Steroidogenic Factor 1 (SF-1) and SP1 Are Required for Regulation of Bovine CYP11A Gene Expression in Bovine Luteal Cells and Adrenal Y1 Cells

Zheng Liu and Evan R. Simpson

Cecil H. and Ida Green Center for Reproductive Biology Sciences, and the Departments of Obstetrics/Gynecology and Biochemistry The University of Texas Southwestern Medical Center Dallas, Texas 75235-9051


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cholesterol side-chain cleavage cytochrome P450 (CYP11A; P450scc) gene expression is regulated by gonadotropins via cAMP in the ovary and by ACTH via cAMP in adrenal cortical cells. Previously, we have characterized a response element located at -118 to -101 bp in the 5'-flanking region of the bovine P450scc gene required for cAMP-stimulated transcription in both mouse adrenocortical Y1 cells and bovine ovarian cells in primary culture. It was shown that this region contains a binding site for the transcription factor Sp1. Deletion of this sequence abolished cAMP-stimulated transcription in both Y1 cells and bovine ovarian luteal cells. Another sequence element located at -57 to -32 bp upstream from the transcription initiation site, which is highly conserved in CYP11A of other species, contains the motif TAGCCTTG, similar to the consensus binding site of steroidogenic factor-1, SF-1 (or Ad4-BP), but in the inverted orientation. In the present study, gel shift analysis using nuclear extracts of either Y1 cells or bovine luteal cells demonstrated that the sequence between -57 and -32 bp bound SF-1. A mutation of the SF-1-binding site that abolished binding of the nuclear protein to DNA reduced markedly the basal transcription of the reporter gene as well as the responsiveness to cAMP, when the mutated fragments containing the region from -186 to +12 bp were cloned into a luciferase construct and transfected into mouse adrenal Y1 cells and bovine luteal cells. The role of SF-1 in P450scc transcription was further confirmed by transactivation of the -186/+12Luc construct employing an SF-1 expression vector after transfection into nonsteroidogenic COS-1 cells. In addition, results obtained employing a double mutation of the Sp1- and SF-1-binding sites, and from a construct containing both Sp1 and SF-1 elements upstream of the CYP11A TATA box, indicated that Sp1 and SF-1 function cooperatively in the transactivation of the bovine CYP11A promoter in both bovine luteal cells and Y1 cells. Finally, a mammalian two-hybrid system was employed to demonstrate that Sp1 and SF-1 can associate in vivo. These results establish that basal and cAMP-stimulated activity of the bovine P450scc promoter in both Y1 cells and bovine luteal cells requires the combined action of at least two transcription factors, Sp1 and SF-1.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The biosynthesis of steroid hormones in the gonads and the adrenal cortex involves at least five distinct steroid hydroxylases that are members of the cytochrome P450 gene superfamily (1). In all steroidogenic pathways the initial and the rate-limiting reaction is the cleavage of the side chain of cholesterol leading to the production of pregnenolone, a mitochondrial reaction catalyzed by cholesterol side-chain cleavage cytochrome P450 (P450scc, the product of the CYP11A gene) (2). During the bovine ovarian cycle, P450scc activity is regulated in a highly coordinated fashion by the gonadotropin, LH, which appears to exert its hormonal effects via cAMP, an activator of protein kinase A (3). In the bovine adrenal cortex, the transcription of this gene is regulated by the peptide hormone ACTH via cAMP (4). Therefore, cAMP is the principal mediator of the hormonal induction of P450scc expression in gonadal and adrenocortical cells.

Studies of the 5'-flanking sequence of CYP11A genes from different species have identified several putative cis-acting elements required for cAMP-dependent transcription, including Sp1, AP-2, cAMP/AP-1, and SF-1 response elements (5, 6, 7, 8, 9, 10, 11). However, no common cis-acting DNA element(s) has been implicated to mediate the cAMP-responsive activity of the P450scc promoter among different species. In previous studies (5, 6, 12), we have reported that a region between -183 and -83 bp of the 5'-flanking sequence of the bovine CYP11A gene is capable of conferring cAMP-dependent regulation of a reporter gene, upon transfection into mouse adrenal Y1 tumor cells, bovine adrenocortical cells, and bovine luteal cells. Further characterization of this region (7, 12) has indicated that a cAMP-response sequence is located within -118 and -100 bp, using transiently transfected mouse Y1 cells and bovine ovarian luteal cells. This element is highly conserved among different species and is similar to the consensus Sp1-binding sequence, except that an ‘A’ replaces the core ‘C’. Later, it was shown that a second sequence element located at -70 to -50 bp is similar to the consensus Sp1-binding site, and that the transcription factor Sp1 mediates cAMP-dependent transcription of a reporter gene through binding to these sequences in Y1 cells (13). However, the mechanism by which Sp1 mediates responsiveness to cAMP is still unknown.

When sequences in the proximal promoter regions of CYP11A from different species are compared, another region between -57 and -32 bp is highly conserved among the bovine, rat, mouse, and human CYP11A genes (13, 14). This region contains the motif TAGCCTTG, similar to the consensus binding site of steroidogenic factor-1, SF-1 (or Ad4-BP), but in the inverted orientation. It was shown that the corresponding region in the rat P450scc gene was required for cAMP-dependent transcription of a reporter gene in rat granulosa cells (9). In the present study, we have investigated the role of this SF-1-like sequence in basal and cAMP-stimulated transcription of the bovine CYP11A gene. We have found that SF-1 binds specifically to the sequence within -57 and -32 bp in the bovine P450scc promoter. Mutations in the TAGCCTTG region abolished binding of SF-1 and markedly reduced both basal and cAMP-dependent transcription in mouse Y1 cells and bovine ovarian luteal cells. The role of this element appears to be mediated by SF-1, as shown by cotransfection experiments in nonsteroidogenic COS-1 cells. In addition, double mutation of both Sp1 and SF-1-like sequences eliminates both basal and cAMP-stimulated transcription. Furthermore, a construct containing both the Sp1 and SF-1 elements shows simultaneous binding of both transcription factors and also exhibits an increase in luciferase activity in response to protein kinase A (PKA). Finally, we provide evidence that Sp1 and SF-1 proteins associate in vivo. These results suggest that the combined action of Sp1 and SF-1 is required for both basal and cAMP-dependent transcription of the bovine CYP11A gene.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Oligonucleotide -57/-32 bp Binding Protein
Examination of the proximal promoter region of the bovine CYP11A gene (Fig. 1Go) showed potential binding sites for the general transcription factor Sp1 (5, 6, 12, 13, 23) and steroidogenic factor-1, SF-1. The region between -57 and -32 bp, which is highly conserved among different species, contains the motif TAGCCTTG, similar to the consensus SF-1-binding sequence, but in the inverted orientation. In the present study, we examined the role of the SF-1-like element in the regulation of the bovine CYP11A promoter activity.



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Figure 1. The Bovine CYP11A Proximal Promoter and 5'-Untranslated Region

The GA box is encircled by a rectangle, the hexameric AGGTCA motif is in bold type, the TATA box is underlined, and the transcriptional start site is indicated by an arrow. The asterisks indicate base changes in the wild type sequence that were introduced in the mutated sequences.

 
The protein that binds to the -57/-32 bp sequence was investigated by gel mobility shift assays. As shown in Fig. 2Go, the -57/-32 bp probe formed a single protein-DNA complex in the presence of either bovine luteal cell nuclear extracts (Fig. 2AGo), or Y1 nuclear extracts (Fig. 2CGo). Formation of this complex was efficiently competed by the unlabeled -57/-32 bp sequence and by an oligonucleotide containing the consensus SF-1 sequence. It also appeared that the same protein-DNA complex was formed from cells whether unstimulated or stimulated by treatment with forskolin, an activator of adenylate cyclase. As shown in Fig. 2BGo, the mutant oligonucleotide -57/-32M, with mutations of GG (CAAGGCTA to CAAtaCTA) within the putative SF-1-binding sequence, lost competition with the wild type oligonucleotides for the formation of the complex. Consistent with this result, no complex was formed in the presence of Y1 nuclear extracts when the -57/-32M oligonucleotide was used as a probe in gel shift assays (Fig. 2CGo). These data indicate that these two bases are important for nuclear protein binding.



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Figure 2. Mobility Shift Analysis Employing Nuclear Extracts from Bovine Luteal Cells or Y1 Cells

A, DNA-protein complexes, formed by the labeled wild type -57/-32 bp fragment in the presence of bovine luteal cell nuclear proteins (10 µg) or COS-1 cell nuclear proteins (10 µg) were analyzed by gel mobility shift assay, as described in Materials and Methods; 500-fold molar excess of unlabeled -57/-32 bp fragment and consensus SF-1 sequence were used as competitors. Nuclear extracts were derived from bovine luteal cells maintained in the absence (-) or presence (+) of 25 µM forskolin for 12 h. B, Labeled -57/-32 bp fragment was bound to 20 µg of bovine luteal cell nuclear proteins, with a 50-fold (lanes 2 and 4) or 100-fold (lanes 3 and 5) molar excess of unlabeled -57/-32 bp (lanes 2 and 3) or mutant oligonucleotide -57/-32M bp (lanes 4 and 5). C, Labeled wild type -57/-32 bp fragment and mutant -57/-32M bp fragment were used as probes in the presence of 2 µg of Y1 nuclear extracts. The nonradiolabeled probes and the consensus SF-1 oligonucleotides were used as competitors. Nuclear extracts were derived from cells maintained in the absence (-) or presence (+) of 25 µM forskolin for 24 h. D, Labeled -57/-32 bp oligonucleotide were incubated with 5 µg of Y1 nuclear proteins or 20 µg of bovine luteal cell nuclear proteins. Before incubation with labeled probe, some of the reactions were first incubated with 1 µg of either bovine SF-1 polyclonal antibody or preimmune serum. The arrowhead indicates the DNA-protein complex formed.

 
The nature of this binding protein was further investigated by antibody supershift experiments. As shown in Fig. 2DGo, the single complex formed between the -57/-32 bp oligonucleotide and Y1 cell nuclear extract protein(s) was displaced, when the extract was incubated with a specific antibody against bovine SF-1, but not with the preimmune serum. This antibody likewise displaced the binding of the bovine luteal cell nuclear protein(s) from the -57/-32 bp oligonucleotide instead of forming an antibody-protein-DNA complex. Thus, the protein from either Y1 or bovine luteal cells that specifically binds to the -57/-32 bp sequence was SF-1 or shared similar binding specificity and antigenicity with SF-1. However, when nuclear extracts from nonsteroidogenic COS-1 cells were incubated with the -57/-32 bp fragment, the binding pattern was different from that observed in the presence of either bovine luteal cell or Y1 cell nuclear extracts (Fig. 2AGo). The formation of these complexes was not competed by excess of unlabeled -57/-32 bp, indicating that they are nonspecific. Since SF-1 mRNA was only detected in steroidogenic tissues, such as testis, adipose tissue, brain, adrenal cortex, and ovary (24, 25), the difference was presumably due to the absence of SF-1 in COS-1 cells, which was confirmed by Western blot analysis (data not shown).

Activation of the Promoter Function of the CYP11A Gene by SF-1
To investigate the role of SF-1 in the activation of the bovine CYP11A promoter, we carried out a transient transfection experiment by expressing SF-1 in nonsteroidogenic COS-1 cells, which lack SF-1 as shown in Fig. 2AGo. The expression vector for SF-1 was cotransfected into COS-1 cells together with the P450scc promoter constructs containing the 5'-flanking sequence of the bovine CYP11A gene fused upstream of the luciferase reporter gene. As illustrated in Fig. 3Go, transcriptional activity of the -186/+12Luc and -101/+12Luc reporter plasmids is about 3- and 2-fold higher, respectively, in the cells cotransfected with SF-1 than in those cotransfected with only vector plasmid DNA. To examine directly whether SF-1 activates the bovine P450scc promoter via the -57/-32 bp sequence, a mutant -186/+12 bp fragment containing the mutation (GG to TA) within the sequence between -57 bp and -32 bp, similar to that used in the gel mobility shift analysis shown in Fig. 2Go, was inserted into the pGL3basic vector and transfected together with the SF-1 expression vector into the COS-1 cells. As shown in Fig. 3Go, expression of SF-1 had no effect on the transcription of the mutant -186/+12SF1mLuc construct. These data indicate that SF-1 is necessary for optimal promoter activity of the bovine CYP11A gene via binding to the -57/-32 bp sequence.



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Figure 3. Transcriptional Activation of Bovine CYP11A Gene Constructs by SF-1

COS-1 cells were cotransfected with 0.5 µg of CYP11A promoter constructs, 0.1 µg of internal reference plasmid pCMVLac, and 0.5 µg of RSV/SF-1 (closed bars) or 0.5 µg of pRC/RSV (open bars) using the lipofectamine method. After transfection for 48 h, the cell lysates were subjected to the luciferase assay. Values for absolute light units range from 10,000 to 100,000. The values of luciferase activity were normalized to ß-galactosidase activity. The results are shown as ± SEM for four independent experiments.

 
Transient Transfection Analysis of the Wild Type and Mutant CYP11A Reporter Gene Plasmids
The initial deletion analysis of the 5'-flanking region of bovine CYP11A showed that the -186/+12 bp chloramphenicol acetyltransferase (CAT) constructs containing the homologous CYP11A promoter exhibited the highest fold induction of promoter activity upon forskolin treatment in both bovine luteal cells (5) and Y1 cells (6). The CAT construct containing -101 bp to +12 bp of the P450scc 5'-flanking region was not expressed at levels higher than the empty CAT construct alone in either bovine luteal cells or Y1 cells, but the -101/+12 bp fragment still exhibited the ability to promote an increase in cAMP-dependent transcription in Y1 cells and bovine luteal cells. This suggested that more than one sequence, in addition to the element at -118 to -101 bp, might be required for optimal bovine P450scc promoter activity.

To test whether the -57/-32 bp sequence contains another element involved in both basal and cAMP-stimulated expression, luciferase reporter plasmids containing the wild type and mutant -186/+12 bp sequences were transfected into bovine luteal cells in primary culture. As illustrated in Fig. 4AGo, forskolin treatment resulted in a 4-fold increase over control levels of luciferase activity with the -186/+12Luc wild type plasmid. A promoter construct containing the mutation between -57 and -32 bp, namely -186/+12SF1mLuc, showed markedly decreased basal and forskolin-induced luciferase activity compared with -186/+12Luc. Similar results were also obtained when the wild type and mutant -186/-32 bp sequences were inserted into the OVEC-ß-globin reporter gene vector containing the heterologous minimal promoter of ß-globin gene and transfected into bovine luteal cells (Fig. 4BGo).



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Figure 4. Analysis of Wild Type and Mutant Bovine CYP11A Promoter Activity in Bovine Luteal Cells

A, Cells were transfected with 75 µg of various CYP11A promoter constructs and 4 µg of internal reference plasmid pCMVLac. Thereafter, the cells were starved for 12 h and treated with (hatched bars) or without (open bars) 25 µM forskolin for 12 h. B, One hundred micrograms of the OVEC constructs as indicated and 2 µg of internal reference vector OVEC-REF were cotransfected into bovine luteal cells. Thereafter the cells were treated with (+) or without (-) 25 µM forskolin for 9 h. RNA was prepared and each sample (10 µg) was analyzed by S1 nuclease protection assay after hybridization using a 32P-labeled 93 bp ß-globin probe (6) followed by electrophoresis and autoradiography. The bands due to the reference OVEC-REF construct indicate the transfection efficiencies of each experiment. C.I., Correctly initiated transcripts; REF., transcripts of OVEC-REF. The data shown are from one of two independent experiments.

 
These results were further corroborated by cotransfection of the PKA catalytic subunit into Y1 cells (Fig. 5Go). The -186/+12Luc construct showed a 4-fold increase in luciferase activity when the free catalytic subunit of PKA was overexpressed in these cells. However, expression of the construct -186/+12SF1mLuc, which has mutations in the -57/-32 bp sequence and cannot bind SF-1, resulted in a marked diminution of basal transcriptional activity as well as activity in response to exogenously supplied PKA catalytic subunit. Thus, the mutation in the -57/-32 bp sequence that eliminated the formation of DNA-protein complexes also markedly reduced basal and cAMP-induced transcription in both bovine luteal cells and Y1 cells.



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Figure 5. Analysis of Wild Type and Mutant Bovine CYP11A Promoter Activity in Y1 Mouse Adrenal Tumor Cells

Y1 cells were cotransfected with 1 µg of CYP11A promoter constructs, 0.25 µg of pCMVLac, and 0.5 µg of the wild type (hatched bars) or mutated (open bars) catalytic subunit of PKA expression vectors. The results are shown as ± SEM for three independent experiments.

 
Previous studies (7, 12) have shown that the sequence located at -118/-101 bp binds the transcription factor Sp1 and supports both basal and cAMP-dependent transcription upon transfection of reporter gene constructs containing the 5'-flanking region of the bovine CYP11A gene linked to the heterologous minimal promoter of the ß-globin gene into bovine luteal cells and Y1 cells. To further confirm the role of this -118/-101 bp sequence in basal and cAMP-dependent transcription, we used the native CYP11A promoter in the transfection experiments employing the luciferase reporter gene. As illustrated in Fig. 4AGo, in bovine luteal cells, the -186/+12Sp1 mLuc construct containing the mutation (GG to CC) between -118 and -101 bp of the Sp1-binding site showed a marked decrease in both basal and forskolin-induced luciferase activity compared with the wild type -186/+12Luc plasmid. In Y1 cells (Fig. 5Go), this mutation also decreased both basal and cAMP-dependent luciferase activity, but to a lesser extent than in luteal cells.

The effect of this mutation (GG to CC) within the -118/-101 bp sequence was further examined employing gel mobility shift assay (Fig. 6Go). Use of the wild type -118/-101 bp sequence as a probe resulted in complexes A and B in the presence of bovine luteal cell (Fig. 6AGo) or Y1 cell nuclear extracts (Fig. 6BGo), typical of Sp1 binding in those cells (12). A supershift complex was also formed when nuclear extracts from either bovine luteal cells or Y1 cells were incubated with the wild type -118/-101 bp sequence in the presence of Sp1 antibody. Formation of two complexes was efficiently competed by the unlabeled -118/-101 bp sequence, the -111/-101 bp sequence, and the consensus Sp1 oligonucleotide, but not by the mutant oligonucleotide, -118/-95M (TGGGAGGAGCT to TGccAGGAGCT). Consistent with the gel competition results, when the -111/-101 bp sequence was used as a probe, a binding pattern similar to that observed with the -118/-101 bp was observed (Fig. 6BGo). Upon employing the -118/-95M sequence as a probe, no DNA-protein complex was formed, indicating that the mutation abolished nuclear protein binding. Thus, these results confirm that Sp1 binds to the region from -111 bp to -101 bp and that the mutation within this region eliminated the formation of DNA-protein complexes and markedly reduced the response to cAMP and PKA in bovine luteal cells and Y1 cells, employing the homologous promoter.



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Figure 6. Gel Mobility Analysis of Binding of Bovine Luteal Cell (A) or Y1 Cell (B) Nuclear Proteins to the Different Bovine CYP11A (Wild Type or Mutated) -118/-101 bp Sequences

A, The bovine luteal cell nuclear extracts were incubated with the labeled -118/-101 bp fragment as described in the legend of Fig. 2Go. The unlabeled -118/-101 bp, -111/-101 bp, the consensus Sp1 oligonucleotide, and CYP11A mutant oligonucleotides (-118/-95M) were used as competitors, respectively. After incubation with labeled probe, some of the reactions were incubated with 1 µg of anti-Sp1 antibody. Nuclear extracts were derived from cells maintained in the absence (-) or presence (+) of 25 µM forskolin for 12 h. B, The nuclear extracts from Y1 cells were incubated with the labeled -118/-101 bp, -111/-101 bp, or mutant -118/-95M sequence, respectively. The nonradiolabeled sequences and the consensus Sp1 oligonucleotide were used as competitors. Supershift assays were conducted as described above. P, Probe; E, extract. The arrows indicate the positions of two DNA-protein complexes A and B.

 
As anticipated from these findings, when the -186/+12 bp fragment containing mutations within both the -111/-101 bp and the -57/-32 bp sequence was inserted into the luciferase vector and transfected into either bovine luteal cells or Y1 cells (Figs. 4Go and 5Go), the double mutation decreased significantly both basal and cAMP-dependent transcription compared with either -186/+12SF1mLuc or -186/+12Sp1mLuc alone. Although the decrease of luciferase activity in bovine luteal cells is less than in Y1 cells, the luciferase activity of the double mutation was comparable to that of the empty vector (pGL3basic) in both cell types. Therefore, optimal bovine CYP11A promoter activity requires both the Sp1- and SF-1-binding sites.

To further characterize the functional relationship between SF-1 and Sp1 elements, a -118/+12{Delta}52Luc construct, which contains the Sp1 element adjacent to the SF-1 site upstream of the endogenous CYP11A TATA box, was created and transfected into Y1 cells. As shown in Fig. 7AGo, this construct exhibited about 4-fold induction in luciferase activity in the presence of overexpressed free catalytic subunit of PKA. Its fold-induction is comparable to that of the -118/+12Luc construct with the native P450scc promoter region from -118 to +12 bp (5-fold). It is known that the region from -118 to +12 bp of the P450scc promoter is required for cAMP responsiveness (7, 12, 13), and the results from Fig. 7AGo suggest that Sp1 and SF-1 are both necessary for this response to cAMP. To investigate whether Sp1 could interact with SF-1, gel mobility shift assay was performed with the probes derived from the -118/+12{Delta}52Luc plasmid. When incubated with Y1 cell nuclear extracts (Fig. 7BGo), in addition to two complexes due to Sp1 and one complex due to SF-1, a new complex was detected. This slower mobility complex was competed by an excess of the -118/-101 bp sequence, the consensus Sp1 element, the -57/-32 bp sequence, and the consensus SF-1 sequence. It was also displaced partially by incubation with anti-Sp1 antibody or totally by the presence of SF-1 antibody. These data suggest that the complex contains both transcription factors. Consistent with this, previous results from DNase I footprint analysis of the bovine CYP11A 5'-flanking sequence have shown that a broad region from -114 to -91 bp and a short sequence at -49 to -33 bp within the fragment from -186 to +12 bp are protected by increasing amounts of Y1 nuclear proteins (7).



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Figure 7. Analysis of the Functional Interaction between Sp1 and SF-1 in Y1 Mouse Adrenal Tumor Cells

A, Y1 cells were cotransfected with 1 µg of CYP11A promoter constructs, 0.25 µg of pCMVLac, and 0.5 µg of the wild type (hatched bars) or mutated (open bars) catalytic subunit of PKA expression vectors. Transfections were conducted in duplicate. Values for absolute light units range from 10,000 to 100,000. The results are shown as ± SEM for five independent experiments. B, The Y1 cell nuclear extracts (25 µg) were incubated with the labeled -114/-35{Delta}52 bp fragment as described in the legend of Fig. 2Go. The unlabeled -114/-35{Delta}52 bp, -118/-101 bp, -57/-32 bp, the consensus Sp1 and SF-1 oligonucleotides were used as competitors, respectively. One microgram of either anti-Sp1 antibody or bovine SF-1 polyclonal antibody was added for the supershift assays. DNA-protein complexes were analyzed by electrophoresis on a native 5% polyacrylamide gel. The relevant Sp1 and SF-1 complexes are indicated. The position of the free probe is also indicated.

 
Two-Hybrid Analysis of Sp1 and SF-1 Interactions in Y1 and COS-1 Cells
To determine whether the functional synergism between Sp1 and SF-1 observed above involves direct protein-protein interaction, we applied the mammalian two-hybrid system, a method to detect in vivo protein-protein interactions, based on the functional reconstitution of the chimeric transcription factor GAL4-VP16 (26, 27). This fusion protein, which contains both the DNA-binding domain of the yeast GAL4 and the transactivation domain of the herpesvirus VP16 protein, strongly activates transcription of reporter genes downstream of the GAL4 sites in mammalian cells (28, 29). Expression vectors were constructed that encode the C-terminal 696 amino acids of Sp1 or the full-length sequence of SF-1 fused to either the GAL4 DNA-binding domain or the VP16 transactivation domain. These hybrid polypeptides should not activate the reporter gene on their own, because they lack either a transactivation domain (GAL4-X) or a proper DNA-binding domain (VP16-Y). However, if Sp1 and SF-1 interact stably in vivo, the protein-protein interaction will reconstitute a functional transcription factor, and expression of the reporter gene regulated by GAL4 would be detected.

The GAL4-Sp1 and VP16-SF-1 expression vectors were transfected singly or pairwise into Y1 cells along with G5Luc, a GAL4-responsive reporter plasmid that contains five GAL4-binding sites upstream of the luciferase gene (19). It has been shown that the GAL-Sp1 fusion protein can activate transcription in a GAL4 binding site-dependent manner (30). While optimal GAL4-Sp1 activation is achieved in Y1 and CV-1 cells with microgram quantities of the activator gene (Ref. 30 and data not shown), we used smaller quantities to optimize for activation by VP16-SF-1. As illustrated in Fig. 8AGo, expression of the VP16-SF-1 polypeptide alone had limited effect on luciferase activity. However, coexpression of both GAL4-Sp1 and VP16-SF-1 polypeptides generated a dramatic increase in luciferase activity to levels 6- or 28-fold higher, respectively, than those observed with GAL4-Sp1 or VP16-SF-1 alone. We also applied this two-hybrid assay in nonsteroidogenic COS-1 cells. As shown in Fig. 8BGo, coexpression of the GAL4-Sp1 and VP16-SF-1 polypeptides generated a marked increase in luciferase activity, to levels 4- or 82-fold higher than those observed with either GAL4-Sp1 or VP16-SF-1 alone, respectively. These data suggest that Sp1 and SF-1 are either capable of a direct association or interact via a common adapter protein in vivo.



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Figure 8. Two-Hybrid Analysis of Interactions between Sp1 and SF-1 in Y1 Cells (A) and in COS-1 Cells (B)

A, Y1 cells were transiently transfected with 1.5 µg of the G5LUC reporter plasmid, 25 ng of a GAL4-hybrid expression vector, 1.5 µg of a VP16-hybrid expression plasmid, and 250 ng of internal reference plasmid pCMVLac. Transfections were conducted in duplicate for each combination of expression plasmids. The values of luciferase activity are normalized to ß-galactosidase activity. The results are shown as ± SEM for four independent experiments. B, COS-1 cells were transiently cotransfected with 150 ng of the G5LUC reporter plasmid, 10 ng of a GAL4-hybrid expression vector, 500 ng of a VP16-hybrid expression plasmid, and 25 ng of pCMVLac. Transfections were conducted in duplicate for each combination of expression plasmids. The values of luciferase activity are normalized to ß-galactosidase activity. The results are shown as ± SEM for three independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although stimulation by cAMP is a pathway of regulation of expression common to all steroidogenic genes, different mechanisms are involved in the regulation of expression of each of the genes. A consensus, however, is emerging, which suggests that a factor common to these pathways is SF-1. However, a role for this component had not previously been determined in the case of bovine CYP11A, because an SF-1-binding site was not apparent in the sequence flanking the transcription start site. Rather, in previous studies (5, 6, 12, 13) the sequence -111/-101 bp, containing a binding site for the transcription factor Sp1, was shown to be involved in basal and cAMP-induced transcription of a heterologous promoter in bovine luteal cells and Y1 cells. The present study confirms that the same sequence can bind to Sp1 or an Sp1-like protein from bovine luteal cells and that the element is required for both basal and cAMP-dependent transcription in bovine luteal cells employing the endogenous CYP11A promoter. However, as a ubiquitous transcription factor, Sp1 protein is detected in steroidogenic bovine luteal cells, Y1 cells, and nonsteroidogenic COS-1 cells (data not shown). Employing the -111/-101 bp sequence as a probe, similar gel mobility shifts were obtained with bovine luteal cell, Y1, or nonsteroidogenic COS-1 cell nuclear extracts (data not shown). Since P450scc is only expressed in steroidogenic tissues, the protein binding to the -111/-101 bp sequence is not sufficient to completely define the regulation of P450scc expression.

In the present study, we have identified another sequence in the region between -57 and -32 bp that is required for basal and cAMP-stimulated expression. This sequence contains an SF-1-binding site in the inverted orientation, which, as a consequence, had previously escaped detection. Gel shift analysis indicates that the -57/-32 bp region is specifically bound by one nuclear protein from bovine luteal cells and Y1 cells, but not from nonsteroidogenic COS-1 cells. Forskolin treatment does not affect the DNA-binding activity of this nuclear protein. The bound nuclear protein is identified by antibody supershift analysis to be the orphan nuclear receptor, SF-1, which is expressed in a tissue-specific fashion (24, 25). Mutations within the SF-1 binding region, which abolished nuclear protein binding, decreased both basal and cAMP-dependent transcription (Figs. 5Go and 6Go), and eliminated activation of the bovine P450scc promoter resulting from expression of SF-1 in COS-1 cells (Fig. 3Go).

From the results of the present study, it is apparent that SF-1 alone is necessary, but not sufficient, for cAMP responsiveness, because mutations within the -57/-32 bp sequence reduced, but did not eliminate, basal and cAMP-stimulated luciferase activity compared with the wild type construct (Figs. 5Go and 6Go). Additionally, the -101/+12Luc construct that contains only the SF-1-binding site was expressed at levels lower than the -186/+12Luc construct including both Sp1- and SF-1-binding sites (Fig. 3Go). To explore the functional synergism between SF-1 and Sp1 in the regulation of bovine CYP11A gene expression, the -186/+12Sp1mSF1mLuc construct that contains double mutations of both the Sp1- and the SF-1-binding sequences was employed in transient transfection assays. The double mutation abolished promoter activity in either unstimulated or stimulated bovine luteal cells, as well as in Y1 cells. In addition, the -118/+12{Delta}52Luc construct containing only Sp1 and SF-1 elements upstream of CYP11A TATA box showed a similar fold increase in the promoter activity, compared with the construct with the native -118 to +12 bp region, although basal activity was less. Furthermore, a complex composed of both Sp1 and SF-1 was detected by gel mobility shift assay (Fig. 7Go). These results suggest that a cooperative relationship exists between SF-1 and Sp1 to mediate basal and cAMP-stimulated expression of the bovine CYP11A gene. To our knowledge, this is the first report to show the cooperative involvement of both Sp1 and SF-1 in the regulation of bovine CYP11A expression.

Recent studies (13) have demonstrated the presence of a second sequence element located at -70 to -50 bp of the bovine CYP11A gene, which also binds Sp1 and supports cAMP-induced transcription when subcloned into the OVEC vector and transfected into Y1 cells. However, the OVEC vector has a potential SF-1-binding site in the reverse orientation adjacent to its TATA box. This SF-1 site was shown to bind the SF-1 protein by gel mobility shift assay (data not shown). These data suggest that the -70/-50 bp sequence may also mediate cAMP responsiveness in cooperation with SF-1. They may explain also why constructs containing mutations within the -111/-101 bp sequence still show modest responsiveness to cAMP in Y1 cells (Fig. 5Go). However, the -118/+12{Delta}15Luc construct with an internal deletion of the -70/-50 bp element exhibited similar level of transcriptional activity to the wild type -118/+12Luc construct (data not shown), and the -118/+12{Delta}52Luc construct showed a similar fold increase in luciferase activity to the -118/+12Luc construct, although basal activity was less. These results indicate that the promoter region lacking the -70/-50-bp sequence is sufficient for basal and cAMP-dependent transcription of bovine CYP11A gene, although the -70/-50 bp sequence might play a complementary role in modulating cAMP responsiveness.

SF-1 is known as a regulator for many steroidogenic genes, such as human (11, 31) and rat (9) CYP11A, human CYP19 (32), bovine CYP11B (31), and rat CYP17 (33). All those studies showed that SF-1 could mediate cAMP responsiveness, but the mechanism for the functional role of SF-1 in the cAMP-PKA signal transduction pathway was not clear. While the role of SF-1 may not be mediated by increased DNA-binding activity, but in part mediated by induced SF-1 expression (32) and/or phosphorylation by PKA, SF-1 was suspected to interact with other nuclear factor(s) that bound to specific DNA sequences in mediating the cAMP-induced transcription of bovine CYP11B (31), human (11, 31), and rat (9) CYP11A. The two-hybrid analysis (Fig. 8Go) further suggests that interaction between Sp1 and SF-1 may be involved in this regulation of the bovine CYP11A promoter activity. Thus, our present study provides evidence for the first time which suggests that the transcription factor Sp1 is able to interact with SF-1 to regulate bovine CYP11A promoter activity.

It has been shown that Sp1 can interact with several other transcription factors, such as p53, bovine papillomavirus type 1 (BPV-1) protein E2, retinoblastoma (RB) protein, and CCAAT/enhancer binding protein ß (C/EBPß), to regulate gene transcription (30, 34, 35, 36). Since Sp1 does not have a PKA phosphorylation site, it was suspected that an unidentified coactivator might be a target for PKA in execution of its role in coupling the ubiquitous transcription factor Sp1 with cAMP-dependent transcription (13). From our studies, it appears that cAMP and PKA might stimulate bovine CYP11A gene expression through an Sp1-SF-1 interaction. However, an attempt to show direct interaction between Sp1 and SF-1 by coimmunoprecipitation was unsuccessful. Furthermore, no significant induction of luciferase activity was produced in the reciprocal two-hybrid experiment involving coexpression of GAL4-SF-1 and VP16-Sp1 (data not shown). It is possible that Sp1 interacts with SF-1 through unidentified adapter protein(s). Perhaps the adapter protein(s) functions like CREB-binding protein (CBP), which serves as a common coactivator required for the function of nuclear receptors such as cAMP response element-binding protein (CREB) and AP-1 (37). Thus, it is not clear, at this point, whether PKA stimulates CYP11A gene expression by stabilizing an SF-1-Sp1 interaction, by potentiating SF-1 activity, or by regulation of the activity of an unknown adapter protein.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Restriction endonucleases were purchased from Bethesda Research Laboratories (Gaithersburg, MD) and used according to the directions supplied by the manufacturer. Forskolin was purchased from Calbiochem (San Diego, CA).

Cell Culture
Bovine ovaries were obtained at a local slaughterhouse, and corpora lutea corresponding to early or midstages of the luteal phase were selected. Luteal cells were prepared as described previously (5, 15), cultured in McCoy’s 5A medium supplemented as described previously (5), and allowed to grow to confluency (5–6 days) before transfection. The Y1 and COS-1 cells were routinely maintained in DMEM supplemented with 10% bovine calf serum at 37 C in a 5% CO2 incubator.

Oligonucleotides
Complementary single-stranded oligonucleotides were synthesized and annealed to generate the following double-stranded oligonucleotides representing both wild type and mutant bovine P450scc promoter elements. The sequences containing consensus GC-rich double-strand oligonucleotide, which binds transcription factor Sp1, and a consensus SF-1 binding site are also shown. a) -118/-101

ACTGAGTCTGGGAGGAGCG

tcgagTGACTCAGACCCTCCTCGCagct b) -111/-101

TGGGAGGAGCG

tcgagACCCTCCTCGCagct c) -118/-95 M

ACTGAGTCTGccAGGAGCTGTGTG

TGACTCAGACggTCCTCGACACAC d) -57/-32

GCTTCTCACTTAGCCTTGAGCTGGTG

CGAAGAGTGAATCGGAACTCGACCAC e) -57/-32 M

GCTTCTCACTTAGtaTTGAGCTGGTG

CGAAGAGTGAATCatAACTCGACCAC f) consensus Sp1

GCGATCGGGGCGGGGCG

tcgaCGCTAGCCCCGCCCCGCagct g) consensus SF-1 motif

CACTCTACCAAGGTCAGAAATG

tcgaGTGAGATGGTTCCAGTCTTTACagct h) -114/-35{Delta}52

AGTCTGGGAGGAGCTTTAGCCTTGAGCTG

TCAGACCCTCCTCGAAATCGGAACTCGAC

DNA Reporter Gene Constructs
The expression systems used are the luciferase reporter gene plasmid, pGL3basic, which is a promoterless vector, purchased from Promega (Madison, WI), and the OVEC vector containing a minimal ß-globin promoter as described previously (6) and generously provided by Dr. W. Schaffner.

Luciferase reporter gene constructs containing -186/+12 bp, -118/+12 bp, and -101/+12 bp of the bovine CYP11A gene were made in the following way: the -186/-9 bp fragment was amplified by PCR using the -896/-32 bp fragment as a template and a primer containing the sequence from -32 to -9 bp. The second PCR reaction was conducted to amplify the -186/+12 bp fragment, using the -186/-9 bp fragment as a template and a primer including the region from -9 to +12 bp. A SalI site and a SacI site were introduced in the 3' and 5'-primer, respectively, to facilitate cloning into the pGL3basic vector. The resultant fragment was cloned into the SacI-SalI sites of the pGL3basic vector, just upstream of the coding region of the luciferase reporter gene. From this plasmid a series of CYP11A gene fragments were deleted by PCR. The construction of plasmids containing the mutated -186/+12 bp fragment was conducted as follows: the -186/+12 bp fragment was mutagenized by overlap extension using PCR (16) and then subcloned into the SacI-SalI sites of the pGL3basic vector. The -118/+12{Delta}52Luc construct was also made by a similar method. All plasmid constructions were confirmed by restriction digestion and dideoxy sequencing. The expression vectors for the wild type and the mutated catalytic subunit of PKA were kindly provided by Dr. R. Maurer.

The OVEC reporter gene constructs containing the wild type -186/-32 bp fragment and mutants thereof inserted upstream of the rabbit ß-globin TATA box were made as described by Ahlgren et al. (6).

Plasmids encoding the GAL4-Sp1 and VP16-Sp1 hybrid polypeptides were constructed by inserting the XhoI fragment from pPacSp1, kindly provided by Dr. R. Tjian, into the SalI site of the pM and pVP16 expression vectors (17), respectively. To produce plasmids encoding GAL4-SF-1 and VP16-SF-1, the fragment including the entire SF-1-coding region was amplified by PCR from the pRc/RSV-SF-1 expression vector, kindly provided by Dr. K. Morohashi. A HindIII site was introduced in both 5'- and 3'-primers. The amplified fragment was cloned into the HindIII site of pM and pVP16 vectors, respectively.

Transient Cell Transfections
Mouse Y1 adrenocortical tumor cells were maintained in DMEM supplemented with 10% bovine calf serum and antibiotics. On the day before transfection, about 2 x 106 cells were seeded in each 60-mm dish. The next morning the medium was changed, and 2 h later the DNA was added to the cells by the calcium-phosphate method (18) using 5 µg of plasmid/60-mm dish. After 4 h of exposure to the DNA precipitates, the cells were shocked with 15% glycerol for 1 min at room temperature. The medium was changed the morning after transfection, and the cells were maintained in the presence of 10% serum.

Confluent bovine luteal cells were removed from the culture dishes using trypsin/EDTA solution and resuspended in McCoy’s 5A medium. Transfection was achieved by electroporation using a cell porator (Bethesda Research Laboratories). Two consecutive discharges (750 V/cm and 1180 µFarads) were applied to 3 x 106 cells in 1 ml of McCoy’s 5A medium containing 75 µg of test plasmid and 4 µg of internal reference plasmid pCMVLac, as reported previously by Lauber et al. (5). After an overnight plating of the transfected cells in McCoy’s 5A medium containing 2.5% bovine calf serum, the treatments were started the day after transfection in McCoy’s 5A medium without serum for 12 h and continued in the presence of 25 µM forskolin for 12 h. For OVEC reporter gene constructs, the transfection procedure was the same as described except 100 µg of test plasmid and 2 µg of internal reference vector OVEC-REF were used, and the cells were exposed to forskolin for 9 h the day after transfection.

COS-1 cells were maintained in DMEM supplemented with 10% bovine calf serum and antibiotics. Plasmid DNA (1 µg) was transfected into COS-1 cells using the lipofectamine method (Bethesda Research Laboratories). After 48 h, the cells were lysed and the cell lysates were used for luciferase assay.

The Two-Hybrid Assay
Approximately 2 x 106 Y1 cells were seeded onto each 60-mm plate and cultured in 5 ml of growth medium. After 1 day, each 60-mm culture was transfected with 1.5 µg of the G5LUC reporter plasmid (19), 25 ng of a GAL4-hybrid expression plasmid, and/or 1.5 µg of a VP16-hybrid expression plasmid, and 0.25 µg of internal reference plasmid pCMVLac. After 48 h of culture the cells were lysed and used for luciferase assay.

Gel Retardation Assay
Nuclear extracts were prepared from mouse Y1 adrenocortical tumor cells and bovine luteal cells in primary culture as described by Dignam et al. (20). The double-stranded oligonucleotides were labeled by T4 polynucleotide kinase using [{gamma}-32P]dATP or by Klenow labeling using [{alpha}-32P]dCTP, and then incubated (10,000 cpm) with nuclear extracts (10 µg of protein) on ice for 10 min (21). For the competition assays, 500-fold molar excess of unlabeled oligonucleotide used as a competitor was added simultaneously with the labeled fragment. The resulting DNA-protein complexes were analyzed by electrophoresis using an 8% polyacrylamide gel with 0.5 x Tris-borate-EDTA as the running buffer (22). Supershift assays were also conducted as described above except 1 µg of anti-Sp1 antibody (Santa Cruz Biotech., Santa Cruz, CA) was added to the DNA-protein complexes after 10 min incubation on ice. Incubation of the DNA-protein complexes with the antibody continued for an additional 30 min at room temperature before electrophoresis. In some experiments, nuclear extract proteins were preincubated with anti-SF-1 polyclonal antibody, provided by Dr. K. Morohashi, or preimmune serum at room temperature for 30 min before addition to the binding reaction.

Luciferase and ß-Galactosidase Assays
Forty-eight hours after the transfection, the cells were lysed with 300 µl of 1% Triton X-100, 0.1 M phosphate buffer, pH 7.8, 2 mM EDTA, and 1 mM dithiothreitol. The luciferase assays were conducted essentially after the protocol provided by Analytical Luminescence Laboratory (San Diego, CA). Fifty microliters of lysate were added to 100 µl of substrate A, and the luciferase reaction was initiated by the injection of 100 µl of substrate B. Light output was measured for 10 sec at room temperature using a monolight luminometer (Analytical Luminescence Laboratory, San Diego, CA). ß-Galactosidase activity was measured using a chemiluminescent reporter assay kit (TROPIX, Bedford, MA). Twenty microliters of lysate were added to 200 µl of reaction buffer and incubated at room temperature for 60 min. After the addition of 300 µl of light emission accelerator, light output was measured for 5 sec using a monolight luminometer.

S1 Nuclease Protection Assay
Total RNA was isolated using a RNAzol B method (Biotecx Laboratories, Inc. Houston, TX). ß-Globin transcripts were detected and quantitated using a single-stranded rabbit ß-globin-specific oligonucleotide (93 mer) (6). After hybridization of the 32P-labeled probe with RNA (10 µg), it was digested with S1 nuclease, and correctly initiated protected fragments (75 nucleotides) were separated from free probe (93 nucleotides) by electrophoresis. Specific bands were visualized by autoradiography.


    ACKNOWLEDGMENTS
 
We thank Carolyn Fisher and Christy Ahsanullah for their technique assistance.


    FOOTNOTES
 
Address requests for reprints to: Evan R. Simpson, Ph.D., Cecil H. and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9051.

This work was supported, in part, by USPHS Grant 5-ROI-HD13234 and by Grant I-1228 from the Robert A. Welch Foundation.

Received for publication September 3, 1996. Revision received November 18, 1996. Accepted for publication November 20, 1996.


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