| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Published online before print June 26, 2008
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Department of Physiology,* the Institute of Basic Medical Sciences, and the Departments of Cell Biology and Anatomy and Obstetrics and Gynecology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
, C/EBPβ, and CREB. Forced expression of either C/EBP or C/EBPβ alone was sufficient to up-regulate StAR promoter activity whereas PGE2 was needed to induce StAR promoter activity in CREB-overexpressed cells. Results from a chromatin immunoprecipitation assay demonstrated that the binding of C/EBPβ to the StAR promoter was increased whereas CREB binding was unchanged after PGE2 treatment. Taken together, PGE2-induced StAR promoter activity appears to be regulated by CREB and C/EBPβ in a cooperative manner in ectopic human endometriotic stromal cells, providing a molecular framework for the etiology of endometriosis.
The biosynthesis of estrogen is controlled by two rate-limiting proteins namely steroidogenic acute regulatory protein (StAR) and aromatase. StAR plays a vital role in steroidogenesis by providing a continuous supply of cholesterol to the P450 side-chain cleavage enzyme. Previously, we reported that expression of StAR in endometriotic stromal cells is induced by PGE2 via EP2-coupled activation of protein kinase A (PKA) and is associated with cyclic adenosine 3',5'-monophosphate (cAMP) responding element-binding protein (CREB) phosphorylation and CREB binding protein (CBP)-mediated histone acetylation.7,8 However, the molecular mechanism responsible for PGE2-induced StAR expression and their corresponding binding elements in StAR promoter were not characterized.
The StAR gene contains binding sites for transcriptional regulators such as steroidogenic factor-1 (SF-1), activator protein-1 (AP-1), GATA, SP1, CCAAT enhancer-binding protein (C/EBP), and dosage-sensitive sex reversal-adrenal hyperplasia congenital critical region on the X-chromosome gene-1 (DAX-1).9,10 Numerous studies have reported the regulation of StAR promoter through binding to these transcription factor-binding sites. For example, it has been shown that SF-1, SP1, GATA-4, C/EBPβ, and members of CREB family play a stimulatory role in basal and cAMP-mediated StAR promoter activity11-15 whereas DAX-1 and Ying Yang-1 (YY-1) inhibit StAR expression.16,17 However, other studies analyzing the activity of StAR promoter in SF-1-expressing cells did not support a role for SF-1 in a cAMP-inducible manner.13 Furthermore, Wang and colleagues18 demonstrated that DAX-1 inhibited the SF-1-mediated transactivation of the P450 aromatase promoter but did not repress the StAR promoter activity. These data suggested that acute regulation of StAR may be mediated via distinct transcription factors in different cells and certain regulators only exert their function in particular cell types under certain circumstance.
An intriguing phenomenon, which remains unresolved, is that in many cases, transcriptional regulation of StAR expression is cAMP/PKA-dependent whereas there is no consensus CRE identified in StAR promoter. Manna and colleagues12
identified three putative half-sites for 5'-canonical CRE sequences (TGAC) in mouse StAR promoter, which could be bound by CREB/CREM to stimulate StAR promoter activity in a mouse Leydig tumor cell line, MA-10. Although similar but less perfect CRE half sites are predicted in human StAR promoter, whether CREB and/or its family members can bind to these half sites and exert a stimulatory effect remains as an open question. Alternatively, cAMP-induced StAR promoter activity may be mediated by C/EBPs via direct or indirect mechanisms. It has been shown that C/EBPβ can be phosphorylated by PKA in its bZIP domain, which can in turn affect its DNA-binding activity.19
However, other members of C/EBP family, C/EBP for example, lack a PKA phosphorylation site but still exert cAMP responsiveness. Thus, it is reasonable to hypothesize that there may be a co-factor that is able to interact with C/EBP when itself being phosphorylated by PKA. For instance, PKA phosphorylates GATA-4, which then dimerizes with C/EBPβ and cooperatively induced StAR promoter activity in MA-10 cells and transiently transfected CV-1 cells.13,20
In the current study, we characterized the involvement of transcription factors in the regulation of StAR promoter activity by PGE2. Considering the overexpression of cyclooxygenase-2 (COX-2) gene leading to elevation of PGE2 in peritoneal fluid of women with endometriosis21,22 and the critical role of estrogen in the development of endometriosis, results from this study may provide a molecular framework that helps explain the etiology of this disease. Herein we demonstrate that CREB and C/EBPβ binds to the same CCAAT cis-element of StAR promoter and the binding is required for activation of StAR transcription in PGE2-treated human endometriotic stromal cells.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Prostaglandin E2, butaprost (EP2 agonist), sulprostone (EP3 agonist), PGE1 alcohol (EP4 agonist), and anti-PGE2 antibody were purchased from Cayman Biochemical Co. (Ann Arbor, MI). Anti-StAR antiserum was a generous gift from Dr. Jerome F. Strauss III (University of Pennsylvania, Philadelphia, PA). Anti-CREB, anti-phosphoCREB (ser133P), and anti-CBP antibodies were from Upstate Inc. (Waltham, MA). Anti-CREB monoclonal antibody (sc-271), anti C/EBP, and anti-C/EBPβ antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). TaqDNA polymerase, fetal bovine serum, Dulbecco’s modified Eagle’s medium/F-12, antibiotics, and 1-kb DNA ladders were from Life Technologies, Inc. (Gaithersburg, MD). Western blot chemiluminescence reagents were from NEN Life Science Products (Boston, MA). The cytomegalovirus-driven renilla reporter system and pGL3-basic vector and S133A CREB plasmid were purchased from Promega Corp. (Madison, WI). The expression plasmid C/EBP and C/EBPβ were provided by Dr. Peter Johnson (National Cancer Institute–Frederick, Frederick, MD).
Stromal Cell Preparation and Culture
Ovarian endometrioma (n = 25) from patients with endometriosis and normal endometria (n = 10) from patients of reproductive age undergoing hysterectomy for leiomyoma or ovarian pathology were collected to process for stromal cell culture as previously described.7,8,22-24 All endometriosis samples were graded according to revised American Society of Reproductive Medicine25 and were histologically confirmed. All ovarian endometrioma samples were derived from advanced endometriosis (moderate and severe phases) with detailed information reported previously.7,8,22-24 All patients were of reproductive age with normal menstrual cycles. Human ethics 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, purified, and validated as previously described.7,8,24 Stromal cells were cultured in Dulbecco’s modified Eagle’s medium/F12 supplemented with 10% fetal bovine serum, penicillin (100 µg/ml), streptomycin (100 U/ml), and fungizone (50 µg/ml) in a humidified atmosphere with 5% CO2 at 37°C. When cells reached 70% confluence, they were placed in serum-free, phenol red-free Dulbecco’s modified Eagle’s medium/F-12 medium for 36 hours and subjected to various treatment regimens.
Promoter Constructs
A previously published sequence of the 5'-flanking region of the human StAR gene was used to clone most of the StAR promoter constructs by a polymerase chain reaction (PCR)-based approach. To generate the –1297/+19 DNA fragment, primers (5'-TGTCTTTTCAAGCTTTCTGCAC-3' and 5'-AGGGTGGTTCTTCGTCCTTC-3') were included in the PCR reaction using human genomic DNA as template. The PCR proceeded with preheating at 95°C for 2 minutes to denature genomic DNA and then 35 cycles of 30 seconds at 95°C, 30 seconds at 55°C, and 1.5 minutes at 72°C, followed by 10 minutes at 72°C. The PCR product was digested with HindIII to release a fragment of 1316 bp (–1297/+19), which was then ligated into the pGL3-basic vector (E1751, Promega). The resulting construct was referred to as –1.3kStAR. Constructs harboring deleted promoters or mutated transcription factor binding sites were generated from the –1.3kStAR plasmid using PCR amplification approach. Each 5' primer (listed in Table 1
) contained the XhoI site and therefore the deletion PCR products amplified by 5' primer and GL primer 3 (counterclockwise primer on pGL3-basic) contained XhoI site at 5' end and HindII site at StAR gene. The mutagenesis was performed by two rounds of PCR. In brief, the primers (mF and mR) used to introduce mutation were designed to be complementary and contain mismatch bases located in the middle of each primer (refer to Table 1
). In the first round PCR, 20 ng of 327 StAR/luc plasmids were amplified in two separate amplification reactions with mF/GLprimer2 (primer on pGL3-basic) and –327StAR/mR primers to generate overlapping DNA fragments. Twenty cycles of amplification reaction was performed (30 seconds denaturation at 95°C, 30 seconds annealing at 50°C, and 30 seconds elongation at 72°C) followed by elongation at 72°C for 10 minutes. The two amplified fragments were separated by 2% agarose gel and purified by gel extraction. In the second round PCR, 20 ng of each amplified fragments were mixed and amplified with –327StAR/GLprimer2 as first round PCR. The PCR products containing mutation bases were purified, digested, and inserted between HindIII/XhoI sites of pGL3-basic as described above.
|
Endometriotic stromal cells were plated on 24-well plates for the luciferase/renilla assays. Plasmids were transfected using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA). Transfections were followed by rinsing and incubation in serum-free Dulbecco’s modified Eagle’s medium/F12 medium for 12 hours. After the medium was changed, cells were then treated with PGE2 (1 µmol/L) for another 12 hours. After treatment, the medium was removed and wells were rinsed with phosphate-buffered saline (PBS) to remove detached cells and residual growth medium. Then 200 µl of 1x passive lysis buffer, provided by a dual-luciferase reporter assay system (Promega Corp.), was added per well. The plate was shaken at 180 rpm for 15 minutes at room temperature. Then cells were detached from the well by pipetting and freeze/thaw cycle to accomplish complete lysis of cells. After centrifugation at 15,000 x g, the cleared cell lysate was transfer to another 1.5-ml microcetrifuge tube. Luciferase assays were performed using a dual luciferase reporter assay system according to the manufacturer’s instructions (Promega). Briefly, 100 µl of luciferase substrate was added to 20 µl of lysate, and luciferase activity was measured using a 20/20 luminometer (Turner Designs, Sunnyvale, CA). Luciferase data were expressed as the means ± SEM. Each luciferase assay experiment was performed in duplicate and repeated for the number of times indicated in the figure legends using different batches of cells.
Preparation of Nuclear Extract
After treatment, cells were washed by cold 1x PBS, and then the cells were scraped with 400 µl of buffer A (10 mmol/L HEPES-KOH, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L phenylmethyl sulfonyl fluoride, 2 µg/ml leupeptin, 10 µg/ml aprotinin, 1 µg/ml pepstatin A, 0.5 mmol/L dithiothreitol, 50 mmol/L NaF, and 1 mmol/L Na3VO4). After incubation on ice for 10 minutes, the cells were vortexed for 20 seconds at high speed, and the nuclei were pelleted by centrifugation for 15 seconds at 12,000 x g and resuspended in buffer C (20 mmol/L HEPES-NaOH, 25% glycerol, 1.5 mmol/L MgCl2, 0.2 mmol/L ethylenediamintetraacetic acid, 420 mmol/L NaCl, 0.5 mmol/L phenylmethyl sulfonyl fluoride, 2 µg/ml leupeptin, 10 µg/ml aprotinin, 1 µg/ml pepstatin A, 0.5 mmol/L dithiothreitol, 50 mmol/L NaF, and 1 mmol/L Na3VO4). After incubation on ice for 20 minutes, the nuclear extract was centrifuged for 2 minutes at 12,000 x g and the supernatant was transferred to a new microcetrifuge tube and stored at –80°C.
Electrophoretic Mobility Shift Assay
Double-stranded oligonucleotides corresponding to C/EBP binding sites (–140 to –118, 5'-6-FAM-CAGCAGTGTGAGAGGCAATCGCTCT-3') in the human StAR promoter were synthesized by Qiagen (Valencia, CA) and annealed in 10 mmol/L Tris-HCl (pH 7.5), 1 mmol/L ethylenediaminetetraacetic acid, 25 mmol/L NaCl, 10 mmol/L MgCl2, and 1 mmol/L dithiothreitol. The oligonucleotide probes were labeled using FAM fluorescence dye. Unlabeled oligonucleotides (50-fold excess) were used as competitors in some experiments, and antibodies against C/EBP, C/EBPβ, and CREB were used for supershift analysis. A total of 10 µg of nuclear extracts were incubated in the presence or absence of competitor or antibodies for 20 minutes at 10°C in binding buffer. The FAM-labeled probe was added, and the incubation was continued for an additional 20 minutes. The DNA/protein complexes were resolved on a 5% nondenaturing acrylamide gel in TGE buffer (0.25 mol/L Tris, 1.9 mol/L glycine, and 10 mmol/L ethylenediaminetetraacetic acid final), which was subsequently dried and exposed to fluorescent Phosphor Imager FLA3000 (Fuji, Tokyo, Japan).
Chromatin Immunoprecipitation-PCR (ChIP) Assay
The protocol used was as described before.26,27
In brief, proteins and DNA in primary culture endometriotic stromal cells (1 x 106 cells) were cross-linked by incubation for 10 minutes at 37°C with a final concentration of 1% formaldehyde. Cells were washed twice with ice-cold PBS containing protease inhibitors, scraped into a conical tube, centrifuged, resuspended in 400 µl of lysis buffer [1% sodium dodecyl sulfate (SDS), 10 mmol/L ethylenediaminetetraacetic acid, 50 mmol/L Tris-HCl, pH 8.1], and placed on ice for 10 minutes. Genomic DNA was sheared to lengths of 0.5 to 1 kb by sonication. Anti-CREB, C/EBP, or C/EBPβ, antibodies, or mouse IgG (for negative control) was added to the supernatant fraction and the mixture was incubated overnight at 4°C with rotation, then incubated with 60 µl of salmon sperm DNA/Protein A agarose slurry for another 1 hour at 4°C with rotation. The pellet was then washed sequentially (5 minutes per wash) on a rotating platform with washing buffers and 1x TE buffer. After the final wash, the pellet was eluted by resuspension in freshly made elution buffer (1% SDS and 50 mmol/L NaHCO3) followed by centrifugation. The DNA was released from protein-DNA complex and 2 µl of ChIP DNA was drawn to perform the first round of PCR with StARP first primer pair (5'-GTTTCTGAGCCTCAATTCCAG-3' and 5'-CGACTTCCGACACGTAGTAG-3'). After a 20-cycle amplification (30 seconds denaturation at 95°C, 30 seconds annealing at 57°C, and 30 seconds elongation at 72°C), the PCR product was diluted and 2 µl of diluted PCR product was subjected to nested PCR amplification with StARP second primer paired (5'-TGGCCCTGTCCTCCCTAC-3' and 5'-TGAAGGCTGTGCATCATCATC-3'). After PCR amplification (30 seconds denaturation at 95°C, 30 seconds annealing at 58°C, and 30 seconds elongation at 72°C followed by elongation at 72°C for 10 minutes), the PCR product was resolved in agarose gel for evaluation.
Western Blot
The detailed procedure of Western blotting was described previously.8,28 In brief, endometriotic stromal cells were stimulated with PGE2 (1 µmol/L) for various times and equal amounts of proteins were resolved on SDS-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and incubated with specific antibodies. After washing and incubating with horseradish peroxidase-conjugated second antibodies, signals were detected by a enhanced chemiluminescence system. The blots were then stripped and reprobed with different antibodies.
Statistical Analysis
All data were expressed as means ± SEM. The data were analyzed by one-way analysis of variance in commercial statistical software (GraphPad Prism 4.02; GraphPad Software, San Diego, CA). Tukey’s procedure was used to test differences between groups found significant using the F-test. Significant differences were accepted when two-tailed analysis yielded P < 0.05.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
To determine the cis-element responsible for PGE2-induced, PKA-dependent StAR promoter activity, a 1.3-kb human StAR gene promoter was cloned and serially deleted constructs were created. PGE2 and its EP2 receptor agonist, butaprost, induced full activity of StAR promoter whereas the EP3 receptor agonist, sulprostone, failed to exert any observable promoter activity (Figure 1A)
. Bioinformatic annotation indicated that there are two SF-1 (–118/–109 and –56/–49), two C/EBP (–132/–124 and –64/–55), two CRE half sites (–112/–109 and –92/–89), one GATA (–77/–72), and one SP-1 (–173/–164) sites within the –327/+19 region of human StAR promoter. In addition, the hairpin structure, which serves as a binding sited for DAX-1 (–61/–27), is also reserved within this –327 StAR promoter (Figure 1B)
. Deletion of the –1297 to –327 of StAR promoter had no substantial effect on basal and PGE2-induced promoter activity (Figure 1C)
. Deletion of –327 to –109 significantly reduced PGE2-induced StAR promoter activity whereas deletion to the –92 bp completely abolished basal and PGE2-induced StAR promoter activity (Figure 1C)
.
|
We next sought to determine which cis-element within this region is important for PGE2-induced StAR gene activity. It has been reported that DAX-1 is an important repressor in regulating aromatase expression in endometriotic stromal cells.29
To characterize whether DAX-1 is involved in PGE2-induced StAR expression in human endometriotic stromal cell, we first determined the expression of DAX-1 in nuclear extract of normal endometrium and ectopic endometriotic implant. Expression of DAX-1 in normal uterine endometrium was greater than that in ectopic endometriotic tissues, which was inversely correlated with the expression of StAR protein (Figure 2A)
. Treatment of endometriotic stromal cells with PGE2, however, failed to affect the nuclear concentration of DAX-1, suggesting that DAX-1 may not be involved in PGE2-induced StAR expression (Figure 2B)
. Transient transfection of human DAX-1 in endometriotic stromal cell decreased basal StAR promoter activity but had no substantial effect on PGE2-induced StAR promoter as evidenced by the similar induction folds (Figure 2C)
. Taken together, these data strongly suggest that PGE2-induced StAR expression in endometriotic stromal cells is DAX-1-independent.
|
Because DAX-1 is not involved in PGE2-induced StAR promoter activity, we then focused on other transcription activators. Site-mutated constructs of several cis-elements were transiently transfected into primary cultured endometriotic stromal cells and the promoter activity induced by PGE2 treatment was determined. Mutation of two SF-1 sites, two CRE half sites, or the GATA-4 site had no substantial effect on PGE2-induced StAR promoter activity (Figure 3)
. In contrast, mutation of C/EBP site (–132/–124) completely abolished PGE2-induced StAR promoter activity (Figure 3)
. These results indicated that the CCAAT site of StAR promoter is critical for transactivation of StAR gene activity by PGE2. Because CREB is indispensable for PGE2-induced StAR expression and only C/EBP mutated element affects StAR promoter activity, it is possible that PGE2-induced StAR expression is mediated via CREB-dependent up-regulation of C/EBP protein. To test this hypothesis, cycloheximide (1 µmol/L) was added to block de novo protein biosynthesis and the effect of PGE2 on StAR mRNA expression was examined. Real-time RT-PCR results demonstrated that PGE2 significantly induced StAR mRNA expression whereas pretreatment with cycloheximide did not inhibit this effect (Figure 4)
.
|
|
To determine which transcription factors bind to the putative C/EBP element identified within the human StAR promoter, electrophoretic mobility shift assay were performed using oligonucleotides corresponding to the middle C/EBP element (–140/–118) with nuclear extracts. Specific bands corresponding to C/EBP and C/EBPβ were identified and could be competed away by adding excess unlabeled consensus C/EBP probe but not mutated probe (Figure 5A)
. Addition of specific antibodies against C/EBP or/and C/EBPβ further confirmed that both C/EBP and C/EBPβ bound to this element (Figure 5B)
. The anti-C/EBP antibody failed to affect the binding of C/EBPβ and vice versa and demonstrated that these two factors bind to the CCAAT site independently (data not shown). Next, we sought to determine whether CREB can bind to this C/EBP cis-element on human StAR promoter. Concordant with the functional role of CREB in PGE2-induced StAR promoter activity, the results demonstrated that CREB also bound to the C/EBP probe, which lacks the consensus CRE half site (Figure 5B)
. This binding was supershifted by incubation with specific CREB antibody (Figure 5B)
indicating the binding is specific. We next tested whether the binding of CREB to the C/EBP site is attributable to formation of heterodimeric CREB-C/EBP complex instead of direct binding by CREB protein. Incubation of double-stranded oligonucleotide corresponding to CCAAT site with nuclear extract obtained from cells that overexpressed C/EBP or C/EBPβ, and CREB clearly showed two distinct bands, which can be supershifted by CREB antibody and C/EBP or C/EBPβ antibody, respectively, without affecting each other’s binding (Figure 5C)
. Co-immunoprecipitation experiments using anti-CREB or anti-C/EBPβ antibody revealed that CREB and C/EBP were not physically associated with each other (data not shown). Taking together, these data show that CREB can bind to CCAAT site without the involvement of C/EBP.
|
Because both CREB and C/EBP bound to the CCAAT site independently, we sought to determine the mechanism of PGE2-induced StAR promoter transactivation. Consistent with the notion that ectopic endometriotic stroma aberrantly expresses StAR protein, a basal binding of CREB, C/EBP, and C/EBPβ to the StAR promoter was observed by ChIP assay (Figure 6A)
. The binding of CREB to the CCAAT site of StAR promoter was not changed after PGE2 treatment and is consistent with the notion that it was regulated at the phosphorylation status. In contrast, the binding of C/EBPβ to this region was markedly increased after the addition of PGE2 (Figure 6A)
. Binding of C/EBP was not as evident as that of C/EBPβ, which is consistent with the result of electrophoretic mobility shift assay.
|
, C/EBPβ, CREB, or S133A-mutated CREB and the promoter activity of human StAR gene was determined. Transfection of CREB did not result in activation of human StAR promoter unless PGE2 was added (Figure 6B)
. In contrast, forced expression of C/EBP and C/EBPβ alone was sufficient to induce StAR promoter activity (Figure 6B)
. That transfection with plasmids containing S133A-mutated CREB abolished PGE2-induced StAR promoter activity suggests again that the presence of functionally competent CREB is a requisite for transactivation of StAR gene activity by PGE2. |
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
. Our results provide a likely explanation of how the CRE-lacking human StAR gene responds to PGE2-mediated promoter activation in ectopic endometriotic stromal cells and may be applicable to other cell types.
|
20% of the cAMP-dependent induction of StAR gene expression in mouse MA-10 cell.32
In the current study, that mutation of GATA-4 binding site in human StAR promoter does not alter the reporter gene expression indicates that GATA-4 is not involved in PGE2-induecd StAR expression in ectopic endometriotic stromal cells. Mutation of the SP-1 site in human StAR promoter also shows no effect on PGE2-induced StAR promoter activity and thus eliminates the role of SP-1 in the aberrant expression of StAR in endometriotic stromal cells. The orphan nuclear receptor DAX-1 has been identified to block steroid synthesis by impairing the expression of StAR gene in adrenal gland, testis, and ovary. The DNA binding site for DAX-1 is a hairpin structure with minimal stem length of 10 nucleotides,16 which is distinct from all other nuclear receptors. It has been reported that binding of DAX-1 resulted in decreasing basal and cAMP-induced StAR promoter activity in Y-1 adrenocortical cells or R2C rat tumor cells.16,33 Our results support part of the previous findings while contradicting with the rest by showing DAX-1 only involves inhibition of basal but not PGE2-induced StAR expression. In the current study, we conclude that DAX-1 inhibits basal StAR expression by direct and indirect evidence. That levels of DAX-1 inversely correlate with StAR expression in normal and endometriotic tissues represents the indirect evidence whereas force expression of DAX-1 in endometriotic stromal cells reduces basal StAR expression provides the direct one. However, PGE2 does not affect DAX-1 expression in endometriotic stromal cells and force expression of DAX-1 also fails to block PGE2-induced StAR expression provide other lines of evidence to demonstrate that DAX-1 is not responsible for up-regulation of StAR by PGE2. The discrepancy between our current finding and previous report is not clear but could be attributable to cell type-specific and ligand-specific effects.
A plethora of evidence has demonstrated that up-regulation of StAR is a cAMP-PKA-dependent event. The intriguing part is that no consensus CRE has been identified in the StAR gene promoter. In mouse StAR promoter, a CRE half-site was identified and proven functional in cAMP-induced StAR gene expression.12
The CRE half-site is not functional in human StAR promoter whereas the C/EBP site provides a different story in regulating StAR gene expression induced by PGE2. In agreement with report by Christenson and colleagues,11
we found this C/EBP element could be bound by C/EBP and C/EBPβ. In addition, our results further demonstrate that CREB also binds to this response element. We have previously shown that phosphorylation of CREB induced by PGE2, which causes CBP recruitment and histone H3 acetylation, is associated with PGE2-induced StAR expression.8
In this study, we further demonstrated that blockage of CREB phosphorylation by overexpression of S133A-mutated CREB abolishes PGE2-induced StAR expression. By identifying that CREB actually binds to the C/EBP site in human StAR promoter, we provide a physical link between PKA-induced CREB phosphorylation and its functional role in StAR promoter activity.
The molecular mechanism of how CREB regulates PGE2-induced, PKA-mediated StAR promoter activity remains unclear. Our current and previous8
results suggest that CREB might play as an initiator to unfold the DNA-histone binding, which then provides space for C/EBP binding to the StAR promoter. CREB constitutively binds to the StAR promoter as has been proposed before.34
On phosphorylation by PKA, phosphorylated CREB recruits CBP, which then causes histone acetylation. Acetylation of histone by CBP may result in its dissociation from DNA, which provides space for C/EBP binding. Although both C/EBP and C/EBPβ can bind to this region and forced expression of both proteins is sufficient to induce StAR promoter activity, the increased binding capacity of C/EBPβ after PGE2 treatment suggests it might play a more prominent role than C/EBP. The identification of CREB binding to the C/EBP site of StAR promoter provides a likely explanation why the conventional CRE-lacking StAR promoter would respond to PKA action. Further investigation is warranted to elucidate whether other stimuli (for example, luteinizing hormone and adrenocorticotropic hormone) that activate StAR gene promoter via PKA-dependent signaling pathway is mediated via a similar molecular paradigm.
Taking together our previous and current findings, we have demonstrated, for the first time, that PGE2-induced up-regulation of StAR gene activity involves phosphorylation of CREB, recruitment of CBP, acetylation of histone, and binding of C/EBPβ to the StAR promoter. Considering the overexpression of COX-2 gene leading to elevation of PGE2 in peritoneal fluid of women with endometriosis21,22 and the critical role of estrogen in the development of endometriosis, our model provides a molecular framework that helps explain the etiology of this disease. These findings also provide novel information for future therapeutic consideration that aims at disruption of steroidogenic capacity of PGE2.
|
Footnotes |
|---|
Supported by the National Science Council of Taiwan (grant NSC 95-2320-B-006-047) and the National Research Program of Genomic Medicine (grant NSC94-3112-B-006-010).
Accepted for publication May 8, 2008.
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
(PGF2) is associated with PGF2 resistance or susceptibility in human granulosa-luteal cells. Mol Hum Reprod 2001, 7:415-423This article has been cited by other articles:
 |
|
 |
|
  P. R. Manna, M. T. Dyson, and D. M. Stocco Regulation of the steroidogenic acute regulatory protein gene expression: present and future perspectives Mol. Hum. Reprod., June 1, 2009; 15(6): 321 - 333. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-W. Chien, S.-C. Lin, Y.-Y. Lai, B.-W. Lin, S.-C. Lin, J.-C. Lee, and S.-J. Tsai Regulation of CD151 by Hypoxia Controls Cell Adhesion and Metastasis in Colorectal Cancer Clin. Cancer Res., December 15, 2008; 14(24): 8043 - 8051. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
