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BOR - Papers in Press, published online ahead of print February 6, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.019646
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BIOLOGY OF REPRODUCTION 70, 1600–1605 (2004)
DOI: 10.1095/biolreprod.103.019646
© 2004 by the Society for the Study of Reproduction, Inc.

Ovary

Insulin Drives Transcriptional Activity of the CYP17 Gene in Primary Culturesof Swine Theca Cells1

Gongqiao Zhang, and Johannes D. Veldhuis2

Division of Endocrinology, Department of Internal Medicine, NIH Specialized Cooperative Center in Reproductive Research, University of Virginia School of Medicine, Charlottesville, Virginia 22908


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Insulin stimulates androgen biosynthesis and the accumulation of CYP17 mRNA and heterogeneous nuclear (hn) RNA in primary cultures of immature swine theca cells. To further assess insulinomimetic transcriptional control, we subcloned 1.007 kilobases (kb) of the 5'-upstream region of the CYP17 gene (–976 to +31 base pairs [bp] to the transcriptional start site) into a firefly-luciferase reporter construct. Insulin drove transcriptional activity of this probe in a time- and dose-dependent fashion, with maximal stimulation of 2.7- to 3.2-fold after insulin exposure (100 ng/ml) for 6 h. Progressive deletional constructs –839, –473, –174, and –75/+31 bp delineated expected reduction in responsiveness, except paradoxical gain of basal CYP17 promoter activity by the –473/+31-bp sequence. The latter suggests a possible intervening inhibitory sequence. Elimination of all sequences 5'-upstream to –174 bp markedly reduced basal transcriptional activity and abolished insulin action. Point mutation of a presumptive Sp1-like element located within –193/–180 bp inhibited basal and insulin-stimulated luciferase activity of the full-length promoter fragment by 40% and 67%, respectively. Disruption of a contiguous presumptive AP-2 site produced a comparable outcome. Combined mutation of the Sp1 and AP-2-like elements eliminated basal and insulin-potentiated CYP17 promoter activity. By Western analysis, insulin augmented cognate receptor phosphoprotein concentrations by 31-fold within 10 min. Chemical inhibitors of MEK-activated ERK1/2 attenuated insulin-enhanced CYP17 transcriptional activity by 76–80%. In summary, insulin drives transcriptional activity of a 5'-upstream regulatory sequence (–976 to +31 bp) of the swine CYP17 gene in primary cultures of theca cells, under a minimal requirement for combined activity of proximal (–193/180 bp) Sp1 and AP-2-like elements.

insulin, luteinizing hormone, steroid hormones, testosterone, theca cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activity of cytochrome P450 17{alpha}-hydroxylase/C17–20 lyase (CYP17) controls entry of progestin precursors into the androgen biosynthetic pathway [1, 2]. The CYP17 enzyme represents a single protein, which catalyzes two biochemical reactions, the 17 alpha-hydroxylation of progesterone or pregnenolone and the subsequent cleavage of the C17–20 bond to yield androstenedione or dehydroepiandrosterone. The latter androgens can be converted to testosterone and 5 alpha-dihydrotestosterone or undergo aromatization to estrogens. Clinical studies suggest that enhanced CYP17 enzyme activity contributes to anovulatory hyperandrogenism in the polycystic ovarian syndrome [3–6].

Insulinomimetic peptides stimulate tyrosine-kinase-dependent signaling and amplify gonadotropin-driven stimulated steroidogenesis in ovarian cells [7, 8]. Insulin also promotes androstenedione and testosterone biosynthesis and increases CYP17 mRNA and heterogeneous nuclear (hn) RNA accumulation in primary cultures of theca cells [9]. The present experiments use transient transfection assays of cloned 5'-upstream regulatory regions of the (porcine) CYP17 gene to test the hypothesis that insulin can transcriptionally regulate this gene.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents

Porcine insulin was obtained from Sigma (St. Louis, MO). Dulbecco modified Eagle medium (DMEM), penicillin/streptomycin, and lipofectamine reagent were obtained from Life Technologies, Inc. (Grand Island, NY). PD98059, U0124, and U0126 were purchased from Calbiochem Corporation (San Diego, CA), and the dual-luciferase reporter assay system was obtained from Promega Corporation (Madison, WI). Insulin receptor antisera was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Porcine Theca-Cell Culture

Ovaries were collected from prepubertal (60- to 70-kg) gilts at an abattoir and transported to the laboratory in iced saline. Theca cells were isolated as described previously [9, 10]. Combined theca-granulosa membranes were removed from immature follicles (diameter <5 mm) using forceps, and granulosa cells were detached by mechanical agitation in tissue culture medium for 3 min. Theca-basal lamina linings were allowed to settle at unit gravity, and the supernatant containing granulosa cells was discarded. Residual tissue was digested with collagenase (1 mg/ml) and DNase (10 µg/ml), and debris removed by filtration (185 µm; Millipore Corp., Billerica, MA). Theca cells were washed three times in F12/DMEM Media (1:1) by centrifugation for 5 min at 1000 rpm and purified further using Percoll density gradient. Cells were resuspended in F-12/DMEM (1: 1) media with 2.5% serum and plated in 96-well plates (Costar, Cambridge, MA) at a density of 1.5 x 105 cells/well and incubated in humidified 95% air, 5% CO2 at 37°C. After initial overnight culture in F-12/ DMEM to allow anchorage, theca cells were exposed to transfection solution (see below) for 4 h and then to insulin for 6 h. Luciferase activity was measured according to the manufacturer's instructions.

Construction of a CYP17 Promoter-Driven Firefly- Luciferase Reporter Plasmid

Based on a porcine CYP17 gene sequence (accession numbers Z11854, S40340) from GenBank, we designed forward (5'-AGATCTAGCTGCCAGCCTATGCCAGA) and reverse (5'-AGATCTAGGCAGGACAGACAGCTCTG) primers targeted to the putative CYP17 promoter region. This pair of primers was engineered with BglII restriction sites (AGATCT) at the 5'-end to facilitate subsequent subcloning of polymerase chain reaction (PCR) products into a firefly-luciferase reporter vector (pGL3 Basic). A PCR product containing 1007 base pairs (bp) of the putative CYP17 promoter region and 6 bp of flanking BglII restriction recognition sites was obtained as follows in a thermal cycler: 95°C for 12 min, 28 cycles at 95°C for 35 sec, 62°C for 30 sec, 67°C for 1 min and 30 sec, and 67°C for 10 min. PCR reaction mixtures of 100-µl final volume contained 2.5 mM MgCl2, 100 pmol each of forward and reverse primers, 0.2 mM of each dNTP, 10 µl of 10x PCR buffer, 1 µg of porcine genomic DNA isolated from theca cells as template, and 2.5 U of AmpliTaq Gold DNA polymerase. The putative CYP17 promoter segment was ligated into the BglII-restricted pGL3 Basic plasmid, according to the protocol provided by the manufacturer. The CYP17-containing pGL3 was sequenced to confirm insert orientation. The sequence was 100% concordant with the published porcine sequence in GenBank (above).

Construction of CYP17 Deletional Fragments

To localize the general region of hormone-sensitive DNA regulatory elements in the putative promoter fragment, four deletional constructs were made. The forward primers (CYP17, F870 bp; CYP17, F504 bp; CYP17, F205 bp; and CYP17, F106 bp) used to amplify four of the porcine CYP17 gene deletional fragments from the full-length (1007 bp) sequence were 5'-GAGTCTACCGGATCCTTAACCCACTG-3,', 5'-GAGTCTTCATTACCGTCTTGTGTGACC-3,', 5'-GAGTCTGCTTGCCTGAAGTTGAGCCA-3', and 5'-GAGTCTAAGGCAAGCGATAACATAAA-3,' respectively. Forward primers were engineered with a SacI restriction site (GAGTCT) at the 5'-end. Each forward primer was used in combination with the single reverse primer (5'-AGATCTAGGCAGGACAGACAGCTCTG-3') with a 5'-end BglII restriction site (AGATCT) to synthesize the 1007-bp full-length promoter fragment (above). The PCR reaction mixture and cycling mode were as noted above, except for including pGL3-CYP17-1007 bp as template instead of genomic DNA. The PCR products containing the deletional fragments were restricted by SacI and BglII and ligated into SacI/BglII cut pGL3 Basic, respectively, and sequenced.

Site-Directed Mutagenesis

To mutate a presumptive Sp1/AP-2 overlap region, 5'-CCTAGCCCCACCCC-3' (–193/–180, Sp1: 5'-CCTAGCCCC-3', and AP-2: 5'-CCCACCCC-3'), three pairs of primers were designed so as to contain one mutation site each (Fig. 1). The preparation of mutants was carried out by using QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) based on the manufacturer's procedure. The latter entailed denaturation of the pGL3-CYP17–1007 bp and annealing with the oligonucleotide primers containing the desired mutation. Using the non-strand- displacing action of Pfu Turbo DNA polymerase, mutagenic primers were extended and incorporated into resulting nicked circular strands. The methylated, nonmutated parental DNA template was digested with DpnI, and the circular, nicked ds DNA was transformed into XL1-Blue supercompetent cells to repair the nicks in the mutated plasmid. Mutated plasmids were partially sequenced to confirm mutation sites.



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  		FIG. 1. Oligonucleotide primers used to mutate the CYP17 promoter

Transfection of Porcine Theca Cells

Transfection conditions were optimized first by exploring a suitable matrix of factors, including preculture with different amounts of serum, nontoxic lipofectamine concentrations, varying plasmid/lipofectamine ratios, transfection duration, duration of insulin exposure, and cell densities. Consistent transfection was achieved after preculture of theca cells at a density of 1.5 x 105 cells/well overnight in 96-well plates in tissue culture medium with 2.5% serum. Transfection used a pGL3-CYP17 DNA to lipofectamine ratio of 0.5 µg:1.5 µl/well (0.2 ml) for 4 h at 37°C in the absence of serum. Following a medium change (F12/DMEM 1:1), cells were exposed to control solvent or insulin for 6 h. Transfections with 5'- deletional fragments were performed at an equimolar concentration to the pGL3-CYP17 full-length construct. A promoterless luciferase construct, pGL3/luc, which exhibited no significant activity in response to any treatment, was used to adjust total DNA to a constant value of 0.5 µg. To normalize for small variations in transfection efficiency across experiments, pRL-TK containing Renilla-driven luciferase activity was cotransfected with pGL3-CYP17. The amount of pRL-TK cotransfected with experimental pGL3-CYP17 plasmid was also optimized to 30 ng within the tested range of 10–100 ng DNA. CYP17 driven firefly or Renilla luciferase activities were measured by adding 100 µl luciferase substrate buffer and Stop & Glow solution (Promega Corporation protocol) in 40 µl of single- thaw cellular lysate in a Turner TD-20e luminometer (Turner Designs, Sunnyvale, CA).

Insulin Receptor Immunoprecipitation

Theca cells were lysed in 1 ml PBS containing 1.5 mM MgCl2, 1 mM EDTA, 1% Triton, and 10% glycerol in the presence of protease inhibitors (34 µg/ml phenylmethylsulfonylfluoride, 0.7 µg/ml pepstatin, and 5 µg/ ml leupeptin) and phosphatase inhibitors (100 mM sodium fluoride, 10 mM sodium pyrophosphate, and 2 mM sodium orthovanadate). Lysates were clarified by centrifugation at 15 000 x g for 10 min at 4°C and supernatants were assayed for protein using the Lowry method (Bio-Rad Laboratories, Hercules, CA). Five-hundred micrograms of sample were incubated overnight at 4°C with phosphotyrosine antibody conjugated to agarose (Santa Cruz Biotechnology), centrifuged at 15 000 x g for 10 min at 4°C, and pellets were washed three times in PBS. Agarose beads were heated for 5 min in hot (90°C) electrophoresis sample buffer and centrifuged three min at 15 000 x g. Supernatants were run on 10% polyacrylamide gels and transferred overnight to nitrocellulose. For immunoblotting, membranes were incubated 1 h in blocking buffer consisting of 10 mM Tris, 150 mM NaCl, 0.05% Tween-20, and 5% nonfat dry milk (pH 8). Membranes were exposed to primary antibody (rabbit polyclonal insulin receptor beta; Santa Cruz Biotechnology) diluted 1:200 in blocking buffer for 2 h at room temperature. Membranes were washed three times in blocking buffer and incubated in horseradish peroxidase-conjugated goat anti-rabbit IgG diluted 1:10 000 in blocking buffer for 1 h. After three washes in blocking buffer, protein bands were visualized using enhanced chemiluminescence (ECL) detection reagents according to the manufacturer's instructions (Amersham Life Sciences, Inc., Arlington Heights, IL) before exposing to x-ray film. Phosphorylated bands were quantified by laser densitometry using ImageQuant software and densitometer (Molecular Dynamics, Sunnyvale, CA).

Statistics

Data are presented in the figures as the mean ± SEM of three or more independent experiments using separate batches of ovaries to confirm reproducibility of results. Data were subjected to the Student two-tailed, two- sample t-test or one-way analysis of variance for three or more comparisons to determine significant interventional effects. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Initial studies were performed to characterize concentration-dependent effects of insulin on full-length (–976 to +31 bp) CYP17-driven firefly-luciferase reporter activity (Fig. 2). Maximal CYP17 transcriptional activity was attained with 100 ng/ml insulin (P < 0.01). Further studies were performed using this hormone concentration. The empty pGL3 plasmid was undetectable (within 1.84 SDs of cell lysate blank).



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  		FIG. 2. Concentration-dependent stimulation by insulin of putative CYP17 promoter transcriptional activity. Primary monolayer cultures of porcine theca cells (1.5 x 105 cells/0.2 ml medium/well) were transfected with a pGL3-CYP17–976/+31 promoter-luciferase reporter plasmid for 4 h and then stimulated with vehicle or insulin for 6 h before measurement of luciferase activity (Methods). Values are normalized for cotransfected pRL-TK Renilla luciferase activity. Data are the mean ± SEM of three different experiments, each conducted with triplicate incubations. The insulin effect was significant at P < 0.05 for >=100 ng/ml insulin. Asterisks indicate P < 0.05 compared with control (0 ng/ml)

Figure 3 depicts the time-course effect of insulin-stimulated luciferase activity on the full-length CYP17-driven reporter over the interval 2–12 h. Basal (control) and insulin-stimulated CYP17 transcriptional activity increased steadily during the initial 2–6 h of treatment, reaching maximal activity by 6 h. At this time point, insulin increased transcription approximately 3-fold compared with untreated (control) cultures. To facilitate the investigation of insulin action on CYP17 gene transcription, further studies were done at the 6-h time point.



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  		FIG. 3. Time course of insulin's stimulation of transcriptional activity of a pGL3-CYP17–976/+31 promoter-luciferase reporter construct. Theca cells were exposed to vehicle or insulin (100 ng/ml) for 0, 2, 4, 6, 8, 10, and 12 h. Values are normalized for cotransfected pRL-TK Renilla luciferase activity. Data are the mean ± SEM of three independent experiments, each performed in triplicate. Asterisks denote P < 0.05 compared with control at a given point

As shown in Figure 4, the –839 to +31 bp CYP17 promoter deletional fragment exhibited an 89% decrease in basal activity (P < 0.01), which rendered expression undetectable over empty plasmid. There was concomitant limited responsiveness to insulin compared with –839/+31 basal activity (albeit values become detectable over empty plasmid, P < 0.05). The –473-bp deletional fragment manifested readily detectable and significantly greater basal luciferase activity than the –839-bp sequence (P < 0.05) but failed to respond to insulin stimulation. The –174-bp and the –75-bp fragments showed lower basal activity and no responsiveness to insulin (P < 0.001 versus full-length promoter). The pGL3 plasmid lacking the CYP17 promoter had no detectable luciferase activity.



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  		FIG. 4. Effects of insulin on transcriptional activity of full-length or successive 5'-upstream deletional fragments of the (–976 bp to +31 bp) CYP17 promoter-luciferase reporter construct compared with empty pGL3 vector. The latter as well as the –839/+31 construct showed no detectable basal expression. Plasmid constructs were transfected into theca cells for 4 h, followed by a 6-h exposure to vehicle or insulin (100 ng/ml). Values are normalized for cotransfected pRL-TK Renilla luciferase activity. Data are the mean ± SEM of three separate experiments, each done in triplicate. Only the full-length promoter fragment retained significant fold responsiveness to insulin. The asterisks indicate P < 0.05 compared with the wild-type control

Based on inspection of the CYP17 promoter sequence between –174 to +31 bp, we considered a possible insulin- responsive cis-acting DNA region associated with putative Sp1 and/or AP-2 sites located within this fragment. Figure 5 highlights the finding that 2-fold mutation of the presumptive Sp1/AP-2 overlap region (Fig. 1) eliminated basal transcriptional activity as well as that stimulated by insulin. Mutations of Sp1 or AP-2 individually resulted in significant loss of basal transcriptional activity and that driven by insulin; namely, reductions of 40% and 67% (Sp1) and 46% and 77% (AP-2).



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  		FIG. 5. Effects of point mutation of putative Sp1 and/or AP-2-like binding sites on presumptive CYP17 promoter transcriptional activity. The wild- type CYP17 (full length, –976/+31 bp) promoter-luciferase reporter or each of three mutant plasmids were transfected into theca cells (Methods). Cultures were then stimulated with vehicle or insulin (100 ng/ml) for 6 h. Luciferase activity was normalized to cotransfected pRL-TK Renilla. Data are the mean ± SEM of three different experiments, each carried out in triplicate. None of the mutants maintained responsiveness to insulin over basal. The asterisk indicates P < 0.05 compared with the full-length control reporter plasmid

Because the mitogenic effects of insulin can be mediated by the mitogen-activated protein (MAP) kinase pathway, we tested whether cotreatment of theca cells with two specific and structurally distinct inhibitors of MAP kinase kinase (MEK) (PD 98059 [50 µM] and U0126 [3 µM] or the latter's inactive derivative, U0124 [3 µM]) would alter CYP17 promoter activity. MEK is an upstream kinase, the substrates of which are ERK1/ERK2 (extracellular signal- regulated kinase 1 and 2). Both active compounds significantly decreased insulin-stimulated CYP17 promoter expression by 75–80% (P < 0.05) (Fig. 6). Basal promoter activity declined 80% in the presence of PD 98059 (P < 0.05 versus control), but was unaltered by U0126, possibly reflecting unequal inhibitor potencies and/or concentrations. The inactive U0124 compound had no effect on insulin action.



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  		FIG. 6. Two inhibitors of the ERK1/ERK2 (MAP kinase) pathway, PD 98059 (50 µM) and U0126 (3 µM), attenuate insulin-stimulated CYP17 reporter gene activity. The full-length (–976 to +31 bp) CYP17 promoter- luciferase reporter construct was transfected into theca cells for 4 h, followed by a 6-h exposure to vehicle, inhibitors, inactive congener (U0124, 3 µM), insulin (100 ng/ml), or insulin plus inhibitors. Data are normalized for cotransfected pRL-TK Renilla luciferase activity and are the mean ± SEM of three separate experiments done in triplicate. Asterisks denote P < 0.05 compared with the vehicle-stimulated control (C)

Western blot analysis of theca-cell lysates demonstrated that both insulin and insulin-like growth factor (IGF)-I induce insulin-receptor phosphoprotein accumulation (Fig. 7). Insulin (100 ng/ml for 10 min) increased phosphorylation of its cognate receptor by 31-fold compared with control (vehicle). IGF-I (100 ng/ml for 10 min) elevated phosphorylated insulin receptor concentrations 10-fold compared with control.



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  		FIG. 7. Theca cell lysate was immunoprecipitated with phosphotyrosine antibody conjugated to agarose, separated by 10% SDS-PAGE, and transferred to a nitrocellulose membrane. The membrane was exposed to insulin-receptor antibody diluted with blocking buffer (1:200) followed by goat anti-rabbit IgG (1:10 000) and visualized by chemiluminescence. This is a representative Western blot, which was repeated three times


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present in vitro analyses establish that insulin can stimulate transcriptional activity of a transiently transfected 5'-upstream (–976/+31 bp) putative regulatory region of the CYP17 gene in a concentration- and time-dependent manner, while initiating rapid phosphorylation of the cognate receptor in primary cultures of swine theca cells. Preliminary signaling studies suggest that the MAP kinase pathway may participate in insulin-regulated CYP17 gene expression in this system. In an earlier study, insulin enhanced the accumulation of CYP17 mRNA and (intronic) hnRNA in this in vitro system [9]. Here, to begin to explore the molecular mechanisms of presumptive insulin-dependent regulation of CYP17 promoter activity, we isolated a 1007-bp gene fragment spanning –976 bp 5'-upstream to +31 bp 3'-downstream from the transcriptional start site, thereby ensuring faithful representation of transcriptional initiation. Transient transfection of the corresponding full- length promoter-luciferase reporter construct into theca cells documented consistent stimulation by insulin concentrations of 100–300 ng/ml within 2 h. Maximal transcriptional responses (2.7- to 3.2-fold stimulation above basal activity) were evident after 4–6 h. Successive 5'-upstream deletional analysis suggested possible operation of an inhibitory region (–839/–473 bp), which restrained basal and insulin-upregulated transcriptional activity in the theca- cell context. Site-directed mutagenesis of the full-length fragment so as to disable both proximal Sp1/AP-2-like response elements (–193/–180 bp) abolished basal and insulin-driven CYP17 promoter activity. Mutation of individual Sp1 or AP-2-like sequences also eliminated insulin stimulation and inhibited basal reporter expression. Accordingly, while not excluding other insulin-responsive elements in the 5'-upstream or other regulatory regions of the CYP17 gene, the present data point to two potential proximal transcriptional control sites that are critical for insulin-induced upregulation of the larger (–976 bp) promoter fragment. The nature of the interaction, if any, between the –193/–180-bp region and more distal (–976/ –839-bp) sequences is not known.

Although Sp1 and AP-2 binding sites would provide a plausible basis for insulin to drive CYP17 gene expression (above), further analyses will be required to document nuclear expression, specific DNA binding, and CYP17 promoter transactivation by one or both of these factors. Sp1 is a widely expressed transcription factor of approximately 100 kDa, which binds to GC-rich DNA sequences via three C-terminal zinc fingers [11]. The transcriptional activity of Sp1 is generally constitutive, but can be regulated developmentally [12, 13]. Sp1 binding appears to be involved in basal and IGF-dependent expression of the CYP11A gene [14, 15]. AP-2 likewise binds to GC-rich DNA sequences and can participate in basal and hormonally stimulated gene expression in other systems [16].

Combined point mutations of AP-2 and Sp1-like elements in theca cells completely disrupted insulin action. In this regard, AP-2 responsive cis-acting DNA elements in the CYP11A gene and the VEGF promoter also bind Sp1 and/or Sp3 [16, 17], and AP-2 and Sp1 protein-protein interactions further facilitate gene transactivation [16]. Other analyses indicate that basal and cAMP-regulated transcription of CYP17 probably involves Sp1 and AP-2 sites in the rat [18] and pig [19]. Cyclic AMP-PKA-dependent stimulation of the CYP17 promoter in other species further requires SF-1 binding sequences in the rat and cow [18, 20, 21] and a Pbx1 site in the cow [22]. The role of the foregoing elements in mediating insulin's facilitative effect on the transcription of the CYP17 gene is not yet known.

Deletional analysis of the 5'-upstream region of the CYP17 promoter fragment revealed loss of basal and insulin-enhanced transcriptional activity in the –839 to +31- bp (compared with the –976 to +31-bp) fragment. Absence of detectable basal activity makes responsiveness to insulin indeterminate for the –839/+31 CYP17 construct. More extensive 5'-upstream deletion to –473 bp reinstated basal reporter expression but did not reconstitute responsiveness to insulin. These data suggest the existence of inhibitory element(s) in the –839-bp to –473-bp region 5'- upstream of the transcriptional start site. In this regard, analysis of cAMP-dependent control of the proximal bovine CYP17 promoter indicates that PKC activators and/or a COUP-TF-like element may confer transcriptional repression [21].

Insulin and IGF-I act via complex tyrosine-kinase signaling cascades [23]. Although the details of insulinomimetic signaling in gonadal cells are not fully understood, insulin can induce rapid receptor autophosphorylation and augment sterol metabolism in swine granulosa cells [24– 26]. Insulin may also exert effects via the inositolglycan pathway in ovarian cells [27, 28]. The present data demonstrate that insulin augments tyrosine kinase-dependent receptor autophosphorylation in theca cells, and IGF-I does so in lesser measure. In addition, putative MEK antagonists, PD98059 and U0126, both suppressed insulin action. PD98059 reduced PDGF-stimulated theca-cell growth but enhanced LH-stimulated steroid secretion [29]. MEK antagonists (presumptively blocking ERK1/2 activation) can reverse the inhibitory effect of PGF2{alpha} on hCG-induced progesterone production in human granulosa-luteal cells [30] and also inhibit insulin and IGF-I's ability to enhance LH- stimulated low density lipoprotein receptor promoter expression in porcine granulosa cells [31].

In summary, the present in vitro experiments demonstrate concentration- and time-dependent stimulation by insulin of transcriptional activity of a –976 to +31-bp cis- acting DNA fragment cloned 5'-upstream of the transcriptional start site of the porcine CYP17 gene in primary cultures of swine theca cells. Unknown elements in the –839/ –473-bp 5'-upstream region appear to restrain basal expression. Sp1/AP-2-like elements (–180 to –193 bp) within the proximal CYP17 promoter are jointly required for maximal transcriptional enhancement by insulin. Insulin-induced transcriptional activity is preceded by rapid homologous receptor autophosphorylation and is inhibited by antagonists of the MAP kinase signal-transduction pathway. This in vitro system provides a model in which to analyze the mechanisms by which insulin coregulates CYP17 gene expression in untransformed theca cells.


   FOOTNOTES
 
1 Supported by NICHD/NIH through cooperative agreement (U54 HD28934) as part of the Specialized Cooperative Centers Program in Reproduction Research and NIH RO1 HD 16393. Back

2 Correspondence: Johannes D. Veldhuis, Division of Endocrinology and Metabolism, Department of Internal Medicine, Mayo Medical and Graduate Schools of Medicine, General Clinical Research Center, Mayo Clinic, 200 First Street Southwest, Rochester, MN 55905. FAX: 507 255 0901; veldhuis.johannes{at}mayo.edu Back

Received: 2 July 2003.

First decision: 24 July 2003.

Accepted: 22 January 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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