Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB), Universidad Nacional del Sur (UNS), Buenos Aires, Argentina.
Invest Ophthalmol Vis Sci. 2011 Aug 22;52(9):6580-8. doi: 10.1167/iovs.10-7065.
PURPOSE. Simple sphingolipids control crucial cellular processes in several cell types. Previous work demonstrated that sphingolipids, such as ceramide, sphingosine, and sphingosine-1-phosphate, are key mediators in the regulation of survival, differentiation, and proliferation of retina photoreceptors. Ceramide-1-phosphate (C1P) regulates growth and survival in several cell types; however, little is known concerning its functions in the retina. Whether C1P also participates in controlling photoreceptor development was also explored. METHODS. Rat retina neuronal cultures were supplemented with 1 to 10 μM C1P. Proliferation was determined by evaluating 5-bromo-2-deoxyuridine (BrdU) uptake and the number of mitotic figures and differentiation by evaluating opsin and peripherin expression by immunocytochemistry and Western blot. Apoptosis was inhibited with the pan caspase inhibitor ZVADFMK and evaluated by TUNEL assay, propidium iodide/annexin V, and DAPI labeling. Preservation of mitochondrial membrane potential was evaluated. RESULTS. C1P enhanced BrdU uptake and increased mitosis in retinal progenitors. C1P addition advanced photoreceptor differentiation, enhancing opsin and peripherin expression and stimulating development of the apical processes in which these proteins were concentrated. In the absence of these trophic factors, photoreceptors degenerated after 4 days in vitro, and at day 6, almost 50% of photoreceptors were apoptotic. C1P decreased photoreceptor apoptosis, reducing this percentage by half. Inhibiting caspase activity reduced photoreceptor apoptosis in the controls, but did not increase opsin expression, implying that C1P has separate effects on differentiation and survival. CONCLUSIONS. These results suggest for the first time that C1P is a novel mediator that has multiple functions in photoreceptors, initially regulating their proliferation and then promoting their survival and differentiation.
目的。简单的神经酰胺控制着几种细胞类型中关键的细胞过程。先前的工作表明，神经酰胺，如神经酰胺、鞘氨醇和鞘氨醇-1-磷酸，是调节视网膜光感受器存活、分化和增殖的关键介质。神经酰胺-1-磷酸（C1P）调节几种细胞类型的生长和存活；然而，关于其在视网膜中的功能知之甚少。C1P 是否也参与控制光感受器发育也进行了探讨。
方法。在大鼠视网膜神经元培养物中添加 1 至 10 μM 的 C1P。通过评估 5-溴-2-脱氧尿苷（BrdU）摄取和有丝分裂数以及通过免疫细胞化学和 Western blot 评估视蛋白和周边蛋白的表达来确定增殖。用泛半胱天冬酶抑制剂 ZVADFMK 抑制细胞凋亡，并通过 TUNEL 测定、碘化丙啶/膜联蛋白 V 和 DAPI 标记评估。评估线粒体膜电位的保存情况。
结果。C1P 增强 BrdU 摄取并增加视网膜祖细胞的有丝分裂。C1P 的添加促进了光感受器的分化，增强了视蛋白和周边蛋白的表达，并刺激了这些蛋白集中的顶端过程的发育。在缺乏这些营养因子的情况下，体外 4 天后光感受器发生退化，第 6 天，近 50%的光感受器发生凋亡。C1P 减少光感受器凋亡，使这一比例减半。抑制半胱天冬酶活性可减少对照组中的光感受器凋亡，但不会增加视蛋白的表达，这表明 C1P 对分化和存活有独立的作用。
结论。这些结果首次表明，C1P 是一种新型介质，在光感受器中具有多种功能，最初调节其增殖，然后促进其存活和分化。
