RESUMO
RECK is a candidate tumor suppressor gene isolated as a gene that induces flat reversion in a cell line transformed by the KRAS oncogene. Since RECK knockout mice die in utero, they are not suitable for studying the effects of RECK on tumor formation. In this study, we found an increased incidence of spontaneous pulmonary adenomas in mice with reduced RECK expression (RECK-Hypo mice). To evaluate the effects of RECK expressed by either tumor cells or host cells on tumor growth, we established a tumorigenic cell line (MKER) from the kidney of a C57BL/6 mouse and performed syngeneic transplantation experiments. Our results indicate that when RECK expression is low in host cells, transplanted MKER cells grow faster and kill the animal more rapidly. Since RECK is required for the formation of proper fibrillin fibers that serve as a tissue reservoir for precursors of TGFß-family cytokines, we assessed the levels of TGFß1 in the peripheral blood. We found a significant increase in TGFß1 in RECK-Hypo mice compared to wild-type mice. We also found that the proportion of FOXP3-positive regulatory T (Treg) cells among splenocytes was higher in RECK-Hypo mice compared to the control mice. Furthermore, the number of FOXP3-positive cells in spontaneous hematopoietic neoplasms in the lungs as well as tumors that formed after MKER transplantation was significantly higher in RECK-Hypo mice compared to the control mice. These findings indicate that RECK-mediated tumor suppression involves a non-cell-autonomous mechanism and that possible roles of TGFß1 and Treg cells in such a mechanism warrant further study.
RESUMO
Fibrillins (FBNs) form mesh-like structures of microfibrils in various elastic tissues. RECK and FBN1 are co-expressed in many human tissues, suggesting a functional relationship. We found that dermal FBN1 fibers show atypical morphology in mice with reduced RECK expression (RECK-Hypo mice). Dermal FBN1 fibers in mice-lacking membrane-type 1-matrix metalloproteinase (MT1-MMP) show a similar atypical morphology, despite the current notion that MT1-MMP (a membrane-bound protease) and RECK (a membrane-bound protease inhibitor) have opposing functions. Our experiments using dermal fibroblasts indicated that RECK promotes pro-MT1-MMP activation, increases cell-associated gelatinase/collagenase activity, and decreases diffusible gelatinase/collagenase activity, while MT1-MMP stabilizes RECK in these cells. Experiments using purified proteins indicate that RECK and its binding partner ADAMTS10 keep the proteolytic activity of MT1-MMP within a certain range. These findings suggest that RECK, ADAMTS10, and MT1-MMP cooperate to support the formation of robust FBN1 fibers.
Assuntos
Fibrilinas/metabolismo , Proteínas Ligadas por GPI/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Proteínas ADAMTS/metabolismo , Animais , Linhagem Celular Tumoral , Colágeno/metabolismo , Elastina/metabolismo , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Proteínas Ligadas por GPI/deficiência , Gelatina/metabolismo , Células HEK293 , Humanos , Integrinas/metabolismo , Metaloproteinase 14 da Matriz/deficiência , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos , Proteólise , Pele/metabolismoRESUMO
Sustained inflammation and matrix metalloproteinase (MMP) activation contribute to vascular occlusive/proliferative disorders. Interleukin-17 (IL-17) is a proinflammatory cytokine that signals mainly via TRAF3 Interacting Protein 2 (TRAF3IP2), an upstream regulator of various critical transcription factors, including AP-1 and NF-κB. Reversion inducing cysteine rich protein with kazal motifs (RECK) is a membrane-anchored MMP inhibitor. Here we investigated whether IL-17A/TRAF3IP2 signaling promotes MMP-13-dependent human aortic smooth muscle cell (SMC) proliferation and migration, and determined whether RECK overexpression blunts these responses. Indeed, IL-17A treatment induced (a) JNK, p38 MAPK, AP-1, NF-κB, and CREB activation, (b) miR-21 induction, (c) miR-27b and miR-320 inhibition, (d) MMP-13 expression and activation, (e) RECK suppression, and (f) SMC migration and proliferation, all in a TRAF3IP2-dependent manner. In fact, gain of TRAG3IP2 function, by itself, induced MMP-13 expression and activation, and RECK suppression. Furthermore, treatment with recombinant MMP-13 stimulated SMC migration in part via ERK activation. Importantly, RECK gain-of-function attenuated MMP-13 activity without affecting its mRNA or protein levels, and inhibited IL-17A- and MMP-13-induced SMC migration. These results indicate that increased MMP-13 and decreased RECK contribute to IL-17A-induced TRAF3IP2-dependent SMC migration and proliferation, and suggest that TRAF3IP2 inhibitors or RECK inducers have the potential to block the progression of neointimal thickening in hyperplastic vascular diseases.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Aorta/citologia , Movimento Celular , Proteínas Ligadas por GPI/metabolismo , Interleucina-17/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Proliferação de Células , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Biológicos , Proteínas Recombinantes/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Transdução de Sinais , Doenças Vasculares/metabolismo , Doenças Vasculares/patologiaRESUMO
Reck encodes a membrane-anchored glycoprotein implicated in the regulation of extracellular metalloproteinases, Notch-signaling, and Wnt7-signaling and shown to play critical roles in embryogenesis and tumor suppression. Precise mechanisms of its actions in vivo, however, remain largely unknown. By homologous recombination, we generated a new Reck allele, ReckCreERT2 (MGI symbol: Reck
Assuntos
Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Alelos , Animais , Técnicas de Introdução de Genes , Engenharia Genética/métodos , Integrases/genética , Camundongos , Transdução de SinaisRESUMO
BACKGROUND: The dystroglycan complex consists of two subunits: extracellular α-dystroglycan and membrane-spanning ß-dystroglycan, which provide a tight link between the extracellular matrix and the intracellular cytoskeleton. Previous studies showed that 43 kDa ß-dystroglycan is proteolytically cleaved into the 30 kDa fragment by matrix metalloproteinases (MMPs) in various non-muscle tissues, whereas it is protected from cleavage in muscles by the sarcoglycan complex which resides close to the dystroglycan complex. It is noteworthy that cleaved ß-dystroglycan is detected in muscles from patients with sarcoglycanopathy, sarcoglycan-deficient muscular dystrophy. In vitro assays using protease inhibitors suggest that both MMP-2 and MMP-9 contribute to the cleavage of ß-dystroglycan. However, this has remained uninvestigated in vivo. METHODS: We generated triple-knockout (TKO) mice targeting MMP-2, MMP-9 and γ-sarcoglycan to examine the status of ß-dystroglycan cleavage in the absence of the candidate matrix metalloproteinases in sarcoglycan-deficient muscles. RESULTS: Unexpectedly, ß-dystroglycan was cleaved in muscles from TKO mice. Muscle pathology was not ameliorated but worsened in TKO mice compared with γ-sarcoglycan single-knockout mice. The gene expression of MMP-14 was up-regulated in TKO mice as well as in γ-sarcoglycan knockout mice. In vitro assay showed MMP-14 is capable to cleave ß-dystroglycan. CONCLUSIONS: Double-targeting of MMP-2 and MMP-9 cannot prevent cleavage of ß-dystroglycan in sarcoglycanopathy. Thus, matrix metalloproteinases contributing to ß-dystroglycan cleavage are redundant, and MMP-14 could participate in the pathogenesis of sarcoglycanopathy.
Assuntos
Distroglicanas/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Músculo Esquelético/metabolismo , Sarcoglicanopatias/genética , Sarcoglicanas/genética , Animais , Deleção de Genes , Humanos , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Knockout , Músculo Esquelético/patologia , Proteólise , Sarcoglicanopatias/metabolismo , Sarcoglicanopatias/patologia , Sarcoglicanas/metabolismo , Regulação para CimaRESUMO
Low sodium levels are strongly associated with poor prognosis in acute heart failure (AHF); however, the prognostic impact of the sodium level trajectory overtime has not been determined. A secondary analysis of the AQUAMARINE study in which patients with AHF and renal impairment were randomized to receive either tolvaptan or conventional treatment was performed. Sodium levels were evaluated at the baseline and at 6, 12, 24, and 48 h. We defined 'sodium dipping' as sodium level falling below the baseline level at any time point. The primary endpoint was the combined event of all-cause death and heart failure rehospitalization during follow-up. The analysis included 184 patients with a median follow-up of 21.1 months. Sodium levels more steeply increased during the 48 h in patients without events as compared to sodium levels in patients with events (P = 0.018 in linear-mixed effect model). The sodium dipping group (n = 100; 54.3%) demonstrated significantly less urine output, less body weight reduction, and poorer diuretic response within 48 h compared to the non-dipping group. The sodium dipping group was also significantly associated with a low combined-event-free survival after adjustment for other prognostic factors (HR 1.97; 95% CI 1.06-3.38; P = 0.033). The trajectory of sodium levels during the acute phase is associated with the prognosis of patients with AHF independently of the baseline sodium level.
Assuntos
Benzazepinas/administração & dosagem , Insuficiência Cardíaca/tratamento farmacológico , Sódio/sangue , Doença Aguda , Idoso , Antagonistas dos Receptores de Hormônios Antidiuréticos/administração & dosagem , Biomarcadores/sangue , Causas de Morte/tendências , Relação Dose-Resposta a Droga , Feminino , Seguimentos , Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/mortalidade , Mortalidade Hospitalar/tendências , Humanos , Hiponatremia , Japão , Masculino , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida/tendências , Fatores de Tempo , TolvaptanRESUMO
The transformation suppressor gene RECK was isolated by cDNA expression cloning (1998), and GPR124/TEM5 was detected as a tumor endothelial marker by differential screening (2000). The importance of Wnt7a/b and Gpr124 in brain angiogenesis was demonstrated by reverse genetics in mice (2008-2010). A series of recent studies using genetically engineered mice and zebrafish as well as luciferase reporter assays in cultured cells led to the discovery of functional interactions among Reck, Gpr124, and Wnt7a/b in triggering canonical Wnt signaling with relevance to embryonic brain angiogenesis and blood-brain barrier formation.
Assuntos
Proteínas Ligadas por GPI/metabolismo , Neovascularização Fisiológica , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Animais , Humanos , Proteínas Proto-Oncogênicas/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
BACKGROUND: More efficacious and/or safer decongestive therapy is clearly needed in acute heart failure (AHF) patients complicated by renal dysfunction. We tested the hypothesis that adding tolvaptan, an oral vasopressin-2 receptor antagonist, to conventional therapy with loop diuretics would be more effective treatment in this population. METHODS AND RESULTS: A multicenter, open-label, randomized control trial was performed, and 217 AHF patients with renal dysfunction (estimated glomerular filtration rate 15-60 mL ⢠min(-1) ⢠1.73 m(-2)) were randomized 1:1 to treatment with tolvaptan (n=108) or conventional treatment (n=109). The primary end point was 48-hour urine volume. The tolvaptan group showed more diuresis than the conventional treatment group (6464.4 vs 4999.2 mL; P <.001) despite significantly lower amounts of loop diuretic use (80 mg vs 120 mg; P <.001). Dyspnea relief was achieved significantly more frequently in the tolvaptan group at all time points within 48 hours except 6 hours after enrollment. The rate of worsening of renal function (≥0.3 mg/dL increase from baseline) was similar between the tolvaptan and conventional treatment groups (24.1% vs 27.8%, respectively; P =.642). CONCLUSIONS: Adding tolvaptan to conventional treatment achieved more diuresis and relieved dyspnea symptoms in AHF patients with renal dysfunction. CLINICAL TRIAL REGISTRATION: URL: http://www.umin.ac.jp/ctr/index/htm/ Unique identifier: UMIN000007109.
Assuntos
Benzazepinas/administração & dosagem , Diurese/efeitos dos fármacos , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Renal/tratamento farmacológico , Doença Aguda , Administração Oral , Idoso , Antagonistas dos Receptores de Hormônios Antidiuréticos/administração & dosagem , Relação Dose-Resposta a Droga , Feminino , Taxa de Filtração Glomerular , Insuficiência Cardíaca/complicações , Humanos , Masculino , Estudos Prospectivos , Insuficiência Renal/complicações , Insuficiência Renal/fisiopatologia , Tolvaptan , Resultado do TratamentoRESUMO
Mutations in the gene encoding the retinoblastoma tumor suppressor predispose humans and mice to tumor development. Here we have assessed the effect of Nras loss on tumor development in Rb1 heterozygous mice. Loss of one or two Nras alleles is shown to significantly reduce the severity of pituitary tumors arising in Rb1(+/-) animals by enhancing their differentiation. By contrast, C-cell thyroid adenomas occurring in Rb1(+/-) mice progress to metastatic medullary carcinomas after loss of Nras. In Rb1(+/-)Nras(+/-) animals, distant medullary thyroid carcinoma metastases are associated with loss of the remaining wild-type Nras allele. Loss of Nras in Rb1-deficient C cells results in elevated Ras homolog family A (RhoA) activity, and this is causally linked to the invasiveness and metastatic behavior of these cells. These findings suggest that the loss of the proto-oncogene Nras in certain cellular contexts can promote malignant tumor progression.
Assuntos
Genes ras/genética , Tumores Neuroendócrinos/patologia , Proteína do Retinoblastoma/deficiência , Neoplasias da Glândula Tireoide/patologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenoma/genética , Adenoma/patologia , Hormônio Adrenocorticotrópico/metabolismo , Amidas/farmacologia , Animais , Carcinoma Medular/genética , Carcinoma Medular/patologia , Inibidores Enzimáticos/farmacologia , Heterozigoto , Camundongos , Camundongos Mutantes , Camundongos Nus , Metástase Neoplásica , Tumores Neuroendócrinos/genética , Proto-Oncogene Mas , Piridinas/farmacologia , Proteína do Retinoblastoma/efeitos dos fármacos , Proteína do Retinoblastoma/genética , Transdução de Sinais , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/secundário , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
A common strategy to identify tumor suppressor genes has been positional cloning, taking advantages of chromosomal abnormalities, linkage to polymorphic markers, etc. We have been taking another approach, based on shotgun cloning with cDNA-expression library, to isolate genes suppressing an aspect of transformed phenotypes. Genes identified in such an artificial system, however, require subsequent validation for their clinical relevance through independent approaches, such as finding mutations and altered expression in human cancers. Whether the parental genes act as oncogenes or tumor suppressors needs to be deduced from available annotations and/or new experimental data. Once validated, such genes, being isolated by virtue of their biological activities, are likely to be useful in developing new strategies for tumor prognosis, tumor stratification, and drug discovery.
Assuntos
Transformação Celular Neoplásica/genética , Genes Supressores de Tumor , Neoplasias/genética , Neoplasias/terapia , Animais , Antígenos de Neoplasias , Biomarcadores Tumorais , Transformação Celular Neoplásica/patologia , Clonagem Molecular/métodos , Descoberta de Drogas , Proteínas Ligadas por GPI , Biblioteca Gênica , Humanos , Lectinas Tipo C , Camundongos , Mutação , Neoplasias/diagnóstico , Neoplasias/patologia , Proteínas Associadas a PancreatiteRESUMO
The pathogenesis of cardiac fibrosis and adverse remodeling is thought to involve the ROS-dependent induction of inflammatory cytokines and matrix metalloproteinases (MMPs), and the activation and migration of cardiac fibroblasts (CF). Here we investigated the role of RECK (reversion-inducing-cysteine-rich protein with Kazal motifs), a unique membrane-anchored MMP regulator, on IL-18-induced CF migration, and the effect of acetylsalicylic acid (ASA) on this response. In a Matrigel invasion assay, IL-18-induced migration of primary mouse CF was dependent on both IKK/NF-κB- and JNK/AP-1-mediated MMP9 induction and Sp1-mediated RECK suppression, mechanisms that required Nox4-dependent H(2)O(2) generation. Notably, forced expression of RECK attenuated IL-18-induced MMP9 activation and CF migration. Further, therapeutic concentrations of ASA inhibited IL-18-induced H(2)O(2) generation, MMP9 activation, RECK suppression, and CF migration. The salicylic acid moiety of ASA similarly attenuated IL-18-induced CF migration. Thus, ASA may exert potential beneficial effect in cardiac fibrosis through multiple protective mechanisms.
Assuntos
Aspirina/administração & dosagem , Fibrose/metabolismo , Proteínas Ligadas por GPI/genética , Interleucina-18/genética , Miocárdio/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Colágeno/química , Combinação de Medicamentos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/patologia , Proteínas Ligadas por GPI/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/metabolismo , Interleucina-18/biossíntese , Laminina/química , Metaloproteinase 9 da Matriz/genética , Camundongos , Miocárdio/patologia , Proteoglicanas/química , Receptores Imunológicos/metabolismoRESUMO
PURPOSE: Over half of all admitted acute decompensated heart failure (ADHF) patients have renal failure. Although diuretics represent the mainstay of treatment strategy even in this population, there are unmet needs for safer and more effective treatment. Tolvaptan is a vasopressin-2 receptor antagonist, and we hypothesized that adding tolvaptan to standard diuretic therapy would be more effective in ADHF patients with renal function impairment. METHODS: The Answering question on tolvaptan's efficacy for patients with acute decompensated heart failure and renal failure (AQUAMARINE) is a multicenter, randomized controlled clinical trial, which will enroll 220 patients from 17 hospitals in Japan. ADHF patients whose estimated glomerular filtration rate is above 15 and below 60 mL/min/1.72 m(2) will be randomly assigned within 6 h after admission to usual care with furosemide or tolvaptan add-on therapy. Primary endpoint is achieved urine output within 48 h. Secondary endpoints include dyspnea relief measured by 7-points Likert scale, incidence of worsening renal function, dose of furosemide used within 48 h, and changes of brain natriuretic peptide. CONCLUSION: This study is the first multicenter study in Japan to evaluate clinical effectiveness of tolvaptan add-on therapy in ADHF patients with renal failure. The results of this study address the treatment strategy of this high-risk population (UMIN Clinical Trial Registry Number: UMIN000007109).
Assuntos
Antagonistas dos Receptores de Hormônios Antidiuréticos , Benzazepinas/uso terapêutico , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Renal/tratamento farmacológico , Doença Aguda , Benzazepinas/administração & dosagem , Diuréticos/administração & dosagem , Diuréticos/uso terapêutico , Quimioterapia Combinada , Furosemida/administração & dosagem , Furosemida/uso terapêutico , Taxa de Filtração Glomerular , Insuficiência Cardíaca/fisiopatologia , Humanos , Japão , Peptídeo Natriurético Encefálico/metabolismo , Estudos Prospectivos , Insuficiência Renal/complicações , Insuficiência Renal/fisiopatologia , Projetos de Pesquisa , TolvaptanRESUMO
Sustained induction and activation of matrixins (matrix metalloproteinases or MMPs), and the destruction and deposition of extracellular matrix (ECM), are the hallmarks of cardiac fibrosis. The reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) is a unique membrane-anchored endogenous MMP regulator. We hypothesized that elevated angiotensin II (Ang II), which is associated with fibrosis in the heart, differentially regulates MMPs and RECK both in vivo and in vitro. Continuous infusion of Ang II into male C57Bl/6 mice for 2weeks resulted in cardiac fibrosis, with increased expressions of MMPs 2, 7, 9 and 14, and of collagens Ia1 and IIIa1. The expression of RECK, however, was markedly suppressed. These effects were inhibited by co-treatment with the Ang II type 1 receptor (AT1) antagonist losartan. In vitro, Ang II suppressed RECK expression in adult mouse cardiac fibroblasts (CF) via AT1/Nox4-dependent ERK/Sp1 activation, but induced MMPs 2, 14 and 9 via NF-κB, AP-1 and/or Sp1 activation. Further, while forced expression of RECK inhibits, its knockdown potentiates Ang II-induced CF migration. Notably, RECK overexpression reduced Ang II-induced MMPs 2, 9 and 14 activation, but enhanced collagens Ia1 and IIIa1 expression and soluble collagen release. These results demonstrate for the first time that Ang II suppresses RECK, but induces MMPs both in vivo and in vitro, and RECK overexpression blunts Ang II-induced MMP activation and CF migration in vitro. Strategies that upregulate RECK expression in vivo have the potential to attenuate sustained MMP expression, and blunt fibrosis and adverse remodeling in hypertensive heart diseases.
Assuntos
Angiotensina II/farmacologia , Movimento Celular/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/enzimologia , Proteínas Ligadas por GPI/metabolismo , Metaloproteinases da Matriz/metabolismo , Miocárdio/citologia , Animais , Colágeno/metabolismo , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Humanos , Masculino , Metaloproteinase 14 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , NADPH Oxidase 4 , NADPH Oxidases/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição AP-1/metabolismoRESUMO
During myocardial infarction (MI), cardiac cells at the infarcted area undergo cell death. In response, cardiac myofibroblasts, which are mainly differentiated from resident fibroblasts upon inflammation, produce extracellular matrix proteins such as collagen to fill the damaged areas of the heart to prevent cardiac rupture. In this study, we identified a cardioprotective role of G-protein-coupled receptor kinase 5 (GRK5) in MI. GRK5 expression was found to increase in the mouse heart after MI and was highly expressed in cardiac fibroblasts/myofibroblasts. In fibroblasts/myofibroblasts, GRK5 promoted the expression of inflammation-related genes through nuclear factor-κB activation, leading to an increase in the expression levels of fibrosis-related genes. Bone marrow transfer experiments confirmed that GRK5 in fibroblasts/myofibroblasts, but not in infiltrated macrophages in the infarcted area, is mainly responsible for GRK5-mediated inflammation in infarcted hearts. In addition, inflammation and fibrosis at the infarcted area were significantly suppressed in GRK5 knockout mice, resulting in increased mortality compared with that in wild-type mice. These data indicate that GRK5 in cardiac fibroblasts/myofibroblasts promotes inflammation and fibrosis to ameliorate the damage after MI.
Assuntos
Infarto do Miocárdio , Miocárdio , Animais , Camundongos , Colágeno/metabolismo , Fibrose , Inflamação/metabolismo , Camundongos Knockout , Infarto do Miocárdio/genética , Miocárdio/metabolismoRESUMO
RECK is downregulated in various human cancers; however, how RECK inactivation affects carcinogenesis remains unclear. We addressed this issue in a pancreatic ductal adenocarcinoma (PDAC) mouse model and found that pancreatic Reck deletion dramatically augmented the spontaneous development of PDAC with a mesenchymal phenotype, which was accompanied by increased liver metastases and decreased survival. Lineage tracing revealed that pancreatic Reck deletion induced epithelial-mesenchymal transition (EMT) in PDAC cells, giving rise to inflammatory cancer-associated fibroblast-like cells in mice. Splenic transplantation of Reck-null PDAC cells resulted in numerous liver metastases with a mesenchymal phenotype, whereas reexpression of RECK markedly reduced metastases and changed the PDAC tumor phenotype into an epithelial one. Consistently, low RECK expression correlated with low E-cadherin expression, poor differentiation, metastasis, and poor prognosis in human PDAC. RECK reexpression in the PDAC cells was found to downregulate MMP2 and MMP3, with a concomitant increase in E-cadherin and decrease in EMT-promoting transcription factors. An MMP inhibitor recapitulated the effects of RECK on the expression of E-cadherin and EMT-promoting transcription factors and invasive activity. These results establish the authenticity of RECK as a pancreatic tumor suppressor, provide insights into its underlying mechanisms, and support the idea that RECK could be an important therapeutic effector against human PDAC.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Hepáticas , Neoplasias Pancreáticas , Animais , Humanos , Camundongos , Caderinas/genética , Carcinoma Ductal Pancreático/genética , Transição Epitelial-Mesenquimal/genética , Proteínas Ligadas por GPI/genética , Neoplasias Hepáticas/genética , Pâncreas , Neoplasias Pancreáticas/genética , Neoplasias PancreáticasRESUMO
RECK encodes a membrane-anchored protease-regulator which is often downregulated in a wide variety of cancers, and reduced RECK expression often correlates with poorer prognoses. In mouse models, forced expression of RECK in tumor xenografts results in suppression of tumor angiogenesis, invasion, and metastasis. RECK mutations, however, are rare in cancer genomes, suggesting that agents that re-activate dormant RECK may be of clinical value. We found a potent RECK-inducer, DSK638, that inhibits spontaneous lung metastasis in our mouse xenograft model. Induction of RECK expression involves SP1 sites in its promoter and may be mediated by KLF2. DSK638 also upregulates MXI1, an endogenous MYC-antagonist, and inhibition of metastasis by DSK638 is dependent on both RECK and MXI1. This study demonstrates the utility of our approach (using a simple reporter assay followed by multiple phenotypic assays) and DSK638 itself (as a reference compound) in finding potential metastasis-suppressing drugs.
Assuntos
Proteínas Ligadas por GPI/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Metástase Neoplásica/prevenção & controle , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Genes Reporter , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a membrane-anchored matrix metalloproteinase regulator, but its functions in cartilage are not fully understood. The aim of the present study was to examine the expression and functions of RECK in human osteoarthritic (OA) cartilage. Quantitative RT-PCR indicated that the expression level of RECK is significantly higher in OA cartilage than in normal cartilage. By immunohistochemical analysis, RECK was localized to chondrocytes in OA cartilage, and the immunoreactivity directly correlated with the Mankin score and degree of chondrocyte cloning and proliferation. In cultured OA chondrocytes, RECK was expressed on the cell surface by glycosylphosphatidylinositol anchoring. The expression was stimulated by insulin-like growth factor-1 and suppressed by interleukin-1 and tumor necrosis factor-alpha. Down-regulation of RECK by small interfering RNA showed reduced spreading and smaller focal adhesions in the chondrocytes. Chondrocyte migration in a monolayer wounding assay was increased by down-regulation of RECK and inhibited by RECK overexpression in an matrix metalloproteinase activity-dependent manner. On the other hand, chondrocyte proliferation was suppressed by RECK silencing, and this was associated with reduced phosphorylation of focal adhesion kinase and extracellular signal-regulated kinase, whereas the proliferation was enhanced by RECK overexpression. These data are the first to demonstrate that RECK is up-regulated in human OA cartilage and suggest that RECK plays a role in chondrocyte cloning probably through suppression and promotion of chondrocyte migration and proliferation, respectively.
Assuntos
Cartilagem Articular/citologia , Condrócitos/metabolismo , Condrócitos/fisiologia , Glicoproteínas de Membrana/metabolismo , Osteoartrite/metabolismo , Idoso , Idoso de 80 Anos ou mais , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Condrócitos/citologia , Citocinas/metabolismo , Proteínas Ligadas por GPI , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , Osteoartrite/patologia , RNA Mensageiro/metabolismo , Regulação para CimaRESUMO
We report that during cortical development in the mouse embryo, reversion-inducing cysteine-rich protein with Kazal motifs (RECK) critically regulates Notch signaling by antagonizing the ectodomain shedding of Notch ligands, which is mediated by a disintegrin and metalloproteinase domain 10 (ADAM10). In the embryonic brain, RECK is specifically expressed in Nestin-positive neural precursor cells (NPCs). Reck-deficient NPCs undergo precocious differentiation that is associated with downregulated Nestin expression, impaired Notch signaling and defective self-renewal. These phenotypes were substantially rescued either by enhancing Notch signaling or by suppressing endogenous ADAM10 activity. Consequently, we found that RECK regulates the ectodomain shedding of Notch ligands by directly inhibiting the proteolytic activity of ADAM10. This mechanism appeared to be essential for Notch ligands to properly induce Notch signaling in neighboring cells. These findings indicate that RECK is a physiological inhibitor of ADAM10, an upstream regulator of Notch signaling and a critical modulator of brain development.
Assuntos
Proteínas ADAM/fisiologia , Secretases da Proteína Precursora do Amiloide/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana/fisiologia , Neurônios/fisiologia , Receptores Notch/fisiologia , Transdução de Sinais/fisiologia , Proteína ADAM10 , Animais , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/metabolismo , Regulação para Baixo/fisiologia , Feminino , Imunofluorescência , Proteínas Ligadas por GPI , Immunoblotting , Imunoprecipitação , Ligantes , Luciferases/biossíntese , Luciferases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Plasmídeos/genética , Gravidez , Interferência de RNA , Proteínas Recombinantes/genética , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Developmental angiogenesis proceeds through multiple morphogenetic events including sprouting, intussusception, and pruning. Mice lacking the membrane-anchored metalloproteinase regulator Reck die in utero around embryonic day 10.5 with halted vascular development; however, the mechanisms by which this phenotype arises remain unclear. RESULTS: We found that Reck is abundantly expressed in the cells associated with blood vessels undergoing angiogenesis or remodelling in the uteri of pregnant female mice. Some of the Reck-positive vessels show morphological features consistent with non-sprouting angiogenesis. Treatment with a vector expressing a small hairpin RNA against Reck severely disrupts the formation of blood vessels with a compact, round lumen. Similar defects were found in the vasculature of Reck-deficient or Reck conditional knockout embryos. CONCLUSIONS: Our findings implicate Reck in vascular remodeling, possibly through non-sprouting angiogenesis, in both maternal and embyonic tissues.