The balance between Gαi-Cdc42/Rac and Gα1/2/13-RhoA pathways determines endothelial barrier regulation by sphingosine-1-phosphate

Nathalie R. Reinhard, Marieke Mastop, Taofei Yin, Yi Wu, Esmeralda K. Bosma, Theodorus W.J. Gadella, Joachim Goedhart, Peter L. Hordijk*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

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-G2i-Gα Rac1 and S1PR1-Gαi-Cdc42 pathways. In parallel, a S1PR2-12/ 13-RhoA pathway is activated that can induce cell contraction and loss of barrier function, but only if Gαi-mediated signaling is suppressed. Our results suggest that Gαq 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-i-Cdc42 pathway.

Original languageEnglish
Pages (from-to)3371-3382
Number of pages12
JournalMolecular Biology of the Cell
Volume28
Issue number23
DOIs
Publication statusPublished - 7 Nov 2017

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