This Article Full Text Full Text (PDF) Data Supplement All Versions of this Article: 104/5/589    most recent CIRCRESAHA.108.183905v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Eisenreich, A. Articles by Rauch, U. Search for Related Content PubMed PubMed Citation Articles by Eisenreich, A. Articles by Rauch, U. Related Collections Cell biology/structural biology Cell signalling/signal transduction Coagulation Endothelium/vascular type/nitric oxide Other Vascular biology

(Circulation Research. 2009;104:589.)
© 2009 American Heart Association, Inc.

Molecular Medicine

Cdc2-Like Kinases and DNA Topoisomerase I Regulate Alternative Splicing of Tissue Factor in Human Endothelial Cells

Andreas Eisenreich, Vladimir Y. Bogdanov, Andreas Zakrzewicz, Axel Pries, Silvio Antoniak, Wolfgang Poller, Heinz-Peter Schultheiss, Ursula Rauch

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 York².

Correspondence to Ursula Rauch, MD, Charitè–Universitätsmedizin Berlin, Campus Benjamin Franklin, Centrum für Herz- und Kreislaufmedizin, Berlin, Germany. 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 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-. Human endothelial cells were pretreated with specific inhibitors or small interfering RNAs against Cdc2-like kinases and DNA topoisomerase 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 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-–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.

Key Words: cardiovascular research • endothelial cells • tissue factor • tumor necrosis factor • vascular biology