Mechanisms of EGF-induced stimulation of sodium reabsorption by alveolar epithelial cells

Spencer I. Danto, Zea Borok, Xiao-Ling Zhang, Melissa Z. Lopez, Paryus Patel, Edward D. Crandall, and Richard L. Lubman

Will Rogers Institute Pulmonary Research Center, Division of Pulmonary and Critical Care Medicine, University of Southern California, Los Angeles, California 90033

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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) alpha -, beta -, and gamma -subunits and Na+ pump alpha 1- and beta 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 alpha 1 and beta 1 Na+ pump subunit mRNA but no increase in rENaC subunit mRNA. EGF-treated monolayers showed parallel increases in Na+ pump alpha 1- and beta 1-subunit protein by immunoblot relative to untreated monolayers. Fixed AEC monolayers demonstrated predominantly membrane-associated immunofluorescent labeling with anti-Na+ pump alpha 1- and beta 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

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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 alpha 1- and beta 1-isoforms of Na+-K+-ATPase (E. Benz, Johns Hopkins University) and the alpha -, beta -, and gamma -subunits of rat ENaC (rENaC; C. Canessa, Yale University and B. Rossier, Université de Lausanne, Switzerland). Probes were labeled with [alpha -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 [gamma -32P]ATP (36). Binding was detected by autoradiography and quantified by densitometry. For the Na+ pump beta 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 alpha 1- and beta 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 alpha 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.

For detection of the beta 1-subunits, cells were first immunoprecipitated with the anti-Na+ pump beta -subunit monoclonal Ab (MAb) IEC 1/48 (37) (A. Quaroni, Cornell University) and deglycosylated as previously demonstrated to be required for immunoblotting (55). Briefly, AEC monolayers or kidney membranes [prepared as previously described (55) and used as a positive control for the Na+ pump beta -subunit] were solubilized in immunoprecipitation lysis buffer [TBS (0.05 M Tris), pH 8.0, 1% Nonidet P-40, 1% BSA] for 1 h on ice and then centrifuged at 10,000 g for 20 min to remove insoluble material. The resulting supernatant was preincubated with both goat IgG-agarose and mouse serum-agarose for 1 h at 4°C to reduce nonspecific binding to primary and secondary Abs before immunoprecipitation. The preclarified supernatants were then incubated with MAb IEC 1/48 overnight at 4°C. After incubation with this primary Ab, the samples were incubated with secondary Ab (goat anti-mouse IgG) conjugated to agarose beads for 1 h. After the bound antigen was washed twice with lysis buffer, once with TBS (pH 8.0), and once with TBS (pH 6.0), the bound antigen was eluted from the goat anti-mouse agarose beads in Na+-PO4 buffer (20 mM, pH 8.0) containing 0.5% SDS for 15 min at 37°C. Deglycosylation was carried out in Na+-PO4 buffer (20 mM, pH 8.0) containing EDTA (8 mM), 1% Nonidet P-40, and 0.4% mercaptoethanol in the presence of N-glycanase (N-glycosidase F, Boehringer Mannheim) (2.5 U/10 µl eluent) for 2 h at 37°C. After this incubation, the samples were processed for SDS-PAGE as described above.

The anti-alpha 1-subunit MAb 6H (24) (M. Caplan, Yale University) was used for detection of Na+ pump alpha 1-subunits. The polyclonal anti-beta 1-subunit Ab FP (A. McDonough, University of Southern California) was used for detection of beta -subunits precipitated by MAb IEC 1/48, since the latter recognizes only the undenatured beta -subunit and is not useful for immunoblotting. Blots were incubated with horseradish peroxidase-linked goat anti-rabbit IgG conjugates for 1 h, and antigen-Ab complexes were visualized by enhanced chemiluminescence (Amersham, Arlington Heights, IL). Relative intensity of protein bands was quantified by densitometry. For the alpha 1-protein, relative intensity of protein bands was determined by comparison to a standard curve of optical density obtained from blots, including serial dilutions of lysates from untreated monolayers by a method similar to that described by Zuege et al. (56). Protein concentrations were determined using the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA), with BSA used as a standard.

35S labeling studies. To quantify newly synthesized alpha 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-beta -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 alpha 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 alpha 1 (6H)- and beta 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.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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 kOmega · 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 alpha -, beta -, and gamma -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 (alpha - and beta -rENaC) or diminished (gamma -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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Fig. 1.   Effects of epidermal growth factor (EGF) on Na+ channel subunit mRNA expression. A: representative Northern blot demonstrates that, following addition of EGF to monolayers on day 4, levels of alpha - and beta -rat epithelial Na+ channel (rENaC) mRNA do not change, whereas levels of gamma -rENaC mRNA decrease by day 5. RNA loading was similar among all conditions, as indicated by equivalence of signal following hybridization with an 18S rRNA oligonucleotide probe. B: bar graph indicates levels of each rENaC subunit mRNA (±SE) for alveolar epithelial cell (AEC) monolayers treated with EGF from days 4 to 5 relative to untreated monolayers (n = 4). * Significantly different from untreated monolayers.

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 alpha 1- and beta 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 alpha 1- and beta 1-subunit mRNA were increased on day 5 (Fig. 2A). As indicated in Fig. 2B, levels of alpha 1- and beta 1-subunit mRNA increased by 42 ± 18% and 52 ± 18%, respectively, on day 5 (n = 4).


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Fig. 2.   Effects of EGF on Na+-K+-ATPase subunit mRNA expression. A: representative Northern blot demonstrates that, following addition of EGF to monolayers on day 4, levels of alpha 1- and beta 1-subunit mRNA are increased on day 5. RNA loading was similar among all conditions, as indicated by equivalence of signal following hybridization with an 18S rRNA oligonucleotide probe. B: bar graph indicates levels of each Na+ pump subunit mRNA (±SE) for AEC monolayers treated with EGF from days 4 to 5 relative to untreated monolayers (n = 4). * Significant difference in alpha 1-subunit mRNA levels. ** Significant difference in beta 1-subunit mRNA levels.

The time course of the effect of EGF on Na+ pump alpha 1-subunit mRNA is illustrated in Fig. 3. A representative Northern blot is shown of Na+ pump alpha 1-subunit mRNA at t = 3, 6, 12, and 24 h for AEC monolayers maintained in the presence of EGF and for untreated monolayers at 0 and 24 h. A bar graph showing average relative densitometric values for three experiments indicates that significant increases in Na+ pump alpha 1-subunit mRNA relative to t = 0 and to untreated monolayers at 24 h occurred by 6 h and were maintained at subsequent time points through 24 h, as assessed by ANOVA (n = 3). There were no differences in Na+ pump subunit mRNA levels between t = 0 and 24 h for untreated monolayers.


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Fig. 3.   Time course of effects of EGF on Na+ pump alpha 1-subunit mRNA. Top: representative Northern blot of Na+ pump alpha 1-subunit mRNA at 3, 6, 12, and 24 h after day 4 AEC monolayers were exposed to EGF and for untreated monolayers at 0 and 24 h. RNA loading was similar among all conditions, as indicated by equivalence of signal following hybridization with an 18S rRNA oligonucleotide probe (not shown). Bottom: bar graph showing average relative densitometric values (±SE) indicates that a significant increase in Na+ pump alpha 1-subunit mRNA [relative to time (t) = 0 and untreated monolayers at 24 h] occurred at 6 h and was sustained through 24 h (n = 3). There were no differences between mRNA levels at t = 0 and 24 h for untreated monolayers. * Significantly different from untreated monolayers at t = 0.

Effects of actinomycin D on Na+ pump alpha 1- and beta 1-subunit levels. A representative Northern blot (Fig. 4A) shows Na+ pump alpha 1- and beta 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 alpha 1- and beta 1-subunit mRNA levels. Na+ pump alpha 1- and beta 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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Fig. 4.   Effects of actinomycin D on Na+ pump alpha 1- and beta 1-subunit mRNA. A: representative Northern blot shows Na+ pump alpha 1- and beta 1-subunit mRNA for monolayers incubated in minimally defined serum-free medium (MDSF) ± EGF for 6 and 12 h in the presence or absence of actinomycin D. RNA loading was similar among all conditions, as indicated by equivalence of signal following hybridization with an 18S rRNA oligonucleotide probe. Actinomycin D prevents the EGF-induced increase in alpha 1- and beta 1-subunit mRNA at 12 h (lanes 7 and 8). B: bar graph of Na+ pump alpha 1-subunit mRNA levels. Indicated are levels of Na+ pump alpha 1-subunit mRNA (±SE) for AEC monolayers incubated under each of the specified conditions, expressed as a percentage of subunit expression in the absence of both EGF and actinomycin D at 6 h (n = 3). alpha 1-Subunit mRNA levels were not significantly different for actinomycin D-treated monolayers grown in either the presence or absence of EGF at either 6 or 12 h. * Significantly different from monolayers incubated in the absence of both EGF and actinomycin D at 6 h. ** Significantly different from monolayers incubated in the absence of both EGF and actinomycin D at 12 h. C: bar graph of Na+ pump beta 1-subunit mRNA levels. Indicated are the levels of Na+ pump beta 1-subunit mRNA (±SE) for AEC monolayers incubated under each of the specified conditions, expressed as a percentage of subunit expression in the absence of both EGF and actinomycin D at 6 h (n = 3). beta 1-Subunit mRNA levels were not significantly different for actinomycin D-treated monolayers grown in either the presence or absence of EGF at either 6 or 12 h. * Significantly different from monolayers incubated in the presence of actinomycin D at 12 h.

Effects of EGF on Na+-K+-ATPase alpha - and beta -subunit protein expression. As shown in the representative Western blot in Fig. 5A (and summarized in the bar graph in Fig. 5C), alpha 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 alpha 1-subunit protein was calculated. In this example, the Na+ pump alpha 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 alpha 1-subunit protein abundance for EGF-treated monolayers. As indicated in Fig. 5C, an average increase of 73 ± 8% in alpha 1-subunit protein abundance was observed for EGF-treated monolayers (n = 6).


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Fig. 5.   Effects of EGF on Na+-K+-ATPase alpha - and beta -subunit protein expression. A: alpha -subunit; calculation of relative abundance using a standard curve. A typical experiment is shown in which serial dilutions of lysate from untreated monolayers were blotted for the Na+ pump alpha 1-subunit together with lysate from EGF-treated monolayers. A standard curve of optical density [arbitrary units (au)] was constructed, and relative abundance of alpha 1-subunit protein in EGF-treated monolayers was calculated. In the experiment illustrated, a relative increase in alpha 1-subunit protein of 85% was observed. B: beta -subunit; detection of core deglycosylated protein. A typical experiment is shown in which lysates from EGF-treated and untreated monolayers were precipitated with the anti-beta 1-subunit monoclonal antibody (MAb) IEC 1/48, divided into 2 equal aliquots, one digested with N-glycanase and both blotted with another anti-beta 1-subunit MAb (FP). Kidney membranes are included as a positive control. Although native beta 1-subunit protein is not detected by the FP antibody in the undigested immunoprecipitate from AEC monolayers, the core protein (~35 kDa) shows a relative increase in EGF-treated vs. untreated monolayers. C: bar graph showing that average increase of 73% in alpha 1-subunit protein (n = 6) and 53% in beta 1-subunit protein abundance (n = 2) was observed for EGF-treated monolayers compared with untreated monolayers. * Significant difference in alpha 1-subunit protein abundance. ** Significant difference in beta 1-subunit protein abundance.

As shown in the representative Western blot in Fig. 5B (and summarized in Fig. 5C), beta 1-subunit protein levels also increased on day 5 following addition of EGF on day 4 relative to untreated monolayers. Figure 5B illustrates a typical experiment in which lysate from EGF-treated and untreated monolayers was precipitated with the anti-beta 1-subunit MAb IEC 1/48. Kidney membranes are included as a positive control. The precipitates were divided into two equal aliquots, one digested with N-glycanase, and both aliquots were blotted with another anti-beta 1-subunit MAb (FP) as indicated in METHODS. Whereas both the native fully glycosylated (~50 kDa) and deglycosylated (~35 kDa) beta 1-subunit protein are detected by the FP antibody in precipitates from kidney membranes, only the deglycosylated protein is detected in precipitates from either EGF-treated or untreated AEC monolayers. These data are in agreement with our previously published findings that native lung Na+ pump beta 1-subunit cannot be reliably detected on immunoblot without first being subjected to deglycosylation (55). On the basis of a comparison of the densitometric values for the deglycosylated beta 1-subunit protein band, an average increase of 53 ± 25% in beta 1-subunit protein abundance was observed for EGF-treated vs. untreated monolayers (n = 2).

The time course of the effect of EGF on Na+ pump alpha 1-subunit protein is illustrated in Fig. 6. A representative Western blot is shown of Na+ pump alpha 1-subunit protein at t = 3, 6, 12, and 24 h for AEC monolayers grown in the presence of EGF and for untreated monolayers at t = 0. A bar graph showing average relative densitometric values for three experiments indicates that significant increases in Na+ pump alpha 1-subunit protein relative to t = 0 occurred by 12 h and were present through 24 h as assessed by ANOVA (n = 3). There was no difference in Na+ pump alpha 1-subunit protein abundance at t = 0 vs. 24 h for monolayers not treated with EGF (data not shown).


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Fig. 6.   Time course of the effects of EGF on Na+ pump alpha 1-subunit protein. Top: a representative Western blot is shown of Na+ pump alpha 1-subunit protein at 3, 6, 12, and 24 h for AEC monolayers grown in the presence of EGF added on day 4 at t = 0 and for untreated monolayers at t = 0. Bottom: a bar graph showing average relative densitometric values (±SE), indicating that a significant increase in Na+ pump alpha 1-subunit protein (relative to t = 0) occurred at 12 h and was present through 24 h (n = 3). *, ** Significantly different from all other conditions.

Effects of Na+ channel inhibition on EGF-induced changes in Na+-K+-ATPase alpha 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 alpha 1-protein abundance. As shown in Fig. 7B, EGF treatment resulted in significant increases in Na+ pump alpha 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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Fig. 7.   Effects of Na+ channel inhibition on short-circuit current (Isc) and Na+ pump alpha 1-subunit. A: Isc is increased in the presence of EGF, in the absence of benzamil. Isc across day 4 AEC monolayers incubated for 24 h with the Na+ channel blocker benzamil (1 µM), with or without EGF, are reduced relative to monolayers grown in MDSF alone. * Significantly different from all other conditions. ** Significantly different from monolayers grown in MDSF without inhibitor present. B: Na+ pump alpha 1-subunit. Western blot (top) shows that Na+ pump alpha 1-subunit protein abundance increases in the presence of EGF relative to MDSF in either the presence or absence of benzamil. Bar graph (bottom) showing average relative densitometric values (±SE) indicates that a significant increase in Na+ pump alpha 1-subunit protein (relative to MDSF) occurred for EGF-treated monolayers grown in either the presence or absence of benzamil (n = 3). * Significantly different from monolayers grown in the absence of EGF.

Effects of EGF on Na+ pump alpha 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 alpha 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 alpha 1-subunit, as demonstrated by transfer to nitrocellulose and immunoblotting with the monoclonal anti-Na+ pump alpha 1-subunit Ab 6H (56).


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Fig. 8.   Effects of EGF on Na+ pump alpha 1-subunit protein synthesis. MAb IEC 1/48 coprecipitates a 97-kDa 35S-labeled protein from both EGF-treated and untreated monolayers that represents the Na+ pump alpha 1-subunit. Band does not appear in lysates from 35S-labeled cells grown in MDSF that were precipitated without 1° Ab (-1° Ab) or with rat IgG (IgG). As shown in the accompanying representative Western blot (n = 3), labeled alpha 1-subunit protein is increased in EGF-treated monolayers relative to untreated monolayers.

Effects of EGF on cell surface expression of alpha 1- and beta 1-subunit proteins. To evaluate the effects of EGF on cell surface-associated expression of alpha 1- and beta 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 alpha 1- and beta 1-subunit proteins are detectable in cell membranes of AEC cultivated in MDSF. Surface immunoreactivity to both alpha 1- and beta 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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Fig. 9.   Effects of EGF on cell surface expression of alpha 1- and beta 1-subunit protein. Surface immunoreactivity to both alpha 1- and beta 1-subunits is increased in EGF-treated monolayers on day 5 compared with untreated monolayers (n = 3). Bar = 20 µm.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 alpha 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 alpha 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 alpha 1 and beta 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 alpha 1-subunit mRNA.

The EGF-induced increase in Na+ pump alpha 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 alpha 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 alpha - and beta -subunit protein levels by both Western blotting and immunofluorescence (Figs. 4 and 9), along with parallel increases in mRNA for both the alpha 1- and beta 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 alpha - and beta -subunit mRNAs and increased rates of both alpha  and beta  gene transcription (26).

The parallel increases in both alpha - and beta -subunit mRNA levels were not predictable, since independent regulation of alpha - and beta -subunit mRNA levels has been reported to occur in some systems. For example, corticosteroid depletion following adrenalectomy reduces expression of alpha 1, but not beta 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 beta -subunit mRNA levels found to be increased despite the accumulation of both newly synthesized alpha - and beta -subunits (30). Expression of either alpha - or beta -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 (alpha  and beta ) or decreased (gamma ) 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-beta ) have been shown to downregulate Na+ pump expression (50). EGF directly induces expression of a protein related to the Na+ pump alpha 1-subunit, the H+-K+-ATPase alpha 1-subunit, in gastric epithelia. Kaise et al. (23) have recently shown that EGF increased levels of H+-K+-ATPase alpha 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 alpha 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 alpha - and beta -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.

    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.

    FOOTNOTES

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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Discussion
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