| Objective:Sphingosine-1-phosphate (SIP) is a bioactive sphingolipid in plasam. SIP is mainly synthesized and secreted by platelets. And other cells such as erythrocytes, neutrophils, macrophages, monocytes, mast cells and endothelial cells are also able to produce and release S1P. Concentration of S1P can be adjusted through a complex metabolic process, and its change in plasma concentration directly regulates its biological effects. Intracellular SIP can interact with regulatory molecules such as enzymes, channel proteins and transcription factors as a second messenger. SIP can also serve as the extracellular ligand binding to SIP membrane receptors existing in almost all cell types. SIP affects cell proliferation, survival, differentiation, migration and morphogenesis through different signal transduction pathways. There are five cognate G-protein-coupled receptors, to which SIP specifically binds, designated S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5, respectively. The SIP receptors most abundantly expressed in endothelial cells are S1PR1, S1PR2 and S1PR3 with S1PR1>S1PR2=S1PR3. Different subtypes of SIP receptor binding different G protein, S1PR1 is coupled to Gi; S1PR2/3 is associated to Gi, Gq, G12/13. Further, S1PR1 and S1PR2/3 have different affinities to S1P. An appropriate or physiological level of SIP causes the activation of S1PR1, resulting in the strengthening of the barrier integrity of endothelial cells by inducing Rac signaling pathway, while the lack of S1P will be harmful in endothelial barrier function. While over-dose S1P will bind to receptor 2 and 3 and the activation of S1PR2/R3 would disrupt the inter-endothelial junctions by evoking the RhoA and ROCK pathway.Some studies have shown that signal pathways induced by S1P are closely related to intracellular calcium signal changes. Alteration of calcium concentration in endothelial cell is mainly influenced by two aspects, one is phospholipase C (PLC) and inositol trisphosphate receptor (IP3R)-mediated endoplasmic reticulum calcium release, the other is store-operated calcium entry (SOC) ion channels.This study is aimed to clarify the SIP receptor binding signal transduction pathways and to understand the role of calcium in regulations of endothelial barrier function by different concentrations of S1P.Methods:Human umbilical vein endothelial cell line (HUVECs) was used in this experiment. Luminescence-based G-LISA assay kits were applied to detect the activity of Racl or RhoA, and immunoblotting was used to detect the phosphorylation of ROCK. Distribution of phospho-ROCK and cytoskeleton in HUVECs were observed with immunocytochemistry under cofocal microscope of Zeiss LSM 780. Trans endothelial electric resistance (TEER) was detected by resistance meter to clarify the alteration of endothelial barrier function.Results:1. S1P induced time-dependent changes of Rac1 activity in HUVECs. Cells were treated with 0.5 μmol/L S1P for 2,5, and 20 min; Racl activity was detected using luminescence-based G-LISATM Racl assay kit. The increasing of Racl activity reached its maximum at 5 min with the relative Rac1 activity increased 2.48 folds.2. Only physiological level of SIP (0.1,0.5,1.0 μmol/L) could induced Racl activation in HUVECs, while high dose of SIP (10.0 μmol/L) has no effect on activation of Racl.3. The pre-treatment of S1PR2 antagonist JET-013 could not inhibit the activation of Racl by 0.5 μmol/L SIP, while the application of S1PR1 antagonist VPC23019 partially abolished 0.5 μmol/L SIP-induced Racl activation. And S1PR1 agonist SEW2871 induced Racl activation in HUVECs. These results indicated that S1PR1 mediates Racl activation induced by 0.5 μmol/L SIP, whereas S1PR2 has no effect on activation of Racl.4. The pre-treatment of PI-PLC inhibitor ET-18-OCH3 and IP3R inhibitor 2-APB partially abolished the activation of Racl by 0.5 μmol/L SIP, while the application of blocker of transmembrane Ca2+ influxes ruthenium red (RR) could not inhibit 0.5 μmol/L S1P-induced Racl activation. These results indicated that intracellular calcium release is involved in mediating Racl activation caused by 0.5 μmol/L S1P, extracellular calcium has no effect on Racl activation.5. The pre-treatment of ET-18-OCH3 or 2-APB partially abolished the activation of Racl by SEW2871, while the application of RR could not inhibit SEW2871-induced Racl activation. These results indicated that intracellular calcium release is involved in mediating Racl activation caused by S1PR1 activation, extracellular calcium has no effect.6. SIP induced time-dependent changes of RhoA activity in HUVECs. Cells were treated with 10.0 μmol/L SIP for 2,5,10 and 30 min; RhoA activity was detected using luminescence-based G-LISATM RhoA assay kit. The increasing of RhoA activity reached its maximum at 2 min with the relative RhoA activity increased 1.91 folds.7. Only high dose of SIP (10.0 μmol/L) could induced RhoA activation in HUVECs, while physiological level of SIP (0.1,0.5,1.0 μmol/L) has no effect on activation of RhoA.8. The pre-treatment of S1PR1 antagonist VPC23019 could not inhibit the activation of RhoA by 10.0 μmol/L SIP, while the application of S1PR2 antagonist JTE-013 partially abolished 10.0 μmol/L S1P-induced RhoA activation. And S1PR1 agonist SEW2871 could not induce RhoA activation in HUVECs. These results indicated that S1PR2 mediates RhoA activation induced by 10.0 μmol/L S1P, whereas S1PR1 has no effect on activation of Racl.9. The pre-treatment of ET-18-OCH3 or 2-APB could not inhibit the activation of RhoA by 10.0 μmol/L S1P, while RR partially abolished 10.0 μmol/L S1P-induced RhoA activation. These results indicated that extracellular calcium is involved in mediating Racl activation caused by 10.0 Lmol/L SIP, while intracellular calcium release has no effect.10. ROCK phosphorylaiton was detected using western bloting. Only high dose of SIP (10.0 μmol/L) could induced ROCK phosphorylaiton in HUVECs in 5 min, while physiological level of SIP (0.1,0.5,1.0 lmol/L) has no effect on ROCK phosphorylaiton.11. The pre-treatment of ET-18-OCH3 or 2-APB could not inhibit the activation of ROCK by 10.0 lmol/L SIP, while RR partially abolished 10.0 kmol/L SIP -induced ROCK activation. These results indicated that extracellular calcium is involved in mediating ROCK activation caused by 10.0 μmol/L SIP, but intracellular calcium release has no effect.12. The administration of high dose SIP also induced a re-distribution of phospho-ROCK from perinuclear area to cytoplasm and this re-distribution might be triggered along with the activaition of above-mentioned signal pathways.13. The physiological level (0.5 μmol/L) of SIP has a protective effect on the structure of ZO-1 and F-actin, while over-dose SIP would disrupt the structure of ZO-1 and F-actin.14. The pre-treatment of S1PR1 antagonist VPC23019 could weaken the protective effect on the structure of ZO-1 and F-actin by 0.5 μmol/L SIP or SEW2871, while the application of S1PR2 antagonist JTE-013 partially abolished 10.0 μmol/L SIP-induced damage of ZO-1 and F-actin. These results indicated that S1PR1 mediates the protective effect on the structure of ZO-1 and F-actin by 0.5 umol/L SIP, and S1PR2 mediates 10.0 μmol/L S1P-induced destruction of ZO-1 and F-actin.15. The pre-treatment of Racl inhibitor NSC23766 could weaken the protective effect on the structure of ZO-1 and F-actin by 0.5 μmol/L SIP, while the application of ROCK inhibitor H1152 partially abolished 10.0 μmol/L S1P-induced destruction of ZO-1 and F-actin. These results indicated that Racl mediates the protective effect on the structure of ZO-1 and F-actin by 0.5 μmol/L SIP, and ROCK mediates 10.0 μmol/L S1P-induced destruction of ZO-1 and F-actin.16. The administration of ET-18-OCH3 or 2-APB disrupted the structure of ZO-1 and F-actin. The pre-treatment of ET-18-OCH3 or 2-APB could weaken the protective effect on the structure of ZO-1 and F-actin by 0.5 μmol/L SIP, while the application of RR partially abolished 10.0 μmol/L S1P-induced destruction of ZO-1 and F-actin. These results indicated that intracellular calcium release mediates the protective effect on the structure of ZO-1 and F-actin by 0.5 μmol/L SIP, and extracellular calcium influx mediates 10.0 μmol/L S1P-induced destruction of ZO-1 and F-actin.17. The administration of physiological level (0.1,0.5,1.0 μmol/L) of SIP induced increased endothelial cell monolayer TEER, while over-dose S1P (10.0 μmol/L) induced reduction of endothelial cell monolayer TEER.18. The pre-treatment of S1PR1 antagonist VPC23019 could reduce increased endothelial cell monolayer TEER by 0.5 μmol/L S1P, while the application of S1PR2 antagonist JTE-013 partially abolished 10.0 μmol/L S1P-induced decrease of endothelial cell monolayer TEER. These results indicated that S1PR1 is involved in the strengthening of the barrier integrity of endothelial cells by 0.5 μmol/L S1P, and S1PR2 mediates 10.0 μmol/L S1P-induced destruction of the inter-endothelial junctions.19. The pre-treatment of Racl inhibitor NSC23766 could reduce increased endothelial cell monolayer TEER by 0.5 μmol/L SIP, while the application of ROCK inhibitor H1152 partially abolished 10.0 μmol/L S1P-induced decrease of endothelial cell monolayer TEER. These results indicated that Racl is involved in the strengthening of the barrier integrity of endothelial cells by 0.5 μmol/L SIP, and ROCK mediates 10.0 μmol/L SIP-induced destruction of the inter-endothelial junctions.20. The pre-treatment of ET-18-OCH3 or 2-APB could reduce increased endothelial cell monolayer TEER by 0.5 μmol/L SIP, while the application of RR partially abolished 10.0 μmol/L S1P-induced decrease of endothelial cell monolayer TEER. These results indicated that intracellular calcium release is involved in the strengthening of the barrier integrity of endothelial cells by 0.5 μmol/L S1P, and extracellular calcium influx mediates 10.0 μmol/L S1P-induced destruction of the inter-endothelial junctions.Conclusions:1. Physiological level of SIP could activate S1PR1, then cause endoplasmic reticulum calcium release, resulting in the strengthening of the barrier integrity of endothelial cells by inducing Rac signaling pathway.2. Over-dose S1P would bind to S1PR2 and the activation of S1PR2 would cause extracellular calcium influx, leading to destruction of the inter-endothelial junctions by evoking the RhoA and ROCK pathway. |
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