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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-1394
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 12 4802-4809
Copyright © 2007 by The Endocrine Society

The Mechanism for Protein Kinase C Inhibition of Androgen Production and 17{alpha}-Hydroxylase Expression in a Theca Cell Tumor Model

Victor E. Beshay, Jon C. Havelock, Rosa Sirianni, Ping Ye, Takashi Suzuki, William E. Rainey and Bruce R. Carr

Department of Obstetrics and Gynecology (V.E.B., J.C.H., B.R.C.), University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9032; Department of Pharmaco-Biology (R.S.), University of Calabria, 87036 Arcavacata di Rende (CS), Italy; Department of Physiology (P.Y., W.E.R.), Medical College of Georgia, Augusta, Georgia 30912; and Department of Pathology (T.S.), Tohoku University School of Medicine, Sendai 980-8575, Japan

Address all correspondence and requests for reprints to: Bruce R. Carr, Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9032. E-mail: Bruce.Carr{at}UTSouthwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Introduction: In polycystic ovary syndrome (PCOS), there is increased formation of androgens by thecal cells. Moreover, PCOS ovaries have been shown to have decreased levels of c-fos transcription factor. We hypothesize that c-fos expression inhibits 17{alpha}-hydroxylase 17,20 lyase (CYP17) activity in the human ovary, and its decreased expression seen in PCOS may lead to elevated CYP17 transcription, resulting in increased androgen production.

Objective: Our objective was to define the role of the activator protein-1 transcription factors, namely c-fos, in the regulation of CYP17 expression in theca cells.

Methods: Human ovarian thecal-like tumor cells were used for all experiments. The following techniques were used: steroid quantification, mRNA extraction, microarray analysis, transfection, small interfering RNA, and immunohistochemistry.

Results: Stimulation of human ovarian thecal-like tumor cells with the protein kinase A pathway activator forskolin resulted in stimulation of C19 androgen production. In contrast, treatment with the protein kinase C pathway activator tetradecanoylphorbol acetate (TPA) resulted in decreased androgen production with a shift toward C21 progesterone production. TPA also led to complete inhibition of CYP17. Microarray data showed a 37-fold increase in c-fos after treatment with TPA. Transfection with steroidogenic factor 1 resulted in an increase in CYP17 promoter activity, which was significantly inhibited in the presence of c-fos. c-fos gene silencing led to an increase in CYP17 mRNA levels. Immunohistochemical staining for c-fos in ovaries demonstrated strong staining in granulosa cells, but not theca.

Conclusions: The activator protein-1 transcription factor c-fos plays a role in the inhibition of CYP17 expression. The decreased levels of c-fos expression in polycystic ovaries may be responsible for increased CYP17 levels in PCOS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE OVARIAN FOLLICLE is characterized by the production of specific steroids based on a distinct pattern of expression of steroid metabolizing-enzymes (1). In particular, the theca produces androgens, mainly androstenedione, which is under the control of circulating LH. Theca cells express high levels of the three enzymes needed for androstenedione production: cholesterol side-chain cleavage, 3β-hydroxysteroid dehydrogenase, and 17{alpha}-hydroxylase 17,20 lyase (CYP17). Androgen from theca cells is used by the adjacent granulosa to make estradiol, which is under regulation by FSH during the follicular phase and LH during the luteal phase of the cycle. The granulosa is characterized by high expression of aromatase during the follicular phase and high 3β-hydroxysteroid dehydrogenase during the luteal phase. The granulosa does not express CYP17, thus leading to the need for two cell types (theca and granulosa) to produce estrogens.

Numerous studies have demonstrated that thecal cell expression of CYP17 is regulated at the transcriptional level through LH activation of the cAMP signaling pathway, leading to increased activity of protein kinase A (PKA). Recent studies have also demonstrated the role of several transcription factors of which the best characterized is steroidogenic factor 1 (SF1) (2, 3, 4). SF1 is an orphan nuclear receptor that increases CYP17 transcription by binding several cis-regulatory elements in its proximal promoter region. Considerable research has focused on the regulation of CYP17 because of its key role in normal androgen production as well as a proposed role in the pathological production of androgens, as is seen in hyperandrogenic conditions such as polycystic ovary syndrome (PCOS).

We and others have previously shown that activation of protein kinase C (PKC) decreases CYP17 mRNA, protein, and activity (5, 6, 7, 8). The PKC family represents a common pathway activated by many growth factors and hormones (9). A number of studies have demonstrated that there are several rapid response genes that increase expression after PKC activation. Perhaps the most studied has been c-fos, which belongs to the activator protein-1 (AP-1) family of transcription factors. PKC selectively increases members of this family, particularly members of the Fos family, which then form heterodimers with members of the Jun family before binding to specific cis-elements in target genes (10). Recent microarray analysis has revealed decreased levels of Jun, and, particularly, Fos transcription factors in PCOS ovaries when compared with normal ovaries (11).

The purpose of this investigation was to determine the role of c-fos in thecal cell steroidogenesis. In this study, we investigated the effect of c-fos on the expression of CYP17 in human ovarian thecal cells, using a human ovarian thecal-like tumor (HOTT) cell model (12, 13). We hypothesize that c-fos plays an inhibitory role on CYP17 transcription in the human ovary, and its decreased expression in PCOS may be involved in elevated CYP17 transcription and increased androgen production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

The descriptions of the HOTT cell culture model have been previously described in detail (12, 13, 14, 15, 16, 17, 18). For experiments, HOTT cells were cultured in DMEM/F12 (Invitrogen, Carlsbad, CA) medium, supplemented with 2% Ultroser G (Pall Biosepra, Cergy-Saint-Christophe, France), 1% ITS+ Premix (BD Biosciences, Bedford, MA), and antibiotics. Cell monolayers were subcultured and used for experiments 48 h after subculture. All experiments were done in triplicates.

Stimulation of steroid secretion and analysis of steroids

Before experiments, cells were maintained overnight in DMEM/F12 medium containing 1% ITS+ Premix and antibiotics (low-serum medium). Cells were untreated (basal conditions), or treated with forskolin (Sigma-Aldrich, St. Louis, MO), tetradecanoylphorbol acetate (TPA) (Sigma-Aldrich), or the combination of TPA and forskolin in fresh low-serum medium and incubated at 37 C for 48 h. Androstenedione and progesterone content of conditioned medium was determined using RIA kits (Diagnostic System Laboratories, Webster, TX). Results of steroid assays were normalized to the cellular protein content in each well and expressed as pmol/mg cell protein.

RNA extraction, cDNA synthesis, and real-time RT-PCR

Before experiments, cells were maintained overnight in low-serum medium. RNA was then isolated, and RT was performed using protocols previously described (19). PCR reactions were performed in the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA), using 0.1 µM of each primer, in a total volume of 30 µl reaction mixture following the manufacturer’s recommendations (Table 1Go). SYBR Green Universal PCR Master Mix (Applied Biosystems) for the dissociation protocol was used for c-fos, and TaqMan Master Mix (Applied Biosystems) was used for CYP17. Each sample was normalized on the basis of its 18S ribosomal RNA content. The 18S quantification was performed using a TaqMan Ribosomal RNA Reagent kit (Applied Biosystems) following manufacturer’s instructions. The relative mRNA expression levels were normalized to a calibrator that was chosen to be the basal, untreated sample. Final results were expressed as n-fold differences in gene expression relative to 18S rRNA and calibrator, calculated following the {Delta}{Delta}CT method, as follows:

Formula
where {Delta}CT values of the sample and calibrator were determined by subtracting the average CT value of the transcript under investigation from the average CT value of the 18S rRNA gene for each sample.


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  		TABLE 1. Oligonucleotide primer and probe sequences used for real-time RT-PCR

 
Microarray analysis

Total RNA from control and TPA treated cells was isolated. The purity and integrity of the RNA were assessed by the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), and its quantity was determined by the NanoDrop spectrophotometers (NanoDrop Technologies, Wilmington, DE). Isolated RNA was used on two genomic expression arrays. RNA was hybridized to an Affymetrix human HG-U133 + 2 oligonucleotide microarray set (Affymetrix, Inc., Santa Clara, CA) containing 54,675 probe sets representing approximately 40,500 independent human genes. The arrays were scanned at high resolution using an Affymetrix GeneChip Scanner 3000 (Codon Biosciences, Houston, TX). Results were analyzed using GeneSpring GX 7.3.1 software (Silicon Genetics, Redwood City, CA) to identify differences in expression of transcription factors between basal and TPA-treated HOTT cells.

Protein assay and Western blot analysis

Cells were lysed in 1x passive lysis buffer (Promega, Madison, WI) for whole cell protein. For nuclear extraction, the Active Motif Nuclear Extract kit (Active Motif, Carlsbad, CA) was used following manufacturer’s directions. The protein content of samples was then determined using the BCA assay kit (Pierce, Rockford, IL). The Western blot protocol used was previously described (19). c-fos (sc-52) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a 1:1000 dilution and CYP17 antibody (provided by Dr. Michael R. Waterman, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN) at 1:1000 dilution were used. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech, Piscataway, NJ), and immunoreactive bands were visualized with the enhanced chemiluminescence Western blotting detection system (Amersham Pharmacia Biotech).

Expression vectors and transfection assay

Expression vectors containing the full-length sequence for c-fos and c-jun were purchased from American Type Culture Collection (Manassas, VA). Coding sequences for the genes were excised from their original vectors and subcloned into pcDNA3.1 zeo(+) (Invitrogen) expression plasmid. Human SF-1 was provided by Dr. Meera Ramayya (Tulane University Hospital for Children, New Orleans, LA). Coding sequence for the SF-1 plasmid was excised from its vector and subcloned into the pcDNA3.1 zeo(+) expression plasmid. The preparation of the CYP17 promoter construct has been described previously (2). Transfections were performed for 6 h using the transfection reagent Fugene 6 (Roche, Indianapolis, IN). For cotransfection experiments, indicated amounts of expression plasmids were included in the transfection reaction, and the total amount of DNA was kept constant by addition of carrier DNA (empty expression vector). To normalize luciferase activity, cells were cotransfected with 50 ng β-galactosidase control vector (Promega). After transfection, cells were incubated with 2.0 ml low-serum medium for 18–24 h to allow for recovery and expression of foreign DNA. Cells were then lysed using passive lysis buffer (Promega), and assayed for enzyme activity using the luciferase (Promega) and β-galactosidase assay systems (Tropix Inc., Bedford, MA). It was noted that our cells have a transfection efficiency of 20%.

RNA interference

Fos SMARTpool small interfering RNA (siRNA) and scrambled (control) siRNA were purchased from Upstate Biotechnology (Charlottesville, VA). Twenty-four hours after plating cells, siRNAs were transfected to a final concentration of 100 nM using the RNAiFect Transfection Reagent (QIAGEN Inc., Valencia, CA) according to manufacturer’s instructions. Twenty-four hours after transfection, the medium was replaced with low-serum medium. Forskolin, TPA, or a combination of forskolin and TPA was added to the cells and treatment allowed for 24 h. Whole cell protein and RNA were then extracted from the cells.

Immunohistochemistry

Normal cycling human ovaries of premenopausal women whose age ranged from 26–49 yr were retrieved from the pathology files of Tohoku University Hospitals, Sendai, Japan. The study protocol was approved by the Ethics Committee. The ovarian tissues were fixed in 10% formalin and embedded in paraffin wax. The rabbit polyclonal antibody for c-fos (Ab-2) was purchased from Calbiochem (San Diego, CA), and the rabbit polyclonal antibody for CYP17 has been described in detail (20). A Histofine Kit (Nichirei, Tokyo, Japan), which uses the streptavidin-biotin amplification method, was used in this study. Antigen retrieval for c-fos immunostaining was performed by heating the slides in an autoclave at 120 C for 5 min in citric acid buffer [2 mM citric acid and 9 mM trisodium citrate dehydrate (pH 6.0)]. c-fos primary antibody was used at a dilution of 1:500, and CYP17 was diluted to 1:2000. The antigen-antibody complex was visualized with 3.3'-diaminobenzidine solution [1 mM 3.3'-diaminobenzidine, 50 mM Tris-HCl buffer (pH 7.6), and 0.006% H2O2], and counterstained with hematoxylin.

Data analysis and statistical methods

Pooled results from triplicate experiments were analyzed using one-way ANOVA with Student-Newman-Keuls multiple-comparison methods, using SigmaStat version 3.0 (SPSS Inc., Chicago, IL). Treatments were considered significantly different when the P value was less than 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PKC effects on thecal cell steroid production and CYP17 expression

To study HOTT cell steroid production, cells were treated for 48 h in the presence or absence of activators of the PKA (forskolin) or PKC (TPA) signaling pathways, followed by assay of medium content of androstenedione and progesterone. Treatment of HOTT cells with forskolin (10 µM) caused a 10-fold increase in androstenedione production above that seen in untreated cells (Fig. 1AGo). In contrast, treatment with the PKC agonist (TPA) inhibited basal and forskolin-stimulated androstenedione production by 57 and 96%, respectively. As opposed to its effects on androgen production, PKC activation significantly stimulated basal and forskolin-stimulated progesterone biosynthesis (Fig. 1BGo).


Figure 1
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  		FIG. 1. Steroid production in HOTT cells after activation of the PKA and PKC pathways. HOTT cells were incubated with basal (control media) forskolin (FSK) (10 µM), TPA (10 nM), or combined forskolin and TPA for 48 h. Androstenedione (A) and progesterone (B) content of the media was determined by RIA. *, P < 0.05 vs. forskolin. These data represent the mean ± SD of three independent experiments.

 
The relative production of androgen and progestin is modulated in part by the expression of the steroid metabolizing enzyme 17{alpha}-hydroxylase (CYP17). We examined CYP17 transcript and protein levels in HOTT cells treated with TPA alone or with forskolin (Fig. 2Go). The results suggest that the shift from androgen to progestin production likely resulted from TPA inhibition of HOTT cell CYP17 transcript level (Fig. 2AGo), which was paralleled by a decrease in CYP17 protein level (Fig. 2BGo). These results confirm previous reports using primary cultures of human thecal cells and HOTT cells, which support the role of the PKC pathway as a modulator of thecal cell androgen production (5, 6, 7, 8).


Figure 2
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  		FIG. 2. Effect of PKA and PKC agonists on CYP17. A, HOTT cells were treated with basal (control media) forskolin (FSK) (10 µM), TPA (10 nM), and combined forskolin and TPA for 24 h. Real-time RT-PCR was used to quantify CYP17 transcript levels. B, Whole cell protein (30 µg) was used for Western blot analysis. *, P < 0.05 vs. basal. These data represent the mean ± SD of three independent experiments.

 
Definition of TPA target genes in HOTT cells

The mechanisms through which PKC inhibits thecal cell androgen production and CYP17 expression are unknown. We hypothesized that PKC activation alters the expression of specific transcription factors that then inhibit CYP17 transcription. To define TPA responsive genes, the HOTT cells were treated with TPA for 1 h, RNA isolated, and used for microarray analysis (Fig. 3Go). Of the 881 transcription factors present on the Affymetrix human HG-U133 + 2 oligonucleotide microarray, 389 transcripts were determined to be present in HOTT cell RNA (with regard to signal intensity) in at least one of the two microarrays. The vast majority of transcription factors (98%) did not show a 30-fold difference between basal and TPA (Fig. 3Go). However, six transcription factors had an increased expression level by greater than 30-fold in TPA vs. basal: early growth response (EGR) 4, nuclear factor (erythroid-derived 2), EGR1, vav1 oncogene, single-minded homolog 2, and FOS. Our attention was drawn toward the AP-1 transcription factor c-fos. In a previous report that compared gene expression between normal and PCOS ovaries, c-fos was noted to be lower in the PCOS samples (11). Furthermore, c-fos has played a modulatory role on the expression of several steroid-metabolizing enzymes (21, 22, 23). Confirmation of array results was accomplished using RNA from three independent HOTT cell experiments in which cells were treated for 1 h with forskolin, TPA, or the combination (Fig. 4AGo). The results demonstrate a small but not significant increase of c-fos mRNA (Fig. 4AGo) and protein expression (Fig. 4BGo) with forskolin treatment, but a 37-fold increase in c-fos expression after TPA treatment (P < 0.001).


Figure 3
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  		FIG. 3. Microarray analysis of PKC stimulated HOTT cells. HOTT cells were treated with basal (control media) TPA (10 nM) for 1 h. mRNA was extracted and hybridized to an Affymetrix human HG-U133 + 2 oligonucleotide microarray set containing 54,675 probe sets representing approximately 40,500 independent human genes. Six transcription factors had an increased expression level by greater than 30-fold in TPA vs. basal. NFE2, nuclear factor (erythroid-derived 2); SIM2, single-minded homolog 2; VAV1, vav1 oncogene.

 

Figure 4
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  		FIG. 4. Effect of PKA and PKC pathway agonists on c-fos. A, HOTT cells were incubated for 1 h with basal (control media) forskolin (FSK) (10 µM), TPA (10 nM), or combined forskolin and TPA. Real-time RT-PCR was used to quantify c-fos. B, Nuclear extracts (15 µg) were used for c-fos Western analysis. *, P < 0.05 vs. basal. These data represent the mean ± SD of three independent experiments.

 
Effect of c-fos and c-jun on CYP17

To determine the effects of c-fos on CYP17 expression, we used promoter constructs prepared from the 5'-flanking regulatory region of the human CYP17 gene. SF1 is a known activator of CYP17 transcription, and c-fos has previously impacted SF1 regulated gene expression (2). We previously have shown this construct to be highly responsive to cotransfection with SF1 containing expression vectors (2). SF1 cotransfection caused a 32-fold increase in luciferase reporter activity (Table 2Go). First, we cotransfected c-fos and SF1 with the CYP17 promoter construct. The result was a 40% inhibition in CYP17 promoter activity. Previous studies have shown that c-fos does not bind DNA as a monomer or homodimer but must form a heterodimer complex with members of the Jun family (24, 25). Therefore, we cotransfected c-fos and c-jun with SF1 and the CYP17 promoter construct. The combination of c-fos with c-jun led to a 60% inhibition in CYP17 promoter reporter activity (Table 2Go).


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  		TABLE 2. Inhibition of SF1 activated transcription of CYP17 by c-fos and c-jun

 
Effect of c-fos gene silencing on CYP17

To determine if PKC-mediated inhibition of CYP17 relies on c-fos, we silenced c-fos expression in HOTT cells using siRNA. Cells were transfected with RNAifect and either c-fos SMARTpool siRNA or, as a control, scrambled siRNA. After 24-h transfection, cells were treated with forskolin, TPA, or the combination of both for another 24 h, followed by RNA isolation and examination of CYP17 mRNA levels. Addition of c-fos siRNA led to an 11-fold increase in CYP17 transcript levels compared with cells transfected with the scrambled siRNA (P < 0.05) (Fig. 5AGo). This was also reflected by a decrease in c-fos at the protein level (Fig. 5BGo). The addition of forskolin to cells transfected with scrambled siRNA increased CYP17 transcript levels by 8-fold over untreated cells (P < 0.05). However, forskolin treatment of c-fos silenced HOTT cells increased CYP17 transcript levels by 28-fold (P < 0.05). As expected, TPA treatment increased c-fos protein levels (Fig. 5BGo) but blocked forskolin induced CYP17 expression by 70% (P < 0.05) (Fig. 5AGo). Furthermore, silencing c-fos expression partially reversed TPA induction of c-fos and inhibition of CYP17 expression. Together, these data support an inhibitory role of c-fos in the regulation of CYP17 expression.


Figure 5
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  		FIG. 5. Effect of c-fos gene silencing. HOTT cells were transfected with RNAifect and either c-fos SMARTpool siRNA or scrambled siRNA for 24 h. After 24-h transfection, cells were treated with basal (control media) forskolin (FSK) (10 µM), TPA (10 nM), or combined TPA and forskolin for 24. A, Real-time RT-PCR was used to quantify CYP17 transcript levels. B, Nuclear extract (15 µg) was used for c-fos Western analysis. *, {Phi}, {Delta}, P < 0.05. These data represent the mean ± SD of three independent experiments.

 
Immunohistochemical staining for c-fos and CYP17 in human ovaries

To localize c-fos in the human ovaries, immunohistochemical staining for c-fos and CYP17 in normal ovaries was performed. c-fos was low in the theca cell layer of follicles (Fig. 6AGo). Instead, c-fos localized to the granulosa cells. As expected, CYP17 staining was noted in the theca cell layer but not the granulosa cells (Fig. 6BGo).


Figure 6
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  		FIG. 6. Immunohistochemical staining for c-fos and CYP17 in normal ovarian follicles. Normal ovaries were stained for c-fos (A) and CYP17 (B). The granulosa cell (G) layer stained positive for c-fos, whereas the theca cell (T) layer stained positive for CYP17. Magnification, x100.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The human ovary consists of two unique steroidogenic cells, the granulosa and the theca. The granulosa secretes primarily estradiol, which is derived from androgens secreted by the theca. The level of thecal cell CYP17 expression plays a critical role in regulating the amount of thecal cell androgen production. However, the molecular mechanisms involved in thecal cell CYP17 expression are not fully defined. We have previously shown using a well-characterized model of human thecal cells (HOTT cell model) that PKA increases CYP17, whereas PKC signaling inhibits CYP17 expression in the ovarian thecal cells (6, 7, 8).

Elevated androgen production is also characteristic of the theca cells of ovaries of women with PCOS. A number of investigations have examined the role of CYP17 in PCOS. CYP17 promoter activity is greater in cells isolated from PCOS ovaries when compared with those from normal ovaries (26). Furthermore, single-base change in the 5' promoter region of CYP17 has been studied and shown to create an additional SP1-type (CCACC box) promoter site, which may cause increased CYP17 expression (27). Other investigators have shown that the calpain 10 gene alleles may play a role in PCOS susceptibility (28, 29).

For our experiments we use HOTT cells as a culture model for theca cells. These cells exhibit functional characteristics of human theca cells (13). Because HOTT cells also exhibit a predominance of 17 {alpha}-hydroxyprogesterone and C19 steroid secretion (14), they appear to behave similarly to isolated theca cells from both normal and polycystic ovaries (30). In addition, HOTT cells respond in a similar manner to primary thecal cells to protein kinase agonists (13). A limitation to this cell model is its lack of LH receptors. Possible reasons include a mutation in the LH receptor or an inactive G-protein coupling system. Despite that, they do respond to other protein kinase agonists. The lack of LH receptors is also a limitation of primary thecal cell cultures because they also lose their LH receptivity rather quickly in culture. This is in addition to the inability to readily obtain human thecal cells.

In the present investigation, we examine the role of AP-1 transcription factors, namely c-fos, in CYP17 transcription and androgen production in theca cells. Our data suggest that c-fos exerts an inhibitory effect on CYP17 transcription, and its decreased expression in PCOS may lead to the excessive CYP17 levels and excess androgen. The family of AP-1 transcription factors is composed of the Jun family members (Jun, JunB, and JunD) and the Fos family members (Fos, FosB, Fra1, and Fra2) (31). Each of the AP-1 transcription factors is differentially expressed and regulated, which means that every cell type has a complex mixture of AP-1 dimers with subtly different functions. Most AP-1 transcription factors are present at low levels in cells but are rapidly induced and activated in response to specific stimuli (32). AP-1 transcription factors regulate the expression of many genes in a variety of cell types. They also participate in cell proliferation, apoptosis, differentiation, and oncogenesis. External stimuli, such as growth factors, hormones, and activators of the cAMP second messenger pathway, have acutely induced Fos and Jun transcription (33, 34). The function of AP-1 proteins is dependent on the cell type in which these transcription factors are expressed. AP-1 transcription factors can either act directly by binding to DNA or indirectly by binding to other transcription factors via protein-protein interactions (35, 36).

AP-1 transcription factors have not been widely studied in the ovary. In the porcine ovary, there were differences in expression and DNA binding activity of the different AP-1 family members depending on the stage of the estrous cycle (37). The AP-1 transcription factors have also been proposed to play a role in the early follicular development in the mouse (38). Other work in the human ovary has concentrated on granulosa cells, in which Jun members were shown to modulate the expression of the aromatase gene (39). To our knowledge no work has been done on AP-1 transcription factors and theca cell function. In the present study, we use the HOTT cell model to investigate the effect of AP-1 transcription factors on CYP17 expression. CYP17 is activated mainly through LH, but because our HOTT cell model lacks functional LH receptors, forskolin was used to activate the PKA pathway, which is the signaling pathway stimulated by LH. The stimulatory effect of PKA in theca cell steroidogenesis is well documented. In the present study, when HOTT cells were incubated with the PKA activator, androgen production was increased significantly. We have previously shown an inhibitory effect on steroidogenesis by PKC (8). The mechanisms through which PKC inhibits CYP17 expression have not been defined. PKC has direct actions through it kinase activity, as well as indirect actions that rely on modifications of other signaling pathways and, in many cases, alterations in gene transcription. To examine the effects of the PKC pathway, we used TPA. PKC activation led to a decrease in androgen production and a shift toward progesterone production. This was due to the repressed transcript levels of CYP17, as we previously demonstrated in both adrenal and ovarian model systems (5, 7, 8).

To study the effect of the AP-1 transcription factors on CYP17 transcription, HOTT cells were transfected with the promoter region of the human CYP17 alone as a control and also with SF1. One of the most potent activators of CYP17 transcription is SF1 (2). SF1 acts by binding to the promoter region of the human CYP17 gene. Addition of SF1 resulted in an expected significant increase in CYP17 promoter activity. This increase was in turn significantly inhibited by c-fos and c-jun. To show the importance of c-fos over c-jun in the control of CYP17 transcription in theca cells, we transfected HOTT cells using either c-fos or a combination of both c-fos and c-jun. c-fos alone was able to cause a significant inhibition in CYP17 transcription. Keeping in mind that c-fos cannot bind DNA alone or as a homodimer, it likely heterodimerized to other transcription factors inherently present in the HOTT cells. However, there was no significant difference between c-fos inhibition alone or in combination with c-jun. Of interest, c-fos has been demonstrated to interact with SF1 and decrease SF1 mediated rat steroidogenic acute regulatory gene expression (21). This was of interest to us because c-fos expression was noted to be low in PCOS ovaries compared with normal ovaries (11). This suggests that suppressed expression of c-fos may regulate the increased androgen production in thecal cells.

To elucidate further the effect of c-fos on CYP17 transcription, we performed c-fos gene silencing experiments, examining c-fos and CYP17 transcript levels before and after silencing, and also examining protein levels of c-fos before and after silencing. The results were clear that once c-fos mRNA levels and protein levels were reduced, CYP17 transcript levels were significantly increased compared with basal conditions. These results further point to an interaction between c-fos and CYP17 transcription. Consistent with our hypothesis of CYP17 inhibition in the presence of c-fos is the intense c-fos staining in the neighboring granulosa cells. The finding of c-fos in these cells may partially explain the lack of granulosa CYP17 expression and, thus, lack of androgen production from granulosa cells. Experiments to define further the role that AP-1 transcription factors play in granulosa CYP17 repression are underway.

Our results suggest that AP-1 transcription factors play a role in CYP17 regulation. Their decreased levels in PCOS ovaries (11) suggest that this may be a mechanism for increased androgen production. Further investigations are needed to evaluate other AP-1 factors and the role they may play in theca androgen production. In addition, further studies need to be done to examine why these transcription factors are present in lower levels in PCOS theca cells. Finally, the role of AP-1 transcription factors in granulosa cell estrogen production remains to be elucidated.


   Footnotes
 
Disclosure Statement: The authors have nothing to declare.

First Published Online September 25, 2007

Abbreviations: AP-1, Activator protein-1; CYP17, 17{alpha}-hydroxylase 17,20 lyase; EGR, early growth response; HOTT, human ovarian thecal-like tumor; PCOS, polycystic ovary syndrome; PKA, protein kinase A; PKC, protein kinase C; SF1, steroidogenic factor 1; siRNA, small interfering RNA; TPA, tetradecanoylphorbol acetate.

Received June 22, 2007.

Accepted September 14, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Suzuki T, Sasano H, Tamura M, Aoki H, Fukaya T, Yajima A, Nagura H, Mason JI 1993 Temporal and spatial localization of steroidogenic enzymes in premenopausal human ovaries–in-situ hybridization and immunohistochemical study. Mol Cell Endocrinol 97:135–143[CrossRef][Medline]
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