Interaction of plasminogen activator inhibitor type-1 (PAI-1) with vitronectin.

The serpin plasminogen activator inhibitor type 1 (PAI-1) plays an important role in physiological processes such as thrombolysis and fibrinolysis, as well as pathophysiological processes such as thrombosis, tumor invasion and metastasis. In addition to inhibiting serine proteases, mainly tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, PAI-1 interacts with different components of the extracellular matrix, i.e. fibrin, heparin (Hep) and vitronectin (Vn). PAI-1 binding to Vn facilitates migration and invasion of tumor cells. The most important determinants of the Vn-binding site of PAI-1 appear to reside between amino acids 110-147, which includes alpha helix E (hE, amino acids 109-118). Ten different PAI-1 variants (mostly harboring modifications in hE) as well as wild-type PAI-1, the previously described PAI-1 mutant Q123K, and another serpin, PAI-2, were recombinantly produced in Escherichia coli containing a His(6) tag and purified by affinity chromatography. As shown in microtiter plate-based binding assays, surface plasmon resonance and thrombin inhibition experiments, all of the newly generated mutants which retained inhibitory activity against uPA still bound to Vn. Mutant A114-118, in which all amino-acids at positions 114-118 of PAI-1 were exchanged for alanine, displayed a reduced affinity to Vn as compared to wild-type PAI-1. Mutants lacking inhibitory activity towards uPA did not bind to Vn. Q123K, which inhibits uPA but does not bind to Vn, served as a control. In contrast to other active PAI-1 mutants, the inhibitory properties of A114-118 towards thrombin as well as uPA were significantly reduced in the presence of Hep. Our results demonstrate that the wild-type sequence of the region around hE in PAI-1 is not a prerequisite for binding to Vn.     

Interaction of plasminogen activator inhibitor type-1 (PAI-1) with vitronectin (Vn): mapping the binding sites on PAI-1 and Vn

The serpin plasminogen activator inhibitor type-1 (PAI-1), as the primary physiological inhibitor of both urokinase-type (uPA) and tissue-type (tPA) plasminogen activator, plays an important role in the regulation of the fibrinolytic system as well as in extracellular remodeling in both physiological and pathophysiological processes. In plasma as well as in the extracellular matrix PAI-1 binds to vitronectin (Vn), an interaction that affects the function of both proteins. As PAl-1/Vn interaction has a significant regulatory function in fibrinolysis, thrombolysis, and cell adhesion in cancer spread, there is a strong interest in defining the binding sites on PAI-1 and Vn as the basis of a rational design of novel drugs that may modulate PAI-1/Vn-mediated effects. In this minireview, we give an overview on the approaches to define the Vn binding site of PAI-1 and vice versa. Although in the case of PAI-1 the region around alpha-helix E and alpha-helix F of PAI-1 has been demonstrated to be important for its interaction with Vn, the precise location of the Vn-binding region has not completely been resolved. The major high-affinity PAI-1 binding region of Vn is localized within the N-terminal somatomedin B (SMB) domain of Vn. There are indications for at least one other low-affinity PAI-1 binding site in the C-terminal region of Vn, which seems to be involved in the formation of larger PAI-1/Vn complexes.     

Abrogation of Plasminogen Activator Inhibitor-1-Vitronectin Interaction Ameliorates Acute Kidney Injury in Murine Endotoxemia

Sepsis-induced acute kidney injury (AKI) contributes to the high mortality and morbidity in patients. Although the pathogenesis of AKI during sepsis is poorly understood, it is well accepted that plasminogen activator inhibitor-1 (PAI-1) and vitronectin (Vn) are involved in AKI. However, the functional cooperation between PAI-1 and Vn in septic AKI has not been completely elucidated. To address this issue, mice were utilized lacking either PAI-1 (PAI-1^−/−) or expressing a PAI-1-mutant (PAI-1^R101A/Q123K) in which the interaction between PAI-1 and Vn is abrogated, while other functions of PAI-1 are retained. It was found that both PAI-1^−/− and PAI-1^R101A/Q123K mice are associated with decreased renal dysfunction, apoptosis, inflammation, and ERK activation as compared to wild-type (WT) mice after LPS challenge. Also, PAI-1^−/− mice showed attenuated fibrin deposition in the kidneys. Furthermore, a lack of PAI-1 or PAI-1-Vn interaction was found to be associated with an increase in activated Protein C (aPC) in plasma. These results demonstrate that PAI-1, through its interaction with Vn, exerts multiple deleterious mechanisms to induce AKI. Therefore, targeting of the PAI-1-Vn interaction in kidney represents an appealing therapeutic strategy for the treatment of septic AKI by not only altering the fibrinolytic capacity but also regulating PC activity.     

Sequences within domain II of the urokinase receptor critical for differential ligand recognition

The receptor for urokinase-type plasminogen activator (uPAR) plays important roles in a number of physiological and pathological processes by virtue of its interactions with urokinase-type plasminogen activator (uPA), vitronectin (Vn), and several other proteins. The uPA binding site spans all three domains (D1 to D3) of uPAR. However, the nature of the Vn binding site within uPAR is still not clear. In this study, we conducted homolog-scanning mutagenesis on uPAR by switching 14 individual segments of 4-8 residues to their counterpart sequences of a uPAR homolog CD59. All 14 mutants were well expressed, reacted with a panel of monoclonal antibodies, and exhibited correct molecular weights. Of these 14 mutants, six mutants were defective in both uPA and Vn binding. Most importantly, we found two unique mutants uPAR(Asn172-Lys175) and uPAR(Glu183-Asn186) within the D2 domain, which displayed differential ligand binding activity: both had high affinity uPA binding, but completely lost Vn binding, indicating that these two sequences constitute a novel Vn binding site. Indeed, two peptides, P1 (153CPGSNGFHNNDTFHFLKC) and P2 (171CNTTKCNEGPILELENLPQ), derived from the sequences of the identified uPA and Vn binding pockets within D2, respectively, behaved like bona fide ligand binding sites: peptide P1 bound uPA but not Vn, whereas peptide P2 bound Vn and inhibited uPAR-mediated cell adhesion, but did not interact with uPA. Altogether, our data demonstrated that uPAR D2 contains two distinct ligand binding sites for uPA and Vn. Such information will help us better understand the complex roles of uPAR in cell adhesion, migration, and tumor metastasis.     

Purified alpha 2-macroglobulin receptor/LDL receptor-related protein binds urokinase.plasminogen activator inhibitor type-1 complex. Evidence that the alpha 2-macroglobulin receptor mediates cellular degradation of urokinase receptor-bound complexes.

Complexes between 125I-labeled urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) bound to purified alpha 2-macroglobulin (alpha 2M) receptor (alpha 2MR)/low density lipoprotein receptor-related protein (LRP). No binding was observed when using uPA. The magnitude of uPA.PAI-1 binding was comparable with that of the alpha 2MR-associated protein (alpha 2MRAP). Binding of uPA.PAI-1 was blocked by natural and recombinant alpha 2MRAP, and about 80% inhibited by complexes between tissue-type plasminogen activator (tPA) and PAI-1, and by a monoclonal anti-PAI-1 antibody. In human monocytes, uPA.PAI-1, like uPA and its amino-terminal fragment, bound to the urokinase receptor (uPAR). Degradation of uPAR-bound 125I-uPA.PAI-1 was 3-4-fold enhanced as compared with uncomplexed uPAR-bound uPA. The inhibitor-enhanced uPA degradation was blocked by r alpha 2MRAP and inhibited by polyclonal anti-alpha 2MR/LRP antibodies. This is taken as evidence for mediation of internalization and degradation of uPAR-bound uPA.PAI-1 by alpha 2MR/LRP.     

Evidence that type 1 plasminogen activator inhibitor binds to the somatomedin B domain of vitronectin.

The interaction between type 1 plasminogen activator inhibitor (PAI-1) and fragments of vitronectin (Vn) was investigated. The PAI-1-binding domain was not destroyed when Vn was cleaved by treatment with either acid or CNBr. Acid-cleaved Vn was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by PAI-1 ligand binding. The smallest fragment (Mr 40,000) that retained PAI-1 binding function was sequenced and shown to contain the NH2 terminus of the molecule. Further cleavage of this fragment by treatment with CNBr generated a Mr 35,000 fragment (Pro52-Asp239) that did not interact with PAI-1, and a Mr 6,000 NH2-terminal fragment (Asp1-Met51) that spanned the somatomedin B domain and contained the RGD (cell binding) sequence. The purified Mr 6,000 fragment competed with immobilized Vn for PAI-1 binding, and formed complexes with activated PAI-1. These complexes could be immunoprecipitated by antibodies to PAI-1. Synthetic peptides containing the RGD sequence had no effect on the binding of this fragment to PAI-1. These results suggest that the cell-binding and PAI-1 binding sequences of Vn occupy distinct regions in the NH2-terminal somatomedin B domain of the molecule.     

The plasminogen activator inhibitor PAI-1 controls in vivo tumor vascularization by interaction with proteases, not vitronectin. Implications for antiangiogenic strategies

The plasminogen (Plg)/plasminogen activator (PA) system plays a key role in cancer progression, presumably via mediating extracellular matrix degradation and tumor cell migration. Consequently, urokinase-type PA (uPA)/plasmin antagonists are currently being developed for suppression of tumor growth and angiogenesis. Paradoxically, however, high levels of PA inhibitor 1 (PAI-1) are predictive of a poor prognosis for survival of patients with cancer. We demonstrated previously that PAI-1 promoted tumor angiogenesis, but by an unresolved mechanism. We anticipated that PAI-1 facilitated endothelial cell migration via its known interaction with vitronectin (VN) and integrins. However, using adenoviral gene transfer of PAI-1 mutants, we observed that PAI-1 promoted tumor angiogenesis, not by interacting with VN, but rather by inhibiting proteolytic activity, suggesting that excessive plasmin proteolysis prevents assembly of tumor vessels. Single deficiency of uPA, tissue-type PA (tPA), uPA receptor, or VN, as well as combined deficiencies of uPA and tPA did not impair tumor angiogenesis, whereas lack of Plg reduced it. Overall, these data indicate that plasmin proteolysis, even though essential, must be tightly controlled during tumor angiogenesis, probably to allow vessel stabilization and maturation. These data provide insights into the clinical paradox whereby PAI-1 promotes tumor progression and warrant against the uncontrolled use of uPA/plasmin antagonists as tumor angiogenesis inhibitors.     

Vimentin exposed on activated platelets and platelet microparticles localizes vitronectin and plasminogen activator inhibitor complexes on their surface.

Type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA), is found in plasma and platelets. PAI-1 circulates in complex with vitronectin (Vn), an interaction that stabilizes PAI-1 in its active conform. In this study, we examined the binding of platelet-derived Vn and PAI-1 to the surface of isolated platelets. Flow cytometry indicate that, like P-selectin, PAI-1, and Vn are found on the surface of thrombin- or calcium ionophore-activated platelets and platelet microparticles. The binding of PAI-1 to the activated platelet surface is Vn-dependent. Vn mediates the binding of PAI-1 to platelet surfaces through a high affinity (K(d) of 80 nm) binding interaction with the NH(2) terminus of vimentin, and this Vn-binding domain is expressed on the surface of activated platelets and platelet microparticles. Immunological and functional assays indicate that only -5% of the total PAI-1 in platelet releasates is functionally active, and it co-precipitates with Vn, and the vimentin-enriched cytoskeleton fraction of activated platelet debris. The remaining platelet PAI-1 is inactive, and does not associate with the cytoskeletal debris of activated platelets. Confocal microscopic analysis of platelet-rich plasma clots confirm the co-localization of PAI-1 with Vn and vimentin on the surface of activated platelets, and platelet microparticles. These findings suggest that platelet vimentin may regulate fibrinolysis in plasma and thrombi by binding platelet-derived Vn.PAI-1 complexes.     

Transforming growth factor-beta1 produced by ovarian cancer cell line HRA stimulates attachment and invasion through an up-regulation of plasminogen activator inhibitor type-1 in human peritoneal mesothelial cells

The processes of ovarian cancer dissemination are characterized by altered local proteolysis, cellular proliferation, cell attachment, and invasion, suggesting that the urokinase-type plasminogen activator (uPA) and its specific inhibitor (plasminogen activator inhibitor type-1 (PAI-1)) could be involved in the pathogenesis of peritoneal dissemination. We showed previously that expression of uPA and PAI-1 in the human ovarian cancer cell line HRA can be down-regulated by exogenous bikunin (bik), a Kunitz-type protease inhibitor, via suppression of transforming growth factor-beta1 (TGF-beta1) up-regulation and that overexpression of the bik gene can specifically suppress the in vivo growth and peritoneal dissemination of HRA cells in an animal model. We hypothesize that the plasminogen activator system in mesothelial cells can be modulated by HRA cells. To test this hypothesis, we used complementary techniques in mesothelial cells to determine whether uPA and PAI-1 expression are altered by exposure to culture media conditioned by HRA cells. Here we show the following: 1) that expression of PAI-1, but not uPA, was markedly induced by culture media conditioned by wild-type HRA cells but not by bik transfected clones; 2) that by antibody neutralization the effect appeared to be mediated by HRA cell-derived TGF-beta1; 3) that exogenous TGF-beta1 specifically enhanced PAI-1 up-regulation at the mRNA and protein level in mesothelial cells in a time- and concentration-dependent manner, mainly through MAPK-dependent activation mechanism; and 4) that mesothelial cell-derived PAI-1 may promote tumor invasion possibly by enhancing cell-cell interaction. This represents a novel pathway by which tumor cells can regulate the plasminogen activator system-dependent cellular responses in mesothelial cells that may contribute to formation of peritoneal dissemination of ovarian cancer.     

Serum-derived vitronectin influences the pericellular distribution of type 1 plasminogen activator inhibitor

Bovine aortic endothelial cells (BAEs) were used as a model system to study the nature and origin of protein(s) in the extracellular matrix that bind to type 1 plasminogen activator inhibitor (PAI-1). Matrix samples were fractionated by SDS-PAGE and analyzed by PAI-1 ligand binding and by immunoblotting using antibodies to vitronectin (Vn). PAI-1 bound primarily to two Vn-related polypeptides of Mr 63,000 and 57,000, and both of these partially degraded polypeptides were present in the culture serum. Radiolabeling experiments failed to detect significant Vn biosynthesis by BAEs (less than 0.03% of total), or by human umbilical vein endothelial cells and HT 1080 cells. The binding of PAI-1 to Vn was relatively specific since direct binding studies failed to demonstrate significant interactions between PAI-1 and other matrix proteins (e.g., fibronectin, type IV collagen, laminin, or matrigel). Kinetic studies indicate that PAI-1 rapidly accumulates in the matrix when BAEs are plated on Vn, appearing in the conditioned medium only after a significant lag period (1-2 h). However, no PAI-1 was detected in the matrix when the cells were plated on fibronectin-coated dishes, and there was no lag period for PAI-1 accumulation in the medium. These results indicate that PAI-1 binds specifically to serum-derived Vn in the matrix, and suggest that the composition of both the matrix and serum itself may influence the pericellular distribution of this important inhibitor.     

Expression of active plasminogen activator inhibitor-1 reduces cell migration and invasion in breast and gynecological cancer cells

Urokinase-type (uPA) plasminogen activator is regulated by serine protease inhibitors (serpins), especially plasminogen activator inhibitor-1 (PAI-1). In many cancers, uPA and PAI-1 contribute to the invasive phenotype. We examined the in vitro migration and invasive capabilities of breast, ovarian, endometrial, and cervical cancer cell lines compared to their plasminogen activator system profiles. We then overexpressed active wild-type PAI-1 and an inactive "substrate" P14 form of PAI-1 (T333R) using stable transfection and adenoviral gene delivery. We also upregulated endogenous uPA and PAI-1 in these cells by treatment with transforming growth factor-beta. Some breast and ovarian cancer cell lines with natural expression of uPA, PAI-1, and urokinase receptor showed substantial migration and invasion compared to other cell lines that lack expression of these proteins. However, overexpression of active wild-type PAI-1, but not P14-PAI-1 (T333R), in these cell lines showed reduced migration and invasion. Since vitronectin binding by both forms of PAI-1 is equivalent, these results imply that PAI-1-vitronectin interactions are less critical in altering migration and invasion. Our results show that the in vitro migratory and invasive phenotype in these breast and ovarian cancer cell lines is reduced by active PAI-1 due to its ability to inhibit plasminogen activation.     

The prognostic value of vascular endothelial growth factor, urokinase plasminogen activator and plasminogen activator inhibitor-1 in node-negative breast cancer

The vascular endothelial growth factor (VEGF) and the plasminogen activator system play an essential role in solid tumor angiogenesis and in tumor invasion and metastasis. In the present study we investigated the relationship between patient outcome and levels of VEGF, urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) in tumor cytosols of 196 node-negative primary invasive breast cancer patients who did not receive any adjuvant therapy. The median follow-up was 65 months. VEGF, uPA and PAI-1 were measured by commercially available enzyme-linked immunosorbent assays. Cox's univariate analysis showed that pT (p = 0.0007), uPA (p = 0.0156) and PAI-1 (p = 0.0015) had a significant impact on relapse-free survival, whereas VEGF did not have any prognostic value (p = 0.18). Bivariate analysis showed significant interactions between uPA and PAI-1 (p = 0.0035) and between VEGF and PAI-1 (p = 0.006). Our study confirms that uPA and PAI-1 cytosol levels can be considered as prognostic factors for relapse-free survival in node-negative breast cancer. Moreover, the interaction between VEGF and PAI-1 warrants further investigation into the relationship between the biomarkers of angiogenesis and those of the protease cascade.     

Ligand binding properties of the very low density lipoprotein receptor. Absence of the third complement-type repeat encoded by exon 4 is associated with reduced binding of Mr 40,000 receptor-associated protein

The very low density lipoprotein receptor (VLDLR) binds, among other ligands, the Mr 40,000 receptor-associated protein (RAP) and a variety of serine proteinase-serpin complexes, including complexes of the proteinase urokinase-type plasminogen activator (uPA) with the serpins plasminogen activator inhibitor-1 (PAI-1) and protease nexin-1 (PN-1). We have analyzed the binding of RAP, uPA.PAI-1, and uPA.PN-1 to two naturally occurring VLDLR variants, VLDLR-I, containing all eight complement-type repeats, and VLDLR-III, lacking the third complement-type repeat, encoded by exon 4. VLDLR-III displayed approximately 4-fold lower binding of RAP than VLDLR-I and approximately 10-fold lower binding of the most C-terminal one of the three domains of RAP. In contrast, the binding of uPA.PAI-1 and uPA.PN-1 to the two VLDLR variants was indistinguishable. Surprisingly, uPA.PN-1, but not uPA.PAI-1, competed RAP binding to both VLDLR variants. These observations show that the third complement-type repeat plays a crucial role in maintaining the contact sites needed for optimal recognition of RAP, but does not affect the proteinase-serpin complex contact sites, and that two ligands can show full cross-competition without sharing the same contacts with the receptor. These results elucidate the mechanisms of molecular recognition of ligands by receptors of the low density lipoprotein receptor family.     

uPA and PAI-1-Related Signaling Pathways Differ between Primary Breast Cancers and Lymph Node Metastases

The supporting role of urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor 1 (PAI-1) in migration and invasion is well known. In addition, both factors are key components in cancer cell-related signaling. However, little information is available for uPA and PAI-1-associated signaling pathways in primary cancers and corresponding lymph node metastases. The aim of this study was to compare the expression of uPA and PAI-1-associated signaling proteins in 52 primary breast cancers and corresponding metastases. Proteins were extracted from formalin-fixed paraffin-embedded tissue samples of the primary tumors and metastases. Protein lysates were subsequently analyzed by reverse phase protein array for the expression of members of the PI3K/AKT (FAK, GSK3-β, ILK, pGSK3-β, PI3K, and ROCK) and the MAPK pathways (pp38, pSTAT3, and p38). A solid correlation of uPA expression existed between primary tumors and metastases, whereas PAI-1 expression did not significantly correlate between them. The correlations of uPA and PAI-1 with signaling pathways found in primary tumors did not persist in metastases. Analysis of single molecules revealed that some correlated well between tumors and metastases (FAK, pGSK3-β, ILK, Met, PI3K, ROCK, uPA, p38, and pp38), whereas others did not (PAI-1 and GSK3-β). Whether the expression of a protein correlated between tumor and metastasis or not was independent of the pathway the protein is related to. These findings hint at a complete deregulation of uPA and PAI-1-related signaling in metastases, which might be the reason why uPA and PAI-1 reached clinical relevance only for lymph node-negative breast cancer tissues.     

Internalization of the urokinase-plasminogen activator inhibitor type-1 complex is mediated by the urokinase receptor.

The role of the urokinase receptor (uPAR) in the internalization of the urokinase-plasminogen activator inhibitor type-1 (uPA.PAI-1) complex has been investigated. First, exploiting the species specificity of uPA binding, we show that mouse LB6 cells (that express a mouse uPAR) were unable to bind or degrade the human uPA.PAI-1 complex. On the other hand, LB6 clone 19 cells, which express a transfected human uPAR, degraded uPA.PAI-1 complexes with kinetics identical to the human monocytic U937 cells. We also show by immunofluorescence experiments with anti-uPA antibodies that in LB6 clone 19 cells, the uPA.PAI-1 complex is indeed internalized. While at 4 degrees C uPA fluorescence was visible at the cell surface, shift of the temperature to 37 degrees C caused a displacement of the immunoreactivity to the cytoplasmic compartment, with a pattern indicating lysosomal localization. If uPA.PAI-1 internalization/degradation is mediated by uPAR, inhibition of uPA.PAI-1 binding to uPAR should block degradation. Three different treatments, competition with the agonist amino-terminal fragment of uPA, treatment with a monoclonal antibody directed toward the binding domain of uPAR or release of uPAR from the cell surface with phosphatidylinositol-specific phospholipase C completely prevented uPA.PAI-1 degradation. The possibility that a serpin-enzyme complex receptor might be primarily or secondarily involved in the internalization process was excluded since a serpin-enzyme complex peptide failed to inhibit uPA.PAI-1 binding and degradation. Similarly, complexes of PAI-1 with low molecular mass uPA (33 kDa uPA), which lacks the uPAR binding domain, were neither bound nor degraded. Finally we also show that treatment of cells with uPA.PAI-1 complex caused a specific but partial down-regulation of uPAR. A similar result was obtained when PAI-1 was allowed to complex to uPA that had been previously bound to the receptor. The possibility therefore exists that the entire complex uPA.PAI-1-uPAR is internalized. All these data allow us to conclude that internalization of the uPA.PAI-1 complex is mediated by uPAR.     

Transforming growth factor beta1 stimulates urokinase plasminogen activator system on prostate cancer cells

The effect of TGFbeta1 on the proliferation and plasminogen activator system (PA) of two prostate carcinoma cell lines, PC3 and DU145, was investigated. PA, particularly urokinase plasminogen activator (uPA), has been implicated in extracellular proteolysis, local invasiveness, metastatic spread and angiogenesis. High levels of uPA and plasminogen activator inhibitor-1 (PAI-1) correlate with poor prognosis in several cancers. TGFbeta1 had no significant effect on the proliferation of either cell line. TGFbeta1 increased the production of uPA in PC3 and DU145 cells. Despite the very low PAI-1 protein levels in both cell lines, TGFbeta1 treatment resulted in a remarkable increase in PAI-1 secretion. PAI-2 protein was also increased by 59% in the PC3 cells. A divergent effect of TGFbeta1 on the uPA enzyme activity was observed (28% decrease in PC3 and 131% increase in DU145 cells). Overall, TGFbeta1 treatment did not affect the invasion of reconstituted basement membrane of PC3 cells. In addition to the uPA:PAI-1 ratio, the presence of PAI-2 may be an important factor in the determination of metastatic sites for prostate cancer cells. In conclusion, the potential contribution of TGFbeta1 to tumor invasion may be considered as positive, based on both loss of growth inhibition and stimulation of components of the invasive system of prostate carcinoma.     

Transforming growth factor-beta1-dependent activation of Smad2/3 and up-regulation of PAI-1 expression is negatively regulated by Src in SKOV-3 human ovarian cancer cells

The net balance between urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) has been implicated in tumor cell invasion and metastasis. To elucidate the mechanism of the transforming growth factor-beta1 (TGF-beta1)-dependent up-regulation of PAI-1 expression, we investigated which signaling pathway transduced by TGF-beta1 is responsible for this effect. Here, we show (1) nontoxic concentrations of TGF-beta1 up-regulates uPA expression in HRA and SKOV-3 human ovarian cancer cells, (2) TGF-beta1 activates Smads (phosphorylation of Smad2 and nuclear translocation of Smad3) and subsequently up-regulates PAI-1 expression in HRA cells, whereas TGF-beta1 neither activates Smads nor up-regulates PAI-1 in SKOV-3 cells, (3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment significantly induces TGF-beta1-dependent activation of Smads, leading to PAI-1 synthesis, compared with controls, in SKOV-3 cells, (4) combination of TGF-beta1 and PP2, which activates PAI-1 expression and reduces uPA expression in SKOV-3, results in decreased invasiveness, (5) pharmacological inhibitors for mitogen-activated protein kinase (MAPK) (PD98059) and phosphoinositide-3-kinase (PI3K) (LY294002 and wortmannin) or AS-PI3K ODN transfection do not affect TGF-beta1-induced Smad signaling and up-regulation of PAI-1 expression in SKOV-3 cells pretreated with PP2, and (6) the induction of PAI-1 protein was partially inhibited by an inhibitor of Sp1-DNA binding, mithramycin, implicating, at least in part, Sp1 in the regulation of this gene by TGF-beta1. In conclusion, TGF-beta1-dependent activation of Smad2/3, leading to PAI-1 synthesis, may be negatively regulated by Src, but not its downstream targets MAPK and PI3K in SKOV-3 cells. These data also reflect the complex biological effect of uPA-PAI-1 system.     

Breast cancer and metabolic syndrome linked through the plasminogen activator inhibitor-1 cycle

Plasminogen activator inhibitor-1 (PAI-1) is a physiological inhibitor of urokinase (uPA), a serine protease known to promote cell migration and invasion. Intuitively, increased levels of PAI-1 should be beneficial in downregulating uPA activity, particularly in cancer. By contrast, in vivo, increased levels of PAI-1 are associated with a poor prognosis in breast cancer. This phenomenon is termed the "PAI-1 paradox". Many factors are responsible for the upregulation of PAI-1 in the tumor microenvironment. We hypothesize that there is a breast cancer predisposition to a more aggressive stage when PAI-1 is upregulated as a consequence of Metabolic Syndrome (MetS). MetS exerts a detrimental effect on the breast tumor microenvironment that supports cancer invasion. People with MetS have an increased risk of coronary heart disease, stroke, peripheral vascular disease and hyperinsulinemia. Recently, MetS has also been identified as a risk factor for breast cancer. We hypothesize the existence of the "PAI-1 cycle". Sustained by MetS, adipocytokines alter PAI-1 expression to promote angiogenesis, tumor-cell migration and procoagulant microparticle formation from endothelial cells, which generates thrombin and further propagates PAI-1 synthesis. All of these factors culminate in a chemotherapy-resistant breast tumor microenvironment. The PAI-1 cycle may partly explain the PAI-1 paradox. In this hypothesis paper, we will discuss further how MetS upregulates PAI-1 and how an increased level of PAI-1 can be linked to a poor prognosis.     

Construction of a plasminogen activator inhibitor-1 variant without measurable affinity to vitronectin but otherwise normal

Vitronectin (VN) and plasminogen activator inhibitor-1 (PAI-1) have important functional interactions: VN stabilises the protease inhibitory activity of PAI-1 and PAI-1 inhibits binding of adhesion receptors to VN. Having previously mapped the PAI-1 binding area for VN, we have now constructed a PAI-1 variant, R103A-M112A-Q125A, without measurable affinity to VN, but with full protease inhibitory activity and endocytosis receptor binding. As a tool for evaluating the physiological and pathophysiological functions of the PAI-1-VN interaction, our new variant is far superior to the previously widely used PAI-1 variant Q125K, which we have found possesses an only about 10-fold reduced affinity to VN.