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 (NFkappaB). 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 72 h before the inhibition experiments were performed. PI3K/Akt pathway inhibition reduced the mRNA expression of asHTF but not flTF. Inhibition of NFkappaB reduced the expression of both isoforms. Moreover, the PI3K/Akt pathway inhibition, but not that of NFkappaB, 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.
BACKGROUND:Tissue factor (TF) is the primary initiator of blood coagulation. In response to tumor necrosis factor (TNF)-alphahuman 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 (NFkappaB). 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 72 h before the inhibition experiments were performed. PI3K/Akt pathway inhibition reduced the mRNA expression of asHTF but not flTF. Inhibition of NFkappaB reduced the expression of both isoforms. Moreover, the PI3K/Akt pathway inhibition, but not that of NFkappaB, 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.
Authors: Richard C Godby; Yascha W Van Den Berg; Ramprasad Srinivasan; Robert Sturm; David Y Hui; Stephen F Konieczny; Bruce J Aronow; Evgeny Ozhegov; Wolfram Ruf; Henri H Versteeg; Vladimir Y Bogdanov Journal: Mol Med Date: 2012-07-18 Impact factor: 6.354
Authors: U Boltzen; A Eisenreich; S Antoniak; A Weithaeuser; H Fechner; W Poller; H P Schultheiss; N Mackman; U Rauch Journal: J Mol Cell Cardiol Date: 2012-01-31 Impact factor: 5.000