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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 11 4338-4346
Copyright © 2000 by The Endocrine Society

Original Studies

Enzymatic Activities of P450c17 Stably Expressed in Fibroblasts from Patients with the Polycystic Ovary Syndrome1

John W. M. Martens, David H. Geller, Wiebke Arlt, Richard J. Auchus, Valeria S. Ossovskaya, Henry Rodriguez, Andrea Dunaif and Walter L. Miller

Departments of Pediatrics (J.W.M.M., D.H.G., W.A., R.J.A., H.R., W.L.M.) and Pathology (V.S.O.), University of California, San Francisco, California 94143; and Department of Medicine, Brigham and Women’s Hospital (A.D.), Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Prof. Walter L. Miller, Department of Pediatrics, Building MR-IV, Room 209, University of California, San Francisco, California 94143-0978.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting approximately 5–10% of women of reproductive age. The clinical features of PCOS include oligo/anovulation, hyperandrogenemia, and hyperinsulinemia. Because P450c17 is the single enzyme catalyzing both 17{alpha}-hydroxylase and 17,20-lyase activities in the ovary and adrenal, some have suggested that defects in P450c17 may cause the hyperandrogenism of PCOS. Previous studies have shown that serine hyperphosphorylation of P450c17 increases the enzyme’s 17,20-lyase activity, thereby favoring androgen production, and that serine phosphorylation of the insulin receptor ß-chain (IR-ß) inhibits IR-ß tyrosine phosphorylation, causing insulin resistance in vitro. We previously suggested that a gain of function mutation in a single serine kinase might cause the hyperandrogenism and insulin resistance observed in PCOS patients by excessive phosphorylation of both P450c17 and IR-ß. To test this hypothesis, we obtained fibroblasts from nine previously studied patients: three controls, three PCOS patients with normal levels of IR-ß serine phosphorylation, and three PCOS patients with increased levels of IR-ß serine phosphorylation. Initial studies showed that such skin fibroblasts could not be transfected effectively by calcium phosphate, diethylaminoethyl-dextran, lipofection or adenovirus procedures. Therefore, we employed a retroviral infection system to stably express human P450c17 in the primary cultures of fibroblast cells from the PCOS patients and controls and measured the resulting 17{alpha}-hydroxylase and 17,20-lyase activity. The cells were analyzed in a blinded fashion until the study was complete. The 17{alpha}-hydroxylase and 17,20-lyase activities in each cell line correlated well with the amount of P450c17 protein expressed, but there was no correlation between either enzymatic activity (or their ratio) with the clinical phenotype of the cells’ donors even when results were corrected for the number of P450c17 complementary DNA inserts per cell line. Overnight incubation with 1 µmol/L insulin also did not affect enzymatic activity. Thus, we were unable to find evidence for the hypothesis that in PCOS a single abnormal kinase hyperphosphorylates both IR-ß, causing insulin resistance, and P450c17, causing hyperandrogenism. However, because fibroblasts do not normally express either P450c17 or the accessory proteins needed for its optimal activity, these results cannot exclude a role for serine phosphorylation in the hyperandrogenism and insulin resistance of PCOS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
POLYCYSTIC OVARY syndrome (PCOS) is the most common endocrine abnormality in women, affecting 5–10% of women of reproductive age (1, 2). The principal features of PCOS are hyperandrogenism and chronic anovulation (1, 2) with hypersecretion of LH (3), but many women also have hyperinsulinemia, acanthosis nigricans, obesity, and dyslipidemia, indicating that PCOS has a major metabolic component (4, 5, 6). In part because of these pleotropic findings, the etiology of PCOS has remained unclear and controversial, and finding mechanistic links between the hyperandrogenism and insulin resistance has been especially challenging. The observation that insulin-sensitizing agents, such as metformin, troglitazone and D-chiro-inositol, ameliorate hyperandrogenism while increasing insulin sensitivity in PCOS suggests a direct link between these two components of the syndrome (7, 8, 9, 10). However, men and women have similar degrees of insulin sensitivity despite the 10-fold higher androgen concentrations in men (11). Although androgen administration decreases insulin sensitivity, it does not do so to the extent seen in PCOS (12), and suppressing the hyperandrogenism of PCOS does not eliminate the insulin resistance (13, 14). Thus, hyperandrogenism alone does not cause hyperinsulinemia. Similarly, although lowering the insulin levels tends to diminish the hyperandrogenism, this effect is seen only in PCOS and not in normal women (2).

Whatever the mechanism linking hyperandrogenism and insulin resistance, it is now generally agreed that both the ovary and the adrenal are sources of the hyperandrogenemia of PCOS. When ovarian androgen synthesis is suppressed with GnRH agonists, PCOS women have higher androgen levels than normal women, indicating an adrenal source (15, 16, 17, 18). Similarly, when adrenal androgen synthesis is suppressed with dexamethasone, PCOS women again have higher androgen levels than normal women, indicating an ovarian source (19, 20). Therefore, we have sought a mechanism that might explain hyperandrogenism and insulin resistance through a single molecular lesion.

P450c17 is a crucial enzyme in the biosynthesis of all sex steroids, as it catalyzes both 17{alpha}-hydroxylase and 17,20-lyase activities (21, 22, 23, 24, 25, 26). The 17,20-lyase activity, which is required for androgen synthesis, is fostered by increased concentrations of its electron-donating redox partner, P450 oxidoreductase (OR) (27, 28), and by the presence of cytochrome b5, which allosterically promotes interactions with OR (29). The androgenic 17,20-lyase activity also requires serine phosphorylation of P450c17 (30). Serine phosphorylation of the ß-chain of the insulin receptor causes insulin resistance in vitro (31, 32), suggesting that a single abnormal serine kinase might hyperphosphorylate both P450c17 and the insulin receptor, accounting for the hyperandrogenism and the hyperinsulinism with a single lesion (30). The simultaneous independent observation that 50% of PCOS women who had both hyperandrogenism and insulin resistance had hyperphosphorylated serine residues on their insulin receptors (33) provides compelling support for the serine kinase hypothesis of PCOS. However, no work has been reported attempting to identify a causal link between the two. In this paper we sought to determine whether the hyperphosphorylating environment in fibroblasts of PCOS patients would hyperphosphorylate ectopically expressed human P450c17 in such a fashion that would increase 17,20-lyase activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Primary skin fibroblast cultures

The diagnostic criteria for PCOS and normal women, the performance of skin biopsies, and the propagation of primary cultures of skin fibroblasts were described previously (33). The clinical characteristics of the nine subjects are shown in Table 1Go. All cell lines had been maintained frozen in liquid nitrogen after five or six passages; cells were thawed, grown to confluence in 25-mL T-flasks, and sent to San Francisco without indication of the origin of the cells (PCOS vs. control). Cell lines from nine different women were studied: three from control women, three from PCOS patients who showed normal IR-ß serine phosphorylation, and three from PCOS patients who had excess IR-ß serine phosphorylation (33). Initial feasibility studies to optimize the transfection protocol (see below) were performed with control fibroblasts prepared, frozen, thawed, and shipped in the same fashion. Primary skin fibroblast cultures were maintained in DMEM with 0.584 g/L glutamine, 3.7 g/L NaHCO3, and 4.5 g/L glucose and supplemented with 10% FCS (HyClone Laboratories, Inc., Logan, UT). Cells were split at 80–90% confluence according to their growth rate and used for experiments between passages 8 and 20.


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  		Table 1. Clinical data

 
Retroviral vectors and packaging cells

The retroviral packaging cell line Phoenix, a derivative of human embryonic kidney HEK293T cells, was a gift from Dr. Gary Nolan (Stanford University, Stanford, CA) and was maintained as described previously (http://www.stanford.edu/group/nolan/phoenix_info. html). These cells express the Moloney murine leukemia virus (MoMuLV) gag and pol genes and the MoMuLV envelope gene 4070A from two different stably integrated plasmids (34). The retroviral shuttle vector pBabe-puro, a derivative of MoMuLV (35), was used for generating human fibroblast cultures expressing human P450c17. This retroviral vector contains a small multiple cloning site downstream from the MoMuLV long terminal repeat (LTR) that drives expression of the foreign gene and also contains a puromycin resistance gene under the control of the simian virus 40 early promoter used to select cells with integrated retrovirus. The retroviral vector pPS-EGFP was derived from pPS-neo (36, 37) by replacing the neomycin resistance gene of pPS-neo with the gene for the enhanced green fluorescent protein (EGFP) from pEGFP-N1 (CLONTECH Laboratories, Inc., Palo Alto, CA). Infection with pPS-EGFP and measurement of EGFP were used to determine the efficiency of gene delivery. To introduce human P450c17 into the retroviral vector, the full-length P450c17 complementary DNA (cDNA) (24) was amplified from pMT2-P450c17 (25) using primers that introduce BamHI and EcoRI sites at the 5'- and 3'-ends, respectively (29). The amplified fragment was subsequently digested with BamHI and EcoRI and introduced into pBabe-puro digested in similar fashion. The insert in the resulting construct, pBabe-c17, was verified by sequencing in its entirety to assure integrity of the human P450c17 cDNA.

Retrovirus-mediated gene transfer of human P450c17 in fibroblasts

Retroviral gene transfer was performed essentially as described previously (38). Briefly, Phoenix cells at 85% confluence were transfected overnight with 10 µg retroviral transfer plasmid, pPS-EGFP, pBabe-puro, or pBabe-c17, using Lipofectamine (Life Technologies, Inc., Gaithersburg, MD). Two days after transfection, the medium containing newly packaged retrovirus was collected and filtered through a 0.2-µm pore size Super Acrodisc 25 filter (Gelman Sciences, Ann Arbor, MI). After supplementation with 4 µg/ml polybrene (Sigma, St. Louis, MO), the augmented medium was applied to each primary fibroblast culture at 50% confluence. Retroviral infection of the primary fibroblasts was allowed to proceed for 24 h. The medium was then replaced with a second batch of medium derived from the transfected Phoenix packaging cells and processed in identical fashion. Puromycin (2 µg/mL; Sigma) was used to select for fibroblasts that had successfully incorporated the retroviral copy of the human P450c17 cDNA.

Enzymatic activity assays and Western blotting for P450c17 and P450 oxidoreductase

Primary skin fibroblasts infected with pBabe-puro or pBabe-c17 were plated in six-well plates. At approximately 80% confluence, the culture medium was replaced by 2 mL fresh medium containing 200,000 cpm [3H]pregnenolone (21 Ci/mmol), 20,000 cpm [14C]progesterone (55.4 mCi/mmol), or 200,000 cpm [3H]17{alpha}-hydroxypregnenolone (21.1 Ci/mmol; NEN Life Science Products, Boston, MA). After the desired incubation interval, steroids were extracted from the medium, concentrated by evaporation, and separated by thin layer chromatography (PE SIL G/UV silica gel plates, Whatman, Maidstone, UK) as previously described (28). Quantification was performed by scintillation counting after autoradiography (28, 29) or by analysis on a Storm 860 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). After removing the medium, the remaining fibroblasts were harvested from the six-well plates for Bradford protein determination (Bio-Rad Laboratories, Inc., Hercules, CA). Equal amounts of total cellular protein (20 µg) were separated on SDS-10% polyacrylamide gels and electrotransferred to polyvinylidene membranes (Millipore Corp., Bedford, MA). Immunodetection was performed on membranes using polyclonal rabbit antisera against human P450c17 (28) or human P450 oxidoreductase (a gift from Dr. C. Roland Wolf, Imperial Cancer Institute, Dundee, UK). A secondary peroxidase-conjugated antibody was used in combination with the ECL chemiluminescent detection method (Amersham International, Arlington Heights, IL). P450 oxidoreductase activity was measured as previously described (39).

Southern blot

Two or three 150-cm2 flasks of confluent primary skin fibroblasts were harvested for each subject. Protein was digested from cell pellets overnight in 500 µL 50 mmol/L Tris-HCl (pH 7.6), 5 mmol/L ethylenediamine tetraacetate, 1% SDS, 0.2 mol/L NaCl, and 1 mg/ml proteinase K, and DNA was extracted twice with phenol/chloroform. Genomic DNA was precipitated in ethanol, dissolved in 10 mmol/L Tris-HCl (pH 7.6) and 1 mmol/L ethylenediamine tetraacetate at a concentration of more than 0.5 µg/µL. Ten micrograms of genomic DNA were digested with 400 U SstI in a total volume of 30 µL, separated by overnight electrophoresis through 0.7% agarose gel, and blotted to a nylon membrane (Hybond N+, Amersham Pharmacia Biotech). The probe was a 623-bp BamHI-XmnI fragment of the human P450c17 cDNA (24) encompassing exons 1–3 and was labeled to a specific activity of more than 108 cpm/µg using [{alpha}-32P]deoxy-CTP and random primers. The blot was probed in 6 x SSC (1 x SSC is 0.15 mol/L NaCl and 0.015 mol/L sodium citrate) with blocking agents at 68 C overnight in the presence of 100 µg/ml salmon sperm DNA, washed once in 1 x SSC-0.5% SDS, then washed twice at 50 C in 0.1 x SSC and 0.5% SDS and analyzed with a Storm 860 PhosphorImager (Molecular Dynamics, Inc.).

Statistical analyses

Enzymatic activity was measured in four or five independent assays for each of the nine cell lines. After the code was broken, the mean results for the three individual cell lines in each group were averaged (three normal controls, three PCOS without IR-ß hyperphosphorylation, and three PCOS with IR-ß hyperphosphorylation). The differences among the three groups were tested by one-way ANOVA followed by the Bonferroni-Dunn post-hoc test for statistical significance. Linear regression analysis was used to test for correlations between the integrated viral copy number and 17{alpha}-hydroxylase and 17,20-lyase activities.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transfection and infection of primary cultures of skin fibroblasts

Serine phosphorylation increases the 17,20-lyase activity of P450c17 (30), and skin fibroblast cultures from about half of PCOS women have excessive serine phosphorylation of the ß-chain of their insulin receptors (IR-ß) (33). To approach the question of whether the same kinase might be responsible for the serine phosphorylation of both P450c17 and IR-ß, we sought to express human P450c17 in cells that exhibit IR-ß hyperphosphorylation. Thus, substantial initial effort was directed toward identifying procedures for expression of P450c17 in primary cultures of human skin fibroblasts. Attempts at transient transfection using calcium phosphate/DNA precipitates, diethylaminoethyl (DEAE)-dextran and lipofection, which all work with human NCI-H295 adrenal cells (40), did not yield useful levels of P450c17 expression. Various modifications of the adenovirus-mediated transfection procedure (41) and live adenovirus infection (42) were also unsuccessful. Therefore, we used a living replication-deficient retroviral system to infect the cells and stably integrate the P450c17 expression cassette into the fibroblast genome.

To determine whether the retroviral infection approach would be effective, we first infected control human fibroblasts with a vector (pPS-EGFP) that expresses the easily monitored EGFP. The replication-deficient pPS-EGFP was packaged into infectious particles in Phoenix cells, and the infectious viral particles were harvested and used to infect primary cultures of human skin fibroblasts. Three days later, examination of the fibroblasts by florescence microscopy showed that about 90% of the cells had been infected and expressed the recombinant EGFP (Fig. 1Go).



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  		Figure 1. Efficiency of gene transfer to primary cultures of skin fibroblasts. Photographs were taken 3 days after infection with the retroviral vector (pPS-EGFP) expressing the GFP. A, Cells photographed under phase contrast microscopy. B, The same microscopic field photographed with florescent light. Note that virtually all of the cells are florescent, indicating the presence of the GFP encoded by the retrovirus.

 
Expression of human P450c17 in primary cultures of human skin fibroblasts

To employ this retroviral approach for the expression of P450c17, a similar replication-deficient retroviral shuttle vector was built by cloning the human P450c17 cDNA into the BamHI/EcoRI site of pBabe-puro so that the P450c17 cDNA would be expressed under control of the viral LTR. After propagation in Phoenix cells, this vector (pBabe-c17) was used to infect control human skin fibroblasts, and its expression of immunodetectable P450c17 protein and assayable 17{alpha}-hydroxylase and 17,20-lyase activities was determined (Fig. 2Go). Fibroblasts infected with pBabe-c17, but not those infected with the empty pBabe-puro vector, expressed immunodetectable P450c17 protein. Furthermore, incubating the cells for 2 h with radiolabeled pregnenolone or progesterone showed that the cells had acquired 17{alpha}-hydroxylase activity, and incubating the cells with 17-hydroxypregnenolone (17OH-pregnenolone) indicated that the cells had acquired 17,20-lyase activity. By contrast, cells infected with pBabe-puro acquired neither activity (Fig. 2Go). Thus, primary cultures of human skin fibroblasts infected with a retrovirus built to express human P450c17 acquired the ability to catalyze both 17{alpha}-hydroxylase and 17,20-lyase activities, demonstrating the efficacy of this retroviral system.



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  		Figure 2. Expression of human P450c17 in primary cultures of human skin fibroblasts. Fibroblast cultures were infected with either the empty retroviral vector pBabe-puro or pBabe-puro containing the full-length human P450c17 cDNA (pBabe-c17), and cells containing the retrovirus integrated into the genome were selected with puromycin. Expression of P450c17 protein could be detected by a Western immunoblot (W-blot) in cells infected with pBabe-c17, but not in cells infected with pBabe-puro. 17{alpha}-Hydroxylase activity (OHase) against both pregnenolone (Preg) and progesterone (Prog) and 17,20-lyase activity (Lyase) against 17OH-pregnenolone (17-Preg) was seen only in cells infected with pBabe-c17, as shown by autoradiography of thin layer chromatograms of radiolabeled steroids incubated with the cells. The identities of the autoradiographic spots were determined by comigration with radiolabeled standards, as previously described (25 28 29 ).

 
To determine the optimal conditions for analyzing the 17{alpha}-hydroxylase and 17,20-lyase activities of P450c17 expressed in fibroblasts, we examined the time course of conversion of pregnenolone to 17OH-pregnenolone, of progesterone to 17-hydroxyprogesterone (17OH-progesterone), and of 17OH-pregnenolone to dehydroepiandrosterone in fibroblasts infected with pBabe-c17. Because human P450c17 converts virtually no 17OH-progesterone to {Delta}4-androstenedione (25, 28, 29), 17{alpha}-hydroxylase activity was measured as conversion of progesterone to 17OH-progesterone, and 17,20-lyase activity was measured as conversion of 17OH-pregnenolone to dehydroepiandrosterone. In control fibroblasts, 17{alpha}-hydroxylase activity was essentially linear with time for at least 2 h, and 17,20-lyase activity was nearly linear for about 4 h before approaching a plateau (Fig. 3Go); similar data were obtained with PCOS fibroblasts (not shown). Based on these data, we assayed 17{alpha}-hydroxylase activity after 30 min and 17,20-lyase activity after 120 min, ensuring that each reaction was in the linear range, before substrate concentrations fell significantly.



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  		Figure 3. Time-course analysis of 17{alpha}-hydroxylase and 17,20-lyase activities in human skin fibroblast cultures infected with pBabe-c17. Control fibroblast cultures containing the integrated retroviral vector pBabe-c17 were incubated for the indicated times with [14C]progesterone for determination of 17{alpha}-hydroxylase activity and with [3H]17OH-pregnenolone for determination of 17,20-lyase activity.

 
Expression of P450c17 in primary cultures of human skin fibroblasts from patients with PCOS

Using the procedures established above, we assessed the 17{alpha}-hydroxylase and 17,20-lyase activities in the primary fibroblast cell cultures of the nine infected cell lines derived from PCOS patients and controls. We also performed Western immunoblots with antisera against P450c17, OR, and cytochrome b5, using a constant amount of protein from each cell line. Neither OR nor cytochrome b5 was detectable by Western blotting in any of the cell lines, but all nine cell lines showed equivalent OR activity, as measured by the cytochrome c reductase assay (39). Coinfection of one control cell line with a pPS-neo vector expressing human OR did not affect the ratio of expressed 17{alpha}-hydroxylase to 17,20-lyase activity (not shown). Thus, OR did not appear to limit P450c17 activity in these fibroblasts, as increasing the intracellular abundance of OR did not affect enzymatic activity. By contrast, P450c17 expressed from the integrated retroviral vector was readily detectable in all nine cell lines, although the level of expression varied considerably (Fig. 4Go). There also were substantial differences among the enzymatic activities seen in the nine cell lines (Fig. 4AGo). The experiment in Fig. 4AGo was repeated four or five times, depending on how rapidly each cell line grew, and the results from the PhosphorImager analysis were used to calculate the 17{alpha}-hydroxylase and 17,20-lyase activities of each cell line, expressed as a function of total cellular protein. Before the code identifying the PCOS/control status of each cell line was revealed, it was clear that the ratio of lyase to hydroxylase activity did not vary significantly, and that the net level of activity correlated with the level of expression of immunodetectable P450c17. The code was then broken: cell lines 4, 6, and 9 were from control women; cell lines 2, 5, and 7 were from PCOS women with normally phosphorylated IR-ß; and cell lines 1, 3, and 8 were from PCOS women with serine hyperphosphorylation of IR-ß (Fig. 4BGo). There were no significant differences among the lyase/hydroxylase ratios calculated for the controls, the PCOS patients with normal IR-ß, and the PCOS patients with IR-ß serine hyperphosphorylation (Table 2Go). Incubation of control and PCOS cells overnight with 1 µmol/L insulin also did not change the lyase/hydroxylase ratio (results not shown).



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  		Figure 4. Human P450c17 activities and expression in primary skin fibroblasts from PCOS patients and control subjects. 17{alpha}-Hydroxylase and 17,20-lyase activities were measured for each of the nine cell lines. Fibroblasts were incubated with either [14C]progesterone or [3H]17{alpha}-OHpregnenolone and assayed for 17{alpha}-hydroxylase and 17,20-lyase activities. A, Results of a representative experiment. Samples 4, 6, and 9 are from normal subjects; samples 2, 5, and 7 are from PCOS patients with normal IR-ß serine phosphorylation; and samples 1, 3, and 8 are from PCOS patients with excess IR-ß serine phosphorylation. B, Mean (±SD) of four or five independent single experiments of the hydroxylase ({blacksquare}) and lyase ({square}) activities of each cell line are expressed per mg cellular protein. The data from the three controls (cell lines 4, 6, and 9) are shown on the left, those from PCOS patients with normally phosphorylated IR-ß (cell lines 2, 5, and 7) are shown in the middle, and those from PCOS patients with IR-ß hyperphosphorylation (cell lines 1, 3, and 8) are shown on the right. The calculated ratio of 17{alpha}-hydroxylase to 17,20-lyase activity (OHase/Lyase) is shown below the bar graph. The abundance of P450c17 relative to total cellular protein is shown below as a Western blot with a constant amount of protein loaded in each lane. These data are from a single blot, which was cut and rearranged to correlate with the groupings of the three classes of cell lines.

 

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  		Table 2. 17{alpha}-Hydroxylase and 17,20-lyase activity (in picomoles per h/mg protein) and 17{alpha}-hydroxylase/17,20-lyase activity ratios (mean ± SEM) in pBabe-c17-infected fibroblast cell lines derived from normal controls (n = 3), PCOS patients with IR-ß hyperphosphorylation (PCOS+; n = 3), and without IR-ß hyperphosphorylation (PCOS-; n = 3)

 
Viral copy number integrated into the cellular genomes

Each patient’s cell line was infected with pBabe-c17 as a population of cells, however, because of the limited number of cell passages possible with primary cultures, it was not possible to isolate clonal lines. Although the puromycin selection procedure should ensure that virtually every cell has incorporated pBabe-c17 into its genome, we considered the possibility that the cells from different individuals incorporated a different average number of copies of the retrovirus. Therefore, to control for the copy number of retroviral incorporation, we performed a Southern blot of total genomic DNA from each cell line and probed for the incorporated P450c17 cDNA (Fig. 5Go). The endogenous gene for P450c17, which has been sequenced in its entirety and is present in a single copy (43), served as an internal control. The number of retroviral copies integrated per genome varied from 0.6–1.7 among the nine cell lines. Linear regression analysis showed a significant correlation between the integrated viral copy number and 17{alpha}-hydroxylase activity (r = 0.359; P = 0.0206; y = 125.3 + 281.7x). The correlation between copy number and 17,20-lyase activity did not reach statistical significance (r = 0.260; P = 0.1014; y = 4.46 + 2.96x). We then corrected the enzymatic activity data for each cell line for the corresponding integrated copy number and analyzed the data by one-way ANOVA (Table 3Go). Neither enzymatic activity nor their ratio differed significantly among the cell lines from the three groups of patients (control, PCOS with normal IR-ß, and PCOS with IR-ß hyperphosphorylation). Although 17,20-lyase activity tended to be lower in PCOS with normal IR-ß compared to that in either control or PCOS with IR-ß hyperphosphorylation, the P values for these comparisons did not reach statistical significance after the Bonferroni-Dunn correction for multiple tests. The comparison of the controls to all PCOS cells combined showed no difference.



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  		Figure 5. Estimation of the number of retroviral copies stably integrated into the skin fibroblast genome. A, Top, Schematic of the pBabe-puro vector. The heavy line represents the pBluescript bacterial cloning vector. LTR designates the MoMuLV long terminal repeat, which drives the expression of downstream sequences. The ATG translational initiation codon of gag has been removed to allow in-frame expression of cDNAs cloned in multiple cloning site. {psi}, Retroviral packaging signal; puroR, puromycin resistance gene; SV40, early promoter of simian virus 40. Middle, Schematic map of pBabe-c17, which is pBabe-puro doubly digested with BamHI and EcoRI with the human P450c17 cDNA inserted in this site. When inserted into the genome of the human fibroblasts and subsequently digested with SstI, the human P450c17 cDNA is liberated as a 1.34-kb fragment. P, PstI; Ss, SstI; BHI, BamHI; BXI, BstXI; SBI, SnaBI; RI, EcoRI; Sal, SalI; X, Xmn I. Bottom, Schematic map of the endogenous human P450c17 gene. Digestion of human fibroblast DNA with SstI liberates most of the gene as a 5.21-kb fragment that is easily distinguished from the 1.34-kb P450c17 cDNA insert. B, Southern blot. Genomic DNA was isolated from the nine different cell lines infected with pBabe-c17 and from two control fibroblast lines; an uninfected wild-type fibroblast line (Fb), and a fibroblast line infected with the empty retroviral shuttle vector pBabe-puro (Vc). Each lane contains 10 µg genomic DNA digested with SstI. The endogenous cellular gene for P450c17 produces a 5.21-kb band in all 11 cell lines, as predicted from the map in A. The nine cell lines infected with pBabe-c17 also contain a 1.34-kb band, representing the P450c17 cDNA in the retroviral vector. The number of P450c17 cDNA inserts per cell (the copy number) was calculated by dividing the number of counts from the 623-bp cDNA probe hybridizing to the 1.34-kb band by the number of counts hybridizing to the 5.21-kb band.

 

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  		Table 3. 17{alpha}-Hydroxylase and 17,20-lyase activity (in picomoles per h/mg protein) and ratio of 17{alpha}-hydroxylase to 17,20-lyase activities (mean ± SEM) in pBabe-c17-infected fibroblast cell cultures derived from normal controls (n = 3), PCOS patients with IR-ß hyperphosphorylation (PCOS+; n = 3) and without IR-ß hyperphosphorylation (PCOS-; n = 3)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The most consistent feature of PCOS is hyperandrogenism, which is involved in disorganized follicular development (1, 44). The qualitative regulator of androgen biosynthesis in both the ovary and the adrenal is P450c17, as the ratio of 17{alpha}-hydroxylase and 17,20-lyase of P450c17 determines the flow of substrate through the steroid biosynthetic pathway and the molar ratio of the steroids produced (26, 29, 45). The hyperandrogenism of PCOS may result from an alteration in this ratio in the ovary (1) and adrenal (46, 47, 48). Proposed mechanisms include a global increase in androgen synthesis (49, 50) or specific posttranslational modifications of the P450c17 molecule that favor the lyase activity (30).

An important subset of women with PCOS have hyperinsulinism and varying degrees of insulin resistance, which are manifested by impaired glucose tolerance, and even type II diabetes (2). The observation that about half of insulin-resistant PCOS women have a 3.7-fold excess of serine phosphorylation of IR-ß provided the first direct evidence of a specific molecular lesion in the insulin signal transduction pathway in PCOS (33). Thus, we sought to determine whether an alteration in P450c17 activity or in the ratio of its two activities contributes to the hyperandrogenism seen in PCOS, and whether there is a correlation between the P450c17 activity and the degree of IR-ß serine phosphorylation.

Because fibroblasts were available from PCOS women and controls whose IR-ß serine phosphorylation had been characterized in detail (33), we sought to express P450c17 in both control and hyperphosphorylating environments to determine whether we could demonstrate that the serine kinase that phosphorylates IR-ß also phosphorylates P450c17. This approach is consistent with the recent observation that insulin action is impaired in primary skin fibroblast cultures from patients with PCOS (51). In addition, skin fibroblasts are readily obtainable, whereas cells from steroidogenic tissues are not, and the use of fibroblasts circumvents the contentious issue of whether the hyperandrogenism is of ovarian or adrenal origin. However, although the logic of the approach is sound, we were unable to detect any differences in normal and PCOS fibroblasts expressing P450c17. The most obvious explanation for these results is that there are no differences in the biology and enzymology of P450c17 in normal women and those with PCOS. We regard this simplistic explanation as being most unlikely, as pharmacological data suggest a role for P450c17 (7, 8, 10), and direct examination of ovarian thecal cells from normal and PCOS women strongly indicate a role for P450c17 (50).

Other work has suggested that a dysregulated 17,20-lyase activity of P450c17 is responsible for the hyperandrogenism of PCOS (7, 16, 18, 52, 53, 54). However, these studies only measured serum androgen levels after patients were treated with dexamethasone, ACTH, or GnRH agonists, and the varying serum concentrations of C19 and C21 steroids were interpreted as indicating changes in the ratio of the 17{alpha}-hydroxylase and 17,20-lyase activities of P450c17; however, P450c17 was not studied. Such serum steroid values also reflect the actions of other steroidogenic enzymes [e.g. the cholesterol side-chain cleavage enzyme (P450scc), 3ß- and 17ß-hydroxysteroid dehydrogenases, and StAR], changes in steroid degradation, changes in peripheral conversion of precursor steroids to C19 androgens, and changes in serum steroid-binding proteins (18, 55). Other studies, both in vitro and in vivo, suggest that there may be global increases in all aspects of ovarian and adrenal C19 steroid biosynthesis in PCOS (49, 50, 56). By contrast, our study directly assayed the two activities of P450c17.

The retroviral gene delivery system provided a suitable approach to deliver comparable amounts of human P450 construct to different primary skin fibroblast lines. However, the infected fibroblasts expressed different amounts of P450c17, as measured by both activity and Western immunoblotting. However, lyase activities did not correlate the PCOS phenotype, with the previously measured extent of IR-ß serine phosphorylation (33) or with the integrated retroviral copy number. Thus, the differences appear to be due to intrinsic differences in the different fibroblast lines that affect retroviral integration. Despite these differences, our study demonstrates the utility of the retroviral system to deliver and express steroidogenic cytochrome P450 enzymes and other foreign proteins in primary cultures of human fibroblasts.

We cannot exclude the possibility that there were important variations in the cellular content or activities of the redox partners, OR and cytochrome b5, that are needed for electron donation to P450c17. Neither OR nor cytochrome b5 was detected by immunoblotting, indicating that their abundance was low. We can infer that each cell line contained comparable amounts of OR, as cytochrome c reductase activity, which constitutes an alternative assay for OR, was equivalent in each cell line. However, no such alternative assay for cytochrome b5 is available; hence, the abundance of cytochrome b5, which is crucial for 17,20-lyase activity (29), in each cell line is unknown. However, we found no evidence to support our hypothesis that the 17,20-lyase activity of P450c17 would be increased in PCOS cells with IR-ß hyperphosphorylation. Finally, the principal variable that this study sought to address, serine phosphorylation, could not be measured directly, as the level of P450c17 expression precluded the direct immunoisolation techniques (30) needed for measuring incorporation of [32P]orthophosphate (data not shown).

Thus, our studies establish that the retroviral infection system is eminently suitable for the expression of foreign sequences in human fibroblasts. However, we were unable to detect any differences in the behavior of P450c17 when expressed in normal fibroblasts vs. those from PCOS patients with or without previously demonstrated serine hyperphosphorylation of the insulin receptor. The reason for this is not clear. It is possible the increased amounts of P450c17 play a role in PCOS, without a change in the ratio of 17{alpha}-hydroxylase to 17,20-lyase activity, as suggested by studies with PCOS thecal cells (50), and that the serine phosphorylations of P450c17 and IR-ß are unrelated. However, because so many different factors participate in the regulation of the ratio of hydroxylase to lyase activity of P450c17 (26), further studies of the role of its serine phosphorylation in PCOS are warranted.


   Acknowledgments
 
We thank Dr. Thea D. Tlsty, Department of Pathology, University of California-San Francisco, for helpful advice and the provision of the retroviral reagents.


   Footnotes
 
1 This work was supported by the National Cooperative Program for Infertility Research (Grant U54-HD-34449) at the University of California-San Francisco (to W.L.M) and at the Brigham and Women’s Hospital (to A.D.), NIH Grants DK-37922 and DK-42154 (to W.L.M.) and DK-40605 (to A.D.), Clinical Investigator Award KO8-DK-02387 (to R.J.A.), and a fellowship from the Deutsche Forschungsgemeinschaft (Ar 310/2–1, to W.A.). Back

Received April 26, 2000.

Revised July 13, 2000.

Accepted August 10, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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