doi:10.1182/blood-2012-08-449181
Prepublished online December 20, 2012;
2013 121: 1436-1445
Joachim Fandrey, Max Gassmann and Ben Wielockx
Gustavo Baretton, Guo-Hua Fong, David R. Greaves, Stefan Bornstein, Triantafyllos Chavakis,
Alex M. Sykes, David M. Poitz, Tatsiana Ripich, Teresa Otto, Sybille Bergmann, Georg Breier,
Weidemann, Vasuprada Iyengar, Steffen Jahn, Kathrin Wieczorek, Kathrin Geiger, Michael Muders,
Kristin Franke, Joanna Kalucka, Soulafa Mamlouk, Rashim Pal Singh, Antje Muschter, Alexander
induced excessive erythropoiesis−αfrom severe HIF-2
is a protective factor in conditional PHD2-deficient mice sufferingαHIF-1
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Regular Article
RED CELLS, IRON, AND ERYTHROPOIESIS
HIF-1␣ is a protective factor in conditional PHD2-deficient mice suffering
from severe HIF-2␣–induced excessive erythropoiesis
*Kristin Franke,1,2 *Joanna Kalucka,1,2 *Soulafa Mamlouk,1,2 *Rashim Pal Singh,1,2 Antje Muschter,1,2
Alexander Weidemann,3 Vasuprada Iyengar,1,2 Steffen Jahn,2 Kathrin Wieczorek,2 Kathrin Geiger,2 Michael Muders,2
Alex M. Sykes,4 David M. Poitz,5 Tatsiana Ripich,6 Teresa Otto,7 Sybille Bergmann,8 Georg Breier,2,9 Gustavo Baretton,2
Guo-Hua Fong,10 David R. Greaves,11 Stefan Bornstein,12 Triantafyllos Chavakis,12 Joachim Fandrey,7 Max Gassmann,13,14
and Ben Wielockx1,2,9
1Emmy Noether Research Group, and 2Institute of Pathology, University of Technology, Dresden, Germany; 3Department of Nephrology and Hypertension,
University Clinic Erlangen, Erlangen, Germany; 4Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany; 5Department of Internal
Medicine and Cardiology, University of Technology, Dresden, Germany; 6Institute of Physiologic Chemistry, University of Technology, Dresden, Germany;
7Department of Physiology, University of Duisburg-Essen, Essen, Germany; 8Institute of Clinical Chemistry and Laboratory Medicine, University of Technology,
Dresden, Germany; 9DFG Research Center and Cluster of Excellence for Regenerative Therapies Dresden, University of Technology, Dresden, Germany;
10Center for Vascular Biology, Department of Cell Biology, University of Connecticut Health Center, Farmington, CT; 11Sir William Dunn School of Pathology,
University of Oxford, Oxford, United Kingdom; 12Department of Medicine III, Carl Gustav Carus University Hospital, Dresden, Germany; 13Institute of Veterinary
Physiology, Vetsuisse Faculty and Zurich Center for Integrative Human Physiology (ZIHP), University of Zu¨rich, Zu¨rich, Switzerland; and 14Universidad Peruana
Cayetano Heredia (UPCH), Lima, Peru
Key Points
• Loss of PHD2 induces HIF-
2␣–induced erythrocytosis.
• HIF-1␣ protects conditional
PHD2-deficient mice.
Erythropoiesis must be tightly balanced to guarantee adequate oxygen delivery to all
tissues in the body. This process relies predominantly on the hormone erythropoietin
(EPO) and its transcription factor hypoxia inducible factor (HIF). Accumulating evidence
suggests that oxygen-sensitive prolyl hydroxylases (PHDs) are important regulators
of this entire system. Here, we describe a novel mouse line with conditional PHD2
inactivation (cKO P2) in renal EPO producing cells, neurons, and astrocytes that
displayed excessive erythrocytosis because of severe overproduction of EPO,
exclusively driven by HIF-2␣. In contrast, HIF-1␣ served as a protective factor, ensuring survival of cKO P2 mice with HCT values up
to 86%. Using different genetic approaches, we show that simultaneous inactivation of PHD2 and HIF-1␣ resulted in a drastic PHD3
reduction with consequent overexpression of HIF-2␣-related genes, neurodegeneration, and lethality. Taken together, our results
demonstrate for the first time that conditional loss of PHD2 in mice leads to HIF-2␣–dependent erythrocytosis, whereas HIF-1␣
protects these mice, providing a platform for developing new treatments of EPO-related disorders, such as anemia. (Blood.
2013;121(8):1436-1445)
Introduction
Erythropoietin (EPO) is the primary regulator of red blood cell
formation and its expression level is extremely responsive to
fluctuations in tissue oxygenation. Dysregulation of EPO production
can lead to either anemia, if levels are inadequately low, such
as in terminal kidney failure or to erythrocytosis if levels are
inappropriately high. Erythrocytosis will develop when EPO is
continuously overproduced, causing the generation of new erythrocytes
at a rate exceeding the removal of senescent red blood cells.1
In adult animals the main physiologic source of EPO is the kidney,
although the liver as well as the central nervous system (CNS) have
also been shown to produce EPO but to a much lesser extent.2,3 The
main physiologic stimulus of EPO production is tissue hypoxia in
the interstitium of the kidney.4 Already 2 decades ago it was shown
that this effect was dependent on binding of the transcription factor
hypoxia inducible factor (HIF) to 5Ј and 3Ј-enhancer regions of the
EPO gene, known as the hypoxia responsive element (HRE).5
HIF is a heterodimeric complex composed of an oxygensensitive
HIF␣ and a constitutive HIF␤ subunit. Of the most
intensively studied HIF-␣ genes, HIF-1␣ has a ubiquitous pattern
of expression in all tissues,6 whereas expression of the paralogue
HIF-2␣ is restricted to certain cell types.7,8 However, both factors
actively promote oxygen delivery and adaptive processes to
hypoxia such as erythropoiesis, angiogenesis, anaerobic glycolysis,
and hematopoiesis.1,9-11 Oxygen-sensing is therefore indispensable
as it enables the cells to instantaneously adapt to an inappropriately
low pO2. This machinery relies on the HIF-prolyl hydroxylases
(PHD1-3), enzymes that hydroxylate, and consequently, lead to the
inactivation of HIF␣ in the presence of oxygen.
Submitted August 21, 2012; accepted December 2, 2012. Prepublished online
as Blood First Edition paper, December 21, 2012; DOI 10.1182/blood-2012-08-
449181.
*K.F., J.K., S.M., and R.P.S. contributed equally to this work.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2013 by The American Society of Hematology
1436 BLOOD, 21 FEBRUARY 2013 ⅐ VOLUME 121, NUMBER 8
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PHD2 is believed to be the crucial oxygen sensor during
normoxia and mild hypoxia,12 which is underscored by the fact that
inactivation of PHD2 severely deregulates normal embryonic
development resulting in embryonic lethality by E14.5, whereas
PHD1Ϫ/Ϫ or PHD3Ϫ/Ϫ mice develop normally.13 Moreover, even
haplodeficient PHD2ϩ/Ϫ mice show normalization of the endothelial
lining during tumor development compared with wild-type
controls.14 In humans, several heterozygous point mutations in the
PHD2 gene have been described, which lead to an absolute
increase in red blood cell mass. These are in some cases associated
with severe clinical conditions, such as hemorrhage, stroke, and
increased risk of thrombosis,15-18 although the latter was shown to
be independent of elevated hemoglobin/HCT.19 PHD2 is therefore
the main HIF prolyl hydroxylase isoform which, next to HIF-2␣
and the von Hippel-Lindau protein (VHL), controls the expression
of EPO in humans.20 Despite this, the role of PHD2 in the etiology
of erythrocytosis and related disorders has not been extensively
studied. Indeed, systemic PHD2 heterozygosity in mice shows only
a very mild induction of hematocrits (HCTs),14 whereas somatic
inactivation shortly before or after birth leads to severe erythrocytosis
and early lethality.21,22
In this report, we describe a new conditional PHD2 mouse line
expressing cre-recombinase under the control of the modified
human CD68 promoter, commonly defined as a monocyte/
macrophage marker.23 In addition, we found PHD2-loss in other
cell lineages, resulting in excessive HIF-2␣–induced EPO production
in kidney and brain, extreme HCTs up to 86%, thrombocytopenia,
and splenomegaly, but survival of the mice in a HIF-1␣–
dependent manner. Additional inactivation of HIF-1␣ (PHD2/
HIF-1␣ double-knockout) leads to early lethality around adulthood,
which is confined to PHD3-reliant HIF-2␣–over-stabilization and
consequent neurodegeneration in the brain.
Taken together, our results establish a principal role for PHD2
as an oxygen sensor in CD68-positive cells for the HIF-2␣–
dependent regulation of EPO and highlight a protective role of
HIF-1␣ in polycythemic mice.
Methods
Generation of a CD68:cre line
The modified hCD68-IVS1 promoter,23 which combines the 2940 bp of
sequence 5Ј to the ATG and the 83-bp first intron of the human CD68 gene
was cloned just in front a codon-improved Cre recombinase sequence
(1091 bp)24 and a polyA signal. Different founders were produced and
tested on expression levels in isolated peritoneal F4/80ϩ cells. Finally,
2 lines were selected of which 1 expressed cre-recombinase already in
sperm as well as egg cells. The other transgenic line was intercrossed with
PHD2f/f mice and used in the current report.
Mice
All mice were housed at the Experimental Center at the University of
Technology Dresden (Medical Faculty, University Hospital Carl-Gustav
Carus), under specific pathogen-free conditions. Experiments were performed
with male and female mice at the age of 8 to 12 weeks or as stated in
the text. All mice described in this report were born in a normal Mendelian
manner. HIF-2␣f/f mice were obtained from The Jackson Laboratory and
originally produced in the research group of Dr C. Simon (Cell and
Developmental Biology, University of Pennsylvania School of Medicine,
Philadelphia, PA).25 All mouse strains were at least 9 generations backcrossed
to C57BL/6. Mice were genotyped using primers described in
supplemental Table 1 (available on the Blood Web site; see the Supplemental
Materials link at the top of the online article). The construction and
identification of the PHD2f/f mouse line will be described elsewhere (R.P.S,
K.F., J.K., S.M., A.M., and B.W., unpublished results, June 2008).
More than 60% of all CD68:cre-PHD2f/f (cKO P2), CD68:cre-PHD2/
HIF-1␣ff/ff (cKO P2/H1) and CD68:cre-PHD2/PHD3ff/ff (cKO P2/P3) mice
showed redness of snout/paws associated with high HCT (HCT; Ͼ ϳ 70%)
and were used for the described experiments. Nonerythrocytotic cKO, cKO
P2/H1 and P2/P3 mice all showed HCTs comparable with WT littermates
(Ͻ 55%), which was always related with lower penetrance of crerecombinase
activity and were only used for breeding. No cKO mice were
found with HCTs between 55 and 70%. Penetrance in cKO P2/H1,
CD68:cre-PHD2/HIF-2␣ff/ff (cKO P2/H2), CD68:cre-PHD2/PHD3ff/ff (cKO
P2/P3), CD68:cre-HIF-1␣f/f (cKO H1) and CD68:cre-HIF-2␣f/f (cKO H2)
mice was defined via qRT-PCR on BM mRNA and/or genomic PCR on ear
biopsies. Knockdown efficiencies for HIF-1␣ and HIF-2␣ in these genotypes
were comparable with expression levels for PHD2. It is noteworthy
that only a low percentage (ϳ 20%) of cKO P2/H2 mice showed cre
activity with high penetrance on both alleles simultaneously. Only these
mice were considered for the presented experiment.All other mice, carrying
CD68:cre-PHD2f/f and HIF-2␣f/f, showed no targeting or only of 1 of the
alleles. All animal experiments were in accordance with the facility
guidelines on animal welfare and were approved by the Landesdirektion
Dresden, Germany.
Blood analysis
Blood parameters (HCT, RBCs, Hb, and thrombocytes) were measured
using a Sysmex automated blood cell counter (Sysmex XE-2100 and
XE-5000). Plasma erythropoietin and thrombopoietin concentrations were
determined using the Quantikine Mouse/Rat EPO immunoassay and the
Quantikine Mouse Tpo immunoassay (R&D Systems).
Blood pressure and heart rate were measured noninvasively with the tail
cuff method (UGO BASILE). After training of the animals for 2 weeks, the
average of at least 2 independent measurements was calculated.
Cells
The human kidney cell line REPC was cultivated as previously described.26
Astrocytes were isolated from the cerebral cortex of 5-day-old pups by
incubation with 0.025% Trypsin for 30 minutes, followed by enrichment
with the biotinylated anti-GLAST (ACSA-1) antibody (Miltenyi Biotec)
and magnetic beads. The highly enriched astrocyte fraction (purity Ͼ 90%)
was confirmed via FACS (data not shown).
Isolation of individual cell types
Primary mouse keratinocytes and fibroblasts were isolated from newborn
mice by incubating the skin with 250 mg/mL Dispase (Roche) overnight at
4°C. The epidermal layer was separated from the dermal layer and
incubated in TrypLE select enzyme (Invitrogen) for 30 minutes at room
temperature. The resulting single-cell suspension was cultured in CnT
medium (Cell-n-Tech). The dermal layer was finely minced and then
incubated in a 0.25% trypsin/EDTA solution (Gibco) for 30 minutes at
37°C. The resulting cell suspension was cultured in DMEM (Lonza)
containing 10% FBS (Biochrom), 1% L-Glutamine (Lonza), and 1%
penicillin/streptomycin (Lonza). Identity of keratinocytes was confirmed
via Keratin6 (K6) staining (data not shown). Enterocytes were isolated from
2 cm of colon starting from the cecum. The colon was cut into 5 to 6 pieces
and incubated in Cell Recovery solution (BD Bioscience) for 1.5 hours at
4°C. After vigorous shaking, the cell suspension was filtered, centrifuged,
and the resulting cell pellet frozen. Identity of the enterocytes (colonocytes)
was confirmed via Villin1 staining (data not shown). Endothelial cells were
isolated from the lung via FACS (CD31ϩCD34ϩ) and confirmed based on
CD31 mRNA expression (data not shown).
Expression analysis
RNA from organs and sorted cells was isolated using RNeasy Mini Kit
(QIAGEN), NucleoSpin RNA XS (Machery-Nagel), Trizol (Invitrogen),
Universal RNA Purification Kit (roboklon), or DNAϩRNAϩPROTEIN
Purification Kit (roboklon). cDNA was synthesized using random primers
DISTINCT REGULATION OF HIF-1␣ AND HIF-2␣ BY PHD2 1437BLOOD, 21 FEBRUARY 2013 ⅐ VOLUME 121, NUMBER 8
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(Roche) and SuperScript II (Invitrogen). Expression levels were determined
by performing quantitative real-time PCR using the Maxima SYBR Green
QPCR Master Mix (Fermentas) on an iCycler iQ (Biorad). Sequences of
primers used are given in supplemental Table 2. Expression levels were
normalized with the ⌬⌬Ct method using primers given in supplemental
Table 3.
Histology and immunofluorescence
For all samples, organs were placed in 4% formaldehyde at 4°C overnight,
dehydrated, embedded in paraffin and cut. Sections were rehydrated and
subjected to hematoxylin and eosin staining or acidic Fuchsin orange
G-staining. For IHC, antigen retrieval (citrate buffer-PH 6.0) at 95°C for
20 minutes was performed on rehydrated sections. Incubation with primary
antibody (Ab; Iba1, WAKO; cleaved-Caspase3,Cell Signaling; NeuN,
Abcam) was 1.5 hours at 37°C. Secondary Abs were Alexa-488 or
Alexa-568 (Molecular Probes) incubated for 30 minutes at room temperature.
Cell nuclei were stained with DAPI. Slides were mounted with
fluorescent mounting media (Dako). For frozen sections, samples were
embedded in OCT, cut and stored at Ϫ20°C. For immunofluorescence
staining, sections were first fixed for 10 minutes in cold acetone and
blocked with 5% goat serum in TNT buffer (20mM Tris pH 7.6, 0.9% NaCl,
0.05% Tween in PBS). Thereafter, sections were incubated with mouse
anti-NeuN and rabbit anti-PHD2 at 37°C for 1 hour (custom-made
polyclonal rabbit anti–mouse antibody against a C-terminal peptide of
PHD2; EKGVRVELKPNSVSKDV) and purified via a peptide sulfoLink
immobilization column (Pierce, ThermoScientific). Secondary antibody as
described.
Microscopy
Fluorescent and light microscopy was done with an Axioplan-2 imaging
microscope and plan Apochromat lenses (Carl Zeiss). The cameras and
acquisition software were either Q Imaging Retiga 2000R and Image pro
MC6.0 (fluo) or Axiocam MRc5 and Axiovision (light). Image processing
and analysis was done using ImageJ 1.45s and the ImageJ distribution Fiji
(http://pacific.mpi-cbg.de/wiki/index.php/Fiji).
Flow cytometry
FACS analysis was performed on LSRII (Becton Dickinson), sorting was
done on Aria II (Becton Dickinson). Cell numbers were counted on MACS
quant (Miltenyi). Data were analyzed on DIVA 7.0 (Becton Dickinson),
MACS quantify Version 3.0 (Miltenyi), or FlowJo Version 9.6.1 (TreeStar)
software. BM single-cell suspensions were made by flushing the marrow
using 23G needles. Single-cell suspensions from spleen and lymph nodes
were prepared by manual disruption using frosted slide ends, additionally
for spleen cells enzymatic digestion was performed with 0.3 U/mL
Collagenase D (Roche) for 40 minutes at 37°C. The lineage cocktail
included CD3 (145-2C11, eBioscience), CD19 (eBio1D3, eBioscience),
NK1.1 (PK136, eBioscience), Ter119 (Terr119, eBioscience), CD11b
(M1/70, eBioscience), Gr1 (RB6-8C5, eBioscience), B220 (RA3-6B2,
eBioscience), CD127 (A7R34, eBioscience), and CD11c (N418, eBioscience).
For MEP analysis, spleen and bone marrow cells along with lineage
cocktail were also incubated with c-kit (APC, 2B8, eBioscience), Sca1
(Pe-Cy7, D7, eBioscience), CD16/32 (A700, 93, eBioscience), CD34
(FITC, RAM34, eBioscience), and SA (PerCP, BD Bioscience). One ϫ 106
cells from bone marrow and 2 ϫ 106 cells from spleen were acquired. For
EB analysis, spleen and bone marrow cells were stained with CD71 (FITC,
R17217, eBioscience) and Ter119 (Pe-Cy7, Ter119, eBioscience). Two
ϫ 105 cells were acquired. For lymphoid cell analysis, spleen, and lymph
node cells were incubated with CD3 (APC, 145-2c11, eBioscience) and
CD19 (FITC, MB19-1, eBioscience). For myeloid cell analysis, spleen cells
were incubated with CD11b (PE, M1/70, eBioscience), F4/80 (PerCPcy5.5,
BM8, eBioscience), and/or Gr1 (A700, RB6-8C5, eBioscience).
Blood volume determination
Blood was collected in EDTA vials, washed twice with 0.9% NaCl, and
stained with 15uM Vybrant DiI Cell-labeling solutions (Invitrogen) for
2.5 hours at room temperature on a tilt shaker. After washing and
resuspension in PBS 100 ␮L was injected intravenously. One hour later a
blood sample was taken from the mouse, diluted, and analyzed for DiI
containing cells.
Statistical analysis
Data and graphs represent mean Ϯ SEM of representative experiments.
Statistical significance was calculated as 2-tailed by the Mann-Whitney
U test (GraphPad Prism Version 5.04), with P Ͻ .05 considered statistically
significant.
Results
Conditional PHD2-deficient mice display severe
HIF-2␣–dependent erythrocytosis
PHD2 is the main HIF prolyl hydroxylase isoform, which controls
the expression of EPO in humans.20 Somatic inactivation of PHD2
shortly before or after birth leads to severe polycythemia probably
in a HIF-1␣–dependent manner but is accompanied by early
lethality and has therefore not been extensively studied.21,22 To
conditionally ablate PHD2, we first generated a PHD2f/f mouse line
by gene targeting using a construct in which exons 2 and 3 are
flanked by loxP sites (R.P.S, K.F., J.K., S.M., A.M., and B.W.,
unpublished results, June 2008). We combined this with a newly
designed mouse line expressing cre-recombinase under the control
of the modified human CD68 promoter, known as a monocyte/
macrophage marker (supplemental Figure 1A). Thorough analyses
revealed that these cKO P2 mice are not only significantly deficient
for PHD2 in macrophages, but in the entire hematopoietic system
(bone marrow; supplemental Figure 1B). In addition, we also found
a profound PHD2 reduction in a few subsets of epithelial cells (eg,
keratinocytes and colonocytes), but not in other cell lineages, such
as endothelial cells (ECs) and fibroblasts (supplemental Figure 1C).
In the lysates of nonhematopoietic organs studied in the current
report, we found no significant reduction of PHD2 expression
(supplemental Figure 1D). Moreover, no significant compensation
by one of the other PHDs could be shown (supplemental Figure 1E)
Interestingly, the majority of these cKO P2 mice showed
obvious redness of paws and snout starting around 4 weeks of age,
which was accompanied by significant growth retardation until
adulthood (Figure 1A-B). This striking phenotype was because of
elevated HCTs which increased with age to Ͼ 85% in some
individuals (Figure 1C). However, although the blood viscosity
was substantially increased, we observed no difference in the blood
pressure, heart rate, or cardiac hypertrophy, as has been shown
before for other erythrocytotic mice (supplemental Figure 2A-B).27
As PHD2 can control the activity of both HIF-1␣ and HIF-2␣ we
determined which of the HIFs contributed to the erythrocytosis.
The notion that HIF-2␣ acts as the main regulator of EPO is based
on histologic and genetic approaches in HIF-2␣–deficient
mice.10,25,28 Moreover, several studies in patients with idiopathic
erythrocytosis revealed the presence of different heterozygous
missense mutation in the coding sequence of HIF-2␣, but not
HIF-1␣, leading to impaired PHD2-induced hydroxylation.29,30 On
the other hand, a somatic knockout of PHD2, suggested that
HIF-1␣, rather than HIF-2␣, contributed to increased renal EPO
synthesis.21 To unequivocally determine the major regulator in the
erythrocytotic cKOs, we therefore generated mice double deficient
for PHD2 and HIF-1␣ or HIF-2␣. Our data demonstrate that the
erythrocytosis phenotype as shown in cKO P2 mice was abolished
in CD68:cre-PHD2/HIF-2␣ff/ff mice (cKO P2/H2), displaying even
significantly lower HCTs and hemoglobin levels than in WT mice
1438 FRANKE et al BLOOD, 21 FEBRUARY 2013 ⅐ VOLUME 121, NUMBER 8
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(Figure 1D-F). On the other hand, HCTs in CD68:cre-PHD2/HIF-
1␣ff/ff mice (cKO P2/H1) were indistinguishable from cKO P2,
showing that PHD2-induced severe erythrocytosis is exclusively
dependent on HIF-2␣ but not on HIF-1␣. These results are
emphasized by the fact that conditional knock-down of HIF-2␣
alone (CD68:cre-HIF-2␣f/f [cKO H2]), but not HIF-1␣ (CD68:creHIF-1␣f/f
[cKO H1]), already resulted in a mild anemia (supplemental
Figure 3A-C).
Erythrocytosis is EPO dependent and associated with
increased EPO expression in kidney and brain
Because excessive secondary erythropoiesis is mainly mediated by
EPO,27 we measured EPO plasma levels in both erythrocytotic
mouse lines (cKO P2 and cKO P2/H1) and found a highly
significant induction of EPO expression in cKO P2 mice compared
with WT littermates. Surprisingly, although cKO P2/H1 mice
showed similar HCTs to cKO P2 mice, they contained on average
2.5-fold higher plasma EPO values than cKO P2 mice (Figure 2A).
The main source of EPO in adult mice is the kidney,3 where we
found a 10-fold and 20-fold increase of EPO in extracts from cKO
P2 and from cKO P2/H1 mice, respectively. In addition, the expression
of EPO in the brain was significantly up-regulated (Figure 2B). In
contrast, hepatic EPO expression levels were very low, and no
increase could be observed in any of the mice (data not shown).
In the kidney, EPO is produced by specialized interstitial cells
localized in the deep cortex between the proximal tubular and
vascular endothelial cells. These renal EPO producing (REP) cells
also express neuronal-specific markers but isolation of these
primary cells has been a real challenge so far.31 Therefore, we
tested the recently described human REP cell line, isolated from the
tumor-free tissue of a male patient26 and demonstrate that these
cells coexpressed both CD68 and EPO, supporting the link between
CD68-driven cre expression, PHD2 inactivation and subsequent
EPO expression in our cKO P2 mice (Figure 2C). In the brain,
astrocytes and neurons are known to be sources of EPO.32 We
therefore isolated astrocytes from the cerebral cortex of 5-day-old
pups and found a significant PHD2 reduction in cKO P2 cells
versus WT (Figure 2D). The latter result was similar to the
knockdown efficiency found in other cell types (supplemental
Figure 1B-C). Via IHC we demonstrate that sections from adult
cKO P2 brains contain a majority of PHD2-negative cells that were
positive for the neuronal marker NeuN (Figure 2E).
Moreover, because cKO P2 mice are also deficient for PHD2 in
the BM, we transplanted BM cells from cKO P2 mice into lethally
irradiated WT mice and vice-versa. However, we found no
significant difference in HCT compared with nontransplanted
animals, thus excluding the possibility that the phenotype could be
transferred with PHD2-deficient hematopoietic cells (Figure 2F).
A
C
B
WT cKO P2
4 5 6 7 8 9
9
12
15
18
WT
cKO P2
*
*
**
**
**
Age (weeks)
Weight(g)
4 5 6 7 8
40
50
60
70
80
**
***
**
Age (weeks)
Hematocrit(%)RBC(x1012
/L)
0
5
10
15 *** ***
N.S.
WT cKO cKO cKO
P2 P2/H1 P2/H2
N.S. (p=0.057)
D
Hematocrit(%)
40
50
60
70
80
90 *
*** N.S.
***
WT cKO cKO cKO
P2 P2/H1 P2/H2
E F
Hb(mmol/L)
0
5
10
15
***
**
*
*
WT cKO cKO cKO
P2 P2/H1 P2/H2
Figure 1. Conditional deficient PHD2 mice display
severe HIF-2␣–induced erythrocytosis. (A) Compared
with WT mice, cKO P2 show profound redness of the
snout and paws. (B) Body weight of WT and cKO P2 mice
was measured on a weekly basis until adulthood. No
significant difference was observed after week 8 (n ϭ 5-11).
(C) Percentage of red blood cell volume per blood volume
(HCT-hct) from mice was measured every 2 weeks until
adulthood, displaying highly significant differences from
week 4 after birth (n ϭ 5-10). (D) HCTs in cKO P2/H1
remained as high as in cKO P2 mice whereas cKO P2/H2
mice showed even slightly but significantly lower HCTs
than WT (n ϭ 6-15). (E-F) RBC count (n ϭ 7-32) and
hemoglobin (n ϭ 6-22) concentration in freshly isolated
blood samples demonstrate that HIF-2␣ but not HIF-1␣
induces erythrocytosis because of loss of PHD2. All data
are mean Ϯ SEM (*P Ͻ .05, **P Ͻ .01, ***P Ͻ .001).
DISTINCT REGULATION OF HIF-1␣ AND HIF-2␣ BY PHD2 1439BLOOD, 21 FEBRUARY 2013 ⅐ VOLUME 121, NUMBER 8
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Taken together, the erythrocytosis phenotype in cKO P2 and
cKO P2/H1 mice is correlated with an enormous induction of EPO
in the kidney and the brain, related to PHD2 inhibition in REP cells,
astrocytes, and neurons.
Erythrocytosis is mainly caused by extramedullary erythropoiesis.
Basal erythropoiesis mainly takes place in the bone marrow,
whereas the spleen can increase its capability to produce RBCs
under conditions of stress erythropoiesis.33 In the cKO P2, polycythemia
resulted in splenomegaly accompanied by loss of splenic
architecture, with an increase in nucleated cells and total weight
(Figure 3A-C). Remarkably, the megakaryocyte-erythroid progenitors
(MEPs) were found to be significantly induced (Ͼ 2.5-fold) in
the spleen of cKO P2 mice but not in BM (Figure 3D-E). In
addition, splenic CD71ϩ/Ter119ϩ erythroblasts (EBs) were increased
more than 10-fold in cKO P2 mice compared with WT
littermates, whereas only a mild increase in EBs was found in BM
(Figure 3F-G). The fraction of B and T cells were markedly decreased
in the cKO P2 spleen, whereas lymph nodes contained an excess of
both populations, suggesting that there is no impairment of their
production but only a difference in localization because of the EB
excess in the spleen (supplemental Figure 4A-D). Splenic
CD11bϩF4/80ϩ macrophages and neutrophils (PMNs) were not
significantly altered, although red pulp macrophages, a distinct
subset of macrophages involved in the removal of senescent
erythrocytes, were highly induced in the cKO P2 spleen (supplemental
Figure 4E-G). These results imply that the spleen is primarily
responsible for the overproduction of RBCs in cKO P2 mice.
Thrombocytopenia is directly related to the higher blood
volume
Further hematologic analysis revealed a significant decrease in
platelet numbers in cKO P2 mice compared with WT mice, as early
as at 4 weeks of age (Figure 4A). This effect was also related to
HIF-2␣ but not HIF-1␣, because the phenotype was rescued only in
the cKO P2/H2 mice (Figure 4B). To examine the molecular
background of this effect we analyzed different parameters but
found no relative difference in thrombopoietin (TPO), the number
of megakaryocytes in the BM or spleen (supplemental Figure
5A-C), nor did we detect microvascular thrombosis, which would
have indicated increased consumption of platelets (Figure 4C-D).
However, using flow cytometry on labeled RBCs, we found that the
total blood volume in cKO P2 mice was significantly increased by
approximately 65% (Figure 4E); strongly suggesting that the
reduced platelet count is a dilution effect.
0
1500
3000
4500
6000
PlasmaEPO(pg/ml)
***
*
WT cKO cKO cKO
P2 P2/H1 P2/H2
N.S.
EPOexpression
Kidney Brain
0
5
10
15
20
25
30 WT
cKO P2
*
cKO P2/H1
N.S.
*
N.S.
cKO P2/H2
N.S.
N.S.
B
C
A
PHD2expression
0.0
0.5
1.0
*
WT cKO
P2
PHD2 PHD2
PHD2/NeuN PHD2/NeuN
WT cKO P2
D
E
0
20
40
60
80
Hematocrit(%)
N.S.
N.S.
WT cKOBM
in WT
WTBM
in cKO
cKO
CD68
EPO
21% O2 1% O2 (24h)
F
Figure 2. Loss of PHD2 in cKO P2 mice leads to
induction of EPO in kidney and brain. (A) EPO concentration
in plasma measured by ELISA in the plasma of
WT, cKO P2, cKO P2/H1, and cKO P2/H2 mice (n ϭ 6-22).
cKO mice contain on average 6 times more EPO in the
circulation than their WT littermates, whereas cKO P2/H1
mice have more than 17 times more EPO. No difference
between WT and cKO P2/H2 was found. (B) mRNA levels
in total extracts from kidney and brain (n ϭ 5-15) is
markedly induced compared with WT but not significantly
different between cKO P2 and cKO P2/H1 mice
because of the high variation between individual samples
(n ϭ 7-11). No difference between WT and cKO P2/H2
could be shown. (C) The renal-derived human cell line
(REPC)26 was tested for the expression of EPO and
CD68 grown under normoxic and hypoxic conditions via
RT-PCR, showing the expected EPO induction and expression
of CD68 under both conditions, suggesting a
potential link between cre-expression, PHD2 inactivation
and subsequent EPO expression in these cell types.
(D) Astrocytes isolated from the cerebral cortex of 5 day
old pups show a significantly reduced PHD2 content in
cKO P2 versus WT mice (n ϭ 3). (E) Typical IHC on brain
sections for PHD2 (red) with or without NeuN (green)
show a majority of PHD2-negative neurons in the brain of
cKO P2 mice (depicted by arrow heads) but not in WT.
(F) WT and cKO P2 mice were lethally irradiated and
received cKO P2 or WT bone marrow (BM) respectively.
HCTs were measured 4 months after transfer. BM from
either genotype did not change HCTs in the recipient
(n ϭ 6-23). Scale bar in (E) represents 50 ␮m. All data
are mean Ϯ SEM (NS indicates not significant; *P Ͻ .05,
***P Ͻ .001).
1440 FRANKE et al BLOOD, 21 FEBRUARY 2013 ⅐ VOLUME 121, NUMBER 8
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cKO P2 mice display a normal life span whereas cKO P2/H1
die early. One common feature of the somatic PHD2-deficient
mice21,22 as well as other EPO-related erythrocytotic mice34,35 is
that these mice die prematurely. Surprisingly, we never observed
any premature death in our cKO P2 mice. Moreover, we did not
encounter any lethality in a group of 9 erythrocytotic cKO P2 mice
that were maintained for more than 20 months. These mice were
bled every 4 months but no significant drop in HCT could be
E
0
2.5 105
5.0 105
*
MEPs/spleen
WT cKO
P2
ckit
Sca1
CD16/32
CD34
WT
cKO P2
D
0
2 100 5
4 100 5
N.S.
MEPs/BM WT cKO
P2
ckit
Sca1
CD16/32
CD34
WT
cKO P2
CD71
Ter119
WT
cKO P2
0
5.0 106
1.0 107
1.5 107
2.0 107 *
EBs/BM
WT cKO
P2
F
CD71
WT
cKO P2
0
2 100 7
4 100 7
6 100 7
8 100 7
**
EBs/Spleen
WT cKO
P2 Ter119
G
WT
A
cKO P2
B
0
50
100
150 WT
cKO P2
**
Spleenweight(g)
C
Figure 3. Erythrocytosis is mainly caused by extramedullary
erythropoiesis in the spleen. (A-B) Representative
sections of WT and cKO P2 spleens (H&E)
show an extensive structural loss and an augmentation
of nucleated cells in erythrocytotic cKO P2 mice.
(C) Spleen weight of WT and cKO P2 mice (n ϭ 10-16).
(D-G) Representative FACS staining profiles of (D and E)
megakaryocyte-erythrocyte progenitors (MEP; n ϭ 6) and
(F-G) erythroblasts (EB; n ϭ 7) in BM and spleen of WT
and cKO P2 showing that the overproduction of RBCs is
mainly executed in the spleen. Scale bars represent
500 ␮m. All data are mean Ϯ SEM (*P Ͻ .05,
***P Ͻ .001).
Age (months)
Hematocrit(%)
6 10 14 18
0
20
40
60
80
A
0 5 10 15 20
0
25
50
75
100
Time (weeks)
Survival(%)
cKO P2/H1
cKO P2
B
Thrombocytes(10^9/l)
0
250
500
750
1000
1250
1500
WT cKO cKO cKO
P2 P2/H1 P2/H2
*** ***
N.S
N.S
4 5 6 7 8
500
750
1000
1250
**
***
**
Age (weeks)
Thrombocytes(10^9/l)
0.5
1.0
1.5
2.0
***
BloodVolume(foldind.)
WT cKO
P2
WT cKO P2
DC
F G
E
Figure 4. cKO P2 mice show thrombocytopenia but
no early lethality, whereas cKO P2/H1 die prematurely.
(A) Thrombocyte concentrations in the blood of
WT and cKO P2 mice until adulthood reveal a significant
reduction of circulating platelets per volume of blood in
cKO P2 mice (n ϭ 5-10). WT is represented by black
squares and cKO P2 by red triangles. (B) Eight- to
10-week-old mice were bled and thrombocytes measured
in a fixed volume of blood showing thrombocytopenia
in cKO and cKO P2/H1 mice compared with WT and
cKO P2/H2 mice (n ϭ 6-7). (C-D) WT and cKO P2
kidneys stained with acid Fuchsin orange-G show no
signs of microvascular thrombosis suggesting that platelets
are not trapped within blood clots. (E) Instead, the
total blood volume of cKO P2 mice was significantly
increased compared with WT mice (n ϭ 9-10). (F) 9 cKO
P2 mice (5 females and 4 males) were kept for 20 months.
Mice were bled every 4 months to measure HCTs. No
significant differences in HCTs were found and none of
the mice died before the end of the experiment. (G) cKO
P2/H1 (n ϭ 17) and cKO P2 (n ϭ 15) mice were
followed for 20 weeks. cKO P2/H1 mice began to die
from week 5 after birth. By week 16, 16 of 17 mice were
deceased. No cKO P2 mice died during the course of this
experiment. Scale bars represent 100 ␮m. All data are
mean Ϯ SEM (**P Ͻ .01, ***P Ͻ .001).
DISTINCT REGULATION OF HIF-1␣ AND HIF-2␣ BY PHD2 1441BLOOD, 21 FEBRUARY 2013 ⅐ VOLUME 121, NUMBER 8
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observed (Figure 4F). Finally, all mice were killed and several
organs examined in great detail. Except for a regressing cyst
associated with the ovary of 1 mouse (supplemental Figure 6A), no
obvious pathologic defects or tumors were detected (supplemental
Figure 6B). On the other hand, the cKO P2/H1 mice started to die
very sudden from week 5 onward, and by week 16, 95% of these
mice were dead (mean survival time: 11 weeks; Figure 4G). In contrast,
neither cKO P2/H2 nor cKO H1 or H2 mice showed any premature
death. Importantly, these results show for the first time that extremely
high HCTs do not necessarily lead to lethality or pathologic
abnormalities in part because of a protective function of HIF-1␣.
Simultaneous loss of PHD2 and HIF-1␣ in the brain leads to
neurodegeneration
To investigate the biologic background of the early lethality of cKO
P2/H1 mice, we conducted a thorough examination of 8 to
10-week-old living as well as recently deceased cKO P2/H1 mice.
No acute cardiovascular complications or obvious evidence for
thrombosis or degeneration in different examined organs were observed
(data not shown). However, via IHC staining we detected a dramatic
induction of activated (Iba1ϩ) microglia cells in the brain of cKO P2/H1
mice compared with WT and cKO P2 mice (Figure 5A-C). This
strongly suggests that pathologic changes in cKO P2/H1 brains led to
the activation of these resident macrophages. Consistent with this, we
found numerous patches of neurons (NeuNϩ) in cKO P2/H1 brain
sections, especially from freshly deceased mice, that showed strong
expression of cytoplasmic cleaved-caspase3 (Cl-Casp3), indicative
of apoptosis (Figure 5D-F). In contrast, no Cl-Casp3 staining could
be detected in cKO P2 or WT brains (data not shown). In line with
the observed phenotype, we found that some (3 of 16) of the cKO
P2/H1 mice even displayed a dome-shaped skull accompanied by a
dramatic hydrocephalus (supplemental Figure 7A-B).
G
WT
Iba1
A
cKO P2
Iba1B
cKO P2/H1
Iba1C
E
NeuN
F
Merge/DAPI
D
Cl-Casp3
ExpressionrelativetoWT(%)
-60
-30
0
30
60
90
120
240
TNF
BNIP3L
IL1
Glut1
VEGFA
*
IL1
*
*
*CXCR4
Adm
cKO P2
cKO P2/H1
*
* *
*
IL12p35
PHD3
PHD2
iNOS
HIF1
TGF1
*
Figure 5. HIF1a serves as a protective factor to
prevent brain damage. (A-C) IHC on brain sections
from WT, cKO P2, and cKO P2/H1 for Iba1 (brown)
showing more activated microglia cells in brain sections
of a cKO P2/H1 mouse. (D-F) IHC on brain sections from
a freshly deceased cKO P2/H1 mouse for (D) cleavedcaspase3
(cl-Casp3; red), (E) neurons (NeuN; green),
and (F) a merged picture combined with DAPI (white
arrows depict typical examples of cl-Casp3ϩ neurons).
No obvious cl-Casp3 staining was detected in WT or cKO
P2 brains (data not shown). (G) Expression profile
(qRT-PCR) of different genes in the lysate of the entire
brain of cKO P2 and cKO P2/H1 mice in relation to their
respective WT littermates (n ϭ 5-9). All data are mean
Ϯ SEM (*P Ͻ .05). Scale bars in panels A through C,
100 ␮m; panels D through F, 50 ␮m.
1442 FRANKE et al BLOOD, 21 FEBRUARY 2013 ⅐ VOLUME 121, NUMBER 8
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To unravel the molecular background of this neurodegeneration,
we compared the gene profiles of cKO and cKO P2/H1 brains.
Remarkably, PHD3, a HIF-1␣–regulated gene and formerly identified
as a very potent negative regulator of HIF-2␣,36 was drastically
down-regulated in cKO P2/H1 brains compared with cKO P2
counterparts. Conversely, we found a significant induction of
proinflammatory cytokines, such as TNF␣, Il-1␣, and IL-1␤, as
well as VEGFA, TGF␤1, CXCR4, and adrenomedullin (Adm;
Figure 5G), many of which have been shown to be HIF-2␣–
dependent genes.37 These data therefore suggest that knocking out
HIF-1␣ in addition to PHD2 in a subset of brain cells (eg,
astrocytes and microglia cells) leads to an additional stabilization
of HIF-2␣ and consequent transcriptional activation of a variety of
genes, including detrimental inflammatory cytokines that can contribute
to neurodegeneration and lethality of the cKO P2/H1 mice.
To evaluate whether HIF-1␣–induced PHD3-expression is
directly related to the protection of cKO P2 mice, we developed
mice lacking PHD2 and PHD313 in CD68ϩ cells (CD68:cre-PHD2/
PHD3ff/ff [cKO P2/P3]). Interestingly, these mice did not only
display severe erythrocytosis (Figure 6A); a majority of them
suddenly died starting shortly after birth (Figure 6B), whereas cKO
P2 as well as PHD3Ϫ/Ϫ mice never died during this period.
Furthermore, we isolated the brains of these cKO P2/P3 mice and
analyzed the same array of genes as we studied for the cKO P2/H1
animals. Interestingly, we found that virtually all differentially
expressed genes in brain lysates from cKO P2/H1 mice are also
significantly changed in the brains of cKO P2/P3 mice, compared
with cKO P2 (Figure 6C). These data strengthen our hypothesis
that PHD3 is a not only a protective factor in the brain of cKO P2
mice, but acts downstream of HIF1␣. Taken together, these results
reveal a tight and essential balance between HIF-1␣ and HIF-2␣
activity in the brain, which also involves the PHD3 regulation.
Discussion
In this study, we used a genetic approach to investigate in detail the
biologic role of PHD2 in CD68-positive cells and the relation of
HIF1
PHD2
Hematocrit(%)
40
50
60
70
80
90
**
WT cKO
P2/P3
PHD2
PHD3, VEGF, Il1, Epo,
Adm,CXCR4,...
HIF1 /HIF2
WT
EPO,VEGF,Adm...
HIF1
PHD3
TNF,Il1,CXCR4...
cKO P2/H1
PHD2
HIF2 HIF2
PHD3
A B
C
0 5 10 15 20
0
25
50
75
100
Time (weeks)
Survival(%)
cKO P2/P3
cKO P2
P3-/cKO
P2
ExpressionrelativetoWT(%)
-60
-30
0
30
60
90
120
240
TNF
IL1
VEGFA
IL1
CXCR4
Adm
cKO P2
PHD3
PHD2
TGF1
cKO P2/P3
*
*
*
*
*
*
*
D
Figure 6. Mice deficient for PHD2 and PHD3 display
erythrocytosis and die prematurely. (A) cKO P2/P3
mice show enhanced HCTs compared with their WT
littermates (n ϭ 5). (B) WT, PHD3Ϫ/Ϫ and cKO P2/P3
mice were followed for 20 weeks. cKO P2/P3 mice began
to die shortly after birth (n ϭ 7). (C) Expression profile
(qRT-PCR) of different genes in the lysate of the entire
brain of cKO P2 and cKO P2/P3 mice in relation to their
respective WT littermates. *P Ͻ .05 is significantly different
from cKO P2 samples (expression relative to their
WT littermates; n ϭ 5-7). (D) Schematic overview of the
different genetic mouse models that were generated in
this study showing nonlethal erythrocytotic cKO P2 mice
that display a perfect balance between the protective
HIF-1␣ activity and detrimental HIF-2␣ activity in the
brain. In cKO P2/H1 mice, this balance is disturbed in
favor of the HIF-2␣ activity leading to early lethality in the
mice. All data are mean Ϯ SEM (*P Ͻ .05, **P Ͻ .01).
DISTINCT REGULATION OF HIF-1␣ AND HIF-2␣ BY PHD2 1443BLOOD, 21 FEBRUARY 2013 ⅐ VOLUME 121, NUMBER 8
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2 of its substrates (HIF-1␣ and HIF-2␣) with erythropoietin
production and during erythropoiesis. We have demonstrated that
conditional PHD2-deficient mice are able to cope with extreme
numbers of RBCs, thrombocytopenia, and splenomegaly through a
discrete interplay between HIF-1␣ and HIF-2␣ involving the
regulation of PHD3.
Although CD68 has been described as a marker mainly limited
to monocytic cells,23 our data demonstrate at least temporary
expression in the hematopoietic compartment as well as in subsets
of epithelial lineages, but not in ECs or fibroblasts. Moreover, our
results demonstrate loss of PHD2 in a subset of renal and brain cells
responsible for the production of EPO, which leads to massive
stress erythropoiesis primarily executed by the adult spleen.
Remarkably and in clear contrast to many other reports,3,21,22,35 our
erythrocytotic cKO P2 mice show normal lifespan, demonstrating
that an unusual high HCT is not per se lethal; a phenomenon that
has also been described in a few healthy cobalt miners at high
altitude that developed HCTs ranging from 75% to 91%.38 Genetically,
several heterozygous PHD2 missense mutations have been
defined in patients, which impair the binding to both HIF-1␣ and
HIF-2␣ resulting in erythrocytosis.15-18 One patient with such a
PHD2 mutation and consequent polycythemia developed a paraganglioma,
suggesting that PHD2 may act as a tumor suppressor.1
However, our results strongly suggest that sustained exposure to
high levels of EPO and permanent loss of PHD2 in different cell
types does not necessarily lead to spontaneous tumor development
over a period of 20 months in mice. This has also been underscored
by recent work from our group showing that silencing of PHD2 in
several tumor lines typically leads to reduced tumor growth.39
In mice, we (K.F. and B.W., unpublished data, October 2011)
and others14 have shown that PHD2 heterozygosity only results in a
very mild form of erythrocytosis whereas global inactivation just
before or after birth leads to severe and lethal polycythemia.21,22
Based on expression levels in the kidney of the latter mice, it was
proposed that HIF-1␣, rather than HIF-2␣, might be the central
erythrocytosis mediator in relation to PHD2.21 However, our
genetic approach (PHD2/HIF␣ double-deficient mice) clearly
shows that this phenotype is driven by HIF-2␣. The notion that
only HIF-2␣ induces EPO is not new,25,28 and is in line with
observations from Percy and colleagues in familial erythrocytosis.29,30
However, to the best of our knowledge this is the first report
that shows that PHD2-induced severe erythrocytosis is exclusively
dependent on HIF2␣ and not on HIF1␣. In addition, the finding that
CD68:cre-HIF-2␣f/f mice were mildly anemic emphasizes the
importance of this HIF-subunit and confirms the, at least temporary,
activity of CD68, in specialized EPO-producing cells.
Simultaneous ablation of PHD2 and HIF-1␣ on the other hand
led to early lethality, clearly demonstrating that HIF-1␣ acts as a
crucial survival factor in the erythrocytotic cKO P2 mice. In
particular, detailed analysis of mature cKO P2/H1 mice showed
pathologic defects only in the brain, a feature that was underlined
by the severe hydrocephalus we observed in a few of the cKO
P2/H1 mice. We found that a majority of cKO P2/H1 mice
contained activated microglia cells and patches of apoptotic
neurons, the latter especially evident in recently deceased mice. A
gene-expression profile of individual brains showed clear induction
of different proinflammatory cytokines (eg, TNF␣ and IL-1␤) in
cKO P2/H1 mice compared with cKO P2 mice. These cytokines are
known as main contributors in several neurodegenerative disorders
and are typically produced by glia cells, including microglia and
astrocytes, as well as neurons.40-43 In addition, the vascular growth
factor VEGFA,44 the chemokine receptor CXCR4,45-47 and the
multifunctional growth factor TGF␤1,48,49 were overexpressed in
cKO P2/H1 brains, and are related to the initiation and/or secondary
phase of neurodegeneration. Interestingly, many of these genes,
including EPO, have been described as HIF-2␣–related genes,25,37
suggesting that loss of HIF-1␣ enhances HIF-2␣ activity, causing
brain damage as well as lethality of these mice. This idea is further
supported by the observation that the HIF-1␣–inducible gene,
PHD3, and known to be an excellent negative regulator of
HIF-2␣,36 is down-regulated in the brain of cKO P2/H1 compared
with cKO P2 mice. In addition, cKO P2/P3 mice also display
severe erythrocytosis, die prematurely, and show a similar induction
of the array of genes induced in the brain of cKO P2/H1. This
strongly suggests that PHD3 is an essential gene downstream of
HIF-1␣ that protects cKO P2 mice and is not just fine-tuning the
HIF response.50 Taken together, this strongly suggests that HIF-1␣
in a subset of cells in the brain, including microglia, neurons, and
astrocytes, protects cKO P2 mice through the induction of PHD3,
and subsequent control of the HIF-2␣-activity. Other evidence for a
prominent role of HIF-2␣ in the PHD2/HIF-1␣–deficient background
was supported by the 2-fold increase of EPO in circulation
in these mice compared with cKO P2 mice. Surprisingly, cKO
P2/H1 mice did not show any significant change in HCT compared
with cKO P2, again implying that the high RBC concentration
alone was probably not the cause of their early death.
In conclusion, we have shown through several loss-of-function
mutations that conditional PHD2 deficiency in kidney and brain
results in an excessive HIF-2␣–driven overproduction of EPO.
However, HIF-1␣ serves as a protective factor in these mice,
providing new insights to a potential interplay with HIF-2␣ and
PHD3 (Figure 6D). In addition, these findings have implications
for pharmacologic strategies that aim at inducing erythropoiesis.
Whereas specific inhibition of PHD2 might be beneficial, compounds
that simultaneously target all PHDs can ultimately be
harmful.
Acknowledgments
The authors thank the teams of Dr Roland Jung and Beate Gnauk
for excellent technical support.
This work was supported by grants from the MeDDriveProgramm
(TU Dresden, Germany) to B.W., the DFG (WI
3291/1-1 and 1-2 to B.W. and FA225/22 to J.F.), and the Swiss
National Science Foundation to M.G. This work was also partially
supported by National Institutes of Health grant 5R01EY019721 to
G-H.F. The work was performed as collaborative project within the
COST Action TD0901 “HypoxiaNet.“ K.F, J.K, R.P.S., S.M., and
A.M. were supported by the Emmy Noether program (the Deutsche
Forschungsgemeinschaft [DFG] Germany). B.W. is an Emmy
Noether fellow.
Authorship
Contribution: K.F., J.K, R.P.S, S.M., and A.M designed and
performed the experiments, analyzed the data, and helped write the
paper; A.W. provided tools and helpful discussions; V.I. performed
experiments and helped write the paper; S.J, K.W., and K.G. did
pathologic analysis and provided helpful discussions; M.M.,A.M.S.,
D.M.P., and T.R provided helpful discussions and analyzed data;
T.O. performed important experiments; S.B. supervised the hematologic
analysis; G. Breier, G.F., D.R.G., and S.B, provided helpful
tools; G. Baretton supervised all pathologic experiments and
1444 FRANKE et al BLOOD, 21 FEBRUARY 2013 ⅐ VOLUME 121, NUMBER 8
For personal use only.at UNIV OF CALIFORNIA DAVIS on June 9, 2013.bloodjournal.hematologylibrary.orgFrom
provided helpful discussions; T.C., J.F., and M.G. provided tools,
helpful discussions, and helped write the paper; B.W. designed the
study, supervised the overall project, performed experiments,
analyzed the data,and wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing
financial interests.
The current affiliation for J.K. is Department of Nephrology and
Hypertension, University Clinic Erlangen, Erlangen, Germany.
Correspondence: Ben Wielockx, Emmy Noether group (DFG)
Inst of Pathology, University of Technology Dresden, Schubertstrasse
15, D-01307 Dresden, Germany; e-mail: ben.wielockx@
uniklinikum-dresden.de.
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DISTINCT REGULATION OF HIF-1␣ AND HIF-2␣ BY PHD2 1445BLOOD, 21 FEBRUARY 2013 ⅐ VOLUME 121, NUMBER 8
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