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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-0330
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 8 4858-4865
Copyright © 2005 by The Endocrine Society

Retinoids and Retinol Differentially Regulate Steroid Biosynthesis in Ovarian Theca Cells Isolated from Normal Cycling Women and Women with Polycystic Ovary Syndrome

Jessica K. Wickenheisser, Velen L. Nelson-DeGrave, Karen L. Hendricks, Richard S. Legro, Jerome F. Strauss, III and Jan M. McAllister

Departments of Cellular and Molecular Physiology (J.K.W., V.L.N.-D., K.L.H., J.M.M.) and Obstetrics and Gynecology (R.S.L., J.M.M.), Pennsylvania State College of Medicine, Hershey, Pennsylvania 17033; and the Center for Research on Reproduction and Women’s Health (J.F.S.), University of Pennsylvania, Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Jan M. McAllister, Ph.D., Pennsylvania State Hershey College of Medicine, Department of Cellular and Molecular Physiology, 500 University Drive, C4723, Hershey, Pennsylvania 17033. E-mail: jmcallister{at}psu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Polycystic ovary syndrome (PCOS) is characterized by ovarian androgen excess and infertility. Recent experiments have suggested that several genes involved in retinoic acid synthesis may be differentially expressed in PCOS theca cells and may contribute to excessive theca-derived androgen production.

Objective: The study was performed to examine whether there are differential effects of retinol and retinoids on normal and PCOS theca cell function.

Design: We used in vitro assays.

Setting: The study was conducted at the university laboratory.

Patients: We studied theca interna cells isolated from normal-cycling women and women with PCOS.

Interventions: Theca cells were treated with all-trans-retinoic acid (atRA), 9-cis retinoic acid (9-cis RA), or the retinoic acid precursor retinol.

Main Outcome Measure(s): We measured dehydroepiandrosterone, testosterone, and progesterone biosynthesis as well as cytochrome P450 17{alpha}-hydroxylase (CYP17), cytochrome P450 cholesterol side-chain cleavage, and steroidogenic acute regulatory protein mRNA abundance and promoter function.

Results: Dehydroepiandrosterone production was increased by atRA and 9-cis RA in normal cells and by atRA, 9-cis RA, and retinol in PCOS. Testosterone production was increased by atRA in normal and by atRA, 9-cis RA, and retinol in PCOS. Progesterone production was not altered by retinoid treatment. Retinoids stimulated mRNA abundance and promoter function for CYP17 and steroidogenic acute regulatory protein in both cell types and cytochrome P450 cholesterol side-chain cleavage in normal cells. Retinol stimulated CYP17 mRNA accumulation and promoter function in PCOS but not normal theca cells. P < 0.05 was considered statistically significant.

Conclusions: Differential responses to retinol and retinoids in normal and PCOS theca suggest that altered retinoic acid synthesis and action may be involved in augmented CYP17 gene expression and androgen production in PCOS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VITAMIN A (RETINOL) and its derivatives, collectively known as retinoids, are important regulators of vision, embryogenesis, reproduction, inflammation, and growth and differentiation (1, 2, 3, 4). Retinol is essential for female reproduction, and retinoids have been suggested to play a role in ovarian steroidogenesis (5, 6, 7), oocyte maturation, and formation of the corpus luteum (3). The pathways for metabolism of retinol into the biologically active ligands all-trans retinoic acid (atRA) and 9-cis retinoic acid (9-cis RA) involve several families of enzymes with overlapping function (8, 9). The rate-limiting conversion of retinol to retinal is performed by alcohol/retinol dehydrogenases as well as short-chain dehydrogenase/reductases (10, 11). The oxidation of retinal to retinoic acid is catalyzed by aldehyde/retinal dehydrogenases as well as cytochrome P450s (11, 12). Cellular binding proteins, cellular retinol-binding proteins, and cellular retinoic acid binding proteins also regulate the storage, metabolism, and cellular levels of retinol and retinoic acid within cells, respectively (8, 13). The major actions of retinoids are transduced through nuclear receptors known as retinoic acid receptors (RARs), which bind atRA and 9-cis RA, and retinoid X receptors (RXRs), which bind 9-cis RA (14, 15). Retinoic acid response elements within the promoters of retinoid-responsive genes are sites for RAR and RXR action (16).

Polycystic ovary syndrome (PCOS) affects 5–10% of reproductive-age women and is associated with increased ovarian androgen production and infertility (17, 18, 19, 20). Additional clinical features (of PCOS patients) may include obesity, hirsutism, hyperinsulinism, and a predisposition for noninsulin-dependent diabetes mellitus. Atherosclerosis, hypertension, dyslipidemia, coronary artery disease, and endometrial carcinoma have also been reported to be associated with PCOS. PCOS and PCOS-associated phenotypes are clustered in families, suggesting that genetic factors are involved in the etiology of the disorder (20, 21).

Unlike the ovary of normal-cycling women, the PCOS ovary is characterized by multiple small follicles 4–7 mm in diameter, with a theca cell compartment that is often hypertrophied. Ovarian theca cells are recognized as one of the primary sources of excess androgen biosynthesis in women with PCOS (22, 23, 24, 25), expressing a variety of genes encoding components of the steroidogenic pathway that are necessary for androgen and progestin biosynthesis (26). Steroidogenic acute regulatory protein (StAR) promotes the translocation of cholesterol from the outer to the inner mitochondrial membrane (27), where cytochrome P450 side chain cleavage enzyme, encoded by CYP11A1 gene, converts cholesterol to pregnenolone in the first step in steroid synthesis. The synthesis of thecal androgens is contingent on the expression of the cytochrome P450 17{alpha}-hydroxylase (CYP17) gene, which encodes a single cytochrome P450 with both 17{alpha}-hydroxylase and C17, 20 lyase activities responsible for the conversion of pregnenolone to 17{alpha}-hydroxypregnenolone, and subsequently dehydroepiandrosterone (DHEA). Alternatively, pregnenolone and 17{alpha}-hydroxypregnenolone can be converted to progesterone and 17{alpha}-hydroxyprogesterone by type II 3ß-hydroxysteroid-{Delta}5-steroid dehydrogenase, {Delta}5,4 isomerase, encoded by the HSD3B2 gene. The final conversion of DHEA to androstenedione and testosterone requires type II 3ß-hydroxysteroid-{Delta}5-steroid dehydrogenase, {Delta}5,4 isomerase and 17ß-hydroxysteroid dehydrogenase.

As a consequence of examining theca cells propagated from normal-cycling women and women with PCOS, we have previously established that increased androgen and progestin production is a stable phenotype of PCOS theca cells in long-term culture (28, 29). This augmented steroid production in PCOS theca cells is associated with increased gene expression of several steroidogenic enzymes important for androgen biosynthesis, including CYP11A1, CYP17, and HSD3B2 (28, 30). Recent microarray analysis comparing normal and PCOS theca cells has revealed that PCOS theca cells have a gene expression profile that is distinct from normal theca cells (31). Furthermore, subsequent experiments have indicated that several genes involved in retinoic acid synthesis/action were differentially expressed in normal and PCOS theca cells, including retinol dehydrogenases RoDH4 and RoDH2, cellular retinoic acid binding protein II, and prostate short-chain dehydrogenase/reductase mRNAs (31). Furthermore, conversion of retinol to retinaldehyde was increased using PCOS cell extracts, compared with normal extracts, suggesting that the enzymes responsible for retinol metabolism are present in theca cells and may be altered in PCOS (31). Treatment of theca cells with atRA increased DHEA production as well as CYP17 and CYP11A1 mRNA accumulation. Together these data suggest that retinoids may regulate androgen biosynthesis and steroidogenic enzyme expression in normal and PCOS theca cells and contribute to the excessive theca-derived androgen production in PCOS (32).

In these studies, we further investigated the extent to which retinoid treatment affects steroid biosynthesis and CYP17, CYP11A1, and StAR gene expression in human theca cells in long-term culture. We evaluated the extent to which normal and PCOS theca cells respond to retinol and retinoid treatment. Our data demonstrate that retinol and retinoids dramatically augment androgen biosynthesis in PCOS theca cells. Furthermore, this increased androgen biosynthesis correlates with differential changes in CYP17, StAR, CYP11A1 mRNA accumulation, and promoter activity. These data support previous findings that altered retinoic acid synthesis/and action may contribute to augmented androgen production in PCOS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Theca cell isolation and propagation

Human theca interna tissue was obtained from follicles of women undergoing hysterectomy, after informed consent, 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 to 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 cell layers were dispersed with 0.05% collagenase I, 0.05% collagenase IA, and 0.01% deoxyribonuclease in medium containing 10% fetal bovine serum (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 Eagle’s Medium (DME) and Ham’s F-12 medium containing 10% FBS, 10% horse serum, 2% UltroSer G, 20 nM insulin, 20 nM selenium, 1 µM vitamin E, and antibiotics. 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 (one 35-mm dish per vial) in culture medium that contained 20% FBS and 10% dimethylsulfoxide. 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 used 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, 35), 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 previously described (28, 36). 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 d, and no clinical signs of hyperandrogenism. Neither PCOS nor 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 using fourth-passage (31–38 population doublings) theca cells isolated from size-matched follicles obtained from age-matched subjects. The theca cells examined in these experiments included stocks of cells isolated and propagated from PCOS and normal women that we have previously examined (37) as well as stocks of cells that we have recently generated from newly characterized patients. 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 HyClone (Logan, UT); UltroSer G was from Reactifs IBF (Villeneuve-la-Garenne, France). atRA, all-trans retinol, and 9-cis RA were purchased from Sigma.

Steroid biosynthesis

For evaluation of steroid production, theca cells were grown until subconfluent and transferred into serum-free medium in the presence or absence of forskolin (20 µM) with and without retinoids, atRA or 9-cis RA, or retinol. At 72 h, the media were collected, and RIAs for DHEA, 17{alpha}-hydroxyprogesterone (17OHP4), testosterone (T), and progesterone (P4) were performed without organic solvent extraction using Diagnostic Products Corp. (Los Angeles, CA) and ICN Biochemicals (Irvine, CA) RIA kits as described previously (28).

Quantitation of CYP17, CYP11A, and StAR mRNA

For quantitative real-time PCR, total mRNA was isolated (28) from fourth passage theca cells that were grown to subconfluence, transferred into serum-free medium, and treated as indicated. RNA (1 µg) samples were then reverse transcribed using oligo (dT), and 200 U Stratascript reverse transcriptase (Stratagene, La Jolla, CA). CYP17, CYP11A1, and StAR mRNA abundance was determined by quantitative real-time PCR as previously described (31, 37). The gene-specific two-step PCR was carried out in triplicate for each cDNA sample and serial diluted cDNA standards in an Mx4000 thermocycler (Stratagene), using the Mx4000 multiplex quantitative PCR system according to the manufacturer’s instructions. An arbitrary value of template was assigned to each serial dilution (i.e. 1000, 300, 100, 30, 10, 3, 1) and plotted against the cycle threshold value (y-axis = cycle threshold; x-axis = value, log scale) to generate a standard curve. Each unknown was assigned an arbitrary value based on the slope and y-intercept of the standard curve. The same process was carried out for TATA box binding protein (TBP), which was used to normalize each reaction. The mean target value for each unknown was divided by the mean TBP value for each unknown to generate a normalized value for the target for each sample. The average normalized value and SE for each target was determined using data from the normal samples with and without treatment for each time point used in the experiment.

Transient transfection analysis

Subconfluent cultures of theca cells were transfected with reporter gene constructs as we have previously described (38) using the modified calcium-phosphate method of Graham and van der Eb (39). The –750 CYP17, –1676 CYP11A1, and –885 STAR promoter luciferase constructs have been previously described (40, 41, 42). These promoter regions have been previously shown to confer both basal and forskolin-stimulated promoter function in human theca cells (37, 38, 40). One hour before transfection the cells were transferred into DME high-glucose medium containing 20 mM HEPES and 2% heat-inactivated calf serum and moved to a 3% CO2, 95% ambient air 37 C incubator. DNA/Ca2PO4 solution containing 20 µg of reporter plasmid, and 1 µg of pSV-ßgal/100 mm dish in HEPES phosphate buffer was added to the media. As we have previously described (38, 40), an expression vector encoding human steroidogenic factor-1 was included in transfections using CYP17 promoter constructs, which is necessary for full induction of the CYP17 promoter. After incubation for 6 h, cells were transferred into 2% calf serum in DME containing 20 mM HEPES and treated as described. Seventy-two hours after forskolin (20 µM) and/or retinoid (5 µM) treatment, the cells were harvested using trypsin/EDTA, pelleted, and resuspended in reporter lysis buffer for luciferase assays. Luciferase assays were performed using the luciferase assay system (Promega, Madison, WI). ß-Galactosidase activities were determined by Galacton Light Plus chemiluminescent assay (Tropix, Bedford, MA) and used to normalize luciferase activities for transfection efficiency.

Statistical analysis

Each experiment was performed using triplicate dishes. Results are presented as the mean ± SEM of steroid levels or mRNA abundance from triplicate theca cell cultures from one normal and one PCOS patients and are representative of experiments performed in four normal and four PCOS theca cell cultures. For determination of statistical significance, two-way ANOVA was performed using PRISM 4.0 (GraphPad Software, San Diego, CA), and P values determined by the Sidak method for multiple comparisons. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
atRA augments thecal steroid biosynthesis

To examine the effects of atRA on steroid biosynthesis, fourth-passage theca cells were treated with increasing concentrations of atRA (0.1–5 µM) in the presence or absence of a maximal dose of forskolin (20 µM) for 72 h. The effects of atRA treatment on basal and forskolin-stimulated DHEA, 17OHP4, and P4 production are presented in Fig. 1Go and are representative of data obtained from theca cells isolated from four different normal patients. Treatment with atRA (0.1–5 µM) produced a dose-dependent increase in DHEA and 17OHP4 production under both control and forskolin-stimulated conditions. In contrast, the full range of atRA had no effect on basal or forskolin-stimulated P4 production.



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  		FIG. 1. The effects of atRA on 17OHP4, DHEA, and P4 production. 17OHP4, DHEA, and P4 production were examined in fourth-passage theca cells that were grown until subconfluent and transferred into serum-free medium containing an increasing concentration of atRA (0.1–5 µM) with and without 20 µM forskolin. After 72 h of treatment, the media were collected, steroid production evaluated by RIA, and the data normalized to cell number. A, Steroid production under control and forskolin-stimulated conditions. B, Steroid production under control conditions (absence of forskolin). Results are presented as the mean ± SEM of steroid levels from triplicate theca cell cultures from four patients.

 
The differential effects of 9-cis RA and retinol on DHEA and T biosynthesis in normal and PCOS theca cells

To evaluate whether normal and PCOS theca cells differentially respond to retinoid treatment, we examined steroid biosynthesis in response to atRA, 9-cis RA, or the retinoic acid precursor retinol. DHEA and T biosynthesis in normal and PCOS theca were examined under basal and forskolin-stimulated conditions in the absence or presence of retinoid (Fig. 2Go). In agreement with our previously published data, both basal and forskolin-stimulated DHEA (31) production was increased approximately 10-fold in PCOS theca cells, compared with normal theca cells. T biosynthesis was also elevated 10-fold in PCOS theca, under forskolin-stimulated conditions.



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  		FIG. 2. The effects of retinoids on basal and forskolin-stimulated steroid biosynthesis in normal and PCOS theca cell cultures. To compare the effect of retinoid treatment on overall steroid biosynthesis normal and PCOS theca cells, DHEA (A) and T (B) production was examined in fourth-passage normal (left) or PCOS (right) theca cells. Cells were grown until subconfluent and transferred into serum-free medium containing vehicle (-), atRA, 9-cis RA, or retinol (5 µM), with and without 20 µM forskolin. After 72 h of treatment, the media were collected, steroid production evaluated by RIA, and the data normalized to cell number. Results are presented as the mean ± SEM of steroid levels from triplicate theca cell cultures from four normal and four PCOS patients. Statistically different steroid biosynthesis are indicated for control (a, P < 0.05) and forskolin-stimulated (b, P < 0.05) conditions. Conditions in which the fold increase in DHEA and T production in response to treatment were greater in PCOS theca cells, compared with normal theca cells are indicated (*).

 
In both normal and PCOS theca cells, atRA treatment stimulated both basal and forskolin-stimulated DHEA production to a similar extent (Fig. 2AGo). In contrast, retinol treatment did not significantly increase either basal or forskolin-simulated DHEA production in normal cells. In PCOS theca cells, the fold-increase in DHEA production in response to retinol was 4.5- and 1.5-fold, under control and forskolin-stimulated conditions, respectively. In normal cells, treatment with 9-cis RA enhanced DHEA biosynthesis 2-fold under control conditions and had no effect under forskolin-stimulated conditions. Treatment of PCOS theca cells with 9-cis RA increased basal and forskolin-stimulated DHEA production by 4- and 2-fold, respectively.

With respect to T production (Fig. 2BGo), treatment of normal theca cells with atRA increased both basal and forskolin-stimulated T biosynthesis, whereas retinol and 9-cis RA had no effect. In PCOS theca cells, T production was increased in response to retinol, atRA, and 9-cis RA in both the absence and presence of forskolin. Overall, the magnitude of retinol and retinoid-stimulated DHEA and T production was markedly increased in PCOS theca, compared with normal theca cells.

The differential effects of 9-cis RA and retinol on CYP17, CYP11A1, and StAR gene expression in normal and PCOS theca cells

The finding that increasing concentrations of atRA stimulate DHEA and 17OHP4 production suggests that retinoids may augment the expression of steroidogenic enzymes required for androgen biosynthesis. Therefore, the time course of CYP17, CYP11A, and StAR mRNA accumulation was examined after treatment with and without 5 µM atRA for 0–48 h using real-time quantitative PCR analysis, as described in Materials and Methods. Treatment with atRA significantly augmented CYP17, CYP11A1, and StAR mRNA accumulation with maximal induction occurring within 16–24 h after treatment (Fig. 3Go).



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  		FIG. 3. The effects of atRA on CYP17, CYP11A1, and StAR mRNA accumulation. CYP17 (A), CYP11A1 (B), and StAR (C) mRNA abundance was evaluated in fourth-passage theca cells that were grown until subconfluent and transferred into serum-free medium with vehicle (control) or 20 µM forskolin, with and without 5 µM atRA for 4–48 h. All data were obtained using quantitative real-time PCR analysis. mRNA accumulation was normalized by TBP mRNA abundance for each sample and is depicted graphically as the mean ± SEM from two normal theca cell cultures performed in triplicate. CYP17, CYP11A, and StAR mRNA abundance was increased in response to atRA treatment.

 
To examine the extent to which retinol and retinoids regulate gene expression in normal and PCOS theca cells, fourth-passage theca cells were grown to subconfluence and transferred into serum-free medium in the presence or absence of atRA, 9-cis RA, or retinol (5 µM), with and without forskolin, for 24 h, and CYP17, CYP11A1, and StAR mRNA accumulation was examined by quantitative real-time PCR analysis (Fig. 4Go). In agreement with previous reports, CYP17 mRNA abundance was increased in PCOS theca cells, compared with normal cells, approximately 10-fold under control conditions and 6-fold under forskolin-stimulated conditions (Fig. 4AGo) (30). In normal theca cells under control conditions, CYP17 mRNA accumulation was significantly increased in response to atRA and 9-cis RA, but no effect was observed in response to retinol. In the presence of forskolin, atRA increased CYP17 mRNA abundance in normal theca cells, whereas 9-cis RA and retinol had no effect. In PCOS cells, both control and forskolin-stimulated CYP17 mRNA accumulation was significantly increased in response to atRA, retinol, and 9-cis RA. Treatment of PCOS theca cells with retinol or 9-cis RA resulted in an increased magnitude and fold increase in CYP17 mRNA abundance, compared with normal cells.



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  		FIG. 4. The effects of retinoids on basal and forskolin-stimulated CYP17, CYP11A1, and StAR mRNA accumulation in normal and PCOS theca. CYP17 (A), CYP11A1 (B), and StAR (C) mRNA abundance was evaluated in fourth-passage theca cells that were grown until subconfluent and transferred into serum-free medium with vehicle (control) or 20 µM forskolin with and without (-) 5 µM atRA, retinol, or 9-cis RA for 24 h. All data were obtained using quantitative real-time PCR analysis. mRNA accumulation was normalized by TBP mRNA abundance for each sample and is depicted graphically as the mean ± SEM of these values. Results represent mRNA accumulation observed in triplicate theca cell cultures from four normal and four PCOS patients. Statistically different mRNA abundance in the presence of retinoid or retinol treatment are indicated for control (a, P < 0.05) or forskolin-stimulated (b, P < 0.05) conditions.

 
CYP11A1 mRNA accumulation was also increased in PCOS theca cells, compared with normal cells, approximately 4-fold under control conditions and 3-fold under forskolin-stimulated conditions (Fig. 4BGo). In normal theca, CYP11A1 mRNA accumulation was significantly elevated in response to atRA or 9-cis RA, under both control and forskolin-stimulated conditions. In PCOS theca, treatment with atRA or 9-cis RA did not significantly alter CYP11A1 mRNA abundance. Retinol treatment had no effect on CYP11A1 mRNA in normal or PCOS cells.

In agreement with our previous reports (28), StAR mRNA abundance was similar in normal and PCOS cells under both control and forskolin-stimulated conditions (Fig. 4CGo). In normal theca, both control and forskolin stimulated StAR mRNA accumulation were increased by atRA and 9-cis RA. In PCOS theca, both control and forskolin-stimulated StAR mRNA accumulation were increased in response to atRA, whereas 9-cis RA increased StAR mRNA only in the presence of forskolin. Retinol treatment had no effect on StAR mRNA accumulation in normal or PCOS cells.

These data indicate that the relative abundance of CYP17, CYP11A1, and StAR mRNAs are increased in response to retinoids in theca cells. Furthermore, retinol treatment increases CYP17 mRNA in PCOS theca but not normal theca cells. Together these data further suggest that altered retinol metabolism and retinoid action in PCOS theca cells may contribute to augmented steroidogenic enzyme gene expression.

The differential effects of atRA, 9-cis RA, and retinol on CYP17, CYP11A1, and StAR promoter function in normal and PCOS theca cells

To investigate whether retinoid treatment affected CYP17, CYP11A1, and StAR promoter activities, fourth-passage theca cells were transiently transfected with reporter constructs containing 5'-flanking sequences of the human CYP17, CYP11A1, or STAR gene previously shown to confer basal and forskolin-stimulated promoter activity in human theca cells (37, 38, 40). The effects of retinol or retinoids (5 µM) on basal and forskolin-stimulated promoter function were examined in theca cells isolated from normal or PCOS patients. CYP17, CYP11A1, and StAR promoter function were increased in response to forskolin treatment (Fig. 5Go).



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  		FIG. 5. Effect of retinoids on basal and forskolin-stimulated CYP17, CYP11A1, and StAR promoter activity. To compare the effect of retinoid treatment on promoter function in normal and PCOS theca cells, fourth-passage normal or PCOS theca cells were transiently transfected with luciferase reporter constructs containing regions of the 5'-flanking sequence of the CYP17, CYP11A1, or StAR gene. After transfection, cells were treated with vehicles (-), atRA, 9-cis RA, or retinol (5 µM), with and without 20 µM forskolin. Seventy-two hours thereafter the cells were harvested, and luciferase (LUC) activity was assayed. Data are presented as relative LUC activity that has been corrected for ß-galactosidase activity. Data represent the mean ± SEM of experiments performed with triplicate cultures of theca cells isolated from four normal and four PCOS patients. Significantly different promoter functions in the presence of retinoid (untreated vs. untreated + retinoid; forskolin vs. forskolin + retinoid) are indicated for control (a, P < 0.05) or forskolin-stimulated (b, P < 0.05) conditions. Promoter function found to be statistically different in PCOS theca, compared with normal theca for each treatment condition, is indicated (*). Treatment with forskolin significantly increased promoter function under all conditions examined, with the exception of –750 CYP17 promoter in normal theca cells in the presence of 9-cis RA.

 
As previously reported, CYP17 promoter function was increased in PCOS theca cells, compared with normal theca cells (Fig. 5AGo), under control and forskolin-stimulated conditions, approximately 2-fold and 1.5-fold, respectively. Under control conditions, CYP17 promoter function was stimulated by atRA and 9-cis RA in normal theca cells, and by atRA, 9-cis RA, and retinol in PCOS theca cells. In the presence of forskolin, only atRA increased CYP17 promoter in normal cells, whereas atRA, retinol, and 9-cis RA stimulated CYP17 promoter function in PCOS theca cells. In the presence of retinol or retinoids, CYP17 promoter function was increased in PCOS cells, compared with normal cells.

CYP11A1 promoter function was also increased in PCOS theca cells under both control and forskolin-stimulated conditions. Compared with normal theca, CYP11A1 promoter function in PCOS was increased 3-fold under control conditions and 2.5-fold in the presence of forskolin (Fig. 5BGo). In normal theca cells, treatment with atRA stimulated both control and forskolin-induced CYP11A1 promoter function, whereas treatment with 9-cis RA increased only CYP11A1 promoter function in the presence of forskolin. No effect on CYP11A1 promoter function was observed in response to retinol or retinoids in PCOS theca cells (Fig. 5BGo). In the presence of retinol and retinoids, CYP11A1 promoter function was increased in PCOS theca cells, compared with normal theca cells.

In agreement with our previously published data (38), StAR promoter function in normal and PCOS theca cells was not significantly different under basal or forskolin-stimulated conditions (Fig. 5CGo). In normal cells, treatment with atRA and 9-cis RA stimulated both control and forskolin-stimulated StAR promoter function. In PCOS cells, treatment with atRA stimulated StAR promoter function in both the absence and presence of forskolin, and 9-cis RA-stimulated STAR promoter function only under control conditions. In both normal and PCOS cells, treatment with retinol had no effect on STAR promoter function.

These data suggest that retinoid treatment augments CYP17, CYP11A1, and StAR transcription in human theca cells. Our analyses also indicate that the CYP17 promoter is the most responsive to retinoid treatment and that PCOS theca cells have the capacity to convert retinol to more active retinoids, which in turn induce CYP17 promoter activity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we have demonstrated that androgen biosynthesis as well as CYP17, CYP11A1, and StAR gene expression is differentially regulated by retinol and retinoids in normal and PCOS theca cells. The data presented here show that retinoids potentiate androgen production and steroidogenic enzyme gene expression in both normal and PCOS theca cells. The increase in the magnitude of retinol- and retinoid-stimulated DHEA and T biosynthesis was observed to be more robust in PCOS cells. In addition, the unique ability of PCOS theca cells, but not normal theca cells, to respond to the retinoic acid precursor retinol with an increase in CYP17 mRNA and promoter function further demonstrates that dysregulation of retinoic acid metabolism and/or action may contribute to augmented androgen biosynthesis in PCOS (31). However, treatment of normal theca cells with atRA or 9-cis RA alone could not recapitulate a full PCOS phenotype at the level of androgen biosynthesis or steroidogenic enzyme gene expression, indicating that additional pathways important for controlling androgen biosynthesis and steroidogenic enzyme expression are likely to be dysregulated in PCOS.

Retinoid-dependent induction of CYP17, CYP11A1, and StAR mRNA abundance and promoter function suggests that retinoids alter steroidogenic enzyme expression at the transcriptional level. Our data suggest that the primary locus of action of retinol, and presumably the endogenous retinoids generated from retinol, is at the level of CYP17. Overall, CYP17 mRNA abundance and promoter function was the most sensitive to retinoids, followed by StAR and CYP11A1, which was induced only by retinoids in normal cells but not PCOS cells. Retinoic acids have been reported to stimulate StAR gene expression and promoter function in mouse Leydig cells; however, the hormone response elements involved in RA-dependent regulation have not been delineated (43). This is the first report of activation of the CYP17 and CYP11A1 promoters by retinoids. Because several putative consensus retinoic acid response elements are found within the promoters of the STAR, CYP11A1, and CYP17 genes (44), regulation by atRA or 9-cis RA may result from direct activation of the promoter by RAR and RXR receptors. We have found no information pertaining to RAR and RXR expression in human theca cells in the literature. Nonquantitative PCR analysis of mRNA from normal and PCOS theca cells indicate that RAR and RXR receptors are expressed in both cell types (data not shown). Whether there is differential expression of RXRs or RARs at the mRNA or protein level in normal and PCOS theca cells is unknown. It is possible that retinoids regulate the expression of other transcription factors that differentially regulate CYP17, CYP11A1, and StAR promoters. In addition, the extent to which RARs and RXRs interact with these transcription factors is also unknown. Future identification of the promoter region(s) and transcription factors required for retinoid-dependent regulation are necessary to determine the mechanism(s) by which retinoids activate the CYP17, CYP11A1, and StAR promoters.

Our previous studies have demonstrated that augmented androgen biosynthesis in PCOS is associated with increased CYP17 mRNA abundance and CYP17 gene transcription (40). The studies presented here demonstrate that normal and PCOS theca also exhibit differential CYP17 gene expression in response to retinol and retinoids. In PCOS theca, retinol and retinoids had a pronounced effect on the magnitude of CYP17 mRNA and promoter function in PCOS theca cells. In normal theca, retinol had no effect on CYP17 gene expression, and 9-cis RA stimulated only basal CYP17 gene expression (Figs. 4AGo and 5AGo). The mechanism by which 9-cis RA enhances forskolin-stimulated CYP17 promoter function in PCOS theca cells but not normal cells is unclear but may involve differential expression and/or regulation of RXRs and RARs. Our studies also indicate a trend for the effects of retinoids on androgen biosynthesis and gene expression to be blunted in the presence of forskolin, perhaps as a consequence of retinoid- and forskolin-dependent induction using overlapping mechanisms to increase CYP17 mRNA. It is possible that the inability of 9-cis RA, and the reduced ability of atRA, to further augment forskolin-stimulated CYP17 promoter function results from use of signaling and/or transcription factors that are tightly regulated in theca cells. The convergence of retinoid and cAMP-dependent regulation is likely because retinoids are known to induce MAPK phosphatase (43, 45), which is also required for induction of CYP17 transcription by cAMP-dependent pathways through regulation of the phosphorylation state of steroidogenic factor-1 (46).

Our studies in this report have further examined the regulation of STAR and CYP11A1 gene expression in theca cells and are the first examination of the transcriptional regulation of the CYP11A1 promoter in normal and PCOS theca cells. The observations of increased CYP11A1 promoter function and CYP11A1 mRNA abundance in PCOS theca cells, demonstrate that CYP11A1 gene expression is increased in PCOS, resulting in part from increased transcription of the CYP11A1 gene. The observation that CYP11A1 mRNA abundance and promoter function are stimulated by retinoids in normal theca cells, but not PCOS theca cells, further supports differential transcriptional regulation of CYP11A1 gene expression in normal and PCOS cells. This lack of a response in PCOS cells to retinoids or retinol at the level of CYP11A1 gene expression may result from increased synthesis of endogenous retinoids by PCOS cells, which maximally induce CYP11A1 gene expression. However, this differential regulation by retinoids in normal and PCOS appears to be specific to CYP11A1 gene expression because both STAR and CYP17 gene expression were regulated by retinoids in both cells types.

These studies demonstrated that retinoids have a major impact on theca cell androgen production and steroidogenic enzyme gene expression. This effect was observed with atRA and 9-cis RA as well as retinol, suggesting that theca cells are capable of converting retinoid precursors to biologically active retinoids. The differential response of CYP17, CYP11A1, and STAR gene expression to retinoids in normal and PCOS theca cells further suggest that PCOS cells may have intrinsic differences in their ability to respond to retinoids as well as synthesize retinoids. Therefore, it is possible that stimulation of androgen production by retinoids may contribute to ovarian hyperandrogenism in PCOS. In general, the pathways involved in cell-specific retinol and retinoid metabolism are not well known, particularly within the ovary. Further studies are necessary to determine the pattern of expression of enzymes involved in retinol metabolism/retinoid synthesis in ovarian cells and their functional significance in retinoid action in PCOS.


   Footnotes
 
This work was supported by National Institutes of Health Grants HD33852 (to J.M.M.) and HD34449 (to J.F.S., J.M.M., R.S.L.).

First Published Online May 24, 2005

Abbreviations: atRA, All-trans retinoic acid; 9-cis-RA, 9-cis-retinoic acid; CYP11A1, gene encoding cytochrome P450 cholesterol side chain cleavage; CYP17, cytochrome P450 17{alpha}-hydroxylase gene; DHEA, dehydroepiandrosterone; FBS, fetal bovine serum; DME, Dulbecco’s Eagle’s Medium; 17OHP4, 17{alpha}-hydroxyprogesterone; P4, progesterone; PCOS, polycystic ovary syndrome; RAR, retinoic acid receptor; RXR, retinoid X receptor; STAR, gene encoding steroidogenic acute regulatory protein gene; T, testosterone; TBP, TATA box binding protein.

Received February 15, 2005.

Accepted May 16, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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