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INTRODUCTION |
The specificity of the glucocorticoid receptor's
(GR)1 transcriptional
activity varies widely between cell types, thus accounting for the
diverse and sometimes opposite physiological effects of glucocorticoid
in different tissues. For example, glucocorticoids have been shown to
promote apoptosis in lymphocytes (1, 2), whereas human mammary
epithelial cells (MECs) (3) and rat hepatoma epithelial cells (4) are
protected from apoptosis after GR activation. Furthermore, studies in
both breast and liver epithelial cells have demonstrated that RU486, a
potent GR antagonist that inhibits GR-mediated transcriptional
activation, reverses the survival effect of glucocorticoids (3, 4). The
antagonistic effects of RU486 on cell survival suggest that
glucocorticoid-mediated survival is regulated specifically through
GR-induced transactivation of downstream genes.
Previous studies using glucocorticoid concomitantly with or without the
GR antagonist RU486 have suggested that the identification of genes
directly induced or repressed by GR activation might reveal important
pathways relevant to epithelial cell survival signaling. One such
GR-inducible gene, serum and glucocorticoid-regulated kinase-1
(sgk), encodes a serine/threonine kinase with 54% homology in its catalytic domain to the well described antiapoptotic kinase AKT.
sgk was originally identified through subtractive cloning of
a serum and glucocorticoid-induced mammary tumor cell cDNA library
(5). More recently, sgk was found to be part of a larger gene family and was designated sgk-1 (6). Interestingly,
sgk has also been shown to be transcriptionally induced
after activation of a variety of steroid receptors including the
mineralocorticoid receptor in kidney epithelial cells (7) and the
follicle-stimulating hormone receptor in ovarian granulosa cells (8).
sgk transcripts have also been shown to be induced after
changes in cell volume (9) and under conditions of extracellular
hyperosmotic stress (10).
In this report, we have extended our original investigation of
GR-mediated survival signaling from the nontumorigenic MEC cell line
MCF10A to breast cancer cell lines. Although only a subset of commonly
studied human breast tumor cell lines undergo significant apoptosis
after growth factor deprivation, most of these growth
factor-dependent cell lines were protected from apoptosis after treatment with physiological concentrations of glucocorticoid. Furthermore, the effect of glucocorticoid appears to be GR-mediated because it can be out-competed by high affinity GR antagonists. We also
demonstrate that the GR survival signal is likely to be transmitted at
least in part as a consequence of the transcriptional activation of
sgk since (i) sgk mRNA is induced in a
GR-dependent fashion immediately after glucocorticoid
treatment of human MECs, (ii) ectopic SGK expression protects MECs from
apoptosis induced by growth factor withdrawal, and (iii) expression of
a kinase-dead SGK inhibits protection from apoptosis. Taken together,
these results suggest the existence of a novel GR-initiated
antiapoptotic pathway that operates, at least in part, through
transcriptional induction of the survival kinase gene,
sgk.
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MATERIALS AND METHODS |
cDNA Constructs--
pOG-hSGK was obtained as a generous
gift from Dr. Siegfried Waldegger (University of Hamburg, Germany). The
human sgk cDNA fragment was amplified from pOG and
cloned into the EcoRI and XhoI sites of the
retroviral vector pLPCX (CLONTECH, Palo Alto, CA)
by applying a polymerase chain reaction-based strategy that incorporated a hemagglutinin (HA) tag in-frame in the amino terminus of
wild type SGK using the following primer (Life Technologies, Inc.):
wild type HA-SGK,
5'-TAATACTCGAGGCTCCATCATGTACCCATATGACGTTCCAGACTACGCTACGGTGAAAACT-3'.
N60 HA-SGK was similarly constructed by inserting an HA tag
in-frame 5' to the coding sequence for amino acid 61 of SGK:
N60
HA-SGK, 5'-GGAATTCCTAAGCGTAGTCTGGAACGTCATATGGGTATACCCCTGCATCAGT-3'. The carboxyl-terminal primer for both constructs was
5'-GGAATTCCTCAGAGGAAAGAGTC-3'. The polymerase chain reaction
mixtures were supplemented with 10% Me2SO and run with
Pfu DNA polymerase (Promega, Madison, WI) with an initial
95 °C, 5 min denaturation followed by 35 cycles at 95 °C for 1 min, 55 °C for 1 min, 72 °C for 3 min, and a final 72 °C for 5 min elongation. Mutations were confirmed using primers provided with
the pLPCX vector by overlapping bidirectional sequencing using an
ABI776 sequencer (PerkinElmer Life Sciences).
Cell Culture and Viral Infection--
All parental cell lines
were obtained from American Type Culture Collection (ATCC, Manassas,
VA). MCF10A and MCF10A-Myc cells (as described in Moran et
al. (3)) were cultured in a 1:1 mixture of Dulbecco's modified
Eagle's medium and Ham's F-12 (BioWhittaker, Walkersville, MD)
supplemented with hydrocortisone (0.5 µg/ml), human recombinant EGF
(10 ng/ml), and bovine insulin (5 µg/ml; Sigma). BT-20, Hs578T,
MDA-MB-231, MDA-MB-468, SK-BR-3, and T-47D cells were cultured in
Dulbecco's modified Eagle's medium (BioWhittaker) supplemented with
10% heat-inactivated fetal calf serum (FCS; Atlanta Biologicals,
Norcross, GA). MCF7 cells were cultured in minimal essential
medium (ATCC) supplemented with 10% heat-inactivated FCS, and
HCC1937 cells were cultured in RPMI 1640 (BioWhittaker) supplemented
with 10% heat-inactivated FCS. Retroviruses were made by transient
transfection of retroviral vectors into amphotropic Phoenix cells (a
gift of Dr. Gary Nolan, Stanford University, Palo Alto, CA) using
either standard calcium phosphate precipitation or Effectene
transfection reagent per manufacturer's instructions (Qiagen, Santa
Clarita, CA). MECs were infected as described previously (3) with
HA-SGK-expressing retroviruses, and clones were selected with puromycin
(400 ng/ml). Individual colonies were tested for HA-SGK expression by
Western analysis using an anti-HA monoclonal antibody.
Apoptosis Assays--
Cells were trypsinized and seeded
subconfluently at 1 × 105 cells/3-cm well in the
appropriate media. Cells were allowed to adhere overnight, rinsed twice
with PBS, and subsequently cultured for 72 h in serum-free media
containing insulin, EGF, hydrocortisone, dexamethasone
(10
6 M), or combinations of these
purified growth factors in the concentrations listed above. In GR
antagonist assays, RU486 (5 × 10
7
M; Sigma), dexamethasone 21-mesylate (DM,
10
7 M; Steraloids Inc, Newport,
RI), and dexamethasone oxetanone (10
5
M; a gift of Dr. Stoney Simons, National Institutes of
Health) were added to media containing glucocorticoid.
In some experiments, cells were pre-treated for 30 min with serum-free
media and 50 µM LY294002 (Calbiochem) or vehicle alone (0.01% Me2SO in PBS) followed by the addition of
appropriate growth factors. After a 72-h incubation, media and floating
debris were gently aspirated from wells and replaced with 2 ml of fresh
serum-free media; cells were then immediately fixed by adding 500 µl
of 37% formaldehyde to each well in a dropwise fashion and incubating at room temperature for 30 min. The fixative solution was subsequently aspirated, and fixed cells were allowed to dry overnight. To score for
apoptosis, cells were then stained with a 1 µM
4,6-diamidino-2-phenylindole/PBS solution as described previously (11).
A Nikon Eclipse E800 microscope with UV illumination at 600×
magnification was used to count at least 200 4,6-diamidino-2-phenylindol -stained cells per well to determine the
percentage of apoptotic cells per experimental condition. All apoptosis
assays were repeated a minimum of three separate times to calculate
averages and S.E.
Western Analysis--
Equal numbers of cells (1 × 105) were cultured in 3-cm dishes overnight. The following
morning, cells were washed twice on ice with ice-cold PBS and lysed
directly in 40 µl of 2× Laemmli buffer. Whole cell lysates were
prepared by scraping cells with a rubber spatula, passing lysates 10 times through an 18-gauge needle, and then boiling for 5 min. Samples
were then electrophoresed in 8 or 9% SDS-polyacrylamide
electrophoresis gels and transferred to nitrocellulose (Osmonics,
Minnetonka, MN). Equal protein loading was confirmed by visual
inspection of Ponceau S staining of the nitrocellulose membrane.
Nitrocellulose was then rinsed with Tris-buffered saline, 0.1% Tween
and incubated with one of the following antibodies: rabbit polyclonal
anti-GR (Affinity Bioreagents, Golden, CO) or rat monoclonal anti-HA
(Roche Molecular Biochemicals, Indianapolis, IN). After primary
antibody incubation and extensive washing with Tris-buffered saline,
0.1% Tween, the appropriate secondary antibody, either anti-rabbit
IgG-horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA)
or anti-rat IgG-horseradish peroxidase (Sigma), was added at a 1:5000
dilution. The nitrocellulose was washed again with Tris-buffered
saline, 0.1% Tween, incubated in ECL substrate according to
manufacturer's instructions (Amersham Pharmacia Biotech), exposed to
film, and developed. Blots were subsequently probed with a mouse
-actin antibody (Sigma) or a mouse GAPDH antibody (Chemicon,
Temecula, CA) as a loading control.
Kinase Assay--
MCF10A-Myc cells stably expressing various SGK
constructs (see above) were lysed as described in Park et
al. (12). Two mgs of lysate, measured by Bradford assay, were then
used to immunoprecipitate SGK. Specifically, lysates were incubated
with rat monoclonal HA antibody-coated 4B Fast Flow protein G-Sepharose
beads (Sigma) for 90 min at 4 °C. Immunoprecipitated SGK was then
used in a kinase assay as described in Park et al. (12).
Incorporation of [
-32P]dATP into SGK-tide was counted
on a Packard 2200CA
counter, and the assay was repeated to obtain
average [
-32P]dATP incorporation.
Northern Analysis--
MCF10A-Myc and MDA-MB-231 cells were
trypsinized, seeded in equal numbers in 10-cm dishes, and allowed to
grow in the appropriate media. When cells reached ~80% confluency,
media was aspirated, and cells were washed twice with PBS and incubated
in serum-deprived (0.5% FCS) media for 72 or 96 h. After serum
deprivation, cells were stimulated for 30 min with either vehicle alone
(ETOH), dexamethasone, dexamethasone/RU486, or 20% FCS. Total RNA was
harvested using the RNeasy mini kit per manufacturer's instructions
(Qiagen). RNA was quantified by spectrophotometry, and 20 µg of RNA
per experimental group was electrophoresed in a 1.0% agarose, 17% formaldehyde gel and then transferred onto Hybond nylon membranes (Amersham Pharmacia Biotech). Membranes were then sequentially hybridized with a full-length human sgk cDNA probe and a
rat gapdh cDNA probe labeled with
[
-32P]dCTP using the Prime-It II random primer
labeling kit (Stratagene, Cedar Creek, TX). Membranes were then washed
and exposed to film. Bands were quantified using a Bio-Rad GS-710
calibrated imaging densitometer so that the relative ratio of
sgk:gapdh signal could be determined. Experiments
were repeated at least two times to calculate average ratios of
sgk:gapdh mRNA intensities and S.E.
Site-directed Mutagenesis--
The kinase-dead mutation (K127M
HA-SGK) and the phosphorylation site mutations (T256A and S422A HA-SGK)
of HA-tagged hSGK were made using the QuikChange site-directed
mutagenesis kit (Stratagene) per the manufacturer's instructions.
Primers were synthesized to alter either the hSGK ATP-binding site,
lysine 127, to methionine or the putative phosphorylation sites
threonine 256 or serine 422 of hSGK to alanine. Primers (Life
Technologies, Inc.) consisted of the following sequences: K127M HA-SGK,
5'-TTCTATGCAGTCATGGTTTTAAAGAAGAAAGCAATC-3'; T256A
HA-SGK, 5'-CACAACAGCACAACATCCGCATTCTGTGGCACGCCGGAG-3';
S422A HA-SGK, 5'-GCCGAGGCTTTCCTGGGCTTTGCCTATGCGCCTCCC-3'
(the mutated codons are underlined). The template DNA used was
HA-tagged pLPCX-hSGK. Mutations were confirmed using primers provided
with the pLPCX vector by bidirectional sequencing using an ABI776
sequencer (PerkinElmer Life Sciences). Mutant pLPCX-SGK vectors were
then used to generate retroviruses and infect MECs as described above.
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RESULTS |
Glucocorticoids Inhibit Apoptosis of Human Breast Cancer Cells via
Activation of the GR--
We previously demonstrated that GR
activation initiates a potent antiapoptotic signal in the
nontumorigenic MEC line MCF10A and its derivative line MCF10A-Myc. In
the current study, we wished to determine whether GR activation might
also inhibit apoptosis in breast cancer cell lines. We therefore
selected eight commonly studied breast cancer cell lines (BT-20,
HCC1937, Hs578T, MCF7, MDA-MB-231, MDA-MB-468, SK-BR-3, and T-47D) and
evaluated them for GR expression. Western blot analysis of whole cell
extracts using affinity-purified anti-human GR antibodies revealed that all eight cell lines expressed the GR protein (Fig.
1A), although as previously
reported, the T-47D line had relatively little GR expression
(13-15).

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Fig. 1.
Glucocorticoid protects MCF10A-Myc, MCF10A,
and a subset of breast tumor cell lines from apoptosis.
A, Western analysis of GR expression in MCF10A cells and a
panel of breast cancer cell lines. B, percentage of
apoptotic MCF10A-Myc, MCF10A, and breast cancer cells after 72 h
of growth factor deprivation in the presence or absence of
dexamethasone (10 6 M) (*,
p < 0.05, one-sided t test, indicating
significantly less apoptosis in the presence of dexamethasone
(10 6 M)). Error bars indicate S.E.
of the mean (S.E.).
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To examine whether glucocorticoid treatment can mediate a survival
pathway in these tumor cell lines, apoptosis was measured after 72 h in the absence of all growth factors (including glucocorticoid) and
in the presence of a physiological concentration
(10
6 M) of the synthetic
glucocorticoid dexamethasone. In the absence of all growth factors, the
average percentage of apoptosis in breast cancer cell lines varied from
10.6% in MDA-MB-231 cells to only 0.6% in MCF7 cells (Fig.
1B). In four of the eight tumor cell lines tested,
MDA-MB-231, BT-20, Hs578T, and MDA-MB-468, treatment with dexamethasone
significantly inhibited apoptosis when compared with complete growth
factor deprivation (p < 0.05).
To determine whether glucocorticoid-mediated survival was functioning
specifically through activation of the GR, MCF10A-Myc and MDA-MB-231
cells were treated concomitantly with dexamethasone and one of three
known antagonists of GR activation and its subsequent transcriptional
activity: RU486 (5 × 10
7 M)
(16), dexamethasone oxetanone (10
5
M) (17), or dexamethasone 21-mesylate
(10
7 M) (18) (Fig.
2A). All three GR antagonists
reversed the antiapoptotic effect of glucocorticoids significantly
(p < 0.05), although to varying degrees depending on
the cell line examined (Fig. 2B). In MCF10A-Myc cells, RU486
co-treatment was the most potent antagonist of glucocorticoid-mediated
survival, resulting in a 4.5-fold increase in apoptosis. In MDA-MB-231
cells, however, DM was the most potent antagonist of survival signaling
by glucocorticoid, resulting in a similar 4.5-fold increase in
apoptosis at 72 h compared with cells protected by glucocorticoid
alone (Fig. 2B). As expected for a receptor-mediated
mechanism of glucocorticoid activation, the effect of all three GR
antagonists could be out-competed in MCF10A-Myc cells (Fig.
2C) and MDA-MB-231 cells (data not shown) with increasing
concentrations of dexamethasone. Taken together, these results suggest
that glucocorticoid-mediated survival in breast cancer cell lines is
mediated specifically through the activation of the GR.

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Fig. 2.
Glucocorticoid-mediated protection from
apoptosis in MECs is abrogated after antagonism of the GR.
A, chemical structures of dexamethasone and the GR
antagonists RU486, dexamethasone 21-mesylate, and dexamethasone
oxetanone. B, percentage of apoptotic MCF10A-Myc and
MDA-MB-231 cells after treatment for 72 h with dexamethasone
(Dex, 10 6 M) and each
one of three GR antagonists (**, p < 0.05 for
MCF10A-Myc; *, p < 0.05 for MDA-MB-231 cells,
indicating significantly more apoptosis with concomitant RU486
(10 7 M), dexamethasone oxetanone
(DexOx, 10 5 M), and DM
(10 7 M) treatment compared with
dexamethasone alone). C, percentage of apoptotic MCF10A-Myc
cells in the presence of GR antagonists and increasing concentrations
of dexamethasone. All error bars indicate S.E.
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Inhibition of Apoptosis after GR Activation Correlates with
Transcriptional Induction of the Immediate Early Response Gene,
sgk--
We next sought to define the survival mechanism downstream of
GR activation. Using a commercial human cancer array blot spotted with
1200 genes (CLONTECH), we compared gene expression
represented by reverse transcription of mRNA from MCF10A cells
treated for 30 min with either vehicle alone (ETOH), dexamethasone
(10
6 M), or both dexamethasone
(10
6 M) and RU486
(10
7 M). We identified several
genes that were induced or repressed at least 2-fold by 30 min of
glucocorticoid treatment but whose expression was not changed after
concomitant treatment with the GR antagonist RU486 (data not shown).
One of the most intriguing GR-induced genes identified from this
differential display of MEC transcripts was sgk, encoding a
putative serine/threonine kinase previously shown to be 54% homologous
in its catalytic domain to the well characterized antiapoptotic
serine/threonine kinase AKT (5). Although sgk had been shown
to be transcriptionally induced by glucocorticoid treatment in rat
mammary carcinoma cells (5), another study found that sgk
transcripts were not induced by glucocorticoid treatment in human
kidney epithelial cells (9). This raised the possibility of a
species-specific difference in sgk promoter activation by
glucocorticoids. Therefore, we first confirmed our array data using
Northern analysis of sgk mRNA transcripts expressed 30 min after glucocorticoid treatment in two glucocorticoid-sensitive human breast cell lines, MCF10A-Myc and MDA-MB-231. Northern blot analysis of total cellular mRNA using an
-32P-labeled full-length human sgk cDNA
probe demonstrated that in MCF10A-Myc cells, GR activation
significantly induced sgk mRNA (Fig.
3A). The GR antagonist RU486
inhibited sgk mRNA induction, whereas gapdh
transcript expression remained relatively constant under all
conditions. MDA-MB-231 cells had a similar, although slightly less
robust, induction of sgk transcripts after glucocorticoid treatment that was also abrogated by concomitant RU486 treatment (Fig.
3B). In both cell types, serum stimulation induced
sgk transcripts. These data suggest that glucocorticoid
stimulation of human MECs appears to directly induce sgk
expression through a GR-dependent mechanism that can be
antagonized by RU486.

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Fig. 3.
Sgk transcripts are immediately induced in
MECs after GR activation. Representative Northern analysis of
sgk mRNA induction in MCF10A-Myc cells (A)
and MDA-MB-231 cells (B) after a 30-min treatment with
dexamethasone (10 6 M),
dexamethasone (10 6 M)/RU486
(10 7 M), or 20% FCS. The average
ratios of sgk mRNA expression to gapdh
mRNA expression are shown as a bar graph below the
Northern analysis, presented as the fold induction over base-line
sgk/gapdh levels. All error bars indicate
S.E.
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Ectopic Overexpression of Wild Type sgk Inhibits Apoptosis--
We
next investigated the biological activity of ectopically expressed SGK
in MCF10A-Myc cells subjected to apoptotic stress. SGK was ectopically
expressed in MCF10A-Myc cells after transduction with retroviruses
encoding either an HA-tagged wild type human SGK (HA-SGK) or a
truncated HA-tagged SGK containing amino acids 61-431 (
N60 HA-SGK).
The truncated
N60 SGK protein had been previously shown to be more
efficiently expressed than wild type SGK in human embryonic kidney 293 cells (6). Individual clones expressing either wild type HA-SGK or
N60 HA-SGK were isolated by puromycin selection, and whole cell
lysates were examined for HA-SGK protein expression by Western analysis
using a monoclonal anti-HA antibody. Similar to previously reported
results in human embryonic kidney 293 cells,
N60 HA-SGK was much
more efficiently expressed than the full-length, wild type HA-SGK,
although both were clearly visible in comparison to cells transduced
with the empty retroviral pLPCX vector alone (Fig.
4A). To determine whether wild
type and
N60 HA-SGK have kinase activity when overexpressed, MCF10A-Myc cells stably expressing these two constructs were deprived of all growth factors overnight, and SGK activity was measured. Fig.
4B presents the kinase activity of SGK immunoprecipitated from wild type HA-SGK,
N60 HA-SGK, and a kinase-dead K127M HA-SGK expressed in MCF10A-Myc cells. Since an efficient substrate for SGK
kinase activity is not known, we used the previously described SGK-tide
as a substrate (12). Elevated kinase activity was seen in both wild
type and
N60 HA-SGK immunoprecipitated from MCF10A-Myc cells when
compared with K127M HA-SGK. Immunoprecipitated wild type and
N60
HA-SGK both appeared as a doublet in Western analysis, suggesting that
phosphorylated forms of SGK exist in MCF10A-Myc cells despite the
absence of growth factors in the culture medium.

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Fig. 4.
Ectopic expression of SGK inhibits cell death
after growth factor withdrawal. A, anti-HA Western
analysis confirming ectopic HA-SGK expression of both wild type
(WT) and N60 HA-SGK clones in MCF10A-Myc cells.
B, relative kinase activity using SGK-tide as a substrate
(12) of both wild type (WT) HA-SGK and N60-HA-SGK
versus K127M (kinase-dead) HA-SGK. Anti-HA
immunoprecipitation followed by Western analysis, confirming ectopic
HA-SGK expression in MCF10A-Myc cells, is shown below the bar
graph. C, percentage of apoptosis after 72 h of
growth factor deprivation in either the absence of all growth factors
or in the presence of insulin and EGF but no glucocorticoid in wild
type (WT) HA-SGK-expressing and N60-HA-SGK-expressing MCF10A-Myc
clones compared with MCF10A-Myc cells expressing only the empty pLPCX
vector. All error bars indicate S.E.
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SGK-overexpressing MCF10A-Myc cell lines were then evaluated for
apoptosis after various conditions of growth factor deprivation. In the
absence of all growth factors (Fig. 4C), both wild type and
N60 HA-SGK-expressing cell lines showed an average of ~60% inhibition of apoptosis compared with control cells expressing only the
empty pLPCX vector. These results suggest that even in the absence of
growth factors, expression of either the wild type or truncated
N60
HA-SGK can protect MECs from apoptosis. Interestingly, the addition of
insulin and EGF (shown previously in MCF10A cells to stimulate the PI
3-kinase pathway sufficiently to induce AKT phosphorylation (3)) did
not significantly increase survival in cells expressing ectopic SGK.
The antiapoptotic activity of SGK in the absence of EGF or insulin
stimulation was somewhat surprising because SGK activation has been
shown previously to be dependent on the insulin-stimulated PI
3-kinase-signaling pathway (12).
To determine whether the survival effect observed after overexpression
of wild type SGK in fact requires SGK kinase activity, we examined the
antiapoptotic activity of two kinase-dead mutant SGK proteins, K127M
HA-SGK and T256A/S422A HA-SGK, a double mutation of the conserved
second messenger phosphorylation residues in SGK (12). These constructs
were used to make stable MCF10A-Myc cell lines (Fig.
5A). Early-passage kinase-dead
mutant HA-SGK cell lines were then evaluated in apoptosis assays. In
contrast to wild type, neither kinase-dead HA-SGK provided significant protection from apoptosis under conditions of serum withdrawal (Fig. 5B). These results suggest that SGK kinase activity is
in fact essential for its antiapoptotic function.

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Fig. 5.
Kinase-dead mutants of wild-type SGK are
defective in protecting from apoptosis. A, anti-HA
Western analysis confirming ectopic mutant HA-SGK expression in
MCF10A-Myc cells. B, percentage of apoptosis in MCF10A-Myc
cells expressing a kinase-dead HA-SGK (either the ATP binding mutant
K127M HA-SGK or the double phosphorylation site mutant T256A/S422A
HA-SGK) in comparison to cells expressing wild type HA-SGK or the empty
pLPCX vector alone. Cells were cultured either without all growth
factors or in the presence of insulin and EGF but no glucocorticoid.
C, fold increase in apoptosis (+/ dexamethasone
(Dex)) of two MCF10A-Myc K127M HA-SGK clones in comparison
to the parental cell line. All error bars indicate
S.E.
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We next asked whether kinase-dead SGK could act as a functional
dominant negative construct and inhibit survival in MECs treated with
glucocorticoid. Cell lines overexpressing K127M HA-SGK were deprived of
all growth factors or treated with dexamethasone
(10
6 M) alone for 72 h. In
K127M HA-SGK-expressing cell lines treated with glucocorticoid, a small
but consistent increase in apoptosis was seen when compared with the
parental cell line (Fig. 5C). These results suggest that the
survival effect seen after glucocorticoid induction of SGK can be
partially abrogated by the presence of kinase-dead SGK, implying a
possible dominant negative function for kinase-dead SGK.
The PI 3-Kinase Pathway Is Required for SGK Survival
Function--
The absence of a requirement for either insulin or EGF
stimulation in conjunction with wild type SGK overexpression suggested that in our system either (a) an alternative pathway to PI
3-kinase signaling was upstream of SGK activation or (b)
endogenous PI 3-kinase activity was sufficient to activate SGK. To
determine whether PI 3-kinase activity was in fact required for SGK
antiapoptotic function, we treated SGK-overexpressing cells with
LY294002 (50 µM), a PI 3-kinase-specific inhibitor.
Pretreatment of MEC lines with LY294002 resulted in a statistically
significant increase in the percentage of apoptosis in both wild type
HA-SGK and
N60 HA-SGK-expressing cells but not in control (pLPCX
alone) cells (Fig. 6). These results
suggest that PI 3-kinase signaling is indeed required for SGK survival
activity and indicate that endogenous PI 3-kinase activity in MECs
appears to be sufficient to activate SGK, even in the absence of
insulin and EGF stimulation.

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Fig. 6.
PI 3-kinase inhibition reverses the
protective effect of ectopically expressed SGK. Percentage of
apoptosis after 72 h of growth factor deprivation in MCF10A-Myc
cells expressing either wild type (WT) HA-SGK,
N60-HA-SGK, or the empty pLPCX vector with or without a 30-min
pretreatment with the specific PI 3-kinase inhibitor LY294002 (50 µM; *, p < 0.05, indicating
significantly more death with LY294002 treatment compared with 0.01%
Me2SO/PBS vehicle alone; N.S., not significant).
All error bars indicate S.E.
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DISCUSSION |
We have identified a novel pathway of mammary cell survival that
operates via GR activation in both nontumorigenic MECs and breast
cancer cell lines. The relationship between GR activation and several
induced and repressed genes was evaluated by array analysis. One of the
GR-induced genes, sgk, encodes a known serum and
glucocorticoid-regulated kinase with strong homology in its catalytic
domain to the catalytic domain of the antiapoptotic kinase AKT (5). In
a panel of MEC cell lines, MCF10A-Myc cells and MDA-MB-231 cells were
found to be most sensitive to growth factor withdrawal-induced
apoptosis and also demonstrated the lowest endogenous levels of SGK
expression (data not shown). Glucocorticoid treatment of these cell
lines significantly induced sgk mRNA and resulted in
survival. Furthermore, ectopically expressed SGK blocked cell death
after growth factor withdrawal, but overexpression of a kinase-dead SGK
did not protect cells from apoptosis. The induction of sgk
mRNA resulting from glucocorticoid treatment suggests an important
transcriptional control mechanism for the activity of this protein.
This finding provides a novel model of kinase activation that appears
partially independent of cell surface growth factor receptor signaling.
However, the downstream targets of SGK kinase activity remain to be identified.
Interestingly, although SGK activity in human embryonic kidney 293 cells has been shown to be regulated by reversible PI
3-kinase-dependent phosphorylation (6, 12), our results
suggest that endogenous PI 3-kinase activity may be sufficient to
activate SGK. One possibility is that endogenous
phosphoinositide-dependent kinase 1 activity, a downstream
target of PI 3-kinase, is particularly high in MECs. Alternatively, a
parallel phosphoinositide-dependent kinase 1-like kinase may be
responsible for SGK activation in these cells. A third possible
explanation is that human MECs may express mutated or low activity
phosphatases that would otherwise reverse endogenous PI 3-kinase activity.
The mechanism through which SGK prevents apoptosis remains unknown.
However, in kidney epithelium, SGK has been demonstrated to be a target
of aldosterone-induced regulation of electrogenic sodium absorption (7,
19, 20), implicating SGK in the control of intracellular fluid volume.
Since cell shrinkage is known to be an early hallmark of apoptosis
(21), SGK expression may counteract the early changes in cell volume
that precede apoptosis by controlling intracellular fluid shifts. The
possibility that SGK may exert its antiapoptotic effects by regulating
cation channels and maintaining intracellular volume is the subject of
future investigation.
In summary, we have identified GR activation as initiating a potent
survival signal in both nonmalignant and malignant human breast
epithelial cells. SGK, a novel member of the second-messenger family of
serine/threonine protein kinases, has been identified as a probable
downstream effector of GR survival signaling. Regulation of SGK's
antiapoptotic activity in breast epithelial cells was found to be
largely independent of insulin or EGF stimulation, although PI 3-kinase
activity is required. Furthermore, transcriptional control of SGK
expression appears to be the dominant mechanism of induction of SGK
activity, suggesting a novel interaction between steroid hormone
receptor activation and serine/threonine kinase-mediated survival.