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 (NFkappaB).


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.
Eisenreich
Andreas
A
Charitè-Universitätsmedizin Berlin, Campus Benjamin Franklin, Centrum für Herz- und Kreislaufmedizin, Berlin, Germany. andreas.eisenreich@charite.de
Malz
Ronny
R
Pepke
Wojciech
W
Ayral
Yunus
Y
Poller
Wolfgang
W
Schultheiss
Heinz-Peter
HP
Rauch
Ursula
U
D017398:Alternative Splicing; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D015398:Signal Transduction; D013450:Sulfones / Q000494:pharmacology; D013925:Thromboplastin / Q000235:genetics*; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D002867:Chromones; D009025:Morpholines; D016328:NF-kappa B; D009570:Nitriles; D009687:Nuclear Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D013450:Sulfones; D013925:Thromboplastin; D014409:Tumor Necrosis Factor-alpha; C434003:3-(4-methylphenylsulfonyl)-2-propenenitrile; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one