(Received for publication, May 17, 1996, and in revised form, December 15, 1996)
From the Department of Medicine and the Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4951
glut1 gene expression and glucose
transport are stimulated in a variety of cells and tissues in response
to hypoxia. glut1 is also up-regulated by inhibitors of
oxidative phosphorylation (such as azide) in the presence of oxygen.
Here, we test the hypothesis that hypoxia stimulates glut1
gene expression independent of its inhibitory effect on oxidative
phosphorylation. We examined the effect of cobalt chloride, a known
stimulator of genes responsive to reduced oxygen concentration
per se, on GLUT1 expression under normoxic conditions and
compared the results with the response to azide. Exposure of a rat
liver cell line (Clone 9) to 250 µM cobalt chloride
increases GLUT1 mRNA content, which becomes evident at 2 h,
reaches a maximal value of ~12-fold at 8 h, and remains elevated
at ~8-fold at 24 h. GLUT1 mRNA was the only GLUT isoform expressed in control cells and in cells exposed to cobalt chloride or
azide. The induction of GLUT1 mRNA by cobalt chloride is associated with a ~10-fold stimulation of cytochalasin B-inhibitable
3-O-methyl-D-glucose transport at 24 h. In
contrast to the rapid decrease in cell ATP levels and the stimulation
of glucose transport in response to azide, cell ATP content and glucose
transport remained unaltered during the initial 1-h period of exposure
to cobalt chloride. The effect of cobalt chloride on GLUT1 mRNA
content is mimicked by Ni(II) or Mn(II) but not by Fe(II). Employing
actinomycin D, we found no increase in the ~1.5-h half-life of GLUT1
mRNA in cobalt chloride-treated cells, suggesting that the effect
of cobalt chloride on GLUT1 mRNA content is largely mediated at the
transcriptional level; in contrast, GLUT1 mRNA half-life increased
to >8 h in azide-treated cells. In transient transfections we found
that ~6 kilobase pairs (kbp) of 5-flanking region of the rat
glut1 promoter confers both cobalt chloride- and
azide-inducibility to a reporter gene. Deletion of ~2,500 base pairs
(bp) from the 5
end of the ~6-kbp DNA fragment results in a
reduction of the response to cobalt chloride and a complete loss of the
response to azide. A 666-bp DNA segment located ~6.0 kbp upstream of
the transcription start site was found to be necessary for the increase in reporter gene expression in response to azide, whereas a 480-bp segment located at approximately
3.5 kbp mediated the response to
cobalt chloride. The 480-bp segment is highly homologous to the
previously reported mouse glut1 enhancer and contains
several potential regulatory elements, including a hypoxia-inducible
element; an additional hypoxia-inducible element is present in
the 666-bp segment. Our results suggest that glut1 gene
expression is regulated in a dual fashion by hypoxia per
se and in response to inhibition of oxidative
phosphorylation.
The expression of a number of genes in mammalian cells is regulated by a decrease in the concentration of oxygen in the extracellular environment. For example, the erythropoietin (EPO1) gene and the gene encoding vascular endothelial growth factor are each stimulated in response to hypoxia, effectively resulting in the activation of processes that enhance blood oxygen-carrying capacity (erythropoiesis) and oxygen delivery (neovascularization), respectively (1-7). Equally important and of fundamental significance to the maintenance of cellular homeostasis is the enhancement of glucose transport by hypoxia (8-11), a process that is largely mediated by glut1, a member of the family of facilitative Na+-independent transporters (12, 13). glut1, which is expressed in a variety of cells and tissues, is thought to mediate a significant fraction of non-insulin-dependent transport of glucose in the organism (13, 14), and its expression is augmented by exposure to serum and growth factors and following transformation (15-19). In addition, GLUT1 mRNA and transporter expression are rapidly and markedly up-regulated in response to hypoxia (9-11). Interestingly, in recent studies employing Clone 9 cells (a nontransformed rat liver cell line), we have shown that GLUT1 expression is also markedly up-regulated by inhibitors of oxidative phosphorylation such as azide or cyanide (20-24). These observations raise the question of whether the induction of GLUT1 mRNA in response to hypoxia results from the attendant inhibition of oxidative phosphorylation or whether reduced oxygen concentration per se, operating through distinct oxygen-sensing mechanisms, also stimulates glut1 gene expression.
Studies performed on the regulation of the EPO gene by oxygen have
suggested that the response to hypoxia is mediated by oxygen-sensing mechanisms (1, 25-27). More specifically it has been shown that exposure to hypoxia leads to the induction of a hypoxia-inducible factor that, when bound to its cognate hypoxia-inducible DNA element (HIE) located in the 3-flanking region of the EPO gene, stimulates EPO
gene transcription (2, 26-29). Although agents such as cobalt chloride
employed in the presence of oxygen mimic the stimulatory effects of
hypoxia on EPO gene expression (1, 5, 26-28, 30, 31), chemical
inhibitors of oxidative phosphorylation such as cyanide or rotenone do
not enhance EPO expression (25, 32). The induction of hypoxia-inducible
factor binding activity by hypoxia and cobalt chloride in a variety of
cell lines (33-35) and the recent identification of hypoxia-inducible
factor binding sites in a number of other genes responsive to hypoxia
(5, 30, 31, 36) suggest that this same oxygen-sensing regulatory mechanism may also be involved in the regulation of the
glut1 gene by hypoxia.
The present study is focused on the question of whether the induction of the glut1 gene by hypoxia is mediated only in response to the attendant inhibition of oxidative metabolism or whether distinct oxygen-sensing mechanisms also mediate the response. To differentiate between mechanisms mediating the response to lowered oxygen concentration per se from those mediating the response to the associated inhibition of oxidative phosphorylation, we examined the effect of cobalt chloride employed under normoxic conditions on glut1 gene expression. We found that the enhancement of GLUT1 expression by cobalt chloride occurred in the absence of any inhibition of oxidative phosphorylation and that the induction was mediated by qualitatively different mechanisms and pathways than those mediating the stimulation of GLUT1 mRNA expression in response to azide. A preliminary report of some of these findings has been presented (37).
Clone 9, C2C12, and
3T3-L1 cells were obtained from the American Type Culture Collection
(Rockville, MD). Calf serum, fetal calf serum, Dulbecco's modified
Eagle's medium (DMEM), minimal essential medium, and Hanks' balanced
salt solution were purchased from Life Technologies, Inc.
Nitrocellulose paper (BAS-85) was obtained from Schleicher & Schuell.
Plastic culture dishes were obtained from Corning Glass Works
(Medfield, MA). Cobalt(II) chloride, nickel(II) chloride, manganese(II)
chloride, sodium azide, anisomycin, actinomycin D, cytochalasin B,
phloretin, and standard chemical compounds were purchased from Sigma.
Qiagen Plasmid Maxi Kit was obtained from Qiagen (Chatsworth, CA).
[-32P]dCTP (3000 Ci/mmol),
[14C]chloramphenicol (0.025 mCi/ml), and
3-O-methyl-D-[3H]glucose
([3H]3-OMG) (3.4 mCi/mmol) were obtained from Amersham
Corp. Thin-layer chromatography sheets (silica gel IB) were obtained
from J. T. Baker Inc. Quickhyb was obtained from Stratagene (La Jolla,
CA). pGL2-Basic plasmid, pRL-TK plasmid, the Dual-luciferase reporter assay system, and the Profection transfection kit were purchased from
Promega (Madison, WI). Luciferase assay kits and Monolight 2010 luminometer were obtained from Analytical Luminescence Laboratory (San
Diego, CA).
Clone 9 cells were passaged in DMEM with 10% calf serum at 37 °C with 9% CO2 and reached confluence after ~4 days. Cells were used between passages 27 and 55. C2C12 and 3T3-L1 cells were grown in DMEM containing 10% fetal calf serum and were used immediately upon reaching confluence prior to differentiation. 24 h prior to initiation of all experiments, the medium was changed to serum-free DMEM.
RNA Isolation and Northern BlotsCytoplasmic RNA was isolated as described previously employing a Nonidet P-40-containing buffer (21, 38). RNA samples were fractionated in 1% formaldehyde-agarose gels, and the resulting blots were probed with ~50 × 106 cpm of full-length rat glut1 cDNA (39) that had been 32P-labeled by the random priming method. To ensure equal RNA loading of the lanes and to control for completeness of RNA transfer, ethidium bromide staining of ribosomal 28S and 18S bands on the gels and on the nitrocellulose paper was monitored throughout.
Measurement of [3H]3-OMG UptakeTriplicate confluent cells grown on 60-mm culture plates were employed as described previously (20, 21). Cytochalasin B-inhibitable [3H]3-OMG uptake was calculated as the difference in uptake in the absence and presence of cytochalasin B assayed in parallel.
DNA Constructs phage EN11 (18) was replicated as
described (38). The ~6-kbp BglII/XhoI DNA
fragment representing the 5
-flanking region of the rat
glut1 gene promoter spanning from ~
6,000 to +138 bp of
the major transcription start site (18) was cloned into the promoterless Photinus (firefly) luciferase reporter vector
pGL2-Basic (Promega) and used as a template to generate various
deletions using mapped restriction enzyme sites (constructs A-J). To
generate constructs K and L, the 480-bp PstI/SacI
fragment (spanning ~
3,500 to ~
3,000 bp), to which flanking
SalI sites had been added, was cloned into the
SalI site immediately upstream of the minimal c-fos promoter vector expressing the luciferase reporter
gene (40). The sequences of the 480- and 666-bp fragments were
determined by the dideoxy method (38). pRL-TK (Promega) is a neutral
constitutive plasmid containing the herpes simplex virus-thymidine
kinase promoter expressing Renilla (sea pansy)
luciferase.
Cells were transfected by the method of calcium phosphate-mediated DNA transfer using Profection (Promega) according to a modification of the manufacturer's protocol. For each construct, 10 µg of test plasmid was transfected into each of four subconfluent (90%) 100-mm culture plates containing Clone 9 cells passaged the day before. Approximately 1 h after the addition of DNA, the plates were washed twice with Hanks' balanced salt solution and incubated for ~5-8 h in DMEM containing 10% calf serum. Cells were then recovered by trypsinization, pooled, and divided equally into 12 10-mm culture wells. After overnight incubation, the medium was changed to DMEM devoid of serum, and after ~6-8 h, azide or cobalt chloride was added and the incubation was continued for 24 h. All experiments were performed in triplicate culture plates with parallel control plates receiving DMEM as diluent.
To compare the basal activity of the various promoter constructs, equimolar amounts of the test vectors were transfected into Clone 9 cells in parallel experiments. 10 µg (per 100-mm plate) of the largest construct was transfected each time. Nonspecific plasmid DNA (pBluescript, Stratagene) was mixed with the smaller test vectors to adjust to a total of 10 µg of DNA. To control for the efficiency of transfection, 10 µg of a chloramphenicol acetyltransferase reporter construct containing an SV40 promoter/enhancer (pCAT-Control, Promega) was also cotransfected. Reporter activity was assayed 48 h posttransfection.
For hypoxic experiments, 6 100-mm culture plates were each
cotransfected with 5 and 1 µg of test vector (see Fig. 6A,
construct A) and control vector (pRL-TK), respectively. ~8
h posttransfection, cells were pooled and split into 12 60-mm plates
and incubated overnight with medium containing 10% calf serum. 8 h prior to exposure to hypoxia, the medium was changed to minimal
essential medium devoid of serum. One set of 6 plates was incubated in
a normoxic chamber (20% O2, 5% CO2) and the
other was exposed to hypoxia (nominal 3% O2, 5%
CO2) for 24 h.
Luciferase Assay
Luciferase assay was performed by a minor modification of the manufacturer's protocol (Analytical Luminescence Laboratory). In brief, cells were washed five times with magnesium- and calcium-free phosphate-buffered saline and lysed directly on each plate by the addition of 200 µl of lysis buffer. The plates were then agitated gently on a gyrator shaker at 4 °C for 20 min. Lysates were centrifuged at 14,000 × g for 1 min, and a 100-µl aliquot was used for measurement of luciferase activity in relative light units. Dual luciferase assay was performed according to the manufacturer's protocol (Promega).
Chloramphenicol Acetyltransferase AssayStandard chloramphenicol acetyltransferase assays were performed using the thin-layer chromatography method. In brief, cells were washed five times with magnesium- and calcium-free phosphate-buffered saline and scraped in 1 ml of magnesium- and calcium-free phosphate-buffered saline. One-fourth of the cells were used for luciferase assay. The remainder of the cells were spun at 4 °C and resuspended in 150 µl of 0.25 M Tris-HCl, pH 8.0, and chloramphenicol acetyltransferase assays were performed as described (38). Chloramphenicol acetyltransferase activity was quantitated as the percentage of butyrylated products.
Statistical AnalysisValues are expressed as means ± S.E. Student's unpaired two-tailed t test was used, and a p value of less than 0.05 was considered to be significant (41).
GenBankTM/EMBLThe nucleotide sequences of the
666-kbp (BglII/PstI) segment and the 480-bp
(PstI/SacI) segment of the rat glut1
5-flanking region have been submitted to
GenBankTM/EMBL.
In previous studies we have shown that inhibition of
oxidative phosphorylation by azide in Clone 9 cells results in the
induction of GLUT1 mRNA (21, 22, 24). To determine whether GLUT1
mRNA is induced by hypoxia as well as by azide, we incubated Clone 9 cells under normoxic conditions in the absence and presence of azide
and under hypoxic conditions (6.5 and 1.2% O2) in the absence of the inhibitor. Compared with control cells incubated under
normoxic conditions, GLUT1 mRNA content in cells incubated at 6.5 and 1.2% O2 increased 4- and 10-fold, respectively (Fig. 1A). In accordance with previous observations
(21, 23), exposure to 5 mM azide also led to an increase in
GLUT1 mRNA content (Fig. 1A), suggesting that the
response to hypoxia, in part or in its entirety, may be secondary to
inhibition of oxidative phosphorylation.
To determine whether induction of GLUT1 mRNA by hypoxia can occur by mechanisms distinct from the attendant inhibition of oxidative phosphorylation, we exposed cells to 250 µM cobalt chloride (Fig. 1B). It is evident that both cobalt chloride and azide enhanced GLUT1 mRNA content in the presence of oxygen. In dose-response studies we found that GLUT1 mRNA increased ~4-fold after 16 h of exposure to 50 µM cobalt chloride, that the response was maximal (~12-fold) following exposure to 250 µM cobalt chloride, and that higher concentrations of cobalt chloride were associated with cell toxicity. Based on these results, cobalt chloride at a concentration of 250 µM was employed in all subsequent studies. It should be noted that mRNAs encoding GLUT2, GLUT3, or GLUT4 isoforms were not detected in Clone 9 cells under basal conditions or after exposure to cobalt chloride or azide (data not shown).
We also measured the effect of azide and cobalt chloride on GLUT1 mRNA content of C2C12 and 3T3-L1 cells (Table I); these cell lines were studied before their differentiation to myotubes and adipocytes, respectively. GLUT1 mRNA content was clearly increased in response to azide and cobalt chloride in both cell lines.
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We next monitored the time course of GLUT1 mRNA induction by 250 µM cobalt chloride over a 24-h interval. We found that upon exposure to cobalt chloride, relative GLUT1 mRNA content remained unchanged at 30 min and 1 h, increased significantly by 3- ± 0.5-fold at 2 h, reached a maximum value of 12- ± 1.0-fold at 8 h, and remained elevated at 8- ± 0.8-fold at 24 h (data not shown). Hence, although both azide and cobalt increased GLUT1 mRNA content after a "delay" period, the effect of cobalt chloride was somewhat more rapid (2 versus 3 h) and was of greater magnitude (12- versus 8-fold) than that of azide (21, 24).
The effect of cobalt chloride on the induction of mRNAs of other
genes responsive to hypoxia has been previously shown to be mimicked by
Ni(II) and Mn(II), presumably by interacting with a putative
heme-containing oxygen-sensing molecule (1). Similarly, an induction of
GLUT1 mRNA content in Clone 9 cells was also observed in response
to Ni(II) and Mn(II) but not to Fe(II) (all employed at 250 µM) (data not shown). Moreover, induction of GLUT1
mRNA by cobalt chloride required de novo protein
synthesis because the increase could be blocked by the simultaneous
presence of anisomycin; it should be noted that anisomycin and other
protein synthesis inhibitors added alone also induce GLUT1 mRNA
expression (Fig. 2).2
If the mechanism of induction of GLUT1 mRNA in response to azide is
different from that of cobalt chloride, then it might be expected that
the stimulatory effect of the two agents employed together would be
additive. Cells were exposed to maximally effective concentrations of
the agents, either singly or in combination (Fig. 3).
Exposure to 250 µM cobalt chloride and 7.5 mM
azide resulted in 5.8- ± 0.6-fold and 2.7- ± 0.4-fold increases in
GLUT1 mRNA content, respectively, whereas the presence of both
agents increased GLUT1 mRNA content by 8.1- ± 0.6-fold.
Effect of Cobalt Chloride on the Rate of Glucose Transport and Cell ATP Content
Incubation of cells in the presence of cobalt
chloride for 24 h resulted in a ~10-fold stimulation in the rate
of cytochalasin B-inhibitable 3-O-methyl
D-glucose transport (Fig. 4). In marked contrast to the early stimulation in response to azide (21, 22), there
was no change in the rate of glucose transport following a 1-h exposure
to cobalt chloride (Fig. 4). Moreover, exposure of Clone 9 cells to 5 mM azide or 0.5 mM cyanide is associated with a
rapid >50% fall in cell ATP, with ATP levels increasing to normal
levels by 1 h despite the continued presence of the inhibitors
(20, 22). Again in contrast to inhibitors of oxidative phosphorylation,
cell ATP content remained unchanged at 15 nmol/mg protein after 15, 30, or 60 min of exposure to cobalt chloride.
Transcriptional Enhancement of GLUT1 mRNA Expression in Response to Cobalt Chloride
To examine the potential role of
GLUT1 mRNA stabilization in the induction of GLUT1 mRNA in
response to cobalt chloride, we measured the half-life of GLUT1
mRNA in control and cobalt chloride-treated cells; azide-treated
cells served as a positive control. Employing actinomycin D, we found
that the ~1.5-h half-life of GLUT1 mRNA in control cells was
unchanged in cobalt chloride-treated cells; in contrast, GLUT1 mRNA
half-life was increased to >8 h in azide-treated cells, as has been
described previously (23) (Fig. 5). These results are
consistent with the possibility that the stimulatory effect of cobalt
chloride on GLUT1 mRNA content is largely mediated at the
transcriptional level.
To identify cis-regulatory elements involved in the transcriptional
stimulation of the glut1 gene in response to cobalt
chloride, we analyzed the rat glut1 promoter in a series of
transient transfection assays. In addition, we extended this analysis
to include our previously reported stimulation of glut1 gene
transcription by azide (23). A series of constructs spanning ~6 kbp
of genomic DNA from the 5-flanking region of the rat glut1
gene was fused to a promoterless luciferase reporter vector
(pGL2-Basic) and transfected into Clone 9 cells. To determine whether
the construct containing ~6 kbp of 5
-flanking region is capable of
being stimulated by hypoxia, we transiently transfected Clone 9 cells
with the construct and exposed the cells to nominal 3% oxygen for
24 h. Results showed a 2.3- ± 0.3-fold increase in luciferase
activity of cells exposed to hypoxia compared with control cells
incubated under normoxic conditions (p < 0.05; Table
II). In parallel experiments, the same hypoxic stimulus
led to a 4.0- ± 0.6-fold increase in the level of endogenous GLUT1
mRNA.
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We next determined the luciferase activity of transfected cells exposed
for 24 h to diluent, 250 µM cobalt chloride, or 5 mM azide, and the results are expressed relative to
luciferase activity in diluent-treated transfected cells (Fig.
6A). Basal expression of constructs A, F, and
H was 20-fold greater than that of construct J, which does not contain
the glut1 TATA box and extends only 15 bp upstream of the
glut1 major transcription start site. Luciferase expression
of the construct containing ~6 kbp of the 5-flanking region
(construct A) and the construct containing a deletion of ~2 kbp
within the 6-kbp region (construct B) was enhanced 6- and 3-fold by
cobalt chloride and azide, respectively. Moreover, treatment of cells
containing either construct A or B with cobalt chloride and azide
together resulted in luciferase expression that was larger than the
response to either agent alone, with the effect being nearly additive
for both constructs (~8-fold; n = 12 for both
constructs). Deletion of ~2,500 bp from the 5
end of the ~6-kbp
DNA (construct C) results in a complete loss of the response to azide.
In contrast, the response of construct C to cobalt chloride, although
reduced to approximately one-half that of constructs A or B, is still
present (Fig. 6A). The positive response of construct C to
cobalt chloride maps to a 480-bp PstI/SacI fragment between ~
3.5 and ~
3.0 kbp from the transcription start site; deletion of this region (constructs D-H) is marked by a loss of
the response to cobalt chloride, and inclusion of the 480-bp fragment
upstream to 200 bp of proximal glut1 promoter reconstitutes
the response to cobalt chloride (compare constructs C, H, and I).
Interestingly, fusion of the 666-bp BglII/PstI
segment, located between ~
6.0 and ~
5.3 kbp of the transcription
start site, upstream to construct C reconstitutes the response to azide (compare construct B with C) and augments the response to cobalt chloride.
To determine whether the 480-bp fragment behaves as an enhancer, we tested whether it can increase the function of a heterologous promoter and confer cobalt chloride-inducibility to it. For these studies, the fragment was cloned upstream of a minimal c-fos promoter (Fig. 6B). Inclusion of the 480-bp fragment led to enhanced basal expression of the minimal c-fos promoter by >20-fold (see legend to Fig. 6). Moreover, this fragment confers cobalt chloride-inducibility on the heterologous c-fos promoter in an orientation-independent manner (constructs K and L). Interestingly and unexpectedly, the 480-bp fragment also confers azide-inducibility on the minimal c-fos promoter (Fig. 6B).
Sequence Analysis of the Rat glut1 EnhancersThe 480-bp
SacI/PstI segment and the 666-bp
BglII/PstI segment were sequenced and analyzed
for the presence of potential binding sites for transcriptional
regulatory elements (Fig. 7, A and
B, respectively). Interestingly, a region in the 3 end of
the 480-bp fragment that shares a high degree of homology to the HIE of
the human or mouse EPO gene was identified (2, 26, 27, 35). In
addition, this fragment contains two phorbol ester response elements
(42), two SP-1s (43), an SRE (44), and three core p53 binding sites
(45). A search of the GenBankTM data base revealed
considerable sequence homology between the 480-bp fragment identified
in this study and the Enhancer-1 region of the mouse glut1
gene located ~3.1 kbp upstream of the transcription start site (46).
By deletion analysis, the SRE homology region has been shown to be
critical to the stimulation of the mouse glut1 Enhancer-1 by
serum or platelet-derived growth factor (46).
The sequence of the 666-bp segment was searched for the presence of potential cis-regulatory elements using a transcription factor data base (Fig. 7B) (43). We found regions matching binding sites for several trans-acting factors, including activator proteins 2 and 3 (AP-2, AP-3), C/EBP, p53, and nuclear factors 1 and 3 (NF-1, NF-3). Of interest is the presence of an HIE site (2). In addition, this region contains a number of cis-elements that have been shown to mediate tissue-specific and developmental gene regulation. The functional significance of these sites remains to be determined.
It is well established that the expression of a number of genes is modified during the adaptive response of cells and tissues to exposure to hypoxic conditions (6, 9, 30, 31, 36, 47). Since hypoxia leads to the inhibition of oxidative phosphorylation, it is important to distinguish between regulatory mechanisms that are controlled by decreased levels of oxygen per se from those that are controlled by the attendant inhibition of oxidative phosphorylation. This distinction can be made through employment of specific chemical agents that selectively mimic the actions of the different components of the hypoxic response. Specifically, cobalt chloride employed in the presence of oxygen mimics many of the effects of lowered oxygen concentration per se on the expression of several genes (1, 2, 5, 26-28, 30, 31), and azide or cyanide lead to an inhibition of oxidative phosphorylation in the absence of hypoxia (11, 20, 21, 32). Employing such a strategy, we have shown that glut1 gene expression is stimulated in response to hypoxia by two distinct pathways. The first pathway appears to involve oxygen-sensing regulatory molecules similar to those regulating the expression of EPO and vascular endothelial growth factor genes (1, 2, 5, 26, 27, 46). This pathway is stimulated by exposure to cobalt chloride in the presence of oxygen, an effect that is shared by Ni(II) and Mn(II) (1). The second pathway leading to the induction of GLUT1 mRNA is activated in response to inhibition of oxidative phosphorylation in the absence of hypoxia.
The pathways leading to induction of GLUT1 mRNA expression in Clone 9 cells in response to hypoxia per se (i.e. by cobalt chloride) versus in response to inhibitors of oxidative phosphorylation (i.e. by azide) differ in a number of important respects. Exposure to cyanide or azide leads to an immediate inhibition of oxidative phosphorylation and a fall in cell ATP content that is associated with a rapid stimulation of glucose transport that reaches ~8-fold control values by 1 h (20-22). In contrast, no change in cell ATP content or in the rate of glucose transport was observed during a 1-h period of exposure to cobalt chloride. Incubation for longer periods in the presence of either azide or cobalt chloride led to increases in GLUT1 mRNA content and a stimulation of glut1-mediated glucose transport, with the effect of cobalt chloride on GLUT1 mRNA content being greater in magnitude and becoming evident somewhat earlier. Azide and cobalt chloride also differed in their effects on GLUT1 mRNA turnover; whereas the half-life of GLUT1 mRNA increased from ~1.5 to >8 h in cells exposed to azide, GLUT1 mRNA half-life remained unaltered in cells incubated in the presence of cobalt chloride. This finding not only suggests that the increase in GLUT1 mRNA content in response to cobalt chloride is mediated primarily at the transcriptional level but also suggests that the previously reported stabilization of GLUT1 mRNA in cells exposed to hypoxia may be due to the attendant inhibition of oxidative phosphorylation (48).
Employing deletion analysis and site-directed mutagenesis, DNA elements in the mouse Enhancer-1 region that mediate the induction by inhibitors of oxidative phosphorylation and by hypoxia have very recently been reported (49). Results of our experiments identifying the region of the rat glut1 enhancer (480-bp fragment) necessary for the response to cobalt chloride are in agreement with this analysis. The mouse Enhancer-1 contains a 7-bp element with high homology to the HIE (49). Moreover, mutation of the core 4-bp consensus nucleotides in the mouse glut1 Enhancer-1 abolishes its positive transcriptional response to cobalt chloride (49). Interestingly, the rat glut1 sequence of this region bears greater homology than that of the mouse gene to the HIE present in either the human or mouse EPO gene (2, 26, 27, 49), and it would be expected that deletion or mutation of this region in the rat glut1 promoter should likewise lead to the elimination of cobalt chloride responsiveness. It is worth emphasizing that although the 480-bp segment was necessary for the response to cobalt chloride, this segment alone was not sufficient for the full response to cobalt chloride. Inclusion of the 666-bp segment located upstream of this region augmented the cobalt chloride response by more than 2-fold (compare constructs C and I with constructs A and B in Fig. 6). Interestingly, within the 666-bp segment we found one region with high homology to the core HIE consensus. Although the function of this region remains to be determined, it is tempting to speculate whether it might cooperate with the downstream HIE site contained within the 480-bp segment to mediate the full cobalt chloride response.
Our results, however, differ markedly from conclusions reached on the region in the mouse glut1 Enhancer-1 that is necessary for glut1 induction in response to inhibition of oxidative phosphorylation (49). Analysis of the mouse Enhancer-1 sequence revealed the presence of an SRE (44, 46); an identical sequence is also present in the 480-bp enhancer segment of the rat glut1 gene (Fig. 7). Deletion or mutation of this latter region in the mouse glut1 Enhancer-1 segment cloned upstream to a heterologous promoter was associated with a loss of induction in response to azide or other inhibitors of oxidative phosphorylation (49). Although we also demonstrated that the 480-bp segment conferred inducibility of the minimal c-fos promoter to azide, we found that the segment was not sufficient to confer such inducibility to the rat glut1 promoter in the context of additional glut1 flanking sequences (Fig. 6, A and B). In keeping with this inference, we found that construct C, which contains the 480-bp segment, showed no positive response to azide. Interestingly, the addition of the 666-bp fragment to construct C reconstituted the full azide response (compare constructs A and B with C). This suggests that the 666-bp region is necessary for the response to azide, although sequences contained in the 480-bp region appear to augment the response to azide (compare constructs A and B with D).
What then is the physiological significance of the 480-bp segment's
response to azide, as described previously (49) and confirmed in this
study (Fig. 6B). Although the answer to this question
remains to be resolved, we would point out that: 1) the response to
azide is observed only in studies examining the role of this region in
isolation by use of minimal heterologous promoters, and that the
response is not observed in the context of the rat glut1
promoter/5-flanking region; and 2) it has been reported that both Fos
and Jun proteins are induced in some cell systems in response to
hypoxia (50, 51, 52). If a similar response occurs in our cells, then
increased activator protein-1 (AP-1) activity may stimulate the SRE
present in the 480-bp segment and lead to the observed response to
azide in the context of the heterologous promoter constructs. However,
a similar series of events may be ineffective at stimulating the gene
in response to inhibition of oxidative phosphorylation because of
additional cis-elements present both upstream and downstream to the
480-bp segment located in its naturally occurring context. Further
studies are required to clarify these issues. Taken together, our
results indicate that the ~6-kbp 5
-flanking region, which includes
sequences in addition to the 480-bp enhancer region, is necessary for
the stimulation of glut1 gene expression in response to
inhibition of oxidative phosphorylation.
Results of the present study demonstrate that glut1 gene expression is dually regulated by hypoxia, namely by a reduction in oxygen concentration per se and by the attendant inhibition of oxidative phosphorylation. Such dual control by hypoxia has not been reported for other hypoxia-responsive genes, whose expression is thought to be regulated only by decreased concentrations of oxygen (1-3, 5, 26, 30, 31, 36, 40). In the case of glut1, we can only speculate that the evolutionary advantage of its dual regulation by hypoxia may reflect the fact that glucose transport is rate-limiting for glucose metabolism in a variety of cells (11, 53); dual control of GLUT1 expression would enable sustained high rates of glycolytic ATP synthesis in the face of reduced mitochondrial ATP production resulting either from exposure to low oxygen concentrations or from exposure to poisons or toxins inhibiting oxidative phosphorylation. It should be noted that the regulation of other known hypoxia-responsive genes by agents such as azide has not been examined rigorously, and it is possible that the expression of some of these genes is also controlled in a dual fashion by hypoxia. Further studies are necessary to identify and characterize other hypoxia-responsive genes that, similar to glut1, might be dually regulated. Moreover, although the sequence of events and mechanisms underlying the regulation of specific genes by hypoxia per se are becoming better understood, virtually nothing is known concerning the regulation of genes responding to inhibition of oxidative phosphorylation. Clearly, the latter constitutes an important area for future investigation.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U82754[GenBank] and U82755[GenBank].
We are grateful to the late Dr. Ora M. Rosen
for supplying full-length rat glut1 cDNA and to Dr.
Morris J. Birnbaum for supplying the EN11 phage containing the rat
glut1 5
-flanking region. We thank Allen Gabriel for help in
preparing RNA.