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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-1860
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 3 1720-1727
Copyright © 2005 by The Endocrine Society

Dysregulation of Cytochrome P450 17{alpha}-Hydroxylase Messenger Ribonucleic Acid Stability in Theca Cells Isolated from Women with Polycystic Ovary Syndrome

Jessica K. Wickenheisser, Velen L. Nelson-DeGrave and Jan M. McAllister

Departments of Cellular and Molecular Physiology (J.K.W., V.L.N.-D., J.M.M.) and Obstetrics and Gynecology (J.M.M.), The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

Address all correspondence and requests for reprints to: Jan M. McAllister, Ph.D., Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, 500 University Drive H166, Hershey, Pennsylvania 17033. E-mail: jmcallister{at}psu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disorder characterized by ovarian hyperandrogenism. Theca interna cells isolated from the ovaries of women with PCOS are characterized by increased expression of cytochrome P450 17{alpha}-hydroxylase (CYP17) [steroid 17{alpha}-hydroxylase/17,20 lyase (P450c17)], a steroidogenic enzyme obligatory for the biosynthesis of androgens. Augmented expression of the gene encoding P450c17 (CYP17) in PCOS theca has been attributed, in part, to differential transcriptional regulation of the CYP17 promoter in normal and PCOS cells. The present studies examine whether CYP17 gene expression is also posttranscriptionally regulated at the level of mRNA stability in normal and PCOS theca cells maintained in long-term culture. Determination of endogenous CYP17 mRNA half-life by pharmacological inhibition of transcription demonstrated that the half-life of CYP17 mRNA increased 2-fold in PCOS theca cells, compared with normal theca cells. Forskolin treatment also prolonged CYP17 mRNA half-life in both normal and PCOS theca cells. In vitro mRNA degradation studies demonstrated that the 5'-untranslated region confers increased stability to CYP17 mRNA in PCOS theca cells and showed that the 5'-untranslated region of CYP17 also confers forskolin-stimulated stabilization of CYP17 mRNA. These studies indicate that a slower rate of CYP17 mRNA decay contributes to increased steady-state mRNA accumulation and augmented CYP17 gene expression in PCOS theca cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
POLYCYSTIC OVARY SYNDROME (PCOS) affects approximately 5–10% of women of reproductive age and is characterized by hyperandrogenism, chronic anovulation, and polycystic ovaries (1, 2, 3, 4, 5, 6). PCOS is associated with elevated levels of circulating testosterone, produced primarily in the ovary by theca interna cells (7, 8, 9, 10, 11). 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 (P450c17) with both 17{alpha}-hydroxylase and C17,20 lyase activities which are responsible for the conversion of pregnenolone to 17{alpha}-hydroxypregnenolone, and subsequently dehydroepiandrosterone. Theca cells isolated from women with PCOS and maintained in long-term culture exhibit a stable phenotype of persistently elevated androgen biosynthesis and augmented CYP17 gene expression (2). Familial clustering of PCOS and PCOS-associated phenotypes suggests that genetic factors are involved in the etiology of the disorder (1, 12).

The expression of steroidogenic enzymes, including CYP17, is primarily regulated by cAMP-dependent mechanisms in response to LH (13). Examination of the CYP17 promoter in normal and PCOS theca cells has demonstrated that increased CYP17 expression in PCOS results, in part, from increased transactivation of the promoter (14, 15), under both basal and cAMP-stimulated conditions. Increased steroid biosynthesis in PCOS theca cells is also associated with augmented gene expression of several steroidogenic enzymes, including cytochrome P450 cholesterol side-chain cleavage (CYP11A1) and 3ß-hydroxysteroid dehydrogenase type II (HSD3B2) (2, 16). In contrast, the abundance of steroidogenic acute regulatory protein (StAR) and 17ß-hydroxysteroid dehydrogenase type V (AKR1C3) mRNA, and the transcriptional activity of the StAR promoter, are not different between normal and PCOS theca cells (14, 16). Therefore, these experiments suggest that expression and activity of only a subset of proteins that are important for progestin and androgen biosynthesis are affected in PCOS theca cells. In addition, microarray analyses comparing normal and PCOS gene expression profiles have demonstrated more global changes in mRNA expression in PCOS theca cells beyond the steroidogenic enzymes (17, 18). The differential expression of several genes in PCOS theca cells could be explained by altered levels or activity of signal transduction proteins and/or transcription factors that regulate genes involved in steroidogenesis, as well as other cellular functions (19).

Numerous studies have examined the transcriptional regulation of steroidogenic gene expression by investigation of specific interactions within/at the promoters of steroidogenic genes. However, few studies have investigated the role of posttranscriptional regulation in steroidogenic tissues (20, 21, 22). Regulation of mRNA turnover is a major mechanism for controlling gene expression involving the interaction of cytoplasmic proteins (trans-acting factors) with regulatory regions (cis-acting sequences) in the mRNA (23). Sequences that control mRNA decay/stability can be localized in the 5'-untranslated region (UTR), coding regions, and/or the 3'-UTR. The association of trans-acting factors with mRNA elements form ribonucleoprotein complexes, which have been identified to stabilize or destabilize mRNA, as well as regulate translational efficiency (23).

Our previous studies have demonstrated that both basal and forskolin-stimulated CYP17 mRNA accumulation were increased in PCOS cells (2). We also reported that CYP17 gene expression in PCOS theca cells is altered at the transcriptional level, and that there is a 2- to 3-fold increase in CYP17 promoter function in PCOS (14, 15). Here, we examined the extent to which posttranscriptional regulation of CYP17 mRNA, at the level of mRNA stability, contributes to increased CYP17 gene expression in PCOS theca cells.


   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, dissected, and dispersed with 0.05% collagenase I, 0.05% collagenase IA, and 0.01% deoxyribonuclease in medium containing 10% fetal bovine serum (FBS), as previously described (24). 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. 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 DMEM 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, primary theca interna cells were grown in 12 35-mm dishes until confluent, removed from the dish with neutral protease (pronase-E; protease type XXIV; Sigma, St. Louis, MO) in DMEM-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% N2-5% 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 (25), 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 (1, 2). 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 cell cultures used in these studies have been described and functionally characterized previously (2, 14, 15, 26). For all studies, theca cell cultures obtained from at least five independent normal and five independent PCOS patients were examined. The use of fourth-passage cells allowed us to perform multiple experiments with well-characterized cells from the same patient population. Sera and growth factors were obtained from the following sources: FBS and DMEM/F12, Irvine Scientific (Irvine, CA); horse serum, Life Technologies (Grand Island, NY); UltroSer G, Reactifs IBF (Villeneuve-la-Garenne, France); and other compounds, Sigma.

Quantitation of CYP17 mRNA

For quantitative real-time PCR (QRT-PCR), total RNA was isolated (2) from fourth-passage theca cells that were grown to subconfluence, transferred into serum-free medium, and treated as indicated. For determination of mRNA half-life, theca cells maintained in serum-free media for 24 h (time zero) were treated with 75 µM of the transcription inhibitor 5,6-dichloro-benzimidazole 1-ß-D-ribofuranoside (DRB) for various time points ranging from 0–48 h. RNA (1 µg) samples were then reverse transcribed using oligo (dT) and 200 U Stratascript Reverse Transcriptase (Stratagene, Cedar Creek, TX). CYP17 abundance was determined by QRT-PCR as previously described (26), using a gene specific two-step PCR and carried out in triplicate for each cDNA sample as well as a series of serial dilutions in an Mx4000 Thermocycler (Stratagene) using the Mx4000 Multiplex Quantitative PCR system. 18S ribosomal and/or TATA-binding protein mRNA abundance was used for data normalization as noted. The rate of decay (k) was assessed by measuring the amount of CYP17 mRNA at each timepoint, over the amount at time zero, and determined by nonlinear regression (Prism 5.0; GraphPad Software, San Diego, CA). Half-life (t1/2) of transcripts/mRNA was calculated from the k based on the equation, t1/2 = (ln 2)/k.

Rapid amplification of cDNA ends (RACE) and construction of CYP17 UTR luciferase (LUC) constructs

Total RNA was isolated from theca cells maintained in serum-free media for 24 h, using TRI reagent (Sigma). Adrenocortical H295 total RNA was used as a control and was generously provided by Diane Thibotout (The Pennsylvania State University College of Medicine, Hershey, PA). Both the 5'- and 3'-UTR were cloned by FirstChoice RNA ligase mediated (RLM)-RACE kit (Ambion Inc., Austin, TX), following the manufacturer’s instructions and using 1 µg (3'-RACE) or 8 µg (5'-RACE) of total theca RNA. The following antisense primer was used in conjunction with the RLM-RACE outer primer for 5' RACE of CYP17, 5'-ACTGTAGTCTTGCTGCCCATACGA-3'. Nested PCR was then performed with the RLM-RACE inner primer and a CYP17 specific primer containing an XbaI site, 5'-GCTCTAGAAGTTGTTATGCATATG-3'. The following sense primer was used for 3' RACE of CYP17, 5'-GAAGGCATCCCCAAGGTGGTCTT-3' in conjunction with the 3' RLM-RACE outer primer. Nested PCR was then performed with the 3' RLM-RACE inner primer and a CYP17 specific primer containing an XbaI site, 5'-TGTCTAGAGGCTGTAACTCACAGC-3'. After PCR, the product was digested with XbaI and BamHI and subcloned into the pGL3 promoter vector for automated DNA sequencing. The 5'-UTR C17/LUC construct was generated by ligating annealed synthetic oligonucleotides corresponding to the 5'-UTR of CYP17 mRNA into NcoI/HindIII sites of the pGL3 control vector (Promega, Madison, WI) located upstream (5') of the LUC coding sequence. The 3'-UTR C17/LUC was generated by insertion of the RACE-PCR product directly into XbaI/BamHI sites of pGL3 control, downstream (3') of the LUC cDNA. All constructs were confirmed by automated DNA sequencing.

Preparation of cytoplasmic extracts

Human theca cells were transferred into serum-free medium containing DMEM/F12, 1.0 mg/ml BSA, 100 µg/ml transferrin, 20 nM insulin, 20 nM selenium, 1.0 µM vitamin E, and antibiotics, in the absence or presence of 20 µM forskolin. At 24 h, cells were harvested with trypsin/EDTA, and cytoplasmic extracts were prepared, as previously described (27), in buffer containing 0.1% Nonidet P-40, 20 mM HEPES (pH 7.9), 20 mM sodium chloride, 1 µM dithiothreitol, 0.5 µM phenylmethylsulfonylfluoride, and 0.2 mM EDTA. In addition, 2 µg/ml leupeptin, 1 µM benzamidine, 1 µM sodium orthovanadate, and 20 µM sodium fluoride were added to inhibit protein phosphatases and proteases. Protein concentrations of extracts were determined by BCA protein assay (Pierce, Rockford, IL).

In vitro degradation of CYP17 mRNA

In vitro mRNA decay reactions were performed as previously described (28), but with the following modifications. Various lengths of the CYP17 cDNA were generated by PCR amplification of a pBS-CYP17 plasmid containing the full-length (1.75 kb) CYP17 cDNA. The PCR products contained a T7 promoter site and were used to synthesize biotinylated CYP17 transcript by the MAXI script kit (Ambion) and 16-biotin-UTP (Roche Diagnostics, Indianapolis, IN). A ß-actin template, provided in the kit, was used to generate the biotinylated 304-bp ß-actin transcript. Biotinylated RNA transcript was purified by multiple ethanol precipitations. Reactions included 0.3 µg cytoplasmic extract and approximately 10 ng biotinylated transcript in RNA degradation buffer comprised of 10 mM Tris-HCl (pH 7.6), 5 mM magnesium acetate, 100 mM potassium acetate, 2 mM dithiothreitol, 10 mM creatine phosphate, 0.7 U creatine phosphokinase (Calbiochem, La Jolla, CA), 1 mM ATP, 0.4 mM GTP, 0.1 mM spermine, and 0.6 U recombinant RNAsin (Promega). Components were combined on ice, mixed, and incubated at 37 C. For comparison of decay rates for transcripts of different length, equamolar amounts of biotinylated RNA were included in the reactions. At each timepoint from 0–60 min, 10 µl of the reaction was removed and immediately precipitated in cold 70% ethanol containing 1 µg yeast tRNA as carrier. After resuspension, the reactions were separated on a 1.5% agarose/formaldehyde gel and transferred to nylon membrane. Biotinylated RNA was detected using the Bright Star BioDetect kit (Ambion) and quantitated using a GeneGnome bioimager and GeneSnap v6.03 and GeneTools v3.03 (Syngene Bioimaging, Cambridge, UK). The half-life was assessed as discussed above.

Transient transfection of normal and PCOS theca cells

Human theca cells, isolated from normal cycling women and women with PCOS, were transfected as previously described (14, 15, 29). One hour before transfection, the cells were transferred into DMEM high-glucose medium containing 20 mmol/liter HEPES and 2% heat-inactivated calf serum (Atlanta Biologicals, Atlanta, GA) and moved to a 3% CO2-95% ambient air, 37 C incubator. The calcium phosphate precipitate contained 20 µg/dish LUC plasmid, and 1 µg/dish of an expression vector for ß-galactosidase, pSVß-gal (Promega). After transfection, the cells were rinsed with 15% glycerol/Hank’s balanced salt solution and PBS, and treated in transfection media containing vehicle or 20 µM forskolin for 48–72 h. Cells were harvested with trypsin, pelleted, and resuspended in reporter lysis buffer (Promega). LUC activity was determined with the Luciferase Assay System (Promega) on a Sirius Luminometer (Zylux Corp., Oak Ridge, TN). ß-Galactosidase activity was measured by the chemiluminescent assay Galacto-Light Plus (Tropix, Bedford, MA) and used for normalization of transfection efficiency. Transfections were performed in triplicate in theca cells isolated from at least five independent normal and five independent PCOS patients.

Statistical analysis

Data are presented and described in the text as the mean ± SEM from mRNA decay analysis (t1/2) or transfections performed in triplicate in five independent normal and five independent PCOS theca cell cultures. The results from mRNA decay analysis (t1/2) and transfection analysis from individual patients were collected, and two-way ANOVA was performed using Prism 3.0c for Macintosh (GraphPad Software). P values were determined by the Sidak method for multiple comparisons when significant differences were indicated by two-way ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CYP17 mRNA abundance in normal and PCOS theca cells

Previous Northern analysis has demonstrated that the level of CYP17 mRNA is increased in PCOS theca cells maintained in long-term culture, compared with normal theca (2). To more accurately determine the abundance of CYP17 mRNA in PCOS and normal theca cells, QRT-PCR was performed on total RNA isolated from cells maintained in serum-free media for 24 h in the absence (untreated) or presence of 20 µM forskolin. As shown in Fig. 1Go, the relative amounts of CYP17 mRNA were increased in PCOS theca cells, compared with normal theca cells, by approximately 9-fold and 5-fold, under untreated and forskolin-stimulated conditions, respectively. In both cell types, CYP17 mRNA was increased in response to forskolin treatment, approximately 7-fold in normal theca cells and 4-fold in PCOS cells (Fig. 1Go). We have previously established that transcriptional regulation of the CYP17 gene is augmented in PCOS, resulting from a 2- to 3-fold increase in CYP17 promoter function (14, 15). However, the increase in transcriptional regulation does not fully account for the 9-fold increase in CYP17 mRNA levels, suggesting that posttranscriptional mechanisms may also contribute to differences in mRNA levels in normal and PCOS theca cells.



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  		FIG. 1. CYP17 mRNA accumulation in normal and PCOS theca cells. CYP17 mRNA abundance was evaluated using QRT-PCR analysis. mRNA accumulation was normalized by TATA-binding protein mRNA abundance and is depicted as the mean ± SEM. An interaction between treatment and cell type was indicated by two-way ANOVA (P < 0.02). Forskolin increased CYP17 mRNA abundance (*, normal, P < 0.0005; PCOS, P < 0.05), compared with untreated. CYP17 mRNA was increased in PCOS theca, under both basal (a, P < 0.05), and forskolin-stimulated conditions (b, P < 0.01).

 
CYP17 mRNA stability in human theca cells

To evaluate whether CYP17 mRNA stability was altered in PCOS theca cells, the decay of endogenous CYP17 mRNA over time was examined under conditions where transcription was pharmacologically blocked. To induce CYP17 mRNA, normal and PCOS theca cells were incubated in serum-free media for 24 h, either in the absence or presence of forskolin, before addition of the transcriptional inhibitor DRB. CYP17 mRNA abundance was measured by QRT-PCR analysis at various timepoints, and the fraction of transcript remaining (Fig. 2AGo) was used to calculate the half-life of the CYP17 mRNA as described in Materials and Methods. Half-life values for CYP17 mRNA were determined from five independent normal and five independent PCOS theca cell cultures (Fig. 2BGo). For normal theca cells, the decay of CYP17 mRNA occurred with a half-life of 11.3 ± 1.0 h under basal conditions, and 20.9 ± 2.0 h after forskolin treatment. For PCOS theca, CYP17 mRNA decay occurred at a slower rate, with a half-life of 20.8 ± 2.5 h under basal conditions, and 30.9 ± 1.3 h after forskolin treatment. The half-life of CYP17 mRNA was extended by approximately 9 h (70%) in PCOS theca cells. In response to forskolin, CYP17 mRNA half-life was prolonged by approximately 10 h in both normal and PCOS cells. In contrast, StAR mRNA half-life, determined from identical samples, indicated that StAR mRNA stabilization was similar (~7.5 h) in normal and PCOS theca and was not significantly affected by forskolin treatment (data not shown).



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  		FIG. 2. Endogenous CYP17 mRNA half-life in normal and PCOS theca cells. The stability of endogenous CYP17 mRNA in normal and PCOS theca cells under untreated and forskolin-stimulated conditions after treatment with the transcriptional inhibitor DRB. A, Graphical representation of the percent of CYP17 mRNA remaining at each time interval, as determined by QRT-PCR. B, The half-life of endogenous CYP17 mRNA presented as the mean ± SEM from independent examinations in five normal and five PCOS theca cell cultures. CYP17 mRNA half-life was increased in PCOS theca, under both untreated (a, P < 0.01) and forskolin-stimulated conditions (b, P < 0.01). Forskolin increased CYP17 mRNA stability (*, P < 0.05), compared with untreated in both cell types.

 
Identification of the 5'- and 3'-UTRs of CYP17 mRNA in human theca cells

Previous reports have identified the transcriptional start site of the CYP17 gene in human adrenal cells (30, 31). Because CYP17 transcriptional regulation is tissue-specific (15, 32), we examined the sequence of the 5'- and 3'-UTRs of CYP17 mRNA in human theca cells by RACE as described in Materials and Methods. By using primers specific for CYP17, we amplified the 5' and 3' ends of capped full-length CYP17 mRNA from normal and PCOS theca cells, as well as adrenocortical H295 cells. The results of these studies demonstrated that the 5'-UTR of CYP17 mRNA in theca and adrenal cells was three nucleotides shorter than previously described, starting at a cytosine residue previously assigned to +4 (30, 31). The 3'-UTR of CYP17 mRNA in theca was identical with previous reports (31).

In vitro CYP17 mRNA decay and examination of 5'- and 3'-UTRs

To further examine CYP17 mRNA stability in normal and PCOS theca cells, in vitro degradation assays were performed. UTP-biotin-labeled CYP17 RNA transcripts corresponding to the full-length CYP17 mRNA were synthesized and incubated with cytoplasmic extracts isolated from either normal or PCOS cells maintained under untreated or forskolin-treated conditions. The fraction of transcript remaining at various timepoints was determined and used to calculate the half-life of each transcript as described in Materials and Methods (Fig. 3Go, A and B).



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  		FIG. 3. In vitro CYP17 mRNA stability in normal and PCOS theca cells. The rate of decay of biotinylated CYP17 mRNA probe was examined by RNA in vitro degradation assays. The full-length 1.75-kb CYP17 mRNA probe was incubated with cytoplasmic extracts (CEs) isolated from normal or PCOS theca cells for various lengths of time (0–60 min). Cytoplasmic extracts were isolated from cells maintained in serum-free media for 24 h, either in the absence (untreated) or presence of forskolin. A, Representative autoradiogram of full-length CYP17 mRNA present in vitro reactions over time using CEs from normal or PCOS theca cells, either in the absence (untreated) or presence of forskolin. "no CE" indicates probe incubated in the absence of CEs. B, Graphical representation of the percent of CYP17 mRNA remaining at each time interval from in vitro reactions performed with CEs from one normal and one PCOS theca cell culture. C, The stability of each transcript (i.e. half-life) is presented as the mean ± SEM of five-independent degradation assays using cytoplasmic extracts from five normal or PCOS theca cell cultures. The stability of CYP17 mRNA was significantly increased in assays using PCOS extracts, compared with normal extracts, under untreated (a, P < 0.01) and forskolin-treated (b, P < 0.05) conditions. The stability of the full-length CYP17 mRNA was significantly increased (*, P < 0.01) in assays using extracts from forskolin-treated, compared with control, in both normal and PCOS theca cells.

 
Using extracts from untreated cells, the half-life of the full-length CYP17 transcript was increased more than 2-fold in PCOS extracts, compared with normal extracts (Fig. 3CGo). Extracts from forskolin-treated cells also resulted in a significantly longer CYP17 transcript half-life (1.3-fold) in PCOS, compared with normal, although this effect was less than that observed using untreated extracts. In both cell types, the half-life of CYP17 transcript was increased in assays using extracts from forskolin-treated cells, compared with untreated cells. This forskolin-dependent increase in half-life was approximately 3-fold using normal theca cell extracts and less than 2-fold in PCOS extracts (Fig. 3CGo). The in vitro half-lives of CYP17 mRNA were observed to be substantially shorter than the endogenous half-lives (Fig. 2Go). However, the in vitro system indicates rank orders of decay (33), which reflect the relative differences in endogenous CYP17 mRNA decay observed between normal and PCOS theca cells, as well as in response to forskolin. In addition, the half-life of a ß-actin transcript was similar using cytoplasmic extracts from normal and PCOS theca cells (data not shown), suggesting that the differential in vitro stability in PCOS was specific for CYP17 mRNA.

To determine the region(s) of the CYP17 mRNA involved in differential regulation of mRNA stability, in vitro degradation assays were performed using labeled CYP17 RNA transcripts corresponding to the full-length CYP17, coding, or 5'-UTR or 3'-UTR and coding region (Fig. 4Go). Removal of the 3'-UTR resulted in a marked stabilization (3-fold) of the transcripts in both normal and PCOS cells but did not affect the increased stability observed in PCOS. Deletion of the 5'-UTR reduced transcript stability in PCOS cells by 2-fold and resulted in similar half-life in normal and PCOS extracts. The stability of the coding region alone was similar in normal and PCOS extracts. These data suggest that both the 5'- and 3'-UTRs contribute to CYP17 mRNA stability. Furthermore, the 5'-UTR is required for the differential CYP17 mRNA stability observed in PCOS theca cells.



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  		FIG. 4. Relative stability of regions of CYP17 mRNA in an in vitro degradation assay. The individual half-life of various CYP17 RNA probes was determined by RNA in vitro degradation assays as described under Materials and Methods. Briefly, CYP17 RNA probes were incubated with cytoplasmic extracts isolated from either untreated (–) or forskolin (20 µM)-treated (+) theca cells for various lengths of time. The stability of each transcript (i.e. half-life) is presented as the mean ± SEM of five independent degradation assays using cytoplasmic extracts from five normal or PCOS theca cell cultures. The stability of CYP17 RNA containing either the full-length, or the 5'+coding region, was significantly increased in assays using PCOS extracts, compared with normal extracts, under untreated (a, full-length, P < 0.01; 5 UTR, P < 0.05) and forskolin-treated (b, full-length, P < 0.01; 5 UTR, P < 0.01) conditions. The stability of the full-length (*, P < 0.05), or the 5'+coding region of CYP17 mRNA (*, P < 0.01), was significantly increased in the presence of extracts from forskolin-treated theca cells.

 
A similar pattern of regulation was observed under forskolin-stimulated conditions (Fig. 4Go). In the presence of forskolin, the half-lives of the full-length and 5'-UTR/coding CYP17 mRNAs were longer in PCOS theca cells, compared with normal. Furthermore, forskolin treatment prolonged the half-life of the full-length and 5'-UTR/coding CYP17 mRNAs in both cell types (Fig. 4Go) by 1.5- to 2-fold. These data demonstrate that the 5'-UTR of the CYP17 mRNA is necessary for forskolin-dependent stabilization of CYP17 mRNA in normal and PCOS theca cells.

Transient transfection analysis of the 5'- and 3'-UTRs of CYP17 mRNA

To examine the extent to which the 5'- and 3'-UTRs regulate reporter function, we transfected normal and PCOS theca cells with reporter constructs containing the 5'-UTR or 3'-UTR of CYP17 mRNA (Fig. 5Go) and examined LUC expression in both untreated and forskolin-treated cells. The empty reporter construct (Ctrl) resulted in similar LUC expression in both cell types and was not stimulated by forskolin. LUC expression by the 5'-UTR construct was significantly increased in PCOS cells, compared with normal theca cells, under both untreated and forskolin-stimulated conditions. Compared with the empty reporter construct, the 5'-UTR construct resulted in a 1.25-fold increase in LUC expression in normal theca and an increase of more than 2-fold in PCOS theca. In normal cells, the LUC expression from the 5'-UTR construct was increased approximately 1.5-fold in response to forskolin. This effect was not observed in PCOS theca cells. Compared with empty expression vector, the 3'-UTR construct exhibited a reduced LUC expression in both cell types and did not result in a differential LUC expression between the two cell types. In addition, forskolin treatment did not significantly affect the 3'-UTR construct. These data further suggest that regulation by the 5'-UTR contributes to increased CYP17 gene expression in PCOS theca cells and confers cAMP-dependent stabilization of the CYP17 mRNA.



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  		FIG. 5. Regulation of reporter gene expression by the 5'- and 3'-UTRs of CYP17 mRNA. Normal and PCOS theca cells were transfected with LUC constructs containing either the 5'-UTR or 3'-UTR of CYP17 mRNA and incubated in the absence (–) or presence (+) of forskolin (20 µM) for 48 h. Data are presented as relative LUC activity after normalization by ß-galactosidase and represent the mean ± SEM from transfections performed in triplicate in four independent normal and four independent PCOS theca cells cultures. Statistically different levels of LUC expression (P < 0.05), compared with empty control vector (*), are indicated. LUC expression of the 5'-UTR constructs was significantly higher in PCOS theca cells, compared with normal cells, in both untreated (a, P < 0.05) and forskolin (b, P < 0.05)-treated cells. LUC expression of the 5'-UTR construct was significantly increased in normal cells in response to forskolin. The expression by the 3'-UTR construct in untreated and forskolin-treated cells was not found to be statistically significant.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Many mRNAs are regulated by differential mRNA stability, and aberrant control of mRNA stability has been implicated in several disease states (23, 34). Specific examples include dysregulation of {alpha}-globin transcript stability in {alpha}-thalassemia (35), amyloid precursor protein transcript stability in Alzheimer’s disease (36), and cyclooxygenase 2 and TNF-{alpha} in cancer and inflammatory disease (23, 37). In this report, we present the first data demonstrating that CYP17 mRNA stability is prolonged from 11.3 h in normal theca cells to 20.8 h in PCOS theca cells. In vitro degradation studies also indicate that increased stabilization of CYP17 mRNA in PCOS, as well as cAMP-dependent regulation, is conferred by the 5'-UTR.

The regulation of steroidogenic enzyme expression in response to cAMP-dependent signaling has been primarily focused on alterations in transcription. Our data suggest that activation of cAMP-dependent signaling cascades also affects CYP17 mRNA stability. Results from in vitro degradation assays and transient transfection of reporter constructs demonstrate that the 5'-UTR of CYP17 mRNA is required for cAMP-dependent stabilization of CYP17 mRNA. Regulation of mRNA stability by cAMP has been observed for a variety of mRNAs, including GLUT5 (38), renin (39), soluble guanylyl cyclase (40), phosphodiesterase 4D (41), nitric oxide synthase (42), and lactate dehydrogenase A (43). It is possible that other trophic factors capable of altering CYP17 gene expression also exert their effects at the level of mRNA stability. Oxidative stress, growth factors, and hormones have been implicated in regulating mRNA stability (23).

The underlying mechanism(s) regulating CYP17 mRNA stability in human theca cells is currently unknown. It is likely that increased association of stabilizing factors with the 5'-UTR of CYP17 mRNA in PCOS, or in response to forskolin-induced increases in cAMP, results in augmented mRNA stability. We believe this to be the first report of forskolin/cAMP-dependent regulation of mRNA stability by a 5'-UTR. At present, the only reports of regulation of mRNA decay by cAMP have been shown to involve adenine uracil (AU)-rich elements in the 3'-UTR of mRNAs as well as forskolin-induced changes in the abundance of the AU-rich element-binding protein Hu-antigen R (HuR) (40, 44). Because the 5'-UTR of CYP17 mRNA does not contain a consensus binding site for HuR, cAMP-dependent stabilization conferred by the 5'-UTR may involve other factors. Examination of the 5'-UTR sequence revealed two consensus sites for polypyrimidine tract-binding protein (PTB). PTB has been implicated in stabilization of the 3'-UTR of insulin mRNA and the 5'-UTR of vascular endothelial growth factor mRNAs (45, 46). The PTB gene resides on 19p13.3, a region associated with PCOS and hyperandrogenism (47, 48). Further examination of the ribonucleoprotein complexes formed by the 5'-UTR of CYP17 mRNA is necessary to determine the mechanisms of cAMP-dependent regulation and increased CYP17 mRNA stability in PCOS. The 3'-UTR does appear to confer instability to the mRNA that is cis-dominant to regulation by the 5'-UTR (Fig. 4Go). Examination of the 3'-UTR of CYP17 mRNA revealed one AU-rich pentamer that could be a putative binding site for HuR, and several C-rich elements char-acteristic of binding sites for poly (C)-binding proteins. However, the 3'-UTR does not appear to be required for cAMP-dependent or differential regulation of CYP17 mRNA half-life in PCOS. The possibility remains for coordinate regulation by the 5'- and 3'-UTRs.

A single-nucleotide polymorphism (T->C) within the 5'-UTR of the CYP17 gene creates a new recognition site for the restriction enzyme MspAI, and these two alleles have been designated the A1 (T) or A2 (C) allele. This transition allele (A2) was initially linked to PCOS (49, 50); however, more recent studies have refuted this association (51, 52). In addition, an approximately equal distribution of the CYP17 A1 and A2 alleles has been reported among the general population (53, 54). With respect to CYP17 gene expression, Lin et al. (55) have reported that the presence of T or C at this position has no effect on transcriptional regulation of the CYP17 gene. The in vitro degradation studies presented here were performed with CYP17 transcript corresponding to the A1 allele and independently suggest differential stability of this variant in normal and PCOS cells. The possibility remains that the A2 variant may also affect CYP17 mRNA stability.

CYP17 stability is increased in PCOS theca under both untreated and forskolin-stimulated conditions, suggesting that cAMP-dependent regulation is not required for differential regulation in PCOS. However, our studies suggest that cAMP-dependent regulation of CYP17 gene expression is altered in PCOS. The fold-change in CYP17 mRNA abundance and CYP17 mRNA half-life in response to forskolin was less robust than observed in normal cells (Figs. 1Go and 2Go). We have also previously reported that forskolin-dependent induction of CYP17 promoter function was less in PCOS cells (15). Microarray analyses have also indicated differences in cAMP-dependent regulation of gene expression in normal and PCOS theca cells (17, 18). The underlying cause of altered cAMP-dependent regulation is unknown; however, the observation of changes at several levels of gene expression (i.e. transcription and mRNA stability) suggests that PCOS theca cells are likely to exhibit alterations in upstream signaling events.

Normal and PCOS theca cells display differential gene expression profiles (17, 18); however, the underlying cause of differential gene expression in PCOS theca cells is unknown. It is likely that alterations in mRNA stability, as well as transcription, are the foundation of differences in gene expression profiles recently reported in normal and PCOS theca cells. The impact of a 2-fold increase in mRNA half-life on CYP17 gene expression is dramatically increased when associated with the 3- to 4-fold increase in CYP17 promoter function we have previously observed in PCOS theca cells. Together, alterations in CYP17 mRNA stability and transcription result in a 9-fold increase in CYP17 mRNA abundance in PCOS (Fig. 1Go) compared with normal. Changes in half-life were specific for CYP17 mRNA, because StAR mRNA stability was not altered in PCOS theca cells compared with normal.

In this report, we present the first data demonstrating that CYP17 gene expression is dysregulated at the level of mRNA stability in PCOS theca cells. Additional studies are required to examine the contribution of transcription and/or mRNA stability to augmented gene expression of other steroidogenic enzymes, such as CYP11A1 and HSD3B2, in PCOS theca cells. Additional examination of altered CYP17 gene transcription and mRNA stability in PCOS theca cells will provide necessary insight regarding the potential mechanisms that may underlie androgen excess and other clinical manifestations in women with PCOS.


   Acknowledgments
 
We thank Dr. Gary Brewer (Department of Molecular Genetics and Microbiology, Robert Wood Johnson Medical School, University of Medicine and Dentistry, Piscataway, NJ) for his expertise in mRNA decay and helpful comments during preparation of the manuscript.


   Footnotes
 
This work was supported by National Institutes of Health Grants HD33852 and HD34449.

First Published Online December 14, 2004

Abbreviations: AU, Adenine uracil; CYP17, cytochrome P450 17{alpha}-hydroxylase gene; DRB, 5,6-dichloro-benzimidazole 1-ß-D-ribofuranoside; FBS, fetal bovine serum; HuR, Hu-antigen R; LUC, luciferase; PCOS, polycystic ovary syndrome; P450c17, steroid 17{alpha}-hydroxylase/17,20 lyase; PTB, polypyrimidine tract-binding protein; QRT-PCR, quantitative real-time PCR; RACE, rapid amplification of cDNA ends; RLM, RNA ligase mediated; StAR, steroidogenic acute regulatory protein gene; UTR, untranslated region.

Received September 20, 2004.

Accepted December 3, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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