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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
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.
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Abstract |
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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
-191 and -101 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
-188/-180 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 cAMP-dependent
expression of the CYP17 gene in the bovine ovary. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, that mediates
both 17-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 P450c17 is under developmental, species-specific, and
tissue-specific control (11). Once tissue differentiation has occurred,
P450c17 activity is regulated at the level of transcription by
pituitary trophic hormones via cAMP as second messenger (12, 13, 14). In
the cow, the CYP17 gene is expressed only in gonads, adrenal cortex,
and placenta (15, 16). In the adrenal cortex, P450c17 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 P450c17 activity and expression
(17, 18). Furthermore, in primary cultures of bovine adrenocortical
cells maintained for several days in the absence of ACTH, P450c17
expression and activity essentially disappear, but can be restored by
treatment with ACTH or agents that increase intracellular levels of
cAMP (19, 20, 21, 22). CYP17 transcriptional regulation in bovine adrenal seems
to involve multiple interactions between two distinct cAMP-responsive
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 P450c17 messenger RNA
(mRNA) is first observed in thecal cells at the time of antrum
formation (26). Changes in 17-hydroxylase/17,20-lyase activities are
regulated at the level of transcription of the CYP17 gene. The capacity
of bovine thecal cells to express P450c17 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 P450c17
expression within 24 h of the LH surge, and no androgen precursors
can be detected in even the earliest corpus luteum (28, 29, 30, 31, 32). Bovine
thecal cells readily lose the ability to produce androstenedione when
placed in culture, indicative of a loss of 17-hydroxylase expression
(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 P450c17 activity and expression for at least 8 days
(35). Using this system, Demeter-Arlotto et al. (36) made
deletion constructs of the 5'-flanking region of the bovine CYP17 gene
that were subcloned into the OVEC vector just upstream of the rabbit
ß-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 5'-flanking CYP17 region on reporter gene expression.
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Materials and Methods |
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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 5'-flanking region of the bovine CYP17 gene
The 5'-flanking region of the bovine CYP17 gene was sequenced up
to -1036 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
5'-flanking bovine CYP17 sequence previously characterized (3).
Construction of bovine CYP17 5'-flanking/luciferase constructs
Reporter gene constructs were prepared by creating 5'-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
(5'-primer) and BglII (3'-primer). The genomic clone
B17-1-PUC18, provided by Dr. M. R. Waterman (Vanderbilt University,
Nashville, TN), was used as DNA template. All of the PCR-amplified
fragments (-866, -709, -437, -293, -243, -191, -179, -101, and
-80/-10 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 (5'-primer)
and BglII (3'-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, 36, 37, 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 µg/ml),
selenium (1 ng/ml), vitamin E (1 µM), 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 µg of the reporter construct and 0.5 µg of the pCMVnlac ß-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 µl Cellfectin reagent for each 35-mm dish of thecal cells or 5 µl/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 µM isobutylmethylxanthine (IBMX) and incubated for 6 h either with or without 10 µM forskolin, whereas luteal cells were changed to serum-free medium containing 0.1% BSA and 100 µM IBMX and incubated for 12 h either with or without 25 µM 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 µg of the reporter construct, 0.5 µg of the pCMVnlac ß-galactosidase expression plasmid, and 0.2 µg of PKA catalytic subunit (ß) or its inactive mutant form (ßm) 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. pGL3-control 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 µM 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 ß-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). ß-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 ß-galactosidase assays were measured by a
Monolight 2010 Luminometer (Analytical Luminescence Laboratory).
Transfection efficiency was assessed by measuring the activity of
ß-galactosidase expressed from the cotransfected ß-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 ß-galactosidase activity or protein concentration, obtained in
samples transfected with the negative reference vector and incubated in
the absence of forskolin.
Electrophoretic mobility shift assay (EMSA)
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 5'-flanking region at -188/-180 bp were used (Fig. 1
, underline; 5'-gtcgacTTACCTAGCCCCTCCCCT-3'
and 3'-AGGGGAGGGGCTAGGTAA-5'), as well as oligonucleotides
corresponding to the sequence of a consensus Sp1-binding site (GC box;
5'-GCGATCGGGGCGGGG-CG-3' and 3'-tcgaCGCTAGCCCCGCCCCGC agct-5').
These oligonucleotides were labeled by Klenow labeling with
[32P]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
(5'-gtcgac-TACCTAGCgCgTCCCCT-3' and
3'-AGGGGAcGcGCTAGGTA-5'; site of mutations
indicated by lowercase letters) was used for competition
assays. When supershift assays were performed, 1 µl 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.
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Results |
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-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 5'-flanking sequence described in the present
study, the region between -865 and +205 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 B17-1-PUC18 clone.
Expression of bovine CYP17 5'-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 5'-flanking
deletion mutations (-866/, -709/, -437/, -293/, -243/, -191/,
-101/, and -80/-10 bp) were created by PCR. All fragments contained
the endogenous bovine CYP17 putative TATA box (TTAAAAA) located at
-27/-21 bp upstream of the transcriptional start site (Fig. 1) as
well as the cAMP-responsive element CRS II, described previously (41),
located at -68/-33 bp (Fig. 1, bold). Inserts from -866
to -243/-10 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 5'-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
5'-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.
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, 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 -437 and -293 bp. There was no
difference in either basal or forskolin-stimulated reporter gene
activity in thecal cells with the deletion of the sequence between
-293 and -191 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 -191 to -101 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 5'-flanking
regions were made (-243 to -107 bp in OVEC or -297 to -100 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 -293 to -80 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 -191 to -101
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 -188 and -180 bp is involved in
transcriptional regulation of the bovine CYP17 gene
As shown in Fig. 2A, deletion of the promoter region between -191
and -101 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 Sp1-binding sequence located at -188/-180 bp (inv
Sp1-bs: CCTAGCCCC). Specific deletion (-179/-10 bp) or mutation
(-191/-10 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
(-179/-10 bp) and the mutated inv Sp1-bs (-191/-10 MUT) constructs
in thecal cells resulted in forskolin-stimulated reporter gene activity
higher than the control value.
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) with luciferase constructs containing the inverted
Sp1-binding site (-191/-10 bp) or its mutated sequence (-191/-10
MUT) and with expression vectors containing the PKA catalytic subunit
(ß) or its inactive mutant form (ßm). The overexpression of the PKA
catalytic subunit increased reporter gene activity of the wild-type
(-191/-10 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 ß. 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 cAMP-dependent expression of
this gene.
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, 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 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 sequence. 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
protein(s) responsible for the formation of complex C might not be
primarily involved in the transcriptional regulation mediated by the
inverted Sp1 sequence.
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, 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 P450c17 expression (29, 30, 31, 32). In the present study,
luciferase reporter gene vectors containing deletion mutations of the
bovine CYP17 promoter and 5'-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 -293/-10 to -191/-10 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 5'-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, 30, 31, 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 -866 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, P450c17 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
P450c17 expression (41, 43, 44, 45, 46). In the bovine ovary, significant
expression of P450c17 is first observed in thecal cells at the time
of antrum formation (26). In thecal cells,
17-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, 48, 49). Previous experiments performed in the mouse adrenal
tumor cell line Y1 and in bovine adrenocortical cells (23, 24, 25) have
shown that two cis-acting elements located in the bovine
CYP17 5'-flanking region, termed CRS I and CRS II, are involved in the
cAMP-dependent 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 ß-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-dependent 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 -297 and -100 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 sequence (-191/-10 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 -191/-10 bp construct to determine its role in cAMP-dependent 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 (-188/-180 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 reporter 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 P450c17 is expressed only in steroidogenic cells, how can Sp1, a
ubiquitously expressed transcription factor, modulate tissue-specific
expression? In the context of the -191/-10 bp construct, when the Sp1
site was mutated, or of the -179/-10 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
P450c17 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.
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Acknowledgments |
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Footnotes |
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Received September 17, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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