RESUMEN
BACKGROUND/AIM: Macrophages prevail in the microenvironment of several tumors, including non-small-cell lung cancer (NSCLC), where they secrete pro-tumorigenic factors that contribute to cancer progression. This study investigated the role of macrophages on the resistance of epidermal growth factor receptor (EGFR)-mutated NSCLC cells to tyrosine kinase inhibitors (TKIs). MATERIALS AND METHODS: EGFR-mutated cell lines PC-9 and HCC827 were cocultured with macrophages and treated with TKIs (erlotinib and gefitinib). The effects of the macrophage-conditioned medium (macrophage CM) on gefitinib resistance and cell migration were also evaluated. RESULTS: Co-culture with macrophages significantly enhanced the resistance to erlotinib and gefitinib in PC-9 and HCC827 cells compared to that in cells cultured independently. Macrophage CM markedly induced gefitinib resistance in PC-9 cells, with maximum resistance observed at 50% CM concentration. This resistance persisted for up to 48 h post-CM removal. Macrophage CM inhibited gefitinib-induced apoptosis, as evidenced by the decreased expression of cleaved caspase-3, PARP, and BIM. Additionally, macrophage CM increased the migration ability of PC-9 cells, as shown by the wound healing and transwell migration assays. Studies have shown that TonEBP is crucial in cancer metastasis and drug resistance; we found that inhibition of TonEBP/NFAT5 expression reduced gefitinib resistance and migration in macrophage CM-induced PC-9 cells, indicating its role as mediator of these effects. CONCLUSION: Macrophages contribute to TKI resistance and enhance the migration of EGFR-mutated NSCLC cells through mechanisms involving TonEBP/NFAT5. Therefore, targeting TonEBP/NFAT5 represents a potential therapeutic strategy for overcoming macrophage-induced TKI resistance in NSCLC cells.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Movimiento Celular , Resistencia a Antineoplásicos , Gefitinib , Neoplasias Pulmonares , Macrófagos , Humanos , Gefitinib/farmacología , Movimiento Celular/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Línea Celular Tumoral , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Receptores ErbB/metabolismo , Receptores ErbB/genética , Apoptosis/efectos de los fármacos , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Antineoplásicos/farmacología , Medios de Cultivo Condicionados/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Técnicas de Cocultivo , Clorhidrato de Erlotinib/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacosRESUMEN
BACKGROUND: Primary cilia on the surface of eukaryotic cells serve as sensory antennas for the reception and transmission in various cell signaling pathways. They are dynamic organelles that rapidly form during differentiation and cell cycle exit. Defects in these organelles cause a group of wide-ranging disorders called ciliopathies. Tonicity-responsive enhancer-binding protein (TonEBP) is a pleiotropic stress protein that mediates various physiological and pathological cellular responses. TonEBP is well-known for its role in adaptation to a hypertonic environment, to which primary cilia have been reported to contribute. Furthermore, TonEBP is involved in a wide variety of other signaling pathways, such as Sonic Hedgehog and WNT signaling, that promote primary ciliogenesis, suggesting a possible regulatory role. However, the functional relationship between TonEBP and primary ciliary formation remains unclear. METHODS: TonEBP siRNAs and TonEBP-mCherry plasmids were used to examine their effects on cell ciliation rates, assembly and disassembly processes, and regulators. Serum starvation was used as a condition to induce ciliogenesis. RESULTS: We identified a novel pericentriolar localization for TonEBP. The results showed that TonEBP depletion facilitates the formation of primary cilia, whereas its overexpression results in fewer ciliated cells. Moreover, TonEBP controlled the expression and activity of aurora kinase A, a major negative regulator of ciliogenesis. Additionally, TonEBP overexpression inhibited the loss of CP110 from the mother centrioles during the early stages of primary cilia assembly. Finally, TonEBP regulated the localization of PCM1 and AZI1, which are necessary for primary cilia formation. CONCLUSIONS: This study proposes a novel role for TonEBP as a pericentriolar protein that regulates the integrity of centriolar satellite components. This regulation has shown to have a negative effect on ciliogenesis. Investigations into cilium assembly and disassembly processes suggest that TonEBP acts upstream of the aurora kinase A - histone deacetylase 6 signaling pathway and affects basal body formation to control ciliogenesis. Taken together, our data proposes previously uncharacterized regulation of primary cilia assembly by TonEBP.
Asunto(s)
Aurora Quinasa A , Centriolos , Cilios , Cilios/metabolismo , Humanos , Aurora Quinasa A/metabolismo , Aurora Quinasa A/genética , Centriolos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Histona Desacetilasa 6/metabolismo , Histona Desacetilasa 6/genética , Animales , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genéticaRESUMEN
Tonicity-responsive enhancer binding protein (TonEBP) is a stress-responsive protein that plays a critical role in the regulation of gene expression and cellular adaptation to stressful environments. Recent studies uncovered the novel role of TonEBP in the DNA damage response, which significantly impacts genomic stability. This review provides a comprehensive overview of the novel role of TonEBP in DNA damage repair, including its involvement in the DNA damage bypass pathway and the recognition and resolution of DNA damage-induced R-loop structures.
Asunto(s)
Daño del ADN , Reparación del ADN , Estructuras R-Loop , Humanos , Animales , Inestabilidad Genómica , ADN/metabolismoRESUMEN
BACKGROUND: Macrophages promote angiogenesis, metastasis, and drug resistance in several cancers. Similarly, TonEBP/NFAT5 induces metastasis in renal carcinoma and colon cancer cells. However, the role of this transcription factor and that of macrophages in lung cancer cells remains unclear. Therefore, this study investigated the effects of macrophages and TonEBP/NFAT5 expression on cisplatin resistance and migration in A549 lung adenocarcinoma cells. RESULTS: A549 cells were cultured alone or indirectly co-cultured with THP-1-derived macrophages using a transwell culture chamber. Cisplatin-induced cell death was markedly decreased and migration increased in co-cultured A549 cells. Macrophage-conditioned media (CM) showed a similar effect on drug resistance and migration. Cisplatin-induced apoptosis, DNA fragmentation, and cleaved apoptotic proteins PARP and caspase-3 were markedly reduced in macrophage CM-induced A549 cells. Here, ERK, p38, JNK, and NF-κB activities were increased by macrophage CM. Furthermore, the proteins involved in cisplatin resistance and cancer cell migration were identified using specific inhibitors of each protein. ERK and NF-κB inhibition considerably reduced cisplatin resistance. The increase in macrophage CM-induced migration was partially reduced by treatment with ERK, JNK, and NF-κB inhibitors. TonEBP/NFAT5 expression was increased by macrophages, resulting in increased cisplatin resistance, cell migration, and invasion. Moreover, RNAi-mediated knockdown of TonEBP/NFAT5 reduced cisplatin resistance, migration, and invasion in macrophage CM-induced A549 cells. CONCLUSIONS: These findings demonstrate that paracrine factors secreted from macrophages can change A549 cells, resulting in the induction of drug resistance against cisplatin and migration. In addition, the TonEBP/NFAT5 ratio, increased by macrophages, is an important regulator of the malignant transformation of cells.
Asunto(s)
Cisplatino , Neoplasias Pulmonares , Humanos , Cisplatino/farmacología , FN-kappa B , Células A549 , Factores de Transcripción , Neoplasias Pulmonares/tratamiento farmacológicoRESUMEN
Interactions between notochord and sclerotome are required for normal embryonic spine patterning, but whether the postnatal derivatives of these tissues also require interactions for postnatal intervertebral disc (IVD) growth and maintenance is less established. We report here the comparative analysis of four conditional knockout mice deficient for TonEBP, a transcription factor known to allow cells to adapt to changes in extracellular osmotic pressure, in specific compartments of the IVD. We show that TonEBP deletion in nucleus pulposus (NP) cells does not affect their survival or aggrecan expression, but promoted cell proliferation in the NP and in adjacent vertebral growth plates (GPs). In cartilage end plates/GPs, TonEBP deletion induced cell death, but also structural alterations in the adjacent NP cells and vertebral bodies. Embryonic or postnatal TonEBP loss generated similar IVD changes. In addition to demonstrating the requirement of TonEBP in the different compartments of the IVD, this comparative analysis uncovers the in vivo interdependency of the different IVD compartments during the growth of the postnatal IVD-vertebral units.
Asunto(s)
Disco Intervertebral , Factores de Transcripción NFATC , Animales , Ratones , Regulación de la Expresión Génica , Disco Intervertebral/metabolismo , Ratones Noqueados , Presión Osmótica , Factores de Transcripción/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismoRESUMEN
Transcription factors within microglia contribute to the inflammatory response following intracerebral hemorrhage (ICH). Therefore, we employed bioinformatics screening to identify the potential transcription factor tonicity-responsive enhancer-binding protein (TonEBP) within microglia. Inflammatory stimuli can provoke an elevated expression of TonEBP in microglia. Nevertheless, the expression and function of microglial TonEBP in ICH-induced neuroinflammation remain ambiguous. In our recent research, we discovered that ICH instigated an increased TonEBP in microglia in both human and mouse peri-hematoma brain tissues. Furthermore, our results indicated that TonEBP knockdown mitigates lipopolysaccharide (LPS)-induced inflammation and the activation of NF-κB signaling in microglia. In order to more deeply comprehend the underlying molecular mechanisms of how TonEBP modulates the inflammatory response, we sequenced the transcriptomes of TonEBP-deficient cells and sought potential downstream target genes of TonEBP, such as Pellino-1 (PELI1). PELI has been previously reported to mediate nuclear factor-κB (NF-κB) signaling. Through the utilization of CUT & RUN, a dual-luciferase reporter, and qPCR, we confirmed that TonEBP is the transcription factor of Peli1, binding to the Peli1 promoter. In summary, TonEBP may enhance the LPS-induced inflammation and activation of NF-κB signaling via PELI1.
Asunto(s)
Hemorragia Cerebral , Microglía , Factores de Transcripción NFATC , Animales , Humanos , Ratones , Hemorragia Cerebral/genética , Hemorragia Cerebral/metabolismo , Inflamación/genética , Inflamación/metabolismo , Lipopolisacáridos/toxicidad , Lipopolisacáridos/metabolismo , Microglía/metabolismo , Enfermedades Neuroinflamatorias , FN-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
INTRODUCTION: This study will test the hypothesis that primary aldosteronism (PA) involves alterations in Na+, K+, and water content in the skin that are corrected by adrenalectomy. AIM AND METHODS: In skin biopsies, we will measure the content of Na+, K+, water, by physical-chemical methods and the osmotic-stress-responsive transcription factor Tonicity-responsive Enhancer Binding Protein (TonEBP, NFAT5) mRNA copy number by droplet digital PCR, in sex-balanced cohorts of 18 -75-year-old consecutive consenting patients with unilateral and bilateral PA, primary (essential) hypertension, and normotension. Before surgery, the patients with unilateral PA will receive the mineralocorticoid receptor antagonist (MRA) canrenone at doses that correct hypokalemia and high blood pressure values. They will be reassessed in an identical way one month after surgical cure, while off MRA. PA patients not selected for adrenalectomy will similarly be assessed at diagnosis and follow-up while on stable MRA treatment. Since a pilot study showed a direct correlation of dry weight (DW) with skin electrolytes and water content and significant differences of biopsy DW between surgery and follow-up, meaningful comparison of the skin cations and water content and TonEBP mRNA copy number, between specimen obtained at different time points, will require DW- and total mRNA-adjustment, respectively. CONCLUSION: This study will provide novel information on the skin Na+, K+ and water content in PA, the paradigm of salt-dependent hypertension, and novel knowledge on the effect of surgical cure of hyperaldosteronism. The TonEBP-mediated regulation of Na+, K+ and water content in the skin will also be unveiled. TRAIL REGISTRY: Trial Registration number: NCT06090617. Date of Registration: 2023-10-19.
Asunto(s)
Hiperaldosteronismo , Hipertensión , Humanos , Adolescente , Adulto Joven , Adulto , Persona de Mediana Edad , Anciano , Proyectos Piloto , Hiperaldosteronismo/diagnóstico , Hiperaldosteronismo/genética , Hiperaldosteronismo/cirugía , Hipertensión/diagnóstico , Hipertensión/tratamiento farmacológico , Hipertensión/genética , Cloruro de Sodio Dietético , Electrólitos/uso terapéutico , ARN Mensajero/uso terapéuticoRESUMEN
Age-related microglial activation is associated with cognitive impairment. Tonicity-responsive enhancer-binding protein (TonEBP) is a critical mediator of microglial activation in response to neuroinflammation. However, the precise role of TonEBP in the middle-aged brain is not yet known. We used TonEBP haploinsufficient mice to investigate the role of TonEBP in middle-aged or amyloid ß oligomer (AßO)-injected brains and examined the effect of TonEBP knockdown on AßO-treated BV2 microglial cells. Consistent with an increase in microglial activation with aging, hippocampal TonEBP expression levels were increased in middle-aged (12-month-old) and old (24-month-old) mice compared with young (6-month-old) mice. Middle-aged TonEBP haploinsufficient mice showed reduced microglial activation and fewer memory deficits than wild-type mice. Electron microscopy revealed that synaptic pruning by microglial processes was reduced by TonEBP haploinsufficiency. TonEBP haploinsufficiency also reduced dendritic spine loss and improved memory deficits in AßO-treated mice. Furthermore, TonEBP knockdown attenuated migration and phagocytosis in AßO-treated BV2 cells. These findings suggest that TonEBP plays important roles in age-related microglial activation and memory deficits.
Asunto(s)
Péptidos beta-Amiloides , Factores de Transcripción NFATC , Animales , Ratones , Péptidos beta-Amiloides/metabolismo , Haploinsuficiencia , Trastornos de la Memoria/metabolismo , Microglía/metabolismo , Factores de Transcripción NFATC/metabolismoRESUMEN
Homeostatic control of intracellular ionic strength is essential for protein, organelle and genome function, yet mechanisms that sense and enable adaptation to ionic stress remain poorly understood in animals. We find that the transcription factor NFAT5 directly senses solution ionic strength using a C-terminal intrinsically disordered region. Both in intact cells and in a purified system, NFAT5 forms dynamic, reversible biomolecular condensates in response to increasing ionic strength. This self-associative property, conserved from insects to mammals, allows NFAT5 to accumulate in the nucleus and activate genes that restore cellular ion content. Mutations that reduce condensation or those that promote aggregation both reduce NFAT5 activity, highlighting the importance of optimally tuned associative interactions. Remarkably, human NFAT5 alone is sufficient to reconstitute a mammalian transcriptional response to ionic or hypertonic stress in yeast. Thus NFAT5 is both the sensor and effector of a cell-autonomous ionic stress response pathway in animal cells.
RESUMEN
PURPOSE: Recent studies, conducted mainly on the rodent model, have demonstrated that regulatory pathway in the skin provided by glycosaminoglycans, nuclear factor of activated T cells 5 (NFAT5), vascular endothelial growth factor C (VEGF-C) and process of lymphangiogenesis may play an important role in extrarenal regulation of sodium (Na+) balance, body water volume, and blood pressure. We aimed to investigate the concentrations and relations among the main factors of this pathway in human skin to confirm that this regulatory axis also exists in humans. PATIENTS AND METHODS: Skin specimens from patients diagnosed with arterial hypertension and from control group were histologically and molecularly examined. RESULTS: The primary hypertensive and control groups did not differ in Na+ âconcentrations in the skin. However, the patients with hypertension and higher skin Na+ concentration had significantly greater density of skin lymphatic vessels. Higher skin Na+concentration was associated with higher skin water content. In turn, skin water content correlated with factors associated with lymphangiogenesis, i.e. NFAT5, VEGF-C, and podoplanin (PDPN) mRNA expression in the skin. The strong mutual pairwise correlations of the expressions of NFAT5, VEGF-C, vascular endothelial growth factor D (VEGF-D) and PDPN mRNA were noted in the skin in all of the studied groups. CONCLUSIONS: Our study confirms that skin interstitium and the lymphatic system may be important players in the pathophysiology of arterial hypertension in humans. Based on the results of our study and existing literature in this field, we propose the hypothetical model which might explain the phenomenon of salt-sensitivity.
Asunto(s)
Hipertensión , Vasos Linfáticos , Humanos , Factor C de Crecimiento Endotelial Vascular/genética , Factor C de Crecimiento Endotelial Vascular/metabolismo , Sodio , Factor D de Crecimiento Endotelial Vascular , Hipertensión/metabolismo , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patología , ARN Mensajero , AguaRESUMEN
High salt intake is a primary cause of over-hydration in chronic kidney disease (CKD) patients. Inflammatory markers are predictors of CKD mortality; however, the pathogenesis of inflammation remains unclear. Sodium storage in tissues has recently emerged as an issue of concern. The binding of sodium to tissue glycosaminoglycans and its subsequent release regulates local tonicity. Many cell types express tonicity-responsive enhancer-binding protein (TonEBP), which is activated in a tonicity-dependent or tonicity-independent manner. Macrophage infiltration was observed in the heart, peritoneal wall, and para-aortic tissues in salt-loading subtotal nephrectomized mice, whereas macrophages were not prominent in tap water-loaded subtotal nephrectomized mice. TonEBP was increased in the heart and peritoneal wall, leading to the upregulation of inflammatory mediators associated with cardiac fibrosis and peritoneal membrane dysfunction, respectively. Reducing salt loading by a diuretic treatment or changing to tap water attenuated macrophage infiltration, TonEBP expression, and inflammatory marker expression. The role of TonEBP may be crucial during the cardiac fibrosis and peritoneal deterioration processes induced by sodium overload. Anti-interleukin-6 therapy improved cardiac inflammation and fibrosis and peritoneal membrane dysfunction. Further studies are necessary to establish a strategy to regulate organ dysfunction induced by TonEBP activation in CKD patients.
Asunto(s)
Insuficiencia Renal Crónica , Sodio , Ratones , Animales , Inflamación/metabolismo , Factores de Transcripción NFATC/metabolismo , Cloruro de Sodio , Cloruro de Sodio Dietético/efectos adversos , Agua , FibrosisRESUMEN
Purpose: Dry eye disease (DED) is characterized by altered ocular surface proinflammatory and antiinflammatory factors. Interferons (IFNs) are a class of pleiotropic cytokines well known for their antimicrobial, inflammatory, and immunomodulatory roles. Hence, this study investigates the ocular surface expression of different types of IFNs in patients with DED. Methods: The cross-sectional, observational study included patients with DED and normal subjects. Conjunctival impression cytology (CIC) samples were obtained from the study subjects (controls, n = 7; DED, n = 8). The mRNA expression levels of type 1 IFN (IFNα, IFNß), type 2 IFN (IFNγ), and type 3 IFN (IFNλ1, IFNλ2, IFNλ3) were measured by quantitative PCR (polymerase chain reaction) in CIC samples. IFNα and IFNγ expression under hyperosmotic stress was also studied in human corneal epithelial cells (HCECs) in vitro. Results: The mRNA expression levels of IFNα and IFNß were significantly lower and that of IFNγ was significantly higher in DED patients compared to healthy controls. The mRNA levels of IFNα, IFNß, and IFNλ were significantly lower compared to IFNγ in DED patients. An inverse association between tonicity-responsive enhancer-binding protein (TonEBP; hyperosmotic stress maker) and IFNα or IFNß expression and a positive association between TonEBP and IFNγ expression was observed in CIC samples. The expression of IFNα was lower than IFNγ in HCECs undergoing hyperosmotic stress compared to HCECs without the stress. Conclusion: The presence of an imbalance between type 1 and type 2 IFNs in DED patients suggests newer pathogenic processes in DED, plausible ocular surface infection susceptibility in DED patients, and potential therapeutic targets in the management of DED.
Asunto(s)
Citocinas , Síndromes de Ojo Seco , Humanos , Estudios Transversales , Citocinas/metabolismo , Interferón-alfa , Síndromes de Ojo Seco/diagnóstico , Síndromes de Ojo Seco/genética , Síndromes de Ojo Seco/metabolismo , Células Epiteliales/metabolismoRESUMEN
Lung cancer has the highest mortality rate among all cancers, in part because it readily metastasizes. The tumor microenvironment, comprising blood vessels, fibroblasts, immune cells, and macrophages [including tumor-associated macrophages (TAMs)], is closely related to cancer cell growth, migration, and invasion. TAMs secrete several cytokines, including interleukin (IL)-1ß, which participate in cancer migration and invasion. p21-activated kinase 1 (PAK1), an important signaling molecule, induces cell migration and invasion in several carcinomas. Tonicity-responsive enhancer-binding protein (TonEBP) is also known to participate in cancer cell growth, migration, and invasion. However, the mechanisms by which it increases lung cancer migration remain unclear. Therefore, in this study, we aimed to elucidate the mechanisms by which IL-1ß and TonEBP affect lung cancer cell migration and invasion. We found that A549 cocultured-MΦ-secreted IL-1ß induced A549 cell migration and invasion via the PAK1 pathway. TonEBP deficiency reduced A549 cell migration and invasion and increased responsiveness to IL-1ß-induced migration and invasion. PAK1 phosphorylation, which was promoted by IL-1ß, was reduced when TonEBP was depleted. These results suggest that TonEBP plays an important role in IL-1ß induction and invasiveness of A549 cells via the PAK1 pathway. These findings could be valuable in identifying potential targets for lung cancer treatment.
Asunto(s)
Neoplasias Pulmonares , Factores de Transcripción NFATC , Humanos , Células A549 , Movimiento Celular , Transformación Celular Neoplásica , Interleucina-1beta/metabolismo , Neoplasias Pulmonares/genética , Factores de Transcripción NFATC/genética , Microambiente TumoralRESUMEN
R-loops are nucleic acid structures containing a DNA-RNA hybrid and the associated non-template single-stranded DNA. R-loops are not only involved in many biological processes but also cause genomic instability when they are abnormally regulated. The R-loop regulation pathway consists of multiple steps associated with diverse proteins. The initial and essential step of the pathway is to recognize R-loops in long DNA of human genome. To elucidate the molecular mechanism underlying R-loop recognition by proteins, we utilize a novel high-throughput single-molecule approach called "DNA curtain" as well as electrophoretic mobility shift assays. Here, we describe the detailed protocols for these techniques that both can be used for studying the R-loop recognition mechanisms.
Asunto(s)
ADN , Estructuras R-Loop , ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Inestabilidad Genómica , Humanos , ARN/genéticaRESUMEN
Multiple sclerosis disproportionally affects women. The present study was undertaken to determine whether NFAT5 contributed to the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, and if it did, whether the impact was sex associated. NFAT5 haplodeficiency reduced the disease severity only in female mice. This effect was associated with significant increases in frequency of T regulatory (Treg) cells in the CNS (from 1.45 ± 0.39% to 3.73 ± 0.94%) and spleen from (0.31 ± 0.06% to 0.94 ± 0.29%) without significantly affecting the CNS CD4+ subsets frequency. NFAT5 haploinsufficiency also significantly reduced the frequency of CD11c+CD8α+ dendritic cells in the female CNS. However, increase of their frequency in the CNS via intraperitoneal Flt3L injection at peak EAE had no significant effect on the disease courses. We conclude that NFAT5 contributes to pathogenesis of EAE in female mice, possibly through decreasing tissue specific frequency of Treg cells.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Linfocitos T Reguladores , Factores de Transcripción , Animales , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple , Bazo , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Diabetic individuals have increased circulating inflammatory mediators which are implicated as underlying causes of neuroinflammation and memory deficits. Tonicity-responsive enhancer-binding protein (TonEBP) promotes diabetic neuroinflammation. However, the precise role of TonEBP in the diabetic brain is not fully understood. METHODS: We employed a high-fat diet (HFD)-only fed mice or HFD/streptozotocin (STZ)-treated mice in our diabetic mouse models. Circulating TonEBP and lipocalin-2 (LCN2) levels were measured in type 2 diabetic subjects. TonEBP haploinsufficient mice were used to investigate the role of TonEBP in HFD/STZ-induced diabetic mice. In addition, RAW 264.7 macrophages were given a lipopolysaccharide (LPS)/high glucose (HG) treatment. Using a siRNA, we examined the effects of TonEBP knockdown on RAW264 cell' medium/HG-treated mouse hippocampal HT22 cells. RESULTS: Circulating TonEBP and LCN2 levels were higher in experimental diabetic mice or type 2 diabetic patients with cognitive impairment. TonEBP haploinsufficiency ameliorated the diabetic phenotypes including adipose tissue macrophage infiltrations, neuroinflammation, blood-brain barrier leakage, and memory deficits. Systemic and hippocampal LCN2 proteins were reduced in diabetic mice by TonEBP haploinsufficiency. TonEBP (+ / -) mice had a reduction of hippocampal heme oxygenase-1 (HO-1) expression compared to diabetic wild-type mice. In particular, we found that TonEBP bound to the LCN2 promoter in the diabetic hippocampus, and this binding was abolished by TonEBP haploinsufficiency. Furthermore, TonEBP knockdown attenuated LCN2 expression in lipopolysaccharide/high glucose-treated mouse hippocampal HT22 cells. CONCLUSIONS: These findings indicate that TonEBP may promote neuroinflammation and cognitive impairment via upregulation of LCN2 in diabetic mice.
Asunto(s)
Disfunción Cognitiva/sangre , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Tipo 2/sangre , Lipocalina 2/sangre , Factores de Transcripción NFATC/sangre , Enfermedades Neuroinflamatorias/sangre , Animales , Cognición/fisiología , Disfunción Cognitiva/etiología , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/psicología , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/psicología , Dieta Alta en Grasa , Aprendizaje por Laberinto/fisiología , Ratones , Enfermedades Neuroinflamatorias/etiología , Células RAW 264.7RESUMEN
The nuclear factor of activated T cells 5 (NFAT5) is well known for its sensitivity to cellular osmolarity changes, such as in the kidney medulla. Accumulated evidence indicates that NFAT5 is also a sensitive factor to stress signals caused by non-hypertonic stimuli such as heat shock, biomechanical stretch stress, ischaemia, infection, etc. These osmolality-related and -unrelated stimuli can induce NFAT5 upregulation, activation and nuclear accumulation, leading to its protective role against various detrimental effects. However, dysregulation of NFAT5 expression may cause pathological conditions in different tissues, leading to a variety of diseases. These protective or pathogenic effects of NFAT5 are dictated by the regulation of its target gene expression and activation of its signalling pathways. Recent studies have found a number of kinases that participate in the phosphorylation/activation of NFAT5 and related signal proteins. Thus, this review will focus on the NFAT5-mediated signal transduction pathways. As for the stimuli that upregulate NFAT5, in addition to the stresses caused by hyperosmotic and non-hyperosmotic environments, other factors such as miRNA, long non-coding RNA, epigenetic modification and viral infection also play an important role in regulating NFAT5 expression; thus, the discussion in this regard is another focus of this review. As the heart, unlike the kidneys, is not normally exposed to hypertonic environments, studies on NFAT5-mediated cardiovascular diseases are just emerging and rapidly progressing. Therefore, we have also added a review on the progress made in this field of research.
Asunto(s)
Enfermedades Cardiovasculares/genética , Epigénesis Genética , MicroARNs/genética , ARN Largo no Codificante/genética , Factores de Transcripción/genética , Virosis/genética , Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Anclaje a la Quinasa A/metabolismo , Animales , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Metilación de ADN , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Respuesta al Choque Térmico/genética , Histonas/genética , Histonas/metabolismo , Humanos , Médula Renal/metabolismo , Médula Renal/patología , MicroARNs/metabolismo , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/metabolismo , Concentración Osmolar , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Largo no Codificante/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Virosis/metabolismo , Virosis/patología , Virosis/virología , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The hypothalamic neuroendocrine system is strongly implicated in body energy homeostasis. In particular, the degree of production and release of arginine vasopressin (AVP) in the hypothalamus is affected by plasma osmolality, and that hypothalamic AVP is responsible for thirst and osmolality-dependent water and metabolic balance. However, the osmolality-responsive intracellular mechanism within AVP cells that regulates AVP synthesis is not clearly understood. Here, we report a role for tonicity-responsive enhancer binding protein (TonEBP), a transcription factor sensitive to cellular tonicity, in regulating osmosensitive hypothalamic AVP gene transcription. Our immunohistochemical work shows that hypothalamic AVP cellular activity, as recognized by c-fos, was enhanced in parallel with an elevation in TonEBP expression within AVP cells following water deprivation. Interestingly, our in vitro investigations found a synchronized pattern of TonEBP and AVP gene expression in response to osmotic stress. Those results indicate a positive correlation between hypothalamic TonEBP and AVP production during dehydration. Promoter and chromatin immunoprecipitation assays confirmed that TonEBP can bind directly to conserved binding motifs in the 5'-flanking promoter regions of the AVP gene. Furthermore, dehydration- and TonEBP-mediated hypothalamic AVP gene activation was reduced in TonEBP haploinsufficiency mice, compared with wild TonEBP homozygote animals. Therefore, our result support the idea that TonEBP is directly necessary, at least in part, for the elevation of AVP transcription in dehydration conditions. Additionally, dehydration-induced reductions in body weight were rescued in TonEBP haploinsufficiency mice. Altogether, our results demonstrate an intracellular machinery within hypothalamic AVP cells that is responsible for dehydration-induced AVP synthesis.
Asunto(s)
Arginina Vasopresina/metabolismo , Regulación de la Expresión Génica , Hipotálamo/metabolismo , Factores de Transcripción NFATC/metabolismo , Neuronas/metabolismo , Animales , Arginina Vasopresina/genética , Haploinsuficiencia , Ratones , Factores de Transcripción NFATC/genética , Concentración Osmolar , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-fos/metabolismo , Privación de AguaRESUMEN
Although numerous studies have confirmed the relationship between high salt intake and elevated blood pressure, the exact molecular mechanisms of this relationship are still unclear. There is growing evidence that skin interstitium, as well as the skin lymphatic system, are important regulators of both sodium (Na+) balance and blood pressure. Skin is in itself a large reservoir of Na+ ions which are stored in an osmotically inactive form on glycosaminoglycans (GAGs). Local hypertonicity due to extensive accumulation of Na+ within the skin as a result of a high-salt diet was demonstrated to induce macrophages to express a transcription factor termed tonicityresponsive enhancer binding protein (TonEBP) and subsequently to secrete vascular endothelial growth factor-C (VEGF-C), activating lymphangiogenesis within the skin. This regulatory axis seems to be adaptive in maintaining blood pressure in high salt-load states. Recent studies have added new insights into the functioning of lymphatic vessels and the pathogenesis of salt-sensitive hypertension as well as questioned the classic view of Na+ homeostasis. This review aims to summarize recent findings pertaining to the involvement of the skin lymphatic system in Na+ and blood pressure regulation.
Asunto(s)
Presión Sanguínea , Homeostasis , Hipertensión/etiología , Hipertensión/metabolismo , Sistema Linfático/metabolismo , Piel/metabolismo , Animales , Biomarcadores , Susceptibilidad a Enfermedades , Humanos , Linfangiogénesis , Sodio/metabolismoRESUMEN
BACKGROUND: Microglia are brain-resident myeloid cells involved in the innate immune response and a variety of neurodegenerative diseases. In macrophages, TonEBP is a transcriptional cofactor of NF-κB which stimulates the transcription of pro-inflammatory genes in response to LPS. Here, we examined the role of microglial TonEBP. METHODS: We used microglial cell line, BV2 cells. TonEBP was knocked down using lentiviral transduction of shRNA. In animals, TonEBP was deleted from myeloid cells using a line of mouse with floxed TonEBP. Cerulenin was used to block the NF-κB cofactor function of TonEBP. RESULTS: TonEBP deficiency blocked the LPS-induced expression of pro-inflammatory cytokines and enzymes in association with decreased activity of NF-κB in BV2 cells. We found that there was also a decreased activity of AP-1 and that TonEBP was a transcriptional cofactor of AP-1 as well as NF-κB. Interestingly, we found that myeloid-specific TonEBP deletion blocked the LPS-induced microglia activation and subsequent neuronal cell death and memory loss. Cerulenin disrupted the assembly of the TonEBP/NF-κB/AP-1/p300 complex and suppressed the LPS-induced microglial activation and the neuronal damages in animals. CONCLUSIONS: TonEBP is a key mediator of microglial activation and neuroinflammation relevant to neuronal damage. Cerulenin is an effective blocker of the TonEBP actions.