Ebbe Rønne

Cell-induced potentiation of the plasminogen activation system is abolished by a monoclonal antibody that recognizes the NH2-terminal domain of the urokinase receptor

We have raised four monoclonal antibodies recognizing different epitopes within the human cell‐surface receptor for urokinase‐type plasminogen activator (u‐PA). One of these antibodies completely abolishes the potentiation of plasmin generation observed upon incubation of the zymogens pro‐u‐PA and plasminogen with U937 cells. This antibody, which is also the only one to completely inhibit the binding of DFP‐inactivated [125I]‐u‐PA to U937 cells, is directed against the u‐PA binding NH2‐terminal domain of u‐PAR, a well‐defined fragment formed by limited chymotrypsin digestion of purified u‐PAR, demonstrating the functional independence of the u‐PA binding domain as well as the critical role of u‐PAR in the assembly of the cell‐surface plasminogen activation system.

The receptor for urokinase plasminogen activator is present in plasma from healthy donors and elevated in patients with paroxysmal nocturnal haemoglobinuria

The urokinase plasminogen activator (uPA) is a proteolytic enzyme which converts the proenzyme plasminogen to the active serine protease plasmin. A cell surface receptor for uPA (uPAR) is attached to the cell membrane by a glycosyl‐phosphatidylinositol anchor. Binding of uPA to uPAR leads to an enhanced plasmin formation and thereby an amplification of pericellular proteolysis. We have shown previously that uPAR is expressed on normal blood monocytes and granulocytes, but is deficient on affected blood monocytes and granulocytes in patients with paroxysmal nocturnal haemoglobinuria (PNH), and that uPAR is present in plasma from these patients. In this study a newly established sensitive enzyme‐linked immunosorbent assay (ELISA) has been applied for quantttation of uPAR in plasma. Unexpectedly, we found that uPAR is not only present in PNH plasma but also in plasma from healthy individuals. In 39 healthy individuals the mean plasma‐uPAR value ±SD was 31 ± 15 pm, median 28 (range 11‐108), and the corresponding value for six PNH patients was 116±67 pm, median 90 (range 61‐228). The elevated uPAR‐level in PNH patients was highly significant (Mann‐Whitney test; P < 0.0001), and may possibly contribute to the propensity for thrombosis in PNH by inhibition of the fibrinolytic system. Binding of pro‐uPA by uPAR in plasma may interfere with the appropriate binding of pro‐uPA to cell‐bound uPAR and therefore inhibit cell‐associated plasmin generation and fibrinolysis. It is likely that the uPAR in normal plasma reflects the overall level of activity of the uPAR‐mediated cell surface proteolysis. The present ELISA may be used for studies of uPAR levels in plasma from patients with conditions in which this activity might be increased, such as cancer and inflammatory disorders. Future studies will determine if uPAR in plasma is a parameter of clinical importance in these diseases.

Urokinase receptor in breast cancer tissue extracts. Enzyme-linked immunosorbent assay with a combination of mono- and polyclonal antibodies

SummaryUrokinase plasminogen activator (uPA) is a proteolytic enzyme involved in degradation of the extracellular matrix during cancer invasion. The levels of uPA and its inhibitor PAI-1 in tumor extracts have previously been demonstrated to be of prognostic value in breast cancer as well as other types of cancer. We have previously characterized a specific cell surface receptor for uPA (uPAR) which strongly enhances the catalytic activity of uPA and is expressed during mammary cancer invasion. In order to quantitate uPAR in breast cancer tissue, we have now developed a sensitive enzyme-linked immunosorbent assay (ELISA), with polyclonal catching antibodies and three monoclonal detecting antibodies. The detection limit of the assay is approximately 0.16 fmol of uPAR in a volume of 100 µl (1.6 pM). There is a linear relationship between signal and uPAR concentration up to at least 6.6 fmol per 100 µl (66 pM). Both free uPAR and uPAR in complex with uPA is detected. The recovery of an internal uPAR standard in breast cancer tissue extracts is above 87%. The intra-assay and inter-assay variation coefficients are 7% and 13%. In order to find a suitable buffer for extraction of various components of the uPA-system from breast cancer tissue, we tested buffers which previously have been used for optimal extraction of estrogen receptor (A), uPA (B), and uPAR (C). Buffer A and B extracted approximately 30% and 50%, respectively, of the amount of uPAR extracted with buffer C. Extracts of samples of breast cancer tissue from 94 patients all contained uPAR in amounts above the detection limit of the present assay, which appears suitable for studies of the potential prognostic value of uPAR in this disease. Significant correlations were found between uPAR, uPA and PAI-1 tumor levels.

Elevated plasma levels of urokinase plasminogen activator receptor in non-small cell lung cancer patients

The urokinase plasminogen activator (uPA) is involved in extracellular matrix degradation during cancer invasion. Binding of uPA to a specific cell surface receptor (uPAR) is a key step in this process. We have previously reported that high levels of uPAR in squamous cell lung cancer tissue extracts are associated with poor prognosis (Pedersen et al., Cancer Res 1994, 54, 4671-4675). Recently we found that uPAR is present in blood plasma from healthy donors as determined by enzyme-linked immunosorbent assay (ELISA) and chemical cross-linking. We now report that uPAR in plasma from 17 patients with non-small cell lung cancer (NSCLC) was significantly higher than in 30 healthy controls (P = 0.0004), while no significant increase was found in plasma from 14 patients with small cell lung cancer (SCLC). The increased levels of uPAR in the plasma from NSCLC patients is likely to be due to release of uPAR from the tumour tissue, and may, therefore, be related to prognosis.

Quantitation of the receptor for urokinase plasminogen activator by enzyme-linked immunosorbent assay

Binding of the urokinase plasminogen activator (uPA) to a specific cell surface receptor (uPAR) plays a crucial role in proteolysis during tissue remodelling and cancer invasion. An immunosorbent assay for the quantitation of uPAR has now been developed. This assay is based on two monoclonal antibodies recognizing the non-ligand binding part of this receptor, and it detects both free and occupied uPAR, in contrast to ligand-binding assays used previously. In a variant of the assay, the occupied fraction of uPAR is selectively detected with a uPA antibody. To be used as a standard, a soluble variant of uPAR, suPAR, has been constructed by recombinant technique and the protein content of a purified suPAR standard preparation was determined by amino acid composition analysis. The sensitivity of the assay (0.6 ng uPAR/ml) is strong enough to measure uPAR in extracts of cultured cells and cancer tissue. Recent studies have shown that a high uPA level in tumor extracts is in some cancers associated with poor prognosis. The present assay will now allow similar prognostic studies of uPAR levels.

Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor: a study based on biosensor technology

Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance or interference with conformational properties of the receptor critical for ligand binding. This distinction is central when employing the antibodies as tools in the elucidation of the structure-function relationship of the protein in question. We have studied the effect of monoclonal antibodies against the urokinase plasminogen activator receptor (uPAR), a protein located on the surface of various types of malignant and normal cells which is involved in the direction of proteolytic degradation reactions in the extracellular matrix. We show that surface plasmon resonance/biomolecular interaction analysis (BIA) can be employed as a highly useful tool to characterize the inhibitory mechanism of specific antagonist antibodies. Two inhibitory antibodies against uPAR, mAb R3 and mAb R5, were shown to exhibit competitive and non-competitive inhibition, respectively, of ligand binding to the receptor. The former antibody efficiently blocked the receptor against subsequent ligand binding but was unable to promote the dissociation of a preformed receptor-ligand complex. The latter antibody was capable of binding the preformed complex, forming a transient trimolecular assembly, and promoting the dissociation of the uPA/uPAR complex. The continuous recording of binding and dissociation, obtained in BIA, is central in characterizing these phenomena. The identification of a non-competitive inhibitory mechanism against this receptor reveals the presence of a determinant which influences the binding properties of a remote site in the molecular structure and which could be an important target for a putative synthetic antagonist.

Cellular receptor for urokinase-type plasminogen activator: protein structure

Publisher Summary This chapter describes the cellular receptor for urokinase-type plasminogen activator (uPAR). The urokinase pathway of plasminogen activation is assumed to play an important role in extracellular proteolytic events in a number of tissue degradation processes, occurring under normal as well as pathological conditions, including cancer invasion. Therefore, the existence of a specific cell surface binding site for the urokinase-type plasminogen activator (uPa) has aroused great interest. A number of in vitro studies have provided clues as to possible roles of the uPAR-uPA interaction. On certain cell types, receptor-bound uPA is discretely localized at cell-cell and focal cell-substratum contact sites. Concomitant binding of the zymogen pro-uPA to uPAR and of plasminogen to unidentified binding sites at cell surfaces leads to a strong enhancement in plasmin generation compared to the liquid-phase situation. These and other observations have led to the speculation that uPAR provides specific areas on surfaces with the potential for preferential plasminogen activation and extracellular proteolysis.

A novel, specific pro-urokinase complex on monocyte-like cells, detected by transglutaminase-catalyzed cross-linking

Radiolabeled pro‐urokinase plasminogen activator (pro‐uPA) was cross‐linked to a specific protein on the surface of human monocyte‐like U937 cells in a reaction catalyzed by tissue transglutaminase. The conjugate formed with this unknown component had a much higher molecular weight (apparent M r, 250,000–300,000) than the complex of pro‐uPA and the urokinase plasminogen activator receptor (uPAR). There was a strong preference for the pro‐form of uPA. The conjugate was recognized by antibodies against uPA but not by anti‐uPAR antibodies. Nevertheless, the blocking of uPAR with a monoclonal antibody abolished the formation of the conjugate, thus showing a role of uPAR in this process.

Confocal fluorescence microscopy of urokinase plasminogen activator receptor and cathepsin D in human MDA-MB-231 breast cancer cells migrating in reconstituted basement membrane.

Using confocal fluorescence microscopy with a monoclonal antibody, we have localized the receptor for urokinase plasminogen activator (uPAR) in MDA-MB-231 human breast cancer cells migrating into a reconstituted basement membrane. Patchy and polarized uPAR immunoreactivity was found at the cell membrane, and strong staining was found both in the ruffled border or leading edge of the cells and at pseudopodia penetrating into the membrane. Intracellular uPAR staining was localized in the paranuclear region and in rounded granule-like structures; some of these were identified as lysosomes by double staining for uPAR and the lysosomal enzyme cathepsin D. Urokinase plasminogen activator (uPA) activity has previously been shown to play a role in migration of cells into basement membranes, and it has been proposed that uPAR also is involved in this process. uPA is known to be internalized and degraded after complex formation with the inhibitor PAI-1. Lysosomal uPAR immunoreactivity may result from concomitant internalization of the receptor.

Urokinase and urokinase receptor expression in somatic cell hybrids

To study the mechanisms that control cell-bound urokinase (u-PA) activity we characterised the expression of u-PA and its cellular receptor (u-PAR) in somatic cell hybrids between mouse L fibroblasts that do not produce u-PA and human HT1080 fibrosarcoma cells that express high levels of u-PA and u-PAR. Although the hybrids possessed a number of copies of the human u-PA and u-PAR genes comparable to that of HT1080 cells, they had levels of human u-PA mRNA and u-PAR mRNA significantly lower than HT1080 cells. Accordingly, the levels of u-PA and u-PAR were lower in the hybrids than in HT1080 cells. However, the hybrid cells surprisingly had a 2- to 6-fold higher u-PA-binding capacity than HT1080 cells. Some hybrid cells expressed glycosilation variants of u-PAR. In addition, in the hybrids, that express very low levels of u-PA, u-PAR was predominantly in the ligand-binding, three-domain form, whereas HT1080 cells, that have a high u-PA level, possessed higher levels of the cleaved, two-domain form of u-PAR devoid of ligand-binding activity. This indicates that the u-PA-binding capacity is controlled not only by u-PAR expression but also depends on receptor affinity and cleavage by u-PA or plasmin.