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Endocrinology Vol. 144, No. 9 3934-3942
Copyright © 2003 by The Endocrine Society

Transactivation of Steroidogenic Acute Regulatory Protein in Human Endometriotic Stromal Cells Is Mediated by the Prostaglandin EP2 Receptor

H. Sunny Sun, Kuei-Yang Hsiao, Chih-Chao Hsu, Meng-Hsing Wu and Shaw-Jenq Tsai

Institute of Molecular Medicine (H.S.S., S.-J.T.), Department of Physiology (K.-Y.H., C.-C.H., S.-J.T.), Department of Obstetrics and Gynecology and Institute of Clinical Medicine (M.-H.W.), National Cheng Kung University Medical College, Tainan 70101, Taiwan, Republic of China

Address all correspondence and requests for reprints to: Shaw-Jenq Tsai, Ph.D., Department of Physiology, National Cheng Kung University Medical College, Tainan 701, Taiwan, Republic of China. E-mail: seantsai{at}mail.ncku.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Steroidogenic acute regulatory protein (StAR) regulates the first committed step in the biosynthesis of steroids, and thus aberrant expression of StAR in endometriotic implants plays a critical role in the etiology of endometriosis. However, the mechanism responsible for abnormal expression of StAR in ectopic endometriotic tissues remains unknown. In the present study, we demonstrate that prostaglandin (PG) E2 stimulates StAR protein expression at the cellular and molecular levels. PGE2 caused a rapid increase in StAR expression that involves activation of the EP2 receptor-coupled protein kinase A pathway. Activation of EP2 receptor-induced phosphorylation of ERK and cAMP response element binding protein (CREB). However, activation of ERK did not involve in CREB phosphorylation or concomitantly StAR expression. Phosphorylation of CREB induced by PGE2 increased the recruitment of CREB binding protein and thus histone H3 acetylation. Chromatin immunoprecipitation experiments showed that acetylated histone H3 bound to the proximal region of the StAR promoter was increased after 30 min treatment with PGE2, and this was mirrored by an increase in nascent StAR RNA transcription. Treatment with the histone deacetylase inhibitor, tricostatin A, enhanced PGE2-induced nascent StAR RNA transcription. We conclude that increased histone H3 acetylation involving the EP2 receptor, protein kinase A, CREB, and CREB binding protein is responsible for PGE2-induced StAR gene activation in endometriotic stromal cells. Our current report may provide new insights in understanding mechanism of abnormally local production of estrogen and the etiology of endometriosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ENDOMETRIOSIS IS THE growth of endometrial stroma and glands outside the uterine cavity. A variety of theories have been proposed to account for this disorder, including a genetic predisposition, an aberrant immunological response, and an altered peritoneal environment (1, 2, 3), but the precise mechanism responsible for the formation and spread within the pelvic cavity of this disease is still unclear. The cells in ectopic endometriotic lesions are histologically similar to their putative eutopic precursors, but there are significant biochemical differences between these two tissues. For example, the aberrant expression of steroidogenic acute regulatory protein (StAR) and P450 aromatase by ectopic endometriotic stromal cells, leading to abnormal production of ovarian steroids, plays an important role in the development of endometriosis because this disorder is highly estrogen dependent (4, 5, 6).

There is a growing body of evidence for a strong association between prostaglandin (PG)E2 levels and the development and severity of endometriosis. Increased PGE2 concentrations in the peritoneal fluid of women with endometriosis have been reported (7, 8). A marked increase in amounts of cyclooxygenase, the enzyme that controls the first committed step of PG production, is seen in peritoneal macrophages from women with endometriosis (8). Increased amounts of cyclooxygenase-2 have also been reported in ectopic and eutopic endometria of women with endometriosis (9). PGE2 is a potent inducer of StAR and aromatase gene expression in ectopic endometriotic stromal cells (4, 10). PGE2-induced aromatase promoter activity is mediated via the protein kinase A (PKA) signaling pathway (10, 11), but the mechanisms responsible for PGE2-stimulated StAR gene activation in endometriotic stromal cells have not been investigated.

PGE2 binds to G protein-coupled plasma membrane receptors. Four distinct PGE2 receptors (EP1–4), encoded by different genes, have been identified in human tissues (12); EP1 and EP3 further undergo alternative mRNA splicing, generating different isoforms (12). Binding of PGE2 to EP2 or EP4 activates adenylyl cyclase and the PKA signaling pathway via Gs activation. EP1 is a Gi-coupled receptor and its activation leads to an increase in the intracellular free calcium levels and/or PKA inhibition. Binding of PGE2 to EP3 causes intracellular calcium mobilization, activation of PKA, protein kinase C (PKC), and MAPK, or inhibition of the PKA-signaling pathway (12). Thus, PGE2 can activate several signaling pathways, depending on the specific EP receptor to which it binds.

In a previous study (4), we reported that endometriosis is associated with aberrant StAR mRNA and protein expression and that PGE2 induces StAR gene expression in stromal cells derived from ectopic endometriotic implants but not from eutopic endometrium; however, we did not characterize the mechanisms responsible for PGE2-induced StAR gene activation. In the present study, we determined the receptor and downstream signaling pathway involved in mediating the action of PGE2 and in the remodeling of chromatin structure at the StAR promoter induced by PGE2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Primers, PGs, agonists, antibodies, and reagents
The primers used, which were designed on the basis of published human gene sequences using Primer3 software (13) or the primer design program of LightCycler (Roche Molecular Biochemicals, Mannheim, Germany), are listed in Table 1Go. PGE2, butaprost (a specific EP2 agonist), sulprostone (an EP3 agonist), PGE1 alcohol (an EP2/EP4 agonist) (14), AH6809 (an EP1/EP2 antagonist), and anti-PGE2 antibody were purchased from Cayman Biochemical Co. (Ann Arbor, MI). The anti-StAR antiserum was a generous gift from Dr. Jerome F. Strauss III (University of Pennsylvania, Philadelphia, PA). The antibodies against cAMP response element binding protein (CREB), phospho-CREB (ser133P), acetylated histone H3, histone H3, and CREB binding protein (CBP) were from Upstate Biotechnology Inc. (Waltham, MA), the anti-CREB monoclonal antibody (sc-271) was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), and the anti-ERK1/2, anti-phospho-ERKs, anti-P38, and antiphospho-P38 antibodies from Cell Signaling Technology (Beverly, MA). The Taq DNA polymerase, fetal bovine serum (FBS), DMEM/F-12, antibiotics, and 1-kb DNA ladders were from Gibco BRL (Gaithersburg, MD), the Magnetight Oligo(dT) particles from Novagen (Madison, WI), the polyvinyl difluoride membranes from Millipore Co. (Bedford, MA), and the Western blot chemiluminescence reagents from NEN Life Science Products (Boston, MA).


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  		TABLE 1. Primer sequences and related information for primer sets used in this study

 
Stromal cell preparation and culture
Tissues from ovarian endometrioma (n = 12; six proliferative and six secretory phase samples) and pelvic endometriotic implants (n = 10; six proliferative and four secretory phase samples) were collected and processed for stromal cell culture. All endometriosis samples were graded according to the revised classification of the American Society of Reproductive Medicine (15) and were histologically confirmed. All patients were of reproductive age, with normal menstrual cycles. Human Ethics Committee approval was obtained from the Clinical Research Ethics Committee at the National Cheng Kung University Medical Center, and informed consent was obtained from each patient.

Endometriotic stromal cells were dissociated and purified using a published procedure (16) with minor modifications (4). Stromal cells were cultured at 37 C in DMEM/F12 supplemented with 10% FBS and streptomycin-penicillin cocktail in a humidified atmosphere with 5% CO2. The medium was changed every other day until the cells reached confluence, when they were trypsinized and subcultured in DMEM/F12 containing 10% FBS and antibiotics either in 24-well culture plates (2 x 104 cells/well) for RNA isolation experiments or in 6-cm petri dishes (1 x 105 cells/dish) for protein analysis. When cells reached 70% confluence, they were placed in serum-free phenol red-free DMEM/F-12 medium for 36 h and then subjected to the different treatment regimens.

In vitro studies
Endometriotic stromal cells were first stimulated with PGE2 (1 µM) for 0, 1, 2, 4, or 8 h to determine the incubation time giving maximal StAR mRNA and protein expression. The time point selected was used for subsequent experiments. After subjection to various treatment regimens, cells in 24-well plates were directly lysed using RNA lysis buffer [4 M guanidinium isothiocyanate; 10 mM Tris-HCl (pH 8.0); 0.5% sodium dodecyl sulfate (SDS); and 1% dithiothreitol] and RNA isolated as described previously (4, 17, 18, 19), or, for Western blot analyses, cells in petri dishes were homogenized in Tris-sucrose-EDTA buffer (10 mM Tris, 250 mM sucrose, and 0.1 mM EDTA, pH 7.4), centrifuged at 600 x g for 30 min at 4 C to remove debris. Protein concentrations were determined by the Lowry method (20). In inhibition studies, the inhibitors were added to the cells 30 min before the inducers.

Determination of steady-state concentrations of StAR mRNA and StAR protein
The detailed procedure for the standard curve quantitative, competitive RT-PCR (QC-RT-PCR) has been described previously (21) and is used routinely in our laboratory (4, 17, 18, 19). For Western blotting, equal amounts of proteins were resolved by SDS-PAGE and transferred to a polyvinyl difluoride membrane, which was then blocked with 5% skim milk and incubated with specific antibodies. After washing and incubation with horseradish peroxidase-conjugated second antibodies, bound antibody was detected using the enhanced chemiluminescence system. The blots were then stripped with stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl, pH 6.7) and reprobed with different antibodies.

Chromatin immunoprecipitation (ChIP) and real-time PCR
The protocol used was that described by Upstate Biotechnology Inc. with modifications. In brief, histones and DNA in vehicle- or PGE2-treated cells (1 x 106 cells) were cross-linked by incubation for 10 min at 37 C with a final concentration of 1% formaldehyde. After aspiration of the formaldehyde, the cells were washed twice with ice-cold PBS containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 1 µg/ml each of aprotinin and pepstatin A) and scraped into a conical tube, centrifuged for 5 min at 500 x g at 4 C, resuspended in 400 µl lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1), and placed on ice for 10 min. Genomic DNA was sheared to lengths of 0.2–1 kb by sonicating the cell lysate, and then debris was removed by centrifugation, and the supernatant diluted with 1.1 ml ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, 16.7 mM NaCl, and proteinase inhibitors, pH 8.0). Five percent (75 µl) of the diluted lysate was kept for input control. The chromatin solution was precleared with salmon sperm DNA and protein A-Sepharose, and then antiacetylated histone H3 antibody was added to the supernatant fraction and the mixture incubated overnight at 4 C with rotation and then with 60 µl salmon sperm DNA/protein A agarose slurry for 1 h at 4 C with rotation. Normal rabbit Ig was used instead of specific antibody in the negative control.

The protein A agarose/antibody/histone complex was pelleted by gentle centrifugation (400 x g at 4 C for 1 min) and the supernatant carefully aspirated for later use as the unbound, nonspecific DNA control. The pellet was washed sequentially (3–5 min per wash) on a rotating platform with 1 ml each of low-salt washing buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, 150 mM NaCl, pH 8.0), high-salt washing buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, 500 mM NaCl, pH 8.0), LiCl washing buffer (0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0), and 1x TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). After the final wash, the pellet was eluted by resuspension in freshly made elution buffer (1% SDS and 50 mM NaHCO3), followed by centrifugation. Twenty microliters of 5 M NaCl were added to the supernatant and the mixture incubated for 4 h at 65 C to reverse the histone-DNA cross-linking, then the DNA was amplified using nested PCR. The first round PCR was performed using 4 µl of bound or nonbound DNA template, 400 nM of each primer (amplification region -34 to -333), and the FastStart DNA Master Mix (Roche). The cycling conditions were 95 C for 10 min, followed by 20 cycles of 95 C for 30 sec, 59 C for 30 sec, and 72 C for 30 sec. Two microliters of diluted PCR products (1:100 dilution) were then subjected to a second round of amplification in the presence of FastStart DNA Master SYBR Green I Mix and nested primers (from -101 to -261) for 45 cycles. The reaction data are expressed as the number of cycle thresholds, which is the PCR cycle number at which the fluorescent signal in each reaction reaches a preset threshold above background. A dissociation curve was created using the built-in melting curve program of LightCycler (Roche) to confirm the presence of a single PCR product. To determine the specificity of histone H3 acetylation induced by PGE2, a second set of primer that amplifies distal region of StAR promoter (from -4157 to -4280) was designed and used to perform the above-mentioned analysis.

Progesterone assay
Progesterone levels in culture supernatants were measured by a competitive ELISA procedure as described previously (4, 19). The sensitivity (80% bound) was 0.16 ng/ml with intra- and interassay coefficients of variation of 4.2 and 9.2%, respectively.

Determination of nascent RNA by real-time RT-PCR
Specific primers amplifying intron-containing nascent RNA were designed according to the human StAR sequence. The forward primer is located across the junction of intron 2 (12 bases) and exon 3 (5 bases), and the reverse primer is within exon 3 (Table 1Go). This approach allowed the specific amplification of newly synthesized, unspliced RNA. To limit the possibility of detecting genomic DNA, total RNA was subjected to DNase I treatment before reverse transcription, and negative controls omitting reverse transcriptase were performed. All RNA samples were quantified by real-time RT-PCR using LightCycler and SYBR Green I Mix. Ribosomal 18S RNA was used as the internal control, and the Star cycle threshold normalized against that of 18S rRNA.

Statistical analysis
All data are expressed as the mean ± SEM and were analyzed by one-way ANOVA, followed by Dunnett’s test if significant differences were found, using the general linear model of the SAS program (22). Differences were classed as significant when two-tailed analysis gave P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time-dependent induction of StAR expression by PGE2
Steady-state concentrations of StAR mRNA in ectopic endometriotic stromal cells were induced by PGE2 in a time-dependent manner, maximal levels being seen after 2 h of treatment (Fig. 1Go, A and B). The concentration of StAR mRNA in the control cells at the various time points did not differ significantly, and all values were therefore combined to give the 0 h control value. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA concentrations were similar in control and PGE2-treated cells at all time points examined (Fig. 1AGo and data not shown). The PGE2-induced increase in StAR mRNA levels was followed by an increase in StAR protein levels (Fig. 1Go, C and D), these being maximal after 4 h of treatment and then falling slightly, but remaining elevated, at 8 h of treatment (Fig. 1DGo).



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  		FIG. 1. PGE2 transactivates StAR gene expression in a time-dependent manner. Cells were treated with 1 µM PGE2 (P) or vehicle control (C) for 1–8 h; 0, zero h control. A, Representative gel pictures for StAR (upper panel) and GAPDH (lower panel) after standard curve quantitative, competitive RT-PCR (QC-RT-PCR) amplification. NC, Negative control without reverse transcriptase. B, Quantification of StAR mRNA levels from four independent experiments using different batches of cells. Asterisks indicate a value significantly different from that for the pooled control (shown as 0 h). C, Representative Western blot, showing marked induction of StAR protein by PGE2 (1 µM). D, Mean StAR proteins levels in four independent experiments using different batches of endometriotic stromal cells. Asterisks indicate a value significantly different from that for the pooled control (shown as 0 h).

 
Induction of StAR expression by PGE2 is mediated by the PKA signaling pathway
As shown in Fig. 2AGo, only three of the four EP receptors, namely EP2, EP3, and EP4, were detected in endometriotic stromal cell preparations by RT-PCR, the EP1 transcript not being detected even after 35 cycles of PCR amplification. To address which receptor-mediated signaling pathway was responsible for PGE2-induced StAR expression, we treated endometriotic stromal cells with the selective EP2 agonist, butaprost (1 or 10 µM); EP3 agonist, sulprostone (1 or 10 µM); or the EP2/EP4 agonist, PGE1 alcohol (1 or 10 µM). PGE1 alcohol and butaprost induced significant StAR expression, whereas sulprostone had no effect (Fig. 2BGo). To clarify which receptor subtype was responsible for PGE1 alcohol-induced StAR expression, an EP1/EP2 antagonist (AH6809, 80 µM) was administrated 30 min before the addition of PGE2, butaprost, or PGE1 alcohol. Pretreatment with AH6809 completely blocked StAR expression stimulated by PGE2, butaprost, and PGE1 alcohol (Fig. 2CGo) indicating EP2 but not EP4 was responsible for PGE1 alcohol-transduced action. Treatment of endometriotic stromal cells with either 8-bromo-cAMP (a PKA activator, 0.5 mM) or forskolin (an adenylyl cyclase activator, 100 µM) resulted in significant induction of StAR expression, but phorbol 12-myristate-13-acetate (a PKC activator, 10 nM) or ionomycin (a calcium ionophore, 1 µM) had no effect (Fig. 3AGo). Inactivation of PKA activity using selective inhibitors, H89 (10 µM) or protein kinase I (PKI) (25 µM), abrogated the induction of StAR expression by either PGE2 or butaprost (Fig. 3BGo).



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  		FIG. 2. EP2 receptor is responsible for PGE2-induced StAR expression. A, Representative gels showing expression of different EP receptors (EP1–4) and GAPDH in five different preparations of endometriotic stromal cells (lanes 1–5) as detected by RT-PCR. Twelve batches of cells were examined with similar results. M, One-kilobase DNA marker; PC, positive control using rat kidney mRNA. B, Western blots showing induction of StAR expression by PGE2 (PG), butaprost (Bu), or PGE1 alcohol (E1OH) but not sulprostone (Sul). Lower panel shows mean StAR proteins levels in four independent experiments using different batches of endometriotic stromal cells. Asterisks indicate a value significantly different from that for the control (C). C, Pretreatment with EP1/EP2 antagonist AH6809 (AH) completely blocked PGE2, butaprost (Buta), and PGE1 alcohol (E1OH) induced StAR expression. This experiment was repeated three times using different batches of cells, and the results were identical.

 


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  		FIG. 3. Expression of StAR by PGE2 is a PKA-dependent event. A, Upper panel, Western blots showing that forskolin (For) and 8-bromo-cAMP (cA) induced StAR expression, whereas phorbol 12-myristate-13-acetate and ionomycin (Ion) had no effect. This experiment was repeated five times using different batches of cells; the quantified results are shown in lower panel. An asterisk indicates a result significantly different from that for the vehicle-treated control (C). B, The specific PKA inhibitors, H89 and PKI, block PGE2- or butaprost-induced StAR expression. Western blots show the effect of H89 (10 µM) and PKI (25 µM) (upper panel). This experiment was repeated five times, and the mean values are shown in the lower panel. An asterisk indicates a significant difference between the inhibitor-treated group and the corresponding control (PGE2 or butaprost only).

 
Induction of StAR expression is MAPK independent
Treatment of endometriotic stromal cells with PGE2 (Fig. 4AGo) or butaprost (data not shown) induced ERK phosphorylation after 5 min, whereas another member of the MAPK family, P38, was not phosphorylated (Fig. 4AGo). To examine the effect of ERK phosphorylation on StAR gene expression, we cotreated endometriotic stromal cells with PGE2 and either H89 or PD98059 [a MAPK kinase (MEK) inhibitor, 5 µM]. As noted previously, H89 blocked PGE2-induced StAR expression, whereas PD98059 had no effect (Fig. 4BGo). In contrast, in parallel experiments, PD98059 completely inhibited both basal and PGE2-induced ERK phosphorylation, but H89 blocked only PGE2-induced ERK phosphorylation (Fig. 4CGo and data not shown). These results show that, although PGE2 caused ERK phosphorylation via the PKA pathway, ERK activation was not involved in PGE2-induced StAR gene expression.



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  		FIG. 4. PGE2-induced StAR expression is independent of the MAPK signaling pathway. Western blots show that treatment with 1 µM PGE2 for 5 or 15 min resulted in phosphorylation of ERK (upper panel) but not of p38 (lower panel) (A). PC, Positive control of fibronectin-treated canine kidney cells (MDCK). B, H89, but not PD98059 (PD, 5 µM), inhibits StAR expression seen after 4 h of PGE2 treatment. This experiment was repeated five times using different batches of cells; the results are summarized in the lower panel. The asterisk indicates a significant difference between the inhibitor/PGE2-treated and the PGE2-treated groups. C, PGE2 induced ERK phosphorylation, and the effect was inhibited by H89, but basal level of ERK phosphorylation was completely blocked by PD98059. This experiment was repeated four times using different batches of cells with similar results.

 
PGE2 induces PKA-dependent CREB phosphorylation and CBP recruitment
When an extended time course for PGE2-induced CREB phosphorylation was examined, CREB phosphorylation peaked after 15 min of treatment, was still high after 120 min of treatment, and then decreased at 240 min (Fig. 5Go, A and B). Phosphorylation of CREB by PGE2 was mimicked by treatment with EP2 agonist but not EP3 agonists (Fig. 5CGo). Pretreatment with H89 inhibited PGE2-induced CREB phosphorylation, but pretreatment with PD98059 had no effect (Fig. 5DGo), showing that this was a PKA-mediated, ERK-independent event.



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  		FIG. 5. Induction of StAR expression by PGE2 is associated with PKA-dependent CREB phosphorylation. A and B, Phosphorylation of CREB was induced by 5 min treatment with PGE2 and was still seen after 120 min of treatment (+ and - indicate presence or absence of PGE2); this experiment was repeated three times using different batches of cells with similar results. C, Specific EP2 agonist, butaprost (Bu), but not EP3 agonist, sulprostone (Sul), induces phosphorylation of CREB. The EP2/EP4 agonist (E1OH) also induces CREB phosphorylation, but the extent is less pronounced. This experiment was repeated three times. D, H89, but not PD98059, blocked PGE2-induced CREB phosphorylation; this experiment was repeated four times using different batches of cells with similar results.

 
To determine whether CREB and CBP were physically associated in PGE2-treated human endometriotic stromal cells, coimmunoprecipitation assays using anti-CBP antibody to pull down CREB, or vice versa, demonstrated that a CREB-CBP complex was seen in cells treated with PGE2 (Fig. 6AGo). Coimmunoprecipitation assay also demonstrated that acetylated histone H3 associates with CREB, and the association was increased by PGE2 treatment (Fig. 6BGo). Further analysis revealed that induction of CREB-recruited CBP by PGE2 was attenuated by pretreatment with PKI (Fig. 6CGo) indicating the involvement of PKA in this interaction. By using antiacetylated histone H3 antibody to pull down the lysate, CBP was detected in the complex and the amounts were increased by treatment with PGE2 (Fig. 6CGo). Inhibition of PKA activity by PKI blocked PGE2-induced CBP-acetylated histone H3 complex formation (Fig. 6CGo).



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  		FIG. 6. Direct interaction of CREB, CBP, and histone H3 in PGE2-treated endometriotic stromal cells. A, Cells were treated with PGE2 for 15 min, lysed, and then either immunoprecipitated with anti-CBP antibody and blotted with monoclonal anti-CREB antibody (Santa Cruz) or immunoprecipitated with anti-CREB polyclonal antibody (Upstate Biotechnology Inc.) and blotted with anti-CBP antibody; + and - indicate whether specific antibody was used in the immunoprecipitation step. B, Association of CREB and acetylated histone H3 is increased by treatment with PGE2. C, PKI inhibits PGE2-induced CREB-CBP and CBP-acetylated histone H3 (AcH3) interaction. Cells were treated with PKI 30 min before the addition of PGE2 or vehicle (C), lysed, immunoprecipitated with anti-CREB or antiacetylated histone H3 polyclonal antibodies, and blotted with anti-CBP antibody. These experiments were repeated three times using different batches of cells with identical results.

 
To further examine effect of PGE2-induced CREB phosphorylation and CBP recruitment on histone acetylation, chromatin immunoprecipitation in conjunction with real-time PCR approach was performed. The proximal StAR promoter region containing a putative CRE (CCAAT/enhancer binding protein) site (-111 to -120) and a CRE half-site (-96 to -99) was amplified (Fig. 7AGo). We found that PGE2 stimulated the acetylation of histone H3 bound to the proximal region of the StAR promoter, a significant increase being seen after 30 min of treatment with a continuing rise to 120 min, followed by a plateau (Fig. 7BGo). The effect of PGE2 on histone H3 acetylation could be mimicked by butaprost and was blocked by PKA inhibitor but not by PD98059 (Fig. 7CGo). In contrast, ChIP analysis of the antiacetylated H3 antibody-immunoprecipitated chromatin for the distal region of the StAR promoter, about 4200 bases upstream of the transcriptional start site, failed to detect differences between the control and PGE2-treated cells (Fig. 7DGo).



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  		FIG. 7. PGE2 induces proximal StAR promoter bound histone H3 acetylation. A, Sequence of StAR promoter annotated TATA box (uppercase letter in a rectangle), putative CRE half-site (bold letter in a rectangle), putative CRE (bold letter in rectangle), and primer sequences used for ChIP assay (underlined). The forward and reverse primers were also indicated by arrows. The CRE site was predicted by transcription element search software on the Internet (http://www.cbil.upenn.edu/tess/). B, Association of acetylated histone H3 with the proximal StAR promoter (-333 to -34) following exposure to PGE2 for 15, 30, 60, 120, and 240 min. The results were expressed as the fold increase over the levels detected in the control cells after correcting for differences in the amount of input chromatin material (n = 6). The levels for the control groups did not differ with time and were combined as 0 min. The asterisks indicate significant differences, compared with the control. C, Representative gel showing that PGE2- (left panel) or butaprost- (right panel) induced proximal promoter bound histone H3 acetylation was blocked by H89 but not by PD98059. This experiment was repeated four times using different batches of cells with similar results. D, Representative gel showing acetylated histone H3 associated with the distal StAR promoter (-4280 to -4157) following exposure to PGE2 for 0, 30, 60,120, and 240 min. This experiment was repeated four times using different batches of cells with similar results.

 
Acetylation of histone H3 bound to the proximal region of the StAR promoter is associated with increase in nascent RNA production
Nascent StAR RNA production after PGE2 treatment was increased after treatment for 30 min, peaked at 120 min of treatment, and returned to baseline by 240 min (Fig. 8AGo). Induction of nascent StAR RNA transcription by PGE2 and butaprost was completely abrogated by pretreatment with PKA inhibitors (Fig. 8BGo and data not shown) but not by PD98059 (Fig. 8BGo). We next examined effect of histone acetylation on PGE2-mediated StAR promoter activity. Treatment with the histone deacetylase inhibitor, tricostatin A (TSA) (1 mM), had no effect on basal StAR RNA transcription but significantly increased PGE2-induced nascent StAR RNA synthesis (Fig. 8CGo).



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  		FIG. 8. Induction of nascent StAR RNA transcription by PGE2. A, Time-dependent increase in nascent RNA synthesis treatment with 1 µM PGE2. The results are expressed as the fold increase over the levels in control cells at the same time point after correcting for differences in the amount of 18S RNA. Asterisks indicate significant differences, compared with time point 0 (n = 7). B, The increase in nascent StAR RNA synthesis induced by PGE2 or butaprost was inhibited by H89 but not by PD98059 (treatment 60 min with PGE2 or butaprost). Asterisks indicate significant differences, compared with cells treated with PGE2 or butaprost alone (n = 7). C, The histone deacetylase inhibitor, TSA (1 mM), augmented PGE2-induced nascent StAR RNA synthesis. The asterisk indicates a significant difference, compared with the PGE2-treated group (n = 4).

 
Finally, we examined the effect of PGE2-induced StAR expression on progesterone production by endometriotic stromal cells. As illustrated in Fig. 9Go, we found that PGE2 treatment led to an increase in progesterone production. This effect was completely blocked by pretreatment with the PKA inhibitors, whereas inhibition of MEK activity by PD98059 had no effect (Fig. 9Go).



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  		FIG. 9. Effect of PGE2-induced StAR expression on progesterone production. Progesterone production induced by 12 h of treatment with PGE2 was inhibited by PKI but not by PD98059. The asterisks indicate significant differences, compared with the PGE2-treated group (n = 4).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we have demonstrated that PGE2 regulates StAR gene expression in endometriotic stromal cells via a mechanism, which requires PKA activation, CREB phosphorylation, CBP recruitment, and histone H3 acetylation. Inhibition of MEK activity did not block induction of StAR expression by PGE2, indicating that this effect was not mediated by the MAPK signaling pathway. The specific EP2 agonist, butaprost, was able to mimic the effects of PGE2, and pretreatment with EP2 antagonist abrogated this effect, suggesting that this regulation is mediated mainly by binding to the EP2 receptor. Herein, we provide the first evidence for the cellular and molecular mechanisms responsible for regulation of StAR gene expression by PGE2.

Endometriosis is highly estrogen dependent, and StAR regulates the first committed step of 17ß-estradiol biosynthesis by promoting the translocation of cholesterol from the cytosol to the inner mitochondrial membrane via a yet-undetermined mechanism (23, 24, 25, 26). Aberrant expression of StAR is seen exclusively in ectopic endometriotic implants and stimulated by PGE2 (4). Previously we reported that PGE2 treatment for 8 h induces dose-dependent StAR gene expression (4). In the present study, we examined the time dependency of StAR expression induced by PGE2 and found that, at concentrations of 1 µM, marked induction of StAR expression was seen after 2 h of treatment and that, although slightly lower, StAR levels were still significantly higher than in controls at 8 h of treatment. This is in concordance with the results of our previous study (4). Moreover, the fact that PGE2 exerts its effect on StAR expression as early as 2 h suggests that this is a direct effect, rather than being mediated through the production of an intermediate protein.

The downstream effector systems of PGE2 signaling are rather complex, compared with those for other prostanoids. Both EP2 and EP4 activate adenylyl cyclase and thus the PKA signaling pathway, whereas EP3 can activate or inhibit the cAMP-PKA pathway and/or increase intracellular calcium and diacylglycerol concentrations (12). Because EP2, EP3, and EP4 were all found to be expressed in human endometriotic stromal cells, we decided to determine which receptor isoform was primarily responsible for the PGE2-mediated induction of StAR expression. Our results demonstrated that the PKA activators, forskolin, and 8-bromo-cAMP, caused significant induction of StAR expression, whereas a PKC activator or calcium ionophore had no effect. Moreover, the specific EP3 agonist, sulprostone did not elicit measurable StAR expression. Although EP2/EP4 agonist (PGE1 alcohol) induced StAR expression, the level was less pronounced and completely blocked by pretreatment with EP2 antagonist. Thus, involvement of EP3- or EP4-coupled signaling in the PGE2-mediated induction of StAR gene expression can therefore be excluded. In contrast, the specific EP2 agonist, butaprost, caused a dose-dependent increase in StAR gene expression, both at the protein and nascent RNA levels (present results and data not shown), the magnitude of the effect being comparable with that induced by PGE2. Inactivation of PKA activity completely blocked butaprost- or PGE2-mediated induction of StAR expression, providing further evidence that PGE2 affects StAR gene activation by binding to the EP2 receptor.

It has been shown that PKA phosphorylates ERK, and ERK activation is associated with StAR gene expression (27, 28). To test whether this notion holds true in endometriotic stromal cells, we examined whether ERK was involved in StAR expression in terms of CREB phosphorylation, histone H3 acetylation, nascent RNA synthesis, protein expression, and progesterone production. Our results consistently demonstrated that PGE2 caused ERK phosphorylation via the EP2-coupled PKA pathway but that ERK activation was not involved in PGE2-induced StAR gene expression. A recent report showed that phosphorylation of ERKs by cAMP induces StAR gene transcription in mouse adrenocortical Y1 cells (27), whereas another report demonstrated that ERK attenuates gonadotropin-induced StAR gene expression in the human immortalized granulosa cell line, rLHR4 (28). Although our present results and those from previous studies agree that ERK1/2 is phosphorylated by PKA, they disagree in terms of the effect of ERK activation on StAR gene regulation. The discrepancy may be due, in part, to differences in the cells or ligands used. However, further investigations are necessary to resolve this disparity.

In many cases, cAMP-PKA-induced transcription is mediated through the interaction of CREB with a consensus cAMP response element (CRE) found in the promoter of target genes. Putative CREs have been identified in the StAR promoter region of many species, including humans (29). Phosphorylation of CREB modulates its transcriptional activity by recruiting CBP, a coactivator with intrinsic histone acetyltransferase activity, to facilitate transcription by directly participating in chromatin remodeling at inducible promoters (30). Furthermore, phosphorylated CREB cooperates with the transcription factor, CCAAT/enhancer binding protein, in modulating genes expression by forming a cAMP response unit that binds to CCAAT element or CRE (11, 31, 32). In this report, we provide several lines of evidence supporting the importance of CREB in PGE2-mediated induction of StAR gene expression. Firstly, putative CRE or CCAAT elements were identified in the proximal region of StAR promoter and the acetylated histone H3 associated with this region of StAR promoter was elevated with PGE2 treatment. Second, PGE2 induced marked and sustained phosphorylation of CREB, and the time course of this effect was in accordance with that for nascent StAR RNA synthesis. Third, coimmunoprecipitation assays demonstrated a direct interaction between CREB, CBP, and acetylated histone H3 in cells stimulated by PGE2, providing a link to histone acetylation. Fourth, blockage of CREB phosphorylation at serine 133, an important step in CBP recruitment, resulted in inhibition of histone H3 acetylation. Finally, phosphorylation of CREB by PGE2 or butaprost was blocked by disruption of the PKA signaling cascade, as was nascent StAR RNA synthesis. Thus, our results are consistent with previous reports that CREB and its family members modulate StAR promoter activity (29).

Histone acetylation and remodeling of chromatin structure is an important regulatory mechanism in the control of gene transcription (33, 34). Acetylation of conserved lysine residues within the amino-terminal region of core histones by histone acetylases neutralizes their positive charges and allows the proteins to dissociate from the negatively charged DNA, thus increasing DNA accessibility to transcription factors and the transcriptional machinery through as yet not completely understood mechanisms. In contrast, histone deacetylases catalyze the opposing deacetylation reaction. Recently it has been reported that acetylation of histone H3, but not of H4, is associated with cAMP-induced StAR gene promoter activation in the mouse Leydig tumor cell line, MA-10 (35). Our current results confirmed the importance of histone H3 acetylation in StAR gene activation and steroid production. In addition, we also showed that transcription of the StAR gene was inhibited by blockage of histone H3 acetylation. More importantly, our data show that CREB phosphorylation and CBP recruitment are required for histone H3 acetylation, providing evidence linking cAMP-PKA signaling to histone acetylation.

Acetylation and dissociation of histone allows the transcription complex to form on the promoter region, and this is followed by initiation of transcription. Treatment with the histone deactylase inhibitor, TSA, increased PGE2-stimulated StAR gene expression providing further support for the involvement of histone acetylation in this process. The time course of nascent RNA synthesis closely matched that of acetylation of StAR promoter-bound histone H3, except at 240 min after treatment. This may be explained by histone acetylation and dissociation from DNA being prerequisites for initiation of transcription but deacetylation/reassembly of the histone complex not being required for promoter inactivation. Acetylation of histones allows the proteins to dissociate from the DNA and increase DNA accessibility to transcription machinery. In contrast, binding of repressors or the removal of activators causes disassembly of the transcription complex and cessation of nascent RNA synthesis. The fact that CREB phosphorylation induced by PGE2 was no longer seen after 240 min of treatment provides evidence to support this notion and further suggests that, in addition to CBP recruitment, CREB may be involved in several other steps in the regulation of StAR expression. As demonstrated repeatedly in this study, stimulation of nascent StAR RNA expression by PGE2 or butaprost was inhibited by H89, but not by PD98059, showing that it is a PKA-mediated, ERK-independent event. Finally, we examined the physiological effect of PGE2-induced StAR expression on progesterone production in endometriotic stromal cells and found that the increase in StAR expression caused by PGE2 led to increased progesterone production and that this effect was blocked by PKA inhibitors but not by MEK inhibitors.

The mechanisms of PGE2-regulated StAR gene expression have never been explored though regulation of the StAR gene promoter by tropic hormones has been studied (28, 29, 35, 36, 37, 38, 39, 40, 41, 42, 43). In the present study, we have characterized the molecular and cellular mechanisms responsible for PGE2-induced StAR gene expression in endometriotic stromal cells. We conclude that the up-regulation of StAR gene expression by PGE2 is mediated by EP2-coupled PKA-induced CREB phosphorylation and histone H3 acetylation through recruitment of CBP/P300. Given the high concentrations of PGE2 in the peritoneal fluid of patients with endometriosis, autonomous de novo synthesis of estrogen by ectopic endometriotic implants may due to the up-regulation of StAR expression. This may also explain, at least in part, the failure of GnRH agonists or danazol, used for the systemic depletion of LH-stimulated ovarian estrogen, to eliminate ectopic endometriotic lesions (44). In contrast, the use of aromatase inhibitors for the systemic and local inhibition of estrogen biosynthesis has shown promising results in the treatment of endometriosis (45). Our present report may provide new insights into the mechanism involved in the abnormal local production of estrogen in, and the etiology of, endometriosis. In addition, it may also provide information for the development of novel treatment strategies for endometriosis by focusing on the molecular targets involved in chromatin remodeling and gene regulation.


   Footnotes
 
This work was supported by grants from the National Science Council of Taiwan NSC90-2321-B-006-001 and NSC91-2320-B-006-061.

Abbreviations: CBP, CREB binding protein; ChIP, chromatin immunoprecipitation; CRE, cAMP response element; CREB, CRE binding protein; EP, PGE2 receptor; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MEK, MAPK kinase; PG, prostaglandin; PKA, protein kinase A; PKC, protein kinase C; PKI, protein kinase I; SDS, sodium dodecyl sulfate; StAR, steroidogenic acute regulatory protein; TSA, tricostatin A.

Received March 5, 2003.

Accepted for publication May 21, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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