1 Department of Internal Medicine, University of Iowa College of Medicine and the 2 Veterans Affairs Medical Center, Iowa City, Iowa 52246
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ABSTRACT |
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In lung and collecting duct epithelia, glucocorticoid (GC)-stimulated Na+ transport is preceded by an increase in the protein kinase sgk1, which in turn regulates the activity of the epithelial Na+ channel (ENaC). We investigated the mechanism for GC-regulated human sgk1 expression in lung and renal epithelia. sgk1 mRNA was increased in these epithelia by GCs, and this was inhibited by actinomycin D and superinduced by cycloheximide, consistent with a transcriptional effect that did not require protein synthesis. To understand the basis for transcriptional regulation, the transcription initiation site was mapped and the 5'-flanking region cloned by PCR. A 3-kb fragment of the upstream region was coupled to luciferase and transfected into A549 cells. By deletion analysis, an imperfect GC response element (GRE) was identified that was necessary and sufficient for GC responsiveness. When tested with cell extracts, a specific protein recognized by an anti-GC receptor (GR) antibody bound the GRE in gel mobility shift assays. We conclude that GCs stimulate sgk1 expression in human epithelial cells via activation of a GRE in the 5'-flanking region of sgk1.
epithelial Na+ channel; glucocorticoid response element; corticosteroids; airway epithelia; gene transcription; gel mobility shift assays
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INTRODUCTION |
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NA+ is absorbed
from the lumen of the collecting duct, the distal colon, airway
and alveolar epithelia, sweat glands, and salivary ducts principally
via an amiloride-sensitive Na+ channel. The molecular
correlate of this Na+ transport pathway is the epithelial
sodium channel (ENaC), composed of a heteromultimer of -,
-, and
-subunits (11, 28). Two of the principal
regulators of Na+ absorption at these sites are the
corticosteroids, aldosterone and cortisol, which act within minutes to
hours to increase Na+ transport. Much recent attention has
been focused on understanding the mechanism of corticosteroid action to
stimulate Na+ transport in each of these sites.
The effects of corticosteroids on Na+ transport can be
substantially reduced by inhibitors of transcription and translation, clearly suggesting that new RNA synthesis is required for the increase
in Na+ transport. Among the putative targets for
corticosteroid action are the ENaC subunits themselves. There is now
considerable evidence that ENaC expression and function can be
regulated by glucocorticoids (GCs) and aldosterone. When given during
the antenatal period to the developing fetus or when infused during
adult life, exogenous GCs increase lung ENaC expression dramatically
(29, 31). Several studies have also reported that either
dexamethasone or aldosterone infusion leads to increased expression of
-ENaC mRNA in the renal cortex and
- and
-ENaC mRNA in the
distal colon (1, 10, 29). The rise in
-ENaC mRNA
expression is due to the trans-activation of a GC response
element (GRE) in the 5'-flanking region of the
-ENaC gene, but the
basis for the corticosteroid-mediated increase in
- and
-ENaC
remains unknown (17, 19, 23, 25).
Although ENaC subunits are regulated by corticosteroids, in some
tissues the increase in Na+ transport precedes the increase
in ENaC, suggesting that there may be other steroid-regulated proteins
that are required for the early increase in Na+ transport.
Two groups simultaneously identified another gene product, the serum-
and GC-regulated serine/threonine protein kinase sgk (now renamed
sgk1), as an aldosterone-induced gene in the amphibian,
rodent, and rabbit kidney (6, 21). Sgk1 was
first described in rat mammary epithelia and rat-2 fibroblasts as an
immediate early response gene that is rapidly induced in vivo by serum
and GCs (36). One of the targets of sgk1 action is ENaC:
co-expression of sgk1 with ,
,
-ENaC mRNA in Xenopus oocytes significantly enhances Na+ current (6, 21,
26, 32). This effect of sgk1 is achieved by increasing the
number of ENaC channels assembled at the cell surface (8,
33) and is the most direct evidence that sgk1 positively
regulates the function of ENaC.
Despite the abundant evidence that GCs regulate sgk1 in lower mammals, an earlier report indicated that GC did not regulate human sgk1 (34). We (14) and others (22) have recently shown that, in human airway epithelia, GCs regulate sgk1. In this study we demonstrate that GCs increase sgk1 expression in human lung and renal cortical epithelial cells via an increase in transcription of the sgk1 gene. We also confirm that an imperfect hormone response element in the human sgk1 5'-flanking region binds the GC receptor (GR) and is required for the GC-mediated transcription of sgk1.
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EXPERIMENTAL PROCEDURES |
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Materials.
Dexamethasone, aldosterone, and cycloheximide were purchased from Sigma
Biochemicals (St. Louis, MO). Actinomycin D was obtained from Roche
Molecular Biochemicals (Indianapolis, IN), and the radionucleotides
[-32P]UTP, [
-32P]ATP, and
[
-32P]dCTP came from NEN Life Science Products
(Boston, MA). RU-38486 was a generous gift from Roussel Uclaf
(Romainville, France), and culture media were obtained from Life
Technologies (Gaithersburg, MD) or from Clonetics Cell Systems
(Cambrex, East Rutherford, NJ). DNA sequencing and synthesis were a
service provided by the University of Iowa DNA core facility. The
monoclonal anti-GR antibody, BuGR, was a gift from Thomas Schmidt,
Department of Physiology, University of Iowa. Iowa City, IA.
Tissue culture, RNA extraction, and ribonuclease protection assay. The H441 and A549 human lung epithelial cells were cultured as previously described (14). The human renal cortical epithelial (HRCE) cell line was obtained from Clonetics cell systems (Cambrex) and maintained in human renal epithelial growth medium supplemented with 10 ng/ml epidermal growth factor, 500 ng/ml hydrocortisone, 500 ng/ml epinephrine, 5 µg/ml insulin, 6.5 ng/ml triiodothyronine, 10 µg/ml transferrin, and 0.5% fetal bovine serum. To examine the effects of dexamethasone or aldosterone on gene expression, cell cultures were switched to serum-free hormone-free medium and then were treated with 100 nM steroids for various time periods or with various concentrations of steroid or vehicle for 2 h. Some experiments were done in the presence of 1 µM actinomycin D or 10 µM cycloheximide or vehicle. Total RNA was extracted from H441, A549, and HRCE cells, as previously described (19).
Steady-state levels of sgk1 mRNA were measured by ribonuclease protection assay (RPA) in H441, A549, and HRCE cells grown as monolayers in six-well plates. To obtain human sgk1 (hsgk1) cDNA, H441 RNA was reverse transcribed using oligo-(dT) and M-MLV reverse transcriptase, and first-strand cDNA was subjected to PCR amplification using primers 5'-ACGTCTTTCTGTCTCCCCG and 5'-GGCTCCACCAAAAGGCTAAC. A 181-bp ApaI-DraI fragment of hsgk1 was then cloned into pCDNA3 (Invitrogen), linearized, and used to synthesize an antisense radiolabeled cRNA probe from the T7 promoter. To measure sgk1 gene expression, 10 µg of RNA samples were hybridized with the sgk1 cRNA probe and an 18S rRNA probe as control for gel loading. The method for solution hybridization, nuclease digestion, and identification of nuclease-protected products by PAGE has been previously described (14, 25).5' Rapid amplification of cDNA ends.
Total RNA was extracted from dexamethasone-treated H441 cells and used
to construct a modified cDNA library by use of the RLM-RACE kit
(Ambion, Austin, TX), following the manufacturer's instructions but
with some modifications. Briefly, 10 µg of RNA were first treated
with calf intestinal phosphatase (CIP) in 1× CIP buffer at 37°C for
60 min, phenol extracted, and then incubated with tobacco acid
pyrophosphatase (TAP) in 1× TAP buffer at 37°C for 60 min. A
single-stranded 5' rapid amplification of cDNA ends (RACE) adapter was
added to decapped mRNA with T4 RNA ligase at 37°C for 60 min, and
modified RNA was stored at 20°C. Two microliters of adaptor-ligated
RNA were then reverse transcribed with oligo-(dT) and M-MLV reverse
transcriptase (RT) in 1× RT buffer with 4 dNTPs and RNAse inhibitor at
42°C for 60 min to create first-strand cDNA. Gene-specific reverse
primers X2: 5'-AGTCGTTCAGACCCATCC [+113 to +94 from the original
initiation codon (see Fig. 4A)] (34) and X1:
5'-AGCAGCCTCAGTTTTCACC (+23 to +5 from the initiation codon) were used
sequentially with adapter-specific primers in PCR reactions by use of
Taq polymerase. The amplification reactions occurred for 35 cycles each at 94°C for 30 s, with annealing at 53 or 58°C for
30 s and extension at 72°C for 3 min. Reaction products were
cloned into pCR-XLTOPO (Invitrogen, Carlsbad, CA) and sequenced.
Primer extension.
Total RNA from dexamethasone-treated H441 cells or yeast RNA was used
in primer extension reactions, with the nested sgk1 primer used in 5'
RACE reactions. Primer (12.5 pmol) was end-labeled with T4
polynucleotide kinase and [-32P]ATP and purified using
a G-25 Sephadex column (Quick Spin, Roche Molecular Biochemicals,
Indianapolis, IN). Labeled primer [100,000 counts/min (cpm)] was
combined with 10 µg of RNA and denatured at 65°C for 5 min and then
added to 15 units of Thermoscript RT (Invitrogen) in a reaction mixture
that included 50 mM Tris-acetate, pH 8.4, 75 mM K-acetate, 8 mM
Mg-acetate, 5 mM DTT, 1.25 mM dNTPs, and 2 µl RNAsin at 60°C for 60 min. Primer extended products were run alongside a labeled 50-bp ladder
on a polyacrylamide gel, as previously described (2).
Cloning of the 5'-flanking region of human sgk1. Sequence information from the 5' end of the locus was found in GenBank (accession no. AL 355881). DNA sequence was analyzed for transcription factor binding motifs with Omiga (Oxford Molecular, Campbell, CA) and for CpG islands with GRAIL DNA sequence analysis software at http://compbio.ornl.gov/Grail-1.3/. To clone the ~3,000 bp of the proximal 5'-flanking region of sgk1 upstream of exon 1, primer F4: 5'-TGCGTGCTGGGGGTGTAATAAA and primer R4: 5'-CCATGCCCCTCATCCTGGAGTA were used to amplify a DNA fragment from human genomic DNA. This fragment, which includes 117 nt of the 5' UTR of sgk1, was directionally subcloned into pGL3basic (Promega, Madison, WI) upstream of the firefly (Photinus pyralis) luciferase coding region. Deletion variants of this construct were created by using internal restriction enzyme sites SpeI, SacI, and MscI.
To test the GRE with a heterologous promoter, a double-stranded oligonucleotide was constructed by annealing primer sgk1_GRE (S): 5'-CGGAACGGACAAAATGTTCTCAGAC and primer sgk1_GRE (AS): 5'-CTAGGTCTGAGAACATTTTGTCCGTTCCGAGCT. This oligonucleotide with AvrII and SacI ends was phosphorylated with T4 polynucleotide kinase and then introduced as two copies at an AvrII site (Transient transfection and analysis of reporter activity.
A549 cells were grown in 24-well plates until subconfluent and were
then transfected, using LipofectAMINE Plus (Life Technologies), with 1 µg of the sgk1 promoter-reporter construct, and as a
control for transfection efficiency, 1 µg of pRL-SV40 (Promega), a
plasmid vector in which the SV40 viral promoter drives sea pansy
luciferase (Renilla reniformis). In some cases, the
-hENaC promoter-luciferase construct (
1388 + 55) that
contains a functional GRE was used as a positive control
(19). On the day after transfection, cells were treated
with 100 nM dexamethasone or vehicle, and 24 h later cell lysates
were prepared, and reporter gene activity was performed with the Dual
Luciferase Assay Kit (Promega) as previously described.
Preparation of whole cell extracts and the gel mobility shift
assay.
A549 cells were grown until nearly confluent, switched to serum-free
media, and treated with 100 nM dexamethasone for 24 h. Cells were
then scraped up into PBS using a rubber policeman and washed twice with
PBS. The cell pellet was resuspended in an extraction buffer containing
25 mM Tris, pH 7.9, 400 mM NaCl, 0.1 mM EDTA, 0.3 mM DTT, 0.5 mM
phenylmethylsulfonyl fluoride, and 1.9 µg/ml aprotinin and was
subjected to three freeze-thaw cycles from 80°C to 4°C. The cell
lysate was centrifuged at 15,000 g for 15 min at 4°C, and
the supernatant was collected. Protein concentration was calculated by
the Bradford method and then stored in aliquots at
80°C.
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RESULTS |
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We have previously shown that H441 cells, an airway epithelial
cell line, and A549 cells, an alveolar epithelial cell line, have
regulated expression of -,
-, and
-ENaC subunits and sgk1 (14, 25). These findings were in contrast to earlier
studies (34) reporting that GC did not stimulate the human
sgk1 transcript in HepG2 cells. To determine the molecular basis for
the increase in sgk1 expression in airway and alveolar
cells, we performed a more detailed study of the characteristics of the
corticosteroid response, focusing on early time points. Dexamethasone
stimulated the expression of sgk1 in H441 and A549 cells in
a time-dependent manner, with the first response seen at 40-60 min
(Fig. 1, A and B).
Whereas aldosterone had no effect in H441 cells, a small effect was
seen in A549 cells at a single time point. In H441 and A549 cells,
dexamethasone increased sgk1 expression in a dose-dependent manner, with stimulation beginning at 3.3 nM (Fig. 1, C and
D). The maximal response in H441 cells and A549 cells was 2- to 5-fold and 4- to 10-fold over basal conditions. We then tested the
effect of actinomycin D, a general transcription inhibitor, and
cycloheximide, a protein synthesis inhibitor on basal and GC-stimulated
sgk1 expression in A549 cells. Actinomycin D blocked both
basal and GC-stimulated sgk1 expression, consistent with a
transcriptional effect of GC on sgk1 gene expression (Fig.
2A). Cycloheximide increased
basal sgk1 expression and further enhanced the GC effect on
sgk1 expression (Fig. 2A), an effect that we have
also seen with
-,
-, and
-ENaC subunits (14, 25)
and is a well described phenomenon for early response genes. These
results indicated to us that the effect of GC on sgk1 gene
transcription was direct and did not involve synthesis of an
intermediary protein. To determine whether the effect of dexamethasone
and aldosterone was via the classic cytosolic GR, we used the GR
blocker RU-38486 with dexamethasone and aldosterone. In the presence of
RU-38486, the effect of dexamethasone and aldosterone on
sgk1 expression was abolished (Fig. 2B).
Collectively, these data suggest that the lack of GC effect on
sgk1 expression, previously reported in HepG2 cells, may
reflect the relative paucity of GR in that cell line or the absence of
cell-specific co-activators (3).
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Because corticosteroids increase Na+ transport in the
collecting duct, we wondered whether GCs could stimulate
sgk1 expression in human collecting duct epithelia. There
are no suitable human collecting duct cell lines with regulated
Na+ transport available, and so we looked at expression of
sgk1 in HRCE cells derived from primary cultures of
dispersed human kidney cortex epithelia. As in human airway epithelia,
GC increased sgk1 expression in these cells in a
time-dependent manner (Fig.
3A). Because aldosterone is
considered to be a more physiological regulator of Na+
transport in the distal nephron, we also looked at the effect of
aldosterone in these epithelia. The results demonstrated that aldosterone increased sgk1 expression in HRCE cells, which
was evident at the first time point selected (Fig. 3B).
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To address the mechanisms of transcriptional regulation of
sgk1, we first identified the transcription start site by
two complimentary techniques: 5' RACE and primer extension analysis.
Because the 5' end of the sgk1 transcript was GC rich, we hypothesized
that this feature may prematurely terminate RT and give a truncated product by primer extension or 5' RACE. To identify the authentic 5'
end of the transcript by 5' RACE, we constructed a cDNA library, which
was designed to include the 5' cap site of mRNAs. RNA from dexamethasone-treated H441 cells was treated with CIP to remove 5'
phosphate from incomplete transcripts that did not include the 5' cap.
RNA was then treated with TAP to remove the 5' cap, and an adaptor
sequence was ligated to decapped RNA. Adaptor-ligated full-length mRNA
was reverse transcribed with oligo-(dT) and M-MLV RT and then subjected
to two rounds of PCR by use of gene-specific primers. A single band was
identified by PCR that on sequence analysis extended the known 5' end
of the sgk1 transcript by 13 bases (Fig.
4A).
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We then performed primer extension analysis in H441 mRNA with the nested primer used for 5' RACE and again identified a single extended product that, by size, matched that seen by 5' RACE (Fig. 4B). To overcome the possible secondary structure in the 5' end of this RNA, primer extension was carried out at 60°C with a thermostable form of RT (Thermoscript RT, Invitrogen, Carlsbad, CA). These results thus confirmed the 5' end of sgk1 mRNA, at least in H441 cells. Compared with genomic DNA sequence, the extended sequence appeared to be within the same exon; furthermore, 23 bp upstream of the 5' end of this gene, a consensus TATA box, TATAA, was identified.
Having mapped the 5' end of the sgk1 mRNA, we cloned ~3 kb of the
5'-flanking region by PCR by use of sequence information available in
GenBank. We directionally inserted this upstream of the firefly
luciferase reporter gene in pGL3basic (Promega, Madison, WI) and then
transiently transfected this into A549 cells along with a plasmid,
pRLSV40, to control for transfection efficiency. As a positive control,
the GC-responsive -ENaC promoter-luciferase construct,
1388 +55,
was used in some wells (19). Twenty-four hours after
transfection, cells were treated with dexamethasone or its vehicle, and
lysates were prepared on the following day to measure luciferase
activity. Our results demonstrated a sixfold increase in luciferase
activity of the sgk1-luciferase construct when exposed to
dexamethasone, confirming that 3 kb of genomic sequence 5' and flanking
the sgk1 gene are sufficient to confer GC responsiveness to
the luciferase gene (Fig. 5). These
results confirm that a GC-responsive enhancer transcriptionally
regulates the sgk1 gene.
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To map the GC-responsive enhancer in the 5'-flanking sequence, we made
a series of deletion constructs with convenient restriction sites (Fig.
6A). When tested in A549
cells, deletions up to 1899 maintained the GC effect, whereas a
deletion to
735 was enough to abolish the effect, pointing to a
GC-responsive element(s) between
1899 and
735 (Fig.
6B). A careful examination of this region
demonstrated an imperfect hormone response element, CGGACAAAATGTTCT, present at
1159 to the transcription start site. To test this as a
candidate element, we transferred this 15-bp sequence as two tandem
copies to the core promoter of
-hENaC-1,
141 +55
-ENaC/luciferase (19). This construct does not contain
the GRE of
-ENaC and hence does not respond to GCs. Consistent with its role as a GC-responsive enhancer, the transferred sgk1
cis-element supported GC stimulation of a heterologous
promoter (Fig. 6C). To demonstrate that the sgk1
cis-element was necessary for the GC effect, a 2-bp
substitution was introduced into the 3' hexameric half-site
(CGGACAAAATCATCT) within the
1899 +117 hsgk1 sequence. This construct was no longer stimulated by GC when
transfected into A549 cells, confirming that the imperfect GRE was
necessary and sufficient to confer GC responsiveness to the
sgk1 gene (Fig. 6D).
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We then asked, by performing gel mobility shift assays, whether GR
could bind this GRE. Whole cell nuclear extracts from
dexamethasone-treated A549 cells were used with sgk1 GRE or
the -ENaC GRE. Compared with probe alone, increasing the amount of
cell extract led to the shift of an increasing fraction of the
sgk1 GRE to a higher position, indicating the presence of a
DNA-binding protein (Fig. 7A).
This binding was specifically competed by a 50-fold excess of cold sgk1
or
-ENaC GRE but not by a nonspecific (NS) oligonucleotide. When
cell extracts were preincubated with an anti-GR antibody, a further
shift or supershift of the GRE to a higher level was noted, confirming
that GR was part of the complex that binds to the sgk1 GRE.
Compared with the sgk1 GRE, there appeared to be enhanced
binding of GR to the
-ENaC GRE, as evidenced by a greater intensity
or mass of the shifted probe with 1 µg of cell extract and a
substantially greater mass of supershifted probe with the anti-GR
antibody (Fig. 7B).
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DISCUSSION |
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Corticosteroids are important physiological regulators of
transepithelial Na+ transport in several sites, such as the
collecting duct of the kidney and throughout the airway epithelia. One
of the consequences of corticosteroid action in collecting duct
epithelia is the increase in mRNA abundance for sgk1 (6, 19,
21). This increase in sgk1 expression precedes the
increased sodium transport in these epithelia, and in heterologous
expression systems, co-expression of sgk1 with ,
,
-ENaC
enhances Na+ transport (8, 33). In vivo, after
aldosterone infusion, cytosolic ENaC subunits are distributed to the
apical membrane of the cortical collecting duct, an effect that
correlates with increasing sgk1 expression
(18). A dominant negative form of sgk1 inhibits ENaC
function when heterologously expressed with
-,
-, and
-ENaC in
Xenopus oocytes, suggesting that an endogenous sgk1 is
required for functional expression of ENaC (33). There is
thus good circumstantial evidence that the increase in sgk1 abundance
is a prerequisite, at least for the early increase in corticosteroid-mediated Na+ transport.
Because there is increasing evidence that sgk1 may be responsible, at
least in part, for the corticosteroid-stimulated increase in
Na+ transport in amphibian and rodent kidney collecting
duct, we asked whether GC could stimulate sgk1 expression in
human epithelia cells. This was not a trivial question, because the
human sgk1 transcript was reported to be unresponsive to GCs
(34). We examined sgk1 expression in H441 and
A549 cells, human lung cell lines where ,
,
-ENaC subunits and
sgk1 are expressed and where Na+ transport occurs via
amiloride-sensitive Na+ channels whose biophysical
properties are those of an
,
,
-ENaC heteromultimer (14,
16). We show that GCs increased sgk1 expression in a
time- and dose-dependent manner and that this effect was via the GR and
could be blocked by actinomycin D. These results suggested that the GC
effect on sgk1 expression in these cells requires gene transcription.
We also determined that GCs and aldosterone stimulate sgk1 expression in HRCE, cells derived from primary culture of renal cortical epithelial cells. The response to aldosterone was less than that seen with GCs. This may reflect the likelihood that these epithelial cells are a mixture of proximal convoluted tubule, distal tubule, and collecting duct cells, in which only the collecting duct cells are expected to be aldosterone responsive. A second possibility that we did not directly test is that these cells in culture no longer express usual levels of the mineralocorticoid receptor as has been observed for many other cell lines derived from the collecting duct (7, 19, 30).
The genomic organization of the sgk1 gene has previously been reported (35). In that study, the 5' end of the sgk1 gene was mapped by 5' RACE, and the upstream genomic DNA was cloned and partially sequenced. The authors noted that the 5'-flanking region did not contain a GRE, corroborating their earlier study demonstrating that the human sgk1 gene was not GC regulated in HepG2 cells, a human hepatocellular carcinoma cell line (34). These findings were in contrast to evidence that a GC stimulated rat sgk1 gene expression via an authentic GRE in its 5'-flanking region (37). When we obtained data demonstrating that sgk1 was GC regulated in two human lung epithelia (Fig. 1, A-D), and that this effect was likely to be transcriptional (Fig. 2A), we decided to reexamine this issue. We wondered whether the earlier study reporting the absence of an authentic GRE in the 5'-flanking region of the human sgk1 gene might have overlooked this cis-element. We reasoned that, if the rat sgk1 gene had a GRE in its 5'-flanking region that was the basis of its GC regulation, and if the human sgk1 gene was also GC regulated, then there was a very high likelihood that the human sgk1 gene was also controlled by a GRE. The previous study may have failed to recognize the GRE in the 5' flanking region of the sgk1 gene for a variety of reasons: 1) the transcription start site had not been properly mapped and that the putative 5'-flanking sequences were, in fact, sequences within an upstream intron; 2) the GRE that regulated the sgk1 gene did not bear any resemblance to the rat sgk1 GRE; or 3) the GRE in the human was not in the proximal 5'-flanking region but, rather, elsewhere in the gene.
Because the characterization of the promoter and other
cis-regulatory elements of any gene is critically dependent
on accurate identification of its transcription start site, we
performed our own experiments to map the start site of sgk1 by use of
5' RACE and primer extension. Our studies identified a single site, 13 bases upstream of that previously identified but still within the same
exon (Fig. 4A). We then cloned the 5'-flanking region upstream of a luciferase reporter gene, and after its transfection into
A549 cells, we were able to identify GC-stimulated transcriptional activity. Although the level of induction was modest compared with the
induction seen with the -ENaC promoter (6-fold vs. 44-fold), it
correlated with the modest increase in endogenous sgk1 gene seen in A549 cells (Fig. 1B). By deletional analysis and by
testing with a heterologous promoter, we identified an imperfect GRE in the 5'-flanking region that is required for this GC effect in A549
cells. The classic GRE (AGAACAnnnTGTTCT) is a bipartite sequence composed of two hexameric nucleotides that are palindromic and separated by three nucleotides. Interestingly, the human
sgk1 GRE is different from the previously reported rat
sgk1 GRE by two bases, one of which is within a hexameric
half-site. In contrast to the rat sgk1 GRE
(AGGACAGAATGTTCT), the human sgk1 GRE
differs from the classic GRE also by two nucleotides, suggesting that
its affinity for or trans-activation by GR may be different,
with resulting effects on the kinetics or amplitude of the GC response
in these two species. We were, however, unable to determine a
difference in binding affinity for A549 extracts between the human and
rat sgk1 GRE in gel mobility shift assays (data not shown).
The sgk1 gene is conserved from yeast to human and is expressed in many tissues in response to a variety of stimuli, including corticosteroids, serum, and inflammatory mediators (5). We have now demonstrated that GC regulation of the human sgk1 gene is mediated via a GRE in its 5'-flanking region. It is interesting that there is a sequence difference within the GRE of the human sgk1 gene compared with the rat. It is difficult to speculate on the direction of change, because both life forms branched off from a common ancestral mammal many millions of years ago. However, an analysis of the mouse genome reveals an imperfect GRE ~1 kb 5' and flanking the mouse sgk1 gene, which is identical to the rat GRE. The level of induction by GCs, at least in cultured cell lines, appears to be considerably less in the human compared with rat or even amphibian epithelial cells, suggesting that the more poorly conserved human GRE may have evolved later (6, 20, 37).
In the collecting duct and in airway epithelial cells, stimulation of sgk1 expression by corticosteroid hormones coincides with an upregulation of Na+ transport. Emerging studies are beginning to provide some insights into how sgk1 may regulate Na+ transport. Activation of sgk1 requires the phosphorylation of residues in the COOH-terminal region of the peptide and is phosphoinositide 3-kinase dependent (12, 15, 24). In turn, sgk1 phosphorylates a number of downstream proteins, including the transcription factor FKHRL1, the cytosolic kinase B-Raf, and Nedd4-2, a WW domain protein that targets membrane ENaC for removal and ubiquitination (4, 9, 27, 38). Phosphorylation of Nedd4-2 at one of three consensus phosphorylation sites reduces its affinity for ENaC subunits, thus providing at least one mechanism for the enhanced expression and function of the channel at the cell surface after GC stimulation.
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ACKNOWLEDGEMENTS |
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We thank Tom Schmidt for the gift of BuGR.
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FOOTNOTES |
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The sgk1 cDNA sequence reported here has been deposited in GenBank with accession number AF460178. Portions of the work were presented in abstract form at the American Thoracic Society meeting in 2002.
This work was supported in part by March of Dimes Birth Defects Foundation Research Grant 6-FY99-444 and by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-54348. C. P. Thomas is an Established Investigator of the American Heart Association.
Address for reprint requests and other correspondence: C. P. Thomas, Dept. of Internal Medicine, E300 GH, Univ. of Iowa, 200 Hawkins Drive, 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.
July 9, 2002;10.1152/ajpendo.00021.2002
Received 25 February 2002; accepted in final form 8 July 2002.
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