Cellular oxygen-sensing through HIF-1α and NF-κB: A therapeutic target for ischemia. Open Access

Ogle, Molly Elizabeth (2012)

Permanent URL: https://etd.library.emory.edu/concern/etds/r207tq22c?locale=en
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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) 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-
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- 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- 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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