The balance between Gi-Cdc 42 / Rac and G 12 / 13-RhoA pathways determines endothelial barrier regulation by sphingosine-1-phosphate

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-Gαi-Rac1 and S1PR1-Gαi-Cdc42 pathways. In parallel, a S1PR2-Gα12/13-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-Gαi-Cdc42 pathway. Monitoring Editor Peter Van Haastert University of Groningen Received: Mar 3, 2017 Revised: Sep 14, 2017 Accepted: Sep 19, 2017 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E17-03-0136) on September 27, 2017. †These authors contributed equally to this work. The authors declare no competing or financial interests. Author contributions: N.R. R. , M.M. , and E.K. B. performed experiments. N.R. R. analyzed the data and wrote the manuscript. Y.W. and T.Y. designed and constructed the DORA-Rho GTPase biosensors. T.W. J.G. , J.G. , and P.L. H. assisted with the experimental design and data interpretation. All authors approved the final manuscript. *Address correspondence to: Peter L. Hordijk (p.hordijk@vumc.nl) or Joachim Goedhart (j.goedhart@uva.nl). Abbreviations used: AJ, adherens junction; BSA, bovine serum albumin; CFP, cyan fluorescent protein; CRIB, Cdc42/Rac interactive binding; EC, endothelial cell; ECIS, Electrical Cell-substrate Impedance Sensing; FA, focal adhesion; FN, Nathalie R. Reinharda,b,c, Marieke Mastopa, Taofei Yind, Yi Wud, Esmeralda K. Bosmaa, Theodorus W. J. Gadella Jr.a, Joachim Goedharta,†,*, and Peter L. Hordijka,b,c,e,†,* avan Leeuwenhoek Centre for Advanced Microscopy, Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, 1098 XH Amsterdam, Netherlands; bMolecular Cell Biology and cUniversity of Amsterdam Academic Medical Centre–Landsteiner Laboratory, Sanquin Research, 1066 CX Amsterdam, Netherlands; dCenter for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, CT 06030; eDepartment of Physiology, Free University Medical Center, 1081 HZ Amsterdam, Netherlands © 2017 Reinhard et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. fibronectin; FRET, fluorescence resonance energy transfer; GAP, GTPase activating protein; GEF, guanine exchange factor; GPCR, G-protein–coupled receptor; HUVEC, human umbilical vein endothelial cell; mAb, monoclonal antibody; mTq2, mTurquoise2; NA, numerical aperture; pAb, polyclonal antibody; PBS, phosphate-buffered saline; PM, plasma membrane; PTX, pertussis toxin; RBD, Rho binding domain; RGS, regulator of G-protein signaling; RhoGEF, Rho guanine exchange factor; S1P, sphingosine-1-phosphate; TEM, transendothelial migration; TJ, tight junction; VE-cadherin, vascular endothelial cadherin protein. INTRODUCTION Endothelial cells (ECs) line blood and lymphatic vessels, forming a semipermeable barrier between the blood and underlying tissue (Aird, 2007a,b). By sensing environmental changes, this cell layer regulates the transport of oxygen and essential nutrients to all organ systems in the human body (Pappenheimer et al., 1951; Del Vecchio et al., 1987; Siflinger-Birnboim et al., 1987). These continuous sensing capabilities are required to maintain vascular homeostasis and form the basis of many physiological and disease-related processes. http://www.molbiolcell.org/content/suppl/2017/09/25/mbc.E17-03-0136v1.DC1 Supplemental Material can be found at: