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Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

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

Authors

Ali M. Ethaeb

Mohammad A. Mohammad

Yahya Madkhali

Anthony Maraveyas

Sophie Featherby

Camille Ettelaie



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.

Journal Article Type Article
Journal Apoptosis
Print ISSN 1360-8185
Electronic ISSN 1573-675X
Publisher Springer (part of Springer Nature)
Peer Reviewed Peer Reviewed
Volume 25
APA6 Citation Ethaeb, A. M., Mohammad, M. A., Madkhali, Y., Maraveyas, A., Featherby, S., Greenman, J., & Ettelaie, C. (in press). Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling. Apoptosis, 25, https://doi.org/10.1007/s10495-019-01576-2
DOI https://doi.org/10.1007/s10495-019-01576-2
Keywords Clinical Biochemistry; Cell Biology; Cancer Research; Pharmacology; Pharmaceutical Science; Biochemistry, medical; Tissue factor; Apoptosis; Src-1; β1-Integrin
Additional Information First Online: 25 October 2019; : ; : The authors do not declare any conflict of interest.

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