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PHYSIOLOGY 25: 272–279, 2010; doi:10.1152/physiol.00029.2010
Ancient Atmospheres and the Evolution
of Oxygen Sensing Via the
Hypoxia-Inducible Factor in Metazoans
Metazoan diversification occurred during a time when atmospheric oxygen
Cormac T. Taylor1 and
Jennifer C. McElwain2
1
UCD Conway Institute, Systems Biology Ireland and School of
Medicine and Medical Science, and 2UCD School of Biology
and Environmental Sciences, University College Dublin, Belfield,
Dublin, Ireland
[email protected]
levels fluctuated between 15 and 30%. The hypoxia-inducible factor (HIF) is a
primary regulator of the adaptive transcriptional response to hypoxia. Although the HIF pathway is highly conserved, its complexity increased during
periods when atmospheric oxygen concentrations were increasing. Thus atmospheric oxygen levels may have provided a selection force on the devel-
Ancient Atmospheres
Changing levels of atmospheric gasses such as oxygen and carbon dioxide, whether due to human
industrial activity or to the processes of nature,
have significant implications for the Earth’s biota
(1, 2, 9, 23, 62). During the 4,500 million year history of the planet, the composition of the thin and
delicate lower terrestrial atmosphere has varied
greatly and played a central role in driving the
evolution of both plants and animals (1, 3, 10, 40,
41). Changes in the composition of the atmosphere
are associated with key events in the evolution of
life on Earth, including the origination and radiation of metazoans, selective and mass extinctions,
and periods of plant and animal gigantism (8, 10,
21, 23, 40). Although the composition of the atmosphere impacted greatly on plant and animal evolution, the converse is also the case with the
evolution of living organisms strongly influencing
the composition of the atmosphere (for example,
the development of photosynthesis was a primary
event in the oxygenation of the atmosphere) (1, 30,
45). Thus the intimate and bidirectional relationship that exists between the composition of the
atmosphere and the evolution of living organisms
has been key to the development of life on Earth.
Following the formation of the planet ⬃4,500
million years ago, the first primitive cellular life
arose from a single source likely within 500 million
years (28). Over the following 1,000 million years,
the evolution of simple single-cell prokaryotic life
included the growth of masses of cyanobacteria in
the Earth’s oceans, which evolved the ability to
harness the energy inherent in sunlight to fuel
metabolism through the process of photosynthesis
(53). This process generated molecular oxygen (O2)
as a highly reactive and toxic by-product. Initially,
the oxygen produced was removed from the atmosphere during the oxidation of inorganic minerals
272
such as iron. After the incorporation of oxygen into
these minerals was saturated, the slow and inexorable rise in atmospheric O2 that occurred over the
following 3,000 million years (FIGURE 1). initially
proved lethal to the vast majority of unicellular
anaerobic life on Earth, which could not survive
the reactive (oxidative) chemical properties of oxygen (20, 59). However, a group of more complex
organisms, the eukaryotes, which formed as a result of a merger (symbiosis) between different families of bacterial cells (28), which had discreet
internal membrane-enclosed compartments, could
not only withstand the toxic effects of oxygen but
utilize its chemical energy to generate cellular ATP
in a manner many times more efficient than anaerobic metabolism through the process of oxidative phosphorylation (50). The consequences of
this ancient endosymbiotic event, mitochondria,
are integral organelles in all eukaryotic cells today
and are responsible for the generation of the majority of cellular ATP in metazoans (28, 38). Once
armed with this new and highly efficient ability to
harness the chemical energy of oxygen matched
with a plentiful supply of the critical fuel in the
atmosphere, the evolution of complex multicellular organisms, which utilized oxidative metabolism, progressed at a breathtaking rate initiated
during the Cambrian era 542– 488 million years
ago and continued throughout the Phanerozoic
eon (from 542 million years ago until the
present) (15, 18).
Although central to the evolution of life on Earth,
oxygen is not the only atmospheric gas to exert a
potent impact on the biota. For example, dramatic
changes in atmospheric CO2 levels over the history
of the planet also likely impacted on plant and
animal evolution (1–3, 40, 41, 49). Indeed, the ratio
of CO2 to O2 may have been an important determinant of evolutionary events. However, the role
1548-9213/10 ©2010 Int. Union Physiol. Sci./Am. Physiol. Soc.
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opment of cellular oxygen-sensing pathways.
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of CO2 in directing evolutionary processes is beyond the scope of the current review.
Matching Atmospheric Oxygen
Levels to Evolution
Hypoxia in Human Health and
Disease
In healthy humans, there is a range of physiological oxygen levels within the tissues of the body,
ranging from PO2 values of ⬃100 Torr in the alveoli
of the lungs to less than 10 Torr in tissues such as
the medulla of the kidney and the retina (61).
Within all of these tissues, the chemical reduction
of molecular oxygen in the mitochondria of individual cells during the process of oxidative phosphorylation is central to oxidative metabolism and
bioenergetic homeostasis. Because of this, any insufficiency in the availability of molecular oxygen
represents a severe threat to continued physiological function and indeed survival. Hypoxia, which
occurs when oxygen levels in the microenvironment of a cell, tissue, or organism are reduced
relative to the normal physiological state, is associated with a range of physiological and pathophysiological processes (53).
Physiological hypoxia is an important microenvironmental signal in a range of processes including new blood vessel formation (angiogenesis)
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Although photosynthesis in the oceans likely began
⬃3,000 million years ago (43), it was not until
⬃1,500 million years ago, when photosynthesisderived oxygen began to accumulate to significant
levels in the atmosphere. While atmospheric oxygen levels continued to increase over the following
1,000 million years, the existing life on Earth during
this time remained as simple singe-cell organisms
(FIGURE 1). However, when atmospheric oxygen
levels reached between 15 and 20% between 550
and 600 million years ago, the first animal body
plans developed, and the evolution of metazoans
started in earnest (9). During the following ⬃550
million years of metazoan evolution, oxygen levels
in the atmosphere have been estimated to have
fluctuated between lows of ⬃15% and highs of
⬎30% (4, 6, 9, 10, 12, 23). During this period, metazoan evolution progressed from simple aquatic
species such as sea sponges and corals to a dramatic array of marine and terrestrial species including Homo sapiens (H. sapiens) (5).
Evidence for fluctuating atmospheric oxygen
concentrations over the last ⬃550 million years
comes mainly from studying the geochemical cycles of carbon and sulfur (6, 9, 12, 23). Independent
models indicate a significant rise in atmospheric
oxygen 300 –260 million years ago to levels as high
as 30 –31%, which has been largely attributed to the
evolution of large land plants and which is evidenced by the burial of woody plants in coal
swamps along with the burial of organic carbon in
the oceans (11, 23). In contrast, oxygen levels
dropped to levels as low as 15% ⬃190 –140 million
years ago (12). How the fluctuating levels of atmospheric oxygen over the last ⬃550 million years
impacted on evolution of metazoans is an area in
need of further investigation (9, 23). However,
existing physiological studies indicate that fluctuations in atmospheric oxygen may have had a significant role to play in helping to shape evolution.
For example, studies in fruit flies (Drosophila) have
indicated that oxygen is a key determinant of body
size in generational studies of D. Melanogaster
bred in atmospheric hypoxia (8, 9, 25, 67). Fluctuating oxygen concentrations have also been linked to
key stages in evolution over the last ⬃550 million
years, including the origin of the first animal body
plans, the conquest of land by animals, gigantism of
various species, and mass extinctions (9).
Because Metazoans evolved to utilize molecular
oxygen as the main electron acceptor in oxidative
metabolism and energy production, the ability to
adapt to and thrive in changing (sometimes excessive and sometimes limiting) oxygen conditions is
a key component of evolutionary selection. Indeed,
it is likely that the evolution of such pathways was
key to the success of the winners of metazoan
evolution. Developing our understanding of how
atmospheric oxygen levels contributed to the
evolution of such pathways may give insight into
the impact of atmospheric oxygen on metazoan
development.
FIGURE 1. Atmospheric oxygen levels during the
history of the planet
The estimated oxygen concentrations in the Earth’s atmosphere since the planet formed ⬃4,550 million years ago
and major events in evolutionary history including the evidence for the first simple cells, the beginning of photosynthesis, the appearance of green plants, and the Cambrian
explosion during which metazoan radiation took place.
PHYSIOLOGY • Volume 25 • October 2010 • www.physiologyonline.org
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Mechanisms of Adaptation to
Hypoxia
Molecular oxygen was central to the evolution of
multicellular life on Earth through the provision of
the fuel to efficiently generate high levels of cellular
ATP during oxidative phosphorylation (53). Consequently, during the course of evolution, many organisms have become dependent on a constant
supply of oxygen to effectively function. Because of
this, it is not entirely surprising that most multicellular organisms also evolved a molecular mech-
FIGURE 2. The HIF pathway in mammalian cells
Under conditions where sufficient oxygen is available, HIF is hydroxylated by a family of HIF prolyl hydroxylases (PHD1–3), leading to ubiquitination by a complex initiated by the VHL E3 ligase and subsequent
degradation. A second asparagine hydroxylation by FIH prevents transactivation of HIF that escapes degradation. These processes are inhibited in hypoxia due to hydroxylase inhibition leading to a rapid
stabilization and transactivation of HIF.
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PHYSIOLOGY • Volume 25 • October 2010 • www.physiologyonline.org
anism by which to respond to conditions where
oxygen demand exceeds supply (hypoxia) with the
induction of genes that encode proteins that increase oxygen supply and modulate metabolic activity in a hypoxic tissue/organism (33, 53, 61).
A central pathway that coordinates this response
in all metazoans involves a transcriptional regulator known as the hypoxia-inducible factor (HIF;
Refs. 53, 54). The HIF pathway is highly conserved
across species and exists in mammals such as humans as well as in more ancient lineages such as
nematodes and corals (27). In humans, HIF consists of one of three oxygen-sensitive alpha subunits (HIF␣) and a constitutively expressed beta
subunit (HIF␤). Under conditions when sufficient
oxygen is available to satisfy bioenergetic requirements, the oxygen not consumed by mitochondrial
oxidative phosphorylation is available to permit
the activity of a family of four HIF hydroxylase
enzymes that hydroxylate defined proline and asparagine residues on the HIF␣ subunit (26, 33).
Three HIF proline hydroxylases (termed PHD1/
2/3) have been described that hydroxylate proline
residues in the NH2-terminal oxygen degradation
domain (NODD; P402) and the COOH-terminal
ODD (CODD: P564) on human HIF-1␣. This results
in targeting of the HIF␣ subunit as a substrate for
an E3 ubiquitin ligase known as the von Hipple
Lindau protein (pVHL), which forms a ubiquitin
ligase complex leading to the ubiquitination and
immediate degradation of HIF␣ through the ubiquitin-proteasome pathway (FIGURE 2). A further
hydroxylation at asparagine 803 [in the COOH-terminal transcriptional activation domain (CTAD)] by
factor inhibiting HIF (FIH) prevents transcriptional
activity of any HIF that has not been degraded.
Thus, under conditions of normoxia, HIF is kept in
a state of repression by the activity of the HIF
hydroxylases (FIGURE 2).
When oxygen demand exceeds supply, the high
affinity of the mitochondrial electron transport
chain for oxygen means that virtually all available
oxygen is consumed by the process of oxidative
phosphorylation leading to a lack of available oxygen for the HIF hydroxylases and their subsequent inhibition (26). Under these conditions,
HIF␣ subunits accumulate within the cytoplasm of
the cell, translocate into the nucleus, and bind to
HIF␤ subunits to form transcriptionally active HIF
heterodimers, which coordinate the expression of
a family of HIF-dependent adaptive genes. HIF can
directly activate the expression of hundreds of direct (primary) target genes and likely impacts on
the expression of thousands of secondary genes
(53). Genes directly under the control of HIF in
mammalian cells include those that contribute to
the control of angiogenesis, vascular tone, metabolism, and erythropoesis. The net effect of the ex-
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during development and wound healing, the regulation of vascular tone, and the response to exercise (48).
However, tissue hypoxia is also associated with a
diverse and wide range of pathophysiological processes including (but not limited to) vascular disease, chronic inflammation, and cancer (33, 48,
61). In vascular diseases such as atherosclerosis
and stroke, vascular occlusion leads to acute or
chronic tissue ischemia with resultant hypoxia. In
chronic inflammatory disease, the greatly increased metabolism of inflamed tissue due to immune cell infiltration matched with vascular
dysfunction leads to tissue hypoxia (17, 24, 44, 60).
In cancer, the growth of a tumor away from the
local blood supply eventually leads to tumor hypoxia. In all of these cases, the induction of a genetic
response to hypoxia leads to the expression of
genes that are essentially adaptive (or maladaptive
in the case of cancer). Seminal discoveries in the
last 20 years have greatly enhanced our understanding of the molecular mechanisms underpinning this critical response (33, 53).
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The Evolution of Components of
the HIF Pathway
Orthologs of various components of the human
PHD/FIH-HIF-VHL pathway are conserved in organisms as simple as the nematode C. elegans
where they also serve hypoxia-sensing functions
(22, 47). Notably, however, the components of the
HIF pathway have developed a higher degree of
complexity possibly because oxygen-sensing requirements became more demanding in larger animals with respiratory/circulatory systems and
atmospheric oxygen levels changed. This complexity is reflected by increasing numbers of isoforms
of the various components of the pathway (see
below). Here, we have examined the conservation
of genes with homology to components of the human HIF pathway (HIF-1␣/2␣/3␣, HIF-1␤, FIH,
PHD1/2/3, pVHL, and CBP/p300) in representative
species from the main metazoan Phyla (mammals,
birds/reptiles, amphibians, fish, arthropods, and
nematodes). Gene homologies were determined
using the Homologene search engine (http://
www.ncbi.nlm.nih.gov/homologene) and individual
papers, which are referenced where appropriate.
HIF
Homologs of the gene encoding human HIF-1␣
(HIF1A) are conserved in the genomes of organisms as primitive as nematodes, coral, and sea
anemones and thus appear to be expressed in all
metazoans (27). This degree of conservation is
striking and underpins the importance of effective
oxygen-sensing pathways for the development of
all metazoans, irrespective of their natural habitat.
Functions of HIF in lower organisms include metabolic switches (e.g., in C. elegans, the HIF pathway
is a regulator of metabolism) and tracheal branching in Drosophila (27).
Metazoans, which evolved relatively early, such
as coral, worms, and fruit flies have a single HIF␣
isoform that is most similar to human HIF1␣. However, some time after the division of arthropods
and fish, which occurred at least 460 million years
ago, a second HIF isoform evolved (termed HIF-2␣,
encoded by the EPAS gene). Although HIF2␣ is
absent in arthropods, it is present in fish species
such as zebrafish (42, 51). Following the division of
mammals and birds/reptiles that occurred at least
312 million yeas ago, a third isoform (HIF-3␣)
evolved that is exclusively found in mammals. This
indicates the development of increasing numbers
of HIF␣ isoforms with increasing complexity of
oxygen-delivery systems during metazoan evolution (27, 33).
As well as increases in the number of HIF␣ isoforms during metazoan evolution, the rate and
mode of molecular evolution has recently been
documented in fishes and mammals and has been
linked to variable environmental oxygen levels (42,
52). Interestingly, the rate and mode of molecular
evolution of HIF-1␣ in fish differs somewhat from
that in mammals, indicating that the different
aquatic oxygen tensions experienced by fish may
have had a differential impact on the evolution of
HIF-1␣ (16, 42). The molecular evolution of HIF in
fish species has been discussed in more detail elsewhere (16, 42, 52).
Thus the early appearance of the HIF1A gene
and its constant expression across species independent of fluctuations, which have occurred in
atmospheric oxygen levels during the evolution of
metazoans, indicates that the HIF pathway appeared early in the evolution of metazoans and is a
primary regulator of oxygen-/hypoxia-dependent
responses. Although a HIF␣ isoform has been
present since the radiation of the metazoans, the
molecular complexity of the molecule has increased over the course of evolution, with primitive
forms of HIF␣ expressing single oxygen-sensitive
regulatory domains and more evolved forms of
HIF␣ containing multiple levels of oxygen dependence (27).
The HIF-1␤ subunit, which is constitutively expressed in mammalian cells and is synonymous
with the ARNT protein, is also highly conserved,
and orthologs for its gene (ARNT) have been identified in fish (52), fruit flies (65), and nematodes
(46), indicating that, like HIF-1␣, this gene evolved
before the divergence of nematodes and mammals
580 –520 million years ago.
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pression of these genes is to increase blood and
oxygen supply to the hypoxic tissue and also induce a metabolic switch favoring the anaerobic
generation of ATP through glycolysis (61). This system is conserved throughout the metazoans in various levels of complexity, as will be discussed
below. The central importance of the HIF pathway
to physiological processes is exemplified by the
fact that homozygous knockout of either pVHL,
PHD2, HIF-1␣, or HIF-2␣ leads to embryonic lethality in mice (33). Furthermore, various conditional knockout studies have revealed important
tissue-specific roles for HIF in processes as divergent as immune cell survival, kidney fibrosis, and
skeletal muscle function during endurance training (19, 29, 39, 57, 63). In subsequent sections of
this review, we map the development of components of the HIF pathway to the radiation of metazoans and to changes that occurred in atmospheric
oxygen levels during the course of the Earth’s history. Our intent is to explore whether the evolving
complexity of the HIF pathway is correlated with
the prevailing atmospheric oxygen levels of the
planet during metazoan evolution.
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Prolyl Hydroxylases
Factor Inhibiting HIF
FIGURE 3. Correlating metazoan evolution, atmospheric oxygen levels,
and development of the HIF pathway during the Phanerozoic Eon
Top: the upper and lower divergence times for major Phyla are indicated [upper and
lower phylogenetic divergence estimates were obtained from The Timetree of Life (5)].
Middle: the atmospheric oxygen concentration during the Panerozoic is plotted (GEOCARBSULF) (7, 12). Bottom: the approximate times of appearance of genes encoding
various components of the PHD/FIH-HIF-VHL pathway are outlined.
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PHYSIOLOGY • Volume 25 • October 2010 • www.physiologyonline.org
FIH is an asparagine hydroxylase that is critical for
the fine tuning of the HIF response (33, 37, 66). The
FIH gene demonstrates a variable conservation
throughout evolution. Functional FIH homologs are
present in mammals but are lacking in some
arthropods and nematodes such as D. melanogaster
and C. elegans, respectively. However, further analyses revealed the presence of FIH in species that
evolved earlier, such as corals, and in species that
evolved between the flies and the worms, such as
beetles (27). Interestingly, the lack of FIH in D.
melanogaster and C. elegans is coupled with a loss
of conservation of the COOH-terminal activation
domain in HIF␣, which harbors the site of hydroxylation by FIH. This variable conservation makes
FIH unique among the proteins involved in oxygen
sensing to the HIF pathway from an evolutionary
perspective.
It has been hypothesized that the variable conservation of the FIH gene is due to its loss from a
number of “mono TAD” invertebrate species including D. melanogaster and C. elegans, whereas
the “dual TAD” regulatory mechanisms are present
in species that evolved before and after (27). In this
work, the authors have have hypothesized a number of possible reasons for the loss of these genes
in some invertebrates, including the possibility
that increased control mechanisms are required
for increased complexity in higher species and the
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Prolyl hydroxylases are a large family of proteins
with diverse functions including intracellular signaling and extracellular matrix remodeling. In
mammals, three isoforms of HIF prolyl hydroxylases with distinct tissue-specific expression profiles, which are key oxygen sensors regulating the
stability of HIF, have been described (33, 36). The
three isoforms of HIF prolyl hydroxylases expressed in humans are called PHD1, PHD2, and
PHD3 (encoded by the EGLN2, EGLN1, and EGLN3
genes, respectively). PHD2 is the isoform mostly
responsible for the regulation of HIF and is thought to be the ancestral form (35). This is supported by
evidence that the PHD isoform expressed in the
parasitic eukaryotic protist Perkinsus olseni (P. olseni) shares a higher degree of homology with
PHD2 than the other isoforms (35). Like HIF, the
PHDs are a very well conserved family of genes,
and orthologs can be found in flies, worms, beetles,
and coral (27). Interestingly, PHD-like enzymes described in P. olseni have been implicated in virulence and pathogenicity, possibly giving a clue to
the early evolutionary history of this gene (35).
However, the number of PHD isoforms is decreased in lower species. For example, Nematodes
and flies have a single PHD isoform known as egl9.
Although beetles have two conserved putative sites
for proline hydroxylation, only a single PHD has
so far been found, leading to the conclusion that
the evolution of multiple PHD isoforms occurred
after the division of the arthropods and the fish.
PHD3 is present in amphibians and fish but not
in arthropods and thus represents the second
member of the PHD family to evolve (31). A third
PHD isoform (PHD1) is present only in mammals
and thus appears to have evolved after the division of the mammals and the birds/reptiles
(FIGURE 3). The conservation of PHD enzymes is
matched by the conservation of target proline
residues for hydroxylation in the HIF␣ proteins
expressed (27). Interestingly, recent work has
also identified a positively selected haplotype of
PHD2 in human populations resident in the
high-altitude Tibetan highlands (56). Thus PHDs,
like HIF, are highly conserved; however, they
show an increased complexity over time, as demonstrated by the appearance of multiple PHD
isoforms in vertebrates vs. a single isoform in
invertebrates.
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possibility that FIH is superfluous in some organisms such as flying insects (27).
The von Hipple Lindau Protein
The von Hipple Lindau gene (VHL), which encodes
the von Hipple Lindau protein (pVHL), was first
cloned in 1993 (34). Genes with a high degree of
similarity to VHL have been described in humans,
primates, rodents, and also nematodes (32, 55).
Indeed, Bishop et al. (13) demonstrated conservation of the HIF-1/VHL-1/EGL-9 pathway in C. elegans (13). Thus, like HIF-1␣ and PHD2, VHL is a
highly conserved gene that is linked to the regulation of HIF in all metazoans.
The transcriptional co-activators CBP and p300,
which interact with HIF in the nucleus to allow the
initiation of transcription of HIF-dependent genes,
are conserved across species, and a CBP/p300 homolog encoded by the cbp-1 gene can be found in
nematodes and fruit flies as well as in mammals
(14). Functional homologs of p300/CBP have also
been identified in plants such as Arabidopsis thaliana, giving strength to the likelihood for a conserved role for CBP/p300 in all metazoans (14).
Mapping Evolutionary Expression of
Components of the HIF Pathway to Ancient
Atmospheric Oxygen Levels
In (FIGURE 3), we have utilized a number of databases to correlate the evolution of components of
the PHD/FIH-HIF-VHL pathway with metazoan
evolution and the estimated atmospheric oxygen
concentrations over the last 550 million years. The
estimations for periods during which evolutionary
divisions between phyla occurred were obtained
from The Timetree of Life (5). The upper time limit
represents the minimum time since the division of
the phyla occurred and the lower limit, which has
been termed the “soft maximum,” represents the
maximum time since the divergence occurred (for
details on how these figures are calculated, see Ref.
5). The atmospheric oxygen levels used were obtained from the updated GEOCARBSULF model (7,
12) and personal communication from Robert
Berner (Yale University). It should be noted that an
alternative model of ancient atmospheric oxygen
levels termed COPSE has also been proposed,
which predicts some common and some differential ancient atmospheric PO2 values (6). The presence of homologs of human genes of the PHD/
FIH-HIF-VHL pathway were obtained from extensive
review of the relevant literature and the use of the
Homologene search engine (http://www.ncbi.nlm.
nih.gov/homologene).
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Transcriptional Co-activators CBP and
p300
From these correlations, it appears that the earliest components of the pathway to evolve were
HIF-1␣, HIF-1␤, PHD2, and VHL, homologs for all
of which can be found in species as early as nematodes. This indicates that these genes evolved earlier than the radiation of the metazoans, were
present in the earliest animal species, and possibly
evolved in response to increasing oxygen concentrations in the pre-Cambrian period (from 4,600 to
542 million years ago), during which time prokaryotic life evolved and atmospheric oxygen rose from
0% to over 20% oxygen (12, 28). The next genes in
this pathway to evolve were those for HIF-2␣ and
PHD3, both of which are present in fish but not in
arthropods, indicating that these genes evolved after the arthropod/fish division but before the fish/
amphibian division between 460 and 421 million
years ago. The most recent additions to the family
are PHD1 and HIF-3␣, which appear only in mammals and thus must have evolved ⬍312 million
years ago.
Using the above approach, we estimate the minimum times at which various components of the
PHD/FIH-HIF-VHL pathway evolved, although the
absence of accurate information regarding the actual time of their evolution makes correlation of
these events with atmospheric oxygen levels speculative. Nevertheless, some intriguing possibilities
can be postulated. First, the evolution of HIF-2␣
and PHD3 occurred after the division of the Fish
and arthropods (460 million years ago) and before
the division of the fish and the amphibians (421.5
million years ago). This period coincides with a
time during which atmospheric oxygen increased
from 17 to 23%. Similarly, HIF-3␣ and PHD1
evolved after the division of the mammals and the
birds/reptiles, which occurred 312 million years
ago. During this period, atmospheric oxygen levels
were also on the increase from 23% 310 million
years ago to their highest point in the course of
history of 32% 280 million years ago.
As outlined above, FIH1 demonstrates selective
conservation and is expressed in primitive organisms including coral and sea anemone (27) and
also fish, amphibians, reptiles, birds, and mammals. However, although the gene is retained in all
species since the fish, it is missing in fruit flies and
nematodes but not in intermediate species such as
beetles. A more in-depth analysis of the phylogenetic digression between beetles and flies reveals
that this division occurred between 414 and 307
million years ago (5), which was a time of relative
stability in terms of atmospheric oxygen concentrations, indicating that this selective conservatism
was not driven by fluctuations in atmospheric oxygen but may have involved other selective forces.
277
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Summary and Conclusions
Work from the author’s laboratories is funded by Science
Foundation of Ireland and the European Union.
No conflicts of interest, financial or otherwise, are declared by the author(s).
References
278
Bergman NM, Lenton TM, Watson AJ. COPSE: a new model
of biogeochemical cycling over phanerozoic time. Am J Sci
304: 397– 437, 2004.
7.
Berner RA. GEOCARBSULF: a combined model for Phanerozoic atmospheric O2 and CO2. Geochimica 70: 5653–5664,
2006.
8.
Berner RA, Beerling DJ, Dudley R, Robinson JM, Wildman RA
Jr. Phanerozoic atmospheric oxygen. Annu Rev Earth Planet
Sci 31: 105, 2003.
9.
Berner RA, Vandenbrooks JM, Ward PD. Evolution. Oxygen
and evolution. Science 316: 557–558, 2007.
10. Berner RA. Atmospheric oxygen over Phanerozoic time. Proc
Natl Acad Sci USA 96: 10955–10957, 1999.
11. Berner RA. The Phanerozoic Carbon Cycle: CO2 and O2.
Oxford, UK: Oxford Univ. Press, 2004.
12. Berner RA. Phanerozoic atmospheric oxygen: new results
using the geocarbsulf model. Am J Sci 309: 603– 606, 2009.
13. Bishop T, Lau KW, Epstein AC, Kim SK, Jiang M, O’Rourke D,
Pugh CW, Gleadle JM, Taylor MS, Hodgkin J, Ratcliffe PJ.
Genetic analysis of pathways regulated by the von HippelLindau tumor suppressor in Caenorhabditis elegans. PLoS
Biol 2: e289, 2004.
14. Bordoli L, Netsch M, Luthi U, Lutz W, Eckner R. Plant orthologs of p300/CBP: conservation of a core domain in metazoan p300/CBP acetyltransferase-related proteins. Nucleic
Acids Res 29: 589 –597, 2001.
15. Budd GE. The earliest fossil record of the animals and its
significance. Philos Trans R Soc Lond B Biol Sci 363: 1425–
1434, 2008.
16. Cao YB, Chen XQ, Wang S, Wang YX, Du JZ. Evolution and
regulation of the downstream gene of hypoxia-inducible factor-1alpha in naked carp (Gymnocypris przewalskii) from lake
Qinghai, China. J Mol Evol 67: 570 –580. 2008.
17. Colgan SP, Taylor CT. Hypoxia: an alarm signal during intestinal inflammation. Nat Rev Gastroenterol Hepatol 7: 281–
287, 2010.
18. Conway MS. Darwin’s dilemma: the realities of the Cambrian
‘explosion’. Philos Trans R Soc Lond B Biol Sci 361: 1069 –
1083, 2006.
19. Cramer T, Yamanishi Y, Clausen BE, Förster I, Pawlinski R,
Mackman N, Haase VH, Jaenisch R, Corr M, Nizet V, Firestein
GS, Gerber HP, Ferrara N, Johnson RS. HIF-1alpha is essential for myeloid cell-mediated inflammation. Cell 112: 645–
657, 2003.
20. Dole M. The natural history of oxygen. J Gen Physiol 49:
5–27, 1965.
21. Dudley R. Atmospheric oxygen, giant Paleozoic insects and
the evolution of aerial locomotor performance. J Exp Biol
201: 1043–1050, 1998.
22. Epstein AC, Gleadle JM, McNeill LA, Hewitson KS, O’Rourke
J, Mole DR, Mukherji M, Metzen E, Wilson MI, Dhanda A, Tian
YM, Masson N, Hamilton DL, Jaakkola P, Barstead R,
Hodgkin J, Maxwell PH, Pugh CW, Schofield CJ, Ratcliffe PJ.
C. elegans EGL-9 and mammalian homologs define a family
of dioxygenases that regulate HIF by prolyl hydroxylation.
Cell 107: 43–54, 2001.
23. Falkowski PG, Katz ME, Milligan AJ, Fennel K, Cramer BS,
Aubry MP, Berner RA, Novacek MJ, Zapol WM. The rise of
oxygen over the past 205 million years and the evolution of
large placental mammals. Science 309: 2202–2204, 2005.
1.
Beerling DJ, Berner RA. Feedbacks and the coevolution of
plants and atmospheric CO2. Proc Natl Acad Sci USA 102:
1302–1305, 2005.
2.
Beerling DJ, Berner RA. Impact of a permo-carboniferous
high O2 event on the terrestrial carbon cycle. Proc Natl Acad
Sci USA 97: 12428 –12432, 2000.
24. Fraisl P, Aragonés J, Carmeliet P. Inhibition of oxygen sensors as a therapeutic strategy for ischaemic and inflammatory
disease. Nat Rev Drug Discov 8: 139 –152, 2009.
3.
Beerling DJ, Osborne CP, Chaloner WG. Evolution of leafform in land plants linked to atmospheric CO2 decline in the
late Palaeozoic era. Nature 410: 352–354, 2001.
25. Frazier MR, Woods HA, Harrison JF. Interactive effects of
rearing temperature and oxygen on the development of
Drosophila melanogaster. Physiol Biochem Zool 74: 641–
650, 2001.
4.
Belcher CM, McElwain JC. Limits for combustion in low O2
redefine paleoatmospheric predictions for the Mesozoic. Science 321: 1197–1200, 2008.
5.
Benton M, Donoghue PCJ, Asher RJ. Calibrating and constraining molecular clocks. In: The Timetree of Life, edited by
Hedges SB, Kumar S. Oxford, UK: Oxford Univ. Press, 2009,
p. 89 –98.
PHYSIOLOGY • Volume 25 • October 2010 • www.physiologyonline.org
26. Hagen T, Taylor CT, Lam F, Moncada S. Redistribution of
intracellular oxygen in hypoxia by nitric oxide: effect on
HIF1alpha. Science 302: 1975–1978, 2003.
27. Hampton-Smith RJ, Peet DJ. From polyps to people: a highly
familiar response to hypoxia. Ann NY Acad Sci 1177: 19 –29,
2009.
Downloaded from http://physiologyonline.physiology.org/ by 10.220.32.247 on June 18, 2017
Metazoans have evolved to use the chemical reduction of molecular oxygen as the primary source
of metabolic energy, making a constant supply of
oxygen key to organism survival. Because of this, at
an early point metazoans began to evolve molecular mechanisms by which to deal with hypoxic
insults that involved the increased expression of
genes that support adaptation to hypoxia. A master
and ancient regulator of this response is the HIF.
Recent work has unveiled the molecular mechanisms linking cellular oxygen sensing to the activation of HIF. Here, we propose that the prevailing
atmospheric oxygen concentrations may have
played a role in shaping the evolution of this pathway. However, some key points need to be considered. First, oxygen is likely not the only gas to drive
evolution, and carbon dioxide levels in the atmosphere were also likely important. Second, HIF is
not the only hypoxia responsive transcription factor; in fact, over 16 other transcriptional regulators
have been demonstrated to possess at least some
degree of hypoxia sensitivity (58). Furthermore, the
absence of complete analysis of the genomes of
species during the relatively long periods between
the absolute minimum (as determined by fossil
evidence) and the soft maximum (as determined
by the molecular clock determinations) for the division time of arthropods and fish and the division
of the arthropods and nematodes means we cannot exclude the possibility of selective conservation of genes involved in the PHD/FIH-HIF-VHL
pathway during these periods. Future studies
should be directed toward taking these important
points into account in forming a global view of the
role of the Earth’s atmosphere on shaping metazoan evolution. 䡲
6.
REVIEWS
28. Hedges Life SB. The Timetree of Life, edited by
Hedges SB, Kumar S. Oxford, UK: Oxford Univ.
Press, 2009, p. 89 –98.
29. Higgins DF, Kimura K, Bernhardt WM, Shrimanker N, Akai Y, Hohenstein B, Saito Y, Johnson RS,
Kretzler M, Cohen CD, Eckardt KU, Iwano M,
Haase VH. Hypoxia promotes fibrogenesis in vivo
via HIF-1 stimulation of epithelial-to-mesenchymal transition. J Clin Invest 117: 3810 –3820,
2007.
30. Igamberdiev AU, Lea PJ. Land plants equilibrate
O2 and CO2 concentrations in the atmosphere.
Photosynth Res 87: 177–194, 2006.
31. Imaoka S, Muraguchi T, Kinoshita T. Isolation of
xenopus HIF-prolyl 4-hydroxylase and rescue of a
small-eye phenotype caused by Siah2 overexpression. Biochem Biophys Res Commun 355:
419 – 425, 2007.
33. Kaelin WG Jr, Ratcliffe PJ. Oxygen sensing by
metazoans: the central role of the HIF hydroxylase pathway. Mol Cell 30: 393– 402, 2008.
34. Latif F, Tory K, Gnarra J, Yao M, Duh FM, Orcutt
ML, Stackhouse T, Kuzmin I, Modi W, Geil L,
Schmidt L, et al. Identification of the von HippelLindau disease tumor suppressor gene. Science
260: 1317–1320, 1993.
35. Leite RB, Brito AB, Cancela ML. An oxygen molecular sensor, the HIF prolyl 4-hydroxylase, in
the marine protist Perkinsus olseni. Protist 159:
355–368, 2008.
36. Lieb ME, Menzies K, Moschella MC, Ni R, Taubman MB. Mammalian EGLN genes have distinct
patterns of mRNA expression and regulation.
Biochem Cell Biol 80: 421– 426, 2002.
37. Mahon PC, Hirota K, Semenza GL. FIH-1: a novel
protein that interacts with HIF-1alpha and VHL to
mediate repression of HIF-1 transcriptional activity. Genes Dev 15: 2675–2686, 2001.
38. Margulis L, Bermudes D. Symbiosis as a mechanism of evolution: status of cell symbiosis theory.
Symbiosis 1: 101–124, 1985.
39. Mason SD, Rundqvist H, Papandreou I, Duh R,
McNulty WJ, Howlett RA, Olfert IM, Sundberg
CJ, Denko NC, Poellinger L, Johnson RS. HIF1alpha in endurance training: suppression of oxidative metabolism. Am J Physiol Regul Integr
Comp Physiol 293: R2059 –R2069, 2007.
40. McElwain JC, Wagner PJ, Hesselbo SP. Science
Fossil plant relative abundances indicate sudden
loss of late Triassic biodiversity in East Greenland. Science 324: 1554 –1556, 2009.
55. Sikora S, Godzik A. Combination of multiple
alignment analysis and surface mapping paves a
way for a detailed pathway reconstruction-The
case of VHL (von Hipple Lindau) protein and angiogenesis regulatory pathway. Protein Sci 13:
786 –796, 2004.
42. Nikinmaa M, Rees BB. Oxygen-dependent gene
expression in fishes. Am J Physiol Regul Integr
Comp Physiol 288: R1079 –R1090, 2005.
56. Simonson TS, Yang Y, Huff CD, Yun H, Qin G,
Witherspoon DJ, Bai Z, Lorenzo FR, Xing J, Jorde
LB, Prchal JT, Ge R. Genetic evidence for highaltitude adaptation in Tibet. Science 329: 72–75,
2010.
43. Nisbett EG, Sleep NH. The habit and nature of
early life. Nature 409:1083–1091, 2001.
44. Oliver KM, Taylor CT, Cummins Hypoxia EP. Regulation of NFkappaB signalling during inflammation: the role of hydroxylases. Arthritis Res Ther
11: 215, 2009.
45. Pagani M, Caldeira K, Berner R, Beerling DJ. The
role of terrestrial plants in limiting atmospheric
CO2 decline over the past 24 million years. Nature 460: 85– 88, 2009.
57. Takeda N, O’Dea EL, Doedens A, Kim JW, Weidemann A, Stockmann C, Asagiri M, Simon MC,
Hoffmann A, Johnson RS. Differential activation
and antagonistic function of HIF-alpha isoforms
in macrophages are essential for NO homeostasis. Genes Dev 24: 491–501, 2010.
58. Taylor CT, Cummins EP. Hypoxia-responsive transcription factors. Pflügers Arch 450: 363–371, 2005.
59. Taylor CT, Pouyssegur J. Oxygen, hypoxia,
stress. Ann NY Acad Sci 1113: 87–94, 2007.
46. Powell-Coffman JA, Bradfield CA, Wood WB.
Caenorhabditis elegans orthologs of the aryl hydrocarbon receptor and its heterodimerization
partner the aryl hydrocarbon receptor nuclear
translocator. Proc Natl Acad Sci USA 95: 2844 –
2849, 1998.
60. Taylor CT. Interdependent roles for hypoxia inducible factor and nuclear factor-kappaB in hypoxic inflammation. J Physiol 586: 4055– 4059,
2008.
47. Powell-Coffman JA. Hypoxia signaling and resistance in C. elegans. Trends Endocrinol Metab 21:
435– 440, 2010.
61. Taylor CT. Mitochondria and cellular oxygen
sensing in the HIF pathway. Biochem J 409: 19 –
26, 2008.
48. Ratcliffe PJ, O’Rourke JF, Maxwell PH, Pugh CW.
Oxygen sensing, hypoxia-inducible factor-1 and
the regulation of mammalian gene expression. J
Exp Biol 201: 1153–1162, 1998.
62. Ward P, Labandeira C, Laurin M, Berner RA. Confirmation of Romer’s Gap as a low oxygen interval constraining the timing of initial arthropod
and vertebrate terrestrialization. Proc Natl Acad
Sci USA 103: 16818 –16822, 2006.
49. Retallack GJ. A 300-million year record of atmospheric carbon dioxide from fossil plant cuticles.
Nature 411: 287–290, 2001.
50. Rich PR. The molecular machinery of Keilin’s respiratory chain. Biochem Soc Trans 31: 1095–
1105, 2003.
51. Rojas DA, Perez-Munizaga DA, Centanin L, Antonelli M, Wappner P, Allende ML, Reyes AE.
Cloning of HIF-1a and HIF-2a and mRNA expression pattern during development in zebrafish.
Gene Expr Patterns 7: 399 –345, 2007.
63. Weidemann A, Johnson RS. Biology of HIF1alpha.
Cell Death Differ 15: 621–627, 2008.
64. Woodward ER, Buchberger A, Clifford SC, Hurst
LD, Affara NA, Maher ER. Comparative sequence
analysis of the VHL tumor suppressor gene.
Genomics 65: 253–265, 2000.
65. Zelzer E, Wappner P, Shilo BZ. The PAS domain
confers target gene specificity of Drosophila
bHLH/PAS proteins. Genes Dev 11: 2079 –2089,
1997.
52. Rytkönen KT, Ryynänen HJ, Nikinmaa M, Primmer CR. Variable patterns in the molecular evolution of the hypoxia-inducible factor-1 alpha
(HIF-1alpha) gene in teleost fishes and mammals.
Gene 420: 1–10, 2008.
66. Zhang N, Fu Z, Linke S, Chicher J, Gorman JJ,
Visk D, Haddad GG, Poellinger L, Peet DJ, Powell
F, Johnson RS. The asparaginyl hydroxylase factor inhibiting HIF-1alpha is an essential regulator
of metabolism. Cell Metab 11: 364 –378, 2010.
53. Semenza GL. Life with oxygen. Science 318: 62–
64, 2007.
67. Zhou D, Visk DW, Haddad GG. Drosophila, a
golden bug, for the dissection of the genetic
basis of tolerance and susceptibility to hypoxia.
Pediatr Res 66: 239 –247.
54. Semenza GL, 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.
PHYSIOLOGY • Volume 25 • October 2010 • www.physiologyonline.org
279
Downloaded from http://physiologyonline.physiology.org/ by 10.220.32.247 on June 18, 2017
32. Iyer NV, Leung SW, Semenza GL. The human
hypoxia-inducible factor 1alpha gene: HIF1A
structure and evolutionary conservation. Genomics 52: 159 –165, 1998.
41. McElwain JC, Willis KJ, Niklas K. Long term fluctuations in atmospheric carbon dioxide concentration influence plant speciation rate. In: Climate
Change and Systematics, edited by Hodkinson T,
Jones M, Parnell J, Waldren S. Cambridge, MA:
Cambridge Univ. Press, 2010.