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Endocrinology, doi:10.1210/en.2008-0481
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Endocrinology Vol. 149, No. 10 5162-5171
Copyright © 2008 by The Endocrine Society

Temporal Recruitment of Transcription Factors at the 3',5'-Cyclic Adenosine 5'-Monophosphate-Response Element of the Human GnRH-II Promoter

Song Ling Poon, Beum-Soo An, Wai-Kin So, Geoffrey L. Hammond and Peter C. K. Leung

Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5

Address all correspondence and requests for reprints to: Peter C. K. Leung, Ph.D., Department of Obstetrics and Gynaecology, University of British Columbia, Room 2H-30, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. E-mail: peleung{at}interchange.ubc.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GnRH-II is a potent GnRH subtype involved in modulating OVCAR-3 cell proliferation and the invasive properties of JEG-3 cells, and an atypical cAMP-response element (CRE) in the human GnRH-II promoter influences its activation. We demonstrated that the GnRH-II promoter is activated by 8-bromoadenosine-cAMP in several cell lines including {alpha}T3, TE671, JEG-3, and OVCAR-3 cells and that cAMP enhances GnRH-II mRNA levels in JEG-3 and OVCAR-3 cells. Moreover, 8-bromoadenosine-cAMP increases cAMP response element-binding protein (CREB) phosphorylation in JEG-3 and OVCAR-3 cells and augments CBP and CCAAT/enhancer-binding protein (C/EBP)-β coimmunoprecipitation with phosphorylated CREB (p-CREB) in a temporally defined manner from nuclear extracts. When CREB, CBP, and C/EBPβ levels were knocked down by small interfering RNA, reductions in any of these transcription factors reduced cAMP-enhanced GnRH-II promoter activity and GnRH-II mRNA levels in JEG-3 and OVCAR-3 cells. Importantly, chromatin immunoprecipitation assay showed that p-CREB bound the CRE within the endogenous GnRH-II promoter within 1 h and that p-CREB association with C/EBPβ occurs within 2 h of cAMP stimulation, coincident with the first appearance of C/EBPβ at the CRE. By contrast, maximum interactions between p-CREB and CBP do not occur until at least 4 h after cAMP stimulation, and this is reflected in the progressive loading of CBP at the CRE at 2–4 h, as demonstrated by chromatin immunoprecipitation. Taken together, these data suggest that p-CREB, C/EBPβ, and CBP are recruited to the CRE of the GnRH-II promoter in a temporarily defined manner to enhance its transcription in JEG-3 and OVCAR-3 cells in response to cAMP.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN HUMANS, THE TWO GnRH genes (GnRH-I and GnRH-II) share the same structural organization but are regulated by unique regulatory elements within their promoter sequences (1) as well as in other regions of the human GnRH-II gene (2, 3). We previously identified a minimal promoter region that includes two enhancer elements (E-boxes) and an V-ets erythroblastosis virus E26 oncogene homolog (ETS) ETS-like element in the untranslated exon 1 of the human GnRH-II gene (3), and others have found an atypical (agacgtca) cAMP-response element (CRE), positioned at nucleotide sequence –860 to –853 bp relative to the translation initiation codon (3) in the GnRH-II promoter, which responds to dibutryl-cAMP in human TE671 neuroblastoma cells (4).

Activation of the cAMP response element binding protein (CREB) via activation of protein kinase A (PKA) is a prerequisite for CRE-mediated alterations in gene expression (5, 6, 7). Through its highly conserved structure (8), unphosphorylated CREB can dimerize and bind DNA, but phosphorylation of CREB at serine 133 appears to increase its affinity for some promoter sequences and notably those with atypical CREs (9). The major effect of CREB phosphorylation at this site is the recruitment of transcriptional coactivators such as the CREB binding protein (CBP) (10), which augment cAMP-induced transcription (11, 12). Other domains of CREB can also recruit other nuclear proteins to modify its transcriptional activity; for example, CCAAT/enhancer binding protein (C/EBP) family members (12, 13) and steroidogenic factor-1 (SF-1) (14, 15) modulate CREB transcriptional activity in different ways (10).

The present study set out to define the transcriptional machinery targeting the CRE of the GnRH-II promoter in JEG-3 and OVCAR-3 cells. These cell lines coexpress GnRH-I and GnRH-II and are valuable models for examining transcriptional regulation of the human GnRH-II gene. We demonstrate that stimulation of JEG-3 or OVCAR-3 cells with 8-bromoadenosine-cAMP (8-bromo-cAMP) increases CREB phosphorylation at serine 133 within 2 h and induces interactions between phosphorylated CREB (p-CREB), CBP and C/EBPβ in a temporally defined manner consistent with the timing of their assembly at the CRE within the human GnRH-II promoter over 1–4 h. These data provide insight into the molecular mechanisms through which the classical cAMP-PKA signaling cascade activates human GnRH-II gene transcription in cancer cell lines of reproductive tissue origin.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and cell culture
Human TE671 neuronal medullablastoma cells, human OVCAR-3 ovarian adenocarcinoma cells, and human JEG-3 choriocarcinoma cells were obtained from American Type Culture Collection (Manassas, VA). The gonadotrope-derived {alpha}T3 cell line was provided by Dr. P. L. Melon (Department of Reproductive Medicine, University of California, San Diego, CA). The cells were maintained in DMEM (Invitrogen Inc., Burlington, Ontario, Canada) supplemented with 10% fetal bovine serum (Hyclone Laboratories Inc., Logan, UT). Cultures were maintained at 37 C in a humidified atmosphere of 5% CO2 in air. The cells were subcultured when they reached about 90% confluence using a trypsin/EDTA solution (0.05% trypsin, 0.5 mM EDTA).

Plasmid construction and reporter gene assays
The full-length human GnRH-II promoter construct (pGL2–2103/+ 1-Luc) was generated by PCR amplification from human genomic DNA using sequence-specific primers followed by subsequent cloning into the promoter-less pGL2-Basic vector (Promega, Madison, WI). Transient transfections were carried out using Lipofectamine 2000 Reagent (Invitrogen) following the manufacturer’s suggested procedures. To correct for transfection efficiencies, the Rous sarcoma virus (RSV)-lacZ plasmid was cotransfected into the cells with the GnRH-II promoter-luciferase construct. Briefly, 5 x 105 cells were seeded into 6-well tissue culture plates the day before transfection. One microgram GnRH-II promoter-luciferase construct and 0.5 µg RSV-lacZ plasmid were cotransfected into cells grown in standard culture medium containing fetal bovine serum. In some experiments, 150 nM small interfering (si) CREB, 150 nM siCBP, and 150 nM siC/EBPβ or their control siRNA oligonucleotides (QIAGEN Inc., Mississauga, Ontario, Canada) were cotransfected with the reporter plasmids. After 6 h, 2 ml of serum-free medium were added, and the cells were further incubated overnight (18 h). The culture medium was then removed, and the cells were treated with 8-bromo-cAMP in serum-free medium for the times indicated. Cellular lysates were collected with 150 µl reporter lysis buffer (Promega) and assayed for luciferase activity The β-galactosidase enzyme assay system (Promega) was used to measure β-galactosidase expression from the (RSV)-lacZ plasmid, and promoter activities were expressed as luciferase activity/β-galactosidase activity.

Real-time PCR
After treatment with 8-bromo-cAMP, medium was removed from the culture dish and RNA was extracted using Trizol (Invitrogen). The RNA concentration was measured based on the absorbance at 260 nm, and its integrity was confirmed by agarose-formaldehyde gel electrophoresis. Total RNA (2.5 µg) was reverse transcribed into first-strand cDNA (GE Healthcare Bio-Science, Piscataway, NJ) following the manufacturer’s procedure. The primers used for SYBR Green real-time RT-PCR were designed using the Primer Express software version 2.0 (Applied Biosystems, Foster City, CA). The primers for GnRH-II mRNA are: sense, 5'-CTGCTGACTGCCCACCTT; and antisense, 5'-GCTTTCCTCCAGGGTACC AG. Real-time PCR was performed using the ABI Prism 7000 Sequence 10 detection system (Applied Biosystems) equipped with a 96-well optical reaction plate. The reactions were set up with 16.5 µl SYBR Green PCR master mix (Applied Biosystems). All real-time experiments were run in triplicate and a mean value was used for the determination of mRNA levels. Negative controls, containing water instead of sample cDNA, were used in each real-time plate. The amount of transcript in each sample was calculated by interpolation using the following formula: (threshold cycle-y intercept)/S. The steady-state concentrations of mRNA for GnRH-II in each cell line were normalized to the amount of glyceraldehyde-3-phosphate dehydrogenase mRNA.

Nuclear protein extraction and immunoprecipitation
Briefly, cells were washed with cold PBS and harvested with 1 ml solution A [10 mM HEPES (pH 7.9), 10 mM KCl, 10 mM EDTA, 0.5 mM dithiothreitol, 1 µg/ml aprotinin, and 1 µg/ml protein inhibitor cocktail]. Cell lysates were transferred to 1.5-ml centrifuge tubes and placed in an orbital rocker for 10 min at 4 C. Nuclear pellets were obtained by centrifugation at 14,000 x g at 4 C for 10 min, and supernatants were collected for cytoplasmic protein. Nuclear pellets were resuspended in solution B [100 mM HEPES (pH 7.9), 2 M NaCl, 5 mM EDTA, 50% glycerol] and placed in an orbital rocker for 2 h at 4 C. After centrifugation at 14,000 x g at 4 C for 5 min, supernatants containing the nuclear protein extracts were removed and stored at –80 C.

Immunoprecipitation was conducted as the manufacturer suggestion (Upstate, Danvers, MA). Briefly, nuclear extracts were incubated with p-CREB antibody (10 µg/ml), CBP antibody (10 µg/ml), and C/EBPβ antibody (10 µg/ml) individually followed by the antibody capture affinity ligand provided by the immunoprecipitation kit at 4 C overnight. The immunoprecipitated proteins were then subjected to electrophoresis on an 8% SDS-PAGE gel and detected with appropriate antibodies.

Chromatin immunoprecipitation (ChIP)
All reagents, buffers, and supplies were included in a ChIP-IT kit (Active Motif, Inc., Carlsbad, CA). Briefly, the cells were cross-linked with 1% formaldehyde for 10 min at room temperature. After washing and treatment with glycine Stop-Fix solution, the cells were resuspended in lysis buffer and incubated for 30 min on ice. The cells were homogenized and nuclei were resuspended in shearing buffer and subjected to preoptimized ultrasonic disruption conditions to yield 100- to 500-bp DNA fragments. The chromatin was precleared with protein G beads and incubated (overnight at 4 C) with 1 µg of the following antibodies: negative control mouse IgG (Active Motif), p-CREB antibody (Cell Signaling Technology Inc., Danvers, MA), CBP antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA), C/EBPβ antibody (Santa Cruz Biotechnology). Protein G beads were then added to the antibody/chromatin incubation mixtures and incubate for 1.5 h at 4 C. After extensive washing, immunoprecipitated DNA/protein complex was removed from the beads by elution buffer. To reverse cross-links and remove RNA, 5 M NaCl and ribonuclease were added to the samples and incubated at 65 C for 4 h. The samples were then treated with proteinase K for 2 h at 42 C, and the DNA was purified using gel exclusion columns. The purified DNA was subjected to PCR amplification (one cycle of 94 C for 3 min; 40 cycles of 94 C for 20 sec; 64 C for 30 sec and 72 C for 30 sec) for the CRE site (–860/–853 bp) within the GnRH-II promoter using specific forward (5'-CCAGCCTAAAGCAAGAGTCC) and reverse (5'-GTCTATAAATCCTGGGGC CA) primers. As an input control, 10% of each chromatin preparation was used. The PCR products (213 bp) were resolved by electrophoresis in a 2.5% agarose gel and visualized by ethidium bromide staining. The ChIP assay was performed at least three times, and consistent data were obtained between experiments.

Data analysis
Reporter gene assays and real time PCR data are shown as the mean ± SEM of three independent experiments. Data were analyzed by one-way ANOVA, followed by a test using the computer software PRISM (GraphPad Software Inc., San Diego, CA). Data were considered significant difference from each other at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Human GnRH-II promoter activity is enhanced by 8-bromo-cAMP in {alpha}T-3, TE671, OVCAR-3, and JEG-3 cells
When {alpha}T-3 (Fig. 1AGo), TE671 (Fig. 1BGo), OVCAR-3 (Fig. 1CGo), and JEG-3 (Fig. 1DGo) cells were transfected with a GnRH-II promoter luciferase reporter gene and treated with 1 mM 8-bromo-cAMP for 8 and 24 h, robust luciferase activity was determined in all cell lines. This indicates that cAMP may be a common second messenger involved in the up-regulation of GnRH-II transcription in these different cell types.


Figure 1
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  		FIG. 1. 8-Bromo-cAMP-induced GnRH-II transcriptional activity. {alpha}T-3 cells (A), TE671 cells (B), OVCAR-3 cells (C), and JEG-3 cells (D) were treated with 1 mM cAMP for 8 and 24 h after transient transfection with a GnRH-II promoter luciferase construct together with a (RSV)-lacZ plasmid. Cell lysates were collected for luciferase assay and measurements of β-galactosidase activity as a control for transfection efficiency. Results are expressed as mean ± SEM luciferase activity/β-galactosidase activity (i.e. relative luciferase activity) of three independent experiments. *, P < 0.05, compared with untreated control (ctrl).

 
Human GnRH-II promoter activity and mRNA levels are enhanced by 8-bromo-cAMP in a time-dependent manner
It is known that GnRH-II has an antiproliferative effect on human ovarian surface epithelial cancer cells (16, 17, 18) as well as a potential to alter the invasiveness of human placental tissues (19, 20). Thus, we chose human JEG-3 and OVCAR-3 cells as in vitro models to further study the molecular controls of GnRH-II promoter activity in response to cAMP. We first examined the activation of the GnRH-II promoter in JEG-3 and OVCAR-3 cells by transfecting them with a GnRH-II promoter-luciferase reporter gene construct and then stimulating them with 1 mM 8-bromo cAMP for increasing times. Figure 2Go shows that 8-bromo-cAMP activates the GnRH-II promoter in a time-dependent manner and that this reached a maximum at 24 h in JEG-3 cells (Fig. 2AGo) and at 36 h in OVCAR-3 cells (Fig. 2BGo). A cAMP dose response was performed and 1 mM cAMP was found to elicit a maximal response in both JEG-3 (Fig. 2CGo) and OVCAR-3 (Fig. 2DGo) cells. In parallel experiments, total RNA preparations of untreated or 8-bromo-cAMP-treated JEG-3 and OVCAR-3 cells for different periods of time were subjected to real-time RT-PCR measurements of GnRH-II mRNA levels. The results showed that 1 mM 8-bromo-cAMP enhanced GnRH-II mRNA levels in a time-dependent manner (Fig. 2Go, E and F). In addition, the PKA inhibitor, H89, attenuated the 8-bromo-cAMP and forskolin-induced GnRH-II promoter activity (Fig. 2Go, G and H), suggesting that the GnRH-II promoter activation involves the classical cAMP/PKA signaling pathway.


Figure 2
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  		FIG. 2. 8-Bromo-cAMP enhanced GnRH- II promoter activity and GnRH-II mRNA levels in a time-dependent manner in JEG-3 and OVCAR-3 cells. JEG-3 (A and C) and OVCAR-3 cells (B and D) were treated with 8-bromo-cAMP for different times or increasing doses of cAMP (C and D) after transient transfection with a GnRH-II promoter luciferase construct together with a (RSV)-lacZ plasmid. In parallel experiments, total RNA was isolated after the administration of 8-bromo-cAMP for 8, 16, and 24 h and subjected for real-time RT-PCR to evaluate the effect of cAMP on GnRH-II mRNA levels expressed as fold changes over control (ctrl) levels in JEG-3 (E) and OVCAR-3 (F) cells. In addition, a similar experiment was performed in which the PKA inhibitor, H89, was cotreated in the presence or absence of 8-bromo-cAMP or forskolin in JEG-3 (G) and OVCAR-3 (H) cells. In A, B, C, D, G, and H, cell lysates were collected for luciferase assay and measurements of β-galactosidase activity as a control for transfection efficiency. Results are expressed as mean ± SEM luciferase activity/β-galactosidase activity (i.e. relative luciferase activity) of three independent experiments. *, P < 0.05, compared with untreated control (ctrl).

 
8-Bromo-cAMP increases CREB phosphorylation in JEG-3 and OVCAR-3 cells
To further verify the involvement of CREB phosphorylation in GnRH-II activation, JEG-3 and OVCAR-3 cells were treated with 1 mM 8-bromo-cAMP for 2, 4, 8, 16, and 24 h. Western blot analysis showed that 8-bromo-cAMP up-regulated the phosphorylation of CREB in a time-dependent manner (Fig. 3Go). Analysis of the p-CREB to CREB ratio indicated that treatment of the cells with 8-bromo-cAMP significantly increased p-CREB levels by 3-fold within 2 h and that this was sustained over the 8- to 16-h treatment time in both JEG-3 (Fig. 3AGo) and OVCAR-3 cells (Fig. 3BGo).


Figure 3
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  		FIG. 3. Regulation of CREB phosphorylation at Ser133 by 8-bromo-cAMP. Upper panel illustrates the levels of p-CREB, total CREB, and β-actin determined by Western blotting after the administration of 8-bromo-cAMP at different time points (2, 4, 8, 16, and 24 h) in JEG-3 (A) and OVCAR-3 cells (B). The lower panel is the integrated OD of p-CREB levels after normalization with total CREB. Results are expressed as mean ± SEM of three independent experiments. *, P < 0.05, compared with untreated control (ctrl).

 
CBP and C/EBPβ interact specifically and in a temporally defined manner with p-CREB in JEG-3 and OVCAR-3 cells after 8-bromo-cAMP stimulation
Immunoprecipitation experiments were conducted to determine the interaction of p-CREB with its potential coactivators including CBP, C/EBP{alpha}, C/EBPβ, and SF-1. Administration of 1 mM 8-bromo-cAMP to both JEG-3 and OVCAR-3 cells enhanced the association of CBP and C/EBPβ with p-CREB in a time-dependent manner, whereas there was no effect on C/EBP{alpha} and SF-1 immunoprecipitations (Fig. 4Go, A and B). More importantly, the interaction between p-CREB and CBP increased progressively from 4 to 16 h after cAMP stimulation, whereas C/EBPβ associates with p-CREB earlier (i.e. within 2 h) and is sustained to 8 h in JEG-3 cells (Fig. 4AGo) and 16 h in OVCAR-3 cells (Fig. 4BGo). Furthermore, reciprocal immunoprecipitations confirmed the interaction of p-CREB with CBP and C/EBPβ (Fig. 4Go, C and D).


Figure 4
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  		FIG. 4. Interaction of CBP and C/EBPβ with p-CREB increases after 8-bromo-cAMP treatment. JEG-3 (A) and OVCAR-3 cells (B) were treated with 8-bromo-cAMP for different times. Cell lysates were immunoprecipitated (IP) with p-CREB antibody. The immunoprecipitates were then probed with CBP, C/EBPβ, C/EBP{alpha}, and SF-1 antibodies. Reciprocal immunoprecipitation (IP) was conducted upon 8-bromo-cAMP treatment of JEG-3 (C) and OVCAR-3 cells (D) for 8 or 16 h, cell lysates were immunoprecipitated with CBP or C/EBPβ antibody, and the IPs were Western blotted probed with p-CREB antibody. Western blots are representative data of IP and reciprocal IP from three independent experiments.

 
CREB, C/EBPβ, and CBP are all required to mediate changes in GnRH-II expression in response to 8-bromo-cAMP stimulation
Specific siRNA oligonucleotides were used to knock down endogenous CREB, C/EBPβ, and CBP protein levels to verify their involvement in GnRH-II expression in OVCAR-3 and JEG-3 cells. In these experiments, 8-bromo-cAMP significantly induced GnRH-II promoter activity (24 h) and GnRH-II mRNA levels (16 h) in cells cotransfected with control siRNA, whereas cotransfection with CREB-, CBP-, or C/EBPβ-specific siRNAs compromised these effects of 8-bromo-cAMP (Figs. 5Go, A–C, and 6Go, A–C).


Figure 5
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  		FIG. 5. Requirement of CREB, CBP, and C/EBPβ in 8-bromo-cAMP-mediated GnRH-II promoter activation. JEG-3 and OVCAR-3 cells were transfected with GnRH-II-luciferase reporter construct together with 150 nM random siRNA controls (s-ctrl) or siRNAs for CREB (A), CBP (B),and C/EBPβ (C), respectively. The cells were then treated with 8-bromo-cAMP for 24 h. The efficiency of the siRNA was tested by immunoblotting for CREB (67.5% knockdown), CBP (88.5% knockdown), or C/EBPβ (70% knockdown), respectively (upper panel). Cell lysates were also assayed for luciferase activity and measurements of β-galactosidase activity as a control for transfection efficiency, the result of which are expressed as mean ± SEM luciferase activity/β-galactosidase activity (i.e. relative luciferase activity) of three independent experiments. *, P < 0.05, compared with cells treated with an siRNA control (si-ctrl); #, P < 0.05, compared with cells treated with respective siRNAs and followed by 8-bromo-cAMP treatment.

 

Figure 6
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  		FIG. 6. Involvement of CREB, CBP, and C/EBPβ in 8-bromo-cAMP-mediated changes in GnRH-II mRNA levels in JEG-3 and OVCAR-3 cells. Cells were transfected with 150 nM random siRNA controls (s-ctrl) or siRNAs for CREB (A), CBP (B), and C/EBPβ (C), respectively, and then treated with 8-bromo-cAMP for 16 h. The efficiency of the siRNAs was tested by immunoblotting for CREB (67.5% knockdown), CBP (88.5% knockdown), or C/EBPβ (70% knockdown), respectively (Fig. 5Go). Total RNA was isolated and cDNA was used in real-time RT-PCR to evaluate the effect of cAMP on GnRH-II mRNA levels expressed as fold changes over control (ctrl) levels in untreated JEG-3 and OVCAR-3 cells. *, P < 0.05, compared with cells treated with a siRNA control (si-ctrl); #, P < 0.05, compared with cells treated with respective siRNAs and followed by 8-bromo-cAMP treatment.

 
8-Bromo-cAMP increases p-CREB, CBP, and C/EBPβ association with the GnRH-II promoter in a temporally defined manner
To determine how 8-bromo-cAMP treatment of JEG-3 and OVCAR-3 cells influences p-CREB, CBP, and C/EBPβ interactions with the CRE of the endogenous GnRH-II promoter, ChIP assays were performed. Cross-linked, sheared chromatin from 1, 2, and 4 h 8-bromo-cAMP-treated JEG-3 and OVCAR-3 cells were immunoprecipitated with p-CREB, CBP, and C/EBPβ antibodies, respectively, and the recovered DNA was subjected to PCR using primers specific to the CRE region (–860/–853) of the GnRH-II promoter. As shown in Fig. 7Go, a 213-bp PCR product was amplified from p-CREB-, CBP-, and C/EBPβ-immunoprecipitated DNA samples in both JEG-3 (Fig. 7AGo) and OVCAR-3 (Fig. 7BGo) cells. In contrast, little or no PCR product was observed with DNA recovered when control IgG was used for the immunoprecipitation. These data reveal the specific associations between p-CREB, CBP, and C/EBPβ at the CRE region of the GnRH-II promoter in these cancer cells after their treatment with 8-bromo-cAMP. Furthermore, in the absence of 8-bromo-cAMP-treatment, ChIP analysis using p-CREB, CBP, or C/EBPβ antibodies indicated no association of these factors with the GnRH-II CRE region. However, a PCR product was observed after 1 h with the p-CREB ChIP, and the apparent abundance of this increased at 2–4 h of cAMP stimulation, whereas no C/EBPβ or CBP or immunoprecipitated products were observed at 1 h in both JEG-3 and OVCAR-3 cells (Fig. 7Go, A and B). At 2 h after cAMP stimulation, PCR products were first observed with both the C/EBPβ and CBP ChIP assays. Although the C/EBPβ ChIP PCR product remained constant between 2 and 4 h, the CBP ChIP PCR product increased further in abundance at 4 h in both cell lines (Fig. 7Go, A and B). Overall, these data suggest that 8-bromo-cAMP promotes p-CREB, C/EBPβ, and CBP associations with the GnRH-II CRE in a temporally defined manner.


Figure 7
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  		FIG. 7. Association of p-CREB, C/EBPβ, and CBP with the CRE region of GnRH-II promoter is increased by 8-bromo-cAMP treatment. ChIP analysis was performed as described in Materials and Methods. JEG-3 (A) and OVCAR-3 cells (B) were treated with 1 mM 8-bromo-cAMP for 1, 2, or 4 h or were untreated (ctrl). Cross-linked, sheared chromatin was immunoprecipitated with p-CREB, C/EBPβ, or CBP antibodies, and recovered chromatin was subjected to PCR analysis using primers spanning the CRE region of the GnRH-II promoter. The IgG lanes are ChIP assays performed using nonspecific IgG. An ethidium bromide-stained gel of PCR products shows a representative of ChIP analysis from three independent experiments.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In extrapituitary tissues, such as the placenta, ovary, and endometrium, GnRH-II mimics and exceeds the activities of GnRH-I (1), and this is manifest in different biological responses. For instance, the antiproliferative effects of GnRH-II in ovarian cancer cells are more potent than those of GnRH-I (21), and GnRH-II is also a more effective regulator of leptin and human chorionic gonadotrophin secretion in human placenta (22, 23). Immunoreactive GnRH-II is abundant in normal ovarian surface epithelial cells, immortalized ovarian surface epithelium cells, primary cultures of ovarian tumors, ovarian cancer cell lines, and various human placental cell types (24, 25). However, little is known about what regulates GnRH-II expression in these extrapituitary cell types. In accordance with the previous finding that (Bu)2cAMP increases GnRH-II but not GnRH-I gene expression in TE671 cells (4), we also found that 8-bromo-cAMP enhanced human GnRH-II promoter activity in a wide range of cell types, including {alpha}T-3 cells, TE671 cells, OVCAR-3 cells, and JEG-3 cells. Given the potential importance of GnRH-II in altering the behavior of reproductive cancer cell types, such as OVCAR-3 and JEG-3 cells (17, 18, 19, 20, 26), we focused our attention on determining the molecular mechanisms that govern the response of the human GnRH-II promoter to cAMP stimulation.

We used a cAMP analog to dissect the transcriptional mechanisms that function via the CRE within the human GnRH-II promoter in JEG-3 and OVCAR-3 cells. It is well known that cAMP regulates a diverse set of genes (27) by phosphorylating a specific subset of nuclear factors, such as CREB, activating transcription factor-1, and cAMP-response element modulator (28, 29), that are all members of the basic region leucine zipper (bZIP) superfamily. However, CREB is the only member of the bZIP family that is phosphorylated in response to cAMP stimulation in JEG-3 and OVCAR-3 cells (data not shown). In these cells, we found that CREB phosphorylation at Ser133 not only occurs very rapidly (within 30 min data not shown) after 8-bromo-cAMP treatment but also remains elevated for up to 16 h. This appears to be an interesting cell-specific effect because a similarly rapid forskolin-induced CREB (Ser133) phosphorylation in NIH 3T3 mouse fibroblast cells is followed by a subsequent attenuation of p-CREB after 2 h via a protein phosphatase 1-dependent mechanism (30, 31). One possibility for this difference may be that the burst-attenuation protein phosphatase 1-dependent mechanism observed in NIH 3T3 cells is either delayed or lacking in OVCAR-3 and JEG-3 cells. Alternatively, the cAMP analog we used may mimic intracellular cAMP levels at higher concentrations and for longer time periods when compared with the forskolin used in the NIH 3T3 studies.

The cAMP-mediated increase in Ser133 phosphorylation of CREB is known to enhance its interaction with other nuclear proteins and their target genes via CREs (32). However, genome-wide analysis of CREB occupancy on target promoters by ChIP-on chip experiments suggested that less than 2% of CREB-occupied genes are responsive to a cAMP elevation (33). Interestingly, this small proportion of cAMP-responsive genes are not regulated by Ser133 p-CREB alone but appear to also require the preferential recruitment of regulatory partners that promote productive interactions with coactivators (33). Thus, it is likely that cAMP-induced GnRH-II promoter activation involves the coordination of a multicomponent complex including p-CREB and its potential coactivators. In this context, both CBP and C/EBPβ have been documented to enhance transcription through interactions with p-CREB and to facilitate activation of the basal and induced transcription machinery (12, 34, 35, 36). Phosphorylation of CREB at Ser133 also triggers the kinase inducible domain (KID) KID-mediated recruitment of the transcriptional coactivator CBP or its paralog p300 via their CREB interaction domain (KIX) domain (37, 38, 39, 40), which reduce the free energy required for p-CREB to bind other coregulatory proteins (41).

The C/EBP members may form heterodimers with both bZIP and non-bZIP factors (42). For instance, CEBP/β may associate through its C-terminal region with the Q1 domain of CREB (10) or it may bind CBP/p300 (43). In addition, C/EBPβ may attract CBP to CREB, thereby creating a stronger CREB-C/EBPβ-CBP transcription complex. The relationship between C/EBPβ and CREB seems to be synergistic, enhancing the activities of both proteins. The C/EBPβ gene promoter contains a CRE motif, and its transcription can be mediated by CREB and enforced by C/EBPβ through its association with CREB (44). Our results suggest that C/EBPβ is a potential coregulator of p-CREB that is recruited robustly to the GnRH-II promoter CRE within 2 h of 8-bromo-cAMP stimulation in JEG-3 and OVCAR-3 cells. By contrast, p-CREB and CBP interactions occur more progressively over a 2- to 16-h time frame, and this is reflected in a slower recruitment of CBP at the CRE region of the GnRH-II promoter after stimulation of the cells by 8-bromo-cAMP.

The critical importance of each of these factors in mediating the 8-bromo-cAMP-induced increases in GnRH-II promoter activity in JEG-3 and OVCAR-3 cells was further demonstrated in specific knockdown experiments and supports the concept that together they are critical components of a multiprotein complex that is assembles at the GnRH-II promoter CRE to mediate cAMP-signaling in these cancer cells.

Both CREB and p-CREB may bind to full-site palindromic (TGACGTCA) or half-site (CGTCA/TGACG) CREs of target genes in a cell-type-dependent manner (5, 29, 33). The CRE within the GnRH-II promoter has been shown to modulate its expression (4), and we have demonstrated that p-CREB, CBP, and C/EBPβ are all tethered at the CRE region of the GnRH-II promoter in OVCAR-3 and JEG-3 cells by using ChIP assays. In addition, our ChIP data indicate that loading of p-CREB onto the CRE of the GnRH-II promoter in unstimulated cells is minimal, and this further supports a dynamic model for p-CREB association with the GnRH-II promoter after cAMP stimulation.

Recruitment of essential coactivators (such as CBP or p300) to the CREB-CRE complex is greatly enhanced by the phosphorylation of CREB (28, 45, 46). Although p-CREB and/or C/EBPβ may bind as homo- or heterodimers to the typical palindromic CRE motif (TGACGTCA) (47, 48), the CRE within the GnRH-II promoter is atypical. It is therefore possible that p-CREB and/or C/EBPβ might occupy different sites within the GnRH-II promoter and then undergo a physical interaction that could lead to further recruitment of other nuclear proteins such as CBP. In support of the latter possibility, p-CREB binds to an atypical CRE within the phosphoenolpyruvate carboxykinase promoter, whereas C/EBPβ is recruited to a separated site and then interacts with the CRE-bound p-CREB (49). Moreover, there is considerable evidence from genome-wide studies of CREB target genes (33, 50) and expression profiling of C/EBPβ target genes (51) that both proteins may recruit additional regulatory proteins to enhance transcription. This is consistent with our observation that rapid binding of p-CREB is followed by a robust association of C/EBPβ at the CRE within the GnRH-II promoter in OVCAR-3 and JEG-3 cells, and that this is followed by the progressive recruitment of CBP to the same site over a longer time frame.

Taken together, our data indicate that the classical cAMP/PKA signal transduction pathway enhances the formation of a p-CREB-C/EBPβ-CBP transcription complex. This complex appears to target the CRE in the human GnRH-II proximal promoter and controls its activity in ovarian and placental carcinoma cells. More importantly, our data suggest that p-CREB, C/EBPβ, and CBP are recruited to the CRE of the GnRH-II promoter in a temporarily defined manner to enhance its transcription in JEG-3 and OVCAR-3 cells in response to cAMP.


   Footnotes
 
This work was supported by an operating grant from the Canadian Institutes of Health Research (to P.C.K.L.). P.C.K.L. is the recipient of a Child and Family Research Institute Senior Investigator Award. G.L.H is a Tier I Canada Research Chair in Reproductive Health.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 3, 2008

Abbreviations: 8-bromo-cAMP, 8-Bromoadenosine-cAMP; bZIP, basic region leucine zipper; C/EBP, CCAAT/enhancer-binding protein; ChIP, chromatin immunoprecipitation; CRE, cAMP-response element; CREB, cAMP response element-binding protein; p-CREB, phosphorylated CREB; PKA, protein kinase A; RSV, Rous sarcoma virus; SF-1, steroidogenic factor-1; si, small interfering.

Received April 4, 2008.

Accepted for publication June 25, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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