A structural basis for differential cell signalling by PAI-1 and PAI-2 in breast cancer cells

Abstract

PAI-1 and PAI-2 (plasminogen-activator inibitor types 1 and 2) are inhibitors of cell surface uPA (urokinase plasminogen activator). However, tumour expression of PAI-1 and PAI-2 correlates with poor compared with good patient prognosis in breast cancer respectively. This biological divergence may be related to additional functional roles of PAI-1. For example, the inhibition of uPA by PAI-1 reveals a cryptic high-affinity site within the PAI-1 moiety for the VLDLr (very-low-density-lipoprotein receptor), which sustains cell signalling events initiated by binding of uPA to its receptor. These interactions and subsequent signalling events promote proliferation of breast cancer cells. Biochemical and structural analyses show that, unlike PAI-1, the PAI-2 moiety of uPA-PAI-2 does not contain a high-affinity-binding site for VLDLr, although uPA-PAI-2 is still efficiently endocytosed via this receptor in breast cancer cells. Furthermore, global protein tyrosine phosphorylation events were not sustained by uPA-PAI-2 and cell proliferation was not affected. We thus propose a structurally based mechanism for these differences between PAI-1 and PAI-2 and suggest that PAI-2 is able to inhibit and clear uPA activity without initiating mitogenic signalling events through VLDLr.

Dependence on Endocytic Receptor Binding via a Minimal Binding Motif Underlies the Differential Prognostic Profiles of SerpinE1 and SerpinB2 in Cancer

“…All of the cell lines were routinely cultured as previously described (21,29). The cells were routinely tested for the absence of mycoplasma contamination and reconfirmed for the presence of VLDLR and absence of LRP1 (MCF-7) or vice versa (PC-3) as well as for the presence of uPAR by immunofluorescence staining as previously described (20,21). Both cell lines also express negligible levels of either Serpin (20,21).…”

“…Additionally, the affinity and mechanism of interaction between uPA-SerpinB2 complex and LDLRs is markedly different from that of uPA-SerpinE1 (20,21,24 1A). Our previous analysis suggested that the decreased affinity of uPASerpinB2 may be due to an incomplete LDLR-binding motif within ␣-helix D of SerpinB2, in which the first hydrophobic/ basic amino acid pair is conserved, whereas the second pair is replaced by serine residues (21) (see Fig.…”

“…Indeed, previous studies using a mutant form of SerpinE1 with an incomplete LDLR-binding motif (R76E) observed decreased LRP1 (low density lipoprotein receptor related protein-1) and VLDLR binding affinity and hence functional outcomes (21,23,25). We hypothesized that SerpinB2 may inhibit and clear cell surface uPA (and hence proteolytic activity) without influencing the promitogenic signaling pathways activated via SerpinE1, thus accounting for the differential prognosis associated with the expression of these serpins in tumors (10,21). Herein, we show that VLDLR and LRP1 binding can be introduced to SerpinB2 by complementation of the LDLR-binding motif within ␣-helix D. This has profound impact on the functionality of SerpinB2.…”

“…Webb et al (23) demonstrated that this promitogenic activity was dependent on the direct, high affinity interaction between the SerpinE1 moiety of the uPASerpinE1 complex and the very low density lipoprotein receptor (VLDLR). We have previously demonstrated that SerpinB2 does not initiate these signaling events following uPA inhibition and VLDLR-mediated endocytosis (21), and SerpinB2 (unlike SerpinE1) does not bind directly to LDLRs (20,21 …”

The Urokinase Plasminogen Activator Receptor Promotes Efferocytosis of Apoptotic Cells

“…In both knockout and transgenic mouse models, overexpression of either uPAR or uPA is associated with enhanced metastasis and poor survival, and efforts aimed at inhibiting the protease activation of uPA are considered promising therapeutic strategies (27). Paradoxically, however, expression of the primary inhibitors of endogenous uPA, the type-1 plasminogen activator inhibitors (28), also correlate with poor prognoses in cancer patients (29,30), suggesting there may be non-proteolysis-mediated uPAR activation events (31). Indeed, uPAR is a saturable receptor for VN, which binds this ligand in an RGDindependent manner via the somatomedin-B domain (14).…”