(Received for publication, July 24, 1995)
From the
Studies of gene regulation by oxygen have recently defined the existence of a widely operative system that responds to hypoxia but not mitochondrial inhibitors and involves the induction of a DNA-binding complex termed hypoxia-inducible factor 1. This system has been implicated in the regulation of erythropoietin, certain angiogenic growth factors, and particular glycolytic isoenzymes. The glucose transporter Glut-1 is induced by both hypoxia and mitochondrial inhibitors, implying the operation of a different mechanism of oxygen sensing. To explore that possibility, we analyzed the cis-acting sequences that convey these responses. An enhancer lying 5` to the mouse Glut-1 gene was found to convey responses both to hypoxia and to the mitochondrial inhibitors, azide and rotenone. However, detailed analysis of this enhancer demonstrated that distinct elements responded to hypoxia and the mitochondrial inhibitors. The response to hypoxia was mediated by sequences that contained a functionally critical, although atypical, hypoxia-inducible factor 1 binding site, whereas sequences lying approximately 100 nucleotides 5` to this site, which contained a critical serum response element, conveyed responses to the mitochondrial inhibitors. Thus, rather than reflecting an entirely different mechanism of oxygen sensing, regulation of Glut-1 gene expression by hypoxia and mitochondrial inhibitors arises from the function of two different sensing systems. One of these responds to hypoxia alone and resembles that involved in erythropoietin regulation, while the other responds to mitochondrial inhibitors and involves activation of a serum response element.
Many biological processes are concerned with adaptation to the availability of oxygen, and oxygen tension is an important regulator of gene expression. A recent advance in the understanding of these processes has been the recognition that a mechanism of oxygen sensing identical or closely similar to that first recognized in the context of erythropoietin regulation is widely operative in different cell types (1, 2, 3) . Production of erythropoietin, the principal humoral regulator of red blood cell production, is stimulated by hypoxia in specific cells within liver and kidney. Characterization of this response has defined a number of distinctive features. In particular, the response to hypoxia is mimicked by transition metal ions such as cobalt (4) and by iron chelating agents (5) but not by inhibitors of mitochondrial respiration(6, 7, 8) . These features have led to the proposal of a specific oxygen sensing system, possibly involving the operation of a heme protein sensor(8) .
Two
types of evidence have been important in implicating this regulatory
system in the control of genes other than erythropoietin: first,
similarities in the characteristics of the inducible response, and,
second, the definition of similar critical elements in the cis-acting
control sequences of the different genes. For certain genes, such as
several of those encoding glycolytic enzymes, the demonstration of
inducible responses to hypoxia, cobalt, and iron chelating agents but
not cyanide, together with the functional definition of cis-acting
sequences that cross-compete with the erythropoietin 3` enhancer for
binding to the hypoxia-inducible factor 1 (HIF-1) ()provide
firm evidence of a common regulatory
mechanism(9, 10, 11) . Similarly, evidence
has recently implicated this mechanism in the regulation of vascular
endothelial growth
factor(12, 13, 14, 15) . For the
genes encoding a number of other vascular growth factors, multiple
similarities with the regulation of the erythropoietin gene have also
been observed(14, 16, 17) , but the
cis-acting sequences mediating the inducible responses have not all
been defined.
For other genes, inducible responses to hypoxia have been described in which there are important differences from erythropoietin regulation. One such gene is that encoding the glucose transporter, Glut-1. Both inhibition of mitochondrial respiration and cellular hypoxia are accompanied by an increase in the rate of glucose uptake, which is largely mediated by increased Glut-1 activity(18, 19, 20, 21, 22, 23) . This occurs by increased transporter synthesis following up-regulation of Glut-1 mRNA and by post-translational mechanisms. Since both hypoxia and mitochondrial inhibitors increase Glut-1 mRNA, it has appeared likely that induction by hypoxia is due to hypoxic compromise of mitochondrial metabolism and is therefore distinct from the mechanism underlying erythropoietin regulation. A further difference in the regulation of the two genes is seen in the responses to phorbol esters; whereas hypoxic induction of the erythropoietin gene is ablated by phorbol 12-myristate 13-acetate (PMA)(24, 25, 26) , this compound induces the expression of Glut-1(27) .
Here we report that, despite these characteristics, there are also similarities between the regulation of Glut-1 and erythropoietin. Induction of Glut-1 mRNA by hypoxia could be mimicked by cobalt and the iron chelator, desferrioxamine (DFO). To characterize further the response of Glut-1 to hypoxia and mitochondrial inhibitors, we analyzed the regulatory sequences that convey these responses. The mouse Glut-1 5` enhancer, lying at -3.3 to -2.7 kb from the transcription initiation site, was found to possess hypoxically inducible activity and also to be responsive to azide, rotenone, cobalt, DFO, and PMA. Deletional and mutational analysis demonstrated that distinct cis-acting sequences within this enhancer conveyed responses to hypoxia and to the mitochondrial inhibitors. Hypoxic stimulation was mediated by a HIF-1 binding site, the operation of which was unaffected by PMA. Responses to mitochondrial inhibitors were mediated by a serum response element (SRE). These findings indicated that rather than operating through an entirely different mechanism of oxygen sensing, the responsiveness of Glut-1 gene expression to hypoxia and mitochondrial inhibitors arises from the integration of two different sensing and transduction systems, one of which is related to that acting on the erythropoietin gene.
In studies of the regulation of Glut-1 mRNA, tissue culture
cells that had reached approximately 70% confluence were exposed to
normoxia, hypoxia, or pharmacological agents for 14-16 h prior to
harvest with trypsin-EDTA. For exposure to hypoxia, cells were
incubated under an atmosphere of 1% O, 5% CO
,
94% N
in a Napco 7301 incubator (Precision Scientific,
Chicago, IL). Cobalt and DFO were dissolved in water and added to the
culture medium to achieve final concentration of 100 µM.
Azide was dissolved in water, and rotenone was dissolved in ethanol and
added to the culture medium to achieve final concentrations of 1
mM and 0.1 µg/ml, respectively. PMA was stored as a 1
mg/ml solution in Me
SO at -20 °C and was used at
a final concentration of 100 nM. All chemicals were obtained
from Sigma.
Progressive 5` and 3` truncations of the Glut-1 5`
enhancer were made by polymerase chain reaction using the plasmid
pG5`E(1-610) as a template. Mutations, exchanging each
pyridimine for the noncomplementary purine and vice versa,
were made using the pALTER-based Altered Sites in vitro mutagenesis system as directed by the manufacturer (Promega).
Four-base pair mutations of putative hypoxia inducible factor-1
(nucleotides 279-282) and serum response elements (nucleotides
171-174) were introduced in the context of
pG5`E(1-326)
, containing nucleotides 1-326 of the
Glut-1 5` enhancer, to create pG5`E(
HIF) and pG5`E(
SRE). All
deletions and mutations were confirmed by dideoxy sequencing.
To test the independent function of subsequences, concatamerized oligonucleotides were cloned into a construct containing the herpes simplex thymidine kinase promoter linked to a human growth hormone reporter gene (pTKGH). Complementary oligonucleotides were synthesized that, when annealed, produced the elements with 5` XbaI and 3` SpeI compatible ends. The sequence of the Glut-1 HIF-1 element was 5`-CACAGGCGTGCCGTCTGACACGCA-3`, and the c-fos SRE was 5`-GGATGTCCATATTAGGACATCT-3`. These elements were then ligated into an XbaI site located 10 bp 5` to the TATA box in the thymidine kinase promoter. Trimers of each element were confirmed by dideoxy sequencing.
To test if, despite the reported induction of Glut-1 mRNA by mitochondrial inhibitors, there might be similarities with erythropoietin regulation, we determined whether Glut-1 mRNA could be induced by cobalt or DFO. HepG2 cells were exposed to cobaltous chloride (100 µM) or to DFO (100 µM) for 16 h. Both of these agents induced Glut-1 mRNA to a similar degree to that observed following hypoxia (Table 2).
Figure 1: Localization of hypoxically inducible cis-acting sequences to the Glut-1 5` enhancer. Sequences with known transcriptional activity from the mouse Glut-1 gene (28) were cloned into reporter constructs as described under ``Materials and Methods.'' The Glut-1 5` enhancer was capable of conferring hypoxic inducibility on the reporter gene, but the intronic enhancer and promoter were not hypoxically inducible. In each transfection, expression was related to that of a co-transfected control plasmid; induction was calculated as the ratio of hypoxic to normoxic expression. The figures shown are the mean values for three independent experiments ± S.E.
Figure 2:
Deletional analysis of the Glut-1 5`
enhancer. Hypoxically inducible cis-acting sequences within the Glut-1
5` enhancer were localized using sequential deletions from the 5` and
3` ends of the Glut-1 5` enhancer. Nucleotides within the enhancer
contained in each deleted construct are given in the plasmid name.
Activity of the sequence in normoxic cells is related to that of
pG5`E(1-610), which was arbitrarily designated 100.
Induction was calculated as the ratio of hypoxic to normoxic
expression. The bars show the mean value for at least three
independent experiments ± S.E.
Figure 3:
Different cis-acting sequences mediate
response to different stimuli. The deleted plasmids
pG5`E(228-610) and pG5`E(1-262)
were compared
with pG5`E(1-326)
(wild-type) in their responses to
different stimuli. Plasmid pG5`E(228-610)
showed induction
by hypoxia, cobalt, and DFO but not azide and rotenone. In contrast,
plasmid pG5`E(1-262)
was induced by azide and rotenone but
not by hypoxia, cobalt, or DFO. The values are related to the
regulation of the wild-type plasmid, pG5`E(1-326)
, which was
arbitrarily designated 100 for each stimulus, and represent the mean
± S.E. of three transfections.
Failure of plasmid pG5`E(228-610) to respond to
azide or rotenone indicated that the sequence lying 5` to nucleotide
228 was responsible for this effect. We therefore tested
pG5`E(108-610)
and found preserved responses to azide (data
not shown). Since the sequence between nucleotides 108 and 228 contains
a conserved serum response element, plasmid pG5`E(
SRE)
was
constructed with a four-nucleotide mutation at this site. This mutation
abolished the response to azide but had little effect the response to
hypoxia or cobalt (Fig. 4).
Figure 4:
Mutation of HIF-1 and SRE sites. HIF-1 and
SRE sites were mutated as described under ``Materials and
Methods.'' Plasmid pG5`E(SRE)
was capable of regulation
by hypoxia and cobalt but not by azide. Plasmid pG5`E(
HIF)
was capable of regulation by azide but not by hypoxia or cobalt. Values
are related to the regulation of pG5`E(1-326)
by each
stimulus, which was arbitrarily set to 100, and represent the mean
± S.E. of three transfections.
Figure 5: A, electrophoretic mobility shift assay with the Glut-1 HIF-1 site. Alternate lanes contain nuclear extract from HepG2 cells incubated for 12 h in normoxia (N) or hypoxia (H). Both hypoxically inducible and constitutive binding activities are observed with the Glut-1 WT probe and are similar to those observed with the Epo WT probe. Binding to the Glut-1 WT probe was competed by a 200-fold excess of unlabeled Glut-1 WT and Epo WT oligonucleotides but not the mutant Epo oligonucleotide, Epo Mut. B, efficiency of different competitors for binding of HIF-1. The Epo WT probe was used with normoxic (in the first lane only) and hypoxic HepG2 nuclear extracts. Each competing oligonucleotide was used as follows: 0, 40-fold excess, and 200-fold excess. The Glut WT competitor is a less efficient competitor than Epo WT or PGK WT. The Epo Mut oligonucleotide does not compete either the inducible or constitutive bands even at the highest concentration.
Plasmid pG5`E(1-326)
responded to both PMA and hypoxia, and the combination produced an
augmented response. Plasmid pG5`E(228-610)
, in which the SRE
and two AP-1 sites are deleted, showed a strikingly reduced response to
PMA but retained the ability to respond to hypoxia. The response to PMA
and hypoxia was similarly reduced, suggesting that it arose from an
interaction between the HIF-1 complex and sequences lying 5` to
nucleotide 228. Though some responsiveness to PMA was retained, very
similar results were obtained with the plasmid pG5`E(
SRE),
indicating that the SRE contributed to the response to PMA. These
results also suggested that PMA did not affect the hypoxically
inducible response conveyed by the HIF-1 binding site itself.
One of the major metabolic consequences of hypoxia is compromise of mitochondrial metabolism, and limitation of mitochondrial electron transport is associated with many biochemical changes that could potentially mediate oxygen-dependent responses. Therefore, in considering mechanisms of oxygen sensing, an important characteristic is whether mitochondrial inhibitors have a similar action to hypoxia. In the case of erythropoietin regulation, this is not the case, indicating that the mechanism of oxygen sensing is not dependent on the consequences of hypoxic limitation of mitochondrial metabolism(6, 7, 8) . However, activation by mitochondrial inhibitors is seen in a number of other systems that respond to hypoxia, suggesting that these responses represent the operation of different types of sensing mechanism(18, 31, 32, 33) .
In this study, we have analyzed the transciptional regulation of the glucose transporter Glut-1 as an example of such a response. In keeping with the induction of Glut-1 mRNA by hypoxia and a number of different mitochondrial inhibitors, we found that an enhancer element lying 5` to the Glut-1 gene could be activated by hypoxia, azide, and rotenone. When the structure of this enhancer was examined in detail, distinct cis-acting sequences were found to convey responses to hypoxia and the mitochondrial inhibitors. Thus, rather than reflecting the operation of a fundamentally different system, these properties reflect the integration of two different inducible responses, one of which is activated by mitochondrial inhibitors but not exposure to 1% oxygen, and the other of which resembles erythropoietin regulation in responding to hypoxia but not mitochondrial inhibitors. Further similarities of this hypoxically inducible response to erythropoietin regulation were demonstrated by induction of Glut-1 mRNA by both cobaltous ions and DFO, and by the definition of the critical cis-acting sequence for these responses as a HIF-1 binding site.
Similar evidence has been assembled to link the regulation of erythropoietin and genes encoding a number of glycolytic enzymes to this common oxygen-sensing mechanism(9, 10, 11) . The current experiments extend these observations to a different, although functionally related, type of gene, and provide further evidence for the importance of this oxygen sensing mechanism, and HIF-1 or related species, in the control of cellular metabolism.
Comparison of the
critical cis-acting sequences at the HIF-1 binding site from these
different genes raises several points. First, although sequence
conservation at the HIF-1 binding site is clear, the Glut-1 site
differs from the previously defined consensus(10) , and the
cross-competition with the erythropoietin enhancer was more complete
for the constitutive species than HIF-1 itself. Affinity purification
of HIF-1 using oligonucleotides derived from the human erythropoietin
3` enhancer has recently enabled the cloning of cDNAs encoding two DNA
binding subunits, HIF-1 and HIF-1
, both of which are members
of the basic helix-loop-helix-PAS group of transcription factors, and
form a heterodimer(34) . It is possible that the complex
binding at the Glut-1 site differs in one or more components of the
heterodimer. Second, it is interesting that all of the functional HIF-1
binding sites defined to date appear to bind both inducible and
constitutive
species(9, 11, 15, 35) , suggesting
the possibility of a functional relevance for the constitutive species.
Third, sequence conservation exists not only at the HIF-1 binding site
but also at a precisely distanced adjacent site shown to be of
functional relevance in other genes regulated by HIF-1 (Fig. 6),
suggesting that the factors that bind at this adjacent site are
important for the operation of
HIF-1(11, 35, 36) .
Figure 6: Comparison of functionally defined HIF-1 sites from different genes that have been subject to detailed mutational analysis. Nucleotides that have been shown to be functionally critical by mutational analysis are underlined(11, 35, 36) .
Whereas activation of the Glut-1 5` enhancer by rotenone and azide was unaffected by mutation or deletion of the HIF-1 binding site, the response to these agents was dependent on the presence of an intact SRE lying 102 nucleotides 5` to this site. Moreover, oligonucleotides derived from the SRE in the c-fos promoter were sufficient to convey responses to these stimuli. Although the mechanisms of sensing and signal transduction underlying these responses were not analyzed in detail, these results indicate that induction by mitochondrial inhibitors involves the activation of factors that bind at this site.
Although the primary
aim of this work was to analyze responses to hypoxia and mitochondrial
inhibitors, activation of the Glut-1 5` enhancer by both PMA and
hypoxia provided an opportunity to study the action of PMA on the
hypoxically inducible response. Following the observation that in
hepatoma cells PMA severely reduces hypoxically inducible
erythropoietin production, it has been argued that some consequence of
exposure to PMA (for instance either activation or depletion of protein
kinase C) has a critical action on the mechanism of oxygen
sensing(24, 25, 26) . Surprisingly, however,
other genes such as vascular endothelial growth factor, whose induction
by hypoxia otherwise resembles that of erythropoietin, are also induced
by PMA (12, 37) . Our studies indicate that the
increased activation of the Glut 5` enhancer by PMA and hypoxia arose
from a positive integration of effects on different cis-acting
sequences, with the operation of the HIF-1 binding site being
substantially unaffected by PMA. This is not a specific property of the
Glut-1 HIF-1 site, since under conditions that abrogate hypoxially
inducible erythropoietin production in hepatoma cells, we found no
effect of PMA on either the functional or protein binding
characteristics of the HIF-1 binding oligonucleotide from the mouse
erythropoietin enhancer (Epo WT). ()The suppressive action
of PMA on hypoxic induction of erythropoietin must therefore presumably
arise from an action of PMA on inducible factors other than HIF-1 or
from an interaction with permissive constraints on erythropoietin gene
expression.
In summary, we have demonstrated that the Glut-1 5` enhancer conveys transcriptional responses to hypoxia and mitochondrial inhibitors through the operation of distinct cis-acting elements and that the response to hypoxia involves the activation of HIF-1. The possibility that the final characteristics of an inducible response arise from the integration of influences from different systems must be considered when comparing the physiological characteristics of a response. In particular, in hypoxically inducible systems, induction by mitochondrial inhibitors does not necessarily indicate that the means of hypoxic sensing involves a signal generated by compromise of mitochondrial metabolism.