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J. Biol. Chem., Vol. 276, Issue 37, 34888-34895, September 14, 2001

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ERKs Regulate Cyclic AMP-induced Steroid Synthesis through Transcription of the Steroidogenic Acute Regulatory (StAR) Gene^*

Shân L. Gyles^§¶, Chris J. Burns^§, Barbara J. Whitehouse, David Sugden, Phil J. Marsh, Shanta J. Persaud, and Peter M. Jones^**

From the  Endocrinology and Reproduction Research Group and The  Randall Centre, Guy's, King's and St. Thomas's School of Biomedical Sciences, King's College London, London SE1 1UL, United Kingdom

Received for publication, March 7, 2001, and in revised form, June 14, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cyclic AMP-dependent expression of the steroidogenic acute regulatory (StAR) protein is thought to be the controlling step for steroid production, but the mechanisms through which external signals are translated into increased transcription of the StAR gene are unknown. We demonstrate that cyclic AMP-induced steroid synthesis is dependent upon the phosphorylation and activation of ERKs and that ERK activation results in enhanced phosphorylation of SF-1 and increased steroid production through increased transcription of the StAR gene. Adenylate cyclase activation with forskolin (FSK) caused a time-dependent increase in ERK activity and translocation from cytoplasm to nucleus, which correlated with an increase in StAR mRNA levels, StAR protein accumulation, and steroidogenesis. Similarly, ERK inhibition led to a reduction in the levels of FSK-stimulated StAR mRNA, StAR protein, and steroid secretion. These effects were attributed to the finding that ERK activity is required for SF-1 phosphorylation, a transcription factor required for the regulation of StAR gene transcription. This conclusion was supported by our demonstration of an ERK-dependent increase in the binding of SF-1 from FSK-treated Y1 nuclei to three consensus double-stranded DNA sequences from the StAR promoter region. These observations suggest that the activation of ERK2/1 by increasing cAMP is an obligatory and regulated stage in the stimulation of steroid synthesis by cyclic AMP-generating stimuli.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Stimulus recognition in steroidogenic cells is dependent initially on the receptor-mediated activation of adenylate cyclase, and subsequent increases in intracellular cyclic AMP that activate the cyclic AMP-dependent protein kinase (PKA)¹ (1, 2). PKA activation stimulates the rapid mobilization of intracellular stores of cholesterol to the outer mitochondrial membrane (OMM) and promotes cholesterol transport to the inner mitochondrial membrane (IMM), where it is converted to pregnenolone by the cytochrome P450 side chain cleavage complex (P450_SCC) (3). The delivery of cholesterol to the IMM is the rate-limiting step in steroid synthesis, thus regulating the rate of secretion of steroid hormones (3). It is now generally accepted that a mitochondrial phosphoprotein, known as steroidogenic acute regulatory (StAR) protein, is essential for cholesterol transport to the IMM, and that the expression of StAR protein is the key regulatory event in steroid synthesis (4, 5). Thus, transcription of the StAR gene is closely coupled to steroidogenesis in a variety of tissues (6-8); the experimental expression of StAR cDNA enables and enhances steroid production (9, 10); and mutations in the StAR gene produce endocrine pathologies with a phenotype of reduced or absent steroidogenesis (11).

The regulation of StAR gene transcription is not fully understood, although the StAR gene contains binding sites for transcriptional regulators such as SF-1 (steroidogenic factor-1, at least three separate binding sites), DAX-1 (dosage-sensitive sex reversal-adrenal hyperplasia congenital critical region on the X-chromosome), and AP-1 (activator protein-1), which have phosphorylation-dependent transcriptional activities (12, 13). Although PKA-mediated protein phosphorylation is undoubtedly important in regulating steroid synthesis, other signaling systems have also been implicated in StAR gene expression (see e.g. Refs. 14-17). The mitogen-activated protein kinase (MAPK) family is a point of convergence for diverse signaling pathways in which extracellular signal-regulated kinases (ERK2/1 or p42/44 MAPKs), c-jun N-terminal kinases (JNKs), and p38 kinases are the terminal kinases in three distinct yet interacting signal transduction cascades (18). These enzymes commonly regulate target gene expression by the activation of downstream transcription factors, and ERK2/1 have been implicated in the regulation of SF-1 and AP-1 activity in human breast cancer MCF-7 cells and in human kidney COS cells (19). We have now demonstrated that cyclic AMP-induced steroid synthesis is dependent upon the activation of the ERK cascade and that ERK2/1 activation leads to increased phosphorylation of SF-1, enhanced SF-1 binding to regions of the StAR promoter, and enhanced steroid production by increasing the availability of StAR protein through increased transcription of the StAR gene.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Tissue culture reagents and plastics were from Life Technologies, Inc. Trilostane was a kind gift from Dr. George Margetts (Stegram Pharmaceuticals, Sussex, UK). PD098059 (PD) and UO126 (UO) were obtained from Calbiochem-Novabiochem Ltd. (Nottinghamshire, UK). A polyclonal antibody to pregnenolone was obtained from Biogenesis (Poole, UK). [7-³H]Pregnenolone (specific activity 22.5 Ci/mmol) for use in radioimmunoassay was obtained from PerkinElmer Life Science Products. The anti-active p42/44 MAPK antibody was from Promega (Southampton, UK), and the anti-p42/44 MAPK antibody was obtained from Transduction Laboratories (Lexington, Kentucky). The anti-SF-1 antibody was obtained from Upstate Biotechnology (Lake Placid, New York). A polyclonal anti-serum against StAR protein was a kind gift from Professor Ian Mason (Edinburgh, UK). Horseradish peroxidase-coupled goat anti-mouse IgG, goat anti-rabbit IgG, and goat anti-sheep IgG were from Pierce. The monoclonal anti-phosphoserine antibody was from Calbiochem-Novabiochem. Alexa fluor-488-conjugated goat anti-rabbit IgG was from Molecular Probes (Poort Gebouw, The Netherlands). Enhanced chemiluminescence (ECL) reagents, Hyperfilm, and x-ray film were from Amersham Pharmacia Biotech International plc (Buckinghamshire, UK). T4 DNA kinase and buffer were obtained from Promega. [³²P]ATP was from Amersham Pharmacia Biotech. Complete protease inhibitor mixture was from Roche Diagnostics (Sandhofer Strasse, Germany). The pCMV6 plasmid containing the full-length StAR cDNA sequence was a kind gift from Prof. Douglas Stocco (Texas-Tech University). PCR primers and electrophoretic mobility shift assay probes were prepared in house (King's College London, Molecular Biology Unit). The QIAquick gel extraction kit was obtained from Qiagen (Crawley, UK). The Dynabeads oligo(dT) ₂₅ kit was obtained from Dynal (Oslo, Norway). Moloney murine leukemia virus-reverse transcriptase (MMLV-RT, Superscript II) was from Life Technologies, Inc. PCR was performed using a LightCycler rapid thermal cycler system from Roche Dignostics Ltd. (Lewes, UK). All other biochemicals were from Sigma.

Cells-- Mouse adrenocortical Y1 cells were obtained from the European Collection of Animal Cell Cultures (Wiltshire, UK) and maintained in DMEM supplemented with 100 µg/ml streptomycin, 100 units/ml penicillin, and 10% (v/v) fetal bovine serum. MA-10 cells were a kind gift from Prof. Mario Ascoli (20) and were maintained in Waymouth MB752/1 supplemented with 20 mM Hepes, 15% horse serum, and 50 µg/ml gentamycin. Both cell lines were cultured at 37 °C in an atmosphere of 5% CO₂.

Steroid Production-- Y1 cells and MA-10 cells were seeded in 96-well microculture plates at a density of 1 × 10⁶ cells/well and incubated overnight at 37 °C in 5% CO₂ to allow the cells to adhere to the plates. The culture medium was replaced with medium alone (control) or medium supplemented with 1 µM forskolin (FSK) with or without PD (50 µM) or UO (10 µM), and the incubation was continued for a further 3 h. Steroid production was measured using a radioimmunoassay for pregnenolone over the range of 0.8 to 100 pmol/ml. The conversion of pregnenolone to other steroids was prevented by the addition of 2 µM trilostane (21, 22), an inhibitor of 3-hydroxysteroid dehydrogenase, to the incubation medium.

Immunoprecipitation of SF-1-- Y1 cells were seeded in 6-well microculture plates at a density of 5 × 10⁶ cells/well. The medium was replaced with fresh growth medium (control) or with medium supplemented with 1 µM FSK with or without PD (50 µM) and UO (10 µM), and the incubation was continued for a further 3 h after which the cells were washed with fresh growth medium. Ice-cold lysis buffer (20 mM Tris, 2 mM EDTA, 0.5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 50 µg/ml leupeptin, 250 µM NaF, 10 µM E64, and 10 µM 1-chloro-3-tosylamido-7-amino-2-heptanone) was added to each well, and the cell extracts were transferred to tubes on ice. Anti-SF-1 antibody (5 µg/tube) was added to the tubes, which were incubated at 4 °C on a rotating mixer. Protein A-agarose (10%) was added to the tubes, and the incubation was continued for a further 3 h at 4 °C. After this time, the tubes were centrifuged at 9000 × g for 3 min, and the pellet was washed three times with lysis buffer. Sample buffer (12.5% Tris, 10% glycerol, 5% -mercaptoethanol, 2% (w/v) SDS, and 0.1% (w/v) bromphenol blue) was added to each pellet, and the tubes were boiled briefly. Immunoreactivity was detected by polyacrylamide gel electrophoresis and immunoblot analysis using a mouse anti-phosphoserine primary antibody and a goat anti-mouse secondary antibody or an anti-SF-1 primary antibody and a goat anti-rabbit secondary antibody. Immunoreactivity was quantified using densitometric scanning (UVP Easy system) of the ECL signal on the film.

Electrophoretic Mobility Shift Assay-- Double-stranded oligonucleotides were generated and end-labeled with [-³²P]ATP according to previously described protocols (12). Y1 nuclear extracts (5 µg) were incubated with 50 fmol of radiolabeled oligonucleotide for 30 min on ice in a binding buffer described by Wooton-Kee et al. (12). Antibody supershift assays were performed with 5 µg of Y1 nuclear extract proteins and 10-25 µg of polyclonal SF-1 antibodies. Antibody and nuclear extract were pre-incubated with all components of the binding reaction, except for radiolabeled probe, for 1 h. Probe was then added, and the incubation was continued for a further 30 min. Binding reactions were resolved on a 6% nondenaturing polyacrylamide gel. The gels were dried, and the radioactive bands were visualized by autoradiography.

Sequences of Oligonucleotides Used for Electrophoretic Mobility Shift Assay Probes-- The following sequences of the mouse StAR promoter region were used (12): SF1-1 (135/83), 5'-CTCCCTCCCACCTTGGCCAGCACT-3'; SF1-2 ([minus51/29), 5'-ATGATGCACAGCCTTCCACGGGA-3'; SF1-3 (105/83), 5'-CATTCCATCCTTGACCCTCTGCA-3'. In each case, SF1 consensus binding sequences are indicated in bold, and positions refer to the number of nucleotides upstream of the transcription start site.

Nuclear Extract Preparation-- A confluent T75 flask of Y1 cells per treatment was incubated for 3 h with growth medium alone or with media supplemented with 1 µM FSK with or without 50 µM PD. The cells were homogenized in 5 volumes of buffer containing 10 mM Hepes, 1.5 mM MgCl₂, 10 mM KCl, 0.25 M sucrose, and 0.5 mM dithiothreitol, pH 7.4, and the nuclei were collected by centrifugation (2400 × g, 5 min, 4 °C). 4 volumes of buffer containing 25% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.64 M KCl, and 1 tablet of Complete protease inhibitor mixture were added to each pellet and centrifuged at 18000 × g, 4 °C for 2 min. The supernatants containing nuclear proteins were stored at 80 °C.

Immunodetection of Proteins-- Y1 cells were incubated in the presence or absence of FSK (1 µM) for 15 min⁶ h at 37 °C in a humidified atmosphere of 5% CO₂. Protein samples were prepared as described in Jones et al. (23). Protein extracts (15 µg) were separated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and probed with an antibody that recognizes the 42- and 44-kDa nonphosphorylated and phosphorylated (total) isoforms of ERK and with an antibody that recognizes only the phosphorylated (active) isoforms. StAR immunoreactivity was detected in mitochondria-enriched Y1 cell fractions using an antibody to the mature 30-kDa form of StAR (23). Antibody binding was detected by enhanced chemiluminescence. Total protein in extracts was measured using the Bradford assay (24).

Confocal Immunohistochemistry-- Y1 cells were seeded onto coverslips in 6-well plates at a density of 5 × 10⁴ cells/coverslip. Cells were incubated overnight to adhere and then incubated with DMEM alone (control) or DMEM supplemented with 1 µM FSK for 3 h at 37 °C, before paraformaldehyde fixation (30 min, 4% paraformaldehyde) followed by incubation (1 h) with 2% goat serum in phosphate-buffered saline, 0.01% Triton-X. Fixed cells were incubated with an antibody to the active (phosphorylated) form of ERK2/1 (1/100, 20 h, 4 °C), washed with phosphate-buffered saline, 0.01% Triton and were further incubated (2 h, 20 °C) with an Alexa fluor-488-conjugated second antibody. Antibody binding was visualized using a Bio-Rad 1024 confocal microscope.

Isolation of Y1 Cell Messenger RNA and Reverse Transcription-- Y1 cells were seeded in 24-well plates at a density of 1 × 10⁵ cells/well. After 24 h to ensure adherence, the cells were incubated for 3 h in the presence of DMEM alone (control) with or without 50 µM PD or in the presence of 1 µM FSK with or without 50 µM PD. Cells that were incubated with PD were pre-incubated for 30 min with the inhibitor. mRNA was isolated from the cells using the Dynabeads Oligo(dT)₂₅ kit. Briefly, cells were lysed by adding 300 µl of lysis buffer (supplied with the kit). 20 µl of Dynabeads (6.6 × 10⁷ beads) was then added to the cell lysate, and the mRNA was allowed to anneal to the beads. After repeated washes, the mRNA was eluted from the beads with 12 µl 10 mM Tris (pH 7.5). cDNA was synthesized simultaneously from all mRNA samples using MMLV-RT, Superscript II. Oligo(dT)₁₈ (1 µg) and random 10-mers (1 µg) were added to the mRNA (10 µl), and the mixture was heated (70 °C, 5 min) to remove secondary RNA structure and then cooled on ice. Dithiothreitol (10 mM), dATP, dCTP, dTTP, and dGTP (all 0.5 mM), recombinant ribonuclease inhibitor (80 units, RNAsin), MMLV-RT (200 units), and diethyl pyrocarbonate-treated water were added to make the final volume 20 µl, and the mixture was incubated at 42 °C for 50 min. MMLV-RT was inactivated by heating at 70 °C for 15 min. The cDNA was diluted 20-fold with tRNA (10 µg/ml) and used immediately in PCR reactions or stored at 20 °C for future use. An aliquot of mRNA was not reverse-transcribed and was diluted with tRNA and stored at 85 °C.

PCR Primers-- Forward and reverse PCR primers designed from the mouse StAR sequence were as follows: sense primer, 5'-CAG CAT GTT CCT CGC TAC GT-3'; antisense primer, 5'-CCT TAA CAC TGG GCC TCA GA-3'. The predicted size of the StAR PCR product was 860 base pairs. Forward and reverse GAPDH PCR primers were: sense primer, 5'-CCC ATC ACC ATC TTC CAG GAG C-3'; antisense primer, 5'-CCA GTG AGC TTC CCG TTC AGC-3'. The predicted size of the GAPDH PCR product was 473 base pairs. Forward and reverse cytochrome P450_SCC primers were: sense primer, 5'-AGT GGC AGT CGT CGG GAC AGT-3'; antisense primer, 5'-TAA TAC TGG TGA TAG GCC ACC-3'. The predicted size of the P450_SCC PCR product was 411 base pairs.

Standard Curves-- The product amplified by the GAPDH or P450_SCC primers was separated by agarose gel electrophoresis (1.8% v/v) and visualized by staining with ethidium bromide (0.5 µg/ml). This product was then cut from the gel and spin column-purified using a Qiaquick gel extraction kit, and 10-fold serial dilutions were prepared as standards. StAR standards were prepared by 10-fold serial dilutions of the pCMV6 plasmid containing the 1.4-kilobase mouse StAR cDNA sequence (standards were used over the range of 3.21 ng/µl to 3.21 fg/µl).

Quantitative PCR-- In initial experiments, StAR mRNA levels in Y1 extracts were quantified by competitive PCR as described previously in Burns et al. (25). In subsequent experiments, real-time PCR was performed using a LightCycler rapid thermal cycler system. Reactions were performed in a 10-µl volume containing nucleotides, Taq DNA polymerase, and buffer (all included in the LightCycler-DNA Master SYBR Green I mix), 3 mM MgCl₂, and 0.5 µM primers. Reactions also included either Y1 cDNA standard or a mRNA/tRNA blank. All PCR protocols included a 10-s denaturation step and then continued for 45 cycles consisting of a 95 °C denaturation for 0 s, annealing for 10 s at 55 °C (GAPDH), 58 °C (P450_SCC), 62 °C (StAR), and a 72 °C extension phase for 19 s (GAPDH), 16 s (P450_SCC), or 34 s (StAR). Fluorescence measurements were taken at the end of the 72 °C extension phase. The amplification product of each primer pair was subjected to melting point analysis and subsequent gel electrophoresis to ensure specificity of amplification.

Data Analysis-- Differences between the means were assessed using Student's t test and considered significant when p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cyclic AMP-induced ERK2/1 Activation, StAR Gene Transcription, StAR Protein Accumulation, and Steroid Synthesis-- The mouse adrenocortical Y1 cell line has a reduced capacity for 21-hydroxylation and 11-hydroxylation reactions compared with primary adrenal cells, but it retains intact the cyclic AMP-responsive early rate-limiting stages of steroid synthesis, including the transport of cholesterol to the IMM and its P450_SCC-mediated conversion to pregnenolone (26, 27). The adenylate cyclase activator forskolin (FSK) stimulated steroid production by Y1 cells (Fig. 1A(iii)); increased steroid production was accompanied by increases in StAR mRNA and protein expression (Fig. 1A (i and ii)). FSK also caused a time-dependent increase in ERK2/1 activities (Fig. 1B(i)) without causing any consistent changes in total ERK2/1 immunoreactivity. The results are expressed as a ratio of active ERK2/1 to total ERK2/1 in cell extracts (Fig. 1B(ii)). The immunoblot measurements of ERK2/1 activation were confirmed by confocal immunohistochemistry of active ERK2/1 (Fig. 2). Control cells contained low amounts of active ERK2/1, and this was localized to discrete areas outside the nucleus but associated with the nuclear membrane. On activation with forskolin there was a marked increase in the overall levels of active ERK2/1, and the immunofluorescence had translocated from the extranuclear accumulations to within the nucleus, giving areas of intense intranuclear fluorescence.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Effects of FSK on Y1 cells. Y1 cells were exposed to FSK (1 µM) for various times (0-6 h). Cell extracts were prepared for polyacrylamide gel electrophoresis, and immunoblotting and mRNA extracts were prepared for quantitative PCR. Steroid synthesis during the incubation period was measured by radioimmunoassay. A, StAR mRNA, StAR protein, and steroid production. i, changes in StAR mRNA in Y1 cell extracts were measured by competitive PCR amplification using primers to mouse StAR cDNA and a competitor sequence generated from Escherichia coli DNA. Exposure to FSK (1 µM, 3 h) caused an ~15-fold increase in StAR mRNA. Data are expressed as attograns of mRNA/10,000 Y1 cells. ii, changes in StAR protein expression were assessed by immunoprobing mitochondria-enriched fractions of Y1 cells. Exposure to FSK (1 µM, 3 h) caused an ~12-fold increase (control, 100%; FSK, 1152%) in the accumulation of a 30-kDa immunoreactive StAR protein, as assessed by scanning densitometry. The blot shown is representative of three similar experiments. iii, pregnenolone production by Y1 cells was stimulated by exposure to FSK (1 µM, 3 h, mean ± S.E., n = 8). B, ERK2/1 immunoreactivity. Polyvinylidene difluoride membranes were probed with an antibody that recognizes the phosphorylated (active) forms of ERK1 and ERK2, migrating with apparent molecular masses of 44 and 42 kDa, respectively (i). The same membrane was then stripped and reprobed with an antibody that recognizes total (phosphorylated and nonphosphorylated) ERK2/1 immunoreactivities. The graph represents active ERK2/1 expressed as a ratio to the total ERK2/1 in each sample (ii). Exposure to FSK caused the activation of ERK2/1 in these cell extracts.

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Effects of FSK on the localization of ERK2/1 in Y1 cells. Y1 cells were seeded onto coverslips and incubated with normal media or medium supplemented with 1 µM FSK for 3 h. Localization of active ERK2/1 in the cells was assessed using a primary antibody to the active forms of ERK2/1 and a fluorescently labeled second antibody that could be visualized by confocal microscopy. Images were taken under a 40× oil objective lens. The figure clearly shows that FSK causes an increase in the activation of ERK2/1, with the movement of the kinases from extranuclear regions to within the nucleus.

Inhibition of ERK2/1 Activation Blocks Cyclic AMP-induced StAR Gene Transcription, StAR Protein Accumulation, and Steroid Synthesis-- ERK2/1 are activated by phosphorylation by a dual specificity tyrosine/threonine kinase, MAPK/ERK kinase (MEK), and therefore ERK2/1 activation can be selectively blocked by compounds that inhibit MEK activity. Fig. 3A demonstrates that cyclic AMP-induced steroid production was fully inhibited by two structurally dissimilar inhibitors of MEK, PD098059 (28) and UO126 (29), when used at concentrations that have been shown to inhibit MEK activity. The inhibition of steroid production by MEK inhibitors was not confined to Y1 cells (Fig. 3A(i)), because both MEK inhibitors totally inhibited FSK-stimulated steroid production by the mouse testicular MA-10 cell line (20) as shown in Fig. 3A(ii). Neither MEK inhibitor had any significant effect on unstimulated steroid production by Y1 cells or MA-10 cells (Y1 cells: basal, 30 ± 4 pmol/10⁵ cells; PD, 21 ± 3 pmol/10⁵ cells; UO, 46 ± 7 pmol/10⁵ cells; MA-10 cells: basal, 16 ± 5.7 pmol/10⁵ cells; PD, 9 ± 3 pmol/10⁵ cells; UO, 15 ± 4 pmol/10⁵ cells). In contrast to their effects on cyclic AMP-dependent steroid synthesis, neither MEK inhibitor had any effect on steroid production from cells supplied with the water-soluble cholesterol analogue 22(R)-hydroxycholesterol (Fig. 3A(iii)). 22(R)-Hydroxycholesterol passes unassisted from the OMM to the IMM, where it is converted to pregnenolone by P450_SCC and thus supports steroid synthesis independently of StAR. These observations demonstrate that MEK inhibitors do not inhibit cyclic AMP-dependent steroid production by interfering with P450_SCC function and identify the site of action of ERK2/1 as proximal to cholesterol delivery to the IMM. FSK-induced accumulation of StAR protein in Y1 cells was assessed by immunoblotting extracts of Y1 cells incubated for 3 h with FSK alone or with FSK in the presence of the MEK inhibitors PD or UO (Fig. 3B(i-iv). MEK inhibition reduced FSK-induced accumulation of StAR protein to near basal levels. StAR mRNA levels in Y1 cell extracts were measured by real-time quantitative RT-PCR using a standard curve generated with a pCMV6 plasmid containing the 1.4-kilobase mouse StAR cDNA sequence (r = 0.99). A standard curve for the amplification of P450_SCC was constructed from product amplified by the primers and recovered and purified from an agarose gel (r = 1.00). In each case the expression of mRNA was normalized against the content of GAPDH mRNA in the same extracts. Agarose gel electrophoresis and melting point analyses of products revealed the presence of a single species in each sample (Fig. 4A(i-iv). FSK caused a marked (~15-fold) increase in the levels of StAR mRNA (Fig. 4B(i)), of a comparable magnitude to that measured by competitive PCR in similar experiments (Fig. 1A(i)). Inhibiting ERK2/1 activation by using the MEK inhibitor PD produced a significant reduction in the effects of FSK on StAR mRNA levels (Fig. 4B(i)). This reduction is consistent with the effects of MEK inhibition on StAR protein expression (Fig. 3B) and on PKA-dependent steroid production (Fig. 3A(i and ii)). FSK also enhanced levels of P450_SCC mRNA in Y1 cells (~3-fold), but MEK inhibition had no effect on the levels of P450_SCC mRNA (Fig. 4B(ii)), suggesting a selective effect of MEK inhibition on the regulation of StAR expression rather than a general reduction in gene transcription.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Effects of ERK2/1 inhibition on steroid synthesis and StAR protein. A, effect of ERK2/1 inhibition on steroid synthesis. iiii, steroid production by adrenocortical Y1 cells (i and iii) or testicular MA-10 cells (ii) was measured after incubation for 3 h at 37 °C. Steroid production was stimulated by the presence of FSK (1 µM) (i and ii) or 22(R)-hydroxycholesterol (22ROHC) (iii) in the presence or absence of the MEK inhibitors PD (50 µM) or UO (10 µM). The results are expressed as % of basal secretion in the absence of any treatment (30 ± 4 pmol of pregnenolone/105 Y1 cells; 16.7 ± 5.7 pmol of pregnenolone/105 MA-10 cells). Bars show mean ± S.E., n = 8; ***, p < 0.01 versus stimulated steroid production. B, effect of MEK inhibition on StAR protein levels. MEK inhibition reduced FSK-induced accumulation of StAR protein in Y1 cells as assessed by immunoblotting extracts of Y1 cells incubated (3 h, 37 °C) under unstimulated conditions (i) in the presence of 1 µM FSK (ii), of 1 µM FSK and 50 µM PD (iii), or of 1 µM FSK and 10 µM UO (iv) (control, 100%; FSK, 1385%; FSK + PD, 333%; FSK + UO, 428%).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effects of ERK2/1 inhibition on mRNA expression in Y1 cells. Real time PCR using SYBR Green I fluorescence to measure product accumulation. A, product analysis. Each of the primer pairs amplified a single product of the appropriate predicted lengths as assessed by agarose gel electrophoresis and ethidium bromide staining (i). Melting point analysis revealed a single and specific product for GAPDH (ii) and P450SCC (iv). However, melting point analysis of StAR product generated with cell cDNA or plasmid gave two peaks (iii), although a single band was observed by gel electrophoresis (i). Sequencing of the full-length StAR plasmid, which generated two peaks, revealed a single sequence, which showed 100% identity to mouse StAR. Double peaks may be caused by a high guanine and cytosine content in an area of the product sequence, however this was not obvious on analysis of this sequence. Another possibility is the formation of secondary structures that may give rise to more than one melting peak. Mouse Y1 cDNA and mouse GAPDH cDNA were compared (ii) to show that there are no melting point differences between these standards. B, mRNA quantification. i, FSK (3 h, 1 µM) caused an ~15-fold increase in the accumulation of StAR mRNA and the effects of FSK were significantly inhibited by the presence of PD (*, p < 0.05, n = 3 separate experiments). ii, in similar experiments, FSK caused an ~3-fold increase in P450SCC mRNA but this effect was not inhibited by the presence of PD.

Effects of ERK2/1 Inhibition on SF-1 Phosphorylation-- The orphan nuclear transcription factor, SF-1, is known to play a major role in regulating the transcription of the StAR gene (15). FSK was seen to cause an increase in the phosphorylation of SF-1 as assessed by SF-1 immunoprecipitation and phosphoserine immunoblotting (Fig. 5). However, MEK inhibition with PD caused a reduction in FSK-stimulated SF-1 phosphorylation levels (Fig. 5). The results were quantified by densitometric scanning and normalized by stripping the membrane and reprobing it for total SF-1 (Fig. 5B). The graph in Fig. 5B represents the level of phosphorylated SF-1 expressed as a ratio to the level of total SF-1 in each sample. In the absence of FSK stimulation, MEK inhibition had no effect on basal SF-1 phosphorylation (data not shown). These results are consistent with the observed reduction in StAR protein accumulation upon MEK inhibition.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   Effect of ERK2/1 inhibition on SF-1 phosphorylation levels. A, Y1 cells were incubated in growth medium (i), 1 µM FSK (ii), and 1 µM FSK + 50 µM PD (iii) for 3 h, and protein fractions were prepared. The 53-kDa SF-1 protein was isolated from these fractions by immunoprecipitation, transferred to a nitrocellulose membrane, and probed with an anti-phosphoserine antibody. The same membrane was stripped and reprobed with an anti-SF-1 antibody to normalize the results. Immunoreactive bands from both the total SF-1 and the phosphorylated SF-1-only blots were quantified by densitometric scanning, and phosphorylated SF-1 was expressed relative to total SF-1 levels. Panel B clearly demonstrates that FSK stimulates SF-1 phosphorylation and that this increase is reduced upon MEK inhibition (control, 100%; FSK, 732%; FSK + PD, 293%).

Effects of ERK2/1 Inhibition on SF-1 Binding-- The DNA binding site of SF-1 recognizes the CAXCCTT motif (where X represents any nucleotide) in genomic DNA in the StAR promoter region. To date, three separate regions encoding this consensus sequence and having high affinity for SF-1 have been identified in the region upstream of the StAR gene transcription start site and have been called SF-1-1, SF-1-2, and SF-1-3 (12, 30). Our results demonstrate that FSK treatment of Y1 cells increases the binding of Y1 cell nuclear proteins to synthetic double-stranded oligonucleotides corresponding to SF-1-1, SF-1-2, and SF-1-3 as shown in Fig. 6. At least part of this nuclear protein binding to SF-1 consensus DNA sequences could be attributed to SF-1 as determined by antibody supershift assays (Fig. 6A). Furthermore, the FSK-induced binding of SF-1 to sequences in the StAR gene promoter region was dependent upon the activation of ERK2/1, because the effects were fully reversed by the presence of the MEK inhibitor, PD (Fig. 6, B-D).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effects of ERK2/1 inhibition on SF-1 binding to the StAR gene promoter. Y1 cells were incubated in growth medium with or without 1 µM FSK and 50 µM PD for 3 h. Electrophoretic mobility shift assays were performed to measure binding of SF-1 in nuclear extracts to each of three (SF-1-1, SF-1-2, and SF-1-3) radiolabeled probes of regions of the StAR promoter that contain consensus SF-1 binding sequences. In each case antibody supershift assays were performed to locate the position of the SF-1-DNA complex. Panel A shows an example of an antibody supershift assay using a probe to the SF-1-2 region of the StAR promoter. Panels B-D show that FSK stimulation caused an increase in binding of SF-1 to each of the three regions of the StAR promoter sequence. Furthermore, this FSK-stimulated increase was ERK-dependent because treatment with PD caused a depletion in binding. The results shown are representative of two separate experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mammalian steroid-producing tissues share a number of common general features. Steroid production is normally regulated by extracellular trophic signals (e.g. LH, FSH, ACTH), which act through specific cell surface receptors to activate intracellular effector systems and thus increase cholesterol metabolism (1, 2). The enzymic events involved in the conversion of cholesterol to the various steroid hormones share common elements that are now fairly well understood (3). Similarly, evidence is accumulating that the control of mitochondrial cholesterol transport in most steroid-producing cells involves the expression of StAR protein and its import into the mitochondria (3).

A number of recent studies have suggested that the MAPK cascade is involved in cellular regulation in steroidogenic tissues, although many of the observations reported to date appear to be contradictory. MAPKs are known to be involved in proliferative responses in many tissues (31), and there is evidence that this is an important mechanism in adrenal tissue (32). Thus, the activation of ERK2/1 has been implicated in the mitogenic responses of human H295R or mouse Y1 adrenocortical cells to angiotensin ll or ACTH, respectively (33-35), although inhibition of other MAPK activities has also been reported during ACTH-induced cell-cycle progression in Y1 cells (36). A similar confusion surrounds the potential role(s) of ERK2/1 as transduction elements in the regulation of steroidogenesis. Thus, for example, the anterior pituitary trophic hormones LH and FSH are reported to activate ERK2/1 and enhance steroid production in ovarian cells (37, 38), but ERK activation has also been associated with the inhibition of agonist-induced steroidogenesis in granulosa-derived cell lines (39). Similarly, the activation of ERK2/1 has been linked to enhanced steroid production in human granulosa-luteal cells, and cAMP-induced steroidogenesis was inhibited by inhibitors of ERK2/1 (40). However, the inhibitory effects of prostaglandin F₂ (41) or gonadotrophin-releasing hormone agonists (42) on steroidogenesis in this tissue have also been attributed to the activation of ERK2/1. There are several likely explanations for these apparently contradictory reports. A prime reason may be the existence of multiple transduction pathways coupled to cell surface receptors, with differences in receptor-effector coupling between tissues, cell lines, and species. One of the major intracellular effector systems in the regulation of steroidogenesis is the adenylate cyclase/cyclic AMP system, although this may not be the sole mechanism through which trophic hormones regulate steroid production in their target tissues (43). In the present study we have circumvented receptor expression, agonist binding, and receptor-effector coupling by using forskolin, an adenylate cyclase activator, to directly elevate intracellular cyclic AMP and thus allow us to focus on the downstream events in the signaling cascade. We chose mouse adrenocortical Y1 cells for these studies because an in vitro cell line can provide the large amount of starting material required for the immunodetection of StAR and for the immunoprecipitation and analysis of the phosphorylation state of transcription factors. In addition, Y1 cells offer an excellent adrenocortical model for the present experiments because they retain intact the cyclic AMP-responsive early rate-limiting stages of steroid synthesis, including the transport of cholesterol to the inner mitochondrial membrane and its P450_SCC-mediated conversion to pregnenolone (26, 27).

An additional reason for disparate conclusions between studies may be the differences in the experimental protocols, particularly with respect to different time courses of the events being measured. Thus, in other tissues, ERK2/1 activation tends to be a rapid and often transient event (44), whereas it normally takes longer for increased steroid production to be detectable, partly because of the requirements for increased expression of the StAR protein (1-6). In the present study we measured cyclic AMP-dependent ERK2/1 activation over the same time course as enhanced steroid production, to demonstrate that the timing of both events is consistent with ERK2/1 activation being involved in initiating the steroidogenic response. Our results clearly demonstrate that cyclic AMP-induced steroidogenesis in Y1 cells is dependent on activation of the ERK2/1 signaling cascade and that steroid production is accompanied by a prolonged activation of ERK2/1. Thus activation of adenylate cyclase induced the activation of ERK2/1 in Y1 cells and the translocation of active ERK2/1 into the nucleus; we were able to link this activation to steroid synthesis because ERK2/1 inhibition reduced cyclic-AMP-dependent steroid production. Furthermore, our demonstration of similar effects in the adrenocortical Y1 cell line and the testicular MA10 cell line suggests that the involvement of ERK2/1 in steroidogenic responses to cyclic AMP may be a general effect rather than a cell type-specific event.

The importance of ERK2/1 activation in steroid production was confirmed by our quantitative measurements of the expression of StAR mRNA and protein in Y1 cells. It is now well established that StAR protein plays a major role in regulating steroid synthesis (1-5) and that the stimulation of steroid production by agonists or by pharmacological elevations in cyclic AMP is dependent upon enhanced transcription of the StAR gene, elevations in StAR mRNA, and the intracellular accumulation of StAR protein (3, 5, 25). Our results confirm the importance of StAR in steroid production, because forskolin caused a rapid and prolonged increase in the levels of StAR mRNA and protein in Y1 cells, associated with increased production of steroid. More importantly, our data show that ERK2/1 activation is required for the effects of cyclic AMP on StAR expression, because preventing the activation of ERK2/1 by inhibiting their upstream activator, MEK-1, was alone sufficient to reduce the cyclic AMP-dependent increases in StAR mRNA and protein. This effect was selective for StAR mRNA, because inhibition of MEK-1, and thus of ERK2/1, did not affect the accumulation of P450_SCC mRNA induced by forskolin. These observations also imply multiple effects of cyclic AMP on the regulation of gene expression in Y1 cells, some of which (e.g. StAR) are mediated through ERK2/1, whereas others (e.g. P450_SCC) do not require the activation of ERK2/1.

Our current results therefore explain the inhibitory effects of two structurally distinct MEK-1 inhibitors on cyclic AMP-induced steroid production in two different cell lines and are consistent with a transduction sequence in which increased intracellular cyclic AMP leads to the phosphorylation and activation of MEK-1, presumably through activation of PKA (45). Activated MEK-1 in turn phosphorylates and activates ERK2/1, which accumulates in the nucleus and increases the transcription of the StAR gene and hence the accumulation of StAR mRNA and protein. Because StAR protein is rate-limiting for cholesterol transport into the mitochondria (1-6), this sequence of events inevitably results in enhanced delivery of cholesterol to the P450_SCC complexes inside the mitochondria and to increased production of steroids. This sequence of events also explains the lack of effect of inhibitors of MEK-1 on steroid production from cells supplied with 22(R)-hydroxycholesterol. This cholesterol analogue is water-soluble and is therefore not dependent on the ERK2/1-driven expression of StAR to enable it to reach the inner mitochondrial membrane and act as a substrate for the P450_SCC.

In other tissues, activated ERK2/1 modify cellular function primarily by regulating the expression of numerous genes through phosphorylating a variety of transcriptional regulators (18, 46). Our measurements of the cellular localization of active ERK2/1 by confocal immunohistochemistry demonstrated that the low level of active ERK2/1 found in unstimulated cells was localized to small extranuclear "caps" associated with specific areas of the nuclear membrane. Activation of Y1 cells by forskolin caused a large increase in active ERK2/1 in the intranuclear compartment, consistent with an intranuclear location for the protein substrates of ERK2/1 in Y1 cells. The complex mechanisms regulating basal and stimulated transcription of the StAR gene are not fully understood, although the cyclic AMP-responsive region is thought to be coded within the first 254 base pairs of the StAR promoter (47), and several activating factors have been implicated in the regulation of StAR gene transcription (48-50). Among these is the 53-kDa SF-1, the presence of which appears to be an absolute requirement for StAR gene expression, because SF-1 knockout mice fail to express StAR mRNA (8). SF-1 contains a serine residue (Ser-203) residing within the ERK2/1 consensus phosphorylation sequence (PX_n(S/T)P) (19) suggesting that the functional status of SF-1 can be modified by ERK2/1; our direct measurements of ERK2/1-dependent SF-1 phosphorylation support this as an important regulatory mechanism in cyclic AMP-induced steroid production.

The promoter region of the StAR gene contains at least three sites encoding potential binding sites for activated SF-1 (12), and our results suggest that SF-1 binding to these sites may be the mechanism through which ERK2/1 activation leads to enhanced steroid production. Thus, we have demonstrated that the ERK2/1-dependent phosphorylation of SF-1 enhances its ability to bind to all three sites in the StAR promoter, supporting a role for ERK2/1-dependent phosphorylation of SF-1 as an important regulator of activational activity. These observations do not rule out an additional regulatory role for direct phosphorylation of SF-1 by PKA (51-53) in the regulation of StAR gene expression nor are they inconsistent with the loss of SF-1 trans-activational activity in PKA-deficient cell lines (54, 55), but they do suggest that the ERK2/1-dependent event is obligatory for cAMP-induced StAR expression and steroid synthesis.

In summary, the current study provides a direct link from PKA activation through the MEK/ERK cascade to the phosphorylation of SF-1, enhanced binding to the StAR promoter region, increased expression of StAR protein, and enhanced steroid production in adrenal cells.

	ACKNOWLEDGEMENTS

We are grateful to Ron Senkus for assisting with the confocal microscopy.

	FOOTNOTES

^* This work was supported by Wellcome Trust Grant 054789/Z/98/Z and a research and development grant from the Guy's and St. Thomas's Charitable Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ These authors share first authorship of this study.

^¶ A Biotechnology and Biological Sciences Research Council postgraduate student.

^** To whom correspondence should be addressed: Endocrinology and Reproduction Research Group, New Hunt's House, 3rd Fl. North, GKT School of Biomedical Sciences, King's College London, London SE1 1UL, UK. Tel.: 44-207-848-6273; Fax: 44-207-848-6280; E-mail: peter.jones@kcl.ac.uk.

Published, JBC Papers in Press, June 15, 2001, DOI 10.1074/jbc.M102063200

	ABBREVIATIONS

The abbreviations used are: PKA, protein kinase A; StAR, steroidogenic acute regulatory protein; ERK, extracellular signal-regulated kinase; SF-1, steroidogenic Factor 1; P450_SCC, cytochrome P450 cholesterol side chain cleavage system; FSK, forskolin; MAPK, mitogen-activated protein kinase; MEK, MAPK-ERK kinase; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane; PD, PD098059; UO, UO126; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ACTH, adrenocorticotropic hormone; LH, luteinizing hormone; FSH, follicle-stimulating hormone.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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