Introduction: FSH acts on its receptor (FSHR) resulting in signal transduction activation, gene expression and steroidogenesis. The FSHR common SNP p.N680S is a marker of gonadal response in vivo. However, in vitro dose–response experiments failed to demonstrate the molecular basis thereof so far. In this study, we systematically investigated whether p.N680S mediates different kinetics of FSH response in vitro. Design: We evaluated the activation kinetics of cAMP, phERK1/2, phCREB by ELISA and western blotting in FSHR homozygous, primary, human granulosa lutein cells (hGLC-680N, -680S) stimulated by 50 nM r-FSH for up to 2 h (short-term stimulation). Following short-term stimulation the expression of target genes was evaluated by real-time PCR after 12 h, and progesterone production kinetics over 24 h. Specific inhibitors/agonists (U0126, PMA) were used in the presence and in the absence of FSH. Results: Intracellular cAMP increased within 5–10 min in hGLC-680N, reaching the plateau in about 45 min. cAMP increase was delayed in hGLC-680S, reaching the plateau in 120 min, revealing different activation kinetics (Mann–Whitney U test; P<0.05; n=4). r-FSH-dependent cAMP stimulation kinetics resulted in different ERK1/2 and CREB phosphorylation, reaching maximal levels in 5–30 min in hGLC-680N, whereas, in hGLC-680S, these were weaker and steady over 2 h (Mann–Whitney U test; P<0.05; n=3). hGLC-680N stimulation resulted in higher expression levels of AREG and StAR (Mann–Whitney U test; P<0.05; n=4) and in subsequently different progesterone production kinetics, achieving overall higher levels in hGLC-680N vs -680S (Mann–Whitney U test; P<0.05; n=3). Interestingly, the different kinetics of progesterone production between hGLC-680N and -680S were interchanged by selective phospho-ERK1/2 blockade/activation through specific inhibitor/agonist, revealing a short-term cross-talk mediated by ERK1/2. Conclusions: This study demonstrates for the first time in vitro, how FSHR p.N680S mediates different response to FSH, resulting in different kinetics of cAMP, phERK1/2 and phCREB activation, and progesterone production.

The FSHR polymorphism p.N680S mediates different response kinetics to FSH in vitro / Casarini, Livio; Moriondo, Valeria; Marino, Marco; Adversi, Francesca; Grisolia, Chiarina; LA MARCA, Antonio; LA SALA, Giovanni Battista; Simoni, Manuela. - (2014) 35:(2014). ((Intervento presentato al convegno 16th European Congress of Endocrinology (ECE) tenutosi a Poland, Wrocław nel 03 May 2014 - 07 May 2014.

The FSHR polymorphism p.N680S mediates different response kinetics to FSH in vitro

CASARINI, Livio;MORIONDO, Valeria;MARINO, MARCO;LA MARCA , Antonio;LA SALA, Giovanni Battista;SIMONI, Manuela
2014

Abstract

Introduction: FSH acts on its receptor (FSHR) resulting in signal transduction activation, gene expression and steroidogenesis. The FSHR common SNP p.N680S is a marker of gonadal response in vivo. However, in vitro dose–response experiments failed to demonstrate the molecular basis thereof so far. In this study, we systematically investigated whether p.N680S mediates different kinetics of FSH response in vitro. Design: We evaluated the activation kinetics of cAMP, phERK1/2, phCREB by ELISA and western blotting in FSHR homozygous, primary, human granulosa lutein cells (hGLC-680N, -680S) stimulated by 50 nM r-FSH for up to 2 h (short-term stimulation). Following short-term stimulation the expression of target genes was evaluated by real-time PCR after 12 h, and progesterone production kinetics over 24 h. Specific inhibitors/agonists (U0126, PMA) were used in the presence and in the absence of FSH. Results: Intracellular cAMP increased within 5–10 min in hGLC-680N, reaching the plateau in about 45 min. cAMP increase was delayed in hGLC-680S, reaching the plateau in 120 min, revealing different activation kinetics (Mann–Whitney U test; P<0.05; n=4). r-FSH-dependent cAMP stimulation kinetics resulted in different ERK1/2 and CREB phosphorylation, reaching maximal levels in 5–30 min in hGLC-680N, whereas, in hGLC-680S, these were weaker and steady over 2 h (Mann–Whitney U test; P<0.05; n=3). hGLC-680N stimulation resulted in higher expression levels of AREG and StAR (Mann–Whitney U test; P<0.05; n=4) and in subsequently different progesterone production kinetics, achieving overall higher levels in hGLC-680N vs -680S (Mann–Whitney U test; P<0.05; n=3). Interestingly, the different kinetics of progesterone production between hGLC-680N and -680S were interchanged by selective phospho-ERK1/2 blockade/activation through specific inhibitor/agonist, revealing a short-term cross-talk mediated by ERK1/2. Conclusions: This study demonstrates for the first time in vitro, how FSHR p.N680S mediates different response to FSH, resulting in different kinetics of cAMP, phERK1/2 and phCREB activation, and progesterone production.
16th European Congress of Endocrinology (ECE)
Poland, Wrocław
03 May 2014 - 07 May 2014
Casarini, Livio; Moriondo, Valeria; Marino, Marco; Adversi, Francesca; Grisolia, Chiarina; LA MARCA, Antonio; LA SALA, Giovanni Battista; Simoni, Manuela
The FSHR polymorphism p.N680S mediates different response kinetics to FSH in vitro / Casarini, Livio; Moriondo, Valeria; Marino, Marco; Adversi, Francesca; Grisolia, Chiarina; LA MARCA, Antonio; LA SALA, Giovanni Battista; Simoni, Manuela. - (2014) 35:(2014). ((Intervento presentato al convegno 16th European Congress of Endocrinology (ECE) tenutosi a Poland, Wrocław nel 03 May 2014 - 07 May 2014.
File in questo prodotto:
Non ci sono file associati a questo prodotto.
Pubblicazioni consigliate

Caricamento pubblicazioni consigliate

Licenza Creative Commons
I metadati presenti in IRIS UNIMORE sono rilasciati con licenza Creative Commons CC0 1.0 Universal, mentre i file delle pubblicazioni sono rilasciati con licenza Attribuzione 4.0 Internazionale (CC BY 4.0), salvo diversa indicazione.
In caso di violazione di copyright, contattare Supporto Iris

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11380/1084163
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact