form (Яm) expression vector (pRC/RSV), provided by Dr. Richard A. Maurer ...... Kramer RE, Rainey WE, Funkenstein B, Dee A, Simpson ER, Waterman MR.
0013-7227/97/$03.00/0 Endocrinology Copyright © 1997 by The Endocrine Society
Vol. 138, No. 5 Printed in U.S.A.
A Putative Binding Site for Sp1 Is Involved in Transcriptional Regulation of CYP17 Gene Expression in Bovine Ovary* RAFFAELLA BORRONI, ZHENG LIU, EVAN R. SIMPSON, MARGARET M. HINSHELWOOD
AND
Cecil H. and Ida Green Center for Reproductive Biology Sciences and the Departments of Obstetrics/ Gynecology and Biochemistry, University of Texas Southwestern Medical Center (R.B., Z.L., E.R.S., M.M.H.), Dallas, Texas 75235-9051; and the Department of Obstetrics and Gynecology, Clinica L. Mangiagalli, University of Milan (R.B.), Milan, Italy ABSTRACT In the bovine ovary, thecal cells are the only cell type capable of expressing the CYP17 gene in response to LH. With the onset of ovulation and luteinization in the cow, there is complete loss of P450c17a expression. To characterize the molecular mechanisms involved in tissue-specific regulation of the CYP17 gene in the bovine ovary, deletion mutations of the bovine CYP17 promoter were ligated into a promoterless luciferase expression vector, and reporter constructs were transiently transfected into primary cultures of bovine thecal and luteal cells. Deletion of the promoter sequences between 2191 and 2101 bp dramatically decreased the levels of reporter gene activity in both thecal and luteal cells. Computer-assisted analysis revealed the presence of a putative inverted Sp1-like binding site at
2188/2180 bp. Deletion or mutation of this sequence caused a decrease in both basal and forskolin-stimulated reporter gene activity. In addition, mutation or deletion of this sequence also decreased reporter gene expression induced by overexpression of the protein kinase A catalytic subunit. Electrophoretic mobility shift assays showed that this sequence binds to a nuclear protein(s) from both thecal and luteal cells that is related to Sp1, as suggested by the results of gel mobility supershift assay employing an antibody raised against Sp1. DNA-binding activity was not increased by the addition of forskolin to thecal or luteal cells. We conclude that this inverted Sp1-like binding sequence is involved in constitutive as well as cAMPdependent expression of the CYP17 gene in the bovine ovary. (Endocrinology 138: 2011–2020, 1997)
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agents that increase intracellular levels of cAMP (19 –22). CYP17 transcriptional regulation in bovine adrenal seems to involve multiple interactions between two distinct cAMPresponsive DNA elements (CRS I and II) and different nuclear factors (23). The bovine CYP17 CRS closest to the promoter, termed CRS II, binds SF1 and other proteins, including chicken ovalbumin up-stream promoter transcription factor (COUP-TF) (24). The upstream bovine CYP17 cAMP-responsive DNA element, namely CRS I, is found to bind four nuclear proteins; two of them are homeodomain proteins from the PBX family, whereas the other two contain unknown peptide sequences (25). This indicates that the sequence is known, but shows no homology to any other database sequences. In the bovine ovary, significant expression of P450c17a messenger RNA (mRNA) is first observed in thecal cells at the time of antrum formation (26). Changes in 17a-hydroxylase/17,20-lyase activities are regulated at the level of transcription of the CYP17 gene. The capacity of bovine thecal cells to express P450c17a mRNA in response to LH seems to vary from the early antral to the preovulatory phase (27). Thecal cells are the only bovine ovarian cell type capable of expressing the CYP17 gene. The surge of gonadotropins initiates the luteinization process. There is a complete disappearance of P450c17a expression within 24 h of the LH surge, and no androgen precursors can be detected in even the earliest corpus luteum (28 –32). Bovine thecal cells readily lose the ability to produce androstenedione when placed in culture, indicative of a loss of 17a-hydroxylase expression
N MAMMALIAN and fish species, the CYP17 gene encodes a single protein (1–7), termed P450c17a, that mediates both 17a-hydroxylase and 17,20-lyase activities in the synthesis of steroid hormones (8, 9). Bovine CYP17 is generally believed to be a single copy gene (3), although a recent report has suggested the presence in the bovine genome of at least three CYP17 genes (10). The expression of P450c17a is under developmental, species-specific, and tissue-specific control (11). Once tissue differentiation has occurred, P450c17a activity is regulated at the level of transcription by pituitary trophic hormones via cAMP as second messenger (12–14). In the cow, the CYP17 gene is expressed only in gonads, adrenal cortex, and placenta (15, 16). In the adrenal cortex, P450c17a expression appears to be essentially ACTH dependent. A transient absence of ACTH occurring during the middle third of gestation or after hypophysectomy induces a substantial decline in P450c17a activity and expression (17, 18). Furthermore, in primary cultures of bovine adrenocortical cells maintained for several days in the absence of ACTH, P450c17a expression and activity essentially disappear, but can be restored by treatment with ACTH or Received September 17, 1996. Address all correspondence and requests for reprints to: Margaret M. Hinshelwood, Ph.D., Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9051. * This work was supported in part by USPHS Grant HD-13234 and in part by the Fondazione Amalia Griffini e Jacopo Miglierina (Varese, Italy).
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(33, 34). Recently, a culture system for maintaining bovine thecal cells has been characterized that employs specific culture conditions to avoid luteinization and allows the maintenance of both basal and cAMP-stimulated P450c17a activity and expression for at least 8 days (35). Using this system, Demeter-Arlotto et al. (36) made deletion constructs of the 59-flanking region of the bovine CYP17 gene that were subcloned into the OVEC vector just upstream of the rabbit b-globin reporter gene and promoter. They demonstrated that the CRS II appears to be the major cAMP response element used in bovine thecal cells in culture. This is in contrast to the results obtained upon transfecting the same reporter constructs into mouse Y1 cells, an adrenal tumor cell line, or into primary cultures of bovine adrenocortical cells, where the CRS I appears to be the major cAMP response element in these cells (23). We were, therefore, interested to continue these studies on the molecular mechanisms involved in cAMP-dependent as well as tissue-specific regulation of CYP17 gene expression in the bovine ovary. In the present experiments, we used CYP17 promoter reporter gene constructs that contain the endogenous CYP17 promoter based upstream of the luciferase reporter gene. These constructs were transfected into bovine theca interna and luteal cells to investigate the effect of deletion or mutation of specific sequences of the 59-flanking CYP17 region on reporter gene expression. Materials and Methods Materials Restriction endonucleases were purchased from either Boehringer Mannheim (Indianapolis, IN) or Promega (Madison, WI) and used according to the directions supplied by the manufacturer. Forskolin was purchased from Calbiochem (San Diego, CA). Radiolabeled nucleoside triphosphates (;3000 Ci/mmol) were purchased from DuPont-New England Nuclear Co. (Boston, MA). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated.
Sequencing the 59-flanking region of the bovine CYP17 gene The 59-flanking region of the bovine CYP17 gene was sequenced up to 21036 bp using the dideoxy chain termination method for a double stranded DNA template (Sequenase 2.0, U.S. Biochemical Corp., Cleveland, OH) and oligonucleotide primers designed on the basis of the 59-flanking bovine CYP17 sequence previously characterized (3).
Construction of bovine CYP17 59-flanking/luciferase constructs Reporter gene constructs were prepared by creating 59-deletion mutations of the bovine CYP17 flanking region by use of PCR amplification and single stranded oligonucleotides as primers synthesized with sequence complementary to the bovine CYP17 regulatory region plus nonannealing ends for the restriction sites SalI (59-primer) and BglII (39-primer). The genomic clone B17a-1-PUC18, provided by Dr. M. R. Waterman (Vanderbilt University, Nashville, TN), was used as DNA template. All of the PCR-amplified fragments (2866, 2709, 2437, 2293, 2243, 2191, 2179, 2101, and 280/210 bp) contained the endogenous bovine CYP17 putative TATA box (TTAAAAA). PCR products were ligated into the pCR 2.0 vector (TA cloning kit, Invitrogen Corp., San Diego, CA) and sequenced to ensure fidelity of the amplified sequences. Inserts were subcloned into the SalI and BglII sites of a promoterless luciferase expression vector (pGL3basicmod, which had been modified; Promega Co.). Site-directed mutagenesis was conducted using PCR and single stranded oligonucleotides containing the desired mutation plus nonannealing ends for the restriction sites SalI (59-primer) and BglII
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(39-primer). Products were characterized and subcloned as described above.
Cell culture and transfection Bovine ovaries were obtained at a local slaughterhouse. Follicles and corpora lutea were dissected from ovaries and theca interna, and luteal cells were prepared as previously described (35–38). Theca interna cells were plated into 35-mm dishes and grown to subconfluence (4 –5 days) in DMEM-Ham’s F-12 medium (1:1, Life Technologies, Gaithersburg, MD) supplemented with sodium bicarbonate, antibiotics, insulin (20 nm), transferrin (1 mg/ml), selenium (1 ng/ml), vitamin E (1 mm), and 2% low protein serum replacement (LPSR). Luteal cells were plated into 35-mm dishes and allowed to grow to subconfluence (5– 6 days) in McCoy’s 5A medium (Life Technologies) supplemented with sodium bicarbonate, HEPES buffer, antibiotics, transferrin (1 mg/ml), selenium (1 ng/ml), and 2.5% bovine calf serum. Transient transfection of bovine theca interna and luteal cells was carried out using the Cellfectin method (Cellfectin reagent, Life Technologies). Cells were transfected with 7.5 mg of the reporter construct and 0.5 mg of the pCMVnlac b-galactosidase expression plasmid as a control for transfection efficiency. For every transfection, a positive control vector (pGL3-control; Promega Co.), as well as the empty vector (pGL3-basicmod) as a negative control were used. Transfection of the cells was performed according to the manufacturer’s protocol for adherent cells, using either 7 ml Cellfectin reagent for each 35-mm dish of thecal cells or 5 ml/35-mm dish of luteal cells. After 5 h of incubation at 37 C in a 5% CO2 incubator, the transfection solution was replaced with supplemented medium, and cells were allowed to recover for 5 (theca interna cells) or 12 h (luteal cells). After recovery in supplemented medium, cells were grown in serum-free medium containing 0.1% BSA for 12 h. Thecal cells were then changed to medium supplemented with 1% LPSR and 100 mm isobutylmethylxanthine (IBMX) and incubated for 6 h either with or without 10 mm forskolin, whereas luteal cells were changed to serum-free medium containing 0.1% BSA and 100 mm IBMX and incubated for 12 h either with or without 25 mm forskolin. IBMX was added to the cell cultures to help potentiate the effects of forskolin; however, we found it had little or no effect on the amount of luciferase activity generated in response to forskolin compared to cell cultures incubated with forskolin alone (data not shown). Each experiment was carried out in duplicate and repeated three times. For protein kinase A (PKA) cotransfection experiments, thecal cells were transfected by means of the cellfectin method, using 7.3 mg of the reporter construct, 0.5 mg of the pCMVnlac b-galactosidase expression plasmid, and 0.2 mg of PKA catalytic subunit (b) or its inactive mutant form (bm) expression vector (pRC/RSV), provided by Dr. Richard A. Maurer, Health Science University (Portland, OR) (39). Cotransfections with the empty pRC/RSV vector were performed as a control. pGL3control and pGL3basicmod vector were transfected separately and considered positive and negative controls, respectively. Cells were incubated for 18 h in a 2% LPSR-supplemented medium, then changed to a 1% LPSR-supplemented medium and incubated for 6 h. Treatment with 10 mm forskolin was carried out during the last 6 h of incubation in some of the control dishes. Control dishes had been transfected with the specific bovine CYP17 construct, the b-galactosidase expression vector, and the empty pRC/RSV vector.
Activity assays The luciferase assay was performed according to the manufacturer’s protocol using the Enhanced Luciferase Assay kit (Analytical Luminescence Laboratory, Ann Arbor, MI). b-Galactosidase activity was measured using the Galacto-Light kit (Tropix, Bedford, MA), also following the protocol provided. Protein concentrations were measured using the BCA protein assay (Pierce Chemical Co., Rockford, IL). Luciferase and b-galactosidase assays were measured by a Monolight 2010 Luminometer (Analytical Luminescence Laboratory). Transfection efficiency was assessed by measuring the activity of b-galactosidase expressed from the cotransfected b-galactosidase expression vector. For PKA cotransfection experiments, luciferase activity was normalized to protein concentrations. All results were normalized to the value of the negative control. An arbitrary value of 1 U was assigned to the luciferase activity, corrected by the value of either b-galactosidase activity or protein concen-
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tration, obtained in samples transfected with the negative reference vector and incubated in the absence of forskolin.
polyclonal Sp1 antiserum (Santa Cruz Biotechnology) and the ECL detection system (Amersham Life Science, Arlington Heights, IL).
Electrophoretic mobility shift assay (EMSA)
Results 59-Flanking sequence of the bovine CYP17 gene
Nuclear extracts were prepared from both cultured bovine theca interna and luteal cells by the method by Dignam et al. (40). Protein concentrations were determined by a modified Bradford assay (Bio-Rad, Hercules, CA). Double stranded oligonucleotides containing the sequence of the inverted Sp1-binding site located in the bovine CYP17 59-flanking region at 2188/2180 bp were used (Fig. 1, underline; 59gtcgacTTACCTAGCCCCTCCCCT-39 and 39-AGGGGAGGGGCTAGGTAA-59), as well as oligonucleotides corresponding to the sequence of a consensus Sp1-binding site (GC box; 59-GCGATCGGGGCGGGGCG-39 and 39-tcgaCGCTAGCCCCGCCCCGC agct-59). These oligonucleotides were labeled by Klenow labeling with [a32P]deoxy-CTP and used as probes. For the competition assays, the unlabeled double stranded oligonucleotides were added simultaneously with the corresponding labeled probes at an approximately 500-fold excess. A double stranded oligonucleotide containing the same mutation in the inverted Sp1-binding site used in transfection experiments (59-gtcgac-TACCTAGCgCgTCCCCT-39 and 39-AGGGGAcGcGCTAGGTA-59; site of mutations indicated by lowercase letters) was used for competition assays. When supershift assays were performed, 1 ml of a rabbit polyclonal Sp1 antiserum (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the nuclear extracts and incubated at room temperature for 30 min before adding the labeled probe. Samples were incubated on ice for an additional 10 min before loading onto an 8% polyacrylamide gel.
Western blotting Nuclear proteins (20 mg) were separated by 10% SDS-PAGE (reduced conditions) and transferred to nitrocellulose (Bio-Rad) with an electrophoretic tank transfer system. Proteins were detected using a rabbit
The bovine CYP17 59-flanking sequence obtained using the genomic clone B17a-1-PUC18 as a template (Fig. 1) has some base pair mismatches with the sequence described previously (3). To verify the fidelity of the CYP17 59-flanking sequence described in the present study, the region between 2865 and 1205 bp of the bovine CYP17 gene was amplified by PCR from genomic DNA obtained from bovine adrenocortical tissue and subsequently sequenced. The sequence derived from the amplified fragment was identical to the sequence that we observed in the genomic B17-a1-PUC18 clone. Expression of bovine CYP17 59-flanking DNA/PGL3 constructs in bovine theca interna and luteal cells
To characterize the genomic elements involved in the regulation of bovine CYP17 expression in bovine ovary, a series of nested 59-flanking deletion mutations (2866/, 2709/, 2437/, 2293/, 2243/, 2191/, 2101/, and 280/210 bp) were created by PCR. All fragments contained the endogenous bovine CYP17 putative TATA box (TTAAAAA) located at 227/221 bp upstream of the transcriptional start site (Fig. 1) as well as the cAMP-responsive element CRS II, described
FIG. 1. Sequence of the 59-untranslated flanking region of the bovine CYP17 gene. This sequence was obtained using the genomic clone B17a-1-PUC18, provided by Dr. M. R. Waterman, as DNA template. Its fidelity was confirmed by sequencing a PCR-amplified DNA fragment (2866/1205 bp) of the bovine CYP17 gene obtained using genomic DNA from bovine adrenocortical tissue as template. CRS I, the inverted Sp1-binding site, and CRS II are presented in bold type. The oligonucleotide used as a probe for EMSA is underlined.
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previously (41), located at 268/233 bp (Fig. 1, bold). Inserts from 2866 to 2243/210 bp also contained the other bovine CYP17 regulatory sequence, CRS I, which was shown to be involved in the cAMP-dependent transcription of the CYP17 gene in mouse Y1 cells (23, 25) (Fig. 1, bold). The amplified fragments were ligated to a promoterless luciferase expression vector, and reporter constructs were transiently transfected into bovine theca interna and luteal cells in primary culture. Bovine CYP17 59-flanking/pGL3 constructs were expressed in both theca interna cells (Fig. 2A) and luteal cells (Fig. 2B), although the luciferase activity was much lower in luteal cells than in thecal cells. In bovine thecal cells, both basal and forskolin-stimulated expression of bovine CYP17 59-flanking/pGL3 constructs were higher after 6 h of treatment than at 9 – 48 h, whereas in luteal cells, the highest basal reporter gene activity was detected after 12 h of incubation. Thus, in thecal cells, a modest stimulation by forskolin was observed, whereas this was not apparent in luteal cells. In the present study, there was an increase in both basal and forskolin-induced reporter gene activity with the deletion of 2866 to 2293 bp of 59-flanking sequence in thecal cells (Fig. 2, A and B). These results are similar to those reported previously in either Y-1 cells (23) or thecal cells (36), indicating that there may be an inhibitory element located between 2437 and 2293 bp. There was no difference in either basal or forskolin-stimulated reporter gene activity in thecal cells with the deletion of the sequence between 2293 and 2191 bp; however, both basal and forskolin-induced reporter gene activities were drastically lowered in thecal (Fig. 2A) and luteal cells (Fig. 2B) when sequence from 2191 to 2101 bp was deleted. Previous experiments with Y-1 cells indicated that there was a drastic decrease in the expression of either the OVEC or chloramphenicol transferase (CAT) reporter constructs when deletions of the 59-flanking regions were made (2243 to 2107 bp in OVEC or 2297 to 2100 bp in CAT) (23). These decreases in activity, however, were attributed to deletion of the CRS I region. In contrast, in bovine thecal cells, there was reported to be no difference in forskolin-induced activity with deletion of 2293 to 280 bp (36), and the researchers concluded that CRS II was important in regulating the response to forskolin in ovarian cells. In the present study, with deletion of 2191 to 2101 bp, the amount of luciferase activity was not different from the activity observed when the empty vector was transfected into thecal cells. The sequence between 2188 and 2180 bp is involved in transcriptional regulation of the bovine CYP17 gene
As shown in Fig. 2A, deletion of the promoter region between 2191 and 2101 bp dramatically decreased (;15- to 20-fold) the levels of basal and forskolin-stimulated reporter gene activity in bovine thecal cells. Data base analysis of this region revealed the presence of a putative inverted Sp1binding sequence located at 2188/2180 bp (inv Sp1-bs: CCTAGCCCC). Specific deletion (2179/210 bp) or mutation (2191/210 MUT; CCTAGCgCg) of this sequence resulted in a dramatic decrease in basal and forskolin-stimulated reporter gene activity in thecal and luteal cells (Fig. 3). However, transfection of both the deleted inv Sp1-bs (2179/
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210 bp) and the mutated inv Sp1-bs (2191/210 MUT) constructs in thecal cells resulted in forskolin-stimulated reporter gene activity higher than the control value. Sp1 is a ubiquitous transcription factor known to be involved in the regulation of a wide variety of genes (42). To determine whether the inverted Sp1-binding site located in the bovine CYP17 59-flanking region could be involved in modulation of transcription via the protein kinase A signaling pathway, we cotransfected bovine thecal cells (Fig. 4) with luciferase constructs containing the inverted Sp1-binding site (2191/210 bp) or its mutated sequence (2191/210 MUT) and with expression vectors containing the PKA catalytic subunit (b) or its inactive mutant form (bm). The overexpression of the PKA catalytic subunit increased reporter gene activity of the wild-type (2191/210 bp) construct to values comparable to those obtained after forskolin treatment, whereas cotransfection of this construct with an expression vector containing the inactive mutant form of PKA catalytic subunit resulted in reporter gene activity equivalent to that obtained in control samples. When the mutated sequence of the inv Sp1-bs was transfected into thecal cells, there was a remarkable decrease in reporter gene expression comparable to that obtained upon transfection of the negative reference vector. Luciferase activity in these cells was not affected by cotransfection with a vector expressing the PKA catalytic subunit b. These results suggest that the inverted Sp1-binding site located in the promoter of the bovine CYP17 gene might be involved in constitutive as well as cAMPdependent expression of this gene. Gel mobility shift analysis
As deletional and mutational analysis of the bovine CYP17 gene identified an inverted Sp1-like sequence (2188/2180 bp) involved in transcriptional regulation, gel mobility shift analyses were performed to determine whether nuclear protein(s) from bovine thecal and luteal cells binds this sequence. A 24-bp double stranded oligonucleotide sequence of the bovine CYP17 promoter containing the inv Sp1-bs (Fig. 1, bold) as well a double stranded oligonucleotide sequence corresponding to a consensus Sp1-binding site (cons Sp1-bs) were used along with nuclear extracts from untreated (control) and forskolin-treated thecal and luteal cells. As shown in Fig. 5, migration of the inv Sp1-bs probe in the presence of nuclear extracts from thecal cells was retarded in three distinct complexes with DNA-binding proteins (complexes A, B, and C). Similar complexes were formed when luteal cell nuclear extract was incubated with the inv Sp1-bs probe; however, the interaction of complex C was decreased compared to that of the complex formed with thecal cell extract. Binding activity was not significantly modified by treatment of either cell type with forskolin. Binding of these thecal cell (Fig. 6) or luteal cell (Fig. 7) nuclear proteins to the 32P-labeled inv Sp1-bs oligonucleotide could be competed with an excess (;500-fold) of nonlabeled oligonucleotide (Fig. 6, lane 3, and Fig. 7, lane 3) as well as with unlabeled cons Sp1-bs oligonucleotide (Fig. 6, lane 4, and Fig. 7, lane 4), but not with an unlabeled oligonucleotide containing a mutated inv Sp1-bs sequence (MUT; Fig. 6, lane 5, and Fig. 7, lane 5). Although the formation of complexes A and B was not competed with
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FIG. 2. Transient expression of bovine CYP17 59-flanking DNA/luciferase reporter gene constructs into bovine thecal cells (A) or luteal cells (B) in primary culture. Cells were transfected with pGL3-basicmod vectors containing 2866/, 2709/, 2437/, 2293/, 2243/, 2191/, 2101/, or 280/210 bp of the bovine CYP17 59-flanking region. A simian virus 40/pGL3 (pGL3-control) construct and the reporter plasmid without insert (pGL3-basicmod) were transfected separately, as positive and negative references, respectively. Thecal cells were treated for 6 h in a 1% LPSRand 100 mM IBMX-supplemented medium in the presence or absence of forskolin (10 mM), whereas luteal cells were treated for 12 h in a 0.1% BSA- and 100 mM IBMX-supplemented medium in the presence or absence of forskolin (25 mM). Cell lysates were assessed for luciferase activity, which was corrected relative to the b-galactosidase activity produced from a cotransfected b-galactosidase expression vector. The experiment was carried out in duplicate and repeated three times. One representative experiment is shown. Results are expressed relative to the value of the negative reference; an arbitrary value of 1 U was assigned to the b-galactosidase-normalized luciferase activity produced by samples transfected with the negative reference vector and incubated in the absence of forskolin. Error bars represent the range.
an excess of unlabeled MUT oligonucleotide, the formation of complex C between nuclear extracts of thecal cells and the radiolabeled inv Sp1-bs was displaced by this mutated se-
quence. As the mutation of the inv Sp1-binding sequence contained in this oligonucleotide was the same as that employed in transfection experiments, we suggest that the pro-
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FIG. 3. The sequence between 2188 and 2180 bp is involved in transcriptional regulation of the bovine CYP17 gene. Primary cultures of bovine thecal and luteal cells were transiently transfected with the wild-type 2191/210 bp/pGL3-basicmod plasmid, containing an inverted Sp1-like binding sequence between 2188 and 2180 bp (CCTAGCCCC), with the 2179/210 bp/pGL3-basicmod plasmid, in which the inv Sp1-bs had been deleted, or with the 2191/210 M/pGL3-basicmod plasmid, containing a mutation of the inv Sp1-bs (CCTAGCgCg). The empty vector was transfected separately, as a negative reference. Thecal and luteal cells were treated as described in Fig. 2. Cell lysates were assessed for luciferase activity as described in Fig. 2. One representative experiment is shown for each cell type. Results are expressed as described in Fig. 2. Error bars represent the range.
tein(s) responsible for the formation of complex C might not be primarily involved in the transcriptional regulation mediated by the inverted Sp1 sequence. Nuclear proteins from both thecal and luteal cells also interacted with a consensus Sp1-binding sequence, resulting in the formation of complexes similar to those observed using the inv Sp1-bs as a probe (complexes A and B), but with much greater affinity. The formation of these complexes could be competed with an excess of cons Sp1-bs unlabeled probe and with an excess of inv Sp1-bs unlabeled oligonucleotide, but not with the mutated inv Sp1-binding sequence (MUT). To determine whether any of these nuclear proteins might be Sp1 or a related protein, we conducted supershift EMSA using polyclonal rabbit antiserum directed against human Sp1. Although a supershift was not observed, complexes A and B formed between nuclear proteins of both thecal and luteal cells and the inverted Sp1-binding sequence were totally displaced by the Sp1 antibody, whereas complex C formed between thecal nuclear extracts and the inv-Sp1-bs was only partially displaced (Fig. 6, lane 6, and Fig. 7, lane 6). By contrast, when Sp1 antibody was added to the binding reaction between a consensus Sp1-binding sequence and nuclear proteins from both thecal and luteal cells, the formation of the complexes was supershifted (Fig. 6, lane 12, and Fig. 7, lane 12). Western blot analysis
Western blot analysis confirmed the presence in both thecal and luteal cells of a 97-kDa protein that immunoreacts
with a polyclonal rabbit antiserum directed against human Sp1 (Fig. 8). The concentration of this protein was higher in luteal cells than in thecal cells. Discussion
The bovine CYP17 gene is known to be expressed in a tissue-specific fashion, as P450c17a mRNA has been detected only in gonads, adrenal cortex, and placenta (15, 16). In the bovine ovary, thecal cells are the only cell type capable of expressing the bovine CYP17 gene, and with the onset of luteinization there is a complete disappearance of P450c17a expression (29 –32). In the present study, luciferase reporter gene vectors containing deletion mutations of the bovine CYP17 promoter and 59-flanking DNA were expressed in both theca interna and luteal cells; however, the level and pattern of activity were different between the two cell types. The overall level of luciferase activity was lower in luteal compared to thecal cells. In addition, there was greater variability in reporter gene activity between the 2293/210 to 2191/210 bp constructs when they were transfected into the luteal vs. thecal cells. This is partially in contrast with the results obtained in a previous study in this laboratory (38), in which both basal and forskolin-stimulated reporter gene expression of OVEC constructs containing sequences of the bovine CYP17 promoter and 59-flanking DNA were undetectable in luteal cells. This could be due in part to a difference in the degree of sensitivity between the two different assays used to measure activity. As the endogenous bovine CYP17 gene is not expressed after in vivo luteinization (29 –
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FIG. 4. PKA cotransfection experiments. Bovine thecal cells were cotransfected with luciferase constructs containing the inverted Sp1-binding site (2191/210 bp/pGL3-basicmod wild type) or its mutated sequence (2191/210 MUT/pGL3-basicmod) and with RSV expression vectors containing the PKA catalytic subunit b or its inactive mutant form bm. Cotransfections of these luciferase plasmids with the empty RSV vector were performed as controls. pGL3control and the pGL3basicmod vector were cotransfected separately with b/RSV vector, bm/RSV vector, or RSV empty vector and considered positive and negative controls, respectively. After transfection, thecal cells were incubated for 18 h in a 2% LPSR-supplemented medium, then changed to a 1% LPSR-supplemented medium and incubated for 6 h. Treatment with 10 mM forskolin was carried out in some of the control dishes (cotransfected with the empty RSV vector) during the last 6 h of incubation. Cell lysates were assessed for luciferase activity, which was corrected relative to the protein concentration. Each experiment was carried out in duplicate and repeated at least three times. One representative experiment is shown for each cell type. Results are expressed as described in Fig. 2. Error bars show the range.
FIG. 5. Nuclear proteins from bovine thecal and luteal cells bind the inverted Sp1-like binding sequence. Nuclear extracts (8 mg) isolated from cultured bovine thecal cells (bTIC) or luteal cells (bLC) treated in the absence (2) or presence (1) of 10 mM (bTIC) or 25 mM (bLC) forskolin were incubated with a 32P-labeled inv Sp1-bs probe (2191/ 2174 bp). DNA-protein complexes were separated on a 8% polyacrylamide nondenaturing gel and visualized by autoradiography. The position of the specific complexes is indicated as A, B, and C. Free probe 5 fp.
32, 35), the results obtained in the current investigation suggest that the trans-acting factors necessary for the regulation of expression of these reporter gene constructs are still
present in luteal cells, although possibly in lower amounts or, perhaps, after luteinization, cis-acting elements in the promoter region are repressed by putative inhibitory trans-acting factors. In addition, putative cis-acting repressor elements important for the lack of expression of the bovine CYP17 gene after luteinization may reside further upstream from 2866 bp. Thus, the mechanisms responsible for the lack of expression of the bovine CYP17 gene after in vivo luteinization may not have been revealed in the present study. In bovine adrenal cortex, P450c17a expression initiates during the early stages of gestation, and it appears to be essentially ACTH dependent (16, 21) via the protein kinase A signaling pathway. Involvement of autocrine or paracrine growth factors and of other signaling pathways has also been suggested in the regulation of P450c17a expression (41, 43– 46). In the bovine ovary, significant expression of P450c17a is first observed in thecal cells at the time of antrum formation (26). In thecal cells, 17a-hydroxylase/17,20-lyase activity is regulated at the level of transcription, in response to LH, via the protein kinase A signaling pathway. An autocrine or paracrine modulation and the activation of alternative signaling pathways have also been suggested as possible mechanisms of regulation of the expression of the CYP17 gene in bovine ovary (47– 49). Previous experiments performed in the mouse adrenal tumor cell line Y1 and in bovine adrenocortical cells (23–25) have shown that two cis-acting elements located in the bovine CYP17 59-flanking region, termed CRS I and CRS II, are involved in the cAMP-depen-
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Sp1-BINDING SITE AND CYP17 GENE EXPRESSION IN OVARY
FIG. 6. Gel mobility shift analysis of binding of thecal nuclear proteins to the inverted Sp1-binding sequence and to a consensus Sp1binding sequence. Nuclear extracts (8 mg) isolated from cultured thecal cells were incubated with a 32P-labeled probe containing the inverted Sp1-binding sequence (inv Sp1-bs probe, lanes 2– 6) or with a 32P-labeled probe corresponding to a consensus Sp1-binding sequence (cons Sp1-bs probe, lanes 8 –12), electrophoresed on a 8% polyacrylamide nondenaturing gel, and visualized by autoradiography. Binding of nuclear proteins to the radiolabeled oligonucleotide could be competed with a 500-fold molar excess of nonradiolabeled homologous competitor (lanes 3 and 9). Binding of nuclear proteins to the inv Sp1-bs probe was competed by a 500-fold molar excess of cold cons Sp1-bs oligonucleotide (lane 4), and binding to the cons Sp1-bs probe was competed by a 500-fold molar excess of nonradiolabeled inv Sp1-bs oligonucleotide (lane 10). The addition of a 500-fold molar excess of a nonradiolabeled oligonucleotide containing a mutation of the inverted Sp1-binding sequence (MUT) could not compete the formation of complexes A and B between nuclear proteins and both inv Sp1-bs probe (lane 5) and con Sp1-bs probe (lane 11). When added to the binding reactions, a rabbit polyclonal Sp1 antiserum (Sp1 Ab; 1 ml) could displace the formation of complexes A and B between thecal nuclear proteins and the inv Sp1-bs probe (lane 6), and it could retard the formation of complexes with the cons Sp1-bs probe by supershift (lane 12). fp, Free probe; fp 1 Sp1 Ab, free probe plus Sp1 antiserum without addition of nuclear extracts.
dent transcriptional regulation of this gene. In studies by Demeter-Arlotto et al. (36), CRS II was shown to be important in cAMP-dependent transcription in thecal cells. In the present study, deletion of the sequence corresponding to CRS I did not remarkably affect basal or cAMP-stimulated reporter gene activity in thecal cells. It is, however, difficult to compare the studies using the OVEC vs. luciferase vectors for two reasons. Firstly, reporter constructs in the OVEC vector used the minimal b-globin TATA box, rather than the endogenous bovine CYP17 TATA box, which was contained in all of the constructs used in the present experiments. Secondly, the OVEC vector contains a potential SF-1 binding site in a reverse orientation near its TATA box, and this SF-1 site was shown to bind the SF-1 protein by EMSA (50). As SF-1 has been shown to be important in regulating cAMP-depen-
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FIG. 7. Gel mobility shift analysis of binding of luteal nuclear proteins to the inverted Sp1-binding sequence and to a consensus Sp1binding sequence. Nuclear extracts (8 mg) isolated from cultured luteal cells were incubated with a 32P-labeled probe containing the inverted Sp1-binding sequence (inv Sp1-bs probe, lanes 2– 6) or with a 32P-labeled probe corresponding to a consensus Sp1-binding sequence (cons Sp1-bs probe, lanes 8 –12), electrophoresed on a 8% polyacrylamide nondenaturing gel and visualized by autoradiography. Binding of nuclear proteins to the radiolabeled oligonucleotide could be competed with a 500-fold molar excess of nonradiolabeled homologous competitors (lanes 3 and 9). Binding of nuclear proteins to the inv Sp1-bs probe was competed by a 500-fold molar excess of cold cons Sp1-bs oligonucleotide (lane 4), and binding to the cons Sp1-bs probe was competed by a 500-fold molar excess of nonradiolabeled inv Sp1-bs oligonucleotide (lane 10). The addition of a 500-fold molar excess of a nonradiolabeled oligonucleotide containing a mutation of the inverted Sp1-binding sequence (MUT) could compete the formation of complexes A and B between nuclear proteins and both inv Sp1-bs probe (lane 5) and con Sp1-bs probe (lane 11). When added to the binding reactions, polyclonal rabbit Sp1 antiserum (Sp1 Ab; 1 ml) could displace the formation of complexes between luteal nuclear proteins and the inv Sp1-bs probe (lane 6), and it could retard the formation of complexes with the cons Sp1-bs probe by supershift (lane 12). fp, Free probe; fp 1 Sp1 Ab, free probe plus Sp1 antiserum without addition of nuclear extracts.
dent transcription of many steroidogenic genes, including the bovine (41) and rat (51) CYP17 genes, the presence of an additional SF-1 cis-acting element in the vector itself makes a comparison of the results of the various experiments difficult. In the experiments by Lund et al. (23), both OVEC and CAT vectors were employed. The CAT vectors used in these studies contain the endogenous bovine CYP17 TATA box, but no deletion constructs were used for transfection experiments intermediate to 2297 and 2100 bp. When we transfected Y-1 cells with the same luciferase reporter constructs previously used in the experiments with thecal cells, we observed a similar pattern of reporter gene activity between Y-1 cells and thecal cells (data not shown). Transfection of constructs containing only the CRS II sequence and the endogenous TATA box resulted in levels of reporter gene activity that were similar to negative control values. Moreover, the activity of the construct containing both the CRS II and the mutation of the inverted Sp1 binding
Sp1-BINDING SITE AND CYP17 GENE EXPRESSION IN OVARY
FIG. 8. Western blot analysis. Western blot analysis of Sp1 in nuclear extracts (20 mg) from cultured bovine thecal (bTIC) and luteal (bLC) cells. The positions of mol wt markers are shown on the left.
sequence (2191/210 MUT), when transfected into thecal cells, was greater than that observed for the negative control. Therefore, our present results do not negate a potential role for the CRS II sequence in cAMP-dependent activation of the CYP17 gene in the bovine ovary (36). However, further studies must be carried out by mutating CRS II in the context of the 2191/210 bp construct to determine its role in cAMPdependent transcriptional regulation of the bovine CYP17 gene in thecal cells. Deletional and mutational analysis of the bovine CYP17 promoter has identified a putative inverted Sp1-like binding sequence (2188/2180 bp) that is implicated in transcriptional regulation of this gene. This sequence binds a nuclear protein(s) from both thecal and luteal cells that is related to Sp1, as complex formation is displaced after incubation with a rabbit polyclonal Sp1 antibody. In addition, Western blotting analysis confirms the presence in nuclear extracts of both thecal and luteal cells of a protein showing immunoreactivity with a Sp1 antibody. Sp1 is an ubiquitous transcription factor known to be involved in the regulation of a wide variety of different genes (42). Although the role of Sp1 in the constitutive expression of a number of genes has been demonstrated (42), the mechanisms by which a possible involvement of Sp1 in cAMP-mediated transcriptional regulation could occur are not yet completely understood. It has recently been suggested that two Sp1-binding sites might modulate cAMP-induced transcription of the bovine CYP11A gene via a protein kinase A signaling pathway (52). Moreover, it has also been shown that SF-1 and Sp1 are required for regulation of the bovine CYP11A gene in luteal cells (50). Therefore, it is not surprising that another gene expressed in the bovine ovary, namely the CYP17 gene, may be modulated by similar trans-acting factors. How might Sp1 modulate bovine CYP17 gene expression? The endogenous bovine CYP17 gene is expressed in thecal cells, but with the onset of luteinization, transcription of this gene is turned off. In our studies, the CYP17 reporter constructs display greater activity in thecal vs. luteal cells. This occurs in the face of a greater abundance of Sp1 protein in the luteal cells compared with thecal cells. Also, the binding of both thecal and luteal cell nuclear proteins to the Sp1-like binding site contained in the bovine CYP17 promoter was not significantly modified after treatment of these cells with forskolin. Nonetheless, mutation of this sequence resulted in a dramatic decrease in both basal and cAMP-stimulated re-
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porter gene activity in transfection experiments and decreased reporter gene expression induced by overexpression of the PKA catalytic subunit. One possible explanation for these observations is the observation that Sp1 can be phosphorylated by a DNA-dependent kinase (53), which may affect the ability of Sp1 to initiate transcription. Therefore, even with the detection of greater amounts of Sp1 protein in luteal vs. thecal cells by Western blot analysis, the phosphorylation status might be a better indicator of potential transcriptional activity. If P450c17a is expressed only in steroidogenic cells, how can Sp1, a ubiquitously expressed transcription factor, modulate tissue-specific expression? In the context of the 2191/ 210 bp construct, when the Sp1 site was mutated, or of the 2179/210 bp construct, where the Sp1 site was deleted, the amount of luciferase activity was still greater than the negative control value. Therefore, one possibility could be the potential role of additional transcription factors in regulating P450c17a expression, perhaps in concert with Sp1. It was shown by Bakke and Lund (41) that SF-1 and COUP-TF might interact to either enhance or suppress expression of the bovine CYP17 gene in Y-1 cells through the CRS II site. Both SF-1 and COUP-TF are expressed in ovarian cells; however, the ratio of these two transcription factors changes throughout the ovarian cycle (54). Either of these transcription factors as well as other transcription factors or adapter proteins may help to modulate both the developmental and tissue-specific expression of the bovine CYP17 gene. To date, the results of the present study suggest that the inverted Sp1-like binding site located in the promoter of the bovine CYP17 gene might be involved in constitutive as well as cAMP-dependent expression of this gene. Additional studies are needed to further elucidate the role of other transcription factors in mediating the expression the CYP17 gene in the bovine ovary. Acknowledgments We thank M. Dodson Michael for his input in this project. We also thank Carolyn Fisher and Christy Ahsanullah for providing excellent technical assistance.
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