PET in vivo generators 134Ce and 140Nd on an internalizing monoclonal antibody probe.

The in vivo-generator radionuclides 140Nd (t1/2 = 3.4 d) and 134Ce (t1/2 = 3.2 d) were used to trace a urokinase-type plasminogen activator (uPA)-targeting mouse monoclonal antibody, ATN-291, in U87 MG xenograft tumor-bearing mice. ATN-291 is known to internalize on the uPA/uPA-receptor pair, making it an appropriate targeting vector for investigating the fate of in vivo generator daughters on internalizing probes. Ante-mortem and post-mortem PET imaging at 120 h post-injection gave no indication of redistribution of the positron emitting daughter nuclides 134La and 140Pr from tumor tissue (p > 0.5). The lack of redistribution indicates that the parent radionuclides 134Ce and 140Nd could be considered as long-lived PET-diagnostic matches to therapeutic radionuclides like 177Lu, 161Tb and 225Ac when internalizing bioconjugates are employed.

Preclinical uPAR-targeted multimodal imaging of locoregional oral cancer.

OBJECTIVES: Establishing adequate resection margins and lymphatic mapping are crucial for the prognosis of oral cancer patients. Novel targeted imaging modalities are needed, enabling pre- and intraoperative detection of tumour cells, in combination with improved post-surgical examination by the pathologist. The urokinase-receptor (uPAR) is overexpressed in head and neck cancer, where it is associated with tumour progression and metastasis. MATERIAL AND METHODS: To determine suitability of uPAR for molecular imaging of oral cancer surgery, human head and neck tumours were sectioned and stained for uPAR to evaluate the expression pattern compared to normal mucosa. Furthermore, metastatic oral squamous carcinoma cell line OSC-19 was used for targeting uPAR in in vivo mouse models. Using anti-uPAR antibody ATN-658, equipped with a multimodal label, the in vivo specificity was investigated and the optimal dose and time-window were evaluated. RESULTS: All human oral cancer tissues expressed uPAR in epithelial and stromal cells. Hybrid ATN-658 clearly visualized tongue tumours in mice using either NIRF or SPECT imaging. Mean fluorescent TBRs over time were 4.3±0.7 with the specific tracer versus 1.7±0.1 with a control antibody. A significant difference in TBRs could be seen between 1nmol (150µg) and 0.34nmol (50µg) dose groups (n=4, p<0.05). Co-expression between BLI, GFP and the NIR fluorescent signals were seen in the tongue tumour, whereas human cytokeratin staining confirmed presence of malignant cells in the positive cervical lymph nodes. CONCLUSION: This study shows the applicability of an uPAR specific multimodal tracer in an oral cancer model, combining SPECT with intraoperative guidance.

Identification of biomarkers for the antiangiogenic and antitumour activity of the superoxide dismutase 1 (SOD1) inhibitor tetrathiomolybdate (ATN-224).

Tetrathiomolybdate (choline salt; ATN-224), a specific, high-affinity copper binder, is currently being evaluated in several phase II cancer trials. ATN-224 inhibits CuZn superoxide dismutase 1 (SOD1) leading to antiangiogenic and antitumour effects. The pharmacodynamics of tetrathiomolybdate has been followed by tracking ceruloplasmin (Cp), a biomarker for systemic copper. However, at least in mice, the inhibition of angiogenesis occurs before a measurable decrease in systemic copper is observed. Thus, the identification and characterisation of other biomarkers to follow the activity of ATN-224 in the clinic is of great interest. Here, we present the preclinical evaluation of two potential biomarkers for the activity of ATN-224: (i) SOD activity measurements in blood cells in mice and (ii) levels of endothelial progenitor cells (EPCs) in bonnet macaques treated with ATN-224. The superoxide dismutase activity in blood cells in mice is rapidly inhibited by ATN-224 treatment at doses at which angiogenesis is maximally inhibited. Furthermore, ATN-224 dosing in bonnet macaques causes a profound and reversible decrease in EPCs without significant toxicity. Thus, both SOD activity measurements and levels of EPCs may be useful biomarkers of the antiangiogenic activity of ATN-224 to be used in its clinical development.

Phase 1 trial of the antiangiogenic peptide ATN-161 (Ac-PHSCN-NH(2)), a beta integrin antagonist, in patients with solid tumours.

To evaluate the toxicity, pharmacological and biological properties of ATN-161, a five -amino-acid peptide derived from the synergy region of fibronectin, adult patients with advanced solid tumours were enrolled in eight sequential dose cohorts (0.1-16 mg kg(-1)), receiving ATN-161 administered as a 10-min infusion thrice weekly. Pharmacokinetic sampling of blood and urine over 7 h was performed on Day 1. Twenty-six patients received from 1 to 14 4-week cycles of treatment. The total number of cycles administered to all patients was 86, without dose-limiting toxicities. At dose levels above 0.5 mg kg(-1), mean total clearance and volume of distribution showed dose-independent pharmacokinetics (PKs). At 8.0 and 16.0 mg kg(-1), clearance of ATN-161 was reduced, suggesting saturable PKs. Dose escalation was halted at 16 mg kg(-1) when drug exposure (area under the curve) exceeded that associated with efficacy in animal models. There were no objective responses. Six patients received more than four cycles of treatment (>112 days). Three patients received 10 or more cycles (> or =280 days). ATN-161 was well tolerated at all dose levels. Approximately, 1/3 of the patients in the study manifested prolonged stable disease. These findings suggest that ATN-161 should be investigated further as an antiangiogenic and antimetastatic cancer agent alone or with chemotherapy.

uPA and uPAR contribute to NK cell invasion through the extracellular matrix.

BACKGROUND: The urokinase plasminogen activator (uPA) system has been implicated in cellular invasiveness of tumor cells and immune cells. Herein we provide evidence for the production by natural killer (NK) cells of both uPA and its receptor (uPAR). MATERIALS AND METHODS: Western blot analysis, RTPCR, casein/plasminogen zymography, and fluorescence microscopy were employed to detect uPA and uPAR on NK cells. NK cell invasiveness was examined using Matrigel invasion assays. RESULTS: NK cell uPA appeared at its characteristic molecular weights, is enzymatically active in casein/plasminogen zymography, and is recognized by monoclonal antibodies. uPAR was detected by RTPCR and fluorescence microscopy. Matrigel invasion assays demonstrated an active role of uPA in NK cell invasion. CONCLUSION: The uPA system contributes to extracellular matrix (ECM) degradation by NK cells, which may be essential for NK cell accumulation into metastases, and may be prerequisite for their killing of tumor cells following NK cell adoptive transfer.

The urokinase plasminogen activator receptor (uPAR) as a target for the diagnosis and therapy of cancer.

The identification and characterization of validated molecular targets for cancer drug and diagnostic development is rapidly changing the way that promising new anti-cancer compounds are developed and evaluated. A significant body of in vitro and in vivo data has established the urokinase plasminogen activator (uPA) system as a promising target for cancer drug development. The uPA system has been demonstrated to have pleiotropic activities in the development of tumors, and in tumor progression and angiogenesis. There are multiple ways to target this system, the most straightforward being the development of small molecule active site inhibitors of the serine protease, uPA. However, compounds of this type have not entered into clinical trials, and issues related to selectivity and specificity of this class of inhibitors have yet to be satisfactorily resolved. Recent evidence suggests that in addition to uPA, its specific cell surface receptor (uPAR) may also be a suitable target for the design and development of cancer therapeutic and diagnostic agents. uPAR is central to several pathways implicated in tumor progression and angiogenesis. The binding of the uPA zymogen (scuPA) to uPAR appears to be a pre-requisite for efficient cell-surface activation of scuPA to the active two-chain form (tcuPA) by plasmin, and simple ligand occupancy of uPAR by scuPA initiates various signaling pathways leading to alterations in cell motility and adhesion. One therapeutic rationale that is currently being investigated is the simple displacement of scuPA or tcuPA from suPAR, which may effectively inhibit both the proteolytic and signal-transducing cascades. In addition, other approaches to the modulation of the activity of this system that may also be useful include blocking the interaction of uPAR with integrins and extracellular matrix proteins as well as strategies to down-regulate the expression of uPA and uPAR in target cells. This review will summarize these approaches, and also describe the targeting of uPAR for diagnosis and imaging.

The role of the plasminogen activation system in angiogenesis and metastasis.

The urokinase plasminogen activator system (uPA) has been demonstrated to be required for the movement of cells through a matrix. These observations have been extended to the migration of endothelial cells during the process of angiogenesis, and recent data suggest that the uPA system is central to this process. uPA is a serine protease that is capable of initiating an extracellular cascade of proteolysis that involves the activation of plasminogen and matrix metalloproteases. These proteolytic cascades remodel extracellular matrix (ECM) and basement membrane (BM), allowing for the movement of cells across and through these barriers. In addition, these proteolytic cascades process and release various growth and differentiation factors that are sequestered on the cell surface or within the ECM, which contribute to the evolution of a migratory or invasive cell phenotype. uPA is also able to modulate signaling and cell adhesion through its specific cell surface receptor, uPAR. Recent data suggest that the nonproteolytic activities of the uPA system are coupled to adhesion, migration and signaling through various integrins. This article reviews the evidence for the role of this system in tumor angiogenesis and metastasis, which suggests that the uPA system initiates multiple cascades that contribute to these processes.

Urokinase-type plasminogen activator receptor (CD87) is a ligand for integrins and mediates cell-cell interaction.

Binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR/CD87) regulates cellular adhesion, migration, and tumor cell invasion. However, it is unclear how glycosyl phosphatidylinositol-anchored uPAR, which lacks a transmembrane structure, mediates signal transduction. It has been proposed that uPAR forms cis-interactions with integrins as an associated protein and thereby transduces proliferative or migratory signals to cells upon binding of uPA. We provide evidence that soluble uPAR (suPAR) specifically binds to integrins alpha4beta1, alpha6beta1, alpha9beta1, and alphavbeta3 on Chinese hamster ovary cells in a cation-dependent manner. Anti-integrin and anti-uPAR antibodies effectively block binding of suPAR to these integrins. Binding of suPAR to alpha4beta1 and alphavbeta3 is blocked by known soluble ligands and by the integrin mutations that inhibit ligand binding. These results suggest that uPAR is an integrin ligand rather than, or in addition to, an integrin-associated protein. In addition, we demonstrate that glycosyl phosphatidylinositol-anchored uPAR on the cell surface specifically binds to integrins on the apposing cells, suggesting that uPAR-integrin interaction may mediate cell-cell interaction (trans-interaction). These previously unrecognized uPAR-integrin interactions may allow uPAR to transduce signals through the engaged integrin without a hypothetical transmembrane adapter and may provide a potential therapeutic target for control of inflammation and cancer.

A peptide derived from the non-receptor-binding region of urokinase plasminogen activator inhibits glioblastoma growth and angiogenesis in vivo in combination with cisplatin.

The urokinase plasminogen activator system is involved in angiogenesis and tumor growth of malignant gliomas, which are highly neovascularized and so may be amenable to antiangiogenic therapy. In this paper, we describe the activity of A6, an octamer capped peptide derived from the non-receptor-binding region of urokinase plasminogen activator. A6 inhibited human microvascular endothelial cell migration but had no effect on the proliferation of human microvascular endothelial cells or U87MG glioma cells in vitro. In contrast, A6 or cisplatin (CDDP) alone suppressed subcutaneous tumor growth in vivo by 48% and 53%, respectively, and, more strikingly, the combination of A6 plus CDDP inhibited tumor growth by 92%. Such combination treatment also greatly reduced the volume of intracranial tumor xenografts and increased survival of tumor-bearing animals when compared with CDDP or A6 alone. Tumors from the combination treatment group had significantly reduced neovascularization, suggesting a mechanism involving A6-mediated inhibition of endothelial cell motility, thereby eliciting vascular sensitivity to CDDP-mediated toxicity. These data suggest that the combination of an angiogenesis inhibitor that targets endothelial cells with a cytotoxic agent may be a useful therapeutic approach.

Urokinase-derived peptides regulate vascular smooth muscle contraction in vitro and in vivo.

We examined the effect of urokinase (uPA) and its fragments on vascular smooth muscle cell contraction. Single-chain uPA inhibits phenylepherine (PE) -induced contraction of rat aortic rings, whereas two-chain uPA exerts the opposite effect. Two independent epitopes mediating these opposing activities were identified. A6, a capped peptide corresponding to amino acids 136-143 (KPSSPPEE) of uPA, increased the EC(50) of PE-induced vascular contraction sevenfold by inhibiting the release of calcium from intracellular stores. A6 activity was abolished by deleting the carboxyl-terminal Glu or by mutating the Ser corresponding to position 138 in uPA to Glu. A single-chain uPA variant lacking amino acids 136-143 did not induce vasorelaxation. A second epitope within the kringle of uPA potentiated PE-induced vasoconstriction. This epitope was exposed when single-chain uPA was converted to a two-chain molecule by plasmin. The isolated uPA kringle augmented vasoconstriction, whereas uPA variant lacking the kringle had no procontractile activity. These studies reveal previously undescribed vasoactive domains within urokinase and its naturally derived fragments.

A peptide derived from the nonreceptor binding region of urokinase plasminogen activator (uPA) inhibits tumor progression and angiogenesis and induces tumor cell death in vivo.

Urokinase plasminogen activator (uPA) plays an important role in the progression of several malignancies including breast cancer. We have identified a noncompetitive antagonist of the uPA-uPAR interaction derived from a nonreceptor binding region of uPA (amino acids 136-143). This 8-mer capped peptide (A6) inhibited breast cancer cell invasion and endothelial cell migration in a dose-dependent manner in vitro without altering cell doubling time. Intraperitoneal administration of A6 resulted in a significant inhibition of tumor growth and suppressed the development of lymph node metastases in several models of breast cancer cell growth and metastasis. Large areas of tumor necrosis and extensive positive staining by TUNEL were observed on histological and immunohistochemical analysis of experimental tumor sections from A6-treated animals. A6 treatment also resulted in a decrease in factor VIII-positive tumor microvessel hot-spots. These results identify a new epitope in uPA that is involved in the uPA-uPAR interaction and indicate that an antagonist based on this epitope is able to inhibit tumor progression by modulating the tumor microenvironment in the absence of direct cytotoxic effects in vivo.

A region in domain II of the urokinase receptor required for urokinase binding.

The urokinase receptor is composed of three homologous domains based on disulfide spacing. The contribution of each domain to the binding and activation of single chain urokinase (scuPA) remains poorly understood. In the present paper we examined the role of domain II (DII) in these processes. Repositioning DII to the amino or carboxyl terminus of the molecule abolished binding of scuPA as did deleting the domain entirely. By using alanine-scanning mutagenesis, we identified a 9-amino acid continuous sequence in DII (Arg(137)-Arg(145)) required for both activities. Competition-inhibition and surface plasmon resonance studies demonstrated that mutation of Lys(139) and His(143) to alanine in soluble receptor (suPAR) reduced the affinity for scuPA approximately 5-fold due to an increase in the "off rate." Mutation of Arg(137), Arg(142), and Arg(145), each to alanine, leads to an approximately 100-fold decrease in affinity attributable to a 10-fold decrease in the apparent "on rate" and a 6-fold increase in off rate. These differences were confirmed on cells expressing variant urokinase receptor. suPAR-K139A/H143A displayed a 50% reduction in scuPA-mediated plasminogen activation activity, whereas the 3-arginine variant was unable to stimulate scuPA activity at all. Mutation of the three arginines did not affect binding of a decamer peptide antagonist of scuPA known to interact with DI and DIII. However, this mutation abolished both the binding of soluble DI to DII-III in the presence of scuPA and the synergistic activation of scuPA mediated by DI and wild type DII-DIII. These data show that DII is required for high affinity binding of scuPA and its activation. DII does not serve merely as a spacer function but appears to be required for interdomain cooperativity.

Novel interactions between urokinase and its receptor.

Urokinase-type plasminogen activator (uPA) binds to its receptor (uPAR) with a K(d) of about 1 nm. The catalytic activity of the complex is apparent at uPA concentrations close to K(d). Other functions of the complex, such as signal transduction, are apparent at much higher concentrations (35-60 nm). In the present study, we show that uPA and recombinant soluble uPAR (suPAR), at concentrations that exceed the K(d) and the theoretical saturation levels (10-80 nm), establish novel interactions that lead to a further increase in the activity of the single-chain uPA (scuPA)/suPAR and two-chain uPA (tcuPA)/suPAR complexes. Experiments performed using dynamic light scattering, gel filtration, and electron microscopy techniques indicate that suPAR forms dimers and oligomers. The three techniques provide evidence that the addition of an equimolar concentration of scuPA leads to the dissociation of these dimers and oligomers. Biacore data show that suPAR dimers and oligomers bind scuPA with decreased affinity when compared with monomers. We postulate that uPAR is present in equilibrium between oligomer/dimer/monomer forms. The binding of uPA to suPAR dimers and oligomers occurs with lower affinity than the binding to monomer. These novel interactions regulate the activity of the resultant complexes and may be involved in uPA/uPAR mediated signal transduction.

Transcriptional induction of the urokinase receptor gene by a constitutively active Src. Requirement of an upstream motif (-152/-135) bound with Sp1.

Since c-src overexpression increases colonic cell invasiveness and because both Src activity and urokinase receptor protein are elevated in invasive colon cancers, the present study was undertaken: 1) to determine if a constitutively active Src regulates urokinase receptor expression and 2) to identify required cis-elements and trans-acting factors. SW480 colon cancer cells transfected with an expression plasmid (c-srcY527F) encoding a constitutively active Src protein manifested increased urokinase receptor gene expression and Src activity. Treatment of the src transfectants with a Src-inhibitor (PD173955) reduced urokinase receptor protein levels and laminin degradation. Inasmuch as we recently implicated an upstream region of the urokinase receptor promoter (-152/-135) in constitutive urokinase receptor expression, we determined its role for the induction by src. Whereas the activity of a CAT reporter driven by this region was stimulated by c-srcY527F, the u-PAR promoter mutated at the Sp1-binding motif in the -152/-135 region was not. Nuclear extracts from the src transfectants demonstrated increased Sp1 binding to region -152/-135 compared with those from SW480 cells. Finally, endogenous urokinase receptor protein amounts in 10 colon cancers and corresponding normal colon correlated with Src specific activity. These data suggest that urokinase receptor gene expression is regulated by Src partly via increased Sp1 binding.

Pulmonary alveolar proteinosis in Israel: ethnic clustering.

BACKGROUND: Pulmonary alveolar proteinosis is a rare disease in which a surfactant-like phospholipid-rich protein accumulates in the lungs. The disease is amenable to effective therapy by total lung lavage. OBJECTIVES: To investigate the prevalence, ethnic distribution and course of PAP in Israel. METHODS: A countrywide survey was conducted during which pulmonologists were questioned about patients with PAP. The patients were examined and their charts, radiological images, pathological slides and physiological data were reviewed. RESULTS: The survey yielded 15 patients (8 females) during the period 1976-98 (14 in the last decade), giving a prevalence of 3.7 x 10(6) and an incidence of 0.36 x 10(6)/year. Mean age of the patients was 33 +/- 13 years (range 0.5-46 years). Seven patients were North African (two were siblings), four were from Iraq and two were Arabs; there was only one Ashkenazi Jew (a child). Symptoms at the onset were dyspnea and chest pain. Spontaneous remission occurred in at least 3 patients, and 10 patients required 1-4 bronchoalveolar lavage treatments. The subjective and physiological response was favorable, but there was less consistent radiological improvement. CONCLUSION: The prevalence of PAP in Israel is approximately 3.7 x 10(6). Most cases occurred in Jews who had immigrated from North Africa or Iraq, and two were siblings. The prevalence among the Arab population appears to be similar. This clustering suggests the existence of a genetic predisposition. The course of the disease appears to be similar to that reported elsewhere.

The urokinase plasminogen activator system in cancer: implications for tumor angiogenesis and metastasis.

Substantial evidence exists which implicates the urokinase plasminogen activator system [urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1)] in the neo-vascularization, invasion and metastasis of many solid tumors. Clinical studies have demonstrated an association between high levels of expression of the components of this system in tumors and poor patient prognosis and outcome. Components of the uPA/uPAR system are differentially expressed or activated on motile cells including invading tumor cells and leukocytes, and migrating endothelial cells. In contrast, there is little or no expression on most normal, quiescent cells. Studies performed in vitro have demonstrated the regulation of the expression of uPA and uPAR by growth and differentiation factors as well as by oncogenes. In this review, we summarize recent findings on the role of the components of the uPA/uPAR system in angiogenesis, invasiveness and tumor metastasis. The activities of this system in endothelial and leukocyte cell biology and the relevance of these activities to angiogenesis and tumor metastasis will be considered. Recent experimental evidence obtained using inhibitors of uPA and uPAR has validated this system as a therapeutic target for the development of anti-angiogenic and anti-metastatic therapeutic agents. These studies, as well as additional therapeutic and diagnostic implications for uPAR targeting, will be discussed.

Stimulation of urokinase-type plasminogen activator receptor expression by PMA requires JNK1-dependent and -independent signaling modules.

The urokinase-type plasminogen activator receptor (u-PAR) has been implicated in tumor progression, and previous studies have shown that the expression of this gene is strongly up-regulated by PMA. Although the signaling mechanism by which PMA modulates u-PAR expression is not known, the effect of this phorbol ester on the expression of other genes has been ascribed to activation of the c-Raf-1-ERK signaling pathway. However, in the current study we examined an alternate possibility that the inductive effect of PMA on u-PAR expression also required a JNK1-dependent signaling cascade usually associated with stress-inducing stimuli. PMA treatment of the u-PAR-deficient OVCAR-3 ovarian cancer cells, which contain low JNK activities, resulted in a rapid (5 min) increase in JNK activity. Maximal JNK activity (12-fold induction) occurred after 30 min; this preceding the earliest detected rise in u-PAR protein (2 h). Dose-response studies with PMA also indicated that the increased JNK activity was tightly correlated with elevated u-PAR protein levels. The stimulation of u-PAR promoter activity by PMA required an intact upstream AP-1 motif (-184) and in PMA-treated cells this motif was bound with c-Jun as indicated from mobility shift assays. PMA up-regulated the c-Jun trans acting activity as indicated by the higher activity of a GAL4-regulated luciferase reporter in phorbol-ester-treated cells co-transfected with an expression vector encoding the c-Jun transactivation domain fused to the GAL4 DNA-binding domain. The ability of PMA to stimulate u-PAR promoter activity was effectively titrated out by the co-expression of either a kinase-defective JNK1 or a dominant negative MEKK1 the latter being an upstream activator of JNK1. Conversely, u-PAR promoter activity was stimulated by the co-expression of a constitutively active MEKK1 and this induction was antagonized by the inclusion of the kinase-defective JNK1 plasmid. We also determined the biological significance of the JNK1-dependent signaling cascade in regulating u-PAR promoter activity by c-Ha-ras since this oncogene is activated and/or overexpressed in a variety of tumors including ovarian cancer. Transfection of an activated c-Ha-ras into OVCAR-3 cells stimulated u-PAR promoter activity over 20-fold and this could be countered by the individual expression of dominant negative expression constructs to Rac-1, MEKK1 or JNK1. Taken together, these data suggest that the PMA- or c-Ha-Ras-dependent stimulation of u-PAR gene expression requires a JNK1-dependent signaling module and that, at least for PMA, the concurrent stimulation of a JNK1-independent signaling module is also required. Thus, caution should be exercised in invoking linear signaling modules to account for the regulation of inducible gene expression.

Binding of high molecular weight kininogen to human endothelial cells is mediated via a site within domains 2 and 3 of the urokinase receptor.

The urokinase receptor (uPAR) binds urokinase-type plasminogen activator (u-PA) through specific interactions with uPAR domain 1, and vitronectin through interactions with a site within uPAR domains 2 and 3. These interactions promote the expression of cell surface plasminogen activator activity and cellular adhesion to vitronectin, respectively. High molecular weight kininogen (HK) also stimulates the expression of cell surface plasminogen activator activity through its ability to serve as an acquired receptor for prekallikrein, which, after its activation, may directly activate prourokinase. Here, we report that binding of the cleaved form of HK (HKa) to human umbilical vein endothelial cells (HUVEC) is mediated through zinc-dependent interactions with uPAR. These occur through a site within uPAR domains 2 and 3, since the binding of 125I-HKa to HUVEC is inhibited by vitronectin, anti-uPAR domain 2 and 3 antibodies and soluble, recombinant uPAR (suPAR), but not by antibody 7E3, which recognizes the beta chain of the endothelial cell vitronectin receptor (integrin alphavbeta3), or fibrinogen, another alphavbeta3 ligand. We also demonstrate the formation of a zinc-dependent complex between suPAR and HKa. Interactions of HKa with endothelial cell uPAR may underlie its ability to promote kallikrein-dependent cell surface plasmin generation, and also explain, in part, its antiadhesive properties.

Induction in human osteoblastic cells (SaOS2) of the early response genes fos, jun, and myc by the amino terminal fragment (ATF) of urokinase.

Previous studies have demonstrated that overexpression of urinary plasminogen activator (uPA) in rat prostate cancer cells results in increased skeletal metastases, which are primarily of the osteoblastic variety. The osseous activation induced by the metastases appears to be mediated through the amino terminal fragment (ATF) of uPA, which lacks the catalytic domain and can act as a growth factor for osteoblasts. To explore further the mechanism of action of uPA in bone cells, we evaluated the effects of ATF on modulating the expression of various proto-oncogenes. Human-osteoblast-derived osteosarcoma cells, SaOS2, were treated with graded doses of ATF for 10-120 min, and effects on early response proto-oncogenes were monitored. ATF increased c-myc, c-jun, and c-fos gene expression in a time-dependent manner for up to 60 min, after which mRNA levels fell. The maximum induction was seen in c-fos gene expression, which was found to be dose dependent. This effect of ATF was localized to its growth-factorlike domain. Examination of the half life of these transcripts in the presence of the transcriptional inhibitor actinomycin D demonstrated that ATF does not alter the stability of c-fos mRNA in these bone cells. Nuclear run-off assays indicated that ATF effects were due to stimulation of c-fos gene transcription. An increase in c-fos protein levels was correlated with the augmentation of its mRNA in ATF-treated SaOS2 cells. Pretreatment of SaOS2 cells with the protein tyrosine kinase inhibitor herbimycin and recombinant soluble uPA receptor (uPAR) caused a significant reduction in the ability of ATF to induce c-fos expression. These results demonstrate a novel role for uPA in activating early response proto-oncogenes, in particular c-fos, which plays an important role in bone cell growth and differentiation and may be a key factor in the signal transduction pathway of ATF.