1 Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206; and 2 Departments of Medicine and Genetics, University of Washington, Seattle, Washington 98195
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ABSTRACT |
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During adaptation to hypoxic and hyperoxic conditions, the genes involved in glucose metabolism are upregulated. To probe involvement of the transcription factor hypoxia-induced factor-1 (HIF-1) in hexokinase (HK) II expression in human pulmonary cells, A549 cells and small-airway epithelial cells (SAECs) were exposed to stimuli such as hypoxia, deferoxamine (DFO), and metal ions. The largest increase in HK-II (20-fold for mRNA and 2.5-fold for enzymatic activity) was observed in A549 cells when exposed to DFO. All stimuli selectively increased the 5.5-kb rather than 4-kb transcript in A549 cells. Cycloheximide and actinomycin D inhibited these responses. In addition, cells were transfected with luciferase reporter constructs driven by the full-length HK-II 5'-regulatory region (4.0 kb) or various deletions of that region. A549 cells transfected with the 4.0-kb construct and exposed to hypoxia or DFO increased their luciferase activity 7- and 10-fold, respectively, indicating that HK-II induction is, at least in part, due to increased gene transcription. Sixty percent of the inducible activity of the 4.0-kb construct was shown to reside within the proximal 0.5 kb. Additionally, cotransfection with a stable HIF-1 mutant and the 4.0-kb promoter construct resulted in increased luciferase activity under normoxic conditions. These results strongly suggest that HK-II is selectively regulated in pulmonary cells by a HIF-1-dependent mechanism.
hypoxia-inducible factor; deferoxamine; glycolytic enzymes; metabolism; cobalt
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INTRODUCTION |
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HIGH CONCENTRATIONS OF OXYGEN are often used to treat respiratory failure. Unfortunately, such treatment can perpetuate or worsen lung disease. One causative agent of lung damage during exposure to high oxygen tensions may be the resultant decrease in respiratory metabolism because the tricarboxylic acid cycle enzymes are a major target of oxidative stress (9, 33, 38). One method of counteracting the loss of respiratory metabolism is to increase glucose metabolism. Hexokinase (HK) is the rate-limiting enzyme for glucose utilization in the lung (31). The transcription factor hypoxia-inducible factor (HIF)-1 has been found to regulate the expression of most glycolytic genes under hypoxic conditions; therefore, we proposed that HK-II could also be upregulated by hypoxia in lung cells.
The mechanisms for the modulation of gene expression in response to hypoxia are incompletely understood but are beginning to be elucidated. At the transcriptional level, three transcription factors, HIF-1, HIF-2 (also referred to as endothelial PAS domain protein-1), and HIF-3, have been identified (25). Both HIF-1 and endothelial PAS domain protein-1 have been demonstrated to bind the hypoxia response element (HRE) (25, 36) in response to hypoxic stimuli. Additionally, a unique pattern of response to various stimuli such as hypoxia, chelators, and transition metals has been associated with the HIF-1 response. Among the genes regulated by HIF-1 are those encoding glycolytic enzymes (13, 36).
There are three HK isoforms expressed in the lung, HK-I, HK-II, and HK-III. HK-I is the predominant isoform in the lung under normoxic conditions. Herein, we report that hypoxia increased the steady-state levels of HK-II mRNA in both primary small-airway epithelial cells (SAECs) and A549 cells but did not increase the steady-state levels of HK-I in either cell type. Other classic inducers of HIF-1, including deferoxamine (DFO) and transition metals (2, 6, 11, 15, 21), increased HK-II but not HK-I mRNA expression. We show that cycloheximide and actinomycin D can inhibit the effect of these substances, consistent with a HIF-1-dependent mechanism. In addition, using luciferase constructs, we found that this increase in mRNA occurs, at least in part, through transcriptional activation. Finally, the transcriptional activation could be stimulated with a HIF-1 overexpression vector, and a significant proportion of this response resided within the basal promoter.
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MATERIALS AND METHODS |
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Reagents and cell culture. All reagents were purchased from Sigma (St. Louis, MO), Fisher Scientific (Pittsburgh, PA), or GIBCO BRL (Life Technologies, Grand Island, NY) unless otherwise specified. Restriction enzymes were purchased from either Promega (Madison, WI) or GIBCO BRL (Life Technologies, Gaithersburg, MD). A549 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA) and were maintained in endotoxin-free F-12K medium (Kaighn's modification) supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 2 mM glutamine. SAECs were purchased from Clonetics (Walkersville, MD) and maintained in small-airway epithelial cell growth medium (Clonetics) as per the supplier's instructions. All treatments were performed on cells at 70-90% confluence on 100-mm polystyrene tissue culture plates. Just before experimental exposures, fresh medium was added to each plate. Hypoxic treatments were performed by incubating the cells in humidified gastight chambers (Billups-Rothenberg, Del Mar, CA) in the presence of 0% O2, 5% CO2, and balance N2 with 20 ml medium/plate. In our hands, such a system creates a severely hypoxic but not an anoxic environment (10). For DFO and concomitant control exposures, additional glucose (20 mM final concentration) was added to the medium to prevent cell death due to glucose starvation (26).
Isolation of RNA and Northern blot analysis. After exposure to
various stimuli, the cells were washed twice with Hanks' balanced salt
solution and harvested in guanidine isothiocyanate solution (32). Total
cell RNA was then purified with the PERFECT RNA isolation kit
(A549 cells; 5 Prime 3 Prime, Boulder, CO) or CsCl
centrifugation (SAECs) (37). Equal amounts of RNA were resolved on a
1% agarose-2.5 M formamide gel in a 20 mM MOPS buffer, pH 7.4, containing 1 mM EDTA. A standard Northern blotting procedure (32) was
used to transfer the RNA to a nylon membrane (Micro Separations,
Westborough, MA). A full-length HK-II cDNA (27) was generously provided
by Dr. Daryl Granner (Vanderbilt University School of Medicine,
Nashville, TN). The full-length HK-I cDNA (phHK15-2) was obtained from
the ATCC. The full-length cDNAs were labeled with a randomly primed
labeling kit (GIBCO BRL) and [32P]dCTP (ICN,
Irvine, CA). Blots were hybridized with the probe following a standard
procedure (32) and autoradiographed. Quantitative analysis of Northern
blots was performed with ImageQuant 1.11 (Molecular Dynamics,
Sunnyvale, CA) after the blots were exposed to a phosphor screen.
Northern blot data were normalized for loading efficiency with a random
prime-labeled 28S rRNA probe (Ambion, Austin, TX).
Protein assays. Electrophoretic separation and detection of HK
isoforms were performed essentially as previously described (1).
Briefly, the extract was prepared by harvesting twice Hanks' balanced
salt solution-washed cells in HK buffer [25 mM Tris, pH 7.0, 1 mM
EDTA, 40 mM KCl, 10 mM glucose, 2 mM glucose 6-phosphate, 10 mM
dithioerythritol, 1% Triton, 20% glycerol, 0.2 U/ml of aprotonin, 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 10 µg/ml of
leupeptin]. One milliliter of buffer was used to harvest 5 million cells. After the cells were harvested, the cell homogenate was
incubated on ice for 30 min followed by centrifugation at 1,000 g at 4°C for 10 min. The supernatants were removed and
stored in aliquots at 80°C for subsequent assay. Activity
was stable for one freeze-thaw. Before electrophoretic separation,
activity of the samples was determined spectrophotometrically. Approximately 10 µl of cell supernatant were added to 1,000 µl of
100 mM Tris · HCl, pH 8.0, 0.5 mM EDTA, 10 mM ATP, 10 mM MgCl2, 2 mM glucose, 0.1 mM NADP, and 0.1 U/ml of
glucose-6-phosphate dehydrogenase. HK activity was determined by
following the glucose 6-phosphate-dependent conversion of NADP to NADPH
spectrophotometrically at 340 nm at 37°C. One activity unit is
defined as micromoles of NADPH per milliliter per minute at 37°C.
Once the overall HK activity was determined, a fresh sample was removed
from the freezer to perform an "in gel" assay. First, the
extracts were incubated for 30 min at 37°C in 2.5 mM
dithioerythritol to ensure maximal recovery of activity. Then, samples
were applied to a Titan III cellulose acetate plate with a Super Z-12
applicator (Helena Laboratories, Beaumont, TX) and separated
electrophoretically at 200 V and 2.5 mA for 20 min at 4°C in 18 mM
Tris, pH 8.0, 10 mM boric acid, 2 mM Na2HPO4, 1 mM EDTA, and 10 mM glucose. HK activity was developed with 40 ml of 1%
ultralow-melt agarose, 100 mM Tris · HCl, pH 8.0, 0.5 mM EDTA, 10 mM MgCl2, 2 mM glucose, 20 mM ATP, 914 µM NADP, 3.2 U/ml of glucose-6-phosphate dehydrogenase, 65.3 µM
phenazine methosulfate, 241 µM thiazolyl blue, and 2 mM KCN. After
application of the solution, the plates were cooled at 4°C until
the agarose solidified and were then developed at room temperature for
30 min. To prevent fading, the plates were rinsed in 5% acetic acid. Estimation of HK activity in the gels was performed by scanning the gel
with a Hewlett-Packard (Palo Alto, CA) ScanJet 4C. Data were quantified
with ImageQuant 1.11 (Molecular Dynamics).
Western blot analysis was performed using standard procedures (32) with the primary antibody for HK-II (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:2,000. The horseradish peroxidase-labeled secondary antibody was purchased from Calbiochem (San Diego, CA) and used at a dilution of 1:20,000. The Western blots were developed with chemiluminescent substrates also from Calbiochem and an enhancer from Pierce (Rockford, IL). The blot was exposed to high-performance chemiluminescent film (Amersham, Piscataway, NJ).
Luciferase assays. A549 cells were transfected when 60-80% confluent with an HK-II-luciferase reporter vector in either the presence or absence of additional vectors that overexpressed a HIF-1 mutant protein. Originally, A549 cells were transfected only with pXP1 (ATCC) or the reporter plasmids p-4kbHK-IILuc, p-1.7HKIILuc, p-0.8HKIILuc, and p-0.5HKIILuc (22) with Fugene-6 (Boehringer Mannheim, Indianapolis, IN) in complete medium. The HK-II reporter plasmid constructs were previously described (22). In this case, after 4-6 h, fresh medium was applied, and the cells were incubated for an additional 24 h to allow for recovery from transfection. At this point, the cells were exposed to either DFO (200 µM) for 48 h or to hypoxia for 24 h.
For the overexpression of the mutant HIF-1 protein in the presence of
the above luciferase reporter plasmids, two additional vectors,
HIF-1(401
603) and pARNT (18), were transfected into A549 cells
with Fugene-6. In this case, the cells were incubated for 36 h to allow
for the full expression and resultant effects of the HIF-1 mutant
protein. In all cases, pSV-
-galactosidase (Promega) cotransfection
was used to control for transfection efficiency. In some experiments,
pGEM4Z (Promega) DNA was used to equalize DNA concentrations in the
transfection mixtures. After the specified exposure times, cells were
harvested in 10 mM PBS, pH 7.5, and 0.5 mM EDTA and frozen for
subsequent assays.
-Galactosidase assays were performed with a
commercially available kit (Stratagene, La Jolla, CA). Luciferase
activities were determined at 22°C with a commercially available
luciferase assay system (PharMingen, San Diego, CA) and a Monolight
3010 Luminometer (Analytical Luminescence Laboratory, Cockeysville, MD).
Statistical analysis. Statistical analysis was done with JMP (SAS Institute, Cary, NC) statistical software. Means were compared by one-way analysis of variance followed by Dunnett's test for comparisons, with a control or Tukey-Kramer (honestly significant difference) test for comparisons between multiple pairs, with P < 0.05 considered significant.
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RESULTS |
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Effect of various stimuli on HK-I and HK-II mRNA expression in A549
cells. Treatment with hypoxia, various transition metals, and
chelators such as DFO can induce the expression of genes through the
HIF-1-transactivating factor (2, 6, 11, 15, 21). To test the hypothesis
that HK-II is upregulated under hypoxic conditions through HIF-1 in
lung cells, we exposed A549 cells to hypoxia (0% O2) or
DFO (200 µM) for various time periods (Fig. 1). In both cases, maximal
induction occurred at 6 h.
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Two transcripts of HK-II mRNA, 4 and 5.5 kb, were detected with the full-length HK-II cDNA probe. Two similarly sized HK-II mRNA transcripts were previously detected in rat adipose tissue and were attributed to the presence of alternate polyadenylation sites and a single transcriptional start site (28). In the present study, the 4-kb transcript was the primary transcript in unstimulated A549 cells (Fig. 1, A and B). However, the stimuli, whether hypoxia or DFO, induced the expression of the 5.5-kb transcript to a greater extent than the 4.0-kb transcript (Fig. 1). After 6 h of hypoxic exposure, the 5.5-kb transcript contributed 85% of the 5.5-fold increase in mRNA intensity of the pooled (added together) intensities of the 4- and 5.5-kb transcripts relative to the pooled intensities for the normoxia-exposed cells. For the DFO-treated cells, the 5.5-kb transcript by itself accounted for 66% of the ~10-fold increase in the pooled intensities for both messages at 6 h.
Figure 1, E-G, shows the intensities of the 5.5-kb transcript. Hypoxia, which maximally induced the 5.5-kb HK-II transcript 7.5-fold relative to that in the normoxia-exposed cells (Fig. 1E), was a less effective stimulus than DFO, which, at maximal induction, increased expression of the 5.5-kb transcript ~20-fold relative to that in control cells (Fig. 1F). Dose-response studies with DFO (Fig. 1, C and G) indicated that 2.5 mM was maximally potent and that concentrations as low as 25 µM were effective in increasing the HK-II message (Fig. 1G). Because DFO can be toxic during prolonged exposure, further study (9) was conducted with 200 µM DFO, which caused minimal toxicity even with prolonged exposure.
In addition to DFO, A549 cells were exposed to a different chelator, o-phenanthroline (OP), to test the specificity of this response to DFO. When cells were exposed to 150 µM OP, HK-II mRNA was induced. Expression of the 5.5-kb transcript was increased 19- and 28-fold in two separate experiments.
Transition metals such as cobalt, manganese, and nickel are also known
to regulate genes mediated by the HIF-1 response, whereas zinc
does not influence the regulation of these same genes (13, 14, 41). To
test the effect of these metals on HK-II mRNA, A549 cells were exposed
to CoCl2 (200 µM), MnCl2 (400 µM), NiCl2 (400 µM), and ZnCl2 (400 µM)
for 16 h at concentrations similar to those that increased
glyceraldehyde-3-phosphate dehydrogenase mRNA levels in endothelial
cells (15). A preliminary experiment showed that maximal induction with
the metal ions was achieved at 16 h rather than at 6 h
(data not shown). As seen in Table 1, cobalt, manganese,
and nickel increased HK-II mRNA levels (~3-, 5-, and 2-fold,
respectively). Zinc did not affect the expression of HK-II.
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Exposure of A549 cells to various stimuli had little effect on HK-I levels (Table 1). DFO treatment increased HK-I levels slightly (1.5-fold); however, hypoxia and metal ions did not increase HK-I mRNA levels.
Effect of various stimuli on HK-I and HK-II mRNA expression in SAECs. Because A549 cells are a carcinoma cell line and increased levels of HK have been indicated as a potential adaptation of cancer cells (24), the levels of HK-I and -II mRNA were determined in primary cultured human SAECs. As seen from the results of these experiments (Table 1), as in A549 cells, the stimuli DFO and hypoxia increased the level of HK-II mRNA while having little effect of the level of HK-I mRNA. Additionally, the amount of HK-II mRNA expressed in SAECs exposed to normoxic conditions is greater than that in A549 cells exposed to normoxic conditions (Fig. 1, D and H). This contrast is most evident in the 5.5-kb transcript that is almost lacking in A549 cells exposed to normoxia.
Effect of protein and RNA synthesis inhibitors on the expression of HK-II in A549 cells. The hypoxic response through HIF-1 is dependent on new protein synthesis (13, 30, 36, 41). To further test the hypothesis that HK-II is regulated by HIF-1, cells were exposed to cycloheximide in the presence of various stimuli. Table 1 shows that cycloheximide (100 µM) alone had little effect on HK-II mRNA levels. However, in the presence of various stimuli, cycloheximide inhibited the ability of A549 cells to increase HK-II mRNA expression.
Actinomycin D inhibits RNA synthesis, and, therefore, inhibition of
increased mRNA expression in its presence indicates a mechanism
dependent on transcriptional activity. Figure
2 shows that when A549 cells
were exposed to actinomycin D, increased expression of HK-II mRNA by
DFO was inhibited.
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Effect of DFO and hypoxia on luciferase activity in transfected
cells. To confirm that the increase in HK-II message was related to
an increase in gene transcription, we used a luciferase construct in
which the entire HK-II promoter (4 kb) was inserted 5' of the luciferase gene. Cells containing the HK-II promoter vector produced a
10-fold increase in luciferase activity in the presence of DFO and an
8-fold increase in luciferase activity in the presence of hypoxia (Fig.
3).
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To further dissect the effect of hypoxia and DFO on the promoter of
HK-II, we tested constructs containing the basal promoter (p-0.5HKIILuc) and 0.8 (p-0.8HKIILuc), and 1.7 (p-1.7HKIILuc) kb of the
promoter 5' from the transcriptional start site (22). Approximately 50 or 60% of the capacity to maximally induce luciferase activity in the presence of hypoxia or DFO, respectively, was manifest
within the 500-bp basal promoter. Extending the promoter length
to 0.8 or 1.7 kb did not recover any additional luciferase activity
beyond that of the basal promoter in the presence of hypoxia or DFO. In
all cases, a -galactosidase-expressing construct was cotransfected
with the luciferase constructs, and the results were used to normalize
for transfection efficiencies.
Effect of a HIF-1 overexpression vector on the expression of HK-II
luciferase in A549 cells. The current studies
indicated that HIF-1 is involved in the expression of
HK-II in lung cells. To further explore the involvement of HIF-1 in the
expression of HK-II, we simultaneously transfected A549 cells with a
HIF-1-overexpressing vector and the full-length HK-II
promoter-luciferase construct (p-4HKIILuc). To avoid stimulation of the
endogenous HIF-1 by exposure to hypoxia, the mutant HIF-1(401
603)
(18), which lacks the oxygen-dependent degradation domain, was used to
cotransfect the cells. Under normoxic conditions, A549 cells were
transfected with equal amounts of p-4HKIILuc and varying amounts of
HIF-1
(401
603). Additionally, amounts of pARNT (HIF-1
) equal to
HIF-1
(401
603) were cotransfected because the active HIF-1
transcription factor is a heterodimer of
- and
-proteins. As
shown in Fig. 4, the relative luciferase
activity increases with increasing doses of HIF-1
(401
603) until
it reaches a saturation point at ~200 ng. Transfection with 2 µg of
HIF-1
(401
603) apparently oversaturated the system and led to
decreased levels of luciferase activity.
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HK-II promoter analysis. The HK-II promoter has been sequenced
(22). A consensus sequence for a HRE has been defined as RCGTG (34). We
analyzed the promoter and identified nine potential HRE sites within
that region (Table 2). Four sites were
found in the sense strand and five in the antisense strand. Six of the potential sites were found within the 500-bp basal promoter. Greater than 50% of the maximal response to hypoxia and DFO was found to
reside within this region (Fig. 3). The two sequences in the 60-bp region are only a few bases apart. Similar tandem sites have been found for other hypoxia-responsive genes such as
erythropoietin, vascular endothelial growth factor, aldolase (Ald) A,
enolase 1, lactate dehydrogenase A, and phosphoglycerate kinase 1 (35).
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Effect of various stimuli on HK-I and HK-II enzymatic expression in
A549 cells and SAECs. To determine whether the increased mRNA
levels resulted in increased HK-II protein activity, we determined the
total HK activity of cells treated with the above stimuli. When A549
cells were exposed to the various metals that increased HK-II mRNA
levels (Table 1), there was very little increase in total
HK activity expressed in HK units per microgram of DNA normalized to
activity in control cells (Mn, 1.22 ± 0.09; Co, 0.94 ± 0.06; Ni,
0.89 ± 0.06; Zn, 1.29 ± 0.08; control, 1.00 ± 0.11;
n = 4 experiments). However, as seen in Table 3,
in A549 cells, both hypoxia and DFO led to significantly increased HK
activity levels. To determine whether this increase in activity was
primarily due to an increase in HK-II, the HK isoforms were separated
on an activity gel (Fig. 5A). Densitometric
analysis (Table 3) showed that although HK-I activities were similar in
untreated and DFO-treated cells, HK-II activity increased ~2.5-fold
after DFO treatment. Neither HK-III nor -IV (glucokinase) activity was
detected. When the hypoxically induced samples from A549 cells were
analyzed, no significant difference could be found in either the HK-I
band or the HK-II band between the control and hypoxic samples (data not shown).
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The levels of HK-II mRNA induction in SAECs were lower than those in
A549 cells. The pattern of activity induction in SAECs matched this
trend. DFO, which most strongly increased total HK and HK-II activity
in A549 cells, significantly increased HK activity in SAECs, apparently
primarily through an increase in HK-II activity (Table 3, Fig.
5B). However, hypoxia, which led to smaller increases in HK
activity in A549 cells, did not significantly increase HK activity
levels in SAECs. Western analysis of the HK-II isoform from SAECs
showed a small but significant increase after exposure to both hypoxia
and DFO compared with that in control cells (Table 3). When analyzed by
Western blotting, the HK-II isoform could not be detected in
unstimulated A549 cells. However, on stimulation with hypoxia, the
HK-II isoform was detectable. Western blotting detected 4.5-fold more
HK-II protein in A549 cells treated with DFO than in cells treated with
hypoxia.
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DISCUSSION |
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Recent reports indicating that both HK-I and HK-II were regulated by HIF-1 in murine ES cells (19) and that A549 cells increase HIF-1 protein levels in response to hypoxia (40) suggested that HIF-1 could modulate HK expression in lung cells. Although the mechanism by which HIF-1 mediates a primarily transcriptional response to hypoxia is not completely understood (13, 34, 41), a distinctive response pattern to hypoxia, metal ions, iron chelation, and cycloheximide has been observed and used to define genes that are modulated by this regulatory mechanism. We tested the effect of two of these stimuli on the HK-I and HK-II mRNA levels in both SAECs and A549 cells. In SAECs, hypoxia and the chelator DFO increased the level of expression of HK-II mRNA approximately twofold but did not alter the level of HK-I mRNA. In A549 cells, HK-II gene expression was maximally induced by the chelators DFO and OP (15- to 30-fold) and less potently by hypoxia and specific transition metals (4- to 10-fold). As in the SAECs, the HK-I mRNA levels in A549 cells were not altered by exposure to hypoxia; however, exposure to DFO did slightly increase the level of HK-I mRNA.
The approximate 20-fold increase in HK-II message is among the largest increases in mRNA reported in response to DFO. In addition, when an alternate chelator, OP, was used, we observed increases of ~30-fold in HK-II message. Other genes involved in glucose metabolism, such as glyceraldehyde-3-phosphate dehydrogenase, Ald-A, phosphofrucotokinase, lactate dehydrogenase, and glucose transporter-1 (GLUT-1), had increases of two- to fourfold in response to DFO over a period of 16 h (5, 6, 11, 15, 26). GLUT-3 and Ald-C mRNA levels increased ~10-fold in response to DFO (6). In one cell type (L6 myoblasts), GLUT-1 mRNA was found to increase 16-fold in response to DFO (26). Similarly, erythropoietin mRNA was reported to increase 15- to 18-fold in response to DFO in cell culture (11, 12) over a 16-h period, although its message was found to increase several hundredfold due to hypoxia in vivo (13). Furthermore, DFO and OP are considerably more potent than any stimuli previously reported to induce HK-II mRNA.
In addition to induction by hypoxia and DFO, we tested the induction by transition metals and inhibition of these responses by cycloheximide in A549 cells. Cobalt, manganese, and nickel generally induce HIF-1-mediated gene expression, whereas zinc usually does not affect such expression (4, 14, 15). HK-II mRNA expression in A549 cells followed this pattern (Table 1), although nickel was a less effective stimulus than cobalt and manganese. Nickel has been reported to be ineffective in some cell lines (11). The induction of HK-II by each of the stimuli associated with hypoxic regulation was eliminated in the presence of cycloheximide (Table 1).
Iyer et al. (19) showed HK-II mRNA levels to be increased in response to hypoxia in Hep3B cells and to be dependent on HIF-1 expression in murine ES cells, although not significantly inducible with exposure to hypoxia. Iyer et al. did not quantitate the increase in HK-II mRNA in Hep3B, but it appears similar to the sevenfold increase in HK-II mRNA in A549 cells. The twofold increase in HK-II mRNA that we observed in SAECs in response to hypoxia is smaller than the increases in messages from both the A549 and Hep3B cells. However, similar to murine ES cells, SAECs basally express a higher level of HK-II mRNA under normoxic conditions than A549 cells (Fig. 1). This increased basal level of mRNA expression is associated with decreased hypoxic responsiveness. There have been several reports relating the increased glycolytic rate of cancerous cells to the increased basal levels of HK-II mRNA (24). This is obviously not the case for A549 cells, which express less HK-II mRNA than SAEC cells in normoxic conditions.
It is of interest that the 5.5-kb mRNA transcript was upregulated preferentially in A549 cells in the current investigation. In A549 cells under normoxic conditions, the predominant isoform is the 4-kb transcript, with little of the 5.5-kb transcript present. In contrast, the 5.5-kb message is the predominant message in SAECs in normoxia. This preferential increase in the 5.5-kb message was seen most significantly on exposure to DFO in A549 cells. Unlike SAECs, which showed a similar increase in HK-II message after exposure to either hypoxia or DFO, A549 cells increased their HK-II mRNA levels to a greater degree after exposure to DFO. There is no ready explanation for this discrepancy. Recently, literature has been published linking various carcinogenic genes (p53 and pVHL) to altered hypoxic responses (24, 25). However, A549 cells have neither mutant p53 nor pVHL genes. Additionally, several factors, including iron-responsive protein-1 and -2 (16, 29), have been indicated in increasing the stability of various mRNAs in response to hypoxic exposure. Analysis of HK-II mRNA showed no iron-responsive elements.
Using stimuli such as hypoxia and DFO, we have shown that HK-II is
likely regulated by an HIF-1 mechanism in A549 cells. Inhibition of
HK-II mRNA induction in the presence of actinomycin D and elevated luciferase activity in cells transfected with HK-II promoter luciferase constructs suggested a primarily transcriptional response.
Additionally, results from cotransfection experiments with
HIF-1/ARNT constructs indicated that HIF-1 is capable of stimulating
HK-II promoter activity in A549 cells (Fig. 4). The slightly smaller
increase in luciferase activity (5.5-fold with HIF-1 cotransfection
compared with 7.5- to 10-fold with hypoxia or DFO exposure) can be
attributed to the lower activation potential of the oxygen-stable
HIF-1
mutant that was used for cotransfection (18).
To further elucidate the transcriptional regulation of HK-II, we analyzed the luciferase expression of several HK-II promoter constructs in response to both hypoxia and DFO. Both stimuli induced expression of luciferase activity in cells transfected with a luciferase construct containing the proximal 4.0 kb of the human HK-II 5' regulatory sequences (Fig. 3). Although the 10-fold induction of luciferase by DFO was less than the 20-fold induction of the 5.5-kb transcript (Fig. 1), it was similar to the multiple of induction of the two transcripts combined. The induction of the two transcripts was a more appropriate comparison for results from the promoter studies because both transcripts share the same transcriptional start site and, therefore, cannot be distinguished in the luciferase assay.
For both stimuli, the majority of the transcriptional activation
occurred either 5' of the 1.7-kb deletion or within the 0.5-kb basal promoter (Fig. 3). When cells containing the basal promoter construct were exposed to either hypoxia or DFO, they produced 50-60% of the luciferase activity expressed in cells containing the full-length promoter construct. When cells containing luciferase constructs with partially truncated promoters (0.8 and 1.7 kb 5'
of the transcriptional start site) were assayed, they did not show any
further increase in luciferase activity over those containing the basal
promoter. The DNA sequence of the promoter was then analyzed to find
potential HREs. Nine potential HRE sites were identified (Table 2). The
promoter sequence of rat HK-II has been published (23). Of the six
sites located within the proximal 500-bp region of the promoter, three
of them (228,
309, and
370 bp) are completely
conserved between the rat and human genes. This strongly suggests
functionality. Further study is needed to fully characterize the
potential HRE sites in the human HK-II gene.
Increased HK-II mRNA levels led to smaller than expected increases in HK activity levels in both A549 cells and SAECs (Table 3). In both cells, DFO exposure increased HK activity levels more than hypoxic exposure and correlated with a specific increase in HK-II activity rather than in HK-I activity. Hypoxia only increased HK activity significantly in A549 cells, not in SAECs. Additionally, hypoxia increased HK activity in A549 cells to a lesser degree than DFO exposure did. SAECs displayed higher basal HK-II activity levels in normoxic conditions than did the A549 cells (Fig. 5), corresponding with their higher basal mRNA levels (Fig. 1). This may be one reason for the lesser induction of HK activity in SAECs. To determine whether the HK-II activity levels directly reflected the HK-II protein levels, we performed Western blots. Interestingly, in both A549 cells and SAECs, the increase in HK-II protein is closer to the increased mRNA levels than to the increase in HK-II activity. HK-II is known (39) to be an extremely labile enzyme, and it is susceptible to various kinetic controls within the cell. However, it seems likely that HK-II is also regulated at the posttranslational level.
We originally became interested in the regulation of HK isozymes in lungs when we were studying an oxidant stress-protective model in which rats were preexposed to 85% oxygen (17). We observed increased HK-II gene expression in the lungs of rats (1) and premature baboons (C. B. Allen, X.-L. Guo, and C. W. White, unpublished observations) and in A549 cells (C. B. Allen, K. Schneider, X.-L. Guo, and C. W. White, unpublished observations) when they were exposed to these adaptive concentrations of hyperoxia. Therefore, we wished to find potential methods for increasing HK activity in lung cells. Other oxidant stress-protective models involved rats preexposed to hypoxia (7, 20, 37) or DFO (3, 8). For this reason, we became interested in the possible regulation of HK-II expression by these stimuli and by HIF-1.
We have shown that in A549 cells and SAECs, HK-II is preferentially upregulated by hypoxia and other stimuli that induce a HIF-1 response. In addition, we have localized the majority of this response to the basal promoter. Future work will need to focus on the further characterization of the precise sites of regulation by HIF-1 of HK-II.
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ACKNOWLEDGEMENTS |
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We thank Yvette Lewis-Molock, Xiao-Ling Guo, Emily Cassidy, and
Stephanie Park for excellent technical assistance, Dr. John Wilson for
helpful discussions, Dr. Daryl Granner for providing the human
hexokinase-II cDNA, and Dr. Franklin Bunn for providing the
hypoxia-inducible factor-1(401
603) and pARNT expression vectors.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-52732, HL-56263, HL-57144, and HL-30068.
S. R. Riddle was supported by National Heart, Lung, and Blood Institute Training Grant HL-07670 and an Arnold and Sheila Aronsen Fellowship in Pediatric Medicine at the National Jewish Medical and Research Center (Denver, CO).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. W. White, Dept. of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson St., J101, Denver, CO 80206 (E-mail: whitec{at}njc.org).
Received 14 April 1999; accepted in final form 10 November 1999.
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