From the Erythropoietin Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom
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ABSTRACT |
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Hypoxia-inducible expression has been
demonstrated for many groups of mammalian genes, and studies of
transcriptional control have revealed the existence of
hypoxia-responsive elements (HREs) in the cis-acting sequences of
several of these genes. These sequences generally contain one or more
binding sites for a heterodimeric DNA binding complex termed
hypoxia-inducible factor-1 (HIF-1). To analyze this response
further, Chinese hamster ovary cells were stably transfected with
plasmids bearing HREs linked to genes encoding immunoselectable cell
surface markers, and clones that showed reduced or absent
hypoxia-inducible marker expression were selected from a mutagenized
culture of cells. Analysis of these cells revealed several clones with
transacting defects in HRE activation, and in one the defect was
identified as a failure to express the -subunit of HIF-1. Comparison
of hypoxia-inducible gene expression in wild type, HIF-1
-defective,
and HIF-1
-complemented cells revealed two types of response. For
some genes (e.g. glucose transporter-1), hypoxia-inducible
expression was critically dependent on HIF-1
, whereas for other
genes (e.g. heme oxygenase-1) hypoxia-inducible expression
appeared largely independent of the expression of HIF-1
. These
experiments show the utility of mutagenesis and selection of mutant
cells in the analysis of mammalian transcriptional responses to hypoxia
and demonstrate the operation of HIF-1
-dependent
and HIF-1
-independent pathways of hypoxia-inducible gene expression in Chinese hamster ovary cells.
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INTRODUCTION |
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Naturally occurring genetic mutations have provided many insights into gene function. In an effort to extend this source of information, a large number of targeted or untargeted means of introducing mutations and selecting by genotype or phenotype have been devised. In somatic cells, one classical approach has been to subject a mutagenized culture to a substance whose toxicity is dependent on the activity of a set of genes involved in a signal transduction or metabolic pathway (1-3). However, the number of properties that are "intrinsically selectable" in this way is limited, leading some workers to explore the possibility of introducing selectable properties by transfection of recombinant plasmids (4, 5). For instance, stable transfection of a plasmid bearing an interferon-responsive promoter linked to a bacterial guanidine phosphoribosyl transferase gene has been used to select cells from the fibrosarcoma line HT1080 that are defective in the response to interferons (4). Similar promoters linked to the gene encoding the cell surface antigen CD2 have been used to select other lines defective in the interferon response using fluorescence-activated cell sorting (6).
The recent recognition of a widespread transcriptional response to
hypoxia (see Refs. 7 and 8; for a review see Ref. 9), mediated by the
activation of a heterodimeric basic-helix-loop-helix PAS1 protein complex termed
hypoxia-inducible factor-1 (HIF-1) (10) presents an important
challenge, in terms of both defining the responsive target genes and
analyzing the underlying mechanism of oxygen sensing and signal
transduction. Here we describe the use of a selection strategy based on
the linking of HIF-1-binding hypoxia-responsive elements (HREs) with
minimal promoters coupled to genes encoding cell surface antigens. We
describe the selection, from a mutagenized culture of Chinese hamster
ovary (CHO-K1) cells, of cells that are deficient in
HRE-dependent transactivation and the identification of one
line that is functionally defective in the -subunit of HIF-1. We
demonstrate the utility of this cell line in characterizing
HIF-1
-dependent and HIF-1
-independent patterns of
hypoxia-inducible gene expression.
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EXPERIMENTAL PROCEDURES |
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Cell Lines, Mutagenesis, and Transfections-- Cells were grown in Ham's F-12 (Sigma) supplemented with 10% fetal calf serum (Globepharm), L-glutamine (2 mM), penicillin (50 IU/ml), and streptomycin sulfate (50 µg/ml).
Transfections were performed by electroporation using a 1-millifarad capacitor array charged at 350 V. Plasmids bearing HREs linked to reporter genes or selectable markers are illustrated in Fig. 1. pHPGK-CD2 was as described previously (11). p(E1-25)5SV-ESel contained five copies of a 25-base pair HRE from the mouse 3'-erythropoietin enhancer coupled to the SV40 early promoter (from p(E1-25)5SV-GH (12)) ligated into pBluescript and linked to the complete E-selectin coding sequence and SV40 splice/poly(A) from pCDM8. pHTK-Luc contained two copies of a 24-base pair HRE from the mouse phosphoglycerate kinase-1 5'-enhancer lying 10 base pairs 5' to the TATA box of the herpes simplex virus thymidine kinase promoter cloned into pGL2 basic (Promega) bearing a firefly luciferase gene. pSV2Neo and pSV2Hyg contained neomycin and hygromycin phosphotransferase genes, respectively, linked to the SV40 early promoter. pCMVAntibody Labeling of Cells and Immunoselection-- For fluorescence-activated cell scanning (FACS) analysis, cells were detached in 2 mM EDTA in HBSS, centrifuged at 1000 rpm at 4 °C, and washed twice in 3 ml of PFA (PBS with 0.1% sodium azide and 1% fetal calf serum). Antibody labeling was performed on ice for 30 min using a phycoerythrin-conjugated mouse IgG2b anti-human CD2 (Serotec) and fluorescein isothiocyanate-conjugated mouse IgG1 anti-human E-selectin (R & D Systems). Cells were then washed and fixed in 1% paraformaldehyde in PBS before FACS analysis (Becton Dickenson).
For immunological selection by panning (16), cells were harvested as above and then washed twice in panning buffer (PBS with 2 mM EDTA, 0.02% sodium azide, and 5% heat-inactivated fetal calf serum) at 4 °C. Antibody labeling (30 min, 4 °C) was performed either with mouse IgG2b anti-human CD2 (Serotec) at a 1:10 dilution or with mouse IgG1 anti-human E-Selectin (Serotec) at 1:100 dilution. The cells were then washed twice in panning buffer, resuspended in 5 ml of panning buffer, and poured onto prepared panning plates. Plates were left at room temperature for 15-60 min, after which the panning buffer was gently aspirated to harvest the nonadherent cells. These cells were washed in culture medium and plated out in selective medium. Panning plates were prepared as follows. Petri dishes were coated with 5 ml of a 10 µg/ml solution of affinity-conjugated goat anti-mouse IgG (Sigma) in 50 mM Tris, pH 9.5, for 1 h at room temperature. The dishes were then washed three times in PBS, blocked by overnight incubation with 5 ml of sterile blocking buffer (2 mg/ml bovine serum albumin in PBS), and stored atCell Fusions--
Parental cells were CHO-K1 cells and an
hypoxanthine phosphoribosyl transferase-deficient (HPRT)
derivative of Ka13 selected in hypoxanthine-free medium containing 10 µM 6-thioguanine. For fusions, equal quantities of CHO-K1
and HPRT
Ka13 cells were mixed and plated such that the
cells were overconfluent. After 4 h, the cells were washed with
serum-free medium, and 0.5 ml of neutralized polyethylene glycol
solution (Mr 1300-1600, 1 g/ml) was added for 1 min. The polyethylene glycol was diluted in 5 ml of serum-free medium
over a further 2 min and then removed, after which the cells were
washed gently in serum-free medium, incubated at 37 °C for 6 h
in complete medium, and then trypsinized and replated. Selective medium
(HAT containing G418) was added 24 h later.
Cell Extracts and Protein Analysis--
Nuclear extract was
prepared using a modified Dignam protocol, and an electrophoretic
mobility shift assay was performed as described in Ref. 17 using a
double-stranded HIF-1 binding oligonucleotide 5'-GCCCTACGTGCTGCCTCGCATGGC-3' from the mouse erythropoietin enhancer (E24). For Western analysis, nuclear extracts were separated by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P membranes. HIF-1 was detected using a polyclonal rabbit antiserum raised against a recombinant immunogen containing amino acids 530-652
of human HIF-1
, a peroxidase-conjugated goat anti-rabbit immunoglobulin (DAKO), and enhanced chemiluminescence.
RNA Analysis-- RNA was extracted by a modified acid/guanidinium thiocyanate/phenol/chloroform method (RNAzol B, Cinna/Biotec Laboratories, Houston), dissolved in hybridization buffer (80% formamide, 40 mM PIPES, 400 mM sodium chloride, and 1 mM EDTA, pH 8), and analyzed by an RNase protection assay or dissolved in diethylpyrocarbonate-treated distilled H2O for Northern blotting.
For Northern blotting, 30 µg of total RNA were fractionated on a denaturing agarose gel and transferred to a nylon membrane. The filters were hybridized with probes labeled with [32P]dCTP using random nonamer priming (Amersham Pharmacia Biotech) for ARNT and by polymerase chain reaction for HIF-1Reporter Gene Assays--
Luciferase activity was determined in
cell lysates using a commercial assay system (Promega) and a TD-20e
luminometer (Turner Designs). -Galactosidase activity in cell
lysates was measured using
o-nitrophenyl-
-D-galactopyranoside as
substrate in a 0.1 M phosphate buffer pH 7.0 containing 10 mM KCl, 1 mM MgSO4, and 30 mM
-mercaptoethanol.
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RESULTS |
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Construction and Characterization of a Cell Line Bearing Surface Markers Controlled by Hypoxia-responsive Elements-- A central difficulty in using selection against exogenous genetic markers introduced by transfection to derive mutants that are functionally defective in components of an interacting transcriptional mechanism is that the transfected gene is generally much less stable than the endogenous genome. As a result, selection against an inducible transfected marker gene will generate a large excess of cells that have simply lost or suppressed expression of the marker over those bearing defects in the endogenous mechanism of gene regulation. We employed two strategies to limit this problem. First, two separately integrated, hypoxically inducible immunoselectable markers were used. This enabled us to limit selection of cells that had simply lost one or the other integration (by alternate selection against each of the two hypoxically inducible antigens) and also allowed us to detect such cells rapidly by FACS analysis. Second, positive selection for co-transfected drug resistance markers was continued throughout the negative immunoselection procedure.
First, to assess the efficiency of various HIF-1-dependent HREs in supporting hypoxia-inducible gene expression in CHO-K1 cells, transient transfections were performed with plasmids bearing different HRE/promoter combinations coupled to cDNAs for cell surface markers. Inducible marker gene expression was measured by mRNA and FACS analysis (data not shown). The two plasmids that showed the highest induction of marker expression were chosen for stable transfection and are illustrated in Fig. 1. CHO-K1 cells were first transfected with pHPGK-CD2 and pSV2Neo, and clones were selected in G418. One such clone was further transfected with p(E1-25)5SV-ESel and pSV2Hyg, and clones were selected in hygromycin B. Several resistant clones were assayed for hypoxia-inducible expression of the transfected cell surface markers, and one (C4.5) was selected on the basis of showing the greatest inducible expression of both markers. Fig. 2A shows FACS analysis of normoxic and hypoxic cultures of C4.5 cells after labeling cells with monoclonal antibodies against human CD2 and E-selectin. Both markers are clearly inducible by hypoxia. Untransfected CHO-K1 cells did not express either marker gene as assessed by RNase protection and FACS.
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Mutagenesis and Selection of Clones with Impaired Responses to Hypoxia-- For mutagenesis, the CHO line C4.5 was exposed to ICR191. In pilot experiments, one exposure to a dose of 1 µg/ml of mutagen for 5 h resulted in nearly 1 in 103 mutants at the HPRT locus per clonable cell (data not shown). Since we anticipated that disabling mutations of hypoxia-inducible transcription would be much less frequent, approximately 7 × 107 C4.5 cells, in 10 separate aliquots, were exposed to ICR191 at 1 µg/ml for 5 h. Previous reports had described a higher frequency of mutation following multiple exposures to ICR191 (19, 20), so cells were allowed to recover and then subjected to a further two exposures of the same dose and duration. The cells were maintained in the 10 separate aliquots throughout these further exposures to ICR191 and the subsequent selection procedure. Cell mortality after one exposure was approximately 55%.
The isolation of cells that were unable to support HRE-dependent induction of gene expression was made by antibody labeling of the HRE-linked cell surface markers and selection by panning. After a 16-h hypoxic exposure, cells were harvested, labeled with primary antibody to CD2 or E-selectin, and then placed on a prepared dish coated with secondary antibody. Cells that failed to adhere to the panning plates were collected, washed, and replated in tissue culture dishes. Once the cells had multiplied to about 5 × 106/pool, they were again made hypoxic, and another round of panning was undertaken. To measure the extent of selection obtained in this way, preliminary experiments were performed in which a marker CHO cell (bearing a constitutively expressed intercellular adhesion molecule-3 gene) was mixed with C4.5 cells in a ratio of 1:1000. After three rounds of panning of this mixed cell pool for nonexpression of E-selectin, the pool was tested for intercellular adhesion molecule-3 expression by FACS. These cells had now reached a prevalence of approximately 10% in the pool (and remained undetectable by FACS in a parallel pool that had simply been passaged without selection). From this, it was estimated that for a mutation frequency likely to be less than 1 in 106, approximately 10 rounds of panning would be required to enrich sufficiently for such a mutant population. Panning of the mutagenized C4.5 cells was therefore performed alternately with anti-CD2 and anti-E-selectin antibodies, to a total of 10 rounds. Throughout the procedure, selection was continued in neomycin and hygromycin B. Each pool was then tested for inducibility of both CD2 and E-selectin and compared with C4.5 cells. Results for one of the pools are shown in Fig. 2B. A clear reduction in the hypoxia inducible expression of both markers was observed in a significant proportion of cells.Isolation of a Clone That Is Functionally Defective for the
-Subunit of HIF-1--
Clonal populations from each pool were
obtained by limiting dilution and then examined for noninducibility of
both surface markers by FACS analysis. 44 clones that showed impaired
regulation of these markers were tested further for defects in HRE
activation by transient transfection with pHTK-Luc, a plasmid bearing
an HRE-dependent luciferase reporter gene. In this assay,
32 of these clones showed levels of HRE activity that were the same or
similar to wild type C4.5. However, 11 showed a clear but incomplete
reduction in the hypoxia-inducible response ranging from 20 to 65% of
that observed in wild type C4.5 cells. For nine of these clones, this change was stable over 12 weeks of continuous passage. One clone, Ka13,
showed an absent response. Fig. 2C illustrates expression of
CD2 and E-selectin in Ka13. Ka13 was stable with respect to this
phenotype and has been subject to detailed further analysis.
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Hypoxia-inducible Gene Expression in Ka13 Cells--
The above
analysis indicates that Ka13 cells manifest a profound defect in
HIF-1 expression. To explore the effect of this on hypoxia-inducible
gene expression, the regulation of particular groups of genes was
examined first in wild type C4.5 cells and Ka13 cells. Table
I lists all of the genes tested. Several
of these genes (gelatinase 92, tissue inhibitor of metalloproteinases 2, transferrin receptor, and ornithine decarboxylase) showed only a low
level or inconsistent regulation by hypoxia in wild type C4.5 cells and
were not analyzed further. Fig.
5A shows results for six genes
selected from different functional groups that showed inducible
responses or were otherwise of particular interest: the VEGF, Glut-1,
GAPDH, AK3, HO-1, and GRP78 genes. Striking differences were observed.
For some genes, a high level of hypoxia-inducible expression was
observed in wild type C4.5 cells that was very much reduced or
completely abolished in Ka13. Surprisingly, little or no induction of
VEGF mRNA by hypoxia was observed in the wild type C4.5 cells, and
VEGF expression was similar in Ka13 cells incubated in hypoxic
conditions, although a small induction was seen in Ka13 cells treated
with desferrioxamine. The largest induction by hypoxia was observed for
Glut-1 mRNA. When cells were exposed to an atmosphere of 1%
oxygen, Glut-1 mRNA expression was induced 5-fold in C4.5 cells but
was not induced in Ka13 cells. For GAPDH and AK3, a similar abrogation
of hypoxia-inducible expression was observed in Ka13 cells. In
contrast, HO-1 and GRP78 showed inducible responses to hypoxia that
were not obviously reduced in Ka13 cells. This suggested that HIF-1
was critical for the induction of certain genes at 1% oxygen but that
independent pathways of hypoxia-inducible gene expression might also
operate. To examine this further, the expression pattern of four genes
was examined after exposure to more severe hypoxia, 0.1% oxygen for
48 h (Fig. 5B). For all four genes, the level of
induction was greater than that observed with 1% oxygen for 16 h.
In Ka13 cells, the response to hypoxia was again abolished for Glut-1
and GAPDH gene expression. The behavior of HO-1 and GRP78 genes
differed from Glut-1 in that the responses were well preserved in Ka13
cells; for instance, HO-1 induction was 17- and 13-fold with 0.1%
oxygen in C4.5 and Ka13 cells, respectively. This suggested that
different pathways of hypoxia-inducible expression were operating on
these genes in CHO cells. Since (like HIF-1), HO-1 and Glut-1 genes
have both been demonstrated to be inducible by cobaltous ions (24-26),
we examined the response of these two contrasting genes to cobaltous ions and other stimuli known to induce HIF-1. Results are shown in Fig.
6. Glut-1 mRNA was induced to a
similar extent by hypoxia, cobaltous ions, and desferrioxamine in C4.5
cells. All of these responses were abolished in Ka13 cells. In
contrast, HO-1 was induced to a much greater extent by cobaltous ions
but was not inducible by desferrioxamine in C4.5 cells, and these
responses were similar in Ka13 cells. When responses to desferrioxamine were compared across the set of genes, two distinct patterns were observed (Fig. 5A). For Glut-1, AK3, and GAPDH genes, the
response to hypoxia was mimicked closely by desferrioxamine, whereas
for HO-1 this was not the case.
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Analysis of Gene Expression in Ka13 Cells Bearing a Stably
Integrated Human HIF-1 Gene--
The above data demonstrate that
the induction of some but not all genes in response to hypoxia is
abrogated in HIF-1
-deficient Ka13 cells, suggesting a critical role
for this transcription factor in these responses. To test this further,
Ka13 cells were transfected with pcDNA3/Neo/HIF-1
and pPur, with
selection in puromycin. Surviving colonies were pooled and checked for
expression of the marker genes. Since expression of the cell surface
markers was only increased in a small proportion of the pool,
immunoselection by panning for high hypoxic expression of E-selectin
was undertaken to augment this population. After four rounds of
panning, clones were obtained by limiting dilution. RNase protection
analysis indicated that these clones expressed varying levels of human but not Chinese hamster HIF-1
RNA. Several clones were analyzed for
Glut-1, GAPDH, and AK3 mRNA expression, and in all clones increased
levels were observed. Results are shown for one clone expressing a
moderate level of human HIF-1
RNA in Fig.
8; restoration of induction by both
hypoxia and desferrioxamine can be seen. In some clones expressing
higher levels of HIF-1
mRNA (as assessed by RNase protection
assays of mRNA), an enhanced level of Glut-1 mRNA was observed
in normoxic cells, suggesting that overexpression of HIF-1
can
increase expression of certain endogenous target genes in normoxic
cells.
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DISCUSSION |
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The purpose of these experiments was to select mutant cells with a
defective transcriptional response to hypoxia. Since we were unaware of
hypoxia-inducible genes with intrinsically selectable properties, we
used stably integrated recombinant plasmids to provide selectable
markers consisting of HREs linked to cell surface antigens. This
strategy was successful in enabling the isolation of several clones
that showed reduced HRE activity as assessed by transient transfection
assays and one with absent HRE activity, which proved to be defective
in the -subunit of the transcriptional complex HIF-1. Nevertheless,
there were some difficulties to be overcome that merit comment.
First, despite high levels of hypoxia-inducible activity observed for some of the HRE/promoter combinations in transient transfection assays, inducibility of the most responsive stably integrated marker was only 5-fold. This suggested that the use of cytotoxic selection markers, where more stringent control of the level of gene expression is required, might present difficulties. Hence, we adopted an immunoselection strategy similar to that which has been described for the selection of interferon unresponsive mutants (6). The technique of panning has been widely used for expression cloning of genes encoding cell surface antigens (16). In these experiments, we measured its efficacy in selecting low expressing cells and demonstrated that with multiple rounds it provided a relatively simple and inexpensive method of achieving the necessary level of selection even with relatively low levels of marker gene induction.
In pilot experiments, the measured mutation frequency of approximately
1 in 103 at the (haploid) HPRT locus was similar to that
previously reported using ICR191, indicating that adequate exposure to
the mutagen was achieved (29). It is clearly not possible to comment
precisely on the frequency of ablative mutations at (presumably
diploid) loci encoding genes involved in the response to hypoxia from
our isolation of one HIF-1-deficient line. Nevertheless, this result is not inconsistent with the relatively low frequency of ablative mutants reported for other loci using similar methodology (4, 20). The
very much higher rate of loss or suppressed expression of the
transfected marker gene that is generally observed when tissue culture
cells are selected in this way will therefore potentially obscure the
selection of such rare events (4, 30). As has been reported by others,
we found that continued positive selection with a co-transfected drug
resistance gene (4) and the use of two separately integrated markers
(5, 6) limited this problem. Nevertheless, a substantial number of
clones (32 out of 44) that showed reduced expression of both the stably
integrated HRE-linked markers used for selection were indistinguishable
from wild type in the hypoxia-inducible response assayed by a
subsequent transient transfection with a third HRE-linked reporter,
suggesting that such effects still occurred quite frequently. Eleven
clones were identified that showed reduced HRE activity when assessed by transient transfection. Nine of these clones showed a partial reduction in HRE activity, which remained stable in tissue culture. Although eight came from one pool and might represent the same mutation, the other must represent an independent mutation. The defect(s) in these cells have not yet been defined, and
hypoxia-inducible expression of the endogenous Glut-1 gene was normal
in all. Presumably, they represent defects that impair the the full
response of the HRE in transfected cells but are not rate-limiting as
far as expression of Glut-1 mRNA is concerned. Since we were
concerned to identify mutations that were critical for the response to
hypoxia, we concentrated our analysis on one line that possessed a much
more striking defect in HRE activity.
Our analysis of this line (Ka13) demonstrated a defect in HIF-1
function that could be complemented by transient or stable transfection
with a human HIF-1
gene. Normal inducibility of a transfected
Gal4/HIF-1
fusion gene indicated that Ka13 cells were not defective
in the oxygen sensing process that activates HIF-1
(21). Southern
blotting showed no genomic rearrangement or deletion at the HIF-1
locus. Based on the known action of ICR191 (31), the defect is most
likely to be a frame-shifting mutation of the HIF-1
gene that
prevents correct translation and leads to an unstable mRNA or a
defect in a transcription factor that is itself essential for HIF-1
expression.
At least two proteins, HIF-1 and EPAS-1, have been shown to dimerize
with ARNT and bind similar DNA sequences (15, 22, 23). Our finding of
absent HRE activity in the HIF-1
-deficient line is not inconsistent
with this, since neither wild type nor mutant cells contained
detectable EPAS-1 mRNA.
Transient co-transfection experiments in several types of cells with
normal endogenous HIF-1 expression have shown that overexpression of
the HIF-1
will drive the transcription of an HRE-linked reporter even in normoxia (32). Ka13 cells provided an opportunity to test this
against a background of absent endogenous HIF-1
expression. The
results show a progressive increase in normoxic HRE activity as more
HIF-1
was transfected into Ka13 cells. Hypoxia greatly increased
activity, so that with high plasmid doses the HRE activity was much
greater than in untransfected wild type cells. A similar result was
obtained with transfection of EPAS-1, indicating that both the HIF-1
and EPAS-1 genes can independently support hypoxia-inducible gene
expression and that sufficient expression of either will drive
substantial HRE activity even in normoxic cells.
Studies of hypoxia-inducible responses among endogenous genes in these
cells showed marked differences from wild type cells. For some genes,
hypoxia-inducible expression was completely abrogated, indicating a
critical dependence on HIF-1. Such a pattern was observed for the
glucose transporter Glut-1, the glycolytic GAPDH gene, and AK3, an
isoenzyme of adenylate kinase (NTP:AMP phosphotransferase), suggesting
an important function for HIF-1
in adapting energy metabolism to
hypoxia. Complete loss of the hypoxia-inducible response for Glut-1
contrasts with the partial loss of inducibility observed in the
ARNT-deficient mutant Hepa-1 line, c4, which also fails to form the
HIF-1 complex (33) and is of interest in relation to the dual
regulation by hypoxia-induced transcription and mRNA stabilization,
which has been observed for Glut-1 in several cell lines (27, 34).
Either the response in CHO cells is entirely transcriptional or both
responses must in some way be dependent on HIF-1
expression.
Stable transfection of Ka13 cells with human HIF-1 restored
hypoxia-inducible expression of this group of genes. Interestingly, some clones with stable overexpression of the human HIF-1
gene also
showed increased expression of Glut-1 mRNA in normoxic cells, indicating that the level of HIF-1
gene expression could have substantial effects on the expression of certain target genes even
under basal conditions.
In contrast with the striking dependence on HIF-1 gene expression
observed for Glut-1 and GAPDH, hypoxia-inducible expression of other
genes appeared to be largely or entirely independent of HIF-1
expression. This was observed most strikingly for heme oxygenase-1,
where marked induction in 1% oxygen, 0.1% oxygen, and after exposure
to cobaltous ions was observed in both wild type and mutant cells. This
independence from HIF-1
was also observed, in least in part, for
GRP78. Both GRP78 and heme oxygenase-1 have been well recognized as
oxygen-regulated proteins in CHO cells (35, 36). The data on HO-1 are
particularly interesting in that a recent analysis of HO-1 expression
defined a functional HIF-1 site in the HO-1 promoter and showed
abrogation of hypoxia-inducible HO-1 expression in the ARNT-deficient
mutant Hepa-1 cell line, c4 (25). Either hypoxia-inducible expression
of HO-1 is differently controlled in CHO cells or there are other
partners for ARNT that confer hypoxia inducibility and operate
specifically on the HO-1 promoter.
Analysis of gene expression in embryonic stem cells bearing a targeted
mutation of the ARNT gene has recently suggested the existence of an
ARNT-dependent transcriptional response to low glucose
(28). We therefore tested responses to low glucose in the CHO cells.
Surprisingly, only HO-1 was induced by low glucose. This response was
preserved in Ka13 cells, indicating independence from HIF-1.
Overall, our experiments have demonstrated the feasibility of selecting mutant CHO cells that are defective in HRE activation and demonstrated the utility of such cells in dissecting pathways of oxygen-regulated gene expression in mammalian cells. In a number of other systems, several different complementation groups of mutants have been defined, corresponding to critical individual components of the response. It seems likely that HIF-1 activation is a complex process involving more than one mechanism of activation (21, 37-39). Further attempts to isolate mutant cells with specific defects in these pathway(s) should prove very useful in understanding oxygen-regulated gene expression in mammalian cells.
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ACKNOWLEDGEMENTS |
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Thanks for kind gifts go to O. Hankinson for
pcDNA1/Neo/mARNT; to S. McKnight for phEP-1; to G. Semenza for
pBluescript/HIF-1 3.2-3T7; D. Simmons for the cDNA encoding
E-selectin; and G. Stark for plasmids pDW9-27CD2 (from which sequences
encoding CD2 were derived), pSV2Neo, and pSV2Hyg.
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FOOTNOTES |
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* This work was supported by grants from the Wellcome Trust and the Medical Research Council (MRC).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
A Wellcome Clinical Training Fellow.
§ Supported by a grant from the German Research Foundation.
¶ An MRC Senior Fellow.
To whom correspondence should be addressed: Room 420, Inst. of
Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United
Kingdom. Tel.: 44 1865 222382; Fax: 44 1865 222500; E-mail: peter.ratcliffe{at}imm.ox.ac.uk.
1 The abbreviations used are: PAS, Per-aryl hydrocarbon receptor-ARNT-Sim; AK3, adenylate kinase-3; ARNT, aryl hydrocarbon receptor nuclear translocator desferrioxamine; EPAS-1, endothelial PAS protein-1; FACS, fluorescence-activated cell scanning; Glut-1, glucose transporter-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRP78, glucose-regulated protein 78; HO-1, heme oxygenase-1; HAT, hypoxanthine-aminopterin-thymidine; HIF-1, hypoxia-inducible factor-1; HRE, hypoxia-responsive element; HPRT, hypoxanthine phosphoribosyl transferase; ICR191, 3-chloro-7-methoxy-9-(3-[chloroethyl]-amino propylamino)-acridine dihydrochloride; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); VEGF, vascular endothelial growth factor; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline.
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REFERENCES |
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