1 Klinik und Poliklinik für Anästhesiologie und Operative Intensivmedizin, Universitätsklinikum Münster, Münster, Germany; and Departments of 2 Critical Care Medicine, 3 Surgery, and 4 Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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Cellular
adaptation to hypoxia depends, in part, on the transcription factor
hypoxia-inducible factor-1 (HIF-1). Normoxic cells exposed to an
inflammatory milieu often manifest phenotypic changes, such as
increased glycolysis, that are reminiscent of those observed in hypoxic
cells. Accordingly, we investigated the effects of cytomix, a mixture
containing IFN-, TNF, and IL-1
on the expression of
HIF-1-dependent proteins under normoxic and hypoxic conditions.
Incubation of intestine-derived epithelial cells (IEC-6) under 1%
O2 increased HIF-1 DNA binding and expression of aldolase
A, enolase-1, and VEGF mRNA. Incubation of normoxic cells with cytomix
for 48 h also markedly increased HIF-1 DNA binding and expression
of mRNAs for these proteins. Incubation of hypoxic cells with cytomix
did not inhibit HIF-1 DNA binding or upregulation of HIF-1-dependent
genes in response to hypoxia. Neither cytomix nor hypoxia increased
steady-state levels of HIF-1
mRNA. Incubation of IEC-6 cells with
cytomix induced nitric oxide (NO·) biosynthesis, which was blocked if
the cultures contained L-NG-(1-iminoethyl)lysine
hydrochloride (L-NIL). Treatment with L-NIL, however, failed to significantly alter aldolase A, enolase-1, and VEGF
mRNA levels in normoxic cytomix-treated cells. Proinflammatory cytokines activate the HIF-1 pathway and increase expression of glycolytic genes in nontransformed rat intestinal epithelial cells, largely through an NO·-independent mechanism.
nitric oxide; glycolysis; epithelium; intestinal; aldolase A; enolase-1; vascular endothelial growth factor; hypoxia-inducible factor-1; deoxyribonucleic acid
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INTRODUCTION |
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IN MAMMALIAN CELLS,
ADAPTATION to reduced oxygen availability includes the
upregulation of specific genes involved in angiogenesis, erythropoiesis, glucose transport, and glycolysis. A number of different transcription factors and signaling molecules have been implicated as being important in this adaptive response, including various Smad proteins (1) and endothelial Per-ARNT-Sim
(PAS) domain protein-1/hypoxia-inducible factor-2
(EPAS-1/HIF-2) (34). The heterodimeric
protein HIF-1, however, is the most thoroughly studied
hypoxia-responsive transcription factor (40). HIF-1, a
member of the basic helix-loop-helix/PAS (PAS homology domain) protein family, binds to a core DNA sequence within hypoxia response elements. HIF-1 consists of HIF-1 and the arylhydrocarbon nuclear translocator (ARNT) (49). ARNT is constitutively
expressed, whereas HIF-1
expression is increased under hypoxic
conditions. HIF-1
stability is regulated posttranscriptionally by
the ubiquitin-proteasome system (22, 36). Under normoxic
conditions, HIF-1
binds to the von Hippel-Lindau tumor suppressor
protein (pVHL) and is targeted for destruction by an E3 ubiquitin
ligase complex (18, 20, 30). When oxygen partial pressure
is low, HIF-1
does not bind pVHL and therefore is not degraded. As a
result, HIF-1
accumulates and forms heterodimers with ARNT that
translocate to the nucleus and upregulate transcription of
HIF-1-responsive genes.
Although signaling through the HIF-1 pathway has been studied
primarily in the context of cellular adaptation to hypoxia, accumulating data suggest that proinflammatory mediators also can
upregulate HIF-1 protein expression and HIF-1 DNA-binding activity.
Cytokines, such as IL-1
and TNF and/or IFN-
, have been shown to
increase HIF-1 DNA-binding activity in human hepatoma cells
(15), human renal tubular epithelial cells
(12), and transformed human intestinal epithelial cells
(7). In addition, several in vitro studies have
demonstrated that another proinflammatory mediator, nitric oxide
(NO·), can promote HIF-1 activation under normoxic conditions
(25, 37).
When normoxic cells are exposed to an inflammatory milieu in vivo or in
vitro, they often manifest phenotypic changes reminiscent of those that
are observed when cells are subjected to hypoxia. For example, Bagby et
al. (4) reported several years ago that the rate of
glycolysis is increased in muscle tissue harvested from endotoxemic
rats. More recently, L'Her and Sebert (26) showed that
the rate of anaerobic glycolysis is significantly increased and the
rate of mitochondrial respiration is significantly decreased in
normoxic myocytes obtained from septic rats compared with sham-operated
controls. Our laboratory recently reported that the cellular
respiration rate is decreased, and the lactate production rate is
increased when cultured normoxic Caco-2 cells are studied after being
incubated for 24 h with cytomix, a mixture of the proinflammatory
cytokines TNF, IL-1, and IFN-
(23). Along similar
lines, Benigni et al. (6) reported that both TNF and
macrophage inhibitory factor promote lactate production by cultured rat
myoblasts, and Albina and co-workers (2, 29) showed that
the rate of glucose flux through the glycolytic pathway is markedly
increased in immunostimulated macrophages.
Because the HIF-1 pathway can be activated by proinflammatory
mediators, we were prompted to hypothesize that HIF-1-mediated signaling is important for the development of a hypoxic phenotype when
normoxic cells are exposed to an inflammatory environment. Remarkably
little is known, however, about changes in the expression of key
glycolytic enzymes in normoxic or hypoxic cells exposed to
proinflammatory cytokines. Accordingly, in the present study, we
investigated the effects of cytomix on the expression of the HIF-1-dependent glycolytic enzymes enolase-1 and aldolase A, as well as
the HIF-1-dependent cytokine vascular endothelial growth factor (VEGF),
under normoxic and hypoxic conditions in nontransformed small
intestine-derived epithelial cells (IEC-6) from rats. We chose a
nontransformed cell line, because constitutive upregulation of HIF-1
has been reported in different malignant tissues, including colon
(45, 52).
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MATERIALS AND METHODS |
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Cell culture and reagents. All reagents were from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. Cell culture media and reagents were from BioWhittaker (Walkersville, MD). The IEC-6 cell line (CRL-1592), a nontransformed small intestine epithelium cell line from rats, was obtained from the American Type Culture Collection (Manassas, VA). Cells (passage 17-52) were grown and maintained as confluent monolayers on Biocoat collagen I-coated plates (Becton Dickinson, Franklin Lakes, NJ) in DMEM supplemented with 10% fetal bovine serum (Life Technologies, Gaithersburg, MD), 45 U/ml penicillin, 9 µg/ml streptomycin, and 2 mM L-glutamine. Cells were fed biweekly and kept in a humidified atmosphere of 5% CO2 in air at 37°C.
Experimental conditions.
Cell culture media were changed immediately before the beginning of an
experiment and, in the case of all of the 48-h experiments, 24 h
later. Cells were left untreated or exposed to a combination of
proinflammatory cytokines (1,000 U/ml IFN-, 10 ng/ml TNF, and 1.0 ng/ml IL-1
; Endogen, Woburn, MA) called cytomix. After 24 h,
the culture medium was changed, and fresh cytomix was added. Some cells
were maintained under 21% O2 (normoxia + cytomix;
NC + NC); others were exposed to hypoxia (H; 1% O2)
for an additional 24 h (NC + HC). Some cells were exposed to
cytomix only during the second 24 h and kept under 21%
O2 (NC) or 1% O2 (HC). For hypoxic
stimulation, dishes were placed in a humidified incubator with an
atmosphere composed of 1% O2, 5% CO2, and
94% N2 for 24 h. The oxygen level was maintained with
a PROOX 100 oxygen-regulating system (Reming Instruments, Redfield,
NY), which purges the chamber with N2 gas and maintains the
PO2 at the set level. Cells not exposed to
cytomix were maintained under 21% O2 (N) or 1%
O2 (H).
RNA isolation.
RNA was isolated from fresh cultures by using TriReagent (Molecular
Research Center, Cincinnati, OH) according to the manufacturer's instructions. RNA was resuspended in 50 µl of RNAsecure (Ambion, Austin, TX), incubated at 60°C for 10 min, and purified with RNeasy columns (Qiagen, Valencia, CA) according to the manufacturer's protocol. To remove potential contamination with genomic DNA, the RNA
was treated with DNAse (DNA-free, Ambion, Austin, TX) according to the
manufacturer's protocol. The RNA concentration was determined by
measuring the optical density (OD260) with a spectrophotometer. OD260/OD280 ratios of >1.8
were obtained for all samples, indicating high purity. RNA was diluted
with nuclease-free water to 40 ng/µl and stored at 80°C. All
solutions were made by using diethyl pyrocarbonate (DEPC)-treated water.
Reverse transcription. Reverse transcription (RT) was carried out in a 100-µl reaction volume containing 1× PCR buffer II (PE Biosystems, Foster City, CA), 125 units of Moloney murine leukemia virus RT (SuperScript II, Life Technologies), 40 units of RNase inhibitor (PE Biosystems), 7.5 mM MgCl2, 1.0 mM each 2-deoxynucleotide 5'-triphosphate (dNTP; Roche Molecular Biochemicals, Indianapolis, IN), 2.5 µM random hexamers (PE Biosystems), and 50-200 ng of total RNA. RT was performed by incubating the samples at 25°C for 10 min, 48°C for 40 min, and 95°C for 5 min in a PerkinElmer GeneAmp PCR System 9700. "No RT" controls were carried out in all cases by using the same RT reaction mixture but substituting DEPC-H2O for the RT. For all quantitative analyses, two RT reactions were carried out for each RNA sample by using 200 and 50 ng/100 µl RT reaction mixture. The "no RT" controls were carried out with 200 ng of RNA.
Real-time quantitative PCR.
Quantitative real-time PCR is based on the detection of a fluorescent
signal that increases linearly with accumulating amplification product
during the PCR reaction. A dual-labeled fluorogenic oligonucleotide probe (TaqMan probe) consists of a short 20-25 base
oligodeoxynucleotide, which anneals to the target sequence between the
forward and reverse primers. The probe is labeled with two different
fluorescent dyes, a reporter dye (6-carboxyfluorescein) and a quencher
dye (6-carboxytetramethylrhodamine), which suppresses the reporter
fluorescence activity by energy transfer within the intact probe.
During the extension phase of PCR, the probe is cleaved by the
endogenous 5'-nuclease activity of AmpliTaq Gold polymerase,
which cleaves the reporter chromophor from the TaqMan probe, leading to
an increase in the intensity of reporter fluorescence. The increase in
fluorescence (Rn) signal is continuously measured and plotted vs.
PCR cycle number, reflecting the amount of PCR amplification products.
The ABI Prism 7700 detection system software calculates the
Rn by
subtracting the fluorescence signal of the baseline emission during
cycles 3 to 6 from the fluorescence signal of the product at any given
time. A threshold was set at the early log phase of product
accumulation. The threshold cycle number value (CT) is the
cycle number at which each sample's amplification plot reaches this threshold.
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Calculation of relative expression.
Relative mRNA expression of target genes was calculated with the
comparative CT method. The amount of target gene was
normalized to the endogenous 18S control gene to control for quantity
of RNA input. Difference in CT values was calculated for
each mRNA by taking the mean CT of duplicate reactions and
subtracting the mean CT of duplicate reactions for
reference RNA measured on an aliquot from the same RT reaction:
CT = CT(target gene)
CT (18S). To compare
gene expression from cells exposed to different conditions to control
cells, the
CT for the samples taken from cells exposed
to the conditions of interest was subtracted from the
CT
for the control cells:
CT =
CT
(test conditions)
CT (controls). Because all RNA
samples were reverse transcribed with two different RNA concentrations,
two
CT (50 and 200 ng) were calculated for each gene
on each RNA sample. By using the mean of the two
CT,
we calculated the fold change in expression of the target gene from the
test conditions relative to control cells: relative expression = 2
CT. This calculation method requires that the
efficiency of the target amplification and the reference amplification
are approximately equal. All PCR efficiencies were measured and found
to be >90%; thus this equation introduces, at most, only a very small error.
Assessment of HIF-1 mRNA expression by using semiquantitative
RT-PCR.
Total RNA was treated with DNAfree (Ambion, Houston, TX) as instructed
by the manufacturer by using 10 units of DNase I/10 µg RNA. The RNA
was reverse transcribed in a 20 µl reaction volume containing 0.5 µg of oligo(dT)15 (Promega, Madison, WI), 1 mM of each
dNTP (Promega), 15 units avian myeloblastosis virus RT (Promega), and 1 U/µl of recombinant RNasin ribonuclease inhibitor (Promega) in 5 mM MgCl2, 10 mM
Tris · HCl (pH 8.0), 50 mM KCl, and 0.1% Triton
X-100. The mixture was incubated at 42°C for 25 min and then heated
to 99°C for 5 min to terminate the reaction. A 20-µl PCR reaction
mixture was assembled by using 2 µl of cDNA template, 10 units
AdvanTaq Plus DNA polymerase (Clontech, Palo Alto, CA), 1.5 mM MgCl2, and 1.0 µM of each primer in 1×
AdvanTaq Plus PCR buffer. The primer pairs for 18S ribosomal
RNA were as follows: upper primer, 5'-CCC GGG GAG GTA GTG ACG AAA
AAT-3'; and lower primer, 5'-CGC CCG CTC CCA AGA TCC AAC TAC-3'. The
HIF-1 primers were as follows: upper primer, 5'-TAC TGG GGT TCA TGA TGA
TTA TTG TGG-3'; and lower primer 5'-ACT TCA GGA ACC GGC GTG GAT TTA-3'.
PCR was performed by heating the samples at 94°C for 2 min, followed
by 94°C for 45 s, 64°C for 45 s, and 68°C for 90 s
for 24 (18S) or 30 (HIF-1) cycles, and then 68°C for 5 min. Fifteen
microliters of each PCR reaction product was electrophoresed on a 2%
agarose gel.
HIF-1 immunoprecipitation and Western blotting. IEC-6 cells were grown in 10-cm dishes then lysed in 1 ml of RIPA buffer (1× PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mg/ml phenylmethylsulfonyl fluoride, 1.0 mM sodium orthovanadate, and 1× mammalian protease inhibitor cocktail). The lysate was transferred to a 1.5-ml Microfuge tube and sonicated for 30 s on ice by using a 0.1-W Fisher Scientific sonic dismembrator fitted with a microtip on power setting 3. The lysate was incubated for 10 min on ice followed by centrifugation at 10,000 g for 15 min at 4°C. The supernatant was transferred to a new tube, and the total protein concentration was determined by using the Bio-Rad (Hercules, CA) protein reagent.
Whole protein lysate (100 µg) was mixed with 0.25 µg of nonimmune murine IgG and 20 µl of protein A/G agarose (resuspended volume; Santa Cruz Biotech, Santa Cruz, CA) and incubated at 4°C for 2 h. The beads were removed by centrifugation at 1,000 g for 5 min at 4°C, and the supernatant was combined with 26 µg anti-mouse HIF-1 monoclonal antibody (NOVUS Biologicals, Littleton, CO) and incubated at 4°C for 2 h. A 20-µl aliquot of suspended agarose A/G beads was added, and the tubes were incubated with rocking overnight at 4°C. The beads were washed five times with 1 ml of RIPA buffer. The final pellet was dissolved by boiling for 10 min in Laemlli buffer, consisting of the following reagents at the indicated final concentrations: 20% glycerol, 10%Measurement of nitrate/nitrite and other biochemical parameters.
Supernatants from cultures of IEC-6 cells were stored at 20°C.
Nitrate/nitrite (NOx) measurements were performed by using a colorimetric assay (Bioxytech NO assay; Oxis International, Portland,
OR) according to the manufacturer's instructions. Briefly, nitrate was
reduced to nitrite by enzymatic reduction with nitrate reductase before
quantification of nitrite by using Griess reagent. Glucose and lactate
concentrations in culture supernatants were measured by using an
auto-analyzer (model ABL-725; Radiometer, Copenhagen, Denmark). Glucose
consumption was determined by subtracting the final glucose
concentration from the concentration of glucose at the beginning of the
incubation period and dividing by 24 h. Lactate production was
determined by subtracting the starting lactate concentration from the
concentration of lactate at the end of the incubation period and
dividing by 24 h.
Nuclear extract preparation.
The day before each experiment, cells were plated at a density of
0.8-1.0 × 106 cells per well in six-well Biocoat
tissue culture plates (Becton Dickinson). Nuclear extracts were
prepared by using the following modification of a previously published
method (11, 43). All steps were performed on ice, and
centrifugation steps were performed in an Eppendorf microcentrifuge
(model 5417R; Brinkmann, Pittsburgh, PA) at 4°C. Cells were removed
from the incubator and immediately placed on ice, washed once with 1 ml
of PBS, and then harvested in 1 ml of PBS by using a rubber policeman.
The cells were transferred to a 1.5-ml tube and centrifuged at 14,000 g for 10 s. The cell pellet was resuspended in 400 µl
of buffer I [(in mM) 10 Tris · HCl
(pH 7.8), 10 KCl, 1.5 MgCl2, 1.0 sodium orthovanadate, 1.0 dithiothreitol, plus 0.3 M sucrose, 500 µM phenylmethylsulfonyl fluoride, and 1× mammalian protease inhibitor cocktail (catalog number P-8340; Sigma-Aldrich)] and incubated for 15 min. We then added
(octylphenoxy)polyethoxyethyl (IGEPAL CA-620; NP-40) to 0.5%
(25 µl of a 10% vol/vol stock) while the tube was vortexed at half
speed for 10 s. Nuclei were isolated by centrifugation at 500 g for 2 min. The supernatant was aspirated, and the nuclear pellet was gently resuspended in 60 µl of buffer II [10
mM Tris · HCl (pH 7.8), 420 mM KCl, 1.5 mM
MgCl2, 20% glycerol]. After a 15-min incubation period,
the nuclear extracts were cleared by centrifugation at 14,000 g for 10 min. The supernatant was transferred to a new tube,
and the protein concentration was determined by using a commercially
available Bradford assay (Bio-Rad protein assay, Hercules, CA). Nuclear
extracts were frozen and stored at 70°C.
EMSA.
Oligonucleotides were synthesized by Life Technologies. The sequence of
the double-stranded oligonucleotide from the human erythropoeitin 3'
enhancer containing the HIF-1 binding sequence was as follows: sense
5'-AGC TTG CCC TAC GTG CTG TCT CAG-3' and
antisense 5'-AAT TCT GAG ACA GCA CGT AGG GCA-3'
(49). Boldface nucleotides indicate the consensus HIF-1
binding site. The double-stranded wild-type oligonucleotide was
end-labeled with -[32P]ATP (New England Nuclear,
Boston, MA) by using T4 polynucleotide kinase (Promega, Madison, WI).
Nuclear proteins (~5 µg/reaction) were incubated with radiolabeled
probe in bandshift buffer [(in mM) 10 Tris · HCl
(pH 7.5), 50 KCl, 50 NaCl, 1.0 MgCl2, 1.0 EDTA, and 5%
glycerol] in the presence of 0.5 ng of calf thymus DNA. For
competition reactions, a 3- to 100-fold molar excess of unlabeled wild-type HIF-1 or NF-
B (Promega) specific oligonucleotide was added
simultaneously with the labeled HIF-1 oligonucleotide probe. Supershift
assays were performed by incubating nuclear extracts with 2 µl of
anti-HIF-1
monoclonal antibody (Novus Biologicals, Littleton, CO)
for 20 min before the addition of the radiolabeled oligonucleotide. An
equivalent amount of mouse anti-human CD25 monoclonal antibody (Caltag,
Burlingame, CA) was used as an isotype control. Reactions were
size-fractionated on 4% nondenaturing polyacrylimide gels at 120 V in
0.25× Tris-borate-EDTA buffer for 1 h at room temperature. The
gels were dried and used to expose Biomax film (Kodak, Rochester, NY)
at
70°C overnight by using an intensifying screen. Figures shown
are representative of experiments repeated on at least five separate occasions.
Data analysis. In one experiment, VEGF mRNA expression was sevenfold greater than the mean value for this parameter measured under the same conditions in numerous other experiments. Accordingly, results from this single experiment were not included in the analyses of the results. Data are expressed as means ± SE. The quantitative real-time RT-PCR assays were carried out on multiple days by using samples collected over a period of many months from multiple cell cultures growing on multiwell plates. In each individual experiment, we always included control wells (i.e., cells grown under normoxia in the absence of cytokines) among the wells used to study the experimental conditions. Accordingly, we used paired-sample t-tests to evaluate contrasts between the various experimental conditions and the control condition. The analyses among the other experimental conditions were carried out by using ANOVA followed by Fisher's protected least-squares difference test. Because the variances among the data for the various experimental groups tended to be proportional to the means, the results were logarithmically transformed before carrying out the statistical analyses to detect differences among groups. Glucose consumption and lactate production rate data were analyzed by using ANOVA followed by Dunnett's test. NOx concentrations in culture supernatants were logarithmically transformed, resulting in a normal distribution of variances, before ANOVA followed by Dunnett's test. The null hypothesis was rejected for P < 0.05.
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RESULTS |
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Glucose consumption and lactate production by cytomix-stimulated
IEC-6 cells.
IEC-6 monolayers were incubated under the following conditions:
normoxia for 24 h in control medium (N); normoxia in the presence of cytomix for 24 h (NC); normoxia with cytomix for 24 h
followed by changing the media and adding fresh cytomix and incubating for an additional 24 h under normoxia (NC + NC); hypoxia (1%
O2) for 24 h (H); hypoxia with cytomix for 24 h
(HC), or normoxia for 24 h in the presence of cytomix followed by
changing the media and then hypoxia for 24 h in the presence of
fresh cytomix (NC + HC). As shown in Fig.
1, glucose consumption more than doubled when IEC-6 cells were stimulated with fresh cytomix-containing medium
twice during successive 24-h periods of incubation (i.e., the NC + NC condition). Glucose consumption also increased when the cells were
incubated under hypoxic conditions, irrespective of whether the cells
were also exposed to cytomix. Although glucose consumption increased
substantially when cells were stimulated with cytomix or incubated
under 1% O2, the glucose concentration in samples of media
at the end of the final incubation period was never <6.5 ± 0.8 mM.
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NO· production by cytomix-stimulated IEC-6 cells.
Our laboratory previously reported that incubation with cytomix
increases inducible NO synthase (iNOS) expression and NO· production
by cultured Caco-2 (transformed human enterocytic) cells
(9). Potoka et al. (35) reported that cytomix
increases iNOS expression and NO· production by cultured
(nontransformed) IEC-6 cells. To confirm and extend these findings,
IEC-6 monolayers were incubated under the following conditions:
normoxia for 24 h in control medium (N), normoxia in the presence
of cytomix for 24 h (NC), normoxia with cytomix for 24 h
followed by changing the media and adding fresh cytomix and incubating
for an additional 24 h under normoxia (NC + NC), hypoxia (1%
O2) for 24 h (H), hypoxia for 24 h in the
presence of cytomix (HC), or normoxia for 24 h in the presence of
cytomix followed by changing the media and then hypoxia for 24 h
in the presence of fresh cytomix (NC + HC). As shown in Fig.
2, the concentration of
NO
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Effect of proinflammatory cytokines on HIF-1 DNA-binding activity
under normoxic and hypoxic conditions.
We used EMSA to assess DNA-binding by HIF-1. The identity of the
HIF-1/DNA complex was confirmed by performing competition studies by
using specific (HIF-1) and nonspecific (NF-B) unlabeled oligonucleotide probes. As expected, HIF-1 DNA binding was not detected
when cells were cultured in normal medium under 21% O2 (N
in Fig. 3). Incubation under 1%
O2 for 24 h (H) induced the formation of an HIF-1 DNA
complex. HIF-1 DNA binding also was induced by incubation with cytomix
for 24 h under normoxic conditions (NC) or by incubating with
cytomix under normoxic conditions for 24 h followed by changing
the media and adding fresh cytomix and incubating for 24 h more
under normoxic conditions (NC + NC). Furthermore, cytomix exposure
did not inhibit formation of an HIF-1 DNA complex in response to
hypoxia. HIF-1 DNA binding was most evident when IEC-6 cells were
pretreated with cytomix for 24 h under 21% O2 and
then after changing the culture media reexposed to cytomix for a
further 24 h under 1% O2 (NC + HC).
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Effect of proinflammatory cytokines on HIF-1 gene expression
under normoxic and hypoxic conditions.
As expected, HIF-1
protein expression increased in IEC-6 cells after
incubation under 1% O2 for 24 h (H) (Fig.
4A). HIF-1
protein levels
also were increased after incubation with cytomix for 48 h under
normoxic conditions (NC + NC) or under normoxic conditions for
24 h followed by hypoxic conditions for 24 h (NC + HC).
In contrast to these observed changes in HIF-1
protein expression,
steady-state levels of HIF-1
mRNA were not appreciably different for
cells incubated under these four conditions (Fig. 4B).
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Effect of cytomix on expression of aldolase A, enolase-1, and VEGF
mRNA.
When IEC-6 cells were incubated with cytomix for 24 h under 21%
O2 (NC in Fig. 5),
steady-state mRNA levels for three HIF-1-responsive genes, aldolase A,
enolase-1, and VEGF, all increased significantly relative to the values
measured in normoxic control cells (N). When cells were stimulated with
cytomix for 24 h under 21% O2 and then incubated for
24 h more with fresh medium containing cytomix under the same
concentration of O2 (NC + NC), there was further
upregulation of the steady-state levels of the mRNAs encoding all three
proteins relative to cells incubated with cytomix under normoxic
conditions for just 24 h (P = 0.026 vs. NC for
aldolase A, P = 0.024 vs. NC for enolase-1,
P = 0.11 vs. NC for VEGF).
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Effect of inhibiting NO· production with L-NIL on the
expression of aldolase A, enolase-1, and VEGF mRNA in
cytomix-stimulated IEC-6 cells.
Because NO· can promote HIF-1 activation under normoxic conditions
(25, 37), and cytomix can induce iNOS expression and NO·
production in IEC-6 cells (see Fig. 2), we sought to determine whether increased NO· production was responsible for cytomix-induced upregulation of aldolase A, enolase-1, and VEGF gene expression in this
cell line. Accordingly, some monolayers were exposed to cytomix for
24 h under normoxic conditions, and after being changed, the media
were reexposed to cytomix for another 24 h (NC + NC). Other
monolayers were treated identically, except during the second 24-h
incubation period with cytomix the cells were also treated with 20 µM
L-NIL. Although treatment with L-NIL markedly
decreased cytomix-induced NO· production (Fig. 2), treatment with
this agent had a relatively small and inconsistent effect on the
upregulation of steady-state aldolase A, enolase-1, and VEGF mRNA
levels after exposure to the cytokine cocktail (Fig.
6). Probability values for the contrasts
between the NC + NC condition and the NCNC + L-NIL condition ranged from 0.12 (aldolase A) to 0.63 (enolase-1).
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Effect of conditioned media from cytomix-stimulated cells on
expression of aldolase A, enolase-1, and VEGF mRNA.
Results presented in Fig. 5 indicate that cytomix increased the
expression of several HIF-1-responsive genes in normoxic IEC-6 cells.
Moreover, these results suggest that this effect was more pronounced
after 48 h of exposure to cytomix (NC + NC) than after 24 h of exposure to the cocktail of proinflammatory cytokines (NC). Although one explanation for the difference between the results
obtained for the NC + NC condition and for the NC condition might
simply be related to the kinetics of the cytokine-induced response in
IEC-6 cells; another possible explanation is that cytomix-stimulated
cells release one or more factors into the medium that act in an
autocrine and/or paracrine fashion to promote upregulation of
HIF-1-responsive genes. To test this hypothesis, conditioned media were
obtained from IEC-6 cells incubated under the NC + NC condition;
that is, the cells were exposed to cytomix for 24 h under 21%
O2, and the cells were then incubated under normoxia for a
second 24-h period in fresh medium containing cytomix. The conditioned
medium was harvested after the second 24-h incubation period. When
naive IEC-6 cells were incubated with the conditioned medium for
24 h under 21% O2 (CM in Fig. 5), they expressed
significantly higher steady-state aldolase A, enolase-1, and VEGF mRNA
levels than did normoxic control cells (N in Fig. 5). Of course,
conditioned medium contained residual cytomix. Accordingly, it is
important to note that steady-state aldolase A, enolase-1, and VEGF
mRNA levels were significantly greater in cells exposed to conditioned medium than in cells incubated with fresh cytomix for the same duration
(24 h) (NC). This latter observation supports the view that conditioned
medium contained a factor or factors in addition to those comprising
cytomix (i.e., IL-1, TNF, and IFN-
) that promoted increased
expression under normoxia of several HIF-1-responsive genes.
Effect of combining hypoxia plus cytomix stimulation on aldolase A,
enolase-1, and VEGF mRNA expression.
Steady-state levels of aldolase A, enolase-1, and VEGF mRNA increased
significantly when IEC-6 cells were incubated under hypoxic conditions
(H vs. N in Fig. 7). When the hypoxic cells were also stimulated with
cytomix, whether just during the period of incubation under 1%
O2 (HC) or before and during the period of incubation under
1% O2 (NC + HC), there was no change in the expression of aldolase A and enolase-1 mRNA relative to that observed for cells challenged with hypoxia alone (Fig. 7, A and
B). In contrast, VEGF mRNA expression, which was increased
by hypoxia alone, was increased still further by prior and concomitant
exposure of cells to cytomix (Fig.
7C).
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DISCUSSION |
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HIF-1, as suggested by its name, was originally identified as a
transactivating factor that regulates the transcription of a number of
genes in response to diminished ambient PO2
(50). Under hypoxic conditions, active HIF-1 heterodimers
accumulate in the nucleus where they bind to cis-acting
elements in the regulatory regions of HIF-1-responsive genes, enhancing
the rate of transcription of these genes. Although the importance of
HIF-1-mediated signaling in the adaptive response of cells to hypoxia
is firmly established, it is becoming increasingly apparent that
certain conditions can lead to HIF-1 DNA binding (12, 14, 15, 39,
46) and, in some cases, expression of HIF-1-responsive genes
(5, 12, 13, 21, 27), even in the absence of cellular
hypoxia. For example, a number of studies have documented HIF-1 DNA
binding in various cell types after incubation with certain
proinflammatory cytokines (notably, TNF and IL-1) (12, 14, 15,
39, 46). Fewer studies, however, have demonstrated increased
expression of hypoxia-responsive genes. Thus El Awad et al.
(12) showed that incubating human renal epithelial cells
with IL-1
produces a small (~50%) but significant increase in
VEGF protein expression. However, these investigators found that VEGF
mRNA expression did not change significantly after exposure of renal
epithelial cells to IL-1
. Other investigators have shown that
IL-1
increases VEGF expression in smooth muscle cells and
fibroblasts (5, 21, 27), TNF increases VEGF expression in
keratinocytes (13), and IL-6 and IL-1
induce VEGF
expression in A-431 epidermoid carcinoma cells (10). In
contrast to these findings, Hellwig-Burgel et al. (15)
reported that incubating HepG2 (human hepatoblastoma) cells with either
IL-1
or TNF decreased production of erythropoietin and had no effect
on the production of VEGF protein or the steady-state level of VEGF
mRNA. Expression of genes encoding both of these proteins is regulated
by HIF-1.
Aldolase A and enolase-1 are important glycolytic enzymes. Aldolase
catalyzes the conversion of fructose-1,6-bisphosphate into
dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. Enolase
catalyzes the penultimate step in glycolysis, which is the dehydration
of 2-phosphoglycerate to form phosphoenolpyruvate. The promoter regions
for the genes that encode these enzymes include hypoxia-responsive
elements that contain functionally active binding sites for HIF-1
(41, 42). Furthermore, these genes are induced by hypoxia
in HIF-1+/+ embryonic stem cells but not in
HIF-1
/
embryonic stem cells (19).
In our study, we observed that incubation of IEC-6 cells under hypoxic
conditions increased cellular levels of HIF-1 protein and HIF-1 DNA
binding. These changes were not accompanied by increased expression of
HIF-1
mRNA, a finding entirely consistent with the accepted notion
that posttranslational events are largely responsible for the
regulation of HIF-1-dependent signaling in response to changes in
oxygen availability (22, 36). When normoxic IEC-6 cells
were incubated with cytomix, a cocktail of cytokines containing TNF,
IL-1
, and IFN-
, we again observed increased cellular levels of
HIF-1
protein and HIF-1 DNA binding, and, as was true for hypoxia,
these changes were not accompanied by increased steady-state levels of
HIF-1
mRNA. Thus as in the case of the HIF-1 response to hypoxia,
the increase in HIF-1 DNA binding induced by cytomix appears to occur
as a result of posttranslational events. When normoxic cells were
incubated with cytomix, we also observed increased steady-state mRNA
levels for three HIF-1-responsive genes: aldolase A, enolase-1, and
VEGF. Therefore, our findings are consistent with observations made by
other investigators, who examined the effects of proinflammatory
cytokines on HIF-1 DNA binding and/or transcriptional activation in
other cell types (12, 14, 15, 39, 46). However, our
observations are also novel, because, to our knowledge, we are the
first to document that proinflammatory cytokines induce transcriptional
activation of the glycolytic HIF-1-responsive genes, aldolase A and
enolase-1. These observations are important for two reasons. First, our
findings may account, at least in part, for the development of a
hypoxic phenotype, when normoxic cells or tissues are exposed to an
inflammatory milieu (2, 6, 23, 26, 29). Second, unlike
many previous studies that focused on changes in expression of VEGF, a
gene that is also regulated by transcription factors other than HIF-1, we studied three different HIF-1-regulated genes. Because the expression of all three genes moved in parallel in all of our studies,
we believe that is likely that the cytomix-induced alterations in the
expression of these genes was mediated through activation of the HIF-1
signaling pathway.
Our data are insufficient to exclude the possibility that although
exposure to cytomix promoted HIF-1 DNA binding and increased expression
of three HIF-1-responsive genes, these cellular events were not
causally related. In other words, it is possible that factors other
than (or in addition to) HIF-1-dependent transcriptional activation may
be important in causing upregulation of aldolase A, enolase-1, and VEGF
expression in cytomix-stimulated IEC-6 cells. An elegant way to
directly test this hypothesis would be to deplete HIF-1 by using an
antisense approach as described recently by Caniggia et al.
(8). We attempted to carry out similar studies. We
prepared the same phosphothioate oligonucleotide described by Caniggia
et al. (8), and successfully introduced it into IEC-6
cells. Unfortunately, we did not observe decreased expression of HIF-1
as revealed by EMSA. Thus we were unable to unambiguously confirm that
increased expression of aldolase A, enolase-1, and VEGF in
cytomix-stimulated IEC-6 cells is mediated via signaling through the
HIF-1 pathway.
Exposure of normoxic cells to compounds that release NO· into
solution, such as
S-nitroso-N-acetyl-penicillamine, NOC5, and S-nitrosoglutathione, has been shown to promote HIF-1 DNA
binding (24, 33, 38, 39) and enhanced transcription of
HIF-1 responsive genes (24). Furthermore, cytomix was
previously shown to induce iNOS expression and NO· biosynthesis by
cultured enterocytes (9), an observation confirmed in the
present series of experiments. Accordingly, we hypothesized that
increased production of NO· is an intermediate step in the process
leading to induction of aldolase A, enolase-1, and VEGF expression when
IEC-6 cells are incubated with cytomix. To test this hypothesis, we
incubated the cells with cytomix in the absence or presence of
L-NIL, a potent isoform selective iNOS inhibitor
(32). Although we confirmed that L-NIL blocked
NO· production by cytomix-stimulated IEC-6 cells, we also showed that
this agent had minimal effects on the induction of aldolase A,
enolase-1, and VEGF mRNA expression after incubation with cytomix.
These findings, which are consistent with recently reported
observations from another laboratory (3), suggest that one
or more NO·-independent pathways are responsible for induction of
these genes when normoxic IEC-6 cells are exposed to cytomix. One such
pathway is suggested by findings obtained by Haddad and Land
(14), who showed that the oxidant species, hydrogen
peroxide and hydroxyl radical, are involved in TNF-dependent activation
of HIF-1 in normoxic cells. Another potential pathway is suggested by
findings reported by Sandau et al. (39). These investigators showed that wortmannin blocked HIF-1 accumulation in
TNF-stimulated normoxic LLC-PK1 (transformed renal) epithelial cells, suggesting that the phosphatidylinositol-3-kinase (PI3-kinase) pathway is involved in this phenomenon (38). This view is
strengthened by studies showing that TNF-induced HIF-1
accumulation
is blocked when cells were transfected with a plasmid encoding a
dominant negative form of the p85 regulatory subunit of PI3-kinase
(39).
The effect of NO· on HIF-1 DNA binding and HIF-1-mediated
transcriptional activation also has been studied in a hypoxic milieu. Various NO· donors have been shown to inhibit HIF-1 DNA-binding activity in response to hypoxia or incubation with CoCl2
(17, 37, 44), the latter condition being an
hypoxia-independent perturbation that stabilizes HIF-1 and promotes
HIF-1 binding to the hypoxia-responsive elements in target genes.
Furthermore, in some studies, incubating hypoxic cells with an NO·
donor was shown to attenuate induction of either a reporter construct
(44, 51) or an HIF-responsive reporter gene (17,
28) in response to hypoxia. Because cytomix-stimulated cells
produce NO·, we hypothesized that incubation of hypoxic IEC-6 cells
with cytomix would blunt the DNA binding of HIF-1 and the upregulation
of several hypoxia-responsive genes normally observed when the cells
are incubated under an atmosphere containing 1% O2.
Contrary to our expectations, however, we observed no diminution in
HIF-1 DNA binding when cells were incubated with cytomix for 24 h
under normoxic conditions and were then incubated for 24 h more
with cytomix under hypoxic conditions. Indeed, if there was any change
at all, it was in the direction of a further increase in HIF-1 DNA
binding relative to that observed with either hypoxia alone or cytomix
alone. One possible explanation for our observations is suggested by
the data shown in Fig. 2. Hypoxia appeared to decrease the production
of NO· by immunostimulated IEC-6 cells. This finding, which is
consistent with previously reported observations from studies that used
other cell types (16, 31), suggests the possibility that
the amount of NO· produced during hypoxia was simply inadequate to
overcome the effects of hypoxia and/or cytomix exposure that promotes
modulation of gene expression through the HIF-1 pathway. Although this
idea is plausible, previously published data regarding the
concentration of NO· that is necessary to inhibit HIF-1 activity in
response to hypoxia are inconsistent; in some studies
(25), inhibition of HIF-1 activity was detected only when
high concentrations of NO· donors were employed, but in other studies
(37), the same effect was observed only at low
concentrations of NO· donors. In any event, it is important to point
out that previous studies of the effect of NO· on hypoxia-induced
HIF-1-dependent gene expression were carried out by using exogenous
sources of NO·. To our knowledge, our study is the first to
investigate the effect of endogenously produced NO· on
HIF-1-dependent gene expression in hypoxia. The effect of endogenously
derived NO· on HIF-1 activity in hypoxia might differ from that
produced by exogenous NO·, possibly because of subtle differences in
the actual molecular species involved (e.g., nitroxyl equivalents vs.
nitrosonium equivalents) (33).
Cytomix treatment for 24 h under normoxic conditions resulted in a
statistically significant, but still relatively minor, increase of
HIF-1 target gene expression. However, when cells were treated with two
24-h applications of cytomix, aldolase A and enolase-1 mRNA levels were
significantly higher than when the cells were exposed to cytomix for
only 24 h. A similar effect was also observed for VEGF mRNA
expression, although in this instance the difference between a single
24-h treatment and two 24-h-long treatments with cytomix did not quite
achieve statistical significance (P = 0.11). As noted
above, Hellwig-Burgel et al. (15) found that incubating
HepG2 cells with either IL-1 or TNF had no effect on steady-state
levels of VEGF mRNA. In view of our findings showing that prolonged
exposure to cytomix is necessary to observed marked upregulation of
VEGF gene expression, the failure of Hellwig-Burgel et al.
(15) to observe this effect was probably due to the short period of incubation (4 h) used in their studies.
Although the greater degree of gene induction after prolonged incubation with cytomix might simply reflect the kinetics of this transcriptional activation pathway, our data suggest that another mechanism might also be important. Specifically, we found that conditioned medium obtained from cytomix-stimulated IEC-6 cells was a potent inducer of aldolase A, enolase-1, and VEGF mRNA expression in naive cells. Because the conditioned medium may have contained one or more of the cytokines present in cytomix, we carried out control experiments in which cells were exposed to cytomix for the same period of time (24 h). Conditioned medium was significantly more potent than cytomix in these assays, suggesting that immunostimulated IEC-6 cells release one or more factors that can initiate transcription of HIF-1-responsive genes. Further studies will be necessary to elucidate the identity of the factor(s) present in conditioned medium.
Two considerations prompted to us to use a combination of three
proinflammatory cytokines (TNF, IL-1, and IFN-
) rather than just
a single cytokine, such as TNF or IL-1
, for the studies presented
here. It is likely that multiple cytokines are involved when a systemic
inflammatory process, such as occurs in sepsis, or a more localized
form of inflammation, such as is the case in Crohn's disease or
ulcerative colitis, affects gut mucosal function (see, for example,
Ref. 47 for a review of intestinal epithelial
hyperpermeability associated with inflammatory conditions). Second, as
noted above, we sought to determine whether endogenous NO· production
contributes to cytokine-induced modulation of expression of the
glycolytic enzymes aldolase A and enolase-1. Although incubating immortalized enterocyte-like cells with IFN-
alone is sufficient to
induce iNOS expression and increased NO· production (9, 48), the effect is considerably more robust when the cells are exposed to IFN-
plus TNF and IL-1
(9).
The overarching rationale for the type of studies described herein was to take advantage of the relative simplicity of an in vitro system to model clinical situations in which the intestinal epithelium is exposed to an inflammatory milieu under normoxic or hypoxic conditions. In studies comparing the effects of hypoxia alone with hypoxia plus cytomix, we observed no differences whatsoever on the expression of aldolase A and enolase-1. In contrast, we observed that VEGF mRNA expression is increased in the HC condition relative to the H condition by ~50% (P = 0.15) and by ~100% in the NC + HC condition (P = 0.047). These findings support the view that the modulation of VEGF expression by the combination of hypoxia plus cytomix is different than the regulation of the other two genes. The data presented herein are insufficient to resolve the molecular mechanism(s) that form the basis for this difference.
In summary, we showed herein that proinflammatory cytokines induce the expression of several HIF-1-dependent genes in normoxic IEC-6 cells. Two of these genes, aldolase A and enolase-1, encode important enzymes in the glycolytic pathway. Thus our findings might explain, at least in part, why cells manifest an hypoxic phenotype when they are subjected to an inflammatory milieu.
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ACKNOWLEDGEMENTS |
---|
This study was supported by Deutsche Forschungsgemeinschaft Grant SCHA915/1-1 and National Institute of General Medical Sciences Grants GM-58484 and GM-37631.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. P. Fink, 616 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261 (E-mail: finkmp{at}ccm.upmc.edu).
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.
First published October 2, 2002;10.1152/ajpgi.00076.2002
Received 20 February 2002; accepted in final form 30 September 2002.
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