The inhibition of tube formation in a collagen-fibrinogen, three-dimensional gel by cleaved kininogen (HKa) and HK domain 5 (D5) is dependent on Src family kinases

Cleaved high molecular weight kininogen (HKa), as well as its domain 5 (D5), inhibits migration and proliferation induced by angiogenic factors and induces apoptosis in vitro. To study its effect on tube formation we utilized a collagen-fibrinogen, three-dimensional gel, an in vitro model of angiogenesis. HKa, GST-D5 and D5 had a similar inhibitory effect of tube length by 90+/-4.5%, 86+/-5.5% and 77+/-12.9%, respectively. D5-derived synthetic peptides: G440-H455 H475-H485 and G486-K502 inhibited tube length by 51+/-3.7%, 54+/-3.8% and 77+/-1.7%, respectively. By a comparison of its inhibitory potency and its sequences, a functional sequence of HKa was defined to G486-G496. PP2, a Src family kinase inhibitor, prevented tube formation in a dose-dependent manner (100-400 nM), but PP3 at 5 microM, an inactive analogue of PP2, did not. HKa and D5 inhibited Src 416 phosphorylation by 62+/-12.3% and 83+/-6.1%, respectively. The C-terminal Src kinase (Csk) inhibits Src kinase activity. Using a siRNA to Csk, expression of Csk was down-regulated by 86+/-7.0%, which significantly increased tube length by 27+/-5.8%. The addition of HKa and D5 completely blocked this effect. We further showed that HKa inhibited Src family kinase activity by disrupting the complex of uPAR, alphavbeta3 integrin and Src. Our results indicate that the anti-angiogenic effect of HKa and D5 is mediated at least in part through Src family kinases and identify a potential novel target for therapeutic inhibition of neovascularization in cancer and inflammatory arthritis.     

Two-chain high molecular weight kininogen induces endothelial cell apoptosis and inhibits angiogenesis: partial activity within domain 5.

We previously reported that the binding of two-chain high molecular weight kininogen (HKa) to endothelial cells may occur through interactions with endothelial urokinase receptors. Since the binding of urokinase to urokinase receptors activates signaling responses and may stimulate mitogenesis, we assessed the effect of HKa binding on endothelial cell proliferation. Unexpectedly, HKa inhibited proliferation in response to several growth factors, with 50% inhibition caused by approximately 10 nM HKa. This activity was Zn(2+) dependent and not shared by either single-chain high molecular weight kininogen (HK) or low molecular weight kininogen. HKa selectively inhibited the proliferation of human umbilical vein and dermal microvascular endothelial cells, but did not affect that of umbilical vein or human aortic smooth muscle cells, trophoblasts, fibroblasts, or carcinoma cells. Inhibition of endothelial proliferation by HKa was associated with endothelial cell apoptosis and unaffected by antibodies that block the binding of HK or HKa to any of their known endothelial receptors. Recombinant HK domain 5 displayed activity similar to that of HKa. In vivo, HKa inhibited neovascularization of subcutaneously implanted Matrigel plugs, as well as rat corneal angiogenesis. These results demonstrate that HKa is a novel inhibitor of angiogenesis, whose activity is dependent on the unique conformation of the two-chain molecule.     

Mapping the interaction between high molecular mass kininogen and the urokinase plasminogen activator receptor

The urokinase plasminogen activator receptor (uPAR) is a multifunctional, GPI-linked receptor that modulates cell adhesion/migration and fibrinolysis. We mapped the interaction sites between soluble uPAR (suPAR) and high molecular mass kininogen (HK). Binding of biotin-HK to suPAR was inhibited by HK, 56HKa, and 46HKa with an IC50 of 60, 110, and 8 nm, respectively. We identified two suPAR-binding sites, a higher affinity site in the light chain of HK and 46HKa (His477-Gly496) and a lower affinity site within the heavy chain (Cys333-Lys345). HK predominantly bound to suPAR fragments containing domains 2 and 3 (S-D2D3). Binding of HK to domain 1 (S-D1) was also detected, and the addition of S-D1 to S-D2D3 completely inhibited biotin-HK or -46HKa binding to suPAR. Using sequential and overlapping 20-amino acid peptides prepared from suPAR, two regions for HK binding were identified. One on the carboxyl-terminal end of D2 (Leu166-Thr195) blocked HK binding to suPAR and to human umbilical vein endothelial cells (HUVEC). This site overlapped with the urokinase-binding region, and urokinase inhibited the binding of HK to suPAR. A second region on the amino-terminal portion of D3 (Gln215-Asn255) also blocked HK binding to HUVEC. Peptides that blocked HK binding to uPAR also inhibited prekallikrein activation on HUVEC. Therefore, HK interacts with suPAR at several sites. HK binds to uPAR as part of its interaction with its multiprotein receptor complex on HUVEC, and the biological functions that depend upon this binding are modulated by urokinase.     

RNA interference-directed knockdown of urokinase plasminogen activator and urokinase plasminogen activator receptor inhibits prostate cancer cell invasion, survival, and tumorigenicity in vivo

The invasive ability of tumor cells plays a key role in prostate cancer metastasis and is a major cause of treatment failure. Urokinase plasminogen activator-(uPA) and its receptor (uPAR)-mediated signaling have been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. This study was undertaken to investigate the biological roles of uPA and uPAR in prostate cancer cell invasion and survival, and the potential of uPA and uPAR as targets for prostate cancer therapy. uPA and uPAR expression correlates with the metastatic potential of prostate cancer cells. Thus, therapies designed to inhibit uPA and uPAR expression would be beneficial. LNCaP, DU145, and PC3 are prostate cancer cell lines with low, moderate, and high metastatic potential, respectively, as demonstrated by their capacity to invade the extracellular matrix. In this study we utilized small hairpin RNAs (shRNAs), also referred to as small interfering RNAs, to target human uPA and uPAR. These small interfering RNA constructs significantly inhibited uPA and uPAR expression at both the mRNA and protein levels in the highly metastatic prostate cancer cell line PC3. Our data demonstrated that uPA-uPAR knockdown in PC3 cells resulted in a dramatic reduction of tumor cell invasion as indicated by a Matrigel invasion assay. Furthermore, simultaneous silencing of the genes for uPA and uPAR using a single plasmid construct expressing shRNAs for both uPA and uPAR significantly reduced cell viability and ultimately resulted in the induction of apoptotic cell death. RNA interference for uPA and uPAR also abrogated uPA-uPAR signaling to downstream target molecules such as ERK1/2 and Stat 3. In addition, our results demonstrated that intratumoral injection with the plasmid construct expressing shRNAs for uPA and uPAR almost completely inhibited established tumor growth and survival in an orthotopic mouse prostate cancer model. These findings uncovered evidence of a complex signaling network operating downstream of uPA-uPAR that actively advances tumor cell invasion, proliferation, and survival of prostate cancer cells. Thus, RNA interference-directed targeting of uPA and uPAR is a convenient and novel tool for studying the biological role of the uPA-uPAR system and raises the potential of its application for prostate cancer therapy.     

Soluble urokinase-type plasminogen activator receptor inhibits cancer cell growth and invasion by direct urokinase-independent effects on cell signaling

The urokinase-type plasminogen activator receptor (uPAR) is released from human cancers and is readily detected in blood. In animal models, soluble uPAR (SuPAR) antagonizes cancer progression; however, the mechanism by which SuPAR functions in vivo remains unclear. It is generally thought that SuPAR scavenges uPA and prevents its interaction with membrane-anchored uPAR. In this study, we demonstrate a novel molecular mechanism by which SuPAR may inhibit cancer progression. We show that SuPAR has the potential to directly and in a uPA-independent manner block the signaling activity of membrane-anchored uPAR. Whether SuPAR inhibits signaling is cell type-specific, depending on the state of the endogenous uPA-uPAR signaling system. In uPAR-deficient cells that lack endogenous uPAR signaling, including uPAR-/-murine embryonic fibroblasts and human embryonal kidney 293 cells, SuPAR functions as a partial signaling agonist that activates ERK/mitogen-activated protein kinase. By contrast, in cells with potent autocrine uPA-uPAR signaling systems, including MDA-MB 231 breast cancer cells and low density lipoprotein receptor-related protein-1-deficient murine embryonic fibroblasts, SuPAR substantially decreases ERK activation. The mechanism probably involves competitive displacement of membrane-anchored uPAR-uPA complex from signaling adaptor proteins. As a result of its effects on cell signaling, SuPAR blocks cell growth and inhibits cellular invasion of Matrigel. Cleavage of SuPAR by proteinases increases its signaling agonist activity and reverses its inhibitory effects on growth and invasion. Thus, proteolytic cleavage represents a molecular switch that neutralizes the anticancer activity of SuPAR.     

活化型高分子量激肽原潜在的抗肿瘤作用及其分子机制

高分子量激肽原是血浆中一种多功能的糖蛋白,与血液凝固的启动、 补体反应及炎症发生等有密切关系.新近的研究显示,活化型高分子量激肽原 具有潜在的抗肿瘤作用.本文综述活化型高分子量激肽原在细胞粘附和血管 生成中发挥的抑制作用及其活性区域,抑制细胞迁移、增殖并诱导细胞凋亡 的作用,及其在细胞表面的作用位点和分子机制.活化型高分子量激肽原作用 机制,包括抑制细胞DNA的从头合成,使细胞周期蛋白D1表达下降,以及通过 影响细胞内信号通路发挥其活性效应等.深入研究活化型高分子量激肽原在 细胞表面作用的信号转导通路可能是今后抗肿瘤研究途径之一.     

Evidence for binding of the ectodomain of amyloid precursor protein 695 and activated high molecular weight kininogen

To identify ligands that bind to the N-terminal portion of human amyloid precursor protein (APP), we sought binding partners for a fragment of the ectodomain of human APP695 (sAPP(695)T). The probe bound to fragments of high molecular weight kininogen (HK) in rat cortical membrane preparations in vitro. Laser confocal microscopy indicated that APP and HK colocalize near cerebral blood vessels, in the neuropil, and in many neurons of rat brain. sAPP(695)T bound to human activated kininogen (HKa) (K(d)=0.3+/-0.1 nM), but not to inactivated or low molecular weight kininogen. Binding was specific for the light chain sequence of HKa. Biotinylated human HKa also bound to sAPP(695) (K(d)=0.3+/-0.5 nM). sAPP(695) and HKa form tight complexes in solution that can be coimmunoprecipitated. These results support the hypothesis that forms of APP and kininogen can interact in brain tissue. Considering the implications of APP in neurite outgrowth, the APP-HKa interaction could modulate neurogenesis.     

Ferritin blocks inhibitory effects of two-chain high molecular weight kininogen (HKa) on adhesion and survival signaling in endothelial cells

Angiogenesis is tightly regulated through complex crosstalk between pro- and anti-angiogenic signals. High molecular weight kininogen (HK) is an endogenous protein that is proteolytically cleaved in plasma and on endothelial cell surfaces to HKa, an anti-angiogenic protein. Ferritin binds to HKa and blocks its anti-angiogenic activity. Here, we explore mechanisms underlying the cytoprotective effect of ferritin in endothelial cells exposed to HKa. We observe that ferritin promotes adhesion and survival of HKa-treated cells and restores key survival and adhesion signaling pathways mediated by Erk, Akt, FAK and paxillin. We further elucidate structural motifs of both HKa and ferritin that are required for effects on endothelial cells. We identify an histidine-glycine-lysine (HGK) -rich antiproliferative region within domain 5 of HK as the target of ferritin, and demonstrate that both ferritin subunits of the H and L type regulate HKa activity. We further demonstrate that ferritin reduces binding of HKa to endothelial cells and restores the association of uPAR with α5β1 integrin. We propose that ferritin blocks the anti-angiogenic activity of HKa by reducing binding of HKa to UPAR and interfering with anti-adhesive and anti-proliferative signaling of HKa.     

Induction of apoptosis in vascular cells by plasminogen activator inhibitor-1 and high molecular weight kininogen correlates with their anti-adhesive properties

Plasminogen activator inhibitor-1 (PAI-1) and two-chain high molecular weight kininogen (HKa) exert anti-adhesive properties in vitronectin-dependent cell adhesion. Here, the hypothesis was tested that these anti-adhesive components promote apoptosis in vascular cells. PAI-1 or HKa induced a 2- to 3-fold increase in apoptosis of human umbilical-vein endothelial cells (HUVEC) and vascular smooth muscle cells (VSMC) adherent to vitronectin, as determined by annexin V-FACS assay, similar to alphav-integrin inhibitor cyclo-(Arg-Gly-Asp-D-Phe-Val)-peptide (cRGDfV). Apoptosis occurred after 12 h incubation and was attributable to caspase 3 activation that in turn induced DNA fragmentation. Induction of apoptosis strongly correlated with the anti-adhesive effect of PAI-1 and HKa on these cells. In contrast, PAI-1 and HKa did not affect fibronectin-dependent adhesion or cell survival. uPA did not influence apoptosis in vitronectin- or fibronectin-adherent cells. In atherosclerotic vessel sections, congruent distribution of vitronectin, PAI-1, HK, and of components of the urokinase plasminogen activator/receptor system with apoptotic cells lining foam cell lesions was demonstrated by immunostaining. These results indicate that inhibition of vitronectin-dependent cell adhesion through PAI-1 and HKa correlates with apoptosis induction in vascular cells mediated through the caspase 3 pathway. Co-distribution of apoptosis with plasminogen activation system components in atherosclerosis exemplifies the significance of anti-adhesive mechanisms and apoptosis for tissue remodeling, such as in neointima development.     

Upregulation of Cdc2 and cyclin A during apoptosis of endothelial cells induced by cleaved high-molecular-weight kininogen

We (8) reported that the cleaved high-molecular-weight kininogen (HKa) and its domain 5 (D5) inhibited angiogenesis. Further studies (15) revealed that D5 could inhibit cell proliferation and induce apoptosis of proliferating endothelial cells, which together may represent a critical part of antiangiogenic activity of HKa and D5. In the present study, we further examined the effect of HKa on cell cycle progression and cell viability. We report that HKa induced a significant upregulation of Cdc2 and cyclin A in proliferating endothelial cells, concurrent with a marked increase of Cdc2 activity. The increased expression of Cdc2 and cyclin A by HKa was not associated with an apparent change in cell cycle profiles of basic fibroblast growth factor-stimulated proliferating cells, but closely correlated with a marked increase of apoptosis, suggesting that the elevated Cdc2 activity is involved in HKa-induced apoptosis of proliferating endothelial cells. Our results support an emerging hypothesis that Cdc2 and cyclin A are important regulators for cell cycle as well as for apoptosis.     

Structural analysis of the interaction between urokinase-type plasminogen activator and its receptor: a potential target for anti-invasive cancer therapy

The ability to degrade the extracellular matrix by controlled proteolysis is an important property of malignant cancer cells, which enables them to invade the surrounding tissue and to gain access to the circulation by intravasation. One proteolytic system thought to be involved in these processes is urokinase-mediated plasminogen activation. Expression of a glycolipid-anchored receptor for urokinase-type plasminogen activator (uPA) targets this system to the cell surface. This receptor (uPAR) is composed of three homologous modules belonging to the Ly-6/uPAR/alpha-neurotoxin protein domain family. Integrity of the three-domain structure of uPAR is required for maintenance of its sub-nanomolar affinity for uPA, but the functional epitope for this interaction is primarily located in uPAR domain I. Using affinity maturation by combinatorial chemistry, we have recently identified a potent 9-mer peptide antagonist of the uPA-uPAR interaction having a high affinity for uPAR (K(d)< 1 nM). Photoaffinity labelling suggests that this peptide interacts with a composite binding site in uPAR involving both domains I and III. When tested in a chicken chorioallantoic membrane assay that was developed to quantify intravasation of human cells, this antagonist was able to reduce the intravasation of HEp-3 cancer cells by approx. 60%.     

Peptide-derived antagonists of the urokinase receptor. affinity maturation by combinatorial chemistry, identification of functional epitopes, and inhibitory effect on cancer cell intravasation

The high-affinity interaction between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays an important role in pericellular plasminogen activation. Since proteolytic degradation of the extracellular matrix has an established role in tumor invasion and metastasis, the uPA-uPAR interaction represents a potential target for therapeutic intervention. By affinity maturation using combinatorial chemistry we have now developed and characterized a 9-mer, linear peptide antagonist of the uPA-uPAR interaction demonstrating specific, high-affinity binding to human uPAR (K(d) approximately 0.4 nM). Studies by surface plasmon resonance reveal that the off-rate for this receptor-peptide complex is comparable to that measured for the natural protein ligand, uPA. The functional epitope on human uPAR for this antagonist has been delineated by site-directed mutagenesis, and its assignment to loop 3 of uPAR domain III (Met(246), His(249), His(251), and Phe(256)) corroborates data previously obtained by photoaffinity labeling and provides a molecular explanation for the extreme selectivity observed for the antagonist toward human compared to mouse, monkey, and hamster uPAR. When human HEp-3 cancer cells were inoculated in the presence of this peptide antagonist, a specific inhibition of cancer cell intravasation was observed in a chicken chorioallantoic membrane assay. These data imply that design of small organic molecules mimicking the binding determinants of this 9-mer peptide antagonist may have a potential application in combination therapy for certain types of cancer.     

cDNA transfection of amino-terminal fragment of urokinase efficiently inhibits cancer cell invasion and metastasis.

Focusing of urokinase-type plasminogen activator (uPA) to the cell surface via binding to its specific receptor (uPAR, CD87) is critical for tumor invasion and metastasis. Consequently, the inhibition of uPA-uPAR interaction on the cell surface might be a promising anti-invasion and anti-metastasis strategy. We examined the effects of cDNA transfection of the human uPA amino-terminal fragment (ATF) on invasion and metastasis of cancer cells. First, a highly metastatic human lung giant-cell carcinoma cell line (PG), used as the target cell for evaluation of this effect, was demonstrated to express both uPA and uPAR. Then, ATF, which contains an intact uPAR binding site but is catalytically inactive, was designed as an antagonist of uPA-uPAR interaction and was transfected into PG cells. [(3)H]-Thymidine incorporation and cell growth curves indicated that expressed ATF did not affect the proliferation of transfected cells. However, analysis by scanning electron microscopy revealed that ATF changed the host cells from the typical invasive phenotype to a noninvasive one. Correspondingly, the modified Boyden chamber test in vitro showed that ATF expression significantly decreased the invasive capacity of transfected cells. Furthermore, in the spontaneous metastasis model, it was confirmed in vivo that expressed ATF remarkably inhibited lung metastasis of implanted ATF-transfected PG cells. In summary, autocrine ATF could act as an antagonist of uPA-uPAR interaction, and ATF cDNA transfection could efficiently inhibit the invasion and metastasis of the cancer cells. Inhibition of uPA-uPAR interaction on the cell surface might be a promising anti-invasion and anti-metastasis strategy.     

Urokinase plasminogen activator induces human smooth muscle cell migration and proliferation via distinct receptor-dependent and proteolysis-dependent mechanisms

In order to define the relative contribution of the proteolytic domain and the receptor-binding domain of urokinase plasminogen activator (uPA) toward its mitogenic properties we studied the effects of different uPA isoforms on migration and proliferation of human aortic smooth muscle cells (hSMC). The isoforms tested included native human glycosylated uPA, and two recombinant uPA forms, namely a recombinant uPA with wild type structure (r-uPA), and a uPA-mutant in which the first 24 N-terminal amino acid residues of the receptor binding domain were replaced by 13 foreign amino acid residues (r-uPAmut). Cell migration was evaluated using a micro-Boyden chamber assay, and cell proliferation assessed by measurement of [3H]-thymidine incorporation into DNA. Competition binding studies on hSMC using 125I-r-uPA as ligand demonstrated that r-uPA and r-uPAmut exhibited equivalent displacement profiles. However, migration of hSMC was promoted by r-uPA and not by r-uPAmut. r-uPA-induced migration occurred at concentrations (half-maximally effective concentration of 2 nM) approximating the Kd for uPA-uPAR binding (1 nM). r-uPA-induced migration was not affected by the plasmin inhibitor aprotinin. In contrast to their differential chemotactic properties, uPA, r-uPA and r-uPAmut, which possess similar proteolytic activities, all stimulated [3H]-thymidine incorporation in hSMC. Since the [3H]-thymidine incorporation response to each isoform occurred at concentrations (> 50 nM) much higher than necessary for uPAR saturation by ligand (1 nM), this mitogenic response may be independent of binding to uPAR. [3H]-thymidine incorporation responses to r-uPA and -uPAmut were sensitive to the plasmin inhibitor aprotinin, and uPA stimulated DNA synthesis was inhibited by plasminogen activator inhibitor. We conclude that hSMC migration in response to uPA depends upon on its binding to uPAR, whereas uPA-stimulated DNA synthesis in these cells requires proteolysis and plasmin generation.     

Urokinase induces basophil chemotaxis through a urokinase receptor epitope that is an endogenous ligand for formyl peptide receptor-like 1 and -like 2

Basophils circulate in the blood and are able to migrate into tissues at sites of inflammation. Urokinase plasminogen activator (uPA) binds a specific high affinity surface receptor (uPAR). The uPA-uPAR system is crucial for cell adhesion and migration, and tissue repair. We have investigated the presence and function of the uPA-uPAR system in human basophils. The expression of uPAR was found at both mRNA and protein levels. The receptor was expressed on the cell surface of basophils, in the intact and cleaved forms. Basophils did not express uPA at either the protein or mRNA level. uPA (10(-12)-10(-9) M) and its uPAR-binding N-terminal fragment (ATF) were potent chemoattractants for basophils, but did not induce histamine or cytokine release. Inactivation of uPA enzymatic activity by di-isopropyl fluorophosphate did not affect its chemotactic activity. A polyclonal Ab against uPAR inhibited uPA-dependent basophil chemotaxis. The uPAR-derived peptide 84-95 (uPAR84-95) induced basophil chemotaxis. Basophils expressed mRNA for the formyl peptide receptors formyl peptide receptor (FPR), FPR-like 1 (FPRL1), and FPRL2. The FPR antagonist cyclosporin H prevented chemotaxis induced by FMLP, but not that induced by uPA and uPAR84-95. Incubation of basophils with low and high concentrations of FMLP, which desensitize FPR and FPRL1, respectively, but not FPRL2, slightly reduced the chemotactic response to uPA and uPAR84-95. In contrast, desensitization with WKYMVm, which also binds FPRL2, markedly inhibited the response to both molecules. Thus, uPA is a potent chemoattractant for basophils that seems to act through exposure of the chemotactic uPAR epitope uPAR84-95, which is an endogenous ligand for FPRL2 and FPRL1.     

Inhibition of cell adhesion by high molecular weight kininogen

An anti-cell adhesion globulin was purified from human plasma by heparin-affinity chromatography. The purified globulin inhibited spreading of osteosarcoma and melanoma cells on vitronectin, and of endothelial cells, platelets, and mononuclear blood cells on vitronectin or fibrinogen. It did not inhibit cell spreading on fibronectin. The protein had the strongest antiadhesive effect when preadsorbed onto the otherwise adhesive surfaces. Amino acid sequence analysis revealed that the globulin is cleaved (kinin-free) high molecular weight kininogen (HKa). Globulin fractions from normal plasma immunodepleted of high molecular weight kininogen (HK) or from an individual deficient of HK lacked adhesive activity. Uncleaved single-chain HK preadsorbed at neutral pH, HKa preadsorbed at pH greater than 8.0, and HKa degraded further to release its histidine-rich domain had little anti-adhesive activity. These results indicate that the cationic histidine-rich domain is critical for anti-adhesive activity and is somehow mobilized upon cleavage. Vitronectin was not displaced from the surface by HKa. Thus, cleavage of HK by kallikrein results in both release of bradykinin, a potent vasoactive and growth-promoting peptide, and formation of a potent anti-adhesive protein.     

Direct binding of occupied urokinase receptor (uPAR) to LDL receptor-related protein is required for endocytosis of uPAR and regulation of cell surface urokinase activity

Low-density lipoprotein receptor-related protein (LRP) mediates internalization of urokinase:plasminogen activator inhibitor complexes (uPA:PAI-1) and the urokinase receptor (uPAR). Here we investigated whether direct interaction between uPAR, a glycosyl-phosphatidylinositol-anchored protein, and LRP, a transmembrane receptor, is required for clearance of uPA:PAI-1, regeneration of unoccupied uPAR, activation of plasminogen, and the ability of HT1080 cells to invade extracellular matrix. We found that in the absence of uPA:PAI-1, uPAR is randomly distributed along the plasma membrane, whereas uPA:PAI-1 promotes formation of uPAR-LRP complexes and initiates redistribution of occupied uPAR to clathrin-coated pits. uPAR-LRP complexes are endocytosed via clathrin-coated vesicles and traffic together to early endosomes (EE) because they can be coimmunoprecipitated from immunoisolated EE, and internalization is blocked by depletion of intracellular K(+). Direct binding of domain 3 (D3) of uPAR to LRP is required for clearance of uPA-PAI-1-occupied uPAR because internalization is blocked by incubation with recombinant D3. Moreover, uPA-dependent plasmin generation and the ability of HT1080 cells to migrate through Matrigel-coated invasion chambers are also inhibited in the presence of D3. These results demonstrate that GPI-anchored uPAR is endocytosed by piggybacking on LRP and that direct binding of occupied uPAR to LRP is essential for internalization of occupied uPAR, regeneration of unoccupied uPAR, plasmin generation, and invasion and migration through extracellular matrix.     

Dynamic assembly of the urokinase-type plasminogen activator signaling receptor complex determines the mitogenic activity of urokinase-type plasminogen activator

The urokinase-type plasminogen activator (uPA) receptor (uPAR) functions in concert with co-receptors, including integrins, FPR-like receptor-1/lipoxin A4 receptor, and the epidermal growth factor receptor (EGFR), to initiate cell signaling. uPAR co-receptors may be dynamically organized into a multiprotein signaling receptor complex. In Chinese hamster ovary-K1 (CHO-K1) cells, uPA-binding to uPAR activates ERK/MAP kinase, even though these cells do not express the EGFR; however, when CHO-K1 cells are transfected to express the EGFR, ERK activation becomes EGFR-dependent. In this study, we demonstrate that ERK activation in response to uPA follows equivalent biphasic kinetics in EGFR-expressing and -deficient CHO-K1 cells. In both cell types, the response is pertussis toxin-sensitive; however, uPA promotes cell proliferation exclusively in the EGFR-expressing cells. uPA-induced mitogenic activity requires activation of both STAT5b and ERK. STAT5b was tyrosine-phosphorylated, in response to uPA, only in EGFR-expressing cells. uPA-induced cell proliferation was blocked by dominant-negative MEK1, dominant-negative STAT5b, and by expression of an EGFR that is mutated at Tyr-845, which is essential for STAT5b activation. In two cell culture models of uPA-stimulated breast cancer growth, MDA-MB 468 cells treated with uPA and MCF-7 cells treated with uPA-plasminogen activator inhibitor-1 complex, proliferation was completely inhibited when EGFR expression or activity was blocked. We conclude that expression and assembly of uPAR co-receptors in a specific cell type determines the response to uPA. The EGFR selectively cooperates with uPAR to mediate mitogenesis.