RESUMEN
The major lysosomal proteases, Cathepsin B (CTSB), Cathepsin D (CTSD) and Cathepsin L (CTSL), are implicated in autophagic activity. To investigate the role of each cathepsin in the exocrine pancreas, we generated mice in which the pancreas was specifically deficient in Ctsb, Ctsd and Ctsl. Each of these gene knockout (KO) and Ctsb;Ctsl and Ctsd;Ctsl double-knockout (DKO) mice were almost normal. However, we found cytoplasmic degeneration in the pancreatic acinar cells of Ctsb;Ctsd DKO mice, similar to autophagy related 5 (Atg5) KO mice. LC3 and p62 (autophagy markers) showed remarkable accumulation and the numbers of autophagosomes and autolysosomes were increased in the pancreatic acinar cells of Ctsb;Ctsd DKO mice. Moreover, these Ctsb;Ctsd DKO mice also developed chronic pancreatitis (CP). Thus, we conclude that both Ctsb and Ctsd deficiency caused impaired autophagy in the pancreatic acinar cells, and induced CP in mice.
Asunto(s)
Autofagia , Catepsina B/deficiencia , Catepsina D/deficiencia , Páncreas/metabolismo , Pancreatitis Crónica/metabolismo , Células Acinares/metabolismo , Animales , Autofagosomas/metabolismo , Catepsina B/genética , Catepsina B/metabolismo , Catepsina D/genética , Catepsina D/metabolismo , Ratones , Páncreas/citología , Pancreatitis Crónica/genéticaRESUMEN
Nonalcoholic steatohepatitis (NASH) is a severe form of nonalcoholic fatty liver disease characterized by fat accumulation and inflammation in liver. Yet, the mechanistic insight and diagnostic and therapeutic options of NASH remain incompletely understood. This study tested the roles of cysteine protease cathepsin B (CatB) in mouse NASH development. Immunoblot revealed increased liver CatB expression in NASH mice. Fructose-palmitate-cholesterol diet increased body weight gain, liver to body weight ratio, blood fasting glucose, plasma total cholesterol and alanine transaminase levels, and liver triglyceride, but decreased plasma high-density lipoprotein in wild-type mice. All these changes were blunted in CatB-deficient (Ctsb-/-) mice. In parallel to reduced expression of genes involved in liver lipid transport and lipogenesis, liver CD36, FABP4, and PPARγ protein levels were also significantly decreased in Ctsb-/- mice, although CatB deficiency did not affect liver gluconeogenesis and fatty acid beta-oxidation-associated gene expression. Mechanistic studies showed that CatB deficiency decreased liver expression of adhesion molecules, inflammatory cytokine, and chemokine, along with reduced liver inflammatory cell infiltration. CatB deficiency also promoted M2 macrophage polarization and reduced liver TGF-ß1 signaling and fibrosis. Together, CatB deficiency improves liver function in NASH mice by suppressing de novo lipogenesis and liver inflammation and fibrosis.
Asunto(s)
Catepsina B/deficiencia , Dieta Alta en Grasa , Inflamación/patología , Metabolismo de los Lípidos , Cirrosis Hepática/complicaciones , Cirrosis Hepática/enzimología , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Animales , Cadherinas , Catepsina B/metabolismo , Polaridad Celular , Hígado/patología , Hígado/fisiopatología , Cirrosis Hepática/fisiopatología , Macrófagos/patología , Masculino , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/enzimología , Enfermedad del Hígado Graso no Alcohólico/fisiopatología , Transducción de Señal , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Aumento de PesoRESUMEN
Lysosomes are known to mediate neurite outgrowth in neurons. However, the principal lysosomal molecule controlling that outgrowth is unclear. We studied primary mouse neurons in vitro and found that they naturally develop neurite outgrowths over time and as they did so the lysosomal cysteine protease cathepsin B (CTSB) mRNA levels dramatically increased. Surprisingly, we found that treating those neurons with CA-074Me, which inhibits CTSB, prevented neurites. As that compound also inhibits another protease, we evaluated a N2a neuronal cell line in which the CTSB gene was deleted (CTSB knockout, KO) using CRISPR technology and induced their neurite outgrowth by treatment with retinoic acid. We found that CTSB KO N2a cells failed to produce neurite outgrowths but the wild-type (WT) did. CA-074Me is a cell permeable prodrug of CA-074, which is cell impermeable and a specific CTSB inhibitor. Neurite outgrowth was and was not suppressed in WT N2a cells treated with CA-074Me and CA-074, respectively. Lysosome-associated membrane glycoprotein 2-positive lysosomes traffic to the plasma cell membrane in WT but not in CTSB KO N2 a cells. Interestingly, no obvious differences between WT and CTSB KO N2a cells were found in neurite outgrowth regulatory proteins, PI3K/AKT, ERK/MAPK, cJUN, and CREB. These findings show that intracellular CTSB controls neurite outgrowth and that it does so through regulation of lysosomal trafficking and remodeling in neurons. This adds valuable information regarding the physiological function of CTSB in neural development.
Asunto(s)
Catepsina B/deficiencia , Lisosomas/metabolismo , Neuritas/metabolismo , Neuronas/metabolismo , Animales , Animales Recién Nacidos , Catepsina B/antagonistas & inhibidores , Catepsina B/genética , Línea Celular Tumoral , Células Cultivadas , Proteasas de Cisteína/deficiencia , Proteasas de Cisteína/genética , Femenino , Técnicas de Inactivación de Genes/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Neocórtex/citología , Neocórtex/metabolismo , Transporte de Proteínas/fisiologíaRESUMEN
During normal aging, innate immunity progresses to a chronic state. However, how oxidative stress and chronic neuroinflammation arise during aging remains unclear. In this study, we found that genetic ablation of cathepsin B (CatB) in mice significantly reduced the generation of reactive oxygen species (ROS) and neuroinflammation and improved cognitive impairment during aging. In cultured microglia, pharmacological inhibition of CatB significantly reduced the generation of mitochondria-derived ROS and proinflammatory mediators induced by L-leucyl-L-leucine methyl ester (LLOMe), a lysosome-destabilizing agent. In the CatB-overexpressing microglia after treatment with LLOMe, which mimicked the aged microglia, CatB leaked in the cytosol is responsible for the degradation of the mitochondrial transcription factor A (TFAM), resulting in the increased generation of mitochondria-derived ROS and proinflammatory mediators through impaired mtDNA biosynthesis. Furthermore, intralateral ventricle injection of LLOMe-treated CatB-overexpressing microglia induced cognitive impairment in middle-aged mice. These results suggest that the increase and leakage of CatB in microglia during aging are responsible for the increased generation of mitochondria-derived ROS and proinflammatory mediators, culminating in memory impairment.
Asunto(s)
Catepsina B/metabolismo , Disfunción Cognitiva/metabolismo , Inflamación/metabolismo , Microglía/metabolismo , Estrés Oxidativo , Envejecimiento/metabolismo , Animales , Catepsina B/deficiencia , Línea Celular , Células Cultivadas , Disfunción Cognitiva/complicaciones , Disfunción Cognitiva/fisiopatología , Citosol/efectos de los fármacos , Citosol/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Hipocampo/patología , Inflamación/complicaciones , Memoria/efectos de los fármacos , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Rotenona/farmacología , Fracciones Subcelulares/metabolismoRESUMEN
Cathepsin B (CtsB) contributes to atherosclerosis and cancer progression by processing the extracellular matrix and promoting angiogenesis. Although CtsB was reported to promote and reduce angiogenesis, there is no mechanistic explanation that reconciles this apparent discrepancy. CtsB cleaves CD18 from the surface of immune cells, but its contribution to angiogenesis has not been studied. We developed an in vivo technique for visualization of immune cell transmigration from corneal vessels toward implanted cytokines. Wild-type (WT) leukocytes extravasated from limbal vessels, angiogenic stalks, and growing tip vessels and migrated toward the cytokines, indicating immune competence of angiogenic vessels. Compared to WT leukocytes, CtsB-/- leukocytes accumulated in a higher number in angiogenic vessels, but extravasated less toward the implanted cytokine. The accumulated CtsB-/- leukocytes in angiogenic vessels expressed more CD18. CD18-/- leukocytes extravasated later than WT leukocytes. However, once extravasated, CD18-/- leukocytes transmigrated more rapidly than their WT counterparts. These results suggest that, although CD18 facilitates efficient extravasation, outside of the vessel CD18 interaction with the extracellular matrix, it reduced transmigration velocity. Our results reveal an unexpected role for CtsB in leukocyte extravasation and transmigration, which advances our understanding of the complex contribution of CtsB to angiogenesis.-Nakao, S., Zandi, S., Sun, D., Hafezi-Moghadam, A. Cathepsin B-mediated CD18 shedding regulates leukocyte recruitment from angiogenic vessels.
Asunto(s)
Antígenos CD18/metabolismo , Catepsina B/metabolismo , Leucocitos/patología , Leucocitos/fisiología , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Animales , Antígenos CD18/deficiencia , Antígenos CD18/genética , Catepsina B/deficiencia , Catepsina B/genética , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Neovascularización de la Córnea/etiología , Neovascularización de la Córnea/metabolismo , Neovascularización de la Córnea/patología , Matriz Extracelular/inmunología , Matriz Extracelular/metabolismo , Leucocitos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica/etiologíaRESUMEN
BACKGROUND & AIMS: Acute pancreatitis is characterized by premature intracellular activation of digestive proteases within pancreatic acini and a consecutive systemic inflammatory response. We investigated how these processes interact during severe pancreatitis in mice. METHODS: Pancreatitis was induced in C57Bl/6 wild-type (control), cathepsin B (CTSB)-knockout, and cathepsin L-knockout mice by partial pancreatic duct ligation with supramaximal caerulein injection, or by repetitive supramaximal caerulein injections alone. Immune cells that infiltrated the pancreas were characterized by immunofluorescence detection of Ly6g, CD206, and CD68. Macrophages were isolated from bone marrow and incubated with bovine trypsinogen or isolated acinar cells; the macrophages were then transferred into pancreatitis control or cathepsin-knockout mice. Activities of proteases and nuclear factor (NF)-κB were determined using fluorogenic substrates and trypsin activity was blocked by nafamostat. Cytokine levels were measured using a cytometric bead array. We performed immunohistochemical analyses to detect trypsinogen, CD206, and CD68 in human chronic pancreatitis (n = 13) and acute necrotizing pancreatitis (n = 15) specimens. RESULTS: Macrophages were the predominant immune cell population that migrated into the pancreas during induction of pancreatitis in control mice. CD68-positive macrophages were found to phagocytose acinar cell components, including zymogen-containing vesicles, in pancreata from mice with pancreatitis, as well as human necrotic pancreatic tissues. Trypsinogen became activated in macrophages cultured with purified trypsinogen or co-cultured with pancreatic acini and in pancreata of mice with pancreatitis; trypsinogen activation required macrophage endocytosis and expression and activity of CTSB, and was sensitive to pH. Activation of trypsinogen in macrophages resulted in translocation of NF-kB and production of inflammatory cytokines; mice without trypsinogen activation (CTSB-knockout mice) in macrophages developed less severe pancreatitis compared with control mice. Transfer of macrophage from control mice to CTSB-knockout mice increased the severity of pancreatitis. Inhibition of trypsin activity in macrophages prevented translocation of NF-κB and production of inflammatory cytokines. CONCLUSIONS: Studying pancreatitis in mice, we found activation of digestive proteases to occur not only in acinar cells but also in macrophages that infiltrate pancreatic tissue. Activation of the proteases in macrophage occurs during endocytosis of zymogen-containing vesicles, and depends on pH and CTSB. This process involves macrophage activation via NF-κB-translocation, and contributes to systemic inflammation and severity of pancreatitis.
Asunto(s)
Catepsina B/metabolismo , Endocitosis , Macrófagos/enzimología , Páncreas/enzimología , Pancreatitis Aguda Necrotizante/enzimología , Tripsinógeno/metabolismo , Traslado Adoptivo , Animales , Catepsina B/deficiencia , Catepsina B/genética , Catepsina L/deficiencia , Catepsina L/genética , Células Cultivadas , Ceruletida , Técnicas de Cocultivo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Activación Enzimática , Predisposición Genética a la Enfermedad , Humanos , Concentración de Iones de Hidrógeno , Mediadores de Inflamación/metabolismo , Macrófagos/inmunología , Macrófagos/patología , Macrófagos/trasplante , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/deficiencia , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Necrosis , Páncreas/inmunología , Páncreas/patología , Pancreatectomía , Pancreatitis Aguda Necrotizante/inducido químicamente , Pancreatitis Aguda Necrotizante/inmunología , Pancreatitis Aguda Necrotizante/patología , Fagocitosis , Fenotipo , Índice de Severidad de la Enfermedad , Factores de TiempoRESUMEN
A critical role for intracellular TLR9 has been described in recognition and host resistance to Leishmania parasites. As TLR9 requires endolysosomal proteolytic cleavage to achieve signaling functionality, we investigated the contribution of different proteases like asparagine endopeptidase (AEP) or cysteine protease cathepsins B (CatB), L (CatL) and S (CatS) to host resistance during Leishmania major (L. major) infection in C57BL/6 (WT) mice and whether they would impact on TLR9 signaling. Unlike TLR9-/-, which are more susceptible to infection, AEP-/-, CatL-/- and CatS-/- mice are as resistant to L. major infection as WT mice, suggesting that these proteases are not individually involved in TLR9 processing. Interestingly, we observed that CatB-/- mice resolve L. major lesions significantly faster than WT mice, however we did not find evidence for an involvement of CatB on either TLR9-dependent or independent cytokine responses of dendritic cells and macrophages or in the innate immune response to L. major infection. We also found no difference in antigen presenting capacity. We observed a more precocious development of T helper 1 responses accompanied by a faster decline of inflammation, resulting in resolution of footpad inflammation, reduced IFNγ levels and decreased parasite burden. Adoptive transfer experiments into alymphoid RAG2-/-γc-/- mice allowed us to identify CD3+ T cells as responsible for the immune advantage of CatB-/- mice towards L. major. In vitro data confirmed the T cell intrinsic differences between CatB-/- mice and WT. Our study brings forth a yet unappreciated role for CatB in regulating T cell responses during L. major infection.
Asunto(s)
Catepsina B/deficiencia , Catepsina B/metabolismo , Leishmania major , Leishmaniasis Cutánea/inmunología , Subgrupos de Linfocitos T/inmunología , Receptor Toll-Like 9/metabolismo , Traslado Adoptivo , Animales , Presentación de Antígeno , Complejo CD3/análisis , Complejo CD3/inmunología , Catepsina B/genética , Catepsina L/deficiencia , Catepsina L/genética , Catepsinas/deficiencia , Catepsinas/genética , Células Dendríticas/inmunología , Endopeptidasas/deficiencia , Pie , Inflamación/inmunología , Interferón gamma/biosíntesis , Leishmania major/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Carga de Parásitos , Transducción de Señal , Células TH1/inmunología , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/inmunologíaAsunto(s)
Catepsina B/deficiencia , Hipertrofia Ventricular Izquierda/prevención & control , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Miocitos Cardíacos/enzimología , Factor de Necrosis Tumoral alfa/metabolismo , Función Ventricular Izquierda , Remodelación Ventricular , Animales , Femenino , Humanos , MasculinoRESUMEN
Cathepsin B (CTSB), a member of the lysosomal cathepsin family that is expressed in both murine and human hearts, was previously shown to participate in apoptosis, autophagy, and the progression of certain types of cancers. Recently, CTSB has been linked to myocardial infarction. Given that cathepsin L, another member of the lysosomal cathepsin family, ameliorates pathological cardiac hypertrophy, we hypothesized that CTSB plays a role in pressure overload-induced cardiac remodeling. Here we report that CTSB was upregulated in cardiomyocytes in response to hypertrophic stimuli both in vivo and in vitro. Moreover, knockout of CTSB attenuated pressure overload-induced cardiac hypertrophy, fibrosis, dysfunction, and apoptosis. Furthermore, the aortic banding-induced activation of TNF-α, apoptosis signal-regulating kinase 1 (ASK1), c-Jun NH2-terminal kinases (JNK), c-Jun, and release of cytochrome c was blunted by CTSB deficiency, which was further confirmed in in vitro studies induced by angiotensin II. In cardiomyocytes pretreatment with SP600125, a JNK inhibitor, suppressed the cardiomyocytes hypertrophy by inhibiting the ASK1/JNK pathway. Altogether, these data indicate that the CTSB protein functions as a necessary modulator of hypertrophic response by regulating TNF-α/ASK1/JNK signaling pathway involved in cardiac remodeling.
Asunto(s)
Catepsina B/deficiencia , Hipertrofia Ventricular Izquierda/prevención & control , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Miocitos Cardíacos/enzimología , Factor de Necrosis Tumoral alfa/metabolismo , Función Ventricular Izquierda , Remodelación Ventricular , Animales , Apoptosis , Catepsina B/antagonistas & inhibidores , Catepsina B/genética , Modelos Animales de Enfermedad , Femenino , Fibrosis , Células HEK293 , Humanos , Hipertrofia Ventricular Izquierda/enzimología , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/patología , Hipertrofia Ventricular Izquierda/fisiopatología , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Masculino , Ratones Noqueados , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , Transducción de Señal , Transfección , Función Ventricular Izquierda/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacosRESUMEN
The osteoclast is an integral cell of bone resorption. Since osteolytic disorders hinge on the function and dysfunction of the osteoclast, understanding osteoclast biology is fundamental to designing new therapies that curb osteolytic disorders. The identification and study of lysosomal proteases, such as cathepsins, have shed light on mechanisms of bone resorption. For example, Cathepsin K has already been identified as a collagen degradation protease produced by mature osteoclasts with high activity in the acidic osteoclast resorption pits. Delving into the mechanisms of cathepsins and other osteoclast related compounds provides new targets to explore in osteoclast biology. Through our anti-osteoclastogenic compound screening experiments we encountered a modified version of the Cathepsin B inhibitor CA-074: the cell membrane-permeable CA-074Me (L-3-trans-(Propylcarbamoyl) oxirane-2-carbonyl]-L-isoleucyl-L-proline Methyl Ester). Here we confirm that CA-074Me inhibits osteoclastogenesis in vivo and in vitro in a dose-dependent manner. However, Cathepsin B knockout mice exhibited unaltered osteoclastogenesis, suggesting a more complicated mechanism of action than Cathepsin B inhibition. We found that CA-074Me exerts its osteoclastogenic effect within 24 h of osteoclastogenesis stimulation by suppression of c-FOS and NFATc1 pathways.
Asunto(s)
Dipéptidos/farmacología , Factores de Transcripción NFATC/antagonistas & inhibidores , Osteoclastos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/antagonistas & inhibidores , Animales , Catepsina B/deficiencia , Sistema de Señalización de MAP Quinasas , Masculino , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Ligando RANKRESUMEN
Cathepsin B has been demonstrated to be involved in several proteolytic processes that support tumor progression and metastasis and neurodegeneration. To further clarify its role, defined monoclonal antibodies are needed. As the primary structure of human cathepsin B is almost identical to that of the mouse, cathepsin B-deficient mice were used in a novel approach for generating such antibodies, providing the chance of an increased immune response to the antigen, human cathepsin B. Thirty clones were found to produce cathepsin B-specific antibodies. Seven of these antibodies were used to detect cathepsin B in MCF10-DCIS human breast cancer cells by immunocytochemistry and immunoblotting. Five different binding sites were identified by epitope mapping giving the opportunity to combine these antibodies in oligoclonal antibody mixtures for an improved detection of cathepsin B.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Catepsina B/deficiencia , Catepsina B/inmunología , Secuencia de Aminoácidos , Animales , Catepsina B/química , Línea Celular Tumoral , Electroforesis en Gel de Poliacrilamida , Mapeo Epitopo , Epítopos/química , Epítopos/inmunología , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Ratones , Datos de Secuencia MolecularRESUMEN
Resistance and susceptibility to Leishmania major infection in the murine model is determined by the capacity of the host to mount either a protective Th1 response or a Th2 response associated with disease progression. Previous reports involving the use of cysteine cathepsin inhibitors indicated that cathepsins B (Ctsb) and L (Ctsl) play important roles in Th1/Th2 polarization during L. major infection in both susceptible and resistant mouse strains. Although it was hypothesized that these effects are a consequence of differential patterns of antigen processing, the mechanisms underlying these differences were not further investigated. Given the pivotal roles that dendritic cells and macrophages play during Leishmania infection, we generated bone-marrow derived dendritic cells (BMDC) and macrophages (BMM) from Ctsb-/- and Ctsl-/- mice, and studied the effects of Ctsb and Ctsl deficiency on the survival of L. major in infected cells. Furthermore, the signals used by dendritic cells to instruct Th cell polarization were addressed: the expression of MHC class II and co-stimulatory molecules, and cytokine production. We found that Ctsb-/- BMDC express higher levels of MHC class II molecules than wild-type (WT) and Ctsl-/- BMDC, while there were no significant differences in the expression of co-stimulatory molecules between cathepsin-deficient and WT cells. Moreover, both BMDC and BMM from Ctsb-/- mice significantly up-regulated the levels of interleukin 12 (IL-12) expression, a key Th1-inducing cytokine. These findings indicate that Ctsb-/- BMDC display more pro-Th1 properties than their WT and Ctsl-/- counterparts, and therefore suggest that Ctsb down-regulates the Th1 response to L. major. Moreover, they propose a novel role for Ctsb as a regulator of cytokine expression.
Asunto(s)
Catepsina B/inmunología , Células Dendríticas/inmunología , Leishmania major/inmunología , Leishmaniasis/inmunología , Macrófagos/inmunología , Células TH1/inmunología , Animales , Presentación de Antígeno , Catepsina B/deficiencia , Catepsina L/inmunología , Modelos Animales de Enfermedad , Femenino , Interleucina-12/biosíntesis , Interleucina-12/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Regulación hacia ArribaRESUMEN
Endolysosomal cysteine cathepsins functionally cooperate. Cathepsin B (Ctsb) and L (Ctsl) double-knockout mice die 4 weeks after birth accompanied by (autophago-) lysosomal accumulations within neurons. Such accumulations are also observed in mouse embryonic fibroblasts (MEFs) deficient for Ctsb and Ctsl. Previous studies showed a strong impact of Ctsl on the MEF secretome. Here we show that Ctsb alone has only a mild influence on extracellular proteome composition. Protease cleavage sites dependent on Ctsb were identified by terminal amine isotopic labeling of substrates (TAILS), revealing a prominent yet mostly indirect impact on the extracellular proteolytic cleavages. To investigate the cooperation of Ctsb and Ctsl, we performed a quantitative secretome comparison of wild-type MEFs and Ctsb (-/-) Ctsl (-/-) MEFs. Deletion of both cathepsins led to drastic alterations in secretome composition, highlighting cooperative functionality. While many protein levels were decreased, immunodetection corroborated increased levels of matrix metalloproteinase (MMP)-2. Re-expression of Ctsl rescues MMP-2 abundance. Ctsl and to a much lesser extent Ctsb are able to degrade MMP-2 at acidic and neutral pH. Addition of active MMP-2 to the MEF secretome degrades proteins whose levels were also decreased by Ctsb and Ctsl double deficiency. These results suggest a degradative Ctsl-MMP-2 axis, resulting in increased MMP-2 levels upon cathepsin deficiency with subsequent degradation of secreted proteins such as collagen α-1 (I).
Asunto(s)
Catepsina B/deficiencia , Catepsina L/deficiencia , Regulación de la Expresión Génica/fisiología , Metaloproteinasa 2 de la Matriz/metabolismo , Animales , Western Blotting , Catepsina B/metabolismo , Catepsina L/metabolismo , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Colágeno Tipo I/metabolismo , Fibroblastos/metabolismo , Citometría de Flujo , Concentración de Iones de Hidrógeno , Ratones , Ratones Noqueados , Proteolisis , Espectrometría de Masas en TándemRESUMEN
When NF-κB activation or protein synthesis is inhibited, tumor necrosis factor alpha (TNFα) can induce apoptosis through Bax- and Bak-mediated mitochondrial outer membrane permeabilization (MOMP) leading to caspase-3 activation. Additionally, previous studies have implicated lysosomal membrane permeability (LMP) and formation of reactive oxygen species (ROS) as early steps of TNFα-induced apoptosis. However, how these two events connect to MOMP and caspase-3 activation has been largely debated. Here, we present the novel finding that LMP induced by the addition of TNFα plus cycloheximide (CHX), the release of lysosomal cathepsins and ROS formation do not occur upstream but downstream of MOMP and require the caspase-3-mediated cleavage of the p75 NDUFS1 subunit of respiratory complex I. Both a caspase non-cleavable p75 mutant and the mitochondrially localized antioxidant MitoQ prevent LMP mediated by TNFα plus CHX and partially interfere with apoptosis induction. Moreover, LMP is completely blocked in cells deficient in both Bax and Bak, Apaf-1, caspase-9 or both caspase-3 and -7. Thus, after MOMP, active caspase-3 exerts a feedback action on complex I to produce ROS. ROS then provoke LMP, cathepsin release and further caspase activation to amplify TNFα apoptosis signaling.
Asunto(s)
Caspasa 3/metabolismo , Permeabilidad de la Membrana Celular/fisiología , Complejo I de Transporte de Electrón/metabolismo , NADH Deshidrogenasa/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Apoptosis , Factor Apoptótico 1 Activador de Proteasas/deficiencia , Factor Apoptótico 1 Activador de Proteasas/metabolismo , Caspasa 3/deficiencia , Caspasa 3/genética , Caspasa 7/deficiencia , Caspasa 7/genética , Caspasa 9/deficiencia , Caspasa 9/metabolismo , Catepsina B/deficiencia , Catepsina B/genética , Catepsina L/deficiencia , Catepsina L/genética , Membrana Celular/metabolismo , Cicloheximida/farmacología , Activación Enzimática , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , NADH Deshidrogenasa/biosíntesis , NADH Deshidrogenasa/genética , Compuestos Organofosforados/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Especies Reactivas de Oxígeno , Ubiquinona/análogos & derivados , Ubiquinona/farmacología , Proteína Destructora del Antagonista Homólogo bcl-2/deficiencia , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/deficiencia , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The molecular mechanism involved in the metastasis of endometrial cancer (EC) remains unclear. The lysosomal cysteine protease Cathepsin B has been implicated in the progression of various human tumors. In the present study, we assessed the expression of Cathepsin B and its functions in EC. Immunohistochemistry was used to examine Cathepsin B expression in 76 paraffin-embedded endometrial tumor tissues. Lentiviral packing short hairpin RNA (shRNA) was transfected into HEC-1A cells to build a stable Cathepsin B knockdown cell line. The cellular levels of Cathepsin B mRNA and protein were detected by real-time PCR and western immunoblotting. The functions of Cathepsin B in EC cells were measured by MTT, migration and invasion assays. In additon, tumorigenicity assays were established in nude mice to study tumor growth in vivo. The results of our study showed that Cathepsin B was overexpressed in EC tissues compared with normal endometrium and endometrial atypical hyperplasia. Depletion of Cathepsin B in vitro inhibited cell proliferation, migration and invasion. Tumor formation assays confirmed that suppression of Cathepsin B inhibited the proliferation potential of HEC-1A cells in vivo, demonstrated by lower proliferation rates. These results suggest that Cathepsin B may act as an oncogene in EC, with the potential to provide a new therapeutic target for treating endometrial malignancy.
Asunto(s)
Catepsina B/deficiencia , Catepsina B/genética , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Animales , Pruebas de Carcinogenicidad/métodos , Catepsina B/biosíntesis , Catepsina B/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Hiperplasia Endometrial/genética , Hiperplasia Endometrial/metabolismo , Hiperplasia Endometrial/patología , Neoplasias Endometriales/metabolismo , Endometrio/metabolismo , Endometrio/patología , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Invasividad Neoplásica , ARN Mensajero/genéticaRESUMEN
Erythropoietin (EPO) and its cell surface receptor (EPOR) are essential for red blood cell production and exert important cytoprotective effects on select vascular, immune, and cancer cells. To discover novel EPO action modes, we profiled the transcriptome of primary erythroid progenitors. We report Serpina3g/Spi2A as a major new EPO/EPOR target for the survival of erythroid progenitors. In knockout mice, loss of Spi2A worsened anemia caused by hemolysis, radiation, or transplantation. EPO-induced erythropoiesis also was compromised. In particular, maturing erythroblasts required Spi2A for cytoprotection, with iron and reactive oxygen species as cytotoxic agents. Spi2A defects were ameliorated by cathepsin-B/L inhibition, and by genetic co-deletion of lysosomal cathepsin B. Pharmacological inhibition of cathepsin B/L enhanced EPO-induced red cell formation in normal mice. Overall, we define an unexpected EPO action mode via an EPOR-Spi2A serpin-cathepsin axis in maturing erythroblasts, with lysosomal cathepsins as novel therapeutic targets.
Asunto(s)
Catepsinas/antagonistas & inhibidores , Eritroblastos/citología , Eritroblastos/metabolismo , Eritropoyesis/fisiología , Eritropoyetina/fisiología , Anemia/genética , Anemia/metabolismo , Animales , Catepsina B/antagonistas & inhibidores , Catepsina B/deficiencia , Catepsina B/genética , Catepsina L/antagonistas & inhibidores , Eritropoyesis/efectos de los fármacos , Lisosomas/metabolismo , Ratones , Ratones Noqueados , Receptores de Eritropoyetina/fisiología , Serpinas/deficiencia , Serpinas/genética , Serpinas/fisiología , Transducción de Señal , TranscriptomaRESUMEN
Numerous studies highlight the fact that concerted proteolysis is essential for skin morphology and function. The cysteine protease cathepsin L (Ctsl) has been implicated in epidermal proliferation and desquamation, as well as in hair cycle regulation. In stark contrast, mice deficient in cathepsin B (Ctsb) do not display an overt skin phenotype. To understand the systematic consequences of deleting Ctsb or Ctsl, we determined the protein abundances of >1300 proteins and proteolytic cleavage events in skin samples of wild-type, Ctsb(-/-), and Ctsl(-/-) mice via mass-spectrometry-based proteomics. Both protease deficiencies revealed distinct quantitative changes in proteome composition. Ctsl(-/-) skin revealed increased levels of the cysteine protease inhibitors cystatin B and cystatin M/E, increased cathepsin D, and an accumulation of the extracellular glycoprotein periostin. Immunohistochemistry located periostin predominantly in the hypodermal connective tissue of Ctsl(-/-) skin. The proteomic identification of proteolytic cleavage sites within skin proteins revealed numerous processing sites that are underrepresented in Ctsl(-/-) or Ctsb(-/-) samples. Notably, few of the affected cleavage sites shared the canonical Ctsl or Ctsb specificity, providing further evidence of a complex proteolytic network in the skin. Novel processing sites in proteins such as dermokine and Notch-1 were detected. Simultaneous analysis of acetylated protein N termini showed prototypical mammalian N-alpha acetylation. These results illustrate an influence of both Ctsb and Ctsl on the murine skin proteome and degradome, with the phenotypic consequences of the absence of either protease differing considerably.
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Catepsina B/deficiencia , Catepsina L/deficiencia , Proteoma/metabolismo , Proteómica/métodos , Piel/metabolismo , Animales , Western Blotting , Catepsina B/genética , Catepsina L/genética , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Cromatografía Liquida , Cistatina B/metabolismo , Cistatina M/metabolismo , Inmunohistoquímica , Ratones , Ratones Noqueados , Péptidos/metabolismo , Proteolisis , Receptor Notch1/metabolismo , Serpinas/metabolismo , Espectrometría de Masas en TándemRESUMEN
Ebola virus (EBOV), family Filoviridae, emerged in 1976 on the African continent. Since then it caused several outbreaks of viral hemorrhagic fever in humans with case fatality rates up to 90% and remains a serious Public Health concern and biothreat pathogen. The most pathogenic and best-studied species is Zaire ebolavirus (ZEBOV). EBOV encodes one viral surface glycoprotein (GP), which is essential for replication, a determinant of pathogenicity and an important immunogen. GP mediates viral entry through interaction with cellular surface molecules, which results in the uptake of virus particles via macropinocytosis. Later in this pathway endosomal acidification activates the cysteine proteases Cathepsin B and L (CatB, CatL), which have been shown to cleave ZEBOV-GP leading to subsequent exposure of the putative receptor-binding and fusion domain and productive infection. We studied the effect of CatB and CatL on in vitro and in vivo replication of EBOV. Similar to previous findings, our results show an effect of CatB, but not CatL, on ZEBOV entry into cultured cells. Interestingly, cell entry by other EBOV species (Bundibugyo, Côte d'Ivoire, Reston and Sudan ebolavirus) was independent of CatB or CatL as was EBOV replication in general. To investigate whether CatB and CatL have a role in vivo during infection, we utilized the mouse model for ZEBOV. Wild-type (control), catB(-/-) and catL(-/-) mice were equally susceptible to lethal challenge with mouse-adapted ZEBOV with no difference in virus replication and time to death. In conclusion, our results show that CatB and CatL activity is not required for EBOV replication. Furthermore, EBOV glycoprotein cleavage seems to be mediated by an array of proteases making targeted therapeutic approaches difficult.
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Catepsina B/metabolismo , Catepsina L/metabolismo , Ebolavirus/fisiología , Fiebre Hemorrágica Ebola/virología , Interacciones Huésped-Patógeno , Replicación Viral , Animales , Catepsina B/deficiencia , Catepsina L/deficiencia , Línea Celular , Chlorocebus aethiops , Modelos Animales de Enfermedad , Ebolavirus/patogenicidad , Fiebre Hemorrágica Ebola/mortalidad , Fiebre Hemorrágica Ebola/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Supervivencia , Internalización del VirusRESUMEN
Interleukin (IL)-1ß and IL-18 play critical roles in the induction of chronic pain hypersensitivity. Their inactive forms are activated by caspase-1. However, little is known about the mechanism underlying the activation of pro-caspase-1. There is increasing evidence that cathepsin B (CatB), a typical lysosomal cysteine protease, is involved in the pro-caspase-1 activation and the subsequent maturation of IL-1ß and IL-18. In this context, CatB is considered to be an important molecular target to control chronic pain. However, no information is currently available about the role of CatB in chronic pain hypersensitivity. We herein show that CatB deficiency or the intrathecal administration of CA-074Me, a specific CatB inhibitor, significantly inhibited the induction of complete Freund's adjuvant-induced tactile allodynia in mice without affecting peripheral inflammation. In contrast, CatB deficiency did not affect the nerve injury-induced tactile allodynia. Furthermore, CatB deficiency or CA-074Me treatment significantly inhibited the maturation and secretion of IL-1ß and IL-18 by cultured microglia following treatment with the neuroactive glycoprotein chromogranin A (CGA), but not with ATP. Moreover, the IL-1ß expression in spinal microglia and the induction of tactile allodynia following the intrathecal administration of CGA depended on CatB, whereas those induced by the intrathecal administration of ATP or lysophosphatidic acid were CatB independent. These results strongly suggest that CatB is an essential enzyme for the induction of chronic inflammatory pain through its activation of pro-caspase-1, which subsequently induces the maturation and secretion of IL-1ß and IL-18 by spinal microglia. Therefore, CatB-specific inhibitors may represent a useful new strategy for treating inflammation-associated pain.
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Catepsina B/metabolismo , Dolor Crónico/etiología , Dolor Crónico/patología , Inflamación/complicaciones , Microglía/metabolismo , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Análisis de Varianza , Animales , Antígeno CD11b/metabolismo , Antígenos CD4/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Catepsina B/deficiencia , Células Cultivadas , Cromogranina A/administración & dosificación , Dolor Crónico/tratamiento farmacológico , Dolor Crónico/genética , Ciclooxigenasa 2/metabolismo , Dipéptidos/administración & dosificación , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/administración & dosificación , Adyuvante de Freund/toxicidad , Lateralidad Funcional , Ganglios Espinales/patología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/fisiopatología , Inflamación/inducido químicamente , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Lisofosfolípidos/toxicidad , Lisosomas/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/metabolismo , Microglía/patología , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Proteína con Dominio Pirina 3 de la Familia NLR , Proteínas del Tejido Nervioso/metabolismo , Umbral del Dolor/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Médula Espinal/patología , TransfecciónRESUMEN
Therapeutic agents that improve the memory loss of Alzheimer's disease (AD) may eventually be developed if drug targets are identified that improve memory deficits in appropriate AD animal models. One such target is ß-secretase which, in most AD patients, cleaves the wild-type (WT) ß-secretase site sequence of the amyloid-ß protein precursor (AßPP) to produce neurotoxic amyloid-ß (Aß). Thus, an animal model representing most AD patients for evaluating ß-secretase effects on memory deficits is one that expresses human AßPP containing the WT ß-secretase site sequence. BACE1 and cathepsin B (CatB) proteases have ß-secretase activity, but gene knockout studies have not yet validated that the absence of these proteases improves memory deficits in such an animal model. This study assessed the effects of deleting these protease genes on memory deficits in the AD mouse model expressing human AßPP containing the WT ß-secretase site sequence and the London γ-secretase site (AßPPWT/Lon mice). Knockout of the CatB gene in the AßPPWT/Lon mice improved memory deficits and altered the pattern of Aß-related biomarkers in a manner consistent with CatB having WT ß-secretase activity. But deletion of the BACE1 gene had no effect on these parameters in the AßPPWT/Lon mice. These data are the first to show that knockout of a putative ß-secretase gene results in improved memory in an AD animal model expressing the WT ß-secretase site sequence of AßPP, present in the majority of AD patients. CatB may be an effective drug target for improving memory deficits in most AD patients.