Improved oxygenation promotes CFTR maturation and trafficking in MDCK monolayers

Zsuzsanna Bebök1,2, Albert Tousson3, Lisa M. Schwiebert2,4, and Charles J. Venglarik2,4

Departments of 1 Medicine, 4 Physiology and Biophysics, and 3 Cell Biology and 2 Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Culturing airway epithelial cells with most of the apical media removed (air-liquid interface) has been shown to enhance cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- secretory current. Thus we hypothesized that cellular oxygenation may modulate CFTR expression. We tested this notion using type I Madin-Darby canine kidney cells that endogenously express low levels of CFTR. Growing monolayers of these cells for 4 to 5 days with an air-liquid interface caused a 50-fold increase in forskolin-stimulated Cl- current, compared with conventional (submerged) controls. Assaying for possible changes in CFTR by immunoprecipitation and immunocytochemical localization revealed that CFTR appeared as an immature 140-kDa form intracellularly in conventional cultures. In contrast, monolayers grown with an air-liquid interface possessed more CFTR protein, accompanied by increases toward the mature 170-kDa form and apical membrane staining. Culturing submerged monolayers with 95% O2 produced similar improvements in Cl- current and CFTR protein as air-liquid interface culture, while increasing PO2 from 2.5% to 20% in air-liquid interface cultures yielded graded enhancements. Together, our data indicate that improved cellular oxygenation can increase endogenous CFTR maturation and/or trafficking.

cystic fibrosis; cellular polarization; hypoxia; cell culture methods


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) is an epithelial anion channel that requires cAMP-dependent phosphorylation plus intracellular ATP to open (19, 46, 50). These channels are normally expressed in the apical membranes of epithelial cells lining the airways, intestines, pancreatic ducts, and kidney tubules (19, 46, 33, 56). Mutations in the gene coding for CFTR impair epithelial Cl- and HCO3- transport and cause cystic fibrosis (CF). Most CF patients (>90%) have an allele coding for a mutant CFTR that lacks phenylalanine at the 508 position (19, 46). Delta F508 CFTR retains function as a protein kinase A- and ATP-regulated anion channel (9) but is absent from the plasma membranes of most cells used for heterologous expression (6, 14). Hence, many CF patients are expected to benefit from increased Delta F508-CFTR expression.

Morris et al. (34, 35) identified an important caveat regarding heterologous expression of CFTR. Specifically, they showed that wild-type CFTR was not present in the plasma membranes of HT-29 intestinal epithelial cells unless the cells were confluent and polarized. Furthermore, the change in CFTR localization to the cell surface was not mediated by a change in transcription or translation, since polarized and nonpolarized HT-29 cells contained similar amounts of CFTR mRNA and protein (34, 35). These data suggest that the trafficking of CFTR to the apical cell surface is highly regulated. There are also a number of reports showing that detectable amounts of endogenous CFTR protein is present at the apical membranes of epithelial cells from Delta F508-homozygous CF patients (14, 15, 28, 44, 49, 62). Thus it is likely that additional factors regulate endogenous CFTR maturation and cell surface localization in epithelial cells. The aims of this study were to test this hypothesis and to gain some insight regarding how endogenous CFTR expression may be increased.

Growing primary or immortalized airway epithelial cells on filters with nearly all of the apical fluid removed has been shown to cause a six- to eightfold increase in CFTR-mediated anion secretion compared with conventional (submerged) controls (30, 52). This maneuver has been referred to as air-liquid interface culture (27, 31, 36, 41, 43) or air interface culture (30, 52). The mediator responsible for the increased Cl- or HCO3- secretory response following air-liquid interface culture and its possible effects on CFTR remains unknown. Since most conventional cell cultures are hypoxic (8, 12, 40, 59), we hypothesized that improved cellular oxygenation may enhance CFTR expression.

We used a subclone of the Madin-Darby canine kidney (MDCK) cell line as a model to test this hypothesis. MDCK cells from American Type Culture Collection (23) consist of at least two strains or types (38, 45). Type I MDCK cells form high-resistance monolayers that demonstrate small Cl- secretory currents following addition of cAMP-mediated agonists, whereas type II MDCK cells form low-resistance monolayers and do not respond to cAMP (5, 32, 45, 51). Recent studies indicate that type I clones endogenously express small amounts of CFTR under conventional culture conditions (32). Thus subclones of type I MDCK cells may provide a good model to test for possible enhancement of CFTR expression.

In the present study, we show that culturing type I MDCK cells with an air-liquid interface caused a 50-fold increase in forskolin-stimulated Cl- secretion over a period of 4-5 days in culture. On the basis of this observation, we then tested the notion that the increased Cl- secretory response was mediated by enhanced CFTR expression. Finally, we exposed submerged or air-liquid interface cultures to varying amounts of O2 to determine whether the effects of air-liquid interface culture were mediated by improved oxygenation. The data presented here show that increasing oxygen from hypoxic to atmospheric levels markedly enhanced CFTR maturation and apical membrane localization. Our observations have important implications since cultured cells (39, 47, 53, 59) and CF patients (18, 24, 57) are often hypoxic.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MDCK-AA7 cells and culture methods. We obtained the type I MDCK subclone, MDCK-AA7 (16, 38), from the University of Alabama at Birmingham Cystic Fibrosis Research Center. Dr. Tom Ecay (Dept. of Physiology, East Tennessee State Univ.) also kindly provided MDCK-AA7 cells for study (16). We grew these cells in MEM containing Earle's salts, L-glutamine, and nonessential amino acids (GIBCO BRL, Grand Island, NY), supplemented with 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA or HyClone, Logan, UT) (23). Medium was aspirated and replaced at 2-day intervals.

Cells were passaged by pretreatment with a Ca2+- and Mg2+-free phosphate-buffered saline (PBS) that contained 1 mM EDTA for 20-30 min, followed by a 10-min incubation in the same PBS plus 0.05% trypsin. Dispersed cells were seeded at a density of 2-7 × 104 cells/cm2 on 0.45-µm Millicell-HA filters (Millipore, Bedford, MA) or 0.40-µm Falcon PET filters (Becton Dickinson, Franklin Lakes, NJ) for use in experiments. MDCK-AA7 cells were maintained with fluid on both sides for 1 wk after seeding to permit monolayer formation. Thereafter, most of the apical media were gently aspirated and not replaced. Previous studies have shown that airway cells grown under these conditions remained hydrated by a thin (~15 µm) fluid layer (27). Some filters were maintained in conventional (submerged) culture to serve as controls. In initial studies, cells were grown in a humidified incubator that contained 5% CO2-95% room air at 37°C. In some later experiments, filters were cultured in sealed plastic chambers that were gassed daily with mixtures of 5% CO2 and 2.5%, 5.2%, 12%, 20%, or 95% O2 plus balance N2 to test for possible effects of cellular oxygenation. Reductions in PO2 were verified by testing the media with an Instrumentation Laboratory 1640 blood gas analyzer.

Transepithelial short-circuit current measurements. Filters were mounted in modified Ussing chambers (Jim's Instruments, Iowa City, IA) and bathed on both sides with identical HEPES-buffered saline solutions that contained (in mM) 130 NaCl, 5 sodium pyruvate, 4 KCl, 1 CaCl2, 1 MgCl2, 5 D-glucose, and 5 HEPES-NaOH (pH 7.4). Bath solutions were stirred vigorously and gassed with room air. Solution temperature was maintained at 37°C. Short-circuit current (Isc) measurements were obtained by using an epithelial voltage clamp (VCC-600; Physiologic Instruments, San Diego, CA). A 10-mV pulse of 1-s duration was imposed every 100 s to monitor the transepithelial resistance (Rt), which was calculated using Ohm's law. Amiloride (100 µM) was routinely added to the apical solutions to abolish Na+ absorption (3). Forskolin (10 µM) was then added to both bathing solutions to stimulate cAMP-mediated Cl- secretion (51). Cl- secretory currents were identified on the basis of activation by forskolin and by sensitivity to basolateral BaCl2 (5 mM), bumetanide (10 µM), or ouabain (100 µM) (1, 5, 10, 51). All drugs were added as a small volume of a concentrated stock solution. Amiloride (10 mM), BaCl2 (0.5 M), and ouabain (10 mM) stocks were made up in water. Forskolin (10 mM) was dissolved in ethanol while bumetanide (3 mM) was dissolved in 0.06 N NaOH.

CFTR immunoprecipitation. The methods used to immunoprecipitate CFTR have been previously described in detail (2, 42). Briefly, cells were washed with ice-cold PBS that contained 1 mM CaCl2 and MgCl2 and lysed in a buffer that contained 150 mM NaCl, 20 mM HEPES, 1 mM EDTA, and 1% Nonidet P-40, supplemented with a protease inhibitor cocktail (Complete mini; Boehringer Mannheim, Indianapolis, IN). Protein concentration was determined using Protein Assay Reagent (Bio-Rad). Lysates that contained 300 µg of total protein were then immunoprecipitated at 4°C for 2 h using an anti-CFTR COOH-terminal monoclonal antibody (Genzyme, Framington, MA) and protein G-agarose beads (Boehringer Mannheim). Immunoprecipitated proteins were phosphorylated using [32P]ATP (DuPont-NEN) plus the catalytic subunit of cAMP-dependent protein kinase (Promega) and separated on 6% polyacrylamide gels (Novex). Gels were placed on a PhosphoScreen and analyzed with a PhosphorImager (Molecular Dynamics). Images were further processed using IPLab Spectrum software (Signal Analytics).

CFTR localization by confocal immunofluorescence microscopy. MDCK-AA7 cells were grown on transparent cyclopore filters (Falcon). Two primary anti-CFTR antibodies were used: a rabbit polyclonal antibody against the first nucleotide binding domain (NBD) that was kindly provided by Dr. Eric Sorscher (Univ. of Alabama at Birmingham) (2, 42) and a monoclonal antibody specific to the regulatory domain (R-domain) from Genzyme. Monolayers probed with anti-NBD antibody were fixed initially in methanol at -20°C. Monolayers probed with anti-R-domain antibody underwent preliminary fixation in methanol:acetic acid (3:1) at -20°C. Formaldehyde (3%) in PBS was used to complete fixation for all specimens. Nonspecific protein binding was blocked with 1% (wt/vol) bovine serum albumin. Oregon green-labeled anti-rabbit IgG or Texas red X-labeled anti-mouse IgG (Molecular Probes) were used as secondary antibodies. Samples were counterstained with the nuclear dye bisbenzimide. Filters were cut and folded cell-side out during mounting to enable cross-sectional views (35). CFTR immunolocalization was examined using an Olympus IX70 inverted epifluorescence microscope equipped with a step motor, filter wheel assembly (Ludl Electronics Products, Hawthorn, NY), and 83,000 filter set (Chroma Technology, Brattleboro, VT). Images were captured with a SenSys-cooled charge-coupled device high-resolution digital camera (Photometrics, Tucson, AZ). Partial deconvolution of images was performed using IPLab Spectrum software (Scanalytics, Fairfax, VA).

Analysis of CFTR mRNA by RT-PCR. Total RNA was isolated from filter-grown MDCK-AA7 monolayers using TRIzol (GIBCO BRL). Contamination of genomic DNA was eliminated using 1 U DNase (GIBCO BRL) per microgram of total RNA. One microgram of the DNase-treated RNA was then reverse transcribed in a reaction that contained 200 U Moloney murine leukemia virus RT (GIBCO BRL), 0.5 µg oligo(dT) primer, 0.5 mM dNTP, and 25 U RNase inhibitor (RNAsin; Promega, Madison, WI). cDNA samples were amplified for CFTR in a reaction that contained 0.2 µg cDNA, 1 U Taq polymerase (Perkin Elmer, Norwalk, CT), 200 µM dNTP, and 20 pmol each of the following PCR primers: 5'-GAG GAC ACT GCT CCT ACA C-3' and 5'-CAG ATT AGC CCC ATG AGG AG-3' (GIBCO BRL). These primers span the region between CFTR nt 531 and 778. Reactions for CFTR were cycled as follows: initial melt at 95°C for 5 min, 40 cycles of 95°C for 1 min, 58°C for 1 min, 72°C for 2 min, and a final extension at 72°C for 10 min. The expected size for the CFTR product is 248 bp.

We amplified the cDNA products for the housekeeping gene beta -actin as a control. Amplification of beta -actin consisted of mixing 0.2 µg of each cDNA sample with 1 U Taq polymerase (Perkin Elmer), 200 mM dNTP, and 20 pmol each of the PCR primers: 5'-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3' and 5'-CTA GAA GCA TTG CGG TGG ACG ATG GAG GG-3' (GIBCO BRL). These primers span nucleotides from 2133 to 2822 of the beta -actin cDNA. PCR reactions for beta -actin were cycled with an initial melt at 95°C for 5 min; 30 cycles of 95°C for 30 s; 46°C for 1 min; 72°C for 1 min; and a final extension at 72°C for 10 min. The expected fragment size for the beta -actin product is 690 bp.

Materials. Salts, buffers, and all other reagents were purchased from Sigma-Aldrich unless otherwise noted. Aqueous solutions were made with MILLI-Q water (Millipore).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Air-liquid interface culture induces a large Cl- secretory response in MDCK-AA7 monolayers. Figure 1 compares representative Isc records from MDCK-AA7 monolayers grown either with the apical side submerged by culture medium (solid line) or with an apical air-liquid interface for 4 days (dashed line). Forskolin (10 µM) was added to both sides to increase cellular cAMP (43). The forskolin-stimulated currents were inhibited by basolateral addition of either barium (5 mM, see Fig. 1), bumetanide (10 µM, not shown), or ouabain (100 µM, not shown). The effects of forskolin, barium, bumetanide, and ouabain indicate that the forskolin-stimulated Isc provides a direct measure of electrogenic Cl- secretion (1, 5, 10, 45, 51, 52, 60). These results also agree with those of previous studies of type I MDCK cells (5, 45, 51). Electrogenic Na+ absorption did not contribute to Isc, since apical amiloride (100 µM) had no effect on Isc when added before (Fig. 1) or after forskolin (not shown). Both filters that produced the records shown in Fig. 1 were seeded with ~5 × 104 cells/cm2, and both were cultured under standard conditions for 1 wk. One monolayer was then exposed to an air-liquid interface for 4 days by having most of the apical fluid removed and not replaced, while the other monolayer remained submerged under >2.5 mm of medium. Thus the imposition of an apical air-liquid interface for 4 days markedly enhanced forskolin-stimulated Cl- secretion.


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Fig. 1.   Comparison of the forskolin-stimulated short-circuit current (Isc) across a Madin-Darby canine kidney (MDCK)-AA7 monolayer cultured for 4 days with an apical air-liquid interface (dashed line) or with a paired medium-submerged control (solid line). The Millicell-HA filters with attached monolayers were bathed on both sides with identical HEPES-buffered saline solutions and voltage clamped to 0 mV as described in MATERIALS AND METHODS. Amiloride (100 µM) was used to abolish possible Isc due to active Na+ absorption (3). Forskolin (10 µM) was then added to both sides to increase cellular cAMP and stimulate electrogenic Cl- secretion (52). We added basolateral BaCl2 (5 mM) to inhibit Cl- secretory currents (1). Amiloride, forskolin, and BaCl2 were present continuously in the bathing solutions following addition. The maximum Isc response was 1 µA/cm2 for the monolayer culture fluid submerged, compared with 47.2 µA/cm2 for the monolayer cultured with an air-liquid interface. This experiment has been repeated >60 times with similar results (also see Fig. 2).

Figure 2 summarizes the relationships between the forskolin-stimulated Isc or basal Rt and number of days that monolayers were grown with an air-liquid interface. All of the time-paired submerged controls (n = 24) behaved similarly and are plotted at day 0. Figure 2 (left) shows that growing MDCK-AA7 cells with an apical air-liquid interface caused a 50-fold increase in forskolin-stimulated Cl- secretion that plateaued between days 4 and 5. In contrast, the control monolayers grown by conventional technique possessed relatively small forskolin-stimulated currents (0.8 ± 0.4 µA/cm2, n = 24). The enhanced response observed in air-liquid interface-grown monolayers compared with cells grown by conventional culture suggests that CFTR Cl- channel activity increased.


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Fig. 2.   The forskolin-stimulated short-circuit current (Delta Isc-forskolin) and basal transepithelial resistance (Rt) plotted as a function of the number of days the monolayers were cultured with most of the apical media removed (air-liquid interface). Delta Isc-forskolin was calculated as the difference between the basal or resting Isc and the maximum Isc that was evoked by forskolin (10 µM). Refer to Fig. 1 and MATERIALS AND METHODS for further details regarding the experimental design and conditions. Rt was calculated from the magnitude of the current pulse arising from a 10-mV change in holding potential using Ohm's law. The basal Rt before forskolin addition provides an estimate of the junctional resistance. All of the time-paired controls (n = 24) behaved similarly, having low forskolin-stimulated Isc (0.8 ± 0.4 µA/cm2) and high Rt (2,250 ± 650 Omega cm2). These data were combined and are plotted as the initial (day 0) points; n = 7 for all other points, and error bars depict SD.

Comparison of the amount, biochemical form, and subcellular localization of CFTR. One possible explanation for the results shown in the previous section is that growth with an air-liquid interface increased the number of CFTR Cl- channels in the apical membranes. As an initial test of this hypothesis, immunoprecipitation was used to determine whether air-liquid interface culture had any effect on the amount and/or biochemical properties of CFTR. Figure 3 compares the immunoprecipitated CFTR derived from conventional, fluid-immersed monolayers (fluid) with the CFTR obtained from air-interface monolayers (air). CFTR immunoprecipitated from filter-grown T84 cells served as a control (1, 10). Submerged MDCK-AA7 monolayers expressed only a small amount of CFTR. Furthermore, much of the protein from submerged monolayers had an electrophoretic mobility corresponding to a 140-kDa molecular weight protein. In contrast, the MDCK-AA7 monolayers grown with an air-liquid interface for 5 days showed an increase in total CFTR protein level as well as a shift toward a higher molecular weight (~170 kDa). Previous studies indicate that a 140-kDa band B form of CFTR represents an immature, core-glycosylated protein, whereas a 170-kDa band C form is fully glycosylated (6, 14). Thus the increase in the total amount of CFTR protein and the increase in molecular weight following air-liquid interface culture suggest that the 50-fold increase in forskolin-stimulated Isc is mediated, at least in part, by enhanced maturation.


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Fig. 3.   Immunoprecipitation of cystic fibrosis transmembrane conductance regulator (CFTR) from lysates of MDCK-AA7 cells grown either fluid submerged (fluid) or with an air-liquid interface (air) for 5 days. 32P-phosphorylated CFTR immunoprecipitates were derived from equivalent amounts of lysates from either MDCK-AA7 monolayers cultured under these 2 conditions or control T84 cells using an anti-CFTR COOH-terminal antibody. We treated the MDCK-AA7 air-liquid interface lysate with preimmune serum as a negative control. Samples were analyzed on a 6% polyacrylamide SDS gel. These data are representative of 4 experiments.

Next, we performed immunocytochemistry and digital confocal microscopy to gain insight regarding possible changes in CFTR staining patterns following air-liquid interface culture. Figure 4 compares monolayers cultured either conventionally (submerged) or exposed to an air-liquid interface for 6 days. On the basis of the immunoprecipitation data shown in Fig. 3 and previous studies (2, 6, 14, 46), we expected CFTR to be located intracellularly in MDCK-AA7 monolayers grown using conventional methodology. Indeed, punctate CFTR immunofluorescence was localized within a perinuclear compartment of the submerged cells. Together, the results shown in Figs. 3 and 4 suggest that CFTR protein is translated but not fully processed and/or trafficked to the cell surface in submerged monolayers. In contrast, Fig. 4 (right; air-liquid interface) shows that considerable amounts of CFTR were present at the apical membranes of MDCK-AA7 monolayers grown without apical media for 6 days.


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Fig. 4.   CFTR immunolocalization in monolayers either grown under conventional conditions (submerged) for 13 days or submerged for 1 wk followed by an apical air-liquid interface for 6 days. We used 2 different primary antibodies raised against CFTR that are labeled R-Domain or Nucleotide Binding Domain (NBD). The anti-CFTR antibodies were then labeled with either Texas red X (R-Domain) or Oregon green 488, and the nuclei counterstained blue with bisbenzimide. Fixed and immunologically stained monolayers were folded to permit the cross-sectional views shown above the en face views. All images were obtained at the same magnification, and the bar = 40 µm.

Analysis of CFTR mRNA between submerged and air-liquid interface monolayers by RT-PCR. We then performed semiquantitative RT-PCR analysis to determine whether air-interface culture augmented CFTR transcription. Figure 5 shows representative results. Triplicate monolayers were assayed for CFTR mRNA after either 2 or 4 days of growth with an apical air-liquid interface (air). These were compared with time-matched triplicate fluid-submerged monolayers (fluid). All MDCK-AA7 monolayers contained low levels of CFTR mRNA compared with control 16HBE14o- airway cells (60). These results are consistent with other studies showing that only low levels of CFTR mRNA can be detected in native epithelial cells (55, 61). Furthermore, the data presented in Fig. 5 show that the 50-fold increase in forskolin-stimulated Isc (Figs. 1 and 2) and the change in CFTR protein (Figs. 3 and 4) observed after air-liquid interface culture were not accompanied by a 50-fold enhancement of CFTR mRNA. These data are consistent with the changes in CFTR mobility and cellular localization shown in the previous section that strongly implicate a posttranscriptional mechanism.


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Fig. 5.   Expression of CFTR mRNA in submerged (fluid) vs. air-liquid interface (air) MDCK-AA7 cultures as determined by RT-PCR. cDNA derived from triplicate MDCK-AA7 monolayers was amplified for CFTR with specific primers spanning the region between nt 531 and 778 (248 bp fragment). cDNA derived from the 16HBE14o- airway cell line was amplified for CFTR as a positive control (lane +); a reaction containing no template was included as a negative control (lane -). Samples were also amplified for beta -actin (690-bp fragment) to control for RNA degradation during DNase treatment and reverse transcription. M, lanes containing 100-bp markers.

Improvements in forskolin-stimulated Isc and CFTR expression are oxygen mediated. Removing the fluid from the apical surface of epithelial cells will give rise to a transepithelial hydrostatic pressure gradient, decrease the luminal volume, possibly dry and irritate the monolayer, increase apical cytokines, and permit more oxygen to reach the cells. To test the hypothesis that improved cellular oxygenation mediates the increase in CFTR maturation and cell surface expression, we cultured submerged MDCK-AA7 monolayers in a humidified atmosphere containing 95% O2 plus 5% CO2 for 5-6 days. Figure 6 compares representative records from monolayers grown under identical conditions except for the culture atmosphere, which contained either 95% O2 (dashed line) or room O2 (solid line). The forskolin-activated current observed following 5-6 days of incubation with 95% oxygen (48 ± 4 µA/cm2, n = 6) was markedly elevated compared with the control. The results with 95% O2 are comparable to the paired control 5- to 6-day responses for air-liquid interface cultures (40.2 ± 8.7 µA/cm2, n = 14). Since both monolayers were grown under identical conditions except for a fivefold increase in PO2, we conclude that increased oxygen mediates the 50-fold increase in Cl- secretion. Immunoprecipitation and immunolocalization of CFTR further revealed that culturing submerged monolayers with 95% oxygen produced similar changes in the mobility and cellular localization of CFTR as air-liquid interface culture (data not shown, also refer to Fig. 8).


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Fig. 6.   A fivefold increase in atmospheric O2 enhances CFTR-mediated Isc across submerged MDCK-AA7 monolayers. These records show 2 representative experiments comparing the effect of forskolin (10 µM) on Isc across submerged filters cultured with either 95% oxygen and 5% CO2 for 5 days (dashed line) or with room air plus 5% CO2 (solid line). Both monolayers were submerged by ~200 µl of media on the apical side corresponding to a depth of ~2.5 mm. This experiment was repeated 6 times with similar results (see text). Additional details regarding the experimental design and methods are provided in the legend for Fig. 1 and MATERIALS AND METHODS.

These data demonstrate that CFTR cell surface expression is increased by improved oxygenation in MDCK-AA7 cells. However, it is not certain from the data shown in Fig. 6. if this enhancement occurs at hypoxic, normoxic, or hyperoxic levels. Thus we grew air-liquid interface cultures in atmospheres containing 2.5%, 5.2%, 12%, or 20% O2 plus 5% CO2 and balance N2 for 4-5 days. Figure 7 summarizes data from functional studies. This plot shows that forskolin-stimulated Isc increased in a graded manner over the range of PO2 tested. Results obtained from submerged monolayers (L) are plotted for comparison. Parallel studies were performed to assay for possible dose-dependent effects on CFTR protein using immunoprecipitation, and a representative gel is presented in Fig. 8 (top). These data show that increasing PO2 enhanced the ~170-kDa form of CFTR. The oxygen dependence of the increase in CFTR glycosylation was quantified by densitometry as shown in Fig. 8 (bottom). Using the change in CFTR function (Fig. 7) or biochemistry (Fig. 8) as a bioassay further suggests that conventional cultures of MDCK-AA7 cells are hypoxic. Finally, these data demonstrate that CFTR maturation and function at the cell surface can be increased over pathophysiological to physiological levels of cellular oxygenation.


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Fig. 7.   Delta Isc-forskolin demonstrates a graded response to increased oxygenation. Air-liquid interface cultures of MDCK-AA7 were exposed to atmospheres that contained 2.5%, 5.2%, 12%, or 20% O2 for 5 days. Each point represents the mean from 5 experiments, and error bars show SD. The response from conventional (submerged) monolayers (L) is plotted for comparison. Refer to MATERIALS AND METHODS and the legend for Fig. 1 for additional information regarding experimental conditions and design.



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Fig. 8.   CFTR protein maturation shows a graded response to increased O2 in air-liquid interface cultures. The top panel shows the CFTR immunoprecipitated from equivalent amounts of lysates from MDCK-AA7 cells cultured with an air-liquid interface in atmospheres that contained 2.5%, 5.2%, 12%, or 20% O2 (refer to Fig. 7). The immunoprecipitated protein from submerged monolayers (L) is plotted for comparison. The control experiment with atmospheric (20%) O2 was performed >7 times, and the increase in the higher mobility form of CFTR in this figure is not typical (refer to the lane with 12% O2 and Fig. 3 for comparison). Densitometry was used to quantify the increase in CFTR protein maturation to the ~170-kDa form as shown (bottom).

Reducing the apical media volume in culture increases forskolin-stimulated Isc. Since CFTR maturation and cell surface expression are augmented by oxygen in MDCK-AA7 cells, we further predict that the forskolin-stimulated Isc should increase as the volume of apical media is decreased. This prediction is based on the notion that the apical media acts as a barrier to oxygen diffusion (refer to DISCUSSION). We tested this hypothesis by growing MDCK-AA7 cells for 6 days with 40, 80, 160, or 300 µl of apical MEM and then assaying for forskolin-stimulated Isc. These data are summarized in Fig. 9. Indeed, the CFTR-mediated Isc (Delta Isc-forskolin) increased with decreasing volumes of media (left), and the plot of Delta Isc-forskolin as a function of volume-1 is suggestive of a linear relationship (right). The minimum depth tested that permitted maximum current was ~0.5 mm (i.e., 40 µl/0.8 cm2). Moreover, these data show that even modest changes in media volume (i.e., <20 µl) markedly altered this measure of CFTR activity. Thus we have identified an important caveat regarding the study of endogenous CFTR expression using conventional cell culture technique.


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Fig. 9.   Relationship between apical fluid volume and CFTR-mediated Cl- secretory response. We grew monolayers with 40, 80, 160, or 300 µl of medium on the apical side for 6 days (surface area = 0.8 cm2). The left panel plots the forskolin-stimulated Isc as a function of apical media volume (). Results from air-liquid interface cultures are shown for comparison (open circle ). To further define this relationship, we plotted the forskolin-stimulated Isc as a function of apical fluid volume-1 as shown (right). The line illustrates the fit of the data by simple linear regression (r = 0.98). Each point represents the mean, and error bars represent SD of 4 experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Hypoxia conditions in culture can have important consequences regarding CFTR trafficking and function. The large unstirred layer of medium overlaying cells in culture limits the amount of oxygen available for cellular respiration. Figure 10 illustrates how the partial pressure of O2 will vary as a linear relation of the media depth (d), given a steady state. This relationship is derived from a simple force-flow relation (i.e., Fick's law)
J<SC>o</SC><SUB><IT>2</IT></SUB><IT>=</IT><FR><NU><IT>k</IT></NU><DE><IT>d</IT></DE></FR><IT>·</IT>[(P<SC>o</SC><SUB><IT>2</IT></SUB>)<SUB>air</SUB><IT>−</IT>(P<SC>o</SC><SUB><IT>2</IT></SUB>)<SUB>cell</SUB>] (1)
where the flow of oxygen (JO2, in units of milliliters per minute per cm2 of cells) is directly proportional to the difference between PO2 at the air-media interface [(PO2)air] and the cells [(PO2)cell] and the O2 diffusion constant of Krough for water at 37°C (k = 3.4 × 10-5) (53), and is inversely related the media depth (d). Since oxygen flow (JO2) and consumption (r) are equivalent at steady state, rearrangement of Eq. 1 yields
d=<FR><NU>k·[(P<SC>o</SC><SUB><IT>2</IT></SUB>)<SUB>air</SUB><IT>−</IT>(P<SC>o</SC><SUB><IT>2</IT></SUB>)<SUB>cell</SUB>]</NU><DE>r</DE></FR> (2)
Stevens (53) derived this equation, estimated the rate of oxygen consumption for freshly isolated, confluent hepatocytes (r = 1.6 × 10-4 ml O2/min), and calculated that the fluid depth should not exceed 0.34 mm to maintain cellular PO2 within the low to normal range (i.e., 40 mmHg). Indeed, monolayers of L cells cultured in excess of this depth were shown to be hypoxic by polarographic analysis (59). More recently, Ostrowski and Nettesheim (41) varied the media overlaying airway cells and found that the maximum depth permitting airway ciliogenesis to be 0.5 mm, which is consistent with the value calculated by Stevens (0.34) as well as our results shown in Fig. 9 (~0.5 mm).


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Fig. 10.   Model illustrating the expected relationship between the partial pressure of oxygen (PO2) in cell culture and the fluid (media) depth (d). This model is based on a linear force-flow relation (e.g., Fick's law) and assumes that oxygen diffusion and utilization have reached steady state.

Maneuvers that improve oxygenation in cultures either by stirring the overlaying fluid layer using a rocking shaker (12, 47) or rolling bottle (20) or by reducing the medium depth with an air-liquid interface (27, 30, 36, 41, 43, 52, 54) enhance cellular growth, proliferation, and differentiation in a variety of cells. Hypoxia can also have important functional consequences. For example, monolayers of kidney proximal tubule epithelial cells are only gluconeogenic (39) and ammoniagenic (8) as observed in vivo when cells are cultured in the presence of increased oxygen levels. Similarly, pancreatic beta -cells have been shown to release insulin only in normoxic cultures (13). The data contained in the present study demonstrate that improved cellular oxygenation markedly augments CFTR function, maturation, and localization in a model cell line.

Assuming that MDCK-AA7 cells consume oxygen at a similar rate as hepatic (53), LLC-PK1 (8), 3T3 (53), or L cells (59), Eq. 2 predicts that confluent MDCK-AA7 monolayers submerged under our conventional conditions (i.e., 200 µl medium/0.8 cm2 area approx  2.5 mm depth) are hypoxic. We further expect that either removing the apical media or culturing these monolayers with an approximate fivefold increase in oxygen should restore cellular PO2 to approximately normoxic levels (53). Indeed, both maneuvers caused similar increases in CFTR-mediated Isc and cell surface expression. However, these data do not exclude the alternative hypothesis that conventional cultures are normoxic and that the effects of 95% O2 were mediated by hyperoxia (58). Thus we exposed air-liquid interface cultures to varying PO2 (Figs. 7 and 8). The use of CFTR as a bioassay indicates that conventional MDCK-AA7 cultures are hypoxic, as predicted by Eq. 2. Furthermore, these data show that CFTR maturation and function varies with physiological oxygenation. Interestingly, the increase in PO2 between arterial and venous blood may cause a two- to threefold enhancement of CFTR expression.

Comparing CFTR expression between cell lines. One question that arises from this study is, Why do conventional culture conditions nearly abolish CFTR expression in MDCK-AA7 cells, whereas T84 cells grown under identical conditions express high levels of mature CFTR protein (refer to Fig. 3)? One possible explanation comes from previous studies by Dickman and Mandel (12) and others showing that the poor oxygenation in culture favors the proliferation and growth of cells with enhanced glycolysis (4, 12, 37, 40). The selection and evolution in vitro is expected to be driven further by media formulated with high glucose (25 mM) and containing few substrates for oxidative phosphorylation. Indeed, T84 cells express few mitochondria (T. Ecay, personal communication). Hence differences in metabolism should be considered in future studies of wild-type and mutant CFTR.

Moreover, the ability of air-liquid interface culture to increase cAMP-dependent Cl- secretion is not limited to MDCK-AA7 cells. An augmented Cl- secretory response following air-liquid interface culture has been observed in three cell lines; Calu-3 (52), MDCK-AA7 (this article), and several T84 clones (Venglarik, unpublished data) as well as in primary cultures of airway epithelial cells (Ref. 30 and Venglarik, unpublished data). The observation that CFTR-expressing epithelial cells derived from airways, kidney, and colon respond similarly to air-liquid interface culture is suggestive of a relationship between oxygen tension and Cl- secretion in vivo. It is also likely that the mediator and cellular mechanisms underlying the enhanced Cl- secretory Isc are the same. Hence, this study provides important new insight regarding a relationship between cellular oxygenation and CFTR maturation and trafficking and identifies a model cell line to further investigate the underlying mechanisms.

Possible role of metabolism in CFTR expression. Previous studies show that vectorial transport is reduced by conditions that decrease oxidative phosphorylation, presumably to preserve cellular integrity (7,17). Therefore, we favor the hypothesis that oxygen-induced increase in CFTR expression in MDCK-AA7 cells may also be related to improved metabolism. We and others have shown that decreasing the cellular ATP/ADP ratio can acutely limit Cl- secretion by reducing the open probability of the CFTR Cl- channels (19, 50). The results presented in this article are suggestive of a more long-term mechanism whereby a decrease in oxygen can reduce the number of CFTR channels present in the apical membrane. Such an adaption may help conserve energy either during prolonged periods of moderate hypoxia or under conditions where ATP is being consumed by either vectorial transport or other metabolic pathways (17). There is already evidence that air-liquid interface culture markedly increases oxidative phosphorylation in primary cultures of bovine airway cells (31). MDCK-AA7 cells should provide a convenient model for future studies investigating the precise relationship between cellular metabolism and CFTR expression.

Relevance to cystic fibrosis. We have identified an important source of variability in CFTR cell surface localization that may explain why some cells have Delta F508 CFTR at the plasma membranes while others have none. Indeed, many freshly isolated epithelial cells and cultured epithelial cells grown with an air-liquid interface have functionally or biochemically detectable levels of Delta F508 CFTR at the cell surface (14, 28, 44, 49, 60, 62), whereas most conventional cultures do not (6, 11, 21, 25). In this regard, Sarkadi et al. (49) were the first to demonstrate that the 180-kDa form of Delta F508 CFTR was expressed in the apical membranes of primary and immortalized CF cell lines (49). On the basis of the oxygen dependence of wild-type CFTR expression in MDCK-AA7 cells, we consider it likely that some of the variation in Delta F508 CFTR cell surface expression is due to hypoxic culture conditions.

Furthermore, although there is evidence that the maturation and trafficking of Delta F508 CFTR may be defective in heterologous systems (6), little is known regarding the processes that normally regulate CFTR biogenesis. Our data show that oxygen can modulate endogenous CFTR expression posttranscriptionally. Oxygen is known to regulate the expression of several other proteins at this level (22, 26, 29, 48). For example, normoxia promotes the translation and/or folding of cytochrome c oxidase (22), while hypoxia abolishes the ubiquitin-mediated proteolysis of hypoxia-inducible factor 1alpha (26, 48) and stabilizes the plasma membrane form of the glucose transporter GLUT-3 (29). Increased synthesis, decreased degradation, or an extended half-life are three possible mechanisms for the O2-enhanced CFTR expression. We have been unable to detect augmented synthesis of CFTR in air-liquid interface cultures of MDCK-AA7 cells using 35S-met pulse chase analysis (data not shown). Hence, we speculate that the increased levels of fully glycosylated (170-kDa) CFTR may be due to increased maturation and/or stability at the apical plasma membrane. There is already some evidence to suggest that normoxia causes apical membranes to differentiate both functionally (47) and morphologically (30, 41). Thus it is possible that the improvement in CFTR biogenesis is mediated by a more global mechanism designed to regulate the composition and/or architecture of the apical membranes during differentiation.

Finally, many CF patients are hypoxic due to mucus plugs in the small airways and progressive loss of lung function (18, 24, 57). If Delta F508 CFTR cell surface expression depends on oxygenation, then there may be some correlation between nasal potential difference and pulmonary function among this subset of CF patients. Indeed, two recent reports show that the basal nasal potential difference was similar to nonaffected controls in a subpopulation of CF patients that have normal pulmonary function (18, 24). Although these results are somewhat controversial, they are consistent with studies localizing mutant CFTR at or near the apical membranes of Delta F508 homozygous patients (14, 44, 49, 60, 62). Thus CF patients with at least one Delta F508 allele (i.e., 92% of the total CF population) may benefit from maneuvers that improve tissue oxygenation or drugs that mimic oxygen signaling pathways in CFTR-expressing epithelial cells.


    ACKNOWLEDGEMENTS

We are grateful to Dr. Eric Sorscher for access to his labs and for support and encouragement. We thank Drs. Tom Ecay, Kim Barrett, and Eric Schwiebert for generously providing the MDCK-AA7, T84, and 16HBE14o- cell lines, respectively, and Drs. Eric Sorscher, Kevin Kirk, and Cathy Fuller for critique of earlier versions of this article. We are also indebted to Dr. Gerhard Giebisch and an anonymous reviewer for suggesting that we vary PO2 in air-liquid interface cultures.


    FOOTNOTES

This work was supported by the Cystic Fibrosis Foundation (VENGI97 and RDP464) and National Heart, Lung, and Blood Institute Grant HL-46943.

Address for reprint requests and other correspondence: C. J. Venglarik, Dept. of Environmental Health Sciences, School of Public Health, Univ. of Alabama at Birmingham, 793 McCallum, Birmingham, AL 35294-0005 (E-mail: cjv{at}uab.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9 April 1999; accepted in final form 24 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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