Cdc2-like kinases and DNA topoisomerase I regulate alternative splicing of tissue factor in human endothelial cells.
Tumor necrosis factor (TNF)-alpha-stimulated human umbilical vein endothelial cells express 2 naturally occurring forms of tissue factor (TF), the primary initiator of blood coagulation: the soluble alternatively spliced isoform and the full-length TF isoform. The regulatory pathways enabling this phenomenon are completely unknown. Cdc2-like kinases and DNA topoisomerase I regulate alternative splicing via phosphorylation of serine/arginine-rich proteins. In this study, we examined effects of serine/arginine-rich protein kinases on TF splicing following stimulation with TNF-alpha. Human endothelial cells were pretreated with specific inhibitors or small interfering RNAs against Cdc2-like kinases and DNA topoisomerase I before stimulation with TNF-alpha. TF levels were determined by semiquantitative RT-PCR, real-time PCR, and Western blotting. Cellular procoagulant activity was analyzed in a chromogenic TF activity assay. All 4 known Cdc2-like kinases forms were expressed in human endothelial cells. Selective inhibition of Cdc2-like kinases and DNA topoisomerase I elicited distinct changes in TF biosynthesis in TNF-alpha-stimulated endothelial cells, which impacted endothelial procoagulant activity. This study is the first to demonstrate that serine/arginine-rich protein kinases modulate splicing of TF pre-mRNA in human endothelial cells and, consequently, endothelial procoagulant activity under inflammatory conditions.
Eisenreich
Andreas
A
Charitè-Universitätsmedizin Berlin, Campus Benjamin Franklin, Centrum für Herz- und Kreislaufmedizin, Berlin, Germany.
Bogdanov
Vladimir Y
VY
Zakrzewicz
Andreas
A
Pries
Axel
A
Antoniak
Silvio
S
Poller
Wolfgang
W
Schultheiss
Heinz-Peter
HP
Rauch
Ursula
U
D017398:Alternative Splicing; D001777:Blood Coagulation / Q000235:genetics; D002478:Cells, Cultured; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D015951:Factor Xa / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012322:RNA Precursors / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D009687:Nuclear Proteins; D047428:Protein Kinase Inhibitors; D012322:RNA Precursors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; C081934:SRSF4 protein, human; D014409:Tumor Necrosis Factor-alpha; D000068103:Serine-Arginine Splicing Factors; D013925:Thromboplastin; C066780:Clk dual-specificity kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D015951:Factor Xa; D004264:DNA Topoisomerases, Type I