Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB), Dept. of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur (UNS) and National Research Council of Argentina (CONICET), Bahía Blanca, Buenos Aires, Argentina.
Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB), Dept. of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur (UNS) and National Research Council of Argentina (CONICET), Bahía Blanca, Buenos Aires, Argentina.
Exp Eye Res. 2022 Nov;224:109222. doi: 10.1016/j.exer.2022.109222. Epub 2022 Aug 27.
Retinal pigment epithelium (RPE) cells, essential for preserving retina homeostasis, also contribute to the development of retina proliferative diseases, through their exacerbated migration, epithelial to mesenchymal transition (EMT) and inflammatory response. Uncovering the mechanisms inducing these changes is crucial for designing effective treatments for these pathologies. Sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) are bioactive sphingolipids that promote migration and inflammation in several cell types; we recently established that they stimulate the migration of retina Müller glial cells (Simón et al., 2015; Vera et al., 2021). We here analyzed whether S1P and C1P regulate migration, inflammation and EMT in RPE cells. We cultured two human RPE cell lines, ARPE-19 and D407 cells, and supplemented them with either 5 μM S1P or 10 μM C1P, or their vehicles, for 24 h. Analysis of cell migration by the scratch wound assay showed that S1P addition significantly enhanced migration in both cell lines. Pre-treatment with W146 and BML-241, antagonists for S1P receptor 1 (S1P1) and 3 (S1P3), respectively, blocked exogenous S1P-induced migration. Inhibiting sphingosine kinase 1 (SphK1), the enzyme involved in S1P synthesis, significantly reduced cell migration and exogenous S1P only partially restored it. Addition of C1P markedly stimulated cell migration. Whereas inhibiting C1P synthesis did not affect C1P-induced migration, inhibiting S1P synthesis strikingly decreased it; noteworthy, addition of C1P promoted the transcription of SphK1. These results suggest that S1P and C1P stimulate RPE cell migration and their effect requires S1P endogenous synthesis. Both S1P and C1P increase the transcription of pro-inflammatory cytokines IL-6 and IL-8, and of EMT marker α-smooth muscle actin (α-SMA) in ARPE-19 cells. Collectively, our results suggest new roles for S1P and C1P in the regulation of RPE cell migration and inflammation; since the deregulation of sphingolipid metabolism is involved in several proliferative retinopathies, targeting their metabolism might provide new tools for treating these pathologies.
视网膜色素上皮 (RPE) 细胞对于维持视网膜内环境稳态至关重要，但它们也通过过度迁移、上皮间质转化 (EMT) 和炎症反应，促进视网膜增殖性疾病的发展。揭示诱导这些变化的机制对于设计这些病变的有效治疗方法至关重要。 1-磷酸鞘氨醇 (S1P) 和 1-磷酸神经酰胺 (C1P) 是生物活性鞘脂，可促进多种细胞类型的迁移和炎症；我们最近证实，它们刺激视网膜 Müller 胶质细胞的迁移 (Simón 等人，2015 年；Vera 等人，2021 年)。我们在这里分析了 S1P 和 C1P 是否调节 RPE 细胞的迁移、炎症和 EMT。我们培养了两种人 RPE 细胞系，ARPE-19 和 D407 细胞，并在其中添加 5 μM S1P 或 10 μM C1P 或其载体，培养 24 小时。划痕实验分析细胞迁移显示，S1P 添加显著增强了两种细胞系的迁移。分别用 S1P 受体 1 (S1P1) 和 3 (S1P3) 的拮抗剂 W146 和 BML-241 预处理，可阻断外源性 S1P 诱导的迁移。抑制鞘氨醇激酶 1 (SphK1)，即 S1P 合成的酶，显著降低细胞迁移，而外源性 S1P 仅部分恢复。C1P 的添加显著刺激细胞迁移。虽然抑制 C1P 合成不影响 C1P 诱导的迁移，但抑制 S1P 合成则显著降低迁移；值得注意的是，C1P 的添加促进了 SphK1 的转录。这些结果表明 S1P 和 C1P 刺激 RPE 细胞迁移，其作用需要内源性 S1P 合成。S1P 和 C1P 均增加 ARPE-19 细胞中促炎细胞因子白细胞介素 6 (IL-6) 和白细胞介素 8 (IL-8) 的转录以及 EMT 标志物α-平滑肌肌动蛋白 (α-SMA) 的转录。综上所述，我们的结果表明 S1P 和 C1P 在调节 RPE 细胞迁移和炎症方面发挥新作用；由于鞘脂代谢失调参与了几种增殖性视网膜病变，因此靶向其代谢可能为治疗这些病变提供新工具。
