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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
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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). 
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 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 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.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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
-actin as a
control. Amplification of -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
-actin cDNA. PCR reactions for -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 -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).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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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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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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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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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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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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
(Isc-forskolin) increased with decreasing
volumes of media (left), and the plot of
Isc-forskolin as a function of
volume1 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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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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>]](/content/vol280/issue1/fulltext/C135/img001.gif)
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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>](/content/vol280/issue1/fulltext/C135/img002.gif)
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(2) |
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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-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 
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 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 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 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 F508 CFTR cell surface expression is due to
hypoxic culture conditions.
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 1
(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 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
F508 homozygous patients (14, 44, 49, 60, 62). Thus CF
patients with at least one 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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.
). Results from air-liquid interface
cultures are shown for comparison (
). To further define
this relationship, we plotted the forskolin-stimulated
Isc as a function of apical fluid
volume