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Molecular Endocrinology, doi:10.1210/me.2006-0226
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Molecular Endocrinology 21 (4): 948-962
Copyright © 2007 by The Endocrine Society

Transcriptional Regulation of the Cholesterol Side Chain Cleavage Cytochrome P450 Gene (CYP11A1) Revisited: Binding of GATA, Cyclic Adenosine 3',5'-Monophosphate Response Element-Binding Protein and Activating Protein (AP)-1 Proteins to a Distal Novel Cluster of cis-Regulatory Elements Potentiates AP-2 and Steroidogenic Factor-1-Dependent Gene Expression in the Rodent Placenta and Ovary

Noa Sher, Natalie Yivgi-Ohana and Joseph Orly

Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

Address all correspondence and requests for reprints to: Dr. Joseph Orly, Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. E-mail: orly{at}vms.huji.ac.il.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The first and key enzyme controlling the synthesis of steroid hormones is cholesterol side chain cleavage cytochrome P450 (P450scc, CYP11A1). This study sought to elucidate overlooked modes of regulation of P450scc transcription in the rodent placenta and ovary. Transcription of P450scc requires two clusters of cis-regulatory elements: a proximal element (–40) known to bind either activating protein 2 (AP-2) in the placenta, or steroidogenic factor 1 in the ovary, and a distal region of the promoter (–475/–447) necessary for potentiation of the AP-2/steroidogenic factor 1-dependent activity up to 7-fold. In primary cultures of mouse trophoblast giant cells and rat ovarian granulosa cells, binding of trans-factors to the distal regulatory sequences generated transcriptional activity in a tissue-specific pattern: in the placenta, cAMP response element (CRE)-binding protein 1 (CREB-1) and GATA-2 binding generates promoter activity in a cAMP-independent manner, whereas in ovarian cells, CREB-1 and GATA-4 are required for FSH responsiveness. However, as ovarian follicles advance toward ovulation, elevated Fra-2 expression replaces CREB-1 function by binding the same CRE1/2 motif. Our findings suggest that upon onset of follicular recruitment, CREB-1 mediates FSH/cAMP signaling, which switches to cAMP-independent expression of P450scc in luteinizing granulosa cells expressing Fra-2. In the placenta, the indispensable role of CREB-1 was demonstrated by use of dominant-negative CREB-1 mutant, but neither cAMP nor Ser133 phosphorylation of CREB-1 is required for P450scc transcription. These observations suggest that placental regulation of P450scc expression is subjected to alternative signaling pathway(s) yet to be found.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PROGESTERONE AND ESTROGEN are essential for successful ovulation, implantation, placentation, and maintenance of pregnancy in humans and other mammals. The first and key enzyme responsible for synthesis of progesterone and all other steroid hormones is the mitochondrial monooxygenase, cholesterol side chain cleavage cytochrome P450 (P450scc, CYP11A1). This study sought to elucidate new aspects related to P450scc regulation of transcription in steroidogenic cells of the female reproductive organs, the mouse placenta and the rat ovary. The comparison between the two organs is interesting due to the fact that the placenta is believed to express many endocrine activities in what seems to be a hormone-independent manner, whereas programmed differentiation of follicular cells in the ovary is cyclical and inducible by the pituitary gonadotropins FSH and LH.

In rodents, progesterone synthesis by the corpus luteum is indispensable throughout pregnancy (1). Nevertheless, two transient rises of steroidogenic gene expression are observed in the pregnant uterus during the first half of gestation. First, unexpected localization of steroidogenic gene products was documented in the embryonic d 4.5 (E4.5)–E8.5 decidua and embryonic endoderm (2, 3, 4, 5). Later, a subsequent local surge of steroidogenic activity is linked to midpregnancy placentation, whereby during E8.5–E10.5 the trophoblast giant cells acquire a marked steroidogenic ability, expressing P450scc as well as steroidogenic acute regulatory protein (StAR, Star), 3ß-hydroxysteroid dehydrogenase/isomerase type IV (3ß-HSDIV, Hsd3b4) and 17{alpha}-hydroxylase-17:20 lyase (Cyp17a1) activities (2, 3, 4, 6, 7). Whereas the relevance of decidual steroidogenesis is not clear at the moment, the midpregnancy placental androgens made in the trophoblast cells serve for estrogen production in the rodent corpus luteum (8). However, live birth of Cyp11a null mouse pups suggests that P450scc expression in the rodent placenta is not essential for completion of apparently normal pregnancy (9).

Little is known about the trans-factors controlling P450scc transcription in rodents. A proximal cis-regulatory element positioned at –40 of the murine P450scc is highly conserved among mammals and was shown to bind isoform {gamma} of the activating protein 2 (AP-2{gamma}, TCFAP2C), a placental trans-factor highly expressed in mouse trophoblast giant cells (10, 11, 15), and the orphan receptor steroidogenic factor-1 (SF-1, Ad4BP, NR5A1) in the ovary (12, 13, 14, 15, 16, 17). The prominent SF-1 binding element at –40 of the P450scc gene was shown to mediate hormone/cAMP-induced promoter activity in ovarian, testicular, and adrenal cells (14, 18, 19, 20, 21, 22, 23).

However, several lines of evidence suggested that AP-2 and SF-1 proteins cannot constitute the sole mechanism of P450scc expression in the pregnant uterus and the ovary: 1) whereas P450scc protein expression is restricted to E8.5–E10.5 trophoblast giant cells (2, 3), spatiotemporal studies of AP-2{gamma} expression in the rodent placenta suggested that rise of this protein already occurs on E6.5 (24), when P450scc is still absent from this cell type; 2) AP-2{gamma} is also expressed in other cell types of the placenta, such as the spongiotrophoblast and labyrinth layers (24), which do not express P450scc (2, 3); 3) unlike the human P450scc promoter (25), no cAMP responsive elements (CREs) have been identified in the promoter of this gene in rodents, despite the ability of the cyclic nucleotide to robustly induce expression of the gene in many cell types; 4) high-dose LH treatment of granulosa cells associates with a marked reduction of SF-1 levels, yet P450scc is strongly up-regulated under such circumstances (26); and 5) SF-1 expression in theca cells is much higher than that observed in the granulosa cell layers, whereas P450scc expression is higher in granulosa cells (27, 28). Finally, other than SF-1, P450scc regulation examined in cell models coexpressing P450scc promoter constructs and trans-factors have suggested that proteins of the GATA family have a role in regulation of P450scc transcription (29). However, the potential role of GATA in these studies remained circumstantial, as no GATA binding elements were identified in the P450scc promoter.

This study shows that P450scc expression in the trophoblast giant cells is not cAMP dependent, whereas regulation of this gene in the ovary begins as hormone/cAMP dependent, and later becomes cAMP independent. We show that the observed variable modes of regulation are addressed at the level of the P450scc promoter by involvement of newly described cis-elements that can bind an array of trans-factors in a modular fashion. To maintain maximal authenticity of our findings, we chose to use primary placental trophoblast giant cells and primary ovarian granulosa cell cultures, instead of using immortalized cell models. The resulting evidence portrays a new paradigm of tissue-specific transcriptional coregulation of P450scc by SF-1, AP-2, tissue-specific GATA proteins, members of the cAMP response element (CRE)-binding protein (CREB)/activating transcription factor (ATF) gene family, and AP-1 proteins.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Distal Promoter Elements Required for P450scc Transcription
To identify novel regulatory elements possibly involved in P450scc transcription, a series of 5'-deletion constructs of the murine P450scc promoter were placed upstream of a chloramphenicol acetyl transferase (CAT) reporter gene and transiently expressed in either E9.5 primary murine trophoblast giant cells or FSH-treated primary granulosa cells expressed from estradiol-primed prepubertal rats (Materials and Methods). In both cell types, a substantial loss of reporter activity was observed in promoter constructs shorter than –497 (Fig. 1Go), suggesting that novel cis-activating elements involved in the regulation of P450scc transcription should be looked for in the –497/–406 promoter region.


Figure 1
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  		Fig. 1. 5'-Deletion Analysis of the –942/+28 Mouse P450scc Promoter

Progressive deletions of the P450scc promoter were subcloned into a CAT reporter gene and transfected by different methods into either E9.5 mouse trophoblast giant cells, or E2-primed rat granulosa cells (see Materials and Methods). Extracts were prepared for CAT analysis as described in Materials and Methods 48 h after transfection of the placental cells incubated in the absence of any inducing agent, and 6 h after FSH treatment (100 ng/ml) of the ovarian cells. CAT activity was determined using 1–10 µg protein for a 16 h assay. The activity results (arbitrary units) are presented as the average ± 95% CI of [14C]chloramphenicol converted to the acetylated products normalized to the –497/+28 construct and to protein concentration. Activity levels were statistically significant when compared with the respective values obtained for the –497/+28 construct: a, P < 0.001.

 
Unlike ovarian steroidogenesis, it is unclear whether cAMP or any trophic hormones control P450scc in the placenta (30, 31, 32). To address this question, we examined the expression of the P450scc promoter constructs in the trophoblast giant cell cultures incubated with or without 8-bromo-cAMP (8-Br-cAMP). Figure 2AGo shows that cAMP did not generate any promoter activity over the basal activities of the examined constructs. These results are consistent with the inability of cAMP to increase the expression of P450scc in the trophoblast giant cells; furthermore, the levels of P450scc protein declined with time in culture (Fig. 2BGo). Interestingly, the indifferent response of P450scc transcription to cAMP did not result from lack of activated CREB-1, because cAMP generated a robust Ser133 phosphorylation of CREB in the trophoblast giant cells, similar to that observed in the granulosa cells (Fig. 2CGo) known to be induced by FSH treatment (33, 34). Also, Fig. 2BGo shows that, in contrast to the granulosa cell model, which does not express P450scc before hormone treatment (35), this cytochrome is already abundantly expressed in E9.5 trophoblast giant cells, suggesting that ongoing transcriptional activation of P450scc in vivo is not dependent on Ser133 phosphorylation of CREB (time zero, Fig. 2CGo). Based on these results, in all subsequent experiments, we compared basal promoter activity in the murine trophoblast cells to FSH-induced responses of the rat granulosa cells.


Figure 2
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  		Fig. 2. Effect of cAMP on Promoter Activity and Downstream Responses in the Trophoblast Giant Cells

A, Several of the deletion constructs described in Fig. 1Go were also examined as luciferase reporter genes transfected into E9.5 mouse trophoblast culture. Cells were further incubated 24 h after transfection with or without 0.5 mM 8-Br-cAMP, and extracts were prepared for luciferase analyses as described in Materials and Methods. The activity results (arbitrary units) are presented as the average ± 95% CI of luciferase activity normalized to the –497/+28 construct and to protein concentration. Activity levels were not statistically significant when compared with the respective values obtained for each of the untreated constructs: a, P > 0.4. B, Trophoblast giant cells were extracted either immediately after isolation (time zero), or after 24 and 48 h in culture, with or without addition of 0.5 mM 8-Br-cAMP added 24 h after plating. Levels of P450scc in 15 µg extract were examined by Western blot analysis. C, Induction of Ser133 phospho-CREB accumulation was assessed in either E9.5 mouse trophoblast giant cells or E2-primed rat granulosa cells. Trophoblast giant cells were extracted either immediately after isolation (time zero), or after 24 h in culture, after a 30-min exposure to 0.5 mM 8-Br-cAMP. Granulosa cells were plated, induced the following morning with 100 ng/ml FSH for 30 min, and 15 µg of the cell extracts were analyzed for the levels of CREB and phospho-CREB by Western blotting and specific antisera. p-CREB, Phospho-CREB.

 
A –475/–470 Conserved GATA Binding Site Is Necessary for Transcription
Seeking putative cis-regulatory binding sites in the –497/–406 region of the P450scc promoter, several in silico transcription factor search engines indicated that a GATA binding site exists at –475/–470, which is conserved in the murine, rat, and human promoters (genomic accession nos. NT_039474, NW_047799, and NT_010194, respectively). To examine whether this GATA site is of functional relevance, we used PCR primers to mutate the –475/–470 element, singly or in conjunction with a mutation in the previously described proximal AP-2/SF-1 binding site at –40 (10). Compared with the wild-type promoter activity, mutation of the GATA element caused a 75% and 50% reduction of the activity examined in the trophoblast giant cells and granulosa cells, respectively (Fig. 3Go). Also, a 2-bp mutation in the –40 overlapping AP-2/SF-1 recognition motifs had a marked effect of 75% and 80% inhibition of the promoter activity in the two cell types, respectively. Double mutations in both the GATA and AP-2/SF-1 sites (–497dmut) did not have a marked effect over the inhibition of each individual mutant.


Figure 3
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  		Fig. 3. A GATA site at –475/–470 of the Mouse P450scc Promoter Enhances P450scc Transcription

PCR-based mutations were created within the context of –497/+28 of P450scc (–497wt), and ligated to pCAT-Basic. Site-directed mutations included the –475/–470 GATA element (GATAGA to GgcAtg; –497mutGATA), the –48/–40 AP-2/SF-1 element (GCCTTCAGC to GtaTTCAGC; –497mutAP2 or –497mutSF1), or double mutant construct (–497dmut). Shown is also a shorter –406/+28 (–406wt) construct. Transient expression and CAT assay examined in murine trophoblast giant cells or FSH-treated rat granulosa cells were conducted as described in Fig. 1Go. The results (arbitrary units) are presented as the average ± 95% CI of [14C]chloramphenicol converted to the acetylated products normalized to the –497/+28 construct and to protein concentration. Activity levels were statistically significant when compared with the respective values obtained for the –497/+28 construct: a, P < 0.001; b, P < 0.01; c, P < 0.05. wt, Wild type.

 
Various GATA Isoforms Bind the –475/–470 Site
To examine the binding characteristics to the putative GATA site at –475/–470, we performed EMSAs using a –488/–457 probe and extracts of trophoblast giant cells. Figure 4AGo shows that a single [32P]DNA-protein complex (lane 1) was readily competed with x50 molar excess of nonlabeled self oligonucleotide (lane 2), or with excess of a GATA consensus element (GATA cons., lane 5), previously identified in the StAR gene (36). Consistent with these observations, a mutated oligonucleotide (GATAGA to GtcAGA) lacked the ability to compete with the radiolabeled wild-type probe (mutGATA, lane 3), as did excess of a nonspecific oligonucleotide (scc1, lane 4) represented by the AP-2/SF1 probe at –60/–34 of the P450scc gene (10). In addition, only a residual, probably nonspecific DNA-protein complex, formed with a mutated 32P-radiolabeled GATA probe (lane 6). Practically identical results were obtained in competition assays using ovarian cell extracts (data not shown).


Figure 4
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  		Fig. 4. Placental GATA-2 and Ovarian GATA-4 Bind to the –475/–470 GATA Motif in Cyp11a1

A, EMSAs were performed using 32P-labeled probes corresponding to either a wild-type –488/–457 sequence of the P450scc gene, or this probe mutated at the GATA site (GATAGA to GtcAGA, mutGATA). Extracts were prepared from trophoblast giant cells isolated from pregnant mice on E9.5 (10 µg). Competition with x50 excess of nonlabeled oligonucleotides included a self sequence, the above mutGATA, the AP-2/SF-1 binding element at –40 [scc1 (10 )] and consensus GATA element (36 ). B, EMSAs were performed using 32P-labeled wild-type –488/–457 of the P450scc gene. Extracts were prepared from the following cells and tissues: trophoblast giant cells isolated from pregnant mice on E9.5, rat ovaries taken 10 h after eCG administration to 25-d-old rats, or ovaries extracted 8 h after hCG administration to eCG-treated rats (see Materials and Methods). Where indicated, extracts were preincubated with antisera to GATA proteins. Arrows denote either the DNA-GATA complexes or band supershifts (ss). C, The levels of tissue-specific GATA proteins were determined by Western blot analyses. Extracts from trophoblast giant cells were prepared from cells freshly isolated (time zero) or after 24 h in culture. Ovarian extracts were prepared from naïve and gonadotropin-treated animals as described above. cons., Consensus.

 
Direct evidence showing that the –475/–470 sequence element serves as a specific binding site to GATA protein(s) was furnished by use of specific GATA antisera as shown in Fig. 4BGo. Extracts of trophoblast giant cells cross-reacted with GATA-2 polyclonal antiserum that caused a supershift and partial ablation of protein binding (lane 2). The residual complex left in the presence of the GATA-2 antiserum is consistent with the minor nonspecific binding observed in Fig. 4AGo, lanes 5–6. Clearly, anti-GATA-3 serum was much less effective (Fig. 4BGo, lane 3), showing only a partial ablation of the DNA-protein signal, although ample amounts of GATA-3 are present in the cells as shown by Western analysis (Fig. 4CGo). Alternatively, in hormone-induced rat ovarian extracts, incubation of extracts with anti-GATA-4 resulted in a complete cross-reactivity of the antibody with the [32P]DNA-GATA signal, suggesting that the GATA-4 isoform is the predominant ovarian protein that binds the –475/–470 GATA element (Fig. 4BGo, lanes 5 and 8). GATA-4 binding remained predominant throughout folliculogenesis as observed using ovarian extracts from gonadotropin-treated animals, either 10 h after equine (e) chorionic gonadotropin (CG) administration (onset of follicular development, lanes 4–6), or during onset of granulosa cell luteinization in ovaries taken 8 h after human (h) CG administration (lanes 7–9). Again, whereas antiserum to GATA-6 did not affect the DNA-protein complex at all (lanes 6 and 9), Western analysis using the same antiserum showed that GATA-6 was highly abundant in the ovarian tissue (Fig. 4CGo). Together, these results suggest that GATA-2 and GATA-4 are the predominant proteins bound to the –475/–470 sequence, although GATA-3 and GATA-6 proteins are also present in the placenta and the ovary as well.

A CRE Element at –450/–447 Is Necessary for Maximal Transcription
Sequence analysis of the –497/–406 region also indicated the presence of a cAMP response element (CRE) half-site, GTCA, at –450/–447 of the promoter. Mutation of this site caused 87% and 92% reduction in promoter activity examined in the placental and the ovarian cells, respectively (Fig. 5AGo). These results demonstrated the functional importance of the –450/–447 CRE half-site. To identify the potential trans-factor involved in binding to this sequence element, we used a –469/–442 EMSA probe that formed two slow migrating complexes (I and II) with protein(s) present in the trophoblast giant cell extracts (Fig. 5BGo, lane 1). Antiserum to CREB-1 (lane 5) supershifted complex I, whereas antisera to AP-1 proteins and ATF-1, both known of being capable to bind a GTCA motif (37, 38, 39, 40), remained ineffective. The minor band indicated as complex II was supershifted by antibodies against ATF-2 (lane 8), but not by the ATF-1 and ATF-3 antisera (lanes 7 and 9). Figure 5CGo shows that a x50 molar excess of nonlabeled wild-type oligonucleotide competed with protein binding (lane 2), whereas a mutated CRE was unable to do so (lane 3). Consistent with this, a [32P]CRE1/2 mutant probe lacked binding activity (lane 6).


Figure 5
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  		Fig. 5. A CREB-1 Binding Site at –450/–447 of Cyp11a1 Is Also Necessary for Maximal P450scc Transcription

A, A PCR-based mutation of the –450/–447 CRE binding site within the context of –497/+28 CAT construct was created, and its activity was assayed by CAT analysis in trophoblast giant cells or FSH-treated granulosa cells as described in Fig. 1Go. The activity values (arbitrary units) are presented as the average ± 95% CI of [14C]chloramphenicol converted to the acetylated products normalized to the –497/+28 construct and to protein concentration. Activity levels were statistically significant when compared with the respective values obtained for the –497/+28 construct: a, P < 0.001. B, EMSAs using a 32P-labeled –469/–442 oligonucleotide (panel D) were performed with murine trophoblast giant cell extracts pre-incubated without or with the indicated antisera (Ab and {alpha}ATFs). Arrows denote either the DNA-protein complexes (I and II), the free probe (F.P.), or an antibody-associated band supershift (ss). C, EMSA were performed using E9.5 trophoblast giant cell extract and 32P-labeled oligonucleotides corresponding to either the wild-type –469/–442 sequence used in panel B, a 32P-labeled probe mutated at the CRE site (GGATAT to GGcaAT, mutCRE), or a [32P]CRE consensus probe (83 ). Competition with x50 excess of nonlabeled oligonucleotides included a self-sequence, mutCRE, mutGATA*, and consensus CRE element. D, Positioning of the –488/–457 oligonucleotide probe nesting the –475/-470 GATA binding site (underlined) and the –469/–442 probe with the CRE half-site examined herein (CRE1/2, framed). Note another putative GATA site (GATA*, underlined) found irrelevant (panel C, lane 4). cons., Consensus; WT, wild type.

 
It should be noted that, in addition to the CRE half-site, the –469/–442 probe also contains a putative consensus GATA element positioned at –458/–453 upstream of the CRE site (GATA*, Fig. 5DGo). However, an oligonucleotide probe in which this putative GATA site was mutated (mutGATA*, GGATAT to GGcaAT) retained its ability to compete with the wild-type 32P-labeled oligonucleotide probe (Fig. 5CGo, lane 4), thus strongly suggesting that the –458/–453 GATA motif is not important for binding of trophoblast proteins.

Ovarian Members of the bZIP Superfamily Bind the –450/–447 CRE Site
Unlike the consistent association of CRE1/2 with trophoblast CREB-1 and ATF-2, binding assays incubating ovarian extracts and the –469/–442 CRE1/2 probe revealed dynamic changes with respect to the proteins that can complex with this DNA. Figure 6AGo shows that incubation with ovarian extract from 25-d-old premature rats (naïve) yields a single predominant complex (lane 1), readily supershifted by CREB-1 antiserum (lane 5). The apparent ineffectiveness of antisera to pan-Fos, pan-Jun, ATF-1 (lanes 2–4, respectively), and ATF-2/3 (data not shown) suggested that such proteins did not bind to this site. When follicles taken 10 h after eCG administration were examined, the ovary extracts exhibited a 3-fold increase of DNA-binding activity (lane 6). Moreover, in addition to a partial cross-reactivity of the extract proteins with the CREB-1 antiserum (lane 10), the pan-Fos antibody ablated most of the complex formation (lane 7), indicating that some member(s) of the Fos family bound the CRE1/2 element, in addition to CREB-1. Upon luteinization examined 8 h after hCG administration to the eCG primed rats, pan-Fos was the only cross-reactive antiserum (lane 12) whereas CREB antibody remained ineffective (lane 15).


Figure 6
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  		Fig. 6. Ovarian CREB-1 and Fos-Related Proteins Bind to the –469/–442 Region of Cyp11a1

A, EMSAs using 32P-labeled –469/–442 oligonucleotide probe and ovarian extracts were performed in the presence of various antisera known to bind members of the CREB/ATF protein family or AP1 binding proteins. Ovarian proteins were extracted before treatment (naïve), or 10 h after administration of eCG, or 8 h after hCG administration to eCG-primed prepubertal rats (Materials and Methods). Arrows denote either the DNA-protein complexes, antibody-associated band supershift (ss), or free probe (F.P.). B, Ovarian Fra-2 binding to the CRE1/2 motif. Ovary extracts from gonadotropin-treated animals were prepared as in panel A and analyzed by EMSA using the –469/–442 32P-labeled oligonucleotide probe incubated in the absence or presence of specific antibodies against members of the AP-1 protein family. Normal IgG and antibodies against members of the Jun family served as negative controls, because the pan-Jun antibody did not display a supershift or ablation (see panel 6A). Arrows denote either the DNA-protein complexes or band supershift (ss).

 
To specifically identify which Fos isoform(s) are responsible for binding to the CRE site, additional EMSAs were performed with antibodies against each member of the AP-1 family (Fig. 6BGo). Jun members (c-Jun, JunB, and JunD) served as negative controls because the pan-Jun antibody did not alter protein binding (Fig. 6AGo, lanes 3, 8, and 13). Fra-2 (FOSL2; Fig. 6BGo, lanes 6 and 16) was the only AP-1 protein responsible for binding of the CRE in developing follicles, both before LH surge (10 h eCG) and after induction of luteinization by hCG.

Effects of Dominant-Negative Variants of GATA, CREB, and Fos
The physiological involvement of GATA, CREB, and Fra-2 in P450scc transcription was examined by efficient expression of dominant-negative and competitor variants of the proteins (41, 42, 43, 44) in the primary cell types. Figure 7AGo shows that dominant-negative GATA (GATA-DN), A-CREB, and inducible cAMP early repressor (ICER) reduced the expression of the –497/+28 CAT plasmid by more than 90% when tested in the trophoblast giant cells, suggesting a critical role for GATA-2 and CREB-1 in the regulation of placental P450scc. A similar trend of activity reduction was observed upon coexpression of GATA-DN, A-CREB, ICER, and A-Fos protein in the ovarian granulosa cells. The profound inhibition of the promoter activity in the presence of A-Fos provides direct evidence implicating Fra-2 as a potential positive regulator of P450scc transcription. EMSA using the DNA sequence nesting the –449/–446 CREB element corroborated the presence of immuno cross-reactive Fra-2 (Fig. 7BGo, lanes 2 and 4), the level of which increased after a 6-h treatment with FSH (lane 3). The spontaneous and hormone induced up-regulation of Fra-2 in granulosa cells put to culture was also corroborated by Western analysis shown in Fig. 7CGo. These findings were somewhat counterintuitive because Fos family members were not observed in naïve granulosa cells in vivo (Fig. 6AGo, lane 2). Therefore, the EMSA findings, as well as the Western, suggest that Fra-2 is spontaneously up-regulated after incubation of the granulosa cells ex vivo, a phenomenon observed previously for expression of CCAAT enhancer binding protein-ß (36). Because the GATA-DN did not reduce P450scc expression levels in granulosa to the extent it did in the giant trophoblast (Fig. 7AGo), we aimed to examine the relevance of GATA proteins in the ovary by overexpressing either GATA-4 or GATA-6 in FSH-induced granulosa cells. Indeed, GATA-4 was capable of significantly up-regulating P450scc expression in these cells (Fig. 7DGo). GATA-6 did not seem to have a significant effect on P450scc promoter expression.


Figure 7
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  		Fig. 7. Effect of GATA-DN, CREB, and Fos/Fra-2 Protein Mutants on the P450scc Promoter Activity

A, The –497/+28 wild-type promoter-CAT construct (–497wt) was coexpressed in the presence of dominant negative mutant proteins including GATA (GATA-DN), CREB (A-CREB), ICER, or A-Fos. An equivalent amount of empty vector was added in the control (–497wt). CAT analysis in trophoblast giant cells or granulosa cells was performed as described in Fig. 1Go. The activity results (arbitrary units) are presented as the average ± 95% CI of [14C]chloramphenicol converted to the acetylated products, normalized to the –497/+28 construct and to protein concentration. Activity levels were statistically significant when compared with the respective values obtained for the –497/+28 construct: a, P < 0.01; b, P <0.001; c, P < 0.05. B, Granulosa cells were extracted in EMSA buffer after 4 h in culture and another 6 h incubation with or without FSH (100 ng/ml). EMSA was conducted using 32P-labeled –469/–442 oligonucleotide probe and antiserum to Fra-2 as shown. Arrows denote the DNA-protein complex formed, antibody-associated band supershift (ss), and free probe (F.P.). C, Western blot analysis using anti-Fra-2 serum or anti-calreticulin serum (equal loading control) was performed with duplicate aliquots of the isolated granulosa cells described in panel B. Cells were extracted either shortly after isolation (Fresh culture) or after FSH treatment in culture (FSH 6 h). D, The –497/+28 wild-type promoter-CAT construct was coexpressed in the presence of either empty vector, or with expression vectors for GATA-4 or GATA-6. CAT analysis in granulosa cells was performed as described in Fig. 1Go. The activity results (arbitrary units) are presented as the average ± 95% CI of [14C]chloramphenicol converted to the acetylated products, normalized to protein concentration. Activity levels were statistically significant when compared with the respective values obtained for the –497/+28 construct: a, P < 0.01; or nonsignificant: b, P > 0.1. wt, Wild type.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Despite a substantial understanding of mechanisms involved in activation of the P450SCC promoter in the human placenta, little is known about the regulation of this gene in rodents. In both species, ovarian and adrenal expression of P450scc is SF-1 dependent (14, 16, 17, 45, 46, 47, 48), whereas the expression of placental P450scc is SF-1 independent (10, 49). This shortcoming is dealt with in a species-specific manner; in humans LBP-1b and LBP-9 mediate placental P450SCC transcription (49, 50), whereas rodents replace the SF-1 function with the AP-2{gamma} transcription factor (10). Focusing on the rodent model, this study shows that, ex vivo, the AP-2/SF-1-dependent transcription of P450scc can be further maximized up to 7-fold by an upstream –497/–406 region of the rodent gene examined in primary cultures of both murine trophoblast giant cells and rat ovarian granulosa cells. In fact, a distal region of the promoter was previously suggested to be of importance for P450scc transcription in the Rcho-1 rat trophoblast cell line (31), but the identification of specific cis-regulatory sequences required for P450scc expression was not pursued. We show that the upstream regulatory sequence nests two elements of functional importance in both the placenta and the ovary; a conserved GATA element at –475/–470 and a CRE half-site (CRE1/2) positioned at –450/–447. Disabling mutations of these binding sites, their truncation, or expression of dominant-negative mutants of the GATA and CREB proteins severely impaired the promoter activity in the placenta and the ovary.

This report is the first to identify a specific cis-GATA element required for transcription of the P450scc gene. In recent years, several lines of study provided indirect evidence suggesting a possible role for GATA proteins in the regulation of P450scc. For example, mice with a targeted mutation in GATA-4 that abrogates its binding to FOG-2 do not express P450scc in the testis (51). Also, overexpression of GATA-6 has recently been shown to play a role in P450scc transcription examined in NCI-H295R human adrenal cells (29). GATA factors are abundantly expressed in the gonads of vertebrates and have been shown to activate transcription of other steroidogenic genes in the reproductive tract (36, 52, 53, 54, 55, 56, 57). Therefore, it is not surprising that P450scc should join a growing list of GATA-regulated genes controlling reproductive functions, differentiation, and development.

The –475/–470 GATA site accommodates binding of GATA proteins in a tissue-specific manner, so that GATA-2 predominantly regulates the Cyp11a1 gene in the placenta and GATA-4 plays a similar role in the ovarian steroidogenic cells. These results are consistent with the fact that GATA-2 and GATA-3 are known to be expressed in rodent trophoblast giant cells (58, 59), whereas GATA-4/6 are present in rodent ovarian granulosa cells (59, 60). Our EMSA and Western blot findings suggest that, whereas GATA-2 and GATA-4 have similar binding specificities to the –475/–470 element (AGATAG), the latter motif apparently has a weaker affinity for the GATA-3 and GATA-6 isoforms abundantly present in the respective cell types. This observation agrees with previous reports showing that the GATA isoform binding specificities do not necessarily overlap (54, 61, 62).

It is widely accepted that the cAMP/protein kinase A signaling pathway is central for the induction of steroidogenesis by trophic hormones in the gonads and adrenal cortex (63, 64). However, the mechanism of cAMP control of the rodent P450scc remained unclear, because no consensus cAMP-responsive elements (CREs) have been identified in its promoter. Therefore, the present identification of CRE-CREB activation of the rodent P450scc promoter uncovers the long-sought mechanism for reception of cAMP signaling at the level of transcription. Still, the mechanism by which CREB-1 is implicated in the placental regulation of P450scc in rodents is not clear at the moment. Although cAMP effects on CRE-like elements were described for the human placental gene (46, 48, 50, 65, 66, 67, 68), in our hands the expression of P450scc in the mouse trophoblast giant cells is clearly cAMP independent, a finding consistent with similar observations previously made (31). Furthermore, because no authentic hormone is known to activate steroidogenesis in the placenta, it is intriguing that 8-Br-cAMP can generate a marked Ser133 phosphorylation of placental CREB, but this does not increase activity of the P450scc promoter.

A unique bifunctionality of the CRE1/2 element at –450/–447 was revealed during progress of follicular development in the ovary; predominant binding of CREB-1 was observed in prepubertal follicles and those at early stages of gonadotropin-induced development, which was progressively replaced by Fra-2 binding to the CRE1/2 in follicles approaching terminal luteinization. Elevated expression of Fra-2 during luteinization is consistent with earlier observations that also suggested a marked luteinization-dependent increase of JunD (69). However, our data do not support any role for JunD in regulation of P450scc. The functional relevance of Fra-2 binding was supported by loss of promoter activation in the presence of dominant negative Fos protein, suggesting that the importance of protein members of the AP-1 family in rodent P450scc regulation was overlooked until now. Credit should be given to previous footprinting assays suggesting a putative involvement of an AP-1 element (–319) in regulation of murine P450scc in Y1 adrenocortical tumor cells, but binding of potential trans-factors to this AP-1 site was not examined (20).

The progressive loss of CREB-1 binding predominance during follicular development may constitute a mechanism for the well-documented regulatory switch of P450scc expression in the rodent granulosa cells, turning from a cAMP-dependent mode in early developing follicles, to a cAMP-independent pattern progressing upon onset of the ovulatory LH-surge (26, 70, 71, 72, 73). Thus, this study suggests that switching from CREB-1 to Fra-2 usage constitutes at least one aspect of terminal differentiation occurring during each cycle of ovarian activity, when the dominant follicle turns to become a secretory gland. In this respect, it is interesting to note that functional pairing of Fra-2 and phospho-CREB action is also known to regulate circadian rhythm by exhibiting circadian fluctuations in melatonin-secreting cells of the pineal gland (74, 75). Therefore, it is not unlikely that pairing of CREB-1 and Fra-2 may represent a general platform regulating genes in organs with a cyclical pattern of functionality.

Overall, it is tempting to suggest that during evolution, the use of a CRE half-site element conferred a functional advantage over the classical CRE palindrome by allowing promiscuous binding of different bZIP superfamily members expressed in different organs or tissues presenting similar, yet distinct, differentiation programs, such as the placenta and the ovary. Expanding transcription modularity controlling the P450scc promoter was further refined by the presence of two clusters of regulatory elements, aiming to accommodate alternative sets of trans-factors expressed in two tissues so distant in origin and function: the placenta representing a unique example of an ephemeral organ of extraembryonic lineage that concludes its role at birth, and the ovary that descends from the intermediate mesoderm lineage and functions in a cyclical manner during the entire fertile lifetime. Figure 8Go illustrates how transcriptional plasticity is thus implemented when a modular sequence of 11 bp allows binding of AP-2 in the placenta or SF-1 in the ovary; then, the dual binding capacities of the CRE1/2 motif, being promiscuous for recognition of CREB/ATF-2 and/or Fra-2; and finally, the GATA element recognizing either GATA-2 or GATA-4 required for P450scc transcription in the placenta and the ovary, respectively.


Figure 8
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  		Fig. 8. Schematized Summary of Transcriptionally Active Proteins Putatively Involved in P450scc Regulation in the Placenta and the Ovary

Regulation of P450scc transcription is based on proximal and distal clusters of nonconsensus cis-regulatory elements that allow tissue-specific variations of trans-factor binding: at –490, AGATAG element selectively binds GATA-2 in the placenta or GATA-4 in the ovary; at –470, a CRE half-site GTCA can either bind CREB-1 in the placenta and cAMP-dependent follicles in the ovary, or Fra-2 in cAMP-independent luteal cells; at –40, largely overlapping base-pairs can bind either placental AP-2 or ovarian SF-1.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Acetyl coenzyme A, deoxyribonuclease I, estradiol, polydeoxyinosinic deoxycytidylic acid, protease inhibitor cocktail, sodium orthovanadate, 8-bromo-cAMP, NaF, DMEM, peroxidase-conjugated goat antirabbit, and peroxidase-conjugated rabbit antigoat sera were obtained from Sigma (St. Louis, MO). BSA was purchased from ICN Biochemicals, Inc. (Cleveland, OH). F-12 medium was from Life Technologies, Inc. (Invitrogen, Paisley, UK). Heat-inactivated fetal calf serum (FCS), trypsin-EDTA (0.25% trypsin, 1:2000 EDTA), and penicillin (10, 000 U/ml)/streptomycin (10 mg/ml) solution were from Biological Industries Israel (Beit Haemek Kibbutz, Israel). Lipofectamine Plus transfection reagents were from Invitrogen (Paisley, UK), and Fugene transfection reagent from Roche Molecular Biochemicals (Mannheim, Germany). Ovine FSH (NIDDK-oFSH-20) was kindly provided by NIDDK’s National Hormone and Pituitary Program and A. F. Parlow (Harbor-UCLA Medical Center, Torrance, CA). eCG (pregnant mare serum gonadotropin) was obtained from Vetimex (Bladel, The Netherlands) and hCG from Organon (Oss, The Netherlands). Restriction enzymes were obtained from New England Biolabs, Inc. (Beverly, MA).

Polyclonal antisera to GATA-2 (sc-9008X), GATA-3 (sc-268X), GATA-4 (sc-1237X), GATA-6 (sc-9055X), pan-Fos (sc-253X), pan-Jun (sc-44X), and ATF-1 (sc-243X) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody against CREB-1 (AHP337) for EMSA was from Serotec (Oxford, UK), antibody against phospho-CREB and CREB for Western blot analyses were from Cell Signaling Technology (Danvers, MA), and antibodies against c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB, and JunD were kindly provided by Douglas Stocco (Texas Tech University Health Science Center, Lubbock, TX). Oligonucleotides and PCR primers were synthesized by Sigma-Genosys (Cambridgeshire, UK).

Animals
Timed pregnant female CB6F1/OlaHsd mice and female Sprague Dawley rats (21 d old) were obtained from Harlan Laboratories (Jerusalem, Israel) and maintained under a schedule of 16 h light, 8 h dark with food and water ad libitum. Noon time on the day after mating was considered d0.5 post coitus, or E0.5. Animals were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All protocols had the approval of the Institutional Committee on Animal Care and Use, The Alexander Silverman Institute of Life Sciences, The Hebrew University of Jerusalem. Preovulatory eCG-hCG-treated ovaries were prepared by administration of 15 IU of eCG to 25-d-old rats, which were further treated with 4 IU of human CG administered sc 50 h later. The animals were killed either before treatment, at 10 h after eCG treatment, or at 8 h after hCG. Ovaries were retrieved for protein extraction for either EMSA or Western blot, as described below.

Promoter Constructs and Expression Plasmids
The –942/+28 region of the murine P450scc promoter was cloned by a PCR-based approach using murine genomic DNA as template. Primers were planned based on NCBI accession no. NW_000353, with 5'-MluI and 3'-BglII cloning sites (lowercase) included in addition to ggcc in both forward and reverse primers. Forward primers used were:

–942: 5'-ggccacgcgtGGATCTTAAAAGCATAATGAAA;

–497: atcgacgcgtCAGACCCGTGCATGATCTTAGG;

–406: atcgacgcgtCGCAAGGAATGGAAGCAAACC;

–307: ggccacgcgtTCTAAGCCTCATTTTCTTCCCA;

–136: ggccacgcgtAAGTTCTTTCTCTGAGTTTGG).

The reverse primer was +28: 5'-ggccagatctTGCCACTTCCTGCTGCACGAGT. Mutations of either or both the GATA, CRE, or AP-2/SF1 elements were obtained by use of a variant forward primer (mutGATA: ATCGACGCGTCAGAC-CCGTGCATGATCTTAGGCcgTacCTC; mutCRE: ATATACGCGTCAGACCC-GTGCATGATCTTAGGCTATCTCTCATCTTAGAGGATAcTtGaagTCACCTA) and/or long reverse primer (mutAP2: gatcagatct TGCCACTTCCTGCTGCACGAG-TGTCTCTGCCCCAAACCTCCAGAGCCACACTTATAACCACCAGCTCAAtaCTAAGAG) in which point mutations (lowercase) were introduced. The PCR products were digested with MluI and BglII before ligation and ligated (T4-DNA ligase; Roche Molecular Biochemicals) into promoterless pGL3-Basic luciferase vector (Promega Corp., Madison, WI) or pCAT3-Basic (Promega) restricted by the same enzymes. All plasmids generated were verified by PCR, restriction analysis, and sequencing.

The dominant negative expression vector, GATA-DN, was kindly provided by Dr. Robert Viger (Chul Research Center and Department of Obstetrics and Gynecology, Laval University, Quebec, Canada) (41), ICER by Paolo Sassone-Corsi (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique-Institut National de la Santé et de la Recherche Médicale Université Louis Pasteur, Strasbourg, France) (76), A-Fos from Eugene Tulchinsky (University of Leicester, Leicester General Hospital, Leicester, UK) (77), and A-CREB by Dr. Richard H. Goodman (Oregon Health Science University, Portland, OR).

Cell Cultures
Trophoblast Giant Cells.
Trophoblast giant cells were prepared from E9.5 mouse uteri as previously described (10) using trypsin dissociation medium (1.7 mg/ml trypsin and 80 µg/ml deoxyribonuclease I) (78), and plated in 12-well plates (~two implantation sites per well) in DMEM supplemented with 10% FCS.

Granulosa Cell Cultures.
Cells were prepared from estradiol-primed animals exactly as described (18, 35) and plated onto serum-coated 12-well plates (~0.8 ovary per well).

Transfection and Reporter Gene Assays
Lipofectamine.
On the morning after seeding, trophoblast giant cells in each well were transfected with 600 ng DNA and Lipofectamine Plus reagent (2 µl Lipofectamine, 5 µl Plus reagent) in serum-free medium according to the basic protocol provided by the manufacturer. The medium was changed, 3 h after onset of transfection, to serum-containing medium. Where indicated, 0.5 mM 8-Br-cAMP was added 24 h after transfection. Extracts for CAT analysis were performed 48 h after transfection.

Electroporation.
Transfection of granulosa cells by electroporation was performed in suspension (4 x 105 cells/ 0.8 ml electroporation cuvette) containing CAT constructs (50 µg DNA) as previously described (18, 36). The treated cells were seeded into two wells (24-well plate), each containing 0.5 ml medium, and extracts for CAT or luciferase activity were made after a 6-h incubation of the cells with FSH (100 ng/ml).

Fugene.
Initially, we were unable to perform dominant negative (DN) experiments using primary granulosa cells, because these have relatively low transfection rates for reliably testing the reduced activities in the presence of the DN proteins. We therefore changed to a new transfection agent using Fugene 6, previously proven to act much more efficiently under similar conditions (79, 80). To this end, granulosa cells were plated in 12-well dishes precoated with 1% serum at a density of 1 x 105 cells/ml in 1 ml of serum-free 4F medium (81). Three hours later, the medium was replaced by DMEM/F12 with 5% FCS without antibiotics, and transfection of granulosa cells was performed by Fugene, with 1.5 µl Fugene per 500 µg DNA, according to the manufacturer’s instructions. We used 500 ng reporter gene plasmid and 100 ng dominant negative or expression vector per well. The following morning, wells were washed twice with serum-free DMEM/F12, fresh 4F was added, and extracts for CAT activity were made after a 6-h incubation of the cells with FSH (100 ng/ml).

CAT Assay.
After the indicated treatments, cell lysates were prepared and CAT activity was analyzed as previously described (18, 36). We found it important to wash cells with the 0.25 M Tris buffer several times before collecting extract, to maximize protein extracted. Data are presented relative to activity of –497/+28 scc construct, normalized per protein and time of assay.

Luciferase Assay.
At 48 h after transfection, trophoblast giant cell lysates were prepared with passive lysis buffer (Promega) and luciferase activity was analyzed using Dual Luciferase Reporter Assay System (Promega) according to manufacturer’s instructions. Data are presented relative to activity of –497/+28 scc construct, normalized per protein and time of assay.

EMSA
Cell and tissue extracts for EMSA were obtained as described previously (36). Briefly, cells from whole tissue were homogenized using a Dounce homogenizer in 3–4 volumes of buffer A (400 mM KCl; 10 mM NaH2PO4, pH 7.4; 10% glycerol; 1 mM EDTA; 1 mM dithiothreitol; 5 mM NaF; 1 mM sodium orthovanadate; and 1% vol/vol protease inhibitor cocktail), and the protein slurry was freeze-thawed three times in liquid nitrogen and a 37 C bath. Cells from culture were washed twice with PBS, scraped off plates, spun at 3000 rpm for 5 min, and then resuspended in Buffer A and freeze-thawed. Finally, the cell lysates were centrifuged for 3 min at 14,000 x g, and the protein content in the supernatants was determined by a modified Bradford assay (82). Extracts were kept at –70 C until use.

Protein extract (10 µg) was incubated with 2 ng of double-stranded DNA previously labeled by a fill-in reaction using Klenow fragment (Promega) and [{alpha}-32P]dCTP (Amersham Pharmacia Biotech, Little Chalfont, UK). Binding assay was performed using a final volume of 30 µl containing 100 mM KCl, 15 mM Tris-HCl (pH 7.5), 10 mM dithiothreitol, 1 mM EDTA, 12% glycerol, and 0.75–4.5 µg polydeoxyinosinic deoxycytidylic acid used for placental and ovarian EMSA, respectively. After incubation for 35 min at room temperature, the binding products were resolved on a native prerun polyacrylamide gel (5%) using 0.5x TBE running buffer (50 mM Tris; 50 mM boric acid; and 10 mM EDTA, pH 8.3). When competition experiments were conducted, the protein extract was added last to the reaction mixture. When antibodies were used for detection of a given protein-DNA complex, the reaction cocktail without the probe was preincubated with 2 µg of the antibody for 25 min at room temperature before addition of the labeled DNA. The dried gels were analyzed using a FLA-3000 Bio-Imaging analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan). Gels were also exposed to Super RX medical x-ray film (Fuji Photo Film Co., Ltd.) for 2–24 h at –70 C and developed by an X-Omat processor (Curix 60; Agfa, Munchen, Germany).

Oligonucleotide Probes.
Oligonucleotides used for EMSA [forward (F) primer is indicated] were:

–488/–457sccF (5'-CTAGATGATCTTAGGCTATCTCTCATCTTAGAGG);

–488/–457mutGATAsccF (5'-CTAGATGATCTTAGGCagTCTCATCTTAGAGG);

–469/–442sccF (5'-CTAGCTCATCTTAGAGGATATTCGTCATCACC);

–469/–442mutCRE-F (5'-CTAGCTCATCTTAGAGGATATTtGaagTCACC);

–469/–442mutGATA-F (5'-CTAGCTCATCTTAGAGGcaATTCGTCATCACC);

scc1-F (–60/–34scc, 5'-GATCGCTCCTCTCTTAGCCTTGAGCTAGTTACCTA) (10); GATA consensus (–75/–42Star, 5'-GGCCAAGCTTGACTTTTTTATCTCAAG-TGATGATGCA-CAGCC) (36); CRE consensus-F (5'-GATCAAATTGACGTCATG GTAA) (83). Synthetic oligonucleotides were annealed at a concentration of 1 µg/µl in annealing buffer (50 mM Tris-HCl, pH 7.5; 250 mM NaCl; 0.5 mM EDTA) at 85 C for 10 min and then cooled slowly to room temperature overnight and stored at –20 C. The annealed oligonucleotides were diluted to 50 µg/µl in double distilled water.

Western Blot Analysis
Total protein from trophoblast giant cells (fresh or cultured, prepared as described above) granulosa cells or ovaries from naive, eCG-, or eCG/hCG-injected rats (as described above) were extracted with radioimmune precipitation assay lysis buffer (RIPA) containing protease-inhibitor cocktail and 10 mM Na-vanadate. The extracts were analyzed by SDS-PAGE (15 µg protein/lane) and electroblotting as previously described (84). Specific signals were detected by chemiluminescence using the LumiGlo substrate (New England BioLabs) and signals recorded on x-ray film.

Data Presentation and Statistical Analyses
CAT activities were normalized to the –497/+28 construct per protein and time of assay. Data are presented as the average ± 95% confidence interval (CI) given in arbitrary units. Multiple independent transfections (n ≥ 3) were performed for each construct. Student’s unpaired two-tailed t test was performed using Excel (Microsoft Corp., Redmond, WA) statistical analysis functions. Differences between the activities of the indicated constructs were considered statistically significant at P < 0.05.


   ACKNOWLEDGMENTS
 
We thank Dr. Douglas M. Stocco of Texas Tech University Health Sciences Center (Lubbock, TX) for sharing antisera and providing expression plasmids; Paolo Sassone-Corsi of Université Louis Pasteur (Strasbourg, France) for providing the ICER; Robert S. Viger of CHUL Research Centre (Quebec City, Canada) for the GATA-DN plasmid; Eugene Tulchinsky of University of Leicester (Leicester, UK) for providing A-Fos plasmid; Richard Goodman of Vollum Institute, Oregon Health and Science University (Portland, OR) for the A-CREB plasmid; Willian Rainey of University of Texas Southwestern Medical Center at Dallas (Dallas, TX) for the GATA-6 expression construct; Venkataraman Sriraman and JoAnne Richards of Baylor College of Medicine (Houston, TX) for useful protocols of Fugene use in granulosa primary cultures.

We wish to dedicate this work in memory of Ruth Orly.


   FOOTNOTES
 
This work was supported by Grant 592/03 from the Israel Science Foundation and Grant 2003/398 from United States-Israel Binational Foundation.

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 9, 2007

Abbreviations: AP-2, Activating protein 2; ATF, activating transcription factor; 8-Br-cAMP, 8-bromo-cAMP; CAT, chloramphenicol acetyltransferase; CG, chorionic gonadotropin; CI, confidence interval; E9.5, embryonic d 9.5; FCS, fetal calf serum; GATA-DN, dominant-negative GATA; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; ICER, inducible cAMP early repressor; P450scc, cholesterol side-chain cleavage cytochrome P450; SF-1, steroidogenic factor-1; StAR, steroidogenic acute regulatory protein.

Received for publication May 25, 2006. Accepted for publication December 29, 2006.


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 DISCUSSION
 MATERIALS AND METHODS
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