| Objective:Sphingosine-1-phosphate (SIP) is a bioactive lysophospholipid capable of inducing a wide spectrum of biological responses. Although S1P has been proposed to act both as an intracellular second messenger and as an extracellular mediator, this bioactive lipid seems to play a more important role as the latter. When it serves as an extracellular mediator, SIP plays a wide range of biological effects by binding to specific SIP receptors and their signal transduction pathway to affect cell survival, differentiation, movement, etc. S1P receptors are a group of G protein-coupled receptors named S1PR1, S1PR2, S1PR3. S1PR4, S1PR5, respectively. S1PR1, R3and a little S1PR2are the main receptors expressed in endothelial cells. It is proposed that the balances of expression and activation of S1PR1, R2and R3in endothelial cells help to maintain the physiological functions, especially the barrier function of endothelial cells. An appropriate or physiological level of S1P 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 SIP will bind to receptor2and3and the activation of S1PR2/R3would disrupt the inter-endothelial junctions by evoking the RhoA and ROCK pathway. The Rho family of small GTPase proteins control a wide variety of cellular processes. RhoA is one of the best-known members of this family and the Rho kinases (ROCK) are the first and the best-characterized RhoA effectors. By modulating the organization of the actin cytoskeleton, RhoA/ROCK signaling regulates a wide range of cellular functions, such as contraction, motility, proliferation, and apoptosis. It has been shown that ROCK-dependent re-arrangement of the actin cytoskeleton and changes of cell contractility are involved in the regulation of endothelial permeability.This study is aimed to clarify the S1P receptor binding signal transduction pathways and to understand the role and mechanisms of different concentrations of S1P, especially the high dose S1P, in regulation of endothelial barrier functon.Methods:Human umbilical vein endothelial cell line (HUVECs) is used in this experiment. HUVECs were grew onto1%gelatin-coated plates in complete DMEM with20%heat-inactivated fetal bovine plasma (FBS),100u/ml penicillin, and125ug/ml streptomycin, maintained at standard culture conditions. Cells of2-4passages were seeded in6cm and10cm culture dishes or Petri dished (1x105/dish) for treatments and RhoA activity measurement, western blotting or immunofluorescence staining.Cells were treated with various concentration of SIP alone or after different interventions with S1PR2antagonist JET-013, ROCK inhibitor Y-27632, a blocker of transmembrane Ca2+fluxes Ruthenium red, PI-PLC inhibitor ET-18-OCH3, or IP3inhibitor2-APB, respectively. SIP agonist SEW2871was used to mimic the activating effect on S1PR1. Luminescence-based G-LISA assay kit was applied to detect the activity of RhoA, immunoblotting was used to detect the phosphorylation of ROCK. Distribution of phospho-ROCK in HUVECs was observed under cofocal microscope of Zeiss LSM780.Result: 1. S1P induced time-dependent changers of RhoA activity in HUVECs. Cells were treated with10μmol/L S1P for2,5,10and30min; RhoA activity was detected using luminescence-based G-LISATM assay. The increasing of RhoA activity reached its maximum at2min with the relative RhoA activity increased1.894folds..2. Only high dose of S1P (10μmol/L) could induced RhoA activation in HUVECs, while physiological level of SIP (0.1,0.5,1.0μmol/L) has no effect on activation of RhoA.3. The pre-treatment of S1PR2antagonist JET-013could not inhibited the activation of RhoA by high dose SIP (10μmol/L), while the application of blocker of transmembrane Ca2+fluxes Ruthenium red partially abolished the high dose S1P-induced RhoA activation. This results indicated that the influx of extracellular Ca2+might invoked the activation of RhoA, but it was not induced by S1PR2. It is possible that in this experiment series, it is S1PR3, rather than S1PR2, that plays a more important role in inducing the activation of RhoA.4. The application of S1PR1agonist SEW2871did not increase the RhoA activity, while the pre-treatments of PI-PLC inhibitor ET-18-OCH3, or IP3inhibitor2-APB did not abolished the high dose SIP-induced RhoA activation. This result further confirmed that the activation of S1PR1does not initiate the activation of RhoA. And the PI-PLC-IP3pathway are not involved in high dose S1P-induced RhoA activation.5. ROCK phosphorylaiton was detected using western blooting. Only high dose of SIP (10μmol/L) could induced ROCK phosphorylaiton in HUVECs in5min, while physiological level of SIP (0.1,0.5,1.0μmol/L) has no effect on ROCK phosphorylaiton.6. The pre-treatment of S1PR2antagonist JET-013could not inhibited the ROCK phosphorylaiton by high dose SIP (10μmol/L), while the application of blocker of transmembrane Ca2+fluxes Ruthenium red partially abolished this high dose SIP-induced ROCK phosphorylaiton. This results indicated that the influx of extracellular Ca2+might invoked the ROCK phosphorylaiton, but it was not induced by S1PR2. It is possible that in this experiment series, it is S1PR3, rather than S1PR2, that plays a more important role in inducing the phosphorylaiton of ROCK.7. The application of S1PR1agonist SEW2871did not increase the phosphorylaiton of ROCK, while the pre-treatments of PI-PLC inhibitor ET-18-OCH3, or IP3inhibitor2-APB did not abolished the high dose SIP-induced ROCK phosphorylaiton. This results further confirmed that the activation of S1PR1does not initiate the phosphorylaiton of ROCK. And the PI-PLC-IP3pathway are not involved in high dose S1P-induced phosphorylaiton of ROCK.8. 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.Conclusion:1. High-dose SIP (10μmol/L) triggered the activation of RhoA and the phosphorylaiton of ROCK, as well as the re-distribution of phospho-ROCK. Physiological level of S1P has no effects on the RhoA/ROCK signal pathway.2. In this experiment series, it is S1PR3, rather than S1PR2, that initiate the activation of RhoA and the phosphorylaiton of ROCK. The influx of extracellular Ca2+might also invoke the RhoA activation and ROCK phosphorylaiton.3. The S1PRI-PI-PLC-IP3pathway are not involved in high dose SIP-induced RhoA activation and ROCK phosphorylaiton. |
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