RESUMEN
Tumour angiogenesis is an independent risk factor for bladder cancer (BCa) progression, but viable and promising antiangiogenic targets are understudied. Secretory autophagy has received increasing interest recently, while the roles and executing mechanisms in the tumour microenvironment (TME) remain unclear. Herein, we found that active cathepsin B (CTSB) was upregulated in tumour tissues and serum EVs of 241 BCa patients from four cohorts and was significantly associated with poor prognosis. Starving TME (STME)-induced conventional autophagy in BCa cells elevated active CTSB levels by facilitating the expression and nuclear translocation of NFATC2. In addition, STME-induced secretory autophagy simultaneously led to markedly increased secretion of LC3-conjugated EVs loaded with active CTSB (EV-CTSB) into the TME. The increased exogenous active CTSB in endothelial cells by directly ingesting EV-CTSB prominently activated the TPX2-mediated phosphorylation of the AURKA-PI3K-AKT axis, increased VEGFA expression, and promoted angiogenesis. Our findings not only verify that EV-CTSB can be a promising target for antiangiogenic strategies in bladder cancer, but also reveal a novel action pattern based on secretory autophagy-induced EV secretion which is enlightening to explore crosstalk in the TME from various perspectives.
Asunto(s)
Autofagia/fisiología , Proteínas de Ciclo Celular/fisiología , Vesículas Extracelulares/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Neovascularización Patológica/etiología , Microambiente Tumoral/fisiología , Neoplasias de la Vejiga Urinaria/irrigación sanguínea , Adulto , Anciano , Animales , Aurora Quinasa A/metabolismo , Catepsina B/fisiología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Invasividad Neoplásica , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive disease harboring significant morbidity and mortality despite recent advances in therapy. Regardless of disease severity acute exacerbations (IPF-AEs) may occur leading to considerable loss of function and are the leading cause of death in IPF. Histologic features of IPF-AE are very similar to acute respiratory distress syndrome (ARDS), but the underlying mechanisms are incompletely understood. We investigated the role of the NLRP3 inflammasome in IPF and IPF-AE. Bronchoalveolar lavage (BAL) cells were sampled from patients with IPF (n = 32), IPF-AE (n = 10), ARDS (n = 7) and healthy volunteers (HV, n = 37) and the NLRP3-inflammasome was stimulated in-vitro. We found the NLRP3 inflammasome to be hyper-inducible in IPF compared to HV with increased IL-1ß and pro-IL-1ß levels on ELISA upon stimulation as well as increased caspase-1 activity measured by caspase-1p20 immunoblotting. In IPF-AE, IL-1ß was massively elevated to an extent similar to ARDS. To evaluate potential mechanisms, we co-cultured BAL cells with radiated A549 cells (a model to simulate apoptotic alveolar epithelial cells), which led to increased NLRP3 mRNA expression and increased caspase-1 dependent IL-1ß production. In the presence of a reactive oxygen species (ROS) inhibitor (diphenyleneiodonium) and a cathepsin B inhibitor (E64D), NLRP3 expression was suppressed indicating that induction of NLRP3 activation following efferocytosis of apoptotic A549 cells is mediated via ROS and cathepsin-B. In summary, we present evidence of involvement of the NLRP3 inflammasome-caspase pathway in the pathogenesis of IPF-AE, similarly to ARDS, which may be mediated by efferocytosis of apoptotic alveolar epithelial cells in IPF.
Asunto(s)
Apoptosis , Caspasa 1/fisiología , Fibrosis Pulmonar Idiopática/complicaciones , Inflamasomas/fisiología , Proteína con Dominio Pirina 3 de la Familia NLR/fisiología , Células A549 , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Catepsina B/fisiología , Femenino , Humanos , Interleucina-1/fisiología , Masculino , Persona de Mediana Edad , Precursores de Proteínas/fisiología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología , Adulto JovenRESUMEN
Myocyte apoptosis and oxidative stress key critical roles in the process of doxorubicin (DOX)induced cardiotoxicity. However, how apoptosis and oxidative stress arise in DOXinduced heart injury remains largely unknown. Cathepsin B (CTSB) is a typical lysosomal cysteine protease that is associated with apoptosis, inflammatory responses, oxidative stress and autophagy. The present study aimed to investigate the role of CTSB in DOXinduced heart injury and its potential mechanism. H9C2 cells were infected with adenovirus or transfected with small interfering RNA to overexpress or knock down CTSB, respectively, and then stimulated with DOX. DOX induced increased CTSB expression levels in H9C2 cells. DOXinduced cardiomyocyte apoptosis and oxidative stress were attenuated by CTSB knockdown but aggravated by CTSB overexpression in vitro. Mechanistically, the present study showed that CTSB activated the NFκB pathway in response to DOX. In summary, CTSB aggravated DOXinduced H9C2 cell apoptosis and oxidative stress via NFκB signalling. CTSB constitutes a potential therapeutic target for the treatment of DOXinduced cardiotoxicity.
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Catepsina B/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Cardiotoxicidad/metabolismo , Catepsina B/fisiología , Línea Celular , China , Doxorrubicina/efectos adversos , Doxorrubicina/farmacología , Lesiones Cardíacas/metabolismo , Lisosomas/metabolismo , FN-kappa B/metabolismo , Estrés Oxidativo/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
Cathepsin B (CatB) is a cysteine proteolytic enzyme widely expressed in various cells and mainly located in the lysosomes. It contributes to the pathogenesis and development of many diseases. However, the role of CatB in viral myocarditis (VMC) has never been elucidated. Here we generated the VMC model by intraperitoneal injection of coxsackievirus B3 (CVB3) into mice. At day 7 and day 28, we found CatB was significantly activated in hearts from VMC mice. Compared with the wild-type mice receiving equal amount of CVB3, genetic ablation of CatB (Ctsb-/-) significantly improved survival, reduced inflammatory cell infiltration, decreased serum level of cardiac troponin I, and ameliorated cardiac dysfunction, without altering virus titers in hearts. Conversely, genetic deletion of cystatin C (Cstc-/-), which markedly enhanced CatB levels in hearts, distinctly increased the severity of VMC. Furthermore, compared with the control, we found the inflammasome was activated in the hearts of wild-type mice with VMC, which was attenuated in the hearts of Ctsb-/- mice but was further enhanced in Cstc-/- mice. Consistently, the inflammasome-initiated pyroptosis was reduced in Ctsb-/- mice hearts and further increased in Cstc-/- mice. These results suggest that CatB aggravates CVB3-induced VMC probably through activating the inflammasome and promoting pyroptosis. This finding might provide a novel strategy for VMC treatment.
Asunto(s)
Catepsina B/fisiología , Infecciones por Coxsackievirus/complicaciones , Enterovirus Humano B/fisiología , Inflamasomas/metabolismo , Miocarditis/virología , Piroptosis/fisiología , Animales , Caspasa 1/metabolismo , Catepsina B/genética , Infecciones por Coxsackievirus/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Activación Enzimática , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocarditis/inmunología , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Miocitos Cardíacos/virologíaRESUMEN
OBJECTIVE: To investigate the role of cathepsin B in hepatic Kupffer cells (KCs) in activating Toll-like receptor 4(TLR- 4)-independent inflammatory pathways in mice with lipopolysaccharide (LPS)-induced sepsis. METHODS: Eighteen wild-type (WT) mice and 18 TLR4-knockout (TLR4-/-) mice were both divided into 3 groups for intraperitoneal injections of a lethal dose (54 mg/kg) of LPS, LPS and CA-074(a cathepsin B inhibitor), or normal saline, and the survival of the mice were observed. Another 36 WT mice and 36 TLR4-/-mice were also divided into 3 groups and subjected to intraperitoneal injections of normal saline, 20 mg/kg LPS, or LPS with CA-074 pretreatment.After the treatments, KCs were collected from the mice for assessing the protein level and activity of cathepsin B.The histopathological changes of the liver were observed with HE staining, and the serum levels of IL-1α, IL-1ß, TNF-α and IL-18 were detected. RESULTS: Compared with the WT mice,TLR4-/-mice receiving the lethal dose of LPS had significantly longer survival time (up to 84 h) after the injection,but were still unable to fully resist LPS challenge.CA-074 pretreatment prolonged the survival time of WT mice and TLR4-/-mice to 60 h and 132 h,respectively.In the mouse models of sepsis,20 mg/kg LPS induced significantly enhanced activity of cathepsin B without affecting its expression level in the KCs (P<0.05) and increased the serum levels of the inflammatory cytokines.CA-074 pretreatment of the mice obviously lessened the detrimental effects of LPS in TLR4-/-mice by significantly lowering cathepsin B activity in the KCs,alleviating hepatocyte apoptosis and reducing the serum levels of inflammatory cytokines. CONCLUSIONS: Cathepsin B plays an important role in activating TLR4-independent inflammatory pathways in mice with LPS-induced sepsis.
Asunto(s)
Catepsina B/fisiología , Macrófagos del Hígado/metabolismo , Sepsis/metabolismo , Receptor Toll-Like 4 , Animales , Catepsina B/antagonistas & inhibidores , Dipéptidos/farmacología , Técnicas de Inactivación de Genes , Hepatocitos , Inflamación/metabolismo , Interleucina-18/sangre , Interleucina-1alfa/sangre , Interleucina-1beta/sangre , Lipopolisacáridos , Hígado/patología , Ratones , Sepsis/etiología , Receptor Toll-Like 4/genética , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Antibody-drug conjugates (ADC) are designed to selectively bind to tumor antigens via the antibody and release their cytotoxic payload upon internalization. Controllable payload release through judicious design of the linker has been an early technological milestone. Here, we examine the effect of the protease-cleavable valine-citrulline [VC(S)] linker on ADC efficacy. The VC(S) linker was designed to be cleaved by cathepsin B, a lysosomal cysteine protease. Surprisingly, suppression of cathepsin B expression via CRISPR-Cas9 gene deletion or shRNA knockdown had no effect on the efficacy of ADCs with VC(S) linkers armed with a monomethyl auristatin E (MMAE) payload. Mass spectrometry studies of payload release suggested that other cysteine cathepsins can cleave the VC(S) linker. Also, ADCs with a nonprotease-cleavable enantiomer, the VC(R) isomer, mediated effective cell killing with a cysteine-VC(R)-MMAE catabolite generated by lysosomal catabolism. Based on these observations, we altered the payload to a pyrrolo[2,1-c][1,4]benzodiazepine dimer (PBD) conjugate that requires linker cleavage in order to bind its DNA target. Unlike the VC-MMAE ADCs, the VC(S)-PBD ADC is at least 20-fold more cytotoxic than the VC(R)-PBD ADC. Our findings reveal that the VC(S) linker has multiple paths to produce active catabolites and that antibody and intracellular targets are more critical to ADC efficacy. These results suggest that protease-cleavable linkers are unlikely to increase the therapeutic index of ADCs and that resistance based on linker processing is improbable. Cancer Res; 77(24); 7027-37. ©2017 AACR.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Antineoplásicos/metabolismo , Catepsina B/fisiología , Inmunoconjugados/metabolismo , Profármacos/metabolismo , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Catepsina B/metabolismo , Línea Celular Tumoral , Células Cultivadas , Citrulina/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Células HEK293 , Humanos , Inmunoconjugados/uso terapéutico , Oligopéptidos , Profármacos/uso terapéutico , Proteolisis , Valina/metabolismoRESUMEN
BACKGROUND & AIMS: Experimental studies in acute pancreatitis (AP) suggest a strong association of acinar cell injury with cathepsin B-dependent intracellular activation of trypsin. However, the molecular events subsequent to trypsin activation and their role, if any, in cell death is not clear. In this study, we have explored intra-acinar events downstream of trypsin activation that lead to acinar cell death. METHODS: Acinar cells prepared from the pancreas of rats or mice (wild-type, trypsinogen 7, or cathepsin B-deleted) were stimulated with supramaximal cerulein, and the cytosolic activity of cathepsin B and trypsin was evaluated. Permeabilized acini were used to understand the differential role of cytosolic trypsin vs cytosolic cathepsin B in activation of apoptosis. Cell death was evaluated by measuring specific markers for apoptosis and necrosis. RESULTS: Both in vitro and in vivo studies have suggested that during AP cathepsin B leaks into the cytosol from co-localized organelles, through a mechanism dependent on active trypsin. Cytosolic cathepsin B but not trypsin activates the intrinsic pathway of apoptosis through cleavage of bid and activation of bax. Finally, excessive release of cathepsin B into the cytosol can lead to cell death through necrosis. CONCLUSIONS: This report defines the role of trypsin in AP and shows that cytosolic cathepsin B but not trypsin activates cell death pathways. This report also suggests that trypsin is a requisite for AP only because it causes release of cathepsin B into the cytosol.
Asunto(s)
Células Acinares/enzimología , Catepsina B/fisiología , Muerte Celular/fisiología , Citosol/enzimología , Pancreatitis/enzimología , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Páncreas/citología , Pancreatitis/patología , Ratas , Ratas Wistar , Tripsina/fisiologíaRESUMEN
Although the global spread of the emerging zoonosis, human angiostrongyliasis, has attracted increasing attention, understanding of specific gene function has been impeded by the inaccessibility of genetic manipulation of the pathogen nematode causing this disease, Angiostrongylus cantonensis. Many parasitic proteases play key roles in host-parasite interactions, but those of A. cantonensis are always expressed as the inactive form in prokaryotic expression systems, thereby impeding functional studies. Hence, a lentiviral system that drives secreted expression of target genes fused to a Myc-His tag was used to obtain recombinant Ac-cathB-1 with biological activity. Although this class of proteases was always reported to function in nutrition and immune evasion in parasitic nematodes, recombinant Ac-cathB-1 was capable of hydrolysis of fibronectin and laminin as well as the extracellular matrix of IEC-6 monolayer, so that the intercellular space of the IEC-6 monolayer increased 5.15 times as compared to the control, while the shape of the adherent cells partly rounded up. This suggests a probable role for this protease in intestinal epithelial penetration. The inhibition of Ac-cathB-1 enzymatic activity with antiserum partly suppressed larval penetration ability in the isolated intestine. Thus, an effective system for heterologous expression of parasite proteases is presented for studying gene function in A. cantonensis; and Ac-cathB-1 was related to larval penetration ability in the host small intestine.
Asunto(s)
Angiostrongylus cantonensis/enzimología , Catepsina B/fisiología , Proteínas del Helminto/fisiología , Parasitosis Intestinales/enzimología , Infecciones por Strongylida/enzimología , Angiostrongylus cantonensis/genética , Angiostrongylus cantonensis/crecimiento & desarrollo , Angiostrongylus cantonensis/fisiología , Animales , Catepsina B/antagonistas & inhibidores , Catepsina B/genética , Catepsina B/inmunología , Catepsina B/aislamiento & purificación , Línea Celular , Activación Enzimática , Células Epiteliales/parasitología , Proteínas de la Matriz Extracelular/metabolismo , Vectores Genéticos/genética , Proteínas del Helminto/antagonistas & inhibidores , Proteínas del Helminto/genética , Proteínas del Helminto/inmunología , Proteínas del Helminto/aislamiento & purificación , Interacciones Huésped-Parásitos , Hidrólisis , Sueros Inmunes , Parasitosis Intestinales/parasitología , Intestinos/parasitología , Larva , Lentivirus/genética , Ratones , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Caracoles/parasitología , Infecciones por Strongylida/parasitologíaRESUMEN
Necrotic cell death triggers a range of biological responses including a strong adaptive immune response, yet we know little about the cellular pathways that control necrotic cell death. Inhibitor studies suggest that proteases, and in particular cathepsins, drive necrotic cell death. The cathepsin B-selective inhibitor CA-074-Me blocks all forms of programmed necrosis by an unknown mechanism. We found that cathepsin B deficiency does not prevent induction of pyroptosis and lysosome-mediated necrosis suggesting that CA-074-Me blocks necrotic cell death by targeting cathepsins other than cathepsin B. A single cathepsin, cathepsin C, drives necrotic cell death mediated by the lysosome-destabilizing agent Leu-Leu-OMe (LLOMe). Here we present evidence that cathepsin C-deficiency and CA-074-Me block LLOMe killing in a distinct and cell type-specific fashion. Cathepsin C-deficiency and CA-074-Me block LLOMe killing of all myeloid cells, except for neutrophils. Cathepsin C-deficiency, but not CA-074-Me, blocks LLOMe killing of neutrophils suggesting that CA-074-Me does not target cathepsin C directly, consistent with inhibitor studies using recombinant cathepsin C. Unlike other cathepsins, cathepsin C lacks endoproteolytic activity, and requires activation by other lysosomal proteases, such as cathepsin D. Consistent with this theory, we found that lysosomotropic agents and cathepsin D downregulation by siRNA block LLOMe-mediated necrosis. Our findings indicate that a proteolytic cascade, involving cathepsins C and D, controls LLOMe-mediated necrosis. In contrast, cathepsins C and D were not required for pyroptotic cell death suggesting that distinct cathepsins control pyroptosis and lysosome-mediated necrosis.
Asunto(s)
Catepsina C/fisiología , Catepsina D/fisiología , Lisosomas/enzimología , Animales , Apoptosis , Catepsina B/antagonistas & inhibidores , Catepsina B/fisiología , Dipéptidos/farmacología , Lisosomas/efectos de los fármacos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , NecrosisRESUMEN
Lung matrix homeostasis partly depends on the fine regulation of proteolytic activities. We examined the expression of human cysteine cathepsins (Cats) and their relative contribution to TGF-ß1-induced fibroblast differentiation into myofibroblasts. Assays were conducted using both primary fibroblasts obtained from patients with idiopathic pulmonary fibrosis and human lung CCD-19Lu fibroblasts. Pharmacological inhibition and genetic silencing of Cat B diminished α-smooth muscle actin expression, delayed fibroblast differentiation, and led to an accumulation of intracellular 50-kDa TGF-ß1. Moreover, the addition of Cat B generated a 25-kDa mature form of TGF-ß1 in Cat B siRNA-pretreated lysates. Inhibition of Cat B decreased Smad 2/3 phosphorylation but had no effect on p38 MAPK and JNK phosphorylation, indicating that Cat B mostly disturbs TGF-ß1-driven canonical Smad signaling pathway. Although mRNA expression of cystatin C was stable, its secretion, which was inhibited by brefeldin A, increased during TGF-ß1-induced differentiation of idiopathic pulmonary fibrosis and CCD-19Lu fibroblasts. In addition, cystatin C participated in the control of extracellular Cats, because its gene silencing restored their proteolytic activities. These data support the notion that Cat B participates in lung myofibrogenesis as suggested for stellate cells during liver fibrosis. Moreover, we propose that TGF-ß1 promotes fibrosis by driving the effective cystatin C-dependent inhibition of extracellular matrix-degrading Cats.
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Catepsina B/fisiología , Diferenciación Celular/fisiología , Cistatina C/fisiología , Pulmón/citología , Factor de Crecimiento Transformador beta1/fisiología , Western Blotting , Catepsina B/genética , Células Cultivadas , Fibroblastos/citología , Silenciador del Gen , Humanos , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Proteínas Smad/metabolismoRESUMEN
Proteases are often overexpressed in tumor cells and/or the stromal compartment and can thus be exploited in tumor therapy to activate cytotoxic prodrugs as, for example, in cytolytic fusion proteins, and for tumor imaging. Specifically, we discuss cathepsin B-activated prodrug conjugates, antibody-directed prodrug therapy, protease-activated peptide-thapsigargin conjugates, protease-activated cytotoxic receptor ligands and other cytotoxic proteins, protease-mediated activation of anthrax toxin, granzyme B as a therapeutic principle in cytolytic fusion proteins, and tumor-imaging based on deregulated proteases.
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Antineoplásicos/metabolismo , Neoplasias/tratamiento farmacológico , Profármacos/metabolismo , Animales , Antineoplásicos/uso terapéutico , Catepsina B/fisiología , Granzimas/fisiología , Humanos , Metaloproteinasas de la Matriz/fisiología , Profármacos/uso terapéutico , Proteolisis , Serina Endopeptidasas/fisiología , Activador de Plasminógeno de Tipo Uroquinasa/fisiologíaRESUMEN
This review focuses on seven aspects of physiology and pathophysiology of the exocrine pancreas that have been intensively discussed and studied within the past few years: (1) the role of neurohormonal mechanisms like melatonin, leptin, or ghrelin in the stimulation of pancreatic enzyme secretion; (2) the initiation processes of acute pancreatitis, like fusion of zymogen granules with lysosomes leading to intracellular activation of trypsinogen by the lysosomal enzyme cathepsin B, or autoactivation of trypsinogen; (3) the role of genes in the pathogenesis of acute pancreatitis; (4) the role of alcohol and constituents of alcoholic beverages in the pathogenesis of acute pancreatitis; (5) the role of pancreatic hypertension, neuropathy, and central mechanisms for the pathogenesis of pain in chronic pancreatitis; (6) the relation between exocrine pancreatic function and diabetes mellitus; and (7) pathophysiology, diagnosis and treatment of pancreatic steatorrhea.
Asunto(s)
Páncreas/fisiopatología , Catepsina B/fisiología , Análisis Mutacional de ADN , Diabetes Mellitus/genética , Diabetes Mellitus/fisiopatología , Ghrelina/fisiología , Leptina/fisiología , Melatonina/fisiología , Jugo Pancreático/metabolismo , Pancreatitis Aguda Necrotizante/genética , Pancreatitis Aguda Necrotizante/fisiopatología , Pancreatitis Alcohólica/genética , Pancreatitis Alcohólica/fisiopatología , Pancreatitis Crónica/genética , Pancreatitis Crónica/fisiopatología , Tripsinógeno/metabolismoRESUMEN
Inflammasome activation has been shown to regulate both innate and adaptive immune responses. It is important to investigate whether immune-enhancing natural products can also activate inflammasome. The current study examined the potential of protein-bound polysaccharide-K (PSK), a hot water extract from Trametes versicolor, to activate inflammasome. Using THP-1 cells, we have demonstrated that PSK induces both pro-IL-1ß and mature IL-1ß in THP-1 cells in a caspase 1- and NLRP3-dependent manner. PSK also induces IL-1ß and IL-18 in human PBMC. Cathepsin B is required for PSK-induced inflammasome activation as CA-074-Me, a cathepsin B inhibitor, significantly decreased PSK-induced IL-1ß. PSK induces NLRP3 at both mRNA and protein level. Comparison of PSK-induced IL-1ß in bone marrow-derived macrophages from wild type C57BL/6 mice, TLR2(-/-), P2X7R(-/-) and NLRP3(-/-) mice demonstrated that PSK-induced IL-1ß is dependent on both TLR2 and NLRP3. P2X7R is not required for PSK-induced inflammasome activation, but enhances PSK-induced caspase-1 activation and IL-1ß induction. Altogether, these results demonstrated that PSK induces inflammasome activation and production of IL-1ß in a TLR2- and NLRP3-dependent mechanism. These results provide novel insights into the mechanisms of the immune modulatory effects of PSK.
Asunto(s)
Proteínas Portadoras/efectos de los fármacos , Inflamasomas/efectos de los fármacos , Interleucina-1beta/biosíntesis , Proteoglicanos/farmacología , Receptor Toll-Like 2/efectos de los fármacos , Animales , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Caspasas/biosíntesis , Catepsina B/fisiología , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Potasio/metabolismo , Receptor Toll-Like 2/biosíntesis , Receptor Toll-Like 2/genéticaRESUMEN
Blastocyst hatching is critical for successful implantation leading to pregnancy. Its failure causes infertility. The phenomenon of blastocyst hatching in humans is poorly understood and the available information on this stems from studies of rodents such as mice and hamsters. We and others showed that hamster blastocyst hatching is characterized by firstly blastocyst deflation followed by a dissolution of the zona pellucida (zona) and accompanied by trophectodermal projections (TEPs). We also showed that embryo-derived cathepsins (Cat) proteases, specifically Cat-L, -B and -P act as zonalysins and are responsible for hatching. In this study, we show the expression and function of one of the potential regulators of embryogenesis, cyclooxygenase (COX)-2 during blastocyst development and hatching. The expression of COX-2 mRNA and protein was observed in 8-cell through hatched blastocyst stages and it was also localized to blastocyst's TEPs. Specific COX-2 inhibitors, NS-398 and CAY-10404, inhibited blastocyst hatching; percentages achieved were only 28.4 ± 5.3 and 32.3 ± 5.4%, respectively, compared with >90% with untreated embryos. Interestingly, inhibitor-treated blastocysts failed to deflate, normally observed during hatching. Supplementation of prostaglandins (PGs)-E2 or -I2 to cultured embryos reversed the inhibitors' effect on hatching and also the deflation behavior. Importantly, the levels of mRNA and protein of Cat-L, -B and -P showed a significant reduction in the inhibitor-treated embryos compared with untreated embryos, although its mechanism remains to be examined. These data provide the first evidence that COX-2 is critical for blastocyst hatching in the golden hamster.
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Blastocisto/fisiología , Desarrollo Embrionario , Animales , Catepsina B/metabolismo , Catepsina B/fisiología , Catepsina L/metabolismo , Catepsina L/fisiología , Catepsina Z/metabolismo , Catepsina Z/fisiología , Cricetinae , Ciclooxigenasa 2/metabolismo , Ciclooxigenasa 2/fisiología , Zona Pelúcida/metabolismo , Zona Pelúcida/fisiología , Zona Pelúcida/ultraestructuraRESUMEN
Although chromogranin A (CGA) is frequently present in Alzheimer's disease (AD), senile plaques associated with microglial activation, little is known about basic difference between CGA and fibrillar amyloid-ß (fAß) as neuroinflammatory factors. Here we have compared the interleukin-1ß (IL-1ß) production pathways by CGA and fAß in microglia. In cultured microglia, production of IL-1ß was induced by CGA, but not by fAß. CGA activated both nuclear factor-κB (NF-κB) and pro-caspase-1, whereas fAß activated pro-caspase-1 only. For the activation of pro-caspase-1, both CGA and fAß needed the enzymatic activity of cathepsin B (CatB), but only fAß required cytosolic leakage of CatB and the NLRP3 inflammasome activation. In contrast, fAß induced the IL-1ß secretion from microglia isolated from the aged mouse brain. In AD brain, highly activated microglia, which showed intense immunoreactivity for CatB and IL-1ß, surrounded CGA-positive plaques more frequently than Aß-positive plaques. These observations indicate differential pathways for the microglial IL-1ß production by CGA and fAß, which may aid in better understanding of the pathological significance of neuroinflammation in AD.
Asunto(s)
Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/fisiología , Cromogranina A/fisiología , Interleucina-1beta/biosíntesis , Microglía/metabolismo , Envejecimiento/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Encéfalo/citología , Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Caspasa 1/metabolismo , Catepsina B/fisiología , Células Cultivadas , Citosol/metabolismo , Inflamasomas/metabolismo , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR , Placa Amiloide/metabolismo , Transducción de Señal/genéticaRESUMEN
Cysteine cathepsins are a family of proteases involved in intracellular protein turnover and extracellular matrix degradation. Cathepsin B (Ctsb) and cathepsin Z (Ctsz) promote tumorigenesis and Ctsb is a known modulator of tumor angiogenesis. We therefore investigated the angiomodulatory function of these cathepsins in vitro as well as in a mouse model of laser-induced choroidal neovascularization (laser-CNV). Ctsb(-/-), Ctsz(-/-), Ctsb/Ctsz double-knockout (Ctsb/z DKO), and wild type (WT) mice underwent argon laser treatment to induce choroidal neovascularization (CNV). The neovascularized area was quantified individually for each lesion at 14 days after laser coagulation. In vitro the effects of cathepsin inhibitors on angiogenesis were analysed by endothelial cell (EC) spheroid sprouting and EC invadosome assays. Retinas from cathepsin KO mice did not show gross morphological abnormalities. In the laser CNV model, however, Ctsb/z DKO mice displayed a significantly reduced neovascularized area compared to WT (0.027 mm(2) vs. 0.052 mm(2); p = 0.012), while single knockouts did not differ significantly from WT. In line, VEGF-induced EC spheroid sprouting and invadosome formation were not significantly altered by a specific cathepsin B inhibitor alone, but significantly suppressed when more than one cathepsin was inhibited. Our results demonstrate that laser-CNV formation is significantly reduced in Ctsb/z DKO mice. In line, EC sprouting and invadosome formation are blunted when more than one cathepsin is inhibited in vitro. These results reveal an angiomodulatory potential of cathepsins with partial functional redundancies between different cathepsin family members.
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Catepsina B/fisiología , Catepsina Z/fisiología , Coroides/irrigación sanguínea , Neovascularización Coroidal/enzimología , Modelos Animales de Enfermedad , Coagulación con Láser , Animales , Catepsina B/antagonistas & inhibidores , Catepsina Z/antagonistas & inhibidores , Neovascularización Coroidal/patología , Inhibidores Enzimáticos/farmacología , Matriz Extracelular/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Láseres de Gas , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Esferoides Celulares , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Cancer-initiating cells comprise a heterogeneous population of undifferentiated cells with the capacity for self-renewal and high proliferative potential. We investigated the role of uPAR and cathepsin B in the maintenance of stem cell nature in glioma-initiating cells (GICs). Simultaneous knockdown of uPAR and cathepsin B significantly reduced the expression of CD133, Nestin, Sox2 and Bmi1 at the protein level and GLI1 and GLI2 at the messenger RNA level. Also, knockdown of uPAR and cathepsin B resulted in a reduction in the number of GICs as well as sphere size. These changes are mediated by Sox2 and Bmi1, downstream of hedgehog signaling. Addition of cyclopamine reduced the expression of Sox2 and Bmi1 along with GLI1 and GLI2 expression, induced differentiation and reduced subsphere formation of GICs thereby indicating that hedgehog signaling acts upstream of Sox2 and Bmi1. Further confirmation was obtained from increased luciferase expression under the control of a GLI-bound Sox2 and Bmi1 luciferase promoter. Simultaneous knockdown of uPAR and cathepsin B also reduced the expression of Nestin Sox2 and Bmi1 in vivo. Thus, our study highlights the importance of uPAR and cathepsin B in the regulation of malignant stem cell self-renewal through hedgehog components, Bmi1 and Sox2.
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Catepsina B/fisiología , Glioma/metabolismo , Células Madre Neoplásicas/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/fisiología , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/fisiología , Antígeno AC133 , Animales , Antígenos CD/metabolismo , Catepsina B/genética , Catepsina B/metabolismo , Línea Celular Tumoral , Proliferación Celular , Separación Celular , Femenino , Citometría de Flujo , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glioma/patología , Glicoproteínas/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Células Madre Neoplásicas/efectos de la radiación , Péptidos/metabolismo , Complejo Represivo Polycomb 1/genética , ARN Interferente Pequeño/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Factores de Transcripción SOXB1/genética , Transducción de Señal , Factores de Transcripción/metabolismo , Activación Transcripcional , Proteína con Dedos de Zinc GLI1RESUMEN
The persistence of memory T lymphocytes confers lifelong protection from pathogens. Memory T cells survive and undergo homeostatic proliferation (HSP) in the absence of Ag, although the cell-intrinsic mechanisms by which cytokines drive the HSP of memory T cells are not well understood. In this study we report that lysosome stability limits the long-term maintenance of memory CD8(+) T cell populations. Serine protease inhibitor (Spi) 2A, an anti-apoptotic cytosolic cathepsin inhibitor, is induced by both IL-15 and IL-7. Mice deficient in Spi2A developed fewer memory phenotype CD44(hi)CD8(+) T cells with age, which underwent reduced HSP in the bone marrow. Spi2A was also required for the maintenance of central memory CD8(+) T cell populations after acute infection with lymphocytic choriomeningitis virus. Spi2A-deficient Ag-specific CD8(+) T cell populations declined more than wild-type competitors after viral infection, and they were eroded further after successive infections. Spi2A protected memory cells from lysosomal breakdown by inhibiting cathepsin B. The impaired maintenance of Spi2A-deficient memory CD8(+) T cells was rescued by concomitant cathepsin B deficiency, demonstrating that cathepsin B was a physiological target of Spi2A in memory CD8(+) T cell survival. Our findings support a model in which protection from lysosomal rupture through cytokine-induced expression of Spi2A determines the long-term persistence of memory CD8(+) T cells.
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Linfocitos T CD8-positivos/inmunología , Catepsina B/fisiología , Memoria Inmunológica , Animales , Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/metabolismo , Catepsina B/antagonistas & inhibidores , Catepsina B/genética , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Proliferación Celular , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Femenino , Homeostasis/genética , Homeostasis/inmunología , Memoria Inmunológica/genética , Lisosomas/enzimología , Lisosomas/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Ratones Transgénicos , Serpinas/deficiencia , Serpinas/fisiologíaRESUMEN
Inflammasomes are increasingly implicated in regulating immunity, but how their activation relates to function of human dendritic cells (DCs) is unknown. Here we show that DC maturation stimuli lead to rapid activation of caspase-1 in human monocyte-derived DCs. RNAi mediated inhibition of the inflammasome component ASC leads to marked inhibition of the capacity of lipopolysachharide (LPS)-matured DCs to stimulate antigen-specific T cells. RNAi mediated inhibition of Cathepsin B (CatB) also similarly inhibits the capacity of human DCs to stimulate immunity. The defective ability of ASC or CatB deficient DCs to stimulate T cells is independent of inflammasome-mediated processing of inflammatory cytokines and also includes DCs loaded with pre-processed peptide. Gene expression profiles of ASC or CatB deficient human DCs show marked overlap with downregulation of genes implicated in DC function. These data demonstrate an important role for ASC and CatB in regulating function of human DCs with overlapping effects on gene expression.
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Presentación de Antígeno/inmunología , Catepsina B/fisiología , Proteínas del Citoesqueleto/fisiología , Células Dendríticas/inmunología , Inflamasomas/genética , Proteínas Adaptadoras de Señalización CARD , Caspasa 1/metabolismo , Catepsina B/genética , Catepsina B/metabolismo , Células Cultivadas , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Células Dendríticas/metabolismo , Activación Enzimática , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Inflamasomas/metabolismo , Interferencia de ARN , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Cathepsin B (CTSB) is a proteolytic enzyme potentially modulating angiogenic processes and extracellular matrix remodeling. While matrix metalloproteinases are shown to be implicated in tissue fibrosis and vasculopathy associated with systemic sclerosis (SSc), the role of cathepsins in this disease has not been well studied. The aim of this study is to evaluate the roles of CTSB in SSc. Serum pro-CTSB levels were determined by enzyme-linked immunosorbent assay in 55 SSc patients and 19 normal controls. Since the deficiency of transcription factor Fli1 in endothelial cells is potentially associated with the development of SSc vasculopathy, cutaneous CTSB expression was evaluated by immunostaining in Fli1(+/-) and wild type mice as well as in SSc and control subjects. The effects of Fli1 gene silencing and transforming growth factor-ß (TGF-ß) on CTSB expression were determined by real-time PCR in human dermal microvascular endothelial cells (HDMECs) and dermal fibroblasts, respectively. Serum pro-CTSB levels were significantly higher in limited cutaneous SSc (lcSSc) and late-stage diffuse cutaneous SSc (dcSSc) patients than in healthy controls. In dcSSc, patients with increased serum pro-CTSB levels showed a significantly higher frequency of digital ulcers than those with normal levels. CTSB expression in dermal blood vessels was increased in Fli1(+/-) mice compared with wild type mice and in SSc patients compared with healthy controls. Consistently, Fli1 gene silencing increased CTSB expression in HDMECs. In cultured dermal fibroblasts from early dcSSc, CTSB expression was decreased compared with normal fibroblasts and significantly reversed by TGF-ß1 antisense oligonucleotide. In conclusion, up-regulation of endothelial CTSB due to Fli1 deficiency may contribute to the development of SSc vasculopathy, especially digital ulcers, while reduced expression of CTSB in lesional dermal fibroblasts is likely to be associated with skin sclerosis in early dcSSc.