Transactivation of the Progesterone Receptor Gene in Granulosa Cells: Evidence that Sp1/Sp3 Binding Sites in the Proximal Promoter Play a Key Role in Luteinizing Hormone Inducibility
Venkataraman Sriraman,
S. Chidananda Sharma and
JoAnne S. Richards
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Dr. JoAnne S. Richards, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030. E-mail: joanner{at}bcm.tmc.edu.
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ABSTRACT
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LH induction of the progesterone receptor (PR) in granulosa cells is a central event in ovulation. To identify critical regions of the mouse PR promoter that confer LH inducibility in granulosa cells, a mouse PR promoter (-384/+680) genomic fragment was ligated to a luciferase reporter construct and transfected into primary cultures of granulosa cells. Forskolin/phorbol myristate (PMA) induced PR promoter-luciferase reporter activity in granulosa cells greater than 15-fold. A deletion construct comprised only of the distal promoter alone (-348/+64) was inactive. Conversely, deletion constructs eliminating putative distal promoter-regulatory elements that bind Sp1, nuclear factor Y, and GATA-4 as well as the transcription start site (+1) failed to reduce forskolin/PMA activation of reporter activity. Additional 5'-deletions identified a minimal promoter region (+420/+680) sufficient to bestow cAMP responsiveness approximately 8- to 10-fold. Two GC-rich regions Sp1(A)[+440/+461] and Sp1(B) [+473/+490] bound Sp1/Sp3. Site-directed mutagenesis of Sp1(A) and Sp1(B) reduced activity of the proximal (+357/+680) promoter reporter construct approximately 50% and 99%, respectively. When the same Sp1(B) mutation was introduced into the intact promoter (-145/+680), forskolin/PMA induction of promoter activity was reduced by 7080%. When the distal GC box as well as the proximal Sp1(B) site were both mutated in the context of the intact promoter, inducibility of the transgene was even more severely reduced. The importance of these Sp1/Sp3 binding regions was confirmed in human MCF-7 cells and Drosophila SL2 cells. Collectively, these results indicate that the Sp1/Sp3 binding sites within the mouse PR proximal promoter are essential for transactivation of the gene by agonists in granulosa cells. The molecular mechanisms by which LH activates Sp1/Sp3 at this region within the PR gene remain unknown but do not involve changes in the binding of Sp1/Sp3 to the critical GC boxes. Rather, Sp1/Sp3 appear to recruit other factors to the promoter.
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INTRODUCTION
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OVULATION IS ESSENTIAL for mammalian reproduction. It is a complex process that occurs only when mature, preovulatory follicles are exposed to an ovulatory surge of LH (1, 2). The maturation of preovulatory follicles has been well studied and involves the combined actions of various hormones and locally produced growth factors that prepare the follicle to respond to LH (3, 4). The LH surge acts to terminate follicular growth and induce genes that are necessary for ovulation. One of these genes encodes the progesterone receptor (PR; Ref. 5) that is obligatory for follicular rupture (6, 7, 8). Two isoforms of PR (PR-A and PR-B) are generated by differential translation of a single transcript (9) with PR-A being more prevalent than PR-B in the ovary (10). Mice null for both PR-A/B (PRKO) fail to ovulate even when supplemented with exogenous hormones and are completely infertile (6, 7, 8), providing convincing evidence to support earlier studies (11). Knowing the molecular mechanisms by which LH induces PR in the ovarian follicle is critical in our understanding of ovulation.
The induction of PR by LH and cAMP in granulosa cells appears to be distinct from the mechanisms by which PR is induced in other tissues (10, 12) where estrogen, via the estrogen receptor (ER), directs PR expression (9, 13). Growth factors also play a role in uterine cells (14). Although ER subtypes, ER
and ERß, are expressed in granulosa cells of preovulatory follicles (15, 16, 17), high endogenous estrogen in these follicles does not induce PR mRNA or protein (7, 10, 12). Rather, surge levels of LH and cAMP-mediated signals are obligatory. Likewise, when immature granulosa cells are cultured with FSH and testosterone (a substrate for aromatase) for 48 h, they differentiate and acquire a preovulatory, estradiol-synthesizing phenotype. In these cells, PR mRNA is not detected until they are exposed to high levels of LH, forskolin, phorbol myristate (PMA), or GnRH (10).
The promoter of the PR gene is complex, and numerous PR mRNA transcripts are present in uterine, mammary, hypothalamic, and ovarian cells (9, 13, 18). Recent studies, however, are beginning to provide some insights into regions of responsiveness. Studies of Kraus et al. (9, 13) identified two putative functional promoters within the rat PR gene. These were designated the distal and proximal promoters corresponding to -131/+65 bp and +461/+675 bp within the 5'-untranslated region, respectively. Each of these regions, when flanked with two GAL-4 DNA binding sites, actively transcribed a chloramphenicol acetyltransferase reporter gene in the presence of cotransfected GAL-VP16. In addition, transcripts specific to the distal and proximal regions of the promoter were observed using probes spanning different regions of the 5'-flanking region. These observations indicate that more than one transcription initiation site is present in this 5'-region of the PR gene. The proximal promoter has an estrogen receptor response element (ERE) half-site in addition to two ERE-like regions (ERE3 and ERE4/5), which, in cotransfection assays with ER expression vectors in 3T3 cells, exhibit E responsiveness. Furthermore, in this 3T3 cell cotransfection system, mutation of the ERE1/2 site or the ERE3 site decreased promoter activity. The ERE3 region competed for the binding of ER
to a consensus ERE probe, albeit at extremely low affinity. In contrast to the proximal promoter, the distal promoter is not estrogen responsive unless two or more copies of PR-proximal ERE-like sequence are ligated to it. In 3T3 cells and MCF-7 cells, cAMP (isobutylmethylxanthine and cholera toxin) has not been shown to activate either the proximal or the distal PR promoter constructs. Moreover, the inducibility of the PR promoter varies based on the context of cell types employed for analysis and could be due to the differential distribution of ER subtypes, other transcription factors, or the phosphorylation status of these factors.
Studies with granulosa cells show that transactivation of chimeric PR distal and proximal promoter constructs is not 17ß-estradiol (E) inducible (12, 19). Rather, forskolin, which directly activates adenylyl cyclase, or forskolin in the presence of PMA, which activates protein kinase C, was obligatory for activation of chimeric PR promoter constructs. These results support the observations that in granulosa cells, LH/cAMP, but not E, is essential for induction of PR mRNA (10). Thus, despite the fact that granulosa cells express both ER
and ERß (15, 16, 20), E alone is not sufficient to transactivate the PR gene. Rather, the phosphorylation of specific factors appears to be required. Because distinct mechanisms appear to induce PR and activate selected PR promoter constructs in granulosa cells, the studies described herein were designed to identify cAMP/PMA-responsive regions of the intact mouse PR promoter rather than use chimeric constructs composed of separate distal and proximal regions as previously reported (12, 19). Specifically, an intact mouse PR promoter region containing both distal and proximal promoters (-368 to +680) was ligated to a luciferase reporter construct. Our goal has been to generate site-specific mutations within this intact promoter region that disrupt cAMP/PMA responsiveness and establish the identity of the factors binding to relevant sites. Based on our original observations (12) and those of others (19), we anticipated that the distal promoter Sp1 binding GC box and a CAAT region would be important for cAMP/PMA inducibility of the intact promoter. However, the results obtained in this study indicate that regions within the proximal promoter, rather than the distal promoter, constitute the essential region of mouse PR promoter that is LH/cAMP responsive.
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RESULTS
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Induction of PR mRNA in Cultured Granulosa Cells
Previous studies have shown that PR mRNA is selectively induced in rat granulosa cells of preovulatory follicles by the LH surge (5, 10). This effect of LH in vivo can be mimicked in primary cultures of rat granulosa cells. Specifically, we have shown that LH rapidly, but transiently, induces PR mRNA and protein in FSH/T-differentiated (preovulatory) granulosa cells in culture (10). Although these differentiated cells provide the most physiological model in which to conduct transfection assays with PR promoter-reporter constructs, they do not transfect easily or efficiently (21). Therefore, to determine the efficacy of using immature granulosa cells as a model for analyzing PR promoter-reporter constructs, it was necessary to determine whether the endogenous PR gene could be activated in these cells, if given the appropriate stimulus. For these experiments, granulosa cells were harvested from E-primed immature rats and cultured overnight in defined medium without serum. At that time (0 h), the granulosa cells were treated with vehicle (control), forskolin (10 µM) to increase cAMP, the phorbol ester PMA (20 nM) as a substitute for diacylglycerol, or the combination for 4 h. PR mRNA was measured by semiquantitative RT-PCR using specific primer pairs, and the ribosomal protein L19 was used as the internal control (Fig. 1A
). As shown, PR mRNA was induced by forskolin alone. Although PMA was less effective than forskolin, PMA greatly enhanced the effects of forskolin. FSH also induced PR mRNA in these cells to the same extent as forskolin (data not shown). These results indicate that maximal induction of the endogenous gene occurred with the combination of forskolin (cAMP) and PMA. These responses are similar to those observed in the more differentiated cells, indicating that the factors required to activate the endogenous PR gene are present in immature granulosa cells, validating this model for analyzing the PR promoter activity.

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Figure 1. PR mRNA and PR Promoter-Reporter Constructs Are Induced in Cultured Immature Granulosa Cells by Treatment with FSH and Fo/PMA that Mimic LH
A, Representative semiquantitative RT-PCR analysis with corresponding autoradiographs reveals that treatment of cultured granulosa cells with Fo/PMA for 4 h induces a 120-fold increase in PR mRNA in contrast to the moderate increase by Fo or PMA alone. RT-PCR was performed multiple times from granulosa cells isolated from estrogen-primed immature rats as described in Materials and Methods. The relative expression was determined by quantifying radioactive PCR products by phosphorimaging and normalizing the PR expression levels to that of L-19. Values are mean ± SD. B, Granulosa cells were obtained from E-primed rats and cultured in defined medium overnight. The cultures were transiently transfected with 1 µg of PR promoter-luciferase reporter construct employing Fugene as described in Materials and Methods. The cells were treated with forskolin (10 µM; Fo), PMA (20 nM), FSH (50 ng/ml), or Fo and PMA for 4 h and harvested for luciferase assays. Data are represented as light specific units (LSU) per µg protein; values are mean ± SD. PR promoter luciferase constructs were transfected in triplicate in each experiment. The schematic in panel B represents the putative functional elements in mouse PR promoter within the -145/+680 construct.
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Transactivation of the Mouse PR Promoter-Luciferase Construct by Forskolin and PMA
To examine the activity of the intact PR promoter in granulosa cells, two murine PR promoter fragments, PRdist-1 (-368/+680) and PRdist-2 (-145/+680) were amplified by PCR cloning and ligated to the luciferase (Luc) reporter gene (see Materials and Methods). These promoter regions contain the previously described distal and proximal promoters but lack the translational start sites for PRA and PRB (9). Immature granulosa cells were harvested and cultured overnight in defined medium, transfected, and stimulated with FSH (50 ng/ml), forskolin (10 µM), PMA (20 nM), or forskolin and PMA as described in Materials and Methods (Fig. 1B
). FSH and forskolin stimulated an 8- to 10-fold increase in transgene activity, whereas PMA had little or no effect (Fig. 1B
). In contrast, the combination of forskolin and PMA induced luciferase activity 15- to 20-fold. Thus, forskolin and PMA acted synergistically to transactivate the intact murine PR promoter luciferase transgene in a manner similar to the expression of the endogenous gene (Fig. 1B
). Similar results were obtained when the PR dist-1 (-368/+680) luciferase construct was transfected (Fig. 2
), indicating that sequences from -368 to -146 do not impact activation of the PR promoter in granulosa cells. E alone had no effect on activation of either of these constructs (Fig. 2
).

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Figure 2. The Proximal Region of PR Promoter Is Critical in Conferring Fo/PMA Responsiveness
Granulosa cells were obtained from E-primed rats and cultured in defined medium overnight. The cultures were transiently transfected with 1 µg of PR promoter-luciferase reporter construct employing Fugene as described in Materials and Methods. The cells were treated with forskolin (10 µM; Fo), PMA (20 nM), Fo+PMA, or E (100 nM) for 4 h and harvested for luciferase assays. Data are represented as light specific units (LSU) per µg protein; values are mean ± SD. PR promoter luciferase constructs were transfected in triplicate in each experiment. The schematic represents the putative functional elements in mouse PR promoter. The length and region of the deletion constructs that it spans are represented in the bars with its activity in the graphs.
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Functional Analysis of the Distal PR Promoter
To analyze the functional activity of specific PR promoter regions, transfections as well as transcription factor binding assays were done. In contrast to the intact promoter constructs, PRdist-1 and PRdist-2, which are highly induced by forskolin and PMA, a construct with the distal promoter alone (PRdist-3; -368/+64) had low basal activity and did not respond to forskolin and PMA (Fig. 2
). These results confirm previous reports with the rat promoter (12, 13) and show that the distal promoter by itself is not functional even though this region contains a CAAT region that binds NFY-B (data not shown), a GC box that binds Sp1/Sp3 (12), and a GATA site that binds GATA-4 (data not shown). Furthermore, when site-specific mutations were made in the GC box (-38/-48), CAAT region (-57/-81), or both regions in the distal promoter of the -145/+680 construct, inducibility by forskolin and PMA was similar to that of the intact construct (data not shown). These results indicate that despite the active binding of nuclear factor Y (NF-Y) and Sp1 in distal promoter, mutation of these sites in the context of the intact promoter did not cause a major change in function and thus are not obligatory for transcriptional activation of the PR gene in this context.
Functional Analyses of the Proximal Promoter
Constructs containing only the proximal (P) promoter region were generated by deleting the distal promoter. These constructs, designated PRprox-1, -2, -3, -4, and -5 contain the regions -1/+680, +43/+680, +357/+680, +420/+680, and +43/+624, respectively (Fig. 2
). In contrast to the absence of activity in distal promoter (PRdist-3), all of the proximal promoter deletion constructs (PRprox-15) showed significant luciferase activity in response to forskolin/PMA (Fo/PMA), although the fold induction was 4050% lower than that of the intact promoter construct PRdist-2 (-145/+680) (Fig. 2
). These results clearly suggest that the transcriptional start site at +1 is not the only site of transcription initiation. Rather, the region between +420/+680 is sufficient to confer cAMP responsiveness to the PR promoter. Previous studies with the rat PR promoter in MCF-7 cells revealed that a site designated as ERE3 is critical for estrogen inducibility and demonstrated ER
can bind to this region (9). Proximal promoter constructs with a mutation at the ERE3 site were not active, and even the addition of two ERE sites upstream could not rescue its activity in MCF-7 cells. Because the induction of PR appears to be estrogen independent in granulosa cells, the role of ERE3 (+620/+632) in activation of PR promoter was investigated by testing a 3'-deletion construct PRprox-2 (+43/+624). Surprisingly, deletion of ERE3 region did not alter the Fo/PMA-induced activation of the promoter-reporter construct (Fig. 2
). These results indicate that the ERE3 region is not critical in cAMP induction of PR promoter activity in this context.
To identify the critical regulatory elements within the +420/+680 region, a computer-based search (TFsearch, Tokyo, Japan) was done. Because this approach did not reveal any specific transcription factor binding sites, we visually inspected the sequences in this region and observed a GC-rich region with putative binding sites for Sp1, designated Sp1 A (+440/+461) and B (+563/+570), as well as a putative GATA-1 site (+563/+570). To verify that these putative Sp1/Sp3 binding sites bound Sp1/Sp3, EMSAs were performed with whole-cell extracts prepared from granulosa cells isolated from ovaries of hormonally primed hypophysectomized (H) rats. Oligonucleotides were designed to wild-type and mutant Sp1(A) and Sp1(B) sites. When Sp1(A) was used as the labeled probe, two major protein/DNA complexes were observed (Fig. 3
). Competition with a 100-fold excess of wild-type cold competitor DNA prevented the complex formation, whereas mutant oligonucleotides failed to compete. The upper band supershifted, in part, upon addition of an Sp1 antibody. The lower band was reduced with an Sp3 antibody, and both the upper and lower bands supershifted completely when both Sp1 and Sp3 antibodies were added to the reaction mix, indicating that Sp1, Sp1/Sp3, and Sp3 complexes are formed. Addition of an early growth response protein 1 (Egr-1) antibody did not affect the formation of these complexes. Similar results were obtained when Sp1(B) was used as the labeled probe; the two complexes could be supershifted by the Sp1 and Sp3 antibodies (Fig. 3
). However, the amount of complex formed with the Sp1(B) probe was comparatively much higher than that observed with the Sp1(A) probe. To determine whether the amount or binding of Sp1/Sp3 in granulosa cell extracts was hormonally regulated, whole-cell extracts (WCE) were prepared from hormonally primed hypophysectomized rats and incubated with labeled Sp1(B) as a probe (Fig. 3
). As shown, the binding of Sp1/Sp3 remained relatively constant in granulosa cells treated with E, FSH, and human chorionic gonadotropin (hCG), indicating that neither the amount or binding activity of Sp1/Sp3 to the PR promoter is hormonally regulated in these cells. Western blot confirmed the stable levels of Sp1 (22) and Sp3 (data not shown). No specific binding was observed for the GATA-1 site (data not shown).

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Figure 3. Sp1/Sp3 binds to Sp1(A) and Sp1(B) Sites within the PR Proximal Promoter
WCE were prepared from granulosa cells obtained from immature hypophysectomized rats treated with E, FSH, and hCG. EMSAs were carried out using 3 µg of protein and a labeled (A) Sp1(A) or (B) Sp1(B) oligonucleotide. Wherever indicated the extracts were preincubated with 100-fold excess (xs) self cold competitor or mutant probe or 1 µg of antibody before the addition of labeled oligonucleotide [Sp1(A)* or Sp1(B)*]. Protein DNA complexes were resolved by PAGE in 0.5x TBE and exposed to autoradiographic film. The complexes formed are indicated by arrows, and the supershifted complexes are depicted by the brackets. The sequences of the wild-type and mutant oligonucleotides employed are indicated in the lower panel. C, Immature hypophysectomized (H) rats were treated with estradiol (E; HE), FSH (F; HEF), and hCG (HEF-hCG) to stimulate follicular growth and luetinization. WCE were prepared from granulosa cells isolated from the ovaries at indicated treatments, and EMSAs were run with 2.5 µg of WCE protein and labeled Sp1(B) oligonucleotide as described earlier.
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To determine the functional activity of these Sp1/Sp3 binding sites, each site was mutated in the context of the PRprox-3 construct for analysis in transfection assays (Table 1
and Fig. 4
). The mutations were identical to those analyzed by the EMSAs described above. Introduction of a mutation in the Sp1(A) site (+440/+661) resulted in a significant reduction in the PR promoter activity (Table 1
and Fig. 4
). On the contrary, mutations at the Sp1(B) site +473/+490 (Table 1
: TT or AA mutant) resulted in total loss of PR promoter activity compared with that of the Sp1(A) site. Altering the downstream GATA site did not have any effect on PR promoter activity in granulosa cells (data not shown).

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Figure 4. Mutations of Sp1(A) and Sp1(B) Sites in the Proximal PR Promoter Luciferase Construct Reduce Transactivation of the Promoter
The functional importance of two Sp1/Sp3 binding motifs designated Sp1(A) and Sp1(B) sites [+440/+461 and +473/+490] in the proximal PR promoter was assessed by transfection of the site-directed mutants to these sites. Mutants were generated using the Gene Editor site-directed mutagenesis kit from Promega Corp. (Madison, WI). Transient transfections were carried out in granulosa cells obtained from E-primed rats employing Fugene as described in Materials and Methods. Data are represented as light specific units (LSU) per µg protein; mean ± SD. PR promoter luciferase constructs were transfected in triplicate in each experiment. The wild-type and mutant constructs are schematically represented with the mutated residues and their functional activity.
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These results clearly demonstrate that the Sp1/Sp3 binding sites are critical for PR-promoter activation in granulosa cells. Because the mutation at the Sp1(B) site was more detrimental than Sp1(A) in the PRprox-3 context, mutation of Sp1(B) was generated in the context of the intact promoter comprised of the proximal and distal promoter regions. Mutation of the Sp1(B) site in PRdist-2 (-145/+680) or PRprox-1 (+1/+680) of the PR promoter resulted in significant reduction of basal activity of the PR promoter but did not completely block Fo/PMA induction (Fig. 5A
). To investigate further the functional roles of Sp1 in the PR promoter, site-specific mutations of the distal GC box as well as the proximal Sp1(B) site were generated in the PR-dist-2 (-145/+680) construct. Transfection studies in granulosa cells showed that mutation of the distal Sp1 site along with the Sp1(B) site reduces the activity even more than that observed with the Sp1(B) site mutant alone (Fig. 5B
). These data clearly demonstrate that the proximal promoter Sp1/Sp3(B) site is a key determinant of transcriptional activation of PR expression in response to cAMP/PMA and that it interacts with the GC-rich Sp1/Sp3 binding region of the distal promoter for full activation of the gene in granulosa cells.

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Figure 5. Sp1(B) Site Is Critical for Transactivation of PR Promoter Luciferase Constructs in Granulosa Cells
A, The role of Sp1(B) [+473/+490] in transactivation of PR promoter was investigated by transient transfection of granulosa cells with varying lengths of wild-type and Sp1(B) site mutant promoters. The mutants were generated by site-directed mutagenesis (Gene Editor, Promega Corp.), and transient transfections in granulosa cells were performed as described in Materials and Methods. Data are represented as light specific units (LSU) per µg protein; mean ± SD. PR promoter luciferase constructs were transfected in triplicate in each experiment. The wild-type and mutant constructs are schematically represented with the mutated residues and their functional activity. B, The importance of Sp1(B) site and its interaction with the Sp1 binding GC box in the distal promoter was also investigated by transient transfection of granulosa cells with constructs containing mutants of both sites. Data are represented as LSU per µg protein; mean ± SD. Transfections were carried out in triplicate for each experiment. The wild-type and mutant constructs are schematically represented with the mutated residues and their functional activity.
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Mutation Analysis of Half-ERE Site in the Proximal Promoter
Studies by Petz and Nardulli (23) suggested that the half-ERE site adjacent to an Sp1 region in the human PR promoter is important in conferring estrogen responsiveness in MCF-7 cells by enhancing the binding of Sp1 to the Sp1 site. Using granulosa cell extracts and oligonucleotides corresponding to the homologous half-ERE site in the mouse PR promoter, no major protein DNA complex was detected, even after prolonged exposure (data not shown). To understand the functional importance of this region, site-directed mutants were generated in the PRdist-2 and PRprox-3 constructs and transfected into granulosa cells. Fo/PMA activation of the mutant constructs was similar to that of the wild-type constructs, indicating that the half-ERE site is not essential for agonist induction of PR promoter in granulosa cells (Fig. 6
).

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Figure 6. A Half-ERE Site in the Proximal Promoter Is Not Essential for cAMP Activation of the PR Promoter
The role of a proximal half-ERE site in transactivation of PR promoter was investigated by transient transfection in granulosa cells with wild-type and half-ERE site mutant promoter constructs (PR dist-2 and PRprox-3). The mutants were generated by site-directed mutagenesis (Gene Editor, Promega Corp.), and transient transfections in granulosa cells were performed as described in Materials and Methods. Data are represented as light specific units (LSU) per µg protein; mean ± SD. PR promoter luciferase constructs were transfected in triplicate in each experiment.
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Comparative Study in MCF-7 Cells
Because MCF-7 cells express estrogen receptor (predominantly ER
) and previous studies have employed this as a model to show that estrogen induces PR (9), we extended our studies to assess the activation of the murine PR promoter by E as well as forskolin and PMA. In the MCF-7 cells, forskolin and PMA induced the mouse PR promoter constructs in a pattern similar to that observed in rat granulosa cells (Fig. 7
). Induction by Fo/PMA was greater than that by estrogen treatment alone (Fig. 7A
). Activation of the PRprox-3 (+357/+680) construct in response to Fo/PMA was approximately 50% of the PRdist-2 (-145/+680) construct. Mutation of the Sp1 B (AA; mutant 2) site in each of these constructs further reduced its inducibility. Transfection of PRprox-3 (+357/+680) Sp1(A) mutant exhibited an even more pronounced reduction in activity (Fig. 7A
). These results contrast with those obtained in rat granulosa cells where the Sp1(B) mutant (AA) showed minimal activity, whereas the Sp1(A) mutant was reduced by 50% (Fig. 5
). Furthermore, a construct containing mutations in both the GC box of the distal promoter as well as the Sp1(B) site in the proximal promoter was inactive in MCF-7 cells as observed in the granulosa cells (Fig. 5
; data not shown).

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Figure 7. The Proximal Sp1/Sp3 Site (B) Is Essential for Activation of PR Promoter Luciferase Construct in MCF-7 Cells
A, The functional activity of Sp1(A) and Sp1(B) mutants of the proximal PR promoter in MCF-7 cells was assessed by transient transfection assays of the site-directed mutants to these sites. Transient transfections were carried out in MCF-7 cells after culturing overnight in defined medium containing 0.5% serum employing Fugene as described in Materials and Methods. Data are represented as light specific units (LSU) per µg protein; mean ± SD. PR promoter luciferase constructs were transfected in triplicate in each experiment. The wild-type and mutant constructs are represented by numbers of PR promoter. B, WCE were prepared from MCF-7 cells cultured overnight in defined medium with 1% serum. Extracts (2 µg of protein) were incubated with labeled Sp1(A) or Sp1(B) oligonucleotide with or without preincubation of unlabeled competitor DNA or mutant DNA or antibody (Sp1, Sp3, Sp1, and Sp3) on ice. Protein DNA complexes were resolved by PAGE in 0.5x TBE and exposed to autoradiographic film. The complexes formed are indicated by arrows, and the supershifted complexes are depicted by the brackets.
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Whole-cell extracts prepared from MCF-7 cells showed that both Sp1 and Sp3 bind to the Sp1(A)- and Sp1(B)-labeled probes and were supershifted with both Sp1 and Sp3 antibodies (Fig. 7B
). As in the rat, the binding of MCF-7 cell Sp1 and Sp3 to the Sp1(A) probe is less intense than to the Sp1(B) probe. In addition, the EMSA demonstrates that a 100-fold excess of Sp1(A) mutant oligonucleotide competed less effectively than the Sp1(A) wild-type probe, indicating that the Sp1(A) mutant does not bind as well to Sp1/Sp3. This loss in binding appears to be sufficient to alter functional activity of the promoter in MCF-7 cells. Furthermore, Sp1(B) mutant 1 (TT) competes for the Sp1/Sp3 binding whereas Sp1(B) mutant 2 (AA) does not, confirming that the Sp1(B) mutant 2 (AA) used for generating the promoter-luciferase constructs is a more complete mutant. These results demonstrate that forskolin and PMA induce promoter activity in MCF-7 cells as in granulosa cells and emphasize the importance of the Sp1/Sp3 sites in the proximal promoter in conferring cAMP responsiveness of the mouse PR promoter in these human cells. The molecular and biochemical reasons for why the +357/+680 Sp1(A) mutant construct exhibited less activity in MCF-7 cells than in granulosa cells is not entirely clear. Although the mouse, rat, and human PR promoters are highly homologous in this proximal region, there are subtle differences (Fig. 8
). The ERE half-site and the Sp1(B) site are conserved in each. However, the Sp1(A) site present in rat and mouse promoters is not conserved in the human promoter; conversely, a proximal Sp1 site within the human promoter is not conserved in rat or mouse. Thus, different coregulators present in rat and human cells may interact differently with sites A and B in the two cell types and mediate subtle differences in the regulatory mechanisms within this region that spans the ERE half-site (Fig. 8
).

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Figure 8. The Nucleotide Sequences of the Sp1(A) and Half-ERE/Sp1(B) Binding Site in the Mouse PR Promoter Is Aligned with Homologous Regions of the Rat, Rabbit, and Human PR Genes
The conserved half-ERE Sp1 site is boxed with number indicating the relative position of the beginning of half -ERE. The Sp1(A) site of the mouse (and rat) PR promoter, the Sp1(B) sites conserved across all species, and the more 3'-Sp1 site of the human promoter (23 ) are indicated separately.
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Sp1 Activates Transcription Driven by PR Promoter
To determine the role of Sp1 on the transcriptional activity of the PR promoter, transient expression experiments were also performed in Drosophila SL2 cells that lack endogenous Sp proteins (24). Wild-type or Sp1(B) mutant PR promoter luciferase reporter constructs (PRdist-2 and PR prox-3) were cotransfected with increasing concentrations of an Sp1 expression plasmid pPacSp1 (0, 100, 200, and 1000 ng). Expression of Sp1 increased luciferase activity of the wild-type constructs in a dose-dependent manner reaching approximately 25-fold with 1 µg Sp1 plasmid (Fig. 9
). In contrast, Sp1(B) mutants exhibited markedly reduced luciferase activity when compared with the wild-type constructs. These results confirm that Sp1 is a potent activator of PR promoter activity.

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Figure 9. Sp1 Activates PR Promoter in SL2 Cells
Drosophila SL2 cells were cotransfected with 1 µg PR promoter luciferase constructs (PRdist-2 or PR prox-3) along with increasing amounts of an Sp1 expression plasmid pPac-Sp1 (0, 100, 200, and 1000 ng) or the empty vector pPac-0 employing Fugene 6 and assayed for luciferase activity after 24 h. Transfections were also carried out with the respective Sp1 B mutant constructs as described above. Luciferase activity was expressed as light specific units (LSU)/µg protein.
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DISCUSSION
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Results of these studies provide significant new information about the regulation of the mouse PR gene by hormones in granulosa cells. First we show that maximal induction of the endogenous PR gene is stimulated by forskolin and PMA, indicating that these two molecules activate kinase-signaling cascades that act synergistically to alter transcription factor activity. Furthermore, PR mRNA was induced markedly in immature cells, indicating that the transcriptional machinery necessary to transactivate the PR gene is present before the time that it is normally induced in response to the LH surge in vivo. Characterization of the intact mouse PR promoter and site-specific mutations revealed that the proximal promoter is essential for basal and agonist-induced activation of the PR promoter; more specifically, two Sp1/Sp3 binding sites confer the hormone responsiveness. In contrast, the distal promoter alone is inactive but does synergize with the proximal promoter for maximal promoter activation of the PR gene.
Although Sp1 and NF-Y have been shown to interact functionally on other promoters (25, 26), mutations of the distal promoter Sp1 binding GC-rich region, the NF-Y binding CAAT region, or both had no major effect on cAMP/PMA inducibility of the intact PR promoter-luciferase construct in granulosa cells. Likewise, deletions of the distal GC region, CAAT region, distal transcription start site, or the GATA-4 binding site downstream of the transcription start site failed to alter cAMP/PMA inducibility. Importantly, the distal promoter PRdist-3 (-368/+64) containing all these transcription factor binding sites was inactive, suggesting that the distal promoter does not function by itself and is not the major site of transcriptional regulation of the PR gene in granulosa cells.
The "proximal promoter" of the PR gene, a region from +457/+680, was sufficient for expression and cAMP/PMA inducibility of the luciferase reporter in granulosa cells. Deletion of the ERE3 at the 3'-end did not reduce activity, indicating that although this region could confer inducibility if placed upstream of the distal promoter (12), it was not essential in the context of the proximal promoter. Within the proximal PR promoter we have identified two regions (+440/+461 and +473/+490) that bind Sp1/Sp3. Specific antibodies to Sp1 and Sp3 supershifted the protein/DNA complex, and excess Sp1 consensus oligonucleotides prevented binding. When either of these Sp1/Sp3 sites was mutated, basal and Fo/PMA activation of the PR promoter was markedly reduced. Thus, Sp1/Sp3 appear to be critical factors for cAMP/PMA activation of the PR gene in granulosa cells. These data support recent observations by others who have shown by in vivo footprinting that Sp1 binds a similar region of the human PR promoter (23). However, no functional data were presented in that study to determine whether this region was functionally active or whether mutations of the Sp1 site altered promoter activity. Interestingly, the Sp1/Sp3 binding sites identified herein span either side of the ERE half-site (+460/+475; GACAGGAGCTGACCAA) within the mouse promoter, whereas the region of the human promoter characterized by Petz and Nardulli (23) reports only the proximal Sp1 site (i.e. site B). These Sp1 binding regions are close to a broad deoxyribonuclease-hypersensitive site previously identified by Hagihara et al. (18). Thus, this region appears to be a hot spot that is highly conserved in mouse, rat, and human PR genes (Fig. 8
). The importance of this region is further highlighted since mutations in the context of the intact mouse PR promoter markedly reduce LH/cAMP and PMA inducibility in granulosa cells, and mutation of this site with distal Sp1 site further reduces its inducibility. The factors that are recruited by Sp1/Sp3 in response to LH/cAMP remain to be determined. It is possible that Sp1/Sp3 recruit ER as suggested by others (27, 28). However, mutation of the half-ERE site adjacent to the Sp1(B) site did not alter Fo/PMA responsiveness of the reporter constructs in granulosa cells, although previous studies had suggested ER may enhance the Sp1 binding to Sp1 site (23). Although Sp1/Sp3 sites have been reported to interact with many factors, the exact nature of those specifically involved in regulating PR induction by LH (cAMP/PMA) in granulosa cells remain to be determined. However, based on our observations, we hypothesize that Sp1 binding to the distal promoter permits interactions in a synergistic manner with proximal promoter binding proteins to augment the inducibility of the promoter (Fig. 10
).

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Figure 10. Schematic of LH Transactivation of the PR Gene
The model depicts the putative interactions of the obligatory Sp1/Sp3 binding sites in the proximal promoter with the regions of the distal promoter that enhance transcription of the PR gene. Presumptive coregulators and coactivators that interact with these regions of the PR promoter and that may confer LH (cAMP)-mediated induction of the endogenous gene are indicated. Phosphorylation of coactivators, corepression, and coregulators, as well as Sp1/Sp3, are indicated by a solid circle.
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The importance of proximal and distal promoters for induction of the mouse PR promoter reporter construct was also assessed in MCF-7 cells since it has been shown that estrogen treatment increases PR mRNA expression in these cells (9). Transfection of MCF-7 cells with our murine promoter-reporter constructs yielded results similar to those observed in the granulosa cells. Specifically, the proximal promoter as well as the intact promoter were induced (10- to 20-fold) by forskolin and PMA but only marginally by E. The absence of E-mediated induction of the murine PR promoter reporter constructs in our studies was unexpected. The lack of E responsiveness could be due to minor differences in the sequences between mouse and human PR promoters or to the shorter duration of E exposure (4 h) used in these studies when compared with the 24-h treatment of MCF-7 cells used by others. Alternatively, the effect of E could be dependent on the presence of serum factors. Our studies were done in serum-free conditions, whereas others have cultured cells in 5% serum. Thus, the induction of PR promoter constructs in granulosa cells and MCF-7 cells shares some marked similarities that have not been observed previously and were not anticipated. Collectively, the data from the granulosa cells and the MCF-7 cells indicate that the Sp1/Sp3 sites are of critical importance for PR expression and that transactivation of the promoter in both cell types requires activation of kinase cascades. The importance of the Sp1/Sp3 site in activation of the PR promoter was further established by studies in Drosophila SL2 cells, which lack endogenous Sp protein. Cotransfection of an Sp1 expression plasmid with PR promoter-reporter constructs (PRdist-2 and PR-Prox3) in SL2 cells increased the activity of these constructs as much as 25-fold. In contrast, activation of the Sp1(B) mutants by Sp1 was markedly reduced.
In granulosa cells Sp1 is expressed constitutively, and only after treatment with hCG in vivo or cAMP/PMA in vitro is the Sp1/Sp3 site activated to promote the transcription of the PR gene. It is important to note at this juncture that many genes regulated in preovulatory granulosa cells by the LH surge have Sp1/Sp3 binding sites and exhibit Sp1/Sp3-dependent transactivation. These genes include Sgk (22), Egr-1 (29), cytochrome P450 side chain cleavage enzyme (30), steroidogenic acute regulatory protein (31), an aldose-reductase-like gene (mouse vas deferens protein) (32), p21CIP (33), and, more recently, the LH- and PR-regulated gene, ADAMTS-1 (34). With the exception of Sgk, which is also induced by FSH, all of the others are induced rapidly by the LH surge. Whereas Egr-1, PR, and ADAMTS-1 are expressed only transiently before ovulation, transcription of cytochrome P450 side chain cleavage enzyme, steroidogenic acute regulatory protein, and Sgk is maintained in corpora lutea. Thus, if Sp1/Sp3 is critical for the regulation of each of these genes, the Sp1/Sp3 binding sites may provide the basal control elements to which different coregulatory factors are recruited and interact with Sp1/Sp3 on each promoter. Known factors that interact with Sp1 include coregulators such as coregulator of Sp1 (CRSP), TATA binding protein associated factor II130 (TAFII 130), and certain corepressors (35, 36) as well as transcription factors such as the NF-Y (25, 26) and steroid receptors (27). It is also clear from these studies that activation of kinase cascades is essential for Sp1/Sp3-regulated induction of the PR promoter. Whether phosphorylation (or other modification) of Sp1/Sp3, a coactivator, or coregulator is involved remains to be determined. It will be critical to determine which of these factors and phosphorylation mechanisms is important for LH (cAMP/PMA) regulation of PR expression in granulosa cells (Fig. 10
).
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MATERIALS AND METHODS
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Reagents
Media and cell culture reagents and materials were purchased from Life Technologies, Inc. (Gaithersburg, MD), Sigma (St. Louis, MO), Research Organics (Cleveland, OH), Fisher Scientific (Pittsburgh, PA), Corning, Inc. (Corning, NY), and HyClone Laboratories, Inc. (Logan, UT). Trypsin, soybean trypsin inhibitor, deoxyribonuclease, PMA, ATP, dithiothreitol, E, and propylene glycol were purchased from Sigma. Electrophoresis and molecular biology-grade reagents were procured from Sigma, Bio-Rad Laboratories, Inc. (Richmond, CA), and Roche Molecular Biochemicals (Indianapolis, IN). Oligonucleotides were purchased from Genosys (The Woodlands, TX). All RT-PCR reagents were from Promega Corp. (Madison, WI). [
32-P]deoxy-CTP was from ICN Biochemicals (Costa Mesa, CA). Reagents for luciferase assays, beetle luciferin protein, and coenzyme A were obtained from Promega Corp. (Madison, WI) and Roche Molecular Biochemicals, respectively. Antibodies to Sp1, Sp3, and Egr-1 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Animals
Intact and hypophysectomized immature (d 23 of age) Holtzman Sprague Dawley females (Harlan Sprague Dawley, Inc., Indianapolis, IN) were housed under a 16-h light and 8-h dark schedule in the Center for Comparative Medicine at Baylor college of Medicine and provided food and water ad libitum. Animals were treated in accordance with the NIH guide for the Care and Use of Laboratory Animals, as approved by the Animal Care and Use Committee at Baylor College of Medicine (Houston, TX).
Granulosa Cell Cultures
Granulosa cells were harvested by needle puncture from untreated or immature (d 26) rats or from immature rats treated with E (1.5 mg E/0.2 ml in propylene glycol) on d 2325 of age as previous described (12, 37) and as indicated in Results and figure legends. Briefly, cells were cultured at a density of 1 x 106 cells per 3 ml serum-free medium (DMEM:F12 containing penicillin and streptomycin) in multiwell (35 mm) dishes that were serum coated. Cells were cultured in defined medium overnight (0 h) followed by the addition of forskolin (10 µM), PMA (20 nM), or both.
RNA Isolation and RT-PCR
Total RNA was isolated from cultured cells using Trizol (Sigma) according to the manufacturers instructions. Each RNA sample was pooled from three duplicate wells. Briefly, the RNA was purified by sequential phenol, phenol-chloroform, and chloroform extraction, followed by ethanol precipitation. The RNA was resuspended in 0.1% diethyl pyrocarbonate-treated water and its concentration was determined by absorbance at 260 nm. RNA samples that showed an A 260/280 ratio more than 1.6 were used for analysis. For determining the relative changes in PR expression, 500 ng of RNA were reverse transcribed and divided into equal aliquots to assess the expression of PR within the linear range amplification (25 cycles) employing L19 as an internal control (20 cycles) with specific primers for rat PR and L19, respectively. Based on the known sequence of the mouse PR cDNA (GenBank accession no. M68915), oligonucleotide primer pairs were designed (forward, 5'-CCCACAGGAGTTTGTCAAGCT-3'; and reverse, 5'-TAACTTCAGACATCATTCCGG-3') and used in the RT-PCRs according to procedures described previously. The amplified cDNA for PR (328 bp) and L19 (196 bp) were resolved by acrylamide gel electrophoresis, and radioactive PCR product bands were quantified by phosphorimage analysis (Betascope 603 Blot Analyzer, Betagen Corp., Mountain View, CA).
PR Promoter Deletion and Mutation Constructs
The mouse PR promoter was ligated to the pGL3 luciferase reporter plasmid using PCR cloning techniques. PCR primers for the distal and proximal promoters were designed to contain KpnI and HindIII sites, respectively. After amplification using the mouse PR14 plasmid, the approximately 1600-bp product of the intact promoter (-380/+1001) was digested with KpnI and HindIII and ligated into the pGL3 vector multiple cloning cassette. Deletional and site-specific mutations within the promoter were generated by restriction digests or by PCR cloning strategies. The authenticity of each was verified by sequence analysis in both directions.
Site-specific mutations in the PR promoter constructs were generated using the Gene Editor Site-directed mutagenesis kit from Promega Corp. Oligonucleotides with site-specific mutations at the critical nucleotides necessary for transcription factor binding to the CCAAT site, distal GC box, and proximal GC regions [Sp1(A) and (B)] used for creating mutants are listed in Table 1
. The mutants obtained were confirmed by sequencing.
Transfections
For transfections, granulosa cells were harvested from E-primed rats and cultured overnight in medium alone. After fresh medium was replaced, the cells were transiently transfected with 1 µg of the respective constructs with Fugene 6 for 4 h. At that time cells were washed and cultured in fresh medium containing forskolin (10 µM), PMA (20 nM), or both and harvested after 4 h with lysis buffer (0.2 M Tris, pH 8.0, containing 0.1% Triton X-100). The protein concentrations were determined by mini-Bradford assay (Bio-Rad Laboratories, Inc.). Luciferase activity in the extracts was analyzed according to a standard protocol. In brief, a 40-µl aliquot of the cell lysate was mixed automatically with 100 µl luciferase assay reagent (20 mM Tris at pH 8.0, containing 4 mM MgSo4, 0.1 mM EDTA, 30 mM dithiothreitol, 0.5 mM ATP, 0.5 mM luciferin, and 0.25 mM coenzyme A), and each reaction was monitored for 20 sec in a luminometer. Data are expressed based on the amount of protein in each sample: light specific units/µg protein (mean ± SD). Transfection of empty pGL3 vector in granulosa cells showed basal values similar to those of the unstimulated wild-type promoter-luciferase construct (PRdist-2), but inducibility of pGL3 by Fo/PMA was never more than 1.5- to 2-fold in each experiment.
Transfections of MCF-7 cells were carried out using a protocol similar to that adopted for granulosa cells. MCF-7 cells (3 x 105) were cultured in 0.5% serum overnight, and the cells were then transfected in serum-free medium with various luciferase constructs (1 µg) employing Fugene 6.
Transfections of SL 2 cells were also carried out using Fugene 6. SL2 cells (8 x 105) were cultured in Schneider medium containing 10% FBS at 25 C in six-well culture plates. On the next day, the cells were transfected with 1 µg of reporter plasmid [PRdist-2 or its Sp1(B) mutant and PR prox-3or its Sp1(B) mutant] with increasing concentrations of pPac-Sp1 expression vector (0, 100, 200, and 1000 ng) adjusted by the addition of pPAC vector to equalize the total DNA transfected. After 24 h of transfection, the cells were harvested and assayed for luciferase activity as described above and expressed as light specific units/µg protein.
EMSA
Oligonucleotides to the CCAAT region, GATA site, Sp1(A) site, Sp1(B) site, and their respective mutants and consensus sequence (Table 1
) were annealed, labeled, and used in EMSAs as described previously (16). 32P-labeled oligonucleotides were incubated with whole-cell extracts prepared from granulosa cells of hypophysectomized (H) rats treated sequentially with E (HE), FSH (HEF), and hCG (HEF, hCG) as described previously (16). After incubation on ice for 30 min the binding reactions were subjected to nondenaturing electrophoresis (0.5% TBE; Tris-borate-EDTA) at 150 V. Where indicated, specific antibodies against Sp1, Sp3, and Egr-1 were added to the reactions for 30 min on ice before the addition of labeled DNA.
Statistics
The values represented are mean ± SD for at least three experiments. Statistical analysis was performed by Students t test and were considered significantly different if P < 0.05.
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ACKNOWLEDGMENTS
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The authors wish to acknowledge Drs. J. P. Lydon and Lakshmi Sivaraman, Baylor College of Medicine, for the genomic PR promoter construct and valuable discussions.
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FOOTNOTES
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This work was supported in part by NIH Grant HD-07495.
Abbreviations: E, 17ß-Estradiol; Egr-1, early growth response protein 1; ER, estrogen receptor; ERE, estrogen receptor response element; Fo/PMA, forskolin/PMA; hCG, human chorionic gonadotropin; PMA, phorbol myristate; PR, progesterone receptor; NF-Y, nuclear factor Y; TBE, Tris-borate-EDTA; WCE, whole-cell extracts.
Received for publication July 17, 2002.
Accepted for publication December 4, 2002.
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