Will Rogers Institute Pulmonary Research Center, Division of Pulmonary and Critical Care Medicine, University of Southern California, Los Angeles, California 90033
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ABSTRACT |
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We investigated the
effects of epidermal growth factor (EGF) on active
Na+ absorption by alveolar
epithelium. Rat alveolar epithelial cells (AEC) were isolated and
cultivated in serum-free medium on tissue culture-treated polycarbonate
filters. mRNA for rat epithelial Na+ channel (rENaC) -,
-,
and
-subunits and Na+ pump
1- and
1-subunits were detected in
day 4 monolayers by Northern analysis
and were unchanged in abundance in day
5 monolayers in the absence of EGF. Monolayers
cultivated in the presence of EGF (20 ng/ml) for 24 h from
day 4 to day
5 showed an increase in both
1 and
1
Na+ pump subunit mRNA but no
increase in rENaC subunit mRNA. EGF-treated monolayers showed parallel
increases in Na+ pump
1- and
1-subunit protein by immunoblot
relative to untreated monolayers. Fixed AEC monolayers demonstrated
predominantly membrane-associated immunofluorescent labeling with
anti-Na+ pump
1- and
1-subunit antibodies, with
increased intensity of cell labeling for both subunits seen at 24 h
following exposure to EGF. These changes in
Na+ pump mRNA and protein preceded
a delayed (>12 h) increase in short-current circuit (measure of
active transepithelial Na+
transport) across monolayers treated with EGF compared with untreated monolayers. We conclude that EGF increases active
Na+ resorption across AEC
monolayers primarily via direct effects on
Na+ pump subunit mRNA expression
and protein synthesis, leading to increased numbers of functional
Na+ pumps in the basolateral
membranes.
alveolar epithelium; epidermal growth factor; gene expression; rat epithelial sodium channel; sodium-potassium-adenosine triphosphatase
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INTRODUCTION |
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THE ALVEOLAR EPITHELIUM provides the major barrier to fluid and solute movement between the alveolar airspaces and pulmonary capillaries. Alveolar epithelial cells (AEC) actively transport Na+ from their apical to basolateral surfaces, thereby creating an osmotic gradient for alveolar fluid reabsorption (5, 16, 17). Current evidence indicates that the primary pathways for transepithelial Na+ transport in AEC are apical amiloride-sensitive epithelial Na+ channels (ENaC) and basolateral Na+ pumps (Na+-K+-ATPase), with Na+-K+-ATPase providing the driving force for active ion transport (5, 11, 47).
Epidermal growth factor (EGF) is a mitogenic polypeptide that exerts a broad range of effects on cell proliferation and tumorigenesis in epithelia, including pulmonary epithelia (4). EGF and its receptor (EGFR) have been shown to be expressed in both fetal and adult lung (44, 46, 48, 54) and in alveolar type II (AT2) cells (43). EGF promotes lung growth and development in utero, stimulates maturation of surfactant metabolism in fetal and newborn AT2 cells (12, 42, 54), and has been shown to attenuate respiratory distress syndrome due to prematurity in rhesus infants (15). EGF stimulates proliferation of fetal and neonatal alveolar epithelia but has little mitogenic activity for adult AEC in vitro in the absence of serum and other growth factors (31-33).
EGF also appears to play a role in recovery from lung injury. EGF activity is present in increased quantity in bronchoalveolar lavage fluid (BALF) from rats exposed to hyperoxia and in conditioned medium from AT2 cells exposed to hyperoxia in vitro (28). EGF is a chemoattractant for AT2 cell migration, suggesting an important role for this growth factor in reepithelialization of the alveolus following injury (34). EGFR abundance increases in bleomycin-injured rat lungs (36) and in hyperplastic AT2 cells following endotoxin instillation in rat lungs (51).
EGF has been shown to stimulate Na+/H+ exchange, Na+-glucose cotransport, and other mechanisms of transcellular water and solute transport in epithelia (20, 21, 25, 41), leading us to investigate its potential to modulate alveolar fluid homeostasis in the adult lung. We have recently demonstrated that EGF decreases paracellular permeability and upregulates transepithelial transport of Na+ across AEC monolayers grown in primary culture (2). The effects of EGF, both on tissue resistance (Rt, a measure of monolayer confluence and intercellular junction integrity) and short-circuit current (Isc, a measure of active transepithelial ion transport), require hours to days before becoming fully expressed. In particular, the effects of EGF on Isc across AEC monolayers are first observed at ~12 h and peak at 36 h after exposure, implicating changes in transport-related gene and protein expression in its effect on active ion transport.
In the present study, we investigated the mechanisms underlying the effects of EGF on active Na+ reabsorption in AEC monolayers. Effects of EGF on expression of Na+ channel subunit mRNA and Na+ pump subunit mRNA and protein were studied using Northern analysis, immunoblotting, and immunofluorescence. Our results indicate that EGF increases active Na+ absorption across AEC monolayers primarily via direct effects on Na+ pump subunit mRNA expression and protein synthesis, leading to increased numbers of functional Na+ pumps in the basolateral cell membranes.
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METHODS |
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Cell isolation and preparation of rat AEC monolayers. AT2 cells were isolated from adult male Sprague-Dawley rats by disaggregation with elastase (2.0-2.5 U/ml) (Worthington Biochemical, Freehold, NJ), followed by differential adherence on IgG-coated bacteriologic plates (9). The enriched AT2 cells were resuspended in a minimally defined serum-free medium (MDSF) consisting of DMEM and Ham's F-12 nutrient mixture in a 1:1 ratio (Sigma Chemical, St. Louis, MO) that was supplemented with 1.25 mg/ml BSA, 10 mM HEPES, 0.1 mM nonessential amino acids, 2.0 mM glutamine, 100 U/ml sodium penicillin G, and 100 µg/ml streptomycin (3). Cells were plated onto tissue culture-treated polycarbonate (Nucleopore) filter cups (Transwell, Corning Costar, Cambridge, MA) at a density of 1.0 × 106 cells/cm2. Cultures were maintained in a humidified 5% CO2 incubator at 37°C. AT2 cell purity (>90%) was assessed by staining freshly isolated cells for lamellar bodies with tannic acid (38). Cell viability (>90%) was measured by trypan blue dye exclusion. Cell number per monolayer was determined by quantifying nuclei as previously described (3).
Media were changed, thereby removing nonadherent cells, on the second day after plating. Monolayers were subsequently fed every other day. Cells were maintained in MDSF until day 4 and in either MDSF or MDSF supplemented with EGF thereafter. Monolayers were treated with EGF at 20 ng/ml, a concentration previously established to give a maximal response with respect to increasing Isc (2). Rt and spontaneous potential difference (SPD) were measured using a rapid screening device (Millicell-ERS, Millipore, Bedford, MA) as previously described (2). Isc was calculated from the relationship Isc = SPD/Rt. RNA and protein were harvested from monolayers maintained in MDSF before addition of EGF on day 4 and from EGF-treated and untreated monolayers at intervals between days 4 and 5. Monolayers maintained in MDSF or in MDSF supplemented with EGF from day 4 were harvested for immunofluorescence studies on day 5. For some experiments, media in the apical fluid compartment were changed on day 4 to MDSF containing either benzamil (1 µM) or benzamil + EGF. In each case, media in the basolateral compartment were of the same composition without the addition of the Na+ transport inhibitor. Bioelectric measurements were performed as described above, and monolayers were harvested for protein on days 4 and 5.RNA isolation and Northern analysis.
Total RNA was isolated from EGF-treated and untreated monolayers by the
acid phenol-guanidinium-chloroform method of Chomczynski and Sacchi
(6). Equal amounts of RNA (5-20 µg) were denatured with
formaldehyde, size-fractionated by agarose gel electrophoresis under
denaturing conditions, and transferred to nylon membranes (Hybond N+,
Amersham Life Science, Cleveland, OH). RNA was immobilized by
ultraviolet cross-linking. Blots were prehybridized for 2 h at 65°C
in 1 M
Na+-PO4
buffer (pH 7), 7% SDS, and 1% BSA. Hybridization was performed for 16 h at 65°C in the same buffer. Blots were studied with
isoform-specific cDNA probes for the
1- and
1-isoforms of
Na+-K+-ATPase
(E. Benz, Johns Hopkins University) and the
-,
-, and
-subunits of rat ENaC (rENaC; C. Canessa, Yale University and B. Rossier, Université de Lausanne, Switzerland). Probes were labeled with
[
-32P]dCTP
(Amersham) by the random-primer method using a commercially available
kit (Boehringer Mannheim, Indianapolis, IN). Blots were washed at high
stringency (0.5× SSC: 75 mM NaCl, 7.5 mM sodium citrate, pH 7.0, with 0.1% SDS at 55°C) and visualized by autoradiography. Differences in RNA loading were normalized using a 24-mer
oligonucleotide probe for 18S rRNA end labeled with
[
-32P]ATP (36).
Binding was detected by autoradiography and quantified by densitometry.
For the Na+ pump
1-subunit, both major
transcripts (2.3 and 2.7 kb) were quantified by scanning densitometry,
and the results are shown as the combined total relative density.
Inhibition of transcription by actinomycin D.
AEC monolayers were grown for 4 days in MDSF and then cultivated in
MDSF ±EGF in either the absence or presence of actinomycin D (1 µg/ml) (an inhibitor of RNA transcription) for up to 12 h. RNA was
extracted from AEC monolayers at 6 and 12 h following addition at time
(t) = 0 of EGF and/or
actinomycin D. Na+ pump
1- and
1-subunit mRNA were quantified
in each condition by Northern analysis as indicated above.
Western analysis.
SDS-PAGE was performed using the buffer system of Laemmli (29), and
immunoblotting was performed using procedures modified from Towbin et
al. (52). For detection of
1-subunits, AEC monolayers were
solubilized directly into 2% SDS sample buffer at 37°C for 15 min.
Equal amounts of cell protein in sample buffer were resolved by
SDS-PAGE under reducing conditions and electrophoretically blotted onto
Immobilon-P (Millipore). The blots were blocked for 2 h with 5% nonfat
dry milk in Tris-buffered saline (TBS) (20 mM Tris, 500 mM NaCl) at pH
7.5 and then incubated with primary antibody (Ab) as indicated below
for detection of Na+ pump subunits
by immunoblot.
35S labeling studies.
To quantify newly synthesized
1-subunits incorporated into
Na+ pump heterodimers, AEC
cultured in MDSF as described in Cell isolation and preparation of
rat AEC monolayers for 4 days were first incubated with
methionine-free medium for 1 h and then incubated for 24 h ±EGF
together with 100 µCi/ml of
[35S]methionine
(Amersham) in methionine-deficient medium. Cell proteins were then
solubilized, and Na+ pump
heterodimers were immunoprecipitated as described in Western analysis
using the anti-
-subunit MAb IEC 1/48. Before electrophoretic separation of the immunoprecipitated proteins, 2-µl samples of the
solubilized proteins precipitated from EGF-treated and untreated monolayers were placed in 10-ml scintillation fluid (Ecoscint, National
Diagnostics, Somerville, NJ) and analyzed for radioactivity in a liquid
scintillation spectrometer (Minaxi TriCarb 4000, Packard, Downers
Grove, IL). Loading of SDS-PAGE gels was adjusted so that approximately
equal amounts of radioactivity were loaded for each condition. For
fluorography of radioactively labeled proteins, unstained slab gels
were fixed in a solution containing isopropanol, water, and acetic acid
in a ratio of 25:65:10 for ~30 min, impregnated with Amplify
(Amersham) for 30 min, and dried under vacuum at 60-80°C. The
gels were then overlaid with Kodak XAR-5 film and exposed at
70°C with a DuPont Cronex intensifying screen. Newly synthesized Na+ pump
1-subunits were detected as a
97-kDa band that had an identity previously established by direct
Western blotting (55).
Immunofluorescence.
On day 5, monolayers maintained in
MDSF ±EGF from day 4 were rinsed
with cold PBS, fixed with 100% methanol at 20°C for 10 min,
rinsed in PBS, and treated with PBS-3% BSA to block nonspecific reactivity. Monolayers were reacted in situ with MAbs to the
1 (6H)- and
1 (IEC 1/48)-subunits of
Na+-K+-ATPase.
After extensive washing, the monolayers were incubated with
fluorescently labeled secondary antibodies. Stained specimens were
viewed with an Olympus microscope equipped with epifluorescence optics.
Chemicals. BSA and EGF were purchased from Collaborative Research (Bedford, MA). Benzamil was obtained from Molecular Probes (Eugene, OR). Cell culture media and all other chemicals were purchased from Sigma and were of the highest commercial quality available.
Statistical analysis. Results are expressed as means ± SE. Significance (P < 0.05) of differences in Rt, Isc, cell number, total protein per monolayer, and specific mRNA and protein per monolayer were determined by Student's t-test, except where indicated in the text. Significance of differences (P < 0.05) among multiple time points were determined by ANOVA as indicated in the text.
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RESULTS |
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Effects of EGF on AEC monolayer bioelectric properties, cell number,
and total protein.
AEC grown in MDSF on polycarbonate filters formed electrically
resistive monolayers by day 4 in
culture. Average
Rt and
Isc for
monolayers maintained in MDSF were 1.57 ± 0.11 k · cm2 and
3.64 ± 0.17 µA/cm2,
respectively, on day 4 (n = 3).
Rt increased by
50 ± 8% and Isc increased by
58 ± 11% (24 h) on day
5 for monolayers treated from day
4 with EGF relative to those grown in MDSF alone
(n = 3, P < 0.05). The EGF-induced increases
in Isc first
appeared at 12 h, with a maximal increase of 80% occurring by 36 h.
EGF had no significant effects on average cell number (51,087 ± 3,510 cells/monolayer in the absence of EGF vs. 51,652 ± 2,233 cells/monolayer in the presence of EGF,
n = 6) or total protein [0.51 ± 0.03 µg/monolayer (n = 16) in
the absence of EGF vs. 0.55 ± 0.06 µg/monolayer
(n = 7) in the presence of EGF].
Effects of EGF on rENaC subunit mRNA expression.
The effects of EGF on expression of mRNA for the -,
-, and
-subunits of the rENaC were evaluated by Northern blotting. In the
representative Northern blot shown in Fig.
1A,
mRNA for all three subunits is detectable in AEC monolayers on
day 4 in culture. In monolayers
exposed to EGF beginning on day 4,
mRNA levels were unchanged (
- and
-rENaC) or diminished
(
-rENaC) on day 5 relative to
monolayers maintained in the absence of EGF (n = 4) (Fig.
1B).
Na+ channel subunit mRNA levels
were similar in day 4 and
day 5 monolayers maintained in the
absence of EGF (data not shown).
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Effects of EGF on
Na+-K+-ATPase
subunit mRNA expression.
Freshly isolated AT2 cells and cultured AEC maintained in MDSF for 4 days express mRNA for the 1-
and
1-isoforms of
Na+-K+-ATPase,
with levels of expression of these isoforms remaining relatively
constant in MDSF between days 4 and
5 in culture (data not shown). A
representative Northern blot demonstrates that, following addition of
EGF to monolayers on day 4, levels of
1- and
1-subunit mRNA were increased
on day 5 (Fig.
2A). As
indicated in Fig. 2B, levels of
1- and
1-subunit mRNA increased by 42 ± 18% and 52 ± 18%, respectively, on day
5 (n = 4).
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Effects of actinomycin D on
Na+ pump
1- and
1-subunit levels.
A representative Northern blot (Fig.
4A)
shows Na+ pump
1- and
1-subunit mRNA at
t = 6 h (lanes
1-4) and 12 h (lanes
5-8) for monolayers in MDSF ±EGF incubated
in the absence (lanes 1, 3, 5,
and 7) or presence
(lanes 2,
4, 6,
and 8) of actinomycin D. Figure 4,
B and
C, shows average relative
densitometric values that have been normalized to 18S rRNA and
expressed as a percentage (±SE) of
Na+ pump subunit expression in the
absence of both EGF and actinomycin D at 6 h
(n = 3). Treatment of AEC monolayers
grown in the presence of EGF with actinomycin D for 12 h (Fig.
4A, lane
8) prevented an increase in
Na+ pump
1- and
1-subunit mRNA levels.
Na+ pump
1- and
1-subunit mRNA levels for
monolayers treated with actinomycin D were not significantly different
in the absence or presence of EGF at both 6 and 12 h. These results
suggest that EGF-induced increases in
Na+ pump subunit mRNA levels
require de novo mRNA synthesis to occur.
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Effects of EGF on
Na+-K+-ATPase
- and
-subunit protein expression.
As shown in the representative Western blot in Fig.
5A
(and summarized in the bar graph in Fig.
5C),
1-subunit protein levels increased on day 5 following addition
of EGF on day 4 relative to untreated
monolayers. Figure 5A illustrates a
typical experiment in which serial dilutions of lysate from untreated
monolayers were blotted together with lysate from EGF-treated
monolayers. A standard curve of optical density (expressed as arbitrary
units) was constructed, and relative abundance of
1-subunit protein was
calculated. In this example, the
Na+ pump
1-subunit band of the
EGF-treated cell lysate, for which 15 µl was loaded, was determined
to have an optical density equivalent to 27.8 µl of lysate from
untreated cells. Dividing this calculated value by 15 µl provides a
ratio of protein abundance for the EGF-treated vs. untreated lysates of
1.85, indicating an increase of 85% in Na+ pump
1-subunit protein abundance for
EGF-treated monolayers. As indicated in Fig.
5C, an average increase of 73 ± 8% in
1-subunit protein
abundance was observed for EGF-treated monolayers
(n = 6).
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Effects of
Na+ channel
inhibition on EGF-induced changes in
Na+-K+-ATPase
1-subunit protein abundance.
AEC monolayers grown for 24 h (day 4 to day 5) in MDSF + benzamil (1 µM), a potent blocker of Na+
entry via high amiloride-affinity
Na+ channels (i.e., rENaC), showed
~60% reduction in
Isc compared with
AEC monolayers at day 5 grown in MDSF
alone (Fig.
7A). In parallel experiments, ~70% reduction in
Isc was seen for
monolayers in MDSF + EGF + benzamil compared with EGF-treated
monolayers without benzamil (n = 3).
Despite the decrease in
Isc, benzamil treatment did not prevent an EGF-induced increase in
Na+ pump
1-protein abundance. As shown
in Fig. 7B, EGF treatment resulted in
significant increases in Na+ pump
1-protein abundance in the
absence and presence of benzamil. No difference was observed between
the EGF-induced increases seen in the absence (72 ± 10%) or
presence (70 ± 30%) of benzamil
(n = 3).
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Effects of EGF on
Na+ pump
1-subunit protein synthesis.
Treatment of AEC monolayers with EGF on day
4 in culture resulted in an increase in newly
synthesized
Na+-K+-ATPase
1-subunits at 24 h. MAb IEC
1/48 coprecipitates a 97-kDa 35S-labeled protein from both
EGF-treated and untreated monolayers that does not appear in lysates
from 35S-labeled cells grown in
MDSF that were precipitated without 1° Ab (
1° Ab) or
with rat IgG (n = 3) (Fig.
8). This protein band represents the
Na+ pump
1-subunit, as demonstrated by
transfer to nitrocellulose and immunoblotting with the monoclonal
anti-Na+ pump
1-subunit Ab 6H (56).
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Effects of EGF on cell surface expression of
1- and
1-subunit proteins.
To evaluate the effects of EGF on cell surface-associated expression of
1- and
1-subunit proteins, monolayers
maintained in MDSF or in MDSF + EGF from day
4 were evaluated by immunofluorescence on
day 5. As shown in Fig.
9, both
1- and
1-subunit proteins are
detectable in cell membranes of AEC cultivated in MDSF. Surface immunoreactivity to both
1- and
1-subunits is increased in
EGF-treated monolayers compared with untreated monolayers. All
photographs are taken at the same magnification and at identical
exposure times. Control antibodies (MF-20, an anti-myosin Ab) give
black immunofluorescence images (not shown). Images are representative of three experiments.
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DISCUSSION |
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We demonstrate in this study that EGF stimulates Na+ reabsorption across AEC monolayers in association with an increase in the abundance of cellular Na+-K+-ATPase protein. The time course of the increase in active ion transport is consistent with a requirement for new mRNA and protein synthesis. The EGF-induced increase in functional Na+ pumps appears most likely to be driven by an increase in Na+ pump subunit transcription, leading to synthesis of additional subunits. The lack of increase in Na+ channel subunit expression in the presence of EGF and the absence of an effect of inhibition of apical Na+ entry on Na+ pump abundance are most consistent with the hypothesis that increased Na+ entry via Na+ channels is not the mechanism driving the increase in Na+ pump expression.
The time courses of EGF-induced increases in
1-subunit mRNA,
Na+ pump abundance, and
Isc form a
temporal sequence that strongly suggests that EGF induces an increase
in transcription of
Na+-K+-ATPase
subunit mRNA, driving the formation of
Na+ pump heterodimers and
augmenting transepithelial Na+
transport. As indicated in Fig. 3, EGF upregulates
Na+ pump
1-subunit mRNA expression
within 3-6 h of exposure. This interval is compatible with the
time required for new mRNA synthesis induced by EGF and extracellular
stimuli in other cells. Our data showing that the increases in
1 and
1 mRNA in the presence of EGF
are prevented by actinomycin D (Fig. 4,
A-C)
also suggest a transcriptional mechanism for the increase in
Na+ pump
1-subunit mRNA.
The EGF-induced increase in Na+
pump 1-subunit mRNA is followed
by an increase in protein at 12-24 h (Figs. 5 and 6) and
concurrently (or shortly thereafter) by an increase in
Isc across the
AEC monolayers (2). The interval between EGF exposure and the increase
in Na+ pump protein is also
compatible with the time required for new protein synthesis. Results of
metabolic labeling studies, in which newly synthesized proteins are
labeled with
[35S]methionine (Fig.
8), confirm that EGF induces an increase in the abundance of labeled
Na+ pump
1-subunits consistent with an
augmented rate of protein synthesis.
Regulation of
Na+-K+-ATPase
expression and activity has been shown in other systems to occur at
multiple levels, including regulation of subunit transcription, mRNA
stability, translation, protein turnover, heterodimer formation, and
membrane insertion. Although various forms of posttranslational
modification have been proposed as regulators of
Na+ pump activity and turnover,
most long-term effects on Na+ pump
regulation are ultimately the result of changes in the number of pumps
present at the plasma membrane (1, 7, 27). In the current study, we
demonstrate that EGF treatment results in increases in - and
-subunit protein levels by both Western blotting and
immunofluorescence (Figs. 4 and 9), along with parallel increases in
mRNA for both the
1- and
1-subunits. Our findings are
similar to previous data in other systems showing concurrent regulation of the two Na+ pump subunits. For
example, treatment of a rat liver cell line (clone 9) with serum
results in increased Na+ pump
-
and
-subunit mRNAs and increased rates of both
and
gene
transcription (26).
The parallel increases in both - and
-subunit mRNA levels were
not predictable, since independent regulation of
- and
-subunit mRNA levels has been reported to occur in some systems. For example, corticosteroid depletion following adrenalectomy reduces expression of
1, but not
1, mRNA in
corticosteroid-sensitive tubular cells in the rat distal nephron (10).
Alternatively,
Na+-K+-ATPase
abundance was increased twofold over control in a renal epithelial cell
line
(LLC-PK1/Cl4)
by 24-h incubation in low K+ in
another study, with only
-subunit mRNA levels found to be increased
despite the accumulation of both newly synthesized
- and
-subunits (30). Expression of either
- or
-subunits can thus
be a limiting factor in regulation of
Na+ pump abundance in a specific
cell or tissue under some conditions, although that did not appear to
be the case in the present study.
A transient rise in intracellular Na+ caused by increased Na+ entry can be a direct stimulus for increased Na+ pump expression in some cells (7, 35, 45). Conversely, extracellular factors may also directly upregulate Na+ pump subunit expression, even in the absence of extracellular Na+ (14). Several lines of evidence suggest that increased Na+ entry is not responsible for the effects of EGF on Na+ pump expression in AEC in this study. If increased Na+ entry via apical Na+ channels preceded increased Na+ pump expression, an increase in Isc would also likely have been observed before the increase in Na+ pump mRNA and protein. This is due to the fact that the Na+ pump is not generally thought to be operating at maximal velocity and that increased Na+ entry should therefore rapidly result in increased transepithelial Na+ flux and increased Isc. An early effect of EGF on AEC monolayers was not seen in the present study, with the EGF-induced rise in Isc taking >12 h to occur (2). Moreover, experiments in which AEC monolayers treated with the Na+ transport inhibitor benzamil still manifest an increase in Na+ pump expression directly support the hypothesis that increased Na+ entry is not required for the EGF-induced stimulation of Na+ pump expression (Fig. 7). Reduction of Isc by ~70% in the presence of apical benzamil, a potent inhibitor of Na+ entry via Na+ channels, fails to block an increase in Na+ pump expression induced by EGF. Together, these data strongly suggest that the EGF-induced increase in Na+ transport is due to a direct effect of EGF on Na+ pump expression and is not secondarily due to an increase in apical Na+ entry.
Further evidence to support the hypothesis that the effects of EGF on
Na+ pump expression are not
mediated via effects on apical Na+
entry is that AEC Na+ channel
expression does not increase in parallel with the increase in
Na+ pump expression in the
presence of EGF (Fig. 2). It is unlikely that increased numbers of
Na+ channel subunits are present
in EGF-treated AEC compared with untreated monolayers at 24 h,
notwithstanding our inability to infer
Na+ channel protein abundance
directly from these mRNA data alone, although it remains possible that
an increase in plasma membrane ENaC occurs despite the same ( and
) or decreased (
) Na+
channel mRNA levels. In any case, a rise in
Na+ entry must accompany the
increase in Isc
across EGF-treated monolayers at >12 h, irrespective of the number of
apical Na+ channel subunits.
Although our data do not directly address either the mechanisms
responsible for the effects of EGF on
Na+ channel subunit expression or
those accounting for the necessary rise in
Na+ channel activity, the former
are most likely to result from some direct effect of EGF on rENaC
expression, whereas the latter most likely occur, at least in part, as
a secondary response to a decline in intracellular
Na+ resulting from the EGF-induced
increase in Na+ pump expression
and activity.
Direct effects of EGF on Na+ pump
gene expression have not previously been reported, although other
growth factors (e.g., transforming growth factor-) have been shown
to downregulate Na+ pump
expression (50). EGF directly induces expression of a protein related
to the Na+ pump
1-subunit, the
H+-K+-ATPase
1-subunit, in gastric
epithelia. Kaise et al. (23) have recently shown that EGF increased
levels of
H+-K+-ATPase
1-subunit mRNA in gastric
parietal cells and that EGF induces increased transcription due to its
interaction with a specific EGF response element and a novel
transcription factor. Similar EGF response elements are known to exist
in genes encoding other proteins (23). With characterization of the
promoter of the Na+ pump
1-subunit (49), the presence of
similar elements responsible for stimulation of
Na+ pump production can now also
be investigated.
EGF was first described as a mitogen that stimulates cell proliferation in a wide variety of cells and tissues (4). Although EGF is important in lung maturation and development and has been shown to stimulate fetal lung cell proliferation (12, 54), EGF does not appear to have significant effects on adult AEC proliferation independent from other growth factors. AEC do not ordinarily divide and proliferate in primary culture, although they may do so to a limited extent under some conditions (e.g., low plating density). Leslie et al. (33) found that DNA synthesis was stimulated by a combination of EGF, cholera toxin, and insulin when adult AT2 cells were cultured on an extracellular matrix prepared from corneal endothelial cells. In subsequent publications, these authors reported that EGF did not augment acidic fibroblast growth factor (aFGF)-stimulated [3H]thymidine incorporation into AT2 cells in serum-free medium (31) and that deletion of EGF from medium containing 2% fetal bovine serum, cholera toxin, aFGF, EGF, transferrin, and BALF did not markedly affect AEC proliferation when cells were plated at low density (32). These latter findings are consistent with our current results, in which we observed no significant effect of EGF on cell number after 24 h for AEC grown in serum-free medium.
EGF interacts directly only with its receptor (EGFR), which possesses tyrosine kinase activity in its cytoplasmic domain and has multiple intracellular substrates (19). In view of the many potential effects of EGF on AEC growth, differentiation, and proliferation, its relatively selective effects on Na+ pump and channel expression appear exceptional. Although there are many instances where EGF stimulates Na+ transport via other mechanisms [e.g., Na+/H+ exchange (25)], we are unaware of other epithelia in which direct stimulation of Na+ pump expression by EGF is postulated to occur. The most likely explanation for this unusual response in AEC is the presence (or absence) of specific elements of the signaling pathway downstream from the EGFR that result in stimulation of transport, but not proliferation, when the receptor is stimulated. The relationships between signaling pathways involved in EGF-induced Na+ pump expression and cell proliferation are currently unknown but will be of great importance if the potential therapeutic effects of EGF on transepithelial transport in the lung are to be exploited.
Augmented active Na+ transport across the alveolar epithelium could stimulate resorption of alveolar edema in congestive heart failure. Intriguingly, EGF has been administered intravenously for 4 days to healthy adult sheep, resulting in a dose-related natriuresis without apparent adverse effects (18). Chronic administration of EGF to pigs for 4 wk resulted in macroscopic enlargement of the ureters, kidneys, and heart, with less pronounced effects on pancreas, lungs, salivary glands, and esophagus. The urothelium was hyperplastic, with intracellular accumulations of glycoproteinaceous material staining with periodic acid-Schiff, but no otherwise ill effects on the adult animal were noted (53). Four-week administration of EGF to adult rats did not alter body weight, tibia length, or liver, heart, and lung weight despite a reduction in circulating total and free insulin-like growth factor I in experimental animals (13). Preliminary data also suggest that administration of EGF increases Na+-K+-ATPase activity in AT2 cells and increases Na+ transport and fluid clearance in isolated rat lungs (22, 40). Whether significant effects on lung fluid balance occurred in any of these in vivo studies, or would occur in animals with alveolar edema, are questions of great clinical interest.
This study demonstrates that the EGF-induced increase in active ion
transport across AEC monolayers is mediated by increases in mRNA
expression for the Na+ pump -
and
-subunits, resulting in increased expression of cellular and
cell-surface associated
Na+-K+-ATPase.
The increases in transepithelial transport and
Na+ pump expression occur in the
absence of cell proliferation, indicating a relatively specific effect
of EGF on AEC transport properties. These results suggest a possible
role for EGF in enhancing alveolar fluid clearance in the setting of
congestive heart failure and other disease states characterized by
alveolar edema.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Alicia McDonough for the generous gift of the FP Ab and for many helpful discussions, Drs. Michael Caplan and Andrea Quaroni for their monoclonal antibody reagents, Dr. Ed Benz for Na+ pump subunit cDNAs, and Drs. Cecilia Canessa and Bernard Rossier for Na+ channel subunit cDNAs. We note with appreciation the expert technical support of Stephanie Zabski, Monica Flores, Martha Jean Foster, Susie Parra, and Jennifer Armstrong.
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FOOTNOTES |
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This work was supported in part by the American Lung Association; the American Heart Association-Greater Los Angeles Affiliate; National Heart, Lung, and Blood Institute Grant Clinical Investigator Development Award HL-02836; National Heart, Lung, and Blood Institute Research Grants HL-03609, HL-38578, HL-38621, and HL-51928; and the Hastings Foundation. E. D. Crandall is Hastings Professor of Medicine and Kenneth T. Norris, Jr., Chair of Medicine.
Address for reprint requests: R. L. Lubman, Division of Pulmonary and Critical Care Medicine, Univ. of Southern California, GNH 11900, 2025 Zonal Ave., Los Angeles, CA 90033.
Received 2 September 1997; accepted in final form 27 March 1998.
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