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Prepublished online as a Blood First Edition Paper on May 31, 2002; DOI 10.1182/blood-2001-12-0169.
Previous Article | Table of Contents | Next Article 
Blood, 1 October 2002, Vol. 100, No. 7, pp. 2623-2628
RED CELLS
Hypoxia-inducible erythropoietin gene expression in human
neuroblastoma cells
Ineke Stolze,
Utta Berchner-Pfannschmidt,
Patricia Freitag,
Christoph Wotzlaw,
Jochen Rössler,
Stilla Frede,
Helmut Acker, and
Joachim Fandrey
From the Institut für Physiologie der
Universität Essen; Max-Planck-Institut für molekulare
Physiologie, Dortmund; and Universitäts-Kinderklinik Freiburg,
Germany.
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Abstract |
Two human neuroblastoma (NB) cell lines, SH-SY5Y and Kelly, were
found to express the gene for erythropoietin (EPO) in an oxygen
(O2)-dependent manner. However, NB cells had maximal
production of EPO with lower partial pressure of O2 values
than the well-characterized hepatoma cell line HepG2. This maximal EPO
expression was preceded by accumulation of the O2-sensitive
subunit of the heterodimeric transcription-factor complex
hypoxia-inducible factor 1 (HIF-1). Western blot analysis revealed that
the amount of the subunit of HIF-1, identical to aryl hydrocarbon
receptor nuclear translocator 1 (ARNT1), and the homolog ARNT2
increased in nuclear extracts from SH-SY5Y cells exposed to anoxia. In
neuronal cells, ARNT1 and ARNT2 can form a heterodimer with HIF-1 ,
generating a functional HIF-1 complex. Using the hypoxia response
element of the human EPO enhancer, we conducted electrophoretic
mobility shift assays that showed accumulation and binding of HIF-1
complexes containing both ARNT1 and ARNT2 in NB cells. In addition to
the HIF-1 complex, hepatocyte nuclear factor 4 (HNF4 ) was found
to be indispensable for hypoxia-induced EPO gene expression in hepatoma
cells. Western blot analysis and polymerase chain reaction assessment
showed that NB cells express neither HNF4 nor the splicing variant
HNF4 7 and thus express EPO in an HNF4 -independent manner.
Together, SH-SY5Y and Kelly cells may provide a new in vitro model for
studying the mechanism of tissue-specific, hypoxia-inducible EPO gene expression.
(Blood. 2002;100:2623-2628)
© 2002 by The American Society of Hematology.
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Introduction |
Hypoxia-inducible expression of the glycoprotein
hormone erythropoietin (EPO) is part of the body's response to
hypoxia, which includes up-regulation of oxygen
(O2)-dependent genes involved in vascular tone and growth,
metabolic adaptation, and O2 delivery.1,2 EPO
is produced primarily by the kidneys and liver, but EPO gene expression
has also been found in several other tissues, including brain
tissue,3 breast cancer cells,4 female genital
tract tissues,5 and rat Sertoli cells.6
EPO gene expression is regulated by the heterodimeric
transcription-factor complex hypoxia-inducible factor 1 (HIF-1), which is composed of a 120-kDa O2-regulated subunit and a 91- to 94-kDa constitutively expressed subunit.7 Under
normoxic conditions, HIF-1 is posttranslationally hydroxylated at
proline residues 402 and 564,8 which tags the protein for
ubiquitination by the E3 ubiquitin ligase complex containing the von
Hippel-Lindau tumor-suppressor protein.9,10 Subsequently,
HIF-1 protein is rapidly degraded by the proteasome
system.11 Under hypoxic conditions, the subunit is
stabilized because of the lack of proline hydroxylation and
accumulates. Stabilized HIF-1 translocates into the nucleus and
forms an HIF-1 complex with the almost ubiquitously expressed HIF-1
(identical to aryl hydrocarbon receptor nuclear translocator 1 [ARNT1]). The HIF-1 complex binds to hypoxia response elements (HREs)
found in enhancers or promoters of hypoxia-inducible genes.12 In addition to ARNT1, kidney and neuronal cells
express the ARNT1 homolog ARNT2, which can form a functional HIF
complex with HIF-1 .13
The HIF-1 binding site (HBS) in the 3' EPO enhancer is one of 3 sites
that are important for hypoxia-induced EPO gene
transcription.14 The HBS is the most upstream element and
is followed by a 4-base-pair (bp) CACA repeat and finally a direct
repeat of 2 steroid-hormone receptor half-sites separated by 2 bp,
termed a DR2 site. In HepG2 and Hep3B cells, the transcription factor
hepatocyte nuclear factor 4 (HNF4 ) can bind to this DR2 site.
HNF4, a member of the nuclear receptor superfamily, is expressed
primarily in the liver but also in the kidney, pancreas, and small
intestine. There are 3 known members of the HNF4 family: (human),
(Xenopus), and (human).15,16 HNF4 is
essential for liver-specific EPO gene expression and seems to act
through the DR2 element in the EPO enhancer.17
One important transcriptional coactivator of the HIF-1 complex is
p300.18 A model was postulated in which an HNF4
homodimer binds constitutively to the DR2 site and interacts with
HIF-1. Hypoxia induces formation of the transcriptional complex in
which p300 is believed to serve as a bridge between the enhancer and the promoter of the EPO gene by binding to HNF4 and HIF-1,
respectively. It is only by means of this interaction that the more
than 50-fold stimulation of EPO transcription is achieved in Hep3B
hepatoma cells.14,19
The human hepatoma cell lines HepG2 and Hep3B are so far the only
established models for partial pressure of oxygen
(pO2)-dependent EPO production.20 In the
current study, we investigated hypoxia-inducible EPO gene expression in
2 human neuroblastoma (NB) cell lines: SH-SY5Y and Kelly. We focused on
HIF-1 and HNF4 , 2 transcription factors previously shown to be key
regulators of hypoxia-induced EPO gene expression in hepatoma cell
lines. We found that NB cells mediate hypoxia-induced EPO gene
expression by HIF-1 but in the absence of HNF4 . These cells showed
maximal production of EPO at lower pO2 values than the
well-characterized hepatoma cell line HepG2. As neuronlike
cells, they expressed ARNT2, which like ARNT1, forms heterodimers with
HIF-1 . Our results indicate that these neuronlike cells can be used
to provide an vitro model for further study of tissue-specific
regulation of the EPO gene.
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Materials and methods |
Cell culture
The human NB cell lines SH-SY5Y and Kelly and the human hepatoma
cell line HepG2 were obtained from the American Type Culture Collection
(Manassas, VA). Cells were grown in RPMI-1640 medium (Bio-Whittaker,
Cambrex, Verviers, Belgium) supplemented with 10% fetal-calf serum,
penicillin (100 U/mL), and streptomycin (100 µg/mL) in a humidified
atmosphere of 5% carbon dioxide (CO2) in air. To achieve
hypoxic conditions, culture dishes were placed in an air-tight Heraeus
incubator (Hanau, Germany) with 5% CO2 and nitrogen
(N2) as balance for different time periods and
O2 concentrations. Hypoxia was defined as incubation with
3% O2 (if not otherwise indicated). Anoxic conditions were
established by using anaerobic-culture jars with hydrogen- and
CO2-generating envelopes (Becton Dickinson, Cockeysville,
MD). A methylene blue anaerobic indicator was used to ensure complete
O2 depletion. Control normoxic cells were placed in an
incubator (5% CO2, 21% O2, and 74%
N2) for the same time period. For reoxygenation
experiments, cells were exposed to hypoxia or anoxia for 4 hours and
then transferred to 21% O2 for different times. Nuclear
extracts were prepared by using the method of Schreiber et
al21 and subjected to Western blot analysis and
electrophoretic mobility shift assays (EMSAs).
To evaluate the effects of hypoxia-mimicking agents, deferoxamine
mesylate (Sigma, St Louis, MO), ciclopirox olamine (Sigma), or cobalt
chloride (Sigma) was added to the human NB cell lines and normoxic
conditions were maintained for 6 to 24 hours.
O2 consumption
O2 consumption was measured as described
previously,22 with minor modifications. About
107 cells/mL (SH-SY5Y and HepG2) were suspended in 2-mL
medium and transferred to a closed water-jacketed chamber (Hansatech
Instruments, King's Lynn, United Kingdom) kept constantly at 37°C.
The O2 pressure in the chamber was measured continuously
with a Clark-type electrode, and O2 consumption was
calculated from the slope of the graph representing decreasing
pO2 values in the chamber. At the end of the experiment,
cells were lysed in sodium hydroxide and sodium dodecyl sulfate for
total protein determination. Five separate experiments were performed
for each cell line, and mean O2 consumption was calculated
as nanomoles of O2 per milligram of total protein and per minute.
RNA preparation and EPO complementary DNA (cDNA) quantification
Total RNA was extracted, and cDNA was prepared from 1 µg total
RNA as described previously.23 For qualitative analysis, EPO forward primer 5'-TCT GGG AGC CCA GAA GGA AGC CAT-3' and reverse primer 5'-CTG GAG TGT CCA TGG GAC AG-3' with an amplification profile
(31 cycles; 94°C for 3 minutes, 60 °C for 1 minute, and 72°C for
1.5 minutes) were used, yielding a polymerase chain reaction (PCR)
product of 301 bp. For EPO quantification, forward primer 5'-CTC CGA
ACA ATC ACT GCT-3' and reverse primer 5'-GGT CAT CTG TCC CCT GTC T-3'
were used in a 2-step real-time PCR with a denaturation step at 95°C
for 10 minutes and then 40 cycles at 95°C for 15 seconds and 60°C
for 1 minute (SYBR-Green, GeneAmp 5700 Sequence Detection System;
Applied Biosystems, Weiterstadt, Germany). In addition, PCR for
-actin was performed by using forward primer 5'-CGG GAA ATC GTG CGT
GAC AT-3' and reverse primer 5'-GAA CTT TGG GGG ATG CTC GC-3' for 25 cycles with an annealing temperature of 57°C. For -actin
quantification, forward primer 5'-TCA CCC ACA CTG TGC CCA TCT ACG A-3'
and reverse primer 5'-CAG CGG ACC CGC TCA TTG CCA ATG G-3' were used.
Human HNF4 was amplified in 35 cycles by using forward primer 5'-GGC
TGA GCG ATC CAG GGA AG AA-3' and reverse primer 5'-CCA GCG GCT TGC TAG
ATA AC-3' with an annealing temperature of 60°C. Human HNF4 7 was
amplified in 35 cycles with an annealing temperature of 63°C by using
forward primer 5'-GGG TGG GCT TGG CCA TGG TCA GCG TG-3' and reverse
primer 5'-TCC GCC TGC AGG AGC GCA TT-3' (provided by G. U. Ryffel,
Essen, Germany). The resulting PCR fragments were visualized on
ethidium bromide-stained 1.5% agarose gels.
Protein-extract preparation and Western blotting
Nuclear protein extracts were prepared from 60-mm dishes of
subconfluent cells by using the method of Schreiber et
al.21 For whole-cell extracts, cells were washed with
ice-cold phosphate-buffered saline, drained, and then lysed on the
plates with 100 µL extract buffer (300 mM sodium chloride, 10 mM Tris
[(tris(hydroxymethyl)aminomethane; pH 7.9], 1 mM EDTA
[ethylenediaminetetraacetic acid], 0.1% NP-40 [nonylphenoxypolyethoxy ethanol], and 1 × protease
inhibitor cocktail; Roche, Basel, Switzerland) for 20 minutes on ice.
The extract was spun down in a microcentrifuge (5000 rpm at 4°C for 5 minutes), and the protein concentration was measured by using a protein assay reagent (Bio-Rad Laboratories, Hercules, CA).
Western blot analysis were performed as described
previously.24 The primary antibodies used were monoclonal
antibodies anti-HIF-1 (diluted 1:250) and anti-HIF-1 (diluted
1:500) (both from Transduction Laboratories, San Diego, CA), a rabbit
polyclonal anti-ARNT2 (diluted 1:500; Santa Cruz Biotechnology, Santa
Cruz, CA), and a rabbit polyclonal anti-HNF4 antibody (diluted
1:500, reactive with HNF4 and, to a lesser extent, with HNF4 ;
Santa Cruz Biotechnology). Anti- -tubulin (diluted 1:500; Santa Cruz
Biotechnology) and antihistone H1 (diluted 1:1000; Biozol, Eching,
Germany) antibodies were used to detect the respective
proteins as loading controls for whole-cell lysate and the nuclear
compartment. Immunoreactive proteins were visualized by using
electrogenerated chemiluminescence detection and x-ray films.
Enzyme-linked immunosorbent assay for EPO
EPO protein in the culture supernatant was measured by
enzyme-linked immunosorbent assay (ELISA; Quantikine IVD EPO; R&D
Systems, Wiesbaden-Nordenstadt, Germany).
Immunofluorescence analysis
HepG2 and SH-SY5Y cells were fixed by applying ice-cold methanol
and acetone (1:1) for 10 minutes at 20°C, blocked, and incubated with the monoclonal anti-HIF-1 antibody (1:50; Transduction
Laboratories) followed by an Alexa Fluor 488-conjugated goat antimouse
IgG antibody (1:400; Molecular Probes, Eugene, OR). The immunostained
cells were visualized in false colors representing different
fluorescence intensities by using a fluorescence microscope (E1000;
Nikon, Düsseldorf, Germany) equipped with a charge-coupled
digital camera (Optronics; Visitron Systems, Puchheim, Germany) and
image-acquisition software (EZ2000; Coord, Utrecht, Netherlands).
EMSAs
Double-stranded oligonucleotides (synthesized by Gibco, Grand
Island, NY) containing the wild-type HBS (EPOWt) or mutated HBS
(EPOMut) from the HRE (5' GCC CTA CGT GCT GTC TCA or 5' GCC CTA AAA GCT
GTC TCA, respectively) of the EPO enhancer were end-labeled with
-phosphorus 32 (32P)-adenosine triphosphate (ICN,
Munich, Germany) and T4 polynucleotide kinase (Fermentas, St Leon-Rot,
Germany) and used as probes. Binding reactions were set up in
a volume of 20 µL, and 5 µg nuclear extract, 30 fmol
32P-labeled oligonucleotide, and a nonspecific competitor
(50 ng calf-thymus DNA; Sigma) were incubated for 30 minutes at room temperature in a buffer with a final concentration of 12 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid; pH 7.9), 4 mM Tris (pH 7.9), 60 mM potassium chloride, 1 mM EDTA, and 1 mM dithiothreitol before the antibody (1 µg) was added
for a final incubation overnight at 4°C. Samples were resolved by
electrophoresis on nondenaturing 5% polyacrylamide gel at 4°C. The
dried gels were exposed to x-ray films overnight.
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Results |
Hypoxic EPO gene expression in human NB cell lines
Human NB cells cultured as a monolayer to a confluence of 50% to
60% were exposed to hypoxic or anoxic conditions for 24 hours. Hypoxia
increased EPO messenger RNA (mRNA) levels in SH-SY5Y (2.3-fold) and Kelly cells (71.6-fold), with a maximal increase after exposure to
anoxia (30-fold and 238.3-fold, respectively; Figure
1). In contrast, 60% confluent hepatoma
cells (HepG2) showed maximal stimulation under hypoxic conditions
(10.4-fold) but had lower EPO mRNA levels under anoxic conditions
(Figure 1).

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| Figure 1.
Hypoxia-inducible EPO gene expression in human NB cell
lines.
SH-SY5Y cells, Kelly cells, and the hepatoma cell line HepG2 were grown
for 24 hours under normoxic (NOX), hypoxic (HOX), or anoxic (AOX)
conditions. Total RNA was prepared, reverse-transcribed into cDNA, and
subjected to real-time PCR for EPO and -actin quantification. EPO
cDNA was normalized to -actin cDNA and expressed as relative
units × 1000. Bars represent mean cDNA values (± SD) from 3 separate experiments in which each cDNA was quantitated in
triplicate.
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Under normoxic conditions, no EPO protein was detectable by ELISA in
the culture supernatant of SH-SY5Y or Kelly cells (50%-60% confluence). Hypoxia increased EPO protein secretion significantly in
Kelly cells (41.5 ± 1.5 mU/mL; n = 6). Maximal EPO protein levels were detected in both cell lines under anoxic conditions (SH-SY5Y, 8.2 ± 1.9 mU/mL, and Kelly, 132.3 ± 10.2 mU/mL;
n = 6). Compared with NB cells, HepG2 cells showed maximal EPO
secretion under hypoxic conditions (16.1 ± 1.4 mU/mL; n = 3),
and secretion returned to baseline levels (6.0 ± 1.0 mU/mL;
n = 3) under anoxic conditions (data not shown).
Exposure to hypoxia-mimicking agents,25 including
ciclopirox olamine (20 µM) for 6 hours, deferoxamine mesylate (100 µM) for 24 hours, and cobalt chloride (100 µM) for 24 hours,
induced EPO mRNA transcription in both cell lines (data not shown).
HIF-1 protein accumulation and nuclear translocation in NB
cells
HIF-1 protein accumulation was analyzed in whole-cell lysates
from subconfluent (50%-60%) SH-SY5Y and Kelly cells incubated under
hypoxic or anoxic conditions for 4 hours. Using the monoclonal anti-HIF-1 antibody, we detected the double band for HIF-1
protein in SH-SY5Y cells under anoxic conditions and in Kelly
cells under hypoxic and anoxic conditions (Figure
2A). Both cell lines showed maximal
HIF-1 protein accumulation (both bands) under anoxic conditions.

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| Figure 2.
Hypoxic HIF-1 protein accumulation and nuclear
translocation in NB cells.
(A) Western blot analysis of HIF-1 was performed on whole-cell
extracts from SH-SY5Y and Kelly cells cultured for 4 hours under
normoxic (N), hypoxic (H), and anoxic (A) conditions. Antibody
against -tubulin was used to ensure equal loading. Gels are
representative of at least 3 experiments. (B) To compare HIF-1
accumulation, HepG2 and SH-SY5Y cells were exposed to different
O2 concentrations for 4 hours and whole-cell lysates (75 µg/lane) were subjected to Western blot analysis. (C) Indirect
immunofluorescence analysis of HIF-1 in SH-SY5Y and HepG2 cells
cultured for 4 hours under different O2 concentrations.
Fluorescent intensities are shown in false colors, from blue
(low fluorescence) to red (high fluorescence), as indicated by a
colored scale bar; white scale bar represents 10 µm.
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To compare HIF-1 protein accumulation and nuclear translocation,
SH-SY5Y and HepG2 cells (50%-60% confluence) were exposed to
different O2 concentrations for 4 hours, and Western blot
and immunofluorescence analyses were performed. Because cellular
pO2 is the result of the O2 supply and
O2 consumption in the culture dish,26
O2 consumption of HepG2 and SH-SY5Y cells was measured. Specific O2 consumption was 35 ± 7 nM
O2/mg protein per minute (n = 5) for HepG2 cells and
35 ± 4 nM O2/mg protein per minute (n = 5) for
SH-SY5Y cells. In addition, culture dishes were shaken gently to avoid
pO2 gradients in the culture supernatant.
In HepG2 cells, very low levels of HIF-1 protein (lower band) were
found under normoxic conditions (Figure 2B). A significant induction of
both bands became visible at 7% O2 and increased gradually
with reduction of the O2 concentration. In SH-SY5Y cells, only small amounts of HIF-1 protein accumulated at 7%
O2 (lower band), but amounts increased significantly under
anoxic conditions.
Indirect immunofluorescence assessment revealed HIF-1 in the nuclear
compartment in a limited number of HepG2 cells under normoxic
conditions (Figure 2C). A substantial number of HepG2 cells showed
nuclear translocation of HIF-1 at 7% O2. In SH-SY5Y cells, nuclear accumulation of HIF-1 was detected at 3%
O2. Nuclear localization at these O2 levels was
heterogeneous in both cell lines and was not detected in all cells.
However, with a further decrease in pO2, more cells with
nuclear HIF-1 localization were observed, as indicated by maximal
immunofluorescence for nuclear HIF-1 protein under anoxic conditions.
To study degradation of HIF-1 protein on reoxygenation, SH-SY5Y
cells were incubated for 4 hours under hypoxic or anoxic conditions and
then transferred to normoxic conditions for different time periods.
Western blot analysis revealed that HIF-1 was rapidly degraded, with
a half-life of 1.5 minutes after exposure to hypoxia and a half-life of
3.5 minutes after exposure to anoxia (data not shown).
Expression of HIF-1 , ARNT1, and ARNT2, but not HNF4 , in
SH-SY5Y cells
In neuronal cell types, HIF-1 can form a heterodimer with
ARNT2, an ARNT1 homolog, generating a functional HIF-1
complex.13 On Western blot analysis, ARNT1 and ARNT2 were
detected in nuclear extracts and whole-cell lysates of SH-SY5Y cells
(Figure 3). Significant expression of
ARNT2 was observed in the nuclei of SH-SY5Y cells under normoxic
conditions. The highest levels of HIF-1 , ARNT1, and ARNT2 were found
in nuclear extracts from SH-SY5Y cells exposed to anoxia for 4 hours.
In nuclear extracts from HepG2 cells, maximal translocation of HIF-1
and ARNT1 was found under hypoxic conditions.

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| Figure 3.
Expression of HIF-1 , ARNT1, and ARNT2 but not HNF4
in SH-SY5Y cells.
Western blot analyses of nuclear extracts (20 µg) and whole-cell
lysates (75 µg) from SH-SY5Y and HepG2 cells exposed to normoxia (N),
hypoxia (H), and anoxia (A) for 4 hours were performed with antibodies
against HIF-1 , ARNT1, ARNT2, and HNF4 . Antibodies against histone
H1 and -tubulin were used as loading controls for nuclear extracts
and total-cell lysates, respectively.
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In hepatoma cells, hypoxia-induced EPO gene expression was enhanced by
the constitutively expressed HNF4 , which was detected in HepG2
extracts by Western blot analysis (Figure 3). Interestingly, the amount
of HNF4 protein decreased in nuclear extracts from HepG2 cells
exposed to anoxia. In contrast, no HNF4 protein was found in SH-SY5Y
cells. An immunoreactive band that appeared in whole-cell lysates from
both SH-SY5Y cells and HepG2 cells represented an unspecific band
rather than HNF4 because the band did not match the size of HNF4 .
We also did not detect any signal of HNF4 or HNF4 7 mRNA (data not
shown), a splicing variant of HNF4 that is expressed in trace
amounts in the brain and several other tissues.27
HIF-1 forms a heterodimer with ARNT1 and ARNT2 in SH-SY5Y
cells
To analyze hypoxia-induced HIF complexes, EMSAs using the EPO 3'
enhancer-derived HIF-1-binding HRE (EPOWt) were performed. Induction
of HIF-1 DNA-binding activity was clearly detected in nuclear extracts
from SH-SY5Y cells exposed to anoxia for 4 hours. To confirm the
identity of the HIF-1 band obtained, supershift experiments using the
monoclonal anti-HIF-1 antibody were performed. Furthermore,
incubation of nuclear extracts from SH-SY5Y cells exposed to anoxia
with the mutated HIF-1 DNA-binding oligonucleotide (EPOMut) abolished
the inducible band (Figure 4). Supershift
analysis with anti-ARNT1 and anti-ARNT2 antibodies revealed
accumulation of both ARNT1-containing and ARNT2-containing HIF
complexes (Figure 4).

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| Figure 4.
Hypoxic-inducible HIF-1 DNA-binding activity in SH-SY5Y
cells.
EMSAs of nuclear extracts prepared from SH-SY5Y cells exposed to anoxia
for 4 hours were performed with the 32P-labeled EPOWt
oligonucleotide. The HIF-1 complex, the constitutive (c), the
unspecific band (u), and the free probe are indicated. Supershifted
bands are indicated by arrows. Supershift analyses were performed with
anti-HIF-1 (lane 1), anti-ARNT1 (lane 2), and anti-ARNT2 (lane 3)
antibodies. Lane 4 represents the inducible HIF-1 band without any
antibody. No inducible HIF-1 band was obtained when the nuclear extract
was incubated with the EPOMut oligonucleotide (lane 5).
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Discussion |
In this study, we investigated EPO gene regulation in 2 human NB
cell lines, SH-SY5Y and Kelly. This is the first report of permanent
neuronlike cells expressing the EPO gene in an O2-dependent manner. NB cells are derived from the sympathetic neuroblasts of the
peripheral nervous system and show features of fetal neuronal cells.
Here, we used undifferentiated, predominantly neuroblastic SH-SY5Y and
Kelly cells, both of which showed expression of the neuronal markers
neuropeptide Y and growth-associated protein 4328 on PCR
(data not shown). In addition, both cell lines expressed ARNT2, which
is found mainly in kidney and neuronal tissue and only at low levels in
embryonic tissues. Therefore, NB cells may provide a new model for
studying hypoxia-inducible EPO gene expression in human neuronlike
cells and may serve to identify tissue-specific factors in the
regulation of the EPO gene.
Two well-characterized hepatoma cell lines, HepG2 and Hep3B, have been
the only in vitro models for studying O2-dependent EPO gene
expression. We found that production of hypoxia-inducible EPO in
SH-SY5Y and Kelly cells appeared to be different from that in HepG2
cells. Maximal stimulation of EPO gene expression was observed
under anoxic conditions, whereas HepG2 cells showed maximal EPO gene
expression under hypoxic conditions and already decreased EPO
production under anoxic conditions. Moreover, it appeared that more EPO
protein per EPO mRNA was made in NB cells. However, this finding may be
misleading because we observed considerable variations in -actin
levels in HepG2 cells. Although -actin was not
O2-regulated in our experiments, it may not be the
appropriate normalization control in HepG2 cells. Nevertheless, maximal
EPO expression in SH-SY5Y and Kelly cells appeared to be shifted to very low pO2 values. We therefore searched for differences
between hepatoma and NB cells with respect to transcription factors
known to control EPO gene expression. In hepatoma cells,
hypoxia-inducible EPO gene expression is regulated mainly by binding of
HIF-1 and HNF4 to regulatory DNA elements.14
On exposure to hypoxia, HIF-1 protein accumulation in NB and hepatoma
cells differed with respect to the appearance of the upper band of
HIF-1 on Western blot analysis. Previous studies had revealed that
the transactivating activity of HIF-1 is increased by phosphorylation
of HIF-1 and that the more slowly migrating HIF-1 (upper band)
represents the phosphorylated form.29,30 Different
kinases, including the phosphatidyl-3 kinase/Akt and the ERK/p42/p44
pathway, have been proposed to be involved in HIF-1 phosphorylation,
causing a doublet of nonphosphorylated (lower band) and phosphorylated
(upper band) HIF-1 .30,31 In SH-SY5Y cells, the lower
band of the HIF-1 doublet accumulated at 7% O2, and
when the O2 concentration was less then 3%, the upper band
became detectable. In Kelly cells, 3% O2 was sufficient to
induce both bands. This was correlated with higher EPO expression in
Kelly cells than in SH-SY5Y cells. In contrast, HepG2 cells accumulated
both forms of HIF-1 at 7% O2 and only gradually
increased the doublet until anoxia occurred. Interestingly, whereas in
NB cells, maximal EPO expression coincided with the highest
phosphorylation status of HIF-1 , anoxic HepG2 cells already had
severely reduced EPO expression again. This was not, however, due to
reduced HIF-1 levels, because they remained as high as they were
under 3% O2. Immunofluorescence analysis revealed a
similar difference between HepG2 and SH-SY5Y cells in the
O2-induced nuclear translocation of HIF-1
protein. It appeared that the induction of nuclear HIF-1 translocation in SH-SY5Y cells was shifted to lower O2
concentrations than in HepG2 cells (Figure 2C). Both cell lines showed
maximal nuclear localization under anoxic conditions, with the
cytoplasm almost "empty" of HIF-1 protein.
With respect to the degradation of HIF-1 on reoxygenation, SH-SY5Y
cells behaved very much as was described previously for HeLa
cells.31 Reoxygenation from a 4-hour period of hypoxia resulted in a shorter half-life of HIF-1 (1.5 minutes) than
reoxygenation from anoxia (3.5 minutes). Different degradation kinetics
could be caused by hypoxic induction of proline hydroxylases that
posttranslationally mark HIF-1 for degradation at a high
pO2. HeLa cells express all 3 proline hydroxylases (PHDs)
identified so far (PHD 1, 2, and 3), but only PHD2 and PHD3 expression
is induced by hypoxia.32 Which PHDs are expressed in NB
cells is currently under investigation, but there is currently no
evidence that anoxia reduces expression of PHDs. Reduced levels of PHDs
would explain the longer half-life after anoxia occurred.
In neuronal cells, different HIF-1 heterodimers seem to exist, because
HIF-1 , ARNT1, and ARNT2 can form functional HIF-1 complexes.13,33 On Western blot analysis, we found maximal levels of HIF-1 , ARNT1, and ARNT2 in nuclear extracts from SH-SY5Y cells exposed to anoxia. Under normoxic conditions, little HIF-1 , ARNT1, or ARNT2 was found in nuclear extracts. In whole-cell lysates, ARNT1 and ARNT2 appeared to be constitutively expressed. These results
are in agreement with data from studies in the rat pheochromocytoma cell line PC12, which constitutively coexpresses HIF-1 , ARNT1, and
ARNT2 under nonhypoxic conditions.34 Interestingly, we
observed increased levels of ARNT1 and ARNT2 under anoxic conditions,
although these dimerization partners for HIF-1 are generally
believed to be constitutively expressed. However, it was previously
shown that heterodimerization of HIF-1 and ARNT1 stabilized both
subunits within the nuclear compartment, whereas single subunits tended to leak from the nuclei into the cytoplasm during preparation of
nuclear extracts.34 This could account for the increased levels of ARNT1 and ARNT2 in nuclei under anoxic conditions. Maximal DNA binding to the HRE from the EPO enhancer was observed in nuclear extracts from SH-SY5Y cells exposed to anoxia. On the basis of supershift analysis of the DNA-bound complexes, it appears that in
SH-SY5Y cells, ARNT1 and ARNT2 generate DNA-binding complexes with
HIF-1 .
In addition to HIF-1, the transcription factor HNF4 plays an
important role in enhancement of the hypoxic induction of the EPO gene
by means of the DR2 element in hepatoma cells.17 The importance of HNF4 for EPO expression in fetal-liver hepatocytes has
been demonstrated.35 On Western blot analysis, we found HNF4 expression in HepG2 cells but not in SH-SY5Y cells. In
addition, using PCR, we excluded expression of HNF4 7, which is
expressed in several tissues typically lacking HNF4 (eg,
brain).28 Although we cannot exclude the possibility that
there is binding of other members of this transcription-factor family
to the DR2 element of the EPO enhancer in NB cells, SH-SY5Y and Kelly
cells appear to regulate hypoxia-inducible EPO gene expression by means
of HIF-1 but in the absence of HNF4 . Together, these cells
may provide a new valuable model for further study of hypoxia-inducible
EPO production in neuronlike cells.
 |
Acknowledgments |
We thank G. U. Ryffel for the gift of the
HNF4 7 primer, G. Endemann for critically reading the manuscript, and
B. Trinidad for excellent technical assistance; we are particularly
grateful to an unknown reviewer whose criticism substantially improved our work.
 |
Footnotes |
Submitted December 5, 2001; accepted May 4, 2002.
Prepublished online
as Blood First Edition Paper, May 31, 2002; DOI
10.1182/blood-2001-12-0169.
Supported by IFORES grant 107 533-0 and BMBF grant 13N7447.
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 U.S.C.
section 1734.
Reprints: Joachim Fandrey, Institut für
Physiologie, Universität Essen, Hufelandstr 55, D-45147 Essen,
Germany; e-mail: joachim.fandrey{at}uni-essen.de.
 |
References |
1.
Jelkmann W.
Erythropoietin: structure, control of production, and function.
Physiol Rev.
1992;72:449-489[Free Full Text].
2.
Semenza GL.
Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1.
Annu Rev Cell Dev Biol.
1999;15:551-578[CrossRef][Medline]
[Order article via Infotrieve].
3.
Dame C, Bartmann P, Wolber E, Fahnenstich H, Hofmann D, Fandrey J.
Erythropoietin gene expression in different areas of the developing human central nervous system.
Dev Brain Res.
2000;125:69-74[Medline]
[Order article via Infotrieve].
4.
Acs G, Acs P, Beckwith SM, et al.
Erythropoietin and erythropoietin receptor expression in human cancer.
Cancer Res.
2001;61:3561-3565[Abstract/Free Full Text].
5.
Yasuda Y, Masuda S, Chikuma M, Inoue K, Nagao M, Sasaki R.
Estrogen-dependent production of erythropoietin in uterus and its implication in uterine angiogenesis.
J Biol Chem.
1998;273:25381-25387[Abstract/Free Full Text].
6.
Magnanti M, Gandini O, Giuliani L, et al.
Erythropoietin expression in primary rat Sertoli and peritubular myoid cells.
Blood.
2001;98:2872-2874[Abstract/Free Full Text].
7.
Wang GL, Jiang BH, Rue EA, Semenza GL.
Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.
Proc Natl Acad Sci U S A.
1995;92:5510-5514[Abstract/Free Full Text].
8.
Masson N, Willam C, Maxwell PH, Pugh CW, Ratcliffe PJ.
Independent function of two destruction domains in hypoxia-inducible factor- chains activated by prolyl hydroxylation.
EMBO J.
2001;20:5197-5206[CrossRef][Medline]
[Order article via Infotrieve].
9.
Ivan M, Kondo K, Yang H, et al.
HIF targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing.
Science.
2001;292:464-468[Abstract/Free Full Text].
10.
Jaakkola P, Mole DR, Tian YM, et al.
Targeting of HIF- to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.
Science.
2001;292:468-472[Abstract/Free Full Text].
11.
Salceda S, Caro J.
Hypoxia-inducible factor 1 (HIF1 ) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions.
J Biol Chem.
1997;272:22642-22647[Abstract/Free Full Text].
12.
Maxwell PH, Pugh CW, Ratcliffe PJ.
Activation of the HIF pathway in cancer.
Curr Opin Genet Dev.
2001;11:293-299[CrossRef][Medline]
[Order article via Infotrieve].
13.
Maltepe E, Keith B, Arsham AM, Brorson JR, Simon MC.
The role of ARNT2 in tumor angiogenesis and the neuronal response to hypoxia.
Biochem Biophys Res Comm.
2000;273:231-238[CrossRef][Medline]
[Order article via Infotrieve].
14.
Ebert BL, Bunn HF.
Regulation of the erythropoietin gene.
Blood.
1999;94:1864-1877[Free Full Text].
15.
Drewes T, Senkel S, Holewa B, Ryffel GU.
Human hepatocyte nuclear factor 4 isoforms are encoded by distinct and differentially expressed genes.
Mol Cell Biol.
1996;16:925-931[Abstract].
16.
Holewa B, Zapp D, Drewes T, Senkel S, Ryffel GU.
HNF4 , a new gene of the HNF4 family with distinct activation and expression profiles in oogenesis and embryogenesis of Xenopus laevis.
Mol Cell Biol.
1997;17:687-694[Abstract].
17.
Bunn HF, Gu J, Huang LE, Park J-W, Zhu H.
Erythropoietin: a model system for studying oxygen-dependent gene regulation.
J Exp Biol.
1998;201:1197-1201[Abstract].
18.
Arany Z, Huang LE, Eckner R, et al.
Participation by the p300/CBP family of proteins in the cellular response to hypoxia.
Proc Natl Acad Sci U S A.
1996;93:12969-12973[Abstract/Free Full Text].
19.
Blanchard KL, Acquaviva AM, Galson DL, Bunn HF.
Hypoxic induction of the human erythropoietin gene: cooperation between the promoter and enhancer, each of which contains steroid receptor response elements.
Mol Cell Biol.
1992;12:5373-5385[Abstract/Free Full Text].
20.
Goldberg MA, Glass GA, Cunningham JM, Bunn HF.
The regulated expression of erythropoietin by two human hepatoma cell lines.
Proc Natl Acad Sci U S A.
1987;84:7972-7976[Abstract/Free Full Text].
21.
Schreiber E, Matthias P, Müller M, Schaffner W.
Rapid detection of octamer binding proteins with `mini-extracts', prepared from a small number of cells.
Nucleic Acids Res.
1989;17:6419[Free Full Text].
22.
Fandrey J, Pagel H, Wolff M, Jelkmann W.
Thyroid hormones enhance hypoxia-induced erythropoietin production in vitro.
Exp Hematol.
1994;22:272-277[Medline]
[Order article via Infotrieve].
23.
Fandrey J, Bunn HF.
In vivo and in vitro regulation of erythropoietin mRNA: measurement by competitive polymerase chain reaction.
Blood.
1993;81:617-623[Abstract/Free Full Text].
24.
Metzen E, Fandrey J, Jelkmann W.
Evidence against a major role for Ca2+ in hypoxia-induced gene expression in human hepatoma cells (Hep3B).
J Physiol.
1999;517.3:651-657.
25.
Wenger RH, Gassmann M.
Oxygen(es) and the hypoxia-inducible factor-1.
Biol Chem.
1997;378:609-616[Medline]
[Order article via Infotrieve].
26.
Metzen E, Wolff M, Fandrey J, Jelkmann W.
Pericellular PO2 and O2 consumption in monolayer cell cultures.
Resp Physiol.
1995;100:101-106[CrossRef][Medline]
[Order article via Infotrieve].
27.
Nakhei H, Lingott A, Lemm I, Ryffel GU.
An alternative splice variant of the tissue specific transcription factor HNF4 predominates in undifferentiated murine cell types.
Nucleic Acids Res.
1998;26:497-504[Abstract/Free Full Text].
28.
Pahlman S, Meyerson G, Lindgren E, Schalling M, Johansson I.
Insulin-like growth factor I shifts from promoting cell division to potentiating maturation during neuronal differentiation.
Proc Natl Acad Sci U S A.
1991;88:9994-9998[Abstract/Free Full Text].
29.
Berra E, Milanini J, Richard DE, et al.
Signaling angiogenesis via p42/44 MAP kinase and hypoxia.
Biochem Pharmacol.
2000;60:1171-1178[CrossRef][Medline]
[Order article via Infotrieve].
30.
Minet E, Michel G, Mottet D, Raes M, Michiels C.
Transduction pathways involved in hypoxia-inducible factor-1 phosphorylation and activation.
Free Radic Biol Med.
2001;31:847-855[CrossRef][Medline]
[Order article via Infotrieve].
31.
Jewell UR, Kvietikova I, Scheid A, Bauer C, Wenger RH, Gassmann M.
Induction of HIF-1 in response to hypoxia is instantaneous.
FASEB J.
2001;15:1312-1324[Free Full Text].
32.
Epstein AC, Gleadle JM, McNeill LA, 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[CrossRef][Medline]
[Order article via Infotrieve].
33.
Drutel G, Kathmann M, Hèron A, et al.
Two splice variants of the hypoxia-inducible factor HIF-1 as potential dimerization partners of ARNT2 in neurons.
Eur J Neurosci.
2000;12:3701-3708[CrossRef][Medline]
[Order article via Infotrieve].
34.
Chilov D, Camenisch G, Kvietikova I, Ziegler U, Gassmann M, Wenger RH.
Induction and nuclear translocation of hypoxia-inducible factor-1 (HIF-1): heterodimerization with ARNT is not necessary for nuclear accumulation of HIF-1 .
J Cell Sci.
1999;112:1203-1212[Abstract].
35.
Makita T, Hernandez-Hoyos G, Chen TH-P, Wu H, Rothenberg EV, Sucov HM.
A developmental transition in definitive erythropoiesis: erythropoietin expression is sequentially regulated by retinoic acid receptors and HNF4.
Genes Dev.
2001;15:889-901[Abstract/Free Full Text].

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