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TABLE OF CONTENTS
Page
List of Figures ............................................................................................................ iii
List of Tables ............................................................................................................. iv
List of Abbreviations................................................................................................... v
Chapter 1: Introduction and Statement of Problem .................................................... 1
cAMP signal transduction ................................................................................ 1
The Phosphodiesterase 8 (PDE8) Family ....................................................... 5
Statement of Problem ..................................................................................... 8
Chapter 2: Phosphodiesterase 8B is a Major Modulator of Adrenal Steroidogenesis
................................................................................................................................. 10
Summary ....................................................................................................... 10
Introduction ................................................................................................... 11
Materials and Methods .................................................................................. 15
Results .......................................................................................................... 21
Discussion ..................................................................................................... 41
Chapter 3: Characterization of a PDE8-selective Inhibitor (PF-04957325) .............. 47
Summary ....................................................................................................... 47
Introduction ................................................................................................... 48
Materials and Methods .................................................................................. 50
Results .......................................................................................................... 53
Discussion ..................................................................................................... 64
Chapter 4: The Role of PDE8B in the Central Nervous System .............................. 66
Summary ....................................................................................................... 66
Introduction ................................................................................................... 67
Materials and Methods .................................................................................. 69
Results .......................................................................................................... 75
Discussion ..................................................................................................... 90
Final Remarks and Future Directions....................................................................... 94
Bibliography ............................................................................................................. 98
Appendix A: Enzyme Kinetic Properties and Selective Inhibitors of PDEs ............. 111
Appendix B: Trilostane Control .............................................................................. 112
Appendix C: shRNA Plasmids Transfection Efficiency in Y-1 Cells ....................... 113
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Appendix D: PDE-selective Inhibitor (PF-04957325) Selectivity Data on PDE
Isoforms ................................................................................................................. 115
Appendix E: The Novel Object Recognition Paradigm ........................................... 116
Appendix F: PDE8B KO Mice Had Same Mobility as WT Controls in Contextual Fear
Conditioning ........................................................................................................... 118
Appendix G: PDE8B KO Mice Showed Normal Swimming Distance in the Morris
Water Maze ........................................................................................................... 119
Appendix H: Training Significantly Decreased the Amount of Restraint-induced
Corticosterone Release ......................................................................................... 120
Appendix I: Preliminary Results from the Adjusted Morris Water Maze ................. 121
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LIST OF FIGURES
Figure Number
Page
1.1. The cAMP signaling pathway. ............................................................................. 3
1.2. Cyclic nucleotide phosphodiesterase (PDEs) families ........................................ 4
2.1. PDE8B is highly expressed in AZF cells ........................................................... 22
2.2 Full-length PDE8B mRNA transcript and PDE8B enzymatic activity are absent in
PDE8B KO adrenal glands ...................................................................................... 24
2.3. PDE8B KO mice exhibit adrenal hypersensitivity in vivo ................................... 27
2.4. Weight of the adrenal glands of PDE8B KO mice compared to WT controls ... 28
2.5. PDE8B gene ablation increases mRNA expressions of p450s ......................... 30
2.6. PDE8 inhibition with inhibitors increases acute adrenal steroid production ...... 34
2.7. shRNA against PDE8B also increases acute adrenal steroid production in Y-1
cells.......................................................................................................................... 36
2.8. PDE8 inhibition increases basal PKA activity and also mRNAs of steroidogenic
enzymes .................................................................................................................. 39
2.9. PDE8 inhibition increases the phosphorylation of HSL ..................................... 40
2.10. A model for modulation of PDEs ..................................................................... 46
3.1. Inhibition curve of PF-04957325 against PDE8B and PDE8A .......................... 55
3.2. PF-04975325 inhibition curve on recombinant PDE4D ..................................... 59
3.3. PF-04957325 potentiates pregnenolone secretion in Y-1 cells ......................... 62
3.4. 100 nM PF-04957325 selectively inhibits PDE8s in adrenal cells ..................... 63
4.1 Elevated plus maze ............................................................................................ 70
4.2. PDE8B expression in the mouse brain ............................................................. 77
4.3. IBMX-insensitive PDE activity found in the pellet of a PDE8B immunoprecipitate
from striatal lysate .................................................................................................... 78
4.4. PDE8B KO mice exhibit anxiety-like behaviors ................................................. 81
4.5. PDE8B KO mice show enhanced freezing behavior in contextual fear
conditioning .............................................................................................................. 83
4.6. PDE8B KO mice have increased generalized fear response after an adverse
stimulus.................................................................................................................... 86
4.7. PDE8B KO mice did not show a significant improvement in performance in the
Morris water maze ................................................................................................... 89
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LIST OF TABLES
Table Number
Page
1. Inhibitor sensitivity of PDE8B ............................................................................... 49
2. Selectivity of PF-04957325 .................................................................................. 56
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LIST OF ABBREVIATIONS
3HSD ------------------------ 3--hydroxysteroid dehydrogenase
5-DHEA ----------------------
5-Dehydroepiandrosterone
5’-AMP ------------------------ adenosine monophosphate
ACTH -------------------------
adrenocorticotropic hormone
ADSD -------------------------
autosomal-dominant striatal degeneration
AKAP -------------------------
A-kinase-anchoring protein
ANP ---------------------------
atrial natriuretic peptide
ATP ---------------------------- adenosine 5’-triphosphate
AZF ---------------------------- adrenal zona fasciculata
BSA ---------------------------
bovine serum albumin
cAMP -------------------------
cyclic adenosine monophosphate
cGMP -------------------------
cyclic guanosine monophosphate
CNG channels --------------
cyclic nucleotide-gated (CNG) ion channels
cpm ---------------------------- count per minute
CRF ---------------------------
corticotropin-releasing factor
DMEM ------------------------
Dulbecco’s modified eagle medium
DMSO ------------------------
dimethyl sulfoxide
DTT ---------------------------- dithiothreitol
EC50 ---------------------------
half maximal effective concentration
EDTA -------------------------
ethylenediamine-N,N,N’,N’-tetraacetic acid
EGTA -------------------------
ethylene glycol (-aminoethyl ether)-N,N,N’,N’tetraacetic acid
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ELISA -------------------------
enzyme-linked immunosorbent assay
EPAC -------------------------
exchange protein activated by cAMP
F12K --------------------------
F-12 Kaighn’s modification
FBS ---------------------------- fetal bovine serum
GFP ---------------------------
cGMP regulated PDEs, Anabaena adenylyl cyclase, E.
coli protein FhIA
green fluorescent protein
HBSS -------------------------
Hank’s balance salt solution
HEPES -----------------------
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HPA axis ---------------------
hypothalamic-pituitary-adrenal axis
GAF ---------------------------
HSL ---------------------------- hormone sensitive lipase
IBMX --------------------------
3-isobutyl-1-methylxanthine
IC50 ----------------------------
half maximal inhibitory concentration
IPTG --------------------------
isopropyl -D-1-thiogalactopyranoside
Km ------------------------------ Michaelis constant
KO -----------------------------
knockout mouse
mA -----------------------------
milliampere
MC2R -------------------------
melanocortin 2 receptor
mg -----------------------------
milligram
mL -----------------------------
milliliter
mM ----------------------------
millimolar
mm ----------------------------
millimeter
MOPS ------------------------- 3-(N-morpholino)propanesultonic acid
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mRNA ------------------------- messenger RNA
nM -----------------------------
nanomolar
p450 --------------------------- cytochrome P450
PAS ---------------------------
Per, ARNT and Sim proteins
PBS ---------------------------
phosphate buffered saline
PCR ---------------------------
polymerase chain reaction
PDEs -------------------------- cyclic nucleotide phosphodiesterases
PFA ---------------------------- paraformaldehyde
PKA ---------------------------
protein kinase A
pM -----------------------------
picomolar
REC ---------------------------
signal receiver
RI ------------------------------
recognition index
RNAi --------------------------
RNA interference
RT-PCR ----------------------
real time polymerase chain reaction
shRNA ------------------------
short hairpin RNA
StAR protein ----------------
steroidogenic acute regulatory protein
Vmax ---------------------------- maximum reaction rate
vol -----------------------------
volume
WT ----------------------------- wildtype mouse
wt ------------------------------
weight
X-gal --------------------------
5-bromo-4-chloro-3-hydroxyindole
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Acknowledgements
I extend immense gratitude to all my teachers who brought the best out of
me, especially my thesis advisor, Professor Joe Beavo, who instilled in me much
knowledge in matters both professional and personal. Joe shaped me into the
scientist that I am today. Without the support and freedom that I received in the
Beavo laboratory, this thesis would not have been possible. Additionally, Joe
exposed me to a broad range of scientific topics and taught me to be critical when
interpreting data. I also thank my committee members for their patience and
insightful feedback on my research. Finally, I would like to thank the members of the
Beavo laboratory for all of the helpful discussions and assistance throughout my
graduate studies.
On a more personal note, I thank my family and friends, especially my
parents and my partner, Lincoln Ballard, for their emotional support and
unconditional love. Without them, the process of pursuing this degree would be
much more challenging and much less fun.
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