Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756-0001
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ABSTRACT |
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To study the role
of serum and glucocorticoid-inducible kinase-1 (SGK1) in mammalian
cells, we compared Na+ transport rates in wild-type (WT) M1
cortical collecting duct cells with M1 populations stably expressing
human full-length SGK1, NH2-terminal truncated (N-60)
SGK1, "kinase-dead" (K127M) SGK1, and cells that have downregulated
levels of SGK1 mRNA (antisense SGK1). Basal rates of transepithelial
Na+ transport were highest in full-length SGK1 populations,
compared among the above populations. Dexamethasone treatment increased Na+ transport in WT and full-length SGK1 cells 2.7- and
2-fold, respectively. Modest stimulation of Na+ absorption
was detected after dexamethasone treatment in
N-60 SGK1 populations.
However,
N-60 SGK1 transport rates remained substantially lower than
WT values. Importantly, a combination of high insulin, dexamethasone,
and serum failed to significantly stimulate Na+ transport
in antisense or K127M SGK1 cells. Additionally, expression of antisense
SGK1 significantly decreased transepithelial resistance values.
Overall, we concluded that SGK1 is a critical component in
corticosteroid-regulated Na+ transport in mammalian
cortical collecting duct cells. Furthermore, our data suggest that the
NH2 terminus of SGK1 may contain a Phox homology-like
domain that may be necessary for effective Na+ transport.
aldosterone; epithelial sodium channel; M1 cell line; serum and glucocorticoid-inducible kinase 1; cortical collecting duct
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INTRODUCTION |
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SERUM AND GLUCOCORTICOID-INDUCIBLE KINASE-1 (SGK1) is a novel member of the serine/threonine family of kinases and is induced in response to a variety of extracellular stimuli (listed in Ref. 18). Implicit in its name, SGK1 was originally identified as a serum and glucocorticoid-induced gene in rat mammary tumor cells by differential display (37). Since then, posttranslational regulation of SGK1 has also been described. For example, components of the phosphoinositide 3-kinase signaling pathway are necessary for the phosphorylation of SGK1, which leads to its activation. Amino acid residues Thr256 (located in the activation loop of SGK1) and Ser422 (located in the COOH-terminal domain of SGK1) must both be phosphorylated by 3-phosphoinositide-dependent protein kinases-1 and -2 (PDK1 and PDK2) for complete activation of SGK1 (16, 26). Recently, our laboratory and others have demonstrated that the level of SGK1 transcript is rapidly upregulated in response to mineralocorticoids, independently of de novo protein synthesis, in target epithelial cells (2, 6, 23, 25, 30).
Mineralocorticoids are the key regulators of transepithelial
Na+ transport. In the absence of 11-hydroxysteroid
dehydrogenase-2 activity, glucocorticoids can also induce
mineralocorticoid-like effects (11, 17, 19, 24).
Mineralocorticoids and glucocorticoids are both effective in
upregulating SGK1 transcript levels in epithelial cells, which implies
a relationship between SGK1 and epithelial Na+ channel
(ENaC) activity in collecting duct cells. This notion is strongly
supported by several heterologous SGK1 expression studies. For
instance, oocytes coexpressing SGK1 and ENaC subunits exhibited higher
Na+ current and more channels localized to the plasma
membrane compared with oocytes expressing ENaC alone (1, 6, 23,
30). Additionally, ectopic expression of SGK1 in Xenopus
laevis A6 cells displayed exceptionally high levels of
Na+ transport under basal conditions, which were sustained
with aldosterone treatment (9). Moreover, inhibition of
the phosphoinositide 3-kinase signaling pathway with LY-294002 was
associated with a substantial decrease in the rate of Na+
transport in A6 cells (28, 36). Together, the above
studies strongly implicate SGK1 as a mediator of aldosterone-stimulated transcellular Na+ reabsorption.
Despite these recent advances, however, the physiological role of SGK1 in mammalian kidney cells has not been definitively established. The goal of our study was to generate stable mouse cortical collecting duct (CCD) cells that either overexpress or downregulate SGK1 activity. Here, we report for the first time that SGK1 is a critical regulator of ENaC activity in mammalian CCD cells.
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MATERIALS AND METHODS |
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Generation of an antisense SGK1 construct.
The antisense SGK1 construct was created by ligating a 708-bp fragment
of cDNA located in the 3'-untranslated region of mouse SGK1 into the
multiple cloning site of TRE vector (Clontech, Palo Alto, CA) in the
reverse (antisense) orientation. Specifically, the DNA fragment is
located in the mouse SGK1 between the BamHI and the
HindIII restriction sites and includes the poly-A tail (Fig.
1A). The newly constructed
antisense TRE construct carries both ampicillin and hygromycin
resistance genes for selection in Escherichia coli and in
mammalian cells, respectively. The subsequent transcription of
antisense RNA downregulates the endogenous expression of the complement
transcript (20, 27, 39).
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Cell culture and construction of M1 CCD cell lines, which overexpress or downregulate SGK1 activity. M1 cells (33) were propagated in PC-1 growth medium (BioWhittaker, Walkersville, MD) containing an abundance of growth supplements. The supplementary growth factors include 15 µg/ml insulin, 5 µM dexamethasone, 5% FBS, 2 mM glutamine, and antibiotics (75 µg/ml penicillin, 100 µg/ml streptomycin, and 12.5 µg/ml tylosin). This medium is hereafter referred to as insulin- and dexamethasone-containing (IDC) medium.
Subconfluent M1 cells were cotransfected with antisense TRE vector concurrently with a Tet-On construct. The Tet-On construct carries neomycin resistance and was designed to facilitate TRE vector expression in the presence of tetracycline. However, it was our experience that Tet-On vector provided strong, constitutive cytomegalovirus promoter activity in trans, which did not require additional tetracycline stimuli. The cotransfections were carried out by using a modified LipofectAMINE protocol (15). M1 cells stably expressing antisense SGK1 cDNA were then selected by using 200 µg/ml hygromycin and 250 µg/ml neomycin. Supernatant from amphotropic Phoenix cells producing retroviruses packaging truncated (Quantitative RT-PCR.
To compare the relative abundance of SGK1 mRNA between wild-type (WT)
M1 cells and populations of M1 cells stably expressing antisense SGK1
construct, we used quantitative RT-PCR methods, previously described in
(23). Briefly, total RNA was extracted from WT and
antisense SGK1 M1 cells by using TRI Reagent per the manufacturer's
protocol (Molecular Research Center, Cincinnati, OH). Then, 2 µg of
isolated total RNA was reverse transcribed by extension of random
primers. SGK1 upper (5'-CTC AGT CTC TTT TGG GCT CTTT-3') and lower
(5'-TTT CTT CTT CAG GAT GGC TTTC -3') PCR primers generated a 450-bp
product. Reactions were performed with AmpliTaq DNA
polymerase (Roche, Indianapolis, IN) under standard conditions with
varying amounts of template (10, 2.5, 0.62, and 0.15 ng of cDNA)
originating from WT or antisense cells cultured in IDC medium. Thermal
cycling conditions included a 2-min denaturing step at 96°C, followed
by 25 cycles of 95°C for 1 min for denaturing, 57°C for 1 min to
reanneal, and 72°C for 1 min for elongation of template, and then a
final extension of 72°C for 8 min. The abundance of -actin mRNA
for each sample was also determined by using primers and PCR conditions
optimized for
-actin, as previously described (34).
Amplified SGK1 PCR products were separated on a 5% polyacrylamide gel,
stained with ethidium bromide, quantified by using a FluoroImager and
ImageQUANT software (Molecular Dynamics, Sunnyvale, CA), and then
normalized for
-actin mRNA levels.
Electrophysiological measurements.
Cells were seeded onto Millicell permeable membranes (Millipore,
Bedford, MA) at a density of 4 × 105 cells/chamber.
After seeding, cells were bathed in IDC medium on the basolateral and
apical sides until they formed confluent monolayers (~3-7 days).
After the cells reached confluency, the bathing medium was changed to
an insulin and steroid hormone-free (I-SterF) medium composed of
DMEM/F-12 (Mediatech, Herndon, VA) supplemented with 5% FBS stripped
twice with charcoal, 15 mM HEPES, 2 mM glutamine, and antibiotics.
Cells were cultured in I-SterF medium for 48 h to establish basal
levels of Na+ transport. Cells were then treated with 1 µM dexamethasone (in I-SterF medium) for a 24-h period to study the
effects of corticosteroids. The transepithelial voltage and
transepithelial resistance (RTE) values of each
population of cells were regularly determined by using an Epithelial
Voltohmmeter (World Precision Instruments, Sarasota, FL), and the
equivalent short-circuit current (Isc) was
calculated. Our laboratory has previously demonstrated that Isc mainly represents transepithelial
Na+ current in this model system (7). Cells
were regarded as confluent monolayers when voltage values remained
steady for two independent readings over a 48-h time period and
RTE values reached threshold levels of 900 · cm2. All membrane resistance
values reported represent the difference between measured
RTE and porous membrane
RTE (~125
· cm2) in which the cells were grown.
Western blot analysis.
Confluent N-60 SGK1, full-length SGK1, K127M SGK1, and WT M1 cells
were rinsed twice with ice-cold PBS before lysing with 500 µl of SDS
solubilization buffer [48.2 mM 2-(N-hexylamino) ethanesulfonic acid, 1% SDS, 10% glycerol, and 1% protease and phosphatase inhibitor cocktail (Sigma, Palo Alto, CA)]. The
protein concentration of cellular lysate was determined by using BCA
Protein Assay Reagent (Pierce Chemical, Rockford, IL). Then, 7 µg of
total protein lysate or immunoprecipitated product concentrated from a
60-mm dish was electrophoresed on a 10% acrylamide gel under denaturing conditions. The proteins were then transferred to
Immobilon-P membrane (Millipore) and blocked in buffer consisting of
5% dry milk, 10 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween, 124 µM
thimerosal, and 1% Mega Block (PGC Scientifics, Frederick, MD) for
1 h. Next, the membrane was incubated in 4 µg of rabbit
polyclonal anti-HA antibody (Upstate Biotechnology, Cleveland, OH)
diluted in 4 ml of blocking buffer at room temperature for 1 h.
After extensive washes, anti-rabbit IgG-horseradish peroxidase
(HRP)-labeled antibody (Cell Signaling Technology, Beverly, MA) was
added at a concentration of 1 µg/ml and incubated for another hour at
room temperature. After thorough washes, HRP signal was detected by
using the ECL substrate (Amersham Pharmacia Biotech, Piscataway, NJ) or
Super Signal West Dura Substrate (Pierce Chemical). The membrane was finally exposed to Kodak X-OMAT AR scientific imaging film (Kodak, Rochester, NY).
Immunoprecipitation.
Confluent cells grown on a 60-mm plate were washed with ice-cold PBS
and then lysed with buffer consisting of 20 mM Tris, pH 7.5, 150 mM
NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, and 1% protease and
phosphatase inhibitor cocktail, further complemented with 1 mM
-glycerol phosphate, 2.5 mM sodium pyrophosphate, 1 mM
Na3VO4, and 1 µg/ml leupeptin. Lysates were
then sonicated for 5-10 s. To immunoprecipitate SGK1 protein, 500 µl of supernatant from hybridomas-producing SGK1 antibody or 7 µg
anti-HA antibody diluted in lysis buffer were added to the cell lysate.
SGK1 monoclonal antibodies were generated in mice immunized with mouse
SGK1 amino acids 147-437, by using standard techniques previously
described (10). Then, the antibody containing lysates was
incubated overnight at 4°C with gentle rocking. The following
morning, 20 µl of protein A-Sepharose beads (Sigma) were added to the
sample and incubated for an additional 2 h at 4°C with gentle
rocking. The sample was then briefly centrifuged at 14,000 g. The precipitated pellet was washed in lysis buffer and
then resuspended in 25 µl SDS solubilization buffer. The
immunoprecipitated complex was analyzed by Western blot technique, as
described above.
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RESULTS |
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Stable integration of SGK1 constructs in the M1 CCD cell line.
To study the role of SGK1 on transepithelial Na+ transport
in mammalian cells, we generated M1 CCD cells stably expressing the
human full-length SGK1, N-60 SGK1, and dominant-negative K127M SGK,
as well as antisense SGK1, which downregulates the endogenous level of
SGK1 transcript.
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Effect of changes in SGK1 expression on transepithelial
Na+ transport in M1 cells.
To observe the effect of expressing full-length, N-60, antisense,
and K127M SGK1 on transepithelial Na+ transport, each cell
line was cultured on permeable filters concurrently with WT M1 cells.
Fig. 3A, illustrates the
steady-state Isc of each cell line grown in IDC
medium. Noticeably, Isc in cells expressing full-length SGK1 was significantly higher than that in WT M1 cells (P < 0.005). In IDC medium, Isc
values of
N60 SGK1 populations were comparable to WT values.
Importantly, the expression of antisense and K127M SGK1 in M1 cells
decreased Isc values markedly, to ~20% of
Isc in WT cells (P < 0.0005) in
the presence of "complete" medium that includes serum and growth
factors.
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Effect of changes in SGK1 expression on RTE.
Generally, high-RTE values characterize cell
types with very tight junctions, whereas low RTE
values are associated with leaky epithelia or multiple open ion
channels. In the present study, as well as in previously
published reports (33), M1 cells predominantly develop
RTE values >900
· cm2. However, antisense
populations incubated in I-SterF medium and 1 µM
dexamethasone-containing medium displayed exceptionally
low-RTE values of 421.2 ± 71.8 and
569.8 ± 87.4
· cm2,
respectively (Fig. 4, B and
C). The apical to basolateral
permeability of Millicell cultures were inspected to determine the
integrity, or "leakiness," of the monolayer. Four hundred
microliters of tissue culture medium were applied to the apical
compartment of the Millicell chamber, and 600 µl of the same medium
were applied to the basolateral compartment. During regular changes of
the culture medium, potential leakage through the monolayer was
determined by measuring fluid volume from each compartment. Under each
experimental growth parameter, there was no significant change in fluid
volumes from each compartment. Additionally, [3H]mannitol
paracellular permeability assays (described in Ref. 41)
were performed to further verify that the monolayer was not leaky in
antisense cell populations cultured on permeable supports. The percent
leakage of radioactively labeled mannitol measured from WT cells
cultured in IDC medium did not differ significantly compared with
antisense cells incubated in I-SterF- and dexamethasone-containing medium (data not shown). Together, these observations are highly suggestive that downregulation of SGK1 alters
RTE values. The resulting epithelial monolayer
is not leaky and therefore is suitable for the determination of
transepithelial Na+ transport.
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DISCUSSION |
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Since the recent identification of SGK1 as an early aldosterone-induced gene, it has been a priority to characterize the physiological role of SGK1 in epithelial Na+ transport. Prior studies utilizing X. laevis oocytes and A6 cell lines are demonstrative of the ability of SGK1 to mediate ENaC activity. However, the role of SGK1 in effecting mineralocorticoid- and corticosteroid-induced Na+ transport in mammalian CCD cells had not been established before this study.
This study contributes to establishing SGK1 as a corticosteroid-regulated mediator of Na+ reabsorption in mammalian collecting duct cells. The overexpression of SGK1 in M1 cell lines increased transepithelial Isc significantly above control levels under basal growth conditions and after dexamethasone treatment. Furthermore, the downregulation of SGK1 transcript using an antisense SGK1 construct and the expression of a dominant-negative SGK1 decreased transepithelial Isc. Because we performed these studies by using a mixed population of cells stably expressing SGK1 constructs, the results cannot be attributed to mere clonal variations. Furthermore, studies published by Faletti et al. (9) similarly ascertain that SGK1 is a necessary component of ENaC-mediated Na+ transport in X. laevis A6 cells. Faletti et al. included overexpression of full-length human and dominant-negative D222A mutant SGK1, which is unresponsive to phosphorylation by PDK2. Basal rates of Na+ transport in A6 cells overexpressing human SGK1 were 3.5 times higher than untransfected control. Conversely, D222A SGK1 clones did not respond to hormone stimulation, which is characteristic of A6 model epithelial cells.
However, our studies also examined populations of cells that
overexpressed truncated SGK1. These cells did not respond to corticosteroid treatment to the extent observed in WT and full-length SGK1 populations. Because N-60 SGK1 is expressed at very high levels
in both M1 cells and other cell types (16, 22) and maintains its phosphorylation (activation) sites, the reason for this
observed effect on ENaC function is not obvious. We speculate that the
first 60 amino acids in SGK1 are necessary for stimulation of ENaC
activity. It is reasonable to propose this, because research within the
past decade has identified several proteins that contain NH2-terminal Phox homology domains, which specifically bind
to phosphoinositides to target to their subcellular site of
activation (5, 8, 14, 32, 40). Interestingly, the
mouse homolog of human SGK3, cytokine-independent survival kinase
(CISK), contains a PX domain in the NH2-terminal region
(21). Mutation of this region inhibited CISK
localization to endosomal compartments and therefore inhibited CISK
function (38). The highly conserved PX sequence motif
identified in CISK [R(R/K)xxLxx(Y/F)] is also conserved within the
NH2-terminal region of SGK1 that was truncated in our
N-60 cell lines. Perhaps, in a manner similar to the Pleckstrin homology domain of PKB and the PX domain of CISK, SGK1 possesses a
targeting motif necessary for PI-3 kinase-dependent activation at the
membrane. We speculate that this may in part explain the lack of effect
on Na+ transport in
N-60 SGK1 cells when they are
treated with corticosteroid.
Additionally, the NH2 terminus of SGK1 may be responsible
for targeting it to the appropriate ENaC-activating substrate. In this
instance, the overexpression of truncated SGK1 in our studies could
possibly be acting as a dominant-negative and thus inhibits full ENaC
activity in M1 cells. Recent evidence from Brickley et al.
(3) supports our contention that the
NH2-terminal region targets SGK1 to the site of
Na+ transport action. Their studies demonstrated a
difference between cellular localization of N-60 SGK1 green
fluorescent protein (GFP) and SGK1 GFP (3). Although
full-length SGK1 GFP is predominantly located in the cytoplasm and
plasma membrane of HEK293T, Cos, SK-BR-3, and Madin-Darby canine kidney
cells, truncated SGK1 GFP is homogenously distributed and fails to
localize to the membrane (3, 29). This group has
previously demonstrated that expression of truncated and WT SGK1
similarly inhibit apoptosis in MCF10A-Myc cell lines. For this
reason,
N-60 SGK1 does not behave as a dominant-negative kinase in
relation to apoptosis (22). We therefore suggest
that the putative SGK1-targeting motif may specifically regulate SGK1 activity in ENaC-mediated Na+ transport.
Analysis of the RTE values obtained in this study implicates SGK1 as a key component in the maintenance of epithelial tight junctions, in addition to its role in mediating corticosteroid-induced Na+ reabsorption. Populations of M1 cells stably expressing antisense SGK1 exhibited significantly lower RTE values compared with WT cells in each experimental medium. We demonstrated that the substantial decrease in RTE values observed did not result in a leaky epithelium by using apical-to-basolateral permeability assays. Additionally, the cells maintained viability, as suggested by a modest rise in RTE values when treated with corticosteroid.
Of particular interest and relevance to the present study, several investigators have reported that steroid hormones positively modulate RTE in other epithelial cells. In these studies, the RTE of rabbit distal colon (12, 13, 35) and mammary epithelial cells (4, 31, 41) revealed a significant increase in RTE values after aldosterone and dexamethasone treatment, respectively. Although the precise molecular mechanism of steroid-regulated tight junction formation is not clearly established, Singer et al. (31) demonstrated that specific serine/threonine kinase inhibitors, such as H7, moderately reduced RTE values in mouse mammary epithelial cells after dexamethasone treatment. Accordingly, the effect of downregulation of SGK1 on RTE values in M1 cells in the present study suggests that SGK1 may be an important regulator of tight junction formation. Given that CCD cells, in vivo, necessarily develop very tight junctions to minimize back-flux of ions, SGK might have dual physiological roles in renal cells. Perhaps aldosterone concurrently modulates RTE and ENaC activity, by way of SGK1, to efficiently reabsorb Na+.
In summary, our data affirms that SGK1 is a critical player in the molecular pathway of ENaC activation. In our mammalian collecting duct cell lines, SGK1 activated transepithelial Na+ transport and ostensibly affected tight junction formation. Our study also suggests that the NH2 terminus of SGK1 is necessary for modulating ENaC activity, possibly through a putative PX domain.
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ACKNOWLEDGEMENTS |
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We thank Dr. Suzanne D. Conzen for providing the HA-tagged
full-length, N-60, and K127M SGK1 constructs.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants T32-DK-7508-17, DK-41841, DK-58898, and DK-55845.
Address for reprint requests and other correspondence: A. Náray-Fejes-Tóth, Dartmouth Medical School, Dept. of Physiology, Borwell Bldg., 1 Medical Center Dr., Lebanon, NH 03756-0001 (E-mail: Aniko.Fejes-Toth{at}Dartmouth.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.
First published November 12, 2002;10.1152/ajprenal.00299.2002
Received 9 August 2002; accepted in final form 1 November 2002.
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