Sphingosine‐1‐phosphate (S1P)‐induced activation of RhoA and enhancement of endothelial barrier integrity

Abstract

While S1P is thought to protect endothelial barrier by activating Rac1, the role of RhoA is less clear, as previous studies indicate that RhoA can promote either increased or decreased endothelial permeability. We hypothesized that S1P activates RhoA at the cell periphery to enhance endothelial barrier function. We used human umbilical vein endothelial cell (HUVEC) monolayers to model the endothelial barrier. Transendothelial electrical resistance (TER) served as an index of barrier function. HUVEC expressing wild-type RhoA FLARE.sc FRET biosensor were used to study local RhoA activation by time-lapse confocal microscopy. S1P-mediated changes in RhoA-GTP over time were also determined by ELISA. We tested the involvement of the RhoA downstream effector mDia using a FH2 effector domain inhibitor, SMIFH2. We discovered that S1P (2 µM) activates RhoA in two locations: at the periphery and perinuclear regions. Pre-treatment of RhoA inhibitors (Rhosin, Y16 or combined) attenuated S1P-mediated endothelial barrier enhancement. ELISA studies confirmed this RhoA activation, which was approximately 4-fold for up to 10 min. Interestingly, blockade of mDia with SMIFH2 (5 µM) failed to abolish S1P-mediated maximum increase in TER. Our data suggests that localized RhoA activation mediates S1P-mediated endothelial barrier enhancement, and the downstream mechanism does not appear to involve mDia. Considering that agents that disrupt the endothelial barrier have been shown to activate RhoA in central areas of the cell, we think the local activation of RhoA at the cell periphery is a key event that promotes local adhesion strength and junctional integrity. Supported by NIH R01HL098215.