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Division of Pediatric Endocrinology and Diabetology, University Children's Hospital Bern, Bern, Switzerland
Received July 16, 2006; accepted November 30, 2006
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Abstract |
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HSDII, key enzymes of androgen biosynthesis. Pioglitazone but not rosiglitazone inhibited the expression of the CYP17 and HSD3B2 genes. Likewise, pioglitazone repressed basal and 8-bromo-cAMP-stimulated activities of CYP17 and HSD3B2 promoter reporters in NCI-H295R cells. However, pioglitazone did not change the activity of a cAMP-responsive luciferase reporter, indicating that it does not influence cAMP/protein kinase A/cAMP response element-binding protein pathway signaling. Although peroxisome proliferator-activated receptor 
(PPAR) is the nuclear receptor for TZDs, suppression of PPAR by small interfering RNA technique did not alter the inhibitory effect of pioglitazone on CYP17 and HSD3B2 expression, suggesting that the action of pioglitazone is independent of PPAR. On the other hand, treatment of NCI-H295R cells with mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) enhanced promoter activity and expression of CYP17. This effect was reversed by pioglitazone treatment, indicating that the MEK/ERK signaling pathway plays a role in regulating androgen biosynthesis by pioglitazone.
HSDII, encoded by the CYP17 and HSD3B2 genes, are key enzymes involved in biosynthesis of androgens. The mature adrenal cortex in humans produces androgens from cholesterol in a multistep pathway. In a first step, cholesterol is converted to pregnenolone by the P450 side-chain cleavage system. Second, pregnenolone then may be converted to 17
-hydroxypregnenolone and to dehydroepiandrostenedione (DHEA) by the 17-hydroxylase and 17,20-lyase activities of P450c17. Third, DHEA is converted to androstenedione by 3-hydroxysteroid dehydrogenase type 2 (3HSDII). Although the fetal adrenals predominantly produce androgens, postnatal androgen production ceases only to resume at the age of 6 to 7 years, after the development of the zona reticularis, the third layer of the mature adrenal cortex.
Thiazolidinediones (TZDs) are a class of oral insulin-sensitizing agents that are generally believed to exert their action as ligands of peroxisome proliferator-activated receptor (PPAR), a member of the nuclear receptor superfamily of transcription factors (Lehmann et al., 1995
). Upon stimulation, PPAR heterodimerizes with the retinoid X receptor to bind to "PPAR-responsive" elements, thereby activating the transcription of specific genes. Although the antihyperglycemic activity of TZDs correlates positively with the binding affinity for PPAR (Willson et al., 1996), there is growing evidence that the biological effects of TZDs expand beyond insulin-sensitizing and may not solely be receptor-mediated (Palakurthi et al., 2001; Feinstein et al., 2005). Apart from their metabolic actions PPAR agonists exhibit antineoplastic effects on several tumor cell lines either through induction of apoptotic cell death or through production of reactive oxygen species. Thus TZD's means of action remain open, and further details on the possible mechanisms of action need to be elucidated.
Pioglitazone and rosiglitazone are the TZDs that are currently in clinical use for the treatment of type 2 diabetes mellitus (T2DM). Several clinical studies have demonstrated that both pioglitazone and rosiglitazone not only are able to improve insulin sensitivity in patients with T2DM but may also decrease plasma androgen concentrations (DHEA, androstenedione) in women with polycystic ovary syndrome (PCOS) (Brettenthaler et al., 2004; Ortega-Gonzalez et al., 2005; Sepilian and Nagamani, 2005). Controversy exists as to whether the antiandrogenic action of TZDs is mediated indirectly through their insulin-sensitizing effect, and it is unknown whether it depends on PPAR.
PCOS is a common disorder of androgen excess. The pathophysiological mechanism(s) underlying PCOS are unknown, as is the exact physiological regulation of androgen biosynthesis. Elevated circulating androgen concentrations in women with PCOS may originate from the ovaries and/or the adrenal cortex (Lachelin et al., 1982; Barnes et al., 1989; Ehrmann et al., 1992). Enhanced androgen biosynthesis in PCOS is caused by an increased expression of the steroidogenic enzymes P450c17 (17-hydroxylase, 17,20-lyase) and 3HSDII, which are essential for the production of androgens (Nelson et al., 1999, 2001).
In this study, we investigated the regulation of human androgen biosynthesis using pioglitazone and rosiglitazone as chemical tools. Whereas previous in vitro studies demonstrated that TZDs may directly inhibit the enzymatic activities of recombinantly produced key enzymes for androgen production (3HSDII and P450c17) (Arlt et al., 2001), we studied the effects of TZDs on cell signaling and gene expression in human adrenocortical NCI-H295R cells. NCI-H295R cells were chosen because this represents a well-established model to study the steroidogenesis of the human adrenal cortex (Gazdar et al., 1990; Staels et al., 1993). NCI-H295R cells express all genes that encode the steroidogenic enzymes present in all three layers of the adult adrenal cortex, including 3HSDII and P450c17, key enzymes of androgen biosynthesis.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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from Upstate (Lake Placid NY), and -actin from Sigma. Goat anti-rabbit horseradish peroxidase-conjugated antibody was obtained from Santa Cruz Biotechnology. Radioactive-labeled [7(N)-3H]pregnenolone (14 Ci/mmol) and [1,2,6,7(N)-3H]DHEA (63 Ci/mmol) were procured from PerkinElmer (Boston MA), and [1,2,6,7(N)-3H]17-hydroxypregnenolone (50 Ci/mmol) was acquired from American Radiolabeled Chemicals (St. Louis, MO). Trilostane was extracted in absolute ethanol from tablets commercially available as Modrenal (Bioenvision, New York, NY).
Cell Culture and Treatment. Human adrenal NCI-H295R cells were ordered from American Type Culture Collection (Manassas, VA). Cells were cultured under standard conditions in Dulbecco's modified Eagle's/Ham's F-12 medium (Invitrogen, Basel, Switzerland) supplemented with 5% NuI serum, 0.1% selenium/insulin/transferrin (SIT) plus, penicillin, and streptomycin (all from Invitrogen). Pioglitazone and rosiglitazone were dissolved in dimethyl sulfoxide (DMSO) at stock concentrations of 10 mM; final concentrations used for treatment were in the range of 2.5 to 10 µM for both drugs. Reported mean effective serum concentrations for the treatment of T2DM patients are 1 µM for rosiglitazone (Cox et al., 2000) and 2.5 µM for pioglitazone (Zhong and Williams, 1996). 8Br-cAMP was diluted in phosphate-buffered saline at a concentration of 100 mM; final concentration for treatment was 0.5 mM. MEK/ERK inhibitor PD98059 was dissolved in DMSO at a concentration of 20 mM, and the final concentration for treatment was 10 µM. For RNA and protein extraction experiments and for steroid labeling experiments, cells were grown in six-well plates. Twenty-four hours after subculturing, medium was replaced, and treatment was added in normal growth medium for 48 h unless indicated differently. Control cells were treated with 0.1% (v/v) DMSO.
RNA Isolation and Semiquantitative RT-PCR. Total RNA from NCI-H295R cells was extracted using a Nucleospin RNA kit (Macherey Nagel, Oensingen, Switzerland). Total RNA from human adrenal tissues was extracted using the TRIzol method as described by the manufacturer (Invitrogen). RNA was reverse-transcribed to cDNA using the Improm RNA Transcriptase kit (Promega, Madison, WI) with either 0.5 µg of oligo(dT) or random hexamers (Promega) per 1 µg of RNA at 42°C for 1 h. Aliquots of cDNA obtained from 100 ng of RNA were taken for PCR reactions in a final volume of 25 µl. For the PCR, GoTaq Polymerase (Promega) and specific primers were used (Table 1) (Fluck and Miller, 2004; Huang et al., 2005). PCR conditions were as follows: 1 min at 94°C, 1 min at 59 to 60°C, 1 min at 72°C, 22 cycles for CYP17 and GAPDH, 25 cycles for HSD3B2 and CYTB5, and 32 cycles for PPAR/PPAR2. Aliquots of PCR products were electrophoresed on a 1.5% agarose gel, visualized with ethidium bromide, scanned, and quantified using an Alpha Imager 3400 (Alpha Innotech, San Leandro, CA). Intensities of specific bands were compared with GAPDH as internal control and expressed as a percentage of the control (DMSO-treated cells).
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Real-Time PCR. The mRNA levels of CYP17 and HSD3B2 were assessed by real-time PCR using an ABI 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). In brief, PCR reactions were performed in 96-well plates (Abgene, Epsom, UK) using cDNA prepared as described above (50 ng/20 µl). We used TaqMan Universal PCR Mix and 1 µl of specific primers and probes obtained as assay-on-demand gene expression products (Applied Biosystems). Relative expression values were determined by the 2–
Ct method using 18S rRNA as an internal control. Amplification curves and the mean Ct (threshold) values were calculated using the ABI Prism Sequence Detection System software (Applied Biosystems). Correction for internal control, Ct, is calculated as Cttarget – Ctreference, and reference is the Ct value for 18S; Ct is expressed as Cttreated –Ctcontrol. Increase in RNA expression of each sample by treatment is then calculated by the formula 2–Ct (Livak and Schmittgen, 2001). Results are expressed as a percentage of DMSO control.
Labeling of Steroidogenesis. NCI-H295R cells were cultivated in normal growth medium in six-well plates and treated for 48 h with TZDs. Steroid metabolism was labeled by adding [3H] pregnenolone, [3H]17-hydroxypregnenolone, or [3H]DHEA (all 220,000 cpm/35-mm well) dissolved in ethanol [maximum 0.5% (v/v) concentration] for 90 min, unless indicated otherwise. In some experiments, cells were pretreated with 1 µM trilostane for 2.5 h before adding labeled steroid. Steroids were extracted from the growth medium as described previously (Arlt et al., 2001), separated on thin-layer chromatography (TLC) plates (Whatman, Clifton, NJ) using chloroform/ethyl acetate (3:1) as solvent system, and then exposed and visualized on a Storm PhosphorImager (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Spots corresponding to specific steroids were densitometrically quantified using ImageQuant Software (GE Healthcare). Steroid conversion was assessed by calculating the percentage of radioactivity found in a specific steroid hot spot compared with total radioactivity added to the reaction. To compare activities of P450c17 and 3HSD with and without pioglitazone treatment, the conversion ratio of substrate to product was calculated (Table 2). Results of probes after treatment were expressed as a percentage of mock-treated controls (DMSO).
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-hydroxypregnenolone and DHEA for 17-hydroxylase activity and as a percentage of total radioactivity incorporated in DHEA for 17,20-lyase activity (Fig. 2).
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expression were performed according to the protocols provided by the manufacturer (Cell Signaling Technology). In brief, cells were treated, washed with ice-cold phosphate-buffered saline, and collected in lysis buffer (200 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease and phosphatase inhibitors). Lysates were passed through 25-gauge needles, centrifuged for 15 min at 12,000g at 4°C, from which supernatants were then collected. Protein concentrations of cell extracts were measured by Bio-Rad protein assay (Bio-Rad Laboratories, München, Germany). Aliquots of protein extracts were resolved by 10% SDS-polyacrylamide gel electrophoresis and blotted on Immobilon P transfer membranes (Millipore, Billerica, MA) by semidry transfer method using the Trans-Blot SemiDry apparatus (Bio-Rad Laboratories, Hercules, CA). Blocking and staining of the membranes with antibodies were performed according to the manufacturer's recommendations. Protein bands were visualized by enhanced chemiluminescence substrate reagent and exposed on ECL Plus films (GE Healthcare). Films were scanned by the Alpha Imager so as to quantify intensities of protein bands. -Actin was used as a control for equal loading.
Plasmid Constructs and Transient Transfection of the Cells. Promoter activities of CYP17 and HSD3B2 were characterized by transient transfection of NCI-H295R cells with luciferase reporter gene constructs. Constructs of the long promoter (–3.7 kb) and the short basal promoter (–227 bp) of CYP17 in pMG3 vector have been described earlier (Fluck and Miller, 2004). The HSD3B2 promoter construct was newly built by amplifying the promoter region (–1050/+55 bp) of the HSD3B2 gene using the following primers: sense, 5'-CCGCTCGAGAGTGGGAACTCTGTGGGAATA-3'; and antisense, 5'-GTCCCAAGCTTAGATTGTTAAAAGCTGGACAGA-3' (restriction sites are underlined). The fragment was cloned into polylinker of the pGL3 basic vector (Promega) using HindIII and XhoI restriction sites. The final construct was verified by direct sequencing (Microsynth, Balgach, Switzerland). Reporter construct pCREluc which is a luciferase expression vector under the control of 16 cAMP-responsive elements has been described elsewhere (Fluck et al., 2002). The mammalian expression vector containing the human PPAR1 cDNA was a generous gift of Dr. Chatterjee (Cambridge, UK).
For the transient transfection, NCI-H295R cells were split into 24-well plates at a density of approximately 200,000 cells/well and incubated in transfection medium (Dulbecco's modified Eagle's/Ham's F-12 medium containing 10% NuI serum, supplemented with 0.1% SIT). The next day, transfection was carried out for 6 h at 37°C using 1.6 µl of Lipofectamine 2000 (Invitrogen, Carlsbad, CA), 10 ng of Renilla reniformis luciferase in a pCMV vector (pRL-CMV, an internal control normalizing for cell number and transfection efficiency), and 0.5 µg of specific firefly luciferase reporter plasmids. After transfection, the medium was replaced by growth medium, and cells were allowed to recover for 2 to 3 h before adding TZDs for 48 h. In some experiments, cells were stimulated with 0.5 mM 8Br-cAMP. Finally, cells were lysed and assayed for dual luciferase activities as described by the manufacturer (Promega).
Silencing of PPAR Expression. Oligonucleotides for silencing of PPAR were designed with Ambion design software (Ambion, Austin, TX). Sequences of oligonucleotides (oligonucleotides 1 and 2) are given in Table 1. Oligonucleotides were annealed and cloned into BglII and HindIII cloning sites of pSUPER basic vector (Oligoengine, Seattle, WA). Correct cloning of constructs was confirmed by direct sequencing (Microsynth). NCI-H295R cells were transiently transfected with either an empty vector or shPPAR constructs in suspension. In brief, cells were detached by trypsinization, collected by centrifugation, and diluted in transfection medium containing 10% NuI serum and 0.1% SIT. Aliquots of this cell suspension were mixed with transfection mixture containing plasmid (4 µg/well) and Lipofectamine (9 µl/well) and then seeded onto six-well plates (1 x 106 cells/well). Cells were allowed to transfect and adhere for 6 h, then they were washed, and normal growth medium was added. Forty-eight hours after transfection, either DMSO or 10 µM pioglitazone was applied to the growth medium for the next 36 h before the cells were collected for RNA extraction. Efficiency of transfection (60–70%) was monitored microscopically using enhanced green fluorescent protein expression vector (Promega) as a control.
Statistics. All data represent the mean of at least three independent experiments. Experimental variation is given as SD. Statistical analysis was performed using the two-tailed Student's t test (Excel; Microsoft Corp., Redmond, WA).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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; Guido et al., 2004; Ortega-Gonzalez et al., 2005). In an in vitro model, TZD troglitazone has been shown to inhibit basal and luteinizing hormone (LH) and insulin-stimulated androgen production in human ovarian theca cells (Veldhuis et al., 2002).
We studied the effect of pioglitazone and rosiglitazone on steroidogenesis in human adrenal NCI-H295R cells. Cells were treated with 2.5 to 10 µM pioglitazone or rosiglitazone for 48 h, and steroidogenesis was assessed with [3H] pregnenolone labeling, which is the main precursor for all steroid synthesis. Overall, we found that both TZDs modified the steroidogenic profile of NCI-H295R cells, resulting in a decrease in androstenedione production and a decrease in the use of pregnenolone under both basal and 8Br-cAMP-stimulated conditions (Fig. 1). Calculations of substrate-to-product conversion ratios suggested a possible inhibition of the enzymatic activities of P450c17 and 3HSDII, which was found to reach statistical significance for pioglitazone (Table 2).
Pioglitazone and Rosiglitazone Inhibit Activities of P450c17 and 3HSDII in NCI-H295R Cells. Incubating NCI-H295R cells with [3H]pregnenolone will label the whole steroid pathway, because pregnenolone is the common precursor. Therefore, to measure the effect of TZDs on P450c17 activities in particular, we blocked 3HSDII in NCI-H295R cells with 1 µM trilostane, a specific 3HSD inhibitor. For the assessment of 17-hydroxylase and 17,20-lyase activities, we treated NCI-H295R cells with trilostane and labeled steroidogenesis with [3H]pregnenolone before quantifying the conversion of pregnenolone to 17-hydroxypregnenolone and DHEA (Fig. 2A). For the assessment of 17,20-lyase activity alone, steroidogenesis was labeled with [3H]17-hydroxypregnenolone, and conversion to DHEA was quantified (Fig. 2B). Treatment of NCI-H295R cells with either 10 µM pioglitazone or rosiglitazone for 48 h decreased both activities of P450c17 significantly (Fig. 2, A and B).
Likewise, to assess the effect of TZDs on 3HSDII activity, we treated NCI-H295R cells with 10 µM pioglitazone or rosiglitazone and labeled steroidogenesis with [3H]DHEA, a specific substrate of 3HSDII that will be converted to androstenedione. In our experiments both TZDs inhibited 3HSDII activity (Fig. 2C).
Pioglitazone Inhibits Expression of Genes Involved in Androgen Production. Because TZDs inhibit the multistep metabolism of pregnenolone through the steroidogenic pathway, they may inhibit steroidogenic enzymes either by repressing the expression of these enzymes or by direct inhibition of enzymatic activities. In fact, pioglitazone and other TZDs have been shown to inhibit enzymatic activities of P450c17 and 3HSDII directly in vitro (Arlt et al., 2001), but this inhibition was observed only at supratherapeutic concentrations of these drugs. Thus, we studied the effect of pioglitazone on steroidogenesis in NCI-H295R cells by assessing the expression of the involved genes at the mRNA level. Analysis of RNA expression by semiquantitative RT-PCR revealed that treatment with 2.5 to 10 µM pioglitazone down-regulated basal expression of CYP17 and HSD3B2 (Fig. 3A). Stimulation of NCI-H295R cells by 8Br-cAMP activated the expression of both CYP17 and HSD3B2 genes, but the inhibitory effect of pioglitazone persisted (Fig. 3A). In contrast, pioglitazone did not influence the expression of genes for microsomal cytochrome b5, a protein that supports 17,20-lyse activity of P450c17, and for sulfotransferase SULT2A1, the enzyme responsible for conversion of DHEA to DHEA-S (data not shown). To better quantify the effect of pioglitazone on gene expression, we performed quantitative real-time PCR. We found that pioglitazone inhibited CYP17 and HSD3B2 expression at concentrations greater than 2.5 µM, down-regulating CYP17 to 40% and HSD3B2 to 
50% at 10 µM concentration. Stimulation with 0.5 mM 8-Br-cAMP for 24 h induced the expression of both genes at the RNA level by 3- to 4.5-fold, but pioglitazone was also able to inhibit the expression of CYP17 (40%) and HSD3B2 (70%) in the stimulated state (Fig. 3B).
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), corroborating our hypothesis. Alternatively, pioglitazone might affect the stability of HSD3B2 mRNA. However, after stimulating NCI-H295R cells with 0.5 mM 8Br-cAMP, the activity of the transiently transfected HSD3B2 promoter reporter increased by 13-fold, and pioglitazone treatment inhibited this activity by approximately 50% (Fig. 4). Thus, pioglitazone showed an inhibitory effect on promoter activities of both CYP17 and HSD3B2 genes.
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Rosiglitazone Does Not Change Gene Expression of CYP17 and HSD3B2. Clinical studies have demonstrated that treatment with pioglitazone and rosiglitazone lowers elevated plasma androgen concentrations in women with PCOS (Brettenthaler et al., 2004; Ortega-Gonzalez et al., 2005; Sepilian and Nagamani, 2005). Analysis of the activities of P450c17 and 3HSDII in NCI-H295R cells treated with rosiglitazone and pioglitazone revealed a similar pattern, although the effect of rosiglitazone seemed to be weaker in quantity (Figs. 1C and 2). Thus, we tested whether rosiglitazone would also change the expression of CYP17 and HSD3B2 genes similar to pioglitazone. Semiquantitative and quantitative real-time PCR experiments were performed on reverse-transcribed RNA extracted from NCI-H295R cells treated with different concentrations of rosiglitazone. In contrast to pioglitazone, rosiglitazone did not change the expression of the CYP17 and HSD3B2 genes significantly, even after stimulation with 0.5 mM 8Br-cAMP (Fig. 5, A and B). Likewise, rosiglitazone did not change the activities of CYP17 and HSD3B2 promoter reporter constructs transfected into NCI-H295R cells (Fig. 5C). These data suggest that unlike pioglitazone, rosiglitazone does not affect androgen production in NCI-H295R cells by modulating expression of genes essential for the biosynthesis of androgens. Therefore, we focused further studies on the regulation of the CYP17 and HSD3B2 genes by pioglitazone.
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Pioglitazone Does Not Signal through the cAMP/PKA/CREBP. Glucocorticoid and androgen production in the human adrenal cortex may be stimulated by the ACTH/cAMP/PKA pathway. Because pioglitazone lowers the ACTH-stimulated androgen production in women with PCOS (Guido et al., 2004), and because we found that pioglitazone treatment repressed 8Br-cAMP-stimulated expression of CYP17 and HSD3B2 in NCI-H295R cells (Figs. 3 and 4), we tested the hypothesis that pioglitazone might interfere with the classic cAMP/PKA/CREBP pathway. We transfected NCI-H295R cells with the luciferase reporter construct pCREluc, which is driven by cAMP-dependent proteins binding to 16 CREs in the promoter (Fluck et al., 2002). Stimulation with 8Br-cAMP activated this luciferase reporter 2.5-fold, and pioglitazone treatment did not alter this stimulation (Fig. 6). Because pioglitazone did not change luciferase activity of pCREluc, we can also exclude that pioglitazone may directly inhibit the enzymatic activity of luciferase. These findings suggest that pioglitazone does not inhibit androgen biosynthesis in NCI-H295R cells by interfering with the regulation of CYP17 and HSD3B2 genes through cAMP/PKA/CREBP signaling.
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). Moreover, theca cells isolated from ovaries of women with PCOS were found to have decreased activity of MEK and ERK1/2 kinases, a finding which is in line with enhanced expression of CYP17 mRNA and increased production of androstenedione observed in these cells (Nelson-Degrave et al., 2005). To study the possible regulation of CYP17 and HSD3B2 by the MAPK pathway in NCI-H295R cells, we compared expression and promoter activities of these genes under the influence of the MEK inhibitor PD98059. Quantitative real-time PCR analysis of gene expression revealed that treatment with 10 µM PD98059 enhanced CYP17 expression 3-fold and HSD3B2 expression 2.5- to 4-fold (Fig. 7A). However, when NCI-H295R cells were treated with pioglitazone, ERK inhibition did not increase CYP17 expression. The observed effect of pioglitazone treatment and ERK inhibition on HSD3B2 expression was similar, but statistical analysis did not reach significance. Likewise, ERK inhibition by PD98059 enhanced the activity of the CYP17 promoter reporter in NCI-H295R cells significantly, and pioglitazone treatment repressed this activation. In contrast, PD98059 treatment alone or in combination with pioglitazone did not change the activity of the HSD3B2 promoter construct significantly when transfected into NCI-H295R cells (Fig. 7B).
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To analyze the phosphorylation status of ERK1/2 in NCI-H295R cells, which directly correlates with ERK activity, we performed a Western blot (Fig. 7, C and D). Pioglitazone slightly increased while 10 µM PD98059 partially decreased the phosphorylation of ERK (P-ERK) in NCI-H295R cells. It is interesting that, when treating NCI-H295R cells with both pioglitazone and the ERK inhibitor PD98059, P-ERK remained moderately up-regulated, similar to pioglitazone treatment alone. Phosphorylation of MEK (P-MEK), the kinase which is responsible for the phosphorylation and thus activation of ERK, was not influenced by pioglitazone or PD98059 (Fig. 7C). Thus, pioglitazone might interfere with MAPK signaling at this level or downstream of the MEK kinase. Stimulation of NCI-H295R cells with 8Br-cAMP markedly induced the amount of P-ERK and slightly of P-MEK, but pioglitazone treatment did not alter this activation, thus confirming our previous suggestion that pioglitazone does not affect cAMP/PKA signaling (Fig. 7C).
To further assess the influence of pioglitazone on ERK-regulated steroidogenesis, we analyzed the activities of P450c17 and 3HSDII in NCI-H295R cells under PD98059 treatment with or without pioglitazone (Fig. 7, E and F). Partial inhibition of ERK resulted in an increase in 17,20-lyase activity of P450c17 and consequently more DHEA production when measured in the presence of 1 µM trilostane. Consistent with previous experiments, pioglitazone inhibited the increase in P450c17 activity (Fig. 7, E, top, and F). In contrast, using [3H]DHEA as a specific substrate, we found that 3HSDII activity was not significantly influenced by PD98059, but pioglitazone still decreased 3HSDII activity in its presence (Fig. 7, E and F).
The Effect of Pioglitazone on CYP17 and HSD3B2 Expression Seems to be PPAR-Independent. TZDs like pioglitazone are believed to be ligands for PPAR, which belong to the large group of nuclear receptors. To evaluate whether the effect of pioglitazone on androgen production might be PPAR-mediated, we first analyzed human adrenal NCI-H295R cells and adult human adrenal tissues for PPAR expression. PPAR has two splice variants, PPAR1, which is known to be expressed in many tissues, and PPAR2, the specific variant for white adipose tissue (Kota et al., 2005). Analysis of reverse-transcribed RNA by PCR revealed that both human adrenals and human adrenal NCI-H295R cells express PPAR1 but not PPAR2 (Fig. 8A).
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expression in NCI-H295R cells, we built two vectors for targeted PPAR silencing (Table 2). Transfection of NCI-H295R cells with these constructs caused a significant decrease in PPAR expression at RNA and protein level regardless of whether cells were treated with 10 µM pioglitazone or with DMSO as a vehicle (Fig. 8, B and C). Pioglitazone treatment slightly increased the expression of PPAR (Fig. 8B). Despite effective silencing of PPAR in NCI-H295R cells, pioglitazone inhibited both CYP17 and HSD3B2 gene expression to the same extent as in NCI-H295R control cells (Fig. 8D), and silencing of PPAR did not influence basal levels of CYP17 and HSD3B2 expression (data not shown). Likewise, transfection of NCI-H295R cells with a pCDNA3-PPAR1 vector for overexpressing PPAR did not change the effect of pioglitazone on CYP17 and HSD3B2 gene expression (data not shown). These data indicate that PPAR is not required for mediating the inhibitory action of pioglitazone on CYP17 and HSD3B2 gene expression.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The cAMP/PKA pathway is the major signaling pathway regulating steroidogenesis (Stocco et al., 2005). cAMP is the main secondary messenger that is generated after stimulation of the gonadotropin receptors in the gonads and the ACTH receptor in the adrenal cortex (Sewer and Waterman, 2003a). Typically, cAMP activates PKA, which then induces gene transcription by phosphorylating and thus activating CREBP. Activities of most steroidogenic enzymes, including P450c17 and 3HSDII, may be stimulated by cAMP in the so-called "long-term" response, a process which seems to require synthesis of new proteins because it was shown to be sensitive to inhibition of translation by cycloheximide (Waterman and Bischof, 1996). However, conflicting with classic cAMP/PKA signaling, promoters of genes for most steroidogenic enzymes do not contain typical CREs. In murine Leydig cells, LH-stimulated expression of CYP17 involves cAMP/PKA but is independent of CREBP (Laurich et al., 2002). In addition, several reports have established the role of a possible cross-talk between cAMP/PKA and other signaling pathways such as MEK1/ERK or phosphatidylinositol 3-kinase/protein kinase B (Richards, 2001). In mouse Leydig cells, LH and 8Br-cAMP were shown to stimulate phosphorylation of ERK by PKA-dependent activation of Ras (Hirakawa and Ascoli, 2003). In human adrenal NCI-H295R cells, cAMP-mediated transcription of the CYP17 gene was demonstrated to be dependent on the activity of dual specificity phosphatase, which was later identified as MAPK phosphatase-1 (Sewer and Waterman, 2002). MAPK phosphatase-1 may directly dephosphorylate kinase ERK1/2 and transcription factor SF-1, which is essential for the expression of almost all steroidogenic genes (Sewer and Waterman, 2003b).
Compared with healthy human theca cells, theca cells in women with PCOS have a significantly altered MEK/ERK phosphorylation status, leading to higher expression of the CYP17 gene and enhanced androstenedione production (Nelson-Degrave et al., 2005). Consistent with this, treatment of normal human theca cells with the MEK inhibitor PD98059 leads to higher abundance of CYP17 mRNA and higher activity of CYP17 promoter constructs in luciferase assays. Likewise, we can demonstrate that partial inhibition of the ERK pathway increases the expression of the CYP17 and HSD3B2 genes and increases the activity of the CYP17 promoter in human adrenal NCI-H295R cells. ERK inhibition seems to predominantly enhance the 17,20-lyase activity. In addition, we show for the first time that these effects may be antagonized by pioglitazone. Moreover, pioglitazone increases phosphorylation of ERK1 in the presence of the inhibitor PD98059 without affecting phosphorylation of MEK, indicating that it may modulate ERK downstream of MEK. Therefore, we suggest that pioglitazone inhibits androgen production, at least in part, through a modulatory effect on ERK phosphorylation.
Although both pioglitazone and rosiglitazone are TZDs and both lower plasma androgen levels in women suffering from PCOS, we find that only pioglitazone inhibits CYP17 and HSD3B2 gene transcription and expression. Thus, in NCI-H295R cells, molecular mechanisms of the androgen-lowering action of pioglitazone and rosiglitazone must be at least partially different. Differing effect of pioglitazone compared with rosiglitazone has been reported in terms of basal and follicle-stimulating hormone-stimulated estradiol production in human ovarian cells (Seto-Young et al., 2005). In that study, pioglitazone seemed to be significantly more potent, but the effect on progesterone production and insulin-induced inhibition of insulin-like growth factor binding protein 1 was similar for both TZDs. In contrast, a former in vitro study showed that both pioglitazone and rosiglitazone repress enzymatic activities of recombinant P450c17 and 3-HSDII directly (Arlt et al., 2001). Direct enzymatic inhibition is strongest for troglitazone at 10 to 20 µM concentrations followed by rosiglitazone (50–100 µM) and pioglitazone at highly supratherapeutic concentrations (>100 µM). Thus, the effect of pioglitazone on androgen production seems to be predominantly at the level of gene regulation, and rosiglitazone may directly modulate the activities of these enzymes. However, from our studies, we cannot exclude that rosiglitazone may also act through other signaling cascades regulating targeted enzymes at the posttranslational level.
TZDs are ligands for PPAR. Therefore, it seems compelling to hypothesize that their influence on androgen production might be mediated through the nuclear receptor PPAR. NCI-H295R cells and human adrenal tissues express PPAR splice variant 1 which is highly expressed in liver, adipose tissue, and macrophages. It is interesting that silencing or overexpressing PPAR in NCI-H295R cells did not change the inhibitory effect of pioglitazone on CYP17 and HSD3B2 expression. Thus, we conclude that pioglitazone influences steroidogenic gene expression independently of its nuclear receptor. For inhibiting gene expression, we used pioglitazone in slightly higher doses (5–10 µM) than is used in the clinical setting of patients with T2DM (1–5 µM) (Zhong and Williams, 1996). But direct comparison of effective drug concentrations in vivo and in vitro may be inappropriate for many reasons such as binding of the drug to proteins or accumulation in tissues. Effective concentrations used in vivo and in vitro greatly exceed reported IC50 affinity constants for TZDs binding to PPAR, which were calculated to be 0.55 µM for pioglitazone and 0.075 µM for rosiglitazone (Willson et al., 1996). Thus, the fact that the antiandrogenic effect of pioglitazone is not PPAR-mediated may also explain why studies of the binding affinity of TZDs and PPAR do not necessarily correlate with their biological activity. Others have reported PPAR-independent effects of TZDs (Feinstein et al., 2005). Perhaps the most interesting findings in line with our results are the observations that PPAR ligands such as ciglitazone, troglitazone, and pioglitazone increase ERK activity in vascular smooth muscle cells (Takeda et al., 2001) and transactivate the epidermal growth factor receptor in rat liver cells independent of PPAR (Gardner et al., 2003).
In summary, pioglitazone inhibits androgen production and the expression of the CYP17 and HSD3B2 genes in NCI-H295R cells, at least in part, through modulation of ERK signaling, and this effect is PPAR-independent. We cannot exclude the possibility that pioglitazone uses additional pathways for modulating the production of androgens. There is growing evidence that TZDs have further mechanisms of actions including direct interactions with mitochondrial metabolism or with the activities of essential transcription factors, which are mostly regulated by (de)phosphorylation. TZDs are widely used for the treatment of common diseases such as T2DM and PCOS. The spectrum of the clinical use of TZDs might even broaden in the near future given their possible anti-inflammatory potential, action as tumor suppressors, or anti-atherogenic effect (Michalik et al., 2004; Feinstein et al., 2005). Thus, our findings underline the importance of characterizing biological effects of each single TZD specifically because their mode of action seems to be compound-specific rather than a characteristic of all TZDs.
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Acknowledgements |
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1 and PPAR2 plasmids. We thank Takeda Pharma AG (Lachen, Switzerland) for providing pioglitazone and GlaxoSmithKline AG, (Münchenbuchsee, Switzerland) for providing rosiglitazone for our studies. |
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: DHEA, dehydroepiandrosterone; ACTH, adrenocorticotropic hormone; 8Br-cAMP, 8-bromo-cAMP; DMSO, dimethyl sulfoxide; ERK1/2, extracellularly regulated kinase 1/2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 3HSDII (HSD3B2), 3-hydroxysteroid dehydrogenase type 2; LH, luteinizing hormone; PCOS, polycystic ovary syndrome; PKA, protein kinase A, PPAR, peroxisome proliferator activated receptor; sh, short hairpin; SIT, selenium/insulin/transferrin supplement; T2DM, type 2 diabetes mellitus; TLC, thin-layer chromatography; TZD, thiazolidinedione; PD98059, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; ROS, rosiglitazone; PIO, pioglitazone; MAPK, mitogen-activated protein kinase; kb, kilobase(s); bp, base pair(s); PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; MEK, mitogen-activated protein kinase kinase; CREBP, cAMP response element-binding protein.
Address correspondence to: Dr. Christa E. Flück, Pediatric Endocrinology and Diabetology, University Children's Hospital Bern, G3 812, Freiburgstrasse 15, 3010 Bern, Switzerland. E-mail: christa.flueck{at}insel.ch
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0.05. D, DMSO treatment; A, androstenedione; Preg, pregnenolone; 17OH Preg, 17
