RESUMO
Notch is an evolutionarily conserved signaling pathway with an important role in development and cell fate determination. Deregulation of Notch signaling has been associated with several pathological conditions, including cancer. Acting as an oncogene in some types of cancers and as a tumor suppressor in other, Notch effects seem to be highly context-dependent in solid tumors. In the present study, we aimed to investigate gene expression levels of Notch pathway constituents, including ligands, receptors, and target genes, during the early stages of inflammation-associated intestinal carcinogenesis. To achieve so, we used our recently developed mouse model, in which colon cancer arises in the absence of urokinase-type plasminogen activator (uPA) due to colitis induced by dextran sodium sulfate (DSS) treatment. Among the cell surface components, ligands Jag1/Jag2 and receptors Notch1/Notch2 were found to be significantly upregulated in the uPA-deficient protumorigenic inflammatory microenvironment. Moreover, several intracellular Notch modulators, i.e. Hes1, Hey1, and Klf4, were also shown to be deregulated with inflammation, yet irrespective of uPA status. Sox9 transcription factor, however, was significantly downregulated in the uPA-deficient/DSS-treated mice that developed colon adenomas as compared to the wild-type/DSS-treated group with no neoplasia identified. The latter finding supports a tumor suppressive role of Sox9 in intestinal carcinogenesis. Our results point towards an early activation of Notch signaling pathway at the receptor-ligand level in inflammation-associated colon neoplasmatogenesis developed in the absence of uPA. Interestingly, such activation may not be accompanied by deregulation of downstream Notch-target genes, possibly due to the effects of other inter-related signaling pathways.
Assuntos
Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Receptor Notch1/biossíntese , Receptor Notch2/biossíntese , Transdução de Sinais , Ativador de Plasminogênio Tipo Uroquinase/deficiência , Animais , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Endogâmicos BALB C , Receptor Notch1/genética , Receptor Notch2/genéticaRESUMO
: Background: The relationship between urokinase-type plasminogen activator (uPA) and the development of type 2 diabetes mellitus (T2DM) was investigated in the study by using mice and cell models, as well as patients with T2DM. METHODS: In mice models, wild-type and uPA knockout (uPA-/-) BALB/c mice were used for induction of T2DM. In cell models, insulin secretion rate and ß cell proliferation were assessed in normal and high glucose after treating uPA siRNA, uPA, or anti-uPA antibody. In our clinical study, patients with T2DM received an oral glucose-tolerance test, and the relationship between uPA and insulin secretion was assessed. RESULTS: Insulin particles and insulin secretion were mildly restored one month after induction in wild-type mice, but not in uPA-/- mice. In cell models, insulin secretion rate and cell proliferation declined in high glucose after uPA silencing either by siRNA or by anti-uPA antibody. After treatment with uPA, ß cell proliferation increased in normal glucose. In clinical study, patients with T2DM and higher uPA levels had better ability of insulin secretion than those with lower uPA levels. CONCLUSION: uPA may play a substantial role in insulin secretion, ß cell regeneration, and progressive development of T2DM. Supplementation of uPA might be a novel approach for prevention and treatment of T2DM in the future.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Regeneração , Ativador de Plasminogênio Tipo Uroquinase/deficiência , Animais , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Células Secretoras de Insulina/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Extracellular proteolysis initiated by the binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR) regulates the development of inhibitory neuronal circuits in the cerebral cortex and tissue remodeling after epileptogenic brain injury. To study the function of different components of the uPA-uPAR system on behavior and epileptogenesis, and to complement our previous studies on naïve and injured mice deficient in the uPA-encoding gene Plau or the uPAR-encoding gene Plaur, we analyzed the behavioral phenotype, seizure susceptibility, and perineuronal nets surrounding parvalbumin-positive inhibitory interneurons in Plau and Plaur (double knockout dKO) mice. In a climbing test, dKO mice showed reduced interest towards the environment as compared with Wt mice (p < 0.01). In a social approach test, however, dKO mice spent more time than Wt mice exploring the compartment containing a stranger mouse than the empty compartment (p < 0.05). Moreover, in a social interaction test, dKO mice exhibited increased contact time (p < 0.01). Compared with Wt mice, the dKO mice also had a longer single contact duration (p < 0.001) with the stranger mouse. In the elevated plus-maze, grooming, and marble burying tests, the anxiety level of dKO mice did not differ from that of Wt mice. Rearing time in an exploratory activity test, and spatial learning and memory in the Morris swim navigation task were also comparable between dKO and Wt mice. In the pentylenetetrazol (PTZ) seizure-susceptibility test, dKO mice had a shorter latency to the first epileptiform spike (p = 0.0001) and a greater total number of spikes (p < 0.001) than Wt mice. The dKO genotype did not affect the number of cortical perineuronal nets. Our findings indicate that Plau/Plaur-deficiency leads to a more social phenotype toward other mice with diminished interest in the surrounding environment, and increased seizure susceptibility.
Assuntos
Regulação da Expressão Gênica/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/deficiência , Convulsões/metabolismo , Comportamento Social , Ativador de Plasminogênio Tipo Uroquinase/deficiência , Animais , Ansiedade/etiologia , Ansiedade/genética , Aprendizagem da Esquiva/fisiologia , Ondas Encefálicas/efeitos dos fármacos , Ondas Encefálicas/genética , Convulsivantes/toxicidade , Modelos Animais de Doenças , Suscetibilidade a Doenças/induzido quimicamente , Suscetibilidade a Doenças/fisiopatologia , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Asseio Animal/fisiologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pentilenotetrazol/toxicidade , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Convulsões/induzido quimicamente , Convulsões/patologia , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Deregulation of the bone morphogenetic protein (BMP) pathway has been documented in colorectal cancer (CRC). Previously, we investigated possible associations between urokinase-type plasminogen activator (uPA) deficiency and expression of extracellular constituents of BMP signaling in a newly developed mouse model of inflammation-driven intestinal neoplasmatogenesis, in which chronic colitis and CRC are induced using dextran sodium sulfate (DSS). In this report, we explored the contribution of intracellular components of Smad-mediated BMP signal transduction using the same model. Interestingly, upon DSS treatment, we noticed an overexpression of Runx1/2/3 transcription factors in both wild-type and uPA-deficient mice. Moreover, Runx1 and Runx2 expression levels exhibited an even higher increase in DSS-treated/uPA-deficient mice as compared to DSS-treated/wild-type animals. In all experimental conditions, in situ investigation of Runx-expressing cell types, revealed detection of all three Runx in the immune cells, yet in the DSS-treated/uPA-deficient mice Runx1 and Runx2 were also identified in the preneoplastic epithelium of advanced high-grade dysplasia and carcinoma in-situ colonic lesions. Finally, the uPA-deficient pro-tumorigenic colitic microenvironment exhibited increased levels of the Runx-induced target genes Snai2, Bim and Claudin1, known to have a role in tumor development and progression. These findings suggest that the absence of uPA correlates with increased levels of Runx transcriptional regulators in a way that promotes inflammation-associated carcinogenesis.
Assuntos
Colite/genética , Neoplasias Colorretais/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Regulação Neoplásica da Expressão Gênica , Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Proteína 11 Semelhante a Bcl-2/genética , Proteína 11 Semelhante a Bcl-2/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Claudina-1/genética , Claudina-1/metabolismo , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Sulfato de Dextrana , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Transdução de Sinais , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Transcrição Gênica , Ativador de Plasminogênio Tipo Uroquinase/deficiênciaRESUMO
Mutations in SLURP1, a secreted protein of keratinocytes, cause a palmoplantar keratoderma (PPK) known as mal de Meleda. Slurp1 deficiency in mice faithfully recapitulates the human disease, with increased keratinocyte proliferation and thickening of the epidermis on the volar surface of the paws. There has long been speculation that SLURP1 serves as a ligand for a receptor that regulates keratinocyte growth and differentiation. We were intrigued that mutations leading to increased signalling through the epidermal growth factor receptor (EGFR) cause PPK. Here, we sought to determine whether reducing EGFR signalling would ameliorate the PPK associated with SLURP1 deficiency. To address this issue, we bred Slurp1-deficient mice that were homozygous for a hypomorphic Egfr allele. The hypomorphic Egfr allele, which leads to reduced EGFR signalling in keratinocytes, did not ameliorate the PPK elicited by SLURP1 deficiency, suggesting that SLURP1 deficiency causes PPK independently (or downstream) from the EGFR pathway.
Assuntos
Antígenos Ly/genética , Antígenos Ly/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Ceratodermia Palmar e Plantar/genética , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Alelos , Animais , Genótipo , Ceratodermia Palmar e Plantar/patologia , Masculino , Camundongos Knockout , Fenótipo , Transdução de Sinais/genética , Ativador de Plasminogênio Tipo Uroquinase/deficiênciaRESUMO
PURPOSE: Recent findings indicate the significant contribution of urokinase and urokinase receptor (uPA and uPAR) in the processes of nerve regeneration, however, their role in axonal growth and branching is unclear. Using a 3D model of mouse Dorsal Root Ganglia (DRG) explants, differentiated into neurons Neuro 2a cells and transgenic mice lacking the urokinase gene, we studied the involvement of the uPA/uPAR system in the neural cell migration, neurite outgrowth, elongation and branching. RESULTS: uPA and uPAR are expressed in the growth cones of axons. Using an ex vivo model of DRG explants in Matrigel we have found that uPA inhibition attenuates neural cell migration and axonal growth, pointing to an important role of urokinase in these processes. Apparently, uPA mediates its effects through its specific receptor uPAR: anti-uPAR antibody, which blocks the uPA binding to uPAR, stimulates axon branching and attenuates neural cell migration from DRG explants. Simultaneous inhibition of uPA and uPAR almost completely prevents the axonal outgrowth from explants into the Matrigels. Experiments in vitro using Neuro 2a cells differentiated into neurons demonstrate that administration of exogenous uPA increases the neurite growth rate (elongation), most likely via the interaction of uPA with uPAR. Blocking of uPAR stimulates neurite formation and enhances branching of preexisting neurites. The results obtained on DRG explants from transgenic mice lacking uPA gene support the assumption that uPA stimulates neurite growth via uPA/uPAR interaction and uPAR role in axons branching and neural cell migration. CONCLUSIONS: The uPA/uPAR system plays an essential role in neural cell migration, axonal growth and branching.
Assuntos
Axônios/fisiologia , Movimento Celular/fisiologia , Neurônios/fisiologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Axônios/metabolismo , Linhagem Celular Tumoral , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Regeneração Nervosa , Crescimento Neuronal/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Técnicas de Cultura de Órgãos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/deficiência , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
The suppression of the bone morphogenetic protein (BMP) signaling pathway has been recently shown to promote adenoma-to-carcinoma transition in sporadic colon cancer. However, its role in the evolution of early preneoplastic changes to neoplasia remains elusive. In the present study, we aimed to investigate the gene expression levels of multiple extracellular BMP family constituents, including BMP ligands/receptors and inhibitors, during the early stages of inflammation-associated colon carcinogenesis. For that, we used the recently developed urokinase-type plasminogen activator (uPA)-deficient mouse model of colonic polypoidogenesis, in which adenomatous polyps arise several months after the induction of dextran sodium sulfate (DSS) colitis. In DSS-treated wild-type mice, the preneoplastic lesions which did not eventually evolve to adenomas resided in a colitic microenvironment characterized by a balanced upregulation of both BMP ligands, i.e., Bmp4/7 and BMP inhibitors, such as chordin, noggin, and gremlin-1. In the uPA-deficient tumor-promoting inflammatory microenvironment, however, there was a clear evidence for BMP pathway suppression. By contrast to DSS-treated wild-type controls, the inflammation-associated Bmp4 upregulation was abolished, and the BMP signaling suppression was further enhanced by a particularly high increase of gremlin-1 expression. These findings propose that BMP pathway suppression in colon cancer could be associated with very early stages of the preneoplasia-to-neoplasia sequence of events.
Assuntos
Adenocarcinoma/patologia , Proteínas Morfogenéticas Ósseas/metabolismo , Transformação Celular Neoplásica/metabolismo , Neoplasias do Colo/patologia , Inflamação/patologia , Adenocarcinoma/metabolismo , Adenoma/metabolismo , Adenoma/patologia , Animais , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Inflamação/metabolismo , Camundongos , Lesões Pré-Cancerosas/patologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/deficiênciaRESUMO
Several components of the urokinase-type plasminogen activator receptor (uPAR)-interactome, including uPAR and its ligand sushi-repeat protein 2, X-linked (SRPX2), are linked to susceptibility to epileptogenesis in animal models and/or humans. Recent evidence indicates that urokinase-type plasminogen activator (uPA), a uPAR ligand with focal proteinase activity in the extracellular matrix, contributes to recovery-enhancing brain plasticity after various epileptogenic insults such as traumatic brain injury (TBI) and status epilepticus. Here, we examined whether deficiency of the uPA-encoding gene Plau augments epileptogenesis after TBI. Traumatic brain injury was induced by controlled cortical impact in the somatosensory cortex of adult male wild-type and Plau-deficient mice. Development of epilepsy and seizure susceptibility were assessed with a 3-week continuous video-electroencephalography monitoring and a pentylenetetrazol test, respectively. Traumatic brain injury-induced cortical or hippocampal pathology did not differ between genotypes. The pentylenetetrazol test revealed increased seizure susceptibility after TBI (p<0.05) in injured mice. Epileptogenesis was not exacerbated, however, in Plau-deficient mice. Taken together, Plau deficiency did not worsen controlled cortical impact-induced brain pathology or epileptogenesis caused by TBI when assessed at chronic timepoints. These data expand previous observations on Plau deficiency in models of status epilepticus and suggest that inhibition of focal extracellular proteinase activity resulting from uPA-uPAR interactions does not modify epileptogenesis after TBI.
Assuntos
Lesões Encefálicas/enzimologia , Epilepsia/enzimologia , Ativador de Plasminogênio Tipo Uroquinase/deficiência , Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Animais , Lesões Encefálicas/complicações , Modelos Animais de Doenças , Suscetibilidade a Doenças , Epilepsia/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The plasminogen activator receptor (uPAR) is required for lung infiltration by innate immune cells in respiratory bacterial infections. In order to verify if this held true for respiratory viruses, wild type (WT) and uPAR knockout (uPAR(-/-)) mice were inoculated intranasally with the human respiratory syncytial virus (HRSV) and the influenza A virus. At several days post-infection (dpi), viral titers in the lungs were determined while cell infiltrates in the bronchoalveolar lavage (BAL) were analyzed by flow cytometry. In the case of influenza A, body weight loss and mortality were also monitored. Only minor differences were observed between infected WT and uPAR(-/-) mice, primarily in influenza virus replication and pathology. These results indicate that uPAR does not play a major role in limiting virus replication or in orchestrating the innate immune response against HRSV or influenza infections in mice. This suggests that there are fundamental differences in the immune control of the viral infections studied here and those caused by bacteria.
Assuntos
Receptores de Ativador de Plasminogênio Tipo Uroquinase/deficiência , Infecções Respiratórias/imunologia , Infecções Respiratórias/virologia , Ativador de Plasminogênio Tipo Uroquinase/deficiência , Animais , Feminino , Humanos , Imunidade Inata/imunologia , Vírus da Influenza A/imunologia , Vírus da Influenza A/patogenicidade , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Ativador de Plasminogênio Tipo Uroquinase/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Vírus Sincicial Respiratório Humano/patogenicidade , Análise de Sobrevida , Ativador de Plasminogênio Tipo Uroquinase/imunologia , Carga Viral , Replicação Viral/fisiologiaRESUMO
Macrophages play crucial roles in repair process of various tissues. However, the details in the role of macrophages during bone repair still remains unknown. Herein, we examined the contribution of the tissue fibrinolytic system to the macrophage functions in bone repair after femoral bone defect by using male mice deficient in plasminogen (Plg-/-), urokinase-type plasminogen activator (uPA-/-) or tissue-type plasminogen activator (tPA-/-) genes and their wild-type littermates. Bone repair of the femur was delayed in uPA-/- mice until day 6, compared with wild-type (uPA+/+) mice. Number of Osterix-positive cells and vessel formation were decreased in uPA-/- mice at the bone injury site on day 4, compared with those in uPA+/+ mice. Number of macrophages and their phagocytosis at the bone injury site were reduced in uPA-/- and Plg-/-, but not in tPA-/- mice on day 4. Although uPA or plasminogen deficiency did not affect the levels of cytokines, including TNF-α, IL-1ß, IL-6, IL-4 and IFN-γ mRNA in the damaged femur, the elevation in CCL3 mRNA levels was suppressed in uPA-/- and Plg-/-, but not in tPA-/- mice. Neutralization of CCL3 antagonized macrophage recruitment to the site of bone injury and delayed bone repair in uPA+/+, but not in uPA-/- mice. Our results provide novel evidence that the tissue fibrinolytic system contributes to the induction of macrophage recruitment and CCL3 at the bone injury site, thereby, leading to the enhancement of the repair process.
Assuntos
Quimiocina CCL3/metabolismo , Fêmur/patologia , Fibrinólise , Macrófagos/metabolismo , Cicatrização , Animais , Anticorpos Neutralizantes/farmacologia , Cartilagem/efeitos dos fármacos , Cartilagem/patologia , Contagem de Células , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL3/genética , Quimiocina CCL4/genética , Quimiocina CCL4/metabolismo , Fêmur/irrigação sanguínea , Fêmur/efeitos dos fármacos , Fibrinólise/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Fagocitose/efeitos dos fármacos , Plasminogênio/deficiência , Plasminogênio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição Sp7 , Fatores de Tempo , Ativador de Plasminogênio Tecidual/deficiência , Ativador de Plasminogênio Tecidual/metabolismo , Fatores de Transcrição/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/deficiência , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
Although pneumonia is one of the most important health problems in children, there is still no widely accepted disease severity score, the data on the correlation between the conventional inflammatory markers or chest X-ray and the disease severity remain disputable, and thus, there is an urgent need for a new pneumonia biomarker. The soluble urokinase plasminogen activator (suPAR) is a soluble form of the urokinase plasminogen activator that plays an important role in the innate host defense in the pulmonary tissue. suPAR levels have been associated with a general activation of the immune system rather than with a particular etiological factor. suPAR has a high prognostic value in critically ill patients, especially with sepsis, but there is a growing number of studies focusing on suPAR in respiratory diseases. The aim of this review is to summarize the knowledge on the role of the suPAR/uPAR in lung pathology and its possible use in pneumonia in children.
Assuntos
Pneumonia/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Criança , Humanos , Pulmão/imunologia , Pneumonia/diagnóstico , Pneumonia/terapia , Ativador de Plasminogênio Tipo Uroquinase/deficiênciaRESUMO
Urokinase-type plasminogen activator (uPA), a serine protease, converts plasminogen to plasmin. Activation of plasmin leads to degradation of the extracellular matrix, which is critical for tissue recovery, angiogenesis, cell migration, and axonal and synaptic plasticity. We hypothesized that uPA deficiency would cause an abnormal neurophenotype and would lead to exacerbated epileptogenesis after brain injury. Wild-type (Wt) and uPA-/- mice underwent a battery of neurologic behavioral tests evaluating general reactivity, spontaneous exploratory activity, motor coordination, pain threshold, fear and anxiety, and memory. We placed particular emphasis on the effect of uPA deficiency on seizure susceptibility, including the response to convulsants (pentylenetetrazol, kainate, or pilocarpine) and kainate-induced epileptogenesis and epilepsy. The uPA-/- mice showed no motor or sensory impairment compared with the Wt mice. Hippocampus-dependent spatial memory also remained intact. The uPA-/- mice, however, exhibited reduced exploratory activity and an enhanced response to a tone stimulus (p<0.05 compared with the Wt mice). The urokinase-type plasminogen activator deficient mice showed no increase in spontaneous or evoked epileptiform electrographic activity. Rather, the response to pilocarpine administration was reduced compared with the Wt mice (p<0.05). Also, the epileptogenesis and the epilepsy phenotype after intrahippocampal kainate injection were similar to those in the Wt mice. Taken together, uPA deficiency led to diminished interest in the environmental surroundings and enhanced emotional reactivity to unexpected aversive stimuli. Urokinase-type plasminogen activator deficiency was not associated with enhanced seizure susceptibility or worsened poststatus epilepticus epilepsy phenotype.
Assuntos
Comportamento Animal/fisiologia , Suscetibilidade a Doenças , Receptores de Ativador de Plasminogênio Tipo Uroquinase/deficiência , Convulsões/fisiopatologia , Ativador de Plasminogênio Tipo Uroquinase/deficiência , Animais , Eletroencefalografia , Potenciais Evocados Auditivos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/fisiologiaRESUMO
Further development in research of bone regeneration is necessary to meet the clinical demand for bone reconstruction. Recently, we reported that plasminogen is crucial for bone repair through enhancement of vessel formation. However, the details of the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) in the bone repair process still remain unknown. Herein, we examined the effects of plasminogen activators on bone repair after a femoral bone defect using tPA-deficient (tPA(-/-)) and uPA-deficient (uPA(-/-)) mice. Bone repair of the femur was delayed in tPA(-/-) mice, unlike that in wild-type (tPA(+/+)) mice. Conversely, the bone repair was comparable between wild-type (uPA(+/+)) and uPA(-/-) mice. The number of proliferative osteoblasts was decreased at the site of bone damage in tPA(-/-) mice. Moreover, the proliferation of primary calvarial osteoblasts was reduced in tPA(-/-) mice. Recombinant tPA facilitated the proliferation of mouse osteoblastic MC3T3-E1 cells. The proliferation enhanced by tPA was antagonized by the inhibition of endogenous annexin 2 by siRNA and by the inhibition of extracellular signal-regulated kinase (ERK)1/2 phosphorylation in MC3T3-E1 cells. Vessel formation as well as the levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) were decreased at the damaged site in tPA(-/-) mice. Our results provide novel evidence that tPA is crucial for bone repair through the facilitation of osteoblast proliferation related to annexin 2 and ERK1/2 as well as enhancement of vessel formation related to VEGF and HIF-1α at the site of bone damage.
Assuntos
Regeneração Óssea , Osteoblastos/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Animais Recém-Nascidos , Anexina A2/antagonistas & inibidores , Anexina A2/genética , Anexina A2/metabolismo , Osso e Ossos/irrigação sanguínea , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Linhagem Celular , Proliferação de Células , Células Cultivadas , Cruzamentos Genéticos , Feminino , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica , Osteoblastos/citologia , Osteoblastos/patologia , Interferência de RNA , Proteínas Recombinantes/metabolismo , Ativador de Plasminogênio Tecidual/deficiência , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tipo Uroquinase/deficiência , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Mice deficient in plasminogen, the precursor of plasmin, show completely arrested healing of tympanic membrane (TM) perforations, indicating that plasmin plays an essential role in TM healing. The activation of plasminogen to plasmin is performed by two plasminogen activators (PAs), urokinase-type PA (uPA) and tissue-type PA (tPA). To elucidate the functional roles of PAs in the healing of TM perforations, we investigated the phenotypes of single gene-deficient mice lacking uPA (uPA(-/-)) or tPA (tPA(-/-)) after TM perforation. Delayed healing of TM perforations was observed in uPA(-/-) mice but not tPA(-/-) mice. The migration of keratinocytes was clearly delayed and seemed to be misoriented in uPA(-/-) mice. Furthermore, fibrin deposition and the inflammatory response were persistent in these mice. Our findings demonstrate that uPA plays a role in the healing of TM perforations. The observed phenotypes in uPA(-/-) mice are most likely due to the reduced generation of plasmin.
Assuntos
Fenótipo , Perfuração da Membrana Timpânica/fisiopatologia , Ativador de Plasminogênio Tipo Uroquinase/deficiência , Cicatrização/fisiologia , Animais , Movimento Celular/fisiologia , Fibrinolisina/biossíntese , Imuno-Histoquímica , Queratinócitos/fisiologia , Queratinas/metabolismo , Camundongos , Camundongos Knockout , Otoscopia , Ativador de Plasminogênio Tecidual/deficiência , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
OBJECTIVE: The urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR) are a potent multifunctional system involved in vascular remodeling. The goal of the study was to unravel the mechanisms of uPA/uPAR-directed vascular smooth muscle cell (VSMC) differentiation. METHODS AND RESULTS: Using cultured human primary VSMCs, we identified a new molecular mechanism controlling phenotypic modulation in vitro and in vivo. We found that the urokinase-type plasminogen activator receptor (uPAR) acts together with the transcriptional coactivator myocardin to regulate the VSMC phenotype. uPAR, a glycosylphosphatidylinositol-anchored cell-surface receptor family member, undergoes ligand-induced internalization and nuclear transport in VSMCs. Platelet-derived growth factor receptor ß and SUMOylated RanGAP1 mediate this trafficking. Nuclear uPAR associates with myocardin, which is then recruited from the promoters of serum response factor target genes and undergoes proteasomal degradation. This chain of events initiates the synthetic VSMC phenotype. Using mouse carotid artery ligation model, we show that this mechanism contributes to adverse vascular remodeling after injury in vivo. We then cultured cells on a microstructured biomaterial and found that substrate topography induced uPAR-mediated VSMC differentiation. CONCLUSIONS: These findings reveal the transcriptional activity of uPAR, controlling the differentiation of VSMCs in a vascular disease model. They also suggest a new role for uPAR as a therapeutic target and as a marker for VSMC phenotyping on prosthetic biomaterials.
Assuntos
Miócitos de Músculo Liso/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Transativadores/metabolismo , Doenças Vasculares/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Células Cultivadas , Endocitose , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/patologia , Fenótipo , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Interferente Pequeno/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/deficiência , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Sumoilação , Ativador de Plasminogênio Tipo Uroquinase/deficiência , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Doenças Vasculares/patologiaRESUMO
OBJECTIVE: Abdominal aortic aneurysms (AAAs) are associated with fragmentation of extracellular matrix during development of aortic dilation and rupture. Therefore, it is important to identify specific protease systems involved in extracellular matrix degradation during AAA formation. The present study determined the contribution of the urokinase system to AAA formation and rupture. METHODS AND RESULTS: Angiotensin II (Ang II)-induced AAAs were associated with increased aortic abundance of both urokinase-type plasminogen activator receptor (uPAR) and urokinase-type plasminogen activator (uPA) proteins. However, this increased presence was unrelated to AAA formation because deficiencies of either uPAR or uPA had no effect on either the incidence or size of Ang II-induced AAAs in both normolipidemic mice and low-density lipoprotein receptor-/- mice fed a saturated fat-enriched diet. Although uPA deficiency did not affect development of AAAs, there was an effect of increasing mortality rate from AAA rupture in hypercholesterolemic mice. Bone marrow transplantation demonstrated that enhanced aneurysmal rupture was attributable to deficiency of uPA in leukocytes. uPA deficiency led to an increased propensity for impaired resolution of the thrombotic material within the aneurysmal tissue. Neither uPAR nor uPA deficiency had any effect on Ang II-induced atherosclerosis in low-density lipoprotein receptor-/- mice. CONCLUSIONS: The uPA-uPAR axis has no effect on the formation of Ang II-induced AAAs, but uPA deficiency promotes aneurysmal rupture.
Assuntos
Aneurisma Roto/fisiopatologia , Angiotensina II/efeitos adversos , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/fisiopatologia , Células da Medula Óssea/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/deficiência , Aneurisma Roto/epidemiologia , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Apolipoproteínas E/fisiologia , Células da Medula Óssea/patologia , Modelos Animais de Doenças , Hipercolesterolemia/metabolismo , Incidência , Leucócitos/metabolismo , Leucócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de LDL/deficiência , Receptores de LDL/genética , Receptores de LDL/fisiologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/deficiência , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/fisiologiaRESUMO
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
Macrophages (Mp) and the plasminogen system play important roles in tissue repair following injury. We hypothesized that Mp-specific expression of urokinase-type plasminogen activator (uPA) is sufficient for Mp to migrate into damaged muscle and for efficient muscle regeneration. We generated transgenic mice expressing uPA only in Mp, and we assessed the ability of these mice to repair muscle injury. Mp-only uPA expression was sufficient to induce wild-type levels of Mp accumulation, angiogenesis, and new muscle fiber formation. In mice with wild-type uPA expression, Mp-specific overexpression further increased Mp accumulation and enhanced muscle fiber regeneration. Furthermore, Mp expression of uPA regulated the level of active hepatocyte growth factor, which is required for muscle fiber regeneration, in damaged muscle. In vitro studies demonstrated that uPA promotes Mp migration through proteolytic and nonproteolytic mechanisms, including proteolytic activation of hepatocyte growth factor. In summary, Mp-derived uPA promotes muscle regeneration by inducing Mp migration, angiogenesis, and myogenesis.
Assuntos
Macrófagos/enzimologia , Músculo Esquelético/enzimologia , Regeneração/imunologia , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Linhagem Celular , Movimento Celular/genética , Movimento Celular/imunologia , Células Cultivadas , Feminino , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/imunologia , Músculo Esquelético/citologia , Músculo Esquelético/imunologia , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/imunologia , Regeneração/genética , Ativador de Plasminogênio Tipo Uroquinase/deficiênciaRESUMO
Plasminogen activators play an important role in synaptic plasticity associated with the crossed phrenic phenomenon (CPP) and recovery of respiratory function after spinal cord injury. A genetic approach using knockout mice lacking various genes in the plasminogen activator/plasmin system has shown that induction of urokinase plasminogen activator (uPA) is required during the first hour after a C2-hemisection for the acquisition of the CPP response. The uPA knockout mice do not show the structural remodeling of phrenic motor neuron synapses characteristic of the CPP response. As shown here uPA acts in a cell signaling manner via binding to its receptor uPAR rather than as a protease, since uPAR knockout mice or knock-in mice possessing a modified uPA that is unable to bind to uPAR both fail to generate a CPP and recover respiratory function. Microarray data and real-time PCR analysis of mRNAs induced in the phrenic motor nucleus after C2-hemisection in C57Bl/6 mice as compared to uPA knockout mice indicate a potential cell signaling cascade downstream possibly involving ß-integrin and Src, and other pathways. Identification of these uPA-mediated signaling pathways may provide the opportunity to pharmacologically upregulate the synaptic plasticity necessary for recovery of phrenic motoneuron activity following cervical spinal cord injury.