Zhang Xun E, Adderley Shaquria P, Breslin Jerome W
Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, Florida, United States of America.
PLoS One. 2016 May 17;11(5):e0155490. doi: 10.1371/journal.pone.0155490. eCollection 2016.
Compromised endothelial barrier function is a hallmark of inflammation. Rho family GTPases are critical in regulating endothelial barrier function, yet their precise roles, particularly in sphingosine-1-phosphate (S1P)-induced endothelial barrier enhancement, remain elusive. Confluent cultures of human umbilical vein endothelial cells (HUVEC) or human dermal microvascular endothelial cells (HDMEC) were used to model the endothelial barrier. Barrier function was assessed by determining the transendothelial electrical resistance (TER) using an electrical cell-substrate impedance sensor (ECIS). The roles of Rac1 and RhoA were tested in S1P-induced barrier enhancement. The results show that pharmacologic inhibition of Rac1 with Z62954982 failed to block S1P-induced barrier enhancement. Likewise, expression of a dominant negative form of Rac1, or knockdown of native Rac1 with siRNA, failed to block S1P-induced elevations in TER. In contrast, blockade of RhoA with the combination of the inhibitors Rhosin and Y16 significantly reduced S1P-induced increases in TER. Assessment of RhoA activation in real time using a fluorescence resonance energy transfer (FRET) biosensor showed that S1P increased RhoA activation primarily at the edges of cells, near junctions. This was complemented by myosin light chain-2 phosphorylation at cell edges, and increased F-actin and vinculin near intercellular junctions, which could all be blocked with pharmacologic inhibition of RhoA. The results suggest that S1P causes activation of RhoA at the cell periphery, stimulating local activation of the actin cytoskeleton and focal adhesions, and resulting in endothelial barrier enhancement. S1P-induced Rac1 activation, however, does not appear to have a significant role in this process.
内皮屏障功能受损是炎症的一个标志。Rho家族GTP酶在调节内皮屏障功能中起关键作用，但其确切作用，特别是在1-磷酸鞘氨醇（S1P）诱导的内皮屏障增强方面，仍不清楚。用人脐静脉内皮细胞（HUVEC）或人真皮微血管内皮细胞（HDMEC）的汇合培养物来模拟内皮屏障。通过使用细胞-基质阻抗传感器（ECIS）测定跨内皮电阻（TER）来评估屏障功能。在S1P诱导的屏障增强中测试了Rac1和RhoA的作用。结果表明，用Z62954982对Rac1进行药理抑制未能阻断S1P诱导的屏障增强。同样，Rac1显性负性形式的表达或用siRNA敲低天然Rac1也未能阻断S1P诱导的TER升高。相反，用抑制剂Rhosin和Y16联合阻断RhoA可显著降低S1P诱导的TER升高。使用荧光共振能量转移（FRET）生物传感器实时评估RhoA激活表明，S1P主要在细胞边缘、靠近连接处增加RhoA激活。这伴随着细胞边缘肌球蛋白轻链-2磷酸化，以及细胞间连接处附近F-肌动蛋白和纽蛋白增加，而这些都可以通过RhoA的药理抑制来阻断。结果表明，S1P导致细胞周边RhoA激活，刺激肌动蛋白细胞骨架和粘着斑的局部激活，从而导致内皮屏障增强。然而，S1P诱导的Rac1激活在这一过程中似乎没有显著作用。
