Molecular competition between plasminogen activator inhibitors type -1 and -2 for urokinase: Implications for cellular proteolysis and adhesion in cancer
Introduction
The urokinase plasminogen activation (uPA) system directly and indirectly controls both proteolytic and non-proteolytic cellular functions, both of which are involved in cell adhesion and migration processes [1], [2], [3]. Control of cell adhesion is crucial for many physiological processes that involve cell migration and dysregulation can lead to pathological outcomes including increased cancer invasion and metastasis – the main cause of death in cancer patients [4].
From a proteolytic point of view, cell surface receptor (uPAR)-bound uPA converts precursor plasminogen into the broad spectrum serine protease plasmin which degrades extracellular matrix (ECM) and activates ECM-associated growth factors [3], [5]. Cell associated plasmin is protected from inhibition by α2-antiplasmin, thus a potentially key regulatory mechanism of cellular invasiveness in vivo is via inhibition of uPA activity by either plasminogen activator inhibitor type -1 or -2 (PAI-1/SERPINE1 and PAI-2/SERPINB2, respectively) [3], [6], as both serpins are very efficient inhibitors of both solution phase and receptor-bound uPA with second-order rate constants of 107–106 and 106–105 M−1 s−1, respectively [7]. Following uPA inhibition, which is executed via a well characterised serpin mechanism resulting in covalent protease:serpin complexes, uPAR/uPA–PAI associate with endocytic receptors of the low-density lipoprotein receptor (LDLR) family resulting in clearance from the cell surface and subsequent uPAR recycling [3], [8], [9], [10].
Despite this shared serpin function, high tumour levels of PAI-1 promote tumour progression, whereas high levels of PAI-2 appear to decrease tumour growth and metastasis [11]. This divergence in function may be related to the complex array of molecular interactions between PAI-1, uPA/uPAR and various ligands and co-receptors, which are not apparent with PAI-2, and that ultimately modulate cell proliferation, adhesion and migration. For example, uPAR is a well characterised adhesion receptor with affinity for the ECM protein vitronectin (Vn) as well as for signalling molecules such as integrins [12], [13], [14]. The binding of uPA to uPAR appears to result in intra- and intermolecular reorganisation which enhances the affinity or avidity of uPAR for these ligands/co-receptors, with subsequent effects on cellular proliferation, adhesion, chemotaxis and migration by mechanisms independent of proteolysis [12]. The relevance of uPA in these processes appears to depend on cellular uPA/uPAR levels, the nature of integrins present and ECM components available for these interactions [14], [15]. PAI-1 can directly regulate the attachment and detachment of migrating cells to Vn by competing with cell-surface integrins and uPAR for Vn binding sites [16], [17]. This may require uPA binding though conflicting reports in the literature confound this issue [17], [18]. In addition, the endocytosis of uPA–PAI-1 via LDLRs is associated with other cell type- and receptor-specific responses such as enhanced mitogenic cell signalling events which promotes proliferation and/or migration [11].
We have previously suggested that the apparent “protective” effect of elevated PAI-2 levels in cancer is associated with the ability of peri/extracellular PAI-2 to compete with PAI-1 for cell surface uPA inhibition and thus prevent or diminish these PAI-1 induced cancer promoting processes [11]. One such mechanism is related to the presence of an LDLR minimal binding motif that we have shown is not conserved in PAI-2 and is associated with the ability of PAI-1, but not PAI-2, to sustain mitogenic signalling events through LDLR binding [8], [9], [11]. As discussed above PAI-1 also contributes to the pro-invasive cancer cell phenotype by modulating cellular adhesion through interactions with uPA/uPAR and ECM. As PAI-2 has no known co-receptor interactions in the pericellular microenvironment, the function of PAI-2 would be limited to protease inhibition in vivo [11], and in this way competition between the serpins for uPA binding may counteract the pro-invasive effects of PAI-1. Furthermore, as free uPA antigen [19] or uPA activity is still detectable and available for inhibition and/or targeting by exogenous inhibitors such as PAI-2 in metastatic breast tumours with high PAI-1 levels [20], [21], there is precedence for considering that PAI-2 can access and inhibit uPA in the presence of PAI-1. In this study we thus examined the potential interplay between the two serpins on uPA inhibition, adhesion/de-adhesion and migration in the background of varying cellular uPA/uPAR levels.
Section snippets
Proteins, antibodies and reagents
Recombinant Hexa-His-tagged human PAI-2 was expressed and purified from Escherichia coli as previously described [22]. PAI-1 (14-1B, stable mutant; CPAI), PAI-1 inhibitory-inactive mutant (HPAI-T333R), PAI-1 vitronectin null binding mutant (HPAI-Q123 K) were from Molecular Innovations, Inc., KI, USA. The second-order rate constants for PAI-1 14-1B or His-tagged PAI-2 ΔCD-loop against uPA in solution are similar to wild-type PAI-1 (8.8 × 106) and PAI-2 (2.40 × 106 M−1 s−1), respectively [23], [24].
PAI-2 inhibits uPA in the presence of PAI-1
Both PAI-1 and PAI-2 are very fast uPA inhibitors. However, the 10-fold faster inhibition kinetics of PAI-1 suggests that if present together in equimolar concentrations PAI-1 would completely out-compete PAI-2 for uPA binding. On the other hand, given the very quick and irreversible mechanism of uPA:serpin complex formation, once a complex is formed one serpin molecule cannot be replaced by another regardless of rate differences. This suggests that uPA:PAI-2 complex formation in the presence
Discussion
In the present study we show that PAI-2 competes with PAI-1 for uPA inhibition both in solution and at the cell surface. The increased (nearly tripled) efficiency of cell-bound uPA binding by PAI-2 in the presence of PAI-1 on adherent cells compared to cells in suspension was likely due to the presence of Vn in the FCS substrate provided to the adherent cells. Within this ECM substrate Vn is present in the “open” conformation to which PAI-1 binds with high affinity [33], [34], and this would
Conflict of interest statement
None declared.
Acknowledgements
The work was supported by the Cancer Institute NSW, Australia. M.R. was a Cancer Institute NSW Fellow. The authors gratefully acknowledge Dr. David R. Croucher for critical reading of the manuscript.
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Present address: Dept. of Applied Mathematics, RSPE, Australian National University Canberra, 0200 ACT, Australia.