively called dioxins and PCBs, including the highly toxic compound 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD). The toxic effects include immune suppression,
endocrine disruption, impaired reproduction and carcinogenesis. In addition, the Ah receptor
regulates expression of several genes, most of which are xenobiotic metabolizing enzymes
such as CYP1A1. Although the high toxicity of these compounds has been known for more
than three decades, the mechanism(s) of action behind the wide spectrum of effects is yet not
known. Moreover, although the Ah receptor was cloned more than ten years ago, a
physiological role or ligand has not unambiguously been identified. A transgenic mouse
model that expresses a constitutively active Ah receptor (CA-AhR) was therefore developed.
The aim of this study was to characterize the functional role of this receptor in these mice.

Homozygous CA-AhR mice were fertile and the CA-AhR was expressed in several tissues
such as thymus, spleen, liver, lungs and heart as well as in all parts of the gastrointestinal
tract. In CA-AhR mice, unexposed to any exogenous Ah receptor ligand, expression of the Ah
receptor target gene CYP1A1 was induced in all of these organs, indicating that the CA-AhR
was transcriptionally active in a constitutive manner. In addition, effects observed in most
experimental animals after TCDD exposure, such as decreased thymus weight and increased
liver weight were also observed in the CA-AhR mice.

Increased mortality was observed from six months of age in the CA-AhR mice. This
correlated with the development of cystic stomach tumors that penetrated all layers of the
glandular stomach. These tumors consisted of differentiated cells, such as foveolar, parietal
and cardio-pyloral type of cells. Immunohistochemical analysis demonstrated expression of
CYP1A1 and the proliferation marker PCNA. Intestinal and squamous metaplasia was also
observed in the tumors. Some tumors were surrounded by a region of connective and fatty
tissue together with lymphatic foci and vessels, reminiscent of hamartomatous lesions
observed in humans. Wild-type animals orally treated with TCDD demonstrated an expansion
of the zone of proliferating cells normally found in the narrow isthmus-region of the stomach
to include the entire parietal-chief cell region. Moreover, the parietal/chief cell region was
decreased in the stomach of CA-AhR mice and this was associated with foveolar hyperplasia.

To identify dysregulated genes in the CA-AhR mouse stomachs, RNA from three months old
animals was subjected suppressive subtraction analysis. Osteopontin gene expression was
found to be down-regulated in the glandular stomach but also in other organs of CA-AhR
mice. TCDD exposure of wild-type and mutant mouse hepatoma cells demonstrated a rapid
but transient decrease in osteopontin expression that was dependent on functional Ah receptor
and Arnt. Moreover, FACS analysis of lymphoid cells revealed that the CD5-expressing
peritoneal population of B cells (B1a) was substantially reduced in CA-AhR mice. Taken
together, these results demonstrate that expression of the CA-AhR in mice resulted in a
number of effects. In particular, the development of well-differentiated stomach tumors
indicates a role of the receptor in regulation of gastric epithelial cell homeostasis.
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List of Publications

This thesis is based on the following papers and will be referred to in the text by their roman
numerals:

I. Andersson P, McGuire J, Rubio C, Petterson S, Hanberg A and Poellinger L. A
constitutively active dioxin/ aryl hydrocarbon receptor induces stomach tumors.
(2002) Proceedings of the National Academy of Sciences USA, 99:9990-9995.
II. Andersson P, Rubio C, Kato Y, Poellinger L and Hanberg A. The histologic
characteristics of stomach tumors in mice expressing a constitutively active dioxin/
aryl hydrocarbon receptor. Manuscript.
III. Andersson P*, Kuznetsov NV*, Gradin K, von Stein P, Dieckmann A, Pettersson S,
Hanberg A and Poellinger L. The dioxin/ aryl hydrocarbon receptor mediates
down-regulation of osteopontin gene expression. Manuscript.
IV. Andersson P, Ridderstad A, McGuire J, Pettersson S, Poellinger L and Hanberg A.
A constitutively active Aryl hydrocarbon receptor causes loss of peritoneal B1
cells. (2003) Biochemical and Biophysical Research Communications, 302: 336-
341, in press.

* These authors contributed equally to this study.
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CONTENTS
ABSTRACT 5
LIST OF PUBLICATIONS 7
INTRODUCTION 11
The dioxin/ Ah receptor 11
Ah receptors in other species 11
Expression pattern of the Ah receptor 12
Ah receptor forms with different sensitivity for TCDD 13
Physiological role for the Ah receptor? (cid:150) Observations in Ah receptor knockout mice 13
The bHLH-PAS protein family 15
Arnt 15
Ah receptor ligands 16
Persistent organohalogen pollutants 16
Polyaromatic hydrocarbons (PAH) 18
Ligands derived from the diet 18
Endogenously formed ligands 18
Ah receptor activation 18
Ligand-independent activation 21
A constitutively active Ah receptor (cid:150) the CA-AhR 21
Ah receptor target genes 21
Ah receptor-mediated toxicity 23
Toxic effects by Ah receptor ligands in the gastrointestinal tract 25
Introducing the B1 cell 27
Transgenic and knockout mice 28
AIMS OF THE PRESENT STUDY 30
METHODOLOGICAL CONSIDERATIONS 31
The CA-AhR mice 31
The expression construct 31
Genetic background 31
Analysis of several founder lines 32
Genotyping 33
Analysis of CA-AhR and target gene expression 33
RT-PCR 33
Northern (RNA) blot 34
In situ hybridisation and Immunohistochemistry 34
SSH vs. Microarrays 38
Using CYP1A1 as a marker of transcriptionally active CA-AhR and Ah receptor 38
TCDD exposure of the mice 38
FACS (cid:150) Fluorescence Activated Cell Sorter 39
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RESULTS AND DISCUSSION 40
CA-AhR founder lines 40
Sex ratios and bodyweight 40
Increased mortality 41
Expression and transcriptional activity of the CA-AhR 41
Stomach tumors 42
Cystic dilatation and penetrating tumoral glands 43
Intestinal metaplasia 44
Influence of sex and founder line 44
Decrease in parietal cells 44
Benign or malignant? 45
Dysregulated gene expression in the glandular stomach (cid:150) osteopontin 46
Suppressive subtraction analysis, SSH 46
Decreased osteopontin expression in the CA-AhR mice 46
Decreased osteopontin expression is dependent on functional AhR and Arnt 46
Effects in the immune system 47
Effects observed on T cell populations 47
Increase in mature bone-marrow derived B cells 48
Decreased peritoneal population of B1 cells 48
CONCLUSIONS 50
ACKNOWLEDGEMENTS 52
REFERENCES 54
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LIST OF ABBREVIATIONS

3-MC 3-Methylcholantrene
Ah Aryl hydrocarbon
AhR Ah receptor
AhRR Ah Receptor Repressor
Arnt Ah Receptor Nuclear Translocator
bHLH Basic Helix-Loop-Helix
CA-AhR Constitutively Active Ah Receptor
CD Cluster of Differentiation
DAB Diaminobenzidine
ES Embryonic Stem Cell
FACS Flourescence Activated Cell Sorter
GAPDH Glyceraldehyde-3-phosphate dehydrogenase
GFP Green Fluorescent Protein
HIF-1α Hypoxia Inducible Factor α
HRP Horse Radish Peroxidase
Hsp90 Heat Shock Protein 90
IRES Internal Ribosomal Entry Site
LBD Ligand Binding Domain
MHC Major Histocompatibility Complex
NES Nuclear Export Signal
NLS Nuclear Localization Signal
NMO1 NAD[P]H:menadione oxidoreductase
OPN Osteopontin
PAS Per-Arnt-Sim
PCB Polychlorinated Biphenyl
PCDD Polychlorinated Dibenzo-p-dioxin
PCDF Polychlorinated Dibenzofuran
PCR Polymerase Chain Reaction
RT-PCR Reverse Transcription PCR
TCDD 2,3,7,8,-tetrachlorodibenzo-p-dioxin
TEF Toxic Equivalence Factor

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