GnRH Antagonists Produce Differential Modulation of the Signaling Pathways Mediated by GnRH Receptors

• Published in: International journal of molecular sciences Vol. 20; no. 22; p. 5548
• Main Authors: Sperduti, Samantha, Limoncella, Silvia, Lazzaretti, Clara, Paradiso, Elia, Riccetti, Laura, Turchi, Sara, Ferrigno, Ilaria, Bertacchini, Jessika, Palumbo, Carla, Potì, Francesco, Longobardi, Salvatore, Millar, Robert P, Simoni, Manuela, Newton, Claire L, Casarini, Livio
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 07.11.2019; MDPI
• Subjects: Accumulation; Activation; Adenylate cyclase; Amino acids; Animals; antagonist; assisted reproduction techniques (art); Biosensors; Calcium (intracellular); Calcium - metabolism; Calcium ions; Calcium Signaling - drug effects; Cell adhesion; Cell Line, Tumor; cetrorelix; Cyclic AMP response element-binding protein; Dose-Response Relationship, Drug; Endocrinology and metabolism; Experiments; follicle-stimulating hormone (fsh); ganirelix; Gene expression; Gonadotropin-releasing hormone; gonadotropin-releasing hormone (gnrh); Gonadotropin-Releasing Hormone - antagonists & inhibitors; Gonadotropin-Releasing Hormone - metabolism; Gonadotropins; HEK293 Cells; Hormone Antagonists - pharmacology; Human health and pathology; Humans; Intracellular; Isoforms; Kinases; Life Sciences; Luteinizing hormone; luteinizing hormone (lh); MAP Kinase Signaling System - drug effects; Mice; Mitogen-Activated Protein Kinase 1 - metabolism; Mitogen-Activated Protein Kinase 3 - metabolism; Nervous system; Nuclei (cytology); Phosphorylation; pituitary; Pituitary (anterior); Protein kinase A; Protein kinase C; Proteins; Receptors, LHRH - metabolism; Regression analysis; Reproductive Biology; Signal transduction; teverelix; Transcription; Wnt protein; β-Catenin; pituitary; Teverelix; Gonadotropin-releasing hormone (GnRH); luteinizing hormone (LH); follicle-stimulating hormone (FSH); assisted reproduction techniques (ART); gonadotropins; antagonist; Cetrorelix; Ganirelix
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms20225548

Commercial gonadotropin-releasing hormone (GnRH) antagonists differ by 1-2 amino acids and are used to inhibit gonadotropin production during assisted reproduction technologies (ART). In this study, potencies of three GnRH antagonists, Cetrorelix, Ganirelix and Teverelix, in inhibiting GnRH-mediated intracellular signaling, were compared in vitro. GnRH receptor (GnRHR)-transfected HEK293 and neuroblastoma-derived SH-SY5Y cell lines, as well as mouse pituitary LβT2 cells endogenously expressing the murine GnRHR, were treated with GnRH in the presence or absence of the antagonist. We evaluated intracellular calcium (Ca ) and cAMP increases, cAMP-responsive element binding-protein (CREB) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation, β-catenin activation and mouse luteinizing-hormone β-encoding gene ( ) transcription by bioluminescence resonance energy transfer (BRET), Western blotting, immunostaining and real-time PCR as appropriate. The kinetics of GnRH-induced Ca rapid increase revealed dose-response accumulation with potency (EC50) of 23 nM in transfected HEK293 cells, transfected SH-SY5Y and LβT2 cells. Cetrorelix inhibited the 3 × EC GnRH-activated calcium signaling at concentrations of 1 nM-1 µM, demonstrating higher potency than Ganirelix and Teverelix, whose inhibitory doses fell within the 100 nM-1 µM range in both transfected HEK293 and SH-SY5Y cells in vitro. In transfected SH-SY5Y, Cetrorelix was also significantly more potent than other antagonists in reducing GnRH-mediated cAMP accumulation. All antagonists inhibited pERK1/2 and pCREB activation at similar doses, in LβT2 and transfected HEK293 cells treated with 100 nM GnRH. Although immunostainings suggested that Teverelix could be less effective than Cetrorelix and Ganirelix in inhibiting 1 µM GnRH-induced β-catenin activation, gene expression increase occurring upon LβT2 cell treatment by 1 µM GnRH was similarly inhibited by all antagonists. To conclude, this study has demonstrated Cetrorelix-, Ganirelix- and Teverelix-specific biased effects at the intracellular level, not affecting the efficacy of antagonists in inhibiting gene transcription.