ABSTRACT
Tazarotene-induced gene 1 (TIG1) is a retinoid acid receptor-responsive gene involved in cell differentiation and tumorigenesis. Aberrant methylation of CpG islands in the TIG1 promoter is found in multiple cancers. Currently, the exact mechanism underlying the anticancer effect of TIG1 is unknown. Here, we show that TIG1 interacts with cathepsin V (CTSV), which reduces CTSV stability and subsequently affects the production of activated urokinase-type plasminogen activator (uPA), an epithelial-mesenchymal transition-associated protein. Ectopic expression of CTSV increased the expression of activated uPA and the number of migrated and invaded cells, whereas ectopic TIG1 expression reversed the effects of CTSV on the uPA signaling pathway. Similar patterns in the production of activated uPA and number of migrated and invaded cells were also observed in TIG1-expressing and CTSV-knockdown cells. The results suggest that CTSV may participate in TIG1-regulated uPA activity and the associated downstream signaling pathway.
Subject(s)
Cathepsins/metabolism , Colorectal Neoplasms/pathology , Cysteine Endopeptidases/metabolism , Receptors, G-Protein-Coupled/metabolism , Cathepsins/deficiency , Cathepsins/genetics , Cell Movement , Cysteine Endopeptidases/deficiency , Cysteine Endopeptidases/genetics , Epithelial-Mesenchymal Transition/genetics , Gene Silencing , HCT116 Cells , Humans , Neoplasm InvasivenessABSTRACT
Cathepsins (CTSs) are ubiquitously expressed proteases normally found in the endolysosomal compartment where they mediate protein degradation and turnover. However, CTSs are also found in the cytoplasm, nucleus, and extracellular matrix where they actively participate in cell signaling, protein processing, and trafficking through the plasma and nuclear membranes and between intracellular organelles. Dysregulation in CTS expression and/or activity disrupts cellular homeostasis, thus contributing to many human diseases, including inflammatory and cardiovascular diseases, neurodegenerative disorders, diabetes, obesity, cancer, kidney dysfunction, and others. This review aimed to highlight the involvement of CTSs in inherited lysosomal storage disorders, with a primary focus to the emerging evidence on the role of CTSs in the pathophysiology of Mucopolysaccharidoses (MPSs). These latter diseases are characterized by severe neurological, skeletal and cardiovascular phenotypes, and no effective cure exists to date. The advance in the knowledge of the molecular mechanisms underlying the activity of CTSs in MPSs may open a new challenge for the development of novel therapeutic approaches for the cure of such intractable diseases.
Subject(s)
Cathepsins/metabolism , Mucopolysaccharidoses/physiopathology , Mucopolysaccharidoses/therapy , Cathepsins/antagonists & inhibitors , Cathepsins/deficiency , Cathepsins/genetics , Humans , Models, Biological , Mucopolysaccharidoses/enzymology , Mutation/genetics , Protease Inhibitors/therapeutic useABSTRACT
Ectromelia virus (ECTV), an orthopoxvirus, undergoes productive replication in conventional dendritic cells (cDCs), resulting in the inhibition of their innate and adaptive immune functions. ECTV replication rate in cDCs is increased due to downregulation of the expression of cathepsins - cystein proteases that orchestrate several steps during DC maturation. Therefore, this study was aimed to determine if downregulation of cathepsins, such as B, L or S, disrupts cDC capacity to induce activating signals in T cells or whether infection of cDCs with ECTV further weakens their functions as antigen-presenting cells. Our results showed that cDCs treated with siRNA against cathepsin B, L and S synthesize similar amounts of pro-inflammatory cytokines and exhibit comparable ability to mature and stimulate alloreactive CD4+ T cells, as untreated wild type (WT) cells. Moreover, ECTV inhibitory effect on cDC innate and adaptive immune functions, observed especially after LPS treatment, was comparable in both cathepsin-silenced and WT cells. Taken together, the absence of cathepsins B, L and S has minimal, if any, impact on the inhibitory effect of ECTV on cDC immune functions. We assume that the virus-mediated inhibition of cathepsin expression in cDCs represents more a survival mechanism than an immune evasion strategy.
Subject(s)
Cathepsins/deficiency , Dendritic Cells/immunology , Ectromelia virus/physiology , Animals , CD4-Positive T-Lymphocytes/immunology , Cathepsins/genetics , Cathepsins/metabolism , Cell Differentiation/immunology , Cell Proliferation , Cells, Cultured , Cytokines/metabolism , Dendritic Cells/metabolism , Dendritic Cells/virology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Th1-Th2 BalanceABSTRACT
BACKGROUND: Given that cathepsin S (CatS) gained attention due to its enzymatic and non-enzymatic functions in signaling, the role of CatS in ischemia-induced angiogenesis of aged mice was explored.MethodsâandâResults:To study the role of CatS in the decline in aging-related vascular regeneration capacity, a hindlimb ischemia model was applied to aged wild-type (CatS+/+) and CatS-deficient (CatS-/-) mice. CatS-/-mice exhibited impaired blood flow recovery and capillary formation and increased levels of p-insulin receptor substrate-1, Wnt5a, and SC35 proteins and decreased levels of phospho-endothelial nitric oxide synthase (p-eNOS), p-mTOR, p-Akt, p-ERK1/2, p-glycogen synthase kinase-3α/ß, and galatin-3 proteins, as well as decreased macrophage infiltration and matrix metalloproteinase-2/-9 activities in the ischemic muscles. In vitro, CatS knockdown altered the levels of these targeted essential molecules for angiogenesis. Together, the results suggested that CatS-/-leads to defective endothelial cell functions and that CatS-/-is associated with decreased circulating endothelial progenitor cell (EPC)-like CD31+/c-Kit+cells. This notion was reinforced by the study finding that pharmacological CatS inhibition led to a declined angiogenic capacity accompanied by increased Wnt5a and SC35 levels and decreased eNOS/Akt-ERK1/2 signaling in response to ischemia. CONCLUSIONS: These findings demonstrated that the impairment of ischemia-induced neovascularization in aged CatS-/-mice is due, at least in part, to the attenuation of endothelial cell/EPC functions and/or mobilization associated with Wnt5a/SC35 activation in advanced age.
Subject(s)
Cathepsins/metabolism , Endothelial Progenitor Cells/enzymology , Ischemia/enzymology , Muscle, Skeletal/blood supply , Serine-Arginine Splicing Factors/metabolism , Wnt-5a Protein/metabolism , Age Factors , Animals , Cathepsins/deficiency , Cathepsins/genetics , Cells, Cultured , Disease Models, Animal , Extracellular Signal-Regulated MAP Kinases/metabolism , Hindlimb , Human Umbilical Vein Endothelial Cells/enzymology , Humans , Ischemia/physiopathology , Male , Mice, Inbred C57BL , Mice, Knockout , Neovascularization, Physiologic , Nitric Oxide Synthase Type III/metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolismABSTRACT
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.
Subject(s)
Antigens, Differentiation, B-Lymphocyte/immunology , B-Lymphocytes/immunology , Genes, MHC Class II , Histocompatibility Antigens Class II/immunology , Animals , Antigens, Differentiation, B-Lymphocyte/metabolism , Aspartic Acid Endopeptidases/deficiency , Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/metabolism , Cathepsins/deficiency , Cathepsins/genetics , Cathepsins/metabolism , Cell Differentiation , Endosomes/immunology , Endosomes/physiology , Histocompatibility Antigens Class II/metabolism , Membrane Proteins/deficiency , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Signal TransductionABSTRACT
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.
Subject(s)
Carotid Artery Injuries/enzymology , Carotid Artery, Common/enzymology , Cathepsins/metabolism , Histone Deacetylases/metabolism , Phosphatidylinositol 3-Kinase/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Toll-Like Receptor 2/metabolism , Wound Healing , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Carotid Artery Injuries/genetics , Carotid Artery Injuries/pathology , Carotid Artery, Common/drug effects , Carotid Artery, Common/pathology , Cathepsins/antagonists & inhibitors , Cathepsins/deficiency , Cathepsins/genetics , Cell Cycle Checkpoints , Cell Movement , Cell Proliferation , Cells, Cultured , Disease Models, Animal , Genotype , Histone Deacetylase 6 , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/genetics , Male , Mice, Knockout , Muscle, Smooth, Vascular/enzymology , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/enzymology , Myocytes, Smooth Muscle/pathology , Neointima , Phenotype , Phosphorylation , Protease Inhibitors/pharmacology , RNA Interference , Signal Transduction , Toll-Like Receptor 2/genetics , Transfection , Vascular Remodeling , Wound Healing/drug effectsABSTRACT
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.
Subject(s)
Cathepsin B/deficiency , Cathepsin B/metabolism , Leishmania major , Leishmaniasis, Cutaneous/immunology , T-Lymphocyte Subsets/immunology , Toll-Like Receptor 9/metabolism , Adoptive Transfer , Animals , Antigen Presentation , CD3 Complex/analysis , CD3 Complex/immunology , Cathepsin B/genetics , Cathepsin L/deficiency , Cathepsin L/genetics , Cathepsins/deficiency , Cathepsins/genetics , Dendritic Cells/immunology , Endopeptidases/deficiency , Foot , Inflammation/immunology , Interferon-gamma/biosynthesis , Leishmania major/immunology , Macrophages/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Parasite Load , Signal Transduction , Th1 Cells/immunology , Toll-Like Receptor 9/genetics , Toll-Like Receptor 9/immunologyABSTRACT
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.
Subject(s)
Caspase 1/metabolism , Cathepsins/metabolism , Inflammation/chemically induced , Lysosomes/drug effects , Macrophages, Peritoneal/drug effects , Particulate Matter/toxicity , Toxicity Tests/methods , Alum Compounds/toxicity , Animals , Biomarkers/metabolism , Cathepsins/deficiency , Cathepsins/genetics , Cells, Cultured , Dipeptides/toxicity , Dose-Response Relationship, Drug , Enzyme Activation , Immunity, Innate/drug effects , Inflammation/enzymology , Inflammation/genetics , Inflammation/immunology , Inflammation/pathology , Interleukin-1beta/metabolism , Kinetics , Lysosomes/enzymology , Lysosomes/immunology , Lysosomes/pathology , Macrophages, Peritoneal/enzymology , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/pathology , Mice, Inbred C57BL , Mice, Knockout , Nanoparticles , Polystyrenes/toxicity , Primary Cell Culture , Proteolysis , Silicon Dioxide/toxicity , Substrate SpecificityABSTRACT
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.
Subject(s)
Antigen-Presenting Cells/enzymology , Cathepsins/metabolism , Escherichia coli Infections/enzymology , Fluorescent Dyes/metabolism , Peptides/metabolism , Spectrometry, Fluorescence , Animals , Antigen-Presenting Cells/drug effects , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/microbiology , Cathepsins/antagonists & inhibitors , Cathepsins/deficiency , Cathepsins/genetics , Cells, Cultured , Cysteine Proteinase Inhibitors/pharmacology , Escherichia coli Infections/immunology , Escherichia coli Infections/microbiology , Hydrolysis , Leucine/analogs & derivatives , Leucine/pharmacology , Mice, Knockout , Reproducibility of Results , Substrate Specificity , Time FactorsABSTRACT
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.
Subject(s)
Carrier Proteins/metabolism , Cathepsins/metabolism , Interleukin-1beta/metabolism , Animals , Cathepsins/antagonists & inhibitors , Cathepsins/deficiency , Cathepsins/genetics , Enzyme Inhibitors/pharmacology , Inflammasomes/metabolism , Macrophages/metabolism , Mice , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein , Phenotype , Signal Transduction/drug effectsABSTRACT
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.
Subject(s)
Cathepsins/metabolism , Cysteine/metabolism , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/metabolism , Animals , Antigen Presentation , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cathepsins/antagonists & inhibitors , Cathepsins/deficiency , Dipeptides/pharmacology , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Encephalomyelitis, Autoimmune, Experimental/genetics , Histocompatibility Antigens Class II/immunology , Macrophages/immunology , Macrophages/metabolism , Male , Mice , Myelin-Oligodendrocyte Glycoprotein/immunology , Myelin-Oligodendrocyte Glycoprotein/metabolism , Sulfones/pharmacologyABSTRACT
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.
Subject(s)
Biological Clocks , Cathepsins/metabolism , Circadian Rhythm , Microglia/metabolism , Synapses/metabolism , Animals , Biological Clocks/genetics , CLOCK Proteins/genetics , CLOCK Proteins/metabolism , Cathepsins/deficiency , Circadian Rhythm/genetics , Dendritic Spines/metabolism , Gene Expression Regulation , Humans , Male , Mice , Mice, Inbred ICR , Motor Activity , Proteolysis , Recombinant Proteins/metabolism , SleepABSTRACT
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.
Subject(s)
Aortic Aneurysm, Abdominal/chemically induced , Aortic Aneurysm, Abdominal/physiopathology , Leukocytes, Mononuclear/pathology , Leukocytes, Mononuclear/physiology , Smoking/adverse effects , Animals , Aorta, Abdominal/pathology , Aorta, Abdominal/physiopathology , Aorta, Abdominal/ultrastructure , Aortic Aneurysm, Abdominal/pathology , Cathepsins/deficiency , Cathepsins/genetics , Cathepsins/physiology , Cell Count , Disease Models, Animal , Doxycycline/pharmacology , Leukocyte Elastase/deficiency , Leukocyte Elastase/genetics , Leukocyte Elastase/physiology , Male , Matrix Metalloproteinase 12/deficiency , Matrix Metalloproteinase 12/genetics , Matrix Metalloproteinase 12/physiology , Matrix Metalloproteinase 9/deficiency , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/physiology , Matrix Metalloproteinases/drug effects , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
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.
Subject(s)
Angiotensin II , Aorta, Abdominal/enzymology , Aortic Aneurysm, Abdominal/prevention & control , Apolipoproteins E/deficiency , Cathepsins/deficiency , Animals , Aorta, Abdominal/immunology , Aorta, Abdominal/pathology , Aortic Aneurysm, Abdominal/chemically induced , Aortic Aneurysm, Abdominal/enzymology , Aortic Aneurysm, Abdominal/genetics , Aortic Aneurysm, Abdominal/immunology , Aortic Aneurysm, Abdominal/pathology , Apolipoproteins E/genetics , Apoptosis , CD3 Complex/metabolism , Cathepsin K/metabolism , Cathepsins/genetics , Cells, Cultured , Collagen/metabolism , Disease Models, Animal , Elastin/metabolism , Inflammation/enzymology , Inflammation/immunology , Inflammation/prevention & control , Male , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Smooth, Vascular/enzymology , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/enzymology , Myocytes, Smooth Muscle/pathology , Neovascularization, Pathologic , T-Lymphocytes/enzymology , T-Lymphocytes/immunology , Time FactorsABSTRACT
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.
Subject(s)
Aneurysm/metabolism , Cathepsins/deficiency , Intracranial Arteriosclerosis/metabolism , Macrophages/cytology , Phagosomes/metabolism , Animals , Autophagy/physiology , Blotting, Western , DNA Primers/genetics , Histological Techniques , Immunohistochemistry , Macrophages/ultrastructure , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron, Transmission , Mitochondria/physiology , NF-kappa B/metabolism , Reactive Oxygen Species/metabolism , Real-Time Polymerase Chain ReactionABSTRACT
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.
Subject(s)
Aortic Diseases/etiology , Complement Activation , Dilatation, Pathologic/etiology , Mucopolysaccharidosis VII/complications , Animals , Aorta/metabolism , Aorta/pathology , Aorta/physiopathology , Aortic Diseases/metabolism , Aortic Diseases/physiopathology , Cathepsins/deficiency , Complement System Proteins/genetics , Complement System Proteins/metabolism , Elastin/metabolism , Gene Expression Profiling , Genetic Therapy , Glucuronidase/biosynthesis , Glucuronidase/blood , Glucuronidase/genetics , Glycosaminoglycans/metabolism , Male , Matrix Metalloproteinase 12/deficiency , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mucopolysaccharidosis VII/physiopathology , Mucopolysaccharidosis VII/therapy , Oligonucleotide Array Sequence Analysis , Pancreatic Elastase/genetics , Pancreatic Elastase/metabolism , Signal Transduction , Tissue Extracts , Up-RegulationABSTRACT
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.
Subject(s)
Biotechnology/methods , Bombyx/genetics , Cellulase/biosynthesis , Cellulose/metabolism , Fungal Proteins/biosynthesis , Larva/genetics , Recombinant Proteins/biosynthesis , Trichoderma/enzymology , Animals , Biodegradation, Environmental , Blotting, Western , Bombyx/metabolism , Bombyx/virology , Cathepsins/deficiency , Cathepsins/genetics , Cell Line , Cellulase/genetics , Chitinases/deficiency , Chitinases/genetics , Fungal Proteins/genetics , Gene Expression , Genetic Vectors , Larva/metabolism , Larva/virology , Nucleopolyhedroviruses/enzymology , Nucleopolyhedroviruses/genetics , Recombinant Proteins/genetics , Trichoderma/chemistry , Trichoderma/geneticsABSTRACT
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.
Subject(s)
Atherosclerosis/metabolism , Cathepsins/metabolism , Cystatin C/metabolism , Obesity/metabolism , Adipose Tissue/drug effects , Adipose Tissue/metabolism , Adipose Tissue/pathology , Animals , Atherosclerosis/enzymology , Cathepsins/antagonists & inhibitors , Cathepsins/deficiency , Cathepsins/genetics , Cystatin C/deficiency , Cystatin C/genetics , Gene Knockout Techniques , Humans , Obesity/drug therapy , Obesity/enzymology , Obesity/pathologyABSTRACT
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.