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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2260-2268
Induction of Endothelial PAS Domain Protein-1 by Hypoxia:
Characterization and Comparison With Hypoxia-Inducible Factor-1
By
M.S. Wiesener,
H. Turley,
W.E. Allen,
C. Willam,
K.-U. Eckardt,
K.L. Talks,
S.M. Wood,
K.C. Gatter,
A.L. Harris,
C.W. Pugh,
P.J. Ratcliffe, and
P.H. Maxwell
From the Institute of Molecular Medicine and the Department of
Cellular Science, John Radcliffe Hospital, Oxford, UK; and Klinikum der
Charité, Humboldt Universtät, Berlin, Germany.
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ABSTRACT |
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 and aryl hydrocarbon nuclear receptor translocator (ARNT). In response to hypoxia, HIF-1 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,
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 responses. Although major differences were observed in the
abundance of EPAS-1 and HIF-1 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 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 transactivation) of the VEGF
promoter than the LDH-A promoter.
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INTRODUCTION |
OXYGEN AVAILABILITY is increasingly
recognized as a major modulator of gene expression.1
Studies of the hematopoietic growth factor erythropoietin have led to
the definition of a widely operative system of gene regulation by
oxygen2 that is dependent on activation of a
transcriptional complex termed hypoxia inducible factor-1 (HIF-1).3 Affinity purification of HIF-1 from Hela cell
nuclear extract led to the definition of the DNA binding complex as a heterodimer of two basic-helix-loop-helix PAS domain (bHLH-PAS) proteins, HIF-1 and a previously identified molecule termed the aryl
hydrocarbon nuclear receptor translocator (ARNT).4 HIF-1 plays an important role not only in the regulation of erythropoietin but also in the hypoxia-inducible expression of many other genes with
diverse functions in relation to the biology of oxygen. These include
glucose transporters, glycolytic enzymes, vascular growth factors, and
nitric oxide synthases.5 Studies of HIF-1 regulation have
indicated that one of the major activation mechanisms involves rapid
nuclear accumulation of the subunit.4,6 Thus, when cells are exposed to an atmosphere of 1% hypoxia striking nuclear accumulation of HIF-1 occurs over a period of minutes to hours, whereas ARNT is present constitutively and increased only modestly (or
not at all) with hypoxic stimulation.6 Current evidence indicates that in normoxic cells HIF-1 is targeted for rapid degradation by the proteasome through the operation of specific domains
in the molecule,6-9 and that accumulation in hypoxia involves a reduction in this degradation.
Recent cloning experiments have greatly expanded the number of
recognized bHLH-PAS proteins,10 raising an important
question as to the possible involvement of other members of this family in the response to hypoxia. Among these a molecule first termed endothelial PAS protein-1 (EPAS-1) shows the closest sequence homology
to HIF-1 (48% identity).11 This molecule was also independently identified and reported as HIF-like factor
(HLF),12 member of PAS super-family 2 (MOP2),13
and HIF-related factor (HRF).14 Although these reports have
shown that EPAS-1, like HIF-1, can dimerize with ARNT and activate
transcription from similar DNA recognition sites, the regulatory
characteristics of the EPAS-1 molecule are not yet clearly defined. In
this report we have used specific antibodies to define and compare the
distribution and regulation of EPAS-1 and HIF-1 in a wide range of
tissue culture cells. We report that both molecules are expressed
widely, although at greatly differing levels within this panel of
cells. Both molecules show high-level induction by hypoxia at the level of protein but not mRNA. Their responses to a variety of
pharmacological agents were also similar, strongly suggesting that they
respond to a similar or identical sensing and signal transduction
system.
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MATERIALS AND METHODS |
Cell culture.
Cell lines were cultured as recommended by the European Collection of
Cell Cultures. The CHO-K1 derivative Ka13 and the endothelial line
HMEC-1 have been described previously.15,16 Cells were plated onto 75-cm2 or 175-cm2 flasks 24 hours
before experiments such that they were approaching confluence. Culture
medium was replaced at the start of each experiment, and when short
hypoxic exposures (<2 hours) were studied this medium was
pre-equilibrated in the hypoxic incubator. Hypoxic exposure was in a
NAPCO 7001 incubator (Precision Scientific, Chicago, IL)
with 1% oxygen, 6% CO2, balance nitrogen. Exposure to
experimental conditions was for 4 hours unless indicated otherwise. The
effect of graded hypoxia was examined in cells cultured on Petriperm
dishes (Heraeus, South Plainfield, NJ) in a set of five purpose-built gas-tight chambers, with continous monitoring of oxygen
tension in each chamber. Culture media and chemicals were from Sigma
(Poole, UK). Diphenylene iodonium chloride was from Calbiochem (Cambridge, MA).
RNA analysis.
Total RNA was extracted using RNAzol B (Biogenesis, Poole,
UK). Ribonuclease protection assays were performed
essentially as described previously,2 with parallel
hybridization using 40 µg for HIF-1, 40 µg for EPAS-1, and 1 µg for U6 small nuclear RNA. 32P-labeled riboprobes were
generated using SP6 or T7 RNA polymerase. The templates used yielded
protected fragments as follows: 221 bp for EPAS-1 (nucleotides 2542 to
2762, accession no. U81984), 255 bp for HIF-1 (nucleotides 764 to
1018, U22431), and 106 bp for U6 (nucleotides 1 to 107, X01366). After
resolution on 8% polyacrylamide gels, quantification was performed
using a PhosphorImager (Molecular Dynamics, Sunnyvale,
CA). Signals for HIF-1 mRNA and EPAS-1 mRNA were
normalized to a value of 100 for EPAS-1 in Hep3B cells, allowing for
the different number of labeled nucleotides in the two protected
fragments.
Bacterial protein expression and immunization.
An Nco 1 restriction fragment encoding amino acids 535 to 631 of human EPAS-1 was cloned in frame into pGEX-4t-1 (Pharmacia Biotech,
St Albans, UK) with a modified polylinker. Expression of
glutathione-S-transferase (GST) fusion protein was induced by exposure
of transformed Escherichia coli DH5 cells to 0.1 mmol/L
isopropyl b-D-thiogalactopyranoside. After bacterial lysis, protein was
affinity purified with glutathione Sepharose 4B (Pharmacia). To
generate polyclonal antisera to EPAS-1, two rabbits were immunized with
200 µg protein in Complete Freund's Adjuvant (Difco, Detroit, MI), followed by six immunizations at 14-day intervals
with 100 µg protein in Incomplete Freund's Adjuvant. To generate
monoclonal antibodies (MoAbs), Balb/c mice were immunized with 50 µg
protein in Complete Freund's Adjuvant followed by three further
immunizations of 50 µg protein in phosphate-buffered saline (PBS) at
10-day intervals. Fusion of mouse splenocytes with myeloma cells was performed using standard techniques 4 days after the last immunization. Hybridoma supernatants were screened by enzyme-linked immunosorbent assay (ELISA) against the GST fusion protein and GST alone, and immunolabelling of COS-1 cells transfected with the human EPAS-1 expression plasmid, phEP1.11 Five MoAbs that reacted with
EPAS-1 were obtained.
Protein extraction and immunoblot analysis.
For whole-cell extracts, adherent cells were washed with ice-cold PBS
and removed by scraping. Cell pellets were homogenized in extraction
buffer (7 mol/L urea/10% glycerol/10 mmol/L Tris-HCl pH 6.8/1% sodium
dodecyl sulfate [SDS]/5 mmol/L dithiothreitol [DTT]/0.5 mmol/L
phenylmethyl sulfonyl fluoride [PMSF] with 1 mg/L aprotinin,
pepstatin, and leupeptin) using an IKA Ultra-Turrax T8 homogenizer
(Janke & Kunkel, Staufen, Germany) for 5 seconds at full
speed. Extracts were quantified using the BioRad DC protein assay
(BioRad, Hemel Hempstead, UK). For differential nuclear and cytoplasmic extraction a modifed Dignam protocol was
used.3 For immunoblotting, proteins were resolved in
SDS/6% polyacrylamide gels and transferred to Immobilon P (Millipore,
Bedford, MA) overnight in 10 mmol/L Tris/100 mmol/L
glycine/10%methanol/0.05% SDS. Membranes were blocked with PBS/5%
fat-free dried milk/0.1% Tween 20. For HIF-1 detection, MoAb 28b
was used at 4 µg/mL. This antibody was from a fusion following
immunization with a GST fusion protein including amino acids 329 to 530 of human HIF-1. For EPAS-1, 190b supernatant was diluted 1:4. For
detection of the Gal4 DNA binding domain in fusion proteins, RK5C1
(Santa Cruz Biotechnology, Santa Cruz, CA) was used at
0.1 µg/mL. Detection was with horseradish peroxidase (HRP)-conjugated
goat anti-mouse Igs (DAKO, Ely, UK) at 1:2,000 and
enhanced chemiluminescence (SuperSignal Ultra; Pierce & Warriner,
Chester, UK). After analysis, membranes were stained with
Ponceau S to verify equal protein loading and transfer. For studies of
antibody specificity and relative sensitivity, protein extracts
containing a high level of EPAS-1 or HIF1 fused to a portion of the
yeast transcription factor Gal4 were obtained by electroporating COS-1
cells with 10 µg pGN/EPAS19-870 or pGN/HIF128-826. These
constructs were based on pcDNA3 (Invitrogen, Carlsbad,
CA) and contained an SV40 origin of replication, the
cytomegalovirus (CMV) promoter, and a bovine growth hormone poly A
signal resulting in expression of in-frame fusions of sequence encoding
amino acids 1-147 of Gal4 and polymerase chain reaction (PCR) products
encoding the specified amino acids of EPAS-1 or HIF-1.
Transient transfections and reporter gene assays.
For functional studies of EPAS-1 and HIF-1, CHO Ka13 cells were
cotransfected with 0.25 µg of expression plasmids for HIF-1 (pcDNA3/Neo/HIF-115) or EPAS-1 (phEP-111) or
pcDNA3 without insert, 3 µg reporter plasmid, and 2 µg
pCMV Gal15 (as a control for transfection efficiency).All
reporter plasmids were based on pGL3 (Promega, Southampton,
UK) but included different promoters as follows. p(24x6)TKLuc contained 6 copies of a 24-bp oligonucleotide (P24) from
the mouse phosphoglycerate kinase-1 5 enhancer,17
placed 10 bp 5 to the TATA box of the herpes simplex thymidine
kinase promoter. pLDH-ALuc contained 233 bp from the mouse LDH-A
promoter, extending from  186 to +47 relative to the
transcriptional start site. pVEGFLuc contained a 1,786-bp BamH1
fragment from the human VEGF promoter, extending from 1288 to
+480. Transfections were performed using DEAE/Dextran in 5-cm dishes
with cells at 70% confluence. After transfection, dishes were
incubated overnight at 37°C in complete medium, and the following
day cells were trypsinized and divided into two 3.5-cm wells on
separate plates for parallel 18-hour incubation in normoxia and
hypoxia. Luciferase activities in cell lysates were determined using a
commercially available luciferase assay system (Promega) and a TD-20e
luminometer (Turner Designs, Sunnyvale, CA). Relative
-galactosidase activity in lysates was measured using
o-nitrophenyl--D-galactopyranoside (0.67 mg/mL) as substrate in a
0.1 mol/L phosphate buffer (pH 7.0) containing 10 mmol/L KCl, 1 mmol/L
MgSO4, and 30 mmol/L -mercaptoethanol for 45 to 90 minutes. The OD420 was determined after stopping the
reaction by the addition of 1 mol/L sodium carbonate.
Immunohistochemistry.
Cells were grown on lysine-coated glass slides (Polysine; BDH, Poole,
UK). After washing in ice-cold PBS, cells were fixed in
methanol at 20°C for 10 minutes and air dried. Slides were incubated with 28b or 190b hybridoma supernatant, followed by HRP-conjugated swine anti-rabbit Igs (DAKO). Detection was with 33diaminobenzidine.
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RESULTS |
Distribution of EPAS-1 and HIF-1 mRNAs in tissue culture cells.
As a first step in this analysis, total RNA from cell lines derived
from diverse human tissues was examined for EPAS-1 gene expression
using RNAse protection (Fig 1 and
Table 1). EPAS-1 mRNA was detected not only in endothelial
cells, but also in fibroblast and epithelial cell lines. The abundance
of EPAS-1 mRNA varied over a wide range (>100-fold) between cell
lines, with cell lines with the highest levels of EPAS-1 mRNA being
HMEC-1 (an endothelial capillary cell line) and MRC5 (fetal lung
fibroblast). In contrast, a line of Epstein-Barr virus-transformed
lymphocytes (RPMI 1788) showed the lowest level of expression among the
cell lines examined, with signal near the limit of detection by RNAse
protection assay. The level of EPAS-1 mRNA was determined both under
normoxic conditions and after 4 hours of exposure to 1% oxygen, and
was not altered by hypoxia in any cell line (Fig 1). Interestingly, two
sublines of HeLa cells showed nearly a threefold difference in the
level of EPAS-1 mRNA. HeLa(A) was a certified line obtained from the European Collection of Animal Cultures. PCR genotyping of both sublines
using six highly polymorphic markers confirmed that the second subline,
HeLa(B), were indeed HeLa cells.
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| Fig 1.
Expression of EPAS-1 and HIF-1 in six human cell
lines. Cells were cultured in parallel for 4 hours in normoxia (upper
three panels) and 1% hypoxia (lower panel). Ribonuclease protection
analysis of total RNA was performed for EPAS-1 and HIF-1 (40 µg
each), and U6 small nuclear RNA (1 µg).
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To compare EPAS-1 and HIF-1 expression we also determined the level
of HIF-1 mRNA in each cell line. The level of HIF-1 mRNA
expression was less variable than that of EPAS-1 mRNA, ranging approximately 20-fold from the lowest expressing lines
(293 cells and Hep3B cells) to the highest expressing lines (MRC5 and
HT1080). HIF-1 mRNA was less abundant than EPAS-1 mRNA in 6 of the
11 cell lines examined (Table 1). We also examined the levels of HIF-1 mRNA after 4 hours of hypoxic exposure and again did not observe modulation in any cell line (data not shown).
Characterization of antibodies reactive against EPAS-1 and HIF-1.
To permit analysis of EPAS-1 regulation at the protein level, and allow
comparison with HIF-1, antibodies were raised against recombinant
immunogens (see Materials and Methods). To detect any cross-reactivity
and determine the approximate sensitivity of these reagents in
immunoblotting procedures, COS-1 cells were transfected with plasmids
expressing chimeric genes in which near full-length cDNAs for EPAS-1 or
HIF-1 were linked to an N-terminal Gal4 DNA binding domain. A total
of five MoAbs and two polyclonal antisera raised against amino acids
535-631 of human EPAS-1 were tested. All seven gave a strong signal
against the Gal4/EPAS-1 fusion protein, but not the Gal4/HIF-1
fusion (data not shown). One MoAb (MoAb 190b) was selected for the
majority of further experiments described
(Fig 2). For detection of HIF-1, MoAb
28b, raised against an immunogen containing amino acids 329-530 of human HIF-1, was tested similarly. This antibody reacted with the
Gal4/HIF-1 fusion protein but not Gal4/EPAS-1 in the transfected COS-1 cells (Fig 2). Using an MoAb to Gal4 protein, the quantity of
extract from each COS-1 cell transfection was adjusted so that equal
amounts of HIF-1 and EPAS-1 fusion proteins were loaded for
detection by either MoAb 28b or MoAb 190b. Under these conditions detection of HIF-1 with MoAb 28b gave approximately fourfold less
signal than detection of EPAS-1 with MoAb 190b
(Fig 2). Detection of the
relevant Gal4 fusion proteins was substantially less sensitive with the
MoAb to Gal4 than with either MoAb 28b or MoAb 190b.
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| Fig 2.
Antibody specificity and relative sensitivity of MoAb
190b (EPAS-1) and MoAb 28b (HIF-1). Immunoblots of whole cell
extracts from COS-1 cells transfected with either pGN/EPAS19-870 or
pGN/HIF-128-826, resulting in expression of fusion proteins with the
N-terminal Gal4 DNA binding domain. Aliquots of the EPAS-1/GAL and
HIF-1/GAL extracts were analyzed in parallel using antibodies to
Gal4 (RK5C1), EPAS-1 (190b), and HIF-1 (28b). After initial analysis
with the Gal4 MoAb (not shown), the amount of each extract loaded was
adjusted to give approximately equal signal with this antibody (left)
indicating similar amounts of EPAS-1 and HIF-1 fusion proteins. The
antibodies to EPAS-1 (middle) and HIF-1 (right) specifically
recognized the appropriate fusion protein. Film exposure times are
shown below each panel, indicating differences in the sensitivity of
detection. The positions of MW markers are shown on the left.
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| Fig 3.
Expression of normoxic and hypoxic EPAS-1 and HIF-1
protein in six human cell lines. Cells were cultured in parallel in
normoxia (N) or 1% oxygen (H) for 4 hours. Whole-cell extracts (50 µg) were analyzed for EPAS-1 (MoAb 190b, top) and HIF-1 (MoAb 28b,
bottom). Exposure time was 30 seconds for EPAS-1 and 120 seconds for
HIF-1, so that sensitivity of detection for the two proteins is
approximately equal. The HeLa(B) hypoxic extract was independently
shown to contain approximately equal amounts of EPAS-1 and HIF-1 by
direct comparison with transfected COS-1 cell extracts.
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EPAS-1 and HIF-1 protein levels in normoxic and hypoxic tissue
culture cells.
Previous studies of nuclear and whole cell extracts have shown striking
increases in HIF-1 protein levels after hypoxic
stimulation.4,6 To determine if EPAS-1 protein levels are
induced by hypoxia and to compare the responses with those of HIF-1
across the range of cell types, whole-cell extracts were prepared
following parallel normoxic and hypoxic incubation for 4 hours. EPAS-1
and HIF-1 were detected by immunoblotting using MoAbs 190b and 28b,
respectively. EPAS-1 was detected as a single species with a somewhat
lower mobility (apparent molecular weight [MW] 115 kD)
than that predicted from the deduced amino acid sequence. In a number
of cells, including endothelial, fibroblast-like, and epithelial lines,
EPAS-1 protein was detectable in extracts of normoxic cells. In all
these cell lines striking induction by hypoxia was observed (Fig. 3).
Overall, there was a broad correlation between the level of mRNA
expression and the hypoxic level of EPAS-1 protein. Thus, the highest
levels of EPAS-1 protein in response to hypoxia were seen in HMEC-1, HepG2, and Hep3B cells. MRC5 cells, which showed the highest levels of
EPAS-1 mRNA, showed an intermediate level of EPAS-1 protein, whereas
EPAS-1 protein was not detected in RPMI 1788, consistent with the very
low level of EPAS-1 mRNA in these cells. Essentially identical results
were obtained using the other MoAbs and polyclonal antisera raised
against EPAS-1 (data not shown).
The same extracts were analyzed in parallel for HIF-1 with MoAb 28b.
As reported previously,4,18 HIF-1 was detected as
several species which migrated more slowly than the predicted MW and
was induced by hypoxia in all cell lines examined (Fig. 3). Overall,
there was a poor correlation between immunodetectable HIF-1 in
hypoxic cell extracts and the level of the mRNA. In particular, hypoxic
HMEC-1 and MRC5 contained relatively low levels of HIF-1 protein,
although these cells expressed the gene at a relatively high level as
assessed by mRNA analysis. The relative amounts of EPAS-1 and HIF-1
were compared by reference to Gal4 fusion proteins. Hypoxic HMEC-1,
Hep3B, and MRC5 cells were found to contain more EPAS-1 than HIF-1.
In hypoxic HeLa(B) and HepG2 cells the amount of HIF-1 and EPAS-1 is
similar, whereas HIF-1 was more abundant than EPAS-1 in HT29, 293, and RPMI 1788 cells.
To characterize the regulation of EPAS-1 in more detail, we compared
the induction of EPAS-1 and HIF-1 by hypoxia in HeLa(B) cells. These
cells were selected because they showed a relatively high level of both
proteins in response to 4 hours' exposure to 1% oxygen. It has
previously been reported that accumulation of HIF-1 after hypoxic
exposure is almost exclusively nuclear.4 Immunoblots of
differential extracts of hypoxic HeLa(B) cells showed accumulation of
both proteins in the nuclear fraction (data not shown), and
immunolabeling of HeLa(B) and HMEC-1 cells grown on glass slides showed
nuclear staining of EPAS-1 after hypoxic exposure, with little
detectable cytoplasmic staining (Fig 4). Interestingly, as illustrated in hypoxic HMEC-1, the intensity of
labeling for EPAS-1 varied from cell to cell; we have also observed
this when labeling for HIF-1.
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| Fig 4.
Immunolabeling for EPAS-1 with MoAb 190b. Cells were
exposed to normoxia (left panels) or 1% oxygen (right panels). Upper
panels show HeLa(B) and lower panels HMEC-1. Nuclear labeling of
variable intensity is seen in the hypoxic cells. Original magnification × 1,000.
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The time course of both HIF-1 and EPAS-1 accumulation was rapid,
with a response being detected within 30 minutes of exposure to 1%
oxygen (Fig 5A). Levels of both EPAS-1 and
HIF-1 increased over the first 2 to 4 hours of exposure (Fig 5A and
B), but were somewhat lower with prolonged hypoxia (Fig 5B). After
return of hypoxic cultures to normoxic incubation (without changing the culture medium), the levels of both EPAS-1 and HIF-1 decreased very
rapidly and were similar to the uninduced level after 30 minutes.
Interestingly, following this, a transient reduction in the level of
each protein below the normoxic level was consistently observed,
suggesting a suppressive effect of reoxygenation (Fig 5C). Figure 5D
and E shows the effect of cell density and graded hypoxia on expression
of the two proteins. Responses were again similar. The normoxic
expression levels were in each case reduced when cells were cultured at
low density (25% of confluence) compared with standard density (at or
approaching confluence). To test the responses to graded hypoxia,
HeLa(B) cells were grown on Petriperm dishes to minimize oxygen
gradients between the hypoxic atmosphere and the monolayer. Parallel
exposure of near-confluent cells to a range of oxygen tensions induced
both proteins, with significant increases in HIF-1 and EPAS-1 at 5%
oxygen compared to normoxia, and further increases at 3% and 1%
oxygen (Fig 5E). Interestingly, exposure to 5% oxygen appeared to
result in a slightly greater increase in EPAS-1 compared to HIF-1.
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| Fig 5.
Characterization of induction of EPAS-1 and HIF-1 by
hypoxia. Immunoblots of HeLa(B) cell whole-cell extracts (100 µg)
with MoAb 190b (upper panels) and MoAb 28b (lower panels). (A) Cells
were exposed to 1% oxygen for 30 to 180 minutes. (B) Cells were
exposed to normoxia or 1% oxygen for 2 to 48 hours. (C) Cells were
incubated in normoxia (N) or 1% oxygen (H) for 16 hours and harvested
at different times following return to normoxia (Reoxygenation). (D)
Cells were seeded at different densities and cultured under normoxic
conditions. (E) Cells on gas-permeable dishes were exposed to different
oxygen concentrations or 100 µmol/L DFO for 6 hours.
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Pharmacological modulation of EPAS-1 and HIF-1 levels.
Important insights into the mechanism of oxygen sensing and/or
signal transduction underlying HIF-1 activation have been gained from
pharmacological interventions which perturb the response as measured
either by HIF-1 or target gene activation. To compare further the
regulation of EPAS-1 with HIF-1, we measured the response of each
protein to several of these interventions.
Cobaltous ions and iron chelators mimic hypoxia in activation of target
genes such as erythropoietin and induce HIF-1 at the protein
level.4,19,20 When Hela(B) cells were exposed to cobalt
chloride (100 µmol/L) or desferrioxamine (100 µmol/L) a marked
increase in EPAS-1 was observed, in parallel with a similar change in
the level of HIF-1 (Fig 6A).
Interestingly, in certain cell types there were differences betweeen
EPAS-1 and HIF-1 in the level of induction achieved by 1% oxygen
when compared with these stimuli. For instance, in Hep3B cells EPAS-1
was induced similarly by 1% hypoxia, cobalt chloride, or
desferrioxamine (DFO), whereas HIF-1 was induced less effectively by
hypoxia than the other stimuli (Fig 6A). In contrast to the effect of
these stimuli, exposure to cyanide does not mimic the effect of hypoxia
on HIF-1-responsive genes21 and was ineffective in
stimulating either EPAS-1 or HIF-1 in the current experiments. In
fact, a reduction in the level of both EPAS-1 and HIF-1 were
observed when Hela(B) cells were exposed to 1 mmol/L potassium cyanide
at either 21% or 1% oxygen (Fig 6B). When cells were exposed to
cyanide for 4 hours, washed with fresh medium, and then exposed to
hypoxia, the level of HIF-1 and EPAS-1 was similar to hypoxic cells
that had not been exposed to cyanide (Fig 6B). This shows that the
effect of this dose of cyanide is reversible, and is not simply caused
by cell death.
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| Fig 6.
Modulation of EPAS-1 and HIF-1 levels by different
chemical agents. Immunoblots of whole-cell extracts (100 µg) with
MoAb 190b (upper panels) and MoAb 28b (lower panels). (A) HeLa(B) and
Hep3B cells were cultured in normoxia (N), 1% oxygen (H), 100 µmol/L
DFO, or 100 µmol/L cobaltous chloride (CoCl2). (B)
HeLa(B) cells were exposed to normoxia (N) or 1% oxygen (H) in the
presence (+) or absence () of 1 mmol/L potassium cyanide (KCN). To
ascertain that the effect of KCN was not caused by cell death, cells in
the right-hand lane [+] were exposed to 1 mmol/L KCN for 4 hours,
washed twice, and exposed to 1% oxygen for 4 hours in fresh medium.
(C) HeLa(B) cells were exposed to the selective proteasomal inhibitor
N-CBZ-Leu-Leu-Norvalinal (CBZ-LLL). (D) HeLa(B) cells were exposed to
the antioxidant N-(2-mercaptopropionyl)-glycine (NMPG).
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Recently two other types of agent have been shown to stimulate HIF-1
expression. Enhanced HIF-1 immunoactivity and DNA binding activity
has been observed in cells treated with peptide aldehyde inhibitors of
the proteasome such as N-acetyl-leucinyl-leucinyl-norleucinal (Calpain
inhibitor 1) and in cells treated with reducing agents such as
N-(2-mercaptopropionyl)-glycine (NMPG).7,9 Although the
effect of Calpain inhibitor 1 or the more selective proteasomal inhibitor N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-norvalinal (CBZ-LLL) on HIF-1 abundance was rather variable, in each experiment the effect on HIF-1 and EPAS-1 was very similar (Fig 6C). Figure 6D
shows that exposure to NMPG resulted in a similar increase in EPAS-1
and HIF-1 protein levels.
Characteristic reductions in HIF-1 or target gene expression in
cells exposed to particular chemicals have also been used to explore
the mechanism of oxygen sensing. Thus, hydrogen peroxide reduces the
induction of HIF-1 target genes by hypoxia22 and destabilizes HIF-1.6 In HeLa(B) cells we observed very
similar responses of both HIF-1 and EPAS-1 to hydrogen peroxide.
Although 100 µmol/L hydrogen peroxide had little effect (data not
shown), cells exposed to 250 µmol/L and 1 mmol/L hydrogen peroxide
showed marked reductions in both EPAS-1 and HIF-1 levels both in
normoxia, and after exposure to hypoxia, cobalt chloride, and DFO
(Fig 7A). Suppression of protein levels by
exposure to hydrogen peroxide was somewhat greater in cells stimulated
by cobalt or DFO when compared with cells stimulated by hypoxia,
although in each case the response was again similar for both HIF-1
and EPAS-1.
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| Fig 7.
Effect of hydrogen peroxide and diphenylene iodonium on
levels of EPAS-1 and HIF-1. Immunoblots of whole-cell extracts (100 µg) from HeLa(B) cells with MoAb 190b (upper panels) and MoAb 28b
(lower panels). (A) Cells were incubated in normoxia (N), 1% oxygen
(H), 100 µmol/L DFO or 100 µmol/L cobalt chloride
(CoCl2). The presence (+) or absence () of 1 mmol/L
hydrogen peroxide (H2O2) is indicated. (B) A
similar experiment examining the effect of 5 µmol/L DPI.
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In previous studies we have shown that diphenylene iodonium (DPI), an
inhibitor of flavoprotein oxidodreductases, will abolish the induction
of HIF-1 target genes by hypoxia.23 Interestingly, we found
that doses of DPI which produced almost complete inhibition of the
response to hypoxia had little effect on the response of the same genes
to cobaltous ions or iron chelation. Therefore, we examined the effects
of DPI on HIF-1 and EPAS-1 protein levels. DPI abolished the
induction of both HIF-1 and EPAS-1 by hypoxia but, in contrast, had
little effect on the induction of either molecule by cobaltous ions or
DFO (Fig 7B).
Transactivation of hypoxia-responsive promoters by HIF-1 and
EPAS-1.
The above results show that despite wide variation in expression,
HIF-1 and EPAS-1 show rather similar inducible responses. We next
compared their ability to transactivate different hypoxia inducible
promoters using a mutant CHO cell line (Ka13) which expresses neither
HIF-1 nor EPAS-1 at detectable levels.15 In keeping with
previous work, the activity of transiently transfected p(24x6)TKLuc,
containing a multimerized hypoxia response element (HRE) adjacent to a
minimal thymidine kinase promoter, was close to absent in the mutant
Ka13 cells, and markedly enhanced by cotransfection of either EPAS-1 or
HIF-1 expression plasmids (Fig 8). In
contrast, two promoters without known hypoxia-inducible activity (the
weak minimal TK promoter and the powerful CMV enhancer/promoter) showed preserved activity in mutant Ka13 cells and no response to
cotransfection of EPAS-1 or HIF-1 (data not shown). We next tested
activity on two hypoxia-inducible native promoters (from the LDH-A and VEGF genes). Interestingly, the LDH-A promoter had almost no activity in the mutant Ka13 cells, indicating that HRE function was critical. Cotransfection of either EPAS-1 or HIF-1 markedly stimulated activity. Responses were similar to those observed with the
multimerized HRE, with EPAS-1 expression showing rather higher activity
than HIF-1, particularly in normoxic cells. In contrast, the VEGF promoter had substantial activity in mutant Ka13 cells. This activity was again considerably enhanced by cotransfection of either EPAS-1 or
HIF-1. However, the relative activities of the two molecules were
quite different on this promoter. The ratio of EPAS-1 to HIF-1
activity with the VEGF promoter was considerably greater than that
observed with either the LDH-A promoter or the simple multimerized HRE.
View larger version (14K):
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| Fig 8.
Transactivation by EPAS-1 and HIF-1 in Ka13 cells.
Histogram showing the relative luciferase expression from transient
cotransfections of CHO Ka13 cells with reporter plasmids containing a
multiple HRE (p24x6TKLuc), the LDH-A promoter (pLDH-ALuc), or the VEGF
promoter (pVEGFLuc) and an expression plasmid for either HIF-1,
EPAS-1, or an empty vector control. Values shown represent averaged
data from seven experiments, with normalization to the normoxic value
for LDH-A (1 arbitrary unit).
|
|
| |
DISCUSSION |
The identification of EPAS-1, a bHLH-PAS protein with a high degree of
homology to HIF-1, has raised the possibility that this might also
be involved in adaptive responses to changes in oxygen tension. The
initial reports showed that in the presence of ARNT the protein can
form an HRE-binding complex, and that EPAS-1 can alter transcription
driven by DNA sequences known to respond to
hypoxia.11-13,15 To date, expression has only been studied
at the mRNA level with several groups reporting analysis of whole-organ
homogenates and in situ hybridization.11-14 These studies
emphasized endothelial expression in developing rodent embryos,
although other cell types including pulmonary epithelium and renal
mesangial cells were also shown to express EPAS-1 mRNA.
Because the functional activation of the related bHLH-PAS family member
HIF-1 involves major changes in the amount of protein,4 which in most studies occur independently of any changes in mRNA levels,6,24 we developed MoAbs allowing specific detection of EPAS-1 protein. We examined expression in a range of cell types both
at the RNA level by RNAse protection, and at the protein level by
immunoblotting. EPAS-1 mRNA was detected in all 11 human cell lines
examined, of which 10 were not endothelial. When cells were exposed to
1% oxygen, a major increase in EPAS-1 protein but not EPAS-1 mRNA was
observed. The accumulation of EPAS-1 in hypoxia clearly supports a role
in inducible responses to changes in oxygen tension, and raises the
question as to the extent to which EPAS-1 and HIF-1 responses are
similar and how they differ.
Therefore, we assayed these two proteins in parallel in extracts of
HeLa(B) cells exposed to a range of experimental conditions known to
activate HIF-1. Overall, we observed very similar responses. Both
proteins showed a similar time course of accumulation after hypoxic
exposure, and a rapid decrease in protein level on return to 20%
oxygen. For both molecules, accumulation also occurred following
exposure of normoxic cells to cobaltous ions, desferrioxamine, the
antioxidant NMPG, and peptide aldehyde inhibitors of the proteasome. Cyanide did not mimic these responses and decreased the levels of both
proteins. Exposure of cells to hydrogen peroxide and to the
flavoprotein inhibitor DPI reduced the levels of both proteins. Thus,
across a range of experimental conditions previously shown to influence
levels of HIF-1, we observed concordance between the HIF-1 and
EPAS-1 responses. Not only do both molecules respond to hypoxia, but it
appears that the same, or strikingly similar, oxygen-sensing and signal
transduction mechanisms (probably involving redox modulation of
proteasomal destruction) regulate the abundance of both transcription
factors.
In addition to providing comparative data on regulation of HIF-1 and
EPAS-1, these experiments also offer some new insights into the
underlying regulatory mechanisms. EPAS-1 protein (and to a lesser
extent HIF-1) could be readily detected in normoxic cells. Such
levels could be suppressed by inhibitors such as hydrogen peroxide, and
following re-oxygenation they were transiently depressed below the
levels in cultures held in normoxia. Because re-oxygenation is
frequently associated with enhanced oxygen radical production, this
could be in keeping with the hypothesis that the oxygen sensitive signal is related to the cellular level of these species.22 In studying the inhibitors of the response we also compared their action on hypoxia-induced protein accumulation with their action on
responses to cobaltous ions and desferrioxamine. As we observed previously for the action of DPI on HIF-1 target gene
expression,23 the action was stimulus specific, with
induction by cobaltous ions and desferrioxamine being relatively
resistant to DPI inhibition. Such an action might be explained if DPI
were to enhance the generation of radicals whose effects on the signal
pathway were blocked by iron chelation for instance, by interference
with Fenton chemistry. Interestingly, although such an argument has
been advanced for interactions between inhibition of erythropoietin
expression by hydrogen peroxide and stimulation by
desferrioxamine,25 we did not find that activation of
EPAS-1 or HIF-1 by desferrioxamine was resistant to hydrogen
peroxide. Rather, we found the reverse, with hydrogen peroxide being
more inhibitory to cells treated with desferrioxamine.
Also of note was the effect of cell density on both EPAS-1 and HIF-1
expression. Cell cultures at higher density consistently expressed
higher levels of both proteinsan effect which might explain previous
observations that certain HIF-1 target genes are expressed at higher
levels as cells approach confluence, and that HRE activity (as assessed
by transiently transfected reporter gene expression) is greater when
transfected cells are grown at higher densities.2
Despite the similarities in HIF-1 and EPAS-1 responses, differences
were observed. First, there were large differences in expression levels
between cell lines. Although an endothelial cell line did express high
levels of EPAS-1, no clear pattern was discernible, with related cell
lines expressing quite different levels of one or other protein.
Second, with the proviso that immunoblotting can give only approximate
quantitation of protein levels, there appeared to be subtle differences
in the inducible response. Overall, in cells that expressed both
proteins the normoxic expression of EPAS-1 was somewhat more pronounced
than that of HIF-1 (particularly the more hypoxically inducible high
MW species). When comparing HIF-1 and EPAS-1 induction in HeLa(B)
cells, it appeared that EPAS-1 may be induced by slightly less severe
hypoxia (Fig 5E). Although the thresholds for target gene activation by HIF-1 and EPAS-1 might differ, these data would be consistent with
EPAS-1 being a more important modulator under normoxic and less
severely hypoxic conditions.
Other differences were observed in the transactivation studies. To
compare the activity of each gene on different target sequences, we
used a cell line deficient in both EPAS-1 and HIF-1 (Ka13), thus
avoiding the potential complexity of interactions between the two
molecules. Consistent with previous reports,11-13,15 both EPAS-1 and HIF-1 transactivated a multimerized minimal HRE, with EPAS-1 showing more activity in normoxic cells and less induction by
hypoxia than HIF-1. Comparison of the relative activities of the two
molecules on the LDH-A and VEGF promoters indicated further differences
in target gene specificity. Although both transcription factors
activated both promoters, EPAS-1 was relatively more active on the VEGF
promoter. Despite near-identical bHLH domains for EPAS-1 and HIF-1,
similar target gene selectivity has been reported by others with EPAS-1
but not HIF-1 activating the Tie-2 enhancer/promoter.11
Consistent with the endothelium-restricted expression of Tie-2 we found
these sequences to be inactive and unresponsive to either EPAS-1 or
HIF-1 in Chinese hamster ovary (CHO) cells (data not shown),
suggesting that cooperative interactions with other cell-specific
proteins are necessary for target-specific transactivation.
Interestingly, domain exchange experiments among Drosophila
bHLH-PAS proteins have indicated a role for the PAS domain in such
interactions.26
In conclusion, EPAS-1 is a second hypoxia-inducible transcription
factor which shares extensive similarities in its mode of regulation
with HIF-1 and the suggestion that this widely expressed protein be
termed HIF-2 seems appropriate.27 Definition of regulatory domains in EPAS-1 and sequence comparison of these with
HIF-1 should be useful in defining features that interact with the
signal transduction mechanism. It is likely that differences in the
cell-specific expression, induction, and target gene specificities of
these two systems will be important in our understanding of the
diversity of adaptive responses to oxygen availability.
| |
FOOTNOTES |
Submitted July 9, 1998;
accepted July 22, 1998.
Supported by research grants from the Wellcome Trust, the Medical
Research Council (UK), the Imperial Cancer Research Fund, the British
Council/German Academic Exchange Service, and the Deutsche
Forschungsgemeinschaft.
Address reprint requests to P.H. Maxwell, DPhil, Wellcome
Trust Centre for Human Genetics, Windmill Rd, Oxford OX3 7BN, UK.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank S. McKnight for pEP-1, E. Raybould for p(24x6)TKLuc,
N. Bacon for the EPAS-1 riboprobe, and R. Bicknell for
HMEC-1.
| |
REFERENCES |
1.
Bunn HF,
Poyton RO:
Oxygen sensing and molecular adaptation to hypoxia.
Physiol Rev
76:839,
1996[Abstract/Free Full Text]
2.
Maxwell PH,
Pugh CW,
Ratcliffe PJ:
Inducible operation of the erythropoietin 3 enhancer in multiple cell lines: Evidence for a widespread oxygen sensing mechanism.
Proc Natl Acad Sci USA
90:2423,
1993[Abstract/Free Full Text]
3.
Semenza GL,
Wang GL:
A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation.
Mol Cell Biol
12:5447,
1992[Abstract/Free Full Text]
4.
Wang GL,
Jiang B-H,
Rue EA,
Semenza GL:
Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.
Proc Natl Acad Sci USA
92:5510,
1995[Abstract/Free Full Text]
5.
Gleadle JM,
Ratcliffe PJ:
Hypoxia and the regulation of gene expression.
Mol Med Today
4:122,
1998[Medline]
[Order article via Infotrieve]
6.
Huang LE,
Arany Z,
Livingston DM,
Bunn HF:
Activation of hypoxia-inducible transcription factor depends primarily on redox-sensitive stabilization of its subunit.
J Biol Chem
271:32253,
1996[Abstract/Free Full Text]
7.
Salceda S,
Caro J:
Hypoxia-inducible factor 1 (HIF-1) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions.
J Biol Chem
272:22642,
1997[Abstract/Free Full Text]
8.
Pugh CW,
O'Rourke JF,
Nagao M,
Gleadle JM,
Ratcliffe PJ:
Activation of hypoxia inducible factor-1; definition of regulatory domains within the subunit.
J Biol Chem
272:11205,
1997[Abstract/Free Full Text]
9.
Huang LE,
Gu J,
Schau M,
Bunn HF:
Regulation of hypoxia-inducible factor 1 is mediated by an oxygen-dependent domain via the ubiquitin-proteasome pathway.
Proc Natl Acad Sci USA
95:7987,
1998[Abstract/Free Full Text]
10.
Crews ST:
Control of cell lineage-specific development and transcription by bHLH-PAS proteins.
Genes Dev
12:607,
1998[Free Full Text]
11.
Tian H,
McKnight SL,
Russell DW:
Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells.
Genes Dev
11:72,
1997[Abstract/Free Full Text]
12.
Ema M,
Taya S,
Yokotani N,
Sogawa K,
Matsuda Y,
Fujii-Kuriyama Y:
A novel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1 regulates the VEGF expression and is potentially involved in lung and vascular development.
Proc Natl Acad Sci USA
94:4273,
1997[Abstract/Free Full Text]
13.
Hogenesch JB,
Chan WK,
Jackiw VH,
Brown RC,
Gu Y-Z,
Pray-Grant M,
Perdew GH,
Bradfield CA:
Characterization of a subset of the basic-helix-loop-helix-PAS superfamily that interacts with components of the dioxin signaling pathway.
J Biol Chem
272:8581,
1997[Abstract/Free Full Text]
14.
Flamme I,
Fröhlich T,
von Reutern M,
Kappel A,
Damert A,
Risau W:
HRF, a putative basic helix-loop-helix-PAS-domain transcription factor is closely related to hypoxia-inducible factor-1 and developmentally expressed in blood vessels.
Mech Dev
63:51,
1997[Medline]
[Order article via Infotrieve]
15.
Wood SM,
Wiesener MS,
Yeates KM,
Okada N,
Pugh CW,
Maxwell PH,
Ratcliffe PJ:
Selection and analysis of a mutant cell line defective in the hypoxia-inducible factor-1 -subunit (HIF-1).
J Biol Chem
273:8360,
1998[Abstract/Free Full Text]
16.
Ades EW,
Candal FJ,
Swerlick RA,
George VG,
Summers S,
Bosse DC,
Lawley TJ:
HMEC-1: Establishment of an immortalized human microvascular endothelial cell line.
J Invest Dermatol
99:683,
1992[Medline]
[Order article via Infotrieve]
17.
Firth JD,
Ebert BL,
Pugh CW,
Ratcliffe PJ:
Oxygen-regulated control elements in the phosphoglycerate kinase 1 and lactate dehydrogenase A genes: Similarities with the erythropoeitin 3 enhancer.
Proc Natl Acad Sci USA
91:6496,
1994[Abstract/Free Full Text]
18.
Kallio PJ,
Pongratz I,
Gradin K,
McGuire J,
Poellinger L:
Activation of hypoxia-inducible factor 1: Posttranscriptional regulation and conformational change by recruitment of the Arnt transcription factor.
Proc Natl Acad Sci USA
94:5667,
1997[Abstract/Free Full Text]
19.
Goldberg MA,
Dunning SP,
Bunn HF:
Regulation of the erythropoietin gene: Evidence that the oxygen sensor is a heme protein.
Science
242:1412,
1988[Abstract/Free Full Text]
20.
Wang GL,
Semenza GL:
Desferrioxamine induces erythropoietin gene expression and hypoxia-inducible factor 1 DNA-binding activity: Implications for models of hypoxia signal transduction.
Blood
82:3610,
1993[Abstract/Free Full Text]
21.
Necas E,
Thorling EB:
Unresponsiveness of erythropoietin-producing cells to cyanide.
Am J Physiol
222:1187,
1972
22.
Fandrey J,
Frede S,
Jelkmann W:
Role of hydrogen peroxide in hypoxia-induced erythropoietin production.
Biochem J
303:507,
1994
23.
Gleadle JM,
Ebert BL,
Ratcliffe PJ:
Diphenylene iodonium inhibits the induction of erythropoietin and other mammalian genes by hypoxia: Implications for the mechanism of oxygen sensing.
Eur J Biochem
234:92,
1995[Medline]
[Order article via Infotrieve]
24.
Wenger RH,
Rolfs A,
Marti HH,
Guénet J-L,
Gassmann M:
Nucleotide sequence, chromosomal assignment and mRNA expression of mouse hypoxia-inducible factor-1.
Biochem Biophys Res Commun
223:54,
1996[Medline]
[Order article via Infotrieve]
25.
Fandrey J,
Frede S,
Ehleben W,
Porwol T,
Acker H,
Jelkmann W:
Cobalt chloride and desferrioxamine antagonize the inhibition of erythropoietin production by reactive oxygen species.
Kidney Int
51:492,
1997[Medline]
[Order article via Infotrieve]
26.
Zelzer E,
Wappner P,
Shilo B-Z:
The PAS domain confers target gene specificity of Drosophila bHLH/PAS proteins.
Genes Dev
11:2079,
1997[Abstract/Free Full Text]
27.
Wenger RH,
Gassmann M:
Oxygen(es) and the hypoxia-inducible factor-1.
Biol Chem
378:609,
1997
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|
|
A.J. Harvey, K.L. Kind, M. Pantaleon, D.T. Armstrong, and J.G. Thompson
Oxygen-Regulated Gene Expression in Bovine Blastocysts
Biol Reprod,
October 1, 2004;
71(4):
1108 - 1119.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. F. Wagner, A.-K. Hellberg, S. Balenger, R. Depping, J. Dodd-O, R. A. Johns, and D. Li
Hypoxia-Induced Mitogenic Factor Has Antiapoptotic Action and Is Upregulated in the Developing Lung: Coexpression with Hypoxia-Inducible Factor-2{alpha}
Am. J. Respir. Cell Mol. Biol.,
September 1, 2004;
31(3):
276 - 282.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Acker and H. Acker
Cellular oxygen sensing need in CNS function: physiological and pathological implications
J. Exp. Biol.,
August 15, 2004;
207(18):
3171 - 3188.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Wellmann, C. Buhrer, E. Moderegger, A. Zelmer, R. Kirschner, P. Koehne, J. Fujita, and K. Seeger
Oxygen-regulated expression of the RNA-binding proteins RBM3 and CIRP by a HIF-1-independent mechanism
J. Cell Sci.,
May 1, 2004;
117(9):
1785 - 1794.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. B. Hough and J. Piatigorsky
Preferential Transcription of Rabbit Aldh1a1 in the Cornea: Implication of Hypoxia-Related Pathways
Mol. Cell. Biol.,
February 1, 2004;
24(3):
1324 - 1340.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C.-J. Hu, L.-Y. Wang, L. A. Chodosh, B. Keith, and M. C. Simon
Differential Roles of Hypoxia-Inducible Factor 1{alpha} (HIF-1{alpha}) and HIF-2{alpha} in Hypoxic Gene Regulation
Mol. Cell. Biol.,
December 15, 2003;
23(24):
9361 - 9374.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. K. Bruick
Oxygen sensing in the hypoxic response pathway: regulation of the hypoxia-inducible transcription factor
Genes & Dev.,
November 1, 2003;
17(21):
2614 - 2623.
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Maxwell
HIF-1: An Oxygen Response System with Special Relevance to the Kidney
J. Am. Soc. Nephrol.,
November 1, 2003;
14(11):
2712 - 2722.
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. M. Sowter, R. Raval, J. Moore, P. J. Ratcliffe, and A. L. Harris
Predominant Role of Hypoxia-Inducible Transcription Factor (Hif)-1{alpha} versus Hif-2{alpha} in Regulation of the Transcriptional Response to Hypoxia
Cancer Res.,
October 1, 2003;
63(19):
6130 - 6134.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Jiang and C. R. Mendelson
USF1 and USF2 Mediate Inhibition of Human Trophoblast Differentiation and CYP19 Gene Expression by Mash-2 and Hypoxia
Mol. Cell. Biol.,
September 1, 2003;
23(17):
6117 - 6128.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. T. Palayoor, P. J. Tofilon, and C. N. Coleman
Ibuprofen-mediated Reduction of Hypoxia-inducible Factors HIF-1{alpha} and HIF-2{alpha} in Prostate Cancer Cells
Clin. Cancer Res.,
August 1, 2003;
9(8):
3150 - 3157.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S.-k. Park, A. M. Dadak, V. H. Haase, L. Fontana, A. J. Giaccia, and R. S. Johnson
Hypoxia-Induced Gene Expression Occurs Solely through the Action of Hypoxia-Inducible Factor 1{alpha} (HIF-1{alpha}): Role of Cytoplasmic Trapping of HIF-2{alpha}
Mol. Cell. Biol.,
July 15, 2003;
23(14):
4959 - 4971.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Haque, D. A. Davis, V. Wang, I. Widmer, and R. Yarchoan
Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Contains Hypoxia Response Elements: Relevance to Lytic Induction by Hypoxia
J. Virol.,
June 15, 2003;
77(12):
6761 - 6768.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Del Toro, K. L. Levitsky, J. Lopez-Barneo, and M. D. Chiara
Induction of T-type Calcium Channel Gene Expression by Chronic Hypoxia
J. Biol. Chem.,
June 13, 2003;
278(25):
22316 - 22324.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z.-Z. Yang, A. Y. Zhang, F.-X. Yi, P.-L. Li, and A.-P. Zou
Redox regulation of HIF-1alpha levels and HO-1 expression in renal medullary interstitial cells
Am J Physiol Renal Physiol,
June 1, 2003;
284(6):
F1207 - F1215.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Khatua, K. M. Peterson, K. M. Brown, C. Lawlor, M. R. Santi, B. LaFleur, D. Dressman, D. A. Stephan, and T. J. MacDonald
Overexpression of the EGFR/FKBP12/HIF-2{alpha} Pathway Identified in Childhood Astrocytomas by Angiogenesis Gene Profiling
Cancer Res.,
April 15, 2003;
63(8):
1865 - 1870.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. N. Seagroves, D. Hadsell, J. McManaman, C. Palmer, D. Liao, W. McNulty, B. Welm, K.-U. Wagner, M. Neville, and R. S. Johnson
HIF1{alpha} is a critical regulator of secretory differentiation and activation, but not vascular expansion, in the mouse mammary gland
Development,
April 15, 2003;
130(8):
1713 - 1724.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. B. Freeburg, B. Robert, P. L. St. John, and D. R. Abrahamson
Podocyte Expression of Hypoxia-Inducible Factor (HIF)-1 and HIF-2 during Glomerular Development
J. Am. Soc. Nephrol.,
April 1, 2003;
14(4):
927 - 938.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A Giatromanolaki, E Sivridis, E Maltezos, D Papazoglou, C Simopoulos, K C Gatter, A L Harris, and M I Koukourakis
Hypoxia inducible factor 1{alpha} and 2{alpha} overexpression in inflammatory bowel disease
J. Clin. Pathol.,
March 1, 2003;
56(3):
209 - 213.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-H. Park, T.-Y. Kim, H.-S. Jong, T. Y. Kim, Y.-S. Chun, J.-W. Park, C.-T. Lee, H. C. Jung, N. K. Kim, and Y.-J. Bang
Gastric Epithelial Reactive Oxygen Species Prevent Normoxic Degradation of Hypoxia-inducible Factor-1{alpha} in Gastric Cancer Cells
Clin. Cancer Res.,
January 1, 2003;
9(1):
433 - 440.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Bernaudin, Y. Tang, M. Reilly, E. Petit, and F. R. Sharp
Brain Genomic Response following Hypoxia and Re-oxygenation in the Neonatal Rat. IDENTIFICATION OF GENES THAT MIGHT CONTRIBUTE TO HYPOXIA-INDUCED ISCHEMIC TOLERANCE
J. Biol. Chem.,
October 11, 2002;
277(42):
39728 - 39738.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Favier, P.-F. Plouin, P. Corvol, and J.-M. Gasc
Angiogenesis and Vascular Architecture in Pheochromocytomas : Distinctive Traits in Malignant Tumors
Am. J. Pathol.,
October 1, 2002;
161(4):
1235 - 1246.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Jesmin, I. Sakuma, Y. Hattori, and A. Kitabatake
In Vivo Estrogen Manipulations on Coronary Capillary Network and Angiogenic Molecule Expression in Middle-Aged Female Rats
Arterioscler Thromb Vasc Biol,
October 1, 2002;
22(10):
1591 - 1597.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. H. WENGER
Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression
FASEB J,
August 1, 2002;
16(10):
1151 - 1162.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. P. Hui, A. T. C. Chan, F. Pezzella, H. Turley, K.-F. To, T. C. W. Poon, B. Zee, F. Mo, P. M. L. Teo, D. P. Huang, et al.
Coexpression of Hypoxia-inducible Factors 1{alpha} and 2{alpha}, Carbonic Anhydrase IX, and Vascular Endothelial Growth Factor in Nasopharyngeal Carcinoma and Relationship to Survival
Clin. Cancer Res.,
August 1, 2002;
8(8):
2595 - 2604.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. O. Fedele, M. L. Whitelaw, and D. J. Peet
Regulation of Gene Expression by the Hypoxia-Inducible Factors
Mol. Interv.,
July 1, 2002;
2(4):
229 - 243.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Zatyka, N. F. da Silva, S. C. Clifford, M. R. Morris, M. S. Wiesener, K.-U. Eckardt, R. S. Houlston, F. M. Richards, F. Latif, and E. R. Maher
Identification of Cyclin D1 and Other Novel Targets for the von Hippel-Lindau Tumor Suppressor Gene by Expression Array Analysis and Investigation of Cyclin D1 Genotype as a Modifier in von Hippel-Lindau Disease
Cancer Res.,
July 1, 2002;
62(13):
3803 - 3811.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Rosenberger, S. Mandriota, J. S. Jurgensen, M. S. Wiesener, J. H. Horstrup, U. Frei, P. J. Ratcliffe, P. H. Maxwell, S. Bachmann, and K.-U. Eckardt
Expression of Hypoxia-Inducible Factor-1{alpha} and -2{alpha} in Hypoxic and Ischemic Rat Kidneys
J. Am. Soc. Nephrol.,
July 1, 2002;
13(7):
1721 - 1732.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. G. Fine and J. T. Norman
The Breathing Kidney
J. Am. Soc. Nephrol.,
July 1, 2002;
13(7):
1974 - 1976.
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Liang, X.-Y. Li, E. J. Rebar, P. Li, Y. Zhou, B. Chen, A. P. Wolffe, and C. C. Case
Activation of Vascular Endothelial Growth Factor A Transcription in Tumorigenic Glioblastoma Cell Lines by an Enhancer with Cell Type-specific DNase I Accessibility
J. Biol. Chem.,
May 24, 2002;
277(22):
20087 - 20094.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. S. Wiesener, M. Seyfarth, C. Warnecke, J. S. Jurgensen, C. Rosenberger, N. V. Morgan, E. R. Maher, U. Frei, and K.-U. Eckardt
Paraneoplastic erythrocytosis associated with an inactivating point mutation of the von Hippel-Lindau gene in a renal cell carcinoma
Blood,
May 15, 2002;
99(10):
3562 - 3565.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. J. P. Beasley, R. Leek, M. Alam, H. Turley, G. J. Cox, K. Gatter, P. Millard, S. Fuggle, and A. L. Harris
Hypoxia-inducible Factors HIF-1{alpha} and HIF-2{alpha} in Head and Neck Cancer: Relationship to Tumor Biology and Treatment Outcome in Surgically Resected Patients
Cancer Res.,
May 1, 2002;
62(9):
2493 - 2497.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. J. Turner, J. W. Moore, A. Jones, C. F. Taylor, D. Cuthbert-Heavens, C. Han, R. D. Leek, K. C. Gatter, P. H. Maxwell, P. J. Ratcliffe, et al.
Expression of Hypoxia-inducible Factors in Human Renal Cancer: Relationship to Angiogenesis and to the von Hippel-Lindau Gene Mutation
Cancer Res.,
May 1, 2002;
62(10):
2957 - 2961.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. D. Leek, K. L. Talks, F. Pezzella, H. Turley, L. Campo, N. S. Brown, R. Bicknell, M. Taylor, K. C. Gatter, and A. L. Harris
Relation of Hypoxia-inducible Factor-2{alpha} (HIF-2{alpha}) Expression in Tumor-infiltrative Macrophages to Tumor Angiogenesis and the Oxidative Thymidine Phosphorylase Pathway in Human Breast Cancer
Cancer Res.,
March 1, 2002;
62(5):
1326 - 1329.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Onita, P. G. Ji, J. W. Xuan, H. Sakai, H. Kanetake, P. H. Maxwell, G.-H. Fong, M. Y Gabril, M. Moussa, and J. L. Chin
Hypoxia-induced, Perinecrotic Expression of Endothelial Per-ARNT-Sim Domain Protein-1/Hypoxia-inducible Factor-2{alpha} Correlates with Tumor Progression, Vascularization, and Focal Macrophage Infiltration in Bladder Cancer
Clin. Cancer Res.,
February 1, 2002;
8(2):
471 - 480.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Alfranca, M. D. Gutierrez, A. Vara, J. Aragones, F. Vidal, and M. O. Landazuri
c-Jun and Hypoxia-Inducible Factor 1 Functionally Cooperate in Hypoxia-Induced Gene Transcription
Mol. Cell. Biol.,
January 1, 2002;
22(1):
12 - 22.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Sato, T. Tanaka, T. Maeno, Y. Sando, T. Suga, Y. Maeno, H. Sato, R. Nagai, and M. Kurabayashi
Inducible Expression of Endothelial PAS Domain Protein-1 by Hypoxia in Human Lung Adenocarcinoma A549 Cells . Role of Src Family Kinases-dependent Pathway
Am. J. Respir. Cell Mol. Biol.,
January 1, 2002;
26(1):
127 - 134.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. C. Vaux, S. M. Wood, M. E. Cockman, L. G. Nicholls, K. M. Yeates, C. W. Pugh, P. H. Maxwell, and P. J. Ratcliffe
Selection of Mutant CHO Cells with Constitutive Activation of the HIF System and Inactivation of the von Hippel-Lindau Tumor Suppressor
J. Biol. Chem.,
November 16, 2001;
276(47):
44323 - 44330.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. M. STROKA, T. BURKHARDT, I. DESBAILLETS, R. H. WENGER, D. A. H. NEIL, C. BAUER, M. GASSMANN, and D. CANDINAS
HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia
FASEB J,
November 1, 2001;
15(13):
2445 - 2453.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Sanchez-Elsner, L. M. Botella, B. Velasco, A. Corbi, L. Attisano, and C. Bernabeu
Synergistic Cooperation between Hypoxia and Transforming Growth Factor-beta Pathways on Human Vascular Endothelial Growth Factor Gene Expression
J. Biol. Chem.,
October 12, 2001;
276(42):
38527 - 38535.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Brusselmans, F. Bono, P. Maxwell, Y. Dor, M. Dewerchin, D. Collen, J.-M. Herbert, and P. Carmeliet
Hypoxia-inducible Factor-2alpha (HIF-2alpha ) Is Involved in the Apoptotic Response to Hypoglycemia but Not to Hypoxia
J. Biol. Chem.,
October 12, 2001;
276(42):
39192 - 39196.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Crowther, N. J. Brown, E. T. Bishop, and C. E. Lewis
Microenvironmental influence on macrophage regulation of angiogenesis in wounds and malignant tumors
J. Leukoc. Biol.,
October 1, 2001;
70(4):
478 - 490.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Blancher, J. W. Moore, N. Robertson, and A. L. Harris
Effects of ras and von Hippel-Lindau (VHL) Gene Mutations on Hypoxia-inducible Factor (HIF)-1{alpha}, HIF-2{alpha}, and Vascular Endothelial Growth Factor Expression and Their Regulation by the Phosphatidylinositol 3'-Kinase/Akt Signaling Pathway
Cancer Res.,
October 1, 2001;
61(19):
7349 - 7355.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. K. Bruick and S. L. McKnight
Building better vasculature
Genes & Dev.,
October 1, 2001;
15(19):
2497 - 2502.
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. C. Vaux, E. Metzen, K. M. Yeates, and P. J. Ratcliffe
Regulation of hypoxia-inducible factor is preserved in the absence of a functioning mitochondrial respiratory chain
Blood,
July 15, 2001;
98(2):
296 - 302.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. S. Wiesener, P. M. Munchenhagen, I. Berger, N. V. Morgan, J. Roigas, A. Schwiertz, J. S. Jurgensen, G. Gruber, P. H. Maxwell, S. A. Loning, et al.
Constitutive Activation of Hypoxia-inducible Genes Related to Overexpression of Hypoxia-inducible Factor-1{{alpha}} in Clear Cell Renal Carcinomas
Cancer Res.,
July 1, 2001;
61(13):
5215 - 5222.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. S. Gross, N. Akeno, T. L. Clemens, S. Komarova, S. Srinivasan, D. A. Weimer, and S. Mayorov
Physiological and Genomic Consequences of Intermittent Hypoxia: Selected Contribution: Osteocytes upregulate HIF-1{alpha} in response to acute disuse and oxygen deprivation
J Appl Physiol,
June 1, 2001;
90(6):
2514 - 2519.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Krones, K. Jungermann, and T. Kietzmann
Cross-Talk between the Signals Hypoxia and Glucose at the Glucose Response Element of the L-Type Pyruvate Kinase Gene
Endocrinology,
June 1, 2001;
142(6):
2707 - 2718.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. C. Clifford, M. E. Cockman, A. C. Smallwood, D. R. Mole, E. R. Woodward, P. H. Maxwell, P. J. Ratcliffe, and E. R. Maher
Contrasting effects on HIF-1{{alpha}} regulation by disease-causing pVHL mutations correlate with patterns of tumourigenesis in von Hippel-Lindau disease
Hum. Mol. Genet.,
May 1, 2001;
10(10):
1029 - 1038.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Jones, C. Fujiyama, C. Blanche, J. W. Moore, S. Fuggle, D. Cranston, R. Bicknell, and Adrian. L. Harris
Relation of Vascular Endothelial Growth Factor Production to Expression and Regulation of Hypoxia-inducible Factor-1{{alpha}} and Hypoxia-inducible Factor-2{{alpha}} in Human Bladder Tumors and Cell Lines
Clin. Cancer Res.,
May 1, 2001;
7(5):
1263 - 1272.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
M. I. Koukourakis, A. Giatromanolaki, J. Skarlatos, L. Corti, S. Blandamura, M. Piazza, K. C. Gatter, and A. L. Harris
Hypoxia Inducible Factor (HIF-1a and HIF-2a) Expression in Early Esophageal Cancer and Response to Photodynamic Therapy and Radiotherapy
Cancer Res.,
March 1, 2001;
61(5):
1830 - 1832.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
C. C. Wykoff, N. Beasley, P. H. Watson, L. Campo, S. K. Chia, R. English, J. Pastorek, W. S. Sly, P. Ratcliffe, and A. L. Harris
Expression of the Hypoxia-Inducible and Tumor-Associated Carbonic Anhydrases in Ductal Carcinoma in Situ of the Breast
Am. J. Pathol.,
March 1, 2001;
158(3):
1011 - 1019.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Rajakumar, K. A. Whitelock, L. A. Weissfeld, A. R. Daftary, N. Markovic, and K. P. Conrad
Selective Overexpression of the Hypoxia-Inducible Transcription Factor, HIF-2{{alpha}}, in Placentas from Women with Preeclampsia
Biol Reprod,
February 1, 2001;
64(2):
499 - 506.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
C. C. Wykoff, N. J. P. Beasley, P. H. Watson, K. J. Turner, J. Pastorek, A. Sibtain, G. D. Wilson, H. Turley, K. L. Talks, P. H. Maxwell, et al.
Hypoxia-inducible Expression of Tumor-associated Carbonic Anhydrases
Cancer Res.,
December 1, 2000;
60(24):
7075 - 7083.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
C. Blancher, J. W. Moore, K. L. Talks, S. Houlbrook, and A. L. Harris
Relationship of Hypoxia-inducible Factor (HIF)-1{{alpha}} and HIF-2{{alpha}} Expression to Vascular Endothelial Growth Factor Induction and Hypoxia Survival in Human Breast Cancer Cell Lines
Cancer Res.,
December 1, 2000;
60(24):
7106 - 7113.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
B. Jiang, A. Kamat, and C. R. Mendelson
Hypoxia Prevents Induction of Aromatase Expression in Human Trophoblast Cells in Culture: Potential Inhibitory Role of the Hypoxia-Inducible Transcription Factor Mash-2 (Mammalian Achaete-Scute Homologous Protein-2)
Mol. Endocrinol.,
October 1, 2000;
14(10):
1661 - 1673.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
C. Willam, P. Koehne, J. S. Jurgensen, M. Grafe, K. D. Wagner, S. Bachmann, U. Frei, and K.-U. Eckardt
Tie2 Receptor Expression Is Stimulated by Hypoxia and Proinflammatory Cytokines in Human Endothelial Cells
Circ. Res.,
September 1, 2000;
87(5):
370 - 377.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. M. Wanner, P. Spielmann, D. M. Stroka, G. Camenisch, I. Camenisch, A. Scheid, D. R. Houck, C. Bauer, M. Gassmann, and R. H. Wenger
Epolones induce erythropoietin expression via hypoxia-inducible factor-1alpha activation
Blood,
August 15, 2000;
96(4):
1558 - 1565.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Ren, K. L. Dorrington, P. H. Maxwell, and P. A. Robbins
Effects of desferrioxamine on serum erythropoietin and ventilatory sensitivity to hypoxia in humans
J Appl Physiol,
August 1, 2000;
89(2):
680 - 686.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Kietzmann, J. Fandrey, and H. Acker
Oxygen Radicals as Messengers in Oxygen-Dependent Gene Expression
Physiology,
August 1, 2000;
15(4):
202 - 208.
[Abstract]
[Full Text]
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K. L. Talks, H. Turley, K. C. Gatter, P. H. Maxwell, C. W. Pugh, P. J. Ratcliffe, and A. L. Harris
The Expression and Distribution of the Hypoxia-Inducible Factors HIF-1{alpha} and HIF-2{alpha} in Normal Human Tissues, Cancers, and Tumor-Associated Macrophages
Am. J. Pathol.,
August 1, 2000;
157(2):
411 - 421.
[Abstract]
[Full Text]
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A. Rajakumar and K. P. Conrad
Expression, Ontogeny, and Regulation of Hypoxia-Inducible Transcription Factors in the Human Placenta
Biol Reprod,
August 1, 2000;
63(2):
559 - 569.
[Abstract]
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J. Peng, L. Zhang, L. Drysdale, and G.-H. Fong
The transcription factor EPAS-1/hypoxia-inducible factor 2alpha plays an important role in vascular remodeling
PNAS,
June 30, 2000;
(2000)
140087397.
[Abstract]
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S. J. Mandriota, C. Pyke, C. Di Sanza, P. Quinodoz, B. Pittet, and M. S. Pepper
Hypoxia-Inducible Angiopoietin-2 Expression Is Mimicked by Iodonium Compounds and Occurs in the Rat Brain and Skin in Response to Systemic Hypoxia and Tissue Ischemia
Am. J. Pathol.,
June 1, 2000;
156(6):
2077 - 2089.
[Abstract]
[Full Text]
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C. Clerici and M. A. Matthay
Hypoxia regulates gene expression of alveolar epithelial transport proteins
J Appl Physiol,
May 1, 2000;
88(5):
1890 - 1896.
[Abstract]
[Full Text]
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G. L. Semenza
HIF-1: mediator of physiological and pathophysiological responses to hypoxia
J Appl Physiol,
April 1, 2000;
88(4):
1474 - 1480.
[Abstract]
[Full Text]
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A. L. Harris
von Hippel-Lindau Syndrome: Target for Anti-Vascular Endothelial Growth Factor (VEGF) Receptor Therapy
Oncologist,
April 1, 2000;
5(90001):
32 - 36.
[Abstract]
[Full Text]
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H. J. H. Marti, M. Bernaudin, A. Bellail, H. Schoch, M. Euler, E. Petit, and W. Risau
Hypoxia-Induced Vascular Endothelial Growth Factor Expression Precedes Neovascularization after Cerebral Ischemia
Am. J. Pathol.,
March 1, 2000;
156(3):
965 - 976.
[Abstract]
[Full Text]
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D. Lando, I. Pongratz, L. Poellinger, and M. L. Whitelaw
A Redox Mechanism Controls Differential DNA Binding Activities of Hypoxia-inducible Factor (HIF) 1alpha and the HIF-like Factor
J. Biol. Chem.,
February 18, 2000;
275(7):
4618 - 4627.
[Abstract]
[Full Text]
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R. Wenger
Mammalian oxygen sensing, signalling and gene regulation
J. Exp. Biol.,
January 4, 2000;
203(8):
1253 - 1263.
[Abstract]
[PDF]
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A. Ouiddir, C. Planès, I. Fernandes, A. VanHesse, and C. Clerici
Hypoxia Upregulates Activity and Expression of the Glucose Transporter GLUT1 in Alveolar Epithelial Cells
Am. J. Respir. Cell Mol. Biol.,
December 1, 1999;
21(6):
710 - 718.
[Abstract]
[Full Text]
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P. W. Conrad, T. L. Freeman, D. Beitner-Johnson, and D. E. Millhorn
EPAS1 trans-Activation during Hypoxia Requires p42/p44 MAPK
J. Biol. Chem.,
November 19, 1999;
274(47):
33709 - 33713.
[Abstract]
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K. Maemura, C.-M. Hsieh, M. K. Jain, S. Fukumoto, M. D. Layne, Y. Liu, S. Kourembanas, S.-F. Yet, M. A. Perrella, and M.-E. Lee
Generation of a Dominant-negative Mutant of Endothelial PAS Domain Protein 1 by Deletion of a Potent C-terminal Transactivation Domain
J. Biol. Chem.,
October 29, 1999;
274(44):
31565 - 31570.
[Abstract]
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D. M. Adelman, E. Maltepe, and M. C. Simon
Multilineage embryonic hematopoiesis requires hypoxic ARNT activity
Genes & Dev.,
October 1, 1999;
13(19):
2478 - 2483.
[Abstract]
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B. L. Ebert and H. F. Bunn
Regulation of the Erythropoietin Gene
Blood,
September 15, 1999;
94(6):
1864 - 1877.
[Full Text]
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E. Metzen, J. Fandrey, and W. Jelkmann
Evidence against a major role for Ca2+ in hypoxia-induced gene expression in human hepatoma cells (Hep3B)
J. Physiol.,
June 15, 1999;
517(3):
651 - 657.
[Abstract]
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Abstract
Introduction
Abstract