Role of the Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway in Regulating Alternative Splicing of Tissue Factor mRNA in Human Endothelial Cells

Background: Tissue factor (TF) is the primary initiator of blood coagulation. In response to tumor necrosis factor (TNF)-alpha human umbilical vein endothelial cells (HUVECs) express 2 TF isoforms: a soluble alternatively spliced isoform (asHTF) and membrane-bound "full length" (fl)TF. How the differential TF isoform expression is regulated is still unknown. This study compared the impact of PI3K/Akt pathway inhibition on alternative splicing of TF in HUVECs, to the influence of transcriptional regulation by inhibiting nuclear factor kappa B (NF kappa B). Methods and Results: The mRNA expression of TF isoforms was assessed by real-time PCR, the thrombogenic activity was measured by a chromogenic TF activity assay and the phosphorylation state of serine/arginine-rich (SR) proteins was analyzed by western blotting. Transfection of HUVECs was done 72h before the inhibition experiments were performed. PI3K/Akt pathway inhibition reduced the mRNA expression of asHTF but not flTF. Inhibition of NF kappa B reduced the expression of both isoforms. Moreover, the PI3K/Akt pathway inhibition, but not that of NF kappa B, modified the phosphorylation of the SR proteins SRp75, SRp55 and SF2/ASF. Additionally, overexpression of SF2/ASF and SRp75 influenced the differential TF-isoform expression in HUVECs. Conclusions: The PI3K/Akt pathway modulates alternative splicing of TF in HUVECs, distinct from transcriptional regulation. (Circ J 2009; 73: 1746-1752)