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

Abstract

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 S1P 1-5 , modulating different signaling pathways. S1P 1 and S1P 3 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 S1PRstimulation in this context. METHODS AND RESULTS. hGLC and hGL5 cells were treated with a fixed dose (0.1 μM) of S1P, or by S1P 1 -and S1P 3 -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, S1Pdependent CREB phosphorylation was dampened by the mitogen-activate protein kinase (MEK) inhibitor U0126 and by the L-type Ca 2+ channel blocker verapamil. The complete inhibition of CREB phosphorylation occurred by blocking either S1P 2 or S1P 3 with the specific receptor antagonists JTE-013 and TY52156, or under PLC/PI3K depletion. 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. 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.

To Be or Not to Be: The Divergent Action and Metabolism of Sphingosine-1 Phosphate in Pancreatic Beta-Cells in Response to Cytokines and Fatty Acids

“…This observation indicates a possible important role of S1P in protection of human beta-cells against cytokine toxicity, since in human beta-cells cytokines exert their toxic effects without induction of the iNOS pathway [50,135,136]. Exposure of INS1E cells to S1P results in an increased cAMP generation [30], extending the earlier observations that S1PR2 activation induces cAMP production in other cell types [137][138][139][140]. It has been demonstrated that HDL, which is enriched in S1P through its binding to apoM, could counteract beta-cell apoptosis induced by cytokines [141].…”

Myosin 1c: A novel regulator of glucose uptake in brown adipocytes

“…Furthermore, phosphorylation of transcription factors CREB and ATF-2 by PClP was similar to isoprenaline activation ( Figure 4 C). Phosphorylation of CREB can involve multiple pathways, some of which are independent of both cAMP induction and PKA activation [ [24] , [25] , [26] ]. We thus further aimed to delineate the mechanism responsible for PClP-mediated phosphorylation on canonical PKA substrates.…”

Downregulation of LHCGR Attenuates COX-2 Expression and Induces Luteinized Unruptured Follicle Syndrome in Endometriosis

“…In a word, the LHCGR-provoked cAMP, which spreads throughout the follicle is critical to identify the mechanisms involved in the pathogenesis of unruptured follicles, especially after LH surge. Previous studies have originally confirmed that cAMP signaling can increase the transcriptional activity of cAMP-response element-binding protein (CREB) ( 46 ), but recent researches provide compelling evidence that C/EBPs also serve as cAMP-responsive transcription factors due to their functionally cAMP-inducible activities ( 47 ). Occupying specific cis-elements in the cAMP response unit (CRU), C/EBPα has proved to play a critical role in this process ( 48 ).…”