An investigation of the regulatory mechanisms of cellular homeostasis and haemostasis initiated by tissue factor

Mohammad, Mohammad Abdullah

Abstract

The signalling arising from tissue factor (TF) has been associated with cellular homeostasis as well as haemostasis and, is particularly pertinent in carcinogenesis and cancer progression. The formation of the TF-FVIIa complex is known to activate protease activated receptor 2 (PAR2) and initiates multiple signalling pathways which can determine the fate of the cell. Among these mechanisms PI3K-Akt is known as a major promoter of cell survival and proliferation. The regulation of Akt is mediated by PTEN, a tumour suppressor that negatively regulates the PI3K-Akt pathway resulting in the loss of downstream proliferative and pro-survival signalling. In this study, seven tumour cell lines from different tissues were activated with PAR 2-activating peptide (SLIGKV), or incubated with a range of concentrations of rec-TF (65- 1300 pg/ml). The outcome on the phosphorylation state and the lipid-phosphatase activity of PTEN, and also Akt activity was assessed. Incubation of cells with rec-TF (65 pg/ml) resulted in de-phosphorylation of PTEN in all the cell lines tested which was concurrent with increased PTEN activity and reduced Akt activity. Moreover, activation of PAR2 reduced PTEN phosphorylation and Akt activity in the majority, but not all of the cell lines tested. However, prolonged exposure of cells to rec-TF over 5 days, resulted in decreased PTEN antigen levels together with enhanced Akt activity and increased cell proliferation in the cell lines tested. To explore the mechanism of regulation of PTEN by PAR2, the association of membrane- associated guanylate kinase-with inverted configuration (MAGI)1-3 proteins with PTEN was assessed by the proximity ligation assay (PLA) and co-IP. The interaction of PTEN with all three MAGI proteins was reduced following PAR2 activation and explains the alterations in PTEN activity and stability. The structure of MAGI proteins contains a guanylate kinase-like (GK) domain, two WW domains and six PDZ domains. This structure allows MAGI to restrain proteins at the cell junction, regulating the activity of the proteins and stabilising cell-cell contacts. In the second part of the study, analysis of a motif within the cytoplasmic domain of TF (ENSPL) indicated the possible interaction with MAGI proteins. PLA and co-IP studies showed that TF may be restrained at the tight junctions through interaction with MAGI-1. Interestingly the activation of PAR2 transiently dissociated TF from MAGI-1. By expressing the individual PDZ domains as tagged-hybrid proteins, the first PDZ domain was identified to be capable of binding TF and competing with MAGI-1. The expression of this hybrid protein also resulted in augmented TF activity, similar to that following PAR2 activation. Analysis of the interactions of the N-terminal region of MAGI-1 with or without PDZ1 further confirmed the role of PDZ1 in the constrainment of TF and regulation of its procoagulant activity. In conclusion, this study has demonstrated that PAR2 activation promotes increased PTEN activity in the short term, allowing for the elimination of severely injured cells. However, prolonged exposure TF reduces the cellular PTEN antigen levels allowing aberrant cell survival and proliferation, through increased Akt activity. This study also demonstrated the interaction of TF with the PDZ1 domain of MAGI-1, suggesting a mechanism for restraining cell-surface TF from contact with the surrounding fluid, and may explain the regulation of both coagulation and signalling mechanisms.

Citation

Mohammad, M. A. (2020). An investigation of the regulatory mechanisms of cellular homeostasis and haemostasis initiated by tissue factor. (Thesis). University of Hull. Retrieved from https://hull-repository.worktribe.com/output/4222998

• Thesis Type: Thesis
• Deposit Date: Mar 26, 2021
• Publicly Available Date: Feb 23, 2023
• Keywords: Biomedical sciences
• Public URL: https://hull-repository.worktribe.com/output/4222998
• Additional Information: Department of Biomedical Sciences, The University of Hull
• Award Date: Nov 1, 2020