Na+-K+-ATPase expression in alveolar epithelial cells: upregulation of active ion transport by KGF

Zea Borok, Spencer I. Danto, Luis L. Dimen, Xiao-Ling Zhang, 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 evaluated the effects of keratinocyte growth factor (KGF) on alveolar epithelial cell (AEC) active ion transport and on rat epithelial Na channel (rENaC) subunit and Na+-K+-adenosinetriphosphatase (ATPase) subunit isoform expression using monolayers of AEC grown in primary culture. Rat alveolar type II cells were plated on polycarbonate filters in serum-free medium, and KGF (10 ng/ml) was added to confluent AEC monolayers on day 4 in culture. Exposure of AEC monolayers to KGF on day 4 resulted in dose-dependent increases in short-circuit current (Isc) compared with controls by day 5, with further increases occurring through day 8. Relative Na+-K+-ATPase alpha 1-subunit mRNA abundance was increased by 41% on days 6 and 8 after exposure to KGF, whereas alpha 2-subunit mRNA remained only marginally detectable in both the absence and presence of KGF. Levels of mRNA for the beta 1-subunit of Na+-K+-ATPase did not increase, whereas cellular alpha 1- and beta 1-subunit protein increased 70 and 31%, respectively, on day 6. mRNA for alpha -, beta -, and gamma -rENaC all decreased in abundance after treatment with KGF. These results indicate that KGF upregulates active ion transport across AEC monolayers via a KGF-induced increase in Na pumps, primarily due to increased Na+-K+-ATPase alpha 1-subunit mRNA expression. We conclude that KGF may enhance alveolar fluid clearance after acute lung injury by upregulating Na pump expression and transepithelial Na transport across the alveolar epithelium.

alveolar epithelium; growth factor; gene expression; sodium transport; rat epithelial sodium channel; keratinocyte growth factor; sodium-potassium-adenosinetriphosphatase

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE ALVEOLAR EPITHELIUM provides a relatively impermeable barrier to the leakage of fluid and solutes from the lung interstitium and vasculature into the alveolar air spaces. Evidence accumulated from studies in isolated type II cells, isolated perfused lungs, and intact animals indicates that, in addition to its barrier properties, the alveolar epithelium actively transports Na in a vectorial fashion from the alveolar lumen to the interstitium (2, 12, 16). The primary pathways for transepithelial Na transport are apical amiloride-sensitive Na channels (ENaC) and basolateral Na pumps [Na+-K+-adenosinetriphosphatase (ATPase)], with Na+-K+-ATPase providing the driving force for active ion transport (10, 38).

Excess alveolar fluid is absorbed from the air space into the blood due to the osmotic gradient established by active vectorial Na transport. Injury to the lung from a number of different causes may result in progressive damage to the alveolar epithelial barrier and the accumulation of fluid in the distal air spaces. Stimulation of transepithelial Na and water absorption via upregulation of either Na channels or pumps could potentially offer a means by which to augment alveolar fluid clearance and accelerate the resolution of alveolar edema.

Na+-K+-ATPase is a heterodimeric protein consisting of an alpha - and beta -subunit. Several isoforms have been cloned, and their physiological properties have been characterized (41). Both alpha - and beta -subunits are expressed in a cell- and tissue-specific fashion. The alpha 1-subunit appears to be the predominant isoform expressed in alveolar epithelium, although low levels of the alpha 2 and alpha 3 isoforms are detectable in lung (34, 40, 49). The beta 1-subunit has been identified in lung and fetal distal lung epithelium by Northern blotting (9, 31, 33, 49) and, more recently, in alveolar epithelial cells (AEC) by immunochemical methods (50).

Na+-K+-ATPase activity is subject to both short-term and long-term hormonal regulation (e.g., aldosterone, thyroid hormone; see Refs. 3 and 22). The long-term effects of these hormones include sustained increases in Na+-K+-ATPase activity that result from increases in Na pump subunit mRNA and protein levels. These effects are exerted in an isoform-specific fashion (19). Relatively little is known concerning the effects of hormones or growth factors on Na+-K+-ATPase in adult lung or alveolar epithelium, which are likely, as is generally the case, to be organ and tissue specific in nature.

Na channels with high affinity for amiloride are located in the apical cell membrane of AEC, where they constitute the major pathway for Na entry into the cells (4, 38). The activity of these channels has been shown to be regulated by a variety of factors, including hormones, intracellular Ca2+, Na+, pH, cytoskeletal elements (e.g., actin filaments), and G proteins (8, 25, 44). Three subunits (alpha , beta , and gamma ) of the rat epithelial Na channel (rENaC) have recently been cloned from colonic epithelium (7). All three subunits are expressed in adult lung and in AEC, and their expression appears to be differentially regulated during lung development (32). Like the Na pump, rENaC is also subject to both short- and long-term regulation by hormones. Expression of mRNA for the different rENaC subunits also appears to be differentially regulated by soluble factors (e.g., corticosteroids and aldosterone), implying the further existence of multiple and potentially complex regulatory mechanisms for Na channel activity (13, 36).

Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, is a polypeptide mitogen secreted by fibroblasts and endothelial cells that acts primarily on epithelial cells. KGF has been shown to ameliorate lung injury from bleomycin (48), radiation (48), hyperoxia (35), and acid instillation (42). In two recent studies, protective effects of KGF were observed in rats after alpha -naphthylthiourea (ANTU)-induced lung injury (17, 28). Pretreatment with KGF significantly attenuated the development of lung leak, an effect reversed by either amiloride or ouabain, suggesting a salutary effect of KGF on alveolar epithelial ion transport.

In this study, we evaluated the effects of KGF on active ion transport in AEC monolayers grown on tissue culture-treated polycarbonate filters. Changes in expression of Na+-K+-ATPase subunit isoforms and Na channel subunits were concurrently evaluated. Our results indicate that KGF upregulates active ion transport across AEC monolayers via a KGF-induced increase in Na pumps, primarily due to increased Na+-K+-ATPase alpha 1-subunit mRNA expression.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell isolation and preparation of rat AEC monolayers. Alveolar type II 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 immunoglobulin G (IgG)-coated bacteriological plates (5). The enriched alveolar type II cells were resuspended in a minimal defined serum-free medium (MDSF) consisting of Dulbecco's modified Eagle's medium and Ham's F-12 nutrient mixture in a 1:1 ratio (Sigma Chemical, St. Louis, MO) supplemented with 1.25 mg/ml bovine serum albumin (BSA; Collaborative Research, Bedford, MA), 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 0.1 mM nonessential amino acids, 2.0 mM glutamine, 100 U/ml sodium penicillin G, and 100 µg/ml streptomycin (5). Cells were plated onto tissue culture-treated polycarbonate (Nuclepore) 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. Alveolar type II cell purity (>90%) was assessed by staining freshly isolated cells for lamellar bodies with tannic acid (29). Cell viability (>90%) was measured by trypan blue dye exclusion. Cell number in the monolayers as a function of time in culture was evaluated by counting cell nuclei as previously described (5).

Media were changed, thereby removing nonadherent cells, on the second day after plating. Cells were grown in MDSF from the time of plating through day 4, at which time media were changed again. The monolayers were fed and refed every second day thereafter with either MDSF or media supplemented with KGF (10 ng/ml except where otherwise indicated). RNA and protein were harvested from monolayers maintained in MDSF before addition of KGF on day 4. RNA was harvested from control and KGF-treated monolayers on days 6 and 8, and protein was harvested from both control and treated monolayers on day 6.

Measurement of monolayer bioelectric properties. Transepithelial resistance (RT; kOmega · cm2) and spontaneous potential difference (SPD; mV) were measured using a rapid screening device (Millicell-ERS; Millipore, Bedford, MA). Equivalent short-circuit current (Isc; µA/cm2) was calculated from the relationship Isc = SPD/RT. To ascertain the time course of these effects, KGF was added to confluent monolayers on day 4, and barrier properties were determined serially over the next 96 h. A dose-response relationship between KGF concentration and bioelectric properties was established for monolayers grown in medium supplemented with KGF (0.001-100 ng/ml) from day 4 in culture, with RT and Isc determined serially over the next 96 h.

For experiments designed to assess the effects of transport inhibitors on KGF-induced changes in Isc, monolayers grown in MDSF with or without KGF were exposed to either amiloride (10 µM) or ouabain (100 µM), and Isc was measured as indicated above. After obtaining baseline measurements, inhibitor was added on day 6 to either the apical (amiloride) or basolateral (ouabain) fluid compartment. Isc was measured at intervals over the next 60 min across AEC monolayers treated with inhibitor (and, in parallel, across monolayers not exposed to either inhibitor).

RNA isolation and Northern analysis. Total RNA was isolated from control and KGF-treated monolayers by the acid phenol-guanidinium-chloroform method of Chomczynski and Sacchi (11). 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. RNA was immobilized by ultraviolet cross-linking (Hybond N+; Amersham Life Science, Cleveland, OH). Blots were prehybridized for 2 h at 65°C in 1 M Na- phosphate buffer (pH 7), 7% sodium dodecyl sulfate (SDS), and 1% BSA. Hybridization was performed for 16 h at 65°C in the same buffer. Blots were probed with isoform-specific cDNA probes for the alpha 1, beta 1, and alpha 2 isoforms of Na+-K+-ATPase (E. Benz, Johns Hopkins University, Baltimore, MD) and the alpha -, beta -, and gamma -subunits of the rENaC (C. Canessa 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 32P-ATP. Northern blots were scanned, and relative differences in mRNA abundance were quantified by densitometry using Scan Analysis software (Biosoft, Cambridge, UK).

Western analysis. SDS-polyacrylamide gel electrophoresis (PAGE) was performed using the buffer system of Laemmli (23), and immunoblotting was performed using procedures modified from Towbin et al. (43). For detection of alpha 1- and alpha 2-subunits, AEC monolayers were solubilized directly into 2% SDS sample buffer. Rat lung and brain membranes were prepared as previously described (50). Equal amounts of cell protein in sample buffer were resolved by SDS-PAGE under reducing conditions and electrophoretically blotted onto Immobilon-P (Millipore, Marlborough, MA). The blotted sheets were blocked for 2 h with 5% nonfat dry milk in tris(hydroxymethyl)aminomethane (Tris)-buffered saline (TBS) at pH 7.5 and then incubated with primary antibody (Ab) for detection of Na pump subunits by immunoblot.

For detection of the beta 1-subunits, cells were first immunoprecipitated with the monoclonal anti-Na pump beta -subunit Ab IEC 1/48 (A. Quaroni, Cornell University; see Ref. 27) and deglycosylated as previously demonstrated to be required (50). Briefly, AEC monolayers or kidney membranes [prepared as previously described (50) and used as a positive control for the Na pump beta -subunit] were solubilized in immunoprecipitation lysis buffer [LB; TBS (0.05 M Tris), pH 8.0, 1% Nonidet P-40 (NP-40), and 1% BSA] for 30 min on ice and then centrifuged at 14,000 g for 20 min to remove insoluble material. The resulting supernatant was incubated with both goat IgG agarose and mouse serum agarose for 1 h at 4°C to preclarify the antigens before immunoprecipitation. This maneuver was performed to reduce nonspecific binding to primary and secondary Abs. The preclarified supernatants were then incubated with IEC 1/48 for 1 h on ice. After incubation with primary Ab, the samples were incubated with a secondary Ab (goat anti-mouse IgG) conjugated to agarose beads for 1 h. After being washed two times with LB, one time with TBS (pH 8.0), and one time with TBS (pH 6.0), the bound antigen was eluted from the goat anti-mouse agarose beads in Na-phosphate buffer (20 mM, pH 8.0) containing 0.5% SDS for 15 min at 37°C. Deglycosylation was carried out in Na phosphate buffer (20 mM, pH 8.0) with EDTA (8 mM), 1% NP-40, and 0.4% mercaptoethanol in the presence of N-glycosidase F (2.5 U/10 µl eluant; Boehringer Mannheim) for 2 h at 37°C. After this incubation, the samples were processed for SDS-PAGE as described.

The monoclonal anti-alpha 1 Ab 6H (M. Caplan, Yale University; see Ref. 21) was used for detection of Na pump alpha 1-subunits. The monoclonal anti-alpha 2 Ab McB2 (K. Sweadner, Harvard University; see Ref. 46) and an anti-alpha 2 polyclonal Ab purchased from Upstate Biotechnology (UBI, Lake Placid, NY) were used for detection of Na pump alpha 2-subunits. The polyclonal anti-beta 1-subunit Ab FP (A. McDonough, University of Southern California) was used for detection of beta -subunits precipitated by Ab 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 chemiluminescence (Amersham, Arlington Heights, IL). Relative intensity of Na pump alpha 1-subunit protein bands was quantified by densitometry and compared after calibration with standard dilutions of control cell lysate. Western blots of immunoprecipitated Na pump beta 1-subunits were scanned, and relative differences in abundance were quantified by densitometry as indicated above for mRNA. Protein concentrations were determined using the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA) with BSA used as a standard.

Materials. Recombinant human KGF was purchased from R&D Systems (Minneapolis, MN). Cell culture media and all other chemicals were purchased from Sigma (St. Louis, MO) and were of the highest commercial quality available.

Statistical analysis. Results are expressed as means ± SE. Significance (P < 0.05) of differences in effects of KGF on mean cell number and Isc were determined by analysis of variance. Significance (P < 0.05) of differences in effects of inhibitors on Isc and effects of KGF on Na channel and pump mRNA levels and Na pump protein abundance were determined by Student's t-test.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effects of KGF on monolayer cell number. Monolayer cell number was serially determined on days 2 through 8 in culture. On day 4, there were 6.3 ± 0.2 × 105 cells/filter for monolayers grown in MDSF. As shown in Fig. 1, the day 6 cell number for monolayers grown in MDSF plus KGF (where KGF was present at 10 ng/ml from day 4) was not significantly different from that for monolayers grown in MDSF alone. Cell number for monolayers grown in MDSF plus KGF for 4 days increased by 29% relative to monolayers grown in MDSF alone by day 8 (n = 3).


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Fig. 1.   Effects of keratinocyte growth factor (KGF) on cell number. Cell number per monolayer fell to 6.3 ± 0.2 × 105 cells/filter by day 4 for monolayers grown in minimal defined serum-free medium (MDSF) and continued to decline slowly on subsequent days. No difference in cell number was observed between KGF-treated and untreated monolayers through day 6. Cell number for monolayers grown in MDSF + KGF increased by 29% relative to monolayers grown in MDSF alone (control) by day 8. * Significantly different from untreated monolayers.

Effects of KGF on AEC monolayer bioelectric properties. AEC grown in MDSF on polycarbonate filters formed electrically resistive monolayers by day 4 in culture. RT and Isc for monolayers grown in MDSF were 2.66 ± 0.31 kOmega · cm2 and 2.91 ± 0.12 µA/cm2, respectively, on day 4. An increase in Isc for monolayers treated with KGF (10 ng/ml) was demonstrable by 24 h (day 5; Fig. 2A), whereas no increase in Isc was seen by 12 h after exposure to KGF (data not shown). Isc increased by 37% for monolayers grown in MDSF plus KGF from day 4 through day 6 compared with MDSF controls over the same time period and by 66% through day 8 in culture. The dose response of Isc to KGF was determined for KGF concentrations from 0.001 to 100 ng/ml (Fig. 2B). Isc measured on day 5 through day 8 increased in a dose-dependent fashion, with a mean effective dose of ~0.5 ng/ml KGF on all four days.


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Fig. 2.   Effects of KGF on short-circuit current (Isc). A: changes in Isc after KGF exposure. KGF (10 ng/ml) induced a relative increase in Isc by day 5, 1 day after addition of KGF to alveolar epithelial cell (AEC) monolayers, with further increases occurring through day 8. Each bar represents the mean difference in Isc for KGF-treated vs. untreated monolayers (±SE). Measurements are from 5 different cell preparations. * Differences in Isc between KGF-treated and untreated monolayers were significant on days 5 - 8. B: dose response of KGF effect on Isc. Isc measured on days 5 - 8 increased in a dose-dependent fashion with mean effective dose of ~0.5 ng/ml. Each point represents mean of measurements from 5 different cell preparations. Delta Isc, change in Isc.

Amiloride, a blocker of Na channels, caused a rapid decline in Isc across AEC monolayers, as previously shown (10). There was no significant difference between the reduction in Isc at 10 min in the presence of amiloride on day 6 AEC monolayers treated (86%) or untreated (78%) with KGF compared with corresponding controls without amiloride (n = 3). Ouabain, an Na pump inhibitor, caused a more gradual decline in Isc that was complete by 1 h. There was no significant difference between the reduction in Isc at 60 min in the presence of ouabain on day 6 AEC monolayers treated (94%) or untreated (91%) with KGF compared with corresponding controls without ouabain (n = 3).

Effects of KGF on rENaC subunit mRNA expression. The effects of KGF on expression of mRNA for the alpha -, beta -, and gamma -subunits of the rENaC were evaluated by Northern blotting. In the representative experiment shown in Fig. 3A, mRNA for all three subunits is detectable in AEC monolayers on day 4 in culture. Reductions in the levels of each subunit mRNA occur in KGF-treated AEC monolayers on days 6 and 8 relative to monolayers grown in the absence of KGF. As indicated in Fig. 3B, significant differences in expression were found when signal intensity was quantified by scanning densitometry. Reductions of alpha -, beta -, and gamma -rENaC subunit mRNA levels of 55, 37, and 42%, respectively, occurred on day 6 in KGF-treated vs. untreated monolayers. Further reductions in alpha -, beta -, and gamma -rENaC subunit mRNA levels (68, 67, and 62%, respectively) were seen on day 8 in treated vs. untreated monolayers (n = 4).


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Fig. 3.   Effects of KGF on Na channel subunit mRNA. A: Northern blot. mRNA for alpha -, beta -, and gamma -subunits of rat epithelial Na channel (rENaC) were readily detectable in AEC by Northern blotting on day 4 in culture. In this representative Northern blot, mRNA levels for all three rENaC subunits declined on days 6 and 8 for KGF-treated monolayers relative to controls grown in MDSF alone. RNA loading was similar among all conditions, as indicated by equivalence of signal after hybridization with an 18S rRNA oligonucleotide probe. B: bar graph. Effects of KGF on each rENaC subunit mRNA level are shown (±SE) for AEC monolayers treated with KGF from days 4 - 6 (day 6) and days 4 - 8 (day 8) relative to untreated monolayers. * Significantly different from untreated monolayers.

Effects of KGF on Na+-K+-ATPase alpha 1- and beta 1-subunit isoform expression. Freshly isolated alveolar type II cells (data not shown) and cultured AEC maintained in MDSF for 4 days express mRNA for the alpha 1 and beta 1 isoforms of the Na+-K+-ATPase (Fig. 4A). Levels of expression of these isoforms remained relatively constant in MDSF between days 4 and 8 in culture. A representative Northern blot demonstrates that, after addition of KGF to monolayers on day 4, levels of alpha 1-subunit mRNA are increased on day 6 and day 8. alpha 1-Subunit mRNA increased by 41% on both day 6 (n = 11) and day 8 (n = 6). Levels of the beta 1 isoform mRNA did not increase significantly in the presence of KGF (n = 3; Fig. 4B).


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Fig. 4.   Effects of KGF on Na+-K+-ATPase alpha - and beta -subunit mRNA. A: Northern blot. This representative Northern blot demonstrates that AEC in culture for 4 days expressed abundant mRNA for the alpha 1 and beta 1 isoforms. After exposure to KGF, alpha 1 mRNA was increased on day 6 and day 8, whereas beta 1 mRNA remained unchanged. RNA loading is similar among all conditions, as indicated by equivalence of signal after reprobing with an 18S rRNA oligonucleotide probe. B: bar graph. Effects of KGF on alpha 1- and beta 1-subunit mRNA levels are shown (±SE) for AEC monolayers treated with KGF from days 4 - 6 (day 6) and days 4 - 8 (day 8) relative to untreated monolayers. * Significantly different from untreated monolayers.

As shown in the representative Western blots in Fig. 5, A and B, both alpha 1- and beta 1-subunit protein are abundantly expressed in cultured AEC on day 4. Na+-K+-ATPase alpha 1-subunit protein levels increased by 70% (n = 3) in KGF-treated AEC monolayers on day 6 relative to monolayers grown in the absence of KGF. A 31% increase in beta 1-subunit protein levels was noted on Western blots of deglycosylated immunoprecipitates from KGF-treated monolayers (n = 4; Fig. 5C).


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Fig. 5.   Effects of KGF on alpha 1- and beta 1-subunit protein. A: Western blot of alpha 1-subunit protein. Exposure of AEC monolayers to KGF from day 4 results in increased levels of Na+-K+-ATPase alpha 1-subunit protein by day 6, as shown in this representative Western blot. Protein loading is similar for each condition. B: Western blot of beta 1-subunit protein. Immunoprecipitates from equal numbers of AEC monolayers were subjected to deglycosylation and blotted with an anti-beta 1-subunit antibody. Deglycosylated immunoprecipitates of kidney membranes are shown as a positive control for Na pump beta 1-subunit. Exposure of AEC monolayers to KGF from day 4 resulted in increased levels of Na+-K+-ATPase beta 1-subunit protein by day 6. C: bar graph. Effects of KGF on alpha 1- and beta 1-subunit protein abundance are shown (+SE) for AEC monolayers treated with KGF from days 4 - 6 (day 6) relative to untreated monolayers. * Significantly different from untreated monolayers.

Effects of KGF on Na+-K+-ATPase alpha 2-subunit isoform expression. The Na+-K+-ATPase alpha 2 isoform is detectable in AEC at extremely low levels compared with the alpha 1 isoform. Figure 6A illustrates a representative Northern blot that includes RNA extracted from AEC on days 4, 6, and 8 in culture (lanes 1, 2, and 4, respectively) and from AEC grown in the presence of KGF from either day 4 through day 6 (lane 3) or day 4 through day 8 (lane 5). The blot was probed for the alpha 2 isoform using a 32P-labeled cDNA probe of approximately equal specific activity compared with that used for probing the alpha 1 isoform. Despite loading three times the total RNA (15 vs. 5 µg) and a 10-fold longer exposure of the autoradiogram (1 wk vs. 16 h) compared with the blot probed for the alpha 1 isoform shown in Fig. 4, the alpha 2 isoform remained only faintly detectable under any of the five conditions shown (n = 3).


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Fig. 6.   Expression of Na+-K+-ATPase alpha 2-subunit in AEC. A: alpha 2-subunit mRNA. This representative Northern blot, including equal amounts of RNA extracted from AEC on days 4, 6, and 8 in culture (lanes 1, 2, and 4, respectively) and from AEC grown in the presence of KGF from either days 4 - 6 (lane 3) or days 4 - 8 (lane 5), was probed for the alpha 2 isoform using a 32P-labeled cDNA probe of approximately equal specific activity compared with that used for probing the alpha 1 isoform. Despite loading 3 times the amount of total RNA (15 vs. 5 µg) and a 10-fold longer exposure of the autoradiogram (1 wk vs. 16 h), the alpha 2 isoform remains only faintly detectable under any of the 5 conditions shown compared with the blot probed for the alpha 1 isoform shown in Fig. 4. B: alpha 2-subunit protein. This representative Western blot, including protein extracted from rat brain and lung membranes (lanes 1 and 2, respectively) from AEC on days 4, 6, and 8 in culture (lanes 3, 4, and 6, respectively) and from AEC grown in the presence of KGF from either days 4 - 6 (lane 5) or days 4 - 8 (lane 7) was probed for the alpha 2 isoform using an anti-alpha 2-subunit monoclonal antibody (McB2). Rat brain and lung are positive for a 97-kDa protein band that represents the alpha 2-subunit, whereas AEC are uniformly negative.

Figure 6B illustrates a representative Western blot including protein extracted from rat brain and lung membranes (lanes 1 and 2, respectively) from AEC on days 4, 6, and 8 in culture (lanes 3, 4, and 6, respectively) and from AEC grown in the presence of KGF from either day 4 through day 6 (lane 5) or day 4 through day 8 (lane 7). The blot was probed for the alpha 2 isoform using an anti-alpha 2-subunit monoclonal Ab (McB2). Rat brain and lung were positive for a 97-kDa protein band that represents the alpha 2-subunit. AEC were uniformly negative despite equivalent loading of protein in all lanes (n = 3). Similar results (data not shown) were obtained using another anti-alpha 2 polyclonal Ab (UBI).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

We demonstrate in this study that KGF, in addition to its previously reported effects on alveolar type II cell proliferation and differentiation, increases transepithelial active ion transport across AEC monolayers. The sustained increase in ion transport represents an increment in active Na transport and correlates with an increase in both alpha 1- and beta 1-subunit protein. Expression of Na+-K+-ATPase alpha 1-subunit mRNA is increased by KGF, whereas mRNA levels for the beta 1 isoform do not change. Na pump alpha 2-subunit mRNA is only marginally detectable in AEC and remains similarly low after treatment with KGF. Levels of mRNA for the epithelial Na channel subunits decrease after treatment with KGF. Taken together, these results indicate that the KGF-induced increase in active ion transport in AEC is attributable principally to an increase in membrane-associated Na pumps. The increase in Na pump abundance appears to be driven by increased expression of Na+-K+-ATPase alpha 1-subunit mRNA.

We have previously demonstrated that AEC grown in primary culture form electrically resistive monolayers that actively transport Na in a vectorial fashion analogous to the movement of salt and water from air space to blood in situ (10). In the current study, we demonstrate that KGF stimulates increases in Isc, an index of active transepithelial ion transport, across AEC monolayers (Fig. 2A). Experiments showing comparable effects of the Na transport inhibitors amiloride and ouabain on Isc across monolayers treated or untreated with KGF indicate that the KGF-induced increases in Isc result primarily, if not exclusively, from effects on active Na transport. These findings are supported by the recent in situ study of Guery et al. (17), in which it was shown that KGF attenuated ANTU-induced lung edema primarily by enhancing alveolar fluid removal, an effect reversed by treatment with either amiloride or ouabain.

KGF is known to be a potent mitogen for epithelial cells in general and for AEC in particular (45). To assist in determining the relationship between the effects of KGF on cell proliferation and Na transport in AEC, monolayer cell number was serially determined after addition of KGF. Cell number in monolayers maintained in MDSF with or without KGF from day 4 were not different from each other on day 6, whereas Isc across monolayers treated with KGF on day 6 had increased by 37% (Figs. 1 and 2). These findings indicate that the initial effects of KGF on Isc do not result from changes in cell number. Changes in Na pump and Na channel subunit mRNA levels were normalized to 18S rRNA. The observed changes, which indicate relative differences in specific mRNA levels compared with total cellular RNA, probably also reflect increases (or decreases) per cell as well. Changes in Na pump alpha 1- and beta 1-subunit protein abundance were normalized to equal amounts of cell protein or equal numbers of monolayers, respectively. Because both cell protein and cell number per monolayer were also equivalent for treated vs. untreated monolayers on day 6, our results also most likely imply increases in Na pump subunits per cell. These data indicate not only that KGF increases transepithelial transport across AEC monolayers but that it specifically affects AEC Na channel and pump expression at 48 h of exposure.

With further time of exposure to KGF, cell number increases by 29% in AEC monolayers exposed to KGF relative to untreated controls on day 8. Over the same interval, Isc across KGF-treated monolayers increases to 66% greater than controls. Further increases in alpha 1-subunit mRNA also are apparent for KGF-treated monolayers on day 8, as shown in Fig. 4, A and B. Because these data are from Northern blots in which lanes were loaded with approximately equal amounts of RNA, it is most likely that they reflect additional increments in alpha 1-subunit mRNA per cell from day 6 to day 8. These results suggest that KGF effects an increase in Isc and Na pump expression in AEC per cell throughout the entire period of exposure (days 4 - 8) and that the additional increase in Isc seen on days 6 - 8 is due in part to an increase in cell number per monolayer.

Effects of KGF on transepithelial transport and transporter expression could represent indirect or secondary effects on other AEC properties. For example, another explanation for the observed KGF-induced increase in Na transport and Na pump expression in AEC could be related to the possibility that KGF promotes transdifferentiation of AEC toward the alveolar type II cell phenotype. We have recently shown that KGF increases expression of alveolar type II cell markers and decreases expression of alveolar type I cell markers in AEC in culture by 48 h, before any increase in cell number (6). If, as some published data suggest (39), alveolar type II cells possess greater numbers of Na pumps per cell than alveolar type I cells, the KGF-induced increase in Na pumps could reflect early changes associated with differentiation rather than a specific effect on Na transport per se. If this were the primary factor, however, changes in expression of Na channels, which also appear to be more abundant in alveolar type II cells than in alveolar type I cells (15), should resemble those for Na pumps. In contrast, our results indicate that expression of all three Na channel subunit mRNAs decreased after exposure to KGF (Fig. 3, A and B).

Upregulation of either Na pump and/or channel activities could potentially result in a net increase in transepithelial Na transport. The relatively slow onset (>12 h) of KGF effects on active ion transport across AEC monolayers, however, is least consistent with a simple or direct effect on Na pump activity (e.g., altered maximal transport rate) or Na channel function (e.g., increased open-time probability or conductance) as the primary mechanism accounting for the observed increase in Isc. Such short-term regulatory effects are most often described as occurring within minutes rather than hours (3). The longer term effects observed for Na pumps in the present study, which result from changes in gene expression and protein synthesis, could account for the KGF-induced increase in active transepithelial Na transport across AEC monolayers. In contrast, the KGF-induced reduction in the relative levels observed for each of the three known rENaC subunit mRNAs (Fig. 3, A and B) make it unlikely that the stimulatory effects of KGF on transepithelial transport are being exerted primarily on Na channels.

KGF appeared to have little effect on the relative mRNA levels among the three rENaC subunits. Differences in rENaC subunit expression in alveolar epithelium, which could affect the functional properties of amiloride-sensitive Na channels, have recently been reported by Farman et al. (15). These authors semiquantitatively assessed the relative expression of alpha -, beta -, and gamma -rENaC along the rat respiratory tract by in situ hybridization, finding equivalent expression of the alpha - and gamma -subunits in alveolar epithelium with substantially lower expression of the beta -subunit. Although the experiments in our current study were primarily designed to assess the effects of KGF on expression of each rENaC subunit, rather than differences among alpha -, beta -, and gamma -rENaC, we can estimate the relative amount of each subunit mRNA based on its average relative densitometric value on Northern blot and the length and specific activity of labeling of the cDNA probes used for detection (33). Based on these latter approximations (data not shown), expression of the alpha - and gamma -subunits appears to be similar, with substantially lower expression of beta -rENaC. These relative levels of expression among the rENaC subunits, which are consistent with those reported previously (15), do not change after KGF exposure. Thus the KGF-induced increase in transepithelial Na transport across AEC monolayers is not related to differential changes in expression of rENaC subunits.

We cannot exclude the possibility that KGF induces an increase in Na channel subunit protein via translational or posttranslational mechanisms, since a suitable quantitative assay for subunit abundance is unavailable to us at this time. Alternatively, KGF could increase ion transport by Na channels by upregulation of some thus far unidentified regulatory protein (e.g., G protein). Nonetheless, the temporal correlations among the increase in Na pump alpha 1-subunit mRNA, Na pump protein abundance, and Isc are most consistent with an increase in Na pumps as the principal mechanism by which KGF increases transepithelial Na transport across AEC monolayers.

In all tissues from which Na+-K+-ATPase has been isolated, it has been found to consist of an alpha - and beta -subunit. Several isoforms of both the alpha - and beta -subunit have been described and have been found to be expressed in a cell- and tissue-specific fashion, which may be important for cell-specific regulation by exogenous factors. Similar to the results of other studies in adult lung and fetal distal lung epithelium (9, 31, 33, 49), we demonstrate that mRNA for the alpha 1 and beta 1 isoforms is abundantly expressed in cultured AEC. Together with data obtained using currently available Ab reagents, our results suggest that new Na pump complexes formed after AEC stimulation by KGF are composed of alpha 1- and beta 1-subunits. We cannot exclude the possibility that the newly formed Na pumps also include the very recently described beta 3-subunit (26), since we do not know whether the anti-beta 1 Abs used in the present study cross-react with the beta 3 protein. This issue can only be definitively addressed by the development of suitable Ab reagents or other methods of specifically detecting the beta 3-subunit.

Regulation of Na+-K+-ATPase expression can occur in an isoform-specific fashion. For example, administration of triiodothyronine to rats results in a preferential increase in abundance of myocardial alpha 2 mRNA and activity relative to alpha 1 (18). Recent studies by Ridge et al. (37) have suggested that significant amounts of mRNA for the alpha 2 isoform are expressed in cultured AEC. Based on the results of Northern blots shown in Figs. 4 and 6A, however, we estimate the relative amount of alpha 2-subunit mRNA to be <1% of the levels of alpha 1-subunit mRNA in AEC under any of the conditions we studied. These results are consistent with those of Orlowski and Lingrel (34) and Young and Lingrel (49), who found very low levels of Na pump alpha 2 isoform in lung. They are also compatible with those of Ingbar et al. (20), who found no evidence of Na pump alpha 2 isoform expression in fetal or neonatal rat lung, and O'Brodovich et al. (33), who found a similar lack of Na pump alpha 2 isoform expression in fetal distal rat lung epithelium. Although Na pump alpha 2-subunit was present at detectable levels in lung membranes on immunoblot, as previously shown by Shyjan and Levenson (40), we were unable to find any alpha 2-subunit protein in AEC by this technique. We do not have any simple explanation for the apparent discrepancies between our data and those described by Ridge et al. (37). Nonetheless, we conclude that expression of the Na pump alpha 2-subunit is minimal or absent in AEC in the presence or absence of KGF and did not contribute significantly to the observed KGF-induced increase in Na pump expression in this study. Although it is possible that other agents could stimulate the expression of the Na pump alpha 2-subunit in AEC, our data are not consistent with a role for this Na pump subunit isoform in transepithelial transport by alveolar epithelium.

In this study, we demonstrate that KGF exposure results in parallel increases in both Na+-K+-ATPase alpha - and beta -subunit protein levels (Fig. 5, A-C), with an increase in mRNA for the alpha 1-subunit alone (Fig. 4, A and B). These findings are consistent with previous data showing independent regulation of Na+-K+-ATPase alpha - and beta -subunit mRNA levels, most often occurring at the level of transcription. For example, Lescale-Matys et al. (24) showed that Na+-K+-ATPase abundance increased twofold over control in a renal epithelial cell line (LLC-PK1/Cl4) after 24 h of incubation in low K+. Only beta -, but not alpha -, subunit mRNA levels increased despite the accumulation of newly synthesized alpha - and beta -subunits. In contrast, Wang et al. (47) found that treatment of infant rats with glucocorticoids resulted in increased Na pump alpha - and beta -subunit mRNAs and increased rates of both alpha  and beta  gene transcription, whereas Farman et al. (14) demonstrated that corticosteroid depletion after adrenalectomy reduced the expression of alpha 1, but not beta 1, mRNA in corticosteroid-sensitive tubular cells in the rat distal nephron. Thus it appears likely that expression of either alpha - or beta -subunits can be a limiting factor in the regulation of Na pump abundance in a specific cell or tissue under a particular set of influences. In the absence of a change in beta -subunit mRNA in the present study, the concurrent increase in beta -subunit protein is best explained by an increase in translational efficiency or a decrease in degradation rate of newly synthesized beta -subunits. The latter may occur due to the increased stability of alpha  /beta heterodimers inserted into the basolateral cell membrane, as previously proposed for Madin-Darby canine kidney cells (30).

KGF is a mitogen that acts specifically on epithelial cells, including those in the skin, liver, gastrointestinal tract, and alveolar epithelium. In the lung, KGF has been shown to stimulate alveolar type II cell proliferation both in vitro and in vivo (45) and, in preliminary studies, has also been found to modulate the AEC phenotype (6). KGF has been shown to ameliorate lung injury after a number of different forms of injury (48). Improved survival was demonstrated in rats after intratracheal administration of KGF before hyperoxic exposure (35). The lungs of KGF-treated animals demonstrated increased numbers of alveolar type II cells, but accumulation of intra-alveolar exudates and pleural effusions was also markedly decreased compared with untreated controls. These observations suggest that some of the beneficial effects of KGF could be attributed to an increase in alveolar fluid clearance. In a recent study, similar protective effects of KGF were observed in a rat model of pulmonary edema induced by ANTU (28). ANTU is associated with increased permeability pulmonary edema thought to be a result of both endothelial and epithelial cell damage. Pretreatment with KGF significantly attenuated the development of lung leak. The mechanisms responsible for this effect have subsequently been explored (17) and may include increased alveolar type II cell proliferation, upregulation of surfactant production, or increased ion transport. The results of our study provide further evidence that upregulation of alveolar epithelial active ion transport by KGF may contribute to its beneficial effects.

In summary, this study demonstrates that exposure to KGF induces a relatively delayed increase in active ion transport in AEC monolayers. These effects appear to be mediated by increases in mRNA expression for the Na pump alpha 1-subunit, resulting in increased expression of cellular and cell surface-associated alpha - and beta -subunits of Na+-K+-ATPase. The observed increase in transepithelial transport appears due to both an increase in cellular Na pumps and, once the cells begin to proliferate, an increase in cell number per monolayer. These results suggest a role for KGF in enhancing alveolar fluid clearance after acute lung injury.

    ACKNOWLEDGEMENTS

We thank Drs. Edward D. Crandall, Hastings Professor of Medicine, and Kenneth Norris, Jr., Chair of Medicine, for thoughtful guidance and encouragement of this work. We also thank Dr. Alicia McDonough for the generous gift of the FP Ab and for many helpful discussions, Drs. Michael Caplan, Kathleen Sweadner, and Andrea Quaroni for monoclonal Ab 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 Monica Flores, Stephanie Zabski, Martha Jean Foster, and Susie Parra.

    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 Clinical Investigator Development Award HL-02836 (to Z. Borok) and Grants HL-03609, HL-38578, HL-38621, and HL-51928, and the Hastings Foundation.

Address for reprint requests: Z. Borok, Div. of Pulmonary and Critical Care Medicine, GNH 11900, Univ. of Southern California, 2025 Zonal Ave., Los Angeles, CA 90033.

Received 1 May 1997; accepted in final form 3 October 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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