RESUMEN
Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disorder in which vasopressin-secreting neurons degenerate over time due to the production of mutant proteins. We have demonstrated therapeutic effects of chemical chaperones in an FNDI mouse model, but the complexity and length of this evaluation were problematic. In this study, we established disease-specific mouse induced pluripotent stem cells (iPSCs) from FNDI-model mice and differentiated vasopressin neurons that produced mutant proteins. Fluorescence immunostaining showed that chemical chaperones appeared to protect vasopressin neurons generated from iPSCs derived from FNDI-model mice. Although KCL stimulation released vasopressin hormone from vasopressin neurons generated from FNDI-derived iPSCs, vasopressin hormone levels did not differ significantly between baseline and chaperone-added culture. Semi-quantification of vasopressin carrier protein and mutant protein volumes in vasopressin neurons confirmed that chaperones exerted a therapeutic effect. This research provides fundamental technology for creating in vitro disease models using human iPSCs and can be applied to therapeutic evaluation of various degenerative diseases that produce abnormal proteins.
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Diabetes Insípida Neurogénica , Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Humanos , Ratones , Animales , Arginina Vasopresina/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Enfermedades Neurodegenerativas/tratamiento farmacológico , Vasopresinas/farmacología , Vasopresinas/metabolismo , Diabetes Insípida Neurogénica/metabolismo , Neurofisinas/genética , Proteínas Mutantes/metabolismo , MutaciónRESUMEN
Apoptosis is one of the hallmarks of MeHg-induced neuronal cell death; however, its molecular mechanism remains unclear. We previously reported that MeHg exposure induces neuron-specific ER stress in the mouse brain. Excessive ER stress contributes to apoptosis, and CHOP induction is considered to be one of the major mechanisms. CHOP is also increased by MeHg exposure in the mouse brain, suggesting that it correlates with increased apoptosis. In this study, to clarify whether CHOP mediates MeHg-induced apoptosis, we examined the effect of CHOP deletion on MeHg exposure in CHOP-knockout mice. Our data showed that CHOP deletion had no effect on MeHg exposure-induced weight loss or hindlimb impairment in mice, nor did it increase apoptosis or inhibit neuronal cell loss. Hence, CHOP plays little role in MeHg toxicity, and other apoptotic pathways coupled with ER stress may be involved in MeHg-induced cell death.
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Encéfalo , Compuestos de Metilmercurio , Neuronas , Animales , Ratones , Apoptosis , Encéfalo/patología , Compuestos de Metilmercurio/toxicidad , Compuestos de Metilmercurio/metabolismo , Ratones Noqueados , Neuronas/patologíaRESUMEN
Lung cancer, primarily non-small-cell lung cancer (NSCLC), is a significant cause of cancer-related mortality worldwide. Cisplatin-based chemotherapy is a standard treatment for NSCLC; however, its effectiveness is often limited due to the development of resistance, leading to NSCLC recurrence. Thus, the identification of effective chemosensitizers for cisplatin is of paramount importance. The integrated stress response (ISR), activated by various cellular stresses and mediated by eIF2α kinases, has been implicated in drug sensitivity. ISR activation globally suppresses protein synthesis while selectively promoting the translation of ATF4 mRNA, which can induce pro-apoptotic proteins such as CHOP, ATF3, and TRIB3. To expedite and economize the development of chemosensitizers for cisplatin treatment in NSCLC, we employed a strategy to screen an FDA-approved drug library for ISR activators. In this study, we identified mifepristone as a potent ISR activator. Mifepristone activated the HRI/eIF2α/ATF4 axis, leading to the induction of pro-apoptotic factors, independent of its known role as a synthetic steroid. Our in vitro and in vivo models demonstrated mifepristone's potential to inhibit NSCLC re-proliferation following cisplatin treatment and tumor growth, respectively, via the ISR-mediated cell death pathway. These findings suggest that mifepristone, as an ISR activator, could enhance the efficacy of cisplatin-based therapy for NSCLC, highlighting the potential of drug repositioning in the search for effective chemosensitizers.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Cisplatino/farmacología , Cisplatino/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mifepristona/farmacología , Reposicionamiento de Medicamentos , Transducción de Señal , Línea Celular Tumoral , Resistencia a AntineoplásicosRESUMEN
Activating transcription factor 6 (ATF6) is an endoplasmic reticulum (ER) stress-regulated transcription factor that induces expression of major molecular chaperones in the ER. We recently reported that ATF6ß, a subtype of ATF6, promoted survival of hippocampal neurons exposed to ER stress and excitotoxicity, at least in part by inducing expression of calreticulin, an ER molecular chaperone with high Ca2+-binding capacity. In the present study, we demonstrate that ATF6ß deficiency in mice also decreases calreticulin expression and increases expression of glucose-regulated protein 78, another ER molecular chaperone, in emotional brain regions such as the prefrontal cortex (PFC), hypothalamus, hippocampus, and amygdala. Comprehensive behavioral analyses revealed that Atf6b-/- mice exhibit anxiety-like behavior in the light/dark transition test and hyperactivity in the forced swim test. Consistent with these results, PFC and hypothalamic corticotropin-releasing hormone (CRH) expression was increased in Atf6b-/- mice, as was circulating corticosterone. Moreover, CRH receptor 1 antagonism alleviated anxiety-like behavior in Atf6b-/- mice. These findings suggest that ATF6ß deficiency produces anxiety-like behavior and hyperactivity via a CRH receptor 1-dependent mechanism. ATF6ß could play a role in psychiatric conditions in the emotional centers of the brain.
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Calreticulina , Receptores de Hormona Liberadora de Corticotropina , Ratones , Animales , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Calreticulina/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Hipotálamo/metabolismo , Ansiedad/metabolismo , Corticosterona/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Sistema Hipófiso-Suprarrenal/metabolismo , Estrés Psicológico/complicaciones , Estrés Psicológico/metabolismo , Factor de Transcripción Activador 6/metabolismoRESUMEN
WNT molecules are the regulators of various biological functions, including body axis formation, organ development, and cell proliferation and differentiation. WNTs have been extensively studied as causative genes for an array of diseases. WNT10A and WNT10B, which are considered to be genes of the same origin, have been identified as causative genes for tooth deficiency in humans. However, the disrupted mutant of each gene does not show a decrease in teeth number. A negative feedback loop, interacting with several ligands based on a reaction-diffusion mechanism, was proposed to be important for the spatial patterning of tooth formation, and WNT ligands have been considered to play a pivotal role in controlling tooth patterning from mutant phenotypes of LDL receptor-related proteins (LRPs) and WNT co-receptors. The Wnt10a and Wnt10b double-mutants demonstrated severe root or enamel hypoplasia. In Wnt10a-/- and Wnt10a+/-;Wnt10b-/- mice, changes in the feedback loop may collapse the modulation of fusion or split a sequence of tooth formation. However, in the double-knockout mutant, a decrease in the number of teeth was observed, including the upper incisor or third molar in both jaws. These findings suggest that there may be a functional redundancy between Wnt10a and Wnt10b and that the interaction between the two genes functions in conjunction with other ligands to control the spatial patterning and development of teeth.
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Odontogénesis , Diente , Proteínas Wnt , Animales , Humanos , Ratones , Proliferación Celular , Mutación , Proteínas del Tejido Nervioso/genética , Fenotipo , Proteínas Proto-Oncogénicas/genética , Proteínas Wnt/genéticaRESUMEN
Brown adipose tissue (BAT) is a highly specialized adipose tissue in its immobile location and size during the entire adulthood. In response to cold exposure and other ß3-adrenoreceptor stimuli, BAT commits energy consumption by nonshivering thermogenesis (NST). However, the molecular machinery in controlling the BAT mass in adults is unknown. Here, we show our surprising findings that the BAT mass and functions can be manipulated in adult animals by controlling BAT adipocyte differentiation in vivo. Platelet-derived growth factor receptor α (PDGFα) expressed in BAT progenitor cells served a signaling function to avert adipose progenitor differentiation. Genetic and pharmacological loss-of-function of PDGFRα eliminated the differentiation barrier and permitted progenitor cell differentiation to mature and functional BAT adipocytes. Consequently, an enlarged BAT mass (megaBAT) was created by PDGFRα inhibition owing to increases of brown adipocyte numbers. Under cold exposure, a microRNA-485 (miR-485) was identified as a master suppressor of the PDGFRα signaling, and delivery of miR-485 also produced megaBAT in adult animals. Noticeably, megaBAT markedly improved global metabolism, insulin sensitivity, high-fat-diet (HFD)-induced obesity, and diabetes by enhancing NST. Together, our findings demonstrate that the adult BAT mass can be increased by blocking the previously unprecedented inhibitory signaling for BAT progenitor cell differentiation. Thus, blocking the PDGFRα for the generation of megaBAT provides an attractive strategy for treating obesity and type 2 diabetes mellitus (T2DM).
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Adipocitos Marrones , Adipocitos , Adipogénesis , Tejido Adiposo Pardo , MicroARNs , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas , Adipocitos/citología , Adipocitos Marrones/metabolismo , Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/metabolismo , Animales , Diabetes Mellitus Tipo 2/terapia , Metabolismo Energético , MicroARNs/genética , MicroARNs/metabolismo , Obesidad/terapia , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Termogénesis/genéticaRESUMEN
Stress-inducible transcription factor ATF4 is essential for survival and identity of ß-cell during stress conditions. However, the physiological role of ATF4 in ß-cell function is not yet completely understood. To understand the role of ATF4 in glucose-stimulated insulin secretion (GSIS), ß-cell-specific Atf4 knockout (ßAtf4KO) mice were phenotypically characterized. Insulin secretion and mechanistic analyses were performed using islets from control Atf4f/f and ßAtf4KO mice to assess key regulators for triggering and amplifying signals for GSIS. ßAtf4KO mice displayed glucose intolerance due to reduced insulin secretion. Moreover, ßAtf4KO islets exhibited a decrease in both the insulin content and first-phase insulin secretion. The analysis of ßAtf4KO islets showed that ATF4 is required for insulin production and glucose-stimulated ATP and cAMP production. The results demonstrate that ATF4 contributes to the multifaceted regulatory process in GSIS even under stress-free conditions.
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Intolerancia a la Glucosa , Células Secretoras de Insulina , Islotes Pancreáticos , Animales , Glucosa/metabolismo , Glucosa/farmacología , Intolerancia a la Glucosa/genética , Intolerancia a la Glucosa/metabolismo , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Perturbation of endoplasmic reticulum (ER) proteostasis is associated with impairment of cellular function in diverse diseases, especially the function of pancreatic ß cells in type 2 diabetes. Restoration of ER proteostasis by small molecules shows therapeutic promise for type 2 diabetes. Here, using cell-based screening, we report identification of a chemical chaperone-like small molecule, KM04794, that alleviates ER stress. KM04794 prevented protein aggregation and cell death caused by ER stressors and a mutant insulin protein. We also found that this compound increased intracellular and secreted insulin levels in pancreatic ß cells. Chemical biology and biochemical approaches revealed that the compound accumulated in the ER and interacted directly with the ER molecular chaperone BiP. Our data show that this corrector of ER proteostasis can enhance insulin storage and pancreatic ß cell function.
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Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Proteostasis , Respuesta de Proteína DesplegadaRESUMEN
The effects of imeglimin, a novel antidiabetes agent, on ß-cell function remain unclear. Here, we unveiled the impact of imeglimin on ß-cell survival. Treatment with imeglimin augmented mitochondrial function, enhanced insulin secretion, promoted ß-cell proliferation, and improved ß-cell survival in mouse islets. Imeglimin upregulated the expression of endoplasmic reticulum (ER)-related molecules, including Chop (Ddit3), Gadd34 (Ppp1r15a), Atf3, and Sdf2l1, and decreased eIF2α phosphorylation after treatment with thapsigargin and restored global protein synthesis in ß-cells under ER stress. Imeglimin failed to protect against ER stress-induced ß-cell apoptosis in CHOP-deficient islets or in the presence of GADD34 inhibitor. Treatment with imeglimin showed a significant decrease in the number of apoptotic ß-cells and increased ß-cell mass in Akita mice. Imeglimin also protected against ß-cell apoptosis in both human islets and human pluripotent stem cell-derived ß-like cells. Taken together, imeglimin modulates the ER homeostasis pathway, which results in the prevention of ß-cell apoptosis both in vitro and in vivo.
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Apoptosis/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Hipoglucemiantes , Células Secretoras de Insulina/fisiología , Triazinas/farmacología , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Glucosa/farmacología , Homeostasis/efectos de los fármacos , Humanos , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/ultraestructura , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Células Madre Pluripotentes , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/fisiología , Factor de Transcripción CHOP/deficiencia , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/fisiología , Triazinas/uso terapéuticoRESUMEN
The eIF2α phosphorylation-dependent integrated stress response (ISR) is a signaling pathway that maintains homeostasis in mammalian cells exposed to various stresses. Here, ISR activation in adipocytes improves obesity and diabetes by regulating appetite in a non-cell-autonomous manner. Adipocyte-specific ISR activation using transgenic mice decreases body weight and improves glucose tolerance and obesity induced by a high-fat diet (HFD) via preferential inhibition of HFD intake. The transcriptome analysis of ISR-activated adipose tissue reveals that growth differentiation factor 15 (GDF15) expression is induced by the ISR through the direct regulation of the transcription factors ATF4 and DDIT3. Deficiency in the GDF15 receptor GFRAL abolishes the adipocyte ISR-dependent preferential inhibition of HFD intake and the anti-obesity effects. Pharmacologically, 10(E), 12(Z)-octadecadienoic acid induces ISR-dependent GDF15 expression in adipocytes and decreases the intake of the HFD. Based on our findings the specific activation of the ISR in adipocytes controls the non-cell-autonomous regulation of appetite.
RESUMEN
OBJECTIVE: Activating transcription factor 4 (ATF4) is a transcriptional regulator of the unfolded protein response and integrated stress response (ISR) that promote the restoration of normal endoplasmic reticulum (ER) function. Previous reports demonstrated that dysregulation of the ISR led to development of severe diabetes. However, the contribution of ATF4 to pancreatic ß-cells remains poorly understood. In this study, we aimed to analyze the effect of ISR enhancer Sephin1 and ATF4-deficient ß-cells to clarify the role of ATF4 in ß-cells under ER stress conditions. METHODS: To examine the role of ATF4 in vivo, ISR enhancer Sephin1 (5 mg/kg body weight, p.o.) was administered daily for 21 days to Akita mice. We also established ß-cell-specific Atf4 knockout (ßAtf4-KO) mice that were further crossed with Akita mice. These mice were analyzed for characteristics of diabetes, ß-cell function, and morphology of the islets. To identify the downstream factors of ATF4 in ß-cells, the islets of ßAtf4-KO mice were subjected to cDNA microarray analyses. To examine the transcriptional regulation by ATF4, we also performed in situ PCR analysis of pancreatic sections from mice and ChIP-qPCR analysis of CT215 ß-cells. RESULTS: Administration of the ISR enhancer Sephin1 improved glucose metabolism in Akita mice. Sephin1 also increased the insulin-immunopositive area and ATF4 expression in the pancreatic islets. Akita/ßAtf4-KO mice exhibited dramatically exacerbated diabetes, shown by hyperglycemia at an early age, as well as a remarkably short lifespan owing to diabetic ketoacidosis. Moreover, the islets of Akita/ßAtf4-KO mice presented increased numbers of cells stained for glucagon, somatostatin, and pancreatic polypeptide and increased expression of aldehyde dehydrogenase 1 family member 3, a marker of dedifferentiation. Using microarray analysis, we identified atonal BHLH transcription factor 8 (ATOH8) as a downstream factor of ATF4. Deletion of ATF4 in ß-cells showed reduced Atoh8 expression and increased expression of undifferentiated markers, Nanog and Pou5f1. Atoh8 expression was also abolished in the islets of Akita/ßAtf4-KO mice. CONCLUSIONS: We conclude that transcriptional regulation by ATF4 maintains ß-cell identity via ISR modulation. This mechanism provides a promising target for the treatment of diabetes.
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Factor de Transcripción Activador 4/metabolismo , Células Secretoras de Insulina/metabolismo , Factor de Transcripción Activador 4/deficiencia , Animales , Estrés del Retículo Endoplásmico , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones TransgénicosRESUMEN
BACKGROUND: The aim of this study was to clarify the effectiveness of a new three-dimensional (3D) culture system for hepatocyte-like cells (HLCs) generated from human adipose-derived mesenchymal stem cells (ADSCs). METHODS: Human ADSCs (2 × 104 ) with or without 0.1 mg/mL human recombinant peptide µ-piece per well were seeded in a 96-well U-bottom plate and then our three-step differentiation protocol was applied for 21 days. At each step, cell morphology and gene expression were investigated. Mature hepatocyte functions were evaluated after HLC differentiation. These parameters were compared between 2D- and 3D-cultured HLCs, and, DNA microarray analysis was also performed. Finally, HLCs were transplanted in to CCl4 induced acute liver failure model mice. RESULTS: Two-dimensional-cultured HLCs at day 21 did not have a spindle shape and had formed spheroids after day 6, which gradually increased in size for 3D-cultured HLCs. Definitive endoderm, hepatoblast, and hepatocyte genes showed significantly higher expression in the 3D culture group. Three-dimensional-cultured HLCs also had higher albumin expression, CYP3A4 activity, urea synthesis, and ammonium metabolism, and much higher expression of ion transporter, blood coagulation, and cell communication genes. HLC transplantation improved serum liver function, especially in T-Bil levels, and engrafted into immunodeficient mice with HLA class I positive staining. CONCLUSION: Our new 3D culture protocol is effective to improve hepatocyte functions. Our HLCs might be promising for clinical cell transplantation to treat metabolic disease.
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Fallo Hepático Agudo , Células Madre Mesenquimatosas , Animales , Diferenciación Celular , Línea Celular , Hepatocitos , Humanos , RatonesRESUMEN
While ATF6α plays a central role in the endoplasmic reticulum (ER) stress response, the function of its paralogue ATF6ß remains elusive, especially in the central nervous system (CNS). Here, we demonstrate that ATF6ß is highly expressed in the hippocampus of the brain, and specifically regulates the expression of calreticulin (CRT), a molecular chaperone in the ER with a high Ca2+-binding capacity. CRT expression was reduced to ~ 50% in the CNS of Atf6b-/- mice under both normal and ER stress conditions. Analysis using cultured hippocampal neurons revealed that ATF6ß deficiency reduced Ca2+ stores in the ER and enhanced ER stress-induced death. The higher levels of death in Atf6b-/- neurons were recovered by ATF6ß and CRT overexpressions, or by treatment with Ca2+-modulating reagents such as BAPTA-AM and 2-APB, and with an ER stress inhibitor salubrinal. In vivo, kainate-induced neuronal death was enhanced in the hippocampi of Atf6b-/- and Calr+/- mice, and restored by administration of 2-APB and salubrinal. These results suggest that the ATF6ß-CRT axis promotes neuronal survival under ER stress and excitotoxity by improving intracellular Ca2+ homeostasis.
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Factor de Transcripción Activador 6/metabolismo , Calreticulina/metabolismo , Neuronas/metabolismo , Animales , Encéfalo , Calreticulina/fisiología , Muerte Celular/fisiología , Supervivencia Celular/fisiología , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Femenino , Hipocampo , Homeostasis , Ácido Kaínico/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/fisiologíaRESUMEN
UTX/KDM6A encodes a major histone H3 lysine 27 (H3K27) demethylase, and is frequently mutated in various types of human cancers. Although UTX appears to play a crucial role in oncogenesis, the mechanisms involved are still largely unknown. Here we show that a specific pharmacological inhibitor of H3K27 demethylases, GSK-J4, induces the expression of transcription activating factor 4 (ATF4) protein as well as the ATF4 target genes (e.g. PCK2, CHOP, REDD1, CHAC1 and TRIB3). ATF4 induction by GSK-J4 was due to neither transcriptional nor post-translational regulation. In support of this view, the ATF4 induction was almost exclusively dependent on the heme-regulated eIF2α kinase (HRI) in mouse embryonic fibroblasts (MEFs). Gene expression profiles with UTX disruption by CRISPR-Cas9 editing and the following stable re-expression of UTX showed that UTX specifically suppresses the expression of the ATF4 target genes, suggesting that UTX inhibition is at least partially responsible for the ATF4 induction. Apoptosis induction by GSK-J4 was partially and cell-type specifically correlated with the activation of ATF4-CHOP. These findings highlight that the anti-cancer drug candidate GSK-J4 strongly induces ATF4 and its target genes via HRI activation and raise a possibility that UTX might modulate cancer formation by regulating the HRI-ATF4 axis.
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Factor de Transcripción Activador 4/agonistas , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Histona Demetilasas/antagonistas & inhibidores , Histona Demetilasas/genética , eIF-2 Quinasa/metabolismo , Animales , Apoptosis , Benzazepinas/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Unión Proteica , Pirimidinas/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
CRISPR/Cas9-mediated gene editing often generates founder generation (F0) mice that exhibit somatic mosaicism in the targeted gene(s). It has been known that Fibroblast growth factor 10 (Fgf10)-null mice exhibit limbless and lungless phenotypes, while intermediate limb phenotypes (variable defective limbs) are observed in the Fgf10-CRISPR F0 mice. However, how the lung phenotype in the Fgf10-mosaic mutants is related to the limb phenotype and genotype has not been investigated. In this study, we examined variable lung phenotypes in the Fgf10-targeted F0 mice to determine if the lung phenotype was correlated with percentage of functional Fgf10 genotypes. Firstly, according to a previous report, Fgf10-CRISPR F0 embryos on embryonic day 16.5 (E16.5) were classified into three types: type I, no limb; type II, limb defect; and type III, normal limbs. Cartilage and bone staining showed that limb truncations were observed in the girdle, (type I), stylopodial, or zeugopodial region (type II). Deep sequencing of the Fgf10-mutant genomes revealed that the mean proportion of codons that encode putative functional FGF10 was 8.3 ± 6.2% in type I, 25.3 ± 2.7% in type II, and 54.3 ± 9.5% in type III (mean ± standard error of the mean) mutants at E16.5. Histological studies showed that almost all lung lobes were absent in type I embryos. The accessory lung lobe was often absent in type II embryos with other lobes dysplastic. All lung lobes formed in type III embryos. The number of terminal tubules was significantly lower in type I and II embryos, but unchanged in type III embryos. To identify alveolar type 2 epithelial (AECII) cells, known to be reduced in the Fgf10-heterozygous mutant, immunostaining using anti-surfactant protein C (SPC) antibody was performed: In the E18.5 lungs, the number of AECII was correlated to the percentage of functional Fgf10 genotypes. These data suggest the Fgf10 gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung. Since dysfunction of AECII cells has been implicated in the pathogenesis of parenchymal lung diseases, the Fgf10-CRISPR F0 mouse would present an ideal experimental system to explore it.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Factor 10 de Crecimiento de Fibroblastos/genética , Edición Génica/métodos , Pulmón/metabolismo , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/metabolismo , Animales , Modelos Animales de Enfermedad , Embrión de Mamíferos/metabolismo , Dosificación de Gen , Genotipo , Pulmón/citología , Pulmón/patología , Enfermedades Pulmonares/metabolismo , Enfermedades Pulmonares/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Ratones TransgénicosRESUMEN
Dickkopf 3 (Dkk3) is a secreted protein belonging to the Dkk family and encoded by the orthologous gene of REIC. Dkk3/REIC is expressed by mouse and human adrenal glands, but the understanding of its roles in this organ is still limited. To determine the functions of Dkk3 in the mouse adrenal gland, we first identified that the mouse Dkk3 protein is N-glycosylated in the adrenal gland as well as in the brain. We performed proteome analysis on adrenal glands from Dkk3-null mice, in which exons 5 and 6 of the Dkk3 gene are deleted. Twodimensional polyacrylamide gel electrophoresis of adrenal proteins from wild-type and Dkk3-null mice revealed 5 protein spots whose intensities were altered between the 2 genotypes. Mass spectrometry analysis of these spots identified binding immunoglobulin protein (BiP), an endoplasmic reticulum (ER) chaperone. To determine whether mouse Dkk3 is involved in the unfolded protein response (UPR), we carried out a reporter assay using ER-stress responsive elements. Forced expression of Dkk3 resulted in the induction of distinct levels of reporter expression, showing the UPR initiated by the ER membrane proteins of activating transcription factor 6 (ATF6) and inositol-requring enzyme 1 (IRE1). Thus, it is possible that Dkk3 is a physiological ER stressor in the mouse adrenal gland.
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Proteínas Adaptadoras Transductoras de Señales , Retículo Endoplásmico/genética , Glándulas Suprarrenales/metabolismo , Animales , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Noqueados para ApoE , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Patients with adult T-cell leukemia (ATL) exhibit a poor prognosis and overall survival rate when treated with standard chemotherapy, highlighting the continued requirement for the development of novel safe and effective therapies for human T-cell leukemia virus type 1 (HTLV-1)-related diseases. In this study, we demonstrated that MK-2048, a second-generation HIV-1 integrase (IN) inhibitor, potently and selectively kills HTLV-1-infected cells. Differential transcriptome profiling revealed significantly elevated levels of gene expression of the unfolded protein response (UPR) PKR-like ER kinase (PERK) signaling pathway in ATL cell lines following MK-2048 treatment. We also identified a significant downregulation in glucose regulated protein 78 (GRP78), a master regulator of the UPR in the CD4+CADM1+ HTLV-1-infected cell population of primary HTLV-1 carrier peripheral blood mononuclear cells (PBMCs) (n = 9), suggesting that HTLV-1-infected cells are hypersensitive to endoplasmic reticulum (ER) stress-mediated apoptosis. MK-2048 efficiently reduced proviral loads in primary HTLV-1 carrier PBMCs (n = 4), but had no effect on the total numbers of these cells, indicating that MK-2048 does not affect the proliferation of HTLV-1-uninfected PBMCs. MK-2048 specifically activated the ER stress-related proapoptotic gene, DNA damage-inducible transcript 3 protein (DDIT3), also known as C/EBP homologous protein (CHOP), in HTLV-1-infected but not uninfected cells of HTLV-1-carrier PBMCs. Our findings demonstrated that MK-2048 selectively induces HTLV-1-infected cell apoptosis via the activation of the UPR. This novel regulatory mechanism of the HIV IN inhibitor MK-2048 in HTLV-1-infected cells provides a promising prophylactic and therapeutic target for HTLV-1-related diseases including ATL.
Asunto(s)
Virus Linfotrópico T Tipo 1 Humano , Leucemia-Linfoma de Células T del Adulto , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Adulto , Molécula 1 de Adhesión Celular , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Humanos , Leucocitos Mononucleares/metabolismo , Respuesta de Proteína DesplegadaRESUMEN
The integrated stress response (ISR) is one of the most important cytoprotective mechanisms and is integrated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α). Four eIF2α kinases, heme-regulated inhibitor (HRI), double-stranded RNA-dependent protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK), and general control nonderepressible 2 (GCN2), are activated in response to several stress conditions. We previously reported that nanosecond pulsed electric fields (nsPEFs) are a potential therapeutic tool for ISR activation. In this study, we examined which eIF2α kinase is activated by nsPEF treatment. To assess the responsible eIF2α kinase, we used previously established eIF2α kinase quadruple knockout (4KO) and single eIF2α kinase-rescued 4KO mouse embryonic fibroblast (MEF) cells. nsPEFs 70 ns in duration with 30 kV/cm electric fields caused eIF2α phosphorylation in wild-type (WT) MEF cells. On the other hand, nsPEF-induced eIF2α phosphorylation was completely abolished in 4KO MEF cells and was recovered by HRI overexpression. CM-H2DCFDA staining showed that nsPEFs generated reactive oxygen species (ROS), which activated HRI. nsPEF-induced eIF2α phosphorylation was blocked by treatment with the ROS scavenger N-acetyl-L-cysteine (NAC). Our results indicate that the eIF2α kinase HRI is responsible for nsPEF-induced ISR activation and is activated by nsPEF-generated ROS.
Asunto(s)
Electricidad/efectos adversos , Fibroblastos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico/fisiología , Acetilcisteína/farmacología , Animales , Línea Celular , Técnicas de Inactivación de Genes , Ratones , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Estrés Fisiológico/efectos de los fármacos , eIF-2 Quinasa/genéticaRESUMEN
MSX1 is a causative gene for oligodontia in humans. Although conventional Msx1-deficient mice die neonatally, a mutant mouse lacking the C-terminus MH6 domain of MSX1 (Msx1ΔMH6/ΔMH6) showed two different phenotypes; newborn homozygotes with cleft palates died neonatally, whereas those with thin palates remained alive and had craniofacial dysplasia and growth retardation compared with wild-type mice, with most mice dying by the age of 4-5 weeks. In a previously reported case of human oligodontia caused by a heterozygous defect of the Msx1 MH6 domain, a small foramen was observed on the occipital bone. The aim of this study was to test the hypothesis that the Msx1 MH6 domain is involved in bone formation in vivo. In Msx1ΔMH6/ΔMH6 mice, cranial suture fusion was delayed at embryonic day 18.5, and the anteroposterior cranial diameter was smaller and long bone length was decreased at 3 weeks of age. The femoral epiphysis showed no change in the trabecular number, but decreased bone mass, bone density, and trabecular width in Msx1ΔMH6/ΔMH6 mice. In addition, cancellous bone mass was reduced and the cartilage layer in the growth plate was thinner in Msx1ΔMH6/ΔMH6 mice. The mRNA expression levels of major osteoblast and chondrocyte differentiation marker genes were decreased in Msx1ΔMH6/ΔMH6 mice compared with wild-type mice. These findings suggest that the C-terminal region including the MH6 domain of MSX1 plays important roles not only in tooth development and palatal fusion, but also in postnatal bone formation.