(Received for publication, February 3, 1997, and in revised form, May 12, 1997)
From the Institute of Physiology, University of Zürich-Irchel, CH-8057 Zürich, Switzerland
Transferrin (Tf) is a liver-derived iron
transport protein whose plasma concentration increases following
exposure to hypoxia. Here, we present a cell culture model capable of
expressing Tf mRNA in an oxygen-dependent manner. A
4-kilobase pair Tf promoter/enhancer fragment as well as the 300-base
pair liver-specific Tf enhancer alone conveyed hypoxia responsiveness
to a heterologous reporter gene construct in hepatoma but not HeLa
cells. Within this enhancer, a 32-base pair hypoxia-responsive element
was identified, which contained two hypoxia-inducible factor-1 (HIF-1)
binding sites (HBSs). Mutation analysis showed that both HBSs function
as oxygen-regulated enhancers in Tf-expressing as well as in
non-Tf-expressing cell lines. Mutation of both HBSs was necessary to
completely abolish hypoxic reporter gene activation. Transient
co-expression of the two HIF-1 subunits HIF-1 and aryl hydrocarbon
receptor nuclear translocator (ARNT)/HIF-1
resulted in enhanced
reporter gene expression even under normoxic conditions. Overexpression
of a dominant-negative ARNT/HIF-1
mutant reduced hypoxic activation. DNA binding studies using nuclear extracts from the mouse hepatoma cell
line Hepa1 and the ARNT/HIF-1
-deficient subline Hepa1C4, as well as
antibodies raised against HIF-1
and ARNT/HIF-1
confirmed that
HIF-1 binds the Tf HBSs. Mutation analysis and competition experiments
suggested that the 5
HBS was more efficient in binding HIF-1 than the
3
HBS. Finally, hypoxic induction of endogenous Tf mRNA was
abrogated in Hepa1C4 cells, confirming that HIF-1 confers oxygen
regulation of Tf gene expression by binding to the two HBSs present in
the Tf enhancer.
Iron is an essential trace metal in all living organisms. Both
iron overload and iron depletion can severely affect physiological processes such as development, erythropoiesis, or biochemical metabolism (reviewed in Refs. 1 and 2). The liver represents the major
organ of iron storage in the body and is most susceptible to injuries
due to iron overload (1). Thus, iron hemostasis has to be tightly
balanced, and, as a consequence, free iron occurs only transiently in
the serum. When iron is absorbed from the small intestine into the
blood, it immediately binds apotransferrin to form transferrin
(Tf)1, which is then
transported by the plasma to all tissues of the vertebrate's body.
Delivery of iron occurs by binding of Tf to the Tf receptor followed by
endocytosis. In erythroblasts, iron is primarily required for heme
synthesis in mitochondria. Tissue-specific expression of the Tf gene is
controlled by distinct positive and negative regulatory elements
located 5 to the transcription initiation site. Apart from the
promoter, the best studied element within this region is the
3600/
3300 enhancer (hereafter referred to as the Tf enhancer). This
cis-acting element enhances the activity of the Tf promoter
in human Hep3B hepatoma cells in a tissue-specific manner (3, 4).
Studies in Hep3B and HeLa cells revealed that multiple liver-enriched
and ubiquitous factors interact with the Tf enhancer (3, 5, 6). The Tf
enhancer, however, is inactive in Tf-expressing neuronal and Sertoli
cells (4, 5).
Hypoxia, a reduction in oxygen concentration, is increasingly recognized as an important regulator of gene expression (reviewed in Ref. 7). The best established example of oxygen-regulated gene expression is provided by the erythropoietic growth factor erythropoietin (Epo, reviewed in Ref. 8). The two human hepatoma cell lines HepG2 and Hep3B are so far the only permanent cell culture models available to investigate oxygen-regulated Epo expression (9). Apart from Epo, we recently demonstrated hypoxic induction of several acute phase genes in HepG2 cells (10). Acute phase reactants are liver-derived serum proteins whose production is induced by proinflammatory cytokines (reviewed in Ref. 11). Tf expression was of particular interest since this protein is one of the rare examples of acute phase reactants that are down-regulated during the acute phase response in both human serum and HepG2 cells (11). In contrast, we found a marked increase in Tf transcription following hypoxic (1% O2) culture of HepG2 cells (10), suggesting that different signaling pathways are mediating the effects of these two stimuli. Given that the erythroid marrow uses more than 80% of plasma iron (2), and considering that hypoxia increases erythropoiesis, it is conceivable that an increase in plasma iron transport capacity is required for hypoxia-induced Epo-mediated erythropoiesis. Indeed, hypoxia was shown to increase iron absorption (12), and hypoxic up-regulation of Tf serum protein concentrations has previously been found in mice (13) and rats (14, 15) exposed to hypobaric hypoxia (0.5 atm) for 1-3 days. Although some of these experiments were established some 40 years ago, the molecular mechanisms leading to hypoxically enhanced Tf expression have not been unraveled so far, mainly due to the lack of a suitable cell culture model.
The hypoxia-inducible factor-1 (HIF-1) was originally identified by its
ability to bind to a hypoxia-responsive cis-element located
3 to the Epo gene (16). HIF-1 is a heterodimer consisting of an
and a
subunit, both belonging to the
basic-helix-loop-helix-Per-AhR/ARNT-Sim family of transcription factors
(17). Whereas the
subunit is a novel member of this family, the
subunit is identical to the aryl hydrocarbon receptor nuclear
translocator (ARNT) known to heterodimerize with the aryl hydrocarbon
receptor/dioxin receptor (AhR) following ligand binding (reviewed in
Refs. 7 and 18).
We have established cell culture models to study oxygen-dependent Tf expression and have subsequently analyzed the regulation of the Tf enhancer. Our results demonstrate the presence of two HIF-1 binding sites (HBSs) within the Tf enhancer and show that binding of HIF-1 to these sites confers oxygen-regulated Tf gene expression.
The human hepatoma cell lines Hep3B and HepG2 were obtained from American Type Culture Collection (ATCC numbers HB-8064 and HB-8065, respectively). The mouse hepatoma cell lines Hepa1 (also termed Hepa1c1c7) and Hepa1C4 (19) were kind gifts of L. Poellinger (Karolinska Institute, Stockholm, Sweden). All cells were cultured in Dulbecco's modified Eagle's medium (high glucose, Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal calf serum (Boehringer Mannheim), 100 units/ml penicillin, 100 µg/ml streptomycin, 1 × non-essential amino acids, 2 mM L-glutamine, and 1 mM sodium pyruvate (all Life Technologies, Inc.) in a humidified atmosphere containing 5% CO2 at 37 °C. Oxygen tensions in the incubator (Forma Scientific, model 3319) were either 140 mm Hg (20% O2, v/v, normoxia) or 7 mm Hg (1% O2, v/v, hypoxia). Cells were subjected to hypoxic induction at a cell density of 2 × 105 cells/cm2. The human epitheloid carcinoma cell line HeLaS3 (ATCC CCL-2.2) was cultured in suspension in Ham's F-12 medium (Life Technologies, Inc.) supplemented as described above. Hypoxic induction was achieved as described elsewhere (20). Briefly, HeLaS3 cells were incubated at a density of 1 × 107 cells/ml in an IL 237 tonometer (Instrumentation Laboratory) under continuous stirring for 4 h at 37 °C using gas mixtures of either 20% O2, 5% CO2, and 75% N2 (normoxia), or 1% O2, 5% CO2 and 94% N2 (hypoxia) at a flow rate of 500 ml/min.
RNA Blot AnalysisImmediately following stimulation, RNA
was isolated as described by Chomczynski and Sacchi (21). Total RNA (10 µg) was denatured in formamide/formaldehyde and electrophoresed
through a 1% agarose gel containing 6% formaldehyde as described
(22). Following pressure blotting (Stratagene) to nylon membranes
(Biodyne A, Pall) and UV cross-linking (Stratalinker, Stratagene), the
filters were hybridized to cDNA probes labeled with
[-32P]dCTP to a specific activity of 1 × 109 dpm/µg using the random-primed DNA labeling method
(22). Hybridization was performed in 50% formamide, 10% dextran
sulfate, 5 × Denhardt's solution, 200 µg/ml sonicated salmon
sperm DNA, 1% SDS, 0.9 M NaCl, 60 mM
NaH2PO4, 6 mM EDTA (pH 7.0) for
14 h at 42 °C. The filters were washed to a final stringency of
55 °C in 0.1 × SSC, 0.2% SDS and the signals recorded using a
PhosphorImager (Molecular Dynamics). The Tf,
1-antitrypsin,
-actin, ribosomal protein L28, and 28 S ribosomal RNA cDNA probes were obtained as described previously
(10, 23). All probes were purified free of vector sequences by
restriction digestion and agarose gel purification.
pGLTf4000 was constructed by insertion
of the 4-kb KpnI-BamHI fragment from the Tf
promoter/enhancer-containing plasmid pTfCAT (kindly provided by M. M. Zakin, Institut Pasteur, Paris, France) into the
KpnI-BglII sites of pGL3Basic (Promega).
Luciferase reporter gene constructs containing the heterologous SV40
promoter were obtained by inserting the DNA sequences of interest into
the BamHI site 3 to the luciferase gene of the pGL3Promoter
plasmid (Promega). The liver-specific Tf enhancer from nucleotide
position
3.6 kb to
3.3 kb relative to the transcriptional start
site (3) was obtained by polymerase chain reaction amplification of
genomic DNA using the oligonucleotide primers 5
-GGTCAGGCAGAGGACACTG-3
and 5
-CAGTTCTAGACCAACCCAAG-3
. The oligonucleotides containing wild
type and mutated HBSs are shown in Fig. 4. Copy number and orientation
were determined using RVprimer4 (Promega) by T7 polymerase-mediated single-stranded DNA sequencing following the manufacturer's
instructions (Pharmacia Biotech Inc.). The
-galactosidase expression
vector pCMVlacZ was a kind gift of S. Kozlov (Institute of
Biochemistry, Zürich, Switzerland). The HIF-1
(pCMVhHIF-1
)
and ARNT/HIF-1
(pCMVhARNT and pCMV
bARNT) expression vectors (19)
were generously provided by L. Poellinger.
Transient Transfections and Reporter Gene Assays
Hep3B,
HepG2, and HeLa cells (0.2-1 × 107 in 350 µl of
medium without fetal calf serum) were co-transfected with 25 µg each of luciferase and -galactosidase reporter gene constructs by electroporation at 250 V and 960 microfarads (Gene Pulser, Bio-Rad). After recovering, the cells were split in two aliquots and incubated for 36 h at 20% or 1% O2, respectively. After
washing twice with phosphate-buffered saline, the cells were lysed in
reporter lysis buffer (Promega) and luciferase and
-galactosidase
activities were determined according to the manufacturer's
instructions (Promega) using a Biocounter M1500 luminometer (Lumac) and
a DU-62 spectrophotometer (Beckman), respectively. Differences in the
transfection efficiency and extract preparation were corrected by
normalization to the corresponding
-galactosidase activities.
Luciferase activities were expressed relative to the empty parental
vector (pGL3Basic or pGL3Promoter) transfectants. For transient
overexpression assays in Hep3B cells, 10 µg of each expression vector
was co-transfected together with equal amounts of the luciferase
reporter construct pTfHBSww and the control plasmid
pCMVlacZ. The unrelated vector plasmid pBluescript
(Stratagene) was added to adjust the total amount of DNA per
electroporation to 50 µg.
Nuclear extracts were prepared as described previously (24). Briefly, 1 × 108 cells were washed twice with ice-cold phosphate-buffered saline and once with buffer A (10 mM Tris-HCl (pH 7.8), 1.5 mM MgCl2, 10 mM KCl). After incubation on ice for 10 min, the cells were lysed by 10 strokes of a Dounce homogenizer, and the nuclei were pelleted and resuspended in buffer C (420 mM KCl, 20 mM Tris-HCl (pH 7.8), 1.5 mM MgCl2, 20% glycerol) and incubated at 4 °C for 30 min with gentle agitation. Immediately before use, buffers A and C were supplemented with 0.5 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, 2 µg/ml each of leupeptin, pepstatin, and aprotinin, and 1 mM Na3VO4 (all obtained from Sigma). The nuclear extract was centrifuged, and the supernatant was dialyzed twice against buffer D (20 mM Tris-HCl (pH 7.8), 100 mM KCl, 0.2 mM EDTA, 20% glycerol). Protein concentrations were determined using the Bradford protein assay (Bio-Rad) with bovine serum albumin as standard.
Electrophoretic Mobility Shift Assay (EMSA)Sequences of
the oligonucleotide probes used for EMSA are shown in Fig. 4. The
EPOHBS oligonucleotides have been described previously (24). All
oligonucleotides (Microsynth) were purified on 10% polyacrylamide gels
prior to 5 end-labeling of the sense strand with
[
-32P]ATP (Hartmann) using T4-polynucleotide kinase
(Fermentas). Unincorporated nucleotides were removed by gel filtration
over Bio-Gel P60 (fine) columns (Bio-Rad). Labeled sense strands were
annealed to a 2-fold molar excess of unlabeled antisense strands.
DNA-protein binding reactions were carried out for 20 min at 4 °C in
a total volume of 20 µl containing 4-5 µg of nuclear extract,
0.1-0.4 µg of sonicated, denatured calf thymus DNA (Sigma), and
1 × 104 cpm of oligonucleotide probe in 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 50 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 5 mM dithiothreitol, and 5% glycerol
and run on 4% non-denaturing polyacrylamide gels. Electrophoresis was
performed at 200 V in TBE buffer (89 mM Tris, 89 mM boric acid, 5 mM EDTA) at 4 °C, and dried
gels were autoradiographed. For supershift analysis, each 1 µl of
rabbit polyclonal antisera derived against HIF-1
or ARNT/HIF-1
(kind gift of L. Poellinger) was added to the completed EMSA reaction
mixture and incubated for 16 h at 4 °C prior to loading. For
competition experiments, a 4-500-fold molar excess of unlabeled
annealed oligonucleotides was added to the binding reaction prior to
addition of labeled probes.
We previously reported on oxygen-regulated mRNA
expression of several acute phase genes in the human hepatoma cell line
HepG2 (10). Regulation of the Tf gene was of special interest since Tf
transcription was down-regulated in response to proinflammatory cytokines (e.g. interleukin-6), but was up-regulated
following exposure to low oxygen concentrations. To test whether
hypoxic Tf induction observed in HepG2 cells might represent a general phenomenon in liver cells, we also exposed Hep3B cells, another human
hepatoma cell line, to 1% O2 for 1-3 days. As shown by
RNA blotting experiments, Tf mRNA was up-regulated about 1.5-fold in Hep3B cells (Fig. 1A) and
up to 4.5-fold in HepG2 cells (Fig. 1B). A similar hypoxic
induction pattern of endogenous gene expression in the two cell lines
was observed for the acute phase reactant 1-antitrypsin,
which was included as positive control (Fig. 1, A and
B). Specificity of hypoxic up-regulation was shown using L28
and 28 S control hybridizations since
-actin mRNA was also slightly up-regulated in both hepatoma cell lines and thus not suitable
as a normalization probe (10).
The Tf Enhancer Is Hypoxia-responsive
In a first attempt to
identify Tf regulatory sequences conveying hypoxia-inducible Tf
transcription, a 4000 to +39 (numbering according to Ref. 3) Tf
promoter/enhancer DNA fragment (Fig. 2A) was inserted upstream of a
promoterless luciferase reporter gene vector. Following transient
transfection into Tf-expressing Hep3B and HepG2 cells, as well as into
non-Tf-expressing HeLa cells, this 4-kb Tf promoter/enhancer induced
basal luciferase expression 10-, 26-, and 8-fold in Hep3B, HepG2 and
HeLa cells, respectively (Fig. 2B, open bars).
Hypoxia (1% O2) stimulated luciferase expression 4.1- and
5.6-fold in Hep3B and HepG2 cells, respectively, but no significant
hypoxic induction could be observed in HeLa cells (Fig. 2B,
filled bars). Thus, hypoxia responsiveness seems to be
coupled to liver-specific cis-acting elements present within
this Tf promoter/enhancer DNA fragment.
In analogy to the liver-specific enhancer and the hypoxia-responsive
element residing in close vicinity in the Epo 3 flanking region (16),
we wondered whether the
3600/
3300 bp liver-specific Tf enhancer
(Fig. 2A) might be responsible for oxygen responsiveness of
the Tf gene. To test this, we subcloned the 300-bp Tf enhancer downstream of a luciferase reporter gene driven by a heterologous SV40
promoter. The Tf enhancer induced normoxic luciferase expression by
8.1-, 1.6-, and 2.0-fold in Hep3B, HepG2, and HeLa cells, respectively (Fig. 3, open bars). This
expression level was further up-regulated by exposing the cells to
hypoxia; luciferase activity in Hep3B, HepG2, and HeLa cells increased
3.1-, 6.8-, and 1.8-fold, respectively (Fig. 3, filled
bars). The weak hypoxic inducibility (1.4-fold) of the
pGL3Promoter plasmid itself has been reported previously (24). Thus,
similar to the observations using the 4-kb Tf promoter/enhancer, the
300-bp Tf enhancer alone conferred hypoxia inducibility in Hep3B and
HepG2 hepatoma cells, but was not significantly active in
non-Tf-expressing HeLa cells.
Two Tandemly Repeated HBSs Confer Hypoxia Responsiveness to the Tf Gene
A computer-assisted search using a HIF-1 consensus
DNA-binding site (24) as query revealed the presence of two tandemly arrayed putative HBSs beginning at nucleotide positions 174 and 191, respectively (Fig. 4), within the 300-bp
Tf enhancer (numbering according to Ref. 3). No other matches to the
HIF-1 query were found in the published nucleotide sequences of the Tf
gene. To test whether these two putative HBSs were functionally
oxygen-responsive, we synthesized oligonucleotides containing both
sites in either the wild type configuration (TfHBSww), or
with one (TfHBSwm or TfHBSmw) or both (TfHBSmm) HBS sites
mutated (Fig. 4). Single copies of these oligonucleotides
were inserted 3 to a luciferase reporter gene driven by a heterologous
SV40 promoter. For comparison, a hypoxia-responsive luciferase
construct (pGLEPOHBS.3) containing three concatamerized copies of the
Epo HBS was included in this study (24). Luciferase activity was
determined following transient transfection of Hep3B and HeLa cells,
splitting in two aliquots and 36 h of normoxic or hypoxic cell
culture. Compared with the normoxic control cells, hypoxia increased
luciferase expression from the control plasmid (pGLEPOHBS.3) 4.1- and
6.8-fold in Hep3B and HeLa cells, respectively (Fig.
5). Hypoxic induction mediated by the two
tandemly arrayed, putative Tf HBSs (pGLTfHBSww) was more effective in
Hep3B cells (9.4-fold) than in HeLa cells (3.7-fold). Although again
less pronounced in HeLa cells compared with Hep3B cells (similar to the
300-bp Tf enhancer; see Fig. 3), the putative Tf HBSs functioned as
hypoxia-dependent enhancer in both cell lines that do or do
not express Tf (Fig. 5). This observation is reminiscent of the Epo
HBS, which has previously been reported to enhance hypoxic gene
expression in Epo-expressing and non-Epo-expressing cell lines (25,
26). Mutation of either one of the two putative Tf HBSs (plasmids
pGLTfHBSwm and pGLTfHBSmw) only partially reduced hypoxic luciferase
expression, and a double mutation of both sites (plasmid pGLTfHBSmm)
was necessary to completely abrogate oxygen responsiveness down to the
basal level observed with the empty vector alone (Fig. 5).
HIF-1 Activates Reporter Gene Expression via the Tf HBS
To
investigate the involvement of the HIF-1 protein complex in Tf
regulation, we performed transient expression experiments using the
HIF-1 and/or ARNT/HIF-1
expression vectors pCMVhHIF-1
and
pCMVhARNT, respectively (19). They were co-transfected into Hep3B cells
together with the reporter gene construct pGLTfHBSww (depicted in Fig.
5), the normalization plasmid pCMVlacZ and the unrelated
plasmid pBluescript used to equalize the total amount of DNA per
transfection. As shown in Fig. 6
(open bars), transient overexpression, under normoxic
conditions, of either of the two HIF-1 subunits weakly (about 2-fold)
induced reporter gene expression, whereas expression of both HIF-1
subunits induced luciferase expression by 5.8-fold (Fig. 6, open
bars). Co-expression with a reporter gene construct containing
mutant HBSs (pGLTfHBSmm) did not result in enhanced luciferase
expression (data not shown), implying that HIF-1 needs to bind to the
Tf HBSs to transactivate reporter gene expression. Hypoxia also
activated the Tf HBSs (Fig. 6, filled bars), and
overexpression of the two HIF-1 subunits further enhanced this effect
1.8-fold. Interestingly, overexpression of a dominant negative
ARNT/HIF-1
mutant (pCMV
bARNT), which lacks the basic domain and
hence is still capable of heterodimerizing with HIF-1
but cannot
bind DNA (19), reduced reporter gene expression by sequestering
endogenous as well as overexpressed HIF-1
(Fig. 6), underlining the
critical role of HIF-1 in hypoxic Tf induction.
DNA Binding of HIF-1 to the Two HBS Sites of the Tf Enhancer
To directly identify the endogenous transcription
factor(s) binding to the HBS of the Tf enhancer, EMSAs were performed
using the TfHBS oligonucleotides shown in Fig. 4 as probes. Following incubation of the TfHBSww probe with nuclear extracts derived from
normoxic or hypoxic Hep3B cells, nonspecific, constitutive, and
hypoxia-inducible factors were detected (Fig.
7A). Using Hep3B nuclear
extracts, mutation of the 3 HBS site (oligonucleotide TfHBSwm) did not
greatly affect binding of the hypoxia-inducible factor. In contrast,
mutation of the 5
HBS site present in the Tf enhancer (oligonucleotide
TfHBSmw) strongly reduced but (as could be seen after prolonged
exposure, data not shown) did not completely abolish protein binding.
Only the double mutation (oligonucleotide TfHBSmm) completely abolished
binding of both the hypoxia-inducible and the constitutive factor,
indicating that both sites are capable of binding these two factors
although with different affinities. Interestingly, in Hep3B cells,
constitutive factor binding was found only with single mutations but
not with the wild type or double mutant probe. To date, we have no
explanation for this observation. In the case of the Epo HBS, we
previously identified the constitutive factor as ATF-1/CREB-1 family
members contacting similar nucleotide residues as HIF-1 itself (24).
Similar constitutive and hypoxia-inducible factors derived from the
non-Tf-expressing human HeLa cervical carcinoma and mouse L929
fibroblast cells, as well as from the Tf-expressing mouse Hepa1
hepatoma cells, also bound to the TfHBSww probe (Fig. 7B).
In contrast to Hep3B cells, constitutive factor binding to the TfHBSww
probe was detectable in all of these extracts.
To demonstrate that the hypoxia-inducible factor binding the TfHBSww
probe is indeed identical with the previously identified HIF-1 (17),
nuclear extracts were prepared from normoxic and hypoxic Hepa1C4 cells
and analyzed by EMSA. The cell line Hepa1C4, a subline of Hepa1 cells,
is deficient in functional ARNT/HIF-1 expression (19) and devoid of
DNA binding activity to the EPOHBS probe as well as of reporter gene
induction with Epo HBS luciferase constructs (19). As shown in Fig.
7B, this cell line also lacked hypoxia-inducible TfHBSww DNA
binding activity, whereas the constitutive and nonspecific factors were
still present. Moreover, rabbit polyclonal antibodies derived against
HIF-1
and ARNT/HIF-1
supershifted the hypoxia-inducible factor
binding to the EPOHBS probe as well as to the TfHBS wild type and
single mutated probes in Hepa1 cells (Fig. 7C), suggesting
that this factor is functionally and immunologically indistinguishable
from HIF-1.
To analyze in more detail the binding of HIF-1 to the two functional Tf
HBSs, competition experiments were performed using the labeled TfHBSww
probe and increasing amounts of unlabeled wild type or mutant
oligonucleotides (depicted in Fig. 4). As shown in Fig.
8, using nuclear extracts derived from
normoxic or hypoxic Hepa1 cells, the 3 single mutant oligonucleotide
(TfHBSwm) competed as efficiently for HIF-1 DNA binding to the TfHBSww
probe as the wild type TfHBSww oligonucleotide itself (i.e.
competition was observed using a 20-fold molar excess). The reduction
in HIF-1 band intensity with 20- and 100-fold molar excesses of the 5
single mutant oligonucleotide (TfHBSmw) was somewhat less prominent, confirming that although both Tf HBSs can bind HIF-1, the 3
site has a
lower affinity for HIF-1. The double mutant TfHBSmm oligonucleotide did
not compete for HIF-1 DNA binding activity (Fig. 8). On the other hand,
oligonucleotide EPOHBS also competed for HIF-1 DNA binding to the
TfHBSww probe, although with a lower efficiency than TfHBSww itself
(i.e. more than a 100-fold molar excess was necessary for
competition), probably due to the presence of two HBSs on the TFHBSww
oligonucleotide compared with only one HBS in the EPOHBS
oligonucleotide.
Tf mRNA Is Not Induced in ARNT/HIF-1
To investigate whether HIF-1 is capable of hypoxically
inducing the endogenous Tf gene, we made use of the
ARNT/HIF-1-deficient Hepa1C4 cell line, which was cultured at 20%
or 1% O2 and analyzed by RNA blotting and hybridization.
In previous experiments, we have demonstrated a lack of hypoxic
aldolase mRNA induction and a reduction in hypoxic VEGF mRNA
induction in this cell line (19). Whereas in the parental
ARNT/HIF-1
-positive Hepa1 cell line hypoxia reproducibly induced Tf
mRNA by a factor of 1.5 over the normoxic control, Hepa1C4 cells
did not show induction of Tf mRNA (Fig. 9). This is in agreement with the lack of
HIF-1 DNA-binding activity to the TfHBSww probe in these cells (shown
in Fig. 6B) and, despite the rather low Tf mRNA
expression levels and hypoxic inducibility in this particular hepatoma
cell line, confirms that HIF-1 is critically involved in the oxygen
responsiveness of the Tf gene.
HIF-1
was originally defined by its capability of binding to a site required
for hypoxic induction of Epo gene transcription (16, 17). Other
examples of HIF-1-dependent oxygen-regulated genes include
those encoding for glycolytic enzymes (27-30), vascular endothelial
growth factor (VEGF, Refs. 31-33), inducible nitric oxide synthase
(34), and glucose transporter-1 (Glut-1, Ref. 35). We have previously
found that Tf gene expression is hypoxically induced in HepG2 hepatoma
cells (10). In this paper, we demonstrate that this effect is also
mediated by HIF-1 via binding of two HBSs present in the Tf enhancer.
We and others recently characterized a previously obtained mouse
hepatoma cell line (Hepa1C4), which lacks functional ARNT expression,
and demonstrated that it is also devoid of functional HIF-1 expression
in terms of DNA binding activity and reporter gene transactivation (19,
33, 36, 37). By using these Hepa1C4 cells, we found that ARNT/HIF-1 is necessary for formation of the hypoxia-inducible complex binding to
the Tf HBSs. Moreover, hypoxic induction of the endogenous Tf gene is
also abrogated in Hepa1C4 cells, suggesting that HIF-1 is critically
involved in oxygen-regulated Tf gene expression.
An interesting feature of the Tf
enhancer is the presence of two functional HBSs in close vicinity to
each other. Such an architecture was not found in the Epo 3 HBS. The
finding that a single HBS derived from the Epo HBS was not sufficient
to convey oxygen responsiveness to a heterologous promoter driving
expression of a reporter gene, although the HIF-1 complex bound to this
site in vitro (24), raised the question as to whether
additional functional cis-acting elements are necessary for
full activation. Such an additional element (CACA) was found close to
the HBSs in the Epo (16) and VEGF genes (33), as well as in the genes encoding several glycolytic enzymes (30). The transacting factors binding to this element, however, still remain to be identified. As
suggested by the hypoxic induction of reporter gene expression by
concatamerized HBSs, such an additional element could also be the HIF-1
site itself (Refs. 24 and 27 and this report). In addition, an
activator protein (AP-1) site in the vicinity of the HIF-1-binding HBS
in the 5
flanking region of the VEGF gene (31) and a cAMP-responsive
element close to the two consensus HBSs in the LDH gene (28) have been
implicated in full hypoxic induction of gene expression. Protein-DNA
interactions of cAMP-responsive element-binding transcription factors
(ATF-1 and CREB-1) were also observed within the HIF-1 site of the Epo
HBS itself (24). In conclusion, it seems to be a common feature of an
HBS that a single HBS in isolation is not sufficient to convey full
hypoxic activation of gene expression. The additional factors required for full activation, however, might differ between the oxygen-regulated genes.
As mentioned above, the hypoxia-inducible Tf enhancer region is composed of two adjacent HBSs, both of which are capable of conveying hypoxic induction to reporter gene expression. The two 8-bp HBSs in the Tf enhancer are spaced by 9 bp only (Fig. 4), raising the question whether two HIF-1 complexes could bind simultaneously to these two sites or whether due to sterical hindrance only one complex can bind at once. As shown by the equal migration properties of TfHBS (wild type and single mutant) and EPO HBS probes in our EMSAs, the predominant protein-DNA complex might consist of only one HIF-1 heterodimer bound to the TfHBSww oligonucleotide. Although EMSA analysis is probably not the ideal method to determine molecular mass differences, we would predict that an increase of more than 200 kDa (the molecular mass of an additional HIF-1 heterodimer) should be detectable.
The two core sequences TACGTGCA and TACGTGCG (note that the
complementary strand to that shown in Fig. 4 is given) conform well
with a previously published (33) consensus HBS (BACGTGSK, where B is C
or G or T, S is C or G, and K is G or T). Strikingly, the presence of
an adenosine residue at position 8 of the 8-bp consensus sequence
(found in the 5 TfHBS) has never been reported in any of the so far
published genes carrying HBSs (18). Moreover, positions 9 and 10 also
did not contain an adenosine residue in these HBSs (not shown). Thus,
one would predict that this lack of adenosine residues in the 3
part
of the HBS is of functional relevance and that the DNA binding affinity
of HIF-1 for the 5
Tf HBS might be decreased. Surprisingly, as shown
with mutated oligonucleotide probes and competition experiments, the 3
rather than the 5
HIF-1 site (which contains the unexpected adenosine residue) constantly produced lower HIF-1 band intensities in EMSAs. On
the functional level, only mutation of the 5
Tf HBS significantly reduced (but did not completely abolish) hypoxic induction of reporter
gene expression, confirming that the 5
HBS is more effective in HIF-1
binding than the 3
HBS. Thus, despite the considerable number of so
far identified HBSs, a conclusive consensus sequence still remains to
be determined.
Having established that HIF-1
is mediating hypoxic induction of Tf gene expression, the question
arises of how HIF-1 itself is activated. So far, little is known about
the mechanisms of oxygen sensing and subsequent conditional regulation
of HIF-1. Initially, it has been reported that HIF-1 (and to a
lesser extent also ARNT/HIF-1
) is regulated at the level of mRNA
expression (17), but work from our and other laboratories could not
confirm this result (19, 38, 39). Hence, HIF-1 must be regulated at the
post mRNA level. Possible mechanisms include translational up-regulation, post-translational protein stabilization (39) or protein
modifications such as phosphorylation (40) or redox modifications (41).
There is good evidence for all of these putative mechanisms and more
than one might turn out to be involved in hypoxic HIF-1 activation
(reviewed in Ref. 18). Interestingly, our transient overexpression
experiments using the Tf HBS demonstrated that forced expression of the
two HIF-1 subunits is sufficient to convey induction to reporter gene
transcription even under normoxic conditions. This observation is in
agreement with recent work using the Epo HBS (19, 42) and VEGF HBS
(33). Since conditional regulation thus does not seem to be of primary
importance for HIF-1 function, we favor the model(s) of translational
up-regulation and/or protein stabilization. However, hypoxia
represented a stronger stimulus than overexpression of HIF-1,
indicating that for a full response conditional regulation of HIF-1 is
required. Further investigations of HIF-1 regulation is crucial for the
elucidation of the signal transduction pathway(s) involved in the
expression of Tf as well as other oxygen-regulated genes.
We are grateful to L. Poellinger, M. M. Zakin, and S. Kozlov for the generous gift of cell lines, antibodies, and plasmids; P. Spielmann and W. Baier-Kustermann for excellent technical assistance; J. Silke for critically reading the manuscript; C. Gasser for the artwork; and C. Bauer for support.