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The tumor suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis



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The tumour suppressor
protein VHL targets
hypoxia-inducible factors for
oxygen-dependent proteolysis
Patrick H. Maxwell*, Michael S. Wiesener*,
Gin-Wen Chang*, Steven C. Clifford, Emma C. Vaux,
Matthew E. Cockman, Charles C. Wykoff,
Christopher W. Pugh, Eamonn R. Maher
& Peter J. Ratcliffe*
*Wellcome Trust Centrefor Human Genetics, WindmillRoad, OxfordOX3 7BN,UK
Section of Medical and Molecular Genetics, Department of Paediatrics and Child
Health, University of Birmingham, Birmingham B15 2TT, UK
Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UK
Hypoxia-inducible factor-1 (HIF-1) has a key role in cellular
responses to hypoxia, including the regulation of genes involved
in energy metabolism, angiogenesis and apoptosis1–4. The a
subunits of HIF are rapidly degraded by the proteasome under
normal conditions, but are stabilized by hypoxia5. Cobaltous ions
or iron chelators mimic hypoxia, indicating that the stimuli may
interact through effects on a ferroprotein oxygen sensor6,7. Here
we demonstrate a critical role for the von Hippel-Lindau (VHL)
tumour suppressor gene product pVHL in HIF-1 regulation. In
VHL-defective cells, HIF a-subunits are constitutively stabilized
and HIF-1 is activated. Re-expression of pVHL restored oxygen-
dependent instability. pVHL and HIF a-subunits co-immunopre-
cipitate, and pVHL is present in the hypoxic HIF-1 DNA-binding
complex. In cells exposed to iron chelation or cobaltous ions,
HIF-1 is dissociated from pVHL. These findings indicate that the
interaction between HIF-1 and pVHL is iron dependent, and
that it is necessary for the oxygen-dependent degradation of
HIF a-subunits. Thus, constitutive HIF-1 activation may underlie
the angiogenic phenotype of VHL-associated tumours. The
pVHL/HIF-1 interaction provides a new focus for understanding
cellular oxygen sensing.
Enhanced glucose metabolism and angiogenesis are classical
features of cancer8,9, involving upregulation of genes that are
normally induced by hypoxia. In addition to stimulation by the
hypoxic microenvironment10, genetic alterations contribute to these
effects8,9. A striking example is von Hippel-Lindau (VHL) disease, a
hereditary human cancer syndrome predisposing sufferers to highly
angiogenic tumours11. Constitutive upregulation of hypoxically
inducible messenger RNAs encoding vascular endothelial growth
factor (VEGF) and glucose transporter 1 (GLUT-1) in these tumour
cells is correctable by re-expression of pVHL. A post-transcriptional
mechanism has been proposed12,13. We studied the involvement of
pVHL in oxygen-regulated gene expression using ribonuclease
protection analysis of two VHL-deficient renal carcinoma lines,
RCC4 and 786-O. Eleven genes encoding products involved in
glucose transport, glycolysis, high-energy phosphate metabolism
and angiogenesis were examined; nine are induced by hypoxia in
other mammalian cells and two (LDH-B and PFK-M) are repressed
by hypoxia. None of these responses was seen in the VHL-defective
cell lines. Responses to hypoxia were restored by stable transfection
of a wild-type VHL gene, with effects ranging from a rather modest
action of hypoxia (PFK-L and LDH-B) to substantial regulation
(Fig. 1 shows results for RCC4 cells; similar effects were seen in 786-
O cells, data not shown). These results indicate that the previously
described upregulation of hypoxia-inducible mRNAs in VHL-
defective cells12,13 extends to a broad range of oxygen-regulated
genes and involves a constitutive ‘hypoxia pattern’ for both posi-
tively and negatively regulated genes.
As a number of these genes (VEGF, GLUT-1, AK-3, ALD-A,
PGK-1, PFK-L and LDH-A) contain hypoxia-response elements
(HREs) which bind HIF-1 and/or show altered expression in cells
lacking HIF-1 (refs 2, 14 and references therein), this survey of
expression in VHL-defective cells prompted us to look for effects of
pVHL on HIF-1 and HRE function. RCC4 cells were co-transfected
with reporter plasmids which did or did not contain HREs from the
mouse phosphoglycerate-kinase-1 or erythropoietin (Epo) enhan-
cers, and either the VHL-expression plasmid pcDNA3-VHL or an
empty vector. pVHL suppressed HRE activity in normoxic cells and
restored induction by hypoxia (Fig. 2a). Similar results were
obtained by sequential stable transfection of RCC4 cells with an
HRE reporter followed by pcDNA3-VHL (data not shown). HIF-1
itself was examined by electrophoretic mobility shift assay (EMSA),
which showed a constitutive HIF-1 DNA-binding species in VHL-
deficient RCC4 cells, with restoration of the normal hypoxia-
inducible pattern in RCC4 cells stably transfected with pcDNA3-
VHL (RCC4/VHL) (Fig. 2b). In other cells, HIF-1 activation by
hypoxia involves a large increase in HIF-1aabundance from low
basal levels in normoxia1,15. Western blotting of whole-cell extracts
showed that RCC4 cells express constitutively high levels of both
HIF-1aand a related molecule, HIF-2a(also termed EPAS-1, HRF,
HLF and MOP2) which is normally regulated in a similar way16
(Fig. 2c). Constitutively high levels of these proteins were found in
eight other VHL-defective cell lines, in contrast to the renal
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VOL 399
20 MAY 1999
carcinoma line Caki-1 (which expresses pVHL normally17) and a
wide range of previously reported cell lines (Fig. 2d and Supple-
mentary Information S2). Certain VHL-defective cells (for example,
786-O, KTCL140) expressed HIF-2aat a high constitutive level but
did not express detectable HIF-1a(Fig. 2c, d and Supplementary
Information S2). Examination of stable transfectants of RCC4 and
786-O cells demonstrated that expression of the wild-type, but not
truncated, VHL gene restored regulation of HIF a-subunits by
oxygen without affecting the levels of mRNA encoding either
subunit (data not shown).
To investigate the role of pVHL in HIF-1 regulation we tested for
interactions between HIF a-subunits and pVHL using a combina-
tion of hypoxia and/or proteasomal blockade to induce HIF a-
subunits. Anti-pVHL immunoprecipitates of extracts from protea-
somally blocked RCC4/VHL cells, but not RCC4 cells, contained
both HIF-1aand HIF-2a(Fig. 3a). Similar results were obtained
with hypoxia in the absence of proteasomal blockade. In the inverse
reactions, immunoprecipitating antibodies to HIF-2aor HIF-1a
co-precipitated pVHL, although a smaller proportion of the total
was captured (Fig. 3b). Anti-pVHL immunoprecipitations also
demonstrated the interaction in HeLa cells, which express pVHL
normally (Fig. 3c). We next tested whether pVHL is incorporated in
the HIF-1 DNA-binding complex. Addition of antibodies raised
against pVHL (but not control antibodies) to nuclear extract from
RCC4/VHL cells and HeLa cells produced a change in mobility,
which was not observed with VHL-defective RCC4 cells (Fig. 3d).
HIF-1 migrated as two species. Only the slower-mobility HIF-1
species was shifted by anti-pVHL, whereas both species were shifted
Figure 1 Effect of pVHL on oxygen-regulated gene expression. mRNA analysis of
RCC4 cells and stable transfectant expressing pVHL (RCC4/VHL). N, normoxia;
H, hypoxia (1% O
, 16 h). VEGF, vascular endoth elial growth factor; GLUT-1,
glucose transporter 1; AK-3, adenylate kinase 3; TGF-b1, transforming growth
factor-b1; ALD-A, aldolase A; PGK-1, phosphoglyceratekinase 1; PFK, phos-
phofructokinase; LDH, lactate dehydrogenase. U6 small nuclear (sn) RNA was
used as an internal control. Also illustrated are two genes not influenced by VHL
status or hypoxia; nuclear respiratory factor 1 (NRF-1) and b-actin. Amount of RNA
analysed is detailed in Table S1 of the Supplementary Information.
Figure 2 Effect of pVHL on HIF-1 and HRE activity. a, Representative transient
transfections of RCC4 cells with VHL expression vector (+) or empty vector, and
luciferase reporter genes containing no HRE, or HREs from the PGK-1 or
erythropoietin (Epo) genes linked to SV40 or TK promoters. N, normoxia; H,
hypoxia (0.1% O
, 24 h —results were similar with 1% O
). b, EMSA using the Epo
HRE. N, normoxia; H, hypoxia (1% O
, 4 h). In HeLa and RCC4/VHL, HIF-1 is a
doublet (S, slower and F, faster mobility). RCC4 cells contain only faster mobility
HIF-1, with equivalent levels in normoxia and hypoxia. Constitutive binding
species are indicated (C). c, Immunoblots of whole cell extracts for HIF a-
subunits. Upper panels; RCC4 and RCC4/VHL cells (+VHL). Lower panel; 786-O
cells stably transfected with vector alone, a full length VHL gene (+VHL), or a
truncated VHL gene (1-115; +Tr). HIF-1awas not detected in 786-O cells. d,
Immunoblot of UMRC2, UMRC3 and KTCL140 (renal carcinoma lines with VHL
mutations30), Caki-1 (renal carcinoma line expressing wild-type pVHL) and the
hepatoma line Hep3B.
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by anti-HIF-1a(Fig. 3e). Similar results were obtained in other cell
lines (Hep3B, Caki-1, MRC5-V2 and 293; data not shown).
Furthermore, whereas RCC4/VHL, HeLa and other cells contained
both HIF-1 species, RCC4 extracts contained only the faster-
mobility species (Figs 3d, Fig. 2b). Thus, VHL-defective cells lack
the slower-mobility species which is restored by re-expression of
pVHL, and shifted by anti-pVHL. This indicates that the DNA-
binding HIF-1 doublet arises from two species containing or not
containing pVHL. Combination supershift analysis confirmed that
the slower-mobility species contained both HIF-1aand pVHL
(Fig. 3e).
HIF-1 activation by hypoxia is mimicked by cobaltous ions and
iron chelation6,7. We therefore tested whether the pVHL/HIF-1
interaction was regulated by these stimuli. Proteasomal blockade
induces an HIF-1 DNA-binding complex in normoxic cells18;
comparison of this normoxic complex with EMSA of hypoxic
cells with or without proteasomal inhibitors showed a similar
shift and anti-pVHL supershift (Fig. 4a and data not shown).
Together with immunoprecipitation data, this indicates that the
interaction with pVHL occurs in both normoxic and hypoxic cells.
In contrast, EMSA analysis of RCC4/VHL cells treated with cobalt
and the iron chelator desferrioxamine (DFO) demonstrated only
the faster mobility HIF-1. This did not supershift with anti-pVHL,
indicating that the pVHL/HIF-1 complex could not form in cells
exposed to these stimuli. Similar results were obtained in other cell
types and are consistent with hitherto unexplained mobility differ-
ences between previous analyses of HIF-1 from cobalt- or DFO-
versus hypoxia-stimulated cells7, indicating that this is a general
effect. Treatment with DFO 4 h before hypoxia prevented the
formation of the pVHL/HIF-1 complex (Fig. 4b). Addition of
iron chelators could not break the pVHL/HIF-1 complex in vitro,
whereas addition of in vitro-translated wild-type pVHL (but not a
truncated pVHL) could restore the slower-mobility species to
nuclear extracts of proteasomally blocked, normoxic and hypoxic
RCC4 cells, but not to cells treated with DFO or cobalt (Fig. 4c, and
Fig. S1a of Supplementary Information). Immunoprecipitation
studies also indicated that the interaction between HIF-1 and
pVHL is iron-dependent. Whereas both HIF-1aand HIF-2awere
contained in anti-pVHL immunoprecipitates from hypoxic RCC4/
VHL cells, neither was contained in precipitates from DFO- or
cobalt-treated cells (Fig. 4d). The iron-dependent interaction
between HIF a-subunits and pVHL may be direct or indirect.
However, in vitro-translated wild-type pVHL did not bind to an
in vitro-translated HIF-1 DNA-binding complex (Fig. S1b in
Supplementary Information), in contrast to the interaction with
RCC4 extracts (Fig. 4c), indicating that an additional factor or
modification of HIF-1 not represented in rabbit reticulocyte lysates
is necessary for the association.
Normally, HIF a-subunits are targeted for rapid degradation in
normoxic cells by a proteasomal mechanism operating on an
internal oxygen-dependent-degradation (ODD) domain5. Our
data suggest that pVHL might be required for this process—a
possibility which would be consistent with recent data that pVHL
forms a multiprotein complex (containing Cul-2 and elongins B
and C) which has homology with ubiquitin-ligase/proteasome-
targeting complexes in yeast19,20. When cells were switched from
Figure 3 Association of pVHL with HIF-1. a, Immunoblots for HIF a-subunits (2a,
1a) of IG32 (VHL ip) and control immunoprecipitates (using VG-7be) of RCC4/VHL
(VHL+) and RCC4 (VHL) cells exposed (4 h) to normoxia or hypoxia (1% O
; H+)
with or without proteasomal inhibition (PI+). Aliquots of selected input lysates
were also immunoblotted. b, Immunoprecipitation of RCC4/VHL (VHL+) and
RCC4 (VHL) extracts with polyclonal antibodies to HIF a-subunits or normal
rabbit immunoglobulin (control ip) followed by immunoblotting for pVHL (V). A
cross-reacting species arose from the HIF-2aantibody (asterisk). c, Immuno-
precipitation of HeLa extracts with IG32 (VHL
) or pAb419 (control) followed by
immunoblotting for HIF a-subunits. d, Anti-pVHL supershifts. IG32 (VHL Ab+) or
VG-7be (Control Ab+) was added to binding reactions of nuclear extracts from
normoxic or hypoxic (1% O
, 4 h; H+) cells. Anti-pVHL supershifted (SS) the slower
HIF-1 species (S) in HeLa and hypoxic RCC4/VHL cells. No supershift was seen
with RCC4 cells, which lack pVHL. e, Anti-pVHL, anti-HIF-1aand combination
supershifts in HeLa cells. Anti-pVHL (IG32, +VHL Ab) supershifted slower mobility
HIF-1. Anti-HIF-1a(clone 54) supershifted both components (SS). Addition of both
antibodies ‘super-super-shifted’ (SSS) slower mobility HIF-1.
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VOL 399
20 MAY 1999
hypoxia to normoxia with addition of cycloheximide, HIF a-
subunits decayed with a half-life of about 5 min in wild-type VHL
transfectants, compared with ,60 min in the VHL-defective RCC4
and 786-O cells, thus confirming a strong effect of pVHL on stability
(Fig. 5a). Moreover, functional studies of Gal4 chimaeras contain-
ing the HIF-1aODD domain demonstrated a striking dependence
of the isolated ODD domain on pVHL (Fig. 5b).
These experiments define a function for pVHL in the regulation
of HIF-1. Given recent demonstrations of the importance of HIF-1
in tumour angiogenesis3,21, constitutive HIF-1 activation is clearly
consistent with the angiogenic phenotype of VHL disease. Whether
it is a sufficient explanation for oncogenesis is less clear. HIF-1
mediates gene activation not only by hypoxia, but also by growth
factors such as insulin and insulin-like growth factor-1 (ref. 22).
HIF-1 targets such as molecules involved in enhanced glucose
metabolism and angiogenesis8,9,14 are classically upregulated (by
different mechanisms) in many forms of cancer, supporting an
important role in tumour progression, although their role in the
initiation of oncogenesis is less clear. pVHL is probably a multi-
functional protein which could have other tumour suppressor
actions11,23,24. One possibility is that other gene products could be
targeted in a similar manner to HIF a-subunits; however, compari-
son of anti-pVHL immunoprecipitates from metabolically labelled
RCC4/VHL cells with and without proteasomal inhibitors has so far
demonstrated only two species: HIF-1aand HIF-2a(G.-W.C.,
unpublished observations).
As both pVHL and HIF-1 are widely expressed, it is likely that the
physiological role of pVHL in HIF-1 regulation is general. It is not
yet clear whether pVHL has actions on oxygen-regulated gene
expression other than through HIF-1. Stabilization of hypoxia-
inducible mRNAs has been reported in VHL-deficient cells12,13. This
might represent an independent action of pVHL. However, regula-
tion of these RNAs is commonly abolished in HIF-1-deficient
cells3,25, so the mRNA stability factors could lie downstream of
Figure 5 Effect of pVHL on HIFastability and ODD domain function. a, Western
analysis of HIF a-subunit stability in cells lacking VHL (RCC4, 786-O) and stable
transfectants expressing pVHL (+VHL). Cells were incubated in hypoxia (4 h),
then moved to normoxia (time 0) with addition of cycloheximide (100 mM) and
harvested up to 80 min later. b, Representative functional assay of the HIF-1aODD
domain. Hep3B or RCC4 cells were transfected with Gal4 reporter pUAS-tk-Luc,
and either pGalVP16 (upper panel) encoding the Gal4 DNA-binding domain fused
to the VP16 activation domain, or pGala344-698VP16 (lower panel) which includes
HIF-1aamino acids 344– 698 (containing the entire ODD domain5). RCC4 cells
were co-transfected with pcDNA3 (), pcDNA3-VHL (VHL) or pcDNA3-VHL.103FS
(TrVHL). After transfection, cells were divided for 24 h incubation in normoxia (N)
or hypoxia (H; 0.1% O
). Corrected luciferase counts are shown, normalized to the
normoxic value with pGalVP16 or pGalVP16+pcDNA3. The HIF-1adomain con-
fers suppression and hypoxic regulationin Hep3B cells but not RCC4 cells, where
re-expression of pVHL restores these properties.
Figure 4 Effect of cobaltous ions and iron chelation on the pVHL/HIF-1
interaction. a, EMSA and supershift analysis of RCC4/VHL cells exposed (4 h)
to normoxia (N), hypoxia (H; 1% O
), DFO (100 mM), cobaltous chloride (Co,
100 mM) or proteasomal inhibition (PI). In lanes 6– 10, IG32 was added (+aVHL). b,
EMSA and supershift analysis of RCC4/VHL cells subjected to hypoxia (H; 1% O
for 8 h) or DFO (100 mM) with hypoxia (D H; cel ls exposed to DFO for 8 h, 4 h
normoxia, then 4 h hypoxia). c, EMSA of RCC4 cells showing only faster mobility
HIF-1 (lanes 1– 4). Addition of in vitro transcribed/translated pVHL alone (lanes 5–
8, +IVTT), or together with IG32 (lanes 9– 12, +IVTT+aVHL); slower mobility HIF-1
forms and supershifts in cells exposed to normoxia, hypoxia and PI, but not DFO.
d, HIFaimmunoblots of anti-pVHL immunoprecipitates of RCC4/VHL cells.
© 1999 Macmillan Magazines Ltd
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| 275
HIF-1. Positive and negative effects on HIF-1 activation by iron
chelators, hydrogen peroxide and redox active agents have indicated
that the underlying oxygen sensing mechanism may involve oxygen-
dependent generation of partially reduced oxygen radical species by
redox active iron centre(s)2,6,7,15,26. Such system(s) could act distantly
on a transduction pathway or might directly modify the ODD
domains of HIFathrough a caged radical system, as proposed for
iron sensing in the regulation of IRP2 (ref. 27). Cobaltous ions
activate HIF-1 in inverse relation to iron availability, indicating that
there may be competition for incorporation into a metal centre28.
Our data support a model in which VHL/HIF complexes form in
normoxic cells and target HIFasubunits for destruction. In
hypoxia, degradation is suppressed despite complex formation,
perhaps because a critical targeting modification of the HIFa
ODD domain cannot occur without oxygen. Cobaltous ions and
desferrioxamine prevent formation of the VHL complex, providing
a different mechanism for stabilization of HIF-1 and potentially
explaining why activation by these stimuli is relatively resistant to
oxygen and certain other radical-generating processes6,16,29. The iron
dependence of complex formation could indicate that an iron-
containing protein is an essential component of the complex,
perhaps involved in local generation of an oxygen-sensing signal. M
Cells and transfections. 786-O cells expressing pVHL, truncated pVHL
(amino acids 1– 115), or empty vector17 were a gift from W. G. Kaelin. RCC4
cells were a gift from C. H. C. M. Buys. Other RCC lines were provided by M.
Lerman. HeLa and Hep3B cells were fromECACC. RCC4/VHL was obtained by
transfection with pcDNA3-VHL and G418 selection. Cells were plated in
medium lacking G418 24 h before experiments, which were performed on
75 cm
dishes approaching confluence. Proteasomal inhibition was with
100 mM calpain inhibitor I and 10 mMN-carbobenzoxyl-L-leucinyl-L-norval-
inal. Transient transfections were by electroporation. Transfected cells were
split for parallel normoxic and hypoxic incubation (Napco 7001, Precision
Scientific). Luciferase reporter gene activity was corrected for transfection
efficiency by assay of b-galactosidase expression from the co-transfected
control plasmid pCMV–bGal.
RNA analysis. Total RNAwas extractedand analysed by ribonuclease protection.
Riboprobe details are given in Table S1 of the Supplementary Information.
Plasmid constructions. pCDNA3-VHL contained nucleotides 214–855 of
GenBank accession no. L15409in pcDNA3 (Invitrogen). pcDNA3-VHL.103FS
was made using site-directed mutagenesis to delete nucleotides 522–523. HRE
reporter genes were based on pGL3-basic (Promega) or pPUR (Clontech) and
contained either a minimal SV40 promoter or a minimal (40 bp) thymidine
kinase promoter linked to a firefly luciferase gene (details of HREs are in
Supplementary Information S3). pGalVP16 encoded the Gal4 DNA-binding
domain (amino acids 1– 147) linked in-frame to the activation domain (amino
acids 410–490) from herpes simplex virus protein 16; pGala344–698VP16
encoded the indicated amino acids of HIF-1abetween those domains. Plasmid
pUAS-tk-Luc contained two copies of the Gal4 binding site linked to a
thymidine-kinase-promoted luciferase reporter gene.
Cell lysis, immunoblotting and immunoprecipitation. Whole cell extracts
were prepared by homogenization in denaturing conditions and aliquots
immunoblotted for HIF a-subunits with 28b (anti-HIF-1a) and 190b (anti-
HIF-2a) as described16, or using clone 54 (anti-HIF-1a, Transduction Labora-
tories). For immunoprecipitation, lysis was performed in 100 mM NaCl, 0.5%
Igepal CA630, 20 mM Tris-HCl (pH 7.6), 5mM MgCl
and 1 mM sodium
orthovanadate with aprotinin (10 mgml
), ‘Complete’ protease inhibitor
(Boehringer) and 1.0 mM 4-(2-aminoethyl)benzene sulphonyl fluoride for
30 min on ice. After clearance by centrifugation, 120mg aliquots of lysate
were incubated for 2 h at 4 8C with 4 mg affinity-purified anti-HIF-2apoly-
clonal antibodies (raised against a bacterially expressed fusion protein includ-
ing amino acids 535– 631) or 4mg ammonium sulphate precipitated anti-HIF-
1apolyclonal antibodies (raised against an immunogen including amino acids
530–652) in parallel with normal rabbit immunoglobulin (control), or alter-
natively with 0.7 mg anti-pVHL antibody (IG32, Pharmingen) or control
(antibody to SV40 T antigen, pAb419, a gift from E. Harlow or antibody to
VEGF, VG-7be, a gift from H. Turley). 10 ml conjugated agarose beads pre-
blocked with 20 mg ml
BSA was added and lysates incubated for 2 h with
rocking. Pellets were washed five times, eluted with sample buffer, and divided
into 2– 6 aliquots for immunoblotting.
Electrophoretic mobility shift and supershift assays. We prepared nuclear
extracts using a modified Dignam protocol and incubated 5mg (HeLa) or
7.5 mg (RCC4) with a
P-labelled 24-bp oligonucleotide probe (sense strand;
59-GCCCTACGTGCTGCCTCGCATGGC-39) from the mouse Epo 39enhan-
cer as described25. For supershift assays, 0.5 mg IG32, VG-7be (isotype and
subclass matched control for IG32) or clone 54 (anti-HIF-1a) was added and
reactions were incubated for 4 h at 4 8C before electrophoresis. In vitro
transcription translations of pcDNA3-VHL and pcDNA3-VHL.103FS were
done using reticulocyte lysate (Promega); 1 ml of a 1:5 dilution in PBS was
added to binding reactions 2 h before electrophoresis or addition of antibody.
Received 11 March; accepted 13 April 1999.
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Supplementary information is available on Nature’s World-Wide Web site ( or
as paper copy from the London editorial office of Nature.
Acknowledgements. We thank W. Kaelin, C. Buys and M. Lerman for cell lines, and N. Proudfoot,
A. Harris, D. Gillespie, J. O’Rourke,Y.-M. Tian and L. Nicholls. Financial support was from the Wellcome
Trust, the Barnes Trust, the Deutsche Forschungsgemeinschaft, the Cancer Research Campaign, Action
Research and the Medical Research Council.
Correspondence and requests for materials should be addressedto P.J.R. (e-mail: peter.ratcliffe@imm.ox.
... 3 Normoxia, a family of iron-and 2-oxoglutarate-dependent dioxygenase HIF prolyl hydroxylases (HIF-PHDs, HIF-PHD1−3), 4 can hydroxylate certain prolines of HIF-α leading to the recognition of HIF-α by Von Hippel− Lindau protein (pVHL) and subsequent degradation via the ubiquitin-proteasome system. 5,6 Concurrently, factor inhibiting hypoxia-inducing factor (FIH), an asparaginyl hydroxylase, can prevent HIF-α from associating with the coactivator p300/CBP and thus results in a reduction of HIF-mediated transcription 7 ( Figure 1A). Among these HIF-related hydroxylases, HIF-PHD2 probably plays the dominant role in controlling HIF-α-mediated endogenous EPO expression. ...
... The inhibition activities against HIF-PHD2 of all the click products were tested by fluorescence polarization (FP) assay. 19 Triazole compound 6e, which was clicked by 4e and benzyl azide (5), reached the best activity of 853.8 nM. Subsequently, we fixed "head" fragment 4e and screened a series of azide "tail" fragments 12a−o, which included electronwithdrawing substituted benzyl azides, electron-donating substituted benzyl azides, and polysubstituted benzyl azides. ...
As a gene associated with anemia, the erythropoi-esis gene is physiologically expressed under hypoxia regulated by †hypoxia-inducing factor-α (HIF-α). Thus, stabilizing HIF-α is a potent strategy to stimulate the expression and secretion of erythropoiesis. In this study, we applied click chemistry to the discovery of HIF prolyl hydroxylase 2 (HIF-PHD2) inhibitors for the first time, and a series of triazole compounds showed preferable inhibitory activity in fluorescence polarization assays. Of particular note was the orally active HIF-PHD inhibitor 15i (IC 50 = 62.23 nM), which was almost ten times more active than the phase III drug FG-4592 (IC 50 = 591.4 nM). Furthermore, it can upregulate the hemoglobin of cisplatin-induced anemia mice (120 g/L) to normal levels (160 g/L) with no apparent toxicity observed in vivo. These results confirm that triazole compound 15i is a promising candidate for the treatment of renal anemia.
... The cell lines used in this study (RCC4, MDA-RCC-62) have been previously described [35,36]. All cells were routinely verified to be free of mycoplasma contamination using the MycoAlert bioluminescence mycoplasma detection kit according to manufacturer's recommended protocol (Lonza, LT07-118). ...
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Background Clear cell renal cell carcinoma (ccRCC), the predominant subtype of kidney cancer, possesses characteristic alterations to multiple metabolic pathways, including the accumulation of cytosolic lipid droplets. However, the pathways that drive lipid droplet accumulation in ccRCC cells and their importance to cancer biology remain poorly understood. Methods We sought to identify the carbon sources necessary for lipid droplet accumulation using Oil red O staining and isotope-tracing lipidomics. The role of the acyl-CoA synthetase (ACSL) family members, an important group of lipid metabolic enzymes, was investigated using siRNA and drug mediated inhibition. CTB and XTT assays were performed to determine the effect of ACSL3 knockdown and lipid starvation on ccRCC cell viability and shRNA was used to study the effect of ACSL3 in an orthotopic mouse model. The relationship between ferroptosis susceptibility of ccRCC and ACSL3 controlled lipid metabolism was examined using CTB and FACS-based assays. The importance of 5-LOX in ferroptosis susceptibility in ccRCC was shown with XTT survival assays, and the expression level and predictive value of 5-LOX in TCGA ccRCC data was assessed. Results We found that ccRCC cells obtain the necessary substrates for lipid droplet accumulation by metabolizing exogenous serum derived lipids and not through de novo lipogenesis. We show that this metabolism of exogenous fatty acids into lipid droplets requires the enzyme acyl-CoA synthetase 3 (ACSL3) and not other ACSL family proteins. Importantly, genetic or pharmacologic suppression of ACSL3 is cytotoxic to ccRCC cells in vitro and causes a reduction of tumor weight in an orthotopic mouse model. Conversely, ACSL3 inhibition decreases the susceptibility of ccRCC cells to ferroptosis, a non-apoptotic form of cell death involving lipid peroxidation. The sensitivity of ccRCC to ferroptosis is also highly dependent on the composition of exogenous fatty acids and on 5-lipoxygenase (5-LOX), a leukotriene producing enzyme which produces lipid peroxides that have been implicated in other cancers but not in ccRCC. Conclusions ACSL3 regulates the accumulation of lipid droplets in ccRCC and is essential for tumor growth. In addition, ACSL3 also modulates ferroptosis sensitivity in a manner dependent on the composition of exogenous fatty acids. Both functions of ACSL3 could be exploited for ccRCC therapy.
... In contrast, under hypoxic conditions, HIF1α is stabilized by limited oxygen as a helper substrate for prolyl hydroxylase domain enzymes (PHDs), and the HIF1α protein hydroxylation rate is reduced by enhancing the transcriptional activation of PHD, HIF1, and CBP-p 300 coactivated complexes. Then, HIFα levels increase, and HIF1α target genes are expressed [34,35]. Recent studies have shown that stimulating angiogenesis plays a key role in the process of increased bone mass while the HIF1α-VEGF pathway is activated [33]. ...
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Bone morphogenetic protein 9 (BMP9) as the most potent osteogenic molecule which initiates the differentiation of stem cells into the osteoblast lineage and regulates angiogenesis, remains unclear how BMP9-regulated angiogenic signaling is coupled to the osteogenic pathway. Hypoxia-inducible factor 1α (HIF1α) is critical for vascularization and osteogenic differentiation and the CBFA1, known as runt-related transcription factor 2 (Runx2) which plays a regulatory role in osteogenesis. This study investigated the combined effect of HIF1α and Runx2 on BMP9-induced osteogenic and angiogenic differentiation of the immortalized mouse embryonic fibroblasts (iMEFs). The effect of HIF1α and Runx2 on the osteogenic and angiogenic differentiation of iMEFs was evaluated. The relationship between HIF1α- and Runx2-mediated angiogenesis during BMP9-regulated osteogenic differentiation of iMEFs was evaluated by ChIP assays. We demonstrated that exogenous expression of HIF1α and Runx2 is coupled to potentiate BMP9-induced osteogenic and angiogenic differentiation both in vitro and animal model. Chromatin immunoprecipitation assays (ChIP) showed that Runx2 is a downstream target of HIF1α that regulates BMP9-mediated osteogenesis and angiogenic differentiation. Our findings reveal that HIF1α immediately regulates Runx2 and may originate an essential regulatory thread to harmonize osteogenic and angiogenic differentiation in iMEFs, and this coupling between HIF1α and Runx2 is essential for bone healing.
... In normoxia, HIF-1a activity is inhibited by the hydroxylation of proline residues within the oxygen-dependent domain of the HIF-1a subunit, which is accomplished by prolyl hydroxylase domain (PHD) proteins (Huang et al., 1998;Ivan et al., 2001;Jaakkola et al., 2001). Following hydroxylation, von Hippel Lindau protein (VHL) polyubiquitinates HIF-1a, targeting it for rapid proteasomal degradation (Wang et al., 1995;Salceda and Caro, 1997;Maxwell et al., 1999). Canonically, the HIF-1a subunit is stabilized in hypoxic environments through inhibition of PHD hydroxylation (Semenza and Wang, 1992;Kaelin and Ratcliffe, 2008). ...
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Osteomyelitis, or bone infection, is a major complication of accidental trauma or surgical procedures involving the musculoskeletal system. Staphylococcus aureus is the most frequently isolated pathogen in osteomyelitis and triggers significant bone loss. Hypoxia-inducible factor (HIF) signaling has been implicated in antibacterial immune responses as well as bone development and repair. In this study, the impact of bone cell HIF signaling on antibacterial responses and pathologic changes in bone architecture was explored using genetic models with knockout of either Hif1a or a negative regulator of HIF-1α, Vhl . Deletion of Hif1a in osteoblast-lineage cells via Osx-Cre ( Hif1a ΔOB ) had no impact on bacterial clearance or pathologic changes in bone architecture in a model of post-traumatic osteomyelitis. Knockout of Vhl in osteoblast-lineage cells via Osx-Cre ( Vhl ΔOB ) caused expected increases in trabecular bone volume per total volume (BV/TV) at baseline and, intriguingly, did not exhibit an infection-mediated decline in trabecular BV/TV, unlike control mice. Despite this phenotype, bacterial burdens were not affected by loss of Vhl . In vitro studies demonstrated that transcriptional regulation of the osteoclastogenic cytokine receptor activator of NF-κB ligand (RANKL) and its inhibitor osteoprotegerin (OPG) is altered in osteoblast-lineage cells with knockout of Vhl . After observing no impact on bacterial clearance with osteoblast-lineage conditional knockouts, a LysM-Cre model was used to generate Hif1a ΔMyeloid and Vhl ΔMyeloid mouse models to explore the impact of myeloid cell HIF signaling. In both Hif1a ΔMyeloid and Vhl ΔMyeloid models, bacterial clearance was not impacted. Moreover, minimal impacts on bone architecture were observed. Thus, skeletal HIF signaling was not found to impact bacterial clearance in our mouse model of post-traumatic osteomyelitis, but Vhl deletion in the osteoblast lineage was found to limit infection-mediated trabecular bone loss, possibly via altered regulation of RANKL-OPG gene transcription.
... pVHL is an inhibitor of HIF-1 which promotes the degradation of HIF-1α protein by the proteasome [32,33]. We noticed that the expression of pVHL was reduced under the TSD serum group. ...
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Background: Vascular damage is a major consequence of bone fracture. Taohong Siwu decoction (TSD) can raise the expression of vascular endothelial growth factor (VEGF) in fracture healing. However, its molecular mechanism in promoting angiogenesis is still unknown. The aim of this study was to investigate the potential mechanisms of TSD in the regulation of osteo-angiogenesis in fracture healing. Methods: A rat tibial fracture model was established. After low- (4.5 g·kg-1), medium- (9 g·kg-1), and high-dose TSD (18 g·kg-1) and panax notoginsenoside (25 mg kg-1) treatment, hematoxylin-eosin staining was employed to visualize pathological changes in bone tissues. The levels of cytokines (interleukin (IL)-2, tumor necrosis factor-α (TNF-α), IL-6, and IL-1β), thromboxane B2 (TXB2), and 6 ketone prostaglandin F1α (6-Keto-PGF1α) were quantified by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence was used to identify the rat aortic endothelial cells (RAECs). Control serum, 10% TSD-containing serum, and 10% TSD-containing serum combined with hypoxia-inducible factor-1α (HIF-1α) inhibitor were used to treat the RAECs and rat osteoblasts. Transwell migration assay was utilized to examine the migration of the RAECs. The Matrigel tubulogenesis assay was used for the assessment of angiogenesis. The expression of angiogenesis- (von Hippel-Lindau tumor suppressor (VHL), HIF-1α, VEGF, angiopoietin-2 (Ang-2), and pVHL) and osteogenesis-related (alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteopontin-1 (OPN-1)) protein and gene was detected by western blot and quantitative real-time PCR (qRT-PCR). Results: Compared with the model group, TSD increased the trabecular bone areas, numbers, and thicknesses in fractured rats. In the plasma, the levels of cytokines and TXB2 in the middle- and high-dose TSD group were significantly lower than those in the model group (P < 0.01). The 6-keto-PGF1α content was increased by middle- and high-dose TSD intervention (P < 0.01). Compared to the control serum group, the angiogenesis and migration of the RAECs were enhanced in the TSD group (P < 0.001). The expression of HIF-1α, VEGF, and Ang-2 in the TSD group upregulated significantly (P < 0.001). VHL and pVHL were inhibited under TSD-containing serum treatment (P < 0.001). ALP, Runx2, and OPN-1 were increased obviously in the TSD group (P < 0.001). Nevertheless, the HIF-1α inhibitor reversed these changes (P < 0.001). Conclusion: TSD promotes angiogenesis and osteogenesis by regulating the HIF-1α signaling pathway. Meanwhile, it can effectively reduce the risk of inflammation and improve blood circulation.
... ------ In particular, binding of HIF restricts the range of the binding affinity of HP4H for molecular oxygen significantly, ∆ ∆ 100 45% (44) Here, the set of conformers of HP4H once bound to HIF ensures that the catalytic activity of HP4H for HIF is significantly reduced in the presence of hypoxia. This will extend the half-life of HIF and facilitate transcription of HIF-responsive genes [36,37]. In contrast, CP4H exhibits no such differential activity. ...
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A ligand when bound to a macromolecule (protein, DNA, RNA) will influence the biochemical function of that macromolecule. This observation is empirical and attributable to the association of the ligand with the amino acids/nucleotides that comprise the macromolecule. The binding affinity is a measure of the strength-of-association of a macromolecule for its ligand and is numerically characterized by the association/dissociation constant. However, despite being widely used, a mathematically rigorous explanation by which the association/dissociation constant can influence the biochemistry and molecular biology of the resulting complex is not available. Here, the ligand-macromolecular complex is modeled as a homo- or hetero-dimer with a finite and equal number of atoms/residues per monomer. The pairwise interactions are numeric, empirically motivated and are randomly chosen from a standard uniform distribution. The transition-state dissociation constants are the strictly positive real part of all complex eigenvalues of this interaction matrix, belong to the open interval $(0, 1)$, and form a sequence whose terms are finite, monotonic, non-increasing and convergent. The theoretical results are rigorous, presented as theorems, lemmas and corollaries and are complemented by numerical studies. An inferential analysis of the clinical outcomes of amino acid substitutions of selected enzyme homodimers is also presented. These findings are extendible to higher-order complexes such as those likely to occur in vivo. The study also presents a schema by which a ligand can be annotated and partitioned into high- and low-affinity variants. The influence of the transition-state dissociation constants on the biochemistry and molecular biology of non-haem iron (Ⅱ)- and 2-oxoglutarate-dependent dioxygenases (catalysis) and major histocompatibility complex (Ⅰ) mediated export of high-affinity peptides (non-enzymatic association/dissociation) are examined as special cases.
Targeted protein degradation using proteolysis-targeting chimeras (PROTACs) has emerged as an effective strategy for drug discovery, given their unique advantages over target protein inhibition. The bromodomain and extra-terminal (BET) family proteins play a key role in regulating oncogene expression and are considered attractive therapeutic targets for cancer therapy. Considering the therapeutic potential of BET proteins in cancer and the marked attractiveness of PROTACs, BET-targeting PROTACs have been extensively pursued. Recently, BET-targeting PROTACs based on new E3 ligases and novel strategies, such as light-activated, macrocyclic, folate-caged, aptamer-PROTAC conjugation, antibody-coupling, and autophagy-targeting strategies, have emerged. In the present review, we provide a comprehensive summary of advances in BET-targeting PROTACs.
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Breast cancer is a kind of malignant tumor disease that poses a serious threat to human health. Its biological characteristics of rapid proliferation and delayed angiogenesis, lead to intratumoral hypoxia as a common finding in breast cancer. HIF as a transcription factor, mediate a series of reactions in the hypoxic microenvironment, including metabolic reprogramming, tumor angiogenesis, tumor cell proliferation and metastasis and other important physiological and pathological processes, as well as gene instability under hypoxia. In addition, in the immune microenvironment of hypoxia, both innate and acquired immunity of tumor cells undergo subtle changes to support tumor and inhibit immune activity. Thus, the elucidation of tumor microenvironment hypoxia provides a promising target for the resistance and limited efficacy of current breast cancer therapies. We also summarize the hypoxic mechanisms of breast cancer treatment related drug resistance, as well as the current status and prospects of latest related drugs targeted HIF inhibitors.
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Gastric and gastro-esophageal junction adenocarcinoma (GEA) remains a considerable major public health problem worldwide, being the fifth most common cancer with a fatality-to-case ratio that stands still at 70%. Angiogenesis, which is a well-established cancer hallmark, exerts a fundamental role in cancer initiation and progression and its targeting has been actively pursued as a promising therapeutic strategy in GEA. A wealth of clinical trials has been conducted, investigating anti-angiogenic agents including VEGF-directed monoclonal antibodies, small molecules tyrosine kinase inhibitors and VEGF-Trap agents both in the resectable and advanced setting, reporting controversial results. While phase III randomized trials testing the anti-VEGFR-2 antibody Ramucirumab and the selective VEGFR-2 tyrosine kinase inhibitor Apatinib demonstrated a significant survival benefit in later lines, the shift of angiogenesis inhibitors in the perioperative and first-line setting failed to improve patients’ outcome in GEAs. The molecular landscape of disease, together with novel combinatorial strategies and biomarker-selected approaches are under investigation as key elements to the success of angiogenesis blockade in GEA. In this article, we critically review the existing literature on the biological rationale and clinical development of antiangiogenic agents in GEA, discussing major achievements, limitations and future developments, aiming at fully realizing the potential of this therapeutic approach.
Background: The von Hippel-Lindau (VHL) gene has two translational initiation sites separated by 53 codons. Both proteins have been detected in cells and have equivalent activity. A mutation in the first 53 codons of the open reading frame has no effect on the structure of the smaller protein. As expected, the vast majority of VHL mutations are downstream of the second initiation site and alter both proteins. However, several candidate mutations have been found in the first 53 codons, including a substitution of leucine for proline at position 25 (P25L) of the larger protein. Methods and Results: DNA sequence analysis showed two VHL gene mutations, P25L and P86R, in an individual with a clinical diagnosis of VHL disease. Both mutations have been reported previously. P25L alters only the upstream protein, whereas P86R alters both VHL proteins. Based on the positions of the mutations, P86R is more likely to be pathogenically significant than the P25L mutation. A survey of anonymized DNAs for P25L, using allele-specific PCR, revealed that it is a variant with an allele frequency of approximately 0.5%. Conclusion: P25L is a rare variant of the VHL gene and cannot be considered a cause of VHL disease. However, this work does not prove that P25L is entirely innocuous.
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The ability of iron to catalyze formation of reactive oxygen species significantly contributes to its toxicity in cells and animals. Iron uptake and distribution is regulated tightly in mammalian cells, in part by iron regulatory protein 2 (IRP2), a protein that is degraded efficiently by the proteasome in iron-replete cells. Here, we demonstrate that IRP2 is oxidized and ubiquitinated in cells before degradation. Moreover, iron-dependent oxidation converts IRP2 into a substrate for ubiquitination in vitro. A regulatory pathway is described in which excess iron is sensed by its ability to catalyze site-specific oxidations in IRP2, oxidized IRP2 is ubiquitinated, and ubiquitinated IRP2 subsequently is degraded by the proteasome. Selective targeting and removal of oxidatively modified proteins may contribute to the turnover of many proteins that are degraded by the proteasome.
Hypoxia results in adaptive changes in the transcription of a range of genes including erythropoietin. An important mediator is hypoxia-inducible factor-1 (HIF-1), a DNA binding complex shown to contain at least two basic helix-loop-helix PAS-domain (bHLH-PAS) proteins, HIF-1α and aryl hydrocarbon nuclear receptor translocator (ARNT). In response to hypoxia, HIF-1α is activated and accumulates rapidly in the cell. Endothelial PAS domain protein 1 (EPAS-1) is a recently identified bHLH-PAS protein with 48% identity to HIF-1α, raising the question of its role in responses to hypoxia. We developed specific antibodies and studied expression and regulation of EPAS-1 mRNA and protein across a range of human cell lines. EPAS-1 was widely expressed, and strongly induced by hypoxia at the level of protein but not mRNA. Comparison of the effect of a range of activating and inhibitory stimuli showed striking similarities in the EPAS-1 and HIF-1α responses. Although major differences were observed in the abundance of EPAS-1 and HIF-1α in different cell types, differences in the inducible response were subtle with EPAS-1 protein being slightly more evident in normoxic and mildly hypoxic cells. Functional studies in a mutant cell line (Ka13) expressing neither HIF-1α nor EPAS-1 confirmed that both proteins interact with hypoxically responsive targets, but suggest target specificity with greater EPAS-1 transactivation (relative to HIF-1α transactivation) of the VEGF promoter than the LDH-A promoter.
Erythropoietin (EPO) gene transcription is activated in kidney cells in vivo and in Hep3B cells exposed to hypoxia or cobalt chloride. Hypoxia- inducible factor 1 (HIF-1) is a nuclear factor that binds to the hypoxia-inducible enhancer of the EPO gene at a site that is required for transcriptional activation. HIF-1 DNA-binding activity is induced by hypoxia or cobalt chloride treatment of Hep3B cells. We report that treatment of Hep3B cells with desferrioxamine (DFX) induced HIF-1 activity and EPO RNA expression with kinetics similar to the induction of HIF-1 by hypoxia or cobalt chloride. Induction by each of these stimuli was inhibited by cycloheximide, indicating a requirement for de novo protein synthesis. DFX appears to induce HIF-1 by chelating iron as induction was inhibited by coadministration of ferrous ammonium sulfate. DFX administration to mice transiently increased EPO RNA levels in the kidney. As previously shown for hypoxia and cobalt treatment, DFX also induced HIF-1 activity in non-EPO-producing cells, suggesting the existence of a common hypoxia signal-transduction pathway leading to HIF-1 induction in different cell types.
This review focuses on the molecular stratagems utilized by bacteria, yeast, and mammals in their adaptation to hypoxia. Among this broad range of organisms, changes in oxygen tension appear to be sensed by heme proteins, with subsequent transfer of electrons along a signal transduction pathway which may depend on reactive oxygen species. These heme-based sensors are generally two-domain proteins. Some are hemokinases, while others are flavohemoproteins [flavohemoglobins and NAD(P)H oxidases]. Hypoxia-dependent kinase activation of transcription factors in nitrogen-fixing bacteria bears a striking analogy to the phosphorylation of hypoxia inducible factor-1 (HIF-1) in mammalian cells. Moreover, redox chemistry appears to play a critical role both in the trans-activation of oxygen-responsive genes in unicellular organisms as well as in the activation of HIF-1. In yeast and bacteria, regulatory operons coordinate expression of genes responsible for adaptive responses to hypoxia and hyperoxia. Similarly, in mammals, combinatorial interactions of HIF-1 with other identified transcription factors are required for the hypoxic induction of physiologically important genes.
Hypoxia results in adaptive changes in the transcription of a range of genes including erythropoietin. An important mediator is hypoxia-inducible factor-1 (HIF-1), a DNA binding complex shown to contain at least two basic helix-loop-helix PAS-domain (bHLH-PAS) proteins, HIF-1 alpha and aryl hydrocarbon nuclear receptor translocator (ARNT), In response to hypoxia, HIF-1 alpha is activated and accumulates rapidly in the cell. Endothelial PAS domain protein 1 (EPAS-1) is a recently identified bHLH-PAS protein with 48% identity to HIF-1 alpha, raising the question of its role in responses to hypoxia. We developed specific antibodies and studied expression and regulation of EPAS-1 mRNA and protein across a range of human cell lines. EPAS-1 was widely expressed, and strongly induced by hypoxia at the level of protein but not mRNA. Comparison of the effect of a range of activating and inhibitory stimuli showed striking similarities in the EPAS-1 and HIF-1 alpha responses. Although major differences were observed in the abundance of EPAS-1 and HIF-1 alpha in different cell types, differences in the inducible response were subtle with EPAS-1 protein being slightly more evident in normoxic and mildly hypoxic cells. Functional studies in a mutant cell line (Ka13) expressing neither HIF-1 alpha nor EPAS-1 confirmed that both proteins interact with hypoxically responsive targets, but suggest target specificity with greater EPAS-1 transactivation (relative to HIF-1 alpha transactivation) of the VEGF promoter than the LDH-A promoter. (C) 1998 by The American Society of Hematology.
The hypoxia-inducible factor 1 transcriptional activator complex (HIF-1) is involved in the activation of the erythropoietin and several other hypoxia-responsive genes. The HIF-1 complex is composed of two protein subunits: HIF-1β/ARNT (aryl hydrocarbon receptor nuclear translocator), which is constitutively expressed, and HIF-1α, which is not present in normal cells but induced under hypoxic conditions. The HIF-1α subunit is continuously synthesized and degraded under normoxic conditions, while it accumulates rapidly following exposure to low oxygen tensions. The involvement of the ubiquitin-proteasome system in the proteolytic destruction of HIF-1 in normoxia was studied by the use of specific inhibitors of the proteasome system. Lactacystin and MG-132 were found to protect the degradation of the HIF-1 complex in cells transferred from hypoxia to normoxia. The same inhibitors were able to induce HIF-1 complex formation when added to normoxic cells. Final confirmation of the involvement of the ubiquitin-proteasome system in the regulated degradation of HIF-1α was obtained by the use ofts20TG R cells, which contain a temperature-sensitive mutant of E1, the ubiquitin-activating enzyme. Exposure of ts20 cells, under normoxic conditions, to the non-permissive temperature induced a rapid and progressive accumulation of HIF-1. The effect of proteasome inhibitors on the normoxic induction of HIF-1 binding activity was mimicked by the thiol reducing agentN-(2-mercaptopropionyl)-glycine and by the oxygen radical scavenger 2-acetamidoacrylic acid. Furthermore,N-(2-mercaptopropionyl)-glycine induced gene expression as measured by the stimulation of a HIF-1-luciferase expression vector and by the induction of erythropoietin mRNA in normoxic Hep 3B cells. These last findings strongly suggest that the hypoxia induced changes in HIF-1α stability and subsequent gene activation are mediated by redox-induced changes.
Hypoxia-inducible expression has been demonstrated for many groups of mammalian genes, and studies of transcriptional control have revealed the existence of hypoxia-responsive elements (HREs) in the cis-acting sequences of several of these genes. These sequences generally contain one or more binding sites for a heterodimeric DNA binding complex termed hypoxia-inducible factor-1 (HIF-1). To analyze this response further, Chinese hamster ovary cells were stably transfected with plasmids bearing HREs linked to genes encoding immunoselectable cell surface markers, and clones that showed reduced or absent hypoxia-inducible marker expression were selected from a mutagenized culture of cells. Analysis of these cells revealed several clones with transacting defects in HRE activation, and in one the defect was identified as a failure to express the α-subunit of HIF-1. Comparison of hypoxia-inducible gene expression in wild type, HIF-1α-defective, and HIF-1α-complemented cells revealed two types of response. For some genes (e.g. glucose transporter-1), hypoxia-inducible expression was critically dependent on HIF-1α, whereas for other genes (e.g. heme oxygenase-1) hypoxia-inducible expression appeared largely independent of the expression of HIF-1α. These experiments show the utility of mutagenesis and selection of mutant cells in the analysis of mammalian transcriptional responses to hypoxia and demonstrate the operation of HIF-1α-dependent and HIF-1α-independent pathways of hypoxia-inducible gene expression in Chinese hamster ovary cells.