Accumulation of tissue factor in endothelial cells induces cell apoptosis, mediated through p38 and p53 activation

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

^* AME and CE contributed equally to this study.

^† Current address: Department of Cardiovascular Sciences, University of Leicester, Clinical Sciences Wing, Glenfield General Hospital, Leicester, LE3 9QP, UK.

• References

• 1 Greeno EW. et al. Apoptosis is associated with increased cell surface tissue factor procoagulant activity. Lab Invest 1996; 75: 281-289.
• 2 Bombeli T. et al. Apoptotic vascular endothelial cells become procoagulant. Blood 1997; 89: 2429-2442.
• 3 Collier ME, Ettelaie C. Regulation of the incorporation of tissue factor into microparticles by serine phosphorylation of the cytoplasmic domain of tissue factor. J Biol Chem 2011; 286: 11977-11984.
• 4 Macey MG. et al. Microparticle formation after exposure of blood to activated endothelium under flow. Cytometry A 2010; 77: 761-768.
• 5 Morel O. et al. Endothelial cell activation contributes to the release of procoagulant microparticles during acute cardiac allograft rejection. J Heart Lung Transplant 2008; 27: 38-45.
• 6 Leroyer AS. et al. Cellular origins and thrombogenic activity of microparticles isolated from human atherosclerotic plaques. J Am Coll Cardiol 2007; 49: 772-777.
• 7 Pradier A, Ettelaie C. The influence of exogenous tissue factor on the regulators of proliferation and apoptosis in endothelial cells. J Vasc Res 2008; 45: 19-32.
• 8 Peng W. et al. The effects of small interfering RNA-targeting tissue factor on an in vitro model of neovascularisation. Mol Vis 2013; 19: 1296-1303.
• 9 Collier ME, Ettelaie C. Induction of endothelial cell proliferation by recombinant and microparticle-tissue factor involves beta1-integrin and extracellular signal regulated kinase activation. Arterioscler Thromb Vasc Biol 2010; 30: 1810-1817.
• 10 Dorfleutner A. et al. Cross-talk of integrin 3 1 and tissue factor in cell migration. Mol Biol Cell 2004; 15: 4416-4425.
• 11 Versteeg HH. et al. Inhibition of tissue factor signaling suppresses tumor growth. Blood 2008; 111: 190-199.
• 12 Collier ME. et al. Influence of exogenous tissue factor on estrogen receptorexpression in breast cancer cells: involvement of 1-integrin, PAR2, and mitogen-activated protein kinase activation. Mol Cancer Res 2008; 06: 1807-1818.
• 13 van den Berg YW. et al. Alternatively spliced tissue factor induces angiogenesis through ligation. Proc Natl Acad Sci USA 2009; 106: 19497-19502.
• 14 Kocatürk B. et al. Alternatively spliced tissue factor promotes breast cancer growth in a 1 integrin-dependent manner. Proc Natl Acad Sci USA 2013; 110: 11517-11522.
• 15 Ettelaie C. et al. p38 phosphorylates serine 258 within the cytoplasmic domain of tissue factor and prevents its incorporation into cell-derived microparticles. Biochim Biophys Acta 2013; 1833: 613-621.
• 16 Nawroth PP, Stern DM. Modulation of endothelial cell hemostatic properties by tumor necrosis factor. J Exp Med 1986; 163: 740-745.
• 17 Noguchi M. et al. Correlation between antigenic and functional expression of tissue factor on the surface of cultured human endothelial cells following stimulation by lipopolysaccharide endotoxin. Thromb Res 1989; 55: 87-97.
• 18 Clauss M. et al. Vascular permeability factor: a tumor-derived polypeptide that induces endothelial cell and monocyte procoagulant activity, and promotes monocyte migration. J Exp Med 1990; 172: 1535-1545.
• 19 Østerud B, Bjorklid E. Tissue factor in blood cells and endothelial cells. Front Biosci (Elite E) 2012; 04: 289-299.
• 20 Yu J. et al. Contribution of host-derived tissue factor to tumor neovascularisation. Arterioscler Thromb Vasc Biol 2008; 28: 1975-1981.
• 21 Collier MEW. et al. Microparticle-associated tissue factor is recycled by endothelial cells resulting in enhanced surface TF activity. Thromb Haemost 2013; 111: 647-655.
• 22 Kawamoto T. et al. Tumor-derived microvesicles induce proangiogenic pheno-type in endothelial cells via endocytosis. PLoS One 2012; 07: e34045.
• 23 Dasgupta SK. et al. Developmental endothelial locus-1 (Del-1) mediates clearance of platelet microparticles by the endothelium. Circulation 2012; 125: 1664-1672.
• 24 Hansen CB. et al. Tissue factor-mediated endocytosis, recycling, and degradation of factor VIIa by a clathrin-independent mechanism not requiring the cytoplasmic domain of tissue factor. Blood 2001; 97: 1712-1720.
• 25 Jesel L. et al. Microparticles in atrial fibrillation: a link between cell activation or apoptosis, tissue remodelling and thrombogenicity. Int J Cardiol 2013; 168: 660-669.
• 26 Morel O. et al. Increased levels of procoagulant tissue factor-bearing microparticles within the occluded coronary artery of patients with ST-segment elevation myocardial infarction: role of endothelial damage and leukocyte activation. Atherosclerosis. 2009; 04: 636-641.
• 27 Martin S. et al. Shed membrane particles from T lymphocytes impair endothelial function and regulate endothelial protein expression. Circulation. 2004; 109: 1653-1659.
• 28 Jung C. et al. Circulating endothelial and platelet derived microparticles reflect the size of myocardium at risk in patients with ST-elevation myocardial infarction. Atherosclerosis 2012; 221: 226-231.
• 29 Chironi GN. et al. Endothelial microparticles in diseases. Cell Tissue Res 2009; 335: 143-151.
• 30 Houston P. et al. Fluid shear stress induction of the tissue factor promoter in vitro and in vivo is mediated by Egr-1. Arterioscler Thromb Vasc Biol 1999; 19: 281-289.
• 31 Ettelaie C. et al. Characterisation of physical properties of tissue factor-containing microvesicles and a comparison of ultracentrifuge-based recovery procedures. J Extracell Vesicles. 2014; Aug 13: 3 doi: 10.3402/jev.v3.23592.
• 32 Ray B. et al. Plasma tissue factor is a predictor for restenosis after femoropopliteal angioplasty. Br J Surg 2007; 94: 1092-1095.
• 33 Nielsen VG. et al. Qualitative thrombelastographic detection of tissue factor in human plasma. Anesth Analg 2007; 104: 59-64.
• 34 Ettelaie C. et al. A. Enhanced binding of tissue factor-microparticles to collagen-IV and fibronectin leads to increased tissue factor activity in vitro. Thromb Hae-most 2013; 109: 61-71.
• 35 Bonnekoh B. et al. Colorimetric growth assay for epidermal cell cultures by their crystal violet binding capacity. Arch Dermatol Res 1989; 281: 487-490.
• 36 Ettelaie C. et al. Differential functions of tissue factor in the trans-activation of cellular signalling pathways. Atherosclerosis 2007; 194: 88-101.
• 37 Komarov PG. et al. A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy. Science 1999; 285: 1733-1737.
• 38 Essmann F. et al. Apoptosis resistance of MCF-7 breast carcinoma cells to ionizing radiation is independent of p53 and cell cycle control but caused by the lack of caspase-3 and a caffeine-inhibitable event. Cancer Res 2004; 64: 7065-7072.
• 39 Wang S. et al. Doxorubicin induces apoptosis in normal and tumor cells via distinctly different mechanisms. intermediacy of H(2)O(2)- and p53-dependent pathways. J Biol Chem. 2004; 279: 25535-25543.
• 40 Livak KG, Schmittgen TD. Analysis of relative gene Expression Data Using Real-Time Quantitative PCR and the 2-ΔΔ^CT. Method Methods 2001; 25: 402-408.
• 41 Albrektsen T. et al. Transcriptional program induced by factor VIIa-tissue factor, PAR1 and PAR2 in MDA-MB-231 cells. J Thromb Haemost 2007; 05: 1588-1597.
• 42 Suen JY. et al. Profiling gene expression induced by protease-activated receptor 2 (PAR2) activation in human kidney cells. PLoS One. 2010; 05: e13809.
• 43 Banfi C. et al. Tissue factor induction by protease-activated receptor 1 requires intact caveolin-enriched membrane microdomains in human endothelial cells. J Thromb Haemost 2007; 05: 2437-2444.
• 44 Frentzou GA. et al. Differential induction of cellular proliferation, hypertrophy and apoptosis in H9c2 cardiomyocytes by exogenous tissue factor. Mol Cell Biochem 2010; 345: 119-130.
• 45 Aharon A. et al. Monocyte-derived microparticle and exosomes induce procoagulant and apoptotic effects on endothelial cells. Thromb Haemost 2008; 100: 878-885.
• 46 Bulavin DV. et al. Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation. EMBO J 1999; 18: 6845-6854.
• 47 Sanchez-Prieto R. et al. A role for the p38 mitogen-acitvated protein kinase pathway in the transcriptional activation of p53 on genotoxic stress by chemo-therapeutic agents. Cancer Res 2000; 60: 2464-2472.
• 48 Kishi H. et al. Osmotic shock induces G1 arrest through p53 phosphorylation at Ser33 by activated p38MAPK without phosphorylation at Ser15 and Ser20. J Biol Chem 2001; 276: 39115-39122.
• 49 El-Deiry WS. et al. WAF1, a potential mediator of p53 tumor suppression. Cell 1993; 75: 817-25.
• 50 Levine AJ. p53, the cellular gatekeeper for growth and division. Cell 1997; 88: 323-331.
• 51 Gottlieb TM, Oren M. p53 in growth control and neoplasia. Biochim Biophys Acta 1996; 1287: 77-102.
• 52 Yoshida K. et al. Protein kinase C delta regulates Ser46 phosphorylation of p53 tumor suppressor in the apoptotic response to DNA damage. J Biol Chem 2006; 281: 5734-5740.
• 53 Saito S. et al. Phosphorylation site interdependence of human p53 post-translational modifications in response to stress. J Biol Chem 2003; 278: 37536-37544.
• 54 Kurihara A. et al. Ser46 phosphorylation of p53 is not always sufficient to induce apoptosis: multiple mechanisms of regulation of p53-dependent apoptosis. Genes Cells 2007; 12: 853-861.
• 55 Bulavin DV. et al. Amplification of PPM1D in human tumors abrogates p53 tumor-suppressor activity. Nat Genet 2002; 31: 210-215.
• 56 Takekawa M. et al. p53-inducible wip1 phosphatase mediates a negative feedback regulation of p38 MAPK-p53 signaling in response to UV radiation. EMBO J 2000; 19: 6517-6526.
• 57 Cohen M. et al. Trophoblastic p53 is stabilised by a cis-trans isomerisation necessary for the formation of high molecular weight complexes involving the N-terminus of p53. Biochimie 2008; 90: 855-862.
• 58 Zacchi P. et al. The prolyl isomerase Pin1 reveals a mechanism to control p53 functions after genotoxic insults. Nature 2002; 419: 853-857.
• 59 Zheng H. et al. The prolyl isomerase Pin1 is a regulator of p53 in genotoxic response. Nature 2002; 419: 849-853.