uPA AND uPAR FUNCTION
Localized Proteolysis
The primary function of uPAR was initially thought to be cell-associated proteolysis through localization of uPA and hence the plasminogen activation cascade to the cell surface.
By polarizing uPA at focal areas of the cell membrane, uPAR participates in cell-associated proteolysis during physiological processes with tissue remodelling and during tumour growth, invasion and metastasis [2,42,63]. Although the primary initiator of the cell-associated PA-system is unknown, pro-uPA itself, tPA, other proteases or autoactivation (specific orientation effects that favour the interaction between cell-bound uPA and cell-bound plasminogen) may initiate the cell-associated cascade [39,42]. However, acceleration of the positive feed back mechanism between plasmin-catalyzed activation of pro-uPA and uPA-catalyzed activation of plasminogen requires not only the binding of pro-uPA to uPAR but also binding of plasminogen to components on the cell surface. The acceleration is therefore most likely due to an effect on both reactions [39,42].In addition to localized proteolysis, uPAR can also modulate cell adhesion and migration, chemotaxis, proliferation and differentiation through intracellular signalling [5-10].
Signal Transduction
Although uPAR lacks a cytoplasmatic domain, binding of uPA (or pro-uPA or ATF) activates several signal transduction pathways involved in adhesion, migration, chemotaxis, cytoskeleton dynamics, proliferation and differentiation [7,77]. Initiation of intracellular signalling may either be induced by a conformational change, by proteolytic cleavage of uPAR or through interaction with molecules containing cytoplasmic domains (transmembrane adaptor molecules) such as integrins, caveolin and a G-protein coupled receptor [7,75,77,78].
Adhesion and Migration
To migrate, cells must adhere to the ECM and activate the integrin-dependent signalling pathway that induces cytoskeleton reorganization and cell-shape changes.
During cell migration, cell-ECM adhesion at the leading edge provides guidance and mechanical force whereas dissociation of the adhesion molecule/ligand complexes allows retraction of the trailing edge [5,79].uPAR mediates the initial binding of cells to a matrix substrate either by direct binding to vitronectin [80] or by integrin interaction/activation [78,79]. uPAR has high lateral mobility in the cell membrane and during cell migration it usually becomes concentrated at focal adhesion sites (cell-cell or cell-ECM contact sites) and at the leading edge, co-localizing with various transmembrane adaptor molecules [7,74,78,79]. The colocalization often occurs in cell surface areas termed lipid rafts which are membrane components enriched in cholesterol, glycosphingolipids, gangliosides and GPI-anchored molecules [7,81].
The cell-associated proteolysis, which occurs upon binding of uPA to uPAR, may also promote migration through degradation of ECM components and adhesion molecules, resulting in release of the trailing edge [5].
uPAR and Integrins
Integrins are transmembrane cell surface receptors that mediate cell-ECM and cell-cell contact and transduce signals from the extracellular environment to the cell interior. Ligand-activated uPAR influences integrin- dependent cell adhesion and migration [74] and these functions are blocked, not only by anti-integrin Ab, but also by anti-uPAR Ab [7]. Co-clustering of the antigen receptor complex with β1 or β2-integrins on T cells increases uPAR mRNA and protein expression and promotes migration of T cells in vitro [56]. In addition, removal of uPAR from the leukocyte surface reduces β2-integrin-mediated leukocyte adhesion to the endothelium in vitro [76] and uPAR-deficient mice have impaired β2-integrin-mediated leukocyte recruitment to sites of inflammation [76,82,83].
Studies of uPAR-deficient mice have demonstrated that uPAR is important for leukocyte recruitment to sites of inflammation/infection and for the antibacterial host defence.
Thus, uPAR-deficient mice have impaired recruitment of neutrophils, monocytes and T cells to inflamed peritoneum [76] and impaired lymphocyte recruitment to inflamed lung [83]. uPAR-deficient mice with pulmonary Pseudomonas (P.) aeruginosa [82] or S. pneumoniae [84] infection have impaired neutrophil [82,84], macrophage and lymphocyte [84] recruitment, impaired pathogen clearance [82,84], enhanced dissemination of the infection and a poor survival [84]. In addition, neutrophil phagocytosis and superoxide generation is impaired in uPAR-deficient mice [85]. Since neutrophil recruitment to the pulmonary parenchyma is β2-integrin dependent in P. aeruginosa infection [82] and β-2-integrin independent in S. pneumoniae infection [84], an abolished uPAR/integrin-interaction cannot alone explain the impaired neutrophil recruitment in uPAR-deficient mice.The uPAR-integrin interaction is most likely mediated by at least two mechanisms: simple interactions at the cell surface (activation of integrins) or interactions resulting in complex formation between integrins, caveolin and uPAR, which function as adhesive and signalling units at the cell surface [78].
The uPAR/integrin interactions have most often been demonstrated within the same cell membrane [74,75,79]. However, it has also been reported that the uPAR/integrin interaction can modulate cell-cell interaction [86]. The interaction most likely requires uPAR(I-III) as cleavage between uPAR(I) and uPAR(II) prevents co-localization of uPAR and integrins [75].
uPAR And Lipid Rafts
Lipid rafts are membrane microdomains that are enriched with lipids and cholesterol, glycolipids, sphingolipids and the cholesterol-binding protein caveolin [7].
GPI-anchored receptors have high affinity towards lipid rafts and uPAR is no exception [100]. Cunningham and co-workers found that uPAR exists in both monomeric and dimeric forms, and that dimeric uPAR partitions preferentially to lipid rafts. The dimerization of uPAR is a prerequisite for the high-affinity interaction between uPAR and vitronectin [51,100].
uPAR-mediated adhesion to vitronectin correlates with the formation of multimeric membrane complexes consisting of integrins, caveolin and uPAR and is associated with the activation of intracellular signalling pathways and the cytoskeleton system involved in cell adhesion and migration [74,78,79].
uPAR and G-Protein Coupled Receptors
The previous finding that chemotaxis induced by the chemotactic epitope in suPAR(II-III) could be blocked by Bordetella pertussis toxin (inhibits some G-proteins), indicated that suPAR (II-III) induced chemotaxis is mediated through a G-protein coupled receptor [87]. This was later shown to be the low-affinity G-protein coupled receptor, FPRL1 [88], and co-expression of uPAR and FPLR1 is required for cell responsiveness to suPAR(II-III) [75,88]. In addition to integrins, caveolin and a G-protein coupled receptor, other transmembrane adaptor molecules also interact with uPAR and may modulate cell adhesion and migration: gp130 (activated upon uPAR clustering), LRP (internalizes the uPAR/uPA/PAI-1 complex), insulin like growth factor II receptor (binds and degrades suPAR(II-III)) [7] and urokinase-receptor-associated protein [39,89].
uPAR, Vitronectin and PAI-1
The interaction uPAR/vitronectin/PAI-1 also regulates cell adhesion and migration. uPAR and PAI-1 share binding regions on vitronectin and thus bind competitively [49,80]. An excess of PAI-1 will replace uPAR from vitronectin resulting in detachment of cells from vitronectin and inhibition of cell migration. Conversely, an excess of uPA will promote attachment through enhanced binding of vitronectin to uPAR and detachment through plasmin-catalyzed disruption of the uPAR-vitronectin binding, thereby promoting cell migration [42,49,78]. Thus, the function of uPA and PAI-1 at the leading edge of migrating cells may not only be to enhance or inhibit plasminogen activation, respectively, but also to modulate cell migration. Overall, uPAR participates in cell migration through proteolytic as well as non-proteolytic functions.
It is possible that proteolytic mechanisms dominate at the trailing edge whereas non-proteolytic mechanisms dominate at the leading edge during normal cell migration [5]. This is opposite tumour cell invasion, in which proteolytic mechanisms seem to be of major importance for degradation of basement membrane barriers at the leading edge [5].Chemotaxis
uPA (or pro-uPA or ATF) binding to uPAR and intra-domain cleavage of uPAR(I-III) both cause a conformational change in uPAR. This can result in exposure of a chemotactic epitope located in the proteasesensitive linker region between uPAR(I) and uPAR(II) (minimum required sequence SRSRY, amino-acid residues 88-92) [87]. Exposure of the chemotactic epitope in vitro not only depends on interaction with uPA as the linker region is sensitive to other proteases and suPAR(I-III)-bound uPA does not expose the chemotactic epitope [8,41,42,64]. Thus, chymotrypsin-catalyzed cleavage of suPAR(I-III) between uPAR(I) and uPAR(II) generates a soluble chemotactic factor, suPAR(II-III) [87,90]. The chemotactic epitope of uPAR interacts with FPRL1 [88] which is expressed by monocytes, lymphocytes and neutrophils and upregulated by various cytokines and growth factors [91]. FPRL1 is also present on many other cells including vascular endothelial cells, epithelial cells, hepatocytes and tumour cells [91] and the widespread expression of FPRL1 is in accordance with the broad range of cells that respond to uPA/uPAR chemotactic signals.
In addition to inducing chemotaxis, suPAR(II-III) decreases the activity of the FPRL1-ligand, fMLP (formyl methionyl leucyl proline) [88] and inhibits chemokine-induced chemotaxis in response to MCP-1 and RANTES [92]. suPAR(II-III) inhibits MCP-1 and RANTES-induced chemotaxis by preventing rapid integrin- dependent cell adhesion.
FPRL1 regulates β2-integrin activities, which is important for monocyte motility. The suPAR(II-III) induced decreased chemotaxis is therefore not due to direct suPAR(II-III)-integrin binding, but is caused by suPAR(II- III) binding to FPRL1.
uPA-uPAR complexes inhibits chemokine induced monocyte chemotaxis in the same manner whereas neither suPAR(I-III) nor suPAR(I) regulate chemotaxis, since they do not expose a chemotactic sequence.The uPAR-derived peptide uPAR84-95, containing the chemotactic epitope, has also been found to chemoattract CD34+ leukaemia cells and CD34+ haematopoietic stem cells (HSCs) and inhibit the stromal- derived factor (SDF) 1-induced migration of CD34+ HSCs by binding to FPRL1. Chemotactically active suPAR hereby seems to be able to mobilise HSCs from the bone marrow and prevent the SDF1-mediated bone marrow retention of HSCs. The authors suggested this to be mediated through heterologous desensitization of the SDF1 receptor CXCR4, but another explanation could be that chemotactically active suPAR inhibits SDF1-induced migration by affecting integrin activation, as observed in monocytes [96,97].
Other Functions of uPAR
uPAR is often over-expressed by human tumour cells, and uPAR-expression and integrin interaction promotes proliferation of certain tumour cells in vitro [7]. Expression of uPAR(I-III) is required for integrin interaction and induction of proliferation as uPAR(II-III) does not interact with integrins [75].
Binding of uPAR to vitronectin is essential for adhesion and hence differentiation of monocytes into macrophages in vitro [7]. In addition, expression of uPAR on maturating monocytes, granulocytes or bone marrow stromal cells may interact with and modify integrin function, thereby contributing to release of monocytes and granulocytes from the bone marrow [63].