Published Time: 2019-11-07
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 2+) 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 (Lhb) transcription by bioluminescence resonance energy transfer (BRET), Western blotting, immunostaining and real-time PCR as appropriate. The kinetics of GnRH-induced Ca 2+ 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 50 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, Lhb 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 Lhb gene transcription.
Gonadotropin releasing hormone (GnRH) is secreted by hypothalamic GnRH-expressing neurons and regulates mammalian reproductive functions. It is a decapeptide released in a pulsatile fashion into the hypophyseal portal blood system and acts on GnRH receptor (GnRHR)-expressing gonadotrope cells of the anterior pituitary, triggering the synthesis and secretion of the luteinizing (LH) and follicle-stimulating (FSH) hormones [1,2,3].
GnRHR is a G-protein coupled receptor (GPCR) [4,5], and its main effector in pituitary cells is the Gα q/11 protein, the activation of which results in phosphoinositide phospholipase Cβ (PLCβ) stimulation and subsequent production of inositol (1,4,5)-trisphosphate (IP 3) and diacylglycerol (DAG) [6]. IP 3 induces intracellular Ca 2+ release by the endoplasmic reticulum, which is linked to gonadotropin secretion and activation of the calmodulin/calcineurin/NFAT- and calmodulin/ Ca 2+ calmodulin-dependent protein kinase II (CaMKII)-pathway, as well as in further Ca 2+ influx through L-type voltage gated calcium channels [2]. However, GnRHR modulates the simultaneous activation of multiple intracellular signaling cascades, depending on cell specific-contexts [7,8]. DAG mediates protein kinase C (PKC) activation and downstream phosphorylation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2), jun-N-terminal kinase (JNK), and p38 mitogen-activated protein kinases (MAPKs) [2,9,10,11]. GnRHR coupling to other heterotrimeric G-protein subunits, such as the Gα s [12,13,14] and Gα i [15], has also been described [6,16]. Gα s protein activation by GnRHR-hormone binding results in adenylyl cyclase and (cAMP) increase, and cAMP response element-binding protein (CREB) phosphorylation via protein kinase A (PKA) activation. cAMP production may also be induced by alternative pathways involving the Gα q/11 protein and specific PKC isoforms [17], implementing the complexity of GnRHR signaling signature. All of these intracellular events precede the expression of LHB and FSHB target genes [13,18]. Finally, another target of GnRH-mediated signal transduction is β-catenin activation [19,20]. β-catenin acts as a dual-function protein, participating in both cell-adhesion, as a member of the adherens junction, and in the regulation of LHB and Wnt-target gene transcription [21,22,23] after translocation into the cell nucleus [19,24].
GnRH agonists and antagonists are useful to control gonadotropin production, in the context of assisted reproduction technologies (ART), as well as for the treatment of certain hormone-dependent diseases [25,26,27]. GnRH antagonists are typically decapeptides structurally similar to GnRH, differing from the native hormone by a few amino acids which results in reversible GnRHR binding without activation [5,28]. The GnRH antagonists Cetrorelix, Ganirelix and Teverelix, share highly similar structure, differing by only two amino acids at position 6 and 8 of the protein chain [5,26]. While the effects of these different GnRH antagonists have never been comprehensively compared in vitro, the use of Cetrorelix and Ganirelix to prevent premature ovulation is considered to lead to similar clinical outcomes [29,30], while Teverelix, although potentially useful for clinical purposes, has not yet been commercially marketed [31,32,33]. Although they share a high degree of similarity, the molecular differences between the antagonists lead to the hypothesis that antagonist-specific, biased effects on GnRHR-dependent pathways may occur upon receptor binding, resulting in ligand-induced selective signaling (LiSS) [34].
In cell lines expressing GnRHR, we compared Cetrorelix, Ganirelix and Teverelix in inhibiting a range of GnRH-induced intracellular signaling cascades, in vitro. This study improves the knowledge of the structure–function relationship of GnRH antagonists and provides results useful to develop drugs for personalized clinical applications.
In order to find the optimal GnRH dose to evaluate the action of antagonists in inhibiting the intracellular Ca 2+ increase, dose-response experiments were performed. Thus, Ca 2+ biosensor-expressing cells were treated by increasing concentrations of GnRH (pM–µM range) and luminescent signals corresponding to the intracellular Ca 2+ concentration were measured by BRET. GnRH-mediated Ca 2+ accumulation was measured in transiently transfected HEK293/GnRHR and SH-SY5Y/GnRHR cells, and in a LβT2 cell line, naturally expressing the murine GnRHR [35].
Upon GnRH injection, intracellular Ca 2+ rapidly increased, achieving the maximal level within about 5 s, before decreasing back to the basal level within about 80 s. No response was observed upon injection of vehicle (negative control). AUCs obtained from Ca 2+ activation kinetics were plotted against the GnRH concentration in a X-Y graph. Data were interpolated by non-linear regression and the potency (EC 50) of GnRH in inducing the intracellular ion increase in HEK293/GnRHR cells was calculated to be 23.26 ± 3.37 nM. GnRH-induced intracellular Ca 2+ accumulation was also observed in both the SH-SY5Y/GnRHR and LβT2 cell lines (SH-SY5Y/GnRHR EC 50 = 5.78 ± 3.04 nM; LβT2 EC 50 = 1.80 ± 2.88 nM). For all cell lines, GnRH potency was similar and fell within the nM range (Kruskal-Wallis test; p ≥ 0.05; n = 3).
Potencies of Cetrorelix, Ganirelix and Teverelix in inhibiting the hormone-induced intracellular Ca 2+ increase were then compared in vitro. Each of the cell lines were treated by a fixed concentration of GnRH corresponding to the three-fold higher dose than the calculated EC 50 (3 × EC 50; a concentration optimized for evaluating signal variations in inhibition experiments), in the presence or absence of increasing antagonist (Cetrorelix, Ganirelix or Teverelix) concentrations. Data representing the kinetics of intracellular Ca 2+ increase, evaluated over 150 s by BRET were compared after AUC calculation.
Analysis revealed that the antagonists have different potencies in inhibiting the GnRH-induced intracellular Ca 2+ increase. In HEK293/GnRHR cells, the highest inhibition of the GnRH-induced signal was observed with 10 nM Cetrorelix, with an area under the curve (AUC) of 21,482 ± 6718. The same concentration (10 nM) of Ganirelix and Teverelix resulted in different inhibitory effects. For Ganirelix, the AUC was 73,164 ± 16,237 while for Teverelix it was 74,321 ± 17,569. The AUC for HEK293/GnRHR cells without GnRH antagonist treatment was 109,340 ± 13,866. When compared with the inhibition induced by Cetrorelix, significant differences were observed for all treatments (p = 0.005). These results were confirmed after comparing the AUCs obtained with a number of antagonist concentrations. Interestingly, a small (1.2-fold) increase in GnRH-induced intracellular Ca 2+ appears to occur in the presence of 10–100 pM antagonist, although this is not statistically significant, compared to treatment by GnRH alone.
SH-SY5Y and LβT2 cell lines were selected to serve as control models in vitro as they have been shown to endogenously express human [36] or mouse [35] GnRHRs, respectively. However, no GnRH-induced intracellular Ca 2+ increase was mediated by the endogenous receptors in either of these cell lines (not shown). Overexpression of the GnRHR-encoding cDNA in the SH-SY5Y cells (SH-SY5Y/GnRHR cells) was required to achieve detectable signals. In this experimental setting, complete efficacy of GnRH antagonists is demonstrated at the 1 µM concentration and no drug-specific differences were observed (Kruskal-Wallis test; p ≥ 0.05). Unfortunately, transfection of LβT2 cells with GnRHR- and Ca 2+ biosensor-encoding cDNA were not successful.
Both HEK293/GnRHR and SH-SY5Y/GnRHR control experiments, in which the effect of increasing antagonist concentrations was examined in the absence of GnRH stimulation, showed no response.
In order to find the optimal GnRH dose necessary to evaluate the action of antagonists in inhibiting the intracellular cAMP increase, dose-response experiments were performed. HEK293/GnRHR and SH-SY5Y cells transiently expressing the CAMYEL cAMP-biosensor were treated with increasing concentrations of GnRH (pM–µM range), in the presence of a phosphodiesterase inhibitor, and the 30 min cAMP accumulation was evaluated by BRET. The appropriate time-point for measurements of this second messenger was established after assessing the kinetics of the GnRH-induced cAMP accumulation, which is maintained at the plateau level between 10 and 50 min. Similarly to what have been seen with Ca 2+ accumulation, in untransfected SH-SY5Y cells, no GnRH-induced cAMP increases were detected (not shown), although these cells are reported to endogenously express the human GnRHR [36], as a likely effect due to cell-specific receptor expression levels and/or intracellular enzymatic milieu. Since no GnRH-induced Ca 2+ accumulation was observed in LβT2 cell
