From the Department of Radiation Oncology, University
of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
19104 and the ¶ Department of Medicine and Molecular Biology and
Microbiology, School of Medicine, Case Western Reserve University,
Cleveland, Ohio 44106
Received for publication, November 7, 2000
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ABSTRACT |
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Oncogenic transformation and hypoxia both induce
glut1 mRNA. We studied the interaction between the
ras oncogene and hypoxia in up-regulating glut1
mRNA levels using Rat1 fibroblasts transformed with
H-ras (Rat1-ras). Transformation with
H-ras led to a substantial increase in glut1
mRNA levels under normoxic conditions and additively increased
glut1 mRNA levels in concert with hypoxia. Using a
luciferase reporter construct containing 6 kilobase pairs of the
glut1 promoter, we showed that this effect was mediated at
the transcriptional level. Promoter activity was much higher in
Rat1-ras cells than in Rat1 cells and could be
down-regulated by cotransfection with a dominant negative Ras construct
(RasN17). A 480-base pair (bp) cobalt/hypoxia-responsive fragment of
the promoter containing a HIF-1 binding site showed significantly
higher activity in Rat1-ras cells than in Rat1 cells,
suggesting that Ras might mediate its effect through HIF-1 even under
normoxic conditions. Consistent with this, Rat1-ras cells
displayed higher levels of HIF1- Oncogenic transformation of mammalian cells is associated with
many alterations in metabolism (see Ref. 1 for review). An increased
rate of glucose transport is among the most characteristic biochemical
markers of the transformed phenotype. The Glut1 glucose transporter is
one of the proteins responsible (reviewed in Ref. 2). A number of
oncogenes, including fps, src, and ras
have been shown to increase glucose transport and to up-regulate
glut1 mRNA and protein levels (3-5). glut1
gene expression and glucose transport are also stimulated in a
variety of cells under hypoxic conditions, a response that is mediated
by the transcription factor HIF-1.1 HIF-1 binds to a
cis-acting binding sites located within the 5'- flanking
region of the glut1 gene (8, 9).
Because hypoxia and oncogenic mutations are both commonly present in
tumors, we set out to examine the interaction between the two in
up-regulating glut1 mRNA levels. Mutations in Ras are seen in a third of human cancers (6); therefore, as our model system we
used Rat1 fibroblasts transformed with H-ras. Transformation with H-ras led to a substantial increase in glut1
mRNA levels under normoxic conditions and additively increased
glut1 mRNA levels in concert with hypoxia. Our results
indicate that a HIF-1 binding site located in the glut1
promoter is important in its regulation by Ras. We further show that
oncogenic H-Ras leads to increased promoter activity by up-regulating
the steady-state level of HIF-1 Cell Culture and Establishment of H-ras-transformed Cell Lines
and Reagents--
Rat1 cells were maintained in culture in DMEM
supplemented with 10% (v/v) fetal bovine serum (Atlanta Biologicals)
and cultured in a humidified 95% air/5% CO2 incubator at
37 °C. To establish stable cell lines, Rat1 cells were transfected
with an H-ras plasmid using Fugene (Roche Molecular
Biochemicals) according to the manufacturer's instructions. Briefly,
Rat1 cells were plated into a 100-mm dish 24 h before
transfection. Cells were transfected using 6 µl of Fugene and 2 µg
of the plasmid pAL8-H-ras, which contains the H-ras genomic DNA and a neomycin-resistance gene.
Transfected cells were selected in DMEM supplemented with 400 µg/ml
G418 (Life Technologies, Inc., Gaithersburg, MD) for 1 week. Colonies
were isolated using cloning cylinders, trypsinized, then expanded as cell lines. The expression of the H-ras gene was confirmed
by Western blotting with an anti-H-Ras monoclonal antibody. Twelve G418-resistant colonies were expanded, all of which expressed high
levels of H-Ras protein compared with the Rat1 parental cells. We used
clone 12, which expressed approximately 10-fold the level of H-Ras as
the parental Rat1 cells (data not shown) for most of our experiments.
Rat1-ras cell lines were maintained in DMEM supplemented
with 10% fetal bovine serum and 400 µg/ml G418. PD098059 and
LY294002 (from Alexis Biochemicals, San Diego, CA) were dissolved in
Me2SO at stock concentrations of 100 mM and 40 mM, respectively.
Northern Blot Analysis--
Total RNA was isolated with TRIzol
(Life Technologies Inc.) following the manufacturer's instructions.
5-10 µg of total RNA was run on a 0.9% agarose gel containing
formaldehyde, transferred to a Duralon-UV membrane (Stratagene, La
Jolla, CA), and UV cross-linked prior to hybridization. Labeling of
radioactive DNA probes was performed using [32P]dCTP and
a Prime-It kit (Stratagene). Hybridization was carried out at 65 °C,
after which the membranes were washed to a stringency of 0.1 × SSC, 0.1% SDS at 65 °C. Autoradiography was carried out at
Western Blot Analysis--
For protein isolation, cells were
washed once with cold phosphate-buffered saline containing 1 mM EDTA, then solubilized by adding lysis buffer (1%
Triton X-100, 20 mM Tris, pH 7.6, 150 mM NaCl,
2 mM EDTA, 10% glycerol, 1 mM dithiothreitol,
1 mM orthovanadate, 2 mM phenylmethylsulfonyl
fluoride) directly on the cells. Lysates were transferred into 1.5-ml
Eppendorf tubes and centrifuged at 12,000 rpm for 10 min at 4 °C.
Supernatants were transferred to a fresh tube and frozen on dry ice.
Protein concentrations were determined using a BCA Protein Assay kit
(Pierce, Rockford, IL). For Western blotting, an equal amount of total
protein was separated by SDS-polyacrylamide gel electrophoresis on a
6% polyacrylamide gel. After completion of gel electrophoresis,
protein was transferred onto a Transblot nitrocellulose membrane
(Bio-Rad, Hercules, CA) using a blotting apparatus. For detection of
H-Ras protein, we used a anti-human H-Ras monoclonal antibody (Quality
Biotech, Camden, NJ) at a dilution of 1:1000 followed by a secondary
goat anti-mouse antibody (Bio-Rad) at a dilution of 1:2000. For
detection of the HIF-1 Hypoxic Conditions--
For hypoxia experiments, 5-7 × 105 cells were plated on day 1 into 60-mm Permanox dishes
(Nunc) and maintained in a 5% CO2 incubator. Permanox
plastic was used because of its high permeability to oxygen, permitting
the efficient evacuation of oxygen from the dishes before hypoxia
induction. On day 2, immediately before the induction of hypoxia,
standard media was replaced with media containing 50 mM
HEPES, 0.15% (w/v) glucose, and 10 mM NaOH to maintain pH.
Dishes were then placed into airtight aluminum chambers. The oxygen
concentration was decreased by sequentially replacing a given
percentage of the total gas within a chamber with 95% nitrogen/5%
CO2 using a precision vacuum gauge. The oxygen
concentration was reduced from 21% to 5% with one gas exchange, from
5% to 1% with a second gas exchange, then with an additional gas
exchange to 0.2%. The aluminum chambers were then placed on an orbital shaker in a warm room maintained at 37 °C.
Plasmid Constructs, Transient Transfections, and Luciferase
Assay--
A series of constructs containing various fragments from
the 5'-flanking region of the rat glut1 gene were used.
Constructs A, C, E, and K are described elsewhere (8). As shown in Fig. 2 (see below), constructs A, C, and E contain, respectively, 6, 3.5, and 3 kbp of the 5'-flanking region of the rat glut1 gene. Construct K contains a 480-base pair sequence from the 5'-region of the
rat glut1 promoter located at
Construct N was made by PCR amplification in the following manner. The
primer pair (1A, 5'-TAATAGGTACCCAACAGAGCCTTTTC-3'; 2B,
5'-TAATAGGCTAGCGAGCTCCCAGGAACTTG-3') was used for amplifying using
construct K as a template. Construct N contains 184 bp of the
glut1 promoter located at approximately
For reporter gene assays, transfections were performed using
LipofectAMINE (Life Technologies Inc.) according to the manufacturer's instructions. Briefly, cells were plated into 60-mm dishes so that
24 h later they were ~50% confluent. Cells in each dish were transfected using 10-15 µl of LipofectAMINE, 2 µg of reporter plasmid, and 1 µg of pSV- H-ras and Hypoxia Additively Increase glut1 mRNA
Levels--
To examine how oncogenic transformation by human
H-ras affects expression of the glut1 gene, we
stably transfected Rat1 cells with an H-ras expression vector. We
compared the expression of glut1 mRNA in Rat1 cells
relative to Rat1-ras cells. Rat1 cells expressed very low
levels of glut1 mRNA but exhibited a 2.8-fold increase
in glut1 mRNA levels after 6 h of hypoxia (Fig.
1; lanes 1 versus
3). In contrast, Rat1-ras cells exhibited
increased levels of glut1 mRNA under normoxic conditions
(2.5-fold over Rat1 cells; lanes 2 versus
1) and showed an additional 2-fold induction after 6 h
of hypoxia (lanes 4 versus 2). Thus,
H-ras overexpression both increases the basal level of
glut1 mRNA and interacts with hypoxia to additively
increase the level of this message. Similar results were obtained using
a second independent H-ras transformed subclone (data not
shown).
Ras and Hypoxia Increase glut1 Promoter Activity--
To
investigate whether the increased expression of glut1
mRNA by H-ras and hypoxia was due to increased promoter
activity, we transfected a rat glut1 promoter-luciferase
construct (construct A; "A" in Fig.
2) into Rat1-ras and Rat1
cells. 24 h after transfection, half the dishes were subjected to
hypoxia (0.2% oxygen) for 6 h and half were maintained in 21%
oxygen. Subsequently cells from all the dishes were harvested and
assayed for luciferase activity. In Rat1 cells there was a 2.2-fold
induction of the glut1 promoter by hypoxia (Fig.
3A). Rat1-ras cells
showed a 9-fold higher luciferase activity than Rat1 cells under
normoxic conditions and exhibited a further 3.3-fold increase when
exposed to hypoxia (Fig. 3A). To demonstrate that the
glut1 promoter activity in Rat1-ras cells was
specifically due to the effect of H-Ras, we cotransfected a construct
expressing dominant negative RasN17 into these cells, which resulted in
a decrease in glut1 promoter activity (Fig. 3B).
Thus, the activity of the glut1 promoter is increased in Rat1-ras cells compared with Rat1 cells, and it can be
further increased by hypoxia.
Analysis of the 5'-Flanking Region of the glut1 Promoter--
To
better characterize the elements in the 5'-flanking region of the rat
glut1 gene responsive to H-Ras, a series of deletion mutants
of the promoter was used in transient transfection experiments in both
Rat1 and Rat1-ras cells under normoxia and hypoxia (Fig. 4). glut1 promoter activity
was weak in the Rat1 parental cells with all of the constructs tested.
There was approximately a 2-fold induction of promoter activity by
hypoxia with constructs A, C, and K, but only a 1.3-fold induction with
construct E, which lacks a potential HIF-1 binding site. Similar to
results shown in Fig. 3, in Rat1-ras cells, the basal level
of glut1 promoter activity with construct A was 5-fold
greater than in Rat1 cells, and hypoxia led to a 3.5-fold induction of
activity compared with normoxia in Rat1-ras cells. Construct
K gave a higher level of expression in Rat1-ras cells under
normoxia or hypoxia that either construct A or C, possibly due to the
fact that construct K contains a minimal c-fos
promoter instead of the native glut1 TATA box and
transcription start site found in constructs A and C. The basal level
of expression of construct E was higher in Rat1-ras cells
than Rat1 cells; the reason for this is unknown. However, as expected,
there was no significant induction of the glut1 promoter
activity by hypoxia with construct E in Rat1-ras cells.
The HIF-1 Binding Site in the glut1 Promoter Is Important for
Up-regulation by Ras under Normoxic Conditions--
The
glut1 promoter is known to contain a HIF-1 binding site,
which is required for its transactivation by hypoxia through the
transcription factor HIF-1 (9). To test whether this site is important
for transactivation of the promoter by H-Ras under normoxic conditions
we made a pair of reporter constructs, construct N, which contains 184 bp of the promoter, including the HIF-1 binding site, and construct M,
which is identical except for a 4-bp mutation, which abolishes the
HIF-1 binding site (Fig. 2). As expected, construct N (Fig.
5A) but not construct M (Fig.
5B) was inducible by hypoxia. We performed transient
transfections of both constructs into Rat1-ras cells with
and without cotransfection of a dominant negative RasN17 expression
vector. Fig. 5C shows that the basal promoter activity using
construct M, which has a mutated HIF-1 binding site, was 37% less than
that of construct N, which has an intact HIF-1 binding site. In two
additional independent experiments we found the promoter activity of
construct M to be 27 and 33% less than the activity of construct N
(data not shown). Fig. 5C also shows that promoter activity
is inhibited by ~40% in cells cotransfected with construct N and
RasN17, whereas RasN17 had no effect on luciferase expression in cells
transfected with construct M. These results support the premise that
H-Ras exerts a positive effect on the glut1 promoter
transcription through the HIF-1 binding site.
Ras Increases HIF-1 Both the MAP Kinase Inhibitor PD098059 and the PI3K Inhibitor
LY294002 Down-regulate glut1 mRNA Expression and Promoter Activity
and HIF-1
Therefore, both PI3K and MAP kinase inhibition have a negative effect
on the glut1 promoter. To determine whether they have an
effect on glut1 mRNA levels, Rat1-ras cells
were treated with either inhibitor under normoxic or hypoxic conditions
before harvesting cells for RNA. Under normoxic conditions, treatment
with either inhibitor led to a decrease in glut1 mRNA
levels (Fig. 8; compare lanes
3 and 4 with lane 2), and treatment with
both drugs led to a greater decrease in the level (Fig. 8; compare
lane 5 with 2). We also treated
Rat1-ras cells with bisindolylmaleimide I, a protein kinase
C inhibitor, and found that this had no effect on VEGF mRNA levels
(data not shown). We then treated Rat1-ras cells with the
inhibitors prior to exposure to hypoxia. Neither drug alone had a
detectable effect on glut1 message level (Fig. 8; compare
lanes 8 or 9 with lane 7); however,
using both drugs together reduced glut1 mRNA levels by
~30% (Fig. 8; compare lane 10 with lane
7).
To determine whether the effect of PD098059 and LY294002 in decreasing
glut1 mRNA levels in normoxia might be mediated through HIF-1 For decades it has been known that tumors display numerous
metabolic changes compared with normal cells. Tumors frequently display
increased glycolytic metabolism even under aerobic conditions, an
effect named after Warburg who originally described it (10). To keep up
with this increased glycolysis, there must be an increased uptake of
the substrate, glucose. Human tumors have been shown to have increased
glucose uptake in vivo compared with normal tissues (see
Ref. 11 and references within). One of the proteins responsible for the
uptake of glucose into cells is Glut1, which was the first of a family
of glucose transporters to be cloned (12). Glut1 is expressed in most
normal tissues and is responsible for basal glucose transport. Numerous
factors have been shown to regulate glut1 mRNA
expression, including phorbol esters, oncogenes, hypoxia, growth
factors, and mitogens (reviewed in Refs. 2 and 13). Over a decade ago
it was observed that transformation of rodent fibroblasts by Fujiyama
sarcoma virus (4) or by the ras or src oncogenes
(3) resulted in a marked increase in glucose uptake, which was
accompanied by an increase in Glut1 protein and mRNA expression. In
the case of Fujiyama sarcoma virus, it was shown that the increase in
glut1 mRNA occurred at the level of transcription (4).
Glut1 levels have been found to be increased in a variety of human
cancers (14, 15). One study found that Glut1 expression increased with
increasing grade of malignancy in human colon cancers and was
associated with a higher proportion of lymph node metastases
(14).
Cells subjected to hypoxia must undergo metabolic adaptations to
survive. Hypoxia induces the expression of many genes to allow for this
to occur (reviewed in Refs. 16 and 17). Part of this adaptation to
hypoxia involves up-regulation of genes that encode the enzymes
required for anaerobic glycolysis, thus allowing cells to switch to
this form of metabolism from oxidative phosphorylation. Under hypoxic
conditions there is also a parallel increase in glucose uptake, which
is facilitated by up-regulation of Glut1 expression. Hypoxia is a
potent stimulus for glut1 mRNA induction in a variety of
tissue types, including endothelial cells (18), hepatic cells (19),
various tumor cell lines (9), and alveolar epithelial cells (20). The
mechanism of glut1 message induction by hypoxia is
complicated and is dually controlled by low oxygen concentrations
per se and by inhibition of oxidative phosphorylation (19).
The former can be mimicked by cobalt chloride and the latter by sodium
azide. The cobalt-responsive element in the rat glut1
promoter has been mapped and is homologous to the mouse Enhancer-1
sequence (9, 19). Transactivation through this element in the promoter
is mediated by HIF-1 (9) a heterodimeric transcription factor that
binds to a specific DNA consensus sequence, 5'-RCGTG-3', found in the
promoters of many hypoxia-inducible genes (for review see Ref. 16). The
HIF-1 transcription factor consists of two subunits, HIF-1 Although the mechanism by which hypoxia increases glut1
mRNA expression through HIF-1 is relatively well understood, it is less clear how oncogenes such as Ras increase glut1 mRNA
levels. The phorbol ester
12-O-tetradecanoylphorbol-13-acetate, which activates
protein kinase C, is known to increase glut1 mRNA levels (23); however, Glut1 induction by growth factors and oncogenes such as
K-ras occurs via a protein kinase C-independent pathway (23,
24). We were interested in understanding how a specific oncogene,
mutant H-ras, led to increased glut1 mRNA
levels and in studying its interaction with hypoxia in regulating
message expression. For our model system we used Rat1 cells transformed with this oncogene. We found that H-ras transformation
increased the basal level of glut1 mRNA under normoxia
compared with Rat1 cells, consistent with what others have found using
other oncogenes (3, 4). We also found that H-ras interacts
additively with hypoxia to increase mRNA expression (Fig. 1).
Furthermore, the effect of Ras on glut1 mRNA levels is
mediated at the level of the promoter, since the promoter activity is
greater in Rat1-ras cells than in Rat1 cells (Fig.
3A).
In analyzing the 5'-flanking region of the glut1 gene using
deletion constructs in transient transfection experiments, we found
that construct K, which contains a HIF-1 binding site, was sufficient
to mediate up-regulation of promoter activity by H-ras. However,
construct K contains a 480-base pair sequence from the 5'-region of the
rat glut1 promoter located between The 480-bp sequence for the rat glut1 5'-region shows
significant homology to the 5'-Enhancer-1 region of the mouse
glut1 gene that was cloned by Murakami et al.
(8). This group found that the mouse glut1 Enhancer-1 was
inducible by H-Ras. They showed that the SRE and two AP-1 sites within
this enhancer were important in promoter activity, although they did
not specifically examine the role that these sites played in regulation
by Ras. However, they found that these three elements were insufficient
for full basal activity of Enhancer-1. Subsequently, the HIF-1 binding site within the mouse glut1 Enhancer-1 was identified (9). Our data indicate that this HIF-1 binding site is important in the
regulation of the rat glut1 promoter by H-Ras. Of note,
activation of the mouse glut1 promoter by oxidative stress
in L6 myotubes was shown to involve both the SRE and the AP-1 binding
sites in Enhancer-1 but was independent of the Ras/MAP kinase pathway
(25).
How does H-Ras regulate activity of the HIF-1 binding site in the
glut1 promoter? One mechanism is by increasing the level of
HIF-1 Both inhibitors of the MAP kinase and the PI3K pathways led to
decreased glut1 promoter activity and glut1
mRNA levels under normoxia. In contrast, neither drug by itself
appeared to block the induction of glut1 mRNA by
hypoxia, although the two together did have an inhibitory effect. This
apparent discrepancy between the effects of these inhibitors on
glut1 message under normoxia and hypoxia might be explained
by the fact that the induction of glut1 mRNA by hypoxia
involves mRNA stabilization in addition to transactivation of the
promoter (19). Therefore, in transfection experiments using promoter
constructs, we are specifically examining transcriptional regulation,
whereas in the Northern blots, the results are complicated by the fact
that RNA stabilization, which may be independent of PI3K and MAP
kinase, also plays a role.
Even though we found that both MAP kinase and PI3K inhibitors decreased
glut1 mRNA promoter activity, only the latter led to a
detectable decrease in HIF-1 Our results suggest a link between H-ras transformation,
HIF-1 protein under normoxic conditions.
In addition, a promoter construct containing a 4-bp mutation in the
HIF1 binding site showed lower activity in Rat1-ras cells
than a construct with an intact HIF1 binding site. The activity of the
latter construct but not the former could be down-regulated by RasN17,
supporting the importance of the HIF1 binding site in regulation by
Ras. The phosphatidylinositol 3-kinase inhibitor LY29004
down-regulated glut1 promoter activity and mRNA levels
under normoxia and also decreased HIF1
protein levels in these
cells. Collectively these results indicate that H-Ras up-regulates the
glut1 promoter, at least in part, by increasing HIF-1
protein levels leading to transactivation of promoter through the HIF-1
binding site.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
protein and that this involves the
PI3K pathway, known to be downstream of Ras (7).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C with intensifying screens. A 400-bp mouse glut1
cDNA fragment (from M. Birnbaum, University of Pennsylvania) was
used to make radioactive probes.
protein, we used an anti-HIF-1
antibody
(clone H1
67, Novus Biologicals, Littleton, CO) at a dilution of
1:1000 followed by a secondary goat anti-mouse antibody (Bio-Rad).
Antibody binding was detected by chemiluminescence using an ECL kit
(Amersham Pharmacia Biotech, Piscataway, NJ).
3.5 to
3.0 kbp relative to
the transcription start site (GenBankTM accession number U82755). This
480-bp enhancer contains a potential HIF-1 binding site at nucleotides
+398 to +401.
3.2 to
3.0 kbp
relative to the transcription start site (from nucleotides +297 to
+480; GenBankTM accession number U82755). These primers were chosen to
eliminate the SRE (at nucleotides +287 to +296), which has been shown
to have a positive role in stimulation of the glut1 promoter
(8), while retaining the potential HIF-1 binding site (nucleotides +398
to +401). Primer 1A has incorporated within it a KpnI
restriction site and primer 2B an NheI restriction site, so
the PCR product could be restricted with these two enzymes and
subcloned into the KpnI/NheI sites of the
reporter plasmid pGL3-Promoter. pGL3-Promoter contains its own SV-40
promoter, which was required because the fragment that was
PCR-amplified lacks the glut1 TATA box and transcription
start site. Construct M was made using a two-step overlapping PCR
strategy. In step one, two separate pairs of primers were used in PCR
amplification, primer 1A (listed above) and primer 1B,
5-TGCGTGTCAGCCAGACATCCTGTG-3' in one reaction, and primer
2A, 5'CACAGGATGTCTGGCTGACACGCA-3' and primer 2B (listed
above) in the second. Primers 1B and 2A overlapped the HIF-1 binding
site but introduced a 4-bp mutation, which is
underlined. After the first PCR amplification with these two sets of primers in separate reactions, the product was then amplified using primers 1A and 2B to generate a 184-bp fragment containing the desired mutation, which was restricted with
KpnI/NheI and subcloned into the pGL3-Promoter
vector. The amplified sequence is shown in Fig. 2B (see below).
-galactosidase (Promega, Madison, WI) to
control for transfection efficiency. 24 h later, cells were harvested by removing the media, washing twice with phosphate-buffered saline, and directly adding 100 µl of lysis buffer per dish. Of this
lysate, an aliquot was used for luciferase assays and another aliquot
for
-galactosidase determination. Luciferase and
-galactosidase assays were performed using the LucLite kit (Packard Instrument Co.)
and the
-galactosidase Enzyme Assay System (Promega, Madison WI),
respectively. Luciferase readings were performed on a TopCount Microplate scintillation and luminescence counter (Packard Instrument Co.).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of H-ras on
glut1 mRNA expression in normoxia and
hypoxia. Rat1 or Rat1-ras cells were subjected to
hypoxia (0.2% oxygen) or left in room air (21% oxygen) for 6 h
before RNA isolation. 5 µg of total RNA were run on an
agarose/formaldehyde gel and subsequently transferred to a nylon
membrane that was probed simultaneously for glut1 and
rpL32 mRNA. The numbers shown at the bottom
of the figure represent the ratio of intensity of the glut1
band to the rpL32 band.
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Fig. 2.
Glut1 promoter constructs. A, a
series of promoter deletion constructs were derived from the
~6-kbp rat GLUT1 promoter. Constructs A, C, E, and K
(letters A, C, E, and K)
have been previously described (19). Construct A contains the entire
6-kb 5'-flanking region of the rat glut1 gene in which there
are two hypoxia-inducible elements, a 660-bp
BglII/PstI azide-responsive segment, located
between approximately 6.0 and
5.3 kbp relative to the transcription
start site and a 480-bp PstI/SacI
cobalt-responsive element, located between approximately
3.5 and
3.0 kbp. Construct C contains a 5'-deletion but still retains the
480-bp cobalt-responsive element, which contains both an SRE and a
HIF-1 binding site. Construct E lacks the 480-bp cobalt-responsive
element. Constructs A, C, and E were made using the luciferase reporter
vector pGL2-Basic. The 480-bp cobalt-responsive element was subcloned
into a luciferase reporter construct containing the c-fos
promoter to make construct K. Construct N was made as described under
"Experimental Procedures" and contains a 184-bp sequence from the
glut1 promoter lying approximately
3.2 to
3.0 kb
relative to the transcription start site (spanning nucleotides +297 to
+480; GenBankTM accession number U82755). Within this 184-bp sequence
is located the HIF-1 binding site (from nucleotide +398 to +402;
GenBankTM accession number U82755); however, the SRE has been deleted.
Construct M is identical to construct N except for a 4-bp mutation in
HIF-1 binding site. Both construct M and N were subcloned into the
reporter pGL3-Promoter, which contains its own SV40 promoter.
B, 184-bp sequence from the glut1 promoter, which
is present in construct M. 4 bases (CGTG) within the HIF-1 binding site
are boxed. These 4 bases were mutated to ATAT in construct
N.
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Fig. 3.
Effect of H-ras on
glut1 promoter activity. A, cells were
plated onto a 60-mm dish the day before transfection. The following
day, cells were transfected with 2 µg of construct A (Fig. 2) and 1 µg of pSV- -galactosidase (Promega). 24 h later, culture media
was replaced with buffered media and the cells were subjected to 6 h of hypoxia (0.2% oxygen) or normoxia before they were collected and
analyzed for luciferase and
-galactosidase. B,
Rat1-ras cells were transfected with 2 µg of construct A
along with 1 µg of pSV-
-galactosidase (Promega). Along with these
plasmids was cotransfected either 0.5 µg of pcDNA3 or 0.2 µg of
pcDNA3 and 0.3 µg of pcDNA3/RasN17. For both A and
B, normalized luciferase levels (ratio of luciferase to
-galactosidase readings) are plotted on the y axis.
Values represent the mean of three independent transfections. The
error bars represent one S.D. from the mean.
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Fig. 4.
Activity of glut1 promoters in Rat1 and
Rat1-ras cells in normoxia and hypoxia.
glut1 promoter constructs (Fig. 2) were tested for hypoxia
responsiveness in Rat1 and Rat1-ras cells. Cells were
transfected with 2 µg of reporter plasmid (either construct A, C, E,
or K) and 1 µg of pSV- -galactosidase, allowed to recover for
24 h, exposed to air or hypoxia for 6 h, then assayed for
luciferase and
-galactosidase activity. Normalized luciferase levels
(ratio of luciferase to
-galactosidase readings) are plotted on the
y axis. Values represent the mean of three independent
transfections. The error bars represent one S.D. from the
mean.
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Fig. 5.
The HIF-1 binding site in the
glut1 promoter is important for regulation by Ras
under normoxic conditions. Rat1-ras cells were
transfected with 2 µg of reporter plasmid (either construct M or N
shown in Fig. 2) and 1 µg of pSV- -galactosidase. In addition, they
were cotransfected with either 0.5 µg of pcDNA3 (control) or 0.5 µg of pcDNA3/RasN17. Normalized luciferase levels (ratio of
luciferase to
-galactosidase readings) are plotted on the
y axis. Values represent the mean of three independent
transfections. The error bars represent one S.D. from the
mean.
Protein Levels and HIF-1
Binding--
Having found that the HIF-1 binding site in the
glut1 promoter was important for its regulation by H-Ras, we
speculated that H-Ras might increase the level of the HIF-1
protein.
We found that Rat1-ras cells contained higher levels of the
protein under both normoxic and hypoxic conditions than did Rat1 cells
(Fig. 6). This figure also shows that
cobalt chloride, which is a hypoxia mimic, leads to higher steady-state
levels of HIF-1
in Rat1-ras cells than Rat1 cells.
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Fig. 6.
HIF-1 levels in Rat1
and Rat1-ras cells. Rat1 and Rat1-ras
cells were subjected to normoxia or hypoxia (0.2% oxygen) for 6 h, then cells were harvested in protein lysis buffer. Samples were run
on a 6% polyacrylamide gel, then Western blotting was performed. Blot
was probed with anti-HIF-1
primary antibody and goat anti-mouse
secondary antibody.
Levels--
Both the PI3K and MAP kinase pathways have
been show to be activated by Ras (7). Therefore, to define the signal
transduction pathways through which expression of Glut1 is regulated by
Ras, we transiently transfected cells with glut1
promoter/luciferase constructs (either construct A or K), then treated
them with either a PI3K inhibitor (LY294002) or an inhibitor of MAP
kinase (PD098059) under normoxic or hypoxic conditions before
harvesting cells for luciferase assays. PD098059 and LY294002 both
down-regulated glut1 promoter activity using construct A in
Rat-ras cells under normoxic conditions, and both inhibited
the induction of glut1 mRNA by hypoxia (Fig.
7). Similar results were obtained using
construct K (Fig. 7).
View larger version (44K):
[in a new window]
Fig. 7.
Effect of PD098059 and LY294002 on
glut1 promoter activity. Cells were transfected
with 2 µg of reporter gene. 24 h later cells were treated with
PD098059 (50 µM) or LY294002 (40 µM) then
subjected to normoxia or hypoxia (0.2% oxygen) for 6 h. Cells
were then collected and assayed for luciferase activity. Normalized
luciferase levels (ratio of luciferase to -galactosidase readings)
are plotted on the y axis. Values represent the mean of
three independent transfections. The error bars represent
one S.D. from the mean.
View larger version (52K):
[in a new window]
Fig. 8.
Effect of PD098059 and LY294002 on
glut1 mRNA levels. Cells were treated with
either PD098059 (50 µM) or LY294002 (40 µM)
for 6 h under normoxic or hypoxic conditions (0.2% oxygen). RNA
was prepared from the cells and then separated on an agarose gel. RNA
was transferred on a hybridization filter and probed for
glut1 and rpL32. The numbers shown at
the bottom of the figure represent the ratio of intensity of
the glut1 band to the rpL32 band.
, we examined HIF-1
levels in Rat1-ras cells
after drug treatment. Fig. 9 shows that
treatment with LY294002 led to a decrease in the level of HIF-1
,
whereas treatment with PD098059 had no such effect. Two additional
independent experiments confirmed this result (data not shown).
View larger version (53K):
[in a new window]
Fig. 9.
LY294002 but not PD098059 decreases
HIF-1 protein levels in Rat1/ras
cells. Rat1-ras cells were treated with
Me2SO, PD098059 (50 µM), LY294002 (40 µM), or both drugs (LY+PD). 4 h later,
cells were harvested and lysed in protein lysis buffer. Samples were
run on a 6% polyacrylamide gel, then Western blotting was performed.
Blot was probed with anti-HIF-1
primary antibody and goat anti-mouse
secondary antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
HIF-1
, both of which are basic helix-loop-helix proteins. HIF-1
is a component of several transcription factors, and its protein level
is not significantly induced by hypoxia. In contrast, HIF-1
protein,
which is unique to the HIF-1 complex, is rapidly degraded in oxygenated
cells by the ubiquitin-proteasome pathway (21). HIF-1
protein is specifically induced by hypoxia in a graded response dependent on the
oxygen concentration (22).
3.5 and
3.0 kbp
relative to the transcription start site. In addition to a HIF-1
binding site (nucleotides +398 to +401; GenBankTM accession number
U82755), this 480-bp stretch contains sites for several other
transcription factors, including AP-1 binding sites (at nucleotides
+248 to +254 and +320 to +327) and an SRE (at nucleotides +287 to
+296), which have been previously implicated in the regulation of this
promoter (8). Therefore, to rigorously test the importance of the HIF-1
binding site, we created reporter constructs M and N, neither of which
contain the SRE. These constructs contain 184 bp of the
glut1 promoter located from approximately
3.2 to
3.0 kb
relative to the transcription start site (from nucleotide +297 to +480;
GenBankTM accession number U82755). Constructs M and N are identical
except that the former contains a 4-base pair mutation of the HIF-1
binding site. Therefore, as expected, construct N was inducible by
hypoxia, but not construct M. Both constructs showed activity in
Rat1-ras cells under normoxic conditions, but a key
difference is that the activity of construct N, but not construct M,
could be down-regulated by mutant RasN17. Therefore, this strongly
suggests the HIF-1 binding site is important in regulation by H-Ras.
Construct M also exhibited lower basal luciferase activity in
Rat1-ras cells than did construct N, between 25 and 40%
less. We speculate that the reason that the difference is not greater
is because both of these constructs (M and N) are driven by an SV40
minimal promoter rather than the TATA box intrinsic to the
glut1 promoter; therefore, there is a high basal level of activity.
protein. Rat1-ras cells contain higher levels of
the protein than do Rat1 cells under both hypoxic and normoxic
conditions. Although HIF-1
was originally described as a
transcription factor, which was undetectable under normoxic conditions
and inducible only under hypoxia (26), there is accumulating evidence
that it is also important in gene regulation under normoxic conditions. Src-transformed cells have been found to contain higher
levels of HIF-1
under normoxic conditions than nontransformed
parental cells (27). HIF-1
has been found to be induced in tumor
cell lines by a variety of hormones and growth factors, including
epidermal growth factor, insulin-like growth factor-1, insulin, and
angiotensin II (28-31).
protein levels. A possible role for the
PI3K pathway in regulating HIF-1
protein levels has been suggested
in the context of PTEN expression in glioblastoma cells (32) and
epidermal growth factor stimulation in prostate carcinoma cells (29).
In regard to MAP kinase, both p42 and p44 MAP kinase have been shown to
phosphorylate HIF-1
in vitro and to stimulate HIF-1
transcription activity without affecting the level of the protein (33).
Therefore, it is conceivable that the drug PD098059 might decrease
glut1 mRNA levels and promoter activity through an
effect on HIF-1
function without affecting the level of the protein itself.
up-regulation, and glut1 mRNA transactivation.
In the future we plan to pursue this path of investigation to
understand how H-Ras leads to HIF-1
up-regulation. Although we have
examined the expression of a specific gene, glut1, as a
downstream target of H-Ras, it is likely that the up-regulation of
other hypoxia-inducible genes such as VEGF occurs by a similar
mechanism. A number of groups have shown that mutant Ras leads to
increased VEGF levels in diverse cell types (34-36). In NIH3T3 cells,
activated H-Ras has been postulated to increase VEGF reporter
expression only when an intact HIF-1 binding site is present (37).
Thus, mutations in H-ras can not only lead to transformation
but may also help cells survive in a hypoxic environment by increasing
the expression of specific genes through up-regulation of HIF-1
.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Don Solomon for technical assistance and Dr. Xiaoling Su for her help with data analysis. We thank Eric Bernhard, Frank Lee, and Ruth Muschel for reading the manuscript and offering helpful comments. We are grateful to Amita Sehgal for use of the TopCount Microplate scintillation and luminescence counter.
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FOOTNOTES |
---|
* This work was supported by the Dept. of Radiation Oncology at the University of Pennsylvania and by National Institutes of Health Grant DK-49450 (to F. I. -B.).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.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed: Rm. 195, John
Morgan Bldg., University of Pennsylvania School of Medicine,
3620 Hamilton Walk, Philadelphia, PA 19104. Tel.: 215-614-0078; Fax: 215-898-0090; E-mail: maity@mail.med.upenn.edu.
Published, JBC Papers in Press, December 18, 2000, DOI 10.1074/jbc.M010144200
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ABBREVIATIONS |
---|
The abbreviations used are: HIF-1, hypoxia-inducible factor-1; SRE, serum-responsive element; PI3K, phosphatidylinositol 3-kinase; DMEM, Dulbecco's modified Eagle's medium; bp, base pair(s); kbp, kilobase pair(s); PCR, polymerase chain reaction; MAP, mitogen-activated protein; VEGF, vascular epidermal growth factor.
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