RESUMO
Cystatin C is a potent cysteine protease inhibitor that plays an important role in various biological processes including cancer, cardiovascular diseases and neurodegenerative diseases. However, the role of CstC in inflammation is still unclear. In this study we demonstrated that cystatin C-deficient mice were significantly more sensitive to the lethal LPS-induced sepsis. We further showed increased caspase-11 gene expression and enhanced processing of pro-inflammatory cytokines IL-1ß and IL-18 in CstC KO bone marrow-derived macrophages (BMDM) upon LPS and ATP stimulation. Pre-treatment of BMDMs with the cysteine cathepsin inhibitor E-64d did not reverse the effect of CstC deficiency on IL-1ß processing and secretion, suggesting that the increased cysteine cathepsin activity determined in CstC KO BMDMs is not essential for NLRP3 inflammasome activation. The CstC deficiency had no effect on (mitochondrial) reactive oxygen species (ROS) generation, the MAPK signaling pathway or the secretion of anti-inflammatory cytokine IL-10. However, CstC-deficient BMDMs showed dysfunctional autophagy, as autophagy induction via mTOR and AMPK signaling pathways was suppressed and accumulation of SQSTM1/p62 indicated a reduced autophagic flux. Collectively, our study demonstrates that the excessive inflammatory response to the LPS-induced sepsis in CstC KO mice is dependent on increased caspase-11 expression and impaired autophagy, but is not associated with increased cysteine cathepsin activity.
Assuntos
Cistatina C/genética , Lipopolissacarídeos/efeitos adversos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Sepse/etiologia , Animais , Autofagia/genética , Caspases Iniciadoras/genética , Caspases Iniciadoras/metabolismo , Cistatina C/deficiência , Inflamassomos/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Leucina/análogos & derivados , Leucina/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismo , Sepse/mortalidade , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais , Taxa de Sobrevida , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Regulação para CimaRESUMO
BACKGROUND AND PURPOSE: Endogenous neuroprotection can be induced by ischemic and nonischemic preconditioning. However, not all subjects that undergo preconditioning exhibit similar favorable outcome. This study is to explore the molecules responsible for this phenomenon and find new therapeutic targets for stroke. METHODS: Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion. High-throughput proteomic technique, isobaric tag for relative and absolute quantification, was used to screen differentially expressed proteins in the rats that developed ischemic tolerance from hyperbaric oxygen (HBO) preconditioning. The proteomic results were verified by Western blot and ELISA. Then, short interfering RNA and gene knockout rats were used to further determine the pivotal role of candidate proteins in HBO preconditioning-induced endogenous neuroprotection. Finally, lysosomal permeability was tested to elaborate the mechanism underlying this intrinsic neuroprotective effect. RESULTS: Nine proteins differentially expressed in the serum of rats, which acquired benefits from HBO preconditioning, were screened and identified. Western blot and ELISA revealed that cystatin C (CysC) and mannose-binding lectin protein C were uniquely changed in rats with smaller infarction after HBO preconditioning and cerebral ischemia. Knockdown and knockout of CysC abolished HBO-induced neuroprotection. Moreover, HBO-induced endogenous CysC elevation preserved lysosomal membrane integrity after stroke in wild-type rats but not in CysC siRNA infusion or CysC-/- rats. Most importantly, exogenous CysC also induced neuroprotection against ischemic/reperfusion injury. CONCLUSIONS: CysC is a crucial determinant contributing to endogenous neuroprotection. It is also a novel candidate for stroke treatment through maintaining lysosomal membrane integrity.
Assuntos
Cistatina C/sangue , Fármacos Neuroprotetores/sangue , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/prevenção & controle , Animais , Biomarcadores/sangue , Cistatina C/deficiência , Cistatina C/genética , Técnicas de Silenciamento de Genes/métodos , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/genéticaRESUMO
Autophagy dysfunction in mouse atherosclerosis models has been associated with increased lipid accumulation, apoptosis and inflammation. Expression of cystatin C (CysC) is decreased in human atheroma, and CysC deficiency enhances atherosclerosis in mice. Here, we first investigated the association of autophagy and CysC expression levels with atheroma plaque severity in human atherosclerotic lesions. We found that autophagy proteins Atg5 and LC3ß in advanced human carotid atherosclerotic lesions are decreased, while markers of dysfunctional autophagy p62/SQSTM1 and ubiquitin are increased together with elevated levels of lipid accumulation and apoptosis. The expressions of LC3ß and Atg5 were positively associated with CysC expression. Second, we investigated whether CysC expression is involved in autophagy in atherosclerotic apoE-deficient mice, demonstrating that CysC deficiency (CysC(-/-) ) in these mice results in reduction of Atg5 and LC3ß levels and induction of apoptosis. Third, macrophages isolated from CysC(-/-) mice displayed increased levels of p62/SQSTM1 and higher sensitivity to 7-oxysterol-mediated lysosomal membrane destabilization and apoptosis. Finally, CysC treatment minimized oxysterol-mediated cellular lipid accumulation. We conclude that autophagy dysfunction is a characteristic of advanced human atherosclerotic lesions and is associated with reduced levels of CysC. The deficiency of CysC causes autophagy dysfunction and apoptosis in macrophages and apoE-deficient mice. The results indicate that CysC plays an important regulatory role in combating cell death via the autophagic pathway in atherosclerosis.
Assuntos
Aterosclerose/metabolismo , Aterosclerose/patologia , Autofagia , Cistatina C/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Idoso , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular , Cistatina C/deficiência , Progressão da Doença , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Permeabilidade , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Proteínas Recombinantes/farmacologiaRESUMO
Neuronal cell culture models have been used to demonstrate the protective effects of cystatin C against a variety of insults, including the toxicity induced by oligomeric and fibrillar amyloid ß (Aß). Here, we describe assays quantifying cystatin C protective effects against cytotoxicity induced by nutrient deprivation, oxidative stress, or cytotoxic forms of Aß. Three methods for the evaluation of either cell death or cell survival are described: measurement of metabolic activity, cell death, and cell division. The cell culture models used are murine primary cortical neurons and murine primary cerebral smooth muscle cells. The effects of exogenously applied cystatin C are studied by comparing the viability of nonstressed control, stressed control, and cystatin C-treated stressed cells. The effect of endogenous level of cystatin C expression is studied by comparing stressed primary cells isolated from brains of cystatin C transgenic, cystatin C knockout, and wild-type mice.
Assuntos
Córtex Cerebral/citologia , Cistatina C/metabolismo , Técnicas Citológicas/métodos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Animais , Técnicas de Cultura de Células , Divisão Celular , Proliferação de Células , Sobrevivência Celular , Cistatina C/deficiência , Cistatina C/genética , Espaço Extracelular/metabolismo , Fluoresceínas/metabolismo , Técnicas de Inativação de Genes , Camundongos , Estresse Oxidativo , Propídio/metabolismo , Coloração e RotulagemRESUMO
Cystatin C (CstC) is a cysteine protease inhibitor of major clinical importance. Low concentration of serum CstC is linked to atherosclerosis. CstC can prevent formation of amyloid ß associated with Alzheimer's disease and can itself form toxic aggregates. CstC regulates NO secretion by macrophages and is a TGF-ß antagonist. Finally, the serum concentration of CstC is an indicator of kidney function. Yet, little is known about the regulation of CstC expression in vivo. In this study, we demonstrate that the transcription factor IFN regulatory factor 8 (IRF-8) is critical for CstC expression in primary dendritic cells. Only those cells with IRF-8 bound to the CstC gene promoter expressed high levels of the inhibitor. Secretion of IL-10 in response to inflammatory stimuli downregulated IRF-8 expression and consequently CstC synthesis in vivo. Furthermore, the serum concentration of CstC decreased in an IL-10-dependent manner in mice treated with the TLR9 agonist CpG. CstC synthesis is therefore more tightly regulated than hitherto recognized. The mechanisms involved in this regulation might be targeted to alter CstC production, with potential therapeutic value. Our results also indicate that caution should be exerted when using the concentration of serum CstC as an indicator of kidney function in conditions in which inflammation may alter CstC production.
Assuntos
Cistatina C/biossíntese , Cistatina C/sangue , Regulação para Baixo/imunologia , Mediadores da Inflamação/fisiologia , Fatores Reguladores de Interferon/biossíntese , Interleucina-10/fisiologia , Animais , Transplante de Medula Óssea/imunologia , Transplante de Medula Óssea/patologia , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Cistatina C/deficiência , Células Dendríticas/classificação , Células Dendríticas/imunologia , Células Dendríticas/patologia , Regulação para Baixo/genética , Mediadores da Inflamação/antagonistas & inibidores , Fatores Reguladores de Interferon/deficiência , Fatores Reguladores de Interferon/fisiologia , Interleucina-10/metabolismo , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos TransgênicosRESUMO
BACKGROUND: Cysteine protease cathepsins are important in extracellular matrix protein degradation, cell apoptosis, and angiogenesis. Mice lacking cathepsins are protected from tumor progression in several animal models, suggesting that the regulation of cathepsin activities controls the growth of various malignant tumors. METHODS AND RESULTS: We tested the role of cathepsins using a mouse model of multistage epithelial carcinogenesis, in which the human keratin-14 promoter/enhancer drove the expression of human papillomavirus type 16 (HPV16) early region E6/E7 transgenes. During the progression of premalignant dysplasia, we observed increased expression of cysteine protease cathepsin S, but concomitantly reduced expression of cathepsin endogenous inhibitor cystatin C in the skin tissue extract. Absence of cystatin C in these transgenic mice resulted in more progression of dysplasia to carcinoma in situ on the face, ear, chest, and tail. Chest and ear skin extract real time PCR and immunoblot analysis, mouse serum sample ELISA, tissue immunohistological analysis, and tissue extract-mediated in vitro elastinolysis and collagenolysis assays demonstrated that cystatin C deficiency significantly increased cathepsin expression and activity. In skin from both the chest and ear, we found that the absence of cystatin C reduced epithelial cell apoptosis but increased proliferation. From the same tissue preparations, we detected significantly higher levels of pro-angiogenic laminin 5-derived γ2 peptides and concurrently increased neovascularization in cystatin C-deficient mice, compared to those from wild-type control mice. CONCLUSION: Enhanced cathepsin expression and activity in cystatin C-deficient mice contributed to the progression of dysplasia by altering premalignant tissue epithelial proliferation, apoptosis, and neovascularization.
Assuntos
Cistatina C/deficiência , Epiderme/metabolismo , Queratina-14/genética , Proteínas Oncogênicas Virais/genética , Animais , Apoptose , Catepsinas/genética , Catepsinas/metabolismo , Proliferação de Células , Cistatina C/genética , Cistatina C/metabolismo , Progressão da Doença , Epiderme/patologia , Feminino , Papillomavirus Humano 16/genética , Humanos , Hiperplasia , Immunoblotting , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas E7 de Papillomavirus/genética , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologiaRESUMO
An imbalance between cysteinyl cathepsins and their principal endogenous inhibitor cystatin C (CystC) may favor proteolysis in the pathogenesis of human abdominal aortic aneurysms (AAA), yet a direct role of CystC in AAA remains unproven. This study used CystC and apolipoprotein E (ApoE) compound mutant (CystC(-/-)ApoE(-/-)) mice to examine directly the role of cysteine protease/protease inhibitor imbalance in AAA formation in angiotensin II-induced AAA. CystC-deficiency increased lumenal diameter and lesion size compared with control mice. CystC(-/-) ApoE(-/-) lesions also demonstrated enhanced inflammatory cell accumulation, more severe elastin fragmentation, and fewer smooth muscle cells in the tunica media. Macrophage content, measured as percent positive area (23.2 +/- 1.4% versus 11.2 +/- 1.4%; P = 0.0003) and number of the CD4(+) T cells (ninefold; P = 0.048), increased significantly in CystC(-/-)ApoE(-/-) lesions. CystC deficiency increased cathepsin activity (5.5 fold; P = 0.001) in AAA, yielding greater elastin degradation and proangiogenic laminin-5 gamma2 peptide production, which may account for increased microvascularization in CystC(-/-)ApoE(-/-) compared with ApoE(-/-) lesions. Increased leukocyte adhesion molecule VCAM-1 expression and leukocyte proliferation might also promote inflammation in CystC-deficient AAA. These data indicate that CystC contributes to experimental AAA pathogenesis and that enhanced cysteine protease activity, due to the lack of CystC, favors inflammation in AAA lesions induced in atherosclerotic mice by promoting microvascularization and smooth muscle cell apoptosis as well as leukocytes adhesion and proliferation.
Assuntos
Angiotensina II/farmacologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/imunologia , Aterosclerose/fisiopatologia , Cistatina C/deficiência , Inflamação/imunologia , Animais , Aneurisma da Aorta Abdominal/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/patologia , Moléculas de Adesão Celular/metabolismo , Proliferação de Células , Cistatina C/genética , Humanos , Leucócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologiaRESUMO
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/patologiaRESUMO
Cystatin C (CC), a cysteine protease inhibitor involved in protein degradation, is a marker of Bunina bodies in lower motor neurons in amyotrophic lateral sclerosis (ALS). TAR-DNA binding protein-43 (TDP-43)-immunoreactive inclusions are also histological hallmarks of ALS but whether CC is found in motor neurons with or without TDP-43-positive inclusions in ALS is not known. To determine whether inclusion body formation affects cytoplasmic CC immunoreactivity, we examined spinal cords from 9 ALS patients and 12 control subjects by immunohistochemistry. Most anterior horn cells (AHCs) showed moderate to intense immunoreactivity in controls, whereas CC immunoreactivity was markedly decreased in AHCs in ALS cases. The proportion of CC-immunolabeled AHCs was reduced regardless of whether they contained Bunina bodies. In contrast, the proportion of CC-immunolabeled AHCs was significantly reduced in those with TDP-43 inclusions. Cystatin C immunoreactivity of astrocytes in the spinal gray matter and white matter in ALS was significantly decreased compared with controls. These findings suggest that the formation of TDP-43 inclusions, but not of Bunina bodies, may be linked to the content of CC in spinal motor neurons and that perturbations in endogenous levels of CC in neuronal and glial cells may be part of the neurodegenerative processes in ALS.
Assuntos
Esclerose Lateral Amiotrófica/imunologia , Astrócitos/imunologia , Cistatina C/imunologia , Neurônios Motores/imunologia , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Astrócitos/metabolismo , Astrócitos/patologia , Cistatina C/deficiência , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Medula Espinal/imunologia , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
The aim of the present investigation was to elucidate possible effects of cystatin C on inflammatory responses mediated by macrophages. Previously it has been shown that in vitro treatment of murine peritoneal macrophages with interferon-gamma (IFN-gamma) causes a down-regulation of cystatin C secretion. To investigate whether such changes in cystatin C expression in turn can affect inflammatory responses mediated by macrophages, we have compared effects of IFN-gamma on macrophages isolated from wild-type (cysC(+/+)) and cystatin C knockout (cysC(-/-)) mice. It was shown that IFN-gamma-primed cysC(-/-) macrophages exhibit significantly higher interleukin-10 (IL-10) but lower tumor necrosis factor-alpha (TNF-alpha) expression, and reduced nuclear factor (NF)-kappaB p65 activation, compared to similarly primed cysC(+/+) cells. Exogenously added cystatin C enhanced IFN-gamma-induced activation of NF-kappaB p65 and increased mRNA levels for inducible NO synthase (iNOS) in cysC(-/-) macrophages as well as levels of nitric oxide and TNF-alpha in the cell culture medium, in agreement with an enhanced pro-inflammatory response. Accordingly, IFN-gamma-induced IL-10 mRNA expression in cysC(-/-) macrophages was down-regulated by exogenously added cystatin C. Taken together, our data provide evidence that changes in cystatin C levels alter macrophage responses to IFN-gamma. The latter down-regulates the production of cystatin C, which leads to a suppressed inflammatory condition with enhanced IL-10 levels and down-regulated TNF-alpha and NF-kappaB. It is concluded that cystatin C through this effect can act as an immunomodulatory molecule.