Apoa-â… Induces S1p Release From Endothelial Cells And Signaling Mechanism.

Objective: Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid generated bythe sphingosine kinase1/2(SphK1/2)-catalysed phosphorylation of sphingosine. Redblood cells, platelet and vascular endothelial cells may be the major cellular sources ofS1P in plasma. More than65%of the S1P in blood is associated with the lipoproteinsLDL, VLDL, and HDL, with the majority of lipoprotein-associated S1P (>54%) bound toHDL. Findings from a growing number of studies indicate that S1P is a mediator formany cardiovascular effects of HDL, including antithrombotic, protection of endothelialfunction and inhibit/reverse atherosclerosis. A key enzyme sphingosine kinase (SphK)can phosphorylate its substrate sphingosine to generate S1P, then S1P is released fromcells through different carriers. The nascent HDL created by ATP-binding cassette A1(ABCA1)-mediated efflux of cellular phospholipid (PL) and free (unesterified)cholesterol (FC) to apolipoprotein A-Ⅰ (apoA-Ⅰ) is a rate-limiting step ofcholesterol-efflux. ApoA-Ⅰ was also allowed to bind onto scavenger receptor class Bmember-Ⅰ (SR-B-Ⅰ), which can promote bidirectional unesterified lipids movement. Wewill discuss an apoA-Ⅰ induce S1P release from endothelial cells and whether ABCA1and SR-B-Ⅰ or their cellar signal participated in this process in this experiment.Methods: HUVECs were treated with20μg/ml apoA-Ⅰ for different time (5,10,20,40,80minutes), or incubated with different concentrations of apoA-Ⅰ (5,10,20,40μg/ml) for5min. Transfected with siRNA of SphK, ABCA1and SR-B148hours later, and HUVECswere treated with20μg/ml apoA-Ⅰ for5min. HUVECs was pretreated withN,N-Dimethylsphingosine (NNDS, SphK inhibitor), U0126(ERK1/2inhibitor),glybenclamide (ABCA1inhibitor), BLT-1(SR-B-Ⅰ inhibitor), AG490(JAK2inhibitor),PP2(Src inhibitor) and PTX (G inhibitor) and then20μg/ml apoA-Ⅰ treated for5min. HUVECs was pretreated with glybenclamide, then20μg/ml apoA-Ⅰ treated for10min.Additional S1PR1agonist, SEW2871to treat HUVECs at5minutes. High performanceliquid chromatography (HPLC) detected S1P levels analyzed in cells or medium. Westernblotting (WB) were conducted to determine the expression of SphK, ABCA1and SR-B-Ⅰand the phosphorylation of ERK1/2.Results: Results showed that apoA-Ⅰ induce S1P release from endothelial cells, the peakbetween5to10minutes. S1P release induced by apoA-Ⅰ is strongly reduced when SphKand ERK1/2are inhibited. The inhibitor and siRNA experiments showed that ATPABCA1and SR-B-Ⅰ was required for the phosphorylation of ERK1/2and S1P releaseinduced by apoA-Ⅰ, but JAK2and Src pathway was not involved in that. However, thephosphorylation of ERK1/2induced by S1P only was restrained by S1PR inhibitor, butnot the inhibitor of ABCA1or SR-B-Ⅰ. The content of S1P in cell and medium were notsignificant change induced by apoA-Ⅰ pretreating glybenclamide. SEW2871increasedS1P level markedly in cell, but S1P realese was not augmented during the inhibition ofABCA1.Conclusions:1. ApoA-Ⅰ is able to promote S1P release from endothelial cells.2. S1PR1/3-ERK1/2-SphK1signal pathway indirectly promotes apoA-Ⅰ induced S1Prelease, and increases S1P level in cells.3. Both ABCA1and SR-B-Ⅰ receptor are involved in S1P release induced by apoA-Ⅰ inendothelial cells.4. A presumable result is that S1PR1/3mediated the activation of ERK1/2-SphK1pathway provides a self-positive-feedback for apoA-Ⅰ promoted S1P release.