Summary
We previously reported that high levels of tissue factor (TF) can induce cellular
apoptosis in endothelial cells. In this study, TF-mediated mechanisms of induction
of apoptosis were explored. Endothelial cells were transfected to express wild-type
TF. Additionally, cells were transfected to express Asp253-substituted, or Ala253-substitued
TF to enhance or prevent TF release, respectively. Alternatively, cells were pre-incubated
with TF-rich and TF-poor microvesicles. Cell proliferation, apoptosis and the expression
of cyclin D1, p53, bax and p21 were measured following activation of cells with PAR2-agonist
peptide. Greatest levels of cell proliferation and cyclin D1 expression were observed
in cells expressing wild-type or Asp253-substituted TF. In contrast, increased cellular
apoptosis was observed in cells expressing Ala253-substituted TF, or cells pre-incubated
with TF-rich microvesicles. The level of p53 protein, p53-phosphorylation at ser33,
p53 nuclear localisation and transcriptional activity, but not p53 mRNA, were increased
in cells expressing wild-type and Ala253-substituted TF, or in cells pre-incubated
with TF-rich microvesicles. However, the expression of bax and p21 mRNA, and Bax protein
were only increased in cells pre-incubated with TF-rich microvesicle and in cells
expressing Ala253-substituted TF. Inhibition of the transcriptional activity of p53
using pifithrin-α suppressed the expression of Bax. Finally, siRNA- mediated suppression
of p38α, or inhibition using SB202190 significantly reduced the p53 protein levels,
p53 nuclear localisation and transcriptional activity, suppressed Bax expression and
prevented cellular apoptosis. In conclusion, accumulation of TF within endothelial
cells, or sequestered from the surrounding can induce cellular apoptosis through mechanisms
mediated by p38, and involves the stabilisation of p53.
Keywords
Tissue factor - serine-phosphorylation - p38-MAP kinase - p53 activation - apoptosis
