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First published online August 6, 2004
Journal of Experimental Biology 207, 3171-3188 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01075
Review Article |
Cellular oxygen sensing need in CNS function: physiological and pathological implications
1 Karolinska Institute, Cellular and Molecular Biology, Stockholm,
Sweden
2 Edinger Institute, Frankfurt, Germany
3 Max Planck Institute for Molecular Physiology, Dortmund,
Germany
* Author for correspondence at address 1 (e-mail: till.acker{at}cmb.ki.se)
Accepted 6 May 2004
 |
Summary |
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 TOP Summary Brain oxygen supplyBrain oxygen sensing needThe HIF transcriptional complexHIF in brain physiology...Putative oxygen sensorsSynopsis of oxygen sensingConclusionsReferences |
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A significant advance in our understanding of the hypoxia response stems from the discovery of the hypoxia inducible factors (HIF), which act as key regulators of hypoxia-induced gene expression. Depending on the duration and severity of the oxygen deprivation, cellular oxygen-sensor responses activate a variety of short- and long-term energy saving and cellular protection mechanisms. Hypoxic adaptation encompasses an immediate depolarization block by changing potassium, sodium and chloride ion fluxes across the cellular membrane, a general inhibition of protein synthesis, and HIF-mediated upregulation of gene expression of enzymes or growth factors inducing angiogenesis, anaerobic glycolysis, cell survival or neural stem cell growth. However, sustained and prolonged activation of the HIF pathway may lead to a transition from neuroprotective to cell death responses. This is reflected by the dual features of the HIF system that include both anti- and proapoptotic components.
These various responses might be based on a range of oxygen-sensing signal cascades, including an isoform of the neutrophil NADPH oxidase, different electron carrier units of the mitochondrial chain such as a specialized mitochondrial, low PO2 affinity cytochrome c oxidase (aa3) and a subfamily of 2-oxoglutarate dependent dioxygenases termed HIF prolyl-hydroxylase (PHD) and HIF asparaginyl hydroxylase, known as factor-inhibiting HIF (FIH-1). Thus specific oxygen-sensing cascades, by means of their different oxygen sensitivities, cell-specific and subcellular localization, may help to tailor various adaptive responses according to differences in tissue oxygen availability.
Key words: glioblastoma, hypoxia inducible factor, HIF prolyl, hydroxylase, iron, ischemia, NADPH oxidase, mitochondria, mitochondriopathy, neurogenesis, neurodegenerative disease, oxygen sensing, preconditioning, reactive oxygen species, stem cell, tumor, VEGF
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Brain oxygen supply |
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TOPSummaryBrain oxygen supplyBrain oxygen sensing needThe HIF transcriptional complexHIF in brain physiology...Putative oxygen sensorsSynopsis of oxygen sensingConclusionsReferences |
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). This heterogeneity is also reflected in regional variations
of hemoglobin O2 saturation, as shown by blood oxygen
level-dependent fMRI-BOLD brain measurements
(Kannurpatti et al., 2003). It
is easy to conceive that cells adjacent to the arterial inflow (high
PO2) have metabolic capacities or electrical
activities different from those in cells located at the venous end (low
PO2). |
Brain oxygen sensing need |
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TOPSummaryBrain oxygen supplyBrain oxygen sensing needThe HIF transcriptional complexHIF in brain physiology...Putative oxygen sensorsSynopsis of oxygen sensingConclusionsReferences |
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Disturbances in oxygen availability have been implicated in the central
nervous system (CNS) pathology of a number of disorders including stroke, head
trauma, neoplasia, vascular malformations and neurodegenerative diseases.
Thus, complex cellular oxygen-sensing systems have evolved to ensure tight
regulation of oxygen homeostasis in the brain to avoid metabolic compromise or
the risk of oxidation toxicity. These induce an elaborate sequence of adaptive
mechanisms in response to variations in PO2
designed to avoid or at least minimize brain damage, including short-term
(seconds, minutes) and long-term (hours, days) responses. Impressive examples
of brain oxygen-sensing systems under various physiological and pathological
conditions are described in the literature: tissue oxygenation is highly
dependent on the regulation of cerebral blood flow. By adjusting the vascular
tone, perfusion is kept constant over a wide range of blood pressures, a
process termed cerebral autoregulation. Autoregulation of cerebral blood flow
provides a powerful mechanism to counteract regional imbalances in oxygen
supply. Thus, increases in brain neural activity that lead to changes in
metabolic demand elicit an increase in local blood supply within the
corresponding brain region. This response is probably triggered by an `initial
dip' of tissue PO2 due to an increased oxygen
consumption, as elegantly shown on the visual cortex
(Thompson et al., 2003).
Neuronal activity and excitability can be directly regulated in response to
O2 availability by altering membrane channel conductivity. Central
neurons contain O2-sensitive potassium channels that are reversibly
inhibited by hypoxia (Jiang and Haddad,
1994). These channels, partly identified as TASK channels, are
also regulated by a number of neurotransmitters, identifying them as key
players in oxygen-sensitive regulation of neuronal excitability
(Brickley et al., 2001;
Maingret et al., 2001).
Neonatal rats survive and avoid brain injury during periods of anoxia up to 25
times longer than adults, most likely due to a hypoxia-suppressed
N-methyl-D-aspartate receptor response to glutamate
excitotoxicity (Bickler et al.,
2003). A further neuroprotective early response lies in the active
suppression of gene transcription (Denko
et al., 2003a) and protein synthesis
(Hata et al., 2000), leading
to a shut-down of non-essential energy-consuming mechanisms that are not
required for immediate cell survival, a process termed oxygen conformance (for
reviews, see Zhou et al.,
2004; Hochachka and Lutz,
2001). Suppression involves inhibiting assembly of the two main
regulators of translation initiation, namely eukaryotic initiation factor
(eIF) 4F (Martin et al.,
2000,
2001;
Arsham et al., 2003) and eIF2
(Sullivan et al., 1999;
Althausen et al., 2001;
Martin et al., 2001;
Koumenis et al., 2002) and
inhibition of the eukaryotic elongation factor (eEF) 2
(Althausen et al., 2001).
Reversible inhibition of translation has been reported to occur at
O2 levels between 0.5% and 1%
(Görlach et al., 2000;
Lang et al., 2002).
Beyond these short-term responses, adaptation to reduction in oxygen
availability necessitates changes in gene expression, which would be predicted
to lead to reduced oxygen consumption and increased oxygen delivery, and
provide a means of counteracting the detrimental effects of hypoxia and
reoxygenation. In particular, reoxygenation encountered after spontaneous or
thrombolytic reperfusion following vessel occlusion, for example, carries a
risk of accumulation of toxic ROS, as antioxidative defense mechanisms in the
cell are perturbed causing additional tissue damage. Specific redox-sensitive
transcription factor systems such as HIF, nuclear factor kB (NF-kB), activator
protein 1 (AP-1) and early growth response protein-1 (EGR-1), have been
described that respond to changes in PO2 or an
excess in ROS by activation of appropriate target gene expression
(Koong et al., 1994;
Yan et al., 1999). The
identification of the HIF transcription system by Wang and Semenza
(1995) was a milestone in our
understanding of oxygen physiology. Since then the HIF system has emerged as a
key regulatory system of responses to hypoxia at both local and systemic
levels. It is believed that approximately 1–1.5% of the genome is
transcriptionally regulated by hypoxia. However, significant heterogeneity in
the transcriptional response to hypoxia is observed between different cell
types (Denko et al.,
2003b).
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The HIF transcriptional complex |
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TOPSummaryBrain oxygen supplyBrain oxygen sensing needThe HIF transcriptional complexHIF in brain physiology...Putative oxygen sensorsSynopsis of oxygen sensingConclusionsReferences |
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). The
HIF complex exists as a heterodimer composed of constitutively expressed
HIF-ß and O2-regulated HIF-
subunits belonging to the
bHLH (basic helix-loop-helix)-PAS (PER-ARNT-SIM) family of transcription
factors. Both HIF- and HIF-ß proteins exist as isoforms
[HIF-1, HIF-2, HIF-3 and ARNT (aryl hydrocarbon receptor
nuclear translocator), ARNT2, ARTN3, respectively] (reviewed in
Wenger, 2002). HIF activity
is tightly regulated throughout the range of physiological and pathological
oxygen concentrations involving multiple mechanisms of control at the levels
of mRNA expression, protein stability, nuclear translocation and
transactivation activity. These combine cooperatively to activate HIF to
maximal levels under decreasing oxygen concentrations. At the molecular level
this is mediated by subjecting HIF- subunits to multiple modes of
post-translational modification, including lysine residue acetylation mediated
by ARD-1 (Jeong et al., 2002),
phosphorylation and two different types of hydroxylation. The dominant control
mechanism occurs via oxygen-dependent proteolysis of HIF-
subunits. Oxygen-dependent enzymatic hydroxylation of proline residues within
HIF- subunits constitutes the critical modification governing protein
stability. Prolyl hydroxylation allows capture by the von Hippel Lindau tumor
suppressor protein (pVHL), which acts as the recognition component of an
E3-ubiquitin ligase enzyme. Subsequent ubiquitination targets the complex for
proteosomal degradation. Consequently, only low levels of HIF- protein
expression can be detected in the presence of oxygen, increasing rapidly and
exponentially as the O2 concentration decreases. A second
oxygen-dependent switch involving hydroxylation of an asparagine residue
within the transactivation domain regulates transcriptional activity, possibly
by interfering with recruitment of the coactivator p300, resulting in reduced
transcriptional activity. A VHL-independent regulation of HIF-
stability was recently suggested by studies reporting that heat shock proteins
Hsp70 and Hsp90 protect HIF-1 from proteosomal degradation by physical
interaction and support HIF- transcriptional activity
(Gradin et al., 1996;
Minet et al., 1999;
Mabjeesh et al., 2002;
Isaacs et al., 2002;
Zhou et al., 2004).
Interestingly, both Hsp70 and Hsp90 have been shown to be induced by hypoxia,
providing an alternative mechanism for hypoxia-induced HIF- activation
(Zhou et al., 2004). Though
oxygen-dependent regulation seems to provide the prevailing control mechanism
of HIF function, receptor-mediated phoshporylation cascades via
binding of various growth factors and cytokines, including angiotensin II,
epidermal growth factor (EGF), platelet derived growth factor (PDGF), tumor
necrosis factor (TNF) , insulin and insulin-like growth factors IGF-1
and -2, represent alternative ways to enhance HIF activity by translational
and post-translational control under normoxia. This induction is generally
less intense than that mediated by reductions in oxygen tension (for a review,
see Bilton and Booker, 2003).
While these pathways do not directly mediate the response to hypoxia,
interactions with HIF signaling suggest that cellular responses to hypoxia can
be fine-tuned by integration into the major signaling systems.
The HIF transcriptional system acts as a master regulator of
oxygen-regulated gene expression, inducing adaptive mechanisms that serve the
common purpose of maintaining oxygen homeostasis. To date more than 60
putative HIF-target genes have been identified, expression of which governs
important processes such as angiogenesis and regulation of vascular tone,
erythropoiesis, iron homeostasis, energy metabolism and pH regulation as well
as cell survival and proliferation (for a review, see
Semenza, 2003). The latter is
especially puzzling, as recent studies support the view that, apart from
inducing pro-proliferative proteins such as IGF-2, IGF-BP (binding proteins)
1–3 and TGF (transforming growth factor) ß3, the HIF pathway
includes responses with adverse effects on cell function by inducing
cell-cycle-arrest-specific and proapoptotic proteins such as DEC (defective
chorion)-1, BNIP (Bcl2/adenovirus E1B 19kD-interacting protein)-3, its
orthologue NIX (Nip3-like protein X) and cyclin G2. In addition, direct
stabilization of the proapoptotic protein p53 has been suggested by studies
demonstrating physical and functional interaction between HIF-1 and p53
(for a review, see Acker and Plate,
2002). Thus, the HIF system transactivates an extended
physiological pathway that encompasses a wide array of physiological responses
to hypoxia, ranging from mechanisms that increase cell survival to those
inducing cell cycle arrest or even apoptosis. Given the range and diversity of
regulated cellular functions it is easy to predict that the HIF system is
likely to be crucially involved in many physiological and pathophysiological
processes within the brain. However, our current understanding of the role of
HIF in these processes is rather limited. Activation of pathways with
seemingly opposing functions on cell survival, e.g. inducing neuroprotection
or neuronal cell death, as outlined in Fig.
1, further complicates elucidation, pointing towards a more
complex and highly cell-context-dependent role of HIF.
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HIF in brain physiology and pathophysiology |
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TOPSummaryBrain oxygen supplyBrain oxygen sensing needThe HIF transcriptional complexHIF in brain physiology...Putative oxygen sensorsSynopsis of oxygen sensingConclusionsReferences |
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; Sun et al.,
2003; Prass et al.,
2003; Zhang et al.,
2003). Further into this line of thinking are HIF-1
loss-of-function studies in KO-mice showing a failure in brain development
that is probably due to impaired vascular development as well as massive cell
death in the cephalic mesenchyme (Iyer et
al., 1998; Ryan et al.,
1998). The early lethal phenotype at embryonic day 11 has so far
prevented a conclusive analysis of HIF-1 function in the adult brain.
Numerous gene and protein expression studies in normal and hypoxic/ischemic
brain, however, have provided indirect experimental evidence for the
importance of HIF-1 activity in the brain.
Cerebral ischemia
The change in HIF-1 activity in response to reduction of
PO2 was analyzed in animal models of hypoxia,
global brain ischemia, focal ischemia and neonatal hypoxia/ischemia.
Widespread constitutive neuronal HIF-1 mRNA, protein and HIF-target
gene expression was reported in different regions of the brain, as shown in
Fig. 2 for the rat cerebral
cortex, which was found to further increase following hypoxic exposure
(Bergeron et al., 1999;
Kietzmann et al., 2001;
Chavez et al., 2000;
Beck et al., 2000).
Interestingly, HIF-1 translation is exempted from the overall
suppression of protein synthesis observed in hypoxic/ischemic conditions,
allowing for efficient and rapid protein accumulation
(Görlach et al., 2000;
Lang et al., 2002).
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The precise role of HIF-2 in the brain following a reduction in
PO2 remains elusive. One report demonstrated
induction of HIF-2 mRNA in endothelial cells following focal cerebral
ischemia (Marti et al.,
2000). However, two studies using different methods to reduce
oxygen availability demonstrated differential regulation of HIF-1 and
HIF-2 subunits according to the experimental conditions and cell type,
suggesting vital non-redundant functions of both isoforms, and warranting
further analysis (Stroka et al.,
2001; Wiesener et al.,
2003).
Concomitant induction of adaptive mechanisms such as glycolytic metabolism
and angiogenesis following hypoxia/ischemia further supports a neuroprotective
role of HIF activation. Subjecting animals to a brief and moderate episode of
hypoxia/ischemia elicits an autoprotective response, which results in
protection against a subsequent prolonged ischemic event, a phenomenon termed
preconditioning (for a review, see Dirnagl
et al., 2003). Preconditioning is likely to involve adaptive
changes in gene expression to activate the endogenous protective mechanism, as
it generally takes several hours to days after a preconditioning episode to
attain a state of tolerance. Preconditioning studies have identified a subset
of neuronal transcripts regulating a number of adaptive processes, including
ROS inactivation, metabolic switch, angiogenesis and neuroprotective
paracrine-acting factors involving known HIF target genes. In particular the
hypoxia-inducible gene products Epo (erythropoietin) and VEGF (vascular
endothelial growth factor), which are commonly expressed by neurons and
astrocytes (Bernaudin et al.,
2002; Beck et al.,
2000) have been identified as potential mediators of hypoxic
preconditioning (Bernaudin et al.,
1999; Prass et al.,
2003; Wick et al.,
2002). Interestingly, hypoxic preconditioning has been found to
increase HIF-1 expression and can also be achieved by known chemical
inducers of HIF- such as CoCl2 and desferrioxamine,
suggesting that the preconditioning phenomenon is mediated by HIF-activity
(Prass et al., 2002;
Bergeron et al., 2000). In
contrast, studies on neonatal mouse cortices revealed that inhibition of
HIF-activity using a dominant negative HIF-1 mutant protected cortical
neurons from delayed cell death following oxygen and glucose deprivation
(Halterman et al., 1999). The
results are consistent with a model in which HIF-1-mediated p53
stabilization promotes neuronal cell death following an ischemic exposure.
Thus, the important question of the role of HIF activation in determining
the outcome of ischemic and oxidative injury may be more difficult to answer
than expected, though essential to a future therapeutic application of HIF
activators or inhibitors. The severity and duration of hypoxia, protein levels
and phosphorylation status of HIF- subunits may be decisive parameters
in governing these fundamentally different responses on cell fate, mediating
the transition from adaptive neuroprotective to cell death responses
(Fig. 1; Piret et al., 2002;
Carmeliet et al., 1998).
Interestingly, VEGF with its pleiotropic effects stimulating angiogenesis and
vascular permeability as well as exerting direct neuroprotective activities,
reveals a similar diverse outcome on cell survival
(Carmeliet and Storkebaum,
2002). It is worth remembering that HIF-regulated prosurvival or
proapoptotic processes in response to ischemia and oxidative stress reflect
common pathophysiological components that are probably shared by many
neuropathological diseases (see below) as, for example, suggested in multiple
sclerosis, an inflammatory, demeyelinating brain lesion
(Aboul-Enein et al., 2003).
Neurogenesis
Recent years have opened an interesting new door with the potential of
restoring and replenishing injured tissue. It is well known that cells are
continuously replaced in various organ systems in the body throughout life.
The CNS has been traditionally viewed for many decades as a system with very
limited capacity for self-renewal and regeneration. This dogma is, however,
challenged by a growing body of evidence supporting the concept that, in
certain brain regions, neural stem cells exist that give rise to new neurons
as part of a general endogenous turnover mechanism, as well as in response to
injury (reviewed by Doetsch,
2003). Various factors that influence neural stem cell function
have subsequently been identified. A number of recent studies suggest that
oxygen-dependent gene expression is of crucial importance in governing
essential steps of neurogenesis such as cell proliferation and renewal,
survival and differentiation. Indirect evidence for this stems from studies
demonstrating increased cell proliferation and neurogenesis after
hypoxic/ischemic episodes (Liu et al.,
1998; Takagi et al.,
1999; Jin et al.,
2001). Low oxygen concentrations have been shown in in
vitro neurosphere assays to induce proliferation, cell survival and
neuronal differentiation (Studer et al.,
2000; Morrison et al.,
2000; Shingo et al.,
2001). Accordingly, a number of hypoxia-inducible growth factors
such as VEGF, Epo, IGF-1 and stem cell growth factor have been implicated as
important regulators of neurogenesis (Jin et al.,
2002a,b;
Shingo et al., 2001;
Aberg et al., 2000). So far no
studies on HIF- function in neural stem cells have been performed, but
analysis of the effects of HIF-1 loss of function in other organ
systems suggests a pivotal role in stem cell homeostasis
(Schipani et al., 2001;
Seagroves et al., 2003).
Interestingly, the redox state of the cell seems to decisively influence cell
fate decision, either supporting self renewal (reduced state) or
differentiation (oxidized state) mechanisms
(Noble et al., 2003). In
support of this concept, hypoxia has been shown to induce and keep cells in an
undifferentiated, immature phenotype (Jogi
et al., 2002). Recent studies have highlighted the endogenous
repair capacity of the brain following injury after focal cerebral ischemia
and the potential for using these inherent regenerative abilities in putative
therapeutic approaches. Considerable regeneration of neurons from the
endogenous neural stem niche occurs within the striatum
(Arvidsson et al., 2002).
Hippocampal regeneration by pyramidal cell replenishment can be even further
enhanced by ventricular application of growth factors known to stimulate
neural stem cell growth (Nakatomi et al.,
2002). A recent study elegantly exploited the pleiotropic and
possibly synergistically acting activities of VEGF to show that
intracerebroventricular VEGF application administered after the insult reduced
infarct size and improved the overall functional outcome, presumably as a
result of enhanced angiogenesis, neuroprotection and neurogenesis
(Sun et al., 2003). Thus,
hypoxia-inducible gene expression may provide crucial cues to stimulate and
direct neurogenesis to areas where cells are lost.
Neurodegenerative disease
Neurodegenerative diseases as diverse as Alzheimer's disease,
frontotemporal dementia, prion diseases, Parkinson's disease, Huntington's
disease or amyotrophic lateral sclerosis are associated with the accumulation
of potentially toxic, aggregation-prone misfolded proteins
(Taylor et al., 2002).
Features of these diseases can be recapitulated in transgenic mouse models
overexpressing mutant proteins (Wong et
al., 2002). Increasing evidence suggests that such protein
assemblies are particularly vulnerable to neurons by triggering cellular and
neuroinflammatory stress responses. Among others, oxidative stress in
particular has been implicated as a causative agent of progressing neuronal
degeneration.
Deposition of ß-amyloid (ßA) peptide, typically present in
extracellular plaques, is thought to be intimately connected to the initiation
of Alzheimer's disease (Bishop et al.,
2002). ßA peptides are believed to act as the primary toxic
agent detrimental to neural cell function and responsible for neural cell
death. They have been shown to directly induce ROS generation, thus causing
oxidative damage. A recent study implicated HIF-1 activation as a
general neuroprotective mechanism against oxidative stress and
ßA-toxicity (Soucek et al.,
2003). Clonal neural cell lines and primary cortical neurons,
which are resistant to ßA-toxicity, revealed an enhanced flux of glucose
through the glycolytic and the pentose-phosphate pathway, due to enhanced
enzymatic activity. Based on this observation the authors went on to show that
this increase in cellular glucose metabolism correlated with elevated
HIF-1 protein levels and activity. Importantly, exposure of neurons to
ßA-peptide was shown to induce HIF-1, though the mechanisms of
induction remained unknown. Interestingly, HIF controls the coordinate
upregulation of genes of the glycolytic pathway, ranging from glucose uptake
to lactate production. HIF-mediated induction of glucose transport and
utilization has been implicated in supporting antioxidative mechanisms, by
generating reducing equivalents in the form of NADH and NADPH via
glycolysis or the pentose-phosphate pathway, as well as synthesis of the
antioxidant pyruvate. Thus, in Alzheimer's disease pathology, HIF may be
involved a feedback mechanism, whereby ßA induces HIF-1 activity,
which in turn upregulates adaptive antioxidative mechanisms to help to combat
the increased oxidative stress inflicted by ßA. Considering the growing
body of evidence that ROS regulate HIF activity (see below), an interesting
question remaining to be answered is whether ßA-mediated HIF induction
operates via ROS generation. This early neuroprotective response
induced by ßA may eventually be overridden by cumulative toxicity
effects. The neuron may finally succumb to the continuous stress afflicted by
neurotoxic peptides such as ß-amyloid and induce a cell death program.
Again, given the pro-apoptotic activity of HIF-1, it is intriguing to
ask to what extent a ßA-mediated sustained and prolonged neural induction
of HIF-1 may participate in this process.
Amyotrophic lateral sclerosis, a progressive motorneuron disease, is
another intriguing example of implicating dysregulated oxygen signaling
pathways in the pathogenesis of neurodegenerative diseases. Motor neurons are
known to be exquisitely susceptible to variations of oxygen concentration.
Hence during transient episodes of ischemia the decrease in oxygen supply or
the oxidative damage caused by free radicals generated during reoxygenation
may prove particularly fatal. Indeed, cumulative oxidative damage due to the
toxic gain-of-function of mutant SOD1 has been linked to the pathogenesis of
amyotrophic lateral sclerosis (ALS;
Cluskey and Ramsden, 2001;
Rakhit et al., 2002). A novel
player in the aetiology of ALS has only recently been identified. To study the
relevance of HIF-mediated induction of the VEGF-gene, transgenic mice with the
hypoxia-responsive element (HRE) deleted were generated
(Oosthuyse et al., 2001).
HRE-deficiency was associated with a decrease in both normoxic baseline and
hypoxia-induced VEGF levels in the central nervous system, which predisposed
the animals to develop an adult-onset, progressive motor neuron disease. Motor
neuron loss in these mice probably resulted from a chronic reduction in neural
vascular perfusion, which in addition may have been exacerbated by an
insufficient VEGF-dependent neuroprotection. These findings prompted the
authors in a recent report to further analyze the contribution of impaired
VEGF regulation in the pathogenesis of human ALS
(Lambrechts et al., 2003).
Interestingly, decreased VEGF protein serum levels were found in all ALS
patients analyzed, which in a subset of patients could be genetically linked
to specific haplotypes within the VEGF promotor/leader sequence being
associated with a reduced transcription, IRES-mediated expression and
translation of VEGF. These data also clearly implicate impaired VEGF
expression as a modulator of ALS pathology in the human situation. No sequence
variations in the HIF-binding site of the VEGF-promotor or functionally
relevant missense mutations in the HIF-1 and HIF-2 genes could
be identified.
Collectively, these studies implicate reduced hypoxia-induced expression of
genes with neurotrophic or neuroprotective effects as novel components in the
pathogenesis of neurodegenerative diseases. In addition, vascular defects and
chronic vascular perfusion deficits, e.g. chronic ischemia, have been
documented in a number of neurodegenerative conditions such as Alzheimer's
disease, Parkinson's disease or ALS (for a review, see
Storkebaum and Carmeliet,
2004). It remains to be elucidated whether and to what extent the
HIF-system may participate in the disease process. Current data would support
a dual, seemingly paradoxical role of the HIF-system, depending on whether it
is the cause or the consequence, i.e. a primary or secondary effect. Again the
opposing effects on cell survival of downstream components of the HIF complex
may be the clue to this paradox (Fig.
1). Impaired activation of the HIF-system would result in reduced
induction of neurotrophic adaptive cascades and thus decreased cell survival.
Support for this notion stems from studies showing that aging, a known risk
factor for neurodegenerative diseases such as Alzheimer's disease, severely
attenuates the capacity to induce HIF-1 activity, thus impairing the
cell's ability to respond adequately to ischemic stress
(Chavez and LaManna, 2003). On
the other hand, chronic and sustained activation of the HIF-system as a
consequence of chronic vascular insufficiency, for example, may end in cell
degeneration. Interestingly, cellular disturbances of proteosomal function
resulting in ubiquitin accumulation have been reported in many
neurodegenerative disorders and are likely also to affect HIF- levels
(Bence et al., 2001). Both
pathomechanisms could even be operative in the same disorder, with the former
contributing to early-stage and the latter to late-stage disease process.
Considering the complexity of neurodegenerative disorders, however, the
process outlined above is likely to be oversimplified and warrants further
analysis. Transgenic animal studies selectively modulating HIF-1 and
HIF-2 expression and function in the different cell types of the brain
may be crucial in further clarifying the role of the HIF system in
neurodegeneration.
Neoplasia
Regions of low oxygen tension are commonly found in malignant tumors and
are associated with increased frequency of tumor invasion and metastasis and a
poor therapy outcome. The ability to initiate homeostatic responses and adapt
to hypoxia represents an important and crucial aspect in solid tumor growth.
In particular, activation of the HIF system has been identified as an
important mediator of these processes. The involvement of HIF in tumor
physiology is covered in more detail in a recent review
(Acker and Plate, 2002). In
comparison to adjacent tissue widespread HIF activation is observed in tumors,
correlating with tumor growth and progression. HIF overexpression in tumors is
related to both hypoxia-dependent (observed close to the necrotic area, as
shown in Fig. 2) and
hypoxia-independent mechanisms, such as oncogene activation and growth factor
signaling pathways. The relevance of the HIF system to tumor growth and
progression is highlighted by the variety of mechanisms regulated by
HIF-target genes, ranging from angiogenesis, to increase tissue oxygenation
over glycolysis, and pH regulation, allowing for energy generation when oxygen
is scarce, to cell proliferation and survival pathways. It is worth
remembering that the HIF pathway exerts its effects not only on the tumor cell
but also on the stromal microenvironment, to enhance and promote tumor
vascularization and growth. In fact, the activation of the HIF system elicits
a cellular and molecular crosstalk leading to the coordinated collaboration
between tumor, endothelial, inflammatory/hematopoietic and circulating
endothelial precursor cells (reviewed by
Acker and Plate, 2003).
The clinical correlation between intratumoral hypoxia and tumor
aggressiveness, which is particularly characterized by tumor invasion and the
ability to metastasize, has found a possible molecular correlate in recent
studies. Thus, crucial steps in these processes, including cell mobility and
migration, tissue invasion and the ability to home into specific organ sites,
are governed by specific signaling pathways known to contain HIF-target genes
such as c-Met, the receptor for HGF (scatter factor/hepatocyte growth factor),
or the chemokine receptor CXCR4
(Pennacchietti et al., 2003;
Schioppa et al., 2003;
Staller et al., 2003). Both
receptor systems have well established functions in tumor invasion and
metastasis, as shown e.g. in breast cancer
(Muller et al., 2001) or
gliomas (reviewed in Lamszus et al.,
1999). In addition, several factors known to determine the
invasive cancer phenotype such as cathepsin D, matrix metalloproteinase 2 and
urokinase plasminogen activator receptor (uPAR) have been shown to be
regulated by HIF (Krishnamachary et al.,
2003). Indeed, inhibition of the HIF pathway by geldanamycin was
shown to diminish glioma cell migration in vitro
(Zagzag et al., 2003).
Interestingly, though a growing body of evidence suggests that HIF-1 is
the major orthologue to convey hypoxia-induced gene expression, both
HIF-1 and HIF-2 seem to be required for hypoxia-induced cell
migration, as shown recently by siRNA-mediated knock-down of each orthologue
in breast cancer cell lines (Sowter et
al., 2003). Thus, low PO2 and
activation of the HIF system causes the tumor cell to migrate away from
hypoxic areas and invade further into the host tissue and organs, thus
supporting tumor spread. It is interesting to note that in highly invasive
glioblastoma multiforme, the most malignant and particular hypoxic brain tumor
entity, single tumor cells with high-level HIF expression have been detected
at the leading tumor invasion front
(Zagzag et al., 2000).
Recent insight into the precise mechanisms of oxygen sensing and signaling
may help to develop novel anti-tumor strategies that specifically target the
PHD-HIF-VHL pathway. Given the widespread HIF activation in tumors, the role
of HIF in transactivating angiogenic factors and the role of angiogenic
factors in tumor growth, interfering with this pathway is particularly
appealing. Its rationale lies in depriving the tumor cell of oxygen and
nutrients by inhibiting angiogenesis while at the same time disabling adaptive
mechanisms that help the cell to survive in this microenvironment. The
feasibility of this approach has been confirmed in different reports
(Maxwell et al., 1997;
Ryan et al., 1998;
Kung et al., 2000;
Kim et al., 2001). However,
given their key regulatory role in various complex physiological pathways
stretching from metabolism, proliferation and differentiation to apoptosis,
general manipulation of the HIF system is likely to show a variable outcome
depending on the cellular context, e.g. tumor cell type
(Carmeliet et al., 1998;
Blancher et al., 2000) or
tumor microenvironment (Blouw et al.,
2003) and should for this reason be employed cautiously. HIF
itself may directly influence the outcome of present therapies such as
chemotherapy and radiotherapy. HIF-1 deficiency in fibroblasts resulted
in increased apoptotic cell death in response to agents such as carboplatin
and ectoposide or ionizing radiation known to induce double-strand breaks,
suggesting that HIF-1 may induce expression of genes involved in the
repair of DNA double-strand breaks (Unruh
et al., 2003).
Manipulation of the putative oxygen sensor system prolyl hydroxylase (PHD;
see below) offers yet another new and challenging strategy to analyze and
influence tumor biology. Interestingly, PHD activity in cancer cells may be
reduced, as suggested by incomplete prolyl hydroxylation of HIF- in
normoxic tissue culture as detected by hydroxylation-specific antibodies
(Chan et al., 2002).
HIF- degradation under these conditions can be enhanced by vitamin C or
iron supplementation (Knowles et al.,
2003), both important cofactors of PHD, suggesting that attenuated
PHD activity may contribute to the general HIF activation in tumor cells. In
line with these observations, ectopic PHD1 overexpression in colon carcinoma
cells suppressed HIF-1 levels and reduced in vivo tumor growth
(Erez et al., 2003).
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Putative oxygen sensors |
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TOPSummaryBrain oxygen supplyBrain oxygen sensing needThe HIF transcriptional complexHIF in brain physiology...Putative oxygen sensorsSynopsis of oxygen sensingConclusionsReferences |
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), arguing
for flexible and highly adaptive systems. These should allow for graded
subsequent cellular reactions, partly requiring changes in gene expression.
Oxygen-sensing heme proteins and PHD have been described as candidate sensor
systems connecting an oxygen-dependent enzymatic activity to the regulation of
hypoxia-inducible responses, as outlined schematically in
Fig. 3.
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Oxygen-sensing heme proteins
Molecules changing their chemical properties as a direct consequence of the
surrounding PO2 may mediate the first step in
oxygen sensing. As for the carotid body, hypoxia leads to an increase in
afferent carotid sinus nerve activity, stimulating ventilation and blood
circulation in the body to avoid hypoxic tissue damage. The primary oxygen
sensor triggering this increase is as yet unknown, but there is large
agreement that it is a heme protein, either a mitochondrial component of the
respiratory chain (Baysal et al.,
2000; Mills and Jöbsis,
1972; Wilson et al.,
1994) or a non-mitochondrial protein like the NAPH oxidase (Nox;
Fu et al., 2000;
Cross et al., 1990).
Light absorption spectra identified very recently a cytochrome
a592 (shown in yellow in
Fig. 4) as a unique component
of carotid body cytochrome c oxidase. In contrast to other
cytochromes, cytochrome a592 revealed an apparent low
PO2 and high CN– affinity,
probably due to a shortcut of electron flow within the cytochrome c
oxidase between CuA and cytochrome
a3–CuB. It was suggested that this specific property
would allow the regulation of intracellular calcium levels under hypoxia
(Streller et al., 2002).
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Furthermore, it was postulated that a NADPH oxidase isoform (shown in dark
green in Fig. 4) within carotid
body type I cell functions as an oxygen sensor to regulate ion channel
conductivity and gene expression (Cross et
al., 1990). In this context, various NADPH oxidase isoforms
comprising complexes between p22phox, gp91phox, p47phox, p40phox, p67phox and
Rac1, 2 components must be considered. Their function may not, however, be
limited to the carotid body, as they show widespread expression throughout the
body, e.g. Nox1 in pulmonary vasculature smooth muscle cells
(Weissmann et al., 2000) or
the neutrophil Nox2 in endothelial cells
(Görlach et al., 2000b)
and neuroepithelial bodies (NEB; Fu et
al., 2000). The isoforms (Nox1–4 and the Duox group) make
use of the gp91phox component, as reviewed by Lambeth et al.
(2000). Gp91phox knock-out
mice showed an impaired hypoxic ventilatory control in neonatal animals due to
a decreased oxygen sensitivity of NEB potassium channel conductivity
(Kazemian et al., 2001;
Fu et al., 2000) indicating an
oxygen sensor function of the NADPH oxidase in this cell type and related
strains (O'Kelly et al.,
2001,
2000). On the other hand,
gp91phox knock-out mice showed unimpaired oxygen-sensing function of pulmonary
vasculature smooth muscle cells (Archer et
al., 1999) or carotid body hypoxic drive
(Roy et al., 2000). However,
p47phox knock-out mice demonstrated an enhanced carotid body hypoxic drive,
suggesting a particular Nox isoform for the carotid body
(Sanders et al., 2002). Taken
together, various NADPH oxidase isoforms may act as part of the oxygen-sensing
system.
Prolyl hydroxylases
Hydroxylation provides a dual mechanism of inhibiting HIF activity,
inducing proteolytic degradation and reducing transcriptional capacity. These
processes are conferred by a recently identified subclass of 2-oxoglutarate
dependent hydroxylases. Interaction of VHL with HIF- requires an
O2- and iron-dependent hydroxylation of specific prolyl residues
(Pro 402, Pro 564) within the HIF- ODD (oxygen-dependent-domain)
carried out by HIF-prolyl hydroxylase (PHD;
Epstein et al., 2001;
Bruick and McKnight, 2001;
Oehme et al., 2002). So far,
four orthologues of PHD have been described (PHD I–IV). A second
oxygen-dependent switch involves hydroxylation of an asparagine residue within
the C-TAD of HIF- subunits by a recently identified HIF asparaginyl
hydroxylase called factor-inhibiting HIF (FIH-1;
Lando et al., 2002).
Asparagine hydroxylation apparently interferes with recruitment of the
coactivator p300, resulting in reduced transcriptional activity. Both PHD and
FIH belong to a superfamily of 2-oxoglutarate dependent hydroxylases, which
employ non-haem iron in the catalytic moiety
(Hewitson et al., 2002). They
require oxygen in the form of dioxygen, with one oxygen atom being
incorporated in the prolyl or asparagyl residue, respectively, and the other
into 2-oxoglutarate, yielding succinate and CO2. Thus, the
hydroxylation reaction is inherently dependent on ambient oxygen pressure,
providing a molecular basis for the oxygen-sensing function of these
enzymes.
Interestingly, PHD are strikingly sensitive to graded levels of oxygen
in vitro, mirroring the progressive increase in HIF- protein
stability and transactivation activity observed when cells are subjected to
graded hypoxia in vitro (Epstein
et al., 2001). In line with this observation, PHD have been found
to have a striking low O2 affinity of 178 mmHg above the
concentration of dissolved O2 in the air
(Hirsilä et al., 2003).
Consequently, taking the regular tissue PO2
distribution as shown in the lower part of
Fig. 3, PHD would operate under
suboptimal, non-equilibrium conditions for HIF- turnover far beyond
their Km. However, given regular Michaelis–Menten
kinetics, this would allow the enzymes to operate in a highly sensitive
manner, in which small changes in oxygen concentration result in pronounced
changes in enzymatic reaction velocity and thus HIF- turnover. In
contrast, collagen prolyl-4-hydroxylases exhibit a Km of
about 28 mmHg, one sixth of the Km of PHD, allowing
optimal hyroxyprolyl-collagen biosynthesis under the low oxygen concentrations
physiologically found in the cell
(Hirsilä et al., 2003).
Recently, FIH was shown to have a Km of around 64 mmHg,
suggesting that this enzyme also acts as a bona fide oxygen sensor,
at least under conditions as found in normoxic tissues in vivo
(Linke et al., 2004).
Immunohistochemical staining of tissues for HIF- subunits is an
indirect method of assessing the activity of the PHD/HIF system in
vivo, and such studies have documented that HIF- levels are
generally low in rodent tissues under physiological conditions and are
substantially increased in response to systemic hypoxia or tissue ischemia
(Stroka et al., 2001;
Wiesener et al., 2003).
Interestingly, HIF- levels remain low even in regions such as the renal
medulla, which are characterized by low oxygen tensions known to enhance
HIF- protein in vitro. In addition, the extent and time course
of induction as well as cell-type-specific expression varies, suggesting that
individual, cell-specific thresholds for activation of the response may exist.
The above mentioned characteristics of the PHD system render it highly
sensitive to alterations of cofactor concentration, such as ferrous iron
(Knowles et al., 2003) or
2-oxoglutarate, substrate concentration, e.g. due to changes in HIF-
synthesis (Wiener et al.,
1996; Zhong et al.,
2000), as well as enzyme concentration, e.g. due to changes in
mRNA expression of PHD orthologues in response to
PO2
(Epstein et al., 2001), being
particularly striking for PHD3 (del Peso
et al., 2003). Consistent with this hypothesis, physiological
concentrations of cofactors such as ascorbate (25–50 µmol
l–1; Knowles et al.,
2003) have been reported to be far below the
Km values of PHD for vitamin C (140–170 µmol
l–1; Hirsilä et al.,
2003), suggesting significant alterations in PHD activity with
changes in cofactor concentrations, as outlined in
Fig. 3A. Our understanding of
the exact interplay of these factors in setting the sensitivity of the PHD/HIF
system is still incomplete, but nevertheless of crucial importance to
understanding the cell- and tissue-specific activity and response of the
oxygen-signaling cascade.
Interestingly, although ubiquitously expressed in various tissues, PHD
orthologues differ in their relative cell-specific abundance of mRNA levels
(Lieb et al., 2002;
Cioffi et al., 2003;
Oehme et al., 2002).
Moreover, PHD2 and PHD3 expression is upregulated by hypoxia, though the
fold-induction apparently varies between cell type and
PO2 analyzed
(Epstein et al., 2001;
D'Angelo et al., 2003;
del Peso et al., 2003;
Berra et al., 2003;
Metzen et al., 2003). The
exact role of four PHD orthologues in the regulation of HIF activity remains
elusive. However, it is evident that the hydroxylation efficiency among PHD1-3
is not identical and in particular differs regarding HIF- orthologue
preference and prolyl hydroxylation pattern, favoring the C-terminal prolyl
residue (Hirsilä et al.,
2003). RNAi (RNA interference)-mediated knock-down studies suggest
that PHD2 is the rate-limiting enzyme controlling the steady state levels of
HIF-1 in normoxia, at least under cell culture conditions
(Berra et al., 2003).
Moreover, the study implicated PHD2 in the initial stages of HIF-1
degradation following reoxygenation. Interestingly, a role for PHD1 in
controlling HIF-1 levels under long-term maintenance of hypoxia
(5–6 days) was proposed, suggesting further non-redundant functions of
each PHD orthologue in other physiological or pathophysiological settings.
The subcellular localization of PHD has been studied by ectopic expression
of chimeric EGFP-fusion proteins and three-dimensional two-photon confocal
laser scanning microscopy (2P-CLSM) analysis
(Metzen et al., 2003). PHD1
was detectable exclusively in the nucleus, PHD3 was distributed more evenly in
cytoplasm and nucleus, while the majority of PHD2 and FIH-1 was found in the
cytoplasm (Fig. 5). The latter
result is of interest because PHD2 and FIH-1 have a calculated molecular mass
of 46 kD and 40.3 kD, respectively. As molecules of up to 60 kD usually have
free access to the nuclear compartment, PHD2 and FIH-1 would be expected to be
present in cytoplasm and nucleus. This finding may suggest that PHD2 and FIH-1
are actively excluded from the nucleus.
|
Little is known about the involvement of PHD in pathological settings such
as neoplasia or neurological disorders. However, PHD may be a target of growth
factor signaling pathways and/or oncogenic transformation. In fact, it has
been shown that certain oncogenes such as ras and src induce HIF under
normoxia by inhibiting prolyl hydroxylation on Pro 564
(Chan et al., 2002). Recent
findings, however, suggest a possible link between PHD function and
tumorigenesis and neurodegeneration. The cosubstrate 2-oxoglutarate as an
intermediate of the Krebs cycle is generated in mitochondria. The two
ubiquitously expressed mitochondrial enzymes, succinate dehydrogenase (SDH)
and fumarate hydratase (FH), catalyze sequential steps in the Krebs cycle.
Interestingly, germline heterozygous mutations in the autosomally encoded
enzyme and enzyme subunits are associated with hereditary predispositions to
various tumors including paraganglioma, pheochromocytoma, benign smooth muscle
cell tumor and RCC (Tomlinson et al.,
2002; Baysal et al.,
2000; Niemann and Muller,
2000; Astuti et al.,
2001). Thus, both SDH and FH act as tumor suppressor genes. In
contrast, homozygous germline mutations in SDH subunits cause
mitochondriopathies such as the Leigh syndrome, which affect the central
nervous system and skeletal muscles. The underlying mechanism through which
loss of function leads to neoplasia or neurodegeneration remains unclear (for
a review, see Eng et al.,
2003). It is nevertheless tempting to speculate that defects in
the Krebs cycle are likely to influence 2-oxoglutarate levels or other
metabolic intermediates compromising PHD function. Further mitochondrial
dysfunction may lead to increased production of ROS which, as described below,
may also signal on the PHD/HIF system. Interestingly, ROS itself have been
shown to interfere with mitochondrial function at the level of
-ketoglutarate dehydrogenase (KGDH) and SDH
(Nulton-Persson and Szweda,
2001).
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Synopsis of oxygen sensing |
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TOPSummaryBrain oxygen supplyBrain oxygen sensing needThe HIF transcriptional complexHIF in brain physiology...Putative oxygen sensorsSynopsis of oxygen sensingConclusionsReferences |
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). When the oxygen concentration decreases this distribution
curve becomes more and more left-shifted, depending on the degree of arterial
blood hypoxia, as shown schematically in
Fig. 3B. Thus, one should
hypothesize the existence of oxygen-sensing systems with high as well as low
PO2 affinities to fit the heterogeneous
PO2 distribution curve. Modulation of the
specific PO2 affinities or oxygen sensor
activities, e.g. by integration into the major signaling pathways, would
efficiently trigger and fine-tune various signal cascades to optimise cellular
function and adaptation over a broad range of
PO2 values, as summarized in
Fig. 3A.
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Oxygen sensing heme proteins
The mitochondrial chain is a classical high affinity system keeping the
electron flux across the cytochrome c oxidase constant below
PO2 values of 1 mmHg
(Arthur et al., 1999).
Consequently, the tissue PO2 range covered by
the oxygen-sensing mitochondrial chain is small. However, with the mutated
cytochrome a592 an affinity modulator was proposed,
lowering the high PO2 affinity of cytochrome
c oxidase to about 30 mmHg
(Streller et al., 2002).
NAD(P)H oxidase with a PO2 affinity of about 20
mmHg seems to be the main ROS donor in many cell systems
(Jones et al., 2000). The low
Km of the NAD(P)H oxidase implies that it may function as
an oxygen sensor operating at low to intermediate
PO2 ranges. Interestingly, NADPH-activity is
exquisitely controlled by rac proteins and various growth factors
(Görlach et al.,
2003).
Prolyl hydroxylases
In contrast, PHD have a strikingly low PO2
affinity above 150 mmHg (Hirsilä et
al., 2003), far beyond the regular tissue
PO2 distribution, allowing efficient regulation
of HIF activity under these conditions (see above). Apart from
PO2, PHD activity is regulated by the amount of
ferrous iron (reduced form) recovered by antioxidants such as vitamin C
(Knowles et al., 2003). Iron
is required for heme formation, being the most common limiting factor for
erythropoiesis. Interestingly, HIF-target genes regulate different steps in
iron homeostasis from iron uptake to iron transport and iron storage
(Rolfs et al., 1997;
Tacchini et al., 1999;
Lok and Ponka, 1999;
Mukhopadhyay et al., 2000).
The implication of iron in oxygen sensing by 2-oxoglutarate-dependent
hydroxylases and the involvement of iron in oxygen toxicity through the Fenton
reaction (Porwol et al.,
2001) give an additional need for tight iron regulation, making
the interaction between oxygen and iron homeostasis physiologically highly
appropriate. A further level of complexity is added as decreasing
O2 concentrations lower PHD activity which, however, may be
partially compensated for by the increased availability of ferrous iron due to
the more reduced state of the cell. Thus, the interplay is rather intricate to
understand and study, and activity of the PHD/HIF system likely to be
influenced by the redox state of the cell. Indeed, HIF is regulated in an
iron- and redox-sensitive fashion. Redox regulatory systems such as
Thioredoxin or Ref-1 have been shown to induce HIF- stabilisation and
transactivation function (Huang et al.,
1996; Ema et al.,
1999; Carrero et al.,
2000; Lando et al.,
2000; Welsh et al.,
2002). It is intriguing to speculate that PHD may thus also act as
an iron- and redox-sensor.
Reactive oxygen species
Apart from toxic accumulation during reoxygenation periods ROS have been
implicated as intracellular second messengers (reviewed in
Lander, 1997;
Nordberg and Arner, 2001).
Various studies have demonstrated their influence on intracellular functions
in oxygen signaling, as shown in Fig.
6. Increased ROS in tissues act on different signal pathways, e.g.
the open probability of potassium channels
(Duprat et al., 1995) or
via enhancement of GATA-2 bi
