Tamborindeguy Maurício Tavares, Matte Bibiana Franzen, Ramos Grasieli de Oliveira, Alves Alessandro Menna, Bernardi Lisiane, Lamers Marcelo Lazzaron
Basic Research Center in Dentistry, Dentistry School, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil.
Center of Biotechnology, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil.
Biol Cell. 2018 Oct;110(10):225-236. doi: 10.1111/boc.201800011. Epub 2018 Sep 16.
Cell migration requires the coordinated activation of structural and signalling molecules, such as the RhoGTPase Rac1. It is known that the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex assembly, which generates reactive oxygen species (ROS) at the cell membrane, also relies on Rac1 activation, indicating a possible effect of ROS during cell migration. In this study, we evaluated the effect of NADPH-oxidase-derived ROS on the migration process.
Using time-lapse videos of CHO.K1 cells plated on fibronectin (2 μg/ml) or collagen (5 μg/cm ), we observed that depletion of ROS by N-acetyl-cysteine (NAC, 10 mM), an unspecific antioxidant, or diphenyliodonium (DPI, 10 μM), a NADPH-oxidase inhibitor, induced a ∼50% decrease in migration speed and severely impacted migration directionality. Then, we analysed the effects of NADPH oxidase on three migratory events: protrusion rate, adhesion process and signalling pathways related to cell migration. DPI induced an increase of ∼3 protrusion/cell, which were 2× faster but had a ∼50% retraction when compared with control. By pull-down assay, we observed no changes on Rac1 activation, indicating that ROS-mediated effects were related to downstream molecules, such as adhesion-related molecules. A reduction of the adhesion marker FAK-Y397 levels in cells treated with NAC and DPI was observed. In order to analyse adhesion dynamics, CHO.K1 cells transfected with paxillin-GFP analysed with total internal reflectance fluorescence (TIRF) indicated that DPI (5 μM) induced larger adhesions when compared with control.
These results indicate that the local generation of NADPH-oxidase-derived ROS can modulate cell migration due to changes on adhesion dynamics and signalling.
This study highlights the physiological requirement of ROS for cell migration and the potential use of these molecules as targets to modulate the cell migration process at different diseases.
细胞迁移需要结构和信号分子的协同激活，如RhoGTPase Rac1。已知在细胞膜上产生活性氧（ROS）的烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶复合物组装也依赖于Rac1激活，这表明ROS在细胞迁移过程中可能具有作用。在本研究中，我们评估了NADPH氧化酶衍生的ROS对迁移过程的影响。
使用接种在纤连蛋白（2μg/ml）或胶原蛋白（5μg/cm）上的CHO.K1细胞的延时视频，我们观察到，非特异性抗氧化剂N-乙酰半胱氨酸（NAC，10mM）或NADPH氧化酶抑制剂二苯基碘鎓（DPI，10μM）消耗ROS会导致迁移速度降低约50%，并严重影响迁移方向性。然后，我们分析了NADPH氧化酶对三个迁移事件的影响：突出率、粘附过程以及与细胞迁移相关的信号通路。DPI诱导每个细胞突出增加约3个，与对照相比，这些突出速度快2倍，但回缩约50%。通过下拉试验，我们观察到Rac1激活没有变化，这表明ROS介导的作用与下游分子有关，如粘附相关分子。在用NAC和DPI处理的细胞中，观察到粘附标记物FAK-Y397水平降低。为了分析粘附动力学，用全内反射荧光（TIRF）分析转染桩蛋白-GFP的CHO.K1细胞表明，与对照相比，DPI（5μM）诱导更大的粘附。
这些结果表明，NADPH氧化酶衍生的ROS的局部产生可因粘附动力学和信号的变化而调节细胞迁移。
本研究强调了ROS对细胞迁移的生理需求，以及这些分子作为调节不同疾病中细胞迁移过程的靶点的潜在用途。
