PI3K/AKT/GSK-3(HUVEC)ICAM-1mRNA PI3K/AKT/GSK-3 signaling pathways regulate ICAM-1 expression at the post transcriptional level

Objective To explore the effect and the mechanism of PI3K/AKT/GSK-3β signaling pathways on the expression of intercellular adhesion molecule-1 (ICAM-1) induced by lipopolysaccharides (LPS) in h uman umbilical vein endothelial cells (HUVECs). Methods 1. HUVECs were treated with LPS(10 μg/ml) for 0 , 6,12 and 24hours , 0, 4, 8 and 12hours, and 0, 30, 60 and 120minutes, the protein and mRNA levels of ICAM-1 were measured by western blot and real-time quantitative PCR ( qPCR) respectively, the expression of PI3K, phospho-PI3K, AKT, phospho-AKT, GSK-3β and pho spho-GSK-3β were measured by western blot; 2. HUVECs were transfected by PI3K, AKT, GSK-3 β and control siRNA respectively, the t ransfection concentration gradient was 40, 80 and 100nM, the expression of PI3K, AKT and GSK-3β were measured by western blot; 3. HUVECs were transfected by PI3K, AKT, GSK-3β and control siRNA respectively, then stimulated with LPS for 30min, 60min, 8hours and 12hours, the expression of phospho-AKT, phospho-GSK-3β and ICAM-1 were measured by western blot, and mRNA levels of ICAM-1 was measured by qPCR; 4. The transfection method is same as above, the transfected cells were stimulated with LPS for 8 hours, then incubated with ActD (5μg/ml ) for 0, 1, 2, 3 and 4hours, mRNA levels of ICAM-1 was measured by qPCR , then calculated t he half-life of mRNAs. Results 1. HUVEC s could be induced the expression of ICAM-1 by LPS, and t he expression of 12h group reached at the peak value, and the difference was statistically significant compared with that of 6h and 24h group (P<0.01, P<0.05); the mRNA transcriptions of ICAM-1 induced by LPS in HUVECs were enhanced, and the expression of 8h group reached at the peak value, and the difference was statistically significant compared with that of 4h and 12h group (P<0.01，P<0.05); the phosphorylation of PI3K, AKT and GSK-3β could be induced by LPS in HUVECs, and the phosphorylation of them were highest at 30min, 60min and 60min respectively; 2. The optimal transfection concentration of PI3K, AKT and GSK-3β siRNA transfect ed group s were 100nM; 3. The PI3K transfected group decreased the expression of p-AKT, p-GSK-3β in HUVECs (P<0.01), the AKT transfected group decreased the expression of p-GSK-3β in HUVECs (P<0.01), PI3K and AKT transfected groups significantly enhanced the protein and mRNA levels of ICAM-1(P<0.05，P<0.01；P<0.01，P<0.01), but the GSK-3β transfected group significantly decreased the protein and mRNA levels of ICAM-1 (P<0.01); 4. The silent expression of PI3K, AKT and GSK-3β significantly reduced the mRNA half-life of ICAM-1 (P<0.01). Conclusion 1. LPS could induce the expression of ICAM-1 in HUVECs; 2. LPS negatively regulates the expression of ICAM-1 by activating the PI3K/AKT/GSK-3β signaling pathway in HUVECs; 3. PI3K , AKT and GSK-3β could regulate the mRNA stability of ICAM-1 at the post-transcriptional level .