Ghislaine Garrel

Chantal Denoyelle

David L'hôte

Jean-Yves Picard

Jose Teixeira

Ursula B Kaiser

Jean-Noël Laverrière

Joëlle Cohen-Tannoudji
email: joelle.cohen-tannoudji@univ-paris-diderot.fr

GnRH transactivates human AMH receptor gene via Egr1 and FOXO1 in gonadotrope cells

Keywords: GnRH, AMHR2, Gonadotrope cells, Egr1, FOXO1, β-catenin

Background/objectives: Anti-Müllerian hormone (AMH) signaling is critical for sexual differentiation and gonadal function. AMH receptor type 2 (AMHR2) is expressed in extragonadal sites such as brain and pituitary and emerging evidence indicates that AMH biological action is much broader than initially thought. We recently reported that AMH signaling enhances FSH synthesis in pituitary gonadotrope cells. However, mechanisms regulating AMHR2 expression in these extragonadal sites remain to be explored. Method/ Results: Here, we demonstrated in perifused murine LβT2 gonadotrope cells that Amhr2 expression is differentially regulated by GnRH pulse frequency with an induction under high GnRH pulsatility. Furthermore, we showed that GnRH transactivates the human AMHR2 promoter in LβT2 cells. Successive deletions of the promoter revealed the importance of a short proximal region (-53/-37 bp) containing an Egr1 binding site. Using site-directed mutagenesis of Egr1 motif and siRNA mediated-knock down of Egr1, we demonstrated that Egr1 mediates basal and GnRH-dependent activity of the promoter, identifying Egr1 as a new transcription factor controlling hAMHR2 expression. We also showed that SF1 and β-catenin are required for basal promoter activity and demonstrated that both factors contribute to the GnRH stimulatory effect, independently of their respective binding sites. Furthermore, using a constitutively active mutant of FOXO1, we identified FOXO1 as a negative regulator of basal and GnRH-dependent AMHR2 expression in gonadotrope cells. Conclusions: This study identifies GnRH as a regulator of human AMHR2 expression, further highlighting the importance of AMH signaling in the regulation of gonadotrope function.

Introduction

The neurohormone Gonadotropin-releasing hormone (GnRH) is the central regulator of the reproductive system. GnRH is secreted by hypothalamic neurons in a pulsatile manner and reaches the anterior pituitary where it controls synthesis and secretion of both gonadotropins, Luteinizing hormone (LH) and Follicle-stimulating hormone (FSH), which are dimeric glycoproteins composed of a common α-subunit and rate limiting β-subunits (LHβ and FSHβ).

GnRH activates a G-protein coupled receptor expressed in gonadotrope cells, which activates a wide array of signaling pathways but predominantly the protein kinase C (PKC)/MAPK signaling pathways [START_REF] Stojilkovic | Gonadotropin-releasing hormone receptors: Structure and signal transduction pathways[END_REF][START_REF] Naor | Interactions of the gnrh receptor with heterotrimeric g proteins[END_REF]. This results in rapid activation of genes belonging to the immediate early gene family and culminates with the increased synthesis of genes coding for gonadotropin subunits i.e. Cga, Lhb and Fshb [START_REF] Counis | Gonadotropin-releasing hormone and the control of gonadotrope function[END_REF][START_REF] Burger | Regulation of gonadotropin subunit gene transcription[END_REF]. Expression of these subunit genes is differentially regulated by GnRH according to the pulse frequency of its stimulation. Fshb expression is optimally induced at low GnRH pulse frequency whereas Lhb expression is favored at high pulse frequency, the common α subunit being considered poorly regulated [START_REF] Dalkin | The frequency of gonadotropin-releasing-hormone stimulation differentially regulates gonadotropin subunit messenger ribonucleic acid expression[END_REF]. GnRH induces Lhb expression through rapid induction of the immediate early gene Egr1 [START_REF] Halvorson | The protein kinase c system acts through the early growth response protein 1 to increase lhbeta gene expression in synergy with steroidogenic factor-1[END_REF][START_REF] Wolfe | Early growth response protein 1 binds to the luteinizing hormone-beta promoter and mediates gonadotropin-releasing hormone-stimulated gene expression[END_REF]. The important role of Egr1 is emphasized by the lack of Lhb in pituitary gonadotrope cells of Egr1 knockout mice [START_REF] Lee | Luteinizing hormone deficiency and female infertility in mice lacking the transcription factor ngfi-a (egr-1)[END_REF][START_REF] Topilko | Multiple pituitary and ovarian defects in krox-24 (ngfi-a, egr-1)-targeted mice[END_REF]. Upon induction by GnRH, Egr1 interacts with other transcription factors such as SF1, Pitx1 or Sp1 to enhance Lhb transcription [START_REF] Tremblay | Egr-1 is a downstream effector of gnrh and synergizes by direct interaction with ptx1 and sf-1 to enhance luteinizing hormone beta gene transcription[END_REF][START_REF] Kaiser | Sp1, steroidogenic factor 1 (sf-1), and early growth response protein 1 (egr-1) binding sites form a tripartite gonadotropin-releasing hormone response element in the rat luteinizing hormone-beta gene promoter: An integral role for sf-1[END_REF]. GnRH regulation of Fshb occurs notably through the induction of immediate early genes belonging to the activator protein-1 family [START_REF] Thackray | Hormones in synergy: Regulation of the pituitary gonadotropin genes[END_REF]. Recently, GnRH was shown to regulate gonadotropin subunit gene transcription by abrogating the repressive effect of FOXO1 [START_REF] Arriola | Foxo1 transcription factor inhibits luteinizing hormone beta gene expression in pituitary gonadotrope cells[END_REF][START_REF] Choi | Foxo1 is a negative regulator of fshbeta gene expression in basal and gnrh-stimulated conditions in female[END_REF][START_REF] Lannes | Rapid communication: A microrna-132/212 pathway mediates gnrh activation of fsh expression[END_REF]. GnRH-induced posttranscriptional modifications of FOXO1 result in FOXO1 export from the nucleus, alleviating FOXO1 repression on the Fshb promoter [START_REF] Choi | Foxo1 is a negative regulator of fshbeta gene expression in basal and gnrh-stimulated conditions in female[END_REF][START_REF] Lannes | Rapid communication: A microrna-132/212 pathway mediates gnrh activation of fsh expression[END_REF]. In addition to GnRH, the coordinated transcription of gonadotropins relies on several signals, including locally produced activin, follistatin or bone morphogenetic proteins, as well as feedback signals originating from the gonads, such as steroids and inhibin [START_REF] Thackray | Hormones in synergy: Regulation of the pituitary gonadotropin genes[END_REF]. We recently demonstrated, by a combination of in vitro and in vivo approaches, that anti-müllerian hormone (AMH) directly and specifically stimulates Fshb and amplifies activin regulation of Fshb, identifying AMH as a new actor regulating gonadotrope activity [START_REF] Garrel | Anti-mullerian hormone: A new actor of sexual dimorphism in pituitary gonadotrope activity before puberty[END_REF].

AMH also called Müllerian inhibiting substance is a homodimeric glycoprotein that belongs to the transforming growth factor-β superfamily. AMH is secreted by Sertoli cells of the fetal testis and plays a crucial role during early male sex differentiation by inducing the regression of Müllerian ducts [START_REF] Josso | Testicular anti-mullerian hormone: History, genetics, regulation and clinical applications[END_REF]. The expression of AMH is however not restricted to embryonic development as AMH can be found in the Sertoli and granulosa cells of postnatal gonads and AMH produced by ovarian granulosa cells is now recognized as an important regulator of folliculogenesis [START_REF] Teixeira | Mullerian inhibiting substance: An instructive developmental hormone with diagnostic and possible therapeutic applications[END_REF][START_REF] Visser | Anti-mullerian hormone: A new marker for ovarian function[END_REF]. In addition, AMH expression was reported in adult mouse brain [START_REF] Wang | Mullerian inhibiting substance acts as a motor neuron survival factor in vitro[END_REF][START_REF] Lebeurrier | Anti-mullerianhormone-dependent regulation of the brain serine-protease inhibitor neuroserpin[END_REF][START_REF] Kow | Development of a sexually differentiated behavior and its underlying cns arousal functions[END_REF] and in pituitaries of immature male and female rats [START_REF] Garrel | Anti-mullerian hormone: A new actor of sexual dimorphism in pituitary gonadotrope activity before puberty[END_REF]. AMH exerts its function by binding to a specific type 2 receptor, AMHR2, that heterodimerizes with a type 1 receptor [START_REF] Di Clemente | Components of the anti-mullerian hormone signaling pathway in gonads[END_REF].

Both receptors are transmembrane serine/threonine receptors that recruit and phosphorylate SMAD proteins that will then translocate into the nucleus to regulate the transcription of several target genes [START_REF] Teixeira | Mullerian inhibiting substance: An instructive developmental hormone with diagnostic and possible therapeutic applications[END_REF][START_REF] Josso | Transduction pathway of anti-mullerian hormone, a sex-specific member of the tgf-beta family[END_REF]. Amhr2 expression was initially found in mesenchymal cells surrounding the Müllerian epithelium in the bipotential urogenital ridge [START_REF] Josso | Testicular anti-mullerian hormone: History, genetics, regulation and clinical applications[END_REF]. Amhr2 mRNA was then localized in Sertoli and granulosa cells of both fetal and adult gonads as well as in testicular Leydig cells [START_REF] Josso | Testicular anti-mullerian hormone: History, genetics, regulation and clinical applications[END_REF][START_REF] Visser | Anti-mullerian hormone: A new marker for ovarian function[END_REF]. In addition, more recent studies highlighted Amhr2 expression in extragonadal tissues, notably in the brain. AMH receptivity has been reported in motor and cortical neurons of rodent brain, in which AMH exerts survival and neuroprotective roles, respectively [START_REF] Wang | Mullerian inhibiting substance acts as a motor neuron survival factor in vitro[END_REF][START_REF] Lebeurrier | Anti-mullerianhormone-dependent regulation of the brain serine-protease inhibitor neuroserpin[END_REF]. Furthermore, accumulating evidence shows that AMH contributes to the central control of gonadal functions. The AMH receptor was indeed identified in a significant subset of hypothalamic GnRH neurons in mice and humans and AMH was found to potently activate the GnRH neuron firing [START_REF] Cimino | Novel role for anti-mullerian hormone in the regulation of gnrh neuron excitability and hormone secretion[END_REF][START_REF] Tata | Elevated prenatal anti-mullerian hormone reprograms the fetus and induces polycystic ovary syndrome in adulthood[END_REF]. Amhr2 transcripts and proteins are also present in rat pituitary gland as well as in the gonadotrope cell line LβT2 [START_REF] Garrel | Anti-mullerian hormone: A new actor of sexual dimorphism in pituitary gonadotrope activity before puberty[END_REF][START_REF] Bedecarrats | Regulation of gonadotropin gene expression by mullerian inhibiting substance[END_REF]. We previously reported a sexually dimorphic expression of Amhr2 transcripts in postnatal rat pituitary with higher steady-state transcript levels in immature female rats than in males [START_REF] Garrel | Anti-mullerian hormone: A new actor of sexual dimorphism in pituitary gonadotrope activity before puberty[END_REF]. In line with this difference, AMH treatment was shown to increase the secretion of the gonadotropin FSH in immature females while being ineffective in age-matched males. Furthermore, AMH was reported to up regulate FSH secretion and pituitary Fshb without affecting LH [START_REF] Garrel | Anti-mullerian hormone: A new actor of sexual dimorphism in pituitary gonadotrope activity before puberty[END_REF].

The mechanisms regulating AMHR2 gene expression have mostly been identified in the context of urogenital ridge or gonadal cell expression. Cloning of AMHR2 genes in humans [START_REF] Di Clemente | Cloning, expression, and alternative splicing of the receptor for anti-mullerian hormone[END_REF][START_REF] Baarends | A novel member of the transmembrane serine/threonine kinase receptor family is specifically expressed in the gonads and in mesenchymal cells adjacent to the mullerian duct[END_REF] and rodents [START_REF] Teixeira | Developmental expression of a candidate mullerian inhibiting substance type ii receptor[END_REF] and studies of their promoters [START_REF] Imbeaud | Insensitivity to anti-mullerian hormone due to a mutation in the human anti-mullerian hormone receptor[END_REF][START_REF] Teixeira | Transcriptional regulation of the rat mullerian inhibiting substance type ii receptor in rodent leydig cells[END_REF][START_REF] Klattig | Wilms' tumor protein wt1 is an activator of the anti-mullerian hormone receptor gene amhr2[END_REF][START_REF] De | Steroidogenic factor-1 regulates transcription of the human anti-mullerian hormone receptor[END_REF][START_REF] Hossain | Synergistic cooperation between the beta-catenin signaling pathway and steroidogenic factor 1 in the activation of the mullerian inhibiting substance type ii receptor[END_REF] have led to the identification of a limited number of regulators and transcription factors responsible for constitutive human or murine AMHR2 gene expression. Among them is the Wnt signaling pathway. Wnt7-a deficient mice are indeed sterile with retained Müllerian ducts and exhibit no Amhr2 mRNA expression [START_REF] Parr | Sexually dimorphic development of the mammalian reproductive tract requires wnt-7a[END_REF]. The canonical Wnt signaling pathway is mediated by stabilization of β-catenin have been identified in the hAMHR2 promoter and β-catenin as well as TCF4 (also designated as TCF7L2) have been reported to transactivate the hAMHR2 promoter [START_REF] Hossain | Synergistic cooperation between the beta-catenin signaling pathway and steroidogenic factor 1 in the activation of the mullerian inhibiting substance type ii receptor[END_REF]. The steroidogenic factor 1 (SF1/NR5A1), which belongs to the nuclear receptor superfamily, was found to transactivate murine and human AMHR2 promoters through its binding to SF1-motifs [START_REF] Teixeira | Developmental expression of a candidate mullerian inhibiting substance type ii receptor[END_REF][START_REF] De | Steroidogenic factor-1 regulates transcription of the human anti-mullerian hormone receptor[END_REF].

and
SF1 has also been described to synergistically cooperate with the β-catenin signaling pathway to activate the hAMHR2 promoter [START_REF] Hossain | Synergistic cooperation between the beta-catenin signaling pathway and steroidogenic factor 1 in the activation of the mullerian inhibiting substance type ii receptor[END_REF]. A handful of hormones regulating AMHR2 expression have been identified so far, mostly from studies on ovarian tissues. FSH and estradiol were reported to down regulate Amhr2 transcripts in rat ovarian follicles [START_REF] Baarends | Anti-mullerian hormone and anti-mullerian hormone type ii receptor messenger ribonucleic acid expression in rat ovaries during postnatal development, the estrous cycle, and gonadotropin-induced follicle growth[END_REF] and an inhibitory effect of LH and estradiol on AMHR2 mRNA levels was reported in human luteinized granulosa cells [START_REF] Pierre | Loss of lh-induced down-regulation of antimullerian hormone receptor expression may contribute to anovulation in women with polycystic ovary syndrome[END_REF][START_REF] Pierre | Dysregulation of the anti-mullerian hormone system by steroids in women with polycystic ovary syndrome[END_REF]. Contrasting with gonadotropins, bone morphogenetic protein 4 (BMP4) and BMP15

were recently demonstrated to up regulate AMHR2 mRNA levels in human granulosa cells through transcriptional activation of the AMHR2 promoter [START_REF] Pierre | The bone morphogenetic protein 15 up-regulates the antimullerian hormone receptor expression in granulosa cells[END_REF]. However, the signaling pathways and mechanisms by which these hormones regulate the transcriptional activity of the AMHR2 gene are still unknown. We recently reported that AMHR2 expression is regulated by GnRH in pituitary gonadotrope cells, adding GnRH to the known regulators of AMHR2 [START_REF] Garrel | Anti-mullerian hormone: A new actor of sexual dimorphism in pituitary gonadotrope activity before puberty[END_REF].

The present study was undertaken to gain further insight into the mechanisms underlying GnRH regulation of AMHR2 gene expression. We demonstrate in perifused LβT2 gonadotrope cells that endogenous Amhr2 expression is differentially regulated by GnRH pulse frequency.

We also show by studying human AMHR2 promoter activity in LβT2 cells that GnRH enhances

AMHR2

Cell cultures

The mouse gonadotrope LβT2 and αT3-1 cell lines and the mouse hypothalamic GnRH neuronal cell line GT1-7, generously provided by Dr P Mellon (University of California, La Jolla, CA) [START_REF] Thomas | The l beta t2 clonal gonadotrope: A model for single cell studies of endocrine cell secretion[END_REF][START_REF] Windle | Cell lines of the pituitary gonadotrope lineage derived by targeted oncogenesis in transgenic mice[END_REF], were maintained in monolayer cultures in DMEM-Glutamax (Gibco, Life Technologies) supplemented with 10% fetal calf serum (FBS) and 0.5% penicillin/streptomycin (P/S) (Sigma-Aldrich). Treatments of cells are indicated below, in the corresponding paragraphs. For Sp1 and Egr1 mRNA quantification, LβT2 cells were plated in 12-well plates (0.8 to 1x10 6 per well) in duplicate and starved overnight in serum-free medium before incubation for 4 h with 10 nM GnRHa.

Pulsatile stimulation of LβT2 cells

The perifusion system was designed according to [START_REF] Bedecarrats | Differential regulation of gonadotropin subunit gene promoter activity by pulsatile gonadotropin-releasing hormone (gnrh) in perifused l beta t2 cells: Role of gnrh receptor concentration[END_REF] with modifications. Briefly, LβT2 cells were plated in perifusion chambers mounted on glass slides coated with poly L-Lysine (Sigma-Aldrich). Cells were incubated in the chambers overnight in a static culture system in DMEM containing 10% FBS and 0.5% P/S and then serum starved for 24 h in DMEM 1% FBS and 0.1% P/S. The chambers were then mounted in the perifusion system at a constant flow rate of 0.3 ml/min and cells were perifused or not with 20 nM GnRH for 21 h in continuous or pulsatile (one pulse every 30 min or one pulse every 120 min) stimulation mode.

Plasmid constructs

A 2252 (-2199/+53)-bp human AMHR2 promoter (Ref-SeqGene NG-015981) [START_REF] Imbeaud | Insensitivity to anti-mullerian hormone due to a mutation in the human anti-mullerian hormone receptor[END_REF][START_REF] Pierre | The bone morphogenetic protein 15 up-regulates the antimullerian hormone receptor expression in granulosa cells[END_REF]

Transient transfections and luciferase assays

For transient transfections, the human AMHR2 promoter luciferase fusion contruct (100 ng/well) together with pRL-SV40 as a control for transfection efficiency (5 ng/well) were mixed with 0.5 µl LipofectAMINE2000 reagent (Life Technologies, Gaithersburg, MD) in 10

µl OptiMEM-1 for 30 min. The transfection mix was then added to 0.9 x 10 5 cells in 30 µl

OptiMEM-1 before plating in 96-well plates (each point in triplicate). Cells were incubated for 5 h before adding 50 µl DMEM supplemented with 5% FBS and 0.25% P/S (Sigma-Aldrich).

The next morning, medium was replaced by 100 µl/well DMEM-1% FBS-0.01% P/S and cells were grown an additional 24 h. Then, cells were treated for 4 h with GnRHa (1, 10 nM), GnRH

Real-time PCR

Total RNA from LβT2 were isolated with a RNeasy-kit (Qiagen, France) and first-strand cDNA was obtained from 1-2 µg RNA with Superscript II reverse transcriptase (Invitrogen) using random primers according to the manufacturer's instructions. Quantification of Amhr2, Lhb, Fshb, Sp1 and Egr1 mRNA was performed by real-time qPCR using Takyon No ROX SYBR master mix (Eurogentec). Real-time qPCR was carried out in LightCycler 480 (Roche Diagnostics) using 5 µl of the mix containing 0.5 µM of each primer and 5 µl of a 1:20 cDNA dilution. Cycling conditions included an initial heat-denaturing step at 95°C for 3 min, followed by 40 cycles at 95°C for 10 sec, 60°C for 10 sec and 72°C for 10 sec. Oligonucleotide primer sequences are indicated in Table 2 and Cyclophilin was used to normalize RNA expression levels. Primer pairs were designed to target cDNA fragments encompassing at least one intron in the gene sequence to prevent amplification of genomic DNA. The specificity of amplification was checked by gel electrophoresis and melting curve analysis. Data were analyzed using the advanced-E-method with standard-curve derived efficiencies obtained from LightCycler 480 software.

Statistical analyses

Experiments were performed in triplicate and all given values are the mean ± SEM of at least 4 independent experiments. Data were analyzed using the Prism 6 Software (GraphPad Software, Inc). When distribution was normal, statistical differences were determined using one-way or two-way ANOVA followed by a Dunnett's t test for multiple comparisons and The GnRH pulsatile pattern is critical for appropriate regulation of gonadotrope activity [START_REF] Dalkin | The frequency of gonadotropin-releasing-hormone stimulation differentially regulates gonadotropin subunit messenger ribonucleic acid expression[END_REF].

To determine whether endogenous Amhr2 expression could be differentially regulated depending on GnRH pulse frequency, LβT2 cells were perifused for 21 h and exposed to vehicle alone, to continuous GnRH stimulation or to pulsatile stimulation at either high (every 30 min) or low (every 2 h) pulse frequency. The pulse frequencies chosen are reported to favor

Lhb expression and Fshb expression respectively [START_REF] Dalkin | The frequency of gonadotropin-releasing-hormone stimulation differentially regulates gonadotropin subunit messenger ribonucleic acid expression[END_REF][START_REF] Thompson | Gnrh pulse frequencydependent stimulation of fshbeta transcription is mediated via activation of pka and creb[END_REF]. Accordingly, maximum Lhb and Fshb transcript levels were found at high and low GnRH pulse frequencies, respectively (Fig

GnRH enhances human AMHR2 promoter activity in gonadotrope cells.

To determine whether GnRH can regulate transcription of the AMHR2 gene in gonadotrope cells, we took advantage of the human AMHR2 (hAMHR2) promoter that we previously cloned [START_REF] Imbeaud | Insensitivity to anti-mullerian hormone due to a mutation in the human anti-mullerian hormone receptor[END_REF] and which shares 70% identity in its proximal region (-375/+78 bp) with the murine promoter. A hAMHR2 promoter/firefly luciferase reporter gene construct was transiently transfected into αT3-1 and LβT2 cell lines that were derived from immature and mature pituitary cells, respectively [START_REF] Alarid | Immortalization of pituitary cells at discrete stages of development by directed oncogenesis in transgenic mice[END_REF][START_REF] Pernasetti | Cellspecific transcriptional regulation of follicle-stimulating hormone-beta by activin and gonadotropinreleasing hormone in the lbetat2 pituitary gonadotrope cell model[END_REF]. Because a significant subset of GnRH neurons, both in mice and human, express AMHR2 [START_REF] Cimino | Novel role for anti-mullerian hormone in the regulation of gnrh neuron excitability and hormone secretion[END_REF], we also expressed this construct in the hypothalamic GnRH neuronal cell line, GT1-7. GnRHa stimulated hAMHR2 promoter activity by ~ 2.5 fold in LβT2 gonadotrope cells (Fig. 2A). This effect was not found in αT3-1 or GT1-7 cells. A significant increase in hAMHR2 promoter activity was also found after a more prolonged treatment (24 h) of LβT2 cells with GnRHa (170 ± 6%, data not shown) and no noticeable increase could still be observed in αT3-1 or GT1-7 cells. Further analyses revealed that luciferase activity can be significantly stimulated by a 10-fold lower concentration of GnRHa and that GnRHa stimulation was completely blocked by the GnRH antagonist antide (Fig. 2B), demonstrating the specificity of the observed effect. The native hormone, GnRH, also enhanced hAMHR2 promoter activity although to a lesser extent than GnRHa, as expected given the higher potency of the agonist triptorelin. In contrast, the neuropeptide PACAP, known to regulate pituitary gonadotrope function, was unable to activate hAMHR2 promoter activity (Fig. 2B). Altogether, these data indicate that GnRH stimulates the 2252-bp hAMHR2

promoter in a cell specific manner, with a significant stimulatory effect in LβT2 gonadotrope cells. We next compared the 2252-bp promoter activity with a shorter 428-bp promoter used for successive cloning of the promoter. As illustrated in Fig. 2C, both promoters exhibited similar basal luciferase activities. Furthermore, GnRHa stimulatory effect did not significantly differ between the two constructs indicating that the cis-acting elements required for GnRH regulation are located within the proximal 428-bp fragment of the hAMHR2 promoter.

A proximal 37-bp promoter region is required for GnRH transactivation of human

AMHR2.

Sequence analysis of the proximal -375/+53 bases of the hAMHR2 promoter using the CIS-BP database (http://cisbp.ccbr.utoronto.ca/index.php) [START_REF] Weirauch | Determination and inference of eukaryotic transcription factor sequence specificity[END_REF] revealed consensus binding sites for several regulatory elements which are indicated in the schematic representation shown in Fig. 3A. Some of them have already been described, including two SF1 binding sites, two TCF binding sites, and one Sp1 binding site ( [START_REF] De | Steroidogenic factor-1 regulates transcription of the human anti-mullerian hormone receptor[END_REF][START_REF] Hossain | Synergistic cooperation between the beta-catenin signaling pathway and steroidogenic factor 1 in the activation of the mullerian inhibiting substance type ii receptor[END_REF], Fig 3B). Furthermore, we also identified two additional motifs that have not yet been reported: a Egr1 motif at -52 bp, identified with a high confidence, which partially overlaps with the Sp1 motif and a non-canonical FOXO binding site in the reverse orientation (CIS-BP Motif ID M5467-1.02, [START_REF] Jolma | DNA-binding specificities of human transcription factors[END_REF]) at nucleotides -113 to -103 (Fig. 3B).

In order to locate the region required for GnRH activation, we performed a panel of gradual deletions of the hAMHR2 promoter. As illustrated in Fig. 3C, the loss of distal SF1 and TCF motifs significantly decreased basal promoter activity. In contrast, the extent of GnRH stimulation was not affected (1.78 ± 0.11 and 2.5 ± 0.39% stimulation for the -375/+53 bp and the -247/+53 bp promoter, respectively). GnRHa-dependent luciferase activity remained unaltered following successive deletions leading to the -53 bp promoter (Fig. 3C). Deletion of the fragment located between -53 and -37 bp of the promoter, which contains the Egr1/Sp1

Egr1 mediates GnRH transactivation of human AMHR2 in gonadotrope cells

To confirm the role of the Egr1/Sp1 binding site in GnRH stimulation of hAMHR2 promoter activity, we performed site-directed mutagenesis. Mutation resulted in a significant decrease in basal as well as in GnRH-dependent luciferase activity as compared to the native promoter fragment (Fig. 4A). Egr1 is highly induced by GnRH in gonadotrope cells and mediates GnRHstimulated expression of Lhb [START_REF] Halvorson | The protein kinase c system acts through the early growth response protein 1 to increase lhbeta gene expression in synergy with steroidogenic factor-1[END_REF][START_REF] Wolfe | Early growth response protein 1 binds to the luteinizing hormone-beta promoter and mediates gonadotropin-releasing hormone-stimulated gene expression[END_REF]. To examine the potential role of Egr1 in GnRH stimulation of AMHR2 expression, we next used a small interfering RNA (siRNA) strategy to decrease Egr1 expression. LβT2 cells were transfected with a pool of siRNAs specific to mouse Egr1 (siRNA Egr1) or with a pool of nontargeting siRNAs (siRNA NT) before GnRHa stimulation.

In siRNA NT-transfected cells, GnRHa stimulated hAMHR2 promoter activity by 218 ± 26% (Fig. 4B). Transfection of cells with siRNA Egr1 reduced basal promoter activity. Furthermore, siRNA Egr1 abrogated the GnRH stimulatory effect indicating that Egr1 is required for GnRH regulation of hAMHR2 expression (Fig. 4B).

FOXO1 represses basal and GnRH-dependent activity of human AMHR2 promoter

To examine the potential involvement of putative FOXO binding site and FOXO transcription factors, we transiently cotransfected LβT2 cells with plasmids expressing wild-type FOXO1 (FOXO1-Wt) or a constitutively active, phosphorylation-defective form of FOXO1 (FOXO1-CAM) [START_REF] Qiang | Uncoupling of acetylation from phosphorylation regulates foxo1 function independent of its subcellular localization[END_REF]. The constitutively active form of FOXO1 was previously reported to localize exclusively into the nucleus of LβT2 cells [START_REF] Choi | Foxo1 is a negative regulator of fshbeta gene expression in basal and gnrh-stimulated conditions in female[END_REF][START_REF] Lannes | Rapid communication: A microrna-132/212 pathway mediates gnrh activation of fsh expression[END_REF]. As illustrated in Fig. 5A, we observed that overexpression of FOXO1-CAM decreased basal hAMHR2 transcriptional activity. Notably, FOXO1-CAM also markedly reduced the GnRHa stimulatory effect on the promoter (Fig. 5A).

Because the phosphoinositide 3-kinase (PI3K) pathway mediates GnRH regulation of FOXO1 export in LβT2 cells [START_REF] Choi | Foxo1 is a negative regulator of fshbeta gene expression in basal and gnrh-stimulated conditions in female[END_REF], we next examined whether blocking the PI3K pathway would decrease GnRH-induced hAMHR2 promoter activity (Fig. 5B). Pretreatment of cells with the PI3K signaling pathway inhibitor LY294002 (20 µM) significantly reduced by 48 ± 8%, (Fig.

5B increased mutated hAMHR2 promoter to a similar extent as that observed with the wild-type promoter. This suggests that although FOXO1 decreases GnRH induction of hAMHR2 activity, it may not act through direct binding to hAMHR2 promoter as already reported for murine Fshb and Lhb promoters [START_REF] Arriola | Foxo1 transcription factor inhibits luteinizing hormone beta gene expression in pituitary gonadotrope cells[END_REF][START_REF] Choi | Foxo1 is a negative regulator of fshbeta gene expression in basal and gnrh-stimulated conditions in female[END_REF][START_REF] Skarra | Forkhead box o1 is a repressor of basal and gnrh-induced fshb transcription in gonadotropes[END_REF].

SF1 and β-catenin are required for basal and GnRH-stimulated human AMHR2 promoter activities.

SF1 and TCF4 have been reported to be necessary for basal hAMHR2 expression [START_REF] De | Steroidogenic factor-1 regulates transcription of the human anti-mullerian hormone receptor[END_REF][START_REF] Hossain | Synergistic cooperation between the beta-catenin signaling pathway and steroidogenic factor 1 in the activation of the mullerian inhibiting substance type ii receptor[END_REF].

Furthermore, both SF1 and β-catenin signaling can be activated by GnRH [START_REF] Haisenleder | Gnrh regulates steroidogenic factor-1 (sf-1) gene expression in the rat pituitary[END_REF][START_REF] Gardner | Nuclear stabilization of beta-catenin and inactivation of glycogen synthase kinase-3beta by gonadotropin-releasing hormone: Targeting wnt signaling in the pituitary gonadotrope[END_REF] and are involved in GnRH regulation of Lhb expression [START_REF] Tremblay | Egr-1 is a downstream effector of gnrh and synergizes by direct interaction with ptx1 and sf-1 to enhance luteinizing hormone beta gene transcription[END_REF][START_REF] Kaiser | Sp1, steroidogenic factor 1 (sf-1), and early growth response protein 1 (egr-1) binding sites form a tripartite gonadotropin-releasing hormone response element in the rat luteinizing hormone-beta gene promoter: An integral role for sf-1[END_REF][START_REF] Salisbury | Maximal activity of the luteinizing hormone beta-subunit gene requires beta-catenin[END_REF]. To better delineate the role of these two transcription factors, we performed site directed mutagenesis and siRNA-mediated knock down experiments. As shown in Fig. 6A, basal luciferase activity of the hAMHR2 promoter carrying mutations of both SF1 binding sites [START_REF] Klattig | Wilms' tumor protein wt1 is an activator of the anti-mullerian hormone receptor gene amhr2[END_REF][START_REF] De | Steroidogenic factor-1 regulates transcription of the human anti-mullerian hormone receptor[END_REF] was significantly reduced compared to the control promoter in LβT2 cells. In contrast, the GnRH stimulatory effect was still observed, suggesting that intact SF1-binding sites are not required for GnRH stimulation.

siRNA-mediated decrease in SF1, leading to a 50 ± 7% reduction in SF1 transcripts levels after 24h transfection (RT-qPCR experiments, data not shown), reduced basal luciferase activity (Fig. 6B) in agreement with results from mutation experiments. Decreasing SF1 expression completely abrogated GnRH stimulation of hAMHR2 promoter activity (Fig. 6B). This indicates that SF1 contributes to GnRH regulation although it may not bind directly to SF1 motifs and suggests an indirect mechanism of control.

To examine the potential role of the β-catenin/TCF pathway in basal and GnRH dependent hAMHR2 activity, we mutated one or both TCF binding sites in the 428-bp hAMHR2 promoter (Fig. 7A). Mutation of the proximal TCF binding site did not affect basal or GnRH stimulated promoter activity. In contrast, basal luciferase activity was reduced by mutation of the distal TCF motif, which was also observed when both TCF sites were mutated (Fig. 7A). The fold increase in GnRH stimulation was unaffected as both basal and GnRH dependent luciferase activities were reduced under these conditions (Fig. 7A). Significant levels of TCF4 transcripts were found in LβT2 cells (data not shown) suggesting that TCF4 transcription factor contributes to basal promoter activity. To further examine the role of β-catenin, we specifically targeted β-catenin expression using siRNAs. LβT2 cells were transfected with a pool of siRNAs specific to mouse β-catenin (siRNA β-catenin) or with a pool of nontargeting siRNAs (Fig. 7B, inset). In a complementary approach, we inhibited activity of the β-catenin pathway using the pharmacological inhibitor, JW74. As shown in Fig. 7C, we observed that JW74 significantly reduced the GnRH stimulatory effect by 44 ± 7%. Activation of β-catenin by GnRH relies on PKC-dependent signaling pathways, including notably the recruitment of c-Jun N-terminal kinases (JNK) [START_REF] Gardner | Nuclear stabilization of beta-catenin and inactivation of glycogen synthase kinase-3beta by gonadotropin-releasing hormone: Targeting wnt signaling in the pituitary gonadotrope[END_REF][START_REF] Wang | Beta-catenin regulates gnrh-induced fshbeta gene expression[END_REF]. Treatment of cells with the PKC inhibitor GF109203X significantly reduced the GnRH-induced increase of hAMHR2 promoter activity by 60 ± 13%.

Furthermore, GnRH-induced activation was fully prevented by the JNK inhibitor, SP600125.

Altogether, these experiments indicate that both SF1 and β-catenin mediate GnRH stimulatory effect on hAMHR2 transcription in LβT2 gonadotrope cells, although they may not bind to their known response elements.

Discussion

First identified in the mesenchyme of Müllerian ducts and in gonads, AMHR2 was subsequently localized in several other organs, including the brain and the pituitary, suggesting that AMH biological effects are far more diverse than initially thought. However, the mechanisms regulating AMHR2 gene expression, notably in extragonadal sites still remain elusive. Here, we demonstrate that gene expression of both human and murine AMHR2 is regulated by the neuroendocrine hormone, GnRH. Indeed, GnRH was shown to increase endogenous AMHR2 transcripts in the gonadotrope cell line LβT2 and to enhance the activity of a human AMHR2 promoter. Transactivation of the promoter was not observed in the immature αT3-1 gonadotrope cell line nor in neuronal GT1-7 cell line, although the latter is receptive to both GnRH and AMH.

We identified several transcription factors required for GnRH regulation of AMHR2 gene expression. We notably uncovered the functional importance of the Egr1 transcription factor.

We localized an Egr1 binding site in the proximal region of the hAMHR2 promoter and provided evidence, by deletion and mutation, that this site is needed for GnRH-mediated induction. Furthermore, because siRNA-inhibited expression of Egr1 mRNA in LβT2 cells do not bind to these sites of the promoter. In line with this result, it was reported in HeLa cells, that SF1 alone does not activate the hAMHR2 promoter but instead binds to β-catenin and synergistically cooperates with β-catenin to activate the promoter [START_REF] Hossain | Synergistic cooperation between the beta-catenin signaling pathway and steroidogenic factor 1 in the activation of the mullerian inhibiting substance type ii receptor[END_REF]. SF1 is known to physically interact with β-catenin in LβT2 cells [START_REF] Salisbury | Maximal activity of the luteinizing hormone beta-subunit gene requires beta-catenin[END_REF]. Furthermore, SF1 has been reported to synergize with Egr1 and Sp1 for transactivation of the Lhb promoter [START_REF] Kaiser | Sp1, steroidogenic factor 1 (sf-1), and early growth response protein 1 (egr-1) binding sites form a tripartite gonadotropin-releasing hormone response element in the rat luteinizing hormone-beta gene promoter: An integral role for sf-1[END_REF]. Interestingly, it was demonstrated in LβT2 cells that SF1 interacts with Egr1 through β-catenin and that β-catenin colocalization with SF1 and Egr1 on the mouse Lhb promoter is increased by GnRH [START_REF] Salisbury | Maximal activity of the luteinizing hormone beta-subunit gene requires beta-catenin[END_REF].

Based on these studies, we can hypothesize from our present findings that, by increasing Egr1 binding to the proximal region of the AMHR2 promoter, GnRH enables the formation of a complex, containing β-catenin and SF1, that cooperates with Egr1 to fully activate the AMHR2 promoter (see the proposed hypothetical model in Fig. 8). In this study, we also uncovered the functional importance of the transcription factor FOXO1 in the regulation of the hAMHR2 [START_REF] Law | Lhcgr expression in granulosa cells: Roles for pka-phosphorylated beta-catenin, tcf3, and foxo1[END_REF], as well as of several genes involved in the steroidogenic biosynthesis pathways [START_REF] Liu | Fsh and foxo1 regulate genes in the sterol/steroid and lipid biosynthetic pathways in granulosa cells[END_REF]. Given the importance of AMH signaling in granulosa cell proliferation and steroidogenesis, it would be of interest to determine whether FOXO1 also represses AMHR2 gene expression in these cells.

It has long been reported that the frequency of GnRH pulses is critical for the appropriate ratio of FSH vs LH synthesis and secretion [START_REF] Dalkin | The frequency of gonadotropin-releasing-hormone stimulation differentially regulates gonadotropin subunit messenger ribonucleic acid expression[END_REF]. This has been proposed as one of the mechanisms allowing gonadotrope cells to interpret the varying pattern of GnRH secretion [START_REF] Burger | Regulation of gonadotropin subunit gene transcription[END_REF].

Gonadotropin genes are not the only genes differentially regulated by GnRH pulse frequency and several other target genes have been identified [START_REF] Lawson | Pulse sensitivity of the luteinizing hormone beta promoter is determined by a negative feedback loop involving early growth response-1 and ngfi-a binding protein 1 and 2[END_REF], including follistatin and nitric oxide synthase 1 [START_REF] Besecke | Gonadotropin-releasing hormone regulates follicle-stimulating hormone-beta gene expression through an activin/follistatin autocrine or paracrine loop[END_REF][START_REF] Garrel | Sustained gonadotropin-releasing hormone stimulation mobilizes the camp/pka pathway to induce nitric oxide synthase type 1 expression in rat pituitary cells in vitro and in vivo at proestrus[END_REF]. Our present study identifies AMHR2 as one of these genes. Indeed, using LβT2 cells in a perifusion system, we showed that GnRH at high pulse frequency increases Amhr2 expression whereas lower frequencies were ineffective. This result as well as the positive regulation of AMHR2 promoter by GnRH is not in agreement with our previous report indicating that GnRHa treatment of LβT2 cells in static cultures decreased Amhr2 transcript levels by ~ 30%, a decrease which was also observed in rat pituitary, 24 h after GnRHa injection [START_REF] Garrel | Anti-mullerian hormone: A new actor of sexual dimorphism in pituitary gonadotrope activity before puberty[END_REF]. Our present data using perifused LβT2 cells strongly suggest that this decrease reflected desensitization of cells to GnRH stimulation. Indeed, we observed that Amhr2 expression is similarly decreased in response to a continuous GnRH stimulation which was associated with a desensitization of the gonadotropin response as reflected by decreased levels of gonadotropin β-subunit transcripts. Furthermore, reevaluation of Lhb and Fshb transcript levels in the pituitaries of GnRHa-treated rats (unpublished data) were also indicative of gonadotrope cell desensitization to GnRH. Altogether, our data thus indicate that GnRH finely regulates Amhr2 expression in gonadotrope cells with a selective increase observed with a high GnRH pulse frequency.

Accumulating evidence highlights that AMH may act at every level of the gonadotrope axis.

Indeed, in addition to gonads, AMHR2 mRNA has recently been localized in rodent and human GnRH neurons, and intracerebroventricular or peripheral injections of AMH in mice was able to increase LH secretion [START_REF] Cimino | Novel role for anti-mullerian hormone in the regulation of gnrh neuron excitability and hormone secretion[END_REF][START_REF] Tata | Elevated prenatal anti-mullerian hormone reprograms the fetus and induces polycystic ovary syndrome in adulthood[END_REF]. Furthermore, AMHR2 was identified in rodent pituitary as well as in gonadotrope cells and AMH was shown to positively regulate FSH [START_REF] Garrel | Anti-mullerian hormone: A new actor of sexual dimorphism in pituitary gonadotrope activity before puberty[END_REF][START_REF] Bedecarrats | Regulation of gonadotropin gene expression by mullerian inhibiting substance[END_REF][START_REF] Kereilwe | Anti-mullerian hormone receptor type 2 is expressed in gonadotrophs of postpubertal heifers to control gonadotrophin secretion[END_REF]. Very recently, AMH regulation on FSH secretion was also reported in a non-rodent species i.e. in heifers [START_REF] Kereilwe | Anti-mullerian hormone receptor type 2 is expressed in gonadotrophs of postpubertal heifers to control gonadotrophin secretion[END_REF]. Because we characterized Amh transcripts in immature rat pituitary and in LβT2 gonadotrope cells [START_REF] Garrel | Anti-mullerian hormone: A new actor of sexual dimorphism in pituitary gonadotrope activity before puberty[END_REF], this suggests that locally produced AMH could contribute to the regulation of FSH secretion as described for pituitary activin or follistatin [66]. It would be of interest to determine whether AMH is coexpressed with its receptor in the pituitary of other mammalian species. Supporting the functional importance of AMH signaling in the pituitary, AMHR2 gene expression has also been identified in the pituitary of non-mammalian species i.e. the teleost Nile Tilapia [START_REF] Poonlaphdecha | Elevated amh gene expression in the brain of male tilapia (oreochromis niloticus) during testis differentiation[END_REF][START_REF] Pfennig | The role of amh signaling in teleost fish -multiple functions not restricted to the gonads[END_REF]. The present work reinforces the intriguing hypothesis that AMH contributes to the pituitary control of gonadal functions by demonstrating that gonadotrope AMH receptivity is regulated by the neuroendocrine hormone GnRH. The physiological implication of Amhr2 up-regulation by high pulse frequency of GnRH remains to be understood. It has been clearly demonstrated that high frequency of GnRH pulses only poorly increases rodent Fshb transcript levels [START_REF] Dalkin | The frequency of gonadotropin-releasing-hormone stimulation differentially regulates gonadotropin subunit messenger ribonucleic acid expression[END_REF]. There are, however, physiological situations where high GnRH pulsatility is associated with elevation of both LH and FSH secretion.

Among them are the cyclic preovulatory period in adults and the so-called "minipuberty" which is characterized by a transient activation of the Hypothalamic-Pituitary-Gonadal axis in immature animals [START_REF] Ojeda | Recent advances in the endocrinology of puberty[END_REF]. We recently detected significant levels of pituitary Amhr2 transcripts in immature but not adult rats suggesting that AMH could play a crucial role in regulating FSH secretion during the immature period. During this period, a marked increase in FSH secretion and pituitary Fshb mRNA levels has been reported in several mammalian species [START_REF] Ojeda | Recent advances in the endocrinology of puberty[END_REF]. Hence, increasing AMH receptivity in pituitary gonadotropes may be an indirect mechanism allowing elevated FSH synthesis despite an increase in GnRH tone that would not favor FSH. In rat as in other mammals, this period is characterized by a sexual dimorphism of FSH secretion with higher circulating FSH concentrations and pituitary Fshb mRNA levels in females than in males [START_REF] Garrel | Anti-mullerian hormone: A new actor of sexual dimorphism in pituitary gonadotrope activity before puberty[END_REF][START_REF] Ojeda | Recent advances in the endocrinology of puberty[END_REF]. Interestingly, we reported that pituitary Amhr2 expression is also higher in female rats during this developmental period [START_REF] Garrel | Anti-mullerian hormone: A new actor of sexual dimorphism in pituitary gonadotrope activity before puberty[END_REF]. A higher GnRH neurons activity has recently been reported in infantile 2-3-week-female mice as compared to males [START_REF] Dulka | Prepubertal development of gonadotropin-releasing hormone neuron activity is altered by sex, age, and prenatal androgen exposure[END_REF]. Based on our present study, we can hypothesize that this contributes to dimorphism of pituitary Amhr2 expression and hence to the sexually dimorphic secretion of FSH during infancy.

In conclusion, our study shed new light on the mechanisms regulating AMHR2 expression.

Moreover, the identification of AMHR2 as a target of GnRH further underlines the role of AMH in the control of pituitary gonadotrope function.

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  its association with nuclear T-cell factor (TCF)/Lymphoid enhancer binding factor (LEF) transcription factors leading to activation of target gene transcription. Four TCF binding sites Downloaded by: Leiden University Medisch Centrum 132.229.13.63 -11/3/2018 3:02:08 AM A c c e p t e d m a n u s c r i p t

  transcriptional activity through the combined stimulatory action of Egr1 together with SF1 and β-catenin and the removal of FOXO1 repression. Downloaded by: Leiden University Medisch Centrum 132.229.13.63 -11/3/2018 3:02:08 AM A c c e p t e d m a n u s c r i p t Materials & Methods Materials GF109203X (Bisindolylmaleimide I), SP600125, LY294002, GnRH, GnRH agonist Triptorelin ([D-Trp 6 ]GnRH; GnRHa) and GnRH antagonist antide were from Sigma (Saint-Quentin Fallavier, France). JW74 were from Tocris (R&D Systems, Lille, France). Pituitary adenylate cyclase-activating polypeptide (PACAP38) was from Calbiochem (VWR International, Strasbourg, France).

1 ,

 1 inset). As shown in Fig 1, a high GnRH pulse frequency significantly increased Amhr2 transcript levels. In contrast, low pulse frequency of GnRH was ineffective. Continuous exposure of cells with GnRH decreased Amhr2 transcript levels by about 33 ± 4.9% as previously observed with the GnRH agonist triptorelin (GnRHa) in static cultures [16]. Under continuous GnRH, Fshb transcript levels were also significantly decreased and Lhb transcript levels were no longer increased (Fig 1, inset), suggesting that cells are desensitized to GnRH stimulation. Altogether, these data indicate that endogenous Amhr2 expression is regulated by GnRH in LβT2 gonadotrope cells and is sensitive to GnRH pulse frequency.

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  inset) the GnRH stimulatory effect on hAMHR2 promoter activity. To examine the requirement of the putative FOXO binding site, we next transiently transfected LβT2 cells with a hAMHR2 promoter with a mutated FOXO motif. As shown in Fig. 5C, GnRHa treatment still Downloaded by: Leiden University Medisch Centrum 132.229.13.63 -11/3/2018 3:02:08 AM A c c e p t e d m a n u s c r i p t

  Centrum 132.229.13.63 -11/3/2018 3:02:08 AM A c c e p t e d m a n u s c r i p t (siRNA NT) before GnRHa stimulation. This led to a significant reduction in β-catenin transcript levels in LβT2 cells as assessed by qRT-PCR (50 ± 3.5% of siRNA NT-transfected cells, data not shown). As expected in NT-transfected cells, GnRHa stimulated the 2252-bp hAMHR2 promoter activity by 187 ± 4% (Fig. 7B). Transfection of cells with siRNA β-catenin reduced basal promoter activity. It also reduced the extent of GnRH stimulation by 48 ± 17%

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  Centrum 132.229.13.63 -11/3/2018 3:02:08 AM A c c e p t e d m a n u s c r i p t bp, base pairs; Egr1, Early growth response protein 1; FOXO, Forkhead Box O; GnRHa, GnRH agonist; JNK, c-Jun N-terminal kinase; PACAP, Pituitary adenylate cyclase-activating polypeptide; PI3K, Phosphatidylinositol 3-kinase; PKC, Protein kinase C; P/S, penicillin/streptomycin; siNT, nontargeting siRNA; TCF/LEF, T-cell factor/Lymphoid enhancer binding factor; SF1, Steroidogenic factor 1; siRNA, small interfering RNA; SMAD, Sma/Mothers against decapentaplegic homolog.

Figure 1 :

 1 Figure 1: Pulsatile GnRH regulates Amhr2 transcript levels in a frequency-dependent manner in perifused LβT2 cells.The effect of different GnRH pulse frequencies on Amhr2 expression was assayed in LβT2 cells in control medium or medium containing GnRH for 21 h in a perifusion chamber under continuous or pulsatile (one pulse every 30 min or every 2 h) stimulation mode. Amhr2 mRNA levels were analyzed by real-time PCR. Levels of Fshb and Lhb transcripts were also measured (inset). Results are the mean ± SEM of four independent experiments and expressed as percent of Amhr2 mRNA level in control cells. *, P ≤ 0.05; **, P ≤ 0.01 compared with control cells, (one-way ANOVA test).

Figure 2 :Figure 3 :

 23 Figure 2: GnRH stimulates the activity of human AMHR2 promoter in LβT2 gonadotrope cells. GnRHa effects on human AMHR2 promoter activity were assayed in gonadotrope LβT2 and αT3-1 and in hypothalamic neuronal GT1-7 cell lines. (A) A promoter/luciferase fusion construct, containing the 2252 (-2199/+53)-bp human AMHR2 promoter, was transiently transfected into LβT2, αT3-1 or GT1-7 cells for 48 h. Cells were then stimulated or not by GnRHa for 4 h. Relative luciferase activity was measured as indicated in Materials and Methods and results are the mean ± SEM of at least four independent experiments performed in triplicate. ***, P ≤ 0.001 compared with respective unstimulated cells (control, unpaired Student's t test). (B) The effects of GnRHa, GnRH and PACAP38 on 2252-human AMHR2 promoter activity in LβT2 cells were compared. LβT2 cells were stimulated with GnRHa, GnRH or with PACAP38 for 4 h. Some cells were pretreated with the GnRH antagonist antide (5 µM) before stimulation with 10 nM of GnRHa. Luciferase activity was expressed as percent of unstimulated promoter activity (control). Data are the mean ± SEM of at least five independent experiments performed in triplicate. *, P ≤ 0.05; **, P ≤ 0.01 compared with control (Mann-Whitney test). (C) The effect of GnRH on a shorter 428-bp hAMHR2 promoter was analyzed. The 2252-hAMHR2 promoter or a 428 (-375/+53)-bp hAMHR2 were transiently transfected in LβT2 cells for 48 h. Cells were then stimulated or not with GnRHa for 4 h and luciferase activities were measured. Results are the mean ± SEM of six independent

Figure 4 :

 4 Figure 4: Egr1 mediates GnRH stimulatory effect on hAMHR2 promoter. (A) Mutation of the Egr1/Sp1 binding site in the 428-bp hAMHR2 promoter was performed as described in Materials and Methods. Wild type and mutated Egr1/Sp1 sequences are shown. Luciferase activities of wild type and mutated 428-hAMHR2 promoters were measured in LβT2 cells stimulated or not with GnRHa for 4 h. Data are the mean ± SEM of four independent experiments performed in triplicate and expressed as percent of unstimulated wild type promoter activity (control). **, P ≤ 0.01 compared with control (two-way ANOVA test). (B) Knockdown of Egr1. LβT2 cells were transiently transfected for 48 h with the 2252-bp hAMHR2 promoter, together with 75 nM siRNA targeting mouse Egr1 (siRNA Egr1) or 75 nM nontargeting siRNA (siRNA NT) and then stimulated or not with 10 nM GnRHa for 4 h. qRT-PCR analysis confirmed that Egr1 siRNAs significantly reduced Egr1 transcript levels in LβT2 cells (55 ± 5% of siRNA NT-transfected cells after 24 h transfection, data not shown).This decrease is probably underestimated as Erg1 mRNAs were measured from the whole population of cells while only a subpopulation was transfected with the siRNA. Luciferase activities are shown as percent of unstimulated siRNA NT-transfected cells (control). Data are

Figure 5 :

 5 Figure 5: FOXO1 represses basal and GnRH-dependent activities of hAMHR2 promoter. (A) Effect of a constitutively active mutant of FOXO1 on GnRH stimulatory effect. The 2252-hAMHR2 was transiently transfected in LβT2 cells together with FOXO1 wild type (FOXO1-Wt), constitutively active mutant of FOXO1 (FOXO1-CAM) or with empty pcDNA3 (pcDNA3) vectors. Cells were then stimulated or not with GnRHa for 4 h. Luciferase activities were measured 48 h later and expressed as percent of unstimulated pcDNA3 transfected cells (control). Data are the mean ± SEM of five independent experiments performed in triplicate. **, P ≤ 0.01 compared with control (two-way ANOVA test). (B) Effect of PI3K pharmacological inhibition on GnRH effect. LβT2 cells expressing the 2252-hAMHR2 promoter were stimulated or not with GnRHa for 4 h in presence or absence of a PI3K specific inhibitor (LY294002, 20 µM). Luciferase activities were expressed as percent of unstimulated promoter activity (control). Data are the mean ± SEM at five independent experiments performed in triplicate. **, P ≤ 0.01 compared with control. GnRH induction of hAMHR2 activity is significantly reduced by 48±8% in presence of LY294002 (inset, unpaired Student's t test). (C) Mutation of the FOXO response element in hAMHR2 promoter. A 428-hAMHR2 promoter with mutation of the FOXO motif was designed as described in Materials and Methods. Wild type (FOXO-Wt) and mutated FOXO sequences extending from -120/-96 bp are shown. Luciferase activities of wild type and mutated hAMHR2 promoters were measured in LβT2 cells stimulated or not with GnRHa for 4 h. Data are the mean ± SEM of six independent experiments performed in triplicate and shown as percent of unstimulated FOXO-Wt transfected cells (control). **, P ≤ 0.01 compared with control (two-way ANOVA test).

Figure 6 :

 6 Figure 6: SF1 contributes to basal and GnRH-stimulated hAMHR2 promoter activities in LβT2 cells. (A) Effect of mutation of the two SF1 binding sites in hAMHR2 promoter. A mutant of the 2252-hAMHR2 promoter with mutation of both SF1-binding sites was designed as described in Materials and Methods. Wild type and mutated SF1 sequences extending from -276/-256 and -216/-196 bp are shown. Luciferase activities of wild type and SF1-mutated hAMHR2 promoters were measured in LβT2 cells stimulated or not with GnRHa for 4 h and expressed

Figure 7 :

 7 Figure 7: β-catenin is involved in GnRH stimulation of hAMHR2. (A) Mutation of the TCF binding sites in the hAMHR2 promoter. Mutants of the 428-hAMHR2 promoter with mutations of one or both TCF motifs were designed as described in Materials and Methods. Wild type and mutated TCF sequences extending from -259/-242 bp and -187/-169 bp are shown. Luciferase activities of wild type and mutated hAMHR2 promoters were measured in LβT2 cells stimulated or not with GnRHa for 4 h. Data are the mean ± SEM of five independent experiments performed in triplicate and expressed as percent of unstimulated wild type promoter activity (control). **, P ≤ 0.01 compared with control (two-way ANOVA test). (B) Knockdown of β-Catenin. LβT2 cells were transiently transfected for 48 h with the 2252-bp hAMHR2 promoter together with 75 nM siRNA targeting mouse Ctnnb1 (siRNA βcatenin) or 75 nM nontargeting siRNA (siRNA NT) and then stimulated or not with GnRHa for 4 h. Luciferase activities are shown as percent of unstimulated siRNA NT-transfected cells (control). Data are the mean ± SEM of four independent experiments performed in triplicate. *, P ≤ 0.05 compared with control cells (two-way ANOVA test). GnRH induction of hAMHR2 is significantly reduced by 48±17% in presence of siRNA β-catenin (inset, unpaired Student's t test). (C) Effects of pharmacological inhibition of β-catenin, PKC and JNK signaling pathways on GnRH effect. LβT2 cells were transiently transfected with the 2252-bp hAMHR2 promoter and then stimulated or not with GnRHa for 4 h in absence or presence of 10 µM JW74 (β-catenin pathway inhibitor), 2 µM GF109203X (GF109, PKC inhibitor) or 20 µM SP600125 (SP6, JNK inhibitor). Luciferase activities are shown as percent of promoter activity in absence of any treatment (control). Data are the mean ± SEM of at least five independent

Figure 8 :

 8 Figure 8: Schematic hypothetical model for GnRH induction of human AMHR2 promoter activity. This schematic drawing illustrates the location of binding sites (in red) and related transcription factors involved in the regulation of AMHR2 promoter in gonadotrope cells. In the insets are illustrated the levels of Egr1 and Sp1 transcripts (expressed relative to basal Sp1 level) in LβT2 cells stimulated or not by GnRH. (A) The constitutive activity of hAMHR2 requires intact TCF, SF1 and overlapping Sp1/Egr1 binding sites. Egr1 is only poorly expressed as compared to Sp1 in basal condition (inset), the transcription factor occupying the Sp1/Egr1 binding site is thus probably Sp1. Significant levels of TCF4 transcripts in LβT2 cells suggest that TCF4 transcription factor contributes to basal promoter activity through binding to TCF site. In addition, FOXO1 exerts a repressive action on the promoter, possibly by counteracting stimulatory transcription factors. (B) GnRH induces high levels of Egr1 while it does not affect Sp1 transcripts (inset). Egr1 binding to the promoter thus probably leads to the competitive displacement of Sp1. The induction of Egr1 may enable the formation of a transcriptional complex containing β-catenin and SF1 that cooperate with Egr1 to fully activate the hAMHR2 promoter. The export of FOXO1 from nucleus induced by GnRH also contributes to the transactivation of the promoter.

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

  binding site, markedly decreased GnRH-dependent luciferase activity. Further deletions did not induce any additional changes in luciferase activity. Altogether, these experiments indicate that the sequence located between -53 and -37 bp of hAMHR2 promoter is required for GnRH regulation.

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Table 2 : Sequences of the primers used for quantification of transcript levels in LβT2 cells.

 2 Cyclophilin is used as reference gene for real-time PCR. Unspecified species mean that the primers are functional for both rat and mouse.

	Target cDNA	Forward primer (5'>3')	Reverse primer (5'>3')
	Mouse Amhr2	CTGAGGTAGCTGTGCTGGTTTTTGC GTGTCTTTGTGCTTCCCCGCACT
	Lhb	ATCACCTTCACCACCAGCAT	GACCCCCACAGTCAGAGCTA
	Mouse Fshb	GACAGCTGACTGCACAGGAC	CAATCTTACGGTCTCGTATACC
	Mouse Ctnnb1 (β-catenin)	CCCAGTCCTTCACGCAAGAG	CATCTAGCGTCTCAGGGAACA
	Cyclophilin	CAAAGTTCCAAAGACAGCAG	CTGGCACATGAATCCTGGAA
	Nr5a1 (SF1)	TTACACGTGCACCGAGAGTC	TGCTTTCTTCTGCTGCTTCA
	Egr1	AGCAGCGCTTTCAATCCTCA	GTCTCCACCAGCGCCTTCTC
	Mouse Sp1	CTCCAGACCATTAACCTCAGTG	ACCAGATCCATGAAGACCAAG

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Acknowledgements

We are grateful to Dr. Pamela Mellon, (University of California, San Diego, CA) for providing us with the LβT2, αT3-1 and GT1-7 cell lines. We thank A Fernandez-Vega for technical assistance in RT-qPCR experiments. We thank Dr N. di Clemente for helpful discussion of the data.

Funding Sources

This study was supported by grants from Paris 7 University, the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale and, in part, by National Institute of Health HD019938 (U. B. K).

Disclosure statement:

A preliminary report of this work was presented in a communication at the 42 nd Annual Meeting of Société de Neuroendocrinologie (SNE), Dijon, France, September 18-21 2017.

The authors have no conflicts of interest to declare.

Author contributions

GG designed and performed the study, interpreted the results and contribute to the writing of the manuscript, CD performed transfection experiments, DL designed and performed perifusion culture experiments and contributed to the interpretation of promoter studies, JYP generated wild type and SF1-mutated hAMHR2 promoter constructs, JT helped with manuscript preparation, UK helped with the design of perifusion culture experiments and manuscript preparation, JNL designed and performed deletion and site-directed mutagenesis experiments, analyzed the data and contributed to the writing of the manuscript, JCT designed the study, interpreted the results and wrote the manuscript.

Abbreviations