Abstract 994: Alternative Splicing of Tissue Factor is Mediated by Clks in Human Endothelial Cells Stimulated with TNF-Alpha
The regulation of alternative splicing provides a powerful mechanism for controlling gene expression. The Clk (Cdc2-like kinase) family has been implicated in splicing control. We recently described alternatively spliced human tissue factor (asHTF), a soluble isoform of tissue factor (TF), the primary initiator of coagulation, to be expressed in endothelial cells in response inflammatory cytokines. AsHTF contributed to the procoagulability and was postulated to be a marker for increased procoagulability. How alternative splicing of TF is regulated is yet unknown. This study examined the effect of TNF-α-induced expression of both TF variants in endothelial cells (HUVEC) and delineates the regulatory impact of Clks on TF splicing. AsHTF and TF mRNA were assessed by real-time PCR, and proteins by Western blot and/or fluorescence microscopy pre and post stimulation with TNF-α (10 ng/mL). In a second step, HUVECs were pre-incubated with TG003 (10μM), an inhibitor of CLks, known to inhibit the phosphorylation of serine/arginine rich proteins. An inhibitor of the NFkappa B pathway (BAY11–7082, 10 μM) was also tested. The procoagulability of cells and supernatant was analysed by a chromogenic assay. We found asHTF mRNA to be 8.7-fold increased 60 min post TNF-α treatment (asHTF/GAPDH ratio 16.76±4.6x10^5 vs. 1.92±0.65x10^5 for controls, p<0.01). Full-length TF was maximally induced 19-fold (TF/GADPH ratio 27.65±10.59x10^3 vs. 1.43±0.56x10^3, p<0.01, respectively). Pre-incubation of HUVECs with the Clk inhibitor reduced the asHTF expression in response to TNF-α to baseline (2.2x10^5±0.81x10^6 for TNF-α with Clk inhibitor vs.16.76±4.6x10^5 for TNF-α without inhibitor, p<0.01). Clk inhibition slightly reduced the full-length TF/GADPH ratio to 13.42±10.59x10^3 compared to 27.65±10.59x10^3 with TNF-α alone (p<0.05). In contrary, pre-incubation of HUVECs with an inhibitior of the NF kappa B pathway completely inhibited the TNF-α induced increase of both TF isoform. In conclusion, the Clk family is involved in the regulation of cytokine-induced alternative splicing of TF. The inhibition of Clks may become a therapeutic tool for reducing procoagulant soluble TF expressed and released from endothelial cells in response to inflammatory cytokines.