Pulmonary and Critical Care, Department of Medicine, The University of Chicago, Chicago, Illinois 60637
HYPOXIA ELICITS A WIDE
RANGE of adaptive responses at the systemic level, at the tissue
level, and at the cellular level. Responses at the systemic level such
as increased alveolar ventilation promote survival of the organism by
maintaining arterial blood hemoglobin saturation and systemic oxygen
transport. Within tissues, hypoxia stimulates the production of
vascular growth factors including vascular endothelial growth factor
(VEGF), which promote capillary growth and sustain local tissue
O2 delivery. At the cellular level, some responses help to
enhance organismal survival. For example, hypoxia elicits an increase
in the expression and secretion of the hormone erythropoietin, which
increases systemic oxygen supply by amplifying the rate of erythrocyte
formation. Other cellular responses promote cellular survival by
enhancing the expression of glycolytic enzymes, cell membrane glucose
transporters, and other genes that tend to protect the cell from more
severe oxygen deprivation. All of these responses occur because a
decrease in O2 has been detected by an oxygen sensor, and a
subsequent signaling pathway has become activated.
From a physiological standpoint, an oxygen sensor needs to be capable
of detecting a wide range of decreases in O2 tension ranging from mild hypoxia to complete anoxia. An ability to detect moderate hypoxia is important because many adaptive responses are
anticipatory to the extent they seek to prevent or delay the onset of
more severe hypoxia. An adaptive response that is only triggered after
the cell has become anoxic is of little benefit in preventing the onset
of that condition in the first place. On the other hand, from a
teleological standpoint it is reasonable to expect the O2
sensor should also continue to function even if anoxia is reached,
because cell death does not occur immediately in the face of
O2 deprivation. From a biophysical perspective, it would be
challenging to design a robust O2 sensor that could detect
and signal hypoxia ranging in severity from mild to severe and that
would continue to function accurately despite physiological variations
in pH and temperature. In fact, no oxygen sensor capable of detecting
moderate decreases in O2 has yet been definitively identified in mammalian cells. The search for a physiological O2 sensor represents an important and exciting area of
research because of its roles in development, in cell survival and in
tumor cell biology.
Although the identity of the oxygen sensors has been elusive, much
recent progress has been made in understanding the molecular systems
activated in response to hypoxia. Most of the genes that are activated
during hypoxia are regulated by the transcription factor hypoxia
inducible factor-1 (HIF-1) or its close relatives HIF-2 or HIF-3. First
identified by Semenza and colleagues (18, 19, 23), this
factor is now recognized as a global regulator of O2
homeostasis in a wide range of multicellular organisms
(16). In mammals, a large number of target genes for HIF
have been identified; the expression of these genes has important
consequences for regulation of cellular metabolism, proliferation,
survival, cardiovascular function, and iron homeostasis and
erythropoiesis. When activated, HIF functions as a heterodimer
comprising an The regulation of HIF-1 has been summarized recently in several
excellent reviews (12, 17). Briefly, both HIF-1 A critical question concerning HIF-1 On the basis of the requirement for iron in the function of prolyl and
asparagine hydroxylases, it is tempting to speculate that
desferrioxamine mimics the effects of hypoxia by chelating the nonheme
iron from the catalytic sites in these hydroxylases. Using similar
reasoning, one might argue that cobalt ions could inhibit these enzymes
by substituting for iron, thereby inactivating enzyme function.
Finally, the requirement for O2 in the hydroxylation reactions suggests that hypoxia inhibits hydroxylation by limiting the
availability of this molecule as a substrate for the reactions. If
true, this would implicate the hydroxylases as functional
O2 sensors in the transcriptional response to hypoxia. As
tantalizing as this explanation may seem, it is prudent to carefully
examine the compatibility of this idea with other data in the field to see whether a consistent picture emerges. Although there is little doubt that these hydroxylases are central participants regulating the
stabilization and function of HIF-1 Several disparate models have been proposed to explain how molecular
oxygen regulates specific processes such as the regulation of HIF-1 The idea that O2-dependent HIF-1 regulation was abolished
in mitochondria-deficient A larger question in the field of oxygen sensing relates to whether
multiple O2 sensors exist in the same cell, whether
different mechanisms of O2 sensing occur in different
cells, or whether a single O2 sensor regulates the diverse
responses to hypoxia among different mammalian cells. In the end, it
could turn out that prolyl and asparagine hydroxylases do function both
during hypoxia as well as during anoxia in the cell. Such a finding
would indicate that multiple O2 sensors must exist, since
many responses to hypoxia, including neurotransmitter release in the
carotid body and smooth muscle cell contraction in the pulmonary
artery, do not require activation of HIF-1. Moreover, other
transcription factors including NF-
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REFERENCES
-subunit and a
-subunit that is identical to the
aryl hydrocarbon receptor nuclear transporter. Transgenic mice with
homologous deletion of either subunit die at midgestation (11,
14), and significant functional abnormalities are even seen in
heterozygous adult mice lacking one allele (10, 20). Given
the critical importance of this factor during development and its role
in both physiological and pathophysiological states, it is not
surprising that much attention has been focused on the mechanisms
regulating its activation during states of O2 deprivation.
and
HIF-1
are constitutively expressed under normoxic as well as hypoxic conditions. Although transcriptional expression of HIF-1
has been reported to be amplified during hypoxia, the primary mechanism of
regulation has been demonstrated to be posttranslational. In this
regard, HIF-1
is rapidly degraded under normoxic conditions as a
result of its ubiquitin labeling and subsequent degradation by the
proteasomal system (7a, 15a). This process is inhibited during
hypoxia, allowing the protein to accumulate and to heterodimerize with
the
-subunit. Oxygen-dependent degradation (ODD) of HIF-1
involves a functional domain of ~200 amino acids, referred to as the
ODD region. This region interacts with the von Hippel-Lindau (VHL)
tumor suppressor protein, which functions as a critical component of a
multiprotein E3 ubiquitin ligase complex (15). Disruption
of the VHL gene in patients with von Hippel-Lindau disease results in a
constitutive stabilization of HIF-1
during normoxia and an
associated activation of HIF-1-regulated genes. Interestingly, these
patients frequently develop highly vascular tumors as a consequence
of dysregulation of vascular growth factors such as VEGF
(5).
degradation relates to how this
process is inhibited during hypoxia. Three experimental tools have long
been employed by investigators seeking to trigger expression of
oxygen-regulated genes. These include hypoxia or administration of
cobaltous chloride or iron chelators such as desferrioxamine during
normoxia. Each is capable of inducing the stabilization of HIF-1
levels and of triggering expression of HIF-1-dependent target genes.
Two recent studies demonstrated that the interaction between VHL
protein and the ODD of HIF-1
was regulated by the hydroxylation of a
conserved proline residue within the ODD region (8, 9).
During normoxia, this proline is hydroxylated by one or more members of
the prolyl hydroxylase family in a reaction requiring iron, oxygen, and
2-oxoglutarate. During hypoxia this process becomes inhibited,
preventing the interaction of VHL protein with the ODD region and
thereby abrogating ubiquitination and degradation. The identification
of prolyl hydroxylase in this system therefore represents an important
step in understanding the pathways regulating gene transcription during
hypoxia. More recently, Lando et al. (13) identified
another O2-dependent posttranslational modification of
HIF-1
involving hydroxylation of a conserved asparagine in the
carboxy-terminal transactivation domain. Like the prolyl hydroxylase
system, the asparagine hydroxylase(s) mediating this reaction requires
iron, oxygen, and 2-oxoglutarate and is inhibited during hypoxia.
Hydroxylation of this asparagine during normoxia interferes with the
interaction of HIF-1
with the p300 coactivator involved in the
transcriptional activation. Therefore, two separate hydroxylation
switches need to be flipped for HIF-1-mediated gene expression to
occur. The involvement of two seemingly distinct hydroxylases in the
regulation of HIF-1
seems to provide a significant safeguard against
inadvertent activation of the hypoxia pathway.
, the factors that regulate these
hydroxylases are not yet clear. For example, it is conceivable that the
activity of these hydroxylases is regulated by some signaling pathway,
making them a downstream target of a separate O2 sensor. Such a system would permit a graded inhibition of the enzymes during
progressive hypoxia and would also suggest how other triggers of HIF-1
activation such as growth factors could trigger the response during
normoxia. Until the apparent Km of the
hydroxylases for O2 in the cell can be determined, the
question of whether these hydroxylases function as the sensor of
hypoxia as opposed to an important downstream effector cannot be known.
.
At least two models have implicated reactive oxygen species (ROS) in
the signal transduction process upstream from HIF-1
stabilization.
Interestingly, these two models appear diametrically opposed in terms
of the hypothesized increases or decreases in ROS production during
hypoxia. In the study by Schroedl and colleagues, one of the current
articles in focus (Ref. 15b, see p. L922 in this
issue), the Chandel group examined HIF-1
stabilization at
low O2 levels in a variety of cell types. Their studies
were motivated by the previous observation that inhibitors such as rotenone, diphenylene iodonium, and
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine that block mitochondrial
complex I also abolished the HIF-1 response to hypoxia (1, 2,
6). By contrast, inhibitors of more distal regions of
the electron transport chain such as cyanide or antimycin A failed to
abolish this response and in some cases actually triggered HIF-1
stabilization during normoxia (3). The HIF-1
response
was also abolished by antioxidants and activated during normoxia by low
concentrations of exogenous H2O2, leading to
the suggestion that mitochondrial ROS production may trigger a signal
transduction pathway leading to HIF-1
stabilization. This idea was
supported by the observation that ROS generation has been found to
increase during hypoxia in cells loaded with oxidant-sensitive
fluorochromes. Other oxidative changes in DNA also occur during
hypoxia, in support of the idea that ROS production increases
(7). Finally,
0 cells depleted of
mitochondrial DNA lost their response to hypoxia, but they still
retained the ability to stabilize HIF-1
in response to cobalt or
desferrioxamine treatment (2, 3). The DNA in mitochondria
encodes specific subunits that are required for a functional electron
transport chain, so these
0 cells cannot respire and are
forced to survive purely by anaerobic glycolysis. Interestingly, they
also fail to increase ROS generation during hypoxia (4).
Collectively, these results tended to implicate the proximal region of
the mitochondrial electron transport chain in the O2
sensing pathway.
0 cells was later challenged
by other groups (21, 22) that studied a collection of
different mutant or
0 cells that exhibited various
defects in mitochondrial electron transport. In contrast to the results
of Chandel et al. (2, 3), they found that
0
cells retained the ability to stabilize HIF-1
in hypoxia. If true, this would seem to rule out mitochondria as a potential site of
O2 sensing. In either case, how can these differences be
resolved? The study by Schroedl et al. (15b) makes an important step in
resolving this discrepancy. Chandel et al. (2, 3) proposed that mitochondria may function as an upstream regulator of
prolyl hydroxylase through the O2-dependent release of ROS under hypoxic conditions (1-3% O2,
PO2 = 7-21 mmHg). On the basis of the
prolyl/asparagine hydroxylase model, one would predict that complete
anoxia (PO2 = 0 mmHg) should inhibit
hydroxylation of HIF-1
because O2 is required as a
substrate for those reactions, regardless of whether mitochondria were
involved in O2 sensing. Careful inspection of the papers by
Vaux et al. (22) and Srinivas et al. (21)
reveals that they measured HIF-1 in
0 cells only under
near-anoxic conditions (e.g., 0.1% O2). Schroedl et al.
(15b) examine this point by comparing HIF-1
responses in
0 cells under both hypoxic and anoxic conditions. They
again report that
0 cells selectively lose the ability
to respond to hypoxia but retain the ability to stabilize HIF-1
in
anoxia. These results suggest that prolyl hydroxylases do not serve as
the primary O2 sensor regulating HIF-1
protein levels
under hypoxic conditions but may indeed serve as the primary
O2 sensor during anoxia. An interesting corollary is that
mitochondria cannot generate ROS during anoxia, due to the lack of
O2 as a substrate for electrons. If mitochondrial ROS
production is required to trigger HIF-1
stabilization during
hypoxia, this response would be lost if the cells became anoxic due to
the inability to generate ROS. It is interesting to note that during
anoxia the hydroxylases also should fail, allowing the transcription of
HIF-1-dependent genes to continue.
B, activator protein-1, and p53
are activated during hypoxia by a pathway that appears to be distinct
from HIF-1 activation and presumably does not involve proline
hydroxylation. Although the prospect of identifying a single unifying
mechanism of O2 sensing that can explain the diverse
responses to hypoxia in multiple cell types is attractive, accumulating
evidence tends to point away from this possibility.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. T. Schumacker, Dept. of Medicine MC6026, Univ. of Chicago, 5841 So. Maryland Ave., Chicago, IL 60637 (E-mail pschumac{at}medicine.bsd.uchicago.edu).
10.1152/ajplung.00205.2002
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REFERENCES |
---|
![]() ![]() ![]() |
---|
1.
Agani, FH,
Pichiule P,
Chavez JC,
and
LaManna JC.
The role of mitochondria in the regulation of hypoxia-inducible factor 1 expression during hypoxia.
J Biol Chem
275:
35863-35867,
2000
2.
Chandel, NS,
Maltepe E,
Goldwasser E,
Mathieu CE,
Simon MC,
and
Schumacker PT.
Mitochondrial reactive oxygen species trigger hypoxia-induced transcription.
Proc Natl Acad Sci USA
95:
11715-11720,
1998
3.
Chandel, NS,
McClintock DS,
Feliciano CE,
Wood TM,
Melendez JA,
Rodriguez AM,
and
Schumacker PT.
Reactive oxygen species generated at mitochondrial complex III stabilize HIF-1- during hypoxia: a mechanism of O2 sensing.
J Biol Chem
275:
25130-25138,
2000
4.
Chandel, NS,
and
Schumacker PT.
Cells depleted of mitochondrial DNA (0) yield insight into physiological mechanisms.
FEBS Lett
454:
173-176,
1999[ISI][Medline].
5.
Clifford, SC,
Cockman ME,
Smallwood AC,
Mole DR,
Woodward ER,
Maxwell PH,
Ratcliffe PJ,
and
Maher ER.
Contrasting effects on HIF-1 regulation by disease-causing pVHL mutations correlate with patterns of tumourigenesis in von Hippel-Lindau disease.
Hum Mol Genet
10:
1029-1038,
2001
6.
Gleadle, JM,
Ebert BL,
and
Ratcliffe PJ.
Diphenylene iodonium inhibits the induction of erythropoietin and other mammalian genes by hypoxia.
Eur J Biochem
234:
92-99,
1995[Abstract].
7.
Grishko, V,
Solomon M,
Breit JF,
Killilea DW,
LeDoux SP,
Wilson GL,
and
Gillespie MN.
Hypoxia promotes oxidative base modifications in the pulmonary artery endothelial cell VEGF gene.
FASEB J
15:
1267-1269,
2001
7a.
Huang, LE,
Gu J,
Schau M,
and
Bunn HF.
Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway.
Proc Natl Acad Sci USA
95:
7987-7992,
1998
8.
Ivan, M,
Kondo K,
Yang H,
Kim W,
Valiando J,
Ohh M,
Salic A,
Asara JM,
Lane WS,
and
Kaelin WG, Jr.
HIF targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing.
Science
292:
464-468,
2001
9.
Jaakkola, P,
Mole DR,
Tian YM,
Wilson MI,
Gielbert J,
Gaskell SJ,
Kriegsheim A,
Hebestreit HF,
Mukherji M,
Schofield CJ,
Maxwell PH,
Pugh CW,
and
Ratcliffe PJ.
Targeting of HIF- to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.
Science
292:
468-472,
2001
10.
Kline, DD,
Peng YJ,
Manalo DJ,
Semenza GL,
and
Prabhakar NR.
Defective carotid body function and impaired ventilatory responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1.
Proc Natl Acad Sci USA
99:
821-826,
2002
11.
Kotch, LE,
Iyer NV,
Laughner E,
and
Semenza GL.
Defective vascularization of HIF-1-null embryos is not associated with VEGF deficiency but with mesenchymal cell death.
Dev Biol
209:
254-267,
1999[ISI][Medline].
12.
Kourembanas, S,
Morita T,
Christou H,
Liu Y,
Koike H,
Brodsky D,
Arthur V,
and
Mitsial SA.
Hypoxic responses of vascular cells.
Chest
114:
25S-28S,
1998
13.
Lando, D,
Peet DJ,
Whelan DA,
Gorman JJ,
and
Whitelaw ML.
Asparagine hydroxylation of the HIF transactivation domain: a hypoxic switch.
Science
295:
858-861,
2002
14.
Maltepe, E,
Schmidt JV,
Baunoch D,
Bradfield CA,
and
Simon MC.
Abnormal angiogenesis and responses to glucose and oxygen deprivation in mice lacking the protein ARNT.
Nature
386:
403-407,
1997[ISI][Medline].
15.
Maxwell, PH,
Wiesener MS,
Chang GW,
Clifford SC,
Vaux EC,
Cockman ME,
Wykoff CC,
Pugh CW,
Maher ER,
and
Ratcliffe PJ.
The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis.
Nature
399:
271-275,
1999[ISI][Medline].
15a.
Salceda, S,
and
Caro J.
Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes.
J Biol Chem
272:
22642-22647,
1997
15b.
Schroedl, C,
McClintock DS,
Budinger GRS,
and
Chandel NS.
Hypoxic but not anoxic stabilization of HIF-1 requires mitochondrial reactive oxygen species.
Am J Physiol Lung Cell Mol Physiol
283:
L922-L931,
2002.
16.
Semenza, GL.
HIF-1: mediator of physiological and pathophysiological responses to hypoxia.
J Appl Physiol
88:
1474-1480,
2000
17.
Semenza, GL.
HIF-1, O2, and the 3 PHDs: how animal cells signal hypoxia to the nucleus.
Cell
107:
1-3,
2001[ISI][Medline].
18.
Semenza, GL,
Nejfelt MK,
Chi SM,
and
Antonarakis SE.
Hypoxia-inducible nuclear factors bind to an enhancer element located 3' to the human erythropoietin gene.
Proc Natl Acad Sci USA
88:
5680-5684,
1991[Abstract].
19.
Semenza, GL,
and
Wang GL.
A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation.
Mol Cell Biol
12:
5447-5454,
1992[Abstract].
20.
Shimoda, LA,
Manalo DJ,
Sham JSK,
Semenza GL,
and
Sylvester JT.
Partial HIF-1 deficiency impairs pulmonary arterial myocyte electrophysiological responses to hypoxia.
Am J Physiol Lung Cell Mol Physiol
281:
L202-L208,
2001
21.
Srinivas, V,
Leshchinsky I,
Sang N,
King MP,
Minchenko A,
and
Caro J.
Oxygen sensing and HIF-1 activation does not require an active mitochondrial respiratory chain electron-transfer pathway.
J Biol Chem
276:
21995-21998,
2001
22.
Vaux, EC,
Metzen E,
Yeates KM,
and
Ratcliffe PJ.
Regulation of hypoxia-inducible factor is preserved in the absence of a functioning mitochondrial respiratory chain.
Blood
98:
296-302,
2001
23.
Wang, GL,
and
Semenza GL.
Purification and characterization of hypoxia-inducible factor 1.
J Biol Chem
270:
1230-1237,
1995