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Endocrinology Vol. 143, No. 1 295-303
Copyright © 2002 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

Activation of cAMP-Dependent Protein Kinase Increases the Protein Level of Steroidogenic Factor-1

Reidun Æsøy, Gunnar Mellgren, Ken-Ichirou Morohashi and Johan Lund

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The orphan nuclear receptor steroidogenic factor 1 (SF-1) is an essential regulator of endocrine organogenesis, sexual differentiation, and steroidogenisis. SF-1 is a transcriptional regulator of cAMP responsive genes, but the exact mechanisms by which cAMP-dependent PKA modulates SF-1 dependent transcription leading to increased steroidogenic output have not been determined. In this report the effects of PKA activation on SF-1 in living cells have been examined by the use of full-length SF-1 cDNA fused to the cDNA encoding green fluorescent protein (GFP). The GFP-SF-1 fusion protein localized to the nucleus of both steroidogenic Y1 cells and nonsteroidogenic COS-1 cells, and the functional properties of wild-type SF-1 were conserved. When the catalytic subunit of PKA was coexpressed with GFP-SF-1, we observed that the fluorescence emission was markedly elevated. These findings were confirmed by Western blot analysis, showing that stimulation of PKA increased SF-1 protein levels. The PKA- induced expression of SF-1 protein was not accompanied by an increase in SF-1 mRNA levels. However, pulse-chase studies showed a decrease in SF-1 degradation rate in response to activation of PKA, indicating that PKA elevates the level of SF-1 by increasing the stability of SF-1 protein.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STEROID HORMONE biosynthesis is mainly under control of the pituitary hormones ACTH, LH, and FSH (1, 2), which act through G-coupled receptors to induce the production of the intracellular messenger cAMP (3). In the adrenal cortex, ACTH has an immediate effect on steroid production by activation of the steroidogenic acute regulatory (StAR) protein, leading to rapid mobilization of the substrate cholesterol (4), as well as a delayed response implicating transcriptional activation of steroidogenic genes.

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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid constructs
The green fluorescent protein (GFP)-SF-1 fusion protein was constructed by insertion of full-length cDNA of bovine SF-1 (1410 bp amplified by PCR) downstream of a full-length humanized GFP sequence, into the SalI site of the plasmid pCMX-GFP-S65A/Y145F kindly provided by Dr. K. Umesono (Nara, Japan). Sequencing confirmed the merge between the GFP C-terminus and the N-terminus of the full-length cDNA of bovine SF-1 and that the SF-1 cDNA was put in frame in pCMX-GFP-SF-1 [S65A/Y145F-bSF-1] through a -Gly-Ser-Thr-Met-Asp-sequence. As wild-type GFP from the jellyfish Aequorea victoria is poorly expressed in mammalian cells, a humanized version of GFP has been designed by substituting preferentially used codons in the human genome for rare or less frequently used codons in the original jellyfish GFP cDNA (26). The humanized GFP is efficiently expressed in mammalian cells and subsequently the fluorescence intensity is increased. Intact GFP is required for fluorescence, but the active chromophore is formed by cyclization and oxidation of the three amino acids Ser65, Tyr66, and Gly67 (27, 28). The formation of the chromophore is a relatively slow process requiring several hours (29), but the humanized GFP used in the present study contains mutations (S65A and Y145F), which increase the rate of chromophore formation (30).

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{alpha} 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 [{gamma}-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{alpha}), 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{alpha} 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cotransfection with PKA increases the emission from a GFP-SF-1 fusion protein
The underlying mechanism(s) by which activation of PKA leads to increased expression of SF-1 target genes has remained elusive. As a tool to study expression and cellular localization of SF-1 in response to various stimuli in living cells, full-length bovine SF-1 cDNA was fused to cDNA encoding GFP from Aequorea victoria. DNA binding studies employing the SF-1 binding site CRS2 from the CYP17 gene as a probe (18) demonstrated that GFP-SF-1 retained its DNA-binding capacity. Interestingly, it was also noted that coexpression of PKA-C{alpha} led to increased complex formation of both wild-type SF-1 and GFP-SF-1 (Fig. 1Go). 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. 2Go, the transactivation of GFP-SF-1 compared with wild-type SF-1 was approximately 50%. However, cotransfection with an expression plasmid encoding PKA-C{alpha} 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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  		Figure 1. DNA binding affinities of wild-type SF-1 and GFP-SF-1 protein. COS-1 cells were transiently transfected with expression plasmids encoding wild-type SF-1 or GFP-SF-1 (1.5 µg) and pCMV5-PKA-C{alpha} (0.1 µg) as indicated. Total cell lysate protein (8 µg) was incubated with a radiolabeled CRS2 oligonucleotide sequence and processed for EMSA as described in Materials and Methods. The figure shows binding of wild-type SF-1 in the absence (lane 2) and presence (lane 3) of coexpressed PKA-C{alpha}, and GFP-SF-1 in the absence (lane 4) and presence of PKA-C{alpha} (lane 5). As a positive control, a nuclear extract of steroidogenic H295 cells was used (lane 1). Lysates of COS-1 cells transfected with empty expression vector in the absence and presence PKA-C{alpha} (100 ng) were used as negative controls (lanes 6 and 7, respectively).

 


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  		Figure 2. Transcriptional activity of GFP-SF-1 and wild-type SF-1. COS-1 cells were cotransfected with the 4CRS2-luc reporter construct (1.5 µg) and expression plasmids encoding wild-type SF-1 (0.1 µg), GFP-SF-1 (0.1 µg), and pCMV5-PKA-C{alpha} (0.1 µg) as indicated. 48 h after transfection luciferase assay were performed. Results are expressed as luciferase activity (mean ± SD) of triplicate transfections from a representative experiment.

 
As determined by the fluorescence pattern after transfection of cells in culture, the GFP-SF-1 fusion protein was predominantly localized to the nucleus of both mouse adrenal Y1 tumor cells and COS-1 cells (Fig. 3Go, 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. 3AGo). Cotransfection of COS-1 cells with PKA-C{alpha} demonstrated that GFP-SF-1 remained predominantly nuclear (Fig. 3CGo, 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{alpha} overexpression (indicated by arrows in Fig. 3CGo, 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{alpha}. 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{alpha}, respectively, compared with cells not transfected with PKA-C{alpha} (P < 0.0001 as determined by the unpaired t test) (Table 1Go). We observed that the fluorescence from cells expressing GFP alone was evenly dispersed throughout the cells in the presence (Fig. 3CGo, i), as well as in the absence (data not shown) of coexpressed PKA-C{alpha}, and that the fluorescence emission from cells expressing GFP alone was not increased by cotransfection of the plasmid encoding PKA-C{alpha} (Table 1Go). 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{alpha}.



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  		Figure 3. Intracellular localization and PKA-mediated regulation of GFP-SF-1. A, Immunofluorescence staining was employed for intracellular localization of endogenous SF-1 in Y1 cells. Y1 cells were grown on slides, fixed, permeabilized and stained as described in Materials and Methods. i, SF-1 was detected using a polyclonal rabbit SF-1 antibody (1:50) together with an antirabbit FITC-conjugated IgG (1:50). ii, For detection of the control protein, ß-tubulin, a monoclonal mouse ß-tubulin antibody (1:25) was used together with an antimouse FITC-conjugated IgG (1:50). B, Y1 cells were transfected with 3 µg of the expression plasmid encoding GFP-SF-1. The figure shows i) green fluorescence from GFP of GFP-SF-1 and ii) red fluorescence from propidium iodide, a DNA specific dye, employed to distinguish the nucleus from the cytoplasm. C, COS-1 cells were transfected with 3 µg of the expression plasmids encoding GFP or GFP-SF-1. Cotransfections with 100 ng of a plasmid encoding PKA-C{alpha} were performed as indicated. The expression pattern of the fluorescent protein was studied 24 h after transfection by confocal laser scanning microscopy, as described in Materials and Methods. The figure shows COS-1 cells expressing GFP (i), GFP-SF-1 in the absence of (ii) and in the presence of overexpressed PKA-C{alpha} (iii). The arrows indicate clusters of GFP-SF-1 proteins.

 

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  		Table 1. Quantitative measurement of the fluorescence emission from COS-1 cells expressing GFP-SF-1 in the presence and absence of PKA-C{alpha} overexpression

 
Activation of PKA leads to increased protein levels of SF-1 and GFP-SF-1
After the fluorescence quantification, the cell cultures were lysed and subjected to Western blot analysis using an anti-GFP antibody. In COS-1 cells, transfection of 50 and 100 ng plasmid encoding PKA-C{alpha} 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{alpha} (Fig. 4AGo). Similarly, we observed increased endogenous expression of SF-1 in Y1 cells after treatment with the cAMP elevating agent forskolin (Fig. 4BGo). 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{alpha} by Northern analysis. The results shown in Fig. 4CGo 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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  		Figure 4. SF-1 protein and mRNA expression in presence of increased PKA activity. A, COS-1 cells were transfected with 3 µg of the expression plasmid encoding GFP-SF-1. Cotransfections with 0, 50, and 100 ng of a plasmid encoding PKA-C{alpha} were performed as indicated. The cells were harvested 48 h after transfection and 30 µg of total cell lysate were processed for Western blot analysis, as described in Materials and Methods, using a polyclonal antibody against GFP. B, Y1 cells were treated with 10 µM forskolin for 24 h before lysis, and 30 µg of total cell lysate were processed for Western blot analysis using a polyclonal antibody raised against bovine SF-1. The effect of the cAMP-elevating agent forskolin on the expression of endogenous SF-1 protein is shown. Detection of SF-1 in Y1 nuclear cell extract (Y1 n.e.) is used as a control. C, COS-1 cells were transiently transfected with an expression vector for wild-type SF-1 (SF-1 WT) and PKA-C{alpha} expression plasmid as indicated. Forty-eight hours after transfection the cells were harvested and processed for Northern blotting and Western blotting as described in Materials and Methods. Twenty micrograms of total RNA were analyzed by Northern blot and 25 µg of cell lysate by Western blot. The wild-type SF-1 (SF-1 WT) and the Ser430Ala mutated form of SF-1 (SF-1 mut) mRNA expression is not affected by coexpression of PKA-C{alpha} (upper panel). Equal loading was determined by stripping the blot and rehybridization with the probe for ß-actin mRNA. SF-1 protein from the same cell extracts was detected with a polyclonal antibody against bovine SF-1, and as shown in the figure, coexpression of PKA-C{alpha} markedly increased the levels of SF-1 WT and SF-1 mut (lower panel). D, COS-1 cells were transfected with 1.5 µg of the reporter plasmid pT81-4CRS2-Luc together with 100 ng of the expression plasmids encoding SF-1 WT or SF-1 mut. Cotransfections with 100 ng of PKA-C{alpha} expression plasmid were performed as indicated. Cells were lysed 48 h after transfection and luciferase assay performed.

 
Mutation of a putative PKA phosphorylation site in SF-1 does not affect the increase of SF-1 protein
SF-1 contains several putative phosphorylation sites for different protein kinases, only one of which is a potential consensus site for PKA-dependent phosphorylation that is located in the ligand-binding domain of SF-1 (8). A plausible hypothesis could therefore be that SF-1 is a direct target for PKA and that SF-1 is regulated by phosphorylation. Using site-directed mutagenesis, we mutated this potential PKA site by substituting Ser430 for Ala. Figure 4CGo (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{alpha} 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. 4CGo (lower panel), cotransfected PKA-C{alpha} 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{alpha} expression plasmid as indicated in Fig. 4DGo. 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. 5AGo). Additionally, one marked and one weak spot with somewhat lower molecular weight were detected (Fig. 5AGo). 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. 5BGo). 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. 5CGo). 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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  		Figure 5. Two-dimensional analysis of SF-1 in adrenocortical H295R cells. Whole cell extracts from cultured H295R cells, which express SF-1 endogenously, were subjected to two-dimensional gel electrophoresis and Western blot analysis using an anti-SF-1 antibody. The figure shows two-dimensional Western blots of lysates from untreated cells (A), lysates from cells treated with 10 µM Forskolin/50 µM 3-isobutyl-1-methylxanthine for 4 h (B), and lysates from cells treated with 300 nM okadaic acid for 1 h (C). The acidic shifts in the distribution of SF-1 isoforms after exposure to the serine/threonine inhibitor okadaic acid are marked. These two-dimensional Western blots were performed separately, and subsequently no quantitative measurements could be performed.

 
PKA increases the stability of SF-1 protein
The varied SF-1 protein expression with and without stimulated PKA activity could result from differences in the rates of protein synthesis or protein degradation. Consistent with the above-mentioned results on the SF-1 mRNA levels in transfected COS-1 cells (Fig. 4CGo), we observed no changes in the synthetic rates of transiently expressed SF-1 in COS-1 cells cotransfected with PKA-C{alpha} (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{alpha} 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{alpha} (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{alpha}. The results shown in Fig. 6Go 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{alpha} overexpression was significantly nonzero (P = 0.012), whereas the slope of the data from cells with coexpressed PKA-C{alpha} 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{alpha} increases the stability of SF-1 protein.



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  		Figure 6. Analysis of SF-1 protein turnover in transfected COS-1 cells. A and B, COS-1 cells were transiently transfected with 100 ng of an expression vector for wild-type SF-1 without (A) or with (B) coexpression with PKA-C{alpha} expression vector (100 ng). Thirty-five hours after transfection cells were labeled with 35S-methionine and chased for different time periods as described in Materials and Methods. Radiolabeled SF-1 was immunoprecipitated using a polyclonal antibody against bovine SF-1 and subjected to 10% SDS-PAGE. C, The gels were exposed to Biomax MS film. The effect of coexpression with PKA-C{alpha} on the SF-1 bands was analyzed by pixel density quantification using a phosphoimager analyzer (Bas-5000, Fuji Photo Film Co., Ltd.). Statistical analyses of the data are given below.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present investigation was initiated to examine the effect of PKA activation on SF-1 in intact cells using a GFP-SF-1 fusion protein. When GFP-SF-1 and PKA-C{alpha} 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{alpha} 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{alpha} (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. 5Go). 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.


   Acknowledgments
 
We thank G. S. McKnight for supplying the pCMV-C{alpha} 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
 
This work was supported by grants from the Norwegian Research Council, the Norwegian Cancer Society, the Novo Nordic Foundation, The Norwegian Academy of Science and Letters, and The Norwegian Endocrine Society.

Abbreviations: CREB, cAMP response element binding protein; CMV, cytomegalovirus; CMV-IEP, cytomegalovirus immediate early promoter/enhancer; CRS2, cAMP responsive sequence 2; C{alpha}, 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.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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