Cellular oxygen-sensing through HIF-1α and NF-κB: A therapeutic target for ischemia. Público
Ogle, Molly Elizabeth (2012)
Abstract
Abstract
Cellular oxygen-sensing through HIF-1α and
NF-κB: A therapeutic target for ischemia.
Stroke is the fourth leading cause of death and the leading cause
of severe disability in
the United States and yet few effective treatments are available to
reduce ischemic brain
damage. The brain has an evolutionarily conserved adaptive response
to low oxygen
which is a potent protective signaling pathway and a novel target
for post-ischemic stroke
therapy. Oxygen deprivation inhibits prolyl hydroxylase (PHD)
enzyme activity and
stimulates a protective oxygen-sensing response in part through the
stabilization and
activation of the Hypoxia Inducible Factor (HIF)
1α transcription factor and stimulation
of the NF-κB
transcription factor family. This dissertation
tested the therapeutic
potential of enhanced activation of oxygen-sensing pathways by
pharmacologic PHD
inhibition after stroke, hypothesizing that post-ischemic PHD
inhibition would reduce
neuronal cell death and require the activation of HIF-1α
and/or the NF-κB family.
The
PHD inhibitor dimethyloxaloylglycine (DMOG) enhanced the
stabilization of HIF-1α
protein and HIF-1-responsive adaptive genes in neural tissues. DMOG
also increased the
activation of the NF-κB family in
cortical neurons. Post-ischemic treatment with DMOG
reduced ischemic damage including peri-infarct apoptosis,
maintained cerebral blood
flow in regions of the ischemic territory that were at risk for
infarction, and improved
functional outcome after MCAO. The beneficial effects of PHD
inhibition after ischemia
required HIF-1α and an intact
NF-κB pathway. Investigation of the
NF-κB family
activation through PHD inhibition in neurons suggests that there is
an interaction between
NF-κB and the expression of
HIF-1α mRNA and protein. Taken
together, the data
presented in this dissertation suggest that supplemental activation
of oxygen-sensing
pathways after stroke may provide a clinically applicable acute
therapeutic intervention
for the promotion of neuronal cell survival after ischemia.
Table of Contents
Chapter I. Background: Ischemic
Stroke............................................................................1
A.
Stroke......................................................................................................................2
1. Prevalence, incidence, and cost
2. Etiology
B. Animal models of ischemic stroke
..................................................................................3
1. Focal ischemia
2. Global ischemia
3. In vitro ischemia
C. Cerebral ischemic injury
............................................................................................11
1. Primary injury: the ischemic core
2. Secondary cell death: the ischemic penumbra
3. Mechanisms and features of cell death
D. Penumbra: the target of neuroprotective therapies
........................................................20
E. Summary and conclusions
..........................................................................................21
Chapter II. Oxygen-sensitive signaling: Prolyl hydroxylases
and Hypoxia-inducible
factor
..........................................................................................................................22
A. Oxygen
..................................................................................................................23
B. Prolyl hydroxylases: Cellular oxygen-sensors
..................................................................24
1. PHD isoform expression, localization, regulation
2. Pathophysiologic inhibition of PHDs
3. Pharmacological inhibition of PHDs
C. The hypoxia-inducible factor (HIF)
...............................................................................29
1. Oxygen-sensing and ischemic stroke
D. Adaptive gene regulation in hypoxia
...........................................................................33
1. Adaptation: Metabolism
2. Adaptation: Cell survival
3. Adaptation: Vascular system
E. Therapeutic use of oxygen-sensing pathways
............................................................36
1. Preconditioning
2. Post-conditioning
F. Summary and conclusions
..........................................................................................41
Chapter III. NF-kB Interaction of oxygen-sensing and
inflammatory pathways........................42
A. NF-kB transcription factor
family................................................................................43
1. NF-kB transcription factor family members.
2. Dimerization and DNA binding specificity.
3. Traditional upstream NF-kB signaling network:
Activation of NF-kBs
4. Additional modulators of NF-kB transcriptional
activity
B. Activation of NF-kB through hypoxia
........................................................................48
C. Inflammation induces HIF-1α
.......................................................................................51
D. Basal regulation of HIF-1α promoter by
NF-kBs.........................................................51
E. Summary and
Conclusions............................................................................................52
Chapter IV. Rationale, Aims, and Experimental Methods
........................................................53
A. Rationale and significance
..........................................................................................53
B. Specific aims
...............................................................................................................54
C. Materials and Methods
................................................................................................55
Chapter V. Inhibition of prolyl hydroxylases by
dimethyloxaloylglycine after stroke reduces
ischemic brain injury and requires HIF-1α
...................................................................................68
A. Introduction
..................................................................................................................69
B. Results
.........................................................................................................................71
1. DMOG induces stabilization of HIF-1α protein
and HIF-1α-responsive genes in cortical
neurons
2. PHD inhibitor pre-treatment or post-treatment attenuates
ischemic cortical neuron cell death in vitro
3. In vitro ischemic neuroprotection by PHD inhibitor requires
HIF-1α.
4. PHD inhibitor DMOG attenuates apoptotic cell death in cortical
neurons.
5. Cell permeable PHD inhibitor DMOG induces stabilization of
HIF-1α protein in vivo.
6. PHD inhibitor post-ischemic treatment reduces focal ischemic
infarct formation.
7. PHD inhibitor post-ischemic treatment reduces loss of local
cerebral blood flow.
8. PHD inhibitor treatment after stroke reduced activation of
caspase-3 in the ischemic cortex.
9. PHD inhibitor post-ischemic treatment reduces behavioral
deficits after stroke.
10. HIF-1α protein and HIF-1-regulated gene
transcription is enhanced after stroke with DMOG post-ischemic
treatment.
11. Digoxin and Acriflavine Hydrochloride inhibit
HIF-1α in the mouse brain and abrogate
DMOG-mediated protective gene expression.
12. Inhibition of HIF-1α abrogates the
post-ischemic DMOG-mediated neuroprotection.
C. Discussion
...................................................................................................................96
Chapter VI. Homozygous knockout of NF-kB p105/p50 disrupts
PHD inhibitor-mediated
postconditiong and HIF-1a expression after
ischemia..................................................................108
A. Introduction
..............................................................................................................109
B. Results
.......................................................................................................................112
1. Hypoxia and PHD inhibition induce nuclear translocation of
NF-κB.
2. Homozygous knockout of NF-kB p105/p50 impairs
DMOG-mediated protection against apoptosis in cortical neurons
3. Homozygous knockout of NF-kB p105/p50 impairs
the pre and post-conditioning response of cortical neurons during
OGD.
4. DMOG post-ischemic treatment reduces focal ischemic infarct
volume in WT but not homozygous p105/p50 KO mice.
5. Knockout of p105/p50 causes disregulation of
HIF-1α in cortical tissue.
6. Knockout of p105/p50 causes disruption of normal
NF-kB signaling and hypoxia responsiveness.
7. HIF-1α promoter has 2 -kB
sites.
8. Protein binding to the kB site -197/188 of the
HIF-1α promoter under normoxia/hypoxia is
dysregulated in KO neurons
C. Discussion
..............................................................................................................128
Chapter VII. Summary and Conclusions
.............................................................................137
Chapter VIII. References
........................................................................................................140
List of figures
Figure 1.1. Mouse cortical vessel anatomy and distal focal MCA model…………………………8
Figure 2.1. Reaction of Prolyl hydroxylase enzymes……………………………….....…………28
Figure 2.2. Regulation of HIF-1α by PHDs………………………………...……….....…………32
Figure 3.1. PHD mediated signaling to NF-κB………………..…………………….....…………50
Figure 5.1. DMOG induces normoxic HIF-1α expression in cortical neuron………...…………72
Figure 5.2. PHD inhibitor attenuates OGD-induced cell death and induces HIF-1α………........74
Figure 5.3. PHD inhibitor requires HIF-1α to attenuate OGD-induced cell death…..…..………76
Figure 5.4. PHD inhibitor DMOG attenuates apoptotic cell death in cortical neurons…………78
Figure 5.5. DMOG intraperitoneal injection stabilizes HIF-1α protein in the brain………….....80
Figure 5.6. PHD inhibitor post-ischemic treatment reduces ischemic infarct formation………..82
Figure 5.7. PHD inhibitor post-ischemic treatment attenuates peri-infarct loss of
cerebral perfusion ………………………………………………….………………...84
Figure 5.8. PHD inhibitor treatment reduces activation of caspase-3 in the ischemic cortex…....86
Figure 5.9. PHD inhibitor post-ischemic treatment reduces sensorimotor behavioral deficits
after stroke.…………………………………………………………..…………...….88
Figure 5.10. Post-ischemic DMOG therapy enhances HIF-1α expression and HIF-1
-responsive gene expression. ……………………………………………….…….90
Figure 5.11. Digoxin and Acriflavine Hydrochloride inhibit HIF-1α in the mouse brain and
reduce DMOG-mediated protective gene expression……………………...………93
Figure 5.12. Inhibition of HIF-1α abrogates the post-ischemic DMOG-mediated
neuroprotection……………………………………………………...……….…….95
Figure 6.1. Hypoxia and PHD inhibition induce nuclear localization of NF-κB in cortical
neurons………………………………………………………………..……………113
Figure 6.2. Homozygous knockout of NF-κB p50 abrogates DMOG-mediated protection
against apoptosis in cortical neurons……………………...……………….………115
Figure 6.3. Homozygous knockout of NF-κB p50 impairs the pre and post-conditioning
response of cortical neurons during OGD…………………………………………117
Figure 6.4. DMOG post-ischemic treatment reduces focal ischemic infarct volume in
WT but not homozygous p50 KO mice……………………………………………119
Figure 6.5 Knockout of p50 causes disregulation of HIF-1α basally and after stroke in
cortical tissue………………………………………………………….…………….121
Figure 6.6. Knockout of p50 causes disruption of normal NF-κB signaling and hypoxia
responsiveness…………………………………………………………..……….…123
Figure 6.7. HIF-1α promoter analysis……………………………………………………….…..125
Figure 6.8. Protein binding to the κB site -197/188 of the HIF-1α promoter under
normoxia/hypoxia is disregulated in KO neurons …………………………………127
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