Urokinase receptor deficiency results in EGFR-mediated failure to transmit signals for cell survival and neurite formation in mouse neuroblastoma cells.

Urokinase-type plasminogen activator uPA and its receptor (uPAR) are the central players in extracellular matrix proteolysis, which facilitates cancer invasion and metastasis. EGFR is one of the important components of uPAR interactome. uPAR/EGFR interaction controls signaling pathways that regulate cell survival, proliferation and migration. We have previously established that uPA binding to uPAR stimulates neurite elongation in neuroblastoma cells, while blocking uPA/uPAR interaction induces neurite branching and new neurite formation. Here we demonstrate that blocking the uPA binding to uPAR with anti-uPAR antibody decreases the level of pEGFR and its downstream pERK1/2, but does increase phosphorylation of Akt, p38 and c-Src Since long-term uPAR blocking results in a severe DNA damage, accompanied by PARP-1 proteolysis and Neuro2a cell death, we surmise that Akt, p38 and c-Src activation transmits a pro-apoptotic signal, rather than a survival. Serum deprivation resulting in enhanced neuritogenesis is accompanied by an upregulated uPAR mRNA expression, while EGFR mRNA remains unchanged. EGFR activation by EGF stimulates neurite growth only in uPAR-overexpressing cells but not in control or uPAR-deficient cells. In addition, AG1478-mediated inhibition of EGFR activity impedes neurite growth in control and uPAR-deficient cells, but not in uPAR-overexpressing cells. Altogether these data implicate uPAR as an important regulator of EGFR and ERK1/2 signaling, representing a novel mechanism which implicates urokinase system in neuroblastoma cell survival and differentiation.

Urokinase receptor regulates nerve regeneration through its interaction with α5ß1-integrin.

PURPOSE: Urokinase receptor (uPAR) promotes extracellular matrix proteolysis, regulates adhesion and cell migration, transduces intracellular signals through interactions with the lateral partners. The expression of uPAR and urokinase (uPA) is significantly upregulated in peripheral nerves after injury, however, little is known about uPAR function in nerve regeneration or the molecular mechanisms involved. The purpose of this study is to investigate the role of uPAR in nerve regeneration after traumatic injury of n. Peroneus communis in uPA-/-, uPAR-/- or control mice (WT) and in neuritogenesis in an in vitro Neuro 2A cell model. RESULTS: Electrophysiological analysis indicates that nerve recovery is significantly impaired in uPAR-/- mice, but not in uPA-/- mice. These data correlate with the reduced amount of NF200-positive axons in regenerating nerves from uPAR-/- mice compared to uPA-/- or control mice. There is an increase in uPAR expression and remarkable colocalization of uPAR with α5 and ß1 integrin in uPA-/- mice in recovering nerves, pointing to a potential link between uPAR and its lateral partner α5ß1-integrin. Using an in vitro model of neuritogenesis and α325 blocking peptide, which abrogates uPAR-α5ß1 interaction in Neuro 2A cells but has no effect on their function, we have further confirmed the significance of uPAR-α5ß1 interaction. CONCLUSION: Taken together, we report evidence pointing to an important role of uPAR, rather than uPA, in peripheral nerve recovery and neuritogenesis.

Molecular modeling as a new approach to the development of urokinase inhibitors.

Proteolytic activity of urokinase plays an important role in negative remodeling of blood vessels, restenosis, tumor angiogenesis, and metastasizing, which necessitates the development of selective urokinase inhibitors. Using methods of computer modeling (docking, post processing, and direct docking) and quantum chemistry, we selected substances from the large compound database, analyzed their structures, and experimentally verified their inhibitor activity. New urokinase inhibitor candidates were proposed based on the theoretical predictions and experimental verification of compound activities. The process of modifying urokinase inhibitors based on (benzothiazol-3-yl)guanidine was developed. A new urokinase inhibitor (5-brom-benzothiazol-3-yl)guanidine, that can be effective for regulation of vascular remodeling and tumor angiogenesis, was created.