Departments of 1 Pediatrics and 2 Medicine, National Jewish Medical and Research Center, Denver, Colorado 80206
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Increased glucose utilization and hexokinase (HK)-II expression are adaptive features of lung cells exposed to hypoxia or hyperoxia. HK-II is the most regulated isoform of HK. Whether its overexpression could be protective against oxidative stress was explored in human lung epithelial-like (A549) cells. HK-II was overexpressed in A549 cells in a tetracycline-repressible retroviral vector system. Elevated expression of HK-II was confirmed by Western blot and activity measurements. Cell death caused by exposure to hyperoxia was decreased in HK-II-overexpressing cells. This effect was reversed when HK-II expression was suppressed with doxycycline. A similar protective effect was observed in HK-II-overexpressing cells after treatment with 1 mM hydrogen peroxide for 48 h. At baseline, fluorescence microscopy showed that overexpressed HK-II was localized to mitochondria. Electron microscopic studies showed that hyperoxia-exposed HK-II overexpressors had better-preserved and quantitatively smaller mitochondria than those in which the HK-II expression was suppressed or in the nontransduced A549 cells. Mitochondrial membrane potential was increased in HK-II-overexpressing cells exposed to hyperoxia compared with the nontransduced control cells under similar conditions. The present study demonstrates that HK-II protects human lung epithelial-like A549 cells against oxidative insults by protecting the mitochondria.
hypoxia; hyperoxia; mitochondria
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
OXIDATIVE STRESS resulting from the production of reactive oxygen species causes lipid peroxidation (21), enzyme inhibition (18), DNA damage (40), and differential expression of certain genes (4). Reactive oxygen species also can act as second messengers in the inflammatory signaling cascade (20). Over time, oxidative stress leads to cell death through either apoptosis or necrosis (9) or some combination of the two (5). Cell death from hyperoxia involves specific reactive oxygen species, especially hydrogen peroxide (H2O2) (25).
Therapeutic use of high concentrations of oxygen (hyperoxia) is common in the treatment of severe lung disease. Prolonged use, often required in treatment of preterm newborns and critically ill patients, results in an increase in reactive oxygen intermediates in the lung (10, 11). This can cause lung injury and can contribute to bronchopulmonary dysplasia (BPD), a pathological condition characterized by lung inflammation, fibrosis, and airway smooth muscle hypertrophy (28). In its contemporary form, BPD is characterized by the presence of larger and fewer alveoli, reflecting impaired lung development (22). Elevated oxygen tension and/or oxidative stress may play a role in this process.
Exposure of the lung to hyperoxia causes an impairment of tricarboxylic acid cycle enzymes (1, 12). Cellular adaptation to hyperoxia also is associated with an increase in glycolysis (36) and total glucose utilization (3). Together, these studies implicate involvement of the glycolytic pathway in adaptation to hyperoxia.
In the lung, hexokinase (HK) is the initial and rate-limiting enzyme in the glycolytic pathway (35). In mammals, there are four isoforms of HK: HK-I, HK-II, HK-III, and HK-IV, or glucokinase. They differ in their affinity for glucose with Km in the order HK-IV > HK-II > HK-I > HK-III (44). Glucokinase is the less-regulated form of HK and is expressed primarily in the liver and pancreas (43). HK I-III are present in lung and are regulated by glucose-6-phosphate and inorganic phosphate. These enzymes differ in their subcellular localization, with HK-III localized to the nuclear membrane and HK-I and HK-II localized to the cytoplasm and mitochondria (38, 44). The extent of HK binding to mitochondria depends on the cell type, with highly glycolytic brain cells having a larger percentage of mitochondrion-bound HK than cells from other organs. In skeletal muscle, nearly 60% of HK-II is bound to mitochondria (7). This binding is suggested to take place at the voltage-dependent anion channels (VDACs), commonly referred to as mitochondrial porins (38).
The importance of HK-II in energy metabolism has been increasingly recognized. HK-II-deficient mice suffer embryonic lethality (19), indicating that HK-II is irreplaceable by the other HK isoforms and showing its vital role in embryonic development. In contrast, glucokinase-deficient mice survive to term (15, 39). Among HK isoforms, only HK-II can be regulated by insulin (31), high glucose and cAMP (32), exercise (29, 30), and ionophores (16). HK-II also is sensitive to altered oxygen concentrations. We have shown previously that HK-II expression is elevated under both by hypoxic (33) and hyperoxic (2) conditions.
Increased glycolysis is perceived as a possible adaptive mechanism during exposure to hyperoxia in which the tricarboxylic acid cycle is impaired. Because HK-II is the dominant HK isoform expressed in highly glycolytic cells and also is the rate-limiting enzyme in glycolysis in the lung (34, 35), its overexpression could enhance adaptation to hyperoxia or other oxidative stresses. It is gradually induced in rat lung during adaptation to sublethal hyperoxia, and this suggests that it may have a role in adaptation to oxidative stress. The present study was undertaken to test this hypothesis. Therefore, we constructed doxycycline-regulated, retrovirally transduced, HK-II-overexpressing, stable A549 cell lines. Herein, we show that overexpression of HK-II protects cells against hyperoxic injury as well as oxidants such as H2O2.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reagents.
The vector pET28c+ containing the human HK-II cDNA was
generously provided by Dr. Samir S. Deeb (University of Washington, Seattle, WA). The retroviral system (23)
containing the bicistronic expression vectors HERMES-HRI-green
flourescent protein (GFP) and the RetroTet RTAb
expressing the transactivator was a gift from Dr. Helen M. Blau (Stanford University School of Medicine, Stanford, CA). The Phoenix amphotropic cell line was obtained from Dr. Gary Nolan (Stanford University Medical Center, Stanford, CA) through ATCC.
Construction of HK-II-overexpressing cells. Human HK-II was PCR amplified from HK-II-containing pET28c+ vector with PfuTurbo DNA polymerase (Stratagene, La Jolla, CA) using the forward primers 5'-GGAATTCGGATCCCGCGGCAGGATGATTGCCTCGCAT-3' for amplification at the 5'-end and the reverse primer 5'-GGAAGTCACGCGTGGGGTTCTATCGCTGTCCAGCCTC-3' for amplification of the 3'-end. The PCR amplified fragment of HK-II was digested with BamHI and MluI, gel purified, and subsequently cloned into the BamHI/MluI site of the tetracycline-inducible bicistronic retroviral expression vector (HERMES-HRI-GFP) by standard cloning techniques.
To generate virus, we transfected 100-mm plates containing 70% confluent Phoenix amphotropic cells with either the HK-II-containing HERMES-HRI-GFP vector or the RetroTet RTAb
|
Western blotting. Cell extracts were prepared as described earlier (1). Samples containing 37 µg of protein were separated on a 4-15% SDS-PAGE gel from Bio-Rad (Hercules, CA) and transferred onto nitrocellulose membrane (HyBond-ECL, Amersham Pharmacia Biotech) in 25 mM Tris, 190 mM glycine, and 20% methanol, pH 8.3, at 90 V for 1 h using a transblot apparatus (Bio-Rad). Human HK-II was detected using polyclonal human HK-II antibody (Santa Cruz Biotechnology, Santa Cruz, CA) raised in goat, and the blots were probed with horseradish peroxidase-conjugated anti-goat antibody. The bands were detected using an enhanced chemiluminescence detection with a supersignal ultradetection reagent (Pierce, Rockford, IL). We scanned the blots on an EPSON expression 800 scanner and subsequently analyzed for the density of the band using an IP Lab Gel Evaluation software (Scanalytics, Fairfax, VA)
HK activity gel and enzyme assay. The cell types expressing different levels of HK-II were harvested in a 50 mM Tris · HCl buffer (pH 7.4) containing 100 mM KCl, 150 mM sucrose, 10 mM glucose, 1 mM glucose-6-phosphate, 1 mM diethylenetriamine pentaacetic acid, 1 mM EDTA, 20% glycerol, 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 2.5 mM dithioerythritol, 10 µg/ml leupeptin, and 0.2 U/ml aprotinin. Cells were lysed by sonication. The HK isoforms were detected, as reported earlier (2), with cellulose acetate plates (Titan III plates; Helena Laboratories, Beaumont, TX) and a Super Z-12 applicator (Helena Laboratories). Thirty-seven micrograms of protein were loaded onto the wells along with heart tissue standards containing HK-II.
The total HK activity in the different cell types was assayed at 37°C in 10 mM Tris · HCl buffer, pH 8.0, containing 0.5 mM EDTA, 10 mM ATP, 10 mM MgCl2, 2 mM glucose, 0.1 mM NADP, and 0.1 U/ml glucose-6-phosphate dehydrogenase at 340 nm (2).Protein assay. We determined protein concentration in the cell lysate using the Bio-Rad DC protein assay kit with a Spectramax 340 microtiter plate reader (Molecular Devices, Sunnyvale, CA) with a 96-well plate and BSA (Bio-Rad) as a standard. Data analysis was done with Softmax Pro1.2 software (Molecular Devices).
Cell culture, transfection, and exposures to oxidants. A549 cells (from ATCC) were cultured in Kaighn's modification (F12K) medium containing 10% tetracycline-free fetal bovine serum (FBS; Clontech, Palo Alto, CA), 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere in 5% CO2 at 37°C. For experiments, custom-made F12K medium (GIBCO-BRL, Life Technologies, Rockville, MD) without L-glutamine was used. It was supplemented with fresh L-glutamine to a final concentration of 2 mM, and additional glucose was added to raise the concentration to 20 mM. A549 cultures were routinely passaged by trypsinization and subcultured at an initial plating density of 0.5 million cells per plate.
The Phoenix amphotropic cell line (a transformed human embryonic kidney cell line) was grown in DMEM (high glucose) containing 10% heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, and 2 mM L-glutamine. Transfection was carried out using Fugene-6 (Roche, Indianapolis, IN) as the transfection reagent in 70-80% confluent 100-mm plates. To 15 µg of DNA, 45 µl of Fugene-6 in 0.2 ml of serum-free medium were added. Initially, 45 µl of Fugene-6 were added to 0.2 ml of serum-free medium, and the mixture was incubated for 5 min before it was slowly added to the DNA. The DNA-containing transfection mixture was incubated for an additional 20 min and added to the plates containing 5 ml of complete medium. Hyperoxic exposure was carried out at Denver's atmospheric pressure (635 mmHg) in an air-tight humidified modular chamber (Billups-Rothenberg, Del Mar, CA) gassed with 95% O2-5% CO2 and incubated at 37°C. Exposures were continued for up to 6 days with daily supply of fresh media. Exposure to H2O2 (1 mM) was continued up to 48 h with media change and fresh addition of H2O2 after 24 h.Mitochondrial membrane potential.
Mitochondrial membrane potential (MMP) was estimated by the uptake of a
fixable dye Mitotracker orange according to the method of Macho et al.
(26). At the end of hyperoxic exposure, the medium was
replaced with gas-conditioned (air- or oxygen-containing) medium
containing 150 nM Mitotracker orange dye and exposed for an additional
30 min to normoxia or hyperoxia in the presence and absence of the 40 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells
were washed once with phosphate-buffered saline (PBS) and harvested
using dye-containing trypsin-EDTA solution in the presence or absence
of the uncoupler CCCP. The cells were pelleted at 200 g for
10 min and resuspended in 1 ml of PBS followed by fixation with the
addition of 1 ml of 8% paraformaldehyde (Electron Microscopy Sciences,
Fort Washington, PA) in PBS (pH 7.4). The cells were incubated in the
dark for 30 min on a shaker at room temperature, after which they were
kept on ice and analyzed on an EPICS XL flow cytometer (Coulter,
Hialeah, FL) operated by Coulter's System II software and
incorporating an argon laser (488 nm, 15 mW) for excitation.
Mitotracker orange fluorescence was assessed in fluorescence
detector-3. Mean fluorescence intensity (MFI) of the cells in
normoxia and hyperoxia was corrected from their respective MFI values
in presence of the uncoupler, CCCP (MFICCCP
MFI+CCCP). The corrected MFI values for each sample were
analyzed by analysis of covariance using JMP software (SAS Institute,
Cary, NC).
PI staining. For the PI staining of nonviable cells, ~106 cells were suspended in 1 ml of PBS, and PI (2 µg/ml, final concentration) was added. After 5 min of incubation on ice in the dark, the flow cytometric analysis was performed.
Glucose assay. Glucose utilization was estimated in cells exposed to either 21 or 95% O2 with the commercially available glucose assay (Trinder) kit from Sigma (St. Louis, MO). After 48 h of exposure, supernatant medium was collected and centrifuged to remove any cellular debris. One milliliter of the assay reagent was mixed with 10 µl of the medium and incubated at 25°C for 18 min, after which the absorbance was recorded at 505 nm on a Shimadzu UV-1601 UV-VIS spectrophotometer. The total glucose consumed was estimated and plotted, expressed as per cell number.
Fluorescence microscopy. Cells were seeded onto no. 1 1/2 glass coverslips in six-well plates at a density of 3 × 105 cells per well and exposed to 21 or 95% O2-5% CO2 for 4 days. Hyperoxic exposures were performed as described in Cell culture, transfection, and exposures to oxidants. After exposure, the cells were fixed in 4% paraformaldehyde for 10 min and then quenched in 1 mM glycine-PBS. The cells were rinsed in PBS and permeabilized with 0.4% nonfluorescent Triton X-100 (Calbiochem, La Jolla, CA) in 10 mM sodium citrate, pH 7.8, for 20 min. After another rinse in PBS, the cells were blocked in 5% normal donkey serum for 20 min, rinsed, and incubated for 1 h with primary antibody [anti-HK-II COOH-terminal goat polyclonal (Santa Cruz Biotechnology)] and anti-inner mitochondrial membrane, mouse monoclonal (Serotec, Raleigh, NC). The cells were thoroughly rinsed in PBS before the 45-min incubation with secondary antibody; Texas red-conjugated, donkey anti-goat IgG; and FITC-conjugated, donkey anti-mouse IgG (Jackson ImmunoResearch Laboratory, West Grove, PA); respectively. After five 10-min rinses in PBS, the coverslips were mounted cell-side down onto glass slides using Prolong, an aqueous antifade mounting medium (Molecular Probes) and allowed to dry overnight. We used an Olympus Vanox-T fluorescence microscope attached to a digital camera (Cooke, Auburn Hills, MI) to examine the fixed cells. Images were recorded using the Slide Book 2.6.5.5 software (Intelligent Imaging Innovations, Denver, CO) on a Macintosh G3 computer.
Quantification of images was performed as follows. After dual-labeling the fixed cells with both the mitochondrial primary antibody-FITC conjugated secondary antibody and HK-II-Texas red, we captured two images per frame, one from each of the two corresponding channels, 490 and 555 nm. Exposure time was kept constant for all slides. The appropriate negative and positive controls also were viewed; minimal crossover between channels was detected. The images were transferred into NIH Image to determine the percentage of HK-II bound to the mitochondria. The gray-scaled images were inverted and subjected to a series of 4-7 smooth/sharpen corrections to increase the contrast between the pixels of the true signal and the background. The images were made binary, and the total number of pixels was measured. At this point, the two binary images, one of mitochondria and one of HK-II, were both multiplied by 0.5, resulting in all positive signal becoming gray. Using the "image math" function, we added the two images together, resulting in black pixels where the HK-II and mitochondria colocalized and gray pixels for all other positive signal. The combined image was thresholded to remove all gray, noncolocalized pixels and was made binary, and the pixels were counted. The percentage of HK-II bound to the mitochondria was determined by dividing the number of pixels of the combined image by the number of pixels from the HK-II image.Electron microscopy. For the electron microscopic analysis, the control and hyperoxic cells were washed twice with 0.1 M cacodylate buffer, pH 7.3, containing 5 mM CaCl2 and 5 mM MgCl2 and then fixed in the same buffer containing 2.5% glutaraldehyde and 3.4% sucrose. The cells were then scraped, and the fixed cells were suspended in fresh fixative and pelleted at 300 g for 10 min. After being dehydrated and embedded in resin, thin sections were cut with a Reichart Ultra Cut E microtome. The sections were collected on 0.4% Formvar-coated, 100-mesh grids and stained in osmium. The sections were examined for mitochondria and other organelles under a Philips CM-10 electron microscope at 8 kV, and the images were photographed.
Ultrathin sections on 100-mesh grids were surveyed in a transmission electron microscope at a magnification of ×3,000 from top to bottom and from left to right until the first grid containing a cell was located. One cell, the one that was closest to the upper left corner per grid in 20 consecutive grid squares, was sampled. Mitochondria in the cells were photographed using a transmission electron microscopy digital camera (Advanced Microscopes Techniques, Danvers, MA) at a magnification of ×16,000 or ×24,500, depending on their sizes. An internal calibration length was created and stored automatically by the camera at the magnification specified. Mitochondrial profile areas were measured using ImagePro software (Media Cybernetics, Silver Spring, MD) by tracing the outline of each mitochondrion. For the control A549 cells, a total of 123 mitochondria in 21% O2 and 158 mitochondria in 95% O2 were analyzed. For the doxycycline-suppressed HK2-10D cells exposed to 21 and 95% O2, a total of 170 mitochondria and 184 mitochondria, respectively, were analyzed. For HK-II-overexpressing HK2-10 cells, a total of 151 mitochondria in 21% O2 and 212 mitochondria in 95% O2 were analyzed. The mitochondrial area was computed by log transforming the area of all the mitochondria in a group, calculating the mean and plotting the mean as ln (area) in nm2 vs. cell type.Statistical analyses. All statistical calculations were performed with JMP software (SAS Institute). Means were compared by one-way analysis of variance followed by a test for comparison between experimental groups vs. the control group using Dunnett's method. A P value of <0.05 was considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Selection of clones expressing GFP and HK-II.
Cells were initially selected by flow cytometry based on the level of
GFP expressed as shown in Fig.
2A. The flow cytometric analysis also was carried out in cells treated with doxycycline (2 ng/ml) for 48 h to suppress their HK-II expression. The
concentration of doxycycline was selected on the basis of dose-response
curves for GFP and HK-II expression, results of which have been omitted for brevity. The minimal concentration of doxycycline providing maximum
suppression of GFP and HK-II was determined to be 2 ng/ml and was used
throughout further experiments. Four clonal populations with different
GFP levels were finally selected and named HK2-5, HK2-10, HK2-20, and
HK2-62. To confirm that the HK-II expression correlated directly to the
levels of GFP expressed downstream in the bicistronic construct, we
further characterized cells for their level of total HK activity using
the spectrophotometric method (Fig. 2B), total HK-II by
Western blot (Fig. 2C), and HK isoforms using activity gel
(Fig. 2D). The fold change in total HK activity from 21 to
95% O2 decreased with the increase in HK-II expression. In
HK2-62, which represents the cell type with maximum HK-II expression,
there was no additional change when cells were exposed to hyperoxia.
Results of Western blot analysis of the different HK-II-expressing
clones are shown in Fig. 2C. There was virtually no HK-II
protein detected in the control A549 cells and the doxycycline-treated
HK-II-suppressed HK2-5D and the HK2-10D cells under the conditions of
the experiment. A densitometric scan of Western blots showed relative
intensity values for A549, HK2-5+Dox, HK2-10+Dox, HK2-5, HK2-10,
HK2-20, and HK2-62 to be 0.04 ± 0.03, 0.24 ± 0.12, 0.10 ± 0.06, 13.11 ± 0.06, 10 ± 0.00, 22.18 ± 1.01, and 25.58 ± 0.20, respectively (n = 3 determinations per condition; representative blot shown in Fig.
2C; values were normalized with respect to HK2-10 as was
done with GFP normalization). There was a slight decrease in the HK-II
levels in the HK2-10 compared with the HK2-5 cells, and this difference
was found to be statistically significant. This is despite the fact
that HK2-10 had a higher GFP content than HK2-5 (Fig. 2A).
This observation is, however, not clearly reflected in Fig.
2B where the total HK specific activity is shown, and that
activity in HK2-5 and HK2-10 in 21% O2 was statistically
not different. HK-II protein values for clones HK2-5, HK2-10, HK2-20,
and HK2-62 were significantly greater than those for A549 and
doxycycline-suppressed cells. HK2-5 and HK2-10 cells contained less
HK-II protein than did HK2-20 and HK2-62 cells. Among these and other
cells expressing elevated levels of HK-II, there was some elevation of
basal HK-II expression, relative to that in A549 cells, even after
maximal suppression with doxycycline. The basal level of expression of
HK-II was based on results of doxycycline dose-response curves followed
by measurements of GFP and HK-II activity levels (data not shown).
Although the doxycycline-suppressed clones HK2-5+Dox and
HK2-10+Dox (Fig. 2B) consistently showed greater total
HK activity than control A549 cells, those differences did not reach
statistical significance. To determine whether changes in the level of
protein also reflect changes in the HK-II activity, we performed an
HK-II activity gel assay. As shown in Fig. 2D, the HK-II
activity correlated well with the level of HK-II protein present in the
cell. In addition, there was no change in the HK-I band intensity
throughout the different clonal populations.
|
Effect of hyperoxia on cell survival in HK-II-overexpressing cells.
To explore the effect of HK-II overexpression on hyperoxia-induced
injury, we exposed A549 cells, those overexpressing HK-II, and those in
which HK-II expression was suppressed to hyperoxia, and the extent of
cell death was determined by PI staining. As shown in Fig.
3, 22% cell death was detected in the
control A549 cells exposed to hyperoxia for 6 days. There was an
approximately twofold decrease in cell death in the HKII-overexpressing
clones. The level of cell death in the HK-II-overexpressing cells
ranged from 8 to 10%. HK2-5, HK2-10, and HK2-20 cells had 10% cell
death upon hyperoxic exposure. HK2-62 had ~8% cell death, which was not statistically different from its baseline in 21% O2.
Cell death was not affected by the presence of doxycycline. In the doxycycline-treated, HK-II-suppressed cells, there was an intermediate level of cell death at ~17% compared with the control A549 cells. The basal level of cell death in the different clones exposed to 21%
O2 was statistically not different from the A549 control cells under similar conditions.
|
Effect of H2O2 on cell survival in
HK-II-overexpressing cells.
To determine whether the effect of HK-II expression on cell survival is
limited to hyperoxia or has a more general protective effect on injury
due to oxidants, we exposed A549 cells, HK-II overexpressing cells, and
these cells treated with doxycycline to 1 mM
H2O2 for 48 h, and cell viability was
assessed with PI (Fig. 4). As is evident,
the HK-II overexpressors were protected, with a twofold decrease in
cell death from 13 to 6%. The results obtained with HK-II-expressing,
doxycycline-suppressed cells were comparable to those found with
control A549 cells. Cell death in HK2-5 cells treated with
H2O2 appears to be less, but this difference is
not statistically significant compared with the rest of the
overexpressing cells, viz HK2-10, HK2-20, and HK2-62 under similar conditions.
|
Effect of hyperoxia on MMP.
Oxidative insults to cells can cause structural and biochemical changes
leading to death. Changes in MMP can precede cell death. The MMP of
control cells and cells overexpressing HK-II after 4 days of hyperoxic
exposure is shown in Fig. 5. After 4 days
of exposure, the MMP of hyperoxia-exposed cells was considerably greater than in cells exposed to 21% O2. Further, there
was a modest increase in the MMP of the HK-II-overexpressing cells in hyperoxia compared with that of the control cells under the same conditions.
|
Glucose consumption in HK-II-overexpressing cells. To determine the effect of HK-II overexpression on glucose consumption in cells exposed to 21 and 95% O2, we measured the glucose consumed from media. Overexpression of HK-II in A549 cells did not lead to an increase in glucose consumption under normoxic conditions. Both the control A549 cells as well as the HK-II-overexpressing and doxycycline-suppressed cells exhibited an approximate threefold increase in glucose consumption upon hyperoxic exposure (data not shown).
Effect of hyperoxia on cell morphology.
Exposure of cells to hyperoxia causes changes in cell morphology. To
gain further perspective on the observed changes in MMP of cells
exposed to hyperoxia, we used electron microscopy to examine the
ultrastructural changes in these cells. Figure
6 shows electron micrographs of cells
exposed to 95% O2 for 6 days. Compared with the control
A549 cells in air, those exposed to hyperoxia appeared flattened and
contained swollen and distorted mitochondria. In addition,
hyperoxia-exposed A549 cells contained more abundant lysosomes than
their counterparts exposed to 21% O2. The
HK-II-overexpressing, hyperoxia-exposed cells had morphology that more
closely resembled their control counterparts in 21% O2
than did doxycycline-suppressed or nontransduced A549 cells in
hyperoxia. The mitochondria of these hyperoxia-exposed cells appeared
more normal, with more defined cristae and less swelling compared with
the doxycycline-suppressed and nontransduced cells. During the exposure
to hyperoxia, the doxycycline-suppressed HK-II cells had a
morphological appearance intermediate between that of control A549
cells and HK-II-overexpressing cells. To get a quantitative estimate of
mitochondrial volume in the different cell types, we measured areas of
mitochondrial profiles in the different cell types under conditions of
21 and 95% O2. As shown in Fig.
7, there was a statistically significant 2.2-fold increase in the mitochondrial area of control A549 cells exposed to 95% O2 when compared with its 21%
O2 control. There was no statistical difference in
mitochondrial area between 21- and 95% O2-exposed
HK-II-overexpressing cells. However, mitochondrial area of
HK-II-suppressed cells in hyperoxia was 1.6-fold higher than the
corresponding cells in 21% O2, and this difference was statistically significant. Other noticeable features of the
HK-II overexpressors were the presence of lamellar bodies
characteristic of type II cells, which were less prominent in
doxycycline-treated HK-II-expressing and control A549 cells. Rough
endoplasmic reticulum and Golgi apparatus were considered more
prominent in HK-II-expressing and in doxycycline-suppressed cells
relative to control A549 cells, both in 95 and 21% O2.
|
|
Immunofluorescence staining of cells exposed to hyperoxia.
To examine the subcellular localization of HK-II after oxidant injury,
we stained cells for both mitochondria and HK-II. We based the
selection of HK2-5 cells for immunofluorescence studies on their
relatively low GFP content, thus avoiding interference with the
staining procedures employed. As seen in Fig.
8, control A549 cells and the
doxycycline-suppressed HK-II cells had a relatively diffuse pattern of
HK-II distribution. After hyperoxic exposure of the
HK-II-overexpressing cells, the mitochondria appeared clustered around
the nuclear membrane, and the HK-II colocalized in a very similar
distribution with the mitochondria. HK-II localization to mitochondria
was minimally detected in doxycycline-treated cells and not detected in
control A549 cells during exposure to hyperoxia. Quantitative estimates
of HK-II bound to mitochondria are shown in Fig.
9. As seen in the figure, there was
7-9% binding of HK-II to the mitochondria in both the control
A549 cells and in HK2-5D cells in which the HK-II expression has been
suppressed by doxycycline. The HK2-5 cells in 21% show a significantly
greater binding of HK-II to mitochondria, which is ~24%. This
binding was further increased to ~49% in the same cells exposed to
95% O2.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Findings of loss of tricarboxylic acid cycle enzyme activities, increased glucose consumption, and increased HK-II expression and reports of increased glycolysis prompted us to speculate that HK-II has an important role in adaptation to hyperoxic stress. The present study demonstrates a protective role of HK-II in hyperoxia-induced cell death in A549 cells. Because lung epithelial cells are the first line of defense against a variety of insults including hyperoxia, a human lung epithelial cell line (A549) was selected. We constructed a doxycycline-regulated, retrovirally transduced HK-II-overexpressing stable A549 cell line. This transformed cell line, unlike many other tumor cells, has a low endogenous HK-II level.
Levels of GFP should reflect a proportionate level of HK-II being expressed from the bicistronic construct. This is largely true. However, HK2-10 cells that had a higher GFP content compared with HK2-5 showed relatively less HK-II compared with HK2-5. This anomaly is clearly reflected in the HK-II Western blot (Fig. 2C) and activity gel assays (Fig. 2D). There was an additional increase in total HK activity of cells exposed to hyperoxia. This increase in activity could be attributed to endogenous increases in HK levels after exposure to hyperoxia. The fold increase in HK activity decreased with increased HK-II expression. The fact that there was no additional increase in HK activity in the HK2-62 cells suggests that production of these levels of HK-II may approach the maximal capacity of these cells.
We observed ~22% cell death in the nontransduced A549 cells exposed to hyperoxia (95% O2) for 6 days. Interestingly, the level of cell death in the HK-II-overexpressing cells under these hyperoxic conditions was comparable with that of A549 cells in 21% O2. However, cells in which HK-II overexpression was suppressed by doxycycline had levels of viability intermediate between those of nontransduced control and HK-II-overexpressing cells. The basal levels of HK-II expression in these cells, even after maximum suppression with doxycycline, were still in excess of levels seen in control A549 cells. The data obtained with the various HK-II expressing cells also suggest that moderate levels of HK-II expression are sufficient to provide partial protection and that increasing the levels of HK-II further does not provide further protection. That protection of cells by HK-II is not limited to hyperoxic oxidative stress was demonstrated by our results with H2O2 (Fig. 4), showing similar protection. Again, moderately increased expression of HK-II was sufficient to provide protection. Unlike the doxycycline-treated HK-II-suppressed cells in hyperoxia, cell death in both HK2-5+Dox and HK2-10+Dox after H2O2 treatment was comparable with the level of death in the control A549 cells under similar conditions. This suggests different minimal HK-II requirements for protection against hyperoxia vs. H2O2 and, in particular, suggests that greater levels of HK-II expression may be required to afford any protection against such an acute challenge with H2O2. Increasing the levels of HK-II expression beyond that seen in clones HK2-5 or HK2-10 did not provide significant additional protection against H2O2 or hyperoxia.
One might anticipate that glucose consumption would increase with elevated expression of HK-II. Notably, glucose consumption increases approximately threefold in A549 cells exposed to hyperoxia (3). Paradoxically, our studies did not demonstrate an additional increase in glucose consumption of the HK-II-overexpressing cells compared with nontransduced control cells or doxycycline-suppressed cells under hyperoxic conditions. The absence of any change in glucose consumption in the HK-II-overexpressing cells is consistent with other reports showing no change in glucose disposal in transgenic mice overexpressing HK-II alone or in combination with elevated expression of GLUT1 (17) or GLUT4 (24).
Cell death by necrosis, apoptosis, or necroptosis is preceded by mitochondrial dysfunction (5, 8). Therefore, we investigated the MMP in these cells at 4 days of hyperoxic exposure, a point in time before cell death had begun. At this stage, the MMP of cells exposed to hyperoxia is significantly higher than that of cells exposed to normoxia (21% O2). Furthermore, the MMP of HK-II-overexpressing cells in hyperoxia was higher than that of nontransduced control cells and the doxycycline-suppressed control cells under the same conditions. It is known that mitochondrial ATP is exchanged for cytosolic ADP to maintain membrane potential and prevent mitochondrial hyperpolarization (41). This ATP/ADP exchange is carried out by the adenine nucleotide translocator in the inner mitochondrial membrane and via the porins, or VDACs, on the outer mitochondrial membrane. Our results on the localization of HK-II in the cells using fluorescence microscopy shows its colocalization to mitochondria to be readily detected in cells expressing elevated levels of HK-II, both in 21 and 95% O2. This is consistent with earlier reports showing localization of HK-I and HK-II to the mitochondria (43, 44). Several studies have suggested that HK is associated with mitochondria at the VDACs (13, 27).
Given the ability of HKs to associate with mitochondria, it was not altogether surprising to find HK-II localized to the mitochondria in cells expressing elevated levels of HK-II, both in 21 and 95% O2. We speculate that this association confers a bioenergetically favorable advantage. Absence of changes in the percentage of bound HK-II to mitochondria in A549 and HK2-5+Dox cells exposed to 21 and 95% O2 may be due to low levels of HK-II present in the cells. Normally HK-I is the predominant HK isoform expressed in lung and lung cells. HK-I also has a higher binding affinity to mitochondria than HK-II. If HK-I and HK-II compete for the same binding sites on mitochondria, it is conceivable that such low levels of HK-II are unable to displace the HK-I already bound. An increase in the percentage of bound HK-II to mitochondria in the HK2-5 cells suggests that increased expression of HK-II increases its probability of binding to mitochondria. In addition, this suggests that binding of HK-II to mitochondria may contribute to protection of cells against oxidative insults. At this location, HK can utilize mitochondrially derived ATP (6). This ATP/ADP exchange is required for the maintenance of MMP.
The potential importance of HK was underscored by the recent findings of Gottlob et al. (14), who showed that the antiapoptotic activity of Akt requires only the first, rate-limiting step of glucose metabolism catalyzed by HK, namely the phosphorylation of glucose. Interestingly, their findings also demonstrate that even the nonmetabolizable but phosphorylatable form of glucose analog 2-deoxyglucose is sufficient to confer protection in their model, in which cell death was caused by ultraviolet irradiation combined with serum deprivation. However, the nonphosphorylatable and nonmetabolizable form of glucose, 5-thioglucose, could not confer this protection. The potential importance of mitochondrial association of HK was suggested in these studies as well. First, mitochondrion-associated HK was decreased by the apoptotic stimulus. Second, elevated expression of HK-I, which also can associate with the mitochondrion, also conferred protection. Thus others also have found that HK can provide cytoprotection even without an apparent impact on total cellular glucose utilization.
Considerable attention has been given to a potential role of the mitochondrial VDAC in modulating the cell death process (37) by interacting with either the proapoptotic proteins Bax and Bak or the antiapoptotic protein Bcl-XL. Bcl-XL also is known to regulate the membrane potential and volume homeostasis of mitochondria (42). Bcl-2 family proteins may modulate the cell death process by either opening or closing of the VDACs. At this point, it is not clear whether the binding of HK-II to mitochondria could mimic or alter the actions of the antiapoptotic or proapoptotic proteins, respectively, through interactions in the vicinity of the VDAC.
In our studies, there was a distinct change in the morphology of mitochondria exposed to hyperoxia, such that A549 cells exposed to hyperoxia had swollen mitochondria and undefined cristae. By contrast, HK-II-overexpressing cells exposed to hyperoxia had mitochondria that were of considerably smaller size, comparable with their counterparts in 21% O2-exposed cells. Given the fact that HK-II-suppressed cells had an intermediate level of protection against hyperoxia compared with HK-II-overexpressing cells and control A549 cells exposed to hyperoxia, it is perhaps not surprising to find a similar pattern in the measurements of mitochondrial area. This further suggested a role for HK-II in protecting mitochondria.
In conclusion, elevated expression of HK-II can provide protection of lung-derived cells against hyperoxia and other oxidative insults such as H2O2. We suggest that HK-II provides this protection, at least in part, by maintaining the membrane potential and volume homeostasis of mitochondria. It is conceivable that the other HK isoform that can associate with mitochondria, HK-I, could have similar actions.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Matthew Strand for help in the statistical evaluation of mitochondrial area data. We are also grateful to Meghan Graves, Kellie Lansberry, and Gretchen Czapla for preparation of the manuscript and Boyd Jacobson for photographic work.
![]() |
FOOTNOTES |
---|
This work was supported by National Heart, Lung, and Blood Institutes Grant HL-52732 and HL-56263.
Address for reprint requests and other correspondence: C. W. White, 1400 Jackson St., Rm. B129, Denver, CO 80206 (E-mail: whitec{at}njc.org).
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.
May 3, 2002;10.1152/ajplung.00410.2001
Received 19 October 2001; accepted in final form 20 April 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ahmad, S,
White CW,
Chang LY,
Schneider BK,
and
Allen CB.
Glutamine protects mitochondrial structure and function in oxygen toxicity.
Am J Physiol Lung Cell Mol Physiol
280:
L779-L791,
2001
2.
Allen, CB,
Guo XL,
and
White CW.
Changes in pulmonary expression of hexokinase and glucose transporter mRNAs in rats adapted to hyperoxia.
Am J Physiol Lung Cell Mol Physiol
274:
L320-L329,
1998
3.
Allen, CB,
and
White CW.
Glucose modulates cell death due to normobaric hyperoxia by maintaining cellular ATP.
Am J Physiol Lung Cell Mol Physiol
274:
L159-L164,
1998
4.
Allen, RG,
and
Tresini M.
Oxidative stress and gene regulation.
Free Radic Biol Med
28:
463-499,
2000[ISI][Medline].
5.
Barazzone, C,
and
White CW.
Mechanisms of cell injury and death in hyperoxia: role of cytokines and Bcl-2 family proteins.
Am J Respir Cell Mol Biol
22:
517-519,
2000
6.
Ceaser, M,
and
Wilson JE.
Further studies on the coupling of mitochondrially bound hexokinase to intramitochondrially compartmented ATP, generated by oxidative phosphorylation.
Arch Biochem Biophys
350:
109-117,
1998[ISI][Medline].
7.
Chang, PY,
Jensen J,
Printz RL,
Granner DK,
Ivy JL,
and
Moller DE.
Overexpression of hexokinase II in transgenic mice. Evidence that increased phosphorylation augments muscle glucose uptake.
J Biol Chem
271:
14834-14839,
1996
8.
Desagher, S,
and
Martinou JC.
Mitochondria as the central control point of apoptosis.
Trends Cell Biol
10:
369-377,
2000[ISI][Medline].
9.
Franek, WR,
Horowitz S,
Stansberry L,
Kazzaz JA,
Koo HC,
Li Y,
Arita Y,
Davis JM,
Mantell AS,
Scott W,
and
Mantell LL.
Hyperoxia inhibits oxidant-induced apoptosis in lung epithelial cells.
J Biol Chem
276:
569-575,
2001
10.
Freeman, BA,
and
Crapo JD.
Biology of disease: free radicals and tissue injury.
Lab Invest
47:
412-426,
1982[ISI][Medline].
11.
Freeman, BA,
and
Crapo JD.
Hyperoxia increases oxygen radical production in rat lungs and lung mitochondria.
J Biol Chem
256:
10986-10992,
1981
12.
Gardner, PR,
Nguyen DDH,
and
White CW.
Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells and in rat lungs.
Proc Natl Acad Sci USA
91:
12248-12252,
1994
13.
Golshani-Hebroni, SG,
and
Bessman SP.
Hexokinase binding to mitochondria: a basis for proliferative energy metabolism.
J Bioenerg Biomembr
29:
331-338,
1997[ISI][Medline].
14.
Gottlob, K,
Majewski N,
Kennedy S,
Kandel E,
Robey RB,
and
Hay N.
Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase.
Genes Dev
15:
1406-1418,
2001
15.
Grupe, A,
Hultgren B,
Ryan A,
Ma YH,
Bauer M,
and
Stewart TA.
Transgenic knockouts reveal a critical requirement for pancreatic beta cell glucokinase in maintaining glucose homeostasis.
Cell
83:
69-78,
1995[ISI][Medline].
16.
Halseth, AE,
O'Doherty RM,
Printz RL,
Bracy DP,
Granner DK,
and
Wasserman DH.
Role of Ca2+ fluctuations in L6 myotubes in the regulation of the hexokinase II gene.
J Appl Physiol
88:
669-673,
2000
17.
Hansen, PA,
Marshall BA,
Chen M,
Holloszy JO,
and
Mueckler M.
Transgenic overexpression of hexokinase II in skeletal muscle does not increase glucose disposal in wild-type or Glut1-overexpressing mice.
J Biol Chem
275:
22381-22386,
2000
18.
Hecht, D,
and
Zick Y.
Selective inhibition of protein tyrosine phosphatase activities by H2O2 and vanadate in vitro.
Biochem Biophys Res Commun
188:
773-779,
1992[ISI][Medline].
19.
Heikkinen, S,
Pietila M,
Halmekyto M,
Suppola S,
Pirinen E,
Deeb SS,
Janne J,
and
Laakso M.
Hexokinase II-deficient mice. Prenatal death of homozygotes without disturbances in glucose tolerance in heterozygotes.
J Biol Chem
274:
22517-22523,
1999
20.
Hensley, K,
Robinson KA,
Gabbita SP,
Salsman S,
and
Floyd RA.
Reactive oxygen species, cell signaling, and cell injury.
Free Radic Biol Med
28:
1456-1462,
2000[ISI][Medline].
21.
Janssen, YMW,
Van Houten B,
Borm PJA,
and
Mossman BT.
Biology of disease: cell and tissue responses to oxidative damage.
Lab Invest
69:
261-274,
1993[ISI][Medline].
22.
Jobe, AJ.
The new BPD: an arrest of lung development.
Pediatr Res
46:
641-643,
1999[ISI][Medline].
23.
Kringstein, AM,
Rossi FM,
Hofmann A,
and
Blau HM.
Graded transcriptional response to different concentrations of a single transactivator.
Proc Natl Acad Sci USA
95:
13670-13675,
1998
24.
Lombardi, AM,
Moller D,
Loizeau M,
Girard J,
and
Leturque A.
Phenotype of transgenic mice overexpressing GLUT4 and hexokinase II in muscle.
FASEB J
11:
1137-1144,
1997
25.
Luo, X,
Christie NA,
McLaughlin MA,
Belcastro R,
Sedlackova L,
Cabacungan J,
Freeman BA,
and
Tanswell AK.
H2O2 mediates O2 toxicity in cultured fetal rat distal lung epithelial cells.
Free Radic Biol Med
26:
1357-1368,
1999[ISI][Medline].
26.
Macho, A,
Decaudin D,
Castedo M,
Hirsch T,
Susin SA,
Zamzami N,
and
Kroemer G.
Chloromethyl-X-Rosamine is an aldehyde-fixable potential-sensitive fluorochrome for the detection of early apoptosis.
Cytometry
25:
333-340,
1996[ISI][Medline].
27.
Nakashima, RA,
Mangan PS,
Colombini M,
and
Pedersen PL.
Hexokinase receptor complex in hepatoma mitochondria: evidence from N,N'-dicyclohexylcarbodiimide-labeling studies for the involvement of the pore-forming protein VDAC.
Biochemistry
25:
1015-1021,
1986[ISI][Medline].
28.
O'Brodovich, HM,
and
Mellins RB.
Bronchopulmonary dysplasia. Unresolved neonatal acute lung injury.
Am Rev Respir Dis
132:
694-709,
1985[ISI][Medline].
29.
O'Doherty, RM,
Bracy DP,
Granner DK,
and
Wasserman DH.
Transcription of the rat skeletal muscle hexokinase II gene is increased by acute exercise.
J Appl Physiol
81:
789-793,
1996
30.
O'Doherty, RM,
Bracy DP,
Osawa H,
Wasserman DH,
and
Granner DK.
Rat skeletal muscle hexokinase II mRNA and activity are increased by a single bout of acute exercise.
Am J Physiol Endocrinol Metab
266:
E171-E178,
1994
31.
Printz, RL,
Koch S,
Potter LR,
O'Doherty RM,
Tiesinga JJ,
Moritz S,
and
Granner DK.
Hexokinase II mRNA and gene structure, regulation by insulin, and evolution.
J Biol Chem
268:
5209-5219,
1993
32.
Rempel, A,
Mathupala SP,
and
Pedersen PL.
Glucose catabolism in cancer cells: regulation of the type II hexokinase promoter by glucose and cyclic AMP.
FEBS Lett
385:
233-237,
1996[ISI][Medline].
33.
Riddle, SR,
Ahmad A,
Ahmad S,
Deeb SS,
Malkki M,
Schneider BK,
Allen CB,
and
White CW.
Hypoxia induces hexokinase II gene expression in human lung cell line A549.
Am J Physiol Lung Cell Mol Physiol
278:
L407-L416,
2000
34.
Salotra, PT,
and
Singh VN.
Regulation of glucose metabolism in rat lung: subcellular distribution, isozyme pattern, and kinetic properties of hexokinase.
Arch Biochem Biophys
216:
758-764,
1982[ISI][Medline].
35.
Salotra, PT,
and
Singh VN.
Regulation of glucose metabolism in the lung: hexokinase-catalyzed phosporylation, a rate-limiting step.
Life Sci
31:
791-794,
1982[ISI][Medline].
36.
Schoonen, WGEJ,
Wanamarta AH,
van der Klei-van Moorsel JM,
Jakobs C,
and
Joenje H.
Respiratory failure and stimulation of glycolysis in Chinese hamster ovary cells exposed to normobaric hyperoxia.
J Biol Chem
265:
11118-11124,
1990
37.
Shimizu, S,
Narita M,
and
Tsujimoto Y.
Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC.
Nature
399:
483-487,
1999[ISI][Medline].
38.
Sui, D,
and
Wilson JE.
Structural determinants for the intracellular localization of the isozymes of mammalian hexokinase: intracellular localization of fusion constructs incorporating structural elements from the hexokinase isozymes and the green fluorescent protein.
Arch Biochem Biophys
345:
111-125,
1997[ISI][Medline].
39.
Terauchi, Y,
Sakura H,
Yasuda K,
Iwamoto K,
Takahashi N,
Ito K,
Kasai H,
Suzuki H,
Ueda O,
Kamada N,
Jishage K,
Komeda K,
Noda M,
Kanazawa Y,
Taniguchi S,
Miwa I,
Akanuma Y,
Kodama T,
Yazaki Y,
and
Kadowaki T.
Pancreatic beta-cell-specific targeted disruption of glucokinase gene. Diabetes mellitus due to defective insulin secretion to glucose.
J Biol Chem
270:
30253-30256,
1995
40.
Ueda, N,
and
Shah SV.
Endonuclease-induced DNA damage and cell death in oxidant injury to renal tubular epithelial cells.
J Clin Invest
90:
2593-2597,
1992[ISI][Medline].
41.
Vander Heiden, MG,
Chandel NS,
Schumacker PT,
and
Thompson CB.
BcL-xL prevents cell death following growth factor withdrawal by facilitating mitochondrial ATP/ADP exchange.
Mol Cell
3:
159-167,
1999[ISI][Medline].
42.
Vander Heiden, MG,
Chandel NS,
Williamson EK,
Schumacker PT,
and
Thompson CB.
Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria.
Cell
91:
627-637,
1997[ISI][Medline].
43.
Wilson, JE.
Hexokinases.
Rev Physiol Biochem Pharmacol
126:
65-198,
1995[ISI][Medline].
44.
Wilson, JE.
An introduction to the isoenzymes of mammalian hexokinase types I-III.
Biochem Soc Trans
25:
103-107,
1997[ISI][Medline].