Activation of PAR2 by tissue factor induces the release of the PTEN from MAGI proteins and regulates PTEN and Akt activities

Abstract

Tissue factor (TF) signalling has been associated with alterations in Akt activity influencing cellular survival and proliferation. TF is also shown to induce signalling through activation of the protease activated receptor (PAR)2. Seven cell lines were exposed to recombinant-TF (rec-TF), or activated using a PAR2-agonist peptide and the phosphorylation state of PTEN, and the activities of PTEN and Akt measured. Furthermore, by measuring the association of PTEN with MAGI proteins a mechanism for the induction of signalling by TF was proposed. Short term treatment of cells resulted in de-phosphorylation of PTEN, increased lipid-phosphatase activity and reduced Akt kinase activity in most of the cell lines examined. In contrast, continuous exposure to rec-TF up to 14 days, resulted in lower PTEN antigen levels, enhanced Akt activity and increased rate of cell proliferation. To explore the mechanism of activation of PTEN by TF, the association of "membrane-associated guanylate kinase-with inverted configuration" (MAGI)1–3 proteins with PTEN was assessed using the proximity ligation assay and by co-immunoprecipitation. The interaction of PTEN with all three MAGI proteins was transiently reduced following PAR2 activation and explains the changes in PTEN activity. Our data is first to show that PAR2 activation directly, or through exposure of cells to TF releases PTEN from MAGI proteins and is concurrent with increases in PTEN phosphatase activity. However, prolonged exposure to TF results in the reduction in PTEN antigen with concurrent increase in Akt activity which may explain the aberrant cell survival, proliferation and invasion associated with TF during chronic diseases.

Factor VIIa Regulates the Level of Cell-Surface Tissue Factor through Separate but Cooperative Mechanisms

“…The comparison of wild-type MDA-MB-231 with MDA-MB-231 TF-KO cells also indicated some association between fVII/fVIIa and PAR2 in both cells lines, although this was significantly lower in the absence of TF (Figure 2A,B). In order to further examine the functional significance of TF in proliferative signalling, MDA-MB-231 TF-KO cells were supplemented with a range of concentrations of recombinant TF (0-4 U/mL) and cell number were examined after 48 h. In support of the above data, supplementation of the MDA-MB-231 TF-KO cells promoted the proliferation of these cells (Figure 2C), although this was at a reduced rate compared to that established for the wild-type MDA-MB-231 cells [32]. Subsequently, siRNA-mediated knock-down of the fVII/fVIIa in all three cell lines was carried out as described before [15,16] and shown to be effective in limiting the expression of cellular fVII/fVIIa in MDA-MB-231 cells, to approximately 10% of the untreated cells (Figure 3A).…”

“…All procedures were carried out using the Duolink reagents (Sigma Chemical Company Ltd., Poole, UK) and adapted from that previously described in detail [31,32]. All cells (10 3 ), were seeded out into 35 mm-glass based µ-dishes (InVitro Scientific/Cellvis, Sunnyvale, CA, USA) in respective media overnight.…”

“…In addition, the association of caveolin-1 with either TF or fVII/fVIIa was examined using a polyclonal rabbit anti-caveolin-1 antibody (10 µg/mL; GenTex, Irvine, CA, USA) together with a mouse anti-TF antibody (10H10; 5 µg/mL; Bio-Rad) or a mouse anti-fVII antibody (321621; 10 µg/mL), respectively. To ensure specificity, the secondary antibodies (probes) were omitted and the assay carried out to ensure specificity [32]. In addition, throughout the various experiments, some of the primary antibodies were substituted in turn, either with rabbit or mouse IgG isotypes as appropriate (New England Biolabs, Hitchin, UK; 20 µg/mL and 10 µg/mL, respectively).…”

“…In this study, the associations between TF, fVII/fVIIa, PAR2 and caveolin-1 were examined and the contributions of TF and fVIIa to these associations investigated. Proximity ligation assay (PLA) has been used by our group and other investigators [29,31,32] to successfully study TF interactions. This procedure, together with studies on co-localisation with cholesterol-rich micro-domains, enabled the analysis of different sets of cell-surface proteins, in situ.…”

A New Story of the Three Magi: Scaffolding Proteins and lncRNA Suppressors of Cancer

“…One interesting point that was not addressed in this study, concerns the impact of MAGI1 expression on celecoxib-induced PTEN targeting to the plasma membrane. In the breast cancer MDA-MB-231 cell line, it has been reported that stimulation of PAR2 by tissue factor, transiently reduces PTEN interaction with MAGI2, this release being associated with enhanced PTEN activity [ 175 ].…”