title: Role of the phosphatidylinositol 3-kinase/protein kinase B pathway in regulating alternative splicing of tissue factor mRNA in human endothelial cells.
abstract: 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.
authors: Charitè-Universitätsmedizin Berlin, Campus Benjamin Franklin, Centrum für Herz- und Kreislaufmedizin, Berlin, Germany. andreas.eisenreich@charite.de
authors: Andreas
authors: A
authors: Eisenreich
authors: Ronny
authors: R
authors: Malz
authors: Wojciech
authors: W
authors: Pepke
authors: Yunus
authors: Y
authors: Ayral
authors: Wolfgang
authors: W
authors: Poller
authors: Heinz-Peter
authors: HP
authors: Schultheiss
authors: Ursula
authors: U
authors: Rauch
mesh_terms: 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
chemical_list: 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