Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves \u03b21-integrin signalling

Ali M Ethaeb,Mohammad A Mohammad,Camille Ettelaie,Yahya Madkhali,Sophie Featherby,Anthony Maraveyas,John Greenman

doi:10.1007/s10495-019-01576-2

Ali M Ethaeb, Mohammad A Mohammad + Show 5 more

Open Access

https://doi.org/10.1007/s10495-019-01576-2

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Abstract

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.

Highlights

The accumulation of tissue factor (TF) within the endothelial cells has been reported to contribute to the progression of chronic pathological disorders including cardiovascular disease [1,2,3,4] and cancer [4,5,6,7]
The activation of HDBEC induced an increase in Src phosphorylation which was comparable to that obtained with Human coronary artery endothelial cell (HCAEC), and is consistent with previous studies regarding the responses of endothelial cells [54]
We used a model for the accumulation of TF in endothelial cells to examine the mechanism of TF-induced cell apoptosis [15, 16]

Summary

Introduction

The accumulation of TF within the endothelial cells has been reported to contribute to the progression of chronic pathological disorders including cardiovascular disease [1,2,3,4] and cancer [4,5,6,7]. This occurs in addition to the procoagulant property of TF and is associated with the ability to regulate cellular processes such as migration and proliferation [8,9,10,11,12,13,14]. The interaction of TF with β1-integrin has recently been suggested to stimulate pro-angiogenic factors and to activate various signalling molecules including focal adhesion kinase (FAK) and p38 [10, 22,23,24]

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