Urokinase-type plasminogen activator

Urokinase-type plasminogen activator

The International Journal of Biochemistry & Cell Biology 39 (2007) 690–694 Molecules in focus Urokinase-type plasminogen activator Massimo P. Crippa...

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The International Journal of Biochemistry & Cell Biology 39 (2007) 690–694

Molecules in focus

Urokinase-type plasminogen activator Massimo P. Crippa ∗ Laboratory of Molecular Genetics, Di.Bi.T., S. Raffaele Scientific Institute, Via Olgettina 58, 20132 Milano, Italy Received 26 September 2006; received in revised form 10 October 2006; accepted 11 October 2006 Available online 21 October 2006

Abstract Urokinase-type plasminogen activator (uPA) is a serine protease involved in tissue remodeling and cell migration. At the gene level, the interplay between a complex enhancer, required for induced and basal transcription, and the minimal promoter finely tunes uPA expression. The active form of uPA is bound to its high affinity receptor on the cell surface, where specific inhibitors modulate its enzymatic activity. Such inhibitors also regulate the cell surface levels of uPA by triggering the internalization of the uPA-receptor–inhibitor complex. The role of uPA is not only linked to its action as an enzyme. In fact, the mere binding of uPA on the cell surface also brings about two events that broaden the spectrum of its biological functions: (1) a conformational change of the receptor, which, in turn, affects its interaction with other proteins; (2) a signal transduction which modulates the expression of apoptosis-related genes. Besides its applications as a thrombolytic agent and as a prognostic marker for tumors, uPA may provide the basis for other therapies, as the structure of the receptor-binding domain of uPA has become a model for the design of anti-cancer molecules. © 2006 Elsevier Ltd. All rights reserved. Keywords: Urokinase-type plasminogen activator; Extracellular matrix; cell migration; Tissue remodeling; Urokinase-type plasminogen activator receptor

1. Introduction The regulation of plasminogen activation involves two proteases with different roles: the tissue-type plasminogen activator (tPA, which will not be discussed in this review), with a strong inclination towards the fibrinolytic process and the urokinase-type plasminogen activator (uPA), which is primarily involved in cell migration and tissue remodeling, two events strictly related to cancer development and spreading. The binding of uPA to its own receptor (urokinase-type plasminogen activator receptor, uPAR) focalizes its proteolytic activity to the cell surface, which is required for some of its functions. On the other hand, binding of ∗

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uPA to uPAR “per se” triggers other functions, brought about by the interaction of the uPA–uPAR complex with other molecules of the cell surface or of the extracellular matrix (ECM). The clinical use of uPA is as a thrombolytic agent. However, it is also a prognostic marker for tumors and its structure is the basis for the design of new anti-cancer drugs and is used for the targeting of cytotoxic agents to uPAR expressing cells (Alfano et al., 2005; Blasi & Carmeliet, 2002; Danø et al., 2005; Sidenius & Blasi, 2003). 2. Structure The 6.4 kb human gene coding for uPA consists of 11 exons and 10 introns (Fig. 1A) (Iba˜nez-Tallon, Caretti, Blasi, & Crippa, 1999; Iba˜nez-Tallon et al., 2002). The first exon and part of the second, which con-

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Fig. 1. (A) Scheme (not to scale) depicting the genomic structure of the uPA gene and its regulatory region. White boxes: untranslated region of exons I and XI. Black boxes: translated exons. The regulatory elements in the 5 flanking region are described in the text. (B) Three-dimensional structure of the trypsin-like catalytic domain of uPA (B chain; PDB 1SQT).

tains the ATG, are untranslated, as is a large portion (about 800 bp) of the last exon. This region, however, is required for the regulation of uPA mRNA stability through the binding of HuR proteins (Irigoyen, MunozCanoves, Montero, Koziczak, & Nagamine, 1999). The 5 flanking sequence contains several features that indicate a tight transcriptional regulation (Iba˜nez-Tallon et al., 1999, 2002). Upstream of the TATA box lies a GCrich region of variable length (depending on the species) containing several canonical and non-canonical binding sites for the transcription factor Sp1, which has a prominent role in the constitutive expression of the gene in PC3 cells and is targeted by a number of signal transduction pathways (Benasciutti et al., 2004; Iba˜nez-Tallon et al., 2002). Approximately 2 kb upstream of the transcription start site lies a well-characterized enhancer. It contains an upstream Ets (PEA3) binding site juxtaposed to an octameric AP-1 binding site and another non-canonical (albeit eptameric) AP-1 binding site about 70 bp downstream. These sites are separated by the cooperativity mediator (COM) region, which has been divided in an upstream and downstream COM (uCOM and dCOM, respectively). The uCOM region contains the binding sites for upstream enhancer factor (UEF) transcription factors 1–4, three of which have been identified as: Prep1/Pbx heterodimers (UEF 2 and 3) and Oct-1 (UEF 4), whereas the fourth (UEF 1) is yet unidentified. dCOM, on the other hand, only contains binding sites for UEF 1–3 (Iba˜nez-Tallon et al., 1999, 2002). Transcriptional activation of the uPA gene can be obtained

through a number of different stimuli (e.g. phorbol esters, growth factors, etc.) through signal transduction pathways, which mostly target the enhancer (Alfano et al., 2005; Iba˜nez-Tallon et al., 1999, 2002). Moreover, the regulatory element acts in a loose chromatin context (Iba˜nez-Tallon et al., 1999) which favors the physical interaction between enhancer and promoter through the looping of the intervening sequence (Ferrai et al., in press). Finally, two more elements have been identified in the regulatory region of the human uPA gene: an NF-␬B binding site, which mediates the transcriptional induction of gene expression by phorbol esters in the absence of the enhancer AP-1 sites (Hansen et al., 1992) and a cell type-specific silencer (Cannio, Rennie, & Blasi, 1991). uPA is secreted as a 411 aminoacid inactive proenzyme (pro-uPA) which undergoes several post-translational modifications and has an apparent molecular weight of 53 kDa. The zymogen binds its own receptor (urokinase-type plasminogen activator receptor, uPAR) and is cleaved by neighboring, membrane-bound plasmin (or other proteases) at K158–K159 to produce the active two-chain form (chains A and B, Fig. 1B) held together by a single disulfide bond. Further cleavage of pro-uPA at K135–K136 releases the amino-terminal fragment (ATF) of uPA, containing an EGF-like and a kringle domain, involved in the binding of the receptor. Chain B contains the catalytic site and maintains the ability to activate plasminogen also when it is not bound to the receptor (Alfano et al., 2005; Irigoyen et al., 1999). In addition the C-terminus of the amino-terminal region

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contains a sequence that interacts with ␣V␤3 integrin and is relevant for cell migration (Franco et al., 2006). 3. Synthesis and degradation The attention of early researchers was drawn by the proteolytic activity of cancer explants as a possible connection to the formation of metastases and by the hope it would be a target to eradicate the disease. However, it was later shown that also normal tissue could synthesize and secrete plasminogen activators (Danø et al., 1985). In particular, uPA was shown to be involved in tissue remodeling events in non-pathological conditions. For instance, a coordinated expression of uPA and uPAR is required for trophoblast implantation. Moreover, uPA is also required for the processes of wound healing and post-lactational involution (Irigoyen et al., 1999). Just as it is now clear that many different cell types can synthesize uPA both in pathological and nonpathological conditions in response to specific external stimuli, it is also evident that uPA degradation is a multi-step process that involves other molecules. The proteolytic activity of uPAR-bound uPA is blocked by a specific inhibitor (plasminogen activato inhibitor 1, PAI-1), which binds two-chain uPA. Subsequently, the uPA–uPAR–PAI-1 complex is internalized, a feat that requires the cooperation of other surface receptors. uPA is then degraded and the receptor (uPAR) is recycled to

the cell surface (Conese & Blasi, 1995). Thus, PAI-1 controls cell-associated uPA in two ways: by suppressing its proteolytic activity and by reducing the amount of surface-bound uPA. 4. Biological functions A number of reviews deal with the many biological functions of uPA, which may or may not require its proteolytic activity (Alfano et al., 2005; Irigoyen et al., 1999; Rabbani, 1998; Sidenius & Blasi, 2003) (Fig. 2). Moreover, extensive studies have been performed on the uPA−/− mouse model, which further clarify its function (Carmeliet et al., 1994). Three aspects of uPA function that do not strictly require proteolytic activity will be mentioned here: chemotaxis, cell adhesion and apoptosis. Cell surface-bound plasmin converts uPAR-bound pro-uPA to the active enzyme, which, in turn, increases the conversion of plasminogen to plasmin and such that they reciprocally enhance their activation. A “side effect” of the proteolytic activity of uPARbound uPA is the cleavage of the receptor itself between domains D1 and D2. This exposes a pentapeptide (SRSRY) with chemotactic activity. Interestingly, the soluble, truncated form of the receptor (suPAR D2D3, exposing the chemotactic epitope) has strong chemokine-like activity and interacts and signals through

Fig. 2. Summary of the proteolytic and non-proteolytic functions of uPA. All of them are indirect in that they are mediated either by its proteolytic products (e.g. plasmin) or by interacting molecules (e.g. uPAR and/or PAI-1).

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the FPRL1/LXA4R receptor (Sidenius & Blasi, 2003), facilitating the signaling to other cells and their recruitment (Irigoyen et al., 1999). However, epitope exposure may also be obtained by the simple binding of a catalytically inactive form of uPA or of an uPA fragment lacking the catalytic domain (ATF), indicating that binding “per se” induces a conformational change in the receptor that exposes the chemotactic epitope (Sidenius & Blasi, 2003). Binding of uPA to uPAR is necessary to expose the vitronectin (Vn)-interacting surface of the receptor and to modulate cell adhesion also through the interaction with receptors of the integrin family. uPA–uPAR interaction with Vn causes the induction of actin cytoskeleton rearrangement, which, together with integrin activation, causes cells in culture to spread on Vn-coated surfaces (Sidenius & Blasi, 2003). The uPA–uPAR/Vn interaction is modulated by PAI-1, which competes with uPAR for the same binding site on Vn. The PAI-1–Vn complex has a two-fold effects: it retains the ability to bind uPAR-bound uPA, forming a uPA–PAI-1 complex (with low affinity for Vn) that is rapidly released, thus “hiding” the Vn-interacting surface of uPAR (Sidenius & Blasi, 2003). Furthermore, PAI-1 blocks the interaction between Vn and integrin ␣V␤3. The net result of both effects is the inhibition of cell spreading (Sidenius & Blasi, 2003). uPA binding to its receptor also plays an important role in protecting cells from apoptosis due to cell detachment (anoikis). This is achieved through signaling of the ligand-bound uPAR to the MEK/ERK and PI3K/Akt pathways, which upregulate transcription of the Bcl-XL gene, coding for an anti-apoptotic protein of the Bcl family (Alfano, Iaccarino, & Stoppelli, 2006). 5. Applications uPA is known as a thrombolytic agent and has been exploited for the treatment of pulmonary embolism, acute myocardial infarction, ophthalmic clot and hemorrage and peripheral arterial occlusion. uPA is also a prognostic marker for human cancers and a target for anti-cancer therapy. As a marker, high uPA levels in primary tumor extracts correlate with high incidence of relapse and poor survival. Recent data from X-ray crystallography and NMR screening have indicated a number of candidate molecules to block the uPA-mediated activation of plasminogen. Moreover, molecules designed on the uPAR-binding domain of uPA are used either alone or fused to inhibiting activities to destroy the uPA–uPAR interaction or to prevent the proteolytic activity. Such

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molecules have also been recently used to selectively target the catalytic domain of toxins to uPAR-expressing cells (Sidenius & Blasi, 2003). Acknowledgements The author would like to thank Prof. F. Blasi and Dr. L. Wrabetz for critically reading the manuscript. Work in the author’s laboratory is supported by grants from the Italian Association for Cancer Research (A.I.R.C.). References Alfano, D., Franco, P., Vocca, I., Gambi, N., Pisa, V., Mancini, A., et al. (2005). The urokinase plasminogen activator and its receptor: Role in cell growth and apoptosis. Thromb. Haemost., 93, 190–191. Alfano, D., Iaccarino, I., & Stoppelli, M. P. (2006). Urokinase signaling through its receptor protects against anoikis by increasing BCL-xL expression levels. J. Biol. Chem., 281, 17758–17767. Benasciutti, E., Pag`es, G., Kenzior, O., Folk, W., Blasi, F., & Crippa, M. P. (2004). Different signal transduction pathways can phosphorylate Sp1 to activate the uPA minimal promoter element and endogenous gene transcription in different cancer cell lines. Blood, 104, 256–262. Blasi, F., & Carmeliet, P. (2002). uPAR: A versatile signalling orchestrator. Nat. Rev. Mol. Cell Biol., 3, 932–943. Cannio, R., Rennie, P. S., & Blasi, F. (1991). A cell-type specific and enhancer-dependent silencer in the regulation of the expression of the human urokinase plasminogen activator gene. Nucl. Acids Res., 19, 2303–2308. Carmeliet, P., Schoonjans, L., Kieckens, L., Ream, B., Degen, J., Bronson, R., et al. (1994). Physiological consequences of loss of plasminogen activator gene function in mice. Nature, 368, 419–424. Conese, M., & Blasi, F. (1995). Urokinase/urokinase receptor system: Internalization/degradation of urokinase–serpin complexes—Mechanism and regulation. Biol. Chem. Hoppe Seyler, 376, 143–155. Danø, K., Andreasen, P. A., Grøndahl-Hansen, J., Kristensen, P., Nielsen, L. S., & Skriver, L. (1985). Plasminogen activators, tissue degradation and cancer. Adv. Cancer Res., 44, 139–266. Danø, K., Behrendt, N., Høyer-Hansen, G., Johnsen, M., Lund, L. R., Ploug, M., et al. (2005). Plasminogen activation and cancer. Thromb. Haemost., 93, 676–681. Ferrai, C., Munari, D., Luraghi, P., Pecciarini, L., Cangi, M. G., Doglioni, C., et al. (in press). A transcription-dependent MNaseresistant fragment of the uPA promoter interacts with the enhancer. J. Biol. Chem. Franco, P., Vocca, I., Carriero, M. V., Alfano, D., Cito, L., LonganesiCattani, I., et al. (2006). Activation of urokinase receptor by a novel interaction between the connecting peptide region of urokinase and ␣V␤5 integrin. J. Cell Sci., 119, 3424–3434. Hansen, S., Nerlov, C., Zabel, U., Verde, P., Johnsen, M., Bauerle, P. A., et al. (1992). A novel complex between the p65 subunit of NF␬B and c-Rel binds to a DNA element involved in the phorbol ester induction of the human urokinase gene. EMBO J., 11, 205–213. Iba˜nez-Tallon, I., Caretti, G., Blasi, F., & Crippa, M. P. (1999). “In vivo” analysis of the state of the human uPA enhancer following induction with TPA. Oncogene, 18, 2836–2845.

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Iba˜nez-Tallon, I., Ferrai, C., Longobardi, E., Facetti, I., Blasi, F., & Crippa, M. P. (2002). Binding of Sp1 to the proximal promoter links constitutive expression of the human uPA gene and invasive potential of PC3 cells. Blood, 100, 3325–3332. Irigoyen, J. P., Munoz-Canoves, P., Montero, L., Koziczak, M., & Nagamine, Y. (1999). The plasminogen activator system: Biology and regulation. Cell Mol. Life Sci., 56, 104–132.

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