Activation of RhoA, but Not Rac1, Mediates Early Stages of S1P-Induced Endothelial Barrier Enhancement

Abstract

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.

Ablation of RhoA impairs Th17 cell differentiation and alleviates house dust mite-triggered allergic airway inflammation

“…Y16 is a specific RhoA inhibitor developed in our laboratory, which fits into a surface groove of the DH‐PH domain of leukemia‐associated Rho GEF (LARG), a G‐protein‐regulated Rho GEF involved in RhoA activation . Y16 has been used for RhoA functional studies of a wide range, from endothelial barrier and macrophage phenotypes to contraction of intrapulmonary artery and airway smooth muscle . The main purpose for using Y16 in this study is to complement genetic deletion of RhoA to demonstrate the role of RhoA in Th17‐cell differentiation in vitro and Th17‐involved allergic airway inflammation in vivo.…”

Mind the gap: mechanisms regulating the endothelial barrier

“…Fluorescence resonance energy transfer (FRET) analysis revealed spontaneous RhoA activity at intact intercellular junctions and in parallel to intercellular gap closure . RhoA via ROCK2b was found to prevent vascular leakage during leucocyte diapedesis and S1P was proposed to enhance barrier function at least in part via RhoA and ROCK . On the other hand, endothelial cells deficient in podocalyxin have delayed barrier recovery and sustained RhoA activation indicating a negative effect of RhoA .…”

Endothelial Protrusions in Junctional Integrity and Barrier Function

“…These include myosin light chain (MLC) kinase (MLCK) and RhoA/ROCK signaling, which control myosin activity (Moy et al , 1993; Sheldon et al , 1993; Garcia et al , 1995; Verin et al , 1995; Yuan et al , 1997; Garcia et al , 1999; Tinsley et al , 2000; Yuan et al , 2002; Huang et al , 2003; Birukova et al , 2004; Breslin & Yuan, 2004; Tinsley et al , 2004b; Breslin et al , 2006; Reynoso et al , 2007; Kumar et al , 2009; Dudek et al , 2010; Beard et al , 2014). The balance of activities among members of the Rho family of small GTPases such as RhoA, Rac1, and Cdc42 have also been shown to be of importance for both disruption and stabilization of the endothelial barrier (Waschke et al , 2004a; Waschke et al , 2004b; Waschke et al , 2006; Baumer et al , 2008a; Schlegel et al , 2009; Schlegel & Waschke, 2009; Spindler et al , 2010; Breslin et al , 2015; Breslin et al , 2016; Zhang et al , 2016). In addition, there is increasing evidence that in addition to phosphorylation, other posttranslational modifications such as S-nitrosation of junctional proteins (Marin et al , 2012; Sánchez et al , 2013; Guequén et al , 2016), or specific palmitoylation of PKCβ by palmitoyl acyltransferase DHHC21 (Beard et al , 2016) can lead to elevated microvascular leakage.…”

“…Endothelial cytoskeleton and actin fiber dynamics are of significant importance in regulating endothelial permeability (Adderley et al , 2015a; Zhang et al , 2016) and they have been studied in the context of brain injury. One study showed that actin filaments are important to maintain the BBB, as treatment with cytochalasin-B, which disrupts actin integrity, caused BBB hyperpermeability (Nag, 1995).…”

“…Rac1 activity appears to correlate with lamellipodia protrusion frequency (Breslin et al , 2015). In addition, application of S1P, which transiently increases local lamellipodia formation (Breslin et al , 2015), also increases RhoA-mediated MLC phosphorylation and recruitment of vinculin near intercellular junctions between endothelial cells (Zhang et al , 2016). Other actin-myosin-independent mechanisms such as microtubule growth may also contribute to the overall mechanism (Ingber, 2003; Birukova et al , 2006).…”