Thibaud Tranchant

Guillaume Durand

Christophe Gauthier

Pascale Crépieux

Alfredo Ulloa-Aguirre

Dominique Royère

Eric Reiter
email: eric.reiter@tours.inra.fr

From

Preferential -arrestin signalling at low receptor density revealed by functional characterization of the human FSH receptor A189V mutation

Keywords: FSH, FSH receptor, A189V mutation, -arrestin, selective signalling

The A189V inactivating mutation of the human FSH receptor (FSHR) leads to subfertility in men and primary ovarian failure in women. This mutation has previously been associated with intracellular retention of the FSHR and impaired cAMP production. Here, we show that the A189V FSHR stably expressed in HEK293N cells provoked ERK MAP kinases phosphorylation through β-arrestins, independently of the canonical cAMP/PKA pathway.

Interesting, both the A189V and wild-type (Wt) FSHRs selectively activated cAMPindependent ERK phosphorylation when expressed at low plasma membrane densities. These data indicate that the selective intracellular signalling triggered by the A189V FSHR resulted from reduced membrane expression rather than by switching receptor coupling. Hence, receptor density at the plasma membrane might control the balance between distinct signal transduction mechanisms. Furthermore, our results help to clarify why mutations of FSH are more deleterious to human fertility than the FSHR A189V mutation which preserves parts of receptor signalling repertoire.

A c c e p t e d M a n u s c r i p t

INTRODUCTION

Follicle-stimulating hormone (FSH) is a glycoprotein synthesized by the pituitary gland. This hormone binds to its cognate receptor (FSHR) in the gonads. The FSHR is expressed by the granulosa cells of the ovarian follicle and by the Sertoli cells of the seminiferous tubules. This gonadotropin promotes folliculogenesis and supports spermatogenesis thereby playing a key role in reproduction.

Knock-out of either the FSHβ-subunit or the FSHR gene in mice suggests that FSH action is fundamental for female reproduction whereas in males, although necessary to achieve normal testis size and sperm production, it is not essential for fertility [START_REF] Kumar | Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility[END_REF][START_REF] Dierich | Impairing follicle-stimulating hormone (FSH) signaling in vivo: targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance[END_REF][START_REF] Krishnamurthy | Qualitative and quantitative decline in spermatogenesis of the follicle-stimulating hormone receptor knockout (FORKO) mouse[END_REF]. In women, inactivating mutations of either the FSHβsubunit or the FSHR lead to primary amenorrhea and infertility, again suggesting that FSH action is crucial for female reproduction [START_REF] Matthews | Primary amenorrhoea and infertility due to a mutation in the beta-subunit of folliclestimulating hormone[END_REF][START_REF] Aittomaki | Mutation in the follicle-stimulating hormone receptor gene causes hereditary hypergonadotropic ovarian failure[END_REF][START_REF] Layman | Delayed puberty and hypogonadism caused by mutations in the follicle-stimulating hormone betasubunit gene[END_REF][START_REF] Layman | FSH beta gene mutations in a female with partial breast development and a male sibling with normal puberty and azoospermia[END_REF][START_REF] Huhtaniemi | Mutations along the pituitary-gonadal axis affecting sexual maturation: novel information from transgenic and knockout mice[END_REF]. In men, the A189V mutation is the only FSHR inactivating mutation reported so far. In agreement with the FSHR knock-out male phenotype, the five Finnish men presenting the A189V mutation displayed variable degrees of oligozoospermia and subfertility [START_REF] Tapanainen | Men homozygous for an inactivating mutation of the follicle-stimulating hormone (FSH) receptor gene present variable suppression of spermatogenesis and fertility[END_REF]. Surprisingly however, men with inactivating mutations of the FSHβ-subunit were completely infertile due to azoospermia [START_REF] Lindstedt | Follitropin (FSH) deficiency in an infertile male due to FSHbeta gene mutation. A syndrome of normal puberty and virilization but underdeveloped testicles with azoospermia, low FSH but high lutropin and normal serum testosterone concentrations[END_REF][START_REF] Phillip | Male hypogonadism due to a mutation in the gene for the beta-subunit of follicle-stimulating hormone[END_REF][START_REF] Layman | FSH beta gene mutations in a female with partial breast development and a male sibling with normal puberty and azoospermia[END_REF]. The reason(s) for this apparent discrepancy between the severity of the phenotypes associated with inactivating mutations in the FSHβ-subunit versus the FSHR has previously been pointed out but still remains to be clarified [START_REF] Huhtaniemi | Mutations along the pituitary-gonadal axis affecting sexual maturation: novel information from transgenic and knockout mice[END_REF].

The FSHR belongs to the superfamily of G protein-coupled receptors (GPCR). It is widely accepted that upon ligand stimulation GPCRs adopt an active conformation which allows the recruitment of one or more Gα subunits of the heterotrimeric G proteins followed A c c e p t e d M a n u s c r i p t by activation of enzyme effectors, production of second messengers, and transduction of intracellular signals [START_REF] Neer | Heterotrimeric G proteins: organizers of transmembrane signals[END_REF]. Until recently, second messengers were thought to be the only mediators for GPCRs to trigger signalling pathways following agonist stimulation.

Therefore, mutations at GPCRs inhibiting second messenger production were classified as inactivating or loss-of-function mutations. Accordingly, the A189V mutation, which dramatically decreases expression of the FSHR at the plasma membrane [START_REF] Rannikko | Functional characterization of the human FSH receptor with an inactivating Ala189Val mutation[END_REF] and suppresses FSH-stimulated cAMP production has been classified as an inactivating mutation [START_REF] Aittomaki | Mutation in the follicle-stimulating hormone receptor gene causes hereditary hypergonadotropic ovarian failure[END_REF]).

However, it has been recently described for many GPCR [START_REF] Luttrell | Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes[END_REF]DeFea et al. 2000a;DeFea et al. 2000b;[START_REF] Luttrell | Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds[END_REF][START_REF] Reiter | GRKs and beta-arrestins: roles in receptor silencing, trafficking and signaling[END_REF][START_REF] Shenoy | beta-arrestin-dependent, G protein-independent ERK1/2 activation by the beta2 adrenergic receptor[END_REF], including the FSHR [START_REF] Kara | A phosphorylation cluster of five serine and threonine residues in the C-terminus of the follicle-stimulating hormone receptor is important for desensitization but not for beta-arrestin-mediated ERK activation[END_REF], that β-arrestins -molecules known for their key role in desensitization and internalisation of GPCRs-are able to activate signalling pathways in response to agonist stimulation. Moreover, it has been demonstrated that G protein-and βarrestin-dependent signalling pathways are independent. A number of GPCR mutants have been reported to selectively trigger β-arrestin-dependent signalling pathways without inducing G protein coupling [START_REF] Wei | Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2[END_REF][START_REF] Reiter | GRKs and beta-arrestins: roles in receptor silencing, trafficking and signaling[END_REF][START_REF] Shenoy | beta-arrestin-dependent, G protein-independent ERK1/2 activation by the beta2 adrenergic receptor[END_REF]). In addition, some GPCR ligands have recently been reported to selectively activate β-arrestinmediated signalling [START_REF] Azzi | Beta-arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors[END_REF][START_REF] Wei | Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2[END_REF][START_REF] Gesty-Palmer | Distinct betaarrestin-and G protein-dependent pathways for parathyroid hormone receptorstimulated ERK1/2 activation[END_REF][START_REF] Wisler | A unique mechanism of beta-blocker action: carvedilol stimulates beta-arrestin signaling[END_REF][START_REF] Drake | beta-arrestin-biased agonism at the beta2-adrenergic receptor[END_REF]. Consistent with this concept, we recently demonstrated that a partially deglycosylated equine LH was able to selectively activate β-arrestin-dependent signalling pathways in the absence of cAMP production at the FSHR (Wehbi et al. 2010a) In light of this emerging paradigm for GPCR signalling, one hypothesis that may explain the discrepancy between subfertility of men carrying the A189V FSHR mutation versus infertility caused by the FSHβ-subunit inactivating mutations would be that the A189V receptor might actually retain some G protein-independent signalling properties. In the present study, we

Reagents

3-isobutyl-1-methylxanthine (IBMX) and forskolin were purchased from Sigma-Aldrich (St Louis, MO, USA). H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-iso-quinolinesulfonamide) was purchased from Calbiochem (San Diego, CA, USA). G418 was from Invivogen (Invivogen, San Diego, CA, USA). Minimum essential medium, fetal bovine serum, penicillin, streptomycin, trypsin-EDTA and bovine serum albumin were obtained from PAA Laboratories GmbH (Pasching, Austria). Rabbit polyclonal anti-ERK2 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), while rabbit polyclonal anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) antibody was from Cell Signaling Technology Inc.

(Danvers, MA, USA). Rabbit polyclonal anti--arrestin 1 and 2 antibody (A1CT) was a kind gift from Dr. R.J. Lefkowitz (Duke University, Durham, NC, USA). The Fugene 6 transfection reagent was purchased from Roche Diagnostics (Basel, Switzerland). The SuperSignal West Pico Chemiluminescent substrate was obtained from Pierce (Pierce Biotechnologies, Rockford, IL, USA). Highly purified porcine FSH (pFSH) was kindly provided by Dr. J. Closset (Université de Liège, Belgium) and Dr. G.R. Bousfield (Department of Biological Sciences, Wichita State University, Wichita, KA, USA).

Plasmids and cells

The plasmid encoding the full-length human FSHR-FLAG fusion protein was generated employing the procedure previously described to construct the rat FSHR-FLAG [START_REF] Reiter | Kinase-inactive G-protein-coupled receptor kinases are able to attenuate folliclestimulating hormone-induced signaling[END_REF]. The original full-length human FSHR cDNA was obtained from the Missouri S&T cDNA Ressource Center (Rolla, MO, USA; www.cdna.org). HEK293N cells were grown in minimum essential medium supplemented with 10% heat-inactivated fetal bovine serum, 100 A c c e p t e d M a n u s c r i p t U/ml penicillin, and 100 µg/ml streptomycin, at 37°C in a humidified 5% CO 2 incubator.

Cells were transfected at 60-70% confluency with 0.018 µg/cm 2 of the plasmid encoding the full-length human FSHR-FLAG using the Fugene 6 transfection reagent. HEK293N cells stably expressing either the Wt or the A189V FSHR were obtained following G418 selection.

To obtain similar levels of plasma membrane expression of the Wt and the mutant A189V FSHRs, HEK293N cells were transiently transfected with 1.05 ng/cm 2 of the full-length human Wt FSHR or 42 ng/cm 2 of full length A189V FSHR. The total amount of DNA transfected was kept constant by adding appropriate amounts of pcDNA 3.1 empty vector to wells transfected with the Wt FSHR.

Binding assays

pFSH was labeled following the IODO-GEN (Pierce, Rockford, IL, USA) method. Total binding of [ 125 I]-labelled pFSH to the Wt or A189V FSHR were measured after 1 hour exposure to intact cells plated in 96-well plates at 37°C. For 125 I-FSH displacement experiments, cells were incubated 1 hour at 37°C with a mix containing 25,000 cpm of 125 I-FSH and increasing doses (0 to 33 nM) of unlabelled pFSH. Binding reactions were carried out in 41.5 l of binding buffer (minimum essential medium, 10 U/ml penicillin, 10 µg/ml streptomycin, 250 mM Hepes and 0.1 % BSA) with 100,000 cpm of 125 I-pFSH. At the end of the incubation period, cell monolayers were washed thrice with 200 µl PBS and then lysed in 200 µl of 0.5 M NaOH. The amount of radiactivity in each lysate was quantitated by spectroscopy using a Wallac Wizard scintillation counter (Amersham Biosciences, Pittsburgh, PA, USA). Each condition was assayed by triplicate.

cAMP assay

A c c e p t e d M a n u s c r i p t HEK293N cells stably expressing either the human Wt or A189V FSHR (cultured on 24-well plates and grown to 50% confluency) were serum-starved in the presence of 100 µM IBMX for 30 min prior to stimulation. Cells were incubated for 1 hour with pFSH in the presence of 100 µM IBMX at 37°C in a humidified 5% CO 2 incubator. Total cAMP was measured using a commercially available cAMP ELISA kit (Biomedical Technologies, Inc., Stoughton, MA, USA) according to the manufacturer's instructions.

FRET assays

HEK293N cells stably or transiently expressing the human Wt or A189V FSHR were transiently transfected with either the cAMP (ICUE) or PKA (AKAR) plasmid-encoded FRET sensor. Both sensors were kindly provided by Dr. Jin Zhang (Johns Hopkins University, Baltimore, MD, USA) (Zhang et al. 2001;[START_REF] Dipilato | Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments[END_REF]). Twenty-four hours after transfection, cells were splitted and seeded on imaging dishes (BD Biocoat, BD Biosciences, San Jose, CA, USA). Forty-eight hours after transfection, cells were serumstarved for 90 min and imaged in the dark on a 37°C temperature-controlled stage. Cells were imaged on a Leica DM IRBE (Leica Microsystems, Wetzlar, Germany) microscope with a CoolSnap fx cooled charge-coupled device camera (Roper Scientific, Ottobrunn, Germany) controlled by METAFLUOR 7.5 (Universal Imaging Corporation, Downingtown, PA, USA).

Dual emission ratio imaging used a 436DF10 excitation filter, a 436-510 DBDR dichroic mirror, and 480AF30 and 550AF30 emission filters for CFP and YFP, respectively. The filters were alternated by a Lambda 10-2 filter changer (Sutter Instruments, Novato, CA, USA). Exposure time was 100 ms, and images were taken every 15 sec. Typically, 10 sensorpositive cells and 4 non-specific areas were chosen in the field of the microscope. Base line signals at the wavelength corresponding to YFP and CFP were recorded for 5 min and then, the hormone was added to the dish. Signals were recorded for the next 40 min before

Reporter gene assay

HEK293N cells stably expressing either the human Wt or A189V FSHR were transiently transfected with the pSOMLuc reporter gene as previously described [START_REF] Lazari | Role of G proteincoupled receptor kinases on the agonist-induced phosphorylation and internalization of the follitropin receptor[END_REF][START_REF] Troispoux | Involvement of G protein-coupled receptor kinases and arrestins in desensitization to follicle-stimulating hormone action[END_REF]. Cells were splited into 12-well plates 24 hours after transfection.

Forty-eight hours after transfection, cells were serum-starved for 24 hours, then stimulated for 6 hours with pFSH. Cell were then washed twice with cold PBS and lysed with passive lysis buffer. The luciferase activity was measured using the luciferase assay system supplied by Promega Corp (Madison, WI, USA). Each sample (20 µl) was mixed with 50 µl of luciferase assay reagent, containing the substrate. The light produced was measured in a luminescence counter (Luminoskan Ascent, Thermo Labsystems, Waltham, MA, USA) and expressed as fold increase over basal.

Small interfering RNA transfection

The siRNA sequence 5'-AAACCUGCGCCUUCCGCUAUG-3' was used to simultaneously target human β-arrestin 1 (positions 172-190 relative to the start codon) and β-arrestin 2 (position 175-193). One small RNA duplex with no silencing effect was used as a control (5'-UUCUCCGAACGUGUCACGU-3'). These chemically synthesized siRNAs (Eurogentec SA, Liège, Belgium) have been previously described and validated [START_REF] Gesty-Palmer | Distinct betaarrestin-and G protein-dependent pathways for parathyroid hormone receptorstimulated ERK1/2 activation[END_REF] A c c e p t e d M a n u s c r i p t [START_REF] Kara | A phosphorylation cluster of five serine and threonine residues in the C-terminus of the follicle-stimulating hormone receptor is important for desensitization but not for beta-arrestin-mediated ERK activation[END_REF]. Early passage HEK293N cells stably expressing human Wt or A189V FSHR at 10-20% confluency in 100 mm dishes were transfected with siRNA using the GeneSilencer transfection reagent following the manufacturer's recommendations (Genlantis, San Diego, CA, USA). Forty-eight hours after transfection, cells were seeded into 12-well plates. All assays were performed 3 days after transfection.

Western blotting

Cells were stimulated with pFSH for various time periods (kinetics) or for 5 minutes with increasing doses of hormone in the presence of vehicle or H89 and the supernatants were removed. Cells were then lysed in 2X Laemmli buffer and equivalent amounts of protein were analysed by Western blot. The membranes were incubated overnight at 4°C with antiphospho-ERK 1/2 (1:3000) and then with secondary anti-mouse IgG horseradish peroxidase conjugate (Biosource International, Armadillo, CA, USA). Signal was developed using the Pierce ECL Western Blotting detection kit (Rockford, IL, USA). Equal protein loading was confirmed in a stripped, washed and reprobed membrane with primary polyclonal antibody against total ERK2 (1:10,000). To monitor β-arrestin protein silencing, endogenous βarrestins 1 and 2 were detected with A1CT antibody (1:3000), as previously described [START_REF] Kara | A phosphorylation cluster of five serine and threonine residues in the C-terminus of the follicle-stimulating hormone receptor is important for desensitization but not for beta-arrestin-mediated ERK activation[END_REF].

Statistical analysis

Statistical analysis was performed with one-way analysis of variance (followed by Bonferroni's multiple comparison tests) to compare results from several samples or with twoway analysis of variance to compare entire curves (GraphPad PRISM Software, San Diego, CA, USA).

A c c e p t e d M a n u s c r i p t 3 Results

The A189V mutation leads to a dramatic reduction of FSHR expression at the plasma membrane in stably transfected HEK293N cells

HEK293N cells were stably transfected using the pcDNA 3.1(+) encoding the full length human FSHR sequence bearing the N-terminus FLAG (DYKDDDDK) and hemaglutinin signal peptide (MKTIIALSYIFCLVFA) sequences (Fig. 1A). Total binding experiments displayed that A189V FSHR is expressed at 1.5% of Wt levels (Fig. 1B). These results are consistent with previous studies showing a dramatic decrease in plasma membrane expression of the A189V FSHR when transiently expressed in MSC-1 or COS7 cell lines. Displacement curves and subsequent Scatchard analysis showed that the calculated Kd for the A189V FSHR (1.3 nM) is similar to that previously reported for the Wt FSHR (0.780 nM) and the A189V FSHR (0.820 nM) (Ranniko et al. 2002) (Figs. 1A and B of supplementary data); the number of cell surface A189V FSHRs per cell was 1500 as disclosed by this analysis.

The A189V mutation suppresses FSHR coupling to the cAMP/PKA pathway.

It has been found that FSH-stimulated cAMP production by MSC-1 or COS7 cell lines transiently expressing the A189V mutation is significantly decreased when compared to the Wt FSHR [START_REF] Aittomaki | Mutation in the follicle-stimulating hormone receptor gene causes hereditary hypergonadotropic ovarian failure[END_REF][START_REF] Rannikko | Functional characterization of the human FSH receptor with an inactivating Ala189Val mutation[END_REF]. Here, we investigated whether this mutation impaired cAMP response to FSH exposure in HEK293N cells stably expressing the FSHR. Dose-response curves of cAMP accumulation in the presence of the phosphodiesterase inhibitor IBMX clearly showed that the A189V mutation completely suppressed the ability of the FSHR to elicit cAMP production after 1 hour of FSH exposure (Fig. 2A). As expected, A c c e p t e d M a n u s c r i p t this response was in sharp contrast with that observed in cells expressing the Wt FSHR. FRET assays using the cAMP sensor ICUE [START_REF] Dipilato | Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments[END_REF]) were then carried out in living cells in order to rule out the possibility of transient intracellular cAMP production that may pass undetected by the cAMP assay. As shown in Fig. 2B, FRET experiments revealed that the robust and rapid agonist-stimulated cAMP production presented by cells expressing the Wt receptor was completely abrogated in cells expressing the A189V mutant. In both conditions, however, adenylyl-cyclase activity remained inducible as revealed by the addition of forskolin (Fig. 2B). To further corroborate this observation, PKA activation was measured in living cells using the FRET sensor AKAR [START_REF] Allen | Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters[END_REF]. Consistent with the results from the cAMP assays, we found that the A189V mutation repressed FSH-induced PKA activation without affecting PKA activity inducible by forskolin (Fig. 2C). Finally, in order to compare the cAMP/PKA transcriptional-associated response elicited by the Wt and mutant FSHRs, cells were transfected with the cAMP-sensitive reporter plasmid pSOMLuc.

The results showed that the Wt FSHR induced a robust dose-dependent response in luciferase activity whereas the A189V mutant FSHR failed to trigger any significant response (Fig. 2D).

The A189V FSHR mutant induces ERK MAPK phosphorylation in response to FSH stimulation

We next examined whether the A189V FSHR, which is unable to activate the cAMP/PKA transduction pathway, retained its ability to trigger ERK 1/2 MAPK phosphorylation, a well known FSHR-mediated signalling event previously reported to be partially cAMP/PKAindependent [START_REF] Kara | A phosphorylation cluster of five serine and threonine residues in the C-terminus of the follicle-stimulating hormone receptor is important for desensitization but not for beta-arrestin-mediated ERK activation[END_REF]. A time-course of ERK phosphorylation provoked by FSH stimulation (3.3 nM) was performed in HEK293N cells stably expressing the Wt or A189V FSHR. When pERK 1 and 2 were analysed by Western blot in whole cell lysates a robust A c c e p t e d M a n u s c r i p t FSH-induced ERK phosphorylation response was detected not only for the Wt FSHR but also for the A189V mutant. Although the mutant FSHR-mediated ERK phosphorylation was clearly lower than that triggered by the Wt FSHR, the response was consistently detected in all experiments despite the low expression levels of the mutant receptor species (Figs. 3A andB). In both FSHRs the increase in pERK signal was rapid, reaching its maximum after 5 minutes of FSH exposure. However, in the case of the Wt receptor FSH-stimulated ERK phosphorylation was detected for over 2 hours whereas for the mutant receptor it only lasted one hour. Dose-response curves at 5 minutes were next carried out (Figs. 4 A andB). The results displayed an unequivocally dose-dependent increase in ERK phosphorylation for both receptors (Figs. 4B and4C), with a lower efficacy for the A189V FSHR (Fig. 4B).

FSH-induced ERK MAPK phosphorylation by the A189V FSHR mutant is PKAindependent

To confirm that the PKA-dependent pathway was not involved in FSH-stimulated ERK MAPK phosphorylation by the A189V FSHR, dose-response experiments in the presence of the PKA inhibitor H89 were performed. The results showed that FSH-induced ERK phosphorylation by the A189V FSHR was recalcitrant to H89 treatment (Fig. 5A and5B).

FSH-induced ERK MAPK phosphorylation by the A189V FSHR mutant is primarily β-arrestin dependent

A c c e p t e d M a n u s c r i p t Previous studies from our group have demonstrated that FSH-induced ERK phosphorylation is triggered by both cAMP/PKA-and β-arrestin-dependent pathways [START_REF] Kara | A phosphorylation cluster of five serine and threonine residues in the C-terminus of the follicle-stimulating hormone receptor is important for desensitization but not for beta-arrestin-mediated ERK activation[END_REF]Wehbi et al. 2010a;[START_REF] Wehbi | Selective Modulation of Follicle-Stimulating Hormone Signaling Pathways with Enhancing Equine Chorionic Gonadotropin/Antibody Immune Complexes[END_REF]). To investigate the contribution of β-arrestin-dependent transduction mechanisms to ERK phosphorylation, HEK293N cells stably expressing the Wt or the A189V FSHR were transfected with siRNA targeting both β-arrestin 1 and 2. As expected, in cells transfected with β-arrestin siRNA endogenous β-arrestin expression was dramatically reduced compared to cells transfected with control siRNA (Fig. 6A). Doseresponse curves demonstrated that ERK phosphorylation induced by the activated Wt receptor was only partially sensitive to β-arrestins depletion (Figs. 6B and6C), whereas ERK phosphorylation induced by the A189V mutant was abrogated by knocking down β-arrestins 1/2 (Figs. 6B and6D). Side-by-side comparison confirmed that the A189V mutant was significantly more sensitive to β-arrestins depletion than the Wt FSHR(Fig. 6E). These results demonstrated that β-arrestins-dependent signalling accounts for most of ERK phosphorylation mediated by the A189V mutant but not by the Wt FSHR, which exhibited a bimodal regulation of ERK phosphorylation.

β-arrestins-dependent ERK phosphorylation predominates at low levels of Wt FSHR plasma membrane expression

We next sought to investigate whether the selective β-arrestin-dependent ERK phosphorylation observed with the A189V FSHR resulted from the impact of the mutation on receptor function, switching its coupling or alternatively from the very low levels of receptor expression at the plasma membrane. To attenuate the expression levels of the Wt FSHR to a minimum, we transiently transfected low amounts of Wt FSHR cDNA (bearing the sequence encoding its natural signal peptide) in HEK293N cells. This procedure dramatically reduced A c c e p t e d M a n u s c r i p t Wt receptor plasma membrane expression at levels comparable with those of the A189V FSHR mutant (Fig. 7A). Under this condition, FSH-stimulated cAMP production was monitored by FRET (Fig. 7B). The results showed that when expressed at low levels, the Wt FSHR is unable to mediate detectable cAMP production in response to a high dose (3.3 nM) FSH challenge. As previously observed, no significant agonist-stimulated cAMP production was detected in cells transfected with the A189V FSHR, and in both conditions adenylylcyclase remained inducible by forskolin. Time-course experiments to monitor ERK phosphorylation following FSH exposure revealed that activation of both Wt and mutant receptors by agonist provoked a comparable transient increase in ERK phosphorylation, with a maximum at 5 minutes (Figs. 7C and7D). Dose-response experiments to monitor ERK phosphorylation at 5 minutes following FSH exposure revealed that stimulation of both receptors increased the pERK signal with comparable efficacies (Figs. 7E and7F).

A c c e p t e d M a n u s c r i p t

DISCUSSION

It has been shown that the AV substitution at position 189 of the FSHR leads to gonadal dysgenesis and hypergonadotrophic hypogonadism in women [START_REF] Aittomaki | Mutation in the follicle-stimulating hormone receptor gene causes hereditary hypergonadotropic ovarian failure[END_REF]. It has also been demonstrated that this mutation provokes intracellular retention of the receptor [due to misfolding of the receptor protein [START_REF] Rannikko | Functional characterization of the human FSH receptor with an inactivating Ala189Val mutation[END_REF]] as well as defective function as disclosed by a marginal or null ability to transduce signals via G proteins [START_REF] Aittomaki | Mutation in the follicle-stimulating hormone receptor gene causes hereditary hypergonadotropic ovarian failure[END_REF][START_REF] Tena-Sempere | Molecular cloning of the mouse follicle-stimulating hormone receptor complementary deoxyribonucleic acid: functional expression of alternatively spliced variants and receptor inactivation by a C566T transition in exon 7 of the coding sequence[END_REF][START_REF] Rannikko | Functional characterization of the human FSH receptor with an inactivating Ala189Val mutation[END_REF]. In fact, this socalled inactivating mutation is well known to prevent agonist-stimulated cAMP production and phosphoinositide turnover [START_REF] Aittomaki | Mutation in the follicle-stimulating hormone receptor gene causes hereditary hypergonadotropic ovarian failure[END_REF][START_REF] Tena-Sempere | Molecular cloning of the mouse follicle-stimulating hormone receptor complementary deoxyribonucleic acid: functional expression of alternatively spliced variants and receptor inactivation by a C566T transition in exon 7 of the coding sequence[END_REF]). In the present study, we provide experimental evidence supporting the notion that the A189V mutation in the FSHR retains its ability to promote G protein-independent signalling. Our results show that this mutant can phosphorylate ERK MAPK despite being essentially uncoupled from the canonical cAMP/PKA pathway. This finding offers a plausible explanation for the mild abnormalities detected in men bearing the A189V mutation (e.g. oligozoospermia) when compared to those carrying FSHβ inactivating mutations [START_REF] Matthews | Primary amenorrhoea and infertility due to a mutation in the beta-subunit of folliclestimulating hormone[END_REF][START_REF] Aittomaki | Mutation in the follicle-stimulating hormone receptor gene causes hereditary hypergonadotropic ovarian failure[END_REF][START_REF] Layman | Delayed puberty and hypogonadism caused by mutations in the follicle-stimulating hormone betasubunit gene[END_REF][START_REF] Layman | FSH beta gene mutations in a female with partial breast development and a male sibling with normal puberty and azoospermia[END_REF]. Thus, whereas FSHβ inactivating mutations lead to complete suppression of all potential FSH-dependent signalling pathways due to inability of the mutant FSH to bind and activate the FSHR, the A189V mutation only impairs Gs-(cAMP/PKA-dependent) and Gq/11-mediated pathways. The overall data suggest that G protein-dependent pathways are essential for female reproduction whereas in men reproduction could still be feasible, though with lower efficiency, independently of G protein-coupling.

The results presented herein additionally demonstrate that β-arrestin-dependent signalling is responsible for ERK phosphorylation triggered by the A189V FSHR. In fact, A c c e p t e d M a n u s c r i p t siRNA-mediated depletion of endogenous β-arrestins 1 and 2 primarily abrogated FSHpromoted ERK phosphorylation by the A189V FSHR but not by the Wt receptor. These data are consistent with our previous studies showing that β-arrestins can induce G proteinindependent signalling at the FSHR [START_REF] Kara | A phosphorylation cluster of five serine and threonine residues in the C-terminus of the follicle-stimulating hormone receptor is important for desensitization but not for beta-arrestin-mediated ERK activation[END_REF]Wehbi et al. 2010a;[START_REF] Wehbi | Selective Modulation of Follicle-Stimulating Hormone Signaling Pathways with Enhancing Equine Chorionic Gonadotropin/Antibody Immune Complexes[END_REF]. Interesting, in contrast to what has been reported for the rat FSHR, which shows a slow and sustained ERK phosphorylation upon agonist exposure [START_REF] Kara | A phosphorylation cluster of five serine and threonine residues in the C-terminus of the follicle-stimulating hormone receptor is important for desensitization but not for beta-arrestin-mediated ERK activation[END_REF], we detected that agonist-provoked activation of the A189V mutant was followed by a rapid and transient β-arrestin-dependent ERK phosphorylation. This apparent discrepancy could be due to the lack of G protein coupling of the mutant receptor, which may potentially abrogate the competition between G proteins and β-arrestins for receptor recruitment. In this scenario, βarrestin coupling to the mutant receptor and subsequent signalling would proceed faster than that presented by Wt receptor. Although physiological roles for β-arrestin-dependent signalling have recently been described for other GPCR [START_REF] Premont | Physiological roles of G protein-coupled receptor kinases and arrestins[END_REF][START_REF] Gesty-Palmer | A beta-arrestin-biased agonist of the parathyroid hormone receptor (PTH1R) promotes bone formation independent of G protein activation[END_REF], it is the first time that β-arrestins are suspected to play a role in reproductive function.

It has been previously reported that the A189V mutation leads to intracellular retention of the FSHR associated with a dramatic decrease in plasma membrane expression level and attenuation of G protein coupling [START_REF] Rannikko | Functional characterization of the human FSH receptor with an inactivating Ala189Val mutation[END_REF]. In the present study, we showed that the preferential -arrestin coupling exhibited by the A189V FSHR is paradoxically due to the low level of plasma membrane expression rather than to the mutation itself. In fact, our data demonstrate that in cells expressing the Wt FSHR at a plasma membrane density comparable to that of the A189V FSHR, FSH exposure promoted ERK phosphorylation without triggering detectable intracellular signalling mediated by the cAMP/PKA pathway. In this vein, [START_REF] Rannikko | Functional characterization of the human FSH receptor with an inactivating Ala189Val mutation[END_REF] showed that overexpression of the A189V FSHR mutant in COS-7 cells resulted in agonist-stimulated cAMP accumulation detectable in the presence of A c c e p t e d M a n u s c r i p t phosphodiesterase inhibitors. Under these conditions, cAMP production per receptor unit was comparable between the Wt and the A189V FSHRs [START_REF] Rannikko | Functional characterization of the human FSH receptor with an inactivating Ala189Val mutation[END_REF]. Based on these observations and depending on the cellular context, it is reasonable to consider that the A189V FSHR can still couple to Gs but that the G protein-independent signalling observed may result from the low cell surface plasma membrane levels of the mutant, rather than from the functional impairment imposed by the A189V mutation. In this setting, FSH-stimulated cAMP production may still be insufficient to promote PKA activation.

The results of the present study, suggest that the A189V FSHR can be phosphorylated by GRKs and recruit β-arrestins as revealed by the receptor's ability to signal through βarrestin scaffolding. A growing body of evidence supports the notion that signalling at GPCR can be selectively triggered or dampened by using either biased ligands or modified receptors (Wehbi et al.;[START_REF] Wei | Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2[END_REF][START_REF] Shenoy | beta-arrestin-dependent, G protein-independent ERK1/2 activation by the beta2 adrenergic receptor[END_REF][START_REF] Violin | Beta-arrestin-biased ligands at seventransmembrane receptors[END_REF]. In this vein, our data establish for the first time that selective signalling may also be achieved by modulating cell surface plasma membrane levels of the receptor, underlining the importance of receptor density as a regulatory mechanism to control cellular signalling. In the present study, induction of β-arrestins-dependent biased signalling was achieved by decreasing the number of functional receptors in HEK293N cells to levels found in primary granulosa cells (1000 receptors per cell; [START_REF] Mcnatty | Binding characteristics of 125I-labelled human FSH to granulosa cells from Booroola ewes which were homozygous, heterozygous or non-carriers of a major gene(s) influencing their ovulation rate[END_REF] or primary Sertoli cells from 19 days old rat [6400 receptors per cell as calculated from our previously published data [START_REF] Musnier | Developmental regulation of p70 S6 kinase by a G protein-coupled receptor dynamically modelized in primary cells[END_REF]]. Since plasma membrane expression of the FSHR varies depending on the developmental and differentiation stages of the gonads [START_REF] Simoni | The follicle-stimulating hormone receptor: biochemistry, molecular biology, physiology, and pathophysiology[END_REF], it is tempting to speculate that varying FSHR levels might lead to differential coupling to effectors and, consequently, to distinct biological responses known to be associated with the gonadotropic signal. Undoubtedly, the cellular context should be considered when trying to understand Gs protein/β-arrestin balance in a given experimental paradigm. In fact, it has been shown that rat A c c e p t e d M a n u s c r i p t Sertoli cells from animals at 2 different developmental stages (5 and 19 days of age ) exhibit similar cell surface membrane levels of FSHR yet differ in coupling to G proteins [START_REF] Musnier | Developmental regulation of p70 S6 kinase by a G protein-coupled receptor dynamically modelized in primary cells[END_REF], Crepieux et al., 2001).

The A189V mutation of the FSHR has been associated with primary ovarian failure in the Finnish population [START_REF] Aittomaki | Mutation in the follicle-stimulating hormone receptor gene causes hereditary hypergonadotropic ovarian failure[END_REF]. Recently, a gene therapy approach using adenoviruses expressing the human FSHR has been proposed as a potential intervention to restore a normal phenotype in women affected by this mutation [START_REF] Ghadami | Toward gene therapy of primary ovarian failure: adenovirus expressing human FSH receptor corrects the Finnish C566T mutation[END_REF]. Since when expressed at high levels the A189V FSHR remains functional a therapeutic approach addressed to improve the expression levels of the A189V FSHR could also be envisioned. In this regard, Janovick and colleagues (2009) recently showed that a membrane permeable allosteric modulator of the FSHR was able to partially rescue plasma membrane expression of the A189V mutant, suggesting that such molecules also represent good candidates for treating patients bearing particular inactivating mutations of the FSHR [START_REF] Janovick | Increased plasma membrane expression of human follicle-A c c e p t e d M a n u s c r i p t stimulating hormone receptor by a small molecule thienopyr(im)idine[END_REF].

A c c e p t e d M a n u s c r i p t

  s c r i p t addressed this hypothesis using HEK293N cells expressing either the wild-type (Wt) or the A189V human FSHR.

  M) was added to the cells as a positive control. The areas that did not respond to forskolin were removed from the analysis. Fluorescent intensity of non-transfected cells was substracted to the intensity of fluorescent cells expressing the sensor in order to quantify the specific signal. The FRET ratio (CFP/YFP for the cAMP sensor and YFP/CFP for the PKA sensor) was calculated for each individual cell. The mean  SEM from at least 20 individual cell responses measured in 3 different experiments were plotted.

Fig. 1 :

 1 Fig. 1: FSH binding to the Wt or A189V human FSH receptor

Fig. 2 :

 2 Fig. 2: FSH-induced cAMP/PKA pathway activation by Wt vs A189V FSHR.

Fig. 3 :

 3 Fig. 3: Kinetics of FSH-induced ERK phosphorylation by the Wt and A189V human

Fig. 4 .

 4 Fig. 4. Doses-response curves of FSH-induced ERK phosphorylation by the Wt and

Fig. 5 .

 5 Fig. 5. Contribution of PKA-dependent transduction mechanism to FSH-induced ERK

Fig. 6 .Fig. 7 .

 67 Fig. 6. Contribution of β-arrestin-dependent transduction mechanism to FSH-induced

  s c r i p t phosphorylated ERK over total ERK2. Each data point represents the mean ± sem of seven individual experiments. (E, F) HEK293N cells transiently transfected with either the Wt or A189V human FSH receptor were serum-starved for 4 hours and then stimulated for 5 minutes with increasing (zero to 10 nM) doses of pFSH. After stimulation, cell extracts were prepared as described in Materials and Methods. Endogenous phosphorylated ERK were revealed using an anti-pERK antibody. Membranes were stripped as described in Materials and Methods and revealed with anti-ERK2 antibody for normalisation. (E) Representative immunoblots of FSH-induced ERK phosphorylation. (F) ERK phosphorylation signals were quantified by densitometry and expressed as the ratio of phosphorylated ERK over total ERK2. The maximal phosphorylation signal of ERK obtained with the Wt receptor was arbitrarily chosen as 100%. Each data point represents the mean ± sem from 5 individual experiments.

  Figure1

Figure

  Figure

Figure

  Figure

Acknowledgments

The authors thank the members of the BIOS group for their support throughout this project. This work was supported by the Action d'Envergure (AE) Institut National de la Recherche en Automatique et en Informatique (INRIA)/INRA Regate, and Region Centre project. BIOS group is a young research team from INRA.

T.T. was granted by

The "Fondation d'Aide à la Recherche d'Organon" (FARO). G.D. was funded by a fellowship from the French MRT. A.U.-A is Le STUDIUM Invited Professor at the INRA.

A c c e p t e d M a n u s c r i p t

A.

B.

A189V + DMSO A189V + H89

C. D.

Figure 6 E.


Harri Makkonen

Miia Kauhanen

Tiina Jääskeläinen

Jorma J Palvimo
email: jorma.palvimo@uef.fi

Androgen receptor amplification is reflected in the transcriptional responses of Vertebral-Cancer of the Prostate cells

Keywords: Androgen receptor, VCaP, prostate cancer cell, antiandrogen, gene expression, chromatin immunoprecipitation (ChIP)

Androgen receptor (AR) is overexpressed in a majority of castration-resistant prostate cancers, but most of the cell model studies addressing AR function have been conducted in LNCaP prostate cancer cells expressing unamplified AR levels. Here, we have compared the responses of various types of AR ligands towards a pattern of AR target genes and chromatin binding sites in Vertebral-Cancer of the Prostate (VCaP) cells and LNCaP cells. In keeping with the AR gene amplification in VCaP cells, our analyses show that these cells contain ≥10-fold receptor mRNA and protein than LNCaP cells. Loading of the agonist-occupied AR onto chromatin regulatory sites and expression of several AR target genes, including their basal expression, were stronger in VCaP cells than LNCaP cells. Bicalutamide displayed a trend towards agonism in VCaP cells. Bicalutamide also evoked AR-chromatin interaction, whereas diarylthiohydantoin antiandrogen RD162 was inert with this respect both in VCaP and LNCaP cells. These results support the notion that the AR protein level translates into augmented occupancy of AR-regulated enhancers and target gene activity in prostate cancer cells.

Introduction

Androgen signaling is essential for both normal and malignant growth of prostate [START_REF] Heinlein | Androgen receptor in prostate cancer[END_REF]. Proliferation of prostate cancer cells can initially be restricted by androgen ablation therapy. However, for reasons that are currently elusive, the therapy eventually fails and prostate cancer turns into a hormone-refractory, i.e. castration-resistant or androgendepletion-independent, state [START_REF] Bonkhoff | From pathogenesis to prevention of castration resistant prostate cancer[END_REF]. The situation has been proposed to result from mutations in AR that liberate its hormone-specificity, altered expression of AR or its coregulator proteins, up-regulation of intraprostatic androgen synthesis during androgen withdrawal, and gene fusions resulting in abnormal androgen regulation of oncogenic transcription factors [START_REF] Han | Mutation of the androgen receptor causes oncogenic transformation of the prostate[END_REF][START_REF] Culig | Expression and function of androgen receptor coactivators in prostate cancer[END_REF][START_REF] Gregory | A mechanism for androgen receptor-mediated prostate cancer recurrence after androgen deprivation therapy[END_REF][START_REF] Shi | Prolonged androgen receptor loading onto chromatin and the efficient recruitment of p160 coactivators contribute to androgen-independent growth of prostate cancer cells[END_REF][START_REF] Montgomery | Maintenance of intratumoral androgens in metastatic prostate cancer: a mechanism for castration-resistant tumor growth[END_REF]. Despite of the apparent hormone-refractory state of the disease, AR remains a critical factor for the growth and survival of the majority of the hormone-refractory tumors. Most of these tumors overexpress AR, and a significant portion of hormone refractory prostate cancers carry an AR gene amplification [START_REF] Koivisto | Androgen receptor gene amplification: a possible molecular mechanism for androgen deprivation therapy failure in prostate cancer[END_REF][START_REF] Linja | Amplification and overexpression of androgen receptor gene in hormone-refractory prostate cancer[END_REF][START_REF] Chen | Molecular determinants of resistance to antiandrogen therapy[END_REF]. Overexpression of AR has been suggested to hypersensitize the cancer cells to minuscule levels of androgens and thereby allow them grow in the apparent absence of androgens [START_REF] Linja | Amplification and overexpression of androgen receptor gene in hormone-refractory prostate cancer[END_REF]Li et al., 2004;[START_REF] Henshall | Altered expression of androgen receptor in the malignant epithelium and adjacent stroma is associated with early relapse in prostate cancer[END_REF][START_REF] Rosner | Higher tumor to benign ratio of the androgen receptor mRNA expression associates with prostate cancer progression after radical prostatectomy[END_REF][START_REF] Waltering | Increased expression of androgen receptor sensitizes prostate cancer cells to low levels of androgens[END_REF].

The AR is a hormone-controlled DNA-binding transcription factor that regulates genes and gene networks in response to androgen exposure [START_REF] Heemers | Androgen receptor (AR) coregulators: a diversity of functions converging on and regulating the AR transcriptional complex[END_REF]. Androgen-occupied AR binds to androgen response elements (AREs) in the regulatory regions of its target genes and interacts with transcriptional coregulatory proteins, which results in remodeling of chromatin structure and activation of RNA polymerase II function on the target gene promoter [START_REF] Heemers | Androgen receptor (AR) coregulators: a diversity of functions converging on and regulating the AR transcriptional complex[END_REF][START_REF] Makkonen | Longrange activation of FKBP51 transcription by the androgen receptor via distal intronic enhancers[END_REF]. Synthetic antiandrogens, such as nonsteroidal bicalutamide, that directly target the AR are often used for the treatment of advanced prostate cancer (Steward et al., 2005). The currently available antiandrogens act as competitive inhibitors for natural androgens, but they do not support the formation of productive AR transcription complexes. Unfortunately, overexpression of AR may convert the bicalutamide (that is under normal cells considered a pure antagonist) to an agonist [START_REF] Chen | Molecular determinants of resistance to antiandrogen therapy[END_REF].

Established cell lines resembling the situation in castration-resistant prostate cancer are instrumental models in improving our understanding of the molecular mechanism of AR action and developing strategies for targeting AR in prostate cancer. However, the majority of single gene and large-scale or genome-wide expression analyses of AR target genes have been performed in LNCaP cells, the most popular cell model for prostate cancer research [START_REF] Dehm | Molecular regulation of androgen action in prostate cancer[END_REF][START_REF] Ngan | Microarray coupled to quantitative RT-PCR analysis of androgen-regulated genes in human LNCaP prostate cancer cells[END_REF][START_REF] Lamont | Androgen regulation of gene expression[END_REF]. The LNCaP cells correspond to a lymph node metastasis, containing a ligand-binding domain-mutated version of the receptor [START_REF] Veldscholte | A mutation in the ligand binding domain of the androgen receptor of human LNCaP cells affects steroid binding[END_REF]. The AR gene locus is not amplified in the LNCaP cells, and the receptor protein is normally expressed in these cells. VCaP (Vertebral-Cancer of the Prostate) cells in turn are derived from a hormone-refractory cancer, possessing amplified AR gene locus encoding otherwise normal AR protein [START_REF] Korenchuk | VCaP, a cell-based model system of human prostate cancer[END_REF]van Bokhoven et al., 2003;[START_REF] Liu | Homozygous deletions and recurrent amplifications implicate new genes involved in prostate cancer[END_REF]. Additionally, this interesting cell line contains the chromosomal rearrangement fusing the androgen-regulated gene TMPRSS2 to the oncogenic ERG transcription factor found in about half of the prostate cancers [START_REF] Liu | Homozygous deletions and recurrent amplifications implicate new genes involved in prostate cancer[END_REF][START_REF] Tomlins | Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer[END_REF]. Therefore, the VCaP cells might better than the LNCaP cells represent the state of androgen signaling in the castration-resistant prostate cancer. To investigate whether the amplification of AR in VCaP cells is reflected in the transcriptional responses of AR target genes, we have in this work compared the regulation and the function of AR between the A c c e p t e d M a n u s c r i p t 4 VCaP and the LNCaP cells. We quantified the expression of AR and that of a pattern of AR target genes in response to various types of AR ligands and compared the loading of AR to a pattern of specific gene loci by using quantitative chromatin immunoprecipitation (ChIP) assays. Our results reveal interesting differences in the AR-target chromatin interaction and the AR target gene expression between these two cell lines.

Materials and methods

Cell culture

VCaP and LNCaP cells were from American Type Culture Collection (ATCC). The cells were maintained as described [START_REF] Makkonen | Longrange activation of FKBP51 transcription by the androgen receptor via distal intronic enhancers[END_REF].

Isolation of RNA and RT-quantitative PCR (RT-qPCR)

VCaP and LNCaP cells were seeded onto 6-well plates (8 x 10 5 and 3.3 x 10 5 cells/well, respectively) and grown for 72 h in transfection medium (VCaP: DMEM containing 2.5% charcoal-stripped FBS (CCS-FBS), LNCaP: RPMI 1640, 10% CCS-FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM Hepes, 25 mM glucose) devoid of steroids. In Fig. 2, the VCaP cells were simultaneously transfected either with negative control siRNA (siSCR, 5'-UUCUCCGAACGUGUCACGUdTdT-3') or siRNA against AR (siAR, 5'-GGAGUUGUGUAAGGCAGUGdTdT-3') using TransIT-siQUEST ® transfection reagent (Mirus Bio Corporation, Madison, WI, USA) according to the manufacturer's instructions. Subsequently, cells were treated with or without R1881 (17β-17-hydroxy-17-methyl-estra-4,9,11-trien-3-one, also known as methyltrienolone or metribolone, Perkin Elmer Inc., Waltham, MA, USA), cyproterone acetate 2]pregna-4,6-diene-3,20-dione, Sigma-Aldrich, St. Louis, MO, USA), bicalutamide (N-[4-cyano-3-(trifluoromethyl)phenyl]-3-[(4-fluorophenyl)sulfonyl]-2hydroxy-2-methylpropanamide, Bidragon Pharmservice LLC, Burlingame, CA, USA), or novel diarylthiohydantoin antiandrogen RD162 (4-[7-(4-cyano-3-trifluoromethyl-phenyl)-8oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-2-fluoro-N-methyl-benzamide, Helix&Bond Pharmaceuticals Inc., Shanghai, China) as indicated. Total RNA was extracted using TRIzol® Reagent (Invitrogen Lifetechnologies, Carlsbad, CA, USA) and converted to cDNA using Transcriptor First Strand cDNA synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany) following manufacturer's instructions. cDNA was used as a template in RT-qPCR, which was carried out using Mx3000P Real-Time PCR System (Stratagene, La Jolla, CA, USA), FastStart SYBR Green Master (Roche) and specific primers for indicated genes (Supplementary Table S1). Analyzed GAPDH mRNA levels were used to normalize the amounts of total RNA between the samples. Fold changes (ligand inductions) in Fig. 6 were calculated using the formula 2 -(ΔΔCt) , where ΔΔCt is ΔCt (ligand) -ΔCt (EtOH) , ΔCt is Ct (gene X) -Ct (GAPDH) and Ct is the cycle at which the threshold is crossed. Relative mRNA levels were calculated in Fig. 2 using the formula 2 -(ΔΔCt) , where ΔΔCt is ΔCt (ligand) -ΔCt (siSCR EtOH) and in Figures 3 and4 using the formula 2 -(ΔΔCt) , where ΔΔCt is ΔCt (ligand) -ΔCt (LNCaP EtOH) .

Immunoblotting

VCaP and LNCaP cells were seeded and grown as for RT-qPCR. The cell monolayers were washed with ice-cold Tris-buffered saline (TBS) and harvested in TBS containing 1:100 protease inhibitor cocktail (PIC) (Sigma-Aldrich, St. Louis, MO, USA). Cell pellets were A c c e p t e d M a n u s c r i p t 5 suspended in SDS-PAGE sample buffer containing 1:100 PIC and lysed by sonication (2 x 10 s). In Fig. 2B, proteins were extracted simultaneously with RNA using TRIzol® reagent following manufacturer's instructions. Samples were heated for 5 min at 95C and separated on 10% SDS-PAGE, proteins were transferred onto nitrocellulose membranes and detected by a rabbit antiserum against AR [START_REF] Karvonen | Interaction of androgen receptors with androgen response element in intact cells. Roles of amino-and carboxyl-terminal regions and the ligand[END_REF] or anti-GAPDH (sc-25778, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The bands were visualized by horseradish peroxidase-conjugated anti-rabbit secondary antibody (Zymax™ Goat Anti-Rabbit IgG (H+L) HRP conjugate (81-6120), Zymed, San Francisco, CA, USA) using the enhanced chemiluminescence detection reagents according to the manufacturer's instructions (Pierce® ECL Western Blotting Substrate, Thermo Scientific, Rockford, IL, USA). For quantification of AR band intensities, goat anti-rabbit IgG (H&L) DyLight™ 800-conjugated secondary antibody (Pierce) was used with Li-COR Odyssey Infrared Imaging System according to manufacturer's instructions (LI-COR Inc., Lincoln, NE, USA). The amount of AR was normalized by the amount of GADPH in the sample.

Chromatin immunoprecipitation (ChIP)

VCaP and LNCaP cells were seeded at ~70% confluence onto 75-cm 2 bottles and allowed to grow in steroid-depleted transfection medium for 72 h prior to ChIP. The experiments were performed essentially as previously described [START_REF] Makkonen | Longrange activation of FKBP51 transcription by the androgen receptor via distal intronic enhancers[END_REF] using the same rabbit antiserum against AR that was used for immunoblotting. Normal rabbit serum (NRS) (Millipore, Billerica, MA, USA) was used as a negative control antibody (IgG). Specific primers for different regions are listed in Supplementary Table S2. Quantitative PCR analyses were carried out with FastStart SYBR Green Master and Mx3000P Real-Time PCR System. In Fig. 5, results were calculated using the formula (E -(ΔCt (AR) ) *10)/(E -(ΔCt (IgG) *10), where E (efficiency of target amplification) is a coefficient of DNA amplification by one PCR cycle for a particular primer pair and ΔCt is Ct (ChIP-template) -Ct (Input) . In Figure 7, results are presented as ligand fold inductions over the value of ethanol treated sample of given cells.

Results and Discussion

AR is overexpressed, but down-regulated by androgens, in VCaP cells in comparison to LNCaP cells

We first compared the AR protein levels in LNCaP and VCaP cells grown in absence and presence of synthetic non-aromatizable ligands methyltrienolone (R1881, pure agonist), cyproterone acetate (CTA, steroidal antiandrogen), bicalutamide (BIC, nonsteroidal antiandrogen) and RD162 (novel arylthiohydantoin antiandrogen). We used saturating ligand concentrations that were adjusted to compensate for differences in their AR binding affinities [START_REF] Gao | Chemistry and structural biology of androgen receptor[END_REF][START_REF] Tran | Development of a second-generation antiandrogen for treatment of advanced prostate cancer[END_REF]. Immunoblotting of the AR bands showed that the VCaP cells contain markedly more receptor protein than the LNCaP cells, with the difference being dependent on the ligand exposure and time (Fig. 1A). Quantification of the AR bands revealed that the difference is 15-fold in the absence of ligand. A short-term (2 h) treatment with R1881, CTA or BIC, but not with RD162, stabilized the receptor protein similarly in both cell lines. Interestingly, a longer (18 h) R1881 or CTA treatment further stabilized the AR in LNCaP cells, but contrastingly reduced the receptor amount in VCaP cells, thus decreasing the difference in the receptor amount between these prostate cancer cell lines to ~4-5.5-fold.

To reveal whether the above differences in the AR protein levels mirror differences in the AR gene expression, AR mRNA levels from VCaP and LNCaP cells exposed to different A c c e p t e d M a n u s c r i p t 6 ligands were quantified by RT-qPCR (Fig. 1B). In accordance with the amplification of the AR gene locus in VCaP cells, the cells expressed ~11-fold more AR mRNA than LNCaP cells. Interestingly, the agonist R1881 significantly down-regulated the AR mRNA level in VCaP cells, resulting in ~85% decrease in the mRNA, whereas in LNCaP cells, the AR mRNA levels were not markedly altered by the agonist exposure. However, longer treatments (≥ 40 h) with agonists have been reported to result in down-regulation of AR mRNA also in LNCaP cells [START_REF] Krongrad | Androgen increases androgen receptor protein while decreasing receptor mRNA in LNCaP cells[END_REF]. The effects of the other ligands on the AR mRNA expression in VCaP cells were smaller than that of R1881, decreasing in the order of CTA > RD162 > BIC (Fig. 1B). Despite of the agonist-mediated down-regulation, the AR mRNA level of VCaP cells exceeded that of LNCaP cells. VCaP cells have been recently reported to contain at least five extra copies of the AR gene [START_REF] Liu | Homozygous deletions and recurrent amplifications implicate new genes involved in prostate cancer[END_REF]. Thus, the AR gene dose is by and large translated into the expression of AR protein in these cells. Androgen-dependent down-regulation of AR expression in VCaP cells resembles the down-regulation that occurs in the normal rodent prostate in vivo [START_REF] Shan | Regulation of androgen receptor protein and mRNA concentrations by androgens in rat ventral prostate and seminal vesicles and in human hepatoma cells[END_REF].

Differential sensitivity of target genes to AR depletion in VCaP cells

Since the AR function has not been widely studied in VCaP cells, we first confirmed the role of AR in the regulation of selected genes that have been previously identified as AR targets in other prostate cancer models, mainly in LNCaP cells. To that end, we set up AR RNAi that silenced ≥ 80% of the AR mRNA, resulting in an efficient depletion of the AR protein (Fig. 2A,B). We next compared the expression of nine AR targets; FKBP51, C6orf81, TMPRSS2, PSA, S100P, ChGn, SLC45A3, SPOCK1, and NKX3.1 [START_REF] Amler | Dysregulated expression of androgen-responsive and nonresponsive genes in the androgen-independent prostate cancer xenograft model CWR22-R1[END_REF][START_REF] Bolton | Celland gene-specific regulation of primary target genes by the androgen receptor[END_REF][START_REF] Lin | Prostate-localized and androgen-regulated expression of the membrane-bound serine protease TMPRSS2[END_REF]Young et al., 1991;[START_REF] Averboukh | Regulation of S100P expression by androgen[END_REF][START_REF] Tomlins | Distinct classes of chromosomal rearrangements create oncogenic ETS gene fusions in prostate cancer[END_REF][START_REF] Nitsche | Differential display RT PCR of total RNA from human foreskin fibroblasts for investigation of androgen-dependent gene expression[END_REF][START_REF] Bieberich | Prostate-specific and androgendependent expression of a novel homeobox gene[END_REF], in control siRNA (siSCR)-and siAR-transfected VCaP cells that were treated with vehicle or R1881 by measuring their mRNAs by RT-qPCR. As shown in Fig. 2C, all of the nine candidate target gene mRNAs were induced in response to androgen exposure in VCaP cells. Depletion of AR blunted or abolished the androgen induction of five target genes; FKBP51, C6orf81, S100P, ChGn, and SPOCK1. In the case of four AR targets, TMPRSS2, SLC45A3, PSA, and NKX3.1, the AR depletion markedly reduced their expression also in vehicle-exposed cells, and therefore their androgen (foldincrease) inductions were not affected. Conversely, the basal expression of C6orf81, S100P and ChGn were interestingly increased upon the AR depletion. All in all, these results indicate the active role of the AR protein in the regulation of these nine genes. However, these genes display interesting differences in their sensitivity towards AR silencing in VCaP cells.

Increased basal expression of several AR target genes in VCaP cells

We next investigated whether the observed difference in the amount of AR between VCaP and LNCaP cells (cf. 3.1.) translates into differences in AR target gene expression. Quantification of the mRNAs for the above-mentioned AR targets in relation to the GAPDH mRNA levels revealed that, except for PSA and S100P, their expression levels in vehicletreated cells were markedly higher in VCaP cells than LNCaP cells (Fig. 3A). The fold difference ranged from 3-(C6orf81) to 660-fold (SPOCK1). R1881 treatment (0.01, 0.1, 1 and 10 nM) for 12 h dose-dependently increased the accumulation of these mRNAs in both cell lines (Fig. 3A). The maximum fold-increases in response to the androgen varied less; FKBP51 (36vs. 26-fold), C6orf81 (7vs. 3-fold), S100P (12vs. 2-fold), TMPRSS2 (9vs. 14-fold), PSA (3vs. 6-fold), ChGn (4vs. 34-fold), SLC45A3 (2vs. 17-fold), NKX3.1 (2vs. 18-fold), and SPOCK1 (3vs. 7-fold), being often higher in LNCaP cells. This is probably A c c e p t e d M a n u s c r i p t 7 derived from the generally lower basal expression of these mRNAs in LNCaP cells. The PSA that has historically been employed as a canonical model for AR target genes contrastingly showed a higher expression in LNCaP cells. However, PSA mRNA does not seem to be a very sensitive marker of AR action, as the PSA mRNA levels were not significantly suppressed in prostatectomy patients in response to androgen deprivation therapy [START_REF] Mostaghel | Intraprostatic androgens and androgen-regulated gene expression persist after testosterone suppression: therapeutic implications for castration-resistant prostate cancer[END_REF]. In addition to the AR, FoxA1 transcription factor plays a central role in the upregulation of the PSA [START_REF] Gao | The role of hepatocyte nuclear factor-3 alpha (Forkhead Box A1) and androgen receptor in transcriptional regulation of prostatic genes[END_REF][START_REF] Wang | A hierarchical network of transcription factors governs androgen receptor-dependent prostate cancer growth[END_REF]. Compared to the PSA mRNA, FKBP51 mRNA has been shown to better reflect the androgen activity in human prostate [START_REF] Mostaghel | Intraprostatic androgens and androgen-regulated gene expression persist after testosterone suppression: therapeutic implications for castration-resistant prostate cancer[END_REF]. Interestingly, both the basal and the androgen-induced expression of FKBP51 were clearly higher in VCaP than LNCaP cells. These findings imply that the increased AR dose in VCaP cells is translated into a higher expression level of several AR target genes. However, since we were also comparing two distinct cell types, not only AR levels, it is likely that also disparities in the cellular milieu, such as overexpression of ERG due to TMPRSS2-ERG gene fusion in VCaP cells [START_REF] Tomlins | Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer[END_REF], contribute to the above differences. ERG was recently reported to occupy AR target genes and repress androgen signaling in VCaP cells (Yu et al., 2010). Even then, depletion of ERG in VCaP cells resulted in only a small increase (<25%) in the androgen-induced expression of genes such as FKBP51, SLC45A3 and TMPRSS2 (Yu et al., 2010). Increased expression of androgen-regulated genes has been previously reported in androgen-independent prostate cancer cells [START_REF] Gregory | Androgen receptor expression in androgen-independent prostate cancer is associated with increased expression of androgen-regulated genes[END_REF][START_REF] Oosterhoff | Expression profiling of androgendependent and -independent LNCaP cells: EGF versus androgen signalling[END_REF][START_REF] Dehm | Molecular regulation of androgen action in prostate cancer[END_REF].

Cyproterone acetate shows gene-selective AR modulator properties both in VCaP and LNCaP cells

Cyproterone acetate (CTA) acts as a near-full agonist in reporter gene assays with the T877to-A-mutated AR originally found from the LNCaP cells [START_REF] Kuil | Deoxyribonucleic acid-binding ability of androgen receptors in whole cells: implications for the actions of androgens and antiandrogens[END_REF][START_REF] Bohl | Crystal structure of the T877A human androgen receptor ligand-binding domain complexed to cyproterone acetate provides insight for ligand-induced conformational changes and structure-based drug design[END_REF]. However, in VCaP cells expressing wild-type AR, the activity of CTA has not been previously reported. We compared the effect of increasing CTA concentrations (0.01, 0.1, 1 and 10 µM) on the expression of the above pattern of AR target genes in VCaP and LNCaP cells. Interestingly, the agonistic activity of CTA varied more between the nine AR target genes than between the two cell lines (Fig. 4). Even in the LNCaP cells and at the highest CTA concentration used, the agonistic activity of CTA on FKBP51, C6orf81, S100P, TMPRSS2, PSA, ChGn, and NKX3.1 however remained clearly below the maximal induction obtained with R1881, showing on the average ca. 55% of the maximal R1881 induction. On the other hand, the CTA acted as a surprisingly potent agonist on SPOCK1 and SLC45A3 expression, equaling the maximum induction obtained with R1881 in both cell lines. In contrast, the agonistic effect of CTA was not evident on S100P and it was poorer than the average on ChGn in both cell lines and on FKBP51 in VCaP cells. These data indicate the CTA acts a partial agonist-antagonist also in the VCaP cells. Moreover, our results imply that the CTA possesses a certain degree of gene-selective activity both in the VCaP and the LNCaP cells.

R1881 and cyproterone acetate dose-dependently trigger loading of AR onto chromatin

We next analyzed the binding of AR to ARE-containing chromatin regions in VCaP and LNCaP cells exposed to increasing R1881 or CTA concentrations by using quantitative chromatin immunoprecipitation (ChIP) assays. The following six distinct AR-binding chromatin regions were examined: an upstream enhancer for TMPRSS2, C6orf81 and PSA, a proximal regulatory region for S100P, an intronic enhancer for SPOCK1 and FKBP51 (Wang , 2007;[START_REF] Paakinaho | Glucocorticoid receptor activates poised FKBP51 locus through long-distance interactions[END_REF][START_REF] Schuur | Prostate-specific antigen expression is regulated by an upstream enhancer[END_REF]Makkonen et al., unpublished data;[START_REF] Makkonen | Longrange activation of FKBP51 transcription by the androgen receptor via distal intronic enhancers[END_REF]. As shown in Fig. 5A, generally more AR occupied these enhancer regions in VCaP than LNCaP cells, and addition of the R1881 brought about dose-dependent increase in the occupancy of AR at these enhancers. Also PSA and S100P enhancers harbored more agonist-bound AR in VCaP cells, although their genes displayed higher expression levels in LNCaP cells. The R1881 concentration responses of AR chromatin binding closely mirrored those of mRNA induction studies, indicating that the availability of the hormone governs the binding of the AR to its cognate chromatin binding sites.

Even though the CTA was weaker than the R1881 in evoking induction of FKBP51, TMPRSS2 and PSA transcription in VCaP cells, the maximum AR loading to these genes' major AR-binding sites did not differ between the CTA-and the R1881-treated cells (Fig. 5B). Moreover, despite of the lack of CTA-induction of S100P, the CTA was interestingly capable of promoting some chromatin loading of the receptor at the two highest CTA concentrations in VCaP cells. In LNCaP cells, the CTA-and R1881-occupied AR showed practically equal loading onto all of the six AR-binding chromatin regions. Collectively, these results imply that although the CTA-bound AR is competent with chromatin binding both in VCaP and LNCaP cells, it fails to fully support the formation of AR transcription complexes in these cells. The CTA-bound AR seems to concomitantly recruit both coactivators and corepressor, which may result in the formation of mixed coactivator/corepressor assemblies [START_REF] Karvonen | Pure antiandrogens disrupt the recruitment of coactivator GRIP1 to colocalize with androgen receptor in nuclei[END_REF][START_REF] Karvonen | Androgen receptor regulates nuclear trafficking andnuclear domain residency of corepressor HDAC7 in a ligand-dependent fashion[END_REF].

RD162 is more potent than bicalutamide in inhibiting AR target gene expression both in VCaP and LNCaP cells

We next compared the gene responses to nonsteroidal antiandrogens bicalutamide (BIC) and RD162, a novel arylthiohydantoin, in VCaP and LNCaP cells. One of the main differences in the behavior of BIC between these two cell lines was that in LNCaP cells, but not in VCaP cells, BIC (10 M) inhibited the expression of SPOCK1 (P<0.001), C6orf81 (P<0.05) and TMPRSS2 (P<0.05) to a transcript level that was significantly below the control (vehicletreated) level. In the VCaP cells, the antiandrogen showed a trend towards a weak agonism, which was significant on the FKBP51 (P<0.001) and the TMPRSS2 (P<0.01) (Fig. 6).

We also examined the capability of BIC (10 M) to block the action of R1881 (1 nM) on the AR target gene expression. Interestingly, the inhibitory effect of BIC varied considerably between different target genes. For example, BIC abolished ≥ 80% of the agonist activity on FKBP51 and S100P in both cell lines as well as that on TMPRSS2, ChGn and SPOCK1 in LNCaP cells, whereas it acted as a relatively poor inhibitor (<40% inhibition) of the androgen induction of PSA in both cells lines as well as that of TMPRSS2, SLC45A3 and SPOCK1 in VCaP cells.

Two diarylthiohydantoins RD162 and MDV3100 were recently reported retain their antagonistic activity under increased AR expression [START_REF] Tran | Development of a second-generation antiandrogen for treatment of advanced prostate cancer[END_REF]. The RD162 proved to be more antagonistic than BIC also in our AR target gene expression assays (Fig. 6). Notably, there was no evidence for a residual agonism in VCaP cells exposed to RD162 (10 M) alone (Fig. 6). The compound was also in VCaP cells capable of repressing the target gene expression to a transcript level that was below the control level. With the curious exception of S100P in LNCaP cells and that of SLC45A3 in VCaP cells, the RD162 also abolished the effect of R1881 on AR target gene expression in both cell types (Fig. 6). These results indicate that, despite of the overexpression of AR, BIC on its own possesses very limited agonistic activity in VCaP cells. Amplified expression level of AR in VCaP cells is thus not sufficient to convert BIC to an efficient agonist, as has been reported for LNCaP and LAPC4 prostate cancer cell models in which about three-fold augmented expression of AR protein A c c e p t e d M a n u s c r i p t 9 was brought about by infecting the cells with a recombinant retrovirus encoding AR cDNA [START_REF] Chen | Molecular determinants of resistance to antiandrogen therapy[END_REF]. However, our results suggest that the AR level contributes to the efficacy of BIC, as it was less efficient in curbing the activity of agonist-bound AR in VCaP cells than in LNCaP cells.

RD162 does not provoke AR-chromatin interaction in LNCaP or VCaP cells as bicalutamide does

We also tested the effect of the nonsteroidal antagonists BIC and RD162 on the ARchromatin interaction at the six chromatin regions studied (cf. 3.5.) by using quantitative ChIP assays. As shown in Fig. 7, BIC promoted a considerable loading of the AR onto the chromatin, especially in VCaP cells. The ability of BIC (10 M) to promote the loading was dependent on the gene enhancer, approaching in VCaP cells the level of R1881-bound AR on C6orf81, TMPRSS2 and PSA enhancer, but being < 80% of the agonist-promoted levels on S100P and SPOCK1 enhancer. Thus, binding of the AR to the regulatory region of a given gene in vivo by no means indicates that the receptor is competent to activate the gene (cf. 3.6.). In contrast to BIC, RD162 (10 M) was inert in promoting loading to any of the six AR chromatin binding sites studied in VCaP or LNCaP cells. In line with the latter finding and agreement with the data in 3.6., the RD162 was also more efficient than BIC in curbing the agonist-exposed AR from interacting with the chromatin. The compound prevented most of the agonist-occupied receptor from interacting with the six AR-regulated enhancers in LNCaP cells, but it still (at 10,000-fold molar excess) allowed a considerable amount of agonist-exposed AR to reside on C6orf81, TMPRSS2 and PSA enhancer in VCaP cells, suggesting that the AR level can modulate the efficiency of antiandrogens in prostate cancer cells in an AR target locus-selective fashion. Our results thus emphasize the importance of investigating AR function, in addition to the popular LNCaP cell model, in prostate cancer cell models, such VCaP cells, showing elevated levels of AR protein.

According to Tran and co-workers, one of the key differences in the molecular properties between the BIC and the RD162 is that the novel compound in contrast to the BIC is inert in inducing the nuclear translocation of AR [START_REF] Tran | Development of a second-generation antiandrogen for treatment of advanced prostate cancer[END_REF]. Our current ChIP data and unpublished immunohistochemical analyses in VCaP cells are in agreement with the latter notion (Rytinki M., unpublished observations). Based on these data, compounds that prevent the nuclear translocation of the AR should block the amplified AR activity in prostate cancer cells more efficiently than the currently widely used antiandrogen BIC.

Contributors

This study was designed by HM and JP; the experimental procedures were performed by HM with assistance from MK and TJ. Yu, J., Yu, J., Mani, R.S., Cao, Q., Brenner, C.J., Cao, X., Wang, X., Wu, L., Li, J., Hu, M., Gong, Y., Cheng, H., Laxman, B., Vellaichamy, A., Shankar, S., Li, Y., Dhanasekaran, S.M., Morey, R., Barrette, T., Lonigro, R.J., Tomlins, S.A., Varambally, S., Qin, Z.S., Chinnaiyan, A.M., 2010. An integrated network of androgen receptor, polycomb, and TMPRSS2-ERG gene fusions in prostate cancer progression. Cancer Cell. 17, 443-454. Columns represent the mean ± SD of three independent experiments. Fig. 2. Effect of AR silencing on the expression of a selection of AR target genes in VCaP cells. The cells were transfected either with a control siRNA (siSCR) or an siRNA against AR (siAR) and subsequently treated with or without 1 nM R1881 for 12 h. (A,C) mRNAs of indicated AR target genes were analyzed by RT-qPCR, relative (rel.) mRNA levels were calculated as described in Materials and methods, and the value of vehicle-treated siSCR sample was set as 1. Columns represent the mean ± SD of three independent experiments. (B) Immunoblotting analysis and AR and GAPDH in siSCR-and siAR-transfected cells grown in the absence (-) and the presence (+) of R1881. Basal expression of a selection of AR target genes in VCaP cells vs. LNCaP cells. mRNAs of indicated AR target genes were analyzed by RT-qPCR. mRNA levels were calculated in relation to GAPDH mRNA levels in the samples, the values of LNCaP samples were set as 1, and the data were expressed as VCaP cell/LNCaP cell ratios. (B) Cells were treated with increasing concentrations of R1881 for 12 h and mRNAs of indicated AR target genes were analyzed by RT-qPCR. Relative (rel.) mRNA levels were calculated as described in Materials and methods, and the value of vehicle-treated LNCaP samples was set as 1. Dots and columns represent the mean ± SD of three independent experiments.

  Supplementary data associated with this article can be found in the online version.

  ., Wheeler, T.,Dai, H., Frolov, A., Thompson, T., Ayala, G., 2004. Highlevel of androgen receptor is associated with aggressive clinicopathologic features and decreased biochemical recurrence-free survival in prostate: Cancer patients treated with radical prostatectomy. Am. J. Surg. Pathol. 28, 928-934. to anti-androgens. Biochem. Biophys. Res. Commun. 173, 534-540.Young, C.Y., Montgomery, B.T., Andrews, P.E., Qui, S.D., Bilhartz, D.L., Tindall, D.J., 1991. Hormonal regulation of prostate-specific antigen messenger RNA in human prostatic adenocarcinoma cell line LNCaP. CancerRes. 51,[3748][3749][3750][3751][3752]

  Figure legendsFig. 1. Androgen receptor (AR) expression is differently regulated by androgens in VCaP and LNCaP cells. (A) Immunoblotting analysis of AR protein in VCaP and LNCaP cells grown for 2 h or 18 h in the presence vehicle (EtOH, -), 10 nM R1881, 10 µM cyproterone acetate (CTA), 10 µM bicalutamide (BIC), or 10 µM RD162 as indicated. AR and GAPDH were detected by respective specific antibodies as described in Materials and methods. The intensities of the AR bands of duplicate blots were measured by Li-COR Odyssey infrared imaging system, and the numbers above the immunoblots depict the amount of AR in relation to AR in vehicle-treated LNCaP cells (=1). The immunoblots are representative examples of three experiments. (B) RT-qPCR analysis of AR mRNA expression in VCaP and LNCaP cells. Cells were treated with indicated ligands for 12 h, and the AR mRNA was quantified as described in Materials and methods. The value of vehicle-treated LNCaP sample was set as 1. Columns represent the mean ± SD of three independent experiments.

Fig. 3 .

 3 Fig. 3. R1881 dose responses of AR target gene expression in VCaP and LNCaP cells. (A)Basal expression of a selection of AR target genes in VCaP cells vs. LNCaP cells. mRNAs of indicated AR target genes were analyzed by RT-qPCR. mRNA levels were calculated in relation to GAPDH mRNA levels in the samples, the values of LNCaP samples were set as 1, and the data were expressed as VCaP cell/LNCaP cell ratios. (B) Cells were treated with increasing concentrations of R1881 for 12 h and mRNAs of indicated AR target genes were analyzed by RT-qPCR. Relative (rel.) mRNA levels were calculated as described in Materials and methods, and the value of vehicle-treated LNCaP samples was set as 1. Dots and columns represent the mean ± SD of three independent experiments.

Fig. 4 .

 4 Fig.4. Cyproterone acetate as an inducer of AR target genes in VCaP and LNCaP cells. Cells were treated with increasing concentrations of cyproterone acetate (CTA) for 12 h and mRNAs of indicated AR target genes were analyzed by RT-qPCR. Relative (rel.) mRNA levels were calculated as described in Materials and methods, and the value of vehicle-treated LNCaP sample was set as 1. Columns represent the mean ± SD of three independent experiments.

Fig. 5 .

 5 Fig. 5. Loading of AR onto chromatin sites in response to R1881 or cyproterone acetate in VCaP and LNCaP cells. VCaP and LNCaP cells were treated with increasing concentrations of R1881 (A) or cyproterone acetate (CTA) (B) for 2 h and ChIP assays were performed using an antiserum against AR or normal rabbit serum (normal IgG). ChIP samples were used as templates in quantitative PCR with primers specific for major AR-binding regions of FKBP51, C6orf81, TMPRSS2, PSA, S100P and SPOCK1. The results are shown as fold over the normal IgG and represent the mean ± SD of three independent experiments.

Fig. 6 .Fig. 7 .

 67 Fig. 6. RD162 inhibits AR target gene expression more efficiently than bicalutamide in VCaP cells and LNCaP cells. Cells were treated with vehicle (EtOH), R1881 (1 nM), BIC (10 µM), RD162 (10 µM) or antagonists in combination with R1881 for 12 h as indicated and AR target gene mRNAs were analyzed by RT-qPCR and calculated as described in Materials and methods. The results are shown as induction by ligand over the vehicle-treated sample (set as 1) and represent the mean ± SD of three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001; ns, nonsignificant; for the difference between BIC-and RD162-treated samples. Fig. 7. Bicalutamide, but not RD162, promotes AR-chromatin interaction in VCaP and LNCaP cells. Binding of AR to the same chromatin regions that were analyzed in Fig. 5 was studied by ChIP assays in LNCaP and VCaP cells that were exposed to vehicle (EtOH), R1881 (1 nM), BIC (10 µM), RD162 (10 µM) or antagonists in combination with R1881 for 2 h as depicted. The results are shown as ligand inductions over the vehicle-treated samples (set as 1) and represent the mean ± SD of three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001; ns, nonsignificant; for the difference between BIC-and RD162treated samples.

  Figure1

Acknowledgements

We thank Merja Räsänen for skilful technical assistance. This work was supported by Academy of Finland, Finnish Cancer Foundation, Sigrid Jusélius Foundation, Ida Montini Foundation, North Savo Cancer Foundation, and The Finnish Medical Foundation.

Conflicts of interest

The authors declare no conflict of interest.