Kisspeptin increases GnRH mRNA expression and secretion in GnRH secreting neuronal cell lines

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Abstract

Kisspeptins, and their G-protein coupled receptor 54 (GPR54), are key components in the regulation of gonadotropin-releasing hormone (GnRH) secretion in humans and other mammals. Several studies demonstrate that the central or systemic administration of kisspeptin increases GnRH and gonadotropin secretion in both prepubertal and adult animals; however, the cellular targets and intracellular mechanisms of action in the central reproductive axis are unclear. In this study, we documented the presence of GPR54 in two GnRH secreting neuronal cell lines (GT1–7 and GN11). Kisspeptin treatment increases GnRH secretion and GnRH mRNA levels in a dose and time dependent manner. 10−9 M kisspeptin maximally stimulated GnRH secretion by 2-fold and GnRH mRNA levels up to 4-fold after 4 h of treatment in both cell lines. Negative regulation by 17β-estradiol of GnRH secretion and GnRH mRNA was antagonized by kisspeptin. Co-treatment with kisspeptin and 17β-estradiol increased GnRH secretion by 2-fold and GnRH mRNA by 4-fold over estradiol alone in both cell lines. Intracellular signaling pathway studies showed that an ERK1/2 MAPK inhibitor (PD98059) and a PI3K inhibitor, LY29402, attenuated the effects of kisspeptin on GnRH mRNA modulation. Furthermore, Western blot analysis showed that phosphorylation of both MAPK and Akt substrates increased with kisspeptin treatment. This work demonstrates that the kisspeptin–GPR54 system plays a significant role stimulating GnRH secretion and positive regulation of GnRH mRNA levels in GnRH neurons in culture, and also, demonstrates the activation of MAPK and Akt signaling pathways by kisspeptin in GT1–7 and GN11 cell lines.

Introduction

The hormonal network responsible for the control of reproduction is composed of three major hierarchical elements: the hypothalamic gonadotropin-releasing hormone (GnRH), the pituitary gonadotropins (LH and FSH) and the products of the gonads, principally, sex steroids. Reproductive capacity is attained at puberty as the end-point of a complex series of developmental and neuroendocrine events that lead to full activation of the GnRH pulse generator, enhanced gonadotropin secretion and complete gonadal maturation and function (Terasawa and Fernandez, 2001, Plant and Witchel, 2006). Since GnRH neurons play a critical central role in the regulation of pubertal development and reproduction, the central and peripheral signals that regulate these cells are important to elucidate.

Gonadal sex steroids are major regulators of GnRH secretion through negative and positive feedback loops. In addition, most recently, the physiological control of the reproductive axis was advanced by the identification of the essential role of kisspeptin, the peptide product of the KiSS-1 gene, and its receptor, G-protein coupled receptor 54 (GPR54), in the neuroendocrine regulation of reproduction (de Roux et al., 2003, Funes et al., 2003, Seminara et al., 2003, Castellano et al., 2005). Investigations by many laboratories over the last 5 years have led to the general concept that kisspeptin neurons activate GnRH neurons (Tena-Sempere, 2006, Kauffman et al., 2007, Caraty and Franceschini, 2008, Roa et al., 2008a, Roa et al., 2009, Seminara and Crowley, 2008).

Navarro et al. (2004) and Shahab et al. (2005) observed an increase of KiSS-1 and GPR54 mRNAs during pubertal development in experiments performed in intact rats and monkeys, suggesting that, these developmental changes in the expression of KiSS-1 mRNA and GPR54 may play a role in the onset of puberty, and could be considered proximal signaling events for pubertal maturation (Han et al., 2005, Gottsch et al., 2006). Central or systemic administration of kisspeptin leads to increased GnRH and gonadotropin secretion in both prepubertal and adult animals (Plant and Barker-Gibb, 2004, Navarro et al., 2005a, Gottsch et al., 2006, Roa et al., 2006, Pielecka-Fortuna et al., 2008, Roa et al., 2008b). Furthermore, GPR54 mutations in humans or targeted deletions in mice produce isolated hypogonadotropic hypogonadism and infertility, thereby demonstrating a required regulatory function in both sexes (Messager et al., 2005, Smith et al., 2006a, Tena-Sempere, 2006, Gottsch et al., 2006, d’Anglemont de Tassigny et al., 2007, Dungan et al., 2007, Clarkson et al., 2008).

Estradiol is one of the most important regulators of GnRH neuronal activity (Herbison, 1998, Wintermantel et al., 2006, Herbison, 2008). Irwig et al. (2004) and Navarro et al. (2004) have provided evidence in rats that kisspeptin-expressing neurons are targets for regulation by sex steroids, furthermore, these neurons are directly regulated by the negative and positive feedback actions of sex steroids in distinct regions of the forebrain (Gottsch et al., 2006, Smith et al., 2006a). These observations suggest that kisspeptin/GPR54 signaling provides tonic stimulatory input to GnRH neurons, which could be governed by the feedback effects of sex steroids acting on kisspeptin secreting neurons (Gottsch et al., 2006). In the female, the estradiol-dependent induction of KiSS-1 mRNA in the anteroventral periventricular nucleus (AVPV) may play a role in mediating the preovulatory GnRH/LH surge, that drives ovulation or the regulation of sexual behavior (Kinoshita et al., 2005, Smith et al., 2005a, Smith et al., 2005b, Smith et al., 2006b, Clarkson et al., 2008, Herbison, 2008). Unlike in the AVPV, kisspeptin neurons in the arcuate nucleus (Arc) play the same role in both sexes (negative feedback regulation of gonadotropin secretion by gonadal steroids). Recently, studies in GPR54 KO mice reported that GPR54 signaling is critical for the maintenance of tonic LH secretion, reflecting a lack of normal follicular development and resulting in infertility (Dungan et al., 2007). In addition to receiving input from estradiol sensitive presynaptic afferents, GnRH neurons have been shown to express estrogen receptors, although the studies have been difficult and controversial. In vivo evidence for the presence of ERβ has been demonstrated, suggesting that the effects of estrogen may be directly transmitted (Skynner et al., 1999, Hrabovszky et al., 2000, Herbison and Pape, 2001, Petersen et al., 2003, Skinner and Dufourny, 2005, Wintermantel et al., 2006). The model GnRH neuronal cell lines, GT1–7 and GN11 have been shown to also express functional ERα and ERβ (Radovick et al., 1991a, Roy et al., 1999, Ng et al., 2009).

The KiSS-1/GPR54 system revealed a fundamental role in the control of puberty and/or maintenance of reproductive function; although the direct targets of kisspeptin, its specific pharmacokinetics and the cellular signaling mechanism remained obscure (Tena-Sempere, 2006). In the present studies both the GN11 and GT1–7 GnRH expressing cell lines were used as models to explore the direct regulation of the GnRH neuron by kisspeptin. The aim of this work is to determine whether the GnRH neuron can be directly regulated by kisspeptin; the potential mechanism of kisspeptin signaling in GnRH neurons, and whether GnRH neurons may be integrators of sex steroid feedback with kisspeptin regulation.

Section snippets

Cell culture

GN11 cells were grown in Dulbecco's modified Eagle's medium (DMEM; Mediatech Inc., Herndon, VA, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 25 mM glucose, 5 mM l-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, Grand Island, NY, USA) in an atmosphere with 5% CO2 at 37 °C. GT1–7 cells were grown in a similar manner, except supplemented with 10% heat-inactivated fetal bovine serum. Cells were placed in media supplemented with 10% dextran and charcoal

GnRH mRNA and protein expression in GT1–7 and GN11 cells

Initial studies using RT-PCR revealed the presence of GPR54 mRNA in both GT1–7 and GN11 neuronal cell lines (Fig. 1A). The detection of a 175 bp band corresponds to amplification of a region between exons 2 and 3 from murine genomic DNA. No product was detected in the lane containing no cDNA (H2O) or performed without RT (RT(−)). Furthermore, studies using immunoprecipitation and Western blot assay revealed the presence of GPR54 protein as a 43 kDa product (Prentice et al., 2007) in these cells (

Discussion

The onset of puberty is heralded by activation of neurons in the forebrain that produce GnRH. Although the central reproductive axis has been studied in many mammalian species, precise identification of the molecular and cellular events in the forebrain that initiate pubertal processes and maintain reproductive competence remains elusive (Plant, 2008).

The goal of our study was to define whether kisspeptin had a direct role in GnRH neuronal function, and if so, the pharmacology of its effect on

Acknowledgements

This research was supported by NICHD/NIH through cooperative agreement [U54 HD 933067 (The Baltimore-Chicago Center for Reproductive Research)] as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research (SCCPIR) and R01 as NIH HD 370246. The authors would like to thank Dr Pamela Mellon for kindly providing the GT1–7 cell line and Dr. Jennifer Mammen for the assistance in the writing of the manuscript.

References (69)

  • Y. Ng et al.

    Estrogen regulation of gene expression in GnRH neurons

    Molecular and Cellular Endocrinology

    (2009)
  • S. Radovick et al.

    Steroid hormone regulation and tissue-specific expression of the human GnRH gene in cell culture and transgenic animals

    Hormones and Behavior

    (1994)
  • J. Roa et al.

    New frontiers in kisspeptin/GPR54 physiology as fundamental gatekeepers of reproductive function

    Frontiers in Neuroendocrinology

    (2008)
  • J. Roa et al.

    Kisspeptins and the control of gonadotropin secretion in male and female rodents

    Peptides

    (2009)
  • C.A. Suarez-Quian et al.

    Receptor-mediated endocytosis of GnRH analogs: differential processing of gold-labeled agonist and antagonist derivatives

    Journal of Steroid Biochemistry

    (1986)
  • T.M. Wintermantel et al.

    Definition of estrogen receptor pathway critical for estrogen positive feedback to gonadotropin-releasing hormone neurons and fertility

    Neuron

    (2006)
  • J.P. Bourguignon et al.

    Pulsatile release of gonadotropin-releasing hormone (GnRH) from the rat hypothalamus in vitro: calcium and glucose dependency and inhibition by superactive GnRH analogs

    Endocrinology

    (1987)
  • S.A. Bustin et al.

    Quantitative real-time RT-PCR—a perspective

    Journal of Molecular Endocrinology

    (2005)
  • A. Caraty et al.

    Basic aspects of the control of GnRH and LH secretions by kisspeptin: potential applications for better control of fertility in females

    Reproduction in Domestic Animals = Zuchthygiene

    (2008)
  • J.M. Castellano et al.

    Changes in hypothalamic KiSS-1 system and restoration of pubertal activation of the reproductive axis by kisspeptin in undernutrition

    Endocrinology

    (2005)
  • J. Clarkson et al.

    Kisspeptin-GPR54 signaling is essential for preovulatory gonadotropin-releasing hormone neuron activation and the luteinizing hormone surge

    The Journal of Neuroscience: the Official Journal of the Society for Neuroscience

    (2008)
  • X. d’Anglemont de Tassigny et al.

    Hypogonadotropic hypogonadism in mice lacking a functional Kiss1 gene

    Proceedings of the National Academy of Sciences of the United States of America

    (2007)
  • X. d’Anglemont de Tassigny et al.

    Kisspeptin can stimulate gonadotropin-releasing hormone (GnRH) release by a direct action at GnRH nerve terminals

    Endocrinology

    (2008)
  • N. de Roux et al.

    Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54

    Proceedings of the National Academy of Sciences of the United States of America

    (2003)
  • H.M. Dungan et al.

    The role of kisspeptin-GPR54 signaling in the tonic regulation and surge release of gonadotropin-releasing hormone/luteinizing hormone

    The Journal of Neuroscience: the Official Journal of the Society for Neuroscience

    (2007)
  • M. El Majdoubi et al.

    Effect of estrogen on hypothalamic transforming growth factor alpha and gonadotropin-releasing hormone gene expression in the female rhesus monkey

    Neuroendocrinology

    (1998)
  • S.K. Han et al.

    Activation of gonadotropin-releasing hormone neurons by kisspeptin as a neuroendocrine switch for the onset of puberty

    The Journal of Neuroscience: the Official Journal of the Society for Neuroscience

    (2005)
  • A.E. Herbison

    Multimodal influence of estrogen upon gonadotropin-releasing hormone neurons

    Endocrine Reviews

    (1998)
  • E. Hrabovszky et al.

    Detection of estrogen receptor-beta messenger ribonucleic acid and 125I-estrogen binding sites in luteinizing hormone-releasing hormone neurons of the rat brain

    Endocrinology

    (2000)
  • M.S. Irwig et al.

    Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat

    Neuroendocrinology

    (2004)
  • J.S. Jacobi et al.

    17-Beta-estradiol directly regulates the expression of adrenergic receptors and kisspeptin/GPR54 system in GT1–7 GnRH neurons

    Neuroendocrinology

    (2007)
  • M. Kinoshita et al.

    Involvement of central metastin in the regulation of preovulatory luteinizing hormone surge and estrous cyclicity in female rats

    Endocrinology

    (2005)
  • X. Liu et al.

    Kisspeptin excites gonadotropin-releasing hormone neurons through a phospholipase C/calcium-dependent pathway regulating multiple ion channels

    Endocrinology

    (2008)
  • A.J. Martinez-Fuentes et al.

    Gonadotropin-releasing hormone (GnRH) receptor expression and membrane signaling in early embryonic GnRH neurons: role in pulsatile neurosecretion.

    Molecular Endocrinology (Baltimore, MD)

    (2004)
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