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1.
J Immunol ; 199(1): 172-185, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28550201

RESUMO

The invariant chain (CD74) mediates assembly and targeting of MHC class II (MHCII) complexes. In endosomes, CD74 undergoes sequential degradation by different proteases, including cathepsin S (CatS) and the intramembrane protease signal peptide peptidase-like 2a (SPPL2a). In their absence, CD74 N-terminal fragments (NTFs) accumulate. In SPPL2a-/- B cells, such an NTF impairs endosomal trafficking and BCR signal transduction. In mice, this leads to a loss of splenic B cells beyond the transitional stage 1. To gain insight into CD74 determinants and the role of MHCII, we compared B cells from CatS-/- , SPPL2a-/- , and SPPL2a-MHCII double-deficient mice. We assessed differentiation of B cells in bone marrow and spleen and analyzed their endosomal morphology, BCR expression, and signal transduction. We demonstrate that MHCII is dispensable for the B cell phenotype of SPPL2a-/- mice, further supporting a CD74-intrinsic effect. Despite significant vacuolization of endosomal compartments similar to SPPL2a-/- B cells, CatS-/- traditional stage 1 B cells show unimpaired degradation of endocytic cargo, have intact BCR signaling, and do not exhibit any relevant defects in maturation. This could indicate that CD74 NTF-induced structural changes of endosomes are not directly involved in these processes. We further found that the block of CD74 degradation in CatS-/- B cells is incomplete, so that NTF levels are significantly lower than in SPPL2a-/- B cells. This suggests a dose dependency and threshold for the CD74 NTF-associated impairment of B cell signaling and maturation. In addition, different functional properties of the longer, MHCII-bound CD74 NTF could contribute to the milder phenotype of CatS-/- B cells.


Assuntos
Antígenos de Diferenciação de Linfócitos B/imunologia , Linfócitos B/imunologia , Genes MHC da Classe II , Antígenos de Histocompatibilidade Classe II/imunologia , Animais , Antígenos de Diferenciação de Linfócitos B/metabolismo , Ácido Aspártico Endopeptidases/deficiência , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Catepsinas/deficiência , Catepsinas/genética , Catepsinas/metabolismo , Diferenciação Celular , Endossomos/imunologia , Endossomos/fisiologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Transdução de Sinais
2.
Arterioscler Thromb Vasc Biol ; 36(8): 1549-57, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27365406

RESUMO

OBJECTIVE: Cathepsin S (CatS) participates in atherogenesis through several putative mechanisms. The ability of cathepsins to modify histone tail is likely to contribute to stem cell development. Histone deacetylase 6 (HDAC6) is required in modulating the proliferation and migration of various types of cancer cells. Here, we investigated the cross talk between CatS and HADC6 in injury-related vascular repair in mice. APPROACH AND RESULTS: Ligation injury to the carotid artery in mice increased the CatS expression, and CatS-deficient mice showed reduced neointimal formation in injured arteries. CatS deficiency decreased the phosphorylation levels of p38 mitogen-activated protein kinase, Akt, and HDAC6 and toll-like receptor 2 expression in ligated arteries. The genetic or pharmacological inhibition of CatS also alleviated the increased phosphorylation of p38 mitogen-activated protein kinase, Akt, and HDAC6 induced by platelet-derived growth factor BB in cultured vascular smooth muscle cells (VSMCs), and p38 mitogen-activated protein kinase inhibition and Akt inhibition decreased the phospho-HDAC6 levels. Moreover, CatS inhibition caused decrease in the levels of the HDAC6 activity in VSMCs in response to platelet-derived growth factor BB. The HDAC6 inhibitor tubastatin A downregulated platelet-derived growth factor-induced VSMC proliferation and migration, whereas HDAC6 overexpression exerted the opposite effect. Tubastatin A also decreased the intimal VSMC proliferation and neointimal hyperplasia in response to injury. Toll-like receptor 2 silencing decreased the phosphorylation levels of p38 mitogen-activated protein kinase, Akt, and HDAC6 and VSMC migration and proliferation. CONCLUSIONS: This is the first report detailing cross-interaction between toll-like receptor 2-mediated CatS and HDAC6 during injury-related vascular repair. These data suggest that CatS/HDAC6 could be a potential therapeutic target for the control of vascular diseases that are involved in neointimal lesion formation.


Assuntos
Lesões das Artérias Carótidas/enzimologia , Artéria Carótida Primitiva/enzimologia , Catepsinas/metabolismo , Histona Desacetilases/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor 2 Toll-Like/metabolismo , Cicatrização , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Artéria Carótida Primitiva/efeitos dos fármacos , Artéria Carótida Primitiva/patologia , Catepsinas/antagonistas & inibidores , Catepsinas/deficiência , Catepsinas/genética , Pontos de Checagem do Ciclo Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Genótipo , Desacetilase 6 de Histona , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Masculino , Camundongos Knockout , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/patologia , Neointima , Fenótipo , Fosforilação , Inibidores de Proteases/farmacologia , Interferência de RNA , Transdução de Sinais , Receptor 2 Toll-Like/genética , Transfecção , Remodelação Vascular , Cicatrização/efeitos dos fármacos
3.
PLoS Negl Trop Dis ; 10(5): e0004716, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27182703

RESUMO

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.


Assuntos
Catepsina B/deficiência , Catepsina B/metabolismo , Leishmania major , Leishmaniose Cutânea/imunologia , Subpopulações de Linfócitos T/imunologia , Receptor Toll-Like 9/metabolismo , Transferência Adotiva , Animais , Apresentação do Antígeno , Complexo CD3/análise , Complexo CD3/imunologia , Catepsina B/genética , Catepsina L/deficiência , Catepsina L/genética , Catepsinas/deficiência , Catepsinas/genética , Células Dendríticas/imunologia , Endopeptidases/deficiência , , Inflamação/imunologia , Interferon gama/biossíntese , Leishmania major/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Carga Parasitária , Transdução de Sinais , Células Th1/imunologia , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologia
4.
Part Fibre Toxicol ; 13: 19, 2016 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-27108091

RESUMO

BACKGROUND: Particulate matter has been shown to stimulate the innate immune system and induce acute inflammation. Therefore, while nanotechnology has the potential to provide therapeutic formulations with improved efficacy, there are concerns such pharmaceutical preparations could induce unwanted inflammatory side effects. Accordingly, we aim to examine the utility of using the proteolytic activity signatures of cysteine proteases, caspase 1 and cathepsin S (CTSS), as biomarkers to assess particulate-induced inflammation. METHODS: Primary peritoneal macrophages and bone marrow-derived macrophages from C57BL/6 mice and ctss(-/-) mice were exposed to micro- and nanoparticulates and also the lysosomotropic agent, L-leucyl-L-leucine methyl ester (LLOME). ELISA and immunoblot analyses were used to measure the IL-1ß response in cells, generated by lysosomal rupture. Affinity-binding probes (ABPs), which irreversibly bind to the active site thiol of cysteine proteases, were then used to detect active caspase 1 and CTSS following lysosomal rupture. Reporter substrates were also used to quantify the proteolytic activity of these enzymes, as measured by substrate turnover. RESULTS: We demonstrate that exposure to silica, alum and polystyrene particulates induces IL-1ß release from macrophages, through lysosomal destabilization. IL-1ß secretion positively correlated with an increase in the proteolytic activity signatures of intracellular caspase 1 and extracellular CTSS, which were detected using ABPs and reporter substrates. Interestingly IL-1ß release was significantly reduced in primary macrophages from ctss(-/-) mice. CONCLUSIONS: This study supports the emerging significance of CTSS as a regulator of the innate immune response, highlighting its role in regulating IL-1ß release. Crucially, the results demonstrate the utility of intracellular caspase 1 and extracellular CTSS proteolytic activities as surrogate biomarkers of lysosomal rupture and acute inflammation. In the future, activity-based detection of these enzymes may prove useful for the real-time assessment of particle-induced inflammation and toxicity assessment during the development of nanotherapeutics.


Assuntos
Caspase 1/metabolismo , Catepsinas/metabolismo , Inflamação/induzido quimicamente , Lisossomos/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Material Particulado/toxicidade , Testes de Toxicidade/métodos , Compostos de Alúmen/toxicidade , Animais , Biomarcadores/metabolismo , Catepsinas/deficiência , Catepsinas/genética , Células Cultivadas , Dipeptídeos/toxicidade , Relação Dose-Resposta a Droga , Ativação Enzimática , Imunidade Inata/efeitos dos fármacos , Inflamação/enzimologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-1beta/metabolismo , Cinética , Lisossomos/enzimologia , Lisossomos/imunologia , Lisossomos/patologia , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nanopartículas , Poliestirenos/toxicidade , Cultura Primária de Células , Proteólise , Dióxido de Silício/toxicidade , Especificidade por Substrato
5.
J Immunol Methods ; 432: 87-94, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26899824

RESUMO

Cathepsin S (CTSS) is a eukaryotic protease mostly expressed in professional antigen presenting cells (APCs). Since CTSS activity regulation plays a role in the pathogenesis of various autoimmune diseases like multiple sclerosis, atherosclerosis, Sjögren's syndrome and psoriasis as well as in cancer progression, there is an ongoing interest in the reliable detection of cathepsin S activity. Various applications have been invented for specific detection of this enzyme. However, most of them have only been shown to be suitable for human samples, do not deliver quantitative results or the experimental procedure requires technical equipment that is not commonly available in a standard laboratory. We have tested a fluorogen substrate, Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH2, that has been described to specifically detect CTSS activities in human APCs for its potential use for mouse samples. We have modified the protocol and thereby offer a cheap, easy, reproducible and quick activity assay to detect CTSS activities in mouse APCs. Since most of basic research on CTSS is performed in mice, this method closes a gap and offers a possibility for reliable and quantitative CTSS activity detection that can be performed in almost every laboratory.


Assuntos
Células Apresentadoras de Antígenos/enzimologia , Catepsinas/metabolismo , Infecções por Escherichia coli/enzimologia , Corantes Fluorescentes/metabolismo , Peptídeos/metabolismo , Espectrometria de Fluorescência , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/microbiologia , Catepsinas/antagonistas & inibidores , Catepsinas/deficiência , Catepsinas/genética , Células Cultivadas , Inibidores de Cisteína Proteinase/farmacologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Hidrólise , Leucina/análogos & derivados , Leucina/farmacologia , Camundongos Knockout , Reprodutibilidade dos Testes , Especificidade por Substrato , Fatores de Tempo
6.
J Immunol ; 195(4): 1685-97, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-26195813

RESUMO

Sterile particles induce robust inflammatory responses that underlie the pathogenesis of diseases like silicosis, gout, and atherosclerosis. A key cytokine mediating this response is IL-1ß. The generation of bioactive IL-1ß by sterile particles is mediated by the NOD-like receptor containing a pyrin domain 3 (NLRP3) inflammasome, although exactly how this occurs is incompletely resolved. Prior studies have found that the cathepsin B inhibitor, Ca074Me, suppresses this response, supporting a model whereby ingested particles disrupt lysosomes and release cathepsin B into the cytosol, somehow activating NLRP3. However, reports that cathepsin B-deficient macrophages have no defect in particle-induced IL-1ß generation have questioned cathepsin B's involvement. In this study, we examine the hypothesis that multiple redundant cathepsins (not just cathepsin B) mediate this process by evaluating IL-1ß generation in murine macrophages, singly or multiply deficient in cathepsins B, L, C, S and X. Using an activity-based probe, we measure specific cathepsin activity in living cells, documenting compensatory changes in cathepsin-deficient cells, and Ca074Me's dose-dependent cathepsin inhibition profile is analyzed in parallel with its suppression of particle-induced IL-1ß secretion. Also, we evaluate endogenous cathepsin inhibitors cystatins C and B. Surprisingly, we find that multiple redundant cathepsins, inhibited by Ca074Me and cystatins, promote pro-IL-1ß synthesis, and to our knowledge, we provide the first evidence that cathepsin X plays a nonredundant role in nonparticulate NLRP3 activation. Finally, we find cathepsin inhibitors selectively block particle-induced NLRP3 activation, independently of suppressing pro-IL-1ß synthesis. Altogether, we demonstrate that both small molecule and endogenous cathepsin inhibitors suppress particle-induced IL-1ß secretion, implicating roles for multiple cathepsins in both pro-IL-1ß synthesis and NLRP3 activation.


Assuntos
Proteínas de Transporte/metabolismo , Catepsinas/metabolismo , Interleucina-1beta/metabolismo , Animais , Catepsinas/antagonistas & inibidores , Catepsinas/deficiência , Catepsinas/genética , Inibidores Enzimáticos/farmacologia , Inflamassomos/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Fenótipo , Transdução de Sinais/efeitos dos fármacos
7.
PLoS One ; 10(6): e0128945, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26075905

RESUMO

The cysteine cathepsins B, S, and L are functionally linked to antigen processing, and hence to autoimmune disorders such as multiple sclerosis. Stemming from several studies that demonstrate that mice can be protected from experimental autoimmune encephalomyelitis (EAE) through the pharmacologic inhibition of cysteine cathepsins, it has been suggested that targeting these enzymes in multiple sclerosis may be of therapeutic benefit. Utilizing mice deficient in cysteine cathepsins both individually and in combination, we found that the myelin-associated antigen myelin oligodendrocyte glycoprotein (MOG) was efficiently processed and presented by macrophages to CD4+ T cells in the individual absence of cathepsin B, S or L. Similarly, mice deficient in cathepsin B or S were susceptible to MOG-induced EAE and displayed clinical progression and immune infiltration into the CNS, similar to their wild-type counterparts. Owing to a previously described CD4+ T cell deficiency in mice deficient in cathepsin L, such mice were protected from EAE. When multiple cysteine cathepsins were simultaneously inhibited via genetic deletion of both cathepsins B and S, or by a cathepsin inhibitor (LHVS), MHC-II surface expression, MOG antigen presentation and EAE were attenuated or prevented. This study demonstrates the functional redundancy between cathepsin B, S and L in EAE, and suggests that the inhibition of multiple cysteine cathepsins may be needed to modulate autoimmune disorders such as multiple sclerosis.


Assuntos
Catepsinas/metabolismo , Cisteína/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Animais , Apresentação do Antígeno , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Catepsinas/antagonistas & inibidores , Catepsinas/deficiência , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Glicoproteína Mielina-Oligodendrócito/imunologia , Glicoproteína Mielina-Oligodendrócito/metabolismo , Sulfonas/farmacologia
8.
Sci Rep ; 3: 2744, 2013 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-24067868

RESUMO

Microglia are thought to play important roles in the maintenance of neuronal circuitry and the regulation of behavior. We found that the cortical microglia contain an intrinsic molecular clock and exhibit a circadian expression of cathepsin S (CatS), a microglia-specific lysosomal cysteine protease in the brain. The genetic deletion of CatS causes mice to exhibit hyperlocomotor activity and removes diurnal variations in the synaptic activity and spine density of the cortical neurons, which are significantly higher during the dark (waking) phase than the light (sleeping) phase. Furthermore, incubation with recombinant CatS significantly reduced the synaptic activity of the cortical neurons. These results suggest that CatS secreted by microglia during the dark-phase decreases the spine density of the cortical neurons by modifying the perisynaptic environment, leading to downscaling of the synaptic strength during the subsequent light-phase. Disruption of CatS therefore induces hyperlocomotor activity due to failure to downscale the synaptic strength.


Assuntos
Relógios Biológicos , Catepsinas/metabolismo , Ritmo Circadiano , Microglia/metabolismo , Sinapses/metabolismo , Animais , Relógios Biológicos/genética , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Catepsinas/deficiência , Ritmo Circadiano/genética , Espinhas Dendríticas/metabolismo , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Atividade Motora , Proteólise , Proteínas Recombinantes/metabolismo , Sono
10.
Arterioscler Thromb Vasc Biol ; 32(12): 2901-9, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23042818

RESUMO

OBJECTIVE: The purpose of this study was to evaluate potential mechanisms promoting abdominal aortic aneurysm development with tobacco smoke (TS) exposure. METHODS AND RESULTS: Experiments used the elastase perfusion model of abdominal aortic aneurysms with smoke-free controls. The effect of TS exposure was evaluated in C57/Bl6 mice, after broad-spectrum matrix metalloproteinase inhibition with doxycycline and in mice deficient in matrix metalloproteinase-9, matrix metalloproteinase-12, Cathepsin-S, and Neutrophil Elastase. Preparations of washed marrow, spleen, and peripheral blood leukocytes were transferred to smoke-free mice from 6-week TS-exposed mice or smoke-free mice. All mice were euthanized 14 days after elastase perfusion, and the percentage of change in aortic diameter (%Δ aortic diameter) was calculated. Electron microscopy of aortic tissue from animals exposed to TS without elastase exposure did not demonstrate any ultrastructural changes. Neither doxycycline nor any specific elastase deficiency was effective at preventing an increase in %Δ aortic diameter in TS-exposed animals. Smoke exposure for 6 weeks increased the %Δ aortic diameter after a smoke-free interval of up to 6 weeks before elastase perfusion. Leukocyte preparations from TS-exposed mice localized to abdominal aortic aneurysms and increased the %Δ aortic diameter in smoke-free mice. CONCLUSIONS: The effect of TS on the development of abdominal aortic aneurysms is not dependent on the activity of elastolytic enzymes and persists for long periods despite cessation of TS. Alterations in leukocyte response to aortic injury appear to mediate this effect.


Assuntos
Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/fisiopatologia , Leucócitos Mononucleares/patologia , Leucócitos Mononucleares/fisiologia , Fumar/efeitos adversos , Animais , Aorta Abdominal/patologia , Aorta Abdominal/fisiopatologia , Aorta Abdominal/ultraestrutura , Aneurisma da Aorta Abdominal/patologia , Catepsinas/deficiência , Catepsinas/genética , Catepsinas/fisiologia , Contagem de Células , Modelos Animais de Doenças , Doxiciclina/farmacologia , Elastase de Leucócito/deficiência , Elastase de Leucócito/genética , Elastase de Leucócito/fisiologia , Masculino , Metaloproteinase 12 da Matriz/deficiência , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/fisiologia , Metaloproteinase 9 da Matriz/deficiência , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/fisiologia , Metaloproteinases da Matriz/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
11.
Cardiovasc Res ; 96(3): 401-10, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-22871592

RESUMO

AIMS: Abdominal aortic aneurysm (AAA) is characterized by extensive aortic wall matrix degradation that contributes to the remodelling and eventual rupture of the arterial wall. Elastinolytic cathepsin S (Cat S) is highly expressed in human aneurysmal lesions, but whether it contributes to the pathogenesis of AAA remains unknown. METHODS AND RESULTS: AAAs were induced in apolipoprotein E (ApoE) and Cat S compound mutant (Apoe(-/-)Ctss(-/-)) mice and in ApoE-deficient Cat S wild-type littermates (Apoe(-/-)Ctss(+/+)) by chronic angiotensin II infusion, and AAA lesions were analysed after 28 days. We found that Cat S expression increased significantly in mouse AAA lesions. The AAA incidence in Apoe(-/-)Ctss(-/-) mice was much lower than that in Apoe(-/-)Ctss(+/+) mice (10 vs. 80%). Cat S deficiency significantly reduced external and luminal abdominal aortic diameters, medial elastin fragmentation, and adventitia collagen content. Cat S deficiency reduced aortic lesion expression and the activity of matrix metalloproteinase (MMP)-2, MMP-9, and Cat K, but not the activity of other major cathepsins, such as Cat B and Cat L. Absence of Cat S significantly reduced AAA lesion media smooth muscle cell (SMC) apoptosis, lesion adventitia microvessel content, and inflammatory cell accumulation and proliferation. In vitro studies proved that Cat S helps promote SMC apoptosis, angiogenesis, monocyte and T-cell transmigration, and T-cell proliferation--all of which are essential to AAA pathogenesis. CONCLUSIONS: These data provide direct evidence that Cat S plays an important role in AAA formation and suggest that Cat S is a new therapeutic target for human AAA.


Assuntos
Angiotensina II , Aorta Abdominal/enzimologia , Aneurisma da Aorta Abdominal/prevenção & controle , Apolipoproteínas E/deficiência , Catepsinas/deficiência , Animais , Aorta Abdominal/imunologia , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/enzimologia , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/imunologia , Aneurisma da Aorta Abdominal/patologia , Apolipoproteínas E/genética , Apoptose , Complexo CD3/metabolismo , Catepsina K/metabolismo , Catepsinas/genética , Células Cultivadas , Colágeno/metabolismo , Modelos Animais de Doenças , Elastina/metabolismo , Inflamação/enzimologia , Inflamação/imunologia , Inflamação/prevenção & controle , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/patologia , Neovascularização Patológica , Linfócitos T/enzimologia , Linfócitos T/imunologia , Fatores de Tempo
12.
PLoS One ; 7(4): e35315, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22558139

RESUMO

BACKGROUND: Cathepsin S (Cat S) is overexpressed in human atherosclerotic and aneurysmal tissues and may contributes to degradation of extracellular matrix, especially elastin, in inflammatory diseases. We aimed to define the role of Cat S in cardiac inflammation and fibrosis induced by angiotensin II (Ang II) in mice. METHODS AND RESULTS: Cat S-knockout (Cat S(-/-)) and littermate wild-type (WT) C57BL/6J mice were infused continuously with Ang II (750 ng/kg/min) or saline for 7 days. Cat S(-/-) mice showed severe cardiac fibrosis, including elevated expression of collagen I and α-smooth muscle actin (α-SMA), as compared with WT mice. Moreover, macrophage infiltration and expression of inflammatory cytokines (tumor necrosis factor α, transforming growth factor ß and interleukin 1ß) were significantly greater in Cat S(-/-) than WT hearts. These Ang II-induced effects in Cat S(-/-) mouse hearts was associated with abnormal accumulation of autophagosomes and reduced clearance of damaged mitochondria, which led to increased levels of reactive oxygen species (ROS) and activation of nuclear factor-kappa B (NF-κB) in macrophages. CONCLUSION: Cat S in lysosomes is essential for mitophagy processing in macrophages, deficiency in Cat S can increase damaged mitochondria and elevate ROS levels and NF-κB activity in hypertensive mice, so it regulates cardiac inflammation and fibrosis.


Assuntos
Aneurisma/metabolismo , Catepsinas/deficiência , Arteriosclerose Intracraniana/metabolismo , Macrófagos/citologia , Fagossomos/metabolismo , Animais , Autofagia/fisiologia , Western Blotting , Primers do DNA/genética , Técnicas Histológicas , Imuno-Histoquímica , Macrófagos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Mitocôndrias/fisiologia , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
13.
Mol Genet Metab ; 104(4): 608-19, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21944884

RESUMO

Mucopolysaccharidosis VII (MPS VII) is due to mutations within the gene encoding the lysosomal enzyme ß-glucuronidase, and results in the accumulation of glycosaminoglycans. MPS VII causes aortic dilatation and elastin fragmentation, which is associated with upregulation of the elastases cathepsin S (CtsS) and matrix metalloproteinase 12 (MMP12). To test the role of these enzymes, MPS VII mice were crossed with mice deficient in CtsS or MMP12, and the effect upon aortic dilatation was determined. CtsS deficiency did not protect against aortic dilatation in MPS VII mice, but also failed to prevent an upregulation of cathepsin enzyme activity. Further analysis with substrates and inhibitors specific for particular cathepsins suggests that this enzyme activity was due to CtsB, which could contribute to elastin fragmentation. Similarly, MMP12 deficiency and deficiency of both MMP12 and CtsS could not prevent aortic dilatation in MPS VII mice. Microarray and reverse-transcriptase real-time PCR were performed to look for upregulation of other elastases. This demonstrated that mRNA for complement component D was elevated in MPS VII mice, while immunostaining demonstrated high levels of complement component C3 on surfaces within the aortic media. Finally, we demonstrate that neonatal intravenous injection of a retroviral vector encoding ß-glucuronidase reduced aortic dilatation. We conclude that neither CtsS nor MMP12 are necessary for elastin fragmentation in MPS VII mouse aorta, and propose that CtsB and/or complement component D may be involved. Complement may be activated by the GAGs that accumulate, and may play a role in signal transduction pathways that upregulate elastases.


Assuntos
Doenças da Aorta/etiologia , Ativação do Complemento , Dilatação Patológica/etiologia , Mucopolissacaridose VII/complicações , Animais , Aorta/metabolismo , Aorta/patologia , Aorta/fisiopatologia , Doenças da Aorta/metabolismo , Doenças da Aorta/fisiopatologia , Catepsinas/deficiência , Proteínas do Sistema Complemento/genética , Proteínas do Sistema Complemento/metabolismo , Elastina/metabolismo , Perfilação da Expressão Gênica , Terapia Genética , Glucuronidase/biossíntese , Glucuronidase/sangue , Glucuronidase/genética , Glicosaminoglicanos/metabolismo , Masculino , Metaloproteinase 12 da Matriz/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mucopolissacaridose VII/fisiopatologia , Mucopolissacaridose VII/terapia , Análise de Sequência com Séries de Oligonucleotídeos , Elastase Pancreática/genética , Elastase Pancreática/metabolismo , Transdução de Sinais , Extratos de Tecidos , Regulação para Cima
14.
Appl Biochem Biotechnol ; 165(2): 728-36, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21625870

RESUMO

Efficient degradation of cellulose needs a synergistic reaction of the cellulolytic enzymes, which include exoglucanases, endoglucanases, and ß-1,4-glucosidase. In this study, we used an improved Bac-to-Bac/BmNPV baculovirus expression system, which lacks the virus-encoded chitinase cathepsin (v-cath) genes of Bombyx mori nucleopolyhedrovirus (BmNPV), to express the endoglucanase V (EG V) gene from Trichoderma viride in silkworm BmN cells and silkworm larvae, and analyzed the characteristics of the recombinant enzyme in silkworm larvae. The result showed that an around 36-kDa protein was visualized in BmN cells at 48 h after the second-generation recombinant mBacmid/BmNPV/EG V baculovirus infection. The crude enzyme extract from the recombinant baculoviruses-infected silkworms exhibited a significant maximum activity at the environmental condition of pH 5.0 and a temperature of 50 °C, and increased 39.86% and 37.76% compared with that from blank mBacmid/BmNPV baculovirus-infected silkworms and normal silkworms, respectively. It was stable at pH range from 5.0 to 10.0 and at temperature range from 40 to 60 °C. The availability of large quantities of EG V that the silkworm provides might greatly facilitate the future research and the potential application in industries.


Assuntos
Biotecnologia/métodos , Bombyx/genética , Celulase/biossíntese , Celulose/metabolismo , Proteínas Fúngicas/biossíntese , Larva/genética , Proteínas Recombinantes/biossíntese , Trichoderma/enzimologia , Animais , Biodegradação Ambiental , Western Blotting , Bombyx/metabolismo , Bombyx/virologia , Catepsinas/deficiência , Catepsinas/genética , Linhagem Celular , Celulase/genética , Quitinases/deficiência , Quitinases/genética , Proteínas Fúngicas/genética , Expressão Gênica , Vetores Genéticos , Larva/metabolismo , Larva/virologia , /genética , Proteínas Recombinantes/genética , Trichoderma/química , Trichoderma/genética
15.
Biochimie ; 92(11): 1580-6, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20417681

RESUMO

Given the increasing prevalence of human obesity worldwide, there is an urgent need for a better understanding of the molecular mechanisms linking obesity to metabolic and cardiovascular diseases. Our knowledge is nevertheless limited regarding molecules linking adipose tissue to downstream complications. The importance of cathepsins was brought to light in this context. Through a large scale transcriptomic analysis, our group recently identified the gene encoding cathepsin S as one of the most deregulated gene in the adipose tissue of obese subjects and positively correlated with body mass index. Other members of the cathepsin family are expressed in the adipose tissue, including cathepsin K and cathepsin L. Given their implication in atherogenesis, these proteases could participate into the well established deleterious relationship between enlarged adipose tissue and increased cardiovascular risk. Here, we review the clinical and experimental evidence relevant to the role of cathepsins K, L and S and their most abundant endogenous inhibitor, cystatin C, in atherosclerosis and in obesity.


Assuntos
Aterosclerose/metabolismo , Catepsinas/metabolismo , Cistatina C/metabolismo , Obesidade/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Aterosclerose/enzimologia , Catepsinas/antagonistas & inibidores , Catepsinas/deficiência , Catepsinas/genética , Cistatina C/deficiência , Cistatina C/genética , Técnicas de Inativação de Genes , Humanos , Obesidade/tratamento farmacológico , Obesidade/enzimologia , Obesidade/patologia
16.
Circulation ; 119(13): 1785-94, 2009 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-19307473

RESUMO

BACKGROUND: Clinical studies have demonstrated that 50% of individuals with chronic renal disease (CRD) die of cardiovascular causes, including advanced calcific arterial and valvular disease; however, the mechanisms of accelerated calcification in CRD remain obscure, and no therapies can prevent disease progression. We recently demonstrated in vivo that inflammation triggers cardiovascular calcification. In vitro evidence also indicates that elastin degradation products may promote osteogenesis. Here, we used genetically modified mice and molecular imaging to test the hypothesis in vivo that cathepsin S (catS), a potent elastolytic proteinase, accelerates calcification in atherosclerotic mice with CRD induced by 5/6 nephrectomy. METHODS AND RESULTS: Apolipoprotein-deficient (apoE(-/-))/catS(+/+) (n=24) and apoE(-/-)/catS(-/-) (n=24) mice were assigned to CRD and control groups. CRD mice had significantly higher serum phosphate, creatinine, and cystatin C levels than those without CRD. To visualize catS activity and osteogenesis in vivo, we coadministered catS-activatable and calcification-targeted molecular imaging agents 10 weeks after nephrectomy. Imaging coregistered increased catS and osteogenic activities in the CRD apoE(-/-)/catS(+/+) cohort, whereas CRD apoE(-/-)/catS(-/-) mice exhibited less calcification. Quantitative histology demonstrated greater catS-associated elastin fragmentation and calcification in CRD apoE(-/-)/catS(+/+) than CRD apoE(-/-)/catS(-/-) aortas and aortic valves. Notably, catS deletion did not cause compensatory increases in RNA levels of other elastolytic cathepsins or matrix metalloproteinases. Elastin peptide and recombinant catS significantly increased calcification in smooth muscle cells in vitro, a process further amplified in phosphate-enriched culture medium. CONCLUSIONS: The present study provides direct in vivo evidence that catS-induced elastolysis accelerates arterial and aortic valve calcification in CRD, providing new insight into the pathophysiology of cardiovascular calcification.


Assuntos
Estenose da Valva Aórtica/metabolismo , Calcinose/metabolismo , Catepsinas/genética , Catepsinas/metabolismo , Falência Renal Crônica/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Estenose da Valva Aórtica/patologia , Estenose da Valva Aórtica/fisiopatologia , Apolipoproteínas E/genética , Calcinose/patologia , Calcinose/fisiopatologia , Doenças das Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/patologia , Doenças das Artérias Carótidas/fisiopatologia , Catepsinas/deficiência , Células Cultivadas , Creatinina/sangue , Cistatina C/sangue , Elastina/metabolismo , Elastina/farmacologia , Humanos , Falência Renal Crônica/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Nefrectomia , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Fosfatos/sangue , Fosfatos/farmacologia , Túnica Íntima/metabolismo , Túnica Íntima/patologia , Túnica Média/metabolismo , Túnica Média/patologia
17.
Calcif Tissue Int ; 84(3): 229-39, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19172215

RESUMO

Cathepsin K deficiency in humans causes pycnodysostosis, which is characterized by dwarfism and osteosclerosis. Earlier studies of 10-week-old male cathepsin K-deficient (knockout, KO) mice showed their bones were mechanically more brittle, while histomorphometry showed that both osteoclasts and osteoblasts had impaired activity relative to the wild type (WT). Here, we report detailed mineral and matrix analyses of the tibia of these animals based on Fourier transform infrared microspectroscopy and imaging. At 10 weeks, there was significant hypercalcification of the calcified cartilage and cortices in the KO. Carbonate content was elevated in the KO calcified cartilage as well as cortical and cancellous bone areas. These data suggest that cathepsin K does not affect mineral deposition but has a significant effect on mineralized tissue remodeling. Since growth plate abnormalities were extensive despite reported low levels of cathepsin K expression in the calcified cartilage, we used a differentiating chick limb-bud mesenchymal cell system that mimics endochondral ossification but does not contain osteoclasts, to show that cathepsin K inhibition during initial stages of mineral deposition retards the mineralization process while general inhibition of cathepsins can increase mineralization. These data suggest that the hypercalcification of the cathepsin K-deficient growth plate is due to persistence of calcified cartilage and point to a role of cathepsin K in bone tissue development as well as skeletal remodeling.


Assuntos
Calcinose/genética , Catepsinas/deficiência , Lâmina de Crescimento/patologia , Tíbia/patologia , Animais , Desenvolvimento Ósseo/genética , Calcificação Fisiológica/genética , Calcinose/patologia , Catepsina K , Catepsinas/antagonistas & inibidores , Catepsinas/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Inibidores Enzimáticos/farmacologia , Lâmina de Crescimento/enzimologia , Masculino , Mesoderma/efeitos dos fármacos , Mesoderma/enzimologia , Mesoderma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteogênese/genética , Tíbia/enzimologia
18.
Bone ; 44(2): 199-207, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18845279

RESUMO

Cathepsin K (CatK) is a cysteine protease expressed predominantly in osteoclasts, that plays a prominent role in degrading Type I collagen. Growing CatK null mice have osteopetrosis associated with a reduced ability to degrade bone matrix. Bone strength and histomorphometric endpoints in young adult CatK null mice aged more than 10 weeks have not been studied. The purpose of this paper is to describe bone mass, strength, resorption, and formation in young adult CatK null mice. In male and female wild-type (WT), heterozygous, and homozygous CatK null mice (total N=50) aged 19 weeks, in-life double fluorochrome labeling was performed. Right femurs and lumbar vertebral bodies 1-3 (LV) were evaluated by dual-energy X-ray absorptiometry (DXA) for bone mineral content (BMC) and bone mineral density (BMD). The trabecular region of the femur and the cortical region of the tibia were evaluated by histomorphometry. The left femur and sixth lumbar vertebral body were tested biomechanically. CatK (-/-) mice show higher BMD at the central and distal femur. Central femur ultimate load was positively influenced by genotype, and was positively correlated with both cortical area and BMC. Lumbar vertebral body ultimate load was also positively correlated to BMC. Genotype did not influence the relationship of ultimate load to BMC in either the central femur or vertebral body. CatK (-/-) mice had less lamellar cortical bone than WT mice. Higher bone volume, trabecular thickness, and trabecular number were observed at the distal femur in CatK (-/-) mice. Smaller marrow cavities were also present at the central femur of CatK (-/-) mice. CatK (-/-) mice exhibited greater trabecular mineralizing surface, associated with normal volume-based formation of trabecular bone. Adult CatK (-/-) mice have higher bone mass in both cortical and cancellous regions than WT mice. Though no direct measures of bone resorption rate were made, the higher cortical bone quantity is associated with a smaller marrow cavity and increased retention of non-lamellar bone, signs of decreased endocortical resorption. The relationship of bone strength to BMC does not differ with genotype, indicating the presence of bone tissue of normal quality in the absence of CatK.


Assuntos
Densidade Óssea/fisiologia , Osso e Ossos/enzimologia , Osso e Ossos/fisiologia , Catepsinas/deficiência , Osteogênese , Animais , Fenômenos Biomecânicos , Reabsorção Óssea/enzimologia , Osso e Ossos/anatomia & histologia , Catepsina K , Catepsinas/metabolismo , Feminino , Fêmur/anatomia & histologia , Fêmur/enzimologia , Vértebras Lombares/enzimologia , Vértebras Lombares/fisiologia , Masculino , Camundongos , Microscopia de Fluorescência , Tamanho do Órgão , Análise de Regressão , Caracteres Sexuais , Propriedades de Superfície
19.
J Biol Chem ; 284(4): 2584-92, 2009 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-19028686

RESUMO

Cathepsin K is responsible for the degradation of type I collagen in osteoclast-mediated bone resorption. Collagen fragments are known to be biologically active in a number of cell types. Here, we investigate their potential to regulate osteoclast activity. Mature murine osteoclasts were seeded on type I collagen for actin ring assays or dentine discs for resorption assays. Cells were treated with cathepsins K-, L-, or MMP-1-predigested type I collagen or soluble bone fragments for 24 h. The presence of actin rings was determined fluorescently by staining for actin. We found that the percentage of osteoclasts displaying actin rings and the area of resorbed dentine decreased significantly on addition of cathepsin K-digested type I collagen or bone fragments, but not with cathepsin L or MMP-1 digests. Counterintuitively, actin ring formation was found to decrease in the presence of the cysteine proteinase inhibitor LHVS and in cathepsin K-deficient osteoclasts. However, cathepsin L deficiency or the general MMP inhibitor GM6001 had no effect on the presence of actin rings. Predigestion of the collagen matrix with cathepsin K, but not by cathepsin L or MMP-1 resulted in an increased actin ring presence in cathepsin K-deficient osteoclasts. These studies suggest that cathepsin K interaction with type I collagen is required for 1) the release of cryptic Arg-Gly-Asp motifs during the initial attachment of osteoclasts and 2) termination of resorption via the creation of autocrine signals originating from type I collagen degradation.


Assuntos
Actinas/metabolismo , Reabsorção Óssea/metabolismo , Catepsinas/metabolismo , Osteoclastos/metabolismo , Animais , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Catepsina K , Catepsina L , Catepsinas/deficiência , Catepsinas/genética , Proliferação de Células , Células Cultivadas , Colágeno/metabolismo , Cisteína Endopeptidases/metabolismo , Ativação Enzimática , Metaloproteases/antagonistas & inibidores , Metaloproteases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Solubilidade
20.
Arterioscler Thromb Vasc Biol ; 29(2): 188-94, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19095996

RESUMO

OBJECTIVE: A dysbalance of proteases and their inhibitors is instrumental in remodeling of atherosclerotic plaques. One of the proteases implicated in matrix degradation is cathepsin-S (CatS). To address its role in advanced lesion composition, we generated chimeric LDLr(-/-) mice deficient in leukocyte CatS by transplantation with CatS(-/-)xLDLr(-/-) or with LDLr(-/-) bone marrow and administered a high-fat diet. METHODS AND RESULTS: No difference in aortic root lesion size could be detected between CatS(+/+) and CatS(-/-) chimeras. However, leukocyte CatS deficiency markedly changed plaque morphology and led to a dramatic reduction in necrotic core area by 77% and an abundance of large foam cells. Plaques of CatS(-/-) chimeras contained 17% more macrophages, 62% less SMCs, and 33% less intimal collagen. The latter two could be explained by a reduced number of elastic lamina fractures. Moreover, macrophage apoptosis was reduced by 60% with CatS deficiency. In vitro, CatS was found to be involved in cholesterol metabolism and in macrophage apoptosis in a collagen and fibronectin matrix. CONCLUSIONS: Leukocyte CatS deficiency results in considerably altered plaque morphology, with smaller necrotic cores, reduced apoptosis, and decreased SMC content and collagen deposition and may thus be critical in plaque stability.


Assuntos
Aorta/enzimologia , Aterosclerose/enzimologia , Catepsinas/metabolismo , Matriz Extracelular/metabolismo , Leucócitos/enzimologia , Animais , Aorta/imunologia , Aorta/patologia , Apoptose , Aterosclerose/etiologia , Aterosclerose/imunologia , Aterosclerose/patologia , Transplante de Medula Óssea , Catepsinas/antagonistas & inibidores , Catepsinas/deficiência , Catepsinas/genética , Movimento Celular , Proliferação de Células , Células Cultivadas , Colesterol/metabolismo , Colágeno/metabolismo , Dieta Aterogênica , Modelos Animais de Doenças , Tecido Elástico/metabolismo , Feminino , Células Espumosas/enzimologia , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Macrófagos Peritoneais/enzimologia , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , Necrose , Inibidores de Proteases/farmacologia , Receptores de LDL/deficiência , Receptores de LDL/genética , Quimeras de Transplante
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