1 Institute of Physiology, University of Lübeck, Ratzeburger Allee 160,
D-23538 Lübeck, Germany
2 Institute of Physiology, University of Essen, Hufelandstr. 55, D-45122 Essen,
Germany
3 Institute of Anatomy, University of Lübeck, Ratzeburger Allee 160,
D-23538 Lübeck, Germany
4 Max-Planck-Institute of Molecular Physiology, Dortmund, Otto-Hahn-Straße
11, D-44227 Dortmund, Germany
* Author for correspondence (e-mail: metzen{at}physio.uni-luebeck.de)
Accepted 12 December 2002
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Summary |
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Key words: Hypoxia, Oxygen sensing, Hypoxia inducible factor, Hydroxylase
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Introduction |
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In normoxia HIF-1 is bound by the von-Hippel-Lindau gene product
(pVHL) (Hon et al., 2002
;
Maxwell et al., 1999
;
Min et al., 2002
), which is
the substrate-recognising component of an E3 ubiquitin ligase complex
(Cockman et al., 2000
;
Ohh et al., 2000
;
Tanimoto et al., 2000
).
HIF-1
is then polyubiquitynated and almost instantaneously degraded by
the proteasome (Huang et al.,
1998
; Salceda and Caro,
1997
). Complete inactivation of pVHL in cultured cells leads to
accumulation of HIF-1
in normoxia, suggesting that oxygen-dependent
degradation of HIF-1
is exclusively controlled by pVHL. The existence
of an oxygen- and pVHL-independent HIF-1
degradation pathway has been
reported very recently (Isaacs et al.,
2002
).
In the presence of oxygen, binding of HIF-1 to pVHL is induced by
hydroxylation of two proline residues of HIF-1
, P564 and P402
(Ivan et al., 2001
;
Jaakkola et al., 2001
;
Masson et al., 2001
;
Yu et al., 2001
). Shortly
after this demonstration, a family of enzymes capable of hydroxylating
HIF-1
subunits was identified
(Epstein et al., 2001
). The
first enzyme shown to have this activity was the Caenorhabditis
elegans protein EGL-9. By database search a group of human enzymes was
found that were termed prolyl hydroxylase domain containing proteins 1, 2, and
3 (PHD1, PHD2, and PHD3). Another group of investigators have independently
identified the same enzymes calling them HPH1, HPH2 and HPH3, where HPH stands
for HIF prolyl hydroxylases (Bruick and
McKnight, 2001
). The existence of a fourth HIF-1
prolyl
hydroxylase has been postulated very recently
(Oehme et al., 2002
). On a
more theoretical basis PHD1, PHD2 and PHD3 were shown to belong to the same
gene family as EGL-9 before a function had been assigned to them. Therefore
they were also named EGLN1, EGLN2 and EGLN3
(Taylor, 2001
).
Very recently oxygen-dependent hydroxylation of Asp803 in the C-terminus of
human HIF-1 has been demonstrated to inhibit the function of the
C-terminal transactivation domain (C-TAD)
(Lando et al., 2002a
). A
protein that was termed the factor inhibiting HIF (FIH-1) was isolated
(Mahon et al., 2001
) and
subsequently shown to abrogate the interaction between HIF-1
and the
transcriptional coactivator p300/CBP
(Hewitson et al., 2002
;
Lando et al., 2002b
). The
existence of FIH-1 and the PHDs suggests that in the presence of oxygen HIF-1
target gene expression is tightly regulated by two separate mechanisms: prolyl
hydroxylases initiate the degradation of HIF-1
and the asparagine
hydroxylase FIH-1 inactivates the C-terminal transactivation domain of
HIF-1
.
As PHD1, PHD2, PHD3 and FIH-1 hydroxylate HIF-1 in an
oxygen-dependent manner they have been postulated to function as oxygen
sensors in vivo. Here we set out to determine the intracellular localisation
of these enzymes. To this end we expressed the hydroxylases fused to enhanced
green fluorescent protein (EGFP) in cultured human osteosarcoma cells and
assessed their effect on the hypoxic induction of endogenous HIF-1
by
immunofluorescence. We also tested the effect of a transient transfection of
PHDs or FIH-1 on an HRE-driven luciferase reporter gene. Furthermore, we
checked for expression of endogenous PHD mRNAs and their responses to hypoxia
and the hypoxia mimics desferrioxamine and cobalt ions.
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Materials and Methods |
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Cell culture and transient transfections
The human osteosarcoma cells (U2OS) were a kind gift from J. M. Gleadle and
P. J. Ratcliffe. U2OS and Hep3B cells (ATCC) were grown in Dulbecco's modified
Eagle's medium (DMEM, Gibco, Karlsruhe, Germany) supplemented with 10% FCS
(Gibco), 50 IU/ml penicillin and 50 µg/ml streptomycin sulfate (Sigma).
Cells were grown in six-well dishes for reporter gene assays and RNA
preparation or on coverslips in 24-well dishes for microscopical analysis.
Transient transfections were performed as described previously
(Masson et al., 2001). In
brief, for each well to be transfected 3 µl Fugene 6 (Roche, Mannheim,
Germany) per µg plasmid DNA were suspended in 100 µl DMEM/well. After 5
minutes at room temperature plasmid DNA was added. The mixture was incubated
for another 30 minutes at room temperature and then added to the cell culture
medium. Cells were incubated for 24 hours and then subjected to the
experimental conditions. Hypoxic incubations were done in an atmosphere of 1%
or 3% O2 in a hypoxia workstation (Ruskinn Technology, Leeds,
UK).
Fluorescence microscopy
U2OS cells were grown on coverslips to 50% density and transfected with the
EGFP- or PK-tagged PHD1, PHD2, PHD3 and FIH-1 as described above. Cells were
incubated for 4 hours in normoxia (20% oxygen) or in hypoxia (1% oxygen) and
fixed by ice cold methanol/acetone (1:1) for 5 minutes. For indirect
immunofluorescence, cells were blocked with 3% BSA in PBS and incubated with a
monoclonal mouse anti-HIF-1 antibody (1:50, Transduction Laboratories)
or with a monoclonal mouse anti-PK antibody (1:200, Serotec). Cells were
washed in PBS prior to an incubation with an Alexa-568-conjugated goat
anti-mouse IgG secondary antibody (1:400, Molecular Probes, Göttingen,
Germany). Coverslips were mounted on slides with Mowiol (Calbiochem, Bad
Soden, Germany).
Images of the immunostained cells were captured using a Nikon E1000 microscope (Nikon, Germany) with Apochromat x60 oil immersion objective lens, equipped with an Optronics digital charge-coupled device (CCD) camera (Visitron Systems, Pucheim, Germany). The images were assessed by using EZ2000 software (Coord, Netherlands). Fluorescence intensities were visualised by using a false color table shading from black (lowest), through blue, green, yellow, orange, red and purple to white (highest intensity).
Two-photon confocal laser scanning microscopy
PHDs fused to EGFP were three dimensionally recorded using two-photon
confocal laser scanning microscopy (2PCLSM) as described previously
(Bestvater et al., 2002). The
two photon pulses were provided by a mode-locked Ti: Sapphire laser [Coherent
Mira 900 F; excitation at 720-910 nm, pulse interval/repetition rate: 13.2
nseconds, 110 fsecond pulses and a high power (5 W, 532 nm) laser (Verdi,
Coherent, Darmstadt); 40 mW at 560 nm]. The light was guided through a grading
dispersion compensator (GDC) to a scan unit (PCM2000, Nikon, Germany) mounted
on an inverted fluorescence microscope (TE300, Nikon). The x,y,z image stacks
(512x512x64 pixels) were obtained by optical sectioning with a
60x water objective (Nikon, Plan Apochromat DIC H; NA 1.2). 850 nm for
excitation was applied for GFP excitation giving an emission peak at 520 nm.
Fluorescence intensities recorded by a photo multiplier were digitised and
visualised by the EZ 2000 software (Version 2.1.4, Coord Automatisering,
Netherlands). The signal-to-noise ratio was determined and the images were
deconvoluted by the Huygens System software (Version 2.2.1, Scientific Volume
Imaging; PS Hilversum, Netherlands) on a Silicon Graphics Octane workstation
using the maximum likelihood estimation (MLE) method. The data were
reconstructed with the Application Visualisation System (AVS Waltham, Mass.,
USA). Calculation of isosurfaces was performed on a Unix-based Octane
workstation (Silicon Graphics, Mountain View) on the basis of the application
visualisation system using a marching cube algorithm (AVS-Express, Waltham) as
described above.
In vitro protein interaction assay
The hydroxylase activity of the PHD-EGFP fusion proteins was demonstrated
in a GalDBD-HIF-1549-582pVHL in vitro interaction assay as
described previously (Jaakkola et al.,
2001
). In brief, we produced GalDBD-HIF-1
549-582, PHD,
PHD-EGFP fusion proteins and 35S-labelled pVHL in a T7-coupled
rabbit reticulocyte lysate in vitro transcription/translation system (Promega)
as recommended by the manufacturer. GalDBD-HIF-1
549-582 was purified
with GalDBD-antibodies conjugated to agarose beads (Santa Cruz Biotechnology,
Heidelberg, Germany). 5 µl GalDBD-HIF-1
549-582 IVTT solution was
then incubated for 30 minutes with either 5 µl unprogrammed reticulocyte
lysate or PHD or PHD-EGFP fusion protein IVTT solution in the presence of 1 mM
ascorbate, 1 mM
-ketoglutarate and 20 µM FeCl2. The beads
were washed several times before 5 µl of 35S-pVHL IVTT solution
was added. The mixture was incubated overnight at 4°C on an end-over-end
rotator. Unbound 35S-pVHL was removed by washing, the beads were
boiled in SDS-PAGE loading buffer and the proteins were separated on a 15%
SDS-polyacrylamide gel. 35S-pVHL was detected by
autoradiography.
Reporter gene assays
Cells were grown in six-well dishes and transfected as detailed above. For
each well, 250 µg of a plasmid containing six copies of a hypoxiaresponsive
element from the PGK promoter in front of a firefly luciferase gene (HRE-luc,
a kind gift of C. Pugh, University of Oxford, Oxford, UK), 250 µg of an
SV40 promoted ß-galactosidase plasmid (Promega) and 500 µg of a
PHD-pcDNA3 construct were used. After 24 hours the cells were given fresh
medium and incubated in the normoxic (20% O2) or in the hypoxic (1%
O2) atmosphere for 24 hours. Cells were harvested and luciferase
assays were done with a commercially available assay kit (Promega) following
the supplier's instructions. ß-galactosidase activity was measured
exactly as published previously (O'Rourke
et al., 1999). HRE luciferase values were divided by
ß-galactosidase values to correct for variations in transfection
efficiency. All transfections were done in three separate wells, data are
given as mean plus standard deviation.
Reverse transcription and quantitative real time PCR
Total RNA (1 µg) was reverse transcribed with oligo (dT) and M-MLV
Reverse Transcriptase (Promega). Gene expression of human PHD1, PHD2, PHD3 and
FIH-1 was quantified using the qPCRTM Mastermix for SYBR Green I
(Eurogentec, Belgium) and the GeneAmp®5700 sequence Detection System (PE
Biosystems, Foster City, CA). The PCR reactions were set up in a final volume
of 25 µl per 0.5 µl cDNA, 1x reaction buffer with SYBR Green I, 10
pmol forward (F) and 10 pmol reverse primer (R). The primer sets used for PHD1
were (F) 5'-GGCGATCCCGCCGCGC-3' and (R)
5'-CCTGGGTAACACGCC-3', for PHD2 (F)
5'-GCACGACACCGGGAAGTT-3' and (R)
5'-CCAGCTTCCCGTTACAGT-3', for PHD3 (F)
5'-GGCCATCAGCTTCCTCCTG-3' and (R)
5'-GGTGATGCAGCGACCATCA-3', for FIH-1 (F)
5'-ACAGTGCCAGCACCCACAA-3' and (R)
5'-GCCCACAGTGTCATTGAGCG-3', for the house keeping gene 60S acidic
ribosomal protein (F) 5'-ACGAGGTGTGCAAGGAGGGC-3' and (R)
5'-GCAAGTCGTCTCCCATCTGC-3'. Agarose gel electrophoresis confirmed
the specificity of the amplification product. Ten-fold dilutions of purified
PCR products (High Pure PCR Product Purification Kit, Roche) starting at 1 pg
to 0.1 fg were used as standards. Amplification conditions were set to 10
minutes at 95°C followed 30 PCR cycles [15 seconds at 95°C, 1 minutes
at 60°C (PHD1, 2 and FIH-1) or 62°C (PHD3 and ribosomal protein)]. The
quantity of cDNA used in each reaction was normalised to the ribosomal protein
cDNA and expressed as cDNA sample/cDNA ribosomal protein.
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Results |
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Effect of hydroxylase expression on endogenous HIF-1
We confirmed by in vitro transcription/translation in rabbit reticulocyte
lysate that all HIF-1 prolyl hydroxylase EGFP fusions could be
expressed as single bands of the expected molecular weight
(Fig. 3A). Next we demonstrated
that the prolyl hydroxylase fusion proteins can modify HIF-1
at Pro564,
enabling pVHL binding. To this end we set up an in vitro protein interaction
assay as described previously (Jaakkola et
al., 2001
). In a control experiment we excluded the possibility
that GalDBD or the agarose beads bind to pVHL non-specifically before or after
treatment with HIF-1
prolyl hydroxylases (data not shown). We then
treated a GalDBD-ODD fusion protein bound to agarose beads with the
hydroxylases or the hydroxylase EGFP fusion protein and analysed pVHL-binding
activity by incubation with 35S-labelled pVHL followed by
autoradiography. Although the activity of the fusion proteins appeared to be
somewhat reduced compared to the native enzymes, the fusion proteins have
clearly retained HIF-1
hydroxylase activity
(Fig. 3B). We then expressed
the PHD-EGFP fusions in U2OS cells and assessed the nuclear accumulation of
endogenous HIF-1
in response to hypoxia by immunohistochemistry. We
found that each of the PHDs reduced HIF-1
induction significantly
(Fig. 4). Interestingly cells
transfected with PHD1 showed a somewhat heterogeneous appearance with some
cells still staining positive for HIF-1
after hypoxic incubation. PHD2
and PHD3 transfection virtually eliminated the HIF-1
accumulation. As
expected, overexpression of FIH-1 did not result in a reduction of hypoxic
HIF-1
levels, as this enzyme is not involved in the HIF-1
degradation pathway. Taken together these results strongly suggest that
degradation of HIF-1
can be initiated by prolyl hydroxylation in the
nucleus as well as in the cytoplasm even under hypoxic conditions.
|
|
Effect of transient overexpression of HIF-1 hydroxylases on
HRE-luciferase expression
To confirm that all enzymes under investigation have a significant impact
on hypoxia-induced gene expression we transiently expressed the hydroxylases
in U2OS cells together with an HRE-driven luciferase reporter gene and
incubated the cells overnight in an atmosphere of 1% oxygen. Each of the PHDs
nearly abrogated hypoxic induction of the reporter gene construct
(Fig. 5). Repetition of the
assay in U2OS and Hep3B cells indicated that there was no reproducible,
statistically significant difference between PHD1, PHD2 and PHD3 with respect
to the inhibition of HRE-luciferase induction. FIH-1 had the same effect
although it does not act by pVHL binding and subsequent degradation but by
hydroxylation of Asp803, which impairs the recruitment of the transcriptional
coactivator p300/CBP.
|
Effect of hypoxia on the expression of endogenous PHDmRNA and
FIH-1mRNA
In HeLa cells, PHD2mRNA and PHD3mRNA are hardly detectable in normoxia but
are induced by hypoxia when PHD1 transcription is independent of ambient
oxygen concentration (Epstein et al.,
2001). In U2OS cells, expression of the PHDs and their response to
hypoxia has not been investigated so far. Quantitative RT-PCR revealed that
the transcripts of PHD1, PHD2 and FIH-1 are easily detectable in normoxia,
whereas PHD3 transcripts were very close to the detection limit. As in HeLa
cells PHD2mRNA and PHD3mRNA showed significant upregulation in U2OS cells in
response to hypoxia. Desferrioxamine (150 µM) induced the transcription of
the two prolyl hydroxylases PHD2 and PHD3 more efficiently than hypoxia or
cobalt ions. PHD1 and FIH-1, however, were expressed constitutively
(Fig. 6).
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Discussion |
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Interestingly, PHD1 is induced by estrogen and stimulates cell
proliferation (Seth et al.,
2002). It is possible that regulation of HIF-1
is not the
primary function of PHD1. From a more general perspective it seems that HIF
prolyl hydroxylases have widespread effects other than the regulation of
HIF-1
. C. elegans lacking the prototype HIF hydroxylase EGL-9
show an egg-laying defect
(Trent et al., 1983
) and are
resistant to an otherwise paralytic Pseudomonas aeruginosa toxin
(Darby et al., 1999
). A rat PHD
homologue named SM-20 takes part in survival/apoptosis decisions in
sympathetic neurons (Lipscomb et al.,
2001
). It is unclear at present whether these phenotypes are
related to loss of HIF-1 regulation or whether they are are caused by the loss
of hydroxylation of other PHD substrates. Certainly characterisation of
further PHD substrates will be important for defining new roles for prolyl
hydroxylation.
It was observed several years ago that the expression of C-TAD fusion
proteins is not oxygen dependent, whereas its transactivating function is
(Ema et al., 1999;
Pugh et al., 1997
). This
function was later found to be hydroxylation sensitive
(Sang et al., 2002
). In our
experiments FIH-1 did not prevent HIF-1
stabilisation, whereas it
markedly inhibited activation of an HRE-driven luciferase reporter gene. Our
results confirm that FIH-1 is not involved in the degradation pathway; instead
it inactivates the C-terminal transactivation domain of HIF-1
. Together
with our finding that FIH-1mRNA is easily detectable in normoxia it seems
likely that FIH-1 has an important function in cellular oxygen sensing.
Two out of three HIF-1 prolyl hydroxylases are hypoxia inducible in
U2OS cells (Fig. 6) and in
Hep3B cells (data not shown). PHD2 and PHD3 showed an upregulation of mRNA
expression after hypoxia or incubation with the hypoxia mimics,
desferrioxamine and cobalt ions. PHD1 was expressed constitutively, confirming
results reported previously for HeLa cells
(Epstein et al., 2001
). The
FIH-1mRNA expression could not be stimulated by hypoxic incubation. Our data
may suggest that PHD2 and PHD3 tightly regulate the HIF level in hypoxia to
avoid excessive nuclear HIF-1
accumulation. Prior to the
characterisation of the HIF-1
prolyl hydroxylases it had been
demonstrated that the half-life of HIF-1
after reoxygenation depends on
the duration of the hypoxic incubation before reoxygenation: a longer hypoxic
period shortens the half-life of HIF-1
(Berra et al., 2001
). This
finding, together with our data, suggests another function for PHD2 and PHD3:
during hypoxia, PHD2 and PHD3 are induced while the actual HIF-1
turnover is low because oxygen supply is limiting. In the case of
reoxygenation the half-life of HIF-1
would then be extremely short
because of a high PHD capacity within the cells.
In conclusion we propose a model in which HIF-1 can be hydroxylated
in the cytoplasm as well as in the cell nucleus. Because of their distinct
intracellular localisation the HIF-1
prolyl hydroxylases PHD1, PHD2,
PHD3 and the asparagine hydroxylase FIH-1 form a cascade of oxygen sensors
that tightly control the expression of HIF-1 target genes.
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Acknowledgments |
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References |
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---|
Berra, E., Richard, D. E., Gothie, E. and Pouyssegur, J. (2001). HIF-1-dependent transcriptional activity is required for oxygen-mediated HIF-1alpha degradation. FEBS Lett. 491, 85-90.[CrossRef][Medline]
Bestvater, F., Spiess, E., Stobrawa, G., Hacker, M., Feurer, T., Porwol, T., Berchner-Pfannschmidt, U., Wotzlaw, C. and Acker, H. (2002). Two-photon absorption and emission spectra of fluorochromes relevant for cell imaging. J. Microsc. 208,108 -115.[CrossRef][Medline]
Bruick, R. K. and McKnight, S. L. (2001). A
conserved family of prolyl-4-hydroxylases that modify HIF.
Science 294,1337
-1340.
Cockman, M. E., Masson, N., Mole, D. R., Jaakkola, P., Chang, G.
W., Clifford, S. C., Maher, E. R., Pugh, C. W., Ratcliffe, P. J. and Maxwell,
P. H. (2000). Hypoxia inducible factor- binding and
ubiquitylation by the von Hippel-Lindau tumor suppressor protein.
J. Biol. Chem. 275,25733
-25741.
Darby, C., Cosma, C. L., Thomas, J. H. and Manoil, C.
(1999). Lethal paralysis of Caenorhabditis elegans by
Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA
96,15202
-15207.
Ema, M., Hirota, K., Mimura, J., Abe, H., Yodoi, J., Sogawa, K.,
Poellinger, L. and Fujii-Kuriyama, Y. (1999). Molecular
mechanisms of transcription activation by HLF and HIF1 in response to
hypoxia: their stabilization and redox signal-induced interaction with
CBP/p300. EMBO J. 18,1905
-1914.
Epstein, A. C. R., Gleadle, J. M., McNeill, L. A., Hewitson, K. S., O'Rourke, J., Mole, D. R., Mukherji, M., Metzen, E., Wilson, M. I., Dhanda, A. et al. (2001). C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107,43 -54.[Medline]
Groulx, I. and Lee, S. (2002). Oxygen-dependent
ubiquitination and degradation of hypoxia-inducible factor requires
nuclear-cytoplasmic trafficking of the von Hippel-Lindau tumor suppressor
protein. Mol. Cell. Biol.
22,5319
-5336.
Hewitson, K. S., McNeill, L. A., Riordan, M. V., Tian, Y. M.,
Bullock, A. N., Welford, R. W., Elkins, J. M., Oldham, N. J., Bhattacharya,
S., Gleadle, J. M. et al. (2002) Hypoxia inducible factor
(HIF) asparagine hydroxylase is identical to Factor Inhibiting HIF (FIH) and
is related to the cupin structural family. J. Biol.
Chem. 277,26351
-26355.
Hofer, T., Desbaillets, I., Hopfl, G., Gassmann, M. and Wenger,
R. H. (2001). Dissecting hypoxia-dependent and
hypoxia-independent steps in the HIF-1alpha activation cascade: implications
for HIF-1alpha gene therapy. FASEB J.
15,2715
-2717.
Hon, W. C., Wilson, M. I., Harlos, K., Claridge, T. D., Schofield, C. J., Pugh, C. W., Maxwell, P. H., Ratcliffe, P. J., Stuart, D. I. and Jones, E. Y. (2002). Structural basis for the recognition of hydroxyproline in HIF-1alpha by pVHL. Nature 417,975 -978.[CrossRef][Medline]
Huang, J., Zhao, Q., Mooney, S. M. and Lee, F. S.
(2002). Sequence determinants in hypoxia inducible
factor-1 for hydroxylation by the prolyl hydroxylases PHD1, PHD2, and
PHD3. J. Biol. Chem.
277,39792
-39800.
Huang, L. E., Gu, J., Schau, M. and Bunn, H. F.
(1998). Regulation of hypoxia-inducible factor 1 is
mediated by an O2-dependent degradation domain via the
ubiquitin-proteasome pathway. Proc. Natl. Acad. Sci.
USA 95,7987
-7992.
Isaacs, J. S., Jung, Y. J., Mimnaugh, E. G., Martinez, A.,
Cuttitta, F. and Neckers, L. M. (2002). Hsp90 regulates a
VHL-independent HIF-1 degradative pathway. J. Biol.
Chem. 277,29936
-29944.
Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M.,
Salic, A., Asara, J. M., Lane, W. S. and Kaelin, W. G., Jr
(2001). HIF targeted for VHL-mediated destruction by
proline hydroxylation: implications for O2 sensing.
Science 292,464
-468.
Jaakkola, P., Mole, D. R., Tian, Y. M., Wilson, M. I., Gielbert,
J., Gaskell, S. J., Kriegsheim, A., Hebestreit, H. F., Mukherji, M.,
Schofield, C. J. et al. (2001). Targeting of HIF- to
the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl
hydroxylation. Science
292,468
-472.
Kallio, P. J., Okamoto, K., O'Brien, S., Carrero, P., Makino,
Y., Tanaka, H. and Poellinger, L. (1998). Signal transduction
in hypoxic cells: inducible nuclear translocation and recruitment of the
CBP/p300 coactivator by the hypoxia-inducible factor-1alpha. EMBO
J. 17,6573
-6586.
Lando, D., Peet, D. J., Whelan, D. A., Gorman, J. J. and
Whitelaw, M. L. (2002a) Asparagine hydroxylation of the HIF
transactivation domain: a hypoxic switch. Science
295,858
-861.
Lando, D., Peet, D. J., Gorman, J. J., Whelan, D. A., Whitelaw,
M. L. and Bruick, R. K. (2002b). FIH-1 is an asparaginyl
hydroxylase enzyme that regulates the transcriptional activity of
hypoxia-inducible factor. Genes Dev.
16,1466
-1471.
Lipscomb, E. A., Sarmiere, P. D. and Freeman, R. S.
(2001). SM-20 is a novel mitochondrial protein that causes
caspase-dependent cell death in nerve growth factor-dependent neurons.
J. Biol. Chem. 276,5085
-5092.
Mahon, P. C., Hirota, K. and Semenza, G. L.
(2001). FIH-1: a novel protein that interacts with HIF-1
and VHL to mediate repression of HIF-1 transcriptional activity.
Genes Dev. 15,2675
-2686.
Masson, N., Willam, C., Maxwell, P. H., Pugh, C. W. and
Ratcliffe, P. J. (2001). Independent function of two
destruction domains in hypoxia-inducible factor- chains activated by
prolyl hydroxylation. EMBO J.
20,5197
-5206.
Maxwell, P. H., Wiesener, M. S., Chang, G. W., Clifford, S. C., Vaux, E. C., Cockman, M. E., Wykoff, C. C., Pugh, C. W., Maher, E. R. and Ratcliffe, P. J. (1999). The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399,271 -275.[CrossRef][Medline]
Min, J. H., Yang, H., Ivan, M., Gertler, F., Kaelin, W. G., Jr
and Pavletich, N. P. (2002). Structure of an
HIF-1-pVHL complex: Hydroxyproline recognition in signaling.
Science 296,1886
-1889.
Oehme, F., Ellinghaus, P., Kolkhof, P., Smith, T. J., Ramakrishnan, S., Hütter, J., Schramm, M. and Flamme, I. (2002) Overexpression of PH-4, a novel putative proline 4-hydroxylase, modulates activity of hypoxia-inducible transcription factors. Biochem. Biophys. Res. Commun. 296,343 -349.[CrossRef][Medline]
Ohh, M., Park, C. W., Ivan, M., Hoffman, M. A., Kim, T. Y., Huang, L. E., Pavletich, N., Chau, V. and Kaelin, W. G. (2000). Ubiquitination of hypoxia-inducible factor requires direct binding to the ß-domain of the von Hippel-Lindau protein. Nat. Cell Biol. 2,423 -427.[CrossRef][Medline]
O'Rourke, J. F., Tian, Y. M., Ratcliffe, P. J. and Pugh, C.
W. (1999) Oxygen-regulated and transactivating domains in
endothelial PAS protein 1: comparison with hypoxia-inducible factor-1alpha.
J. Biol. Chem. 274,2060
-2071.
Pugh, C. W., O'Rourke, J. F., Nagao, M., Gleadle, J. M. and
Ratcliffe, P. J. (1997). Activation of hypoxia-inducible
factor-1; definition of regulatory domains within the subunit.
J. Biol. Chem. 272,11205
-11214.
Quimby, B. B. and Corbett, A. H. (2001). Nuclear transport mechanisms. Cell. Mol. Life Sci. 58,1766 -1773.[Medline]
Ruas, J. L., Poellinger, L. and Pereira, T.
(2002). Functional analysis of hypoxia-inducible
factor-1-mediated transactivation identification of amino acid
residues critical for transcriptional activation and/or interaction with CBP.
J. Biol. Chem. 277,38723
-38730.
Salceda, S. and Caro, J. (1997).
Hypoxia-inducible factor 1 (HIF-1
) protein is rapidly degraded
by the ubiquitin-proteasome system under normoxic conditions. Its
stabilization by hypoxia depends on redox-induced changes. J. Biol.
Chem. 272,22642
-22647.
Sang, N., Fang, J., Srinivas, V., Leshchinsky, I. and Caro,
J. (2002). Carboxyl-terminal transactivation activity of
hypoxia-inducible factor 1 is governed by a von Hippel-Lindau
protein-independent, hydroxylation-regulated association with p300/CBP.
Mol. Cell. Biol. 22,2984
-2992.
Semenza, G. L. (1998). Hypoxia-inducible factor 1: master regulator of O2 homeostasis. Curr. Opin. Genet. Dev. 8,588 -594.[CrossRef][Medline]
Semenza, G. L. (1999). Perspectives on oxygen sensing. Cell 98,281 -284.[Medline]
Seth, P., Krop, I., Porter, D. and Polyak, K. (2002). Novel estrogen and tamoxifen induced genes identified by SAGE (Serial Analysis of Gene Expression). Oncogene 21,836 -843.[CrossRef][Medline]
Tanimoto, K., Makino, Y., Pereira, T. and Poellinger, L.
(2000). Mechanism of regulation of the hypoxia-inducible
factor-1 by the von Hippel-Lindau tumor suppressor protein.
EMBO J. 19,4298
-4309.
Taylor, M. S. (2001). Characterization and comparative analysis of the EGLN gene family. Gene 275,125 -132.[CrossRef][Medline]
Trent, C., Tsung, N. and Horvitz, H. R. (1983).
EGG-laying defective mutants of the nematode Caenorhabditis elegans.Genetics 104,619
-647.
Wang, G. L., Jiang, B. H., Rue, E. A. and Semenza, G. L. (1995). Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc. Natl. Acad. Sci. USA 92,5510 -5514.[Abstract]
Wenger, R. H. (2000). Mammalian oxygen sensing,
signalling and gene regulation. J. Exp. Biol.
203,1253
-1263.
Wenger, R. H. (2002). Cellular adaptation to
hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible
transcription factors, and O2-regulated gene expression.
FASEB J. 16,1151
-1162.
Yu, F., White, S. B., Zhao, Q. and Lee, F. S.
(2001). HIF-1 binding to VHL is regulated by
stimulus-sensitive proline hydroxylation. Proc. Natl. Acad. Sci.
USA 98,9630
-9635.