Published online before print January 22, 2009, doi: 10.1161/CIRCRESAHA.108.183905
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Submitted on July 23, 2008
Revised on January 12, 2009
Accepted on January 14, 2009
Cdc2-Like Kinases and DNA Topoisomerase I Regulate Alternative Splicing of Tissue Factor in Human Endothelial Cells
Andreas Eisenreich ;
From Centrum für Herz- und Kreislaufmedizin (A.E., S.A., W.P., H.-P.S., U.R.) and Institute of Physiology (A.Z., A.P.), Campus Benjamin Franklin, Charitè–Universitätsmedizin Berlin, Germany; and the Department of Medicine, Division of Hematology and Medical Oncology (V.Y.B.), Mount Sinai School of Medicine, New York.
* To whom correspondence should be addressed. E-mail: ursula.rauch{at}charite.de.
Tumor necrosis factor (TNF)-
–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 (asHTF), and the "full-length" isoform (flTF). The regulatory pathways enabling this phenomenon are completely unknown. Cdc2-like kinases (Clks) and DNA topoisomerase I (DNA topo I) regulate alternative splicing via phosphorylation of serine/arginine-rich (SR) proteins. In this study, we examined effects of SR protein kinases on TF splicing following stimulation with TNF-. Human endothelial cells (ECs) were pretreated with specific inhibitors or small interfering RNAs against Clks and DNA topo I before stimulation with TNF-. 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 Clk forms were expressed in human ECs. Selective inhibition of Clks and DNA topo I elicited distinct changes in TF biosynthesis in TNF-–stimulated ECs, which impacted endothelial procoagulant activity. This study is the first to demonstrate that SR protein kinases modulate splicing of TF pre-mRNA in human ECs and, consequently, endothelial procoagulant activity under inflammatory conditions.
Key words: cardiovascular research • endothelial cells • tissue factor • tumor necrosis factor • vascular biology