Urokinase plasminogen activator (uPA) is thought to exert its effects on cell growth, adhesion, and migration by mechanisms involving proteolysis and interaction with its cell surface receptor (uPAR). The functional properties of uPA and the significance of its various domains for chemotactic activity were analyzed using human airway smooth muscle cells (hAWSMC). The wild-type uPA (r-uPAwt), inactive urokinase with single mutation (His 204 to Gln) (r-uPA H/Q ), urokinase with mutation of His 204 to Gln together with a deletion of growth factor-like domain (r-uPA H/Q -GFD), the catalytic domain of urokinase (r-uPA LMW ), and its kringle domain (r-KD) were expressed in Escherichia coli. We demonstrate that glycosylated uPA, r-uPAwt, r-uPA H/Q , and r-uPA H/Q -GFD elicited similar chemotactic effects. Half-maximal chemotaxis (EC 50 ) were apparent at approximately 2 nM with all the uPA variants. The kringle domain induced cell migration with an EC 50 of about 6 nM, whereas the denaturated r-KD and r-uPA LMW were without effect. R-uPAwt-induced chemotaxis was dependent on an association with uPAR and a uPA-kringle domain-binding site, determined using a monoclonal uPAR antibody to prevent the uPA-uPAR interaction, and a monoclonal antibody to the uPA-kringle domain. The binding of iodinated r-uPAwt with hAWSMC was due to interaction with a high affinity binding site on the uPAR, and a lower affinity binding site on an unidentified cell surface target, which was mediated exclusively through the kringle domain of urokinase. Specific binding of r-uPA H/Q -GFD to hAWSMC involved an interaction with a single site whose characteristics were similar to those of the low affinity site of r-uPAwt binding to hAWSMC. uPAR-deficient HEK 293 cells specifically bound r-uPAwt and r-uPA H/Q -GFD via a single, similar type of binding site. These cells migrated when stimulated by r-uPA H/Q -GFD and uPAwt, but not r-uPA LMW . HEK 293 cells transfected with the uPAR cDNA expressed two classes of sites that bound r-uPAwt; however, only a single site was responsible for the binding of r-uPA H/Q -GFD. Together, these findings indicate that uPA-induced chemotaxis is dependent on the binding of the uPA-kringle to the membrane surface of cells and the association of uPA with uPAR.

Summary Spontaneous pneumothoraces due to lung cyst rupture afflict patients with the rare disease Birt-Hogg-Dubé (BHD) syndrome caused by mutations of the tumor suppressor gene folliculin (FLCN) by unknown mechanism. BHD lungs exhibit increased alveolar epithelial cell apoptosis. We show that Flcn deletion in lung epithelium leads to cell apoptosis, alveolar enlargement and impaired lung function. FLCN loss also impairs alveolar epithelial barrier function. Flcn-null epithelial cell apoptosis is the result of impaired AMPK activation and increased cleaved caspase-3. AMPK activator LKB1 and E-cadherin are downregulated by Flcn loss and restored by its expression. Flcn-null cell survival is rescued by AICAR or constitutively active AMPK. AICAR also improves lung condition of Flcnf/f:SP-C-Cre mice. Our data show that Flcn regulates lung epithelial cell survival and alveolar size and suggest that lung cysts in BHD may result from an underlying defect in alveolar epithelial cell survival attributable to FLCN regulation of the E-cadherin-LKB1-AMPK axis.

Plasminogen activators (PAs) are used to treat life-threatening thrombosis, but not for thromboprophylaxis because of rapid clearance, risk of bleeding, and central nervous system (CNS) toxicity. We describe a novel strategy that may help to overcome these limitations by targeting a thrombin-activated PA pro-drug to circulating red blood cells (RBCs). We fused a single chain antibody (scFv Ter-119) that binds to mouse glycophorin A (GPA) with a variant human single-chain low molecular weight urokinase construct that can be activated selectively by thrombin (scFv/uPA-T). scFv/uPA-T bound specifically to mouse RBCs without altering their biocompatibility and retained its zymogenic properties until converted by thrombin into an active 2-chain molecule. IntroductionPlasminogen activators (PAs; tissue-type, tPA; urokinase, uPA) can provide urgent thrombolysis within a narrow therapeutic window of time in the setting of life-or limb-threatening thrombosis. 1,2 However, their efficacy is limited by plasma inhibitors (eg, PAI-1) and inadequate delivery into impermeable occlusive clots, a situation that is exacerbated by delays in initiating treatment. Endowing tPA derivatives with higher affinity for fibrin 3,4 further impairs clot permeation, 5 while increased dosing and constitutive lytic activity enhances the risk of bleeding and central nervous system (CNS) toxicity.Coupling tPA to carrier red blood cells (RBCs) followed by re-infusion of the RBC/tPA conjugates in animals provides protracted thromboprophylaxis in arteries and veins, including the vulnerable cerebrovascular circulation. 6-8 RBC carriage prolongs the half-life of tPAs in the circulation from minutes to many hours and prevents drug extravasation and access to hemostatic plugs, thereby reducing the risk of bleeding episodes. 7 In the prophylactic setting where tPAs lacked efficacy but caused bleeding and CNS toxicity, RBCs/tPAs mediated timely reperfusion, reducing morbidity and mortality. 8 Translation of RBC/PA thromboprophylaxis into the clinical domain could improve management of patients known to be at high risk of thrombosis or rethrombosis, including after acute myocardial infarction (AMI), transient ischemic attack, pulmonary embolism, angioplasty, and abdominal or other surgical procedures such as knee replacements, where the efficacy of thromboprophylaxis is low and/or the risk of bleeding is high. The goal of this study was to modify an existing prototypic approach (ex vivo coupling of PAs to RBCs followed by RBC/PA re-infusion) into a more clinically applicable approach to thromboprophylaxis. To anchor the injected PAs to circulating RBCs, we used a scFv fragment of the monoclonal antibody Ter-119 that recognizes mouse glycophorin A (GPA), an abundant RBC-specific surface molecule (ϳ 10 6 copies/ RBC, 9,10 similar to its human homologue 11 ). We fused scFv Ter-119 to a recombinant low molecular weight single chain uPA lacking the kringle and growth factor-like domains (lmw scuPA). Lmw scuPA is a zymogen that is naturally activated by plasmi...

Urokinase-type plasminogen activator (uPA) participates in diverse (patho)physiological processes through intracellular signaling events that affect cell adhesion, migration, and proliferation, although the mechanisms by which these occur are only partially understood. Here we report that upon cell binding and internalization, single-chain uPA (scuPA) translocates to the nucleus within minutes. Nuclear translocation does not involve proteolytic activation or degradation of scuPA. Neither the urokinase receptor (uPAR) nor the low-density lipoprotein-related receptor (LRP) is required for nuclear targeting. Rather, translocation involves the binding of scuPA to the nucleocytoplasmic shuttle protein nucleolin through a region containing the kringle domain. RNA interference and mutational analysis demonstrate that nucleolin is required for the nuclear transport of scuPA. Furthermore, nucleolin is required for the induction smooth muscle ␣-actin (␣-SMA) by scuPA. These data reveal a novel pathway by which uPA is rapidly translocated to the nucleus where it might participate in regulating gene expression. (Blood. 2008;112: 100-110) IntroductionUrokinase-type plasminogen activator (uPA) is a multifunctional protein that has been implicated in several physiological and pathological processes, including cell proliferation and migration during angiogenesis, tissue regeneration, inflammatory responses, and tumor growth/metastases. These complex processes all involve intracellular signal transduction and regulation of gene transcription in addition to proteolysis (see Alfano et al 1 for review). uPA is secreted as a single-chain protein (scuPA) that consists of an N-terminal EGF-like domain (GFD), a kringle domain (KD), and a serine protease domain. Binding of uPA to its high-affinity receptor CD87 (uPAR) is mediated by the GFD. 2 Plasmin converts scuPA into a proteolytically active 2-chain enzyme (tcuPA) 3 that is rapidly inhibited primarily by plasminogen activator inhibitor-1 (PAI-1). tcuPA-PAI-1 complexes are internalized with the aid of lipoprotein receptor-related protein (LRP) 4 by clathrin-mediated endocytosis. The tcuPA-PAl-1 complexes traffic to lysosomes and are degraded, while unoccupied uPAR and LRP recycle back to the cell surface. 5 uPA-induced signal transduction occurs via uPAR-dependent and uPAR-independent pathways (reviewed in Alfano et al 1 ; Kjoller 6 ; Blasi and Carmeliet 7 ). Among the latter, we have shown that cleavage of scuPA by plasmin releases the GFD fragment, generating a form of uPA unable to bind to uPAR, 8 but that stimulates migration of smooth muscle cells (SMCs). 9 Signal transduction by this scuPA fragment may be mediated in part by LRP 10 and certain integrins. 11 However, there is limited information as to the mechanism by which uPA modifies gene transcription, [12][13][14][15] and our previous studies have provided reason to hypothesize that cells express additional uPA-binding proteins that possess distinct signal-transducing activities involved in cell contractility, migration, an...