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Original Studies |
-Hydroxylase and Steroidogenic Acute Regulatory Protein Gene Promoters in Normal and Polycystic Ovary Syndrome Theca Cells1
Departments of Cellular and Molecular Physiology (J.K.W., P.G.Q., V.L.N., J.M.M.) and Obstetrics and Gynecology (R.S.L.), Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033; and Center for Research on Reproduction and Women’s Health (J.F.S.), University of Pennsylvania, Philadelphia, Pennsylvania 19104
Address correspondence and requests for reprints to: Jan M. McAllister, Ph.D., Department of Cellular and Molecular Physiology, Pennsylvania State University, Hershey Medical Center, 500 University Drive, Hershey, Pennsylvania 17033.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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-Hydroxylase (CYP17) expression in propagated theca cells isolated
from the ovaries of women with polycystic ovary syndrome (PCOS) is
persistently elevated, compared with theca cells isolated from normal
ovaries. To investigate the mechanism for increased CYP17 messenger RNA
accumulation in PCOS theca cells, we examined CYP17 and steroidogenic
acute regulatory protein (StAR) promoter activities in normal and PCOS
theca cells. Conditions were established to transiently transfect human
theca cells with reporter gene constructs containing 5' truncations of
the human CYP17 and StAR promoters. Cotransfection of a steroidogenic
factor-1 expression plasmid was found to be required for
detection of basal and forskolin-stimulated CYP17 promoter activity but
not for StAR promoter activity. However, cotransfection with a
steroidogenic factor-1 expression plasmid augmented both basal and
forskolin-stimulated StAR promoter activity. CYP17 reporter activity
was compared in theca cells isolated from normal and PCOS patients.
Basal and forskolin-stimulated CYP17 promoter activity was 4-fold
greater in PCOS cells than in theca cells isolated from normal ovaries.
In contrast, StAR promoter activity, and the activity of a reporter
construct containing three copies of a cAMP response element (3xCRE),
were similar in normal and PCOS theca cells. The results of these
studies document: 1) that basal and cAMP-dependent CYP17 gene
transcription is increased in PCOS theca cells; 2) that there is
differential regulation of promoters of genes required for
steroidogenesis in PCOS theca cells; and 3) that passaged normal and
PCOS theca cells provide a model system for studying tissue-specific
regulation of genes encoding steroidogenic enzymes and identifying the
molecular mechanisms involved in increased androgen production in PCOS. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In the human ovarian follicle, the androgen-secreting theca cells
express the 17-hydroxylase (CYP17) gene that encodes a cytochrome
P450 with 17-hydroxylase and C17, 20 lyase activities, which
are necessary for the conversion of pregnenolone to
17-hydroxypregnenolone and dehydroepiandrosterone and for the
conversion of progesterone to 17-hydroxyprogesterone (5, 14, 15).
These represent obligatory and key steps involved in the synthesis of
androgens. Theca cells also express steroidogenic acute regulatory
protein (StAR), which promotes the translocation of cholesterol to the
inner mitochondrial membrane (16), as well as cytochrome P450
cholesterol side chain cleavage enzyme (CYP11A) and 3ß-hydroxysteroid
dehydrogenase (3ß-HSD), each of which is required for androgen
biosynthesis (17).
Although a number of theories have been proposed to explain the etiology of excess androgen production by PCOS ovaries, few studies have focused on the regulation of steroidogenic enzyme expression in isolated theca interna cells. We recently reported conditions to examine the regulation of androgen production at the metabolic and molecular level using normal and PCOS theca interna cells isolated from size-matched follicles and propagated for multiple population doublings (5, 18). Using these conditions, we established that increased androgen production is a stable phenotype of PCOS theca cells that not only results from preferentially increased CYP17 expression but involves the up-regulation of other steroidogenic enzymes, including CYP11A, 3ß-HSD, and 17ß-HSD (5). These studies also revealed increased basal and cAMP-stimulated steady-state CYP17 and CYP11A messenger RNA (mRNA) levels in PCOS theca cells, as compared with normal cells. In marked contrast, basal and forskolin-stimulated StAR mRNA accumulation were similar in normal and PCOS theca cells, suggesting that there are selective abnormalities in the expression of the steroidogenic machinery in PCOS theca cells.
To our knowledge, no one has defined the promoter elements required for CYP17 or StAR gene expression in human theca cells. The CYP17 gene has been shown to be regulated by cAMP in a variety of steroidogenic tissues and species, and it involves several different cis-regulatory elements and trans-acting factors (19, 20, 21, 22, 23). Examination of the cis-regulatory elements involved in basal and cAMP-stimulated CYP17 gene expression has revealed distinct tissue- and species-specific regulation of the gene (see Refs. 30, 32). In contrast, StAR gene expression, in a variety of steroidogenic tissues and species, is regulated by similar cis-regulatory elements (24, 25). The transcription factor steroidogenic factor-1 (SF-1) has been identified to be a common regulator involved in the expression of CYP17 and StAR in adrenocortical and gonadal tissues (26, 27, 28). SF-1 is expressed in theca and granulosa cells of the mouse (26), rat (29), and human (30) ovary. In the human ovary, SF-1 expression is first observed in theca cells of the preantral follicle, preceding CYP17 expression, and it increases throughout follicular development (30). An examination of the 5' flanking sequence of the human CYP17 gene reveals that there are multiple copies of the consensus SF-1 binding motif (27). The human StAR promoter has three functional SF-1 response elements involved in basal and cAMP-responsiveness (31, 32).
Although we have reported that steady-state CYP17 mRNA levels are increased in PCOS theca cells, we did not determine whether this increase results from altered transcriptional regulation of the CYP17 gene. In this report, we have begun to examine the molecular mechanisms involved in the differential regulation of CYP17 and StAR gene transcription in normal and PCOS theca cells. Here we established conditions to transiently transfect human theca cells, with reporter gene constructs containing 5' truncations of the human CYP17 and StAR promoters. Transient transfection assays were then performed in normal and PCOS theca cells to determine whether differences in CYP17 and StAR mRNA accumulation could be explained by differences in transcriptional activation.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Human theca interna tissue was obtained from follicles of women undergoing hysterectomy, under a protocol approved by the Institutional Review Board of the Pennsylvania State University College of Medicine. Individual follicles were dissected away from ovarian stroma. The isolated follicles were size-selected for diameters ranging from 3–5 mm so that theca cells derived from follicles of similar size from normal and PCOS subjects could be compared. The dissected follicles were placed into serum-containing medium and bisected. Under a dissecting microscope, the theca interna was stripped from the follicle wall, and the granulosa cells were removed with a platinum loop. The cleaned theca shells were dispersed with 0.05% collagenase I, 0.05% collagenase IA, and 0.01% deoxyribonuclease in medium containing 10% FBS (33). Dispersed cells were placed in culture dishes that had been precoated with fibronectin by incubation at 37 C with culture medium containing 5 µg/mL human fibronectin. The growth medium used was a 1:1 mixture of Dulbecco’s Eagles Medium and Hams F-12 medium containing 10% FBS, 10% horse serum, 2% UltroSer G, 20 nmol/L insulin, 20 nmol/L selenium, and 1 µmol/L vitamin E and antibiotics (33). From each follicle, 12 35-mm dishes of primary theca interna cells were grown until confluent, removed from the dish with neutral protease (pronase-E; protease type XXIV, Sigma, St. Louis, MO) in DME-F12 (1:1), frozen, and stored in liquid nitrogen (1 35-mm dish per vial) in culture medium that contained 20% FBS and 10% dimethylsulfoxide (33). In all experiments, cells were thawed and propagated in the growth medium described above. To obtain successive passages of normal and PCOS theca cells, cells were thawed, propagated, and frozen at consecutive passages. The cells were grown in 5% O2, 90% N2, and 5% CO2. Reduced oxygen tension and supplemental antioxidants (vitamin E and selenium) were employed to prevent oxidative damage.
The PCOS and normal ovarian tissue came from age-matched women, 38–40 yr old. The diagnosis of PCOS was made according to established guidelines (34), including hyperandrogenemia, oligoovulation and the exclusion of 21-hydroxylase deficiency, Cushing’s syndrome, and hyperprolactinemia. All of the PCOS theca cell preparations studied came from ovaries of women with fewer than six menses per year and elevated serum total testosterone or bioavailable testosterone levels, as we previously described (5, 11). Each of the PCOS ovaries contained multiple subcortical follicles of less than 10 mm in diameter. The control (normal) theca cell preparations came from ovaries of fertile women with normal menstrual histories, menstrual cycles of 21–35 days, and no clinical signs of hyperandrogenism. Neither PCOS or normal subjects were receiving hormonal medications at the time of surgery. Indications for surgery were dysfunctional uterine bleeding, endometrial cancer, and pelvic pain. The passage conditions and split ratios for all normal and PCOS cells were identical. Experiments comparing PCOS and normal theca were performed utilizing 4th-passage (31–38 population doublings) theca cells isolated from size-matched follicles obtained from age-matched subjects.
Sera and growth factors were obtained from the following sources: FBS and DME/F12 were obtained from Irvine Scientific (Irvine, CA): horse serum was obtained from Life Technologies(Grand Island, NY); UltroSer G was from Reactifs IBF (Villeneuve-la-Garenne, France): other compounds were purchased from Sigma.
Transient transfections
Subconfluent theca cultures were transfected with reporter gene constructs using the calcium-phosphate method of Gram and Vander Eb (35). One hour before transfection, the cells were transferred into DME high-glucose medium containing 20 mmol/L HEPES and 2% heatinactivated calf serum and moved to a 3% CO2, 95% ambient air, 37-C incubator. DNA/Ca2P04 solution containing 20 µg of reporter plasmid and 5 µg of pSV-ßgal/100-mm dish in HEPES phosphate buffer was added to the media. After incubation for 6 h, cells were transferred into 2% heat-inactivated calf serum in DME containing 20 mM HEPES and treated as described. Cells were harvested 24–72 h after forskolin treatment using trypsin/EDTA, pelleted, and resuspended in 250 mmol/L Tris-HCL (pH 7.9) for chloramphenicol acetyltransferase (CAT) assays, and reporter lysis buffer for luciferase assays. The initial characterization of CYP17 and StAR reporter constructs was performed in theca cells propagated from several different PCOS patients, to facilitate analysis. Transfections were performed in triplicate, at least three times in theca cells isolated from various individual patients, using reporter plasmid isolated from several different plasmid preparations. Calf serum was obtained from Atlanta Biologicals (Atlanta, GA); other compounds were from Sigma.
CAT and luciferase assays
CAT activity was determined using the 3H-chloramphenicol method (36), employing preferential extraction of acetylated chloramphenical with mixed xylenes. Luciferase assays were performed using the Luciferase assay system from Promega Corp. (Madison, Wisconsin). ßgalactosidase (ß-gal) activity was determined by Galacton-Light Plus chemiluminescent assay from Tropix (Bedford, MA),and used to normalize CAT and luciferase activities. Chemiluminescence was measured with a Monolight 3010 Luminometer from Analytical Luminescence Laboratory (San Diego, CA). To control for transfection efficiency, relative CAT or luciferase activity was calculated by dividing the observed reporter activity by the corresponding ß-gal activity.
Plasmids and construction of CYP17 promoter reporter constructs
The 5' flanking sequence of CYP17 was generously provided by Michael Waterman (Vanderbilt University, Nashville, TN). Reporter gene plasmids containing sequentially smaller fragments of the 5' flanking region of the human CYP17 gene were constructed using existing restriction sites. -1800/+44 (NcoI/NaeI) -750/+44 (NaeI/NaeI), -235/+44 bp (SacI/NaeI), and -109/+44 bp (SacI/NaeI) fragments of the CYP17 promoter were isolated and subcloned into the CAT reporter plasmid, PQrCAT (37), to produce -1800, -750, -235, and -109 CYP17/CAT. The -235/+44 bp (SacI/NaeI) fragment of the CYP17 promoter was also subcloned into the luciferase vector pGL3 basic (Promega Corp.), to produce -235 CYP17/LUC.
The human StAR promoter luciferase constructs have been previously described (31, 32). The SF-1 expression plasmid, generously provided by Keith Parker (University of Texas Southwestern University, Dallas, TX), contains the full-length cDNA encoding mouse SF-1 in pCMV5. The ß-gal control vector, pSV-bgal, was obtained from Promega Corp.
Statistical analysis
Each experiment was performed using triplicate dishes. After combining the results from individual patients, unpaired two-tailed t tests were performed using StatView 5.0 from SAS Institute, Inc. (Cary, NC).
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Results |
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A series of promoter constructs containing successive truncations
of the 5' flanking sequence of the human CYP17 gene were generated
(Fig. 1A
). -1800, -750, -235, and
-109 CYP17 CAT reporter vectors (20 µg/dish) were transiently
transfected into human theca cells isolated from PCOS patients in the
presence or absence of a pCMV5 expression vector (SF-1pCMV5) expressing
mouse SF-1 (1 µg/dish). In the absence of cotransfected SF-1 (Fig. 1B), CAT activity was barely detectable after transfection of the
-1800, -750, -235, and -109 CYP17 reporter constructs, under both
control and forskolin-stimulated conditions. Cotransfection with SF-1
increased basal -1800 and -750 CYP17 reporter activity
approximately 8-fold, and -235 CYP17 reporter activity approximately
4-fold over baseline values; whereas forskolin-stimulated -1800, -750
CYP17, and -235 CYP17 reporter activity was increased approximately
22, approximately 20, and approximately 15-fold, respectively, over
baseline levels. Forskolin-stimulated -1800, -750, and -235 CYP17
reporter activities were increased approximately 2- to 3-fold above
basal values, in the presence of cotransfected SF-1. In contrast, after
cotransfection with SF-1, -109 CYP17 reporter activity was not
increased above baseline values in the presence or absence of
forskolin. Forskolin-stimulated CYP17 reporter activity was found to be
maximal after cotransfection with 1 µg/dish of SF-1 expression
plasmid (data not shown). To control for possible vector-specific
interactions, these observations were confirmed after transfection of
theca cells with a series of luciferase (pGL3) reporter constructs
containing successive truncations of -750/+44 of the CYP17 promoter
(data not shown).
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, under basal conditions, reporter
activity was only marginally increased after transfection of -1800 or
-235 CYP17/CAT over 72 h. After 24 h of forskolin treatment,
-1800 and -235 CYP17 reporter activity was unchanged.
Forskolin-stimulated -1800 CYP17 reporter activity was increased
approximately 2-fold at 48 h and approximately 7-fold at 72
h, over respective control values. -235 CYP17 reporter activity was
also increased approximately 2-fold at 48 h and increased 4-fold
at 72 h, over respective control values, in response to forskolin.
As shown in Fig. 2, 72 h of treatment is required for maximal
induction of forskolin-stimulated -1800 and - 235 CYP17 reporter
activity. These data are in agreement with the time course of
forskolin-stimulated CYP17 mRNA accumulation in passaged human theca
cells (data not shown), showing that cAMP-dependent increases in CYP17
mRNA require a lag time of 24–48 h.
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Transient transfection studies were performed using a series of
human StAR constructs (31, 32) containing deletion mutants of the 5'
flanking sequence of the human StAR gene fused to the luciferase
reporter pGL2 (Fig. 3A). -1300, -885,
-150, and -43 StAR reporter vectors (20 µg/dish) were transiently
transfected into human theca cells isolated from PCOS patients in the
presence or absence of SF-1pCMV5 (1 µg/dish) (Fig. 3B). After 72
h of treatment, in the absence or presence of 20 µmol/L forskolin,
the cells were harvested and luciferase activity quantitated. As shown
in Fig. 3B, in the absence of cotransfected SF-1, forskolin-stimulated
-1300, -885, and -150 StAR reporter activity was increased
approximately 3-fold over basal conditions. In contrast, there was no
increase in forskolin-stimulated -43 StAR reporter activity in the
absence of SF-1. Upon cotransfection of SF-1, -1300, -850, and -150
StAR reporter activity increased 3-fold above values observed in the
absence of SF-1 cotransfection, under control conditions.
Forskolin-stimulated -1300 StAR reporter activity increased 6-fold,
whereas -885 and -150 StAR reporter activity increased 3-fold, and
-43 StAR reporter activity, which was very low, increased 2-fold over
basal values after cotransfection with SF-1. The lack of a requirement
for SF-1 cotransfection for detection of StAR reporter activity in
human theca cells is in agreement with our previous observations in
human granulosa cells transfected under identical conditions (31).
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To determine whether increased steady-state CYP17 mRNA
accumulation in PCOS theca cells is associated with increased
transcriptional activation of the CYP17 gene, basal and
forskolin-stimulated CYP17 reporter activity was examined in theca
cells isolated from three normal and three PCOS patients. -1800,
-750, -235, and -109 CYP17 CAT reporter constructs (Fig. 1A
) were
transiently transfected into normal and PCOS theca cells in the
presence of SF-1pCMV5. In contrast to the data presented in
Figs. 1–3,
reporter activity is presented as fold increase above values for the
empty PQrCAT reporter vector, to control for patient-to-patient
variation in transfection efficiency. As shown in Fig. 4, the fold increase in both basal and
forskolin-stimulated CAT activities of -1800, -750, and -235 CYP17
reporter constructs was greater in PCOS theca cells than in normal
theca cells. The basal reporter activities in PCOS theca cells were, on
average, approximately 4-fold greater than respective reporter
activities in normal theca cells (P < .001). The fold
increase in forskolin-stimulated reporter activity in PCOS theca cells
was approximately 10-times greater than respective reporter activities
in normal theca cells (P < .001). In the absence of
cotransfected SF-1, CYP17 reporter activity was not detectable in
normal or PCOS theca cells.
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, forskolin-stimulated -235
CYP17/LUC reporter activity increased more than 2-fold above basal
values in both normal and PCOS theca cells (P < .01).
As with the CYP17/CAT reporter constructs, CYP17/LUC reporter activity
was not detectable in normal or PCOS theca cells in the absence of
cotransfected SF-1. The fold-increase in basal and forskolin-stimulated
luciferase activity in PCOS theca cells was approximately 3-fold
greater than respective activities in normal theca cells
(P < .01). These data are in agreement with those
presented in Fig. 4, and they confirm that increased CYP17 promoter
activity is increased in PCOS. These data support the notion that basal
and forskolin-stimulated CYP17 gene transcription are increased in PCOS
theca cells. Moreover, the observed increases in basal and
forskolin-stimulated CYP17 reporter activities are in agreement with
our previous report of increased CYP17 enzyme activity/cell and
increased steady-state CYP17 mRNA accumulation in propagated PCOS theca
cells (5).
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To determine whether the cAMP signal transduction pathway is
altered in PCOS theca cells, we transiently transfected normal and PCOS
theca cells with a vector containing three copies of a cAMP-response
element (TGACGTCA) (38) fused to a luciferase reporter, 3xCREpGL3 (39).
Theca cells, isolated from three normal and three PCOS patients, were
transiently transfected with 3xCREpGL3 (20 µg/dish) and cultured, in
the absence or presence of 20 µmol/L forskolin, for 24 h. To
control for patient-to-patient variation in transfection efficiency,
reporter activity is presented as fold increase above values for the
empty pGL3 reporter vector. As shown in Fig. 6, the fold-increase in basal and
forskolin-stimulated 3xCREpGL3 reporter activities were not
significantly different in PCOS theca cells, as compared with normal
theca cells. In normal and PCOS theca cells, 3xCREpGL3 reporter
activity increased approximately 4-fold above basal values, in response
to F treatment (P < .01). These data are in agreement
with previous observations that the ED50 for
forskolin-stimulated CYP17 activity/expression is identical in normal
and PCOS theca cells (5). These data also suggest that adenylate
cyclase/cAMP levels and the signal transduction cascades mediating cAMP
stimulation of gene transcription are not altered in PCOS theca
cells.
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To determine whether transcriptional regulation of the StAR
promoter is altered in PCOS theca cells as compared with normal theca
cells, theca cells isolated from three individual and three PCOS
patients were transiently transfected with the -885 StAR luciferase
construct (20 µg/dish) and cultured, in the absence or presence of 20
µmol/L forskolin, for 72 h. To control for patient-to-patient
variation in transfection efficiency, reporter activity is presented as
fold-increase above values for the empty pGL3 reporter vector. In both
normal and PCOS theca cells, -885 StAR reporter activity increased
approximately 3-fold above basal values, in response to forskolin
treatment, with and without SF-1 cotransfection (P <
.01). Although cotransfection with SF-1 proportionally increased basal
and forskolin-stimulated StAR reporter activity in both cell types
(Fig. 7), the fold-increases in basal and
forskolin-stimulated StAR luciferase activities were not significantly
different in PCOS theca cells, as compared with normal theca cells.
These data are consistent with our previous Northern blot data
demonstrating the lack of a significant difference in basal or
forskolin-stimulated StAR mRNA accumulation in normal and PCOS theca
cells (5).
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Discussion |
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In this report, theca cell preparations that had previously been characterized for their steroidogenic potential (5) were transfected with DNA plasmids containing the 5' flanking sequence of the human CYP17 or StAR genes fused to reporters. In theca cells transfected with a series of CYP17 reporter constructs, cotransfection of SF-1 was required for the detection of both basal and stimulated promoter activity. Both basal and forskolin-stimulated -750 and -235 CYP17 reporter activities were increased after cotransfection with SF-1. In contrast, -109 CYP17 reporter activity was not increased in response to forskolin or SF-1. These data indicate that the elements involved in cAMP-dependent regulation of the CYP17 gene in human theca cells lie within -750/-109 bp of the transcription start site, with the primary element(s) lying between -235/-109 bp. The relative increases in SF-1-dependent CYP17 reporter activity is consistent with the number of putative SF-1 response elements in the 5' flanking sequence of the CYP17 gene (27). The -750 CYP17 construct contains more SF-1 consensus binding motifs than the -235 CYP17 construct, and exhibits greater activity. Also, the -235 CYP17 construct contains SF-1 binding motifs not present in the -109/CAT vector.
It is noteworthy that the region of the CYP17 promoter that confers cAMP inducibility in theca cells (-235/-109) is distinct from that previously reported for regulation of the human CYP17 gene in human H295 adrenal cells (23, 40). In human adrenal NCIH295 cells, the regulatory sequences involved in cAMP-dependent expression of CYP17 reporter constructs lie within the first -63 bp upstream of the start site of transcription (23). Moreover, the DNA sequences that confer cAMP-inducibility of the CYP17 gene in human theca cells are evidently different from elements that seem to be their functional equivalents in human NCI-H295 adrenal cells (23), mouse adrenal Y1 cells (19, 41), and mouse leydig MA10 cells (22, 23, 41). In addition, the -235/-109 CYP17 sequences we found necessary for cAMP-regulation in human theca cells have no sequence homology to the -80/-40 region of the bovine CYP17 gene ,which was shown to be necessary for cAMP-regulation in bovine theca cells (21). Thus, our data are consistent with those of Rodriguez et al. (23) and confirm that the promoter elements that confer transcriptional activation of the CYP17 gene are not only tissue-specific but are species-specific as well.
SF-1- and cAMP-dependent regulation of human StAR gene expression has been examined in a variety of cell systems (24, 31, 32). Transient transfection studies of StAR reporter constructs in human granulosa cells identified several SF-1 binding sites involved in basal, as well as cAMPdependent, regulation (31). However, the transcriptional regulation of the human StAR promoter has never been examined in human theca cells. In this report, we established that, in human theca cells -1300, -885, and -150, StAR reporter activity is increased in response to forskolin treatment in the absence of SF-1 cotransfection. Cotransfection of SF-1 augmented both basal and forskolin-stimulated StAR promoter function. In transient transfections of human granulosa- lutein cells in long-term culture, SF-1 cotransfection was not required for StAR reporter activation. The reduction in forskolin-stimulated -885 StAR reporter activity, as compared with -1300 StAR reporter activity we observed in human theca cells, may be attributed to the deletion of an SF-1 binding site at -926 to -918, which has been shown to be required for maximal StAR reporter activity in human granulosa-lutein cells (31, 32). Similarly, the deletion of a SF-1 binding site at -105 to -95 (31, 32) may also explain the reduction in forskolin-stimulated -43 StAR reporter activity, as compared with the activity of the -150 StAR construct.
The finding that SF-1 cotransfection is required for detection of CYP17 promoter activity, but not for StAR promoter activity, raises the possibility that the mechanism by which CYP17 gene transcription is regulated by SF-1 in theca cells is distinct from that controlling StAR gene transcription. We initially believed that the requirement for SF-1 cotransfection for expression of our CYP17 reporter constructs resulted from insufficient endogenous SF-1 under conditions where reporter plasmid DNA was in excess. However, the fact that human StAR reporter activity, which has been previously shown to require functional SF-1 response elements (24, 31, 32) does not require SF-1 cotransfection under identical conditions, indicates that endogenous theca cell SF-1 concentrations are sufficient to drive StAR gene transcription. Similarly, our preliminary experiments indicate that endogenous SF-1 levels are sufficient for the expression of CYP11A promoter activity in human theca cells. These data demonstrate that the requirement for SF-1 cotransfection in the examination of basal and cAMP-stimulated CYP17 reporter activity is promoter-specific and is not an artifact of our culture or transfection conditions. Given that the human CYP17 gene has numerous SF-1 response elements in its 5' flanking sequence (27), it is conceivable that CYP17 promoter demands greater concentrations of SF-1 than the human CYP11A or StAR promoters. It is also possible that the intracellular mechanism(s) involved in SF-1-dependent activation of the CYP17 gene involves additional transcription factors that may not affect CYP11A or StAR gene transcription. For instance, the orphan nuclear receptor, COUP-TF (42), has been shown to compete for overlapping binding sites on both the human and bovine CYP17 promoters (40, 43, 44). It has been reported that cAMP-dependent transactivation of the mouse and bovine CYP17 promoter constructs requires SF-1-dependent displacement of COUP from the CYP17 reporter (43, 44). Hence, it is possible that SF-1 cotransfection in theca cells is required for displacement of COUP from the CYP17 reporter. In contrast, the mechanism for increased StAR and CYP11A expression may not involve displacement of COUP, and therefore would require less SF-1 for transactivation. Alternatively, it is possible that cAMP-dependent StAR and CYP11A promoter regulation involves additional transcription factors (or cofactors) that act cooperatively with endogenous SF-1, thereby negating the requirement for SF-1.
Transfection analysis of CYP17 promoter constructs in normal and PCOS theca cells has confirmed that increased steady-state levels of CYP17 mRNA in PCOS theca cells results, at least in part, from increased CYP17 gene transcription. Both basal and forskolin-stimulated CYP17 reporter activity is markedly increased in PCOS theca cells. The observed increases in basal and forskolin-stimulated CYP17 reporter activities are in agreement with our previous report of increased CYP17 enzyme activity/cell and increased steady-state CYP17 mRNA accumulation in propagated PCOS theca cells (5). In contrast, -885 StAR promoter activity was not different in normal and PCOS theca cells. These data are also in agreement with previous Northern blot data showing that basal and forskolin-stimulated StAR mRNA accumulation are not significantly different in normal and PCOS theca cells (5).
Increased CYP17 reporter activity in PCOS does not seem to result from
differences in forskolin-stimulated adenylate cyclase activity in
normal and PCOS theca cells, because there were no differences in
reporter activity in normal and PCOS theca cells transfected with a
plasmid containing three classical cAMP-response elements (3xCRE) or
the -885 StAR reporter construct. Similarly, increased CYP17 mRNA
accumulation does not seem to result from increased adenylate cyclase
activity, because StAR mRNA accumulation is unaffected. Furthermore,
although progesterone and 17-hydroxyprogesterone production are
markedly increased in PCOS theca cells, the ED50
for forskolin-stimulated production of these steroids is identical in
normal and PCOS theca cells (5).
In conclusion, our data suggest that dysregulation of the processes involved in CYP17 transcription in PCOS theca cells may, in part, account for increased ovarian androgen production in patients with PCOS. Examination of the cis-regulatory sequences and trans-acting factors involved in cAMP- and SF-1-dependent regulation of the CYP17 gene in normal and PCOS theca cells will further our understanding of the molecular mechanisms underlying increased CYP17 transcription in PCOS. Furthermore, identification of the signal transduction pathways involved in increased CYP17 expression, and overall androgen production in PCOS theca cells, may provide valuable information that can be applied to other clinical manifestations of PCOS, such as follicular growth arrest, insulin resistance, obesity, and endometrial cancer.
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Footnotes |
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Received September 24, 1999.
Revised January 13, 2000.
Accepted February 15, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-Hydroxyprogesterone response to leuprolide and serum androgens in
obese women with and without polycystic ovary syndrome after dietary
weight loss. J Clin Endocrinol Metab. 82:556–559.-hydroxylase/lyase activities in proliferating theca interna cells
in long term monolayer culture. Endocrinology. 125:1959–1966.-hydroxylase/c17–20 lyase). Mol Endocrinol. 10:147–158.-hydroxylase gene expression in the bovine ovary:
mechanisms regulating expression differ from those in adrenal cells. J
Steroid Biochem Mol Biol. 59:21–29.[CrossRef][Medline]
-hydroxylase/C17–20-lyase
(CYP17): transcriptional regulation of the gene by cyclic AMP in MA-10
cell. Mol Endocrinol. 6:927–934.This article has been cited by other articles:
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S. Catteau-Jonard, S. P. Jamin, A. Leclerc, J. Gonzales, D. Dewailly, and N. di Clemente Anti-Mullerian Hormone, Its Receptor, FSH Receptor, and Androgen Receptor Genes Are Overexpressed by Granulosa Cells from Stimulated Follicles in Women with Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., November 1, 2008; 93(11): 4456 - 4461. [Abstract] [Full Text] [PDF]
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V. E. Beshay, J. C. Havelock, R. Sirianni, P. Ye, T. Suzuki, W. E. Rainey, and B. R. Carr The Mechanism for Protein Kinase C Inhibition of Androgen Production and 17{alpha}-Hydroxylase Expression in a Theca Cell Tumor Model J. Clin. Endocrinol. Metab., December 1, 2007; 92(12): 4802 - 4809. [Abstract] [Full Text] [PDF]
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 F. Taniguchi, J. F. Couse, K. F. Rodriguez, J. M. A. Emmen, D. Poirier, and K. S. Korach Estrogen receptor-{alpha} mediates an intraovarian negative feedback loop on thecal cell steroidogenesis via modulation of Cyp17a1 (cytochrome P450, steroid 17{alpha}-hydroxylase/17,20 lyase) expression FASEB J, February 1, 2007; 21(2): 586 - 595. [Abstract] [Full Text] [PDF]
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 S. H. Mellon, S. R. Bair, C. Depoix, J.-L. Vigne, N. B. Hecht, and P. B. Brake Translin Coactivates Steroidogenic Factor-1-Stimulated Transcription Mol. Endocrinol., January 1, 2007; 21(1): 89 - 105. [Abstract] [Full Text] [PDF]
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C. K. M. Ho, J. R. Wood, D. R. Stewart, K. Ewens, W. Ankener, J. Wickenheisser, V. Nelson-Degrave, Z. Zhang, R. S. Legro, A. Dunaif, et al. Increased Transcription and Increased Messenger Ribonucleic Acid (mRNA) Stability Contribute to Increased GATA6 mRNA Abundance in Polycystic Ovary Syndrome Theca Cells J. Clin. Endocrinol. Metab., December 1, 2005; 90(12): 6596 - 6602. [Abstract] [Full Text] [PDF]
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 E. Diamanti-Kandarakis and C. Piperi Genetics of polycystic ovary syndrome: searching for the way out of the labyrinth Hum. Reprod. Update, November 1, 2005; 11(6): 631 - 643. [Abstract] [Full Text] [PDF]
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J. K. Wickenheisser, V. L. Nelson-DeGrave, K. L. Hendricks, R. S. Legro, J. F. Strauss III, and J. M. McAllister Retinoids and Retinol Differentially Regulate Steroid Biosynthesis in Ovarian Theca Cells Isolated from Normal Cycling Women and Women with Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., August 1, 2005; 90(8): 4858 - 4865. [Abstract] [Full Text] [PDF]
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 H. F. Escobar-Morreale, M. Luque-Ramirez, and J. L. San Millan The Molecular-Genetic Basis of Functional Hyperandrogenism and the Polycystic Ovary Syndrome Endocr. Rev., April 1, 2005; 26(2): 251 - 282. [Abstract] [Full Text] [PDF]
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 C. Glister, S. L. Richards, and P. G. Knight Bone Morphogenetic Proteins (BMP) -4, -6, and -7 Potently Suppress Basal and Luteinizing Hormone-Induced Androgen Production by Bovine Theca Interna Cells in Primary Culture: Could Ovarian Hyperandrogenic Dysfunction Be Caused by a Defect in Thecal BMP Signaling? Endocrinology, April 1, 2005; 146(4): 1883 - 1892. [Abstract] [Full Text] [PDF]
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J. K. Wickenheisser, V. L. Nelson-DeGrave, and J. M. McAllister Dysregulation of Cytochrome P450 17{alpha}-Hydroxylase Messenger Ribonucleic Acid Stability in Theca Cells Isolated from Women with Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1720 - 1727. [Abstract] [Full Text] [PDF]
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 J. R. Wood, V. L. Nelson-Degrave, E. Jansen, J. M. McAllister, S. Mosselman, and J. F. Strauss III Valproate-induced alterations in human theca cell gene expression: clues to the association between valproate use and metabolic side effects Physiol Genomics, February 10, 2005; 20(3): 233 - 243. [Abstract] [Full Text] [PDF]
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V. L. Nelson-Degrave, J. K. Wickenheisser, K. L. Hendricks, T. Asano, M. Fujishiro, R. S. Legro, S. R. Kimball, J. F. Strauss III, and J. M. McAllister Alterations in Mitogen-Activated Protein Kinase Kinase and Extracellular Regulated Kinase Signaling in Theca Cells Contribute to Excessive Androgen Production in Polycystic Ovary Syndrome Mol. Endocrinol., February 1, 2005; 19(2): 379 - 390. [Abstract] [Full Text] [PDF]
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E. Jansen, J. S. E. Laven, H. B. R. Dommerholt, J. Polman, C. van Rijt, C. van den Hurk, J. Westland, S. Mosselman, and B. C. J. M. Fauser Abnormal Gene Expression Profiles in Human Ovaries from Polycystic Ovary Syndrome Patients Mol. Endocrinol., December 1, 2004; 18(12): 3050 - 3063. [Abstract] [Full Text] [PDF]
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S. Jonard and D. Dewailly The follicular excess in polycystic ovaries, due to intra-ovarian hyperandrogenism, may be the main culprit for the follicular arrest Hum. Reprod. Update, March 1, 2004; 10(2): 107 - 117. [Abstract] [Full Text] [PDF]
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J. K. Wickenheisser, V. L. Nelson-DeGrave, P. G. Quinn, and J. M. McAllister Increased Cytochrome P450 17{alpha}-Hydroxylase Promoter Function in Theca Cells Isolated from Patients with Polycystic Ovary Syndrome Involves Nuclear Factor-1 Mol. Endocrinol., March 1, 2004; 18(3): 588 - 605. [Abstract] [Full Text] [PDF]
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V. L. Nelson-DeGrave, J. K. Wickenheisser, J. E. Cockrell, J. R. Wood, R. S. Legro, J. F. Strauss III, and J. M. McAllister Valproate Potentiates Androgen Biosynthesis in Human Ovarian Theca Cells Endocrinology, February 1, 2004; 145(2): 799 - 808. [Abstract] [Full Text] [PDF]
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V. De Leo, A. la Marca, and F. Petraglia Insulin-Lowering Agents in the Management of Polycystic Ovary Syndrome Endocr. Rev., October 1, 2003; 24(5): 633 - 667. [Abstract] [Full Text] [PDF]
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 J. R. Wood, V. L. Nelson, C. Ho, E. Jansen, C. Y. Wang, M. Urbanek, J. M. McAllister, S. Mosselman, and J. F. Strauss III The Molecular Phenotype of Polycystic Ovary Syndrome (PCOS) Theca Cells and New Candidate PCOS Genes Defined by Microarray Analysis J. Biol. Chem., July 11, 2003; 278(29): 26380 - 26390. [Abstract] [Full Text] [PDF]
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 S. Jonard, Y. Robert, C. Cortet-Rudelli, P. Pigny, C. Decanter, and D. Dewailly Ultrasound examination of polycystic ovaries: is it worth counting the follicles? Hum. Reprod., March 1, 2003; 18(3): 598 - 603. [Abstract] [Full Text] [PDF]
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V. L. Nelson, K.-n. Qin, R. L. Rosenfield, J. R. Wood, T. M. Penning, R. S. Legro, J. F. Strauss III, and J. M. McAllister The Biochemical Basis for Increased Testosterone Production in Theca Cells Propagated from Patients with Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., December 1, 2001; 86(12): 5925 - 5933. [Abstract] [Full Text] [PDF]
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R. M. Koivunen, L. C. Morin-Papunen, A. Ruokonen, J. S. Tapanainen, and H. K. Martikainen Ovarian steroidogenic response to human chorionic gonadotrophin in obese women with polycystic ovary syndrome: effect of metformin Hum. Reprod., December 1, 2001; 16(12): 2546 - 2551. [Abstract] [Full Text] [PDF]
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R. M. Calvo, M. Asunción, D. Tellería, J. Sancho, J. L. San Millán, and H. F. Escobar-Morreale Screening for Mutations in the Steroidogenic Acute Regulatory Protein and Steroidogenic Factor-1 Genes, and in CYP11A and Dosage-Sensitive Sex Reversal-Adrenal Hypoplasia Gene on the X Chromosome, Gene-1 (DAX-1), in Hyperandrogenic Hirsute Women J. Clin. Endocrinol. Metab., April 1, 2001; 86(4): 1746 - 1749. [Abstract] [Full Text]
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