title: The activation of RhoC in vascular endothelial cells is required for the S1P receptor type 2-induced inhibition of angiogenesis.
abstract: Sphingosine-1-phosphate (S1P) is a multifunctional phospholipid inducing a variety of cellular responses in endothelial cells (EC). S1P responses are mediated by five G protein coupled receptors of which three types (S1P1R-S1P3R) have been described to be of importance in vascular endothelial cells (EC). Whereas the S1P1R regulates endothelial barrier function by coupling to Gαi and the monomeric GTPase Rac1, the signaling pathways involved in the S1P-induced regulation of angiogenesis are ill defined. We therefore studied the sprouting of human umbilical vein EC (HUVEC) in vitro and analyzed the activation of the RhoGTPases RhoA and RhoC. Physiological relevant concentrations of S1P (100-300nM) induce a moderate activation of RhoA and RhoC. Inhibition or siRNA-mediated depletion of the S1P2R preferentially decreased the activation of RhoC. Both manipulations caused an increase of sprouting in a spheroid based in vitro sprouting assay. Interestingly, a similar increase in sprouting was detected after effective siRNA-mediated knockdown of RhoC. In contrast, the depletion of RhoA had no influence on sprouting. Furthermore, suppression of the activity of G proteins of the Gα12/13 subfamily by adenoviral overexpression of the regulator of G protein signaling domain of LSC as well as siRNA-mediated knockdown of the Rho specific guanine nucleotide exchange factor leukemia associated RhoGEF (LARG) inhibited the S1P-induced activation of RhoC and concomitantly increased sprouting of HUVEC with similar efficacy. We conclude that the angiogenic sprouting of EC is suppressed via the S1P2R subtype. Thus, the increase in basal sprouting can be attributed to blocking of the inhibitory action of autocrine S1P stimulating the S1P2R. This inhibitory pathway involves the activation of RhoC via Gα12/13 and LARG, while the simultaneously occurring activation of RhoA is apparently dispensable here.
authors.affiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, Mannheim Medical Faculty, Heidelberg University, Maybachstrasse 14, 68169 Mannheim, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Del Galdo
authors.forename: Christiane
authors.initials: C
authors.lastname: Vettel
authors.forename: Dagmar Meyer Zu
authors.initials: DM
authors.lastname: Heringdorf
authors.forename: Thomas
authors.initials: T
authors.lastname: Wieland
mesh_terms: D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D008246:Lysophospholipids / Q000378:metabolism*; D018919:Neovascularization, Physiologic; D049349:Receptors, Lysosphingolipid / Q000378:metabolism*; D064067:Rho Guanine Nucleotide Exchange Factors / Q000378:metabolism; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D000075024:rhoC GTP-Binding Protein
chemical_list: C577222:ARHGEF12 protein, human; D008246:Lysophospholipids; D049349:Receptors, Lysosphingolipid; D064067:Rho Guanine Nucleotide Exchange Factors; C060506:sphingosine 1-phosphate; C066690:RHOC protein, human; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D000075024:rhoC GTP-Binding Protein; D013110:Sphingosine
keywords: Angiogenesis; Leukemia associated Rho guanine nucleotide exchange factor; RhoC; RhoGTPases; S1P(2) receptor; Sphingosine-1-phosphate