Elevated expression of hexokinase II protects human lung epithelial-like A549 cells against oxidative injury

Aftab Ahmad1, Shama Ahmad1, B. Kelly Schneider1, Corrie B. Allen1, Ling-Yi Chang2, and Carl W. White1

Departments of 1 Pediatrics and 2 Medicine, National Jewish Medical and Research Center, Denver, Colorado 80206


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

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.

Propidium iodide (PI) and Mitotracker orange (CMTMRos) dyes were obtained from Molecular Probes (Eugene, OR).

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- vector (expressing the tetracycline transactivator) using Fugene-6 as transfection reagent in a ratio of 1 µg of DNA to 3 µl of Fugene-6. A total of 15 µg of DNA were transfected per plate. Twenty-four hours posttransfection, the medium was changed, and 5.0 ml of fresh medium were added. The medium containing virus was collected 48 h posttransfection, filtered, and stored at -80°C.

Next, 50% confluent A549 cells in 100-mm plates were co-infected with 5.0 ml each of the virus-containing media obtained earlier from HK2-HERMES-HRI-GFP and RetroTet RTAb--transfected cell supernatants in the presence of 6 µg/ml of polybrene. After 51/2 h, 10 ml of fresh complete F12K medium were added. At 24 and 48 h after the first co-infection, second and third subsequent coinfections were carried out, respectively. Each time, fresh virus-containing medium was used. Then, 24 h after the last coinfection, the cells were split and allowed to grow in the absence of doxycycline for 2 additional days before they were sorted on a flow cytometer [fluorescence-activated cell sorter (FACS)]. Cells were sorted by FACS for content of GFP, and the GFP-positive population was replated. A general outline of the method is shown in Fig. 1. We repeated this selection process three additional times, each time selecting for cells expressing different levels of GFP. The cells expressing different levels of GFP were then analyzed for HK-II by activity gel and by Western blotting in the presence and absence of the suppressor doxycycline. Total HK activity also was determined by the spectrophotometric method.


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Fig. 1.   Flow diagram outlining method for generating hexokinase (HK)-II overexpressing cells. IRES, internal ribosome entry site; GFP, green fluorescent protein; gfp, enhanced GFP.

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 (MFI-CCCP - 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

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.


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Fig. 2.   Characterization of cells overexpressing HK-II. A: the cells were sorted for different HK-II levels on the basis of GFP content. Peak A shows untreated A549 cells. Peaks B and C represent the cells treated with doxycycline (Dox, 2 ng/ml) to suppress expression of HK-II/GFP. Peaks D-G show GFP expression in cells overexpressing HK-II in absence of Dox. B: the specific activity of total HK determined spectrophotometrically. HK2-5+Dox and HK2-10+Dox were treated with Dox (2 ng/ml) to suppress the expression of HK-II. * and triangle , Cell types with statistically significant (P < 0.05) difference by ANOVA from the control A549 cells in hyperoxia and in 21% O2, respectively. C: the same cells probed with HK-II COOH-terminal antibody in a Western blot (representative blot shown; for data see Selection of clones expressing GFP and HK-II). D: the activity of HK-II compared with the other HK isoforms in an activity gel.

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.


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Fig. 3.   Effect of hyperoxia on viability of cells expressing different levels of HK-II. A549 cells and cells overexpressing HK-II were exposed to 95% O2-5% CO2 for 6 days at Denver's altitude, and cell viability was assessed with propidium iodide. HK2-5+Dox and HK2-10+Dox were treated with Dox at a final concentration of 2 ng/ml to suppress their HK-II expression. A549 cells were also treated with Dox as a control. *Statistically significant (P < 0.05) difference by ANOVA from the hyperoxia-exposed A549 cells (n = 3/condition).

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.


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Fig. 4.   Effect of hydrogen peroxide (H2O2) on viability of cells expressing different levels of HK-II. A549 cells and cells overexpressing HK-II were exposed to 1 mM H2O2 for 48 h, and cell viability was assessed using propidium iodide by flow cytometry. HK2-5+Dox and HK2-10+Dox were treated with Dox (2 ng/ml) to suppress HK-II expression. A549 cells also were treated with Dox as a control. *Statistically significant (P < 0.05) difference by ANOVA from the H2O2-treated A549 cells (n = 3/condition).

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.


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Fig. 5.   Effect of hyperoxia on mitochondrial membrane potential of cells expressing different levels of HK-II. Cells overexpressing HK-II and control A549 cells were exposed to 95% O2-5% CO2 for 4 days at Denver's altitude. HK2-10D cells were treated with Dox (2 ng/ml) to suppress HK-II expression. The mitochondrial membrane potential of cells were determined with the dye Mitotracker orange (CMTMRos) and analyzed using flow cytometer as described in Mitochondrial membrane potential. *Statistically significant (P < 0.05) difference by ANOVA from the control A549 cells exposed to hyperoxia (n = 3/condition). MFI, mean fluorescence intensity; CCCP, carbonyl cyanide m-chlorophenylydrozone.

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.


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Fig. 6.   Electron micrographs showing A549 (A), Dox-treated (2 ng/ml) HK2-10 (B), and HK2-10 (C) cells exposed to 21% O2 for 144 h. Also shown are A549 (D), Dox-treated HK2-10 (E), and HK2-10 (F) cells exposed to 95% O2 for 144 h. Magnification, ×17,500. Bars at bottom center of B, E, and F indicate 1 µm. m, mitochondria; mi, inner mitochondrial membrane; g, Golgi apparatus; n, nucleus; lb, lamellar body.



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Fig. 7.   Effect of hyperoxia on the mitochondrial profile area of cells expressing different levels of HK-II. Cells overexpressing HK-II, those in which HK-II was suppressed by Dox, and control A549 cells were exposed to either 21% O2 (Air) or 95% O2 (Oxygen) for 6 days at Denver's altitude. #Statistically significant (P < 0.05) difference by ANOVA from the hyperoxia-exposed A549 cells; *statistically significant (P < 0.05) difference by ANOVA from the respective control cells in 21% O2 (n = 168-212/condition).

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.


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Fig. 8.   Fluorescence microscopy demonstrating distribution of HK-II protein and mitochondria [inner mitochondrial membrane (IMM)] in A549 cells (1, a-c: 21% O2; 4, a-c: 95% O2), HK2-5 cells treated with Dox (2, a-c: 21% O2; 5, a-c: 95% O2), and HK2-5 cells without Dox treatment (3, a-c: 21% O2; 6, a-c: 95% O2). HK-II staining (red) is shown in all "a" panels, IMM staining (green) in those labeled "b", and staining for both in "c." Convergence of red and green staining produces a yellow color indicating colocalization of HK-II and mitochondrial membranes. Colocalization was very minimally detected in 5c and readily detected in 2c, 3c, and 6c.



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Fig. 9.   Quantitation of HK-II bound to mitochondria. Cells overexpressing HK-II, those in which HK-II was suppressed by Dox, and control A549 cells were exposed to either 21 (Air) or 95% O2 (Oxygen) for 4 days at Denver's altitude. The percent HK-II bound to mitochondria was analyzed as described in Immunofluorescence staining of cells exposed to hyperoxia. *Statistically significant (P < 0.05) difference by ANOVA from all groups. triangle , Statistical difference from the control A549 cells in 21% O2 (n = 3-6/condition).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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