cAMP-dependent Protein Kinase Enhances CYP17 Transcription via MKP-1 Activation in H295R Human Adrenocortical Cells*

Marion B. SewerDagger § and Michael R. Waterman

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 3beta -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.

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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 -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.

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 [alpha -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.

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-gamma -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.

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 gamma  [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.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


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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.

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.


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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.

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 [gamma -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 [gamma -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 + [gamma -32P]ATP; lane 3, MKP-1-GST + [gamma -32P]ATP + 1 unit of PKA; lane 4 MKP-1-GST + [gamma -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.

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.


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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.

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).


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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.

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).


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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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    FOOTNOTES

* 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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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