Accumulation of tissue factor (TF) within cells leads to cellular apoptosis mediated through p38 and p53 pathways. In this study, the involvement of Src1 in the induction of TF-mediated cell apoptosis, and the mechanisms of Src1 activation were investigated. Human coronary artery endothelial cell (HCAEC) were transfected with plasmids to express the wild-type TF (TFWt-tGFP), or a mutant (Ser253 → Ala) which is incapable of being released from cells (TFAla253-tGFP). The cells were then activated with PAR2-agonist peptide (SLIGKV-NH) and the phosphorylation of Src and Rac, and also the kinase activity of Src were assessed. Transfected cells were also pre-incubated with pp60c Src inhibitor, FAK inhibitor-14, or a blocking anti-β1-integrin antibody prior to activation and the phosphorylation of p38 as well as cellular apoptosis was examined. Finally, cells were co-transfected with the plasmids, together with a Src1-specific siRNA, activated as above and the cellular apoptosis measured. Activation of PAR2 lead to the phosphorylation of Src1 and Rac1 proteins at 60 min regardless of TF expression. Moreover, Src phosphorylation and kinase activity was prolonged up to 100 min in the presence of TF, with a significantly higher magnitude when the non-releasable TFAla253-tGFP was expressed in HCAEC. Inhibition of Src with pp60c, or suppression of Src1 expression in cells, reduced p38 phosphorylation and prevented cellular apoptosis. In contrast, inhibition of FAK had no significant influence on Src kinase activity or cellular apoptosis. Finally, pre-incubation of cells with an inhibitory anti-β1-integrin antibody reduced both Src1 activation and cellular apoptosis. Our data show for the first time that the over-activation of Src1 is a mediator of TF-induced cellular apoptosis in endothelial cells through a mechanism that is dependent on its interaction with β1-integrin.

中文翻译：

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内皮细胞中组织因子的积累通过Src1的过度激活促进细胞凋亡，并涉及β1-整合素信号传导。

细胞内组织因子（TF）的积累导致通过p38和p53途径介导的细胞凋亡。在这项研究中，研究了Src1在诱导TF介导的细胞凋亡中的作用，以及Src1激活的机制。用质粒转染人冠状动脉内皮细胞（HCAEC）以表达野生型TF（TFWt-tGFP）或无法从细胞释放的突变体（Ser253→Ala）（TFAla253-tGFP）。然后将细胞用PAR2-激动剂肽（SLIGKV-NH）激活，将Src和Rac磷酸化，并评估Src的激酶活性。转染的细胞也应在激活前与pp60c Src抑制剂，FAK抑制剂-14或阻断性抗β1-整合素抗体一起预孵育，然后检查p38的磷酸化以及细胞凋亡。最后，将细胞与质粒以及Src1特异性siRNA一起共转染，如上激活并测量细胞凋亡。不管TF表达如何，PAR2的激活都会在60分钟时导致Src1和Rac1蛋白的磷酸化。此外，在TF存在下，Src磷酸化和激酶活性被延长至100分钟，当不可释放的TFAla253-tGFP在HCAEC中表达时，Src的磷酸化和激酶活性显着更高。用pp60c抑制Src或抑制细胞中Src1的表达可减少p38磷酸化并防止细胞凋亡。相反，抑制FAK对Src激酶活性或细胞凋亡无明显影响。最后，将细胞与抑制性抗β1-整合素抗体进行预孵育可降低Src1激活和细胞凋亡。