RESUMO
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.
Assuntos
Catepsina B/fisiologia , Catepsina Z/fisiologia , Corioide/irrigação sanguínea , Neovascularização de Coroide/enzimologia , Modelos Animais de Doenças , Fotocoagulação a Laser , Animais , Catepsina B/antagonistas & inibidores , Catepsina Z/antagonistas & inibidores , Neovascularização de Coroide/patologia , Inibidores Enzimáticos/farmacologia , Matriz Extracelular/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Lasers de Gás , Inibidores de Metaloproteinases de Matriz/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esferoides Celulares , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Autodigestion of the pancreas by its own prematurely activated digestive proteases is thought to be an important event in the onset of acute pancreatitis. The mechanism responsible for the intrapancreatic activation of digestive zymogens is unknown, but a recent hypothesis predicts that a redistribution of lysosomal cathepsin B (CTSB) into a zymogen-containing subcellular compartment triggers this event. To test this hypothesis, we used CTSB-deficient mice in which the ctsb gene had been deleted by targeted disruption. After induction of experimental secretagogue-induced pancreatitis, the trypsin activity in the pancreas of ctsb(-/-) animals was more than 80% lower than in ctsb(+/+) animals. Pancreatic damage as indicated by serum activities of amylase and lipase, or by the extent of acinar tissue necrosis, was 50% lower in ctsb(-/-) animals. These experiments provide the first conclusive evidence to our knowledge that cathepsin B plays a role in intrapancreatic trypsinogen activation and the onset of acute pancreatitis.
Assuntos
Catepsina B/metabolismo , Pâncreas/enzimologia , Pâncreas/patologia , Pancreatite/enzimologia , Tripsinogênio/metabolismo , Doença Aguda , Amilases/sangue , Animais , Apoptose/efeitos dos fármacos , Catepsina B/deficiência , Catepsina B/genética , Ceruletídeo/farmacologia , Modelos Animais de Doenças , Edema/patologia , Ativação Enzimática , Deleção de Genes , Marcação de Genes , Humanos , Lipase/sangue , Camundongos , Camundongos Knockout , Necrose , Pâncreas/efeitos dos fármacos , Pancreatite/induzido quimicamente , Pancreatite/etiologia , FenótipoRESUMO
The impairment of the complexes of the respiratory chain was studied in isolated rat liver mitochondria under the conditions of an iron/ascorbate-mediated oxidative stress. Using blue native electrophoresis technique the NADH-ubiquinone oxidoreductase, ubiquinol-cytochrome-c oxidoreductase, cytochrome oxidase and ATP-synthetase were separated from mitochondrial samples at different stages of peroxidation and quantified by densitometry. In the second dimension the protein complexes were separated into their individual subunits by Tricine/SDS-electrophoresis. In relation to the time course of lipid peroxidation protein losses were moderate in the exponential phase and enhanced towards plateau phase of TBARS formation, when the intensity of staining for the native complexes became reduced by 84%, 69%, 63% and 24% for complexes I, III, V and IV, respectively, and a high molecular aggregation band as a putative marker of oxidative stress was formed. The decline of overall staining by 23%, a decrease in trichloroacetic acid precipitable protein and the formation of acid soluble primary amines suggest the occurrence of fragmentation or degradation processes. Apparently, the impairment of the respiratory chain complexes during peroxidation was not reflected in altered electrophoretic mobilities or specific losses of protein subunits of these innermitochondrial membrane components.
Assuntos
Transporte de Elétrons/fisiologia , Mitocôndrias Hepáticas/enzimologia , Complexos Multienzimáticos/metabolismo , Estresse Oxidativo/fisiologia , Oxirredutases/metabolismo , Animais , Ácido Ascórbico/farmacologia , Eletroforese em Gel Bidimensional , Compostos de Ferro/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Fosforilação Oxidativa , Estresse Oxidativo/efeitos dos fármacos , ATPases Translocadoras de Prótons/metabolismo , Ratos , Substâncias Reativas com Ácido Tiobarbitúrico/análiseRESUMO
Glucose-6-phosphate dehydrogenase (G6PD) was treated with various concentrations of hypochlorite, which is produced by myeloperoxidase and is one of the most important oxidants during inflammatory processes. Inhibition of enzymatic activity, protein fragmentation, and proteolytic susceptibility toward the isolated 20S proteasome of G6PD were investigated. With rising hypochlorite concentrations, an increased proteasomal degradation of G6PD was measured. This occurred at higher hypochlorite concentrations than G6PD inactivation and at lower levels than G6PD fragmentation. The proteolytic activities of the 20S proteasome itself was determined by degradation of oxidized model proteins and cleavage of the synthetic proteasome substrate suc-LLVY-MCA. Proteasome activities remained intact at hypochlorite concentrations in which G6PD is maximally susceptible to proteasomal degradation. Only higher hypochlorite concentrations could decrease the proteolytic activities of the proteasome, which was accompanied by disintegration and fragmentation of the proteasome and proteasome subunits. Therefore, we conclude that the 20S proteasome can degrade proteins moderately damaged by hypochlorite and could contribute to an increased protein turnover in cells exposed to inflammatory stress.
Assuntos
Cisteína Endopeptidases/metabolismo , Inibidores Enzimáticos/farmacologia , Glucosefosfato Desidrogenase/metabolismo , Ácido Hipocloroso/farmacologia , Complexos Multienzimáticos/metabolismo , Cumarínicos/metabolismo , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Eritrócitos/enzimologia , Glucosefosfato Desidrogenase/química , Humanos , Oligopeptídeos/metabolismo , Complexo de Endopeptidases do ProteassomaRESUMO
Free radical-mediated injury is believed to play a key role in the pathogenesis of acute pancreatitis (AP). Therefore, oxidative damage of proteins may be an important event in the development of AP. The present study was performed to investigate oxidative protein modification, quantified as 2,4-dinitrophenylhydrazine-reactive protein-carbonyls, during the time course of taurocholate-induced pancreatitis of the rat and to analyze oxidatively modified proteins by Western blotting. Protein modification in pancreatic homogenates was found as early as 30 min after induction of severe AP with 3% taurocholate preceding the elevation of serum amylase activity and the increase of malondialdehyde in the tissue. A correlation of protein-carbonyl contents to a score of pancreatic macroscopic alterations (r = .69) and to the wet weight/dry weight ratio (r = .65) was found. Infusion of 5% taurocholate resulted in fulminant AP with high lethality during the 24 h of the experiment. However, rats surviving showed significantly lower level of protein-carbonyls than animals that died between 20-24 h after AP induction. The quantitative data were confirmed by the intensity of immunostained protein-carbonyls. The present data show a rather uniform increase in the staining pattern not revealing single, selectively damaged proteins. The aldehydic product of lipid peroxidation 4-hydroxynonenal (HNE) is known for its reactivity towards proteins. Interestingly, an antibody raised against protein-bound HNE did not indicate an increased protein modification by this aldehyde. In conclusion, experimental AP is characterized by an early oxidative protein modification, possibly contributing to functional impairment of the pancreas. This protein alteration may not be mediated by HNE.
Assuntos
Pancreatite/induzido quimicamente , Proteínas/química , Doença Aguda , Aldeídos/química , Amilases/sangue , Animais , Western Blotting , Cinética , Peroxidação de Lipídeos , Masculino , Malondialdeído/metabolismo , Oxirredução , Pancreatite/metabolismo , Fenil-Hidrazinas/química , Proteínas/análise , Ratos , Ratos Wistar , Ácido TaurocólicoRESUMO
The NMDA-type glutamate receptor agonist quinolinic acid (QA), which causes tissue lesions in the rat brain as well as cell loss in neuronal cultures, is widely used in models of glutamate excitotoxicity. The aim of this study was to evaluate the alterations in gene expression in a primary hippocampal cell culture after exposure to QA. By means of differential mRNA display, we were able to pinpoint as many as 23 bands which appeared to be upregulated after a 6-h treatment with quinolinic acid. The differential expression of 13 cDNAs could be confirmed by dot blot and/or Northern analysis. Of the cDNAs, the p112 regulatory subunit of the 26S proteasome, a PDGF-associated protein and the glia-derived protease nexin PN-1 could be identified. The results provide emphasis to the participation of proteolysis and protease inhibition in neurodegenerative processes.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/metabolismo , Neurônios/metabolismo , Neurotoxinas/toxicidade , Complexo de Endopeptidases do Proteassoma , Ácido Quinolínico/toxicidade , Transcrição Gênica/efeitos dos fármacos , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Células Cultivadas , Feto , Ácido Glutâmico/toxicidade , Hipocampo/citologia , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/efeitos dos fármacos , Peptídeo Hidrolases/genética , Fator de Crescimento Derivado de Plaquetas/genética , Nexinas de Proteases , RNA Mensageiro/biossíntese , Ratos , Receptores de Superfície Celular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Dedos de ZincoRESUMO
Oxidative stress is known to cause oxidative protein modification and the generation of reactive aldehydes derived from lipid peroxidation. Extent and kinetics of both processes were investigated during oxidative damage of isolated rat liver mitochondria treated with iron/ascorbate. The monofunctional aldehydes 4-hydroxynonenal (4-HNE), n-hexanal, n-pentanal, n-nonanal, n-heptanal, 2-octenal, 4-hydroxydecenal as well as thiobarbituric acid reactive substances (TBARS) were detected. The kinetics of aldehyde generation showed a lag-phase preceding an exponential increase. In contrast, oxidative protein modification, assessed as 2,4-dinitrophenylhydrazine (DNPH) reactive protein-bound carbonyls, continuously increased without detectable lag-phase. Western blot analysis confirmed these findings but did not allow the identification of individual proteins preferentially oxidized. Protein modification by 4-HNE, determined by immunoblotting, was in parallel to the formation of this aldehyde determined by HPLC. These results suggest that protein oxidation occurs during the time of functional decline of mitochondria, i.e. in the lag-phase of lipid peroxidation. This protein modification seems not to be caused by 4-HNE.
Assuntos
Aldeídos/metabolismo , Peroxidação de Lipídeos , Mitocôndrias Hepáticas/metabolismo , Estresse Oxidativo , Proteínas/metabolismo , Aldeídos/química , Aldeídos/imunologia , Animais , Anticorpos , Western Blotting , Cromatografia , Ácidos Graxos/metabolismo , Glutationa/metabolismo , Cinética , Fenil-Hidrazinas/imunologia , Fenil-Hidrazinas/metabolismo , Proteínas/química , Ratos , Ratos Wistar , Espectrofotometria , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
The combination of UVA and 8-methoxypsoralen (8-MOP) is known for the ability to produce reactive oxygen species (ROS) that react subsequently with DNA, lipids and proteins. In most studies concerned with UVA effects mediated by free radicals, UVA doses higher than those exhibiting beneficial clinical results in extracorporeal photoimmunotherapy (ECPI) were used. The present study was undertaken to determine markers of oxidative stress in plasma and cells from the buffy coat using conditions relevant for ECPI (cumulative UVA dose at the sample level < or = 2 J/cm2). Plasma exposed to UVA of 20 J/cm2 resulted in protein oxidation as well in crosslinking and fragmentation revealed by electrophoresis. Exposure of the buffy coat and plasma to considerably lower doses of UVA (up to 2 J/cm2) combined with various 8-MOP concentrations resulted neither in an increase of malondialdehyde as a marker of lipid peroxidation nor in a changed electrophoretic protein pattern. In these same experiments the total antioxidative capacity decreased to 65% of the initial value, suggesting that the antioxidative defense of plasma is able to cope with oxidative stress under ECPI conditions. These results were confirmed by data from 10 patients with scleroderma or cutaneous T-cell lymphoma during ECPI treatment. The present results suggest that, although ROS are formed during ECPI, gross oxidative damage does not occur. It is, however, possible, that specific effects mediated by oxygen radicals may co-trigger the photoimmunomodulatory effects of ECPI.
Assuntos
Proteínas Sanguíneas/efeitos da radiação , Imunoterapia/métodos , Lipídeos/efeitos da radiação , Estresse Oxidativo , Fotoquimioterapia/métodos , Raios Ultravioleta , Relação Dose-Resposta à Radiação , Estudos de Avaliação como Assunto , Humanos , Peroxidação de Lipídeos/efeitos da radiação , Linfoma de Células T/sangue , Linfoma de Células T/terapia , Escleroderma Sistêmico/sangue , Escleroderma Sistêmico/terapiaRESUMO
The phosphatidylinositol 3-kinase (PI3K) regulatory subunits p55α and p50α are coordinately transcriptionally upregulated by signal transducer and activator of transcription 3 (Stat3) at the onset of mammary gland involution, a process that requires Stat3. Deletion of both p55α and p50α subunits in vivo abrogated mammary epithelial cell death during involution. This was associated also with reduced cytosolic levels and activity of the cysteine protease cathepsin L, which is implicated in lysosomal-mediated programmed cell death (LM-PCD) and is upregulated in involution. Furthermore, involution is delayed in cathepsin L-deficient mice suggesting that the p55α/p50α subunits mediate cell death in part by elevating the level of cathepsin L resulting in increased cytosolic activity. Surprisingly, we found that p55α/p50α localize to the nucleus where they bind to chromatin and regulate transcription of a subset of inflammatory/acute phase genes that are also Stat3 targets. Our findings reveal a novel role for these PI3K regulatory subunits as regulators of LM-PCD in vivo.
Assuntos
Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Animais , Morte Celular/genética , Feminino , Masculino , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/deficiência , Fosfatidilinositol 3-Quinases/genética , Subunidades Proteicas/deficiência , Subunidades Proteicas/genéticaRESUMO
Lysosomal cysteine cathepsins contribute to proteolytic events promoting tumor growth and metastasis. Their enzymatic activity, however, is tightly regulated by endogenous inhibitors. To investigate the role of cathepsin inhibitor stefin B (Stfb) in mammary cancer, Stfb null mice were crossed with transgenic polyoma virus middle T oncogene (PyMT) breast cancer mice. We show that ablation of Stfb resulted in reduced size of mammary tumors but did not affect their rate of metastasis. Importantly, decrease in tumor growth was correlated with an increased incidence of dead cell islands detected in tumors of Stfb-deficient mice. Ex vivo analysis of primary PyMT tumor cells revealed no significant effects of ablation of Stfb expression on proliferation, angiogenesis, migration and spontaneous cell death as compared with control cells. However, upon treatment with the lysosomotropic agent Leu-Leu-OMe, cancer cells lacking Stfb exhibited a significantly higher sensitivity to apoptosis. Moreover, Stfb-ablated tumor cells were significantly more prone to cell death under increased oxidative stress. These results indicate an in vivo role for Stfb in protecting cancer cells by promoting their resistance to oxidative stress and to apoptosis induced through the lysosomal pathway.
Assuntos
Apoptose/genética , Neoplasias da Mama/patologia , Cistatina B/genética , Neoplasias Mamárias Experimentais/patologia , Estresse Oxidativo/genética , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Catepsinas/antagonistas & inibidores , Movimento Celular/genética , Proliferação de Células , Inibidores de Cisteína Proteinase/genética , Dipeptídeos/farmacologia , Progressão da Doença , Feminino , Imunossupressores/farmacologia , Lisossomos/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Knockout , Metástase Neoplásica/genética , Neovascularização Patológica/genéticaRESUMO
Signal transducer and activator of transcription 3 (STAT3) is altered in several epithelial cancers and represents a potential therapeutic target. Here, STAT3 expression, activity and cellular functions were examined in two main histotypes of esophageal carcinomas. In situ, immunohistochemistry for STAT3 and STAT3-Tyr705 phosphorylation (P-STAT3) in esophageal squamous cell carcinomas (ESCC, n=49) and Barrett's adenocarcinomas (BAC, n=61) revealed similar STAT3 expression in ESCCs and BACs (P=0.109), but preferentially activated P-STAT3 in ESCCs (P=0.013). In vitro, strong STAT3 activation was seen by epidermal growth factor (EGF) stimulation in OE21 (ESCC) cells, whereas OE33 (BAC) cells showed constitutive weak STAT3 activation. STAT3 knockdown significantly reduced cell proliferation of OE21 (P=0.0148) and OE33 (P=0.0243) cells. Importantly, STAT3 knockdown reduced cell migration of OE33 cells by 2.5-fold in two types of migration assays (P=0.073, P=0.015), but not in OE21 cells (P=0.1079, P=0.386). Investigation of transcriptome analysis of STAT3 knockdown revealed a reduced STAT3 level associated with significant downregulation of cell cycle genes in both OE21 (P<0.0001) and OE33 (P=0.01) cells. In contrast, genes promoting cell migration (CTHRC1) were markedly upregulated in OE21 cells, whereas a gene linked to tight-junction stabilization and restricted cell motility (SHROOM2) was downregulated in OE21 but upregulated in OE33 cells. This study shows frequent, but distinct, patterns of STAT3 expression and activation in ESCCs and BACs. STAT3 knockdown reduces cell proliferation in ESCC and BAC cells, inhibits migration of BAC cells and may support cell migration of ESCC cells. Thereby, novel STAT3-regulated genes involved in ESCC and BAC cell proliferation and cell migration were identified. Thus, STAT3 may be further exploited as a potential novel therapeutic target, however, by careful distinction between the two histotypes of esophageal cancers.
Assuntos
Adenocarcinoma/metabolismo , Esôfago de Barrett/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/biossíntese , Adenocarcinoma/genética , Adenocarcinoma/patologia , Esôfago de Barrett/genética , Esôfago de Barrett/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Ciclo Celular/genética , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação para Baixo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Técnicas de Silenciamento de Genes , Humanos , Fosforilação , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
The cysteine protease cathepsin B (CTSB) is frequently overexpressed in human breast cancer and correlated with a poor prognosis. Genetic deficiency or pharmacological inhibition of CTSB attenuates tumor growth, invasion and metastasis in mouse models of human cancers. CTSB is expressed in both cancer cells and cells of the tumor stroma, in particular in tumor-associated macrophages (TAM). In order to evaluate the impact of tumor- or stromal cell-derived CTSB on Polyoma Middle T (PyMT)-induced breast cancer progression, we used in vivo and in vitro approaches to induce human CTSB overexpression in PyMT cancer cells or stromal cells alone or in combination. Orthotopic transplantation experiments revealed that CTSB overexpression in cancer cells rather than in the stroma affects PyMT tumor progression. In 3D cultures, primary PyMT tumor cells showed higher extracellular matrix proteolysis and enhanced collective cell invasion when CTSB was overexpressed and proteolytically active. Coculture of PyMT cells with bone marrow-derived macrophages induced a TAM-like macrophage phenotype in vitro, and the presence of such M2-polarized macrophages in 3D cultures enhanced sprouting of tumor spheroids. We employed a doxycycline (DOX)-inducible CTSB expression system to selectively overexpress human CTSB either in cancer cells or in macrophages in 3D cocultures. Tumor spheroid invasiveness was only enhanced when CTSB was overexpressed in cancer cells, whereas CTSB expression in macrophages alone did not further promote invasiveness of tumor spheroids. We conclude that CTSB overexpression in the PyMT mouse model promotes tumor progression not by a stromal effect, but by a direct, cancer cell-inherent mode of action: CTSB overexpression renders the PyMT cancers more invasive by increasing proteolytic extracellular matrix protein degradation fostering collective cell invasion into adjacent tissue.
Assuntos
Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Catepsina B/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Macrófagos/metabolismo , Células Estromais/transplante , Animais , Antígenos Transformantes de Poliomavirus/genética , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Catepsina B/genética , Progressão da Doença , Doxiciclina/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos TransgênicosRESUMO
Elevated expression of the cysteine protease cathepsin B (CTSB) has been correlated with a poor prognosis for cancer patients. In order to model high CTSB expression in mammary cancer, transgenic mice expressing human CTSB were crossed with transgenic polyoma virus middle T oncogene breast cancer mice (mouse mammary tumor virus-PymT), resulting in a 20-fold increase in cathepsin B activity in the tumors of double-transgenic animals. CTSB expression did not affect tumor onset, but CTSB transgenic mice showed accelerated tumor growth with significant increase in weight for end-stage tumors, as well as an overall worsening in their histopathological grades. Notably, the lung metastases in the CTSB transgenic animals were found to be both significantly larger and to occur at a significantly higher frequency. Ex vivo analysis of primary PymT tumor cells revealed no significant effects from elevated CTSB levels on tumor cell characteristics, that is, the formation of tumor cell colonies and the sprouting of invasive strands from PymT cell spheroids. However, tumors from CTSB-overexpressing mice showed increased numbers of tumor-associated B cells and mast cells. In addition, more CD31+ endothelial cells were detected in these tumors, correlating with higher levels of vascular endothelial growth factor (VEGF) being present in the tumor and serum. We conclude that elevated proteolytic CTSB activity facilitates progression and metastasis of PymT-induced mammary carcinomas, and is associated with increased immune cell infiltration, enhanced VEGF levels and the promotion of tumor angiogenesis.
Assuntos
Catepsina B/genética , Neoplasias Mamárias Experimentais/enzimologia , Animais , Antígenos Transformantes de Poliomavirus/genética , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Catepsina B/biossíntese , Catepsina B/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Neovascularização Patológica/enzimologia , Neovascularização Patológica/patologiaRESUMO
Apoptotic stimuli have been shown to trigger lysosomal membrane permeability (LMP), leading to the release of cathepsins, which activate death signaling pathways in the cytosol. However, it is unknown whether this process is an initiating or amplifying event in apoptosis. In this study, we used fibroblasts and monocytes exposed to etoposide, ultraviolet light, FasL or deprived of interleukin-3 (IL-3) to show that LMP and the cytosolic release of cathepsins B, L and D consistently depends on Bax/Bak and components of the apoptosome. Neither Bax nor Bak resided on the lysosomes, indicating that lysosomes were not directly perforated by Bax/Bak but by effectors downstream of the apoptosome. Detailed kinetic analysis of cells lacking cathepsin B or L or treated with the cysteine protease inhibitor, E64d, revealed a delay in these cells in etoposide- and IL-3 deprivation-induced caspase-3 activation and apoptosis induction but not clonogenic survival, indicating that cathepsins amplify rather than initiate apoptosis.
Assuntos
Apoptose , Catepsinas/metabolismo , Lisossomos/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptossomas/metabolismo , Caspase 3/metabolismo , Catepsinas/genética , Permeabilidade da Membrana Celular , Inibidores de Cisteína Proteinase/farmacologia , Etoposídeo/farmacologia , Proteína Ligante Fas/farmacologia , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Interleucina-3/genética , Interleucina-3/metabolismo , Leucina/análogos & derivados , Leucina/farmacologia , Camundongos , Monócitos/metabolismo , Raios UltravioletaRESUMO
To define a functional role for the endosomal/lysosomal cysteine protease cathepsin L (Ctsl) during squamous carcinogenesis, we generated mice harboring a constitutive Ctsl deficiency in addition to epithelial expression of the human papillomavirus type 16 oncogenes (human cytokeratin 14 (K14)-HPV16). We found enhanced tumor progression and metastasis in the absence of Ctsl. As tumor progression in K14-HPV16 mice is dependent on inflammation and angiogenesis, we examined immune cell infiltration and vascularization without finding any effect of the Ctsl genotype. In contrast, keratinocyte-specific transgenic expression of cathepsin V, the human orthologue of mouse Ctsl, in otherwise Ctsl-deficient K14-HPV16 mice restored the phenotype observed in the control HPV16 skin. To better understand this phenotype at the molecular level, we measured several oncogenic signal transduction pathways in primary keratinocytes on stimulation with keratinocyte-conditioned cell culture medium. We found increased activation of protein kinase B/Akt and mitogen-activated protein kinase pathways in protease-deficient cells, especially if treated with media conditioned by Ctsl-deficient keratinocytes. Similarly, the level of active GTP-Ras was increased in Ctsl-deficient epidermis. We conclude that Ctsl is critical for the termination of growth factor signaling in the endosomal/lysosomal compartment of keratinocytes and, therefore, functions as an anti-tumor protease.
Assuntos
Carcinoma de Células Escamosas/patologia , Catepsina L/deficiência , Epitélio/patologia , Neoplasias Cutâneas/patologia , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Catepsina L/genética , Células Cultivadas , Progressão da Doença , Epitélio/metabolismo , Feminino , Papillomavirus Humano 16/genética , Humanos , Imuno-Histoquímica , Queratina-14/genética , Queratinócitos/metabolismo , Queratinócitos/patologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Transdução de Sinais , Pele/metabolismo , Pele/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Fatores de TempoRESUMO
Expression levels of the papain-like cysteine protease cathepsin B (Ctsb) have been positively correlated with mammary tumour progression and metastasis; however, its roles in the hallmark processes of malignant growth remain poorly defined. Using Ctsb-deficient mice we investigated tumour cell differentiation, proliferation and apoptosis in the Tg(MMTV-PyMT) mouse mammary cancer model. Absence of Ctsb significantly impaired development of high-grade invasive ductal carcinomas and reduced the metastatic burden in the lungs. Mice lacking Ctsb exhibited reduced cell proliferation in mammary carcinomas and their lung metastases. Notably, intravenous injection of primarily isolated, Ctsb-expressing tumour cells into congenic Ctsb-deficient mice revealed impaired cell proliferation in the resulting experimental lung metastases, providing evidence for the involvement of Ctsb in paracrine regulation of cancer cell proliferation. No Ctsb genotype-dependent difference in tumour cell death was observed in vivo or by treatment of isolated PyMT cancer cells with tumour necrosis factor-alpha. However, cancer cells lacking Ctsb exhibited significantly higher resistance to apoptosis induction by the lysosomotropic agent Leu-Leu-OMe. Thus, our results indicate an in vivo role for Ctsb in promoting cellular anaplasia in mammary cancers and proliferation in lung metastases.
Assuntos
Carcinoma/genética , Catepsina B/genética , Proliferação de Células , Imunidade Inata/genética , Neoplasias Mamárias Animais/genética , Carga Tumoral/genética , Animais , Carcinoma/patologia , Morte Celular/genética , Progressão da Doença , Feminino , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Knockout , Transplante de Neoplasias , Fatores de Tempo , Células Tumorais CultivadasRESUMO
In cerebral amyloidoses, such as Alzheimer's disease, proteolytic processing of the precursor protein is a fundamental mechanism of the disease, since it generates the amyloid protein. However, the putative significance of proteases in extracerebral amyloidoses is less well defined. In this study, we investigated the biological significance of cathepsin (Cath) B, CathK, and CathL in the pathology and pathogenesis of extracerebral amyloidoses by using the murine model of reactive or secondary AA amyloidosis with three different cathepsin-deficient mouse strains. Extracerebral AA amyloid was induced by injecting amyloid-enhancing factor and silver nitrate into CathB(-/-), CathK(-/-), and CathL(-/-) mice. Wild-type mice served as a control. CathK(-/-) mice deposited over 90% more amyloid and CathL(-/-) mice 60% less amyloid than the control (p < 0.0001). The amyloid load in CathB(-/-) mice did not differ from that in wild-type mice. In vitro degradation experiments with recombinant human and murine serum amyloid A (SAA) 1.1 and CathK and CathL showed that CathL generates a large number of differently sized SAA cleavage products. One of these fragments spans the heparin/heparan sulphate binding site and the neutral cholesterol ester hydrolase activating region of SAA. CathK showed only endoproteolytic activity and did not generate any AA amyloid-like peptides. This study provides unequivocal evidence that proteases modulate amyloid load in extracerebral amyloidosis. CathL was identified as an amyloid-promoting and CathK as an amyloid-retarding cysteine protease. CathB may only modulate the primary structure of the amyloid peptide without affecting amyloid load.
Assuntos
Amiloidose/metabolismo , Catepsinas/metabolismo , Cisteína Endopeptidases/metabolismo , Reação de Fase Aguda/metabolismo , Animais , Catepsina B/metabolismo , Catepsina K , Catepsina L , Feminino , Imuno-Histoquímica/métodos , Camundongos , Camundongos Endogâmicos , Monócitos/metabolismo , Desnaturação Proteica , Proteínas Recombinantes/metabolismo , Proteína Amiloide A Sérica/metabolismo , Baço/metabolismoRESUMO
Bone resorption by osteoclasts depends on the activity of various proteolytic enzymes, in particular those belonging to the group of cysteine proteinases. Next to these enzymes, tartrate-resistant acid phosphatase (TRAP) is considered to participate in this process. TRAP is synthesized as an inactive proenzyme, and in vitro studies have shown its activation by cysteine proteinases. In the present study, the possible involvement of the latter enzyme class in the in vivo modulation of TRAP was investigated using mice deficient for cathepsin K and/or L and in bones that express a high (long bone) or low (calvaria) level of cysteine proteinase activity. The results demonstrated, in mice lacking cathepsin K but not in those deficient for cathepsin L, significantly higher levels of TRAP activity in long bone. This higher activity was due to a higher number of osteoclasts. Next, we found considerable differences in TRAP activity between calvarial and long bones. Calvarial bones contained a 25-fold higher level of activity than long bones. This difference was seen in all mice, irrespective of genotype. Osteoclasts isolated from the two types of bone revealed that calvarial osteoclasts expressed higher enzyme activity as well as a higher level of mRNA for the enzyme. Analysis of TRAP-deficient mice revealed higher levels of nondigested bone matrix components in and around calvarial osteoclasts than in long bone osteoclasts. Finally, inhibition of cysteine proteinase activity by specific inhibitors resulted in increased TRAP activity. Our data suggest that neither cathepsin K nor L is essential in activating TRAP. The findings also point to functional differences between osteoclasts from different bone sites in terms of participation of TRAP in degradation of bone matrix. We propose that the higher level of TRAP activity in calvarial osteoclasts compared to that in long bone cells may partially compensate for the lower cysteine proteinase activity found in calvarial osteoclasts and TRAP may contribute to the degradation of noncollagenous proteins during the digestion of this type of bone.
Assuntos
Fosfatase Ácida/biossíntese , Ossos do Braço/enzimologia , Isoenzimas/biossíntese , Ossos da Perna/enzimologia , Osteoclastos/enzimologia , Crânio/metabolismo , Fosfatase Ácida/deficiência , Fosfatase Ácida/genética , Animais , Catepsina K , Catepsina L , Catepsinas/metabolismo , Cisteína Endopeptidases/metabolismo , Ativação Enzimática , Isoenzimas/deficiência , Isoenzimas/genética , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fosfatase Ácida Resistente a TartaratoRESUMO
Exposure to various forms of mild oxidative stress significantly increased the intracellular degradation of both "short-lived" and "long-lived," metabolically radiolabeled, cell proteins in cultures of Clone 9 liver cells (normal liver epithelia). The oxidative stresses employed were bolus H2O2 addition; continuous H2O2 flux; the redox cycling quinones, menadione and paraquat; and the aldehydic products of lipid peroxidation, 4-hydroxynonenal, malonyldialdehyde, and hexenal. In general, exposure to more severe oxidative stress produced a concentration-dependent decline in intracellular proteolysis, in some cases to below baseline levels. Oxidatively modified "foreign" proteins (superoxide dismutase and hemoglobin) were also selectively degraded, in comparison with untreated foreign proteins, when added to lysates of Clone 9 liver cells. As with intracellular proteolysis, the degradation of foreign proteins added to cell lysates was greatly increased by mild oxidative modification, but depressed by more severe oxidative modification. The proteinase activity was recovered in > 300-kDa cell fractions, and inhibitor profiles and immunoprecipitation studies indicated that the multicatalytic proteinase complex, proteasome, was responsible for most of the selective degradation observed with mild oxidative stress; up to approximately 95% for intracellular proteolysis and 65-80% for degradation of foreign modified proteins. Seven days of daily treatment with an antisense oligodeoxynucleotide, directed against the initiation codon region of the proteasome C2 subunit gene, severely depressed the intracellular levels of several proteasome subunit polypeptides (by Western blot analysis), and also depressed the H2O2 induced increase in intracellular proteolysis by approximately 95%, without significantly affecting baseline proteolytic rates. Extensive studies revealed only small or no increases in the overall capacity of oxidatively stressed cells to degrade oxidatively modified protein substrates; a finding supported by both Western blot and Northern blot analyses which revealed no significant increase in the levels of proteasome subunit polypeptides or mRNA transcripts. We conclude that mild oxidative stress increases intracellular proteolysis by modifying cellular proteins, thus increasing their proteolytic susceptibility. In contrast, severe oxidative stress diminishes intracellular proteolysis, probably by generating severely damaged cell proteins that cannot be easily degraded (e.g. cross-linked/aggregated proteins), and by damaging proteolytic enzymes. We further conclude that the multicatalytic proteinase complex proteasome is responsible for most of the recognition and selective degradation of oxidatively modified proteins in Clone 9 liver cells.