BACKGROUND AND AIMS: Sphingosine-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.
CONCLUSIONS: This 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.