Role of the phosphatidylinositol 3-kinase/protein kinase B pathway in regulating alternative splicing of tissue factor mRNA in human endothelial cells

Circ J. 2009 Sep;73(9):1746-52. doi: 10.1253/circj.cj-99-0225. Epub 2009 Jul 13.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing*
  • Cells, Cultured
  • Chromones / pharmacology
  • Dose-Response Relationship, Drug
  • Endothelial Cells / drug effects
  • Endothelial Cells / enzymology*
  • Humans
  • Morpholines / pharmacology
  • NF-kappa B / antagonists & inhibitors
  • NF-kappa B / metabolism
  • Nitriles / pharmacology
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Phosphatidylinositol 3-Kinases / metabolism*
  • Phosphoinositide-3 Kinase Inhibitors
  • Phosphorylation
  • Protein Kinase Inhibitors / pharmacology
  • Proto-Oncogene Proteins c-akt / antagonists & inhibitors
  • Proto-Oncogene Proteins c-akt / metabolism*
  • RNA, Messenger / metabolism*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • Serine-Arginine Splicing Factors
  • Signal Transduction*
  • Sulfones / pharmacology
  • Thromboplastin / genetics*
  • Time Factors
  • Transfection
  • Tumor Necrosis Factor-alpha / metabolism


  • 3-(4-methylphenylsulfonyl)-2-propenenitrile
  • Chromones
  • Morpholines
  • NF-kappa B
  • Nitriles
  • Nuclear Proteins
  • Phosphoinositide-3 Kinase Inhibitors
  • Protein Kinase Inhibitors
  • RNA, Messenger
  • RNA-Binding Proteins
  • Sulfones
  • Tumor Necrosis Factor-alpha
  • Serine-Arginine Splicing Factors
  • 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
  • Thromboplastin
  • Proto-Oncogene Proteins c-akt