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INTRACELLULAR SIGNAL SYSTEMS |
Department of Anatomy and Cell Biology (R.A., G.M., J.L.), The Hormone Laboratory, Department of Clinical Biochemistry, (G.M.), University of Bergen, N-5009 Bergen, Norway; and Department of Developmental Biology, National Institute for Basic Biology, and Core Research for Evolutional Science and Technology, Japan Science and Technology (K.-I.M.), Okazaki, 444-8585 Japan
Address all correspondence and requests for reprints to: Reidun Aesøy, Department of Anatomy and Cell Biology, University of Bergen, Aarstadveien 19, N-5009 Bergen, Norway. E-mail: reidun.asoy{at}pki.uib.no
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Introduction |
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The nuclear receptor steroidogenic factor 1 (SF-1/Ad4BP/NR5A1), which is essential for adrenal development and sexual differentiation (5), is expressed in the adrenal cortex, the gonads, pituitary gonadotrope cells, hypothalamus, and spleen (6, 7). SF-1 is an orphan member of the nuclear receptor superfamily (8) and contains a characteristic zinc finger DNA-binding domain, an intervening hinge region, and a carboxy-terminal putative ligand-binding domain (8, 9). SF-1 regulates the cell specific expression of a variety of different proteins involved in steroidogenesis, reproduction and male gonadal differentiation (10, 11). Although oxysterols have been reported to activate SF-1 in certain cell types (12), no obligatory SF-1 ligand has been identified so far (13). In the adrenal cortex, SF-1 regulates genes encoding cytochrome P450 steroid hydroxylases (14), 3ß-hydroxysteroid dehydrogenase, the ACTH receptor (15), StAR (16) and the high density lipoprotein receptor SR-B1 (17) by binding to cAMP-responsive sequences. However, the mechanisms by which PKA leads to increased SF-1-dependent transcription (18) is not fully defined (19). It has been shown that SF-1 mRNA levels are largely unaffected by pituitary hormones or agents that stimulate cAMP production (20, 21, 22, 23). Transcriptional regulation by PKA may involve posttranslational modification of SF-1, and some reports have suggested that SF-1 is directly phosphorylated by PKA (20, 24). However, it has not been possible to show that PKA actually leads to phosphorylation of SF-1 in vivo (19, 25).
In this report, we investigated the role of PKA in the regulation of SF-1-dependent transcription. It is demonstrated that activation of PKA leads to elevated levels of SF-1 in both transiently transfected nonsteroidogenic COS-1 cells and adrenocortical Y1 cells. This is not caused by stimulation of SF-1 mRNA expression, but rather by increased stabilization of the SF-1 protein. Our findings suggest that the expression of SF-1 target genes can be regulated by PKA through increased levels of SF-1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The QuickChange Site-directed Mutagenesis kit
(Stratagene, La Jolla, CA) was used for mutagenesis
of the putative PKA site, Ser430. Briefly,
double-stranded plasmid pCMV5-SF-1 and two synthetic
oligonucleotide primers with the desired mutation, each complementary
to opposite strands, were annealed and extended by means of Pfu DNA
polymerase. Following temperature cycling, the product was treated with
DPN I to digest the parental plasmid. The DNA vector with the desired
mutation was transformed into Escherichia coli (Epicurian
Coli XL1-supercompetent cells), and the Ser430
Ala mutation was confirmed by sequencing. The luciferase
reporter plasmid pT81–4CRS2-Luc, containing a minimal TK-promoter and
4 repeats of the SF-1-binding site CRS2 (cAMP responsive sequence 2)
from the bovine CYP17 gene, and the expression plasmid pCMV5-SF-1 are
described in (18). The pCMV5-C
expressing the catalytic
subunit of PKA was a gift from Dr. G. S. McKnight (Seattle,
WA).
EMSA
Double-stranded synthetic oligonucleotides containing the
SF-1-binding site from the bovine CYP17 gene (CRS2) (18)
were labeled with [
-32P]ATP using T4 polynucelotide
kinase for detection of SF-1 binding. Total cell lysates (8 µg) from
COS-1 cells transiently transfected with pCMV5-SF-1 or
pCMX-GFP-SF-1, with or without cotransfected pCMV5-PKA-catalytic
subunit (C), were incubated with the radiolabeled oligonucleotide
sequence. The reaction conditions are described previously
(31). Protein-DNA complexes and unbound probe were
separated by nondenaturing electrophoresis on a 5% polyacrylamide gel
in 0.5x TBE (0.045 M Tris borate, 0.001 M
EDTA) and the binding proteins were detected by autoradiography.
Cell culture and transfections
In our experimental setup, mouse adrenocortical Y1 tumor cells,
kindly provided by Dr. B. Schimmer (Toronto, Canada) (32)
were maintained in DMEM (high glucose) supplemented with 10% FCS, 10
U/ml penicillin and 10 µg/ml streptomycin. COS-1 (African green
monkey kidney) cells (ATCC CRL-1650) were maintained in DMEM (low
glucose) supplemented as described above. Human adrenocortical H295R
cells were selected from NCI-H295 cells obtained from the
American Type Culture Collection (Manassas, VA) as
described previously (33) and cultured in a 1:1 mixture of
DMEM and Ham’s F-12 medium supplemented with insulin (6.25 µg/ml),
transferrin (6.25 µg/ml), selenium (6.25 ng/ml), linoleic acid (5.35
µg/ml), 2% NU-Serum (Collaborative Research, Bedford, MA), and
antibiotics as described above. For transfection, cells were plated on
six-well dishes at a density of 2.5 x 105
cells/well, and transiently transfected the following day using the
calcium phosphate method (Y1 cells) (31) or by the
SuperFect transfection procedure (COS-1 cells) according to the
manufacturer’s protocol (QIAGEN, Valencia, CA).
The total amount of plasmid was kept constant by compensating with an
empty expression vector. The cells were lysed in 200 µl lysis buffer
[25 mM Tris-Acetate-EDTA; pH 7.8, 1 mM EDTA,
10% (vol/vol) glycerol, 1% (vol/vol) Triton-X-100 and 2
mM DTT (freshly added)] 48 h after transfection. The
luciferase assay was performed in accordance with the protocol of the
Luciferase Assay Kit (BIO Thema AB, Dalarö, Sweden).
Immunofluorescence and propidium iodide (PI) staining
Immunofluorescence staining was employed for localization of
endogenous SF-1 in Y1 cells. The method was optimized for SF-1
detection based on procedures by (34, 35). The day before
staining, 5.0 x 103 cells were plated in
6-mm wells on immunofluorescent-printed slides. Cells were washed with
PBS and fixed for 15 min with 2% (wt/vol) paraformaldehyde (pH 7.4).
The cells were washed with 0.02 M glycerin/PBS for 10 min
and 3% (wt/vol) BSA/PBS for 2 x 10 min. Cell membranes were
permeabilized by incubation in 0.5% (vol/vol) Triton X-100 in 3%
(wt/vol) BSA/PBS for 15 min and then washed with 3% (wt/vol) BSA/PBS
for 3 x 10 min. For localization of an endogenous protein, cells
were incubated with a specific primary antibody in 3% (wt/vol) BSA/PBS
for 2 h. The cells were washed three times, and slides containing
cells were incubated for 1 h with appropriate, fluorescein
isothiocyanate (FITC)-conjugated secondary antibody in 3%
(wt/vol) BSA/PBS. After the incubation, the slides were washed one time
and mounted in 10% (vol/vol) glycerol. A plastic-slide was placed over
the immunofluorescent-printed slide, and nail polish used to seal the
edges. All incubations, including fixation and permeabilization
procedures, were performed at room temperature, whereas incubations
with primary and secondary antibodies were performed at 37 C in a
humidified chamber. An FITC filter set was used for fluorescence
microscopy.
PI was employed for detection of the nucleus in Y1 cells. PI is an intercalating dye with an affinity to bind DNA. After PI-staining, the cells emit red fluorescence from the nucleus, while the cytoplasm remains unstained (36). The day after transfection, the cells were washed three times with PBS, then fixed with 2% (wt/vol) paraformaldehyde (pH 7.4) for 10 min. The cell membranes were permeabilized by incubation with 0.1% (vol/vol) Triton X-100 in cell medium for 10 min followed by incubation with RNase A (1 mg/ml) for 2 min. After these incubations, the cells were washed three times with PBS, and incubated with PI for 30 min. All incubations were performed at room temperature.
Image analysis and microscopic photometry of fluorescent
cells
Fluorescent cells were examined by confocal laser scanning
microscopy, employing a Leica Corp. (Wetzlar,
Germany) DM IRBE microscope, using a FITC/TRITC filter set for
fluorescence detection. Fluorescence emission was measured by employing
an inverted microscope equipped for epi- fluorescence connected to
an excitation light source and a D-104B/C Microscope Photometer (Photon
Technology International, Lawrenceville, NJ). The excitation
light source was a Super high-pressure mercury lamp. The
photomultiplier was set to 900 V. Fluorescence emission was collected
by the microscope optics and directed to the D-104B/C microscope
photometer. A parfocal eyepiece was used for viewing the sample and the
region of interest was selected by manually adjusting the four control
knobs. Fluorescence was measured by passing the emission light into the
photon-counting photomultiplier and a PC analyzer program, FELIX
(37), was used for detection.
Western blot analysis and two-dimensional gel electrophoresis
Y1, COS-1, and H295R cells were grown to subconfluent phase,
washed with PBS, and lysed in 200 µl lysis buffer as described above.
Samples were subjected to electrophoresis on 10% SDS-polyacrylamide
gels and transferred onto nitrocellulose membranes. The membranes were
incubated with anti-SF-1 antibody (polyclonal antiserum raised against
bovine SF-1) (38) or anti-GFP antibody (CLONTECH Laboratories, Inc., Palo Alto, CA) and subsequently with
an HRP-conjugated goat antirabbit IgG secondary antibody (Pierce Chemical Co.), using the SuperSignal chemiluminescent substrate
(Pierce Chemical Co.) for detection. For visualization of
the immunostaining patterns, the membrane was immediately exposed in a
Luminescent Image analyzer (Las-1000, Fuji Photo Film Co., Ltd., Tokyo, Japan) for 1–30 min.
For two-dimensional gel electrophoresis (2-DG) cell lysates were prepared by quickly washing the cells in conditioned medium before the addition of lysis buffer (9.8 M urea; 100 mM dithioerytrol; 1.5% Pharmalyte, pH 3.5–10; 0.5% Pharmalyte, pH 5–6; 4% (3-[(3-cholamidopropyl)dimethylammino]-1-propane sulfonate; 0.2% SDS). Sample separation was by isoelectric focusing (80,000 Volt-hours) in linear immobilized pH gradients (IPG strips, pH range 4.0–7.0; Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK). After completion of the run, the strips were equilibrated for 12 min in a solution [50 mM Tris-HCl, pH 6.8; 2% (wt/vol) SDS, 6.5 M urea, 26% (wt/vol) glycerol] with 100 mM dithioerytrol and thereafter for 5 min in the same solution with 0.24 M iodoacetamide. The strips were then subjected to SDS-electrophoresis (13.5% (wt/vol) polyacrylamide separation gel).
Northern analysis
Total RNA was isolated from COS-1 cells using the TRIzol reagent
according to the manufacturer’s protocol (Life Technologies, Inc., Gaithersburg, MD). The RNA samples were electrophoresed in
1% agarose gel in the presence of 1.2% formaldehyde, blotted onto
nylon membranes and hybridized with a
32P-deoxy-CTP-labeled probe made by random
priming of a 1.0-kb PCR-fragment of the carboxy-terminal part of SF-1.
A cDNA probe for human actin (a 1.4-kb SmaI fragment of
ß-actin) was also prepared as an internal control. The membranes were
analyzed by exposure to x-ray film, then stripped and reprobed with
ß-actin cDNA. The molecular weight of the RNA bands was estimated
using a RNA ladder and the position of 18 S and 28 S ribosomal RNA.
Pulse-chase analysis of protein turnover
COS-1 cells were transiently transfected with SF-1 expression
vector with or without cotransfection with PKA-C expression vector.
34.5 h post transfection, the cells were washed 2x with PBS and
incubated with methionine-free medium (DMEM) for 30 min. The cells were
then pulse labeled with [35S]-methionine
(50–60 µCi, Amersham Pharmacia Biotech) for 60
min. At the end of the pulse period, the medium was rapidly removed by
aspiration, and the cells washed two times with PBS. After the final
wash, 3 ml of chase medium (DMEM with 10% FCS containing 50x excess
methionine) was added to the wells, and cells were chased for various
time periods. At the desired time points, cells were washed two times
with PBS, lysed in 200 µl buffer containing 50 mM
Tris-HCl (pH 7.5), 100 mM NaCl, 0.5% SDS, 1%
Triton-X-100, and 0.2 mM phenylmethylsulfonyl fluoride
(PMSF), and snap-frozen in liquid nitrogen. After collection of all
samples, cell lysates were incubated on a spinning wheel for 1 h
at 4 C followed by centrifugation at 13,000 rpm for 2 min. The
supernatants were transferred to new tubes and supplemented with 4x
volume of wash solution A [50 mM Tris-HCl (pH 7.5), 100
mM NaCl, 0.2 mM PMSF], followed by addition of
polyclonal rabbit antiserum raised against bovine SF-1. The samples
were then incubated at 4 C with shaking overnight. Fifty microliters of
protein A-Sepharose (Amersham Pharmacia Biotech)
were added and incubation continued for 2 h. The samples were then
centrifuged at 13,000 rpm at 4 C for 2 min and the precipitated
Sepharose was washed four times with wash solution B [50
mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1% SDS,
0.2% Triton-X-100, 0.2 mM PMSF]. Thirty micro-liters of
µl SDS-PAGE (2x) sample buffer was added to the Sepharose pellets
and the samples were incubated at 95 C for 5 min and subjected to 10%
SDS-PAGE. The gels were exposed to Biomax MS film (Kodak,
Rochester, NY) or a phosphoimager analyzer (Bas-5000, Fuji Photo Film Co., Ltd., Tokyo, Japan) for pixel density quantification.
The background pixel densities of each of the gel-lanes were subtracted
and quantitative densitometry of radiolabeled immunoprecipitated SF-1
from transfected COS-1 cells were performed. Determination of the
background pixel densities were based on measurements from four
separate parts of each lane. Statistical analysis of the data were
carried out using the PRISM program.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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led to increased complex formation of both
wild-type SF-1 and GFP-SF-1 (Fig. 1
). To
ensure that the GFP-SF-1 fusion protein was functional, we performed
transient transfection experiments using a reporter plasmid in which
four cAMP-responsive and SF-1 binding elements from the bovine CYP17
gene (CRS2) are placed upstream of luciferase. As SF-1 is a strong cAMP
inducible activator of CRS2-dependent transcription (18),
we also compared the effects of PKA activation on the transcriptional
activity of wild-type SF-1 and GFP-SF-1. As shown in Fig. 2, the transactivation of GFP-SF-1
compared with wild-type SF-1 was approximately 50%. However,
cotransfection with an expression plasmid encoding PKA-C led to a
similar stimulation of the wild-type SF-1- and GFP-SF-1 transcriptional
activation (6-fold), indicating that the PKA-stimulated transactivation
capacity of GFP-SF-1 remained similar to wild-type SF-1.
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, B and C), as was the distribution of
endogenous SF-1 in Y1 cells as shown by immunocytochemistry using a
polyclonal antibody against SF-1 (Fig. 3A). Cotransfection of COS-1
cells with PKA-C demonstrated that GFP-SF-1 remained predominantly
nuclear (Fig. 3C, iii), indicating that increased PKA activity does not
change the intracellular localization of SF-1. However, in several
cells we observed cluster formations of GFP-SF-1 proteins in the
presence of PKA-C overexpression (indicated by arrows in Fig. 3C, iii). This suggests a preferential assembly of SF-1 proteins at certain
foci within the nucleus as a result of increased PKA activity. The
nucleoli demonstrated no fluorescence in COS-1 cells transfected with
GFP-SF-1 plasmid and examination of several images suggested that this
was also the case in Y1 cells. Thus, nucleolar structures
appeared to be devoid of GFP-SF-1 in both cell types. Interestingly, it
was noted that the fluorescence emission from GFP-SF-1 expressing cells
was markedly enhanced in the presence of PKA-C. To obtain
quantitative data we employed microscope photometry. The fluorescence
emission from COS-1 cells expressing GFP-SF-1 was increased 4.6- and
5.7-fold when cotransfected with 50 and 100 ng of the plasmid encoding
PKA-C, respectively, compared with cells not transfected with
PKA-C (P < 0.0001 as determined by the unpaired
t test) (Table 1). We observed
that the fluorescence from cells expressing GFP alone was evenly
dispersed throughout the cells in the presence (Fig. 3C, i), as well as
in the absence (data not shown) of coexpressed PKA-C, and that the
fluorescence emission from cells expressing GFP alone was not increased
by cotransfection of the plasmid encoding PKA-C (Table 1). As PKA
activation induced rounding of Y1 cells (39), it was not
possible to examine the fluorescence pattern in these cells after
treatment with forskolin or overexpression of PKA-C.
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clearly increased the amount of immunoreactive
GFP-SF-1 protein, compared with the levels of immunoreactive GFP-SF-1
in the absence of PKA-C (Fig. 4A).
Similarly, we observed increased endogenous expression of SF-1 in Y1
cells after treatment with the cAMP elevating agent forskolin (Fig. 4B). Previous studies on Y1 cells and other steroidogenic cells have
uniformly demonstrated that SF-1 mRNA levels are not affected by the
cAMP- signaling cascade (20, 21, 22, 23). Therefore, these
results point to the effect(s) of PKA on SF-1 in Y1 cells occuring at
the posttranscriptional level. To rule out the possibility that the
effect we observed on SF-1 protein levels in transfected COS-1
cells was simply due to activation of the promoter that directed
the ectopic expression of SF-1, we compared the SF-1-encoding mRNA
levels in the absence or presence of coexpressed PKA-C by Northern
analysis. The results shown in Fig. 4C are from a representative
experiment based on total RNA samples (upper panel) and
protein samples (lower panel) prepared from the same cell
cultures. These data show that the regulation of SF-1 protein
expression by PKA is not at the level of transcription.
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(upper panel) shows that there are no differences with
respect to PKA-modified increase in SF-1 mRNA levels when the potential
PKA site was mutated. The effect of PKA-C on the protein levels of
the Ser430Ala mutated form of SF-1 and the
wild-type SF-1 were examined, and, as shown in Fig. 4C (lower
panel), cotransfected PKA-C stimulated the protein levels of
mutated SF-1 and wild-type SF-1 equally. We further determined whether
this mutation affected the transactivation capacity of SF-1. COS-1
cells were cotransfected with the 4CRS2 reporter plasmid and expression
plasmid encoding SF-1 or the mutated form of SF-1 together with the
PKA-C expression plasmid as indicated in Fig. 4D. The results
suggested that the mutated form of SF-1 transactivated CRS2 reporter
gene activity equally to wild-type SF-1. Thus, it appears that
Ser430 is not the site of PKA action.
To further examine whether SF-1 is a direct target for PKA-dependent
phosphorylation, two-dimensional gel electrophoresis and Western blot
analysis using an anti-SF-1 antibody were performed on lysates from
human adrenocortical (H295R) cells, which express SF-1 endogenously. In
lysates from nontreated H295R cells the anti-SF-1 antibody detected
four marked and one weak spot with equal molecular weights and
different iso-electric points corresponding to endogenous SF-1 (Fig. 5A). Additionally, one marked and one
weak spot with somewhat lower molecular weight were detected (Fig. 5A).
These spots may represent different forms of SF-1, and the difference
in iso-electric points may be caused by phosphorylation at a different
number of residues. To study whether PKA activation modulated the SF-1
protein, cells were treated with the cAMP elevating agents Forskolin
(10 µM) and 3-isobutyl-1-methylxanthine (50
µM) for 4 h followed by two-dimensional analysis.
Interestingly, we detected no significant difference in the
distribution of SF-1 isoforms after activation of PKA (Fig. 5B). In
contrast, two-dimensional analysis of SF-1 from H295R cells treated
with the serine/threonine phosphatase inhibitor okadaic acid (300
nM) for 1 h, revealed several changes in the
distribution of SF-1 isoforms (Fig. 5C). Thus, it appears that SF-1 can
be phosphorylated at several serine/threonine residues after exposure
to okadaic acid. However, our results did not indicate that activation
of PKA led to phosphorylation of SF-1.
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), we observed no changes in the synthetic rates of
transiently expressed SF-1 in COS-1 cells cotransfected with PKA-C
(data not shown). To assess the possibility that changes in SF-1
expression were due to altered protein degradation, we used pulse-chase
experiments to examine the rate of SF-1 turnover. COS-1 cells
transiently expressing SF-1 were incubated with
35S-methionine for 60 min, followed by a chase
for different lengths of time. Cell lysates were prepared and
radiolabeled SF-1 was immunoprecipitated as described previously.
Comparisons of the intensities of labeled SF-1 were performed at each
time point using phosphorimager analysis. The combined results of
several experiments (n = 4) showed a significant reduction in SF-1
protein amount in transfected COS-1 cells in the absence of PKA-C
overexpression measured 4 h post chase (mean ±
SEM = 23.7 ± 4.64%, P = 0.0022, as
determined by the unpaired t test), compared with the amount
of SF-1 protein after 1 h, which was set to 100%. In contrast, no
significant reduction in SF-1 protein amount was observed after 4
h in cells cotransfected with PKA-C (mean ±
SEM = 77.2 ± 9.5%,
P = 0.143, as determined by the
unpaired t test). Thus, it appears that the turnover of
transiently expressed SF-1 was decreased by coexpression of PKA-C.
The results shown in Fig. 6 are from a
representative experiment, and the above data were corroborated by
linear regression analysis of the logarithmic values of SF-1 protein
amounts at 1, 2, 3, 4, and 6 h post chase. The slope of the data
from the SF-1 protein amounts in absence of PKA-C overexpression was
significantly nonzero (P = 0.012), whereas the slope of
the data from cells with coexpressed PKA-C did not significantly
deviate from zero (P = 0.349). This confirms that the
half-life of SF-1 is increased in cells with increased PKA activity.
Taken together, the present results suggest that increased SF-1 protein
caused by activation of PKA can be attributed to a decrease in the
degradation rate of SF-1 and that coexpression of PKA-C increases
the stability of SF-1 protein.
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Discussion |
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were coexpressed in COS-1 cells, an
interesting observation was made; the fluorescence emission from
GFP-SF-1 was stronger in the presence of overexpressed PKA-C than in
its absence. Microscopic photometry measurements confirmed that the
GFP-SF-1 fluorescence emission intensity was elevated by activation of
PKA. Because PKA activation in Y1 cells led to altered cell morphology
(39), we were not able to perform microscopic photometry
of the GFP-SF-1 fluorescence and examine the effects of PKA in these
cells. However, the results from COS-1 cells transfected with GFP-SF-1
were corroborated by Western blot analysis of SF-1 in Y1 cells and
transfected COS-1 cells, demonstrating that the increase in
fluorescence emission observed after activation of PKA most likely
reflected an increase in the amount of SF-1 protein. Compared with
COS-1 cells, the increase of SF-1 protein expression was less
pronounced in Y1 cells. However, this can be explained by the vast
amount of SF-1 normally present in the steroidogenic Y1 cells.
The finely granular nuclear distribution of the GFP-SF-1 fusion protein
against a field of diffuse distribution indicates an assembly of SF-1
proteins at certain foci within the nucleus of COS-1 cells. It is
becoming clear that the eukaryotic nucleus is dynamically organized
with respect to particular activities, such as RNA transcription, RNA
processing or DNA replication (40, 41). Cellular
organization of transcription-related factors can be important in
facilitating or regulating gene expression (42, 43).
Induction by ligands have been shown to change the subnuclear
localization of the steroid receptors ER (44, 45),
glucocorticoid receptor (GR) (46), mineralocorticoid
receptor (MR) (47), and AR (48) from nuclear
diffuse to discrete foci, suggesting that such fluorescence foci
correspond to transcriptionally active areas. Our results, together
with previous data showing that the cAMP-dependent transcription of the
bovine CYP17 gene is SF-1 dependent (18), may indicate
that activation of PKA leads to increased transcription of SF-1 target
genes by assembling SF-1 at active sites in the nucleus. This should be
confirmed by further investigations, e.g. by colocalization
studies with RNA polymerase II, and the exact nature of the fluorescent
foci in the nucleus remains to be elucidated.
In this study, we have examined the mechanisms underlying PKA-mediated
regulation of SF-1 protein expression. Previous reports by others have
shown that pituitary hormones or cAMP-elevating agents do not affect
the mRNA levels for SF-1 in Y1 cells, MA-10 Leydig cells, or bovine
luteal cells, nor in the late-gestation ovine fetus
(20, 21, 22, 23). We confirm that this is also the case in
transiently transfected COS-1 cells, demonstrating that the regulation
of SF-1 by PKA is not at the transcriptional level. It has been
reported that SF-1 interacts with cAMP response element binding protein
(CREB), leading to recruitment of CREB binding protein and increased
histone acetylation (49). CREB is phosphorylated by PKA
and the interaction between SF-1 and phosphorylated CREB has been
suggested as a mechanism for the synergism between the SF-1 and cAMP
(50). Our data suggest that PKA acts to increase the
amount of SF-1 by increasing its stability. The exact mechanism
underlying the increased stability of SF-1 protein is not clear.
Recently, it has been demonstrated that certain members of the nuclear
receptor (NR) family are degraded through the ubiquitin-proteasome
pathway in a ligand-dependent manner (51, 52). The
ubiquitin-proteasome pathway is the major system in eukaryotes for
selective degradation of cellular proteins (53).
Phosphorylation may serve as a signal for ubiquitination and subsequent
proteosomal degradation, and it has been shown that MAPK-induced
phosphorylation of a single site in the progesterone receptors signals
their degradation by the 26S proteasome (54). On the
other hand, RXR, which is also a substrate for MAPK, is destabilized by
inhibition of the MAPK-signaling pathway (55). PKA has
been reported to phosphorylate SF-1 in vitro
(20), and the primary structure of SF-1 has predicted a
potential PKA phosphorylation site Ser430
(8). However, mutation of this site clearly demonstrates
that Ser430 is not essential for PKA-mediated
regulation of SF-1 protein expression or for PKA-stimulated SF-1
transcriptional activity. Here we present data suggesting that
endogenous SF-1 in adrenocortical H295R cells is phosphorylated after
treatment with okadaic acid, whereas SF-1 remained unchanged after PKA
activation as determined by two-dimensional Western blot analysis of
H295R cell extracts (Fig. 5). These results are in accordance with a
previous report showing that treatment with 8-Br-cAMP failed to
significantly increase the phosphorylation level of SF-1 in transfected
MCF-7 and COS-1 cells (19). Interestingly, it has been
shown that SF-1 is phosphorylated at Ser203 by
activation of the MAPK pathway (19). In some cell types,
elevation of cAMP leads to inhibition of the MAPK pathway (27, 56), and one possibility could be that PKA acts indirectly
through modulation of the MAPK pathway to stabilize SF-1. This issue
should be a subject for further investigation. However, mutation of the
MAPK phosphorylation site (Ser203Ala) in SF-1 did
not affect the ability of PKA to stimulate SF-1 transcriptional
activity (Børud, B., T. Hoang, M. Bakke, A. Jacob, J. Lund, and G.
Mellgren, submitted for publication). Activation of PKA has been shown
to stimulate the transcriptional activity of other NRs such as ER
(57, 58) and MR (59). Whether this in part
could be caused by increased protein stability also remains to be
shown.
The mechanisms for NR down-regulation is not well understood. In addition to ligand binding and phosphorylation, coactivator recruitment has been suggested as a signal for initiation of NR degradation (60, 61). SF-1 interacts with several transcriptional coregulators such as SRC-1/NcoA-1 (62), TIF2/GRIP1/NcoA-2 (19), p/CIP/SRC-3/ACTR/AIB1 (Børud, B., T. Hoang, M. Bakke, A. Jacob, J. Lund, and G. Mellgren, submitted for publication). Specific amino acids within the AF-2 domain of SF-1 are important for interaction with these proteins. One possibility is that PKA modulates the interaction with a coactivator and subsequently increases SF-1 stability, and mutation analysis of amino acids within the AF-2 domain of SF-1 may elucidate whether coactivator recruitment is required for the degradation of SF-1.
This study was based on the use of GFP-SF-1 that allowed us to examine the expression and distribution of transfected SF-1 in living cells. We present results showing that PKA activation elevates the level of SF-1, and this may indicate that the expression of SF-1 target genes can be regulated by PKA through increased levels of SF-1.
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Acknowledgments |
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plasmid
and A. L. Jacob for the Ser430Ala mutated
form of the pCMV5-SF-1 plasmid. Technical assistance from T. Ellingsen,
C. Fladeby, C. Cook, and A. M. Sellevold is highly
appreciated. We also thank Marit Bakke and Thor E. Thorsen for
reviewing the manuscript before publication. |
Footnotes |
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Abbreviations: CREB, cAMP response element binding protein;
CMV, cytomegalovirus; CMV-IEP, cytomegalovirus immediate early
promoter/enhancer; CRS2, cAMP responsive sequence 2; C, PKA
catalytic subunit; FITC, fluorescein isothiocyanate; GFP, green
fluorescent protein; NR, nuclear receptor; PI, propidium iodide; SF-1,
steroidogenic factor 1; StAR, steroidogenic acute regulatory.
Received June 20, 2001.
Accepted for publication September 27, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase/c17–20 lyase). Mol Endocrinol 10:147–158) gene. Identification of two cAMP regulatory regions
lacking the consensus cAMP-responsive element (CRE). J Biol Chem 265:3304–3312 and steroid receptor coactivator-1. Mol Endocrinol 14:518–534 reveals a role
for ligand in the nuclear distribution of the receptor. Mol Biol Cell 10:471–486-promoter by steroidogenic
factor-1 and cyclic adenosine 3',5'-monophosphate. Mol Endocrinol 14:66–81 and through the ubiquitin-proteasome
pathway. J Biol Chem 275:33280–33288 (RAR)
and oncogenic RAR fusion proteins. Proc Natl Acad Sci USA 96:14807–14812 and coactivator
turnover and for efficient estrogen receptor- transactivation. Mol
Cell 5:939–948[CrossRef][Medline]
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