Sphingosine-1 phosphate induces cAMP/PKA-independent phosphorylation of the cAMP response element-binding protein (CREB) in granulosa cells

Elia Paradiso,Clara Lazzaretti,Samantha Sperduti,Francesco Antoniani,Giulia Fornari,Giulia Brigante,Giulia Di Rocco,Simonetta Tagliavini,Tommaso Trenti,Daria Morini,Angela Immacolata Falbo,Maria Teresa Villani,Jerzy-Roch Nofer,Manuela Simoni,Francesco Potì,Livio Casarini

doi:10.1016/j.mce.2020.111082

Abstract

Background and aimsSphingosine-1 phosphate (S1P) is a lysosphingolipid present in the ovarian follicular fluid. The role of the lysosphingolipid in gonads of the female is widely unclear. At nanomolar concentrations, S1P binds and activates five specific G protein-coupled receptors (GPCRs), known as S1P1-5, modulating different signaling pathways. S1P1 and S1P3 are highly expressed in human primary granulosa lutein cells (hGLC), as well as in the immortalized human primary granulosa cell line hGL5. In this study, we evaluated the signaling cascade activated by S1P and its synthetic analogues in hGLC and hGL5 cells, exploring the biological relevance of S1PR-stimulation in this context. METHODS AND RESULTS. hGLC and hGL5 cells were treated with a fixed dose (0.1 μM) of S1P, or by S1P1- and S1P3-specific agonists SEW2871 and CYM5541. In granulosa cells, S1P and, at a lesser extent, SEW2871 and CYM5541, potently induced CREB phosphorylation. No cAMP production was detected and pCREB activation occurred even in the presence of the PKA inhibitor H-89. Moreover, S1P-dependent CREB phosphorylation was dampened by the mitogen-activate protein kinase (MEK) inhibitor U0126 and by the L-type Ca2+ channel blocker verapamil. The complete inhibition of CREB phosphorylation occurred by blocking either S1P2 or S1P3 with the specific receptor antagonists JTE-013 and TY52156, or under PLC/PI3K depletion. S1P-dependent CREB phosphorylation induced FOXO1 and the EGF-like epiregulin-encoding gene (EREG), confirming the exclusive role of gonadotropins and interleukins in this process, but did not affect steroidogenesis. However, S1P or agonists did not modulate granulosa cell viability and proliferation in our conditions. ConclusionsThis study demonstrates for the first time that S1P may induce a cAMP-independent activation of pCREB in granulosa cells, although this is not sufficient to induce intracellular steroidogenic signals and progesterone synthesis. S1P-induced FOXO1 and EREG gene expression suggests that the activation of S1P–S1PR axis may cooperate with gonadotropins in modulating follicle development.

Highlights

Sphingosine-1-phosphate (S1P) is a lysosphingolipid present at high concentrations in plasma and lymph
As determined in dose-response experiments (Fig. S2), the optimal concentration for cell treatment with native S1P as well as SEW2871 and CYM5541, two selective S1P1 and S1P3 agonists, respectively, was 0.1 μM. This concentration was subsequently used for the temporal evalu ation of ERK1/2, AKT and CREB phosphorylation. human primary granulosa lutein cells (hGLC) and hGL5 cells were exposed for various times (0–120 min) to each agonist and protein phosphorylation was detected by Western blotting
S1P markedly elevated the levels of phosphorylated CREB in these cells with the time course resembling primary granulosa cells, while CYM5541 and SEW2871 exerted no effect on CREB phosphorylation in hGL5 cells

Summary

Introduction

Sphingosine-1-phosphate (S1P) is a lysosphingolipid present at high concentrations in plasma and lymph. While implicated in several intracellular processes such as cell growth, survival, motility and migration (Strub et al, 2010), S1P can be transported out of the cell, where it acts in autocrine and paracrine mode via specific membrane receptors. S1P binds and activates five specific G protein-coupled receptors (GPCRs), known as S1P1-5, modulating different signaling pathways. S1P and, at a lesser extent, SEW2871 and CYM5541, potently induced CREB phosphorylation. S1P-dependent CREB phosphorylation induced FOXO1 and the EGF-like epiregulinencoding gene (EREG), confirming the exclusive role of gonadotropins and interleukins in this process, but did not affect steroidogenesis. Conclusions: This study demonstrates for the first time that S1P may induce a cAMP-independent activation of pCREB in granulosa cells, this is not sufficient to induce intracellular steroidogenic signals and pro gesterone synthesis. S1P-induced FOXO1 and EREG gene expression suggests that the activation of S1P–S1PR axis may cooperate with gonadotropins in modulating follicle development

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