Unit of Endocrinology, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy; International PhD School in Clinical and Experimental Medicine (CEM), University of Modena and Reggio Emilia, Modena, Italy.
Unit of Endocrinology, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy; Center for Genomic Research, University of Modena and Reggio Emilia, Modena, Italy.
Mol Cell Endocrinol. 2021 Jan 15;520:111082. doi: 10.1016/j.mce.2020.111082. Epub 2020 Nov 12.
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, modulating different signaling pathways. S1P and S1P 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.
hGLC and hGL5 cells were treated with a fixed dose (0.1 μM) of S1P, or by S1P- and S1P-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 Ca channel blocker verapamil. The complete inhibition of CREB phosphorylation occurred by blocking either S1P or S1P 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.
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.
鞘氨醇-1-磷酸（S1P）是一种存在于卵巢卵泡液中的溶血神经鞘脂。这种溶血神经鞘脂在女性性腺中的作用尚不清楚。在纳摩尔浓度下，S1P 结合并激活五种特定的 G 蛋白偶联受体（GPCR），称为 S1P，调节不同的信号通路。S1P 和 S1P 在人原代颗粒黄体细胞（hGLC）以及永生化人原代颗粒细胞系 hGL5 中高度表达。在这项研究中，我们评估了 S1P 及其合成类似物在 hGLC 和 hGL5 细胞中激活的信号级联，探索了在这种情况下 S1PR 刺激的生物学相关性。
用固定剂量（0.1 μM）的 S1P 或 S1P 和 S1P 特异性激动剂 SEW2871 和 CYM5541 处理 hGLC 和 hGL5 细胞。在颗粒细胞中，S1P 以及在较小程度上 SEW2871 和 CYM5541 强烈诱导 CREB 磷酸化。未检测到 cAMP 产生，即使在 PKA 抑制剂 H-89 的存在下，pCREB 激活也会发生。此外，S1P 依赖性 CREB 磷酸化被丝裂原激活蛋白激酶（MEK）抑制剂 U0126 和 L 型钙通道阻滞剂维拉帕米抑制。通过用特异性受体拮抗剂 JTE-013 和 TY52156 阻断 S1P 或 S1P，或在 PLC/PI3K 耗竭的情况下，完全抑制 CREB 磷酸化。S1P 依赖性 CREB 磷酸化诱导 FOXO1 和 EGF 样表皮生长因子样基因（EREG）的表达，证实了促性腺激素和白细胞介素在这一过程中的独特作用，但不影响类固醇生成。然而，在我们的条件下，S1P 或激动剂并未调节颗粒细胞的活力和增殖。
这项研究首次表明，S1P 可能在颗粒细胞中诱导 cAMP 非依赖性 pCREB 激活，尽管这不足以诱导细胞内甾体生成信号和孕酮合成。S1P 诱导的 FOXO1 和 EREG 基因表达表明，S1P-S1PR 轴的激活可能与促性腺激素一起调节卵泡发育。
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