RESUMEN
Lung macrophages are the first line of defense against invading respiratory pathogens including SARS-CoV-2, yet activation of macrophage in the lungs can lead to hyperinflammatory immune response seen in severe COVID-19. Here we used human M1 and M2 polarized macrophages as a surrogate model of inflammatory and regulatory macrophages and explored whether immune complexes (IC) containing spike-specific IgG can trigger aberrant cytokine responses in macrophages in the lungs and associated lymph nodes. We show that IC of SARS-CoV-2 recombinant S protein coated with spike-specific monoclonal antibody induced production of Prostaglandin E2 (PGE2) in non-polarized (M0) and in M1 and M2-type polarized human macrophages only in the presence of D-dimer (DD), a fibrinogen degradation product, associated with coagulopathy in COVID-19. Importantly, an increase in PGE2 was also observed in macrophages activated with DD and IC of SARS-CoV-2 pseudovirions coated with plasma from hospitalized COVID-19 patients but not from healthy subjects. Overall, the levels of PGE2 in macrophages activated with DD and IC were as follows: M1â«M2>M0 and correlated with the levels of spike binding antibodies and not with neutralizing antibody titers. All three macrophage subsets produced similar levels of IL-6 following activation with DD+IC, however TNFα, IL-1ß, and IL-10 cytokines were produced by M2 macrophages only. Our study suggests that high titers of spike or virion containing IC in the presence of coagulation byproducts (DD) can promote inflammatory response in macrophages in the lungs and associated lymph nodes and contribute to severe COVID-19.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Complejo Antígeno-Anticuerpo/metabolismo , Mediadores de Inflamación/metabolismo , Dinoprostona/metabolismo , COVID-19/metabolismo , Macrófagos/metabolismo , Citocinas/metabolismoRESUMEN
An overreactive inflammatory response and coagulopathy are observed in patients with severe form of COVID-19. Since increased levels of D-dimer (DD) are associated with coagulopathy in COVID-19, we explored whether DD contributes to the aberrant cytokine responses. Here we show that treatment of healthy human monocytes with DD induced a dose dependent increase in production of pyrogenic mediator, Prostaglandin E2 (PGE2) and inflammatory cytokines, IL-6 and IL-8. The DD-induced PGE2 and inflammatory cytokines were enhanced significantly by co-treatment with immune complexes (IC) of SARS CoV-2 recombinant S protein or of pseudovirus containing SARS CoV-2 S protein (PVCoV-2) coated with spike-specific chimeric monoclonal antibody (MAb) containing mouse variable and human Fc regions. The production of PGE2 and cytokines in monocytes activated with DD and ICs was sensitive to the inhibitors of ß2 integrin and FcγRIIa, and to the inhibitors of calcium signaling, Mitogen-Activated Protein Kinase (MAPK) pathway, and tyrosine-protein kinase. Importantly, strong increase in PGE2 and in IL-6/IL-8/IL-1ß cytokines was observed in monocytes activated with DD in the presence of IC of PVCoV-2 coated with plasma from hospitalized COVID-19 patients but not from healthy donors. The IC of PVCoV-2 with convalescent plasma induced much lower levels of PGE2 and cytokines compared with plasma from hospitalized COVID-19 patients. PGE2 and IL-6/IL-8 cytokines produced in monocytes activated with plasma-containing IC, correlated well with the levels of spike binding antibodies and not with neutralizing antibody titers. Our study suggests that a combination of high levels of DD and high titers of spike-binding antibodies that can form IC with SARS CoV-2 viral particles might accelerate the inflammatory status of lung infiltrating monocytes leading to increased lung pathology in patients with severe form of COVID-19.
Asunto(s)
COVID-19 , Monocitos , Animales , Complejo Antígeno-Anticuerpo , COVID-19/terapia , Citocinas/metabolismo , Dinoprostona/metabolismo , Productos de Degradación de Fibrina-Fibrinógeno , Humanos , Inmunización Pasiva , Factores Inmunológicos/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Ratones , Glicoproteína de la Espiga del Coronavirus/metabolismo , Sueroterapia para COVID-19RESUMEN
Severe dry mouth in patients with Sjögren's Syndrome, or radiation therapy for patients with head and neck cancer, significantly compromises their oral health and quality of life. The current clinical management of xerostomia is limited to palliative care as there are no clinically-proven treatments available. Previously, our studies demonstrated that mouse bone marrow-derived mesenchymal stem cells (mMSCs) can differentiate into salivary progenitors when co-cultured with primary salivary epithelial cells. Transcription factors that were upregulated in co-cultured mMSCs were identified concomitantly with morphological changes and the expression of acinar cell markers, such as α-amylase (AMY1), muscarinic-type-3-receptor(M3R), aquaporin-5(AQP5), and a ductal cell marker known as cytokeratin 19(CK19). In the present study, we further explored inductive molecules in the conditioned media that led to mMSC reprogramming by high-throughput liquid chromatography with tandem mass spectrometry and systems biology. Our approach identified ten differentially expressed proteins based on their putative roles in salivary gland embryogenesis and development. Additionally, systems biology analysis revealed six candidate proteins, namely insulin-like growth factor binding protein-7 (IGFBP7), cysteine-rich, angiogenetic inducer, 61(CYR61), agrin(AGRN), laminin, beta 2 (LAMB2), follistatin-like 1(FSTL1), and fibronectin 1(FN1), for their potential contribution to mMSC transdifferentiation during co-culture. To our knowledge, our study is the first in the field to identify soluble inductive molecules that drive mMSC into salivary progenitors, which crosses lineage boundaries.
Asunto(s)
Transdiferenciación Celular , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Proteoma/metabolismo , Glándulas Salivales/citología , Transducción de Señal , Animales , Forma de la Célula/efectos de los fármacos , Transdiferenciación Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Ontología de Genes , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacosRESUMEN
Fever and inflammatory responses were observed in some subjects in early clinical trials of vaccines adjuvanted with muramyl dipeptide (MDP), a NOD2 agonist. Biosynthesis of Prostaglandin E2 (PGE2) that transmits febrile signals to the brain is controlled by an inducible enzyme, Cyclooxygenase 2 (COX-2). MDP alone was not sufficient to induce expression of COX-2 and PGE2 production in vitro. Conditioned medium prepared from Peripheral Blood Mononuclear Cells (PBMCs)-derived CD3-bead purified human T cells (TCM) dramatically increased COX2 gene transcription, COX-2 protein expression, and PGE2 production in MDP-treated monocytes. We explored epigenetic changes at the COX2 promoter using Chromatin Immunoprecipitation assay (ChIP). Increase in COX2 transcription correlated with increased recruitment of RNA polymerase II (Pol II) and p300 histone acetyl transferase (HAT) to the COX2 promoter in monocytes activated with MDP and TCM. The role of p300 HAT was confirmed by using C646, an inhibitor of p300, that reduced binding of acetylated H3 and H4 histones at the COX2 promoter, COX2 transcription, and PGE2 production in monocytes. Binding of p300, Nuclear Factor Kappa B (NF-κB), and Pol II to the COX2 promoter was also sensitive to inhibitors of Mitogen-Activated Protein Kinase (MAPK) pathway and to antibodies against Macrophage-1 (Mac-1) integrin in MDP/TCM-treated monocytes. Importantly, recombinant Glycoprotein Ib alfa (GPIbα), the recently identified factor in TCM, increased binding of NF-κB, p300, and of Pol II to the COX2 promoter and COX2 transcription in MDP-treated monocytes. Our findings suggest that a second signal through Mac-1 and MAPK is triggered by a T cell derived soluble GPIbα protein leading to the assembly of the transcription machinery at the COX2 promoter and production of PGE2 in human monocytes in response to MDP/NOD2 activation.
Asunto(s)
Dinoprostona/metabolismo , Proteína p300 Asociada a E1A/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Monocitos/metabolismo , Transducción de Señal/fisiología , Linfocitos T/metabolismo , Acetilmuramil-Alanil-Isoglutamina/farmacología , Adyuvantes Inmunológicos/farmacología , Células Cultivadas , Ciclooxigenasa 2/metabolismo , Fiebre/metabolismo , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Antígeno de Macrófago-1/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Monocitos/efectos de los fármacos , FN-kappa B/metabolismo , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/fisiología , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Células TH1/efectos de los fármacos , Células TH1/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/fisiologíaRESUMEN
Sjögren's syndrome (SjS) is an autoimmune disease that destroys the salivary glands and results in severe dry mouth. Mesenchymal stem cell (MSC) transplantation has been recently proposed as a promising therapy for restoring cells in multiple degenerative diseases. We have recently utilized advanced proteomics biochemical assays to identify the key molecules involved in the mesenchymal-epithelial transition (MET) of co-cultured mouse bone-marrow-derived MSCs mMSCs with primary salivary gland cells. Among the multiple transcription factors (TFs) that were differentially expressed, two major TFs were selected: muscle, intestine, and stomach expression-1 (MIST1) and transcription factor E2a (TCF3). These factors were assessed in the current study for their ability to drive the expression of acinar cell marker, alpha-salivary amylase 1 (AMY1), and ductal cell marker, cytokeratin19 (CK19), in vitro. Overexpression of MIST1-induced AMY1 expression while it had little effect on CK19 expression. In contrast, TCF3 induced neither of those cellular markers. Furthermore, we have identified that mMSCs express muscarinic-type 3 receptor (M3R) mainly in the cytoplasm and aquaporin 5 (AQP5) in the nucleus. While MIST1 did not alter M3R levels in mMSCs, a TCF3 overexpression downregulated M3R expressions in mMSCs. The mechanisms for such differential regulation of glandular markers by these TFs warrant further investigation.
Asunto(s)
Amilasas/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células Madre Mesenquimatosas/metabolismo , Glándulas Salivales/metabolismo , Animales , Acuaporina 5/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Técnicas de Cocultivo/métodos , Regulación hacia Abajo/fisiología , Regulación de la Expresión Génica/fisiología , Humanos , Queratina-19/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteómica/métodos , Síndrome de Sjögren/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Lipid droplet accumulation has been related to salivary gland hypofunction in diabetes. In this study, the effect of laser irradiation on the parotid glands (PGs) of diabetic rats was analyzed with regard to its effect on lipid droplet accumulation, intracellular calcium concentration and calmodulin expression. The animals were distributed into 6 groups: D0, D5, D20 and C0, C5, C20, for diabetic (D) and control animals (C), respectively. Twenty-nine days following diabetes induction, PGs of groups D5 and C5; D20 and C20 were irradiated with 5 and 20 J/cm2 of a red diode laser at 100 mW, respectively. After 24 hours, PGs were removed for histological, biochemical, and western blotting analysis. The diabetic animals showed lipid droplet accumulation, which was decreased after irradiation. Ultrastructurally, the droplets were nonmembrane bound and appeared irregularly located in the cytoplasm. Moreover, diabetic animals showed an increased intracellular calcium concentration. In contrast, after laser irradiation a progressive decrease in the concentration of this ion was observed, which would be in agreement with the results found in the increased expression of calmodulin in D20. These data are promising for using laser to decrease lipid droplet accumulation in PGs, however, more studies are necessary to better understand its mechanisms. Micrographs showing decreased lipid accumulation after laser irradiation in light micrographs (LM), and morphology of lipid droplet in transmission electron microscopic (TEM). LM: (A) PGs from nondiabetic rats that did not receive Laser irradiation (LI), (B) PGs from nondiabetic rats that received a dose of 20 J/cm2 , (C) lipid accumulation (arrows) in the secretory cells from diabetic rats that did not receive irradiation, (D) reduction of lipid accumulation in the secretory cells from diabetic rats that received a dose of 20 J/cm2 and TEM: (E) scale bar = 5 µm, (F) scale bar = 1 µm, and (G) scale bar = 0.5 µm.
Asunto(s)
Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/terapia , Gotas Lipídicas/metabolismo , Gotas Lipídicas/efectos de la radiación , Terapia por Luz de Baja Intensidad , Glándula Parótida/patología , Glándula Parótida/efectos de la radiación , Animales , Calcio/metabolismo , Femenino , Espacio Intracelular/metabolismo , Espacio Intracelular/efectos de la radiación , Ratas , Ratas WistarRESUMEN
Severe xerostomia (dry mouth) compromises the quality of life in patients with Sjögren's syndrome or radiation therapy for head and neck cancer. A clinical management of xerostomia is often unsatisfactory as most interventions are palliative with limited efficacy. Following up our previous study demonstrating that mouse BM-MSCs are capable of differentiating into salivary epithelial cells in a co-culture system, we further explored the molecular basis that governs the MSC reprogramming by utilizing high-throughput iTRAQ-2D-LC-MS/MS-based proteomics. Our data revealed the novel induction of pancreas-specific transcription factor 1a (PTF1α), muscle, intestine and stomach expression-1 (MIST-1), and achaete-scute complex homolog 3 (ASCL3) in 7 day co-cultured MSCs but not in control MSCs. More importantly, a common notion of pancreatic-specific expression of PTF1 α was challenged for the first time by our verification of PTF1 α expression in the mouse salivary glands. Furthermore, a molecular network simulation of our selected putative MSC reprogramming factors demonstrated evidence for their perspective roles in salivary gland development. In conclusion, quantitative proteomics with extensive data analyses narrowed down a set of MSC reprograming factors potentially contributing to salivary gland regeneration. Identification of their differential/synergistic impact on MSC conversion warrants further investigation.
Asunto(s)
Diferenciación Celular/efectos de la radiación , Proteoma/metabolismo , Glándulas Salivales/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Western Blotting , Diferenciación Celular/genética , Células Cultivadas , Análisis por Conglomerados , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Proteómica , Calidad de Vida , Glándulas Salivales/fisiología , Espectrometría de Masas en Tándem , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Anti-muscarinic type 3 receptor autoantibodies (anti-M3R) are reported as potential inhibitors of saliva secretion in Sjögren's syndrome (SjS). However, despite extensive efforts to establish an anti-M3R detection method, there is no clinical test available for these autoantibodies. The purpose of this study was to propose inclusion of anti-M3R testing for SjS diagnosis through investigation of their prevalence using a modified In-Cell Western (ICW) assay. A stable cell line expressing human M3R tagged with GFP (M3R-GFP) was established to screen unadsorbed and adsorbed plasma from primary SjS (n=24), rheumatoid arthritis (RA, n=18), systemic lupus erythematosus (SLE, n=18), and healthy controls (HC, n=23). Anti-M3R abundance was determined by screening for the intensity of human IgG interacting with M3R-GFP cells by ICW assay, as detected by an anti-human IgG IRDye800-conjugated secondary antibody and normalized to GFP. Method comparisons and receiver-operating-characteristic (ROC)-curve analyses were performed to evaluate the diagnostic value of our current approaches. Furthermore, clinical parameters of SjS were also analyzed in association with anti-M3R. Anti-M3R was significantly elevated in SjS plasma in comparison with HC, SLE, or RA (P<0.01). SjS anti-M3R intensities were greater than two-standard deviations above the HC mean for both unadsorbed (16/24, 66.67%) and adsorbed (18/24, 75%) plasma samples. Furthermore, anti-M3R was associated with anti-SjS-related-antigen A/Ro positivity (P=0.0353). Linear associations for anti-M3R intensity indicated positive associations with focus score (R(2)=0.7186, P<0.01) and negative associations with saliva flow rate (R(2)=0.3052, P<0.05). Our study strongly supports our rationale to propose inclusion of anti-M3R for further testing as a non-invasive serological marker for SjS diagnosis.
Asunto(s)
Autoantígenos/inmunología , ARN Citoplasmático Pequeño/inmunología , Receptor Muscarínico M3/inmunología , Ribonucleoproteínas/inmunología , Pruebas Serológicas/métodos , Síndrome de Sjögren/diagnóstico , Adulto , Anciano , Artritis Reumatoide/diagnóstico , Autoanticuerpos/sangre , Femenino , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Lupus Eritematoso Sistémico/diagnóstico , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Curva ROC , Receptor Muscarínico M3/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Tooth derived stem cells or dental stem cells are categorized according to the location from which they are isolated and represent a promising source of cells for regenerative medicine. Originally, as one kind of mesenchymal stem cells, they are considered an alternative of bone marrow stromal cells. They share many commonalties but maintain differences. Considering their original function in development and the homeostasis of tooth structures, many applications of these cells in dentistry have aimed at tooth structure regeneration; however, the application in other than tooth structures has been attempted extensively. The availability from discarded or removed teeth can be an innate benefit as a source of autologous cells. Their origin from the neural crest results in exploitation of neurological and numerous other applications. This review briefly highlights current and future perspectives of the regenerative applications of tooth derived stem cells in areas beyond tooth regeneration.
RESUMEN
Sjögren's syndrome (SjS) is an autoimmune condition that primarily affects salivary and lacrimal glands, causing loss of secretion. We have previously shown that microRNA-146a (miR-146a) is over-expressed in the salivary glands and peripheral blood mononuclear cells (PBMC) of SjS-prone mice (C57BL/6.NOD-Aec1Aec2, B6DC) and in PBMC of SjS patients. The purpose of this research was to identify a target molecule of miR-146a and identify subpopulations of cells affected by altered miR-146a in the salivary glands of SjS-prone mice. In silico analyses identified costimulatory molecule CD80 as a potential target of miR-146a. Luciferase assay of the human CD80 3'untranslated region demonstrated miR-146a directly inhibited CD80 protein expression as indicated by reduced luciferase reporter expression and an examination of B6DC salivary glands revealed a reduction in CD80 protein. More interestingly, the specific reduction in CD80 protein was detected from the salivary gland epithelial cell population and in interstitial dendritic cells in the glands as well. The reduction in CD80 protein levels in salivary gland epithelial cells were negatively associated with elevated miR-146a expression. Therefore, this study provides the first indication that salivary gland epithelial cells may be critically involved in SjS progression by altering CD86:CD80 protein ratio in response to miR-146a upregulation.
Asunto(s)
Antígeno B7-1/biosíntesis , Antígeno B7-2/biosíntesis , Regulación de la Expresión Génica/inmunología , MicroARNs/genética , Síndrome de Sjögren/genética , Animales , Antígeno B7-1/genética , Antígeno B7-1/inmunología , Antígeno B7-2/inmunología , Western Blotting , Modelos Animales de Enfermedad , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/genética , Humanos , Ratones , Ratones Endogámicos C57BL , MicroARNs/inmunología , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Síndrome de Sjögren/inmunología , Transfección , Regulación hacia ArribaRESUMEN
Patients with Sjögren's syndrome or head and neck cancer patients who have undergone radiation therapy suffer from severe dry mouth (xerostomia) due to salivary exocrine cell death. Regeneration of the salivary glands requires a better understanding of regulatory mechanisms by which stem cells differentiate into exocrine cells. In our study, bone marrow-derived mesenchymal stem cells were co-cultured with primary salivary epithelial cells from C57BL/6 mice. Co-cultured bone marrow-derived mesenchymal stem cells clearly resembled salivary epithelial cells, as confirmed by strong expression of salivary gland epithelial cell-specific markers, such as alpha-amylase, muscarinic type 3 receptor, aquaporin-5, and cytokeratin 19. To identify regulatory factors involved in this differentiation, transdifferentiated mesenchymal stem cells were analyzed temporarily by two-dimensional-gel-electrophoresis, which detected 58 protein spots (>1.5 fold change, p<0.05) that were further categorized into 12 temporal expression patterns. Of those proteins only induced in differentiated mesenchymal stem cells, ankryin-repeat-domain-containing-protein 56, high-mobility-group-protein 20B, and transcription factor E2a were selected as putative regulatory factors for mesenchymal stem cell transdifferentiation based on putative roles in salivary gland development. Induction of these molecules was confirmed by RT-PCR and western blotting on separate sets of co-cultured mesenchymal stem cells. In conclusion, our study is the first to identify differentially expressed proteins that are implicated in mesenchymal stem cell differentiation into salivary gland epithelial cells. Further investigation to elucidate regulatory roles of these three transcription factors in mesenchymal stem cell reprogramming will provide a critical foundation for a novel cell-based regenerative therapy for patients with xerostomia.
Asunto(s)
Células Epiteliales/metabolismo , Células Madre Mesenquimatosas/metabolismo , Glándulas Salivales/citología , Animales , Biomarcadores/metabolismo , Transdiferenciación Celular , Técnicas de Cocultivo , Biología Computacional , Células Epiteliales/citología , Masculino , Células Madre Mesenquimatosas/citología , Ratones , Proteoma , Proteómica/métodos , Factores de Transcripción/metabolismoRESUMEN
Sjögren's syndrome (SjS) is a chronic autoimmune disease that mainly targets the salivary and lacrimal glands. It has been controversial whether anti-muscarinic type 3 receptor (α-M3R) autoantibodies in patients with SjS inhibit intracellular trafficking of aquaporin-5 (AQP5), water transport protein, leading to secretory dysfunction. To address this issue, GFP-tagged human AQP5 was overexpressed in human salivary gland cells (HSG-hAQP5) and monitored AQP5 trafficking to the plasma membrane following carbachol (CCh, M3R agonist) stimulation. AQP5 trafficking was indeed mediated by M3R stimulation, shown in partial blockage of trafficking by M3R-antagonist 4-DAMP. HSG-hAQP5 pre-incubated with SjS plasma for 24 hours significantly reduced AQP5 trafficking with CCh, compared with HSG-hAQP5 pre-incubated with healthy control (HC) plasma. This inhibition was confirmed by monoclonal α-M3R antibody and pre-absorbed plasma. Interestingly, HSG-hAQP5 pre-incubated with SjS plasma showed no change in cell volume, compared to the cells incubated with HC plasma showing shrinkage by twenty percent after CCh-stimulation. Our findings clearly indicate that binding of anti-M3R autoantibodies to the receptor, which was verified by immunoprecipitation, suppresses AQP5 trafficking to the membrane and contribute to impaired fluid secretion in SjS. Our current study urges further investigations of clinical associations between SjS symptoms, such as degree of secretory dysfunction, cognitive impairment, and/or bladder irritation, and different profiles (titers, isotypes, and/or specificity) of anti-M3R autoantibodies in individuals with SjS.
Asunto(s)
Acuaporina 5/metabolismo , Receptor Muscarínico M3/genética , Glándulas Salivales/metabolismo , Síndrome de Sjögren/metabolismo , Adulto , Anciano , Acuaporina 5/genética , Autoanticuerpos/metabolismo , Calcio/metabolismo , Carbacol/farmacología , Línea Celular , Membrana Celular/efectos de los fármacos , Femenino , Humanos , Aparato Lagrimal/citología , Aparato Lagrimal/metabolismo , Persona de Mediana Edad , Transporte de Proteínas , Receptor Muscarínico M3/antagonistas & inhibidores , Receptor Muscarínico M3/inmunología , Glándulas Salivales/citología , Síndrome de Sjögren/patologíaRESUMEN
Loss-of-function mutations in the PARK7/DJ-1 gene cause early onset autosomal-recessive Parkinson disease. DJ-1 has been implicated in protection of neurons from oxidative stress and in regulation of transcriptional activity. However, whether there is a relationship between the subcellular localization of DJ-1 and its function remains unknown. Therefore, we examined the subcellular localization of DJ-1 during dopaminergic neurodegeneration induced by various insults. Immunoblotting and immunocytochemistry showed that the nuclear pool of DJ-1 dramatically increased in both MN9D dopaminergic neuronal cells and primary cultures of mesencephalic dopaminergic neurons after 6-hydroxydopamine (6-OHDA) treatment. This was paralleled by a corresponding decrease in its cytosolic level, indicating drug-induced nuclear translocation of DJ-1. The same phenomenon was detected in other cell death paradigms induced by pro-oxidants including hydrogen peroxide and cupric chloride. Consequently, cotreatment with the antioxidant N-acetyl-l-cysteine blocked the translocation of DJ-1 into the nucleus. However, mutation at cysteine 106 had no effect on the translocation of DJ-1 into the nucleus, suggesting that reactive oxygen species-mediated downstream signaling and/or modifications other than oxidative modification are involved in its nuclear translocation. Ectopic expression of nucleus localization signal (NLS)-tagged DJ-1 prevented cell death from 6-OHDA. We investigated whether nuclear DJ-1 was involved in transcriptional regulation and found that DJ-1 was localized in promyelocytic leukemia bodies, and this localization increased upon 6-OHDA treatment. We also confirmed that binding of DJ-1 and promyelocytic leukemia bodies indeed increased after 6-OHDA treatment. Consequently, expression levels of acetylated p53 and PUMA were downregulated in cells overexpressing DJ-1 or NLS-tagged DJ-1. Taken together, our data suggest that nuclear translocation of DJ-1 may protect neurons from cell death after oxidative stress.
Asunto(s)
Apoptosis , Neuronas Dopaminérgicas/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Oncogénicas/metabolismo , Estrés Oxidativo , Acetilcisteína/farmacología , Transporte Activo de Núcleo Celular , Animales , Antioxidantes/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasa 3/metabolismo , Células Cultivadas , Cobre/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Humanos , Ratones , Oxidantes/farmacología , Oxidopamina/farmacología , Cultivo Primario de Células , Proteína Desglicasa DJ-1 , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Loss-of-function mutations in the DJ-1/PARK7 gene are responsible for early-onset autosomal-recessive Parkinson's disease. DJ-1 is implicated in the protection of neurons from oxidative stress by scavenging hydrogen peroxide and regulating the transcriptional activity of multiple pathways. Here, we attempted to identify the protein profiles modulated by DJ-1 in MN9D dopaminergic neurons following 6-hydroxydopamine (6-OHDA) treatment. We found that reactive oxygen species (ROS) levels increased in DJ-1-deficient cells that were either untreated or subjected to 6-OHDA treatment. The incidence of apoptosis after 6-OHDA treatment was increased in DJ-1 knockdown cells. Using these cells, we then performed two-dimensional gel electrophoresis in conjunction with mass spectrometry to assess changes in protein profiles before and after 6-OHDA treatment. Several protein spots were positively or negatively altered in DJ-1-deficient cells with or without 6-OHDA. Among the altered proteins, immunoblot analysis confirmed an increase in galectin-7 and a decrease in peroxiredoxin-6 in DJ-1 knockdown cells. Moreover, transcriptional levels of putative p53 target proteins, including selenophosphate synthetase 1 and glycogen phosphorylase, were increased in the DJ-1 knockdown cells. Taken together, our data suggest that increases in pro-apoptotic proteins and decreases in anti-apoptotic proteins render DJ-1 knockdown cells more susceptible to oxidative stress.
Asunto(s)
Apoptosis/fisiología , Citoprotección , Neuronas Dopaminérgicas/fisiología , Proteínas Oncogénicas/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Técnicas de Silenciamiento del Gen , Ratones , Proteínas Oncogénicas/genética , Oxidopamina/farmacología , Peroxirredoxinas , Proteína Desglicasa DJ-1 , Proteómica , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The mouse model is the one of the most frequently used and well-established animal models, and is currently used in many research areas. To date, various mouse models have been utilized to elucidate underlying causes of multifactorial autoimmune conditions, including pathological immune components and specific signaling pathways. This review summarizes the more recent mouse models for Sjögren's syndrome, a systemic autoimmune disease characterized by lymphocytic infiltration in the exocrine glands, such as the salivary and lacrimal glands, and loss of secretory function, resulting in dry mouth and dry eyes in patients. Although every Sjögren's syndrome mouse model resembles the major symptoms or phenotypes of Sjögren's syndrome conditions in humans, the characteristics of each model are variable. Moreover, to date, there is no single mouse model that can completely replicate the human conditions. However, unique features of each mouse model provide insights into the roles of potential etiological and immunological factors in the development and progression of Sjögren's syndrome. Here, we will overview the Sjögren's syndrome mouse models. Lessons from these mouse models will aid us to understand underlying immune dysregulation in autoimmune diseases in general, and will guide us to direct future research towards appropriate diagnostic and therapeutic strategies.
Asunto(s)
Enfermedades Autoinmunes/etiología , Enfermedades Autoinmunes/inmunología , Modelos Animales de Enfermedad , Síndrome de Sjögren/etiología , Síndrome de Sjögren/inmunología , Animales , Enfermedades Autoinmunes/patología , Humanos , Síndrome de Sjögren/patologíaRESUMEN
In this study, alkaline phytase was added to whole-wheat bread and the phytate content and mineral profiles were compared to commercially available acidic phytase. At neutral pH, some phytate (approximately 20%) was degraded by endogenous phytase in wheat flour, while 40% of phytate was hydrolyzed by alkaline phytase DS11 and a 35% reduction was observed with acidic phytase. Most of the enzymatic activity occurred during the proofing stage, and the rate of reaction depended on pH. DS11 phytase effectively degraded the phytate level within a 30 min treatment at pH 7; however, at least 60 min was needed with acidic phytase to achieve the same hydrolysis level. Mineral profiles were also dramatically affected by the phytate reduction. The biggest increase was observed in Fe²âº by the phytase treatment. The Fe²âº content increased 10-fold at pH 7 and 8-fold at pH 5 with alkaline phytase DS11. Alkaline phytase DS11 was shown to be effective at phytate reduction in whole-wheat bread preparation. Additionally, phytate degradation enhanced the mineral availability of bread.
Asunto(s)
6-Fitasa/metabolismo , Proteínas Bacterianas/metabolismo , Pan/análisis , Manipulación de Alimentos , Alimentos Funcionales/análisis , Minerales/química , Ácido Fítico/análisis , Bacillus/enzimología , Cromatografía Líquida de Alta Presión , Fibras de la Dieta/análisis , Fibras de la Dieta/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Hierro de la Dieta/análisis , Minerales/análisis , Oxidación-Reducción , Ácido Fítico/metabolismo , Solubilidad , Espectrofotometría Atómica , Factores de TiempoRESUMEN
Mycophenolic acid (MPA), widely used to prevent organ transplant rejection, may induce toxicity and impair function in beta-cells. Mechanisms of MPA-induced cell death have not been fully explored. In this study, we examined gene expression patterns in INS-1E cells and isolated primary rat islets following MPA treatment using the Illumina-cDNA microarray. The MPA treatment decreases RhoGDI-alpha gene expression, which points to apoptosis by JNK activation through a MAPKs-dependent pathway. A strong association between RhoGDI-alpha and Rac1 activation during MPA-induced apoptosis is also consistent with apoptosis through JNK. Suppression of RhoGDI-alpha using siRNA and gene over-expression both affected the cell death rate, consistent with Rac1 activation and downstream activation of MAPKs signaling. We confirmed that Rac1 protein mediates the interaction between RhoGDI-alpha and JNK signaling. We conclude that MPA-induced cell death in primary beta-cells and an insulin-secreting cell line proceeds through RhoGDI-alpha down-regulation linked to Rac1 activation, with subsequent activation of JNK. The RhoGDI-alpha/Rac1/JNK pathway may present a key to intervention in MPA-induced islet apoptosis.
Asunto(s)
Apoptosis , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Ácido Micofenólico/toxicidad , Animales , Regulación de la Expresión Génica , Inhibidores de Disociación de Guanina Nucleótido/genética , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Islotes Pancreáticos/efectos de los fármacos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Proteína de Unión al GTP rac1/metabolismo , Inhibidores de la Disociación del Nucleótido Guanina rho-EspecíficoRESUMEN
Mycophenolic acid (MPA)-induced beta-cell toxicity is an important factor for islet graft function. The signal transduction mechanisms underlying this process have not been fully explored. Using a proteomics approach, we examined protein expression patterns in MPA-treated RIN-5 cells and found that RhoGDI-alpha expression is altered by MPA-treatment. We examined the relationship between RhoGDI-alpha expression and activated JNK during MPA-induced apoptosis. Cells were treated with N-acetyl-cysteine (NAC), caspase inhibitor, JNK inhibitor, guanosine or GTP for 1 h before being treated with MPA. To investigate the regulatory effects of RhoGDI-alpha on JNK activity, we examined cells showing either elevated or reduced expression of RhoGDI-alpha as a result of transfection with cDNA or siRNA constructs, respectively. MPA significantly increased cell death, caspase-3 expression and JNK activation, but it decreased the expression of a protein spot 25 observed by two-dimensional electrophoresis. This protein 25 was identified as RhoGDI-alpha by mass spectrometry. MPA-induced cell death and down-regulation of RhoGDI-alpha were prevented by guanosine, GTP or a JNK inhibitor. However, MPA-induced cell death was partially restored by treatment with a caspase inhibitor, but not by NAC treatment. RhoGDI-alpha expression was not affected by treatment with NAC or caspase inhibitor. Over-expression of RhoGDI-alpha increased cell viability and decreased activated JNK expression following exposure to MPA, whereas knockdown of RhoGDI-alpha enhanced MPA-induced cell death and increased the activation of JNK. In conclusion, MPA induces significant apoptosis in insulin-secreting cells via down-regulation of RhoGDI-alpha linked with increased JNK expression. This RhoGDI-alpha/JNK pathway might be the focus of therapeutic target for the prevention of MPA-induced islet apoptosis.
Asunto(s)
Apoptosis , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ácido Micofenólico/farmacología , Análisis de Varianza , Animales , Apoptosis/efectos de los fármacos , Secuencia de Bases , Caspasa 3/metabolismo , Línea Celular , Electroforesis en Gel Bidimensional , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/enzimología , Proteómica , Ratas , Transducción de Señal/efectos de los fármacos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Inhibidores de la Disociación del Nucleótido Guanina rho-EspecíficoRESUMEN
The aim of this study was to investigate changes in protein profiles during the early phase of dopaminergic neuronal death using two-dimensional gel electrophoresis in conjunction with mass spectrometry. Several protein spots were identified whose expression was significantly altered following treatment of MN9D dopaminergic neuronal cells with 6-hydroxydopamine (6-OHDA). In particular, we detected oxidative modification of thioredoxin-dependent peroxidases (peroxiredoxins; PRX) in treated MN9D cells. Oxidative modification of PRX induced by 6-OHDA was blocked in the presence of N-acetylcysteine, suggesting that reactive oxygen species (ROS) generated by 6-OHDA induce oxidation of PRX. These findings were confirmed in primary cultures of mesencephalic neurons and in rat brain injected stereotaxically. Overexpression of PRX1 in MN9D cells (MN9D/PRX1) exerted neuroprotective effects against death induced by 6-OHDA through scavenging of ROS. Consequently, generation of both superoxide anion and hydrogen peroxide following 6-OHDA treatment was decreased in MN9D/PRX1. Furthermore, overexpression of PRX1 protected cells against 6-OHDA-induced activation of p38 MAPK and subsequent activation of caspase-3. In contrast, 6-OHDA-induced apoptotic death signals were enhanced by RNA interference-targeted reduction of PRX1 in MN9D cells. Taken together, our data suggest that the redox state of PRX may be intimately involved in 6-OHDA-induced dopaminergic neuronal cell death and also provide a molecular mechanism by which PRX1 exerts a protective role in experimental models of Parkinson disease.