Will Rogers Institute Pulmonary Research Center, Division of Pulmonary and Critical Care Medicine, University of Southern California, Los Angeles, California 90033
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ABSTRACT |
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We 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
1-subunit mRNA abundance was
increased by 41% on days 6 and
8 after exposure to KGF, whereas
2-subunit mRNA remained only
marginally detectable in both the absence and presence of KGF. Levels
of mRNA for the
1-subunit of
Na+-K+-ATPase
did not increase, whereas cellular
1- and
1-subunit protein increased 70 and 31%, respectively, on day 6. mRNA
for
-,
-, and
-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
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
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INTRODUCTION |
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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 - and
-subunit.
Several isoforms have been cloned, and their physiological properties
have been characterized (41). Both
- and
-subunits are expressed
in a cell- and tissue-specific fashion. The
1-subunit appears to be the
predominant isoform expressed in alveolar epithelium, although low
levels of the
2 and
3 isoforms are detectable in lung (34, 40, 49). The
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
(,
, and
) 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
-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
1-subunit mRNA expression.
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METHODS |
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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;
k · 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
1,
1, and
2 isoforms of
Na+-K+-ATPase
(E. Benz, Johns Hopkins University, Baltimore, MD) and the
-,
-,
and
-subunits of the rENaC (C. Canessa and B. Rossier, Université de Lausanne, Switzerland). Probes were labeled with [
-32P]dCTP
(Amersham) by the random-primer method using a commercially available
kit (Boehringer Mannheim, Indianapolis, IN). Blots were washed at high
stringency [0.5× SSC (75 mM NaCl-7.5 mM sodium citrate, pH
7.0) with 0.1% SDS at 55°C] and visualized by
autoradiography. Differences in RNA loading were normalized using a
24-mer oligonucleotide probe for 18S rRNA end labeled with
32P-ATP. 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
1- and
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
1-subunits, cells were first
immunoprecipitated with the monoclonal anti-Na pump
-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
-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-1 Ab 6H (M. Caplan, Yale University; see Ref. 21) was used for detection of Na pump
1-subunits. The monoclonal
anti-
2 Ab McB2 (K. Sweadner,
Harvard University; see Ref. 46) and an
anti-
2 polyclonal Ab purchased
from Upstate Biotechnology (UBI, Lake Placid, NY) were used for
detection of Na pump
2-subunits. The polyclonal
anti-
1-subunit Ab FP (A. McDonough, University of Southern California) was used for detection of
-subunits precipitated by Ab IEC 1/48, since the latter recognizes only the undenatured
-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
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
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.
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RESULTS |
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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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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 k · 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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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 -,
-, and
-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
-,
-, and
-rENaC subunit mRNA
levels of 55, 37, and 42%, respectively, occurred on day
6 in KGF-treated vs. untreated monolayers. Further
reductions in
-,
-, and
-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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Effects of KGF on
Na+-K+-ATPase
1- and
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
1 and
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
1-subunit mRNA are increased on
day 6 and day
8.
1-Subunit
mRNA increased by 41% on both day 6 (n = 11) and day
8 (n = 6). Levels of
the
1 isoform mRNA did not
increase significantly in the presence of KGF
(n = 3; Fig. 4B).
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Effects of KGF on
Na+-K+-ATPase
2-subunit isoform
expression.
The
Na+-K+-ATPase
2 isoform is detectable in AEC
at extremely low levels compared with the
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
2 isoform using a
32P-labeled cDNA probe of
approximately equal specific activity compared with that used for
probing the
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
1 isoform shown
in Fig. 4, the
2 isoform
remained only faintly detectable under any of the five conditions shown
(n = 3).
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DISCUSSION |
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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 1- and
1-subunit protein. Expression of
Na+-K+-ATPase
1-subunit mRNA is increased by
KGF, whereas mRNA levels for the
1 isoform do not change. Na
pump
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
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 1- and
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 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
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 -,
-, and
-rENaC along the rat respiratory tract
by in situ hybridization, finding equivalent expression of the
- and
-subunits in alveolar epithelium with substantially lower expression
of the
-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
-,
-, and
-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
- and
-subunits appears to be similar, with substantially lower expression of
-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
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 - and
-subunit. Several isoforms of both the
- and
-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
1 and
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
1- and
1-subunits. We cannot exclude
the possibility that the newly formed Na pumps also include the very
recently described
3-subunit
(26), since we do not know whether the
anti-
1 Abs used in the present
study cross-react with the
3
protein. This issue can only be definitively addressed by the
development of suitable Ab reagents or other methods of specifically
detecting the
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 2 mRNA and activity relative to
1 (18). Recent studies by Ridge
et al. (37) have suggested that significant amounts of mRNA for the
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
2-subunit
mRNA to be <1% of the levels of
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
2 isoform
in lung. They are also compatible with those of Ingbar et al. (20), who
found no evidence of Na pump
2
isoform expression in fetal or neonatal rat lung, and O'Brodovich et
al. (33), who found a similar lack of Na pump
2 isoform expression in fetal
distal rat lung epithelium. Although Na pump
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
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
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
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
- and
-subunit protein levels (Fig. 5, A-C), with
an increase in mRNA for the
1-subunit alone (Fig. 4,
A and
B). These findings are consistent
with previous data showing independent regulation of
Na+-K+-ATPase
- and
-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
-, but not
-,
subunit mRNA levels increased despite the accumulation of newly
synthesized
- and
-subunits. In contrast, Wang et al. (47) found
that treatment of infant rats with glucocorticoids resulted in
increased Na pump
- and
-subunit mRNAs and increased rates of
both
and
gene transcription, whereas Farman et al. (14)
demonstrated that corticosteroid depletion after adrenalectomy reduced
the expression of
1, but not
1, mRNA in
corticosteroid-sensitive tubular cells in the rat distal nephron. Thus
it appears likely that expression of either
- or
-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
-subunit mRNA in the present study, the concurrent
increase in
-subunit protein is best explained by an increase in
translational efficiency or a decrease in degradation rate of newly
synthesized
-subunits. The latter may occur due to the increased
stability of
/
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 1-subunit,
resulting in increased expression of cellular and cell
surface-associated
- and
-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.
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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.
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