Keywords: oxygen sensing; hypoxia; hydroxylation; transcriptional regulation; hypoxia-inducible factor HIF.. Fax: + 61 8 8303 4348, E-mail: daniel.peet@adelaide.edu.au Abbreviations: ARN
Trang 1R E V I E W A R T I C L E
Oxygen-dependent regulation of hypoxia-inducible factors by prolyl and asparaginyl hydroxylation
David Lando1, Jeffrey J Gorman2,*, Murray L Whitelaw1and Daniel J Peet1
1
Department of Molecular BioSciences (Biochemistry) and the Centre for Molecular Genetics of Development,
University of Adelaide, Australia;2CSIRO Health Sciences and Nutrition, Parkville, Victoria, Australia
To sustain life mammals have an absolute and continual
requirement for oxygen, which is necessary to produce
energy for normal cell survival and growth Hence,
main-tainingoxyg en homeostasis is a critical requirement and
mammals have evolved a wide range of cellular and
phy-siological responses to adapt to changes in oxygen
avail-ability In the past few years it has become evident that the
transcriptional protein complex hypoxia-inducible factor
(HIF) is a key regulator of these processes In this review we
will focus on the way oxygen availability regulates HIF proteins and in particular we will discuss the way oxygen-dependent hydroxylation of specific amino acid residues has been demonstrated to regulate HIF function at the level of both protein stability and transcriptional potency.
Keywords: oxygen sensing; hypoxia; hydroxylation; transcriptional regulation; hypoxia-inducible factor (HIF).
Introduction
The development of complex cardiovascular, respiratory
and hemopoietic systems in mammals provides a means to
efficiently capture and deliver oxygen (O2) from the
environment to every cell of the body While a sufficient
supply of oxygen is essential for energy production, too
much oxygen in the form of free radicals (i.e superoxide,
OH–) can be detrimental [1] Therefore to maximize oxygen
use, as well as at the same time minimize the impact of
oxygen free radicals, cells have developed mechanisms to
maintain oxygen concentrations within a narrow
physiological range To achieve this mammals regulate
oxygen consumption and levels by a combination of both
cellular and systemic processes For example, when oxygen is limiting(hypoxia) individual cells decrease oxidative phos-phorylation and rely on glycolysis as the primary means of ATP production To facilitate this switch to glycolysis cells up-regulate the expression of a select set of genes, such as those encoding glycolytic enzymes and glucose transporters [2] Other hypoxic responses monitor global oxygen levels and effect system wide changes in tissue oxygen availability For instance, the hypoxic induction of the hormone erythropoietin (Epo) by the kidney stimulates red blood cell production to increase the oxygen carrying capacity of the blood [2] Tissues and cells experiencingreduced oxyg en supply, like those associated with wound healing, increase the levels of the angiogenic cytokine vascular endothelial growth factor (VEGF) VEGF then acts on endothelial cells
to stimulate the proliferation of new blood vessels, which in turn help maintain an adequate supply of oxygen [3] However, in many disease states such as cancer, stroke and heart attack these same oxygen delivery systems can become misregulated and hypoxia becomes a major component of the pathophysiology of these diseases [4].
For many years the Epo system was used to study the molecular mechanisms associated with the induction of hypoxia responsive genes and from these investigations the hypoxia-inducible factor (HIF) was identified as a key transcriptional hypoxic regulator of Epo [5,6] Subsequent research has now found that a large number of other hypoxia-inducible genes (Fig 1) are also induced by HIF under hypoxic conditions, revealingthat HIF functions as a master transcriptional regulator of the adaptive response to hypoxia [7–53].
Hypoxia-inducible factor
The HIF transcriptional complex is a heterodimer consist-ingof one of three alpha subunits (HIF-1a, HIF-2a or HIF-3a) and a beta subunit called ARNT [6,54–57].
Correspondence toD J Peet, Department of Molecular
BioSciences (Biochemistry) and the Centre for Molecular Genetics
of Development, University of Adelaide, Adelaide, South Australia,
5005 Australia Fax: + 61 8 8303 4348,
E-mail: daniel.peet@adelaide.edu.au
Abbreviations: ARNT, aryl hydrocarbon nuclear translocator;
bHLH, basic helix-loop-helix; CAD, carboxy-terminal transactivation
domain; CBP, CREB bindingprotein; CH, cysteine-histidine;
CO, carbon monoxide; CREB, cyclic AMP-response element binding
protein; DMOG, dimethyloxalylglycine; Dsfx, desferrioxamine;
Epo, erythropoietin; FIH-1, factor inhibitingHIF-1; HIF,
hypoxia-inducible factor; HPH, HIF prolyl-4-hydroxylase; MAPK, mitogen
activated protein kinase; NAD, amino-terminal transactivation
domain; NO, nitric oxide; ODD, oxygen-dependent degradation
domain; PAS, Per-ARNT-Sim; PHD, prolyl hydroxylase
domain-containingprotein; RLL, arginine-dileucine; VEGF, vascular
endothelial growth factor; VHL, von Hippel-Lindau
*Present address: Institute for Molecular Bioscience, University of
Queensland, St Lucia, Queensland, 4067, Australia
(Received 15 October 2002, revised 13 December 2002,
accepted 3 January 2003)
Trang 2Both the alpha and ARNT subunits belongto the basic
helix-loop-helix (bHLH)/Per-ARNT-Sim (PAS) family of
transcription factors The bHLH domain contains the basic
DNA bindingregion and HLH primary dimerization
inter-face The adjacent PAS domain is comprised of
approxi-mately 300 amino acids and is subdivided into two semi
conserved repeat regions designated PAS A and PAS B The
role of the PAS domain is to mediate protein–protein
interactions and act as a second dimerization interface in
conjunction with the HLH motif [58] ARNT is a general
partner protein and is known to heterodimerize with a
number of other bHLH-PAS proteins to form
transcrip-tionally active complexes [59] Biochemical comparison of
the HIF-1a and HIF-2a subunits have revealed that these
proteins share very similar biochemical properties (i.e.
dimerize with ARNT to recognize the same DNA
recogni-tion sequence) but, surprisingly, each subunit controls quite
distinct biological functions during embryo development (i.e.
HIF-1a for vascularization, HIF-2a for catecholamine
production; for a comprehensive review see [60,61]).
Regulation of HIF proteins by hypoxia
One of the major challenges facing the HIF research field
has been to understand the molecular mechanism by which
cells are able to sense oxygen levels and transduce the
physiological signal of reduced oxygen levels to HIF It has
been reported that oxygen levels can affect the protein
stability, subcellular localization, DNA bindingcapacity
and transcriptional potency of the HIFa subunits, whereas the ARNT subunit is constitutively expressed and its activity not affected by hypoxia (reviewed in [60,61]) While the HIFa subunits may be subject to numerous levels of regulation by oxygen, it has been the recent analysis of HIF-1a and HIF-2a protein stability and transactivation potency that have provided the greatest insights into oxygen sensing and regulation.
HIF protein stability Initial biochemical analysis of HIF-1a revealed that this protein was subject to rapid turnover and degradation at normoxia, whereas hypoxia blocked degradation leading to the accumulation of the HIF-1a protein [62,63] Treatment with proteasomal inhibitors and mutation of the ubiquitin activatingenzyme E1 revealed that HIF-1a was being degraded by the ubiquitin proteasome pathway under normoxic conditions [64] Subsequent studies mapped the instability region of HIF-1a to a domain of approximately
200 amino acids located carboxy-terminal to the PAS domain [65] This region was subsequently called the oxygen-dependent degradation domain (ODD) and removal of the entire ODD rendered HIF-1a stable at normoxia Likewise, analysis of HIF-2a revealed that it was also subject to proteasomal degradation at normoxia [66] via a similar ODD like region [67].
A hallmark of von Hippel-Lindau (VHL) disease is the high degree of vascularization, which is due to the
Fig 1 Hypoxia-inducible factor (HIF) target genes and their roles in oxygen homeostasis Hypoxia activates the HIF complex which binds to hypoxia response elements (HREs) containingthe core recognition sequence 5¢-RCGTG found in numerous genes involved
in a variety of cellular and system wide responses to low oxygen stress
Trang 3constitutive expression of a large number of hypoxia
inducible genes such as VEGF (reviewed in [68]) Because
VEGF and other hypoxia inducible genes are known HIF
targets, these observations raised the question of whether
VHL disease and HIF were somehow related Usingvarious
cell lines deficient in VHL, Maxwell and coworkers
demon-strated that VHL–/– cells expressed increased levels of
endogenous HIF-1a and HIF-2a protein [69] Moreover,
the protein levels of HIF-1a and HIF-2a could not be further
induced by hypoxia in VHL–/–cells Reintroduction of VHL
back into these VHL deficient cell lines resulted in a reduction
of normoxic endogenous HIF-1a and HIF-2a protein that
could now be induced by hypoxia [69] Further analysis
demonstrated that VHL could physically interact with the
HIFa subunits via the ODD [69], and the VHL complex
functioned as an ubiquitin ligase capable of ubiquitylating the
HIFa subunits at normoxia and targeting them for
destruc-tion by the proteasome [70–73] Alongwith hypoxia, iron
chelatingagents such as desferrioxamine (Dsfx) were also
able to block VHL interaction, suggesting a requirement
for iron in the normoxic degradation of HIFa subunits [69].
HIF transactivation
Deletion analysis of HIF-1a protein revealed that HIF-1a
contained two transactivation regions, termed the
amino-terminal transactivation domain (NAD) and the
carboxy-terminal transactivation domain (CAD) [74,75] Functional
analysis revealed that the activity of both the NAD and
CAD were enhanced by hypoxia treatment Since the NAD
overlaps with the ODD its increase in transcriptional
activity at hypoxia was largely attributed to increased
protein stability [75] However, the increase in
transcrip-tional activity of the CAD was not attributed to changes in
protein level [75] Instead, hypoxia was suggested to
promote the recruitment of transcriptional coactivator
proteins such as CBP/p300 [76–78], steroid receptor
coac-tivator-1 (SRC-1), and transcription intermediary factor 2
(TIF2) [77] to the CAD region Likewise, analysis of
HIF-2a revealed that it also contained two transactivation
domains with similar organization [67,79] and, as with
HIF-1a, the CAD of HIF-2a was also inducible by hypoxia.
While the transactivation capability of HIF-3a is poorly
characterized, sequence alignment suggests that HIF-3a
lacks an analogous inducible CAD region [57].
Therefore, the transactivation potency of HIF-1a and
HIF-2a CADs is negatively regulated by oxygen
independ-ent of protein stability, revealingthat alongwith the ODD
there exists a second oxygen sensing region near the
carboxy-terminus Moreover, like the ODD the CAD is also sensitive
to iron antagonists (i.e cobalt chloride, Dsfx) suggesting that
the mechanism of regulation of both domains involves a
common iron dependent process [74,75,79].
Regulation of HIFa subunits
by oxygen-dependent prolyl and asparaginyl
hydroxylation
A variety of oxygen sensors have been described for
prokaryotes and yeast [80]; however, for many years the
nature of the cellular oxygen sensor in higher organisms
remained elusive A number of interestingmodels have been
proposed to explain how mammalian cells could sense oxygen, including those that involve the hemoprotein, NADPH oxidoreductase, members of the mitochondrial electron transport chain [81], or oxygen-regulated potassium channels [82] Disappointingly, however, when investigated
in more detail none of these models could clearly demon-strate how HIF activation was beinguniversally regulated.
To better understand the mechanism of oxygen sensing and signal transduction, considerable effort over the last few years has focused on decipheringthe biochemical param-eters by which the ODD and CAD were beingregulated by low oxygen levels.
By employingboth protein interaction and ubiquitylation assays the major VHL bindingregion was narrowed down
to a 20 amino acid stretch within the ODD of both HIF-1a and HIF-2a [70,71,83] Treatment with hypoxia was able to induce the dissociation of VHL from HIF-1a, sug g esting that some cellular activity in normoxic cells maybe respon-sible for promotingVHL association [83] In support of this hypothesis, a synthetic peptide comprisingof the minimal VHL bindingmotif of HIF-1a was unable to interact with VHL unless pretreated with normoxic cell extracts [84–86] Likewise, similar biochemical experiments with the CAD demonstrated that a cellular activity was targeting the CAD for repression at normoxia, and hypoxia blocked this cellular activity, thereby promotingthe recruitment of coactivator proteins such as CBP/p300 [78].
Then, in an elegant set of experiments, a number of groups concurrently demonstrated that the cellular activity responsible for targeting HIFa for degradation was the enzymatic hydroxylation of a specific proline residue within the ODD Hydroxylation of this proline residue was shown
to promote high affinity binding of VHL protein [84–86] Subsequently, a second proline hydroxylation site was identified within the ODD and was also shown to promote VHL bindingin a hydroxylation dependent manner [87] Surprisingly, in a similar but distinct mechanism, an enzymatic hydroxylase activity was also found to specifically modify the CAD at normoxia to block p300 binding[88,89] However, in contrast to the ODD, the hydroxylation activity targeting the CAD was found to occur on an asparagine residue [88] While the CAD contains a number
of proline residues no evidence of hydroxylation of these proline sites has ever been found (D Lando, J J Gorman,
M L Whitelaw & D J Peet, unpublished observations) Hydroxyproline and hydroxyasparagine therefore control HIFa activity by regulating protein–protein interactions; hydroxyproline provides a dockingsite for VHL binding while hydroxyasparagine prevents binding of the coacti-vator p300 Finally, the longsoug ht after links between oxygen availability and iron in the regulation of HIFa protein stability and transactivation potential were realized when it was demonstrated that hypoxia and iron chelators could block hydroxylation of both the proline and aspara-gine residues, regulating the association of VHL with the ODD [84–87] and p300 with the CAD [88], respectively HIF prolyl and asparaginyl hydroxylases
Prior to the discovery of the HIF hydroxylases, the best characterized prolyl and asparaginyl hydroxylases were those that modify proline residues in collagen [90] and
Trang 4asparagine or aspartic acid residues in epidermal growth
factor (EGF)-like domains [91] The structures of the
catalytic domains of some of these iron dependent
hydroxylases have been solved and reveal a conserved
double-stranded b-helix enzymatic core commonly referred
to as a jellyroll Within this enzymatic core is a critical
2-His1-carboxylate motif (His-X-Asp/Glu…His),
respon-sible for coordinatingthe bindingof the iron atom [92] By
usinga combination of protein database mining , and
genetic and biochemical assays, three novel HIF prolyl
hydroxylase enzymes (designated prolyl hydroxylase
do-main-containingproteins (PHDs) 1, 2 and 3 [93], or HIF
prolyl hydroxylases (HPHs) 3, 2 and 1, respectively [94]), and one HIF asparaginyl hydroxylase enzyme called factor-inhibitingHIF-1 (FIH-1) [88,95] were identified and shown to hydroxylate the key proline and asparagine residues in HIFa The enzymatic reactions carried out by the PHD/HPHs and FIH-1 revealed that the hydroxyla-tion reachydroxyla-tion requires oxygen (in the form of dioxygen O2), iron (Fe2+) and the cofactor 2-oxoglutarate The hydroxy-lation reaction is inherently dependent on ambient oxygen because the oxygen atom used to form the proline and asparagine hydroxyl groups is derived directly from molecular oxygen [95,96] The cofactor 2-oxoglutarate is required because it undergoes a decarboxylation reaction, consumingthe remainingoxygen atom to form succinate and CO2(Fig 2).
Therefore, the rapid turnover and transcriptional silen-cingof the HIFa protein subunits involves oxygen-depend-ent prolyl and asparaginyl hydroxylation by the PHD/ HPHs and FIH-1 proteins, respectively These modifica-tions then serve as signals for either VHL binding and polyubiqutylation which targets the HIFa subunits for proteasomal degradation, or blocking coactivator proteins such as p300 from bindingthe CAD (Fig 3) The import-ance of the PHD/HPH-HIFa-VHL pathway in the oxygen response is further confirmed by the findingthat compo-nents of this pathway are functionally conserved in Caenorhabditis elegans [93] and Drosophila [94,97] Cur-rently FIH-1 homologues have been predicted to exist in
C elegans and Drosophila [98] but their functionality awaits further confirmation.
Oxygen sensing
By conductingreactions in a controlled oxygen environment
it has been demonstrated that the activity of the PHD/ HPHs are sensitive to graded oxygen levels [93] Moreover,
Fig 2 General reaction scheme for oxygen-dependent hydroxylation by
PHD/HPH and FIH-1 hydroxylases The hydroxylation of target
substrates requires dioxygen (O2), iron [Fe(II)] and the cofactor
2-oxoglutarate During catalysis the substrate accepts one oxygen
atom while 2-oxoglutarate undergoes a decarboxylation reaction
consumingthe remainingoxygen atom to form succinate and CO2
Fig 3 Regulation of hypoxia inducible factors (HIF) by oxygen-dependent hydroxylation In oxygenated conditions (normoxia) the asparaginyl and HIF prolyl hydroxylases (FIH-1 and PHD/HPH) hydroxylate (OH) HIFa on specific asparagine (Asn) and proline (Pro) residues, blocking transactivation and targeting HIFa for destruction by ubiquitin proteasome pathway, respectively Hypoxia and iron antagonists block both PHD/HPH and FIH-1 activity, then HIFa escapes destruction and recruits coactivators (CBP/p300) to induce hypoxia target genes Oxygen-dependent degradation domain (ODD), carboxy-terminal activation domain (CAD), von Hippel Lindau protein (VHL), cobalt chloride (Co), desferrioxamine (Dfrx), 2¢-2-dipyridyl (DP)
Trang 5the decreased hydroxylation seems to mirror the progressive
cellular increase in HIFa protein levels observed when cells
are subjected to similar oxygen gradients [99] Therefore, it
has been speculated that the PHD/HPHs represent primary
oxygen sensors Likewise, it has been suggested, but not yet
demonstrated, that FIH-1 catalytic activity is also sensitive
to oxygen gradients [95] Evidence so far suggests that while
the PHD/HPHs may act more as gross regulators of HIF
activity, FIH-1 may play a more subtle role For example,
when HIFa subunits are fully stabilized, as observed in
VHL-deficient cells [69], then FIH-1 activity is saturated by
the large excess of HIF protein, rendering the CAD active at
normoxia However, it is likely that FIH-1 plays a crucial
role in regulating the transcriptional activity of relatively
smaller, yet physiologically relevant, amounts of stabilized
HIFa, such as that produced in response to growth factors.
Interestingly, reporter assays have demonstrated that
overexpression of FIH-1 is still able to partially inhibit CAD
activity under hypoxic conditions [98,100] As
hydroxyla-tion of the CAD under hypoxic condihydroxyla-tions is essentially
absent [88] this inhibition is unlikely to be mediated by
hydroxylation It may be due to direct competition for p300
bindingto the CAD, the recruitment of other factors such as
histone deacetylases or VHL [98], or may well be an artefact
of overexpression.
Structural implications
Synthetic peptides composed of the minimal VHL binding
motif of HIF-1a chemically synthesized with a
4-hydroxy-proline at the critical 4-hydroxy-proline position were shown to bind
VHL protein [84–86] Subsequent structural analysis
dem-onstrated that the tight binding of VHL to the hydroxylated
peptide was due to the 4-hydroxyproline residue forming
critical hydrogen bonds with residues in VHL [101,102].
Taken together, these observations suggest that VHL can
specifically recognize a 4-hydroxyproline.
Recently the solution structures of the CH1 domains of
p300 or CBP bound to the CAD of HIF-1a were solved
[103,104] Analysis of the bound complex revealed that the
CAD remains relatively extended, wrappingitself around
the globular structure of the CH1 domain in a hand grasp or
vice like manner The critical asparagine residue (Asn803) in
the CAD of HIF-1a is found buried deep within the
molecular interface and nearly 45% of its surface is
concealed in the interface Asparagine 803 forms two side
chain hydrogen bonds with aspartic acid residues in the
CAD (Asp799) and the CH1 domain that help to stabilize
the complex Analysis of the asparagine and aspartic acid
hydroxylation products in the EGF like domains of other
hydroxylated proteins has revealed that the hydroxyl group
is attached to the b carbon in the erythro isoform [105] If
the asparagine in the CAD is also hydroxylated on the
b-carbon, either erythro or threo isoforms are predicted to
destabilize the p300/CBP- HIFa complex formation
[103,104] Apart from hydroxylatingthe asparagine on the
b carbon, FIH-1 could also potentially hydroxylate the
asparagine on the side chain amide nitrogen to form a
hydroxyamic acid However, it has been recently reported
that the asparagine 803 in HIF-1a is indeed hydroxylated on
the b-carbon Surprisingly, this hydroxylation is in the threo
isoform [106], unlike the previously characterized EGF-like
domain asparaginyl hydroxylases, which hydroxylate exclusively in the erythro position [105] Also, unlike other asparaginyl hydroxylase enzymes, which can hydroxylate both asparagine and aspartic acid residues, the FIH-1 enzyme was shown to have a clear preference for asparagine
in the CAD of HIF-1a [95] If the asparagine in the CAD was substituted with an aspartic acid residue, FIH-1 hydroxylase activity for the aspartic acid residue was only 7% of that obtained with asparagine This clear difference
in amino acid specificity and the production of threo rather than erythro isomers suggests that FIH-1 belongs to a new subfamily of 2-oxoglutarate-dependent asparaginyl hydroxylases.
Substrate specificity The three PHD/HPH enzymes have been shown to hydroxylate specific proline residues within the context of two strongly conserved LXXLAP* motifs (P* indicates hydroxy proline acceptor) within the ODD [87,93] While in-vitro substrate analysis has revealed that the three PHD/ HPHs have differinghydroxylatingactivity towards the proline residue, they also unfortunately report conflicting evidence showingdifferent enzymes as havinghig hest activity (i.e PHD-2/HPH-2 [107] vs PHD-3/HPH-1 [94]) Nevertheless, it will now be interestingto determine which PHD/HPH enzymes are the main regulator of HIFa hydroxylation in the cell under physiological conditions Expression analysis of the three PHD/HPHs in HeLa cells has revealed that all three mRNAs are expressed at normoxia with PHD-1/HPH-3 exhibitingthe g reatest expression [93] Interestingly, the expression of PHD-2/ HPH-2 and PHD-3/HPH-1, but not PHD-1/HPH-3 are induced by hypoxia [93], suggesting a possible role for these inducible enzymes in a negative feedback pathway respon-sible for enhanced degradation of HIFa after re-oxygen-ation.
Intriguingly, interaction assays have shown that FIH-1 interacts with the CAD of HIF-1a in a region that does not contain the hydroxylated asparagine residue [98] This asparagine residue is actually located approximately 20–30 residues carboxy-terminal to the putative FIH-1 binding region This suggests that for FIH-1 to efficiently hydroxy-late the asparagine residue in HIF-1a it may need to bind to
a reg ion in HIF-1a adjacent to the asparagine motif To support this notion it has been demonstrated that bindingof p300 is not enhanced by the hydroxylase inhibitor dime-thyloxalylglycine (DMOG), or iron antagonists, when the putative FIH-1 bindingregion is removed [89] A region spanningthe FIH-1 bindingsite in HIF-1a contains an arginine-dileucine (RLL) motif that has previously been shown to be critical for the normal silencingof the HIF-1a CAD at normoxia [79] An analogous RLL motif-contain-ingreg ion also operates in a similar silencingfashion in HIF-2a [79], suggesting that this region of both HIF-1a and HIF-2a may contain important elements for targeting FIH-1 The findingthat FIH-1 must bind to HIF-1a in a region away from the critical asparagine residue for efficient hydroxylation may help explain the longknown phenom-enon that uncouplingthe HIF-1a CAD containingresidues 786–826 from the adjacent inhibitory domain results in a highly active CAD under normoxic conditions [74,75] that
Trang 6binds strongly to CBP/p300 irrespective of hypoxia
treat-ment [78].
As well as interactingwith the CAD region FIH-1 has
also been shown to interact with VHL via its b domain [98].
Initially this interaction was thought to be important for the
repressive activity of FIH-1 on CAD function [98]; however,
a more recent analysis in VHL null cells has shown that
VHL is not critical for FIH-1 repressive activity [89] It is
possible that FIH-1 and VHL complexes may operate in
additional oxygen regulated processes that affect the
transcriptional response of other pathways Interestingly,
both FIH-1 and VHL have been shown to interact with
chromatin modifyinghistone deacetylase (HDAC)
enzymes, which are known to play an important role in
gene repression [98] Together these observations raise the
possibility that FIH-1 may have multiple roles other than
just regulating the CAD of HIF-1a and HIF-2a.
Other mechanisms of activation
The induction of HIF activity by well established agents
such as hypoxia, cobaltous ions and iron chelators can be
easily explained by the findingthat the PHD/HPHs and
FIH-1 are members of the 2-oxoglutarate-dependent family
of hydroxylase enzymes that utilize iron and oxygen to
modify their target amino acid residues However, it has
also been reported that HIF activity is influenced by
particular gas molecules (i.e NO, CO), reactive oxygen
species (i.e H2O2), and phosphorylation events (i.e p38
MAPK), although the understanding of how these
proces-ses may influence PHD/HPHs and FIH-1 function and HIF
activity is less clear (reviewed in [7]) NO is a known
analogue of dioxygen and analysis of the non heme iron
(Fe2+) dependent isopenicillin N synthase enzyme, a closely
related oxygenase to the 2-oxoglutarate family, has
dem-onstrated that NO can bind to the iron centre of this enzyme
[108] Because NO has only one available oxygen atom for
use in catalysis and 2-oxoglutarate-dependent dioxygenases
normally require two oxygen atoms for completing the
hydroxylation of their substrates (Fig 2), the binding of NO
to the catalytic core of PHD/HPHs and FIH-1 may block
enzymatic activity, explainingthe reported positive effects of
NO on HIF activity [109] The hydroxylation of substrates
by the collagen prolyl-4-hydroxylase has been shown to be
inhibited by the artificial generation of radicals at the
enzyme active site [110] Thus, the effects of reactive oxygen
species on HIF stability [64] and transactivation [62] may
relate to the alteringof the redox balance of the cell, which
then may affect the catalytic activity of PHD/HPHs and
FIH-1 Finally, it is possible that these other reported agents
that regulate HIF activity may target components of the
VHL ubiquitin ligase or CBP/p300 coactivator complex, or
even the PHD/HPHs and FIH-1 enzymes directly.
Therapeutic benefits
Hypoxia constitutes a major component of many disease
states and can have both a proliferative (cancer) or
damaging affect (stroke, heart attack) on disease
pathogene-sis [4] Therefore, it has been suggested that inducing HIF
activity may be beneficial for stroke and heart attack victims
as this would help promote vascularization of damaged tissue Conversely, blockingHIF activity may be advant-ageous in inhibiting cancer progression as this would help starve growing tumours of oxygen and nutrient supply Coupled with previous studies that have provided Ôproof of principleÕ that targeting HIF stability [111] and transactiva-tion [78] can enhance oxygen delivery and inhibit cancer progression, respectively, it is reasoned that HIF is an attractive target for pharmaceutical manipulation With the discoveries that HIF stability and transcriptional activity are controlled by two distinct modifications (prolyl and aspar-aginyl hydroxylation) the development of small molecule drugs to selectively target HIF to differentially modulate its activity should be possible For instance, while it has been demonstrated that the biological activity of FIH-1 requires 2-oxoglutarate [95,100], an unusual feature of FIH-1 is that
it lacks an arginine or lysine residue located on the eighth
b strand of the enzymatic core These conserved residues have previously been demonstrated to be involved in binding5-carboxylate of 2-oxog lutarate in many other 2-oxoglutarate dependent enzymes [92] Because these 2-oxoglutarate binding residues are conserved in the PHD/HPHs [93,94], it provides further evidence that
FIH-1 represents a new structural submember of the 2-oxoglu-tarate dependent enzyme family, and raises the possibility that selective agonists and antagonists for PHD/HPHs and FIH-1 can be developed Furthermore, a preliminary study has found that certain well-established inhibitors of the collagen prolyl hydroxylase enzymes do not inhibit PHD/ HPH activity, suggesting that it may be possible to design pharmacological inhibitors that can selectively target the HIF prolyl hydroxylases [112].
Global oxygen sensing by protein hydroxylation? Apart from the HIF pathway and HIFa subunits, the regulation and activity of a large number of other cellular processes and proteins have also been demonstrated to be influenced by oxygen availability For example, chronic hypoxia is known to extend the replicative life span of certain cell types such as vascular smooth muscle cells [113] A recent study attributed this increase in long-term proliferation to enhanced hypoxic phosphorylation of the telomerase cata-lytic component TERT [114] Apart from the putative hypoxia regulated kinase that phosphorylates TERT, the activity of a number of other protein kinases have also been shown to be regulated by hypoxia These include p44/ p42MAPK [115], p38 MAPK [116,117] and diacylglycerol kinase [118] Likewise, the stability of certain messenger RNAs, such as VEGF [119,120], are also known to be increased under hypoxic stress, while the splicingof specific alternative mRNA transcripts has recently been shown to be influenced by low oxygen tension [121] While the mechanism
by which low oxygen stress controls these other processes is unknown it will now be of great interest to determine whether the PHD/HPHs or FIH-1 are involved, or if additional oxygen sensing proteins exist that utilize hydroxylation to modify their target substrates The use of pharmacological inhibitors such as DMOG should now allow quick and easy analysis of the contribution of post-translational hydroxy-lation in other oxygen sensitive processes.
Trang 7To date three prolyl and one asparaginyl hydroxylase
enzymes have been discovered that can target different
domains of the HIFa subunits, affectingdistinct steps in the
induction of the HIF complex The numerous enzymes and
their various targets may have evolved to help manipulate
the magnitude of the HIF transcriptional response by
providinga variable mechanism to g radually alter the
activity of the HIFa subunits in response to subtle changes
in oxygen levels Furthermore, other studies have suggested
that the nuclear accumulation of HIFa subunits may also be
oxygen regulated [76], and it will now be interesting to
establish if this or other components of HIF regulation are
also influenced by the above or other hydroxylation
mediated events.
Acknowledgements
D J P is the W Bruce Hall Cancer Research Fellow supported by the
Cancer Council of South Australia, and this work was also supported
by the National Heart Foundation and National Health and Medical
Research Council of Australia
References
1 Storz, G & Imlay, J.A (1999) Oxidative stress Curr Opin
Microbiol 2, 188–194
2 Webster, K.A & Murphy, B.J (1988) Regulation of
tissue-specific glycolytic isozyme genes: coordinate response to oxygen
availability in myogenic cells Can J Zool 66, 1046–1058
3 Ferrara, N (1999) Molecular and biological properties of
vascular endothelial growth factor J Mol Med 77, 527–543
4 Semenza, G.L (2000) HIF-1 and human disease: one highly
involved factor Genes Dev 14, 1983–1991
5 Semenza, G.L & Wang, G.L (1992) 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
6 Wang, G.L & Semenza, G.L (1995) Purification and
characterization of hypoxia-inducible factor 1 J Biol Chem
270, 1230–1237
7 Semenza, G.L (1999) Regulation of mammalian O2
homeostasis by hypoxia-inducible factor 1 Annu Rev Cell
Dev Biol 15, 551–578
8 Feldser, D., Ag ani, F., Iyer, N.V., Pak, B., Ferreira, G &
Semenza, G.L (1999) Reciprocal positive regulation of
hypoxia-inducible factor 1alpha and insulin-like growth factor 2 Cancer
Res 59, 3915–3918
9 Tazuke, S.I., Mazure, N.M., Sugawara, J., Carland, G., Faessen,
G.H., Suen, L.F., Irwin, J.C., Powell, D.R., Giaccia, A.J &
Giudice, L.C (1998) Hypoxia stimulates insulin-like growth
factor bindingprotein 1 (IGFBP-1) gene expression in HepG2
cells: a possible model for IGFBP-1 expression in fetal hypoxia
Proc Natl Acad Sci USA 95, 10188–10193
10 Bhattacharya, S., Michels, C.L., Leung, M.K., Arany, Z.P.,
Kung, A.L & Livingston, D.M (1999) Functional role of p35srj,
a novel p300/CBP bindingprotein, duringtransactivation by
HIF-1 Genes Dev 13, 64–75
11 Zaman, K., Ryu, H., Hall, D., O’Donovan, K., Lin, K.I., Miller,
M.P., Marquis, J.C., Baraban, J.M., Semenza, G.L & Ratan,
R.R (1999) Protection from oxidative stress-induced apoptosis
in cortical neuronal cultures by iron chelators is associated with
enhanced DNA bindingof hypoxia-inducible factor-1 and ATF-1/
CREB and increased expression of glycolytic enzymes, p21 (waf1/cip1), and erythropoietin J Neurosci 19, 9821–9830
12 Bruick, R.K (2000) Expression of the gene encoding the proapoptotic Nip3 protein is induced by hypoxia Proc Natl Acad Sci USA 97, 9082–9087
13 Bazan, N.G & Lukiw, W.J (2002) Cyclooxygenase-2 and presenilin-1 gene expression induced by interleukin-1beta and amyloid beta 42 peptide is potentiated by hypoxia in primary human neural cells J Biol Chem 277, 30359–30367
14 Lukiw, W.J., Gordon, W.C., Rogaev, E.I., Thompson, H & Bazan, N.G (2001) Presenilin-2 (PS2) expression up-regulation
in a model of retinopathy of prematurity and pathoangiogenesis Neuroreport 12, 53–57
15 Estes, S.D., Stoler, D.L & Anderson, G.R (1995) Anoxic induction of a sarcoma virus-related VL30 retrotransposon is mediated by a cis-actingelement which binds hypoxia-inducible factor 1 and an anoxia-inducible factor J Virol 69, 6335–6341
16 Oikawa, M., Abe, M., Kurosawa, H., Hida, W., Shirato, K & Sato, Y (2001) Hypoxia induces transcription factor ETS-1 via the activity of hypoxia-inducible factor-1 Biochem Biophys Res Commun 289, 39–43
17 Miyazaki, K., Kawamoto, T., Tanimoto, K., Nishiyama, M., Honda, H & Kato, Y (2002) Identification of functional hypoxia response elements in the promoter region of the DEC1 and DEC2 genes J Biol Chem 277, 47014–47021
18 Takahashi, Y., Takahashi, S., Shiga, Y., Yoshimi, T & Miura,
T (2000) Hypoxic induction of prolyl 4-hydroxylase alpha (I) in cultured cells J Biol Chem 275, 14139–14146
19 Ebert, B.L., Firth, J.D & Ratcliffe, P.J (1995) Hypoxia and mitochondrial inhibitors regulate expression of glucose transporter-1 via distinct cis-actingsequences J Biol Chem
270, 29083–29089
20 Chen, C., Pore, N., Behrooz, A., Ismail-Beig i, F & Maity, A (2001) Regulation of glut1 mRNA by hypoxia-inducible factor)1 Interaction between H-ras and hypoxia J Biol Chem 276, 9519–9525
21 Zelzer, E., Levy, Y., Kahana, C., Shilo, B.Z., Rubinstein, M & Cohen, B (1998) Insulin induces transcription of target genes through the hypoxia-inducible factor HIF-1a/ARNT EMBO J
17, 5085–5094
22 O’Rourke, J.F., Pugh, C.W., Bartlett, S.M & Ratcliffe, P.J (1996) Identification of hypoxically inducible mRNAs in HeLa cells usingdifferential-display PCR Role of hypoxia-inducible factor-1 Eur J Biochem 241, 403–410
23 Semenza, G.L., Jiang, B.H., Leung, S.W., Passantino, R., Concordet, J.P., Maire, P & Giallongo, A (1996) Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1 J Biol Chem 271, 32529–32537
24 Semenza, G.L., Roth, P.H., Fang , H.M & Wang , G.L (1994) Transcriptional regulation of genes encoding glycolytic enzymes
by hypoxia-inducible factor 1 J Biol Chem 269, 23757–23763
25 Mathupala, S.P., Rempel, A & Pedersen, P.L (2001) Glucose catabolism in cancer cells: identification and characterization of
a marked activation response of the type II hexokinase gene to hypoxic conditions J Biol Chem 276, 43407–43412
26 Riddle, S.R., Ahmad, A., Ahmad, S., Deeb, S.S., Malkki, M., Schneider, B.K., Allen, C.B & White, C.W (2000) Hypoxia induces hexokinase II gene expression in human lung cell line A549 Am J Physiol Lung Cell Mol Physiol 278, L407–L416
27 Firth, J.D., Ebert, B.L., Pugh, C.W & Ratcliffe, P.J (1994) Oxygen-regulated control elements in the phosphoglycerate kinase 1 and lactate dehydrogenase A genes: similarities with the erythropoietin 3¢ enhancer Proc Natl Acad Sci USA 91, 6496–6500
Trang 828 Minchenko, A., Leshchinsky, I., Opentanova, I., Sang, N.,
Srinivas, V., Armstead, V & Caro, J (2002) Hypoxia-inducible
factor-1-mediated expression of the 6-phosphofructo-2-kinase/
fructose-2,6-bisphosphatase-3 (PFKFB3) gene Its possible role
in the Warburgeffect J Biol Chem 277, 6183–6187
29 Graven, K.K., Yu, Q., Pan, D., Roncarati, J.S & Farber, H.W
(1999) Identification of an oxygen responsive enhancer element
in the glyceraldehyde-3-phosphate dehydrogenase gene
Biochim Biophys Acta 1447, 208–218
30 Wykoff, C.C., Beasley, N.J., Watson, P.H., Turner, K.J.,
Pastorek, J., Sibtain, A., Wilson, G.D., Turley, H., Talks, K.L.,
Maxwell, P.H., Pugh, C.W., Ratcliffe, P.J & Harris, A.L (2000)
Hypoxia-inducible expression of tumor-associated carbonic
anhydrases Cancer Res 60, 7075–7083
31 Jelkmann, W (1992) Erythropoietin: structure, control of
production, and function Physiol Rev 72, 449–489
32 Wang, G.L & Semenza, G.L (1993) Desferrioxamine induces
erythropoietin gene expression and hypoxia-inducible factor 1
DNA-bindingactivity: implications for models of hypoxia signal
transduction Blood 82, 3610–3615
33 Rolfs, A., Kvietikova, I., Gassmann, M & Wenger, R.H (1997)
Oxygen-regulated transferrin expression is mediated by
hypoxia-inducible factor-1 J Biol Chem 272, 20055–20062
34 Lok, C.N & Ponka, P (1999) Identification of a hypoxia
response element in the transferrin receptor gene J Biol Chem
274, 24147–24152
35 Bianchi, L., Tacchini, L & Cairo, G (1999) HIF-1-mediated
activation of transferrin receptor gene transcription by iron
chelation Nucleic Acids Res 27, 4223–4227
36 Tacchini, L., Bianchi, L., Bernelli-Zazzera, A & Cairo, G (1999)
Transferrin receptor induction by hypoxia HIF-1-mediated
transcriptional activation and cell-specific post-transcriptional
regulation J Biol Chem 274, 24142–24146
37 Mukhopadhyay, C.K., Mazumder, B & Fox, P.L (2000) Role
of hypoxia-inducible factor-1 in transcriptional activation of
ceruloplasmin by iron deficiency J Biol Chem 275, 21048–
21054
38 Norris, M.L & Millhorn, D.E (1995) Hypoxia-induced protein
bindingto O2-responsive sequences on the tyrosine hydroxylase
gene J Biol Chem 270, 23774–23779
39 Levy, A.P., Levy, N.S., Wegner, S & Goldberg, M.A (1995)
Transcriptional regulation of the rat vascular endothelial growth
factor gene by hypoxia J Biol Chem 270, 13333–13340
40 Liu, Y., Cox, S.R., Morita, T & Kourembanas, S (1995)
Hypoxia regulates vascular endothelial growth factor gene
expression in endothelial cells Identification of a 5¢ enhancer
Circulat Res 77, 638–643
41 Forsythe, J.A., Jiang, B.H., Iyer, N.V., Agani, F., Leung, S.W.,
Koos, R.D & Semenza, G.L (1996) Activation of vascular
endothelial growth factor gene transcription by
hypoxia-inducible factor 1 Mol Cell Biol 16, 4604–4613
42 Gerber, H.P., Condorelli, F., Park, J & Ferrara, N (1997)
Differential transcriptional regulation of the two vascular
endothelial growth factor receptor genes Flt-1, but not Flk-1/
KDR, is up-regulated by hypoxia J Biol Chem 272, 23659–
23667
43 Eckhart, A.D., Yang, N., Xin, X & Faber, J.E (1997)
Characterization of the a1B-adrenergic receptor gene promoter
region and hypoxia regulatory elements in vascular smooth
muscle Proc Natl Acad Sci USA 94, 9487–9492
44 Lee, P.J., Jiang, B.H., Chin, B.Y., Iyer, N.V., Alam, J., Semenza,
G.L & Choi, A.M (1997) Hypoxia-inducible factor-1 mediates
transcriptional activation of the heme oxygenase-1 gene in
response to hypoxia J Biol Chem 272, 5375–5381
45 Melillo, G., Musso, T., Sica, A., Taylor, L.S., Cox, G.W &
Varesio, L (1995) A hypoxia-responsive element mediates a
novel pathway of activation of the inducible nitric oxide synthase promoter J Exp Med 182, 1683–1693
46 Palmer, L.A., Semenza, G.L., Stoler, M.H & Johns, R.A (1998) Hypoxia induces type II NOS gene expression in pulmonary artery endothelial cells via HIF-1 Am J Physiol 274, L212– L219
47 Hu, J., Discher, D.J., Bishopric, N.H & Webster, K.A (1998) Hypoxia regulates expression of the endothelin-1 gene through a proximal hypoxia-inducible factor-1 bindingsite on the antisense strand Biochem Biophys Res Commun 245, 894–899
48 Minchenko, A & Caro, J (2000) Regulation of endothelin-1 gene expression in human microvascular endothelial cells by hypoxia and cobalt: role of hypoxia responsive element Mol Cell Biochem 208, 53–62
49 Kietzmann, T., Roth, U & Jung ermann, K (1999) Induction of the plasminogen activator inhibitor-1 gene expression by mild hypoxia via a hypoxia response element bindingthe hypoxia-inducible factor-1 in rat hepatocytes Blood 94, 4177–4185
50 Cormier-Reg ard, S., Ng uyen, S.V & Claycomb, W.C (1998) Adrenomedullin gene expression is developmentally regulated and induced by hypoxia in rat ventricular cardiac myocytes
J Biol Chem 273, 17787–17792
51 Nguyen, S.V & Claycomb, W.C (1999) Hypoxia regulates the expression of the adrenomedullin and HIF-1 genes in cultured HL-1 cardiomyocytes Biochem Biophys Res Commun 265, 382–386
52 Furuta, G.T., Turner, J.R., Taylor, C.T., Hershberg , R.M., Comerford, K., Narravula, S., Podolsky, D.K & Colgan, S.P (2001) Hypoxia-inducible factor 1-dependent induction of intestinal trefoil factor protects barrier function during hypoxia J Exp Med 193, 1027–1034
53 Ambrosini, G., Nath, A.K., Sierra-Honigmann, M.R & Flores-Riveros, J (2002) Transcriptional activation of the human leptin gene in response to hypoxia: involvement of hypoxia-inducible factor 1 J Biol Chem 277, 34601–34609
54 Tian, H., McKnight, S.L & Russell, D.W (1997) Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells Genes Dev 11, 72–82
55 Ema, M., Taya, S., Yokotani, N., Sogawa, K., Matsuda, Y & Fujii-Kuriyama, Y (1997) A novel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1a regulates the VEGF expression and is potentially involved in lungand vascular development Proc Natl Acad Sci USA 94, 4273–4278
56 Flamme, I., Frohlich, T., von Reutern, M., Kappel, A., Damert,
A & Risau, W (1997) HRF, a putative basic helix-loop-helix-PAS-domain transcription factor is closely related to hypoxia-inducible factor-1a and developmentally expressed in blood vessels Mech Dev 63, 51–60
57 Gu, Y.Z., Moran, S.M., Hog enesch, J.B., Wartman, L & Bradfield, C.A (1998) Molecular characterization and chromosomal localization of a third a-class hypoxia inducible factor subunit, HIF3a Gene Expr 7, 205–213
58 Huang, Z.J., Edery, I & Rosbash, M (1993) PAS is a dimerization domain common to Drosophila period and several transcription factors Nature 364, 259–262
59 Crews, S.T (1998) Control of cell lineage-specific development and transcription by bHLH-PAS proteins Genes Dev 12, 607–620
60 Fedele, A.O., Whitelaw, M.L & Peet, D.J (2002) Regulation of gene expression by the hypoxia-inducible factors Mol Interventions 2, 229–243
61 Wenger, R.H (2002) Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression FASEB J 16, 1151–1162
62 Huang , L.E., Arany, Z., Living ston, D.M & Bunn, H.F (1996) Activation of hypoxia-inducible transcription factor depends
Trang 9primarily upon redox-sensitive stabilization of its alpha subunit.
J Biol Chem 271, 32253–32259
63 Kallio, P.J., Pongratz, I., Gradin, K., McGuire, J & Poellinger,
L (1997) Activation of hypoxia-inducible factor 1a:
posttranscriptional regulation and conformational change by
recruitment of the ARNT transcription factor Proc Natl Acad
Sci USA 94, 5667–5672
64 Salceda, S & Caro, J (1997) 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
65 Huang, L.E., Gu, J., Schau, M & Bunn, H.F (1998) Regulation
of hypoxia-inducible factor 1a is mediated by an O2-dependent
degradation domain via the ubiquitin-proteasome pathway
Proc Natl Acad Sci USA 95, 7987–7992
66 Wiesener, M.S., Turley, H., Allen, W.E., Willam, C., Eckardt,
K.U., Talks, K.L., Wood, S.M., Gatter, K.C., Harris, A.L.,
Pugh, C.W., Ratcliffe, P.J & Maxwell, P.H (1998) Induction of
endothelial PAS domain protein-1 by hypoxia: characterization
and comparison with hypoxia-inducible factor-1a Blood 92,
2260–2268
67 Ema, M., Hirota, K., Mimura, J., Abe, H., Yodoi, J., Sog awa,
K., Poellinger, L & Fujii-Kuriyama, Y (1999) Molecular
mechanisms of transcription activation by HLF and HIF1alpha
in response to hypoxia: their stabilization and redox signal–
induced interaction with CBP/p300 EMBO J 18, 1905–1914
68 Kaelin, W.G Jr & Maher, E.R (1998) The VHL
tumour-suppressor gene paradigm Trends Genet 14, 423–426
69 Maxwell, P.H., Wiesener, M.S., Chang, G.W., Clifford, S.C.,
Vaux, E.C., Cockman, M.E., Wykoff, C.C., Pugh, C.W., Maher,
E.R & Ratcliffe, P.J (1999) The tumour suppressor protein
VHL targets hypoxia-inducible factors for oxygen-dependent
proteolysis Nature 399, 271–275
70 Tanimoto, K., Makino, Y., Pereira, T & Poelling er, L (2000)
Mechanism of regulation of the hypoxia-inducible factor-1 alpha
by the von Hippel-Lindau tumor suppressor protein EMBO J
19, 4298–4309
71 Cockman, M.E., Masson, N., Mole, D.R., Jaakkola, P., Chang,
G.W., Clifford, S.C., Maher, E.R., Pugh, C.W., Ratcliffe, P.J &
Maxwell, P.H (2000) Hypoxia inducible factor-alpha binding
and ubiquitylation by the von Hippel-Lindau tumor suppressor
protein J Biol Chem 275, 25733–25741
72 Kamura, T., Sato, S., Iwai, K., Czyzyk-Krzeska, M., Conaway,
R.C & Conaway, J.W (2000) Activation of HIF1a
ubiquitination by a reconstituted von Hippel-Lindau (VHL)
tumor suppressor complex Proc Natl Acad Sci USA 97,
10430–10435
73 Ohh, M., Park, C.W., Ivan, M., Hoffman, M.A., Kim, T.Y.,
Huang, L.E., Pavletich, N., Chau, V & Kaelin, W.G (2000)
Ubiquitination of hypoxia-inducible factor requires direct
bindingto the b-domain of the von Hippel-Lindau protein
Nat Cell Biol 2, 423–427
74 Jiang, B.H., Zheng, J.Z., Leung, S.W., Roe, R & Semenza, G.L
(1997) Transactivation and inhibitory domains of
hypoxia-inducible factor 1a Modulation of transcriptional activity by
oxygen tension J Biol Chem 272, 19253–19260
75 Pugh, C.W., O’Rourke, J.F., Nagao, M., Gleadle, J.M &
Ratcliffe, P.J (1997) Activation of hypoxia-inducible factor-1;
definition of regulatory domains within the a subunit J Biol
Chem 272, 11205–11214
76 Kallio, P.J., Okamoto, K., O’Brien, S., Carrero, P., Makino, Y.,
Tanaka, H & Poellinger, L (1998) Signal transduction in
hypoxic cells: inducible nuclear translocation and recruitment of
the CBP/p300 coactivator by the hypoxia-inducible factor-1a
EMBO J 17, 6573–6586
77 Carrero, P., Okamoto, K., Coumailleau, P., O’Brien, S., Tanaka, H
& Poellinger, L (2000) Redox-regulated recruitment of the transcriptional coactivators CREB-bindingprotein and SRC-1 to hypoxia-inducible factor 1alpha Mol Cell Biol 20, 402–415
78 Kung, A.L., Wang, S., Klco, J.M., Kaelin, W.G & Livingston, D.M (2000) Suppression of tumor growth through disruption of hypoxia-inducible transcription Nat Med 6, 1335–1340
79 O’Rourke, J.F., Tian, Y.M., Ratcliffe, P.J & Pugh, C.W (1999) Oxygen-regulated and transactivating domains in endothelial PAS protein 1: comparison with hypoxia-inducible factor-1alpha J Biol Chem 274, 2060–2071
80 Bunn, H.F & Poyton, R.O (1996) Oxygen sensing and molecular adaptation to hypoxia Physiol Rev 76, 839–885
81 Semenza, G.L (1999) Perspectives on oxygen sensing Cell
98, 281–284
82 Seta, K.A., Spicer, Z., Yuan, Y., Lu, G & Millhorn, D.E (2002) Respondingto hypoxia: lessons from a model cell line Sci STKE 146, RE11
83 YuF., White, S.B., Zhao, Q & Lee, F.S (2001) Dynamic, site– specific interaction of hypoxia-inducible factor-1alpha with the von Hippel-Lindau tumor suppressor protein Cancer Res 61, 4136–4142
84 Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Asara, J.M., Lane, W.S & Kaelin, W.G Jr (2001) HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2sensing Science 292, 464– 468
85 Jaakkola, P., Mole, D.R., Tian, Y.M., Wilson, M.I., Gielbert, J., Gaskell, S.J., Kriegsheim, A., Hebestreit, H.F., Mukherji, M., Schofield, C.J., Maxwell, P.H., Pugh, C.W & Ratcliffe, P.J (2001) Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation Science 292, 468–472
86 YuF., White, S.B., Zhao, Q & Lee, F.S (2001) HIF-1alpha bindingto VHL is regulated by stimulus-sensitive proline hydroxylation Proc Natl Acad Sci USA 98, 9630–9635
87 Masson, N., Willam, C., Maxwell, P.H., Pugh, C.W & Ratcliffe, P.J (2001) Independent function of two destruction domains in hypoxia-inducible factor-a chains activated by prolyl hydroxylation EMBO J 20, 5197–5206
88 Lando, D., Peet, D.J., Whelan, D.A., Gorman, J.J & Whitelaw, M.L (2002) Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch Science 295, 858– 861
89 Sang, N., Fang, J., Srinivas, V., Leshchinsky, I & Caro, J (2002) Carboxyl-terminal transactivation activity of hypoxia-inducible factor 1a is governed by a von Hippel-Lindau protein-independent, hydroxylation–regulated association with p300/ CBP Mol Cell Biol 22, 2984–2992
90 Kivirikko, K.I & Myllyharju, J (1998) Prolyl 4-hydroxylases and their protein disulfide isomerase subunit Matrix Biol 16, 357–368
91 Stenflo, J (1991) Structure–function relationships of epidermal growth factor modules in vitamin K-dependent clotting factors Blood 78, 1637–1651
92 Schofield, C.J & Zhang, Z (1999) Structural and mechanistic studies on 2-oxoglutarate-dependent oxygenases and related enzymes Curr Opin Struct Biol 9, 722–731
93 Epstein, A.C., Gleadle, J.M., McNeill, L.A., Hewitson, K.S., O’Rourke, J., Mole, D.R., Mukherji, M., Metzen, E., Wilson, M.I., Dhanda, A., Tian, Y.M., Masson, N., Hamilton, D.L., Jaakkola, P., Barstead, R., Hodg kin, J., Maxwell, P.H., Pug h, C.W., Schofield, C.J & Ratcliffe, P.J (2001) C elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation Cell 107, 43–54
Trang 1094 Bruick, R.K & McKnight, S.L (2001) A conserved family of
prolyl-4-hydroxylases that modify HIF Science 294, 1337–1340
95 Hewitson, K.S., McNeill, L.A., Riordan, M.V., Tian, Y.M.,
Bullock, A.N., Welford, R.W., Elkins, J.M., Oldham, N.J.,
Bhattacharya, S., Gleadle, J.M., Ratcliffe, P.J., Pugh, C.W &
Schofield, C.J (2002) Hypoxia-inducible factor (HIF)
asparagine hydroxylase is identical to factor inhibiting HIF
(FIH) and is related to the cupin structural family J Biol Chem
277, 26351–26355
96 McNeill, L.A., Hewitson, K.S., Gleadle, J.M., Horsfall, L.E.,
Oldham, N.J., Maxwell, P.H., Pugh, C.W., Ratcliffe, P.J &
Schofield, C.J (2002) The use of dioxygen by HIF prolyl
hydroxylase (PHD1) Bioorg Medical Chem Lett 12, 1547–1550
97 Lavista-Llanos, S., Centanin, L., Irisarri, M., Russo, D.M.,
Gleadle, J.M., Bocca, S.N., Muzzopappa, M., Ratcliffe, P.J &
Wappner, P (2002) Control of the hypoxic response in
Drosophila melanogaster by the basic helix-loop-helix PAS
protein similar Mol Cell Biol 22, 6842–6853
98 Mahon, P.C., Hirota, K & Semenza, G.L (2001) FIH-1: a novel
protein that interacts with HIF-1a and VHL to mediate
repression of HIF-1 transcriptional activity Genes Dev 15,
2675–2686
99 Jiang, B.H., Semenza, G.L., Bauer, C & Marti, H.H (1996)
Hypoxia-inducible factor 1 levels vary exponentially over a
physiologically relevant range of O2tension Am J Physiol 271,
C1172–C1180
100 Lando, D., Peet, D.J., Gorman, J.J., Whelan, D.A., Whitelaw,
M.L & Bruick, R.K (2002) FIH-1 is an asparaginyl hydroxylase
enzyme that regulates the transcriptional activity of
hypoxia-inducible factor Genes Dev 16, 1466–1471
101 Hon, W.C., Wilson, M.I., Harlos, K., Claridge, T.D., Schofield,
C.J., Pugh, C.W., Maxwell, P.H., Ratcliffe, P.J., Stuart, D.I &
Jones, E.Y (2002) Structural basis for the recognition of
hydroxyproline in HIF-1a by pVHL Nature 417, 975–978
102 Min, J.H., Yang, H., Ivan, M., Gertler, F., Kaelin, W.G Jr
& Pavletich, N.P (2002) Structure of an HIF-1a-pVHL
complex: hydroxyproline recognition in signaling Science 296,
1886–1889
103 Dames, S.A., Martinez-Yamout, M., De Guzman, R.N., Dyson,
H.J & Wright, P.E (2002) Structural basis for Hif-1a/CBP
recognition in the cellular hypoxic response Proc Natl Acad
Sci USA 99, 5271–5276
104 Freedman, S.J., Sun, Z.Y., Poy, F., Kung, A.L., Livingston,
D.M., Wagner, G & Eck, M.J (2002) Structural basis for
recruitment of CBP/p300 by hypoxia-inducible factor-1a Proc
Natl Acad Sci USA 99, 5367–5372
105 Przysiecki, C.T., Staggers, J.E., Ramjit, H.G., Musson, D.G.,
Stern, A.M., Bennett, C.D & Friedman, P.A (1987) Occurrence
of beta-hydroxylated asparagine residues in non-vitamin
K-dependent proteins containingepidermal growth factor-like
domains Proc Natl Acad Sci USA 84, 7856–7860
106 McNeill, L.A., Hewitson, K.S., Claridge, T.D., Seibel, J.F.,
Horsfall, L.E & Schofield, C.J (2002) Hypoxia-inducible factor
asparaginyl hydroxylase (FIH-1) catalyses hydroxylation at the
b-carbon of asparagine-803 Biochem J 367, 571–575
107 Huang, J., Zhao, Q., Mooney, S.M & Lee, F.S (2002) Sequence
determinants in hypoxia inducible factor-1alpha for
hydroxylation by the prolyl hydroxylases PHD1, PHD2, and PHD3 J Biol Chem 277, 39792–39800
108 Roach, P.L., Clifton, I.J., Hensgens, C.M., Shibata, N., Scho-field, C.J., Hajdu, J & Baldwin, J.E (1997) Structure of isopenicillin N synthase complexed with substrate and the mechanism of penicillin formation Nature 387, 827–830
109 Huang, L.E., Willmore, W.G., Gu, J., Goldberg, M.A & Bunn, H.F (1999) Inhibition of hypoxia-inducible factor 1 activation
by carbon monoxide and nitric oxide Implications for oxygen sensing and signaling J Biol Chem 274, 9038–9044
110 Wu, M., Moon, H.S., Begley, T.P., Myllyharju, J & Kivirikko, K.I (1999) Mechanism-based inactivation of the human prolyl-4-hydroxylase by 5-oxaproline-containingpeptides: evidence for
a prolyl radical intermediate JACS 121, 587–588
111 Elson, D.A., Thurston, G., Huang, L.E., Ginzinger, D.G., McDonald, D.M., Johnson, R.S & Arbeit, J.M (2001) Induction of hypervascularity without leakage or inflammation
in transgenic mice overexpressing hypoxia-inducible factor-1a Genes Dev 15, 2520–2532
112 Ivan, M., Haberberger, T., Gervasi, D.C., Michelson, K.S., Gunzler, V., Kondo, K., Yang, H., Sorokina, I., Conaway, R.C., Conaway, J.W & Kaelin, W.G Jr (2002) Biochemical purification and pharmacological inhibition of a mammalian prolyl hydroxylase actingon hypoxia-inducible factor Proc Natl Acad Sci USA 26, 26
113 Kourembanas, S., Morita, T., Liu, Y & Christou, H (1997) Mechanisms by which oxygen regulates gene expression and cell–cell interaction in the vasculature Kidney Int 51, 438–443
114 Minamino, T., Mitsialis, S.A & Kourembanas, S (2001) Hypoxia extends the life span of vascular smooth muscle cells through telomerase activation Mol Cell Biol 21, 3336–3342
115 Conrad, P.W., Freeman, T.L., Beitner-Johnson, D & Millhorn, D.E (1999) EPAS1 trans-activation duringhypoxia requires p42/p44 MAPK J Biol Chem 274, 33709–33713
116 Conrad, P.W., Rust, R.T., Han, J., Millhorn, D.E & Beitner-Johnson, D (1999) Selective activation of p38a and p38c by hypoxia Role in regulation of cyclin D1 by hypoxia in PC12 cells J Biol Chem 274, 23570–23576
117 Hirota, K & Semenza, G.L (2001) Rac1 activity is required for the activation of hypoxia-inducible factor 1 J Biol Chem 276, 21166–21172
118 Aragones, J., Jones, D.R., Martin, S., San Juan, M.A., Alfranca, A., Vidal, F., Vara, A., Merida, I & Landazuri, M.O (2001) Evidence for the involvement of diacylglycerol kinase in the activation of hypoxia-inducible transcription factor 1 by low oxygen tension J Biol Chem 276, 10548–10555
119 Levy, A.P., Levy, N.S & Goldberg, M.A (1996) Post-transcriptional regulation of vascular endothelial growth factor
by hypoxia J Biol Chem 271, 2746–2753
120 Shima, D.T., Deutsch, U & D’Amore, P.A (1995) Hypoxic induction of vascular endothelial growth factor (VEGF) in human epithelial cells is mediated by increases in mRNA stability FEBS Lett 370, 203–208
121 Makino, Y., Kanopka, A., Wilson, W.J., Tanaka, H & Poel-linger, L (2002) Inhibitory PAS domain protein (IPAS) Is a hypoxia-inducible splicingvariant of the hypoxia-inducible factor-3a locus J Biol Chem 277, 32405–32408