The balance between Gα_i-Cdc42/Rac and Gα₁₂/₁₃-RhoA pathways determines endothelial barrier regulation by...

The bioactive sphingosine-1-phosphatephosphate (S1P) is present in plasma, bound to carrier proteins, and involved in many physiological processes, including angiogenesis, inflammatory responses, and vascular stabilization. S1P can bind to several G-protein-coupled receptors (GPCRs) activating a number of different signaling networks. At present, the dynamics and relative importance of signaling events activated immediately downstream of GPCR activation are unclear. To examine these, we used a set of fluorescence resonance energy transfer-based biosensors for different RhoGTPases (Rac1, RhoA/B/C, and Cdc42) as well as for heterotrimeric G-proteins in a series of live-cell imaging experiments in primary human endothelial cells. These experiments were accompanied by biochemical GTPase activity assays and transendothelial resistance measurements. We show that S1P promotes cell spreading and endothelial barrier function through S1PR₁-Gα_i-Rac1 and S1PR₁-Gα_i-Cdc42 pathways. In parallel, a S1PR₂-Gα_12/13-RhoA pathway is activated that can induce cell contraction and loss of barrier function, but only if Gα_i-mediated signaling is suppressed. Our results suggest that Gα_q activity is not involved in S1P-mediated regulation of barrier integrity. Moreover, we show that early activation of RhoA by S1P inactivates Rac1 but not Cdc42, and vice versa. Together, our data show that the rapid S1P-induced increase in endothelial integrity is mediated by a S1PR₁-Gα_i-Cdc42 pathway.