Effect Of Sphingosine 1-Phosphate Receptors On Monolayer Hyper-permeability Induced By LPS And TNF-α

Sphingosine 1-phosphate (SIP) is a bioactive sophospholipid capable of inducing a wide spectrum of biological responses, including but not limited to cell survival, growth, migration, and contraction. Although SIP 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. Many cells express some of a family of G protein-coupled SIP receptors named S1P1R,2R,3R,4Rand 5R, originally referred to as EDG-1,5,3,6 and 8, respectively. It had been revealed that different cells from different species have different SIP expression profiles. S1P1R,3R and a little S1P2R were the main receptors expressed in endothelial cells. The balances of expression and activation of S1P1R,2R and 3R in endothelial cells help to maintain the physiological functions, especially the barrier function of endothelial cells. For example, the activation of S1P1R could strengthen the barrier integrity of endothelial cells by inducing the Rac signal, while the activation of S1P2R/3R would disrupt the inter-endothelial junctions by evoking the RhoA and ROCK pathway. It had been quite clear about the expression profiles and their roles of SIP receptors played in physiological conditions. But it is still unknown about the the alteration of expression and activation of SIP receptors under pathological conditions. As important inflammatory mediators, both LPS and TNF-a could increase the vascular permeability through destroying the intercellular junctions of endothelia. It is possible that LPS and TNF-a would weaken the endothelial barrier function by affecting the expression and activity of SIP receptors in endothelial cell.In this study, we first explored the SIP receptor expression profiles in ECV 304 and human dermal microvascular endothelial cells (HMVEC) using RT-PCR and western blot. The effects of LPS and TNF-a on SIP receptor expression were also investigated. Then, the relationship between the regulation of S1PRs and endothelial hyper-permeability induced by LPS or TNF-a was studied by blocking selected SIP receptor using a method of detecting the leakage of fluorescence-labeled albumin from endothelial monolayer in Transwell.Results1. Expression of S1P receptors in cultured ECV 304 and HMVECThe RT-PCR results showed that the mRNA of S1P1R, S1P2R and S1P3R were all appeared in both ECV 304 and HMVEC under quiescent condition. But the western blot results revealed that there was only S1P2R protein expressed in ECV 304. But in HMVECs, S1P1R and S1P3R were strongly expressed, while S1P2R was slightly expressed in protein level.2. The effects of LPS stimulation on S1P receptors' expression in ECV 304 and HMVECThe results demonstrated that the stimulation of LPS did not alter the mRNA and protein expression of S1P1R and S1P3R in both ECV 304 and HMVECs. The administration of 500 ng/ml LPS could strongly enhance the mRNA and protein expression of S1P2R in both ECV 304 and HMVECs in 12 h and this up-regulation of S1P2R sustained for at least 24 h. 3. The effects of TNF-a intervention on SIP receptors'expression in ECV 304 and HMVECThe data in this study showed that 50 or 100 ng/ml TNF-αapplication enhanced the mRNA expression of S1P1R in EVC 304 with a pattern of time-and dose-dependency in 12-24 h, while the enhancement of S1P1R mRNA expression in HMVECs only happened when TNF-αconcentration reached 100 ng/ml at 24 h later. The administration of 50 ng/ml TNF-αcould significantly enhance the mRNA and protein expression of S1P2R in both ECV 304 and HMVECs in 12 h and these up-regulations of S1P2R were dose-and time-dependent. The addition of TNF-a did not change the mRNA and protein expression level of S1P3R in both ECV 304 and HMVECs.4. The effects of SIP receptor blocking in LPS or TNF-a induced endothelial monolayer hyper-permeability responsesAccording to the above mentioned results, a specific inhibitor of S1P2R, JTE-013 was chosen to block the binding of SIP with S1P2R induced by LPS or TNF-a. The stimulation of LPS or TNF-αsignificantly increased the endothelial monolayer permeability, while the application of JTE-013 could dose-dependently attenuate these hyper-permeability responses in both ECV 304 and HMVECs.Conclusion:1. Different endothelial cells from varied tissues have distinct SIP receptor expression profiles. While both ECV 304 and HMVECs express mRNA of S1P1R, S1P2R, and S1P3R, there is only S1P2R protein presented in ECV 302, and S1P1R and S1P3R are strongly expressed in HMVECs.2. The intervention of inflammatory mediators, such as LPS and TNF-α, could alter the SIP receptor expression profiles. While LPS enhances the presentation of S1P2R in endothelial cells, TNF-αcould up-regulate the expression of both S1P1R and S1P2R.3. Since S1P2R is known as a permeability increasing receptor, the blockage of S1P2R could attenuate the hyper-permeability responses induced by LPS and TNF-α. These results indicate that the up-regulation of S1P2R expression might be involved in these inflammatory mediator evoked endothelial barrier dysfunction.