Abstract 805: Differential Regulation Of Cytokine-induced Alternative Splicing Of The TF Gene By Serine/Arginine Rich Protein Kinases
Background: Higher eukaryotes control gene expression and increase protein diversity by alternative splicing of pre-mRNA. The Cdc2-like kinase (Clk) family, DNA topoisomerase I (DNA topo I) or Akt kinase are involved in splicing control by regulating the phosphorylation state of serine/arginine rich (SR) proteins. We recently showed that alternatively spliced human tissue factor (asHTF), a soluble isoform of tissue factor (TF), the primary initiator of coagulation, is expressed in HUVECs in response to inflammatory cytokines. This study investigated the role of Clks, DNA topo I and the PI3K-Pathway in regulation of TF-splicing in TNF-α induced HUVECs.
Methods: HUVECs were incubated with inhibitors of Clks, DNA-topo I or PI3K and were then stimulated with TNF-α. The SR protein phosphorylation state was determined 2 min post induction. The full length (fl) TF and asHTF mRNA were assessed 60 min post induction by Real-Time PCR. Proteins were measured 5 and 8 hours after stimulation by Western blots and the cell thrombogenicity was analyzed via a chromogenic assay.
Results: TNF-α inceased the mRNA expression of asHTF and flTF in HUVECs. The Clk-inhibitor completely inhibited the TNF-α induced expression of asHTF and reduced flTF by 30 %. Inhibition of DNA topo I increased asHTF expression and reduced the flTF expression. Inhibition of the PI3K/Akt-pathway had no effect on TF mRNA expression. Reduced Clk-inhibition the TF activity by 50 % whereas DNA topo I inhibition significantly decreased the procoagulant TF activity 8 hours post TNF-α induction. The Clk- and DNA-topo I-inhibitors altered the SR-protein phosphorylation pattern post TNF-α-induction. Additionally resulted inhibition of Clks in the generation of a third TF mRNA-splice variant, TF-A.
Conclusion: Selective inhibition of Clks or DNA topo I leads to alterations of SR-protein phosphorylation and affects the differential expression of TF isoforms, thereby modulating the thrombogenicity of HUVECs. The inhibition of Clks contributes to the generation of a third TF splice variant. The inhibition of these kinases gives new insights into the regulation of the TF gene splicing process, which may result in new therapeutic strategies for modulating cellular thrombogenicity.