Hyperosmotic Stress Stimulates Promoter Activity and Regulates
Cellular Utilization of the Serum- and Glucocorticoid-inducible Protein
Kinase (Sgk) by a p38 MAPK-dependent Pathway*
Lisa M.
Bell
§,
Meredith L. L.
Leong,
Brian
Kim,
Edward
Wang,
Jongsun
Park¶,
Brian A.
Hemmings¶, and
Gary L.
Firestone
From the Department of Molecular and Cell Biology and
The Cancer Research Laboratory, University of California, Berkeley,
California 94720-3200 and the ¶ Friedrich Miescher-Institut,
Maulbeerstrasse 66, CH-4056 Basel, Switzerland
Received for publication, March 10, 2000, and in revised form, May 26, 2000
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ABSTRACT |
We have established that the serum- and
glucocorticoid-inducible protein kinase (Sgk) is a new component of the
hyperosmotic stress response. Treatment of NMuMg mammary epithelial
cells with the organic osmolyte, sorbitol, caused the stable
accumulation of Sgk transcripts and protein after an approximately 4-h
lag. Transient transfection of a series of sgk-CAT reporter
plasmids containing either 5' deletions or continuous 6-base pair
substitutions identified a hyperosmotic stress-regulated element that
is GC-rich and is necessary for the sorbitol stimulation of
sgk gene promoter activity. Gel shift analysis
identified four major DNA-protein complexes in the hyperosmotic
stress-regulated element that, by competition with excess consensus
wild type and mutant oligonucleotides and by antibody supershifts,
contains the Sp1 transcription factor. Several lines of evidence
suggest that the p38 MAPK signaling pathway mediates the
hyperosmotic stress stimulation of sgk gene expression.
Treatment with pharmacological inhibitors of p38 MAPK or with a
dominant negative form of MKK3, an upstream regulator of p38 MAPK,
significantly reduced or ablated the sorbitol induction of
sgk promoter activity or protein production. Using an
in vitro peptide transphosphorylation assay, sorbitol
treatment activates either endogenous or exogenous Sgk that is
localized to the cytoplasmic compartment. Thus, we propose that the
stimulated expression of enzymatically active Sgk after sorbitol
treatment is a newly defined component of the p38 MAPK-mediated
response to hyperosmotic stress.
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INTRODUCTION |
The extracellular milieu is highly dynamic, and cells adapt to
changes in their environment by altering their patterns of gene
transcription and protein modification and their cytoskeletal structure. The ability of a cell to sense and appropriately respond to
adverse conditions is determined by an integrated network of intracellular signaling pathways that trigger adaptive and survival responses or mediate events leading to cell death (1-3). Environmental stresses as divergent as osmotic shock, ionizing radiation, and nutrient deprivation activate intracellular protein kinase cascades that are generally conserved between metazoans and mammals, culminating in the transcriptional control of specific sets of target genes. The
best characterized osmoregulatory signaling pathways have been
described in Saccharomyces cerevisiae, in which solute
imbalances in the environment are detected by two different receptors,
the histidine kinase receptor Sln1 and the four-pass transmembrane receptor Sho1p. The activation of kinase cascades by these receptors leads to the phosphorylation and activation of the
HOG11 proline-directed
protein kinase (4, 5). The HOG1 gene is necessary for the maintenance
of osmotic gradients in hyperosmolar environments and allows the
proliferation of yeast under osmotic stress (6). HOG1 has been shown to
phosphorylate various members of the Msn family of transcription
factors, resulting in their binding to and transactivation of
stress-responsive elements located in the upstream promoter regions of
osmotically regulated genes (7-10). One class of osmoregulatory genes
targeted by HOG1 encode proteins that have osmoprotective functions,
such as the glycerol-3-phosphate dehydrogenase gene that is responsible
for the accumulation of the yeast osmolyte, glycerol (10, 11).
In mammalian cells, however, much less is known about the signal
transduction pathways that control gene transcription in response to
environmental insults. The mammalian homologue of the yeast HOG1 is p38
MAPK, which is a member of the mitogen-activated protein kinase (MAPK)
family of serine/threonine protein kinases, has been shown to
play a role in transducing stress signals in a variety of cell systems
(1, 12-14). Activation of p38 MAPK results in the phosphorylation of a
variety of targets, a subset of which regulates transcription of
stimulus-specific genes (15). For example, the p38 MAPK phosphorylation
of the ATF2, CHOP, Elk1, MEF2C, and SAP1 transcription factors has been
shown to induce their DNA binding and transactivation activity
(16-19). A number of protein kinases (such as MAPKAP kinase-2, MAPKAP
kinase-3, Mnk-1, PRAK, and RSK-B) have also been shown to be targets of p38 MAPK. These studies suggest the existence of additional hitherto unidentified downstream targets of p38 MAPK signaling that may be
responsible for mediating transcription of mammalian osmoregulatory genes (20-25). Although, in some cases, p38 MAPK-initiated
phosphorylation events can trigger programmed cell death (26, 27), the
functional connections between the immediate targets of p38 MAPK, its
downstream effectors, and the cellular response to hyperosmotic stress
in mammalian cells have yet to be elucidated.
Conceivably, important downstream targets of the p38 MAPK stress
cascade may themselves be cell-signaling molecules that help trigger
and mediate the selectivity of the stress response to environmental
cues. We have reported the isolation and characterization of a
serine/threonine serum- and glucocorticoid-inducible protein kinase
gene, sgk, by subtractive cloning from a mammary tumor cell
cDNA library that is under acute transcriptional control by both
serum and glucocorticoids (28, 29). sgk is approximately 45-55% homologous to Akt/PKB, protein kinase A, protein kinase C, and
the rat p70S6K/p85S6K. In serum-treated cells, Sgk is phosphorylated and shuttles between the nucleus and cytoplasm in a cell
cycle-dependent manner (30). We have recently shown that
Sgk enzymatic activity, phosphorylation and subcellular localization is
regulated by PI 3-kinase signaling through PDK1, suggesting a possible
role for Sgk in a cell survival pathway (31). Moreover, the
physiological stress hormones, glucocorticoids, stimulate
sgk promoter activity through a glucocorticoid response
element, and sgk is a transcriptional target of the
p53 tumor suppressor gene, a known target of genotoxic stress (28, 32).
Consistent with a role for Sgk in the cellular stress response,
sgk transcripts have been shown to be elevated in response
to ischemic injury of the brain, changes in cell volume, and
inflammatory disease and in a screen for transcripts involved in wound
repair in fibroblasts (33-36). Therefore, to determine whether
transcription of the sgk gene is a component of the stress response in mammalian cells, the hyperosmolar regulation of Sgk was
examined in mammary epithelial cells. We demonstrate that an increase
in extracellular solute levels stimulates sgk promoter activity and gene expression through the p38 MAPK-dependent pathway.
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EXPERIMENTAL PROCEDURES |
Cells and Materials--
NMuMg nontransformed mouse
mammary epithelial cells were originally derived from normal glandular
tissue of an adult NAMRU mouse (37). Cells were regularly cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, 10 µg/ml insulin, 50 units/ml penicillin, and 50 µg/ml streptomycin. Cells were propagated at 37 °C in humidified
air containing 5% CO2. Cells were passaged every 2-3
days. Cell culture reagents such as Dulbecco's modified Eagle's
medium, fetal bovine serum, calcium- and magnesium-free
phosphate-buffered saline, and trypsin/EDTA were supplied by
BioWhittaker, Inc. (Walkersville, MD). Insulin and
D-sorbitol were purchased from Sigma.
[3H]Acetyl coenzyme A (200 mCi/mmol) and
[
-32P]dATP were obtained from NEN Life Science
Products. To induce hyperosmotic stress, cells received equal volumes
of Dulbecco's modified Eagle's medium (control) or 0.3 M
sorbitol (Sigma) in Dulbecco's modified Eagle's medium for the
indicated time. For treatment with the LY294002 PI 3-kinase inhibitor,
cells were treated with 50 µM LY294002 for 16 h,
half of the cell cultures received serum-free medium with
LY294002, and the other half received 0.3 M sorbitol
dissolved in serum-free medium containing LY294002. Cells were
harvested 24 h later and lysed, and whole cell extracts were
analyzed for Sgk protein expression as described below.
Plasmid Constructions--
The construction of the 
4.0
sgk-CAT plasmid, which contains sgk promoter
sequences (3560 to + 51) cloned upstream of the chloramphenicol
acetyltransferase (CAT) gene in the vector pBLCAT3, has been described
previously (29). The various sgk promoter-CAT deletions down
to 236 sgk-CAT were generated utilizing the Erase-a-Base system (Promega, Madison, WI) and are further described (32). The 78
sgk-CAT was generated as described (38). For the 67 wild
type and mutant sgk-CAT vectors, sense and antisense
oligonucleotides of the region between 67 and 35 were synthesized
as per usual techniques (Microchemical Facility, Cancer Research
Laboratory, University of California, Berkeley), and then 800 pmol of
each strand were mixed and annealed. The wild type and mutant
double-stranded oligonucleotides were then subcloned into the pCAT
Basic vector (Promega) containing the region between 35 and +55 of
the sgk promoter. The 67 wild type and mutant vectors were
confirmed by DNA sequencing.
Transfection Methods and CAT Reporter Gene Assays--
NMuMg
mammary epithelial cells from 60-70% confluent cultures in 100-mm
tissue culture plates were transfected by the calcium phosphate
precipitation method as described previously (32). The total amount of
DNA used in calcium phosphate transfections for CAT assays was held
constant at 20 µg, and in appropriate transfections, the total DNA
was adjusted to this amount using the empty CAT vector plasmid pBLCAT3
or pCAT Basic. Following transfection for at least 4 h, cells were
exposed to a 15% glycerol shock for 3 min at 37 °C. Transfections
were performed in triplicates and repeated at least three times. At
36-40 h post-transfection, the cells were harvested for CAT assays,
and the protein content of the cell extracts was estimated with the
Bradford procedure (39). A quantitative nonchromatographic assay (40)
was used to measure CAT activity in the cell extracts as detailed
elsewhere (28)
Isolation of RNA and Northern Blot Analysis--
NMuMg cells
were plated on 10-cm dishes so they were 80-90% confluent on the day
of harvest. Cells were scraped on ice using a rubber policeman, washed
in 1× PBS, and then pelleted. To each plate, 100 µl of TSM buffer
(10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40, 1.5 mM
MgCl2) plus 1 mM dithiothreitol was added, and
the cells were incubated for 5 min on ice. Nuclei were pelleted by
spinning at 14,000 rpm in an Eppendorf centrifuge at 4 °C. The
supernatant was transferred to new tubes, and 100 µl of digestion
buffer (0.2 M Tris-Cl, pH 8.0, 25 mM EDTA, 0.3 M NaCl, 2% SDS) plus 100 µg/ml proteinase K was added.
The samples were incubated at 37 °C for 30 min and then extracted
two times with 1:1 phenol/chloroform. RNA was precipitated with ethanol overnight at 70 °C, and pellets were resuspended in diethyl
pyrocarbonate-treated water plus 0.1% SDS.
Approximately 10-13 µg of RNA isolated from each sample was
separated on a 1.1% agarose/formaldehyde gel run at 3-4 V/cm for approximately 3 h. The RNA was transferred overnight by blotting onto MSI Magnagraph nitrocellulose. The RNA was covalently cross-linked onto the membrane using a Stratagene Stratalinker, and then transfer was confirmed by staining the blot with 0.02% methylene blue dye. The
blot was prehybridized for a minimum of 2 h as described
previously (28). The blot was then probed using a
SspI/PstI fragment of murine sgk
cDNA that was labeled by [-32P]dCTP random primed
labeling (Roche Molecular Biochemicals). A total of 10 × 106 cpm/ml of probe in hybridization buffer was added to
the blot and allowed to incubate at 42 °C overnight. The blot was
washed in 2× SSC, 0.1% SDS twice for a total of 1.5 h at room
temperature. The blot was then exposed to a Molecular Dynamics
PhosphorImager screen for 24 h.
Gel Mobility Shift Assays--
Preparation of nuclear extracts
from NMuMg mammary cells was based on the method of Dignam
et al. (41). The protein contents in the nuclear extracts
were determined by the Bradford procedure (39). The sequences of the
various oligonucleotides (sense) used for DNA binding studies and cold
competition experiments are as follows (mutations indicated in boldface
type): sgk 67/35, 5'-GGTCCCGCCTGCCCCGCCCCCTGGAGGCTC-3';
-67/35 mutant 1, 5'-TTCGAAGCCTGCCCCGCCCCCTGGAGGCTC-3'; 67/35 mutant 2, 5'-GGTCCCTTCGAACCCGCCCCCTGGAGGCTC-3'; 67/35 mutant 3, 5'-GGTCCCGCCTGCTTCGAACCCTGGAGGCTC-3'; 67/35 mutant 4, 5'-GGTCCCGCCTGCCCCGCCTTCGAAAGGCTC-3'; 67/35 mutant 5, 5'-GGTCCCGCCTGCCCCGCCCCCTGGTTCGAA-3'. The Sp1 consensus oligonucleotide sequence is 5'-cgatGCCCCGCCCCagtc-3'. The mutant Sp1-binding sequence is 5'-cgatGCCCTTTCCCagtc-3'. The core CGC nucleotides have been changed to TTT, and Sp1 is unable to
bind to this sequence (42). The EGR1 consensus oligonucleotide sequence
is 5'-cgatTCGCCCCCTCtgac-3'. All oligonucleotides were synthesized by a
model 394 synthesizer in the Cancer Research Laboratory Microchemical
Facility of the University of California, Berkeley. Radiolabeling of
5'-ends of the appropriate oligonucleotides was carried out in the
presence of equal amounts (10 pmol) of sense and antisense strands,
[-32P] ATP (6000 Ci/mmol; ICN Biomedicals Inc.), and
T4 polynucleotide kinase (Roche Molecular Biochemicals) at 37 °C for
30 min, followed by annealing of the labeled strands. The annealing
procedure for generating either labeled or unlabeled DNA involved
adding 0.1 M NaCl (0.1 volume) to equal amounts of sense
and antisense strands, heating for 10 min at 70 °C, and gradually
cooling to room temperature. The free unincorporated nucleotides and
single-stranded DNA were separated from the end-labeled double-stranded
DNA by native polyacrylamide gel electrophoresis (8% gel). The labeled
double-stranded oligonucleotide was excised and eluted in 400 µl of
TE buffer (10 mM Tris, 1 mM EDTA) and 40 µl
of 3 M sodium acetate, pH 5.0, for 3 h, followed by
ethanol precipitation, rinsing in 70% ethanol, and resuspension in TE
buffer. The radioactive oligonucleotide probes were stored at
70 °C. The DNA binding reactions (18 µl) contained nuclear extract proteins (7 µg), 0.5 ng of 32P-labeled
(approximately 2 × 106 cpm) DNA probe, poly(dI-dC) (2 µg), and 6 µl of 3× binding buffer (21.6% glycerol, 36 mM Hepes, pH 7.5, 126 mM KCl, 9 mM
MgCl2, 0.9% Nonidet P-40, 180 mM
ZnCl2, 0.9 mM dithiothreitol) and were allowed
to incubate at room temperature for 20 min. For competition experiments, a 200-fold excess (unless otherwise noted) of the indicated unlabeled double-stranded oligonucleotides was added prior to
the addition of radiolabeled DNA probe. In some cases, the reaction
mixtures were incubated at 4 °C with either 5 µl of specific
anti-Sp1 polyclonal antibodies (a generous gift from the R. Tjian
laboratory, University of California, Berkeley) or an equal volume
(equivalent to 100 ng) of anti-p110 PI 3-kinase polyclonal antibody
(Santa Cruz Biotechnology, Inc.) for 2 h before the
addition of radiolabeled probes. The protein-DNA complexes were
resolved on a 4% native polyacrylamide gel (19:1
acrylamide/bisacrylamide) in 1× gel shift running buffer (25 mM Tris base, 190 mM glycine, 1.3 mM EDTA) at 4 °C, 170 V. The gels were prerun for 0.5-1
h at 4 °C. The protein-DNA complexes in the dried gels were
visualized by autoradiography using Amersham Pharmacia Biotech Hyperfilm.
Western Blot Analysis--
Soluble whole cell extracts (20-50
µg of protein) were electrophoretically resolved on 7.8%
SDS-polyacrylamide gel electrophoresis, and the proteins were
transferred to Nytran Plus membranes (Schleicher & Schuell). The
membrane was probed with a 1:2500 dilution of affinity-purified
anti-Sgk antibody in 50 mM Tris, pH 8.0, 150 mM
NaCl, 0.05% Tween 20 with 1% nonfat dry milk. The goat anti-rabbit IgG horseradish peroxidase-conjugated antibody (Bio-Rad) secondary antibody was used at a dilution of 1:10,000. The Western blot was
developed by using the Renaissance developing kit (NEN Life Science
Products) and exposed to x-ray film.
Immunofluorescent Detection--
Cells were plated onto
eight-well LabTek chamber slides and then treated as described (30).
The next day, the cells were treated with 0.3 M sorbitol
for approximately 4-6 h. Following washes with 1× PBS, cells were
fixed using 4% paraformaldehyde in PBS for 30 min at room temperature.
They were then permeabilized in 0.1% Triton X-100, 0.1% sodium
citrate for 2 min on ice. Affinity-purified anti-Sgk antibody diluted
1:150 in PBS was added to samples and allowed to incubate for 1 h
at room temperature or overnight at 4 °C. Goat anti-rabbit
fluorescein isothiocyanate-conjugated secondary antibody was added at a
dilution of 1:150 in PBS and allowed to incubate for 1 h at room
temperature. The cells were then washed with 1× PBS, and coverslips
were mounted using Antifade (Molecular Probes, Inc., Eugene, OR).
Results were visualized on a Nikon Optiphot fluorescence microscope.
Images were captured using Adobe Photoshop 3.0.5 (Adobe Systems, Inc.,
Mountain View, CA) and a Sony DKC-5000 digital camera. Nonspecific
fluorescence was determined by incubation with the secondary antibody
alone and shown to be negligible.
Immunoprecipitation and in Vitro Kinase Assay of Sgk--
HEK
293 cells were transfected as described (31) and then treated with
either serum-free media or 0.3 M sorbitol for 5 min. Cells were placed on ice and extracted with lysis buffer containing 50 mM Tris-HCl, 25 mM NaF, 120 mM
NaCl, 40 mM 
-glycerol phosphate, 1% Nonidet P-40, 0.1 mM sodium orthovanadate, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 2 nM
microcystin (Calbiochem). Lysates were centrifuged for 15 min at
12,000 × g, and the HA-Sgk protein was
immunoprecipitated from 400 µg of cell-free extracts with the anti-HA
epitope 12CA5 monoclonal antibody coupled to protein A-Sepharose
(Amersham Pharmacia Biotech). The immune complexes were washed once
with lysis buffer containing 0.5 M NaCl and then with lysis
buffer and finally with kinase buffer (50 mM Tris-HCl, 0.1% -mercapoethanol). In vitro kinase assays were
performed for 60 min at 30 °C in a reaction volume of 50 µl
containing 20 µl of immunoprecipitate in kinase buffer, 0.1 mM Sgktide peptide substrate (KKRNRRLSVA), 10 mM MgCl2, 50 µM ATP, 1 mM protein kinase A inhibitor (Calbiochem), and 5 µCi of
[-32P]ATP (NEN Life Science Products). The kinase
reaction was terminated with 0.55 mM EDTA. The 20 µl of
the reaction was spotted onto P-81 cation exchanger chromatography
paper (Whatman), which was then washed four times in 1% phosphoric
acid and dried with acetone. The amount of 32P-labeled
Sgktide was quantitated by scintillation counting.
Kinase assays performed using endogenous Sgk protein were performed as
above except for the following modifications. NMuMg cells treated with
either serum-free media or 0.3 M sorbitol for 8 h were harvested and then lysed as described above. Lysates were
centrifuged for 15 min at 15,000 × g and precleared
with rabbit IgG and protein A-Sepharose beads (Amersham Pharmacia
Biotech) for 40 min. The Sgk protein was immunoprecipitated from
precleared supernatants using affinity-purified anti-Sgk antibodies and
protein A beads for 2 h at 4 °C. A control set
immunoprecipitations employed nonimmune serum. The Sgk-specific
transphosphorylation was determined by subtracting the filter-bound
radioactivity observed with the nonimmune antibodies from that observed
with the Sgk-specific antibodies.
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RESULTS |
Stimulation of sgk Transcript and Protein Levels in Response to
Hyperosmotic Stress--
To initially test whether sgk gene
expression is targeted by the mammalian hyperosmotic stress pathway,
the levels of Sgk protein and mRNA were determined in NMuMg (normal
murine mammary gland) cells at various times after treatment with 0.3 M of the organic osmolyte sorbitol to induce a hyperosmotic
stress. Western blot analysis of whole of cell extracts using
affinity-purified anti-Sgk polyclonal antibodies (30) revealed that
after an approximately 4-h time lag, sorbitol treatment induced a
significant elevation in Sgk protein levels (Fig.
1A). A high level of Sgk
protein was still maintained 24 h after the hyperosmotic stress.
Northern blot analysis of total cellular mRNA of the same time
course using a -32P-labeled murine sgk
cDNA probe demonstrated a striking sorbitol stimulation in
expression of the 2.2-kb sgk transcript (Fig.
1B). Consistent with the protein data, sgk
transcripts were not detected until 4 h after sorbitol treatment,
and sgk mRNA levels continued to accumulate 24 h
following treatment. In the absence of sorbitol, the level of Sgk
protein and mRNA remained at a low basal level through out the
entire time course.
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Fig. 1.
Effect of 0.3 M sorbitol
treatment on Sgk protein and transcript expression. NMuMg mammary
epithelial cells were treated with or without 0.3 M
sorbitol in serum-free media, and at the indicated times the
cells were harvested. A, Western blots of
electrophoretically fractionated total cell extracts were probed for
the production of Sgk protein using affinity-purified Sgk-specific
primary antibodies. B, total RNA was isolated and separated
on a 1.1% agarose gel, transferred to a nitrocellulose blot, and
hybridized with a radiolabeled murine sgk cDNA probe as
described under "Experimental Procedures." As a loading control, 18 S ribosomal RNA stained with methylene blue is shown above
the sgk transcript band.
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To examine whether the sorbitol stimulation of sgk gene
expression is a direct response or requires de novo protein
synthesis, NMuMg cells were pretreated for 1 h with or without
cycloheximide (10 µg/ml), a protein synthesis inhibitor, and then
each set of cells was incubated in the presence or absence of 0.3 M sorbitol for 24 h. In the cycloheximide-treated
cells, protein synthesis was inhibited by greater than 95% without any
significant effects on mRNA synthesis (data not shown). Northern
blot analysis revealed that pretreatment of the cells with
cycloheximide had no effect on the inducibility of the sgk
transcripts in response to sorbitol (Fig.
2). Because the hyperosmotic stress
induction of sgk mRNA occurred in the absence of
de novo protein synthesis, we suggest that the regulation of
sgk gene expression is mediated by one or more preexisting
cellular components that are likely to be involved in the
transcriptional control of this gene. As we have previously reported
(29), exposure to cycloheximide stabilized sgk transcript
turnover, which caused both the sorbitol-treated and -untreated cells
to produce a higher level of sgk mRNA (Fig. 2). Our
results suggest that a hyperosmotic stress-induced signaling pathway
stimulates sgk gene transcription, which accounts for the
increased production of Sgk protein after sorbitol treatment.
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Fig. 2.
Induction of sgk transcripts
in response to hyperosmotic stress does not require new protein
synthesis. NMuMg mammary cells were pretreated with 10 µg/ml of
cycloheximide or buffer carrier for 30 min. Each pair of cell cultures
was then treated with or without 0.3 M sorbitol in
serum-free media for 24 h. Total RNA was isolated,
separated on an agarose gel, and transferred to a nitrocellulose blot
and probed for sgk transcript expression as described in the
legend to Fig. 1. As a loading control, 18 S ribosomal RNA stained with
methylene blue is shown in the lower panel.
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Identification of a Hyperosmotic Stress-responsive Region of the
sgk Promoter--
To functionally dissect the region within the
sgk gene promoter that is responsible for the hyperosmotic
stress regulation of sgk expression, NMuMg cells were
transfected with a series of sgk-CAT reporter genes
containing a series of 5'-progressive deletions starting at 1350 bp
upstream of the sgk gene and terminating at +55 bp
downstream of the transcriptional initiation site of the sgk
gene. Cells were then treated with or without 0.3 M
sorbitol, and cell lysates were assayed for CAT activity 24 h
after the hyperosmotic stress. As shown in Fig.
3, sorbitol stimulated transcriptional activity of sgk-CAT constructs containing the three
largest sgk promoter regions with deletions ending at 1350
bp, 236 bp, and 78 bp. Maximum stimulation of sgk-CAT
activity by hyperosmotic stress (5-8-fold) was observed with these
constructs. Sorbitol inducibility of the sgk promoter was
lost in deletions beyond 35 bp, indicating that the promoter fragment
between nucleotides 78 and 35 bp of the upstream region of the
sgk gene is a hyperosmolar stress-responsive region. In
additional experiments, this hyperosmotic regulated region was further
narrowed to between 67 and 35 base pairs (data not shown, and see
below). These results suggest that the region between 67 and 35 of
the sgk promoter contains transcription factor binding sites
involved in the stimulation of sgk gene transcription in
response to hyperosmotic stress.
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Fig. 3.
Identification of a hyperosmotic
stress-responsive region within the sgk promoter.
A series of sgk-CAT reporter plasmids containing the
indicated 5'-deletions of sgk promoter fused to the
bacterial CAT reporter gene were transfected by the calcium phosphate
precipitate method into NMuMg cells. Cells received 10 µg of reporter
plasmid and 10 µg of the promoterless CAT vector, pBLCAT3, and then
were incubated for 24 h with (+) or without () 0.3 M
sorbitol in serum-free media. CAT activity was assayed by
quantification of the conversion of [3H]acetyl-CoA into
[3H]acetylchloramphenicol by the two-phase fluor
diffusion assay described under "Experimental Procedures," and the
values were normalized to protein levels to determine the CAT-specific
activity. Each set of assays was performed in triplicate, and the
reported values are the mean and S.D. from three independent sets of
experiments.
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Functional Analysis of the Hyperosmolar Stress-responsive Region of
the sgk Promoter--
To test whether the hyperosmolar
stress-responsive region defined by the deletion analysis is sufficient
to mediate the sorbitol responsiveness of the sgk gene, an
oligonucleotide corresponding to the wild type 67 to 35 bp fragment
was cloned into pCAT-Basic that already contained the 34 to +55
region of the sgk promoter (38) and linked to the bacterial
CAT gene. NMuMg cells were transiently transfected with 67/35 wild
type CAT reporter plasmid, and CAT activity was monitored in cell
extracts isolated from cells treated with or without sorbitol. As shown
in Fig. 4, sorbitol induced CAT
expression by 3.7-fold compared with the unstressed mammary cells.
Reporter gene activity in cells transfected with the minimal promoter
tkCAT alone was low and unaffected by sorbitol treatment (data not
shown). This result establishes that the hyperosmolar stress responsive
region of the sgk promoter in transfected mammary epithelial
cells is located between 67 and 35 bp upstream of the RNA start
site.
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Fig. 4.
Mutational analysis of the 67/35
hyperosmotic stress-regulated region of the sgk
promoter. For the mutagenic fine mapping of the hyperosmotic
stress-regulated region, a series of six base pair substitutions of
TTCGAA were sequentially placed throughout the 67 to 35 bp region
of the sgk promoter. These five mutant oligonucleotides are
designated M1-M5. CAT reporter plasmids containing the indicated wild
type and mutant oligonucleotides were transfected into NMuMg cells,
incubated for 24 h with (+) or without () 0.3 M
sorbitol, and the resulting CAT activity and determination of
CAT-specific activity was assayed as described in the legend to Fig. 3.
The numbers display the -fold induction of CAT-specific
activity observed with each reporter plasmid. Each set of assays was
performed in triplicate, and the reported values are the mean and S.D.
from three independent sets of experiments.
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Analysis of the hyperosmotic stress-regulated region of the
sgk promoter using transcription factor consensus binding
site algorithms such as TFSEARCH and MatInspector revealed the presence of a highly conserved GC-rich region (43, 44). Therefore, to further
characterize the promoter region necessary for the sorbitol induction
of sgk gene expression, five mutants of this region were
synthesized in which each mutant contained a different set of six base
pairs that were mutated to the same sequence, TTCGAA, which is a
BstBI restriction enzyme site (shown in Fig. 4). Annealed
oligonucleotides (sense plus antisense) for the indicated five mutants
(M1-M5) of the 67/35 region of the sgk promoter were
cloned into the same pCAT-Basic vector used to generate the wild type
CAT reporter plasmid. The CAT reporter activities were monitored in
transiently transfected NMuMg cells treated with or without sorbitol.
As shown in Fig. 4, the M1, M2, and M5 mutants were fully inducible by
hyperosmolar stress at a level comparable with the wild type
sgk promoter fragment. In contrast, the M3 and M4 were
unable to respond to sorbitol treatment. The M3 and M4 mutations
overlapped with and ablated the GC-rich region of the sgk
promoter, suggesting that the corresponding transcription factor is a
target of the hyperosmotic stress response in mammary epithelial cells.
Electrophoretic Mobility Shift Assay Analysis of the
Transcriptionally Responsive Region of the sgk Promoter--
A
competitive gel shift analysis was employed to characterize the
DNA-binding protein complexes associated with the hyperosmolar stress
responsive region of the sgk promoter. The double-stranded wild type 67/35 oligonucleotide was radiolabeled and incubated with
nuclear extracts that were prepared from 4-h sorbitol-treated or
control NMuMg cells along with an excess of one of the mutant oligonucleotides (M1-M5). The gel shift pattern of the protein-DNA complexes was visualized by electrophoretic fractionation in a 6%
SDS-polyacrylamide gel. As shown in Fig.
5, without any competing oligonucleotides, the 67/35 region of the sgk promoter
forms the same four major protein-DNA complexes in the presence of cell extracts from untreated control or sorbitol treated cells (defined as
complexes A, B, C, and D). Incubation with a 200-fold excess of
unlabeled M1, M2, or M5 mutant oligonucleotides, which are fully
sorbitol-responsive, quantitatively competed with the wild type
oligonucleotide for formation of each of the protein-DNA complexes
using extracts from either the control or hyperosmolar stressed cells
(Fig. 5). A similar complete competition was observed with the wild
type oligonucleotide (data not shown). In contrast, mutant
oligonucleotides M3 and M4, which effectively ablate the GC-rich
sequence and are not sorbitol-responsive, were ineffective as
competitors in this gel shift assay (Fig. 5). These results suggest
that the GC-rich sequence located at 55 to 46 bp mediates the
hyperosmotic stress regulation of the sgk promoter through the function of the corresponding protein-DNA complex in this region of
the promoter.
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Fig. 5.
Competitive gel shift analysis of the
67/35 hyperosmotic stress-regulated region using the M1-M5 mutant
oligonucleotides. Cytoplasmic extracts (7 µg) were prepared from
NMuMg mammary cells that had been treated for 4 h with or without
0.3 M sorbitol in serum-free media. Cell extracts
were then preincubated for 20 min on ice with a 200-fold excess of each
of the unlabeled mutant oligonucleotides (M1-M5) shown at the
top of this figure and in Fig. 4. The
radiolabeled wild type 67/35 probe (see Fig. 4) was added to each
reaction mixture for 20 min, and the resulting protein-DNA complexes
were electrophoretically resolved in low ionic strength native 6%
polyacrylamide gels. The arrows show the migration of the
four major protein-DNA complexes (A, B,
C, and D) that were observed under these
conditions.
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Identification of the Sp1 Transcription Factor as a Component of
the Protein-DNA Complex That Binds to the Hyperosmolar Stress-regulated
Region of the sgk Promoter--
It is well established that the Sp1
transcription factor binds to a GC-rich DNA site that is identical to
the GCCCCGCCCC sequence located between 55 and 46 of the
sgk promoter (42, 45). The competitive gel shift assays were
used to initially test whether the hyperosmolar stress-responsive
region binds to a transcription factor with Sp1 specificity. Nuclear
extracts from control and sorbitol-treated cells were preincubated with
a 50-, 100-, or 200-fold excess of unlabeled competitor
oligonucleotides corresponding to a consensus Sp1 site. As a control, a
mutant Sp1 oligonucleotide, in which the middle three base pairs of the
Sp1 consensus have been changed in a manner that abrogates Sp1 (42),
was used as the competitor DNA. Each of these mixtures, as well as a
reaction without any added competitor, were incubated with the
radiolabeled wild type oligonucleotides encoding the 67 to 35
region of the sgk promoter, and the protein-DNA complexes
were visualized after electrophoretic fractionation. As shown in Fig.
6A, increasing amounts of the
consensus Sp1 oligonucleotide strongly competed for formation of each
of the four major protein-DNA complexes, whereas the mutant Sp1
oligonucleotide was unable to compete. In contrast, an unlabeled
oligonucleotide containing the consensus 5'-TCGCCCCCTC-3' sequence
corresponding to the EGR1 transcription factor, which also binds to a
GC-rich DNA site, failed to compete in the same assay (data not
shown).
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Fig. 6.
Evidence for the presence of Sp1 activity in
the gel-shifted complex formed with the hyperosmotic stress-regulated
region of the sgk promoter. Cytoplasmic extracts
(7 µg) were prepared from NMuMg mammary cells that had been treated
for 4 h with or without 0.3 M sorbitol in serum-free
media and then preincubated for 20 min on ice with either 50-, 100-, or 200-fold excess of unlabeled Sp1 consensus oligonucleotide
(SP1) or of a mutant Sp1 oligonucleotide
(MutSP1). The radiolabeled wild type 67/35 probe (see
Fig. 4) was added to each reaction mixture for 20 min, and the
resulting protein-DNA complexes were electrophoretically resolved in
low ionic strength native 6% polyacrylamide gels. B,
isolated cell extracts were incubated for 2 h on ice with either
polyclonal anti-Sp1 antibodies or as a negative control with polyclonal
anti-p110 antibodies. The radiolabeled wild type 67/35 probe (see
Fig. 4) was added to each reaction mixture for 20 min, and the
resulting protein-DNA complexes were electrophoretically resolved in
low ionic strength native 6% polyacrylamide gels. The
arrows show the migration of the four major protein-DNA
complexes (A-D), and the asterisks show the
migration on the new protein-DNA complex that is formed only in the
presence of anti-Sp1 antibodies.
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In a complementary approach, the presence of Sp1 in the gel-shifted
complexes was tested by assessing the ability of anti-Sp1 polyclonal
antibodies to alter the wild type gel shift complex. Nuclear extracts
isolated from control or sorbitol-treated cells were incubated for
2 h with either Sp1 antibodies (SP1Ab) or control antibodies directed against the phosphatidylinositol 3-kinase p110
subunit (p110Ab). The resulting formation of protein-DNA complexes on the wild type Sgk 67/35 double-stranded
oligonucleotide was visualized after electrophoretic fractionation. As
also shown in Fig. 6B, the anti-Sp1 antibodies caused a
specific loss of band B with a corresponding increase in a new lower
molecular weight protein-DNA complex (indicated by the
asterisk), whereas no reproducible changes were detected in
the gel mobility of bands A, C, and D. Conceivably, the Sp1 antibodies
either sequestered the available Sp1 protein in the extracts, thereby
preventing it from binding to the sgk wild type gel shift
probe to form band B, or prevented the binding of other transcription
factors that are normally tethered to the Sp1-containing gel shift
complex. By comparison, incubation with the control anti-p110 antibody had no effect on the gel-shifted protein complexes. Incubation of the
nuclear extracts with higher concentrations of the anti-Sp1 antibody
did result in a more pronounced loss of band B, although there was no
noticeable effect on the presence of any of the other protein-DNA
complexes (data not shown). These results, along with the functional
studies described above, implicate Sp1 as a target or a member of a
multiprotein complex that is involved in hyperosmotic stress regulation
of the sgk promoter.
Hyperosmotic Stress Regulation of sgk Transcript and Protein
Expression Is Mediated by the p38 MAPK Pathway--
It is well
documented that p38 MAPK is phosphorylated and activated by the dual
specificity MKK3/6 kinases at a TGY motif in response to various forms
of stress, including hyperosmotic stress (6, 16, 46). To test whether
p38 MAPK is activated in NMuMg mammary cells in response to sorbitol, a
Western blot of extracts from cells treated at various times with or
without 0.3 M sorbitol was probed with anti-phospho-p38
MAPK antibodies. The phosphorylated form of p38 MAPK was detected in
the mammary epithelial cells between 1 and 2 h after hyperosmotic
stress and then decreased to near basal levels by 4 h
poststress (Fig. 7A). Interestingly, p38 MAPK phosphorylation occurs before the induction of
Sgk protein expression, suggesting a causal relationship between these
cellular processes. To directly determine whether the hyperosmotic stress stimulation of Sgk protein expression requires p38 MAPK activity, NMuMg cells were treated with the p38 MAPK pharmacological inhibitors SB203580 (20 µM) or SB202190 (10 µM) for 30 min prior to the addition of either serum-free
media (untreated control) or 0.3 M sorbitol for
6 h. Western blot analysis of Sgk protein levels revealed a
striking reduction in the amount of Sgk protein induced in hyperosmolar
stressed cells treated with either p38 MAPK inhibitor (Fig.
7B).
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Fig. 7.
Inhibitors of the p38 MAPK suppresses the
hyperosmotic stress stimulation of Sgk protein and promoter
activity. A, NMuMg cells were treated with (+) or
without () 0.3 M sorbitol in serum-free media and
at the indicated time points. Cell extracts were electrophoretically
fractionated in SDS-polyacrylamide gels. Western blots were probed with
phospho-p38 MAPK-specific antibodies to analyze the production of
active p38 MAPK. B, cells were treated with the p38 MAPK
pharmacological inhibitors SB203580 (20 µM) or SB202190
(10 µM) for 30 min, and then the cells were treated with
(+) or without () 0.3 M sorbitol in serum-free
media for 6 h. Total cell extracts were
electrophoretically fractionated in SDS-polyacrylamide gels and Western
blots probed for the production of Sgk as described under
"Experimental Procedures." C, cells were transfected
with the 1.3 sgk-CAT reporter plasmid, as described in the
legend to Fig. 3. 24 h post-transfection, the cells were
pretreated for 30 min with or without SB202190 (10 µM).
The cells were then treated with or without 0.3 M sorbitol
in serum-free media for 24 h, harvested, and lysed, and
CAT activity was assayed by quantification of the conversion of
[3H]acetyl-CoA into
[3H]acetyl-chloramphenicol by the two-phase fluor
diffusion assay described under "Experimental Procedures." The
resulting CAT activities were normalized to protein levels to determine
the CAT-specific activity, and the -fold induction was calculated as a
ratio of the CAT-specific activity observed in sorbitol-treated cells
to that observed in control cells. Three independent assays (data
points) were performed in triplicate, and the bar
graphs show the mean from these experiments.
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The p38 MAPK pathway targets and phosphorylates several transcription
factors in response to hyperosmotic stress (16), and the attenuation of
Sgk protein levels in the presence of p38 pharmacological inhibitors
suggested a functional link between p38 MAPK function and the
stimulation of sgk promoter activity. To determine whether the p38 MAPK cascade is involved in the hyperosmotic stress stimulation of the sgk promoter, NMuMg cells transfected with the 1.3
sgk-CAT reporter plasmid were pretreated with or without the
p38 MAPK inhibitor SB202190 and then incubated in the presence or
absence 0.3 M sorbitol to induce the hyperosmolar stress.
In the absence of SB202190, the 1.3 sgk-CAT construct was
induced on average 4-fold in response to sorbitol treatment (Fig.
7C), and in cells pretreated with SB202190 the hyperosmolar
stress induction of sgk promoter was abrogated. There are
several p38 MAPK gene family members, and based on the selectivity of
the SB203580 and SB202190 pharmacological inhibitors, our results
suggest that the p38-- or p38--mediated signaling pathway (47)
contributes to the hyperosmotic stress stimulation of sgk
promoter activity and protein production.
One of the upstream kinases that activates p38 MAPK in response to
cellular stress is MKK3 (16, 46). As a complementary approach to
demonstrate the involvement of the p38 MAPK pathway in the hyperosmolar
stress stimulation of the sgk promoter, cells were
cotransfected with the 236 sgk-CAT reporter construct and with expression plasmids for either the wild type MKK3 kinase (MKK3) or
the kinase-dead form of MKK3 (MKK3AL) that has been shown to act as a
dominant negative inhibitor of the p38 MAPK pathway (48). As shown in
Fig. 8, co-transfection with the wild type MKK3 leads to a high level of sorbitol-stimulated reporter activity, whereas the co-transfection of the dominant negative acting
MKK3AL plasmid abolished the sorbitol induction of sgk promoter activity. These results show that the hyperosmotic stress stimulation of sgk promoter activity requires the
MKK3-mediated activation of p38 MAPK kinase. Because treatment with
either of the p38 MAPK pharmacological inhibitors fails to completely
dampen the hyperosmotic stress stimulation of sgk promoter
activity, MKK3 may have one or more as yet unidentified targets in
addition to the p38- or p38- MAPK isoforms that play a role in
the sgk transcriptional response.
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Fig. 8.
Effects of wild type and dominant negative
MKK3 on the hyperosmotic stress stimulation of sgk
promoter activity. NMuMg mammary epithelial cells were
transiently co-transfected with the 236 sgk-CAT reporter
plasmid along with either the wild type (MKK3) or kinase
dead dominant negative MKK3 (MKK3AL) expression plasmid.
Another set of cells were transfected only with the 236
sgk-CAT reporter plasmid. The cells were then treated with
(+) or without () 0.3 M sorbitol for 24 h, and the
CAT-specific activity was determined in the isolated cell extracts as
described in Fig. 7. Each set of assays was performed in triplicate,
and the reported values are the mean and S.D. from three independent
sets of experiments.
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Hyperosmotic Stress Induces a Cytoplasmically Localized and
Enzymatically Active Sgk Protein--
We have previously established
that the nuclear/cytoplasmic subcellular distribution of Sgk can be
regulated in a stimulus-dependent manner (30). For example,
in serum-treated cells Sgk shuttles between the nucleus and cytoplasm
in synchrony with the cell cycle, whereas in glucocorticoid-treated
cells Sgk is exclusively localized to the cytoplasmic compartment.
Therefore, to characterize the effects of hyperosmotic stress on the
subcellular distribution of Sgk, NMuMg cells were grown to
confluency and treated with 0.3 M sorbitol for 4 h,
and the fixed cells were permeabilized and stained with anti-Sgk
polyclonal antibody followed by fluorescein isothiocyanate-conjugated
goat anti-rabbit secondary antibody. Indirect immunofluorescence
revealed that hyperosmotic stress stimulates the production of a
cytoplasmic localized Sgk (Fig. 9).
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Fig. 9.
Hyperosmotic stress induction of the
endogenous Sgk protein localizes to the cytoplasmic compartment.
NMuMg mammary cells were treated with (+) or without () 0.3 M sorbitol in serum-free media for 4 h. The
subcellular distribution of Sgk was examined by indirect
immunofluorescence microscopy using affinity-purified rabbit polyclonal
antibodies to Sgk followed by fluorescein isothiocyanate-conjugated
goat anti-rabbit secondary antibodies. No immunofluorescence staining
is observed when preimmune antiserum is used as the primary antibodies
(data not shown).
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We have recently established that Sgk protein kinase activity is
activated in response to serum growth factors through a
phosphatidylinositol 3-kinase-mediated pathway (31). Furthermore, other
groups have shown that sorbitol inhibits other PI 3-kinase downstream
targets, such as Akt/PKB and p70 S6 kinase (49-51). Interestingly,
this inhibition is not due to the inhibition of PI 3-kinase or PDK1 activity but instead through the activity of the PP2A phosphatase. Therefore, to determine whether the sorbitol induced the slower migrating phosphorylated form of Sgk as a result of signaling through
PI 3-kinase, cells were pretreated with or without the pharmacological
inhibitor for PI 3-kinase, LY294002. This drug renders the PI 3-kinase
molecule inactive by preventing ATP binding (52). As shown in Fig.
10B, in the absence of
LY294002, a Sgk protein doublet (49 and 51 kDa) is induced in response
to hyperosmotic stress. Our previous work has demonstrated that these
two protein species represent hyperphosphorylated and
hypophosphorylated forms of Sgk (30). We also previously documented
that an inhibition of PI 3-kinase activity prevented the appearance of
the hyperphosphorylated form of Sgk (31). Consistent with these
results, LY294002 ablated production of the slower migrating (and
hyperphosphorylated) form of Sgk (Fig. 10B), suggesting that
the Sgk protein, which is produced after sorbitol treatment, is
targeted by the PI 3-kinase signaling pathway.
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Fig. 10.
Effect of hyperosmotic stress on the
phosphorylation state and enzymatic activity of endogenous Sgk
protein. A, affinity-purified anti-Sgk antibodies
(Sgk) or nonimmune antibodies (NI) were
added to total cell extracts from cells either treated with (+) or
without () 0.3 M sorbitol for 8 h. As a positive
control for Sgk enzymatic activity, Sgk was immunoprecipitated from
48-h serum-starved cells that had been incubated with (+) or without
() 10% serum for 24 h. Transphosphorylation activity in the
immunoprecipitated protein was monitored by quantifying the formation
of 32P-labeled Sgktide peptide in each reaction.
Sgk-specific transphosphorylation activity was determined by
subtracting the filter-bound radioactivity observed with the nonimmune
antibodies from that observed with the anti-Sgk antibodies.
B, NMuMg cells were pretreated with either 50 mM
LY294002 or Me2SO carrier for 16 h and then incubated
with (+) or without () 0.3 M sorbitol in serum-free
media. After 8 h, cells were harvested, and Western blots
of electrophoretically fractionated total cell extracts were probed for
the expression of Sgk protein.
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To biochemically assess whether hyperosmotic stress had any effect on
endogenous Sgk protein kinase activity, NMuMg cells were treated with
or without sorbitol for 8 h, and the isolated cell extracts were
incubated with affinity-purified anti-Sgk antibody or nonimmune
antibodies as a negative control. As a positive control for this assay,
Sgk was immunoprecipitated from serum-treated and -untreated cells. The
Sgktide peptide (KKRNRRLSVA) was utilized as a substrate in an in
vitro transphosphorylation assay, and the Sgk-specific activity
was determined by quantitating the level of 32P-labeled
Sgktide in the anti-Sgk immunoprecipitated compared with those from
nonimmune antibodies. As shown in Fig. 10A, the endogenous
Sgk immunoprecipitated expressed in hyperosmotically stressed cells is
an active kinase with a transphosphorylation activity comparable with
that observed in serum-treated cells.
As a complementary approach, the effects of hyperosmotic stress on Sgk
enzymatic activity in the absence of the transcriptional stimulation
were examined by expression of an exogenous sgk gene driven
by the constitutive RSV mammalian expression vector. To assess the
activity of exogenous wild type Sgk protein, human embryo kidney 293 cells (HEK 293) were transfected with the pCDNA3 HA-sgk
expression vector and then treated with either isosmotic or
hyperosmotic media. The immunoprecipitated HA-Sgk protein, using
antibodies directed against the HA epitope tag, was assayed for
transphosphorylation activity using the Sgktide substrate. As shown
in Fig. 11, hyperosmotic stress
stimulated the kinase activity of exogenously expressed Sgk protein
approximately 3-fold over isosmotic treatment. After the peak
production of active Sgk within the first 5-15 min after sorbitol
treatment, the level of Sgk transphosphorylation activity returned to
basal levels during the next 60 min. Although activation of Sgk is
transient, this result suggests that hyperosmotic stress regulates Sgk
expression at the transcriptional level and also posttranslationally
regulates Sgk enzymatic activity.
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Fig. 11.
Effect of hyperosmotic stress on the
enzymatic activity of exogenous Sgk protein. HEK 293 cells
overexpressing HA epitope-tagged Sgk (HA-Sgk) were treated
with either isosmotic (Iso) or hyperosmotic (0.3 M sorbitol) media for the indicated times. HA-Sgk
was immunoprecipitated from each cell extract using anti-HA-specific
antibodies and assayed for kinase activity using Sgktide as the
substrate. The anti-HA-Sgk immunoblot analysis is shown in the
lower panel.
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DISCUSSION |
The evolutionarily conserved ability of an organism to adapt to
extreme changes in nutrient levels, temperature, or osmolarity is
crucial to the survival of unicellular prokaryotes as well as the
eukaryotic cells that constitute mammalian tissues. Several signaling
cascades have been shown to play a necessary role in the detection and
response to environmental stress. One such evolutionarily conserved
pathway employs the activation of the p38 MAPK/HOG1 family of protein
kinases (14), which can be triggered by osmotic imbalances, UV light
radiation, heat shock, DNA-damaging agents, FAS-induced cell death, and
exposure to the protein synthesis inhibitor anisomycin (46, 48, 53).
Several transcription factors and protein kinases have been identified
as cellular targets of p38 MAPK in mammalian cells; however, relatively
little is known about their respective gene targets and protein
substrates. We and others have shown that an important cellular feature
of sgk is its acute transcriptional control in rodent and
human cell lines (28, 29, 32, 33, 38, 54-57), and our study shows for
the first time that the transcriptional regulation of sgk can be directly linked to a specific stress signaling pathway involving
p38 MAPK in mammalian cells.
As shown in Fig. 12, we propose that
exposure of mouse NMuMg mammary epithelial cells to hyperosmotic
medium activates the p38 MAPK cascade, which in turn stimulates
sgk transcription by targeting a GC-rich hyperosmotic
stress-regulated element in the sgk promoter that is
recognized by the Sp1 transcription factor. Activation of this pathway
induces an increase in the production of an active Sgk protein kinase
that maintains its dependence on the PI 3-kinase pathway for its
phosphorylation and activity. Several lines of evidence demonstrated a
role for p38 MAPK in the stimulation of sgk transcription.
The robust stimulation of sgk transcription and protein
production can be disrupted by treatment with p38 MAPK-specific
pharmacological inhibitors or by exogenous expression of dominant
negative MKK3, the immediate upstream regulator of p38 MAPK. Moreover,
ectopic expression of wild type MKK3 further stimulated the magnitude
of the sorbitol-induced sgk promoter activity. Our results
also suggest that the activated p38 MAPK cascade either directly or
indirectly targets a Sp1 transcription factor protein complex on the
sgk promoter to stimulate sgk
transcription (Fig. 12). Sp1 is a Cys2His2 zinc
finger-binding protein that has been shown to bind specifically to the
GC-rich GCCCCGCCCC sequence (42, 45) that we defined as the
hyperosmotic stress-regulated element in the sgk
promoter. Our studies are generally consistent with previous
observations in the yeast S. cerevisiae in which the Msn
family of transcription factors are targeted by the yeast p38 MAPK
homologue HOG1 and bind to a CCCCT DNA sequence that is similar to the
Sp1 binding core sequence (7, 8, 10). In earlier studies, Sp1 was
viewed as a component of the basal transcriptional machinery. However,
recent studies have shown that Sp1 transactivation activity is also
subject to regulation by several different extracellular cues including
oxidative and shear stress (58, 59). Our results now implicate the Sp1
transcription factor as a component in the hyperosmotic stress response
that leads to the stimulated expression of sgk.
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Fig. 12.
Model summarizing the regulation of Sgk by
hyperosmotic stress. We propose that hyperosmotic stress
stimulates sgk promoter activity through the activation of
p38 MAPK. The activation of sgk transcription can be
inhibited by the overexpression of a dominant negative kinase-dead form
of MKK3 (dnMKK3) and by the p38 pharmacological inhibitors
(SB202190 or SB203580). In addition to ATF2, CHOP, Elk-1, and other
known substrates, the activated p38 MAPK pathway targets a GC-rich
hyperosmotic-regulated element located at 55 to 46 base pairs in
the sgk promoter that includes the Sp1 transcription factor
and most likely at least one other nuclear target. Hyperosmotic stress
leads to the production of a phosphorylated and enzymatically active
form of Sgk that resides in the cytoplasm. Sgk is enzymatically
activated through phosphorylation by the PI 3-kinase-PDK1 signaling
pathway, which we have previously shown to regulate Sgk. We propose
that the transcriptionally induced and active Sgk plays a key role in
the cellular response to hyperosmotic stress.
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A deletion analysis initially identified a hyperosmotic
stress-responsive region between 67 and 35 bp of the sgk
promoter that accounts for the striking sorbitol stimulation of
sgk gene expression. Fine mapping of this 32-bp region
defined a GCCCCGCCCC sequence at 54 to 44 bp as being necessary for
the hyperosmotic shock induction. Competitive electrophoretic mobility
shift assays and mutational analyses demonstrated the presence of Sp1
in the transcription factor complex that interacts with the
hyperosmotic stress-responsive region and thereby implicates the Sp1
transcriptional regulator as a necessary factor for this response. For
example, inclusion of Sp1-specific antibodies in the gel shift reaction caused a specific loss of one of the predominant protein-DNA complex bands with a corresponding increase in a new lower molecular weight protein-DNA complex. Also, the consensus Sp1 oligonucleotide strongly competed for formation of each of the four major protein-DNA complexes observed with the 67 to 35 hyperosmotic stress-regulated region of
the sgk promoter. In contrast, a mutant Sp1 oligonucleotide, which is incapable of binding Sp1, and a wild type consensus
oligonucleotide corresponding to the EGR1 transcription factor, which
also binds to a GC-rich DNA site, were unable to compete in this assay.
It is likely that the Sp1 transcription factor functions in the
hyperosmotic stress response through an interaction with another
unidentified nuclear factor, because transfection of a reporter plasmid
containing the consensus Sp1 binding site with or without an expression
plasmid for Sp1 (60) failed to be sorbitol-responsive (data not shown). Consistent with this concept, we observed multiple protein-DNA complexes in an electrophoretic mobility gel shift assay using an
oligonucleotide corresponding to the 67 to 35 hyperosmotic stress-responsive region. We are currently examining whether one or
more of the protein factors in these DNA-protein complexes may be
transcriptional targets of the hyperosmotic stress cascade that
interact with Sp1.
The four major protein-DNA complexes that form on the hyperosmotic
stress-regulated element in the sgk promoter remained
unaffected by sorbitol treatment. There are several precedents for
promoters being activated through a p38 MAPK cascade that do not show
obvious changes in protein-DNA complexes. For example, the
lipopolysaccharide induction of a NF-
B consensus binding site
reporter construct was inhibited by the p38 MAPK pharmacological
inhibitor SB203580 and by co-expression of a dominant negative p38 MAPK
under conditions that did not change the observed gel-shifted
DNA-protein complexes (61). We speculate that sgk
transcription may be stimulated by the recruitment of specific
coactivators or altered interactions with components of the basal
transcriptional machinery that can potentially be phosphorylated by p38
MAPK in response to hyperosmotic stress. For example, the TFIID
component of the basal transcription complex has been shown to be
phosphorylated by p38 MAPK and also to interact with Sp1 in other cell
systems (61, 62). Conceivably, other cellular components that are known
to be phosphorylated by p38 MAPK, such as transcription factors and
protein kinases (16-18, 20-22, 25, 63, 64), could potentially
interact with Sp1 in a p38 MAPK-dependent manner and play a
role in the hyperosmotic stress response.
We propose that the Sgk mediates certain cellular responses to osmolar
imbalances, because after hyperosmotic stress of mammary epithelial
cells, Sgk protein levels are robustly induced and maintained at a high
level throughout the time course of sorbitol treatment. As predicted by
the promoter studies, pretreatment of cells with the p38 MAPK
inhibitors SB203580 and SB202190 resulted in the significant
attenuation of Sgk protein levels. Although one other study has
reported that anisosmotic changes regulate sgk transcript
levels (34), our study is the first to define the sgk
promoter elements that mediate the hyperosmotic stress stimulation of
transcripts, to uncover a connection between Sgk and the p38 MAPK
stress signaling cascade, and to demonstrate an increase in the
production of endogenous Sgk protein and Sgk-specific serine/threonine
protein kinase activity. In response to hyperosmotic stress, the Sgk
protein is localized throughout the cytoplasm that presumably includes
the cytoplasmic surface of the plasma membrane. One potential role of
Sgk in mediating the cellular hyperosmotic stress response may be to
regulate cell volume through activation of the epithelial sodium
channel (55, 56). Our results suggest that the enzymatic activity of
Sgk is important for this response, and we are attempting to define the
membrane interactions between endogenous Sgk and the epithelial sodium channel that are necessary for channel activity that probably account
for the increased channel activity that is observed in response to
hyperosmotic stress (65).
Following treatment of cells with serum and/or growth factors, PI
3-kinase activity is induced, which initiates a PDK-1 signaling pathway
(66). These events are responsible for the activation of the downstream
kinases Akt/PKB, p70 S6 kinase, and Sgk (31, 67-70). A well documented
consequence of the activation of Akt/PKB is the phosphorylation of the
proapoptotic protein Bad, which regulates the cell survival
response (71-73). Both Sgk and Akt/PKB are downstream targets of the
PI 3-kinase-dependent activation of PDK1. Treatment of
3T3L1 preadipocytes with sorbitol has been shown to inhibit Akt/PKB as
well as p70 S6 kinase activity (49-51). It has been demonstrated that
PI 3-kinase and PDK1 are active under this hyperosmolar condition, and
consequently the decrease in the cellular pool of phospho-Akt/PKB is
attributable to the PP2A phosphatase (49, 50). We observed that
sorbitol induced a phosphorylated and kinase-active form of endogenous
Sgk that is sensitive to the LY294002 PI 3-kinase inhibitor. It is
therefore possible that in the mammary epithelial cells used in our
study, the activity of the PP2A phosphatase remains low enough to
maintain the activity of endogenous Sgk. In this regard, ectopic
expression of sgk in the human embryo kidney 293 cells (HEK
293) only transiently produced an active Sgk enzyme, suggesting that
the constitutive PP2A phosphatase activity plays a counter regulatory
role on Sgk in these cells. We have previously shown that a lack of
phosphorylation of Sgk at both the critical Thr256 and
Ser422 residues results in an inactive Sgk kinase (31).
Therefore, as a alternative explanation, it is possible that in the
presence of sorbitol, Sgk is only phosphorylated on one of these
residues and as a result cannot sustain its activity in the HEK 293 cells.
Several groups have reported that perturbations of the PI 3-kinase
signaling pathway can affect insulin-dependent glucose uptake by the GLUT4 transporter and other insulin responses (74-78). Conceivably, Sgk, which is expressed in the kidney (55, 56) and
expressed at high levels in the kidney glomeruli and distal tubules,2 may be involved in
the hyperosmotic response to high plasma glucose levels in which p38
MAPK activity has been shown to be induced and Akt/PKB activity
inhibited (49, 50, 79-81). Moreover, one other report suggests that
Sgk may also play a role in the nephropathy that is associated with the
diabetic disease state (82). Further studies are in progress to
identify the physiological substrates of Sgk under high osmolarity
conditions, which will add greatly to the understanding of the role of
Sgk in cellular stress signaling pathways.
TOP
ABSTRACT
INTRODUCTION
