Pia Ragno

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.

Expression and functions of the vascular endothelial growth factors and their receptors in human basophils.

Angiogenesis is a multistep complex phenomenon critical for several inflammatory and neoplastic disorders. Basophils, normally confined to peripheral blood, can infiltrate the sites of chronic inflammation. In an attempt to obtain insights into the mechanism(s) underlying human basophil chemotaxis and its role in inflammation, we have characterized the expression and function of vascular endothelial growth factors (VEGFs) and their receptors in these cells. Basophils express mRNA for three isoforms of VEGF-A (121, 165, and 189) and two isoforms of VEGF-B (167 and 186). Peripheral blood and basophils in nasal polyps contain VEGF-A localized in secretory granules. The concentration of VEGF-A in basophils was 144.4 ± 10.8 pg/106 cells. Immunologic activation of basophils induced the release of VEGF-A. VEGF-A (10–500 ng/ml) induced basophil chemotaxis. Supernatants of activated basophils induced an angiogenic response in the chick embryo chorioallantoic membrane that was inhibited by an anti-VEGF-A Ab. The tyrosine kinase VEGFR-2 (VEGFR-2/KDR) mRNA was expressed in basophils. These cells also expressed mRNA for the soluble form of VEGFR-1 and neuropilin (NRP)1 and NRP2. Flow cytometric analysis indicated that basophils express epitopes recognized by mAbs against the extracellular domains of VEGFR-2, NRP1, and NRP2. Our data suggest that basophils could play a role in angiogenesis and inflammation through the expression of several forms of VEGF and their receptors.

Soluble and cleaved forms of the urokinase-receptor: degradation products or active molecules?

The urokinase-mediated plasminogen activation (PA) system is involved in many physiological and pathological events that include cell migration and tissue remodelling, such as embryogenesis, ovulation, inflammation, wound healing, angiogenesis, and tumor invasion and metastasis. The urokinase receptor (uPAR) is a key molecule of this system and can bind extracellular and cell membrane molecules such as urokinase (uPA), vitronectin (VN), integrins and chemotaxis receptors. These multiple interactions can be modulated by the shedding or the cleavage of the cell membrane receptor. Indeed, cleaved forms of uPAR, lacking the N-terminal D1 domain, have been detected on the surface of cells and in tissues, while soluble forms have been found in biological fluids. Cleaved and soluble forms could represent the intermediary products of the uPAR metabolism or active molecules with precise and distinct functional roles. Here, we review the data concerning the in vitro and in vivo identification of these uPAR forms, their origin and functions, and the role that uPAR shedding and cleavage could play in biological processes.

In vivo Activity of the Cleaved Form of Soluble Urokinase Receptor: A New Hematopoietic Stem/Progenitor Cell Mobilizer

Cleaved forms of soluble urokinase receptor (c-suPAR) have been detected in body fluids from patients affected by various tumors. We recently reported increased c-suPAR levels in sera of healthy donors during granulocyte colony-stimulating factor (G-CSF)-induced mobilization of CD34(+) hematopoietic stem cells (HSC). In vitro, c-suPAR or its derived chemotactic peptide (uPAR(84-95)) stimulated migration of human CD34(+) HSCs and inactivated CXCR4, the chemokine receptor primarily responsible for HSC retention in bone marrow. These results suggested that c-suPAR could potentially contribute to regulate HSC trafficking from and to bone marrow. Therefore, we investigated uPAR(84-95) effects on mobilization of mouse CD34(+) hematopoietic stem/progenitor cells (HSC/HPC). We first showed that uPAR(84-95) stimulated in vitro dose-dependent migration of mouse CD34(+) M1 leukemia cells and inactivated murine CXCR4. uPAR(84-95) capability to induce mouse HSC/HPC release from bone marrow and migration into the circulation was then investigated in vivo. uPAR(84-95) i.p. administration induced rapid leukocytosis, which was associated with an increase in peripheral blood CD34(+) HSCs/HPCs. In vitro colony assays confirmed that uPAR(84-95) mobilized hematopoietic progenitors, showing an absolute increase in circulating colony-forming cells. uPAR(84-95) mobilizing activity was comparable to that of G-CSF; however, neither synergistic nor additive effect was observed in combining the two molecules. These findings show for the first time in vivo biological effects of c-suPAR. Its capability to mobilize HSCs suggests potential clinical applications in HSC transplantation.

Multiple activities of a multifaceted receptor: roles of cleaved and soluble uPAR.

The urokinase-type plasminogen activator receptor (uPAR) is a GPI-anchored cell-surface receptor involved in many physiological and pathological events that include cell migration and tissue invasion. uPAR traditional role was considered the focusing of uPA proteolytic activity on the cell surface; however, different uPAR activities have been demonstrated in the last years. In fact, cell surface uPAR functionally interacts with integrins, fMLP-receptors (fMLP-Rs) and growth factor receptors, thus regulating cell adhesion, migration and proliferation. uPAR also exists in a soluble form (suPAR) that has been detected in human body fluids. Both cell surface and suPAR can be proteolytically cleaved, thus generating truncated forms lacking the N-terminal domain and exposing the specific sequence able to interact with the fMLP-Rs. The cleaved form of suPAR binds and activates the fMLP-Rs and regulates the activity of MCP-1, RANTES and SDF1 receptors. Here, we review the role that shedding and cleavage could play in regulating uPAR structural/functional interaction with other cell-surface receptors and in uPAR-mediated biological and pathological processes.

Urokinase-mediated posttranscriptional regulation of urokinase-receptor expression in non small cell lung carcinoma.

The urokinase‐type plasminogen activator (uPA) and its cellular receptor (uPAR) are involved in the proteolytic cascade required for tumor cell dissemination and metastasis, and are highly expressed in many human tumors. We have recently reported that uPA, independently of its enzymatic activity, is able to increase the expression of its own receptor in uPAR‐transfected kidney cells at a posttranscriptional level. In fact, uPA, upon binding uPAR, modulates the activity and/or the level of a mRNA‐stabilizing factor that binds the coding region of uPAR‐mRNA. We now investigate the relevance of uPA‐mediated posttranscriptional regulation of uPAR expression in non small cell lung carcinoma (NSCLC), in which the up‐regulation of uPAR expression is a prognostic marker. We show that uPA is able to increase uPAR expression, both at protein and mRNA levels, in primary cell cultures obtained from tumor and adjacent normal lung tissues of patients affected by NSCLC, thus suggesting that the enzyme can exert its effect in lung cells. We investigated the relationship among the levels of uPA, uPAR and uPAR‐mRNA binding protein(s) in NSCLC. Lung tissue analysis of 35 NSCLC patients shows an increase of both uPA and uPAR in tumor tissues, as compared to adjacent normal tissues, in 27 patients (77%); 19 of these 27 patients also show a parallel increase of the level and/or binding activity of a cellular protein capable of binding the coding region of uPAR‐mRNA. Therefore, in tumor tissues, a strong correlation is observed among these 3 parameters, uPA, uPAR and the level and/or the activity of a uPAR‐mRNA binding protein. We then suggest that uPA regulates uPAR expression in NSCLC at a posttranscriptional level by increasing uPAR‐stability through a cellular factor that binds the coding region of uPAR‐mRNA.

Role of uPA/uPAR in the Modulation of Angiogenesis

Blood vessels connect all districts of the body and allow blood oxygen and nutrients to reach every cell in the organism. Dysregulation of blood vessel formation or functionality is the origin of a large number of diseases. During new vessel formation, endothelial cells degrade their basement membrane, migrate into the interstitial matrix and proliferate. Migrating endothelial cells need to be polarized, to focus at their leading edge the proteolytic machinery, which is essential for extracellular matrix degradation; thus, proteases and their receptors play a crucial role in angiogenesis. The urokinase-mediated plasminogen activation system is a complex system of serine proteases strongly involved in angiogenesis. The plasminogen activation system includes plasminogen/plasmin, activators, inhibitors and cell receptors. In the last decades, a large body of evidence has clearly indicated that the role of this system is not limited to extracellular matrix proteolysis but can contribute to all phases of the angiogenic process.

PED is overexpressed and mediates TRAIL resistance in human non-small cell lung cancer

PED (phosphoprotein enriched in diabetes) is a death‐effector domain (DED) family member with a broad anti‐apoptotic action. PED inhibits the assembly of the death‐inducing signalling complex (DISC) of death receptors following stimulation. Recently, we reported that the expression of PED is increased in breast cancer cells and determines the refractoriness of these cells to anticancer therapy. In the present study, we focused on the role of PED in non‐small cell lung cancer (NSCLC), a tumour frequently characterized by evasion of apoptosis and drug resistance. Immunohistochemical analysis of a tissue microarray, containing 160 lung cancer samples, indicated that PED was strongly expressed in different lung tumour types. Western blotting performed with specimens from NSCLC‐affected patients showed that PED was strongly up‐regulated (>6 fold) in the areas of tumour compared to adjacent normal tissue. Furthermore, PED expression levels in NSCLC cell lines correlated with their resistance to tumour necrosis factor related apoptosis‐inducing ligand (TRAIL)‐induced cell death. The involvement of PED in the refractoriness to TRAIL‐induced cell death was investigated by silencing PED expression in TRAIL‐resistant NSCLC cells with small interfering (si) RNAs: transfection with PED siRNA, but not with cFLIP siRNA, sensitized cells to TRAIL‐induced cell death. In conclusion, PED is specifically overexpressed in lung tumour tissue and contributes to TRAIL resistance.

Discovery of new inhibitors of Cdc25B dual specificity phosphatases by structure-based virtual screening.

Cell division cycle 25 (Cdc25) proteins are highly conserved dual specificity phosphatases that regulate cyclin-dependent kinases and represent attractive drug targets for anticancer therapies. To discover more potent and diverse inhibitors of Cdc25 biological activity, virtual screening was performed by docking 2.1 million compounds into the Cdc25B active site. An initial subset of top-ranked compounds was selected and assayed, and 15 were found to have enzyme inhibition activity at micromolar concentration. Among these, four structurally diverse inhibitors with a different inhibition profile were found to inhibit human MCF-7, PC-3, and K562 cancer cell proliferation and significantly affect the cell cycle progression. A subsequent hierarchical similarity search with the most active reversible Cdc25B inhibitor found led to the identification of an additional set of 19 ligands, three of which were confirmed as Cdc25B inhibitors with IC(50) values of 7.9, 4.2, and 9.9 μM, respectively.

Cleavage of urokinase receptor regulates its interaction with integrins in thyroid cells

The urokinase‐type plasminogen activator uPA‐R can regulate integrin functions by associating with several types of β‐subunit. We have recently shown that normal thyroid TAD‐2 cells express both a native and a cleaved form of uPA‐R which lacks the binding domain for uPA. We found this cleaved form to be present in reduced amounts in papillary and follicular thyroid carcinoma cells and completely absent in cells derived from an anaplastic thyroid carcinoma (ARO). We now report that in normal thyroid cells the intact form of uPA‐R strongly associates with β‐1 integrins, whereas its cleaved form does not. uPA‐R expressed by ARO cells shows a stronger resistance to the cleavage mediated by uPA, plasmin and chymotrypsin than does uPA‐R expressed by normal thyroid cells. This resistance to cleavage correlates with the higher level of glycosylation of uPA‐R of ARO cells as compared to that of cleavable uPA‐R of normal thyroid cells. These results suggest that uPA‐R cleavage, which occurs in several cell types, represents a mechanism regulating the interactions of uPA‐R with integrins and, possibly, the subsequent integrin‐mediated cell adhesion. Moreover we hypothesize that glycosylation regulates uPA‐R cleavage and, indirectly, its interaction with integrins.