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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2006.00174.x |
Differential and Reciprocal
Regulation between Hypoxia-inducible Factor-a
Subunits and Their Prolyl Hydroxylases in Pulmonary Arteries of Rat with
Hypoxia-induced Hypertension
Yun-Rong CHEN, Ai-Guo DAI*,
Rui-Cheng HU, and Yong-Liang JIANG
Department of Respiratory Medicine, Hunan
Institute of Gerontology,
Received:
January 25, 2006
Accepted:
March 27, 2006
This
work was supported by the grants from the National Natural Science Foundation
of China (No. 30570815 and 30270581), the Hunan Province Natural Science
Foundation of China (No. 05JJ30073), the Major Science and Technology
Foundation of the Ministry of Education of China (No. 03091) and the
Postdoctorate Science Foundation of China (No. 2003033436)
*Corresponding
author: Tel, 86-731-4762793; Fax, 86-731-4735215; E-mail, [email protected]
Abstract Hypoxia-inducible factor (HIF)-a subunits (HIF-1a,
HIF-2a and HIF-3a), which play a pivotal role during the
development of hypoxia-induced pulmonary hypertension (HPH), are regulated
through post-translational hydroxylation by their three prolyl hydroxylase
domain-containing proteins (PHD1, PHD2 and PHD3). PHDs could also be regulated
by HIF. But differential and reciprocal regulation between HIF-a and PHDs during the development of HPH
remains unclear. To investigate this, a rat HPH model was established. Mean
pulmonary arterial pressure increased significantly after 7 d of hypoxia.
Pulmonary artery remodeling index and right ventricular hypertrophy became
evident after 14 d of hypoxia. HIF-1a and
HIF-2a mRNA increased a little after
7 d of hypoxia, but HIF-3a increased significantly after
3 d of hypoxia. The protein expression levels of all three HIF-a were markedly upregulated after exposure
to hypoxia. PHD2 mRNA and protein expression levels were upregulated after 3 d
of hypoxia; PHD1 protein declined after 14 d of hypoxia without significant
mRNA changes. PHD3 mRNA and protein were markedly upregulated after 3 d of
hypoxia, then the mRNA remained at a high level, but the protein declined after
14 d of hypoxia. In hypoxic animals, HIF-1a
proteins negatively correlated with PHD2 proteins, whereas HIF-2a and HIF-3a
proteins showed negative correlations with PHD3 and PHD1 proteins,
respectively. All three HIF-a proteins were positively
correlated with PHD2 and PHD3 mRNA. In the present study, HIF-a subunits and PHDs showed differential
and reciprocal regulation, and this might play a key pathogenetic role in
hypoxia-induced pulmonary hypertension.
Key words hypoxia-inducible factor; prolyl
hydroxylase; hypertension; pulmonary; hydroxylation
Hypoxia-inducible factor (HIF) is an ab-heterodimer consisting of
one of three HIF-a subunits (HIF-1a, HIF-2a or HIF-3a), and the aryl hydrocarbon receptor nuclear translocator, also
known as HIF1-b. HIF is one of the most important factors in the cellular response
to hypoxia, which might regulate the transcription of more than 5% of all human
genes (over 70 genes have so far been identified) in pulmonary artery
endothelial cells [1]. Heterozygous HIF-1a deficient (Hif-1a+/–) mice have impaired
pulmonary vascular remodeling after exposure to 10% O2 for 3 weeks, whereas
heterozygous HIF-2a deficient (Hif-2a+/–)
mice show a complete absence of pulmonary vascular remodeling after exposure to
10% O2 for 4 weeks [2]. Our previous studies showed
that the three HIF-a subunits, with differential dynamic expression [3], play a critical
role during the development of rat hypoxia-induced pulmonary hypertension (HPH)
together with its target genes such as heme oxygenase-1, vascular endothelial
growth factor and inducible nitric oxide synthase [4–6].
HIF-b subunits are constitutive nuclear proteins, whereas HIF-a subunits are
inducible by hypoxia in animals and cultured cells. HIF-a subunits are
translated constitutively but have a very short half-life under normal oxygen
concentration. Regulation of the half-life and activity of the HIF-a subunits is
dependent on their post-translational hydroxylation by hydroxylases [7]. Prolyl
hydroxylation of HIF-a subunits is required for binding of the von Hippel-Lindau tumor
suppressor protein, which is the recognition component of an E3
ubiquitin-protein ligase that targets HIF-1a for proteasomal
degradation [8]. Three prolyl hydroxylase domain-containing proteins (PHD1,
PHD2 and PHD3) that are responsible for this modification have been identified
[9]. They are all Fe2+– and
2-oxoglutarate-dependent dioxygenases with an absolute requirement for
molecular oxygen, allowing HIF to escape degradation in hypoxia. The enzymatic
process splits dioxygen, with one oxygen atom creating the hydroxylated amino
acid, and the other oxidizing 2-oxoglutarate to succinate with the release of
CO2 [9]. PHDs are critical oxygen
sensors regulating the degradation of oxygen-dependent HIF-a subunits either
in normoxia or following exposure to hypoxia, but their roles in HPH have so
far not been studied.
To evaluate the differential role of the three PHDs with respect to
HIF-a hydroxylation and expression in pulmonary hypertension, we studied
the expression patterns of PHDs and HIF-a subunits in pulmonary
arteries of rats at different phases of HPH development.
Materials and Methods
Materials
Hypoxia and normoxia rat models were set up as described previously
[10]. Briefly, 40 Wistar rats (male, 270±
mPAP and RVHI measurement
mPAP was measured as described
previously [10]. After rats were anesthetized with pentobarbital sodium (40
mg/kg intraperitoneally), a specially designed single lumen catheter was
inserted into the main pulmonary artery through the right jugular vein. The
injecting position was confirmed by the waveform of pressure. Through this
catheter, mPAP was measured using a Medlab bio-signal operating system
(Nanjing MedEase Science and Technology,
Eq. 1
Morphometric analysis
Lung slices of 5 mm were embedded with paraffin, stained with hematoxylin-eosin, then
by elastic fiber staining, and examined under a light microscope. At least five
representative pulmonary arteries (approximately 100 mm in outer diameter) chosen
from three different sections of each animal were independently examined. To
evaluate hypoxic remodeling by calculating the parameters of pulmonary vascular
cross-sections, the ratio of vascular wall area to total vascular area (WA)
and pulmonary artery media thickness (PAMT) were measured. The images of
the arteries were captured and analyzed using PIPS-2020 image software
(Chongqing Tianhai Company,
In situ hybridization of HIF-a subunits and PHD mRNA
In situ hybridization was carried out
using an In situ hybridization detection kit (Wuhan Boster Biological
Technology,
5‘-TGCGGTACTATGGTATCTGCGTCAAGGACAACTTC-3‘; and 5‘-TTCAAGTACCCGTGTCACAGCCAGCTACACCTACC-3‘.
The sequences of probes against PHD2 mRNA were: 5‘-AGCAGATCGGCGACGAGGTGCGCGCCCTGCACGAC-3‘;
5‘-ATGAGCAGCATGGACGACCTGATCCGCCACTGCAG-3‘; and 5‘-GTTGAACTCAAGCCCAATTCAGTCAGCAAAGACGT-3‘.
The sequences of probes against PHD3 mRNA were: 5‘-ATCTCCAAAAGGGGCCCTCCGACTTCTCACTGGGC-3‘;
5‘-CACGAGGTCGGTTTCTGCTACCTGGACAACTTCCT-3‘; and 5‘-TTCAGGAATCTAACTAGAAAAACTGAATCTGCTCT-3‘.
Hybridization was carried out on serial lung tissue slices in
paraffin fixed by formalin containing 0.1% diethypyrocarbonate according to the
manufacturer’s instructions. Briefly, slices were digested with pepsin for 15
min at 37 ºC. After 2 h of pre-hybridization, slices underwent hybridization
with digoxin-labeled single-stranded oligonucleotide probes for 16 h at 38 ºC.
In negative control studies, labeled oligonucleotide probes were substituted by
phosphate-buffered saline. After washing off unbound probes, slices were
incubated firstly with rabbit antibodies against digoxin, then with
biotinylated goat antibodies against rabbit. Slices were then incubated with
streptavidin-biotin-peroxidase. Peroxidase activity was visualized by a color
reaction using diaminobenzidine (Wuhan Boster Biological Technology) as the
substrate. Brown and yellow colors indicated positive results (mainly in
cytoplasm). Finally, the sections were counterstained with hematoxylin
(resulting in blue nuclei) and mounted. Expression levels of mRNA were
quantified by a pathology image analysis system (PIPS-2020; Chongqing Tianhai)
using absorbance of positive signal.
Reverse
transcription-polymerase chain reaction (RT-PCR) analysis for HIF-a subunits and PHD genes
Total RNA was extracted using Trizol reagent (Sangon,
Immunohistochemistry for HIF-a subunits and PHD proteins
A commercial streptavidin-biotin complex kit (Wuhan Boster
Biological Technology) was used for immunohistochemistry, which was carried out
similar to that described previously [3]. Briefly, serial sections of
formalin-fixed paraffin-embedded lung tissues were digested with 3% H2O2 for 20 min at room
temperature, then preincubated with 10% nonimmunized serum. Sections were
incubated with goat anti-HIF-1a, anti-HIF-2a and anti-HIF-3a (Santa Cruz Biotechnology, Santa Cruz, USA) specific polyclonal
antibodies (1:200) or rabbit anti-PHD1, anti-PHD2 and anti-PHD3 (Novus
Biological, Littleton, USA) specific polyclonal antibodies (1:300) overnight at
4 ºC respectively. Phosphate-buffered saline was used as the negative control
in this study. After unbound antibodies were washed off, the sections were
incubated with corresponding biotinylated secondary antibodies against rabbit
and thereafter incubated with streptavidin peroxidase. Subsequently, peroxidase
activity was visualized by a color reaction with diaminobenzidine similar to
that of in situ hybridization.
Western blot analysis for HIF-a subunits and PHD proteins in lung tissue
Proteins from whole tissue samples were extracted using a modified
RIPA homogenization buffer [50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5%
deoxycholic acid, 1 mM EDTA (1%), 1% NP-40, 0.1 mg/ml phenylmethylsulphonyl
fluoride and 2 mg/ml leupeptin]. Protein concentrations were determined by the
Statistical analysis
Data are expressed as mean±standard deviation. One-way ANOVA was used to determine
statistically significant difference in more than two groups, and the
Newman-Keuls test was used to analyze statistical significance between two
groups. P<0.05 was considered as a statistically significant
difference.
Results
Chronic hypoxia increases mPAP
In hypoxic animals, mPAP changed as expected: pulmonary
hypertension was increased after 7 d of exposure to hypoxia (P<0.05),
reached its peak after 14 d of hypoxia, then remained unchanged (Table 2).
Chronic hypoxia-induced
hypoxic pulmonary vascular remodeling and right ventricular hypertrophy
The wall area and medial thickness of pulmonary arterioles were
increased after 7 d of hypoxic exposure (Table 2). Quantification of
these changes, compared with normoxic controls, revealed that PAMT increased
significantly in hypoxic animals after 7 d of hypoxia and the increase of WA%
became significant after 14 d of hypoxia. They both increased further with
prolonged hypoxia. Right ventricular hypertrophy is a hallmark of pulmonary
hypertension resulting from right ventricle pressure overload. After 14 d of
hypoxia, RVHI was significantly increased compared with the control, and
increased further after 21 d of hypoxia. These data indicated that right
ventricular hypertrophy developed after 14 d of exposure to hypoxia (Table 2).
mRNA and protein levels of three HIF-a subunits in pulmonary arterial walls during normoxia and chronic hypoxia
conditions
All three HIF-a mRNA were detected using RT-PCR, but HIF-3a was at a lower level in
lungs of control rats (Fig. 1). HIF-3a mRNA increased markedly
after 3 d of hypoxia, reached its peak at hypoxia day 7, then stabilized. HIF-1a mRNA was
slightly upregulated after 7 d of hypoxia. HIF-2a mRNA levels were
significantly increased in lungs of hypoxic rats after 7 d and 14 d, but were
comparable to that of control rats at hypoxia day 21 (Fig. 1). The
results of in situ hybridization showed the same dynamic expression of
HIF-a mRNA in pulmonary arterioles (Figs. 2–4).
All three HIF-a proteins were hypoxia-inducible in rat lung. In the Western blot
results, even in normoxia, the expression of all three HIF-a proteins in the
lung tissue could be clearly detected. Expression was enhanced after 3 d of
systemic hypoxia and increased further with the prolonged duration of hypoxia,
except that HIF-3a proteins declined slightly after 14 d of hypoxia (Fig. 5).
In normoxic rat lung, all three HIF-a proteins were stained weakly in wall of
artery but strongly in the smooth muscle and epithelium of airways. HIF-1a and HIF-2a were positive
after 3 d and 7 d of hypoxia. HIF-1a then lessened but HIF-2a increased
further till hypoxia day 14, then remained unchanged (Figs. 6 and 7).
HIF-3a staining became strong after 7 d of hypoxia, and remained stable
thereafter (Fig. 8).
mRNA and protein levels of PHD1, PHD2 and
PHD3 in pulmonary arterial walls during normoxia and chronic hypoxia
conditions
Although PHD1 mRNA showed little change (P>0.05) according
to in situ hybridization (Fig. 9) and RT-PCR analysis (Fig. 1),
in hypoxic animals over time, PHD1 protein showed a gradual decrease in hypoxic
animals compared with controls. This decrease became significant at hypoxia day
7 (Fig. 5). Immunohistochemical analysis revealed that PHD1 was highly
expressed in the pulmonary arterial wall of control rats. No significant
changes were observed through 3 d and 7 d of hypoxia. However, PHD1-specific
staining appeared to be relatively weak in both smooth muscle and endothelium
of small pulmonary arteries in rats suffering hypoxic stress for 14 d and 21 d
compared with controls (Fig. 10).
The result of in situ hybridization (Fig. 11) and
RT-PCR (Fig. 1) analysis showed that PHD2 mRNA was clearly expressed in
rat lung in normorxia. PHD2 mRNA increased gradually until hypoxia day 14, then
remained unchanged. Western blot showed that PHD2 protein expression in rats
was increased with prolonged hypoxia. Compared with control values, an increase
in PHD2 protein expression was evident at hypoxia day 3 and was significant
after hypoxia day 7 (Fig. 5). Immunohistochemical analysis revealed that
PHD2 was clearly expressed in the pulmonary arterial wall of control rats.
Increased staining of PHD2 was found in the smooth muscle layer and endothelium
of small pulmonary artery after 7 days of hypoxia (Fig. 12).
In normorxic rat lung, in situ hybridization (Fig. 13)
and RT-PCR (Fig. 1) analysis showed that PHD3 mRNA was expressed at
relatively low levels compared with PHD1 and PHD2, but was upregulated more
dramatically after exposure to hypoxia. PHD3 protein was also detectable in
normoxic rats, and its level was relatively lower than PHD1 or PHD2. PHD3
protein levels were markedly increased in the lung of rats at hypoxia day 3 and
day 7 (Fig. 5). The levels decreased significantly after 14 d and 21 d
of hypoxia, but were still higher than that in the lung of control rats (Fig.
5). Immunohistochemical analysis showed that PHD3 was weakly expressed in
the lung of control rats. PHD3 staining appeared to be much more intense in
both smooth muscle and endothelium of small pulmonary arteries after 3 d and 7
d of hypoxia compared with controls. At hypoxia day 14 and 21, PHD3 level was
significantly lower than at hypoxia day 3 and day 7, but significantly higher
than in normoxia (Fig. 14).
Analysis of linear correlation
Linear correlation analysis showed that there was a significant
negative correlation between HIF-1a and PHD2. HIF-2a and HIF-3a showed
significant negative correlations with PHD3 and PHD1, respectively, in hypoxic
animals. All three HIF-a proteins were positively correlated with PHD2 and PHD3 mRNA, but no
correlation was observed between PHD1 mRNA and HIF-a proteins.
Discussion
Chronic hypoxia, due to various obstructive and restrictive lung diseases,
is well known to elicit remodeling of the pulmonary vasculature that is
characterized by structural and functional changes in the intima, media and
adventitia of the pulmonary artery. These changes cause chronic pulmonary
arterial hypertension and subsequent cor pulmonale. HIF plays a pivotal role in
vascular remodeling by regulating the expression of its target genes.
Recent studies have defined post-translational modification by PHDs
as a key regulatory event that targets HIF-a for proteasomal destruction
through the von Hippel-Lindau ubiquitylation complex [7,11]. The present study
showed that, when exposed to hypoxia, HIF-a proteins increased
dramatically, whereas HIF-a mRNA was upregulated only a little in rat lung. It suggested the
post-translational mechanism in rat lung. The Km values of the three PHDs for
O2 are slightly above atmospheric concentration,
so that any decrease in O2 will result in a reduction in the rate of
hydroxylation and an accumulation of HIF protein [12]. This might be why
moderate hypoxia (10%) leads to marked accumulation of HIF-a proteins in rat
lung. In cultured cells, Appelhoff et al. [13] found that HIF-a levels were
induced maximally early after hypoxic exposure, then declined significantly due
to hypoxia-induced PHD3 and PHD2 levels. The effect was more marked for HIF-2a than for HIF-1a. Interestingly,
our data showed, after 14 d of hypoxia, a decline in HIF-1a levels but
constant high levels of HIF-2a. The disparity in these results might be attributed to the different
expression patterns of PHDs between their study and ours. In our study, PHD1
protein was downregulated after hypoxia exposure and PHD3 protein, in spite of
upregulation at earlier stages (hypoxia for 3–7 d), was decreased
significantly, compared with hypoxia day 3–7, at later stages (hypoxia
for 14 and 7 d).
Although all three PHD proteins contributed to the regulation of
HIF-1a and HIF-2a, relative selectivity has also been reported [12–15]. The
induction of HIF-2a was affected less than HIF-1a by specific gene silencing
of PHD2 using short interfering RNA (siRNA) [13,15]. However, PHD3 suppression
alone led to induction of HIF-2a but not HIF-1a [13]. These results indicate that PHD2 has the highest specific
activity toward the primary hydroxylation site of HIF-1a, and PHD3 has the highest
specificity for hydroxylation of HIF-2a [13,14]. There was also a significant
negative correlation between HIF-1a protein and PHD2 protein. In hypoxic rats
(control was excluded), HIF-2a and HIF-3a proteins showed significant negative correlations with PHD3 and
PHD1 proteins, respectively. These results suggest that the same relative
selectivity exists in rat lung after hypoxia exposure. This selectivity
contributes, at least in part, to the differential expression patterns of the
three HIF-a proteins. It also implies that PHDs retain significant activity in
hypoxic rat lungs.
Many studies of cultured cells have shown that the expression of
PHD2 and PHD3, not PHD1, is inducible by hypoxia [16–19]. Our data showed that
PHD2 mRNA was increased moderately and PHD3 mRNA was upregulated dramatically
after exposure to hypoxia. In cell lines with well defined deficiencies in the
activation of HIF, no change of PHD3 or PHD2 mRNAs was observed upon exposure
to low oxygen tension; and inactivation of pVHL, a protein necessary for
oxygen-dependent degradation of HIF-1a, is sufficient to induce PHD3 mRNA in
normoxia [17]. Furthermore, Metzen et al. [20] have identified the
dominant promoter of the phd2 gene, which contains a functional
hypoxia-responsive element and confers hypoxic inducibility. These studies
suggested that the hypoxia-induced expression of PHDs is in a HIF-a dependent
manner. In the present study, linear correlation analysis showed that HIF-a proteins were
positively correlated with PHD2 and PHD3 mRNA, but there was no correlation
between PHD1 mRNA and HIF-a protein. This suggested that PHD2 and PHD3 might also be directly
upregulated by HIF-a.
Silence of HIF-1a by siRNA results in significant downregulation of hypoxia-induced
PHD2 and PHD3 levels [16,19]. Knockdown of HIF-2a by siRNA reduced hypoxic
induction of PHD3, but not PHD2 [19]. This indicated relative selectivity for
the regulation of PHD isoforms by HIF-a isoforms, but whether this selectivity is
applicable in the rat model needs to be investigated further. One published
study also showed that PHD1 expression is reduced under hypoxic conditions
[21], but the same reduction of PHD1 mRNA could not be observed in the lung of
our HPH model. Taken together, in the lung of our HPH model, PHDs and HIF-a would form a
feedback loop when exposed to hypoxia: the hypoxia-induced accumulation of HIF-a leads to the
increase in the total amount of PHD1 and PHD3 enzymes after a long time. As a
result, at least under moderate hypoxia, PHD induction should result in
augmented HIF-a degradation and provide a feedback control on HIF signaling.
In the present study, the mRNA expression levels of PHD1 and PHD3
did not parallel their proteins. PHD1 proteins declined gradually without
significant changes after the rats were exposed to hypoxia. After 7 d and 14 d
of hypoxia, although PHD3 mRNA increased further, PHD3 protein was decreased
significantly. This indicated that PHDs might be downregulated by some
post-transcriptional mechanism. Nakayama et al. recently documented that
Siah1a/2, mammalian homologues of the Drosophila seven-in-absentia,
targets PHD1 and PHD3 for proteasome-dependent degradation under hypoxic
conditions [22]. But further research is needed to clarify whether Siah1a/2
contributes to the regulation of PHDs during the development of HPH.
Considering that HIF-a signaling is fundamental for the homeostasis
of pulmonary arteries, we propose that alteration of PHD expression levels and
activity, due to decreased availability of oxygen, and subsequent accumulation
of HIF-a, is likely to play an important role in the development of the
pulmonary vascular remodeling in our model of pulmonary hypertension secondary
to increased systemic hypoxic stress. Further understanding of these mechanisms
could lead to potential new therapies for the management of hypoxic pulmonary
hypertension.
References
1 Manalo DJ, Rowan A, Lavoie T, Natarajan L,
Kelly BD, Ye SQ, Garcia JG et al. Transcriptional regulation of vascular
endothelial cell responses to hypoxia by HIF-1. Blood 2005, 105: 659–669
2 Brusselmans K, Compernolle V, Tjwa M,
Wiesener MS, Maxwell PH, Collen D, Carmeliet P. Heterozygous deficiency of
hypoxia-inducible factor-2a protects mice against
pulmonary hypertension and right ventricular dysfunction during prolonged
hypoxia. J Clin Invest 2003, 111: 1519–1527
3 Li QF, Dai AG. Differential expression of
three hypoxia-inducible factor-a subunits in pulmonary
arteries of rat with hypoxia-induced hypertension. Acta Biochim Biophys Sin
2005, 37: 665–672
4 Hu R, Dai AG, Tan S. Hypoxia-inducible factor
1 alpha upregulates the expression of inducible nitric oxide synthase gene in
pulmonary arteries of hypoxic rat. Chin Med J 2002, 115: 1833–1837
5 Li QF, Dai AG. Hypoxia inducible factor-1 a correlates the
expression of heme oxygenase 1 gene in pulmonary arteries of rat with
hypoxia-induced pulmonary hypertension. Acta Biochim Biophys Sin 2004, 36: 133–140
6 Li QF, Dai AG. Hypoxia-inducible factor-1a regulates the role
of vascular endothelial growth factor on pulmonary arteries of rats with
hypoxia-induced pulmonary hypertension. Chin Med J 2004, 117: 1023–1028
7 Jaakkola P, Mole DR, Tian YM, Wilson MI,
Gielbert J, Gaskell SJ, Kriegsheim A et al. Targeting of HIF-a to the von
Hippel–Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 2001, 292:
468–472
8 Kaelin WG. Proline hydroxylation and gene
expression. Annu Rev Biochem 2005, 74115–74128
9 Epstein AC, Gleadle JM, McNeill LA, Hewitson
KS, O’Rourke J, Mole DR, Mukherji M et al. C. elegans EGL-9 and
mammalian homologs define a family of dioxygenases that regulate HIF by prolyl
hydroxylation. Cell 2001, 107: 43–54
10 Jiang YL, Dai AG, Li QF, Hu RC. Transforming
growth factor-b1 induces transdifferentiation of fibroblasts into
myofibroblasts in hypoxic pulmonary vascular remodeling. Acta Biochim Biophys
Sin 2006, 38: 29–36
11 Brahimi-Horn C, Mazure N, Pouyssegur J.
Signalling via the hypoxia-inducible factor-1alpha requires multiple
posttranslational modifications. Cell Signal 2005, 17: 1–9.
12 Hirsila M, Koivunen P, Gunzler V, Kivirikko
KI, Myllyharju J. Characterization of the human prolyl 4-hydroxylases that
modify the hypoxia-inducible factor. J Biol Chem 2003, 278: 30772–30780
13 Appelhoff RJ, Tian YM, Raval RR, Turley H,
Harris AL, Pugh CW, Ratcliffe PJ et al. Differential function of the
prolyl hydroxylases PHD1, PHD2, and PHD3 in the regulation of hypoxia-inducible
factor. J Biol Chem 2004, 279: 38458–38465
14 Huang J, Zhao Q, Mooney SM, Lee FS. Sequence
determinants in hypoxia-inducible factor-1a for hydroxylation
by the prolyl hydroxylases PHD1, PHD2, and PHD3. J Biol Chem 2002, 277: 39792–39800
15 Berra E, Benizri E, Ginouves A, Volmat V, Roux
D, Pouyssegur J. HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low
steady-state levels of HIF-1a in normoxia. EMBO J 2003, 22:
4082–4090
16 Aprelikova O, Chandramouli GV, Wood M,
Vasselli JR, Riss J, Maranchie JK, Linehan WM et al. Regulation of HIF prolyl
hydroxylases by hypoxia-inducible factors. J Cell Biochem 2004, 92: 491–501
17 del Peso L, Castellanos MC, Temes E,
Martin-Puig S, Cuevas Y, Olmos G, Landazuri MO. The von Hippel
Lindau/hypoxia-inducible factor (HIF) pathway regulates the transcription of
the HIF-proline hydroxylase genes in response to low oxygen. J Biol Chem 2003,
278: 48690–48695
18 D’Angelo G, Duplan E, Boyer N, Vigne P, Frelin
C. Hypoxia up-regulates prolyl hydroxylase activity: A feedback mechanism that
limits HIF-1 responses during reoxygenation. J Biol Chem 2003, 278: 38183–38187
19 Marxsen JH, Stengel P, Doege K, Heikkinen P,
Jokilehto T, Wagner T, Jelkmann W et al. Hypoxia-inducible factor-1
(HIF-1) promotes its degradation by induction of HIF-a-prolyl-4-hydroxylases.
Biochem J 2004, 381: 761–767
20 Metzen E, Stiehl DP, Doege K, Marxsen JH,
Hellwig-Burgel T, Jelkmann W. Regulation of the prolyl hydroxylase domain
protein 2 (phd2/egln-1) gene: Identification of a functional hypoxia-responsive
element. Biochem J 2005, 387: 711–717
21 Erez N, Stambolsky P, Shats I, Milyavsky M,
Kachko T, Rotter V. Hypoxia-dependent regulation of PHD1: Cloning and
characterization of the human PHD1/EGLN2 gene promoter. FEBS Lett 2004, 567:
311–315
22 Nakayama K, Frew IJ, Hagensen M, Skals M,
Habelhah H, Bhoumik A, Kadoya T et al. Siah2 regulates stability of
prolyl-hydroxylases, controls HIF1a abundance, and modulates
physiological responses to hypoxia. Cell 2004, 117: 941–952