1 Department of Internal Medicine, University of Iowa College of Medicine, Iowa City 52242; 3 Veterans Affairs Medical Center, Iowa City, Iowa 52246; and 2 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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H441
cells, a bronchiolar epithelial cell line, develop a
glucocorticoid-regulated amiloride-sensitive Na+ transport
pathway on permeable supports (R. Sayegh, S. D. Auerbach, X. Li,
R. Loftus, R. Husted, J. B. Stokes, and C. P. Thomas.
J Biol Chem 274: 12431-12437, 1999). To understand
its molecular basis, we examined the effect of glucocorticoids (GC) on
epithelial Na+ channel (ENaC)-, -
, and -
and
sgk1 expression and determined the biophysical properties of
Na+ channels in these cells. GC stimulated the expression
of ENac-
, -
, and -
and sgk1 mRNA, with the first
effect seen by 1 h. These effects were abolished by actinomycin D,
but not by cycloheximide, indicating a direct stimulatory effect on
ENaC and sgk1 mRNA synthesis. The GC effect on transcription
of ENaC-
mRNA was accompanied by a significant increase in ENaC-
protein levels. GC also stimulated ENaC-
, -
, and -
and
sgk1 mRNA expression in A549 cells, an alveolar type II cell
line. To determine the biophysical properties of the Na+
channel, single-channel currents were recorded from cell-attached H441
membranes. An Na+-selective channel with slow kinetics and
a slope conductance of 10.8 pS was noted, properties similar to
ENaC-
, -
, and -
expressed in Xenopus
laevis oocytes. These experiments indicate that
amiloride-sensitive Na+ transport is mediated through
classic ENaC channels in human lung epithelia and that GC-regulated
Na+ transport is accompanied by increased transcription of
each of the component subunits and sgk1.
epithelial sodium channel; amiloride; short-circuit current; patch clamp; adenosine 3',5'-cyclic monophosphate; corticosteroids; airway epithelia; alveolar type II cells
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INTRODUCTION |
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THE DEVELOPING ALVEOLI in the fetal
lung are filled with liquid that arises, in part, from fluid secreted
in the alveolar lumen coupled to Cl secretion
(49). At the time of birth, net fluid secretion ceases and
absorption occurs to establish pulmonary gas exchange. It is now clear
that this transition from secretion to absorption coincides with the
loss of Cl
secretion and the active reabsorption of
Na+ across the luminal surface of alveolar and bronchiolar
epithelia (7, 36).
The leading molecular candidate to effect Na+ absorption by
the lung is the epithelial Na+ channel (ENaC). Three
subunits termed ,
, and
have been identified by several
laboratories (reviewed in Refs. 3 and 19). When expressed
together, these subunits reconstitute an amiloride-sensitive Na+-selective ion channel with properties similar to that
recorded in various epithelia, including fetal distal lung epithelial
cells (FDLE) and alveolar type II cells (23, 56). In the
developing rat fetal lung, ENaC-
, -
, and -
mRNA are expressed
around the time of birth, coinciding with the phenotype switch that
occurs to reabsorb liquid from the alveolar lumen (52,
63).
There is considerable evidence that ENaC expression and function can be
regulated by glucocorticoids. Glucocorticoids induce amiloride-sensitive Na+ transport in the immature fetal
lung and increase Na+ and fluid transport in the adult lung
(4, 17, 60). Whether given during the antenatal period to
the developing fetus or during adult life, exogenous glucocorticoids
increase ENaC- mRNA dramatically (48, 52). In addition,
the increase in lung ENaC mRNA abundance in the immediate perinatal
period correlates closely with the increase in circulating endogenous
glucocorticoids (51, 63). This effect of glucocorticoid
hormones on ENaC expression may be a previously unrecognized mechanism
of action of glucocorticoid therapy on lung maturation when given to
the preterm infant (37).
The serum- and glucocorticoid-regulated serine/threonine protein kinase
sgk was first described as an immediate early response gene
in rat mammary epithelia and rat-2 fibroblasts (64). The sgk transcript (now renamed sgk1) is rapidly
induced in vivo by glucocorticoids or aldosterone in a variety of rat
tissues, and similar responses are seen in epithelial cells derived
from the rabbit, amphibian, and canine kidney collecting duct (9,
13, 33, 34, 46). The stimulated kinase may have a direct impact on corticosteroid-regulated epithelial Na+ transport,
as coexpression of sgk1 with ENaC-, -
, and -
ENaC mRNA in Xenopus oocytes significantly enhances the
Na+ current (13, 34).
Alveolar type II cells and airway epithelial cells are thought to be
the primary sites for reabsorption of Na+ in the lung.
These cells express all ENaC mRNAs, but the biophysical profile of
Na+ channels expressed in these cells may be different from
that of the kidney collecting duct (31). Because channels
made of ENaC- alone and ENaC-
and -
or ENaC-
and -
subunits have properties that are different from the heteromultimer
(32), it has been proposed that the alveolar and airway
Na+ channel may have a different stoichiometry of ENaC
subunits. Alternatively, Na+ transport in the alveolar and
airway epithelia may occur, at least partly, via non-ENaC
Na+ channels.
In this paper, we describe human airway and alveolar cell lines with
glucocorticoid-regulated expression of ENaC-, -
, and -
and
sgk1 mRNA and amiloride-sensitive Na+ transport.
We demonstrate that one of these cell lines shows cAMP-stimulated
Na+ transport and has Na+ channels with
biophysical properties predicted for an ENaC-
, -
, and -
heteromultimer. We also determine that the glucocorticoid effects on
all ENaC subunits and sgk1 are likely to be transcriptional.
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METHODS |
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Materials.
Dexamethasone, amiloride, cycloheximide, forskolin, and IBMX were
purchased from Sigma Biochemicals (St. Louis, MO). Actinomycin D was
obtained from Roche Molecular Biochemicals (Indianapolis, IN); benzamil
was from Research Biochemical International (Natick, MA), and
[-32P]UTP was from NEN Life Science Products (Boston,
MA). Culture media were obtained from Life Technologies
(Gaithersburg, MD), and DNA sequencing and synthesis was a
service provided by the University of Iowa DNA core facility.
Tissue culture and RNA analysis. H441 cells were cultured in RPMI 1640, as described previously (53). A549 cells (American Tissue Culture Collection, Manassas, VA) and HEK-293 cells (Gene Transfer Vector Core, University of Iowa) were cultured, respectively, in MEM and Ham's F-12 supplemented with 10% FBS. To examine the effects of dexamethasone on gene expression, cell cultures were switched to serum-free medium and then treated with various concentrations of steroid or vehicle for 24 h in the presence or absence of actinomycin D or cycloheximide. To determine the time course for gene expression on permeable supports, H441 cells were grown on 30-mm Millicell PCF filters (Millipore, Bedford, MA), switched to serum-free steroid-free media for 24 h, and then exposed to 100 nM dexamethasone for various times. Total RNA was extracted from H441, A549, and HEK-293 cells as previously described (33).
Measurement of short-circuit current. To measure short-circuit current (Isc), H441 cells were grown for 4-7 days in RPMI 1640 with 6% serum and 100 nM dexamethasone on 12-mm Millicell PCF filters (44). To determine the effect of glucocorticoids and cAMP on epithelial Na+ transport, cells on filters were switched to steroid-free media for 24 h and then exposed to 100 nM dexamethasone or to 10 µM forskolin and 100 µM IBMX for various times. Isc and transepithelial resistance were measured at various time points in an Ussing chamber with or without 10 µM apical benzamil, and the cells were returned to normal culture conditions between measurements.
Cloning of hsgk1, -2, and -3. Total RNA prepared from H441 cells that had been treated with 100 nM dexamethasone for 24 h was reverse transcribed using oligo(dT) and Moloney murine leukemia virus reverse transcriptase as previously described (33). Briefly, 2 µl of first-strand cDNA was subjected to PCR amplification for 25 cycles using gene-specific primers and Taq DNA polymerase (Promega, Madison, WI) to obtain hsgk1, -2, and -3 cDNA fragments. To clone hsgk1, the primers 5'-CTCCTGCAGAAGGACAGGA and 5'-GGACAGGCTCTTCGGTAAACT were used with an anneal step at 55°C; to clone hsgk2, the primers 5'-TGTATCTCTCTGCCCTGCCAACC and 5'-CATTTCCCAGCCTCCATTCC were used with an anneal step at 55°C; and to clone hsgk3, the primers 5'-CCACTTACAAAGAGAACGGTCC and 5'-CATACAGAACAGCCCCAAGG used were with an anneal step at 62°C. Amplified fragments were cloned into pCRXLTOPO (Invitrogen, Carlsbad, CA), and individual clones were sequenced.
Ribonuclease protection assay for ENaC and sgk.
Steady-state levels of ENaC-, -
, and -
mRNA were measured by
ribonuclease protection assay (RPA) in H441 and A549 cells grown either
as monolayers on 75-cm2 polystyrene flasks (Corning) or on
30-mm Millicell PCF filters. To measure ENaC-
, -
, and -
mRNA,
10 µg RNA samples were hybridized with individual radiolabeled
antisense cRNA probes along with 18S rRNA as a control. Templates for
synthesis of human ENaC-1
and human ENaC-
cRNA probes have been
previously described, as has solution hybridization, nuclease
digestion, and identification of nuclease-protected products by PAGE
(44, 54). To measure human ENaC-
mRNA levels, one of
two templates was used. In some experiments, a 537-bp human ENaC-
fragment in pCR3.1 (gift from Paul McCray, University of Iowa) was used
to create a linearized template, and a 182-bp cRNA probe was then
synthesized from the T7 promoter to protect a 102-bp ENaC-
cRNA
fragment from ribonuclease digestion. In later experiments, a human
ENaC-
fragment in pCRII (Invitrogen) was used to create a linearized
template, and a 358-bp cRNA probe synthesized from the SP6 promoter was
used to protect a 177-bp cRNA fragment from ribonuclease digestion.
Immunoblotting of ENaC-.
H441 cells exposed to 100 nM dexamethasone or vehicle for 24 h
were directly lysed in 1× Laemmli buffer (1.5% SDS, 6% glycerol, and
50 mM Tris, pH 6.8), and protein concentration was determined by
spectrofluorometry (2). Protein lysates (30 µg
protein/lane) were heated to 60°C for 15 min and resolved by SDS-PAGE
on 10% polyacrylamide minigels (Bio-Rad, Hercules, CA). Gels were
transferred electrophoretically to nitrocellulose membranes and blocked
with 5 g/dl nonfat dry milk. Membranes were then incubated with an anti-ENaC-
antibody [ENaC-
antibody 3560-2(4);
IgG concentration = 0.518 µg/ml] overnight at 4°C in
a diluent containing 150 mM NaCl, 50 mM sodium phosphate, pH 7.5, 10 mg/dl sodium azide, 50 mg/dl Tween 20, and 1 g/dl BSA
(30). After a series of washes, membranes were exposed to
anti-rabbit IgG conjugated to horseradish peroxidase (Pierce, Rockford,
IL) at 0.16 µg/ml. Luminol-based enhanced chemiluminescence (LumiGLO;
Kirkegaard and Perry Laboratories, Gaithersburg, MD) was used to detect
antibody-antigen binding upon exposure to light-sensing film.
Appropriate bands were then analyzed using densitometry (Molecular
Dynamics, San Jose, CA).
Transient transfection and analysis of reporter activity.
The organization of the 5'-end of the human ENaC- gene has been
described previously (33, 53). A portion of the
5'-flanking region of the human ENaC-
gene (
487 +55), which
contains the functional glucocorticoid response element (GRE), was
cloned upstream of the firefly luciferase gene in the plasmid pGL3basic
(Promega). The GRE of human ENaC-
, AGAACAgaaTGTCCT, was mutated
within this plasmid to AGTCTAgaaTGTCCT using the Quikchange
Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) and primers
5'-CAGTGTAAAGAAGTCTAGAATGTCCTAGGGCCC and
5'-GGGCCCTAGGACATTCTAGACTTCTTTACACTG. Briefly,
the
487 +55 construct in pGL3basic was annealed with the above
primers and extended with Pfu DNA polymerase, the parental
plasmid was then digested with Dpn I, and the extended
circular double-stranded DNA molecule (
487 +55 mutGRE) was recovered
by transformation into bacteria.
Patch clamp of H441 cells. H441 cells were grown on a Transwell-clear filter (Costar, Cambridge, MA) for 4 days in RPMI 1640 containing 6% FBS and 100 nM dexamethasone and then for 2 days in serum-free RPMI containing 100 nM dexamethasone. Before patch-clamp analysis, the Isc for each filter was measured in an Ussing chamber and ranged between 5 and 7 µA/cm2. Single channel currents were measured in cell-attached patches while cells were superfused at 37°C, using an Axopatch 2B voltage-clamp amplifier under the control of the pClamp software suite (Axon Instruments, Burlingame, CA), as described earlier (57). The bathing solution contained (in mM) 140 NaCl, 4.5 KCl, 2.5 CaCl2, 1 MgCl2, 10 HEPES, and 5 D-glucose, pH 7.35, and the pipette solution contained (in mM) 140 LiCl, 3 MgCl2, and 10 HEPES, pH 7.35. Slope conductance was calculated using pClamp software (Axon Instruments).
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RESULTS |
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We have previously shown, by Isc
measurements, that H441 cells express a glucocorticoid-regulated
Na+ transport pathway (44). To begin to
determine the basis for regulation of Na+ transport, we
first measured the time course for glucocorticoid-mediated stimulation
of Isc in H441 cells grown on permeable
supports. Isc begins to increase after 4 h
of glucocorticoid exposure and is clearly elevated by 6 h (Fig.
1A). The current continues to increase over the next 18 h, and, as we have previously reported, almost all of the current is inhibited by 10 µM benzamil, an ENaC inhibitor (44). In the control cells, there was a slow
decline in Isc over 24 h, which was not
related to a change in resistance and may have been secondary to the
continued serum and steroid deprivation. The results confirm that
glucocorticoids increase benzamil-sensitive Na+ transport,
consistent with an increase in ENaC function.
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We next determined, by RPA, if ENaC subunit mRNAs were
regulated by glucocorticoids in H441 cells. The effect of 100 nM
dexamethasone on ENaC mRNA from H441 cells grown on filters was
examined at different time points. The results demonstrate that
dexamethasone increased the expression of all three subunits in a
time-dependent manner, with a different profile for each of the three
subunits. Under basal conditions, although there was abundant
expression of ENaC-, ENaC-
and -
expression was not
identifiable (Fig. 1, B and C). In the presence
of dexamethasone, ENaC-
mRNA levels increased significantly by
4 h and continued to increase over the next several hours. The
ENaC-
subunit mRNA was substantially increased as early as 2 h,
the earliest time point tested, and continued to increase up to 24 h, whereas the ENaC-
subunit was only increased at 24 h. When
coupled with the Na+ transport data in Fig. 1A,
the results show that the increase in ENaC-
and -
mRNA levels
occurs before an increase in Na+ transport and suggests
that the increase in transport may be accompanied by an increase in
some subunit proteins. The data also indicate that an increase in
expression of ENaC-
mRNA may not be required for the early
glucocorticoid effect on Na+ transport in H441 cells. To
assess if the glucocorticoid regulation of ENaC-
, -
, and -
mRNA was qualitatively different if the cells were grown on solid
supports, the expression of these three subunits was also determined in
H441 cells grown in polystyrene cell culture flasks. Although a careful
time course analysis was not performed, the data showed that
glucocorticoids stimulate ENaC-
, -
, and -
expression in a
similar fashion (data not shown). We then determined the dose-response
curve for dexamethasone at 24 h for each of the three subunits
when H441 cells were grown on solid supports. Under steroid-free
conditions, ENaC-
expression was easily detectable, whereas ENaC-
and -
expression was difficult to identify, and dexamethasone
increased the expression of each of the three mRNAs in a dose-dependent
manner (Fig. 2, A-C).
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To determine if the increase in ENaC mRNA is accompanied by an increase
in ENaC protein, cell lysates from glucocorticoid- and vehicle-treated
H441 cells were immunoblotted with specific polyclonal antibodies
raised against the rat ENaC- and -
proteins (30).
The results clearly demonstrate that there is a two- to threefold
increase in ENaC-
protein after exposure to 100 nM dexamethasone for
24 h (Fig. 3). We were unable to
detect ENaC-
or -
protein in these cells using our antibody,
which may relate to abundance of the protein or the affinity of the
antibodies for the human protein.
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The finding that all three ENaC subunits are regulated by
glucocorticoids in H441 cells prompted us to examine ENaC mRNA
expression in A549 cells. Recently, Lazrak et al. (26, 27)
reported that A549 cells, a human type II alveolar epithelial
cell line, have glucocorticoid-stimulated amiloride-inhibitable
Na+ transport with regulated expression of some ENaC
subunits. Our results show that all three subunits are not detectable
by RPA under basal conditions but are induced by stimulation with 100 nM dexamethasone. Although ENaC- mRNA is evident as early as 2 h after stimulation, ENaC-
and -
expression was only evident at
later time points (Fig. 4,
A-C).
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There is increasing evidence that sgk1 may be responsible,
at least in part, for the corticosteroid-mediated increase in
Na+ transport in amphibian, rabbit, and rat kidney
(13, 34). The mRNA for sgk1 is rapidly induced
by glucocorticoids and aldosterone, and coexpression of sgk1
with ENaC-, -
, and -
subunits in Xenopus oocytes
leads to an increase in Na+ transport. This effect of
sgk is achieved by increasing the number of ENaC channels
assembled at the cell surface (16). The increase in
sgk1 mRNA by glucocorticoids was first noted in rat mammary epithelia and has also been reported in the canine collecting duct and
in some human epithelial cell lines (33, 35, 64), although, when first cloned, the human sgk1
(hsgk1) transcript did not appear to be regulated by
glucocorticoids (58). Recently, two related transcripts,
sgk2 and sgk3 have been cloned from human tissues, and these gene products also possess similar kinase activity (25). We first asked if any of the sgk isoforms
were expressed in H441 cells. We were able to clone each of the
hsgk isoforms by RT-PCR, confirming that all sgk
isoforms are expressed in these cells (Fig.
5A). To examine their
regulation, we measured steady-state levels of hsgk
transcripts by RPA in H441 cells. Our results clearly demonstrate that
hsgk1 is increased by corticosteroid treatment, with a
maximal effect seen at 1 h (Fig. 5B). Hsgk3
is not induced by corticosteroids (data not shown). In addition,
hsgk2 was not identifiable by RPA in H441 cells, and its
regulation was not examined further.
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We next asked if the effects of glucocorticoids on ENaC subunits and
sgk1 in H441 cells were at a transcriptional level and if
protein synthesis was required. We have previously shown that the
glucocorticoid and mineralocorticoid effect on ENaC- expression on
human and canine ENaC-
expression was transcriptional (33, 44), so these experiments were restricted to ENaC-
and -
and sgk1 transcripts. The effect of dexamethasone on
ENaC-
and -
and sgk1 expression was abolished by
simultaneous treatment with actinomycin D, providing strong supportive
evidence that glucocorticoids increase transcription of ENaC-
and
-
subunits (Fig. 6, A and B). Cycloheximide, a protein synthesis inhibitor, had no
effect on basal levels of ENaC-
and -
and appeared to augment
dexamethasone-induced ENaC-
and -
expression (Fig. 6,
A and B), suggesting that a labile intermediary
protein expressed in H441 cells may inhibit the glucocorticoid effects
on these genes. In contrast to the results seen with ENaC-
and -
,
cycloheximide superinduced basal and corticosteroid-stimulated
sgk1 mRNA expression (Fig. 6C).
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Because hsgk1 was transcriptionally regulated by
glucocorticoids in one human epithelial cell line, we asked if other
human epithelial cells would also show similar regulation. We evaluated hsgk1 expression by RPA in A549 cells and in a human
embryonic kidney cell line (HEK-293). Hsgk1 was rapidly
increased by dexamethasone in A549 cells but not in HEK-293 cells
(Fig. 7, A and B).
To further explore the differential glucocorticoid response in these
cell lines, we expressed a luciferase-coupled human ENaC-
promoter-enhancer in A549 cells. This promoter-enhancer
construct contains the functional GRE of the human ENaC-
gene
(44), and the data show that reporter gene activity was
robustly stimulated by dexamethasone in A549 cells (Fig.
7C). This response was predictable, since the human ENaC-
transcript, at least in our studies, is induced by dexamethasone (Fig.
4A). Consistent with our previous studies, a targeted
mutation of the ENaC-
GRE abolished the dexamethasone response,
confirming that the GRE in the ENaC-
gene is necessary and
sufficient for glucocorticoids to stimulate ENaC-
transcription in
A549 cells. The inability of glucocorticoids to stimulate
hsgk1 expression in HEK-293 cells is not because of the
absence of a functional glucocorticoid receptor (GR), since the plasmid
TAT3-luc is fully responsive to glucocorticoids and likely indicates
that unidentified cofactors may modulate glucocorticoid regulation of
sgk in specific epithelia.
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Given that all three ENaC subunits were regulated by corticosteroids,
we hypothesized that the corticosteroid-stimulated Na+
transport in H441 cells occurred via a classic ENaC heteromultimeric complex. To determine the biophysical properties of H441
Na+ channels, glucocorticoid-stimulated cells grown on
permeable supports were subjected to patch-clamp analysis at 37°C
with Li+ in the pipette. All patches were made on the
apical membrane in the cell-attached mode, and channels were rarely
seen. When channels were occasionally identified, the single channel
traces showed very long open and close times (several hundred
milliseconds, usually), a well-known characteristic of ENaC channels.
The open-channel current amplitude for various voltages was measured,
and a current-voltage plot was generated (Fig.
8). Linear regression analysis of these points gives a slope conductance of 10.8 pS. Extrapolation of the
conductance line indicates a very positive reversal potential indicative of an Na+-selective channel. These
characteristics are indistinguishable from ENaC channels heterologously
expressed in Xenopus oocytes (53).
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Another signaling pathway with direct effects on Na+
transport in airway epithelial cells involves the stimulation of cAMP, as occurs, for example, with epinephrine stimulation (38).
To determine if the H441 cell is a model to study cAMP regulation of
Na+ transport, we used forskolin, a direct activator of
adenylyl cyclase, and IBMX, a phosphodiesterase inhibitor, to elevate
intracellular cAMP levels. When grown on permeable supports, cAMP
stimulation led to a substantial increase in Isc
after 24 h in these cells (Fig.
9A). To confirm that the
increase in current was the result of Na+ transport and not
Cl secretion, the effect of 10 µM benzamil on basal and
stimulated Isc was examined. The results
demonstrate that almost all of the current is benzamil-sensitive, thus
excluding a significant contribution from Cl
secretion
(Fig. 9A). To examine the effect of corticosteroids and cAMP
stimulation together, the effect of these agents on
Isc was measured. The results show that
forskolin/IBMX stimulation further enhanced the effect of
corticosteroids on Isc and that the effect
appeared to be more than additive (Fig. 9B). Finally, we
examined the time course of the forskolin/IBMX effect on
Isc. cAMP stimulation on
Isc was fairly rapid with an effect that was obvious within 5 min, and this Isc remained
persistently elevated (Fig. 9C). When 10 µM benzamil was
added to the apical surface, the current was completely abolished, thus
confirming that almost all electrogenic ion transport could be
accounted for by Na+ entry at the apical membrane.
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DISCUSSION |
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In this paper, we report that glucocorticoids regulate the
expression of ENaC-, -
, and -
, and sgk1 mRNA in two
human lung cell lines. The H441 cell line, established from the
pericardial fluid of a patient with papillary adenocarcinoma of the
lung, expresses the Clara cell 10-kDa (CC-10) protein, the surfactant proteins-A, -B, and -D, and has the morphological characteristics of a
bronchiolar epithelial cell line of Clara cell lineage (20, 39,
43, 50). We have recently reported that glucocorticoids stimulate amiloride-sensitive Na+ transport in this cell
line and that this correlates with the regulated expression of ENaC-
(44), similar to that reported in primary cultures of
fetal and adult rat lung epithelial cells (11, 15). We now
report that glucocorticoids increase the expression of ENaC-
and
-
and sgk1 in this cell line. The A549 cell line, also
established from a lung adenocarcinoma, displays characteristics that
are more typical of alveolar type II cells, yet they do not express any
of the surfactant genes (8, 47). Recently, the biophysical
properties of Na+ channels and the expression profile of
ENaC mRNA in A549 cells and their modulation by glucocorticoids were
reported (26, 27). In this study, 24-48 h after
stimulation with 1 µM dexamethasone the authors demonstrated a
17-fold increase in ENaC-
mRNA, a 1.6-fold increase in ENaC-
mRNA, and no increase in ENaC-
mRNA by RT-PCR. To our knowledge,
this is the first report of a lung epithelial cell where ENaC-
mRNA
is not regulated by glucocorticoids. Our results are different and
clearly show a substantial and early increase in ENaC-
mRNA in A549
cells when stimulated with 100 nM dexamethasone (Fig. 4A).
This result is in agreement with studies done by others and us,
demonstrating that the glucocorticoid-responsive enhancer of the human
ENaC-
gene is functional in A549 cells (Fig. 7C and Ref.
62). The studies reported in this paper provide clear
evidence that glucocorticoids regulate expression of all three subunits
in these human lung epithelial cell lines. The reason for the apparent
discrepancy from the previously published work is not clear but may
result from dissimilar culture conditions and/or different methods for
measurement of RNA levels.
The finding that all three ENaC subunits are regulated by
glucocorticoids in these cell lines was, at first, a little surprising, since many investigators have reported that corticosteroids increase expression of ENaC- but not -
and -
mRNA in the fetal and
mature rodent lung (48, 52). Developmental studies in the
rat lung demonstrate that, although ENaC-
mRNA expression shows a
dramatic increase at the time of birth, coinciding with the perinatal
glucocorticoid surge, expression of ENaC-
and -
mRNA is either
not evident or increases modestly before birth (52, 63).
Analysis of the available literature, however, suggests that
developmental expression and glucocorticoid regulation of ENaC subunits
may be different in the human lung and in derived epithelia. Human (21 wk gestation) fetal lung explants express ENaC-
, -
, and -
mRNA
in culture, and all three subunits are further regulated by
glucocorticoids (55). Using specific polyclonal antisera
against ENaC-
and -
subunits, Gaillard et al. (18)
recently demonstrated ENaC-
and -
subunit protein expression as
early as 17 wk of gestation in human bronchial and bronchiolar
epithelium and by 30 wk of gestation in a pattern similar to adult
airways. Further evidence that the fetal and perinatal regulation of
ENaC expression is different in humans compared with rodents is the
difference in lung phenotype between patients who have homozygous loss
of function mutations in the ENaC-
subunit and mice in which the
ENaC-
subunit has been inactivated. Although the human mutation
causes severe renal disease, pseudohypoaldosteronism type 1 with salt
wasting, hypotension, and hyperkalemia, the lung phenotype is milder,
with a tendency to increased airway fluid and a chronic cough
(12, 24, 45). By contrast, ENaC-
knockout mice die
within a few hours of birth from inadequate lung liquid absorption
(22).
Glucocorticoids increase the mRNA levels for ENaC-, -
and -
subunits and sgk1 in a cell- and tissue-specific fashion. An imperfect palindromic GRE in the 5'-flanking region of the human and
rat ENaC-
gene is necessary and sufficient for glucocorticoid regulation of the ENaC-
subunit (28, 33, 40, 44).
Similarly, a GRE in the 5'-flanking region of the rat sgk1
gene is required for steroid regulation of sgk1
(65). The temporal profile of expression of
sgk1 and ENaC-
after glucocorticoid stimulation is quite
different, with sgk1 transcript levels that peak within 1 h, although increases in ENaC-
mRNA levels are only evident by 2 h and then continue to increase for 24-48 h. These
differences probably arise, in part, from complex regulation by
additional transcription factors that modulate the rate of
glucocorticoid-dependent transcription of individual genes and, in
part, from differences in mRNA stability. Furthermore, all tissues that
express GR do not show glucocorticoid regulation of ENaC-
and
sgk1, indicating that cell-specific coactivators and/or
repressors determine spatial expression of these genes. The lack of
regulation of hsgk1 in the Hep G2 cell line, a cell line in
which the GR is clearly expressed, was interpreted by Waldegger et al.
(58) as indicating that the human sgk1
transcript, in contrast to amphibian and rodent sgk1, was
not regulated by corticosteroids. In support of this hypothesis, the
authors were unable to locate a GRE in the proximal 2.4 kb of the
5'-flanking region of the hgsk1 gene (59). Our studies with two human lung cell lines clearly indicate that
hsgk1 is regulated by glucocorticoids, and, at least in the
H441 cell line, this effect is transcriptional. These studies are in
agreement with recently published studies demonstrating that
sgk1 is a glucocorticoid-regulated transcript in several
human cell lines (35). Our findings suggest that
hsgk1 may be regulated by a GRE within the transcriptosome of the hsgk1 gene but that this element may be further 5'
and flanking, 3' and flanking, or elsewhere within the gene.
Glucocorticoids also regulate ENaC- and -
mRNA levels in H441 and
A549 cells, and, based on the ability of actinomycin D to abolish
glucocorticoid-dependent expression, this effect is likely to be at the
level of gene transcription. We have cloned and characterized the
5'-flanking region of human ENaC-
and -
genes and have not yet
identified a functional glucocorticoid-responsive enhancer
(1, 54a). This could indicate that
the glucocorticoid-dependent regulation of ENaC-
and -
is not
transcriptional, although it is more likely that the enhancer elements
are located elsewhere in the genome. At the present time, we can only
conclude that, although ENaC-
and sgk1 are regulated by
GREs, the molecular basis for glucocorticoid regulation of ENaC-
and
-
remains unknown.
The biophysical properties of Na+ channels in alveolar and
airway epithelial cells have been studied by single channel analysis. Several types of channels have been identified, including
calcium-activated nonselective and Na+-selective cation
channels and a calcium-insensitive Na+-selective channel
(for review, see Ref. 31). The calcium-insensitive Na+ channel identified in rat FDLE cells has a conductance
of 4.4 pS, is highly Na+ selective, and has long open and
slow times very similar to the properties of ENaC-, -
, and -
reconstituted Na+ channels in Xenopus oocytes
(10, 56). Na+-selective channels were also
identified by patch-clamp analysis of A549 cells, where dexamethasone
increased channel open time and open probability and altered channel
conductance from 8.6 to 4.4 pS (26, 27). In this paper, we
report that H441 cells express an Na+-selective channel
with a conductance of 10.8 pS when measurements were performed at
37°C and Li+ was used as the charge carrier. The kinetic
properties of the channel seen in H441 cells are very typical of ENaC
channels. When heterologously expressed in Xenopus oocytes
and when measurements were made at 22°C with Li+ in the
pipette, the human ENaC subunits reconstitute an
Na+-selective channel with a slope conductance of ~7 pS
(53). We believe that these channels cannot be
distinguished from the 4.4-pS channel seen in FDLE and
corticosteroid-treated A549 cells (27, 56). The disparity
in channel conductance between the H441 channel and those reported from
Na+-selective channels in FDLE and A549 probably reflect
differences in the temperature at which measurements were made and the
use of Li+ rather than Na+ as the charge
carrier (41, 42). Our results also suggest that the ENaC
heteromultimer is the ion channel responsible for Na+
transport in H441 cells, at least under glucocorticoid-treated conditions. We are unable to comment on the properties of
Na+ channels in H441 cells not stimulated with
glucocorticoids, since these channels were very difficult to identify.
Recently, Jain and colleagues (23) demonstrated that
alveolar type II cell expression of a highly selective Na+
channel with ENaC-type properties was substantially enhanced when these
cells were exposed to corticosteroids and grown on permeable supports
in the presence of an air-liquid interface. The significance of the
Ca2+-activated and nonselective cation channels that have
been previously reported from a variety of lung epithelial cells
is not entirely clear but could be attributed to the substrate on which
the cells are grown, the culture conditions, and the patch
configuration in those studies.
In addition to glucocorticoids, amiloride-inhibitable Na+
transport in airway epithelia can be regulated by arginine vasopressin and by catecholamines (6, 14, 21). Catecholamines and
arginine vasopressin are thought to act via their second messenger
cAMP, since their effects can be mimicked by membrane-permeant analogs of cAMP (5, 61). We use forskolin and IBMX to increase
cAMP levels and show that amiloride-sensitive Na+ transport
is increased in H441 cells. This increase is seen even in the absence
of glucocorticoids, and, more importantly, cAMP stimulation potentiates
the effect seen with glucocorticoids, suggesting that these agonists
activate distinct pathways. In contrast to the effect of
glucocorticoids on Na+ transport, which takes hours, the
effect of forskolin/IBMX is seen within minutes and persists for at
least 24 h, suggesting that posttranscriptional and
transcriptional mechanisms are likely to play a part in this effect. In
comparison, cAMP stimulation of the amiloride-sensitive Na+
current in fetal rat alveolar epithelial cells was seen at 8 h,
the first time point reported, and there was no additive effect with
glucocorticoids (15). The increase in Na+
transport seen with cAMP in these primary cultures correlated with an
increase in ENaC- mRNA expression, similar to results we have seen
in H441 cells (data not shown). The H441 cell line thus appears to be a
good model to study glucocorticoid- and cAMP-regulated Na+
transport mediated by ENaC.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Kang Liu for excellent technical support, Paul McCray for
the gift of a human ENaC- cDNA clone, and David Pearce and Keith
Yamamoto for the TAT3-luc cDNA clone and acknowledge the DNA synthesis
and sequencing services provided by the University of Iowa DNA core facility.
![]() |
FOOTNOTES |
---|
Portions of the work submitted here were presented in abstract form at the American Thoracic Society meeting in 2000.
This work was supported in part by March of Dimes Birth Defects Foundation Research Grant 6-FY99-444, National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54348 and DK-52617, by a grant from the Department of Veteran's Affairs, and by a Career Investigator Award from the American Lung Association to C. P. Thomas.
Address for reprint requests and other correspondence: C. P. Thomas, Dept. of Internal Medicine, E300 GH, Univ. of Iowa, 200 Hawkins Dr., Iowa City, IA 52242 (E-mail: christie-thomas{at}uiowa.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajplung.00085.2001
Received 7 March 2001; accepted in final form 10 October 2001.
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