Urokinase receptor expression involves tyrosine phosphorylation of phosphoglycerate kinase

The interaction of urokinase-type plasminogen activator (uPA) with its receptor, uPAR, plays a central role in several pathophysiological processes, including cancer. uPA induces its own cell surface receptor expression through stabilization of uPAR mRNA. The mechanism involves binding of a 51 nt uPAR mRNA coding sequence with phosphoglycerate kinase (PGK) to down regulate cell surface uPAR expression. Tyrosine phosphorylation of PGK mediated by uPA treatment enhances uPAR mRNA stabilization. In contrast, inhibition of tyrosine phosphorylation augments PGK binding to uPAR mRNA and attenuates uPA-induced uPAR expression. Mapping the specific peptide region of PGK indicated that its first quarter (amino acids 1–100) interacts with uPAR mRNA. To determine if uPAR expression by uPA is regulated through activation of tyrosine residues of PGK, we mutated the specific tyrosine residue and tested mutant PGK for its ability to interfere with uPAR expression. Inhibition of tyrosine phosphorylation by mutating Y76 residue abolished uPAR expression induced by uPA treatment. These findings collectively demonstrate that Y76 residue present in the first quarter of the PGK molecule is involved in lung epithelial cell surface uPAR expression. This region can effectively mimic the function of a whole PGK molecule in inhibiting tumor cell growth.     

The receptor for the plasminogen activator of urokinase type is up-regulated in transformed rat thyroid cells.

Five rat thyroid cell lines were tested for the expression of the cell surface receptor for urokinase type plasminogen activator (uPA). All tested lines were found to bind uPA, but transformed 1-5G and Ki-Mol cells, which are also high uPA producers, bound at least ten times more uPA, as compared to non-producers, 'normal' TL5 cells. Moreover, it was possible to remove membrane-bound uPA by treating the cells with phosphatidylinositol-specific phospholipase C, suggesting that rat uPAR, like its human counterpart, is linked to the membrane by a glucosyl-phosphatidylinositol anchor. The specificity of the binding was tested by competition with three different synthetic peptides corresponding to amino acids 14-37 of human, rat and mouse uPA. The results indicate also that the receptor binding region of rat uPA is located within the growth factor domain of the molecule and that its expression may be dependent on the transformed state of the cells.     

Urokinase induces expression of its own receptor in Beas2B lung epithelial cells

Interaction between the urokinase-type plasminogen activator (uPA) and its receptor (uPAR) localizes cellular proteolysis and promotes cellular proliferation and migration. The interaction between uPA and uPAR at the surface of epithelial cells thereby contributes to the pathogenesis of lung inflammation and neoplasia. In this study, we sought to determine if uPA itself alters uPAR expression by lung epithelial cells. uPA enhanced uPAR expression as well as (125)I-uPA binding in Beas2B lung epithelial cells in a time- and concentration-dependent manner. The uPA-mediated induction of uPAR is not accomplished through its receptor and requires enzymatic activity. The low molecular weight fragment of uPA, lacking the receptor binding domain, was as potent as intact two-chain uPA in inducing expression of uPAR at the cell surface. Plasmin, the end product of plasminogen activation, did not alter uPA-mediated uPAR expression. Induction of uPAR by uPA represents a novel pathway by which epithelial cells can regulate uPAR-dependent cellular responses that may contribute to stromal remodeling in lung injury or neoplasia.     

The cleavage of the urokinase receptor regulates its multiple functions

The urokinase-type plasminogen activator (uPA) is able to cleave its cell surface receptor (uPAR) anchored to the cell membrane through a glycophosphatidylinositol tail. The cleavage leads to the formation of cell surface truncated forms, devoid of the N-terminal domain 1 (D1) and unmasks or disrupts, depending on the cleavage site, a sequence in the D1-D2 linker region (residues 88-92), which in the soluble form is a potent chemoattractant for monocyte-like cells. To investigate the possible role(s) of the cleaved forms of cell surface glycophosphatidylinositol-anchored uPAR, uPAR-negative human embrional kidney 293 cells were transfected with the cDNA of intact uPAR (uPAR-293) or with cDNAs corresponding to the truncated forms of uPAR exposing (D2D3-293) or lacking (D2D3wc-293) the peptide 88-92 (P88-92). Cell adhesion assays and co-immunoprecipitation experiments indicated that the removal of D1, independently of the presence of P88-92, abolished the lateral interaction of uPAR with integrins and its capability to regulate integrin adhesive functions. The expression of intact uPAR induced also a moderate increase in 293 cell proliferation, which was accompanied by the activation of ERK. Also this effect was abolished by D1 removal, independently of the presence of P88-92. The expression of intact and truncated uPARs regulated cell directional migration toward uPA, the specific uPAR ligand, and toward fMLP, a bacterial chemotactic peptide. In fact, the uPA-dependent cell migration required the expression of intact uPAR, including D1, whereas the fMLP-dependent cell migration required the expression of a P88-92 containing uPAR and was independent of the presence of D1. Together these observations indicate that uPA-mediated uPAR cleavage and D1 removal, occurring on the cell surface of several cell types, can play a fundamental role in the regulation of multiple uPAR functions.     

Recycling of the urokinase receptor upon internalization of the uPA:serpin complexes.

The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA) but readily internalizes and degrades uPA:serpin complexes in a process that requires the alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2MR-LRP). This process is accompanied by the internalization of uPAR which renders it resistant to phosphatidylinositol-specific phospholipase C (PI-PLC). In this paper we show that during internalization of uPA:serpins at 37 degrees C, analysed by FACScan, immunofluorescence and immunoelectron microscopy, an initial decrease of cell surface uPAR was observed, followed by its reappearance at later times. This effect was not due to redistribution of previously intracellular receptors, nor to the surface expression of newly synthesized uPAR. Recycling was directly demonstrated in cell surface-biotinylated, uPA:PAI-1-exposed cells in which biotinylated uPAR was first internalized and subsequently recycled back to the surface upon incubation at 37 degrees C. In fact, uPAR was resistant to PI-PLC after the 4 degrees C binding of uPA:PAI-1 to biotinylated cells, but upon incubation at 37 degrees C PI-PLC-sensitive biotinylated uPAR reappeared at the cell surface. Binding of uPA:PAI-1 by uPAR, while essential to initiate the whole process, was, however, dispensable at later stages as both internalization and recycling of uPAR could be observed also after dissociation of the bound ligand from the cell surface.     

Physical association of uPAR with the alphaV integrin on the surface of human NK cells

The urokinase-type plasminogen activator receptor (uPAR) serves as a receptor for urokinase plasminogen activator (uPA) and plays a role in invasion and migration of certain immune cells, including NK cells. Although uPAR is anchored to the plasma membrane via a glycosylphosphatidylinositol lipid moiety, we have previously shown that uPAR crosslinking results in MAP kinase signaling and increased integrin expression on the surface of the human NK cell line, YT. We report, herein, that the binding of uPA to uPAR also activates the MAP kinase signaling cascade. Furthermore, we show the physical association between uPAR and integrins on YT cells using cocapping and fluorescence microscopy. These results suggest that signaling initiated by either uPAR binding to uPA or by uPAR clustering may depend on the physical association of uPAR with integrins, a process that may be a prerequisite for NK cell accumulation within established tumor metastases during adoptive therapy.     

Urokinase plasminogen activator/urokinase-specific surface receptor expression and matrix invasion by breast cancer cells requires constitutive p38alpha mitogen-activated protein kinase activity

Overexpression of urokinase plasminogen activator (uPA) and its receptor (uPAR) has been well documented in a wide variety of tumor cells. In breast cancer, expression of uPA/uPAR is essential for tumor cell invasion and metastasis. However, the mechanism responsible for uPA/uPAR expression in cancer cells remains unclear. In the studies reported here, we show that endogenous p38 MAPK activity correlates well with breast carcinoma cell invasiveness. Treatment of highly invasive BT549 cells with a specific p38 MAPK inhibitor SB203580 diminished both uPA/uPAR mRNA and protein expression and abrogated the ability of these cells to invade matrigel, suggesting that p38 MAPK signaling pathway is involved in the regulation of uPA/uPAR expression and breast cancer cell invasion. We also demonstrated that SB203580-induced reduction in uPA/uPAR mRNA expression resulted from the de- stabilization of uPA and uPAR mRNA. Finally, by selectively inhibiting p38alpha or p38beta MAPK isoforms, we demonstrate that p38alpha, rather than p38beta, MAPK activity is essential for uPA/uPAR expression. These studies suggest that p38alpha MAPK signaling pathway is important for the maintenance of breast cancer invasive phenotype by promoting the stabilities of uPA and uPAR mRNA.     

Urokinase receptor cleavage: a crucial step in fibroblast-to-myofibroblast differentiation

Fibroblasts migrate into and repopulate connective tissue wounds. At the wound edge, fibroblasts differentiate into myofibroblasts, and they promote wound closure. Regulated fibroblast-to-myofibroblast differentiation is critical for regenerative healing. Previous studies have focused on the role in fibroblasts of urokinase plasmingen activator/urokinase plasmingen activator receptor (uPA/uPAR), an extracellular protease system that promotes matrix remodeling, growth factor activation, and cell migration. Whereas fibroblasts have substantial uPA activity and uPAR expression, we discovered that cultured myofibroblasts eventually lost cell surface uPA/uPAR. This led us to investigate the relevance of uPA/uPAR activity to myofibroblast differentiation. We found that fibroblasts expressed increased amounts of full-length cell surface uPAR (D1D2D3) compared with myofibroblasts, which had reduced expression of D1D2D3 but increased expression of the truncated form of uPAR (D2D3) on their cell surface. Retaining full-length uPAR was found to be essential for regulating myofibroblast differentiation, because 1) protease inhibitors that prevented uPAR cleavage also prevented myofibroblast differentiation, and 2) overexpression of cDNA for a noncleavable form of uPAR inhibited myofibroblast differentiation. These data support a novel hypothesis that maintaining full-length uPAR on the cell surface regulates the fibroblast to myofibroblast transition and that down-regulation of uPAR is necessary for myofibroblast differentiation.     

Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor

The high-affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays a regulatory role for both extravascular fibrinolysis and uPAR-mediated adhesion and migration on vitronectin-coated surfaces. We have recently proposed that the adhesive function of uPAR is allosterically regulated via a "tightening" of its three-domain structure elicited by uPA binding. To challenge this proposition, we redesigned the uPAR structure to limit its inherent conformational flexibility by covalently tethering domains DI and DIII via a non-natural interdomain disulfide bond (uPAR(H47C-N259C)). The corresponding soluble receptor has 1) a smaller hydrodynamic volume, 2) a higher content of secondary structure, and 3) unaltered binding kinetics towards uPA. Most importantly, the purified uPAR(H47C-N259C) also displays a gain in affinity for the somatomedin B domain of vitronectin compared with uPAR(wt), thus recapitulating the improved affinity that accompanies uPA-uPAR(wt) complex formation. This functional mimicry is, intriguingly, operational also in a cellular setting, where it controls lamellipodia formation in uPAR-transfected HEK293 cells adhering to vitronectin. In this respect, the engineered constraint in uPAR(H47C-N259C) thus bypasses the regulatory role of uPA binding, resulting in a constitutively active uPAR. In conclusion, our data argue for a biological relevance of the interdomain dynamics of the glycolipid-anchored uPAR on the cell surface.     

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.     

Down‐regulation of uPA and uPAR by 3,3′‐diindolylmethane contributes to the inhibition of cell growth and migration of breast cancer cells

3,3′‐Diindolylmethane (DIM) is a known anti‐tumor agent against breast and other cancers; however, its exact mechanism of action remains unclear. The urokinase plasminogen activator (uPA) and its receptor (uPAR) system are involved in the degradation of basement membrane and extracellular matrix, leading to tumor cell invasion and metastasis. Since uPA‐uPAR system is highly activated in aggressive breast cancer, we hypothesized that the biological activity of B‐DIM could be mediated via inactivation of uPA‐uPAR system. We found that B‐DIM treatment as well as silencing of uPA‐uPAR led to the inhibition of cell growth and motility of MDA‐MB‐231 cells, which was in part due to inhibition of VEGF and MMP‐9. Moreover, silencing of uPA‐uPAR led to decreased sensitivity of these cells to B‐DIM indicating an important role of uPA‐uPAR in B‐DIM‐mediated inhibition of cell growth and migration. We also found similar effects of B‐DIM on MCF‐7, cells expressing low levels of uPA‐uPAR, which was due to direct down‐regulation of MMP‐9 and VEGF, independent of uPA‐uPAR system. Interestingly, over‐expression of uPA‐uPAR in MCF‐7 cells attenuated the inhibitory effects of B‐DIM. Our results, therefore, suggest that B‐DIM down‐regulates uPA‐uPAR in aggressive breast cancers but in the absence of uPA‐uPAR, B‐DIM can directly inhibit VEGF and MMP‐9 leading to the inhibition of cell growth and migration of breast cancer cells. J. Cell. Biochem. 108: 916–925, 2009. © 2009 Wiley‐Liss, Inc.     

Urokinase-type plasminogen activator binding to its receptor stimulates tumor cell migration by enhancing integrin-mediated signal transduction.

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) participate in matrix degradation and cell migration by focusing proteolysis and functioning as a signaling ligand/receptor complex. uPAR, anchored by a lipid moiety in the membrane, is thought to require a transmembrane adapter to transduce signals into the cytoplasm. To study uPAR signaling, we transfected the prostate carcinoma cell line LNCaP, which does not express endogenous uPA or uPAR, with a uPAR encoding cDNA, resulting in high-level surface expression. We studied migration of these cells on fibronectin, which is mediated by the integrin alpha5beta1. Ligation of uPAR with uPA or its amino-terminal fragment enhanced haptotactic migration to fibronectin. In cells on fibronectin, but not on poly-l-lysine, ligation of uPAR also resulted in tyrosine phosphorylation of several proteins, including two proteins involved in integrin signaling, focal adhesion kinase and the crk-associated substrate p130(Cas). Furthermore, after uPAR ligation, uPAR was co-immunoprecipitated with beta1 integrins from the detergent-insoluble fraction of cell lysates. Thus, our data suggest that uPAR occupancy results in an interaction between uPAR and integrins and a potentiation of integrin-mediated signaling, which leads to enhanced cell migration.     

In vivo paracrine interaction between urokinase and its receptor: effect on tumor cell invasion

Numerous studies have linked the production of increased levels of urokinase type plasminogen activator (uPA) with the malignant phenotype. It has also been shown that a specific cell surface receptor can bind uPA through a domain distinct and distant from the proteolytic domain. In an in vivo model of invasion, consisting of experimentally modified chorioallantoic membrane (CAM) of a chick embryo, only cells that concurrently expressed both uPA and a receptor for uPA, and in which the receptor was saturated with uPA, were efficient in invasion. To test whether uPA produced by one cell can, in a paracrine fashion, affect the invasive capacity of a receptor-expressing cell, we transfected LB6 mouse cells with human uPA (LB6[uPA]), or human uPA-receptor cDNA (LB6[uPAR]). LB6(uPA) cells released into the medium 1-2 Ploug units of human uPA per 10(6) cells in 24 h. The LB6(uPAR) cells expressed on their surface approximately 12,000 high affinity (Kd 1.7 x 10(-10) M uPA binding sites per cell. Unlabeled LB6(uPA) and 125-IUdR-labeled LB6(uPAR) cells were coinoculated onto experimentally wounded and resealed CAMs and their invasion was compared to that of homologous mixtures of labeled and unlabeled LB6(uPAR) or LB6(uPA) cells. Concurrent presence of both cell types in the CAMs resulted in a 1.8-fold increase of invasion of the uPA-receptor expressing cells. A four-fold stimulation of invasion was observed when cells were cocultured in vitro, prior to in vivo inoculation. Enhancement of invasion was prevented in both sets of experiments by treatment with specific antihuman uPA antibodies, indicating that uPA was the main mediator of the invasion-enhancing, paracrine effect on the receptor-expressing cells.