Sphingosine-1 phosphate (S1P) is a lysosphingolipid present in the ovarian follicular fluid at the nanomolar concentration. The physiological functions of S1P are mediated through five specific G protein-coupled receptors (GPCRs), known as S1PR1-5. S1PR1 and S1PR3 are expressed in human primary granulosa lutein cells (hGLC), as well as in the immortalized human primary granulosa cell line hGL5. S1PRs are activated at nanomolar concentrations inducing specific activation or inhibition of different signaling pathways. This in vitro study aims to characterize the role of S1P in the ovarian follicle. hGLC and hGL5 cells were cultured and treated by a fixed dose (100 nM) of S1P, or by S1PR1 and S1PR3 specific antagonists SEW2871 and CYM5541, after dose-finding experiments. cAMP production, ERK1/2, AKT and cAMP-responsive element binding protein (CREB) phosphorylation, intracellular Ca2+ increase, gene expression, cell viability and progesterone synthesis were evaluated by ELISA, Western blotting, bioluminescence resonance energy transfer (BRET), real-time PCR, MTT-assay and immunoassay, respectively. Specific inhibitor/antagonists were also used. In granulosa cells, S1P and, to a lesser extent, SEW2871 and CYM5541 induce cAMP/PKA-independent activation of pCREB via a PLC/PI3K-dependent mechanism. Indeed, no cAMP production was detected (unstimulated=2.6±0.2; agonists 2.51.9±0.8 range pmol/ml; means±S.D. two-way ANOVA; P≥0.05; n=2) and pCREB activation occurred even in the presence of the PKA inhibitor H-89 (n=3). This is relevant because both cAMP and PKA activation are known to precede the phosphorylation of CREB, inducing steroidogenesis. Moreover, S1P-dependent CREB phosphorylation is dampened by preventing pERK1/2 activation using the inhibitor U0126 and by the L-type Ca2+-channel blocker verapamil. The complete inhibition of CREB phosphorylation occurred by antagonizing either S1PR2 or S1PR3 by the receptor-specific compound JTE-013 and TY52156, or under PLC/PI3K depletion, detected by Western blotting (n=3). CREB phosphorylation is not linked to expression of genes encoding steroidogenic enzymes and pro/anti-apoptotic molecules in granulosa cells, while induces FOXO1 and the EGF-like epiregulin-encoding gene (EREG) expression (two-way ANOVA; P<0.05; n=4). However, cell viability and proliferation is not modulated upon treatment by S1P or agonists. Most importantly, we demonstrated that S1P-dependent CREB phosphorylation does not induce steroidogenic signals (basal progesterone=30.3±3.8; agonist-induced progesterone=28.7-26.1±3.2 ng/ml range; means±SD; two-way ANOVA; P≥0.05; n=4). We demonstrated a novel cAMP/PKA-independent activation of pCREB not inducing intracellular steroidogenic signals and progesterone synthesis in granulosa cells. However, S1P-induced FOXO1 and EREG gene expression suggest that lysosphingolipids may act synergistically with gonadotropins in modulating follicle development.
18 May 2019 - 21 May 2019