RESUMO
SIGNIFICANCE STATEMENT: Proteinuria predicts accelerated decline in kidney function in CKD. The pathologic mechanisms are not well known, but aberrantly filtered proteins with enzymatic activity might be involved. The urokinase-type plasminogen activator (uPA)-plasminogen cascade activates complement and generates C3a and C5a in vitro / ex vivo in urine from healthy persons when exogenous, inactive, plasminogen, and complement factors are added. Amiloride inhibits uPA and attenuates complement activation in vitro and in vivo . In conditional podocin knockout (KO) mice with severe proteinuria, blocking of uPA with monoclonal antibodies significantly reduces the urine excretion of C3a and C5a and lowers tissue NLRP3-inflammasome protein without major changes in early fibrosis markers. This mechanism provides a link to proinflammatory signaling in proteinuria with possible long-term consequences for kidney function. BACKGROUND: Persistent proteinuria is associated with tubular interstitial inflammation and predicts progressive kidney injury. In proteinuria, plasminogen is aberrantly filtered and activated by urokinase-type plasminogen activator (uPA), which promotes kidney fibrosis. We hypothesized that plasmin activates filtered complement factors C3 and C5 directly in tubular fluid, generating anaphylatoxins, and that this is attenuated by amiloride, an off-target uPA inhibitor. METHODS: Purified C3, C5, plasminogen, urokinase, and urine from healthy humans were used for in vitro / ex vivo studies. Complement activation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and ELISA. Urine and plasma from patients with diabetic nephropathy treated with high-dose amiloride and from mice with proteinuria (podocin knockout [KO]) treated with amiloride or inhibitory anti-uPA antibodies were analyzed. RESULTS: The combination of uPA and plasminogen generated anaphylatoxins C3a and C5a from intact C3 and C5 and was inhibited by amiloride. Addition of exogenous plasminogen was sufficient for urine from healthy humans to activate complement. Conditional podocin KO in mice led to severe proteinuria and C3a and C5a urine excretion, which was attenuated reversibly by amiloride treatment for 4 days and reduced by >50% by inhibitory anti-uPA antibodies without altering proteinuria. NOD-, LRR- and pyrin domain-containing protein 3-inflammasome protein was reduced with no concomitant effect on fibrosis. In patients with diabetic nephropathy, amiloride reduced urinary excretion of C3dg and sC5b-9 significantly. CONCLUSIONS: In conditions with proteinuria, uPA-plasmin generates anaphylatoxins in tubular fluid and promotes downstream complement activation sensitive to amiloride. This mechanism links proteinuria to intratubular proinflammatory signaling. In perspective, amiloride could exert reno-protective effects beyond natriuresis and BP reduction. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Increased Activity of a Renal Salt Transporter (ENaC) in Diabetic Kidney Disease, NCT01918488 and Increased Activity of ENaC in Proteinuric Kidney Transplant Recipients, NCT03036748 .
Assuntos
Nefropatias Diabéticas , Ativador de Plasminogênio Tipo Uroquinase , Humanos , Camundongos , Animais , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Plasminogênio/metabolismo , Amilorida/farmacologia , Fibrinolisina/metabolismo , Inflamassomos , Camundongos Endogâmicos NOD , Proteinúria/metabolismo , Ativação do Complemento , Anafilatoxinas , FibroseRESUMO
Genetic absence of the urokinase-type plasminogen activator (uPA) reduces arthritis progression in the collagen-induced arthritis (CIA) mouse model to an extent just shy of disease abrogation, but this remarkable observation has not been translated into therapeutic intervention. Our aim was to test the potential in mice of an Ab that blocks the proteolytic capacity of uPA in the CIA model and the delayed-type hypersensitivity arthritis model. A second aim was to determine the cellular origins of uPA and the uPA receptor (uPAR) in joint tissue from patients with rheumatoid arthritis. A mAb that neutralizes mouse uPA significantly reduced arthritis progression in the CIA and delayed-type hypersensitivity arthritis models. In the CIA model, the impact of anti-uPA treatment was on par with the effect of blocking TNF-α by etanercept. A pharmacokinetics evaluation of the therapeutic Ab revealed target-mediated drug disposition consistent with a high turnover of endogenous uPA. The cellular expression patterns of uPA and uPAR were characterized by double immunofluorescence in the inflamed synovium from patients with rheumatoid arthritis and compared with synovium from healthy donors. The arthritic synovium showed expression of uPA and uPAR in neutrophils, macrophages, and a fraction of endothelial cells, whereas there was little or no expression in synovium from healthy donors. The data from animal models and human material provide preclinical proof-of-principle that validates uPA as a novel therapeutic target in rheumatic diseases.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Artrite Experimental/patologia , Artrite Reumatoide/patologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Membrana Sinovial/patologia , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Animais , Anticorpos Monoclonais/imunologia , Modelos Animais de Doenças , Progressão da Doença , Células Endoteliais/imunologia , Etanercepte/farmacologia , Feminino , Humanos , Hipersensibilidade Tardia/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Neutrófilos/imunologia , Membrana Sinovial/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
BACKGROUND AND OBJECTIVES: The aim was to evaluate the prognostic biomarker potential of the soluble urokinase-type plasminogen activator receptor (suPAR) in plasma samples collected pre- and postoperatively from patients resected for colorectal cancer (CRC). METHODS: Patients with CRC were recruited prospectively at six centers from 2006 to 2008. Preoperative plasma samples were available from 494 patients and from 328 of these patients at 6 months postoperatively. Determinations of intact soluble uPAR (suPAR) suPAR(I-III) and the cleaved forms suPAR(I-III) + (II-III) and uPAR(I) were performed. Clinical data were retrieved retrospectively. RESULTS: In a multivariable model based on preoperative plasma samples suPAR(I-III) + (II-III) and uPAR(I) showed an independent statistically significant association to long term survival. When including the change in biomarker level between the pre- and postoperatively samples the hazard ratios were 3.06 (95% confidence interval [CI], 1.78-5.28; P < .0001) and 2.24 (95% CI, 1.59-3.16; P < .0001) for suPAR(I-III) + (II-III) and uPAR(I), respectively. A one-unit decrease in biomarker levels between the pre- and postoperative levels resulted in a 55% and 34% reduction in the risk estimate of death for suPAR(I-III) + (II-III) and uPAR(I), respectively. CONCLUSION: This study validates previously findings regarding the prognostic significance of suPAR in preoperative samples. The inclusion of postoperative samples added further prognostic information.
Assuntos
Neoplasias Colorretais/sangue , Neoplasias Colorretais/mortalidade , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Neoplasias Colorretais/cirurgia , Dinamarca/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Estudos Prospectivos , Estudos RetrospectivosRESUMO
Urokinase plasminogen activator (uPA) converts plasminogen to plasmin, resulting in a proteolytic cascade that has been implicated in tissue destruction during inflammation. Periodontitis is a highly prevalent chronic inflammatory disease characterized by destruction of the tissue and bone that support the teeth. We demonstrate that stimulation of macrophages with the arginine- and lysine-specific cysteine protease complex (RgpA-Kgp complex), produced by the keystone pathogen Porphyromonas gingivalis, dramatically increased their ability to degrade matrix in a uPA-dependent manner. We show that the RgpA-Kgp complex cleaves the inactive zymogens, pro-uPA (at consensus sites Lys(158)-Ile(159) and Lys(135)-Lys(136)) and plasminogen, yielding active uPA and plasmin, respectively. These findings are consistent with activation of the uPA proteolytic cascade by P. gingivalis being required for the pathogen to induce alveolar bone loss in a model of periodontitis and reveal a new host-pathogen interaction in which P. gingivalis activates a critical host proteolytic pathway to promote tissue destruction and pathogen virulence.
Assuntos
Adesinas Bacterianas/metabolismo , Cisteína Endopeptidases/metabolismo , Macrófagos/enzimologia , Periodontite/enzimologia , Porphyromonas gingivalis/enzimologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Adesinas Bacterianas/genética , Animais , Células Cultivadas , Cisteína Endopeptidases/genética , Ativação Enzimática , Feminino , Cisteína Endopeptidases Gingipaínas , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Periodontite/genética , Periodontite/microbiologia , Porphyromonas gingivalis/genética , Ligação Proteica , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
The urokinase plasminogen activator system plays a key role in tissue degradation during cancer invasion. The linker region between domains I and II of the intact, three domain urokinase receptor uPAR(I-III) is highly susceptible to proteolytic cleavage and the resulting cleaved uPAR forms are strong prognostic biomarkers in several types of cancer, i.e., high levels of the cleaved uPAR forms indicate poor survival. To better understand the role of uPAR cleavage in cancer, we have designed immunoassays for specific quantification of intact mouse uPAR [muPAR(I-III)] and mouse uPAR domain I [muPAR(I)]. The level of muPAR(I) is significantly increased in mammary tumor-bearing mice compared to controls and, notably, there is a strong correlation to tumor volume. In contrast, the tumor volume is only weakly correlated to the level of intact muPAR(I-III), indicating that cleavage of muPAR is a more specific marker for cancer than increased expression of muPAR per se. The levels of the muPAR forms are dramatically affected by in vivo challenge with a urokinase -blocking antibody, demonstrating a functional role of uPA in uPAR cleavage. The levels of the muPAR forms are, however, unaffected by uPA-deficiency, suggesting that redundant proteases maintains the task of cleaving uPAR(I-III) when uPA is absent. Our findings emphasize the significance of the cleaved uPAR forms as cancer biomarkers. The strong correlation between muPAR(I) and the tumor volume in our experimental setup may motivate investigations of human uPAR(I) as biomarker for response to oncological treatment.
Assuntos
Neoplasias Mamárias Experimentais/patologia , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Feminino , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Transgênicos , Carga Tumoral , Ativador de Plasminogênio Tipo Uroquinase/químicaRESUMO
Circulating forms of the urokinase plasminogen activator receptor (uPAR) are associated with prognosis in patients with colorectal cancer. Preclinical studies have shown that uPAR can influence the state of phosphorylation and signalling activity of the epidermal growth factor receptor (EGFR) in a ligand-independent manner. The purpose of the study was to evaluate whether plasma soluble intact and cleaved uPAR(I-III)+(II-III) levels could identify a subpopulation of patients with metastatic colorectal cancer (mCRC) where treatment with cetuximab would have a beneficial effect. Plasma samples were available from 453 patients treated in the NORDIC VII study. Patients were randomized between FLOX and FLOX + cetuximab. The levels of uPAR(I-III)+(II-III) were determined by time-resolved fluorescence immunoassay. We demonstrated that higher baseline plasma uPAR(I-III)+(II-III) levels were significantly associated with shorter progression-free survival (PFS) (HR = 1.30, 1.14-1.48, p = 0.0001) and overall survival (OS) (HR = 1.75, 1.52-2.02, p < 0.0001). Multivariate Cox analysis showed that plasma uPAR(I-III)+(II-III) was an independent biomarker of short OS (HR = 1.45, 1.20-1.75, p = 0.0001). There were no significant interactions between plasma uPAR(I-III)+(II-III) levels, KRAS mutational status and treatment either PFS (p = 0.43) or OS (p = 0.095). However, further explorative analyses indicated that patients with low levels of circulating suPAR and a KRAS wild-type tumor have improved effect from treatment with FLOX + cetuximab as compared to patients with KRAS wild-type and high levels of suPAR. These results thus support the preclinical findings and should be further tested in an independent clinical data set.
Assuntos
Antineoplásicos/administração & dosagem , Cetuximab/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/mortalidade , Compostos Organoplatínicos/administração & dosagem , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Biomarcadores Tumorais/sangue , Cetuximab/uso terapêutico , Neoplasias Colorretais/sangue , Feminino , Humanos , Masculino , Metástase Neoplásica , Compostos Organoplatínicos/uso terapêutico , Oxaliplatina , Análise de Sobrevida , Resultado do TratamentoRESUMO
Serine protease catalytic activity is in many cases regulated by conformational changes initiated by binding of physiological modulators to exosites located distantly from the active site. Inhibitory monoclonal antibodies binding to such exosites are potential therapeutics and offer opportunities for elucidating fundamental allosteric mechanisms. The monoclonal antibody mU1 has previously been shown to be able to inhibit the function of murine urokinase-type plasminogen activator in vivo. We have now mapped the epitope of mU1 to the catalytic domain's 37- and 70-loops, situated about 20 Å from the S1 specificity pocket of the active site. Our data suggest that binding of mU1 destabilizes the catalytic domain and results in conformational transition into a state, in which the N-terminal amino group of Ile16 is less efficiently stabilizing the oxyanion hole and in which the active site has a reduced affinity for substrates and inhibitors. Furthermore, we found evidence for functional interactions between residues in uPA's C-terminal catalytic domain and its N-terminal A-chain, as deletion of the A-chain facilitates the mU1-induced conformational distortion. The inactive, distorted state is by several criteria similar to the E* conformation described for other serine proteases. Hence, agents targeting serine protease conformation through binding to exosites in the 37- and 70-loops represent a new class of potential therapeutics.
Assuntos
Serina Endopeptidases/imunologia , Inibidores de Serina Proteinase/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/imunologia , Regulação Alostérica , Animais , Anticorpos Monoclonais , Domínio Catalítico , Ativação Enzimática/efeitos dos fármacos , Epitopos/metabolismo , Fibrinolisina/metabolismo , Cinética , Camundongos , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Ressonância de Plasmônio de Superfície , Ativador de Plasminogênio Tipo Uroquinase/efeitos dos fármacosRESUMO
The catalytic activity of serine proteases depends on a salt-bridge between the amino group of residue 16 and the side chain of Asp194. The salt-bridge stabilizes the oxyanion hole and the S1 specificity pocket of the protease. Some serine proteases exist in only partially active forms, in which the amino group of residue 16 is exposed to the solvent. Such a partially active state is assumed by a truncated form of the murine urokinase-type plasminogen activator (muPA), consisting of residues 16-243. Here we investigated the allosteric interconversion between partially active states and the fully active state. Both a monoclonal antibody (mU3) and a peptidic inhibitor (mupain-1--16) stabilize the active state. The epitope of mU3 is located in the 37- and 70-loops at a site homologous to exosite I of thrombin. The N-terminus((Ile16)) of muPA((16--243)) was less exposed upon binding of mU3 or mupain-1--16. In contrast, introduction of the mutations F40Y or E137A into muPA((16--243)) increased exposure of the N-terminus((Ile16)) and resulted in large changes in the thermodynamic parameters for mupain-1--16 binding. We conclude that the distorted state of muPA((16--243)) is conformationally ordered upon binding of ligands to the active site and upon binding of mU3 to the 37- and 70-loops. Our study establishes the 37- and 70-loops as a unique site for binding to compounds stabilizing the active state of serine proteases.
Assuntos
Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Regulação Alostérica , Animais , Domínio Catalítico , Ativação Enzimática , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Peptídeos Cíclicos/metabolismo , Mutação Puntual , Conformação Proteica , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP9, gelatinase B) have separately been recognized to play important roles in various tissue remodeling processes. In this study, we demonstrate that deficiency for MMP9 in combination with ablation of either uPA- or tissue-type plasminogen activator (tPA)-catalyzed plasminogen activation is critical to accomplish normal gestation in mice. Gestation was also affected by simultaneous lack of MMP9 and the uPA receptor (uPAR). Interestingly, uPA-deficiency additionally exacerbated the effect of MMP9-deficiency on bone growth and an additive effect caused by combined lack in MMP9 and uPA was observed during healing of cutaneous wounds. By comparison, MMP9-deficiency combined with absence of either tPA or uPAR resulted in no significant effect on wound healing, indicating that the role of uPA during wound healing is independent of uPAR, when MMP9 is absent. Notably, compensatory upregulation of uPA activity was seen in wounds from MMP9-deficient mice. Taken together, these studies reveal essential functional dependency between MMP9 and uPA during gestation and tissue repair.
Assuntos
Metaloproteinase 9 da Matriz/deficiência , Gravidez/fisiologia , Fenômenos Fisiológicos da Pele , Ativador de Plasminogênio Tipo Uroquinase/deficiência , Cicatrização/fisiologia , Animais , Western Blotting , Pesos e Medidas Corporais , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Feminino , Técnicas Histológicas , Hibridização In Situ , Camundongos , Cicatrização/genéticaRESUMO
BACKGROUND: The liberated domain I of the urokinase plasminogen activator receptor [uPAR(I)] is a significant prognostic marker in lung and ovarian cancer, although the uPAR(I) concentration is below the limit of quantification (LOQ) in a substantial proportion of patient samples (Lung Cancer 2005;48:349-55; Clin Cancer Res 2008;14:5785-93; APMIS 2009;117:755-61). This study was undertaken to design an immunoassay with improved functional sensitivity for measuring uPAR(I) and to evaluate the prognostic value of uPAR(I) for colorectal cancer (CRC) patients. METHODS: Surface plasmon resonance analysis identified 2 monoclonal antibodies, R3 and R20, that simultaneously bind to the liberated uPAR(I) but not to intact uPAR. We used R3 for capture and Eu-labeled R20 for detection in designing a 2-site sandwich time-resolved fluorescence immunoassay (TR-FIA 4) for measuring liberated uPAR(I). TR-FIA 4 was validated for use with citrated plasma. The prognostic value of the uPAR(I) concentration was evaluated in 298 CRC patients. The Cox proportional hazards model was used for the uni- and multivariate survival analyses. RESULTS: The LOQ was 0.65 pmol/L. Liberated uPAR(I) was measurable in all patient samples with TR-FIA 4. In the multivariate analysis that included sex, age, tumor stage, tumor localization, and adjuvant treatment, the uPAR(I) concentration measured with TR-FIA 4 (hazard ratio, 1.72; 95% CI, 1.15-2.57; P = 0.009), as well as the concentration of intact soluble uPAR plus the cleaved uPAR fragment containing domains II and III, tumor stage, and age were independent predictors of prognosis. CONCLUSIONS: TR-FIA 4 has a functional sensitivity improved 4-fold over that of the previous uPAR(I) assay. The uPAR(I) concentration measured with TR-FIA 4 is an independent predictor of prognosis in CRC patients.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias Colorretais/diagnóstico , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/sangue , Neoplasias Colorretais/mortalidade , Feminino , Humanos , Imunoensaio , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Estrutura Terciária de Proteína , Sensibilidade e EspecificidadeRESUMO
Matrix metalloproteinase-9 (MMP-9) is a 92-kDa soluble pro-enzyme implicated in pathological events including cancer invasion. It is therefore an attractive target for therapeutic intervention studies in mouse models. Development of inhibitors requires sufficient amounts of correctly folded murine MMP-9. Constructs encoding zymogens of full-length murine MMP-9 and a version lacking the O-glycosylated linker region and hemopexin domains were therefore generated and expressed in stably transfected Drosophila S2 insect cells. After 7 days of induction the expression levels of the full-length and truncated versions were 5 mg/l and 2 mg/l, respectively. The products were >95% pure after gelatin Sepharose chromatography and possessed proteolytic activity when analyzed by gelatin zymography. Using the purified full-length murine MMP-9 we raised polyclonal antibodies by immunizations of rabbits. These antibodies specifically identified pro-MMP-9 in incisional skin wound extracts from mice when used for Western blotting. Immunohistochemical analysis of paraffin embedded skin wounds from mice showed that MMP-9 protein was localized at the leading-edge keratinocytes in front of the migrating epidermal layer. No immunoreactivity was observed when the antibody was probed against skin wound material from MMP-9 deficient mice. In conclusion, we have generated and purified two proteolytically active recombinant murine MMP-9 protein constructs, which are critical reagents for future cancer drug discovery studies.
Assuntos
Drosophila/genética , Expressão Gênica , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/isolamento & purificação , Murinae/genética , Animais , Anticorpos/imunologia , Linhagem Celular , Cromatografia de Afinidade , Drosophila/citologia , Metaloproteinase 9 da Matriz/análise , Metaloproteinase 9 da Matriz/imunologia , Camundongos , Estrutura Terciária de Proteína , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Pele/metabolismoRESUMO
Background Diabetic nephropathy is a common diabetes mellitus complication associated with hypertension, proteinuria, and excretion of urinary plasmin that activates the epithelial sodium channel, ENaC, in vitro. Here we hypothesized that the deletion of plasminogen and amiloride treatment protect against hypertension in diabetes mellitus. Methods and Results Male plasminogen knockout (plasminogen-deficient [Plg-/-]) and wild-type mice were rendered diabetic with streptozotocin. Arterial blood pressure was recorded continuously by indwelling catheters before and during 10 days of angiotensin II infusion (ANGII; 30-60 ng/kg per minute). The effect of amiloride infusion (2 mg/kg per day, 4 days) was tested in wild-type, diabetic ANGII-treated mice. Streptozotocin increased plasma and urine glucose concentrations and 24-hour urine albumin and plasminogen excretion. Diabetic Plg-/- mice displayed larger baseline albuminuria and absence of urine plasminogen. Baseline mean arterial blood pressure did not differ between groups. Although ANGII elevated blood pressure in wild-type, diabetic wild-type, and Plg-/- control mice, ANGII did not change blood pressure in diabetic Plg-/- mice. Compared with ANGII infusion alone, wild-type ANGII-infused diabetic mice showed blood pressure reduction upon amiloride treatment. There was no difference in plasma renin, ANGII, aldosterone, tissue prorenin receptor, renal inflammation, and fibrosis between groups. Urine from wild-type mice evoked larger amiloride-sensitive current than urine from Plg-/- mice with or without diabetes mellitus. Full-length γ-ENaC and α-ENaC subunit abundances were not changed in kidney homogenates, but the 70 kDa γ-ENaC cleavage product was increased in diabetic versus nondiabetic mice. Conclusions Plasmin promotes hypertension in diabetes mellitus with albuminuria likely through the epithelial sodium channel.
Assuntos
Amilorida/uso terapêutico , Angiotensina II/efeitos adversos , Diabetes Mellitus Tipo 1/complicações , Bloqueadores do Canal de Sódio Epitelial/uso terapêutico , Hipertensão/prevenção & controle , Plasminogênio/deficiência , Animais , Diabetes Mellitus Experimental , Canais Epiteliais de Sódio/efeitos dos fármacos , Hipertensão/diagnóstico , Hipertensão/etiologia , Masculino , CamundongosRESUMO
(1) Background: Persistent Helicobacter pylori infection is the most important risk factor for gastric cancer. The urokinase receptor (uPAR) is upregulated in lesions harboring cancer invasion and inflammation. Circumstantial evidence tends to correlate H. pylori colonization with increased uPAR expression in the human gastric epithelium, but a direct causative link has not yet been established in vivo; (2) Methods: In a mouse model of H. pylori-induced gastritis, we investigated the temporal emergence of uPAR protein expression in the gastric mucosa in response to H. pylori (SS1 strain) infection; (3) Results: We observed intense uPAR immunoreactivity in foveolar epithelial cells of the gastric corpus due to de novo synthesis, compared to non-infected animals. This uPAR induction represents a very early response, but it increases progressively over time as do infiltrating immune cells. Eradication of H. pylori infection by antimicrobial therapy causes a regression of uPAR expression to its physiological baseline levels. Suppression of the inflammatory response by prostaglandin E2 treatment attenuates uPAR expression. Notwithstanding this relationship, H. pylori does induce uPAR expression in vitro in co-cultures with gastric cancer cell lines; (4) Conclusions: We showed that persistent H. pylori colonization is a necessary event for the emergence of a relatively high uPAR protein expression in murine gastric epithelial cells.
RESUMO
AIM: Activation of sodium reabsorption by urinary proteases has been implicated in sodium retention associated with nephrotic syndrome. The study was designed to test the hypothesis that nephrotic proteinuria in mice after conditional deletion of podocin leads to urokinase-dependent, amiloride-sensitive plasmin-mediated sodium and water retention. METHODS: Ten days after podocin knockout, urine and faeces were collected for 10 days in metabolic cages and analysed for electrolytes, plasminogen, protease activity and ability to activate γENaC by patch clamp and western blot. Mice were treated with amiloride (2.5 mg kg-1 for 2 days and 10 mg kg-1 for 2 days) or an anti-urokinase-type plasminogen activator (uPA) targeting antibody (120 mg kg-1 /24 h) and compared to controls. RESULTS: Twelve days after deletion, podocin-deficient mice developed significant protein and albuminuria associated with increased body wt, ascites, sodium accumulation and suppressed plasma renin. This was associated with increased urinary excretion of plasmin and plasminogen that correlated with albumin excretion, urine protease activity co-migrating with active plasmin, and the ability of urine to induce an amiloride-sensitive inward current in M1 cells in vitro. Amiloride treatment in podocin-deficient mice resulted in weight loss, increased sodium excretion, normalization of sodium balance and prevention of the activation of plasminogen to plasmin in urine in a reversible way. Administration of uPA targeting antibody abolished urine activation of plasminogen, attenuated sodium accumulation and prevented cleavage of γENaC. CONCLUSIONS: Nephrotic range glomerular proteinuria leads to urokinase-dependent intratubular plasminogen activation and γENaC cleavage which contribute to sodium accumulation.
Assuntos
Amilorida/farmacologia , Glomérulos Renais/metabolismo , Síndrome Nefrótica/metabolismo , Proteinúria/metabolismo , Sódio/metabolismo , Animais , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Peptídeo Hidrolases/urina , Plasminogênio/urina , Ativador de Plasminogênio Tipo Uroquinase , Água/metabolismo , Redução de Peso/efeitos dos fármacosRESUMO
The cellular receptor for urokinase, uPAR, localizes its ligand, uPA, and thereby the plasminogen activation, to the cell surface. uPA also cleaves uPAR, liberating the ligand-binding domain I, and thereby inactivates the binding potential of uPAR for both uPA and vitronectin. The uPA-catalyzed cleavage of uPAR is fast on the cell surface, when uPA is bound to a neighboring uPAR molecule. uPAR can be shed from the cell surface. However, the soluble form cannot be cleaved by uPA. Glycolipid-anchored and soluble forms of intact, uPAR(I-III), and cleaved receptor, uPAR(II-III) and uPAR(I), have been identified in tissue and body fluids. It is well-established, that the total amount of all uPAR forms is a strong prognostic marker in different types of cancer. Using immunoassays, measuring the individual uPAR forms, has revealed that the cleaved uPAR forms are even stronger prognostic markers and have diagnostic utility. This review will focus on the mechanism of uPAR cleavage and the functional consequences, as well as the clinical applicability of cleaved uPAR forms.
Assuntos
Neoplasias/diagnóstico , Receptores de Superfície Celular/análise , Ativação Enzimática , Fibrinólise , Humanos , Neoplasias/fisiopatologia , Fragmentos de Peptídeos/análise , Plasminogênio/metabolismo , Prognóstico , Receptores de Superfície Celular/metabolismo , Receptores de Ativador de Plasminogênio Tipo UroquinaseRESUMO
The urokinase plasminogen activator receptor (uPAR) is a versatile three-domain GPI-anchored protein, which binds urokinase plasminogen activator (uPA) and thereby focalises plasminogen activation on the cell surface. Generation of a proteolytic potential is essential in both normal physiological and pathological extracellular tissue remodelling processes. uPA can also cleave uPAR, resulting in liberation of the amino-terminal domain I, which encompasses binding sites for both uPA and the adhesion molecule, vitronectin. In order to localise the different uPAR forms on the plasma membrane of murine monocyte macrophage-like P388D.1 cells, we have now generated and characterised two high-affinity murine mAbs, mR3 and mR4, raised against murine uPAR. mR3 was found to recognise an epitope located in domain I of uPAR. Surface plasmon resonance analyses and cell binding studies revealed that this mAb was able to bind preformed complexes of murine pro-uPA and murine uPAR. In contrast, mR4 recognises domains II-III in uPAR and does not bind preformed pro-uPA-uPAR complexes in similar analyses. Immunofluorescence microscopy of P388D.1 cells revealed that mR3 stained the cells equally well in the presence or absence of saturation with the amino-terminal fragment of uPA, ATF. However, the signal intensity obtained using another uPAR domain I specific mAb, mR1, was significantly reduced upon ATF saturation. Furthermore, when adding ATF, mR4 selectively stained the cleaved receptor. Applying these newly generated mAbs, we additionally demonstrated that cleaved and intact uPAR was evenly distributed on the surface of these cells.
Assuntos
Anticorpos Monoclonais/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Animais , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/imunologia , Sítios de Ligação de Anticorpos/imunologia , Linhagem Celular , Membrana Celular/química , Membrana Celular/imunologia , Humanos , Camundongos , Estrutura Terciária de Proteína , Receptores de Ativador de Plasminogênio Tipo Uroquinase/imunologia , Ressonância de Plasmônio de Superfície/métodos , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/imunologiaRESUMO
The catalytic activity of trypsin-like serine proteases is in many cases regulated by conformational changes initiated by binding of physiological modulators to exosites located distantly from the active site. A trypsin-like serine protease of particular interest is urokinase-type plasminogen activator (uPA), which is involved in extracellular tissue remodeling processes. Herein, we used hydrogen/deuterium exchange mass spectrometry (HDXMS) to study regulation of activity in the catalytic domain of the murine version of uPA (muPA) by two muPA specific monoclonal antibodies. Using a truncated muPA variant (muPA16-243), containing the catalytic domain only, we show that the two monoclonal antibodies, despite binding to an overlapping epitope in the 37s and 70s loops of muPA16-243, stabilize distinct muPA16-243 conformations. Whereas the inhibitory antibody, mU1 was found to increase the conformational flexibility of muPA16-243, the stimulatory antibody, mU3, decreased muPA16-243 conformational flexibility. Furthermore, the HDXMS data unveil the existence of a pathway connecting the 70s loop to the active site region. Using alanine scanning mutagenesis, we further identify the 70s loop as an important exosite for the activation of the physiological uPA substrate plasminogen. Thus, the data presented here reveal important information about dynamics in uPA by demonstrating how various ligands can modulate uPA activity by mediating long-range conformational changes. Moreover, the results provide a possible mechanism of plasminogen activation.
Assuntos
Plasminogênio/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Ligantes , Camundongos , Conformação Proteica , Ativador de Plasminogênio Tipo Uroquinase/químicaRESUMO
Binding of urokinase plasminogen activator (uPA) to its cellular receptor, uPAR, potentiates plasminogen activation and localizes it to the cell surface. Focal plasminogen activation is involved in both normal and pathological tissue remodeling processes including cancer invasion. The interaction between uPA and uPAR therefore represents a potential target for anti-invasive cancer therapy. Inhibitors of the human uPA-uPAR interaction have no effect in the murine system. To enable in-vivo studies in murine cancer models we have now generated murine monoclonal antibodies (mAbs) against murine uPAR (muPAR) by immunizing uPAR-deficient mice with recombinant muPAR and screened for antibodies, which inhibit the muPA-muPAR interaction. Two of the twelve mAbs obtained, mR1 and mR2, interfered with the interaction between muPAR and the amino-terminal fragment of muPA (mATF) when analyzed by surface plasmon resonance. The epitope for mR1 is located on domain I of muPAR, while that of mR2 is on domains (II-III). In cell binding experiments using radiolabelled mATF, the maximal inhibition obtained with mR1 was 85% while that obtained with mR2 was 50%. The IC(50) value for mR1 was 0.67 nM compared to 0.14 nM for mATF. In an assay based on modified anthrax toxins, requiring cell-bound muPA activity for its cytotoxity, an approximately 50% rescue of the cells could be obtained by addition of mR1. Importantly, in-vivo efficacy of mR1 was demonstrated by the ability of mR1 to rescue mice treated with a lethal dose of uPA-activatable anthrax toxins.
Assuntos
Anticorpos Monoclonais/farmacologia , Macrófagos/efeitos dos fármacos , Plasminogênio/metabolismo , Receptores de Superfície Celular/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Reações Antígeno-Anticorpo , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Antígenos de Bactérias/toxicidade , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/toxicidade , Sítios de Ligação de Anticorpos , Ligação Competitiva , Linhagem Celular , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Mapeamento de Epitopos , Feminino , Humanos , Hibridomas/metabolismo , Imunização , Radioisótopos do Iodo/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Fatores de TempoRESUMO
AIM: Urokinase plasminogen activator receptor (uPAR) plays a central role during cancer invasion by facilitating pericellular proteolysis. We initiated the prospective 'Copenhagen uPAR Prostate Cancer' study to investigate the significance of uPAR levels in prostate cancer (PCa) patients. METHODS: Plasma samples and clinical data from patients with newly diagnosed PCa have been collected prospectively. The uPAR forms have been measured in plasma using time-resolved fluorescence immunoassays. RESULTS: The level of intact uPAR(I-III) did not differ. Plasma uPAR(I-III) + uPAR(II-III) levels and uPAR(I) levels were significantly higher in hormone-naive and castrate-resistant patients compared with patients with localized disease (both: p < 0.0001). CONCLUSION: Our results show that cleaved uPAR forms are significantly increased in patients with advanced PCa.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias de Próstata Resistentes à Castração/sangue , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Idoso , Bases de Dados Factuais , Dinamarca/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Proteólise , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
BACKGROUND: High levels of circulating forms of the urokinase-type plasminogen activator receptor (uPAR) are significantly associated to poor prognosis in cancer patients. Our aim was to determine biological variations and reference intervals of the uPAR forms in blood, and in addition, to test the clinical relevance of using these as cut-points in colorectal cancer (CRC) prognosis. METHODS: uPAR forms were measured in citrated and EDTA plasma samples using time-resolved fluorescence immunoassays. Diurnal, intra- and inter-individual variations were assessed in plasma samples from cohorts of healthy individuals. Reference intervals were determined in plasma from healthy individuals randomly selected from a Danish multi-center cross-sectional study. A cohort of CRC patients was selected from the same cross-sectional study. RESULTS: The reference intervals showed a slight increase with age and women had ~20% higher levels. The intra- and inter-individual variations were ~10% and ~20-30%, respectively and the measured levels of the uPAR forms were within the determined 95% reference intervals. No diurnal variation was found. Applying the normal upper limit of the reference intervals as cut-point for dichotomizing CRC patients revealed significantly decreased overall survival of patients with levels above this cut-point of any uPAR form. CONCLUSIONS: The reference intervals for the different uPAR forms are valid and the upper normal limits are clinically relevant cut-points for CRC prognosis.