From the School of Biology, Georgia Institute of
Technology, Atlanta, Georgia 30332-0230 and the ¶ Department of
Biochemistry and Center in Toxicology, Vanderbilt University School of
Medicine, Nashville, Tennessee 37232-0146
Received for publication, October 7, 2002, and in revised form, December 11, 2002
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ABSTRACT |
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Steroid hormone biosynthesis in the adrenal
cortex is controlled by adrenocorticotropin (ACTH), which increases
intracellular cAMP, resulting in the activation of
cAMP-dependent protein kinase(PKA) and
subsequent increase in steroidogenic gene transcription. We have found
that a dual-specificity phosphatase is essential for conveying
ACTH/cAMP-stimulated transcription of several steroidogenic genes in
the human adrenal cortex. In the present study, the role of
mitogen-activated protein kinase phosphatase-1 (MKP-1), a nuclear dual-specificity phosphatase, in the transcriptional activation of
human CYP17 (hCYP17) in H295R human adrenocortical cells is established. Stimulation of H295R cells with dibutyryl-cAMP
(Bt2cAMP) induces MKP-1 mRNA and protein expression
within 30 min of exposure. In transient-transfection studies,
transcriptional activity of an hCYP17 promoter-reporter construct was
increased by Bt2cAMP and by overexpression of PKA or MKP-1.
Furthermore, PKA phosphorylated an MKP-1-glutathione
S-transferase fusion protein in in vitro assays and Bt2cAMP increased 32P associated
with MKP-1 that was immunoprecipitated from H295R cells. Finally,
silencing MKP-1 expression using antisense oligonucleotides attenuated
cAMP-stimulated hCYP17 expression, whereas silencing of ERK1/2
increased hCYP17 expression. These findings demonstrate integral roles
for MKP-1 and ERK1/2 via regulation of the phosphorylation state of
steroidogenic factor-1 (SF-1) in mediating
ACTH/cAMP-dependent transcription of hCYP17, thereby
maintaining the balance between transcriptional activation and repression.
The role of ACTH1 is to
assure that optimal steroidogenic capacity is maintained in the adrenal
cortex. This is achieved, via the actions of cAMP, by maintaining
transcriptional pressure on the genes encoding the steroid hydroxylases
(CYPs) and 3 MAPKs are regulated through reversible phosphorylation, where
activation is catalyzed by their cognate dual-specificity MAPK kinases
(e.g. MEK), and inactivation is primarily achieved by a
group of MKPs (12). Ten MKPs have been identified (13-17), one of
which (MKP-1) is induced in pheochromocytoma PC12 cells by agents that
increase intracellular cAMP (18).
Based on our previous studies suggesting a role for the
dephosphorylation of SF-1 in ACTH/cAMP-mediated steroidogenic gene expression (10, 11), the findings of other laboratories implicating a
role for protein phosphatase activity in steroid hormone biosynthesis (19-22) and other studies suggesting cross-talk between the MAPK and
ACTH/cAMP pathways (23, 24), we hypothesized that a DSP, via
dephosphorylation of SF-1, played an integral role in
cAMP-dependent hCYP17 gene expression. Thus, we
characterized the effect of cAMP stimulation on MKP-1 expression and
the role of MKP-1 in activating hCYP17 gene expression. We show that
MKP-1 mRNA and protein levels are induced by Bt2cAMP
and that overexpression of MKP-1 stimulates hCYP17 reporter gene
activity. Moreover, we demonstrate that PKA phosphorylates MKP-1.
Finally, using antisense technology to suppress MKP-1 and ERK1/2
expression, we show that MKP-1 is essential for ACTH/cAMP-dependent hCYP17 expression, whereas silencing
ERK1/2 expression increased basal hCYP17 mRNA levels, suggesting an
essential role for ERK1/2 in maintaining low constitutive hCYP17
expression. Our studies demonstrate a central role for the MKP-1/ERK1/2
system in conveying ACTH/cAMP-dependent transcriptional
activation of steroidogenic gene expression via
dephosphorylation/phosphorylation of SF-1.
Reagents--
Bt2cAMP, cycloheximide (CHX), and
hydrogen peroxide were obtained from Sigma. Okadaic acid (OA), PD98059,
endothall (ETL), bpv(phen), dephostatin (DPN), cyclosporine A
(CyA), and sodium orthovanadate were obtained from Calbiochem (San
Diego, CA). Peroxyvanadate (PV) was prepared by mixing equal
concentrations (12 mM) of H2O2 with
sodium orthovanadate prior to treatment. OA and ETL are inhibitors of
the serine/threonine phosphatases PP1 and PP2A, CyA inhibits serine/threonine phosphatase PP2B (calcineurin) and PV, bpv(phen) and
DPN inhibit phosphotyrosine phosphatase activity. PD98059 inhibits MEK activity.
Cell Culture--
H295R adrenocortical cells (25, 26) were
generously donated by Dr. William E. Rainey (University of Texas
Southwestern Medical Center, Dallas, TX) and cultured in Dulbecco's
modified Eagle's/F12 medium (Invitrogen, Carlsbad, CA) supplemented
with 10% Nu-Serum I (BD Biosciences, Palo Alto, CA), 0.5% ITS Plus (BD Biosciences, and antibiotics.
Transient Transfection and Reporter Gene Analysis--
Cells
were subcultured onto 12-well plates and 24 h later transfected
with a hCYP17 57-pGL3 reporter plasmid (27). The hCYP17 57-pGL3
construct was generated by ligating double-stranded oligonucleotides corresponding to the region RNA Antisense--
Phosphorothioate-modified oligonucleotides
(28-32) were obtained from Qiagen/Operon (Alameda, CA). H295R cells
were transfected with MKP-1 antisense (G*G*A*ACTCAG
TGGAACTC*A*G*G) or scrambled negative control (A*G*G*TCCTGA
AAGCGAAG*T*C*G) oligonucleotides, as previously described (33). For ERK
gene silencing, cells were transfected with ERK antisense
(A*T*G*GCGGCGGCGGCGGCG*G*C*T) or ERK negative-control
(G*C*A*CAGCCGCCTGCCGCC*G*C*C) oligonucleotides (34). The asterisks
denote locations of phosphorothioate modifications. MKP-1
oligonucleotides were added to the cells at concentrations of 0.3, 1, or 3 µM, and ERK1/2 oligonucleotides were added to cells
at 1 µM using the Effectene transfection reagent
(Qiagen). After 6 h, the medium was removed and cells were treated
for 1 h (for maximal MKP-1 induction) or 12 h (maximal hCYP17
induction) with 1 mM Bt2cAMP.
RNA Isolation, Northern Blotting, and Hybridization
Conditions--
Cells were harvested and total RNA was prepared from
treated cells by acid-phenol extraction (TRIzol, Invitrogen). RNA
concentrations were determined by absorbance at 260 nm. For Northern
blot assays (35), RNA (10 µg) was fractionated by agarose (1%) gel
electrophoresis in the presence of 5% formaldehyde and transferred to
nylon transfer membrane filters (HybondTM-N+, Amersham
Biosciences). A 500-bp cDNA fragment obtained by digestion
of MKP-1pCMV5 with PstI was used to detect MKP-1 mRNA expression. A 1.2-kb hCYP17 cDNA fragment was used to detect hCYP17 mRNA expression. Results were normalized to the content of
glyceraldehyde-3-phosphate dehydrogenase (GAP) mRNA. cDNA
probes were labeled with [ Western Blotting--
H295R nuclear extracts (10 µg
protein/lane) were harvested from cells treated with 1 mM
Bt2cAMP for periods ranging from 30 min to 4 h,
subjected to SDS-PAGE (10%), and transferred onto a polyvinylidene
difluoride membrane (Immobilon-P, Millipore, Bedford, MA). Nuclear
extracts were prepared according to the method of Dignam et
al. (36). Immunoblotting was carried out using anti-MKP-1 (1:5000
dilution) or anti-ERK2 (1:10,000 dilution) obtained from Santa Cruz
Biotechnology. Protein expression was detected by the ECL+ Western blot
detection kit (Amersham Biosciences) using a protein
G-peroxidase-conjugated protein (Calbiochem, San Diego, CA).
Metabolic Labeling and Immunoprecipitation--
For in
vivo labeling, H295R cells were cultured in 100-mm dishes. The
media was changed to either phosphate-free Dulbecco's modified
Eagle's media containing 0.5 mCi/ml of
[32P]orthophosphate (PerkinElmer Life Sciences) or
cysteine- and methione-free media containing 0.5 mCi/ml
[35S]methionine/[35S]cysteine (RedivueTM
in vitro cell labeling mix; Amersham Biosciences). Cells
were metabolically labeled for 4 h followed by stimulation with 1 mM Bt2cAMP for 2 h prior to harvest.
Radiolabeled cells were harvested, and nuclear extracts isolated as
described above.
For immunoprecipitation assays, nuclear proteins were incubated with
anti-MKP-1 antiserum and protein A/G agarose beads (Santa Cruz
Biotechnology) overnight at 4 °C with rotation. The mixture was then
centrifuged, and the supernatant was removed. Beads were washed three
times with RIPA buffer (150 mM NaCl, 50 mM
Tris-Cl, pH 8.0, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS, 0.2 mM phenylmethylsulfonyl
fluoride, 10 µg/ml aprotinin, 10 µg/ml pepstatin, and 10 µg/ml
leupeptin), twice with PBS, and resuspended in SDS-PAGE loading buffer
for SDS-PAGE (10% gel). Gels were stained with Coomassie blue, dried,
and subjected to audioradiography to detect 32P- or
35S-labeled proteins.
GST Fusion Protein Preparation and in Vitro
Phosphorylation--
MKP-1 was PCR-amplified using a 5' primer
containing a BamH1 site linked to the first codon of MKP-1
and a 3' primer containing a HindIII site. The
BamH1-HindIII digest of the PCR products were cloned into BamH1-HindIII-digested pET42b(+)
(Novagen, Milwaukee, WI). For preparation of GST fusion proteins in
Eschirichiacoli, 5 ml of overnight cultures were diluted 100 times, induced with 1 mM
isopropyl-
For in vitro phosphorylation assays, 40 µl of a 50%
slurry of MKP-1/GST-glutathione-agarose beads were incubated for 30 min at 30 °C in a reaction buffer containing 20 mM Tris-Cl,
pH 7.5, 50 mM KCl, 10 mM MgCl2, 2 mM dithiothreitol, 10 µCi Statistical Analysis--
Data from transfection assays were
expressed as the percentage of the mean of the control group in each
experiment. One-way analysis of variance and the Newman-Keuls test were
used to determine differences among treatment groups. Asterisks denote
significant difference from control, p < 0.05.
cAMP Induces MKP-1 mRNA and Protein Expression--
Agents
that increase intracellular cAMP have been found to induce the mRNA
expression of MKP-1 in PC12 cells (18). Moreover, we have previously
shown that both serine/threonine and tyrosine phosphatase activities
are required for cAMP-dependent gene expression of
steroidogenic genes in the human adrenal cortex (10, 11). Thus, we
first examined whether the DSP MKP-1 was expressed in H295R cells and
was responsive to Bt2cAMP. Cells were treated with 1 mM Bt2cAMP for 30 min to 4 h and harvested
for analysis of MKP-1 mRNA expression. As shown in Fig.
1A, MKP-1 mRNA expression was significantly induced by cAMP within 30 min. This induction was
maximal after 1 h and decreased by 4 h (Fig. 1A).
Furthermore, unlike the cAMP-dependent mRNA expression
of steroidogenic genes, which is CHX-sensitive (3, 26, 37, 38), the
induction of MKP-1 mRNA by cAMP was not attenuated by CHX (Fig.
1B). MKP-1 protein expression levels were also measured over
the same time course (Fig. 1C). An increase in MKP-1 protein
expression was observed within 30 min, was maximal after 2 h, and
began to decline after 4 h of exposure to Bt2cAMP
(Fig. 1C).
MKP-1 Overexpression Activates hCYP Reporter Gene Activity--
To
determine the effect of MKP-1 on the transcriptional activity of
hCYP17, we performed transient-transfection assays. H295R cells were
transfected with hCYP17 57-pGL3, PKA-pCMV, and MKP-1-pCMV. As shown in
Fig. 2 administration of 1 mM
Bt2cAMP significantly increased transcriptional activity of
hCYP17-pGL3. Co-expression of either PKA or MKP-1 also significantly
increased transcriptional activity of the hCYP17 57-bp construct (Fig.
2), indicating that MKP-1 mimics cAMP/PKA-stimulated transcription of
hCYP17 reporter gene expression.
cAMP/PKA-dependent Phosphorylation of
MKP-1--
Once we established that cAMP induces MKP-1 mRNA and
protein levels, we next wanted to determine whether PKA can directly phosphorylate MKP-1 in vitro. An MKP-1-GST fusion protein
was prepared and incubated with the catalytic subunit of PKA and
[ SF-1 Transactivation Ability Depends on Phosphatase
Activity--
SF-1 is essential for increased steroidogenic gene
expression. Previous studies have shown that both basal and
cAMP-dependent hCYP17 transcriptional activity is dependent
on complex formation of SF-1, p54nrb, and PSF (27).
Moreover, SF-1 was found to be dephosphorylated in response to cAMP
stimulation (10). To further characterize the role of phosphatase
activity in SF-1-mediated steroidogenic gene transcription, H295R cells
were transfected with the hCYP17-pGL3 reporter construct and a SF-1
expression plasmid. Cells were treated for 12 h with OA, ETL, PV,
bpv(phen), DPN, or CyA and harvested for luciferase activity assays. As
shown in Fig. 4, SF-1 significantly increased the transcriptional activity of the hCYP17 57-bp construct. Administration of both serine-threonine (OA, ETL) and phosphotyrosine (PV, bpv(phen), DPN) phosphatase inhibitors completely attenuated SF-1-mediated transcriptional activity. OA and ETL significantly reduced SF-1-mediated transcriptional activity of the hCYP17 57-pGL3 construct below basal levels. CyA, a PP2B
(calcineurin) inhibitor, had no significant effect on
SF-1-mediated luciferase activity.
Effect of MKP-1 Gene Silencing on hCYP17 Gene Expression--
Thus
far, the data presented in this paper demonstrate that the synthesis of
MKP-1 is stimulated by cAMP in H295R cells and is an in
vitro target for PKA. However, these findings provide only
indirect evidence for its role in hCYP17 transcription. To determine
whether MKP-1 is the phosphatase essential for
ACTH/cAMP-dependent hCYP17 expression in vivo,
we used antisense oligonucleotides to block MKP-1 expression in H295R
cells. The oligonucleotides were made as phosphorothioate derivatives,
which have been shown to enhance nuclease resistance and support RNase
H cleavage of hybridizing RNA (31, 32). Sequences used have been
previously described (33). H295R cells were incubated for 6 h with
oligonucleotides at concentrations ranging from 0.3 to 3 µM in serum-free media containing the nonliposomal lipid
transfection Effectene. After oligonucleotide uptake, cells were
stimulated for 1 h or 12 h with Bt2cAMP, and
Northern blot analysis was performed to measure the steady-state levels
of endogenous MKP-1 and hCYP17 mRNA. As shown in Fig.
5A, 1 µM MKP-1
antisense oligonucleotide completely abolished cAMP-stimulated MKP-1
mRNA expression. Maximal induction of MKP-1 mRNA and protein
expression occurred after 1 h and 2 h of cAMP stimulation,
respectively (Fig. 1, A and C). To determine whether this immediate rise in MKP-1 expression is consistent with the
time course of cAMP-stimulated hCYP17 mRNA expression, the effect
of Bt2cAMP on hCYP17 mRNA expression over time was measured. As shown in Fig. 5B, cAMP increases hCYP17
mRNA expression within 4 h, and this increase is maximal after
12 h. Taken together with the time course of MKP-1 induction by
cAMP (Fig. 1, A and C), these data suggest that
MKP-1 plays a direct role in cAMP-dependent hCYP17
expression. However, these findings do not rule out the possibility
that another protein(s) may also play intermediary roles in
cAMP-stimulated hCYP17 expression. MKP-1 antisense oligonucleotides were also used to determine the effect of MKP-1 suppression on cAMP-stimulated hCYP17 mRNA expression. H295R cells were
transfected with the oligoncleotides and stimulated with 1 mM Bt2cAMP for 12 h. The 12 h time
point was chosen because it is the point at which cAMP maximally
increases hCYP17 mRNA levels (Fig. 5B). When the
transfected cells were incubated with Bt2cAMP for 12 h, cAMP failed to increase hCYP17 mRNA (Fig. 5, C and
D). In contrast, the scrambled negative control
oligonucleotide had no effect on cAMP-stimulated MKP-1 and hCYP17
mRNA expression (Fig. 5, A and C).
Effect of ERK1/2 Gene Suppression on hCYP17
Expression--
We have previously found that inhibiting ERK1/2
activation using the MEK inhibitor PD98059 induces hCYP17 mRNA
expression (10). Based on these findings, we postulated that activation of ERK1/2 results in constitutive phosphorylation of SF-1 and subsequent decreased hCYP17 expression. To determine whether ERK1/2 plays a role in maintaining low basal hCYP17 levels, we used antisense oligonucleotides to suppress ERK1/2 RNA expression. H295R cells were
transfected with either antisense oligonucleotides or a scrambled negative control oligonucleotide for 24 h and harvested for RNA analysis by Northern blotting. As shown in Fig.
6A, suppression of ERK1/2
increases basal hCYP17 mRNA expression, with no significant effect
with the ERK1/2 scrambled negative control oligonucleotide. Western
blotting for ERK2 confirmed that the antisense oligonucleotides effectively targeted and decreased ERK1/2 in the H295R cells (Fig. 6B).
ACTH exerts its stimulatory action on steroid hormone biosynthesis
by evoking both a rapid, acute response and a long-term, chronic
response, each regulated by PKA. During the acute response, an
essential site of phosphorylation by PKA is cholesterol ester hydrolase, activating the conversion of cholesterol esters to free
cholesterol (39). In our studies of the mechanism of chronic ACTH-dependent steroiodogenesis in the human adrenal cortex
(H295R cells), we have demonstrated that cAMP-inducible binding of a SF-1, p54nrb, polypyrimidine tract-binding protein-associated
splicing factor (PSF) complex to the cAMP-responsive sequence of the
hCYP17 promoter occurs via changes in the phosphorylation state of the
orphan nuclear receptor, SF-1 (27). cAMP stimulation of H295R cells led
to the dephosphorylation of SF-1, as measured by immunoprecipitation of
SF-1 after in vivo labeling with
[32P]orthophosphate (10). Moreover, inhibiting ERK1/2
activation by treatment with PD98059 mimicked the stimulatory effects
of cAMP on endogenous hCYP17 mRNA expression and on hCYP17
promoter-reporter gene activity (10), indicating a role for the MAP
kinase pathway in conveying constitutive transcription of steroidogenic
genes in the adrenal cortex.
In the present study, we have clearly demonstrated that MKP-1 is the
essential phosphatase in regulating hCYP17 in human adrenal cortex.
Cross-talk between the MAPK and ACTH/cAMP pathways has been described
in human adrenal cortex by our laboratory (10, 11) and by others (23,
24). We demonstrate that MKP-1 mRNA and protein levels are rapidly
induced by Bt2cAMP in H295R cells and that overexpression
of MKP-1 stimulates reporter gene activity driven by the hCYP17 CRS.
This increase in MKP-1 expression precedes increases in hCYP17 mRNA
expression, temporally confirming the role of MKP-1 in the
cAMP-dependent transcription of hCYP17. However, it is
possible that another unidentified intermediary protein(s) are involved
in the pathway between PKA and increased hCYP17 gene expression.
As shown in Fig. 3, PKA phosphorylates MKP-1 in vitro and
that in vivo Bt2cAMP treatment enhances
phosphorylation of MKP-1, suggesting that PKA-dependent
activation of MKP-1 stimulates the ability of this protein to
dephosphorylate SF-1. Based on the data presented, we cannot conclude
that the amino acid residue(s) targeted by PKA in vitro are
the same as those phosphorylated after stimulating the cells with
Bt2cAMP. Studies are now underway to compare the
phosphorylation sites of in vitro-phosphorylated MKP-1 and
MKP-1 that has been immunoprecipitated from H295R cells exposed to
Bt2cAMP. If phosphorylation sites are the same, it will
indicate that PKA directly targets MKP-1. Our present finding of the
central role of MKP-1 in hCYP17 gene expression has also warranted
future study on the role of this DSP in the regulation of other
steroidogenic genes.
Recently it was found that phosphorylation of SF-1 at serine 203 enhances its stability and transcriptional activity (40). It was also
shown that the levels of phosphorylated SF-1 (as detected by the use of
a phosphoserine 203 SF-1 antibody) decrease when MKP-1 is overexpressed
in Y1 cells (40), indicating that serine 203 may be a direct target for
MKP-1. These findings showing MKP-1-stimulated decreased
phosphorylation of SF-1 are in agreement with our present data
demonstrating that cAMP induces MKP-1 expression and our previous data
showing that cAMP stimulation results in decreased levels of
phosphorylated SF-1. However, the role that phosphorylated serine 203 SF-1 plays in ACTH/cAMP-dependent transcription of a
steoroidogenic gene is unclear because we have found no effect of
mutating serine 203 to alanine on the transactivation activity of SF-1
in H295R cells.2
Our data (10, 11, 27, herein) leads to a proposed mechanism by which
ACTH regulates hCYP17 transcription in human adrenal cortex that
involves activation of adenylyl cyclase, elevation of cAMP levels, and
subsequent activation of PKA. There are two possible mechanisms by
which PKA may act to stimulate dephosphorylation of SF-1: 1) activation
of MKP-1 and/or 2) inhibition of ERK1/2. Activation of MKP-1 can result
in dephosphorylation of SF-1 directly by MKP-1 and also by MKP-1
inactivating ERK1/2. It is well established that DSPs directly
inactivate MAP kinases (41-44), and, based on the studies presented,
we speculate that SF-1 may be a direct target of MKP-1. In addition to
MKP-1 inactivating ERK1/2, it is possible that PKA may inhibit ERK1/2.
Studies in fibroblasts and vascular smooth muscle cells have
demonstrated that PKA inhibits Raf-1 activity, thus inactivating the
ERK1/2 pathway (24). This inactivation has been mapped to PKA
phosphorylation of Raf-1 kinase on serine 259 (45). Other studies have
found that cAMP stimulates ERK1/2, via B-raf activation in several cell
types, including preadipocytes, neuronal cells, ovarian granulose
cells, and pituitary cells (24). Raf-1 and B-raf are upstream kinases
in the ERK1/2 pathway. Collectively, these studies show that the effect
of PKA on ERK1/2 is cell-type-specific and consequently detailed
examination of the effect of PKA on the activation state of ERK1/2 in
H295R cells is needed.
The coordinated activation/repression of hCYP17 expression is mediated
via dephosphorylation (MKP-1)/phosphorylation (ERK1/2), suggesting that
direct targets of PKA in human adrenal steroidogenesis are MKP-1 and a
member of the ERK1/2 cascade. Elevated ACTH levels shift this
steroidogenic ying-yang mechanism in favor of increased steroid hormone
production and a decline in ACTH levels has the opposite effect. One
feature of the chronic response of steroidogenesis to ACTH/cAMP that we
revealed 20 years ago and has remained unexplained is that the
transcriptional activation of steroidogenic genes by ACTH (cAMP) can be
inhibited by the protein synthesis inhibitor CHX (46). The rapid
increase in activation of the immediate early gene MKP-1, resistance of
this process to CHX, and the rapid translation of this mRNA (Fig.
1) suggest that translation of MKP-1 is the CHX-sensitive event
required for enhancement of hCYP17 transcription. Whether MKP-1 is the
CHX-sensitive factor essential for ACTH/cAMP-dependent
transcription of other steroidogenic genes awaits further study.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-hydroxysteroid dehydrogenase (1, 2). cAMP activates
PKA, ultimately leading to increased gene transcription via the binding
of various transcription factors to cAMP-responsive sequences that lie
within the promoters of steroidogenic genes (1, 3-9). The direct
target of PKA has yet to be determined; however, we have demonstrated
that the cAMP-stimulated gene expression of multiple steroidogenic
genes in the human adrenal cortex is mediated by phosphatase activity (10, 11). Using various selective inhibitors of either serine/threonine or tyrosine phosphatase activities, we found that the mRNA levels of both mitochondrial and microsomal steroidogenic gene products depend
on the activity of a dual-specificity phosphatase (DSP) (10, 11). cAMP
stimulation of H295R cells resulted in the dephosphorylation of SF-1,
as measured by immunoprecipitation of SF-1 after in vivo
labeling with [32P]orthophosphate (10). Moreover,
inhibition of the extracellular signal-regulated kinase (ERK) pathway
mimicked cAMP-dependent transcriptional activation of
hCYP17 expression in H295R cells, indicating a role for a
mitogen-activated protein kinase (MAPK) pathway in regulating
transcription of steroidogenic genes in the adrenal cortex (10). Taken
together, these findings lead us to postulate that the target of PKA is
a DSP, which leads to the dephosphorylation of SF-1.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
57/
2 of the hCYP17 5' flank upstream of
the luciferase gene in the pGL3 vector (Promega, Madison, WI). The
expression vector for SF-1 was generously provided by Dr. K. Morohashi
(National Institute for Basic Biology, Okazaki, Japan) and the
expression plasmid for MKP-1 by Dr. J. Dixon (University of Michigan,
Ann Arbor, MI). Reporter (250 ng) and expression (100 ng) plasmids were
transfected into H295R cells using the Effectene nonliposomal lipid
transfection reagent (Qiagen) for 24 h. Cells were then treated
with Bt2cAMP in the presence or absence of phosphatase
inhibitors for periods ranging from 6 to 12 h. Phosphatase
inhibitors were administered at the following concentrations: 25 nM OA, 100 µM ETL, 25 µM PV, 20 µM DPN, and 500 nM CyA. Cells were harvested
and cellular extracts prepared for luciferase assays (Promega), and
protein concentration was determined (Pierce, Rockford, IL). Data
obtained from luciferase assays (relative luciferase light units) were
normalized to Renilla (pRL, Promega) luciferase activity of
each sample.
-32P]dCTP (PerkinElmer Life
Sciences) by random primer labeling (Prime It II, Stratagene, La Jolla,
CA). Blots were hybridized overnight at 42 °C in a 2× sodium
citrate-sodium chloride (SSC) solution containing 50% formamide, 5×
Denhardt's solution, 2% sodium dodecyl sulfate (SDS), 100 µg/ml
denatured single-stranded salmon sperm DNA, and 32P-labeled
cDNA probes. The hybridized membranes were sequentially washed for
2 × 10 min in 2x SSC, 0.2% SDS, and 2 × 5 min in 0.2× SSC, 0.2% SDS at 42 °C. The amount of probe bound to the filter was
quantitated using a Molecular Dynamics (Sunnyvale, CA) PhosphorImager and ImageQuant software.
-thiogalactopyranoside (IPTG), and grown further for
20 h. Bacteria were pelleted and resuspended in 20 ml of PBS containing 50 mM EDTA (pH 8.0), 1 mM
dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml pepstatin, and 10 µg/ml leupeptin. The
bacteria were incubated with lysozyme (1 mg/ml) for 15 min on ice, and
the cells were disrupted using a sonicator. The suspension was
centrifuged (39,000 × g) for 25 min, and the supernatant was incubated with 500 µl of a 50% slurry of
glutathione-agarose beads (Novagen) for 30 min at 25 °C. The beads
were then washed three times with PBS and suspended in 250 µl of PBS
containing 2 mM dithiothreitol and 50% glycerol and stored
at
20 °C.
[32P]ATP
(PerkinElmer Life Sciences), and 5 units of PKA catalytic subunit (from
bovine heart; Calbiochem). The reactions were stopped by the addition
of EDTA (2 mM final concentration). The beads were then
washed three times with PBS and resuspended in SDS-PAGE gel loading
buffer for SDS-PAGE (10% gel). Gels were stained with Coomassie blue,
dried, and subjected to audioradiography to detect 32P-labeled protein.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
cAMP induces MKP-1 mRNA and
protein expression in H295R cells. A,
Northern blot showing time-dependent increase in MKP-1
mRNA expression after Bt2cAMP treatment. H295R cells
were treated with 1 mM Bt2cAMP for 30 min,
1 h, 2 h, or 4 h and harvested for isolation of total
RNA. Northern blotting and hybridization to cDNAs for MKP-1 and GAP
were performed as described under "Materials and Methods."
B, cells were treated with 1 mM
Bt2cAMP ± 40 µM CHX for 1 h.
Northern blot shows no effect of CHX on Bt2cAMP-stimulated
MKP-1 mRNA expression. C, Western blot showing MKP-1
protein levels in the nuclear extracts isolated from H295R cells
stimulated with Bt2cAMP over time. Cells were treated for
30 min, 1 h, 2 h, or 4 h with 1 mM
Bt2cAMP then harvested for analysis of MKP-1 protein
expression by SDS-PAGE and Western blotting.
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Fig. 2.
Effect of cAMP, PKA, and MKP-1 on
hCYP17 57pGL3 transcriptional activity. Cells were
transfected with the hCYP17 57-pGL3, the catalytic subunit of PKA or
MKP-1 via the Effectene nonliposomal delivery system. Twenty-four hours
after transfection, cells transfected with only hCYP17 57-pGL3 were
stimulated with 1 mM Bt2cAMP. After 12 h,
all cells were harvested for isolation of cellular extracts and
determination of luciferase activity. Data is expressed as -fold
increase in firefly luciferase activity normalized to
Renilla luciferase activity and represents the mean of at
least 3 separate experiments, each performed in triplicate. *,
statistically different from control, p < 0.05.
-32P]ATP. As shown in Fig.
3A, MKP-1 can be directly
phosphorylated by PKA. Moreover, stimulation of metabolically labeled
cells with Bt2cAMP results in increased MKP-1
phosphorylation (Fig. 3B). These findings suggest that MKP-1
is a direct target of PKA in the human adrenal cortex, however, further
studies aimed at mapping the phosphorylation sites of MKP-1 after cAMP
stimulation are necessary to establish for certain that the action of
PKA on MKP-1 is direct.
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Fig. 3.
In vitro and in vivo
phosphorylation of MKP-1. A, MKP-1 was expressed as a GST
fusion protein, purified, and incubated with [ -32P]ATP
and the catalytic subunit of PKA for 30 min at 30 °C. Reaction was
terminated by the addition of SDS-PAGE loading buffer and analyzed by
SDS-PAGE followed by autoradiography. Lane 1, MKP-1-GST;
lane 2, MKP-1-GST + [
-32P]ATP; lane
3, MKP-1-GST + [
-32P]ATP + 1 unit of PKA;
lane 4 MKP-1-GST + [
-32P]ATP + 0.5 unit of
PKA. B, cells were metabolically labeled in vivo
with [32P]orthophosphate or
[35S]methionine/cysteine followed by stimulation with
Bt2cAMP for 2 h. Immunoprecipitation of MKP-1 was
carried out using a MKP-1 antibody and protein A/G-Sepharose beads.
SDS-PAGE showing 32P- and 35S-labeled MKP-1.
, control; +, Bt2cAMP.
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Fig. 4.
Effect of protein phosphatase inhibitors on
SF-1-stimulated hCYP17 57-pGL3 transcriptional
activity. Cells were transfected with 250 ng of hCYP17
57-pGL3 reporter plasmid, 25 ng of Renilla-pRL control
plasmid, and 100 ng of SF-1-pCR3.1 as described under "Materials and
Methods." After 24 h, cells were incubated with 25 nM okadaic acid (OA), 100 µM endothall (ETL),
25 µM PV, 5 µM bpv(phen) (bpV), 20 µM dephostatin (DPN), or 500 nM cyclosporine
A (CyA) for 12 h. Cell lysates were harvested, and luciferase
activity was determined using a dual-luciferase reporter assay system
(Promega). Data represent the mean of at least 3 separate experiments,
each performed in triplicate. *, statistically different from control,
p < 0.05.
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Fig. 5.
MKP-1 is required for enhanced hCYP17
mRNA expression in response to cAMP. A,
cells were transfected with 1 µM antisense
oligonucleotides as previously described and subsequently treated with
1 mM Bt2cAMP for 1 h. Panel A
shows a representative Northern blot of MKP-1 mRNA expression. Two
samples are shown for each treatment group. Treatment groups: control,
Bt2cAMP, Bt2cAMP + 1 µM MKP-1
antisense oligonucleotide, Bt2cAMP + 1 µM
scrambled MKP-1 oligonucleotide. B, time course of
cAMP-dependent hCYP17 mRNA expression. Cells were
treated for 2, 4, 8, 12, 18, 24, 36, or 48 h with 1 mM
Bt2cAMP. Northern blots were probed for hCYP17 and GAP
mRNA expression. The graph represents quantitative analysis of
hCYP17 mRNA expression from three experiments (n = 4 per experiment), generated from PhosphorImager scanning and
normalization to GAP content of each sample. *, statistically different
from control, p < 0.05. C, representative
Northern blot showing hCYP17 (upper panel) and
GAP (lower panel) mRNA expression. Antisense or
scrambled MKP-1 oligonucleotides were administered at a final
concentration of 1 µM. After 6 h, cells were treated
for 12 h with 1 mM Bt2cAMP. Two samples
are shown for each treatment group (control, Bt2cAMP,
Bt2cAMP + 1 µM MKP-1 antisense
oligonucleotide, Bt2cAMP + 1 µM scrambled
MKP-1 oligonucleotide). D, H295R cells were transfected with
0.3, 1, or 3 µM antisense oligonucleotides as described
under "Materials and Methods" using the Effectene transfection
delivery system (Qiagen). After 6 h, the media was changed and
cells were treated for 12 h with 1 mM
Bt2cAMP. RNA was isolated for Northern blot analysis. The
graph represents quantitative analysis of hCYP17 mRNA expression
from four experiments, each performed in triplicate generated from
PhosphorImager scanning and normalization to GAP content of each sample
(n = 12 per dosage group). Untreated control was set at
100% and is not displayed. *, statistically different from control,
p < 0.05.
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Fig. 6.
Suppressing ERK1/2 expression increases
constitutive hCYP17 mRNA levels. Cells were
transfected with 1 µM antisense or scrambled
oligonucleotides as described under "Materials and Methods" using
the Effectene transfection delivery system (Qiagen). After 6 h,
the media was changed and untransfected cells were treated for 12 h with 1 mM Bt2cAMP. RNA (A) or cell
lysates (B) were harvested for Northern and Western
blotting, respectively. A, Northern blot showing hCYP17
mRNA expression in cells transfected with ERK1/2 antisense or
scrambled oligonucleotides. Lane 1, control; lane
2, Bt2cAMP; lane 3, ERK1/2 antisense;
lane 4, ERK1/2 scrambled oligonucleotide. B,
Western blot of ERK2 protein levels in cell lysates isolated from
control (lane 1), Bt2cAMP-treated (lane
2), or cells transfected with ERK1/2 antisense oligonucleotides
(lane 3).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK28350 and ES00267 (to M. R. W.), a UNCF/Merck Science Initiative Postdoctoral Fellowship (to M. B. S.), and National Institutes of Health Postdoctoral Training Grant CA09582 (to M. B. S.).
§ To whom correspondence should be addressed: School of Biology, Georgia Institute of Technology, 310 Ferst Dr., Atlanta, GA 30332-0230. Tel.: 404-385-4211; Fax: 404-894-0519; E-mail: marion.sewer@biology.gatech.edu.
Published, JBC Papers in Press, December 27, 2002, DOI 10.1074/jbc.M210264200
2 M. B. Sewer and M. R. Waterman, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: ACTH, adrenocorticotropin; PKA, cAMP-dependent protein kinase; MKP-1, mitogen-activated protein kinase phosphatase-1; Bt2cAMP, dibutyryl-cAMP; hCYP17, human CYP17; DSP, dual-specificity phosphatase; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; CHX, cycloheximide; OA, okadaic acid; ETL, endothall; DPN, dephostatin; CyA, cyclosporine A; PV, peroxyvanadate; PBS, phosphate-buffered saline; GAP, glyceraldehyde-3-phosphate dehydrogenase.
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