RESUMEN
Generalized lymphatic anomaly (GLA) and kaposiform lymphangiomatosis (KLA) are rare congenital disorders that arise through anomalous embryogenesis of the lymphatic system. A somatic activating NRAS p.Q61R variant has been recently detected in GLA and KLA tissues, suggesting that the NRAS p.Q61R variant plays an important role in the development of these diseases. To address this role, we studied the effect of the NRAS p.Q61R variant in lymphatic endothelial cells (LECs) on the structure of the lymphatics during embryonic and postnatal lymphangiogenesis applying inducible, LEC-specific NRAS p.Q61R variant in mice. Lox-stop-Lox NrasQ61R mice were crossed with Prox1-CreERT2 mice expressing tamoxifen-inducible Cre recombinase specifically in LECs. Whole-mount immunostaining of embryonic back skin using an antibody against the LEC surface marker VEGFR3 showed considerably greater lymphatic vessel width in LEC-specific NRAS p.Q61R mutant embryos than in littermate controls. These mutant embryos also showed a significant reduction in the number of lymphatic vessel branches. Furthermore, immunofluorescence staining of whole-mount embryonic back skin using an antibody against the LEC-specific nuclear marker Prox1 showed a large increase in the number of LECs in LEC-specific NRAS p.Q61R mutants. In contrast, postnatal induction of the NRAS p.Q61R variant in LECs did not cause abnormal lymphatic vessel morphogenesis. These results suggest that the NRAS p.Q61R variant in LECs plays a role in development of lymphatic anomalies. While this model does not directly reflect the human pathology of GLA and KLA, there are overlapping features, suggesting that further study of this model may help in studying GLA and KLA mechanisms.
Asunto(s)
Células Endoteliales , Linfangiogénesis , Vasos Linfáticos , Animales , Ratones , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patología , Vasos Linfáticos/embriología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Linfangiogénesis/genética , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Mutación , Morfogénesis/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Humanos , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética , Embrión de Mamíferos/metabolismo , Proteínas de Homeodominio , Proteínas Supresoras de TumorRESUMEN
Costello syndrome (CS) is an autosomal-dominant disorder characterized by distinctive facial features, hypertrophic cardiomyopathy, skeletal abnormalities, intellectual disability, and predisposition to cancers. Germline variants in HRAS have been identified in patients with CS. Intragenic HRAS duplications have been reported in three patients with a milder phenotype of CS. In this study, we identified two known HRAS variants, p.(Glu63_Asp69dup), p.(Glu62_Arg68dup), and one novel HRAS variant, p.(Ile55_Asp57dup), in patients with CS, including a patient with craniosynostosis. These intragenic duplications are located in the G3 domain and the switch II region. Cells expressing cDNA with these three intragenic duplications showed an increase in ELK-1 transactivation. Injection of wild-type or mutant HRAS mRNAs with intragenic duplications in zebrafish embryos showed significant elongation of the yolk at 11 h postfertilization, which was improved by MEK inhibitor treatment, and a variety of developmental abnormalities at 3 days post fertilization was observed. These results indicate that small in-frame duplications affecting the G3 domain and switch II region of HRAS increase the activation of the ERK pathway, resulting in developmental abnormalities in zebrafish or patients with CS.
Asunto(s)
Anomalías Múltiples , Síndrome de Costello , Anomalías Múltiples/genética , Animales , Síndrome de Costello/genética , Humanos , Sistema de Señalización de MAP Quinasas , Fenotipo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Pez Cebra/genéticaRESUMEN
Noonan syndrome (NS) is characterized by distinctive craniofacial appearance, short stature, and congenital heart disease. Approximately 80% of individuals with NS harbor mutations in genes whose products are involved in the RAS/mitogen-activating protein kinase (MAPK) pathway. However, the underlying genetic causes in nearly 20% of individuals with NS phenotype remain unexplained. Here, we report four de novo RRAS2 variants in three individuals with NS. RRAS2 is a member of the RAS subfamily and is ubiquitously expressed. Three variants, c.70_78dup (p.Gly24_Gly26dup), c.216A>T (p.Gln72His), and c.215A>T (p.Gln72Leu), have been found in cancers; our functional analyses showed that these three changes induced elevated association of RAF1 and that they activated ERK1/2 and ELK1. Notably, prominent activation of ERK1/2 and ELK1 by p.Gln72Leu associates with the severe phenotype of the individual harboring this change. To examine variant pathogenicity in vivo, we generated zebrafish models. Larvae overexpressing c.70_78dup (p.Gly24_Gly26dup) or c.216A>T (p.Gln72His) variants, but not wild-type RRAS2 RNAs, showed craniofacial defects and macrocephaly. The same dose injection of mRNA encoding c.215A>T (p.Gln72Leu) caused severe developmental impairments and low dose overexpression of this variant induced craniofacial defects. In contrast, the RRAS2 c.224T>G (p.Phe75Cys) change, located on the same allele with p.Gln72His in an individual with NS, resulted in no aberrant in vitro or in vivo phenotypes by itself. Together, our findings suggest that activating RRAS2 mutations can cause NS and expand the involvement of RRAS2 proto-oncogene to rare germline disorders.
Asunto(s)
Mutación con Ganancia de Función , Mutación de Línea Germinal , Proteínas de la Membrana/genética , Proteínas de Unión al GTP Monoméricas/genética , Síndrome de Noonan/etiología , Pez Cebra/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Niño , Preescolar , Exoma , Femenino , Humanos , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/metabolismo , Síndrome de Noonan/patología , Fenotipo , Conformación Proteica , Proto-Oncogenes Mas , Homología de Secuencia , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Long-term administration of some antiepileptic drugs often increases blood lipid levels. In this study, we investigated its molecular mechanism by focusing on the nuclear receptors constitutive active/androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα), which are key transcription factors for enzyme induction and lipid metabolism, respectively, in the liver. Treatment of mice with the CAR activator phenobarbital, an antiepileptic drug, increased plasma triglyceride levels and decreased the hepatic expression of PPARα target genes related to lipid metabolism. The increase in PPARα target gene expression induced by fenofibrate, a PPARα ligand, was inhibited by cotreatment with phenobarbital. CAR suppressed PPARα-dependent gene transcription in HepG2 cells but not in COS-1 cells. The mRNA level of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), a coactivator for both CAR and PPARα, in COS-1 cells was much lower than in HepG2 cells. In reporter assays with COS-1 cells overexpressing PGC1α, CAR suppressed PPARα-dependent gene transcription, depending on the coactivator-binding motif. In mammalian two-hybrid assays, CAR attenuated the interaction between PGC1α and PPARα Chemical inhibition of PGC1α prevented phenobarbital-dependent increases in plasma triglyceride levels and the inhibition of PPARα target gene expression. These results suggest that CAR inhibits the interaction between PPARα and PGC1α, attenuating PPARα-dependent lipid metabolism. This might explain the antiepileptic drug-induced elevation of blood triglyceride levels. SIGNIFICANCE STATEMENT: Constitutive active/androstane receptor activated by antiepileptic drugs inhibits the peroxisome proliferator-activated receptor α-dependent transcription of genes related to lipid metabolism and upregulates blood triglyceride levels. The molecular mechanism of this inhibition involves competition between these nuclear receptors for coactivator peroxisome proliferator-activated receptor γ coactivator-1α binding.
Asunto(s)
Anticonvulsivantes/farmacología , PPAR alfa/metabolismo , PPAR gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Triglicéridos/sangre , Animales , Línea Celular Tumoral , Receptor de Androstano Constitutivo , Inducción Enzimática/efectos de los fármacos , Fenofibrato/farmacología , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fenobarbital/farmacología , Factores de Transcripción/metabolismo , Activación Transcripcional/efectos de los fármacosRESUMEN
RASopathies are a group of developmental disorders caused by mutations in genes that regulate the RAS/MAPK pathway and include Noonan syndrome (NS), Costello syndrome, cardiofaciocutaneous syndrome and other related disorders. Whole exome sequencing studies recently identified LZTR1, PPP1CB and MRAS as new causative genes in RASopathies. However, information on the phenotypes of LZTR1 mutation-positive patients and functional properties of the mutations are limited. To identify variants of LZTR1, PPP1CB, and MRAS, we performed a targeted next-generation sequencing and reexamined previously analyzed exome data in 166 patients with suspected RASopathies. We identified eight LZTR1 variants, including a de novo variant, in seven probands who were suspicious for NS and one known de novo PPP1CB variant in a patient with NS. One of the seven probands had two compound heterozygous LZTR1 variants, suggesting autosomal recessive inheritance. All probands with LZTR1 variants had cardiac defects, including hypertrophic cardiomyopathy and atrial septal defect. Five of the seven probands had short stature or intellectual disabilities. Immunoprecipitation of endogenous LZTR1 followed by western blotting showed that LZTR1 bound to the RAF1-PPP1CB complex. Cells transfected with a small interfering RNA against LZTR1 exhibited decreased levels of RAF1 phosphorylated at Ser259. These are the first results to demonstrate LZTR1 in association with the RAF1-PPP1CB complex as a component of the RAS/MAPK pathway.
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Biomarcadores/análisis , Mutación , Síndrome de Noonan/genética , Proteína Fosfatasa 1/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Factores de Transcripción/metabolismo , Adolescente , Adulto , Niño , Preescolar , Exoma , Femenino , Estudios de Seguimiento , Humanos , Masculino , Síndrome de Noonan/metabolismo , Síndrome de Noonan/patología , Fenotipo , Pronóstico , Unión Proteica , Proteína Fosfatasa 1/genética , Proteínas Proto-Oncogénicas c-raf/genética , Factores de Transcripción/genética , Adulto JovenRESUMEN
Pregnane X receptor (PXR), a xenobiotic-responsive nuclear receptor, plays key roles in drug disposition. PXR activation induces liver hypertrophy in rodents, but the molecular mechanism of this effect remains unclear, although the PXR-mediated induction of cytochrome P450s (P450s) is proposed to be involved. Since yes-associated protein (YAP), an effector protein of the Hippo pathway, functions as a transcriptional cofactor that controls organ size via TEA domain family members (TEADs) or other transcription factors, we investigated the functional interaction of PXR with YAP in liver hypertrophy and drug metabolism in this study. The treatment of mice with a PXR activator induced liver hypertrophy, promoted nuclear YAP accumulation, and increased the expression of YAP/TEAD target genes in the liver, suggesting the coactivation of PXR and YAP. Through chronological analyses of this in vivo model, no clear association between PXR-dependent liver hypertrophy and P450 induction was observed. In reporter assays, ligand-activated PXR enhanced YAP-mediated gene transcription, whereas YAP overexpression inhibited PXR-dependent gene transcription. No clear species differences in these transcriptional interactions between humans and mice were observed. Furthermore, in human hepatocarcinoma and primary hepatocyte-like cells, YAP suppressed the expression of liver-enriched transcription factors, including hepatocyte nuclear factor 4α, PXR, the constitutive androstane receptor, and their target genes. These results suggest that YAP is involved in PXR-induced liver hypertrophy and that YAP activation interferes with gene expression associated with various liver functions. SIGNIFICANCE STATEMENT: We have investigated the functional interaction between PXR and YAP, an effector protein of the Hippo pathway. PXR plays central roles in various liver functions including drug metabolism, and the Hippo pathway and YAP regulate organ size through interacting with several transcription factors, including TEADs. Our results suggest that YAP is involved in PXR-mediated liver hypertrophy and that YAP activation interferes with the expression of liver-enriched transcription factors and thus drug-metabolizing enzymes.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Hepatomegalia/metabolismo , Hígado/metabolismo , Receptor X de Pregnano/metabolismo , Xenobióticos/farmacología , Animales , Células Cultivadas , Sistema Enzimático del Citocromo P-450/metabolismo , Células Hep G2 , Humanos , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Receptor X de Pregnano/efectos de los fármacos , Proteínas Señalizadoras YAPRESUMEN
Perfluorocarboxylic acids (PFCAs) including perfluorooctanoic acid (PFOA) are environmental pollutants showing high accumulation, thermochemical stability and hepatocarcinogenicity. Peroxisome proliferator-activated receptor α is suggested to mediate their toxicities, but the precise mechanism remains unclear. Previous reports also imply a possible role of constitutive androstane receptor (CAR), a key transcription factor for the xenobiotic-induced expression of various genes involved in drug metabolism and disposition as well as hepatocarcinogenesis. Therefore, we have investigated whether PFCAs activate CAR. In wild-type but not Car-null mice, mRNA levels of Cyp2b10, a CAR target gene, were increased by PFOA treatment. PFCA treatment induced the nuclear translocation of CAR in mouse livers. Since CAR activators are divided into two types, ligand-type activators and phenobarbital-like indirect activators, we investigated whether PFCAs are CAR ligands or not using the cell-based reporter gene assay that can detect CAR ligands but not indirect activators. As results, neither PFCAs nor phenobarbital increased reporter activities. Interestingly, in mouse hepatocytes, pretreatment with the protein phosphatase inhibitor okadaic acid prevented an increase in Cyp2b10 mRNA levels induced by phenobarbital as reported, but not that by PFOA. Finally, in human hepatocyte-like HepaRG cells, PFOA treatment increased mRNA levels of CYP2B6, a CAR target gene, as did phenobarbital. Taken together, our present results suggest that PFCAs including PFOA are indirect activators of mouse and human CAR and that the mechanism might be different from that for phenobarbital. The results imply a role of CAR in the hepatotoxicity of PFCAs.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Caprilatos/toxicidad , Familia 2 del Citocromo P450/metabolismo , Contaminantes Ambientales/toxicidad , Fluorocarburos/toxicidad , Receptores Citoplasmáticos y Nucleares/metabolismo , Esteroide Hidroxilasas/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Receptor de Androstano Constitutivo , Citoplasma/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , PPAR alfa/genética , PPAR alfa/metabolismo , Cultivo Primario de Células , Transporte de Proteínas , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genéticaRESUMEN
Growth factor-mediated hepatocyte proliferation is crucial in liver regeneration and the recovery of liver function after injury. The nuclear receptor, pregnane X receptor (PXR), is a key transcription factor for the xenobiotic-induced expression of genes associated with various liver functions. Recently, we reported that PXR activation stimulates xenobiotic-induced hepatocyte proliferation. In the present study, we investigated whether PXR activation also stimulates growth factor-mediated hepatocyte proliferation. In G0 phase-synchronized, immortalized mouse hepatocytes, serum or epidermal growth factor treatment increased cell growth and this growth was augmented by the expression of mouse PXR and co-treatment with pregnenolone 16α-carbonitrile (PCN), a PXR ligand. In a liver regeneration model using carbon tetrachloride, PCN treatment enhanced the injury-induced increase in the number of Ki-67-positive nuclei as well as Ccna2 and Ccnb1 mRNA levels in wild-type (WT) but not Pxr-null mice. Chronological analysis of this model demonstrated that PCN treatment shifted the maximum cell proliferation to an earlier time point and increased the number of M-phase cells at those time points. In WT but not Pxr-null mice, PCN treatment reduced hepatic mRNA levels of genes involved in the suppression of G0/G1- and G1/S-phase transition, e.g. Rbl2, Cdkn1a and Cdkn1b. Analysis of the Rbl2 promoter revealed that PXR activation inhibited its Forkhead box O3 (FOXO3)-mediated transcription. Finally, the PXR-mediated enhancement of hepatocyte proliferation was inhibited by the expression of dominant active FOXO3 in vitro. The results of the present study suggest that PXR activation stimulates growth factor-mediated hepatocyte proliferation in mice, at least in part, through inhibiting FOXO3 from accelerating cell-cycle progression.
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Proliferación Celular , Factor de Crecimiento Epidérmico/metabolismo , Factores de Transcripción Forkhead/metabolismo , Hepatocitos/citología , Receptores de Esteroides/metabolismo , Animales , Ciclo Celular , Células Cultivadas , Ciclina A1/genética , Ciclina A1/metabolismo , Ciclina A2/genética , Ciclina A2/metabolismo , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Hepatocitos/metabolismo , Hígado/citología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Receptor X de Pregnano , Carbonitrilo de Pregnenolona/metabolismo , Receptores de Esteroides/genéticaRESUMEN
Leucine zipper-like transcriptional regulator 1 (LZTR1), a substrate adaptor of Cullin 3 (CUL3)-based E3 ubiquitin ligase, regulates proteostasis of the RAS subfamily. Mutations in LZTR1 have been identified in patients with several types of cancer. However, the role of LZTR1 in tumor metastasis and the target molecules of LZTR1, excluding the RAS subfamily, are not clearly understood. Here, we show that LZTR1 deficiency increases tumor growth and metastasis. In lung adenocarcinoma cells, LZTR1 deficiency induced the accumulation of the RAS subfamily and enhanced cell proliferation, invasion, and xenograft tumor growth. Multi-omics analysis to clarify the pathways related to tumor progression showed that MAPK signaling, epithelial-mesenchymal transition (EMT), and extracellular matrix (ECM) remodeling-related gene ontology terms were enriched in LZTR1 knockout cells. Indeed, LZTR1 deficiency induced high expression of EMT markers under TGF-ß1 treatment. Our search for novel substrates that interact with LZTR1 resulted in the discovery of a Kelch-like protein 12 (KLHL12), which is involved in collagen secretion. LZTR1 could inhibit KLHL12-mediated ubiquitination of SEC31A, a component of coat protein complex II (COPII), whereas LZTR1 deficiency promoted collagen secretion. LZTR1-RIT1 and LZTR1-KLHL12 worked independently regarding molecular interactions and did not directly interfere with each other. Further, we found that LZTR1 deficiency significantly increases lung metastasis and promotes ECM deposition around metastatic tumors. Since collagen-rich extracellular matrix act as pathways for migration and facilitate metastasis, increased expression of RAS and collagen deposition may exert synergistic or additive effects leading to tumor progression and metastasis. In conclusion, LZTR1 deficiency exerts high metastatic potential by enhancing sensitivity to EMT induction and promoting collagen secretion. The functional inhibition of KLHL12 by LZTR1 provides important evidence that LZTR1 may be a repressor of BTB-Kelch family members. These results provide clues to the mechanism of LZTR1-deficiency carcinogenesis.
Asunto(s)
Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Humanos , Transición Epitelial-Mesenquimal/genética , Colágeno , Matriz Extracelular , Adenocarcinoma del Pulmón/genética , Neoplasias Pulmonares/genética , Proteínas Adaptadoras Transductoras de Señales , Factores de TranscripciónRESUMEN
Radioulnar synostosis with amegakaryocytic thrombocytopenia (RUSAT) is an inherited bone marrow failure syndrome characterized by the congenital fusion of the forearm bones. RUSAT is largely caused by missense mutations that are clustered in a specific region of the MDS1 and EVI1 complex locus (MECOM). EVI1, a transcript variant encoded by MECOM, is a zinc finger transcription factor involved in hematopoietic stem cell maintenance that induce leukemic transformation when overexpressed. Mice with exonic deletions in Mecom show reduced hematopoietic stem and progenitor cells (HSPCs). However, the pathogenic roles of RUSAT-associated MECOM mutations in vivo have not yet been elucidated. To investigate the impact of the RUSAT-associated MECOM mutation on the phenotype, we generated knockin mice harboring a point mutation (translated into EVI1 p.H752R and MDS1-EVI1 p.H942R), which corresponds to an EVI1 p.H751R and MDS1-EVI1 p.H939R mutation identified in a patient with RUSAT. Homozygous mutant mice died at embryonic day 10.5 to 11.5. Heterozygous mutant mice (Evi1KI/+ mice) grew normally without radioulnar synostosis. Male Evi1KI/+ mice, aged between 5 and 15 weeks, exhibited lower body weight, and those aged ≥16 weeks showed low platelet counts. Flow cytometric analysis of bone marrow cells revealed a decrease in HSPCs in Evi1KI/+ mice between 8 and 12 weeks. Moreover, Evi1KI/+ mice showed delayed leukocyte and platelet recovery after 5-fluorouracil-induced myelosuppression. These findings suggest that Evi1KI/+ mice recapitulate the bone marrow dysfunction in RUSAT, similar to that caused by loss-of-function Mecom alleles.
Asunto(s)
Proteínas de Unión al ADN , Factores de Transcripción , Masculino , Animales , Ratones , Proteínas de Unión al ADN/genética , Proteína del Locus del Complejo MDS1 y EV11/genética , Factores de Transcripción/genética , Células Madre Hematopoyéticas , MutaciónRESUMEN
Auditory studies in animals benefit from quick and accurate audiometry. The auditory brainstem response (ABR) and prepulse inhibition (PPI) have been widely used for hearing assessment in animals, but how well these assessments predict subjective audiometry still remains unclear. Human studies suggest that subjective audiometry is consistent with the ABR-based audiogram, not with the PPI-based audiogram, likely due to top-down processing in the cortex that inhibits PPI. Here, we challenged this view in Wistar rats, as rodents exhibit less complexity of cortical activities and thereby less influence of the cerebral cortex on PPI compared to humans. To test our hypothesis, we investigated whether subjective audiometry correlates with ABR- or PPI-based audiograms across the range of audible frequencies in Wistar rats. The subjective audiogram was obtained through pure-tone audiometry based on operant conditioning. Our results demonstrated that both the ABR-based and PPI-based audiograms significantly correlated to the subjective audiogram. We also found that ASR strength was information-rich, and adequate interpolation of this data offered accurate audiometry. Thus, unlike in humans, PPI could be used to predict subjective audibility in rats.
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Audiometría de Tonos Puros , Umbral Auditivo/fisiología , Audición/fisiología , Inhibición Prepulso/fisiología , Estimulación Acústica/métodos , Animales , Potenciales Evocados Auditivos del Tronco Encefálico , Estudios de Factibilidad , Masculino , Modelos Animales , Ratas , Ratas Wistar , Especificidad de la EspecieRESUMEN
A 31-year-old woman who was clinically diagnosed with Silver-Russell syndrome (SRS) in childhood was admitted with complaints of dyspnea. She had hypercapnic respiratory failure accompanied by nocturnal hypoventilation. Computed tomography revealed systemic muscle atrophy and superior mesenteric artery syndrome; however, the bilateral lung fields were normal. She was treated with nocturnal noninvasive positive pressure ventilation and showed improvement of respiratory failure. In this case, loss of methylation on chromosome 11p15 and maternal uniparental disomy of chromosome 7, which are the common causes of SRS, were not detected. This is a rare case of adult SRS manifesting as chronic hypercapnic respiratory failure.
Asunto(s)
Insuficiencia Respiratoria , Síndrome de Silver-Russell , Adulto , Femenino , Humanos , Insuficiencia Respiratoria/etiología , Insuficiencia Respiratoria/genética , Síndrome de Silver-Russell/complicaciones , Síndrome de Silver-Russell/diagnóstico , Síndrome de Silver-Russell/genética , Disomía UniparentalRESUMEN
Pregnane X receptor (PXR) is a liver-enriched xenobiotic-responsive transcription factor. Although recent studies suggest that PXR shows anti-inflammatory effects by suppressing nuclear factor kappa B (NF-κB), the detailed mechanism remains unclear. In this study, we aimed to elucidate this mechanism. Mice were treated intraperitoneally with the PXR agonist pregnenolone 16α-carbonitrile (PCN) and/or carbon tetrachloride (CCl4). Liver injury was evaluated, and hepatic mRNA levels were determined via quantitative reverse transcription polymerase chain reaction. Reporter assays with wild-type and mutated mouse Cxcl2 promoter-containing reporter plasmids were conducted in 293T cells. Results showed that the hepatic expression of inflammation-related genes was upregulated in CCl4-treated mice, and PCN treatment repressed the induced expression of chemokine-encoding Ccl2 and Cxcl2 among the genes investigated. Consistently, PCN treatment suppressed the increased plasma transaminase activity and neutrophil infiltration in the liver. In reporter assays, tumor necrosis factor-α-induced Cxcl2 expression was suppressed by PXR. Although an NF-κB inhibitor or the mutation of an NF-κB-binding motif partly reduced PXR-dependent suppression, the mutation of both NF-κB and activator protein 1 (AP-1) sites abolished it. Consistently, AP-1-dependent gene transcription was suppressed by PXR with a construct containing AP-1 binding motifs. In conclusion, the present results suggest that PXR exerts anti-inflammatory effects by suppressing both NF-κB- and AP-1-dependent chemokine expression in mouse liver.
Asunto(s)
Quimiocina CXCL2/genética , Inflamación/genética , FN-kappa B/metabolismo , Receptor X de Pregnano/metabolismo , Factor de Transcripción AP-1/metabolismo , Animales , Antiinflamatorios , Tetracloruro de Carbono/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Carbonitrilo de Pregnenolona/farmacología , Unión Proteica , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Leucine zipper-like transcriptional regulator 1 (LZTR1) encodes a member of the BTB-Kelch superfamily, which interacts with the Cullin3 (CUL3)-based E3 ubiquitin ligase complex. Mutations in LZTR1 have been identified in glioblastoma, schwannomatosis, and Noonan syndrome. However, the functional role of LZTR1 in carcinogenesis or human development is not fully understood. Here, we demonstrate that LZTR1 facilitates the polyubiquitination and degradation of RAS via the ubiquitin-proteasome pathway, leading to the inhibition of the RAS/MAPK signaling. The polyubiquitination and degradation of RAS was also observed in cells expressing MRAS, HRAS, NRAS, and KRAS as well as oncogenic RAS mutants and inhibited the activation of ERK1/2 and cell growth. In vivo ubiquitination assays showed that MRAS-K127 and HRAS-K170 were ubiquitinated by LZTR1 and that the polyubiquitinated-chains contained mainly Ub-K48, K63, and K33-linked chains, suggesting its possible involvement in autophagy. Immunoprecipitation analyses showed the interaction of LZTR1 and RAS-GTPases with autophagy-related proteins, including LC3B and SQSTM1/p62. Co-expression of LZTR1 and RAS increased the expression of lipidated form of LC3B. However, long-term treatment with chloroquine had little effect on RAS protein levels, suggesting that the contribution of autophagy to LZTR1-mediated RAS degradation is minimal. Taken together, these results show that LZTR1 functions as a "RAS killer protein" mainly via the ubiquitin-proteasome pathway regardless of the type of RAS GTPase, controlling downstream signal transduction. Our results also suggest a possible association of LZTR1 and RAS-GTPases with the autophagy. These findings provide clues for the elucidation of the mechanisms of RAS degradation and regulation of the RAS/MAPK signaling cascade.
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Poliubiquitina/metabolismo , Proteolisis , Factores de Transcripción/metabolismo , Ubiquitinación , Proteínas ras/metabolismo , Autofagia , Secuencia de Bases , Proteínas Cullin/metabolismo , Células HEK293 , Humanos , Lisina/metabolismo , Modelos Biológicos , Multimerización de ProteínaRESUMEN
Costello syndrome is an autosomal dominant disorder that is caused by germline HRAS mutations. Patients with Costello syndrome present craniofacial abnormalities, cardiac defects, and cancer predisposition, as well as skin abnormalities, including papillomas, keratosis pilaris, and eczematous dermatitis. However, the mechanisms underlying the dermatological abnormalities remain unclear. Here, we demonstrated that knock-in mice expressing an Hras G12S mutation (HrasG12S/+ mice) are susceptible to develop atopic dermatitis (AD)-like skin lesions, including eczema, pruritus, elevated serum IgE levels, acanthosis, and the infiltration of mast cells, basophils, and type-2 innate lymphoid cells in the dermis, after stimulation with house dust mite allergens (Dermatophagoides farinae, Dfb). Reduced skin barrier function, increased proliferation of phosphorylated ERK (p-ERK)-positive epidermal cells, and increased Th2-type cytokines as well as epithelial cell-derived cytokines, including IL-33, were observed in the skin tissue of HrasG12S/+ mice compared with Hras+/+ mice. Cultured HrasG12S/+ keratinocytes exhibited increased IL-33 expression after Dfb stimulation. PD0325901, an MEK inhibitor, ameliorated AD-like symptoms in HrasG12S/+ mice, showing decreased proliferation of p-ERK-positive epidermal cells and decreased expression of IL-33. Our findings indicate that the epidermis of HrasG12S/+ mice stimulated by Dfb strongly induced IL-33 expression and type-2 innate lymphoid cells, resulting in AD-like skin lesions. These results suggest that the epidermis of HrasG12S/+ mice are prone to development of eczematous dermatitis stimulated with house dust mite allergens.
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Síndrome de Costello/genética , Dermatitis Atópica/genética , Dermatitis Atópica/parasitología , Mutación/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Pyroglyphidae/fisiología , Animales , Benzamidas/farmacología , Proliferación Celular/efectos de los fármacos , Síndrome de Costello/complicaciones , Síndrome de Costello/patología , Citocinas/metabolismo , Dermatitis Atópica/complicaciones , Dermatitis Atópica/patología , Difenilamina/análogos & derivados , Difenilamina/farmacología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Oído/patología , Epidermis/efectos de los fármacos , Epidermis/parasitología , Epidermis/patología , Mediadores de Inflamación/metabolismo , Interleucina-33/metabolismo , Masculino , Ratones Endogámicos C57BL , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Modelos Biológicos , Inhibidores de Proteínas Quinasas/farmacología , Prurito/complicaciones , Prurito/patología , Pyroglyphidae/efectos de los fármacosRESUMEN
Samples of Paramecium caudatum are observed by means of a scanning electron microscope (SEM) and a projection X-ray microscope (XRM) with computer tomography (CT) function. The samples are fixed with two kinds of fixatives, glutaraldehyde and osmium-tetra oxide acid. After the fixation and replacement procedure with t-buthyl alcohol, the samples followed by a freeze drying, well retain their structures. Surface structures, cilia and microfibrillar systems including infraciliary lattice structures, are clearly depicted by SEM observation. On the other hand, XRM images give quite different information, namely, in the case of osmium oxide fixation, the structures of internal organelles like the macronucleus placed in the central part of cell body and trichocysts located under the cell membrane of a whole body are visible. In the case of glutaraldehyde fixation, the surface structures and internal structures are both visible but their image contrast is fairly weak. In order to examine toxicological effect, Paramecium caudatum samples treated in the environmental condition containing nano-particles of Ag (17 nm across) and Co-ferrite (300 nm across) are observed with results of certain morphological differences, namely, inner vacuoles increase in number and in volume in Co-ferrite treated cells as compared with Ag treated ones. But then, cilia-less areas increase on the surface of the body of Ag treated cells. In the case of Co-ferrite treated cells, cilia-less areas are not clearly detected. Whether these morphological differences observed in Ag and Co-ferrite treated cells are caused by the differences of materials or particle sizes remain to be examined in future.
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Microscopía Electrónica de Rastreo/métodos , Microscopía/métodos , Paramecium/ultraestructura , Preservación Biológica/métodos , Animales , Aumento de la Imagen/métodos , Rayos XRESUMEN
Andrastins are meroterpenes isolated from Penicillium sp. FO-3929 that display highly potent inhibitory activities toward protein farnesyltransferase. Structurally, they possess a unique steroidal tetracyclic skeleton (the ABCD-ring) with three contiguous quaternary stereocenters on the C-ring. Herein, we describe our nitrile cyclization-based approach to the stereoselective construction of the BCD-ring system of andrastins, which contains three contiguous quaternary stereocenters on the C-ring and the correct oxidation states of the D-ring.
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Androstadienos/síntesis química , Androstadienos/química , Ciclización , Estructura Molecular , Penicillium/química , EstereoisomerismoRESUMEN
Constitutive androstane receptor (CAR) is a xenobiotic-responsive nuclear receptor that is highly expressed in the liver. CAR activation induces hepatocyte proliferation and hepatocarcinogenesis in rodents, but the mechanisms remain unclear. In this study, we investigated the association of CAR-dependent cell proliferation with Yes-associated protein (YAP), which is a transcriptional cofactor controlling organ size and cell growth through the interaction with various transcriptional factors including TEA domain family member (TEAD). In mouse livers, 1,4-bis-(2-[3,5-dichloropyridyloxy])benzene (TCPOBOP) (a mouse CAR [mCAR] activator) treatment increased the nuclear YAP accumulation and mRNA levels of YAP target genes as well as cell-cycle related genes along with liver hypertrophy and verteporfin (an inhibitor of YAP/TEAD interaction) cotreatment tended to attenuate them. Furthermore, in cell-based reporter gene assays, CAR activation enhanced the YAP/TEAD-dependent transcription. To investigate the role of YAP/TEAD activation in the CAR-dependent hepatocyte proliferation, we sought to establish an in vitro system completely reproducing CAR-dependent cell proliferation. Since CAR was only slightly expressed in cultured mouse primary hepatocytes compared with mouse livers and no proliferation was observed after treatment with TCPOBOP, we overexpressed CAR using mCAR expressing adenovirus (Ad-mCAR-V5) in mouse primary hepatocytes. Ad-mCAR-V5 infection and TCPOBOP treatment induced hepatocyte proliferation. Similar results were obtained with immortalized normal mouse hepatocytes as well. In the established in vitro system, CAR-dependent proliferation was strongly inhibited by Yap knockdown and completely abolished by verteporfin treatment. Our present results obtained in in vivo and in vitro experiments suggest that YAP/TEAD activation plays key roles in CAR-dependent proliferation of murine hepatocytes.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proliferación Celular , Hepatocitos/metabolismo , Hígado/metabolismo , Fosfoproteínas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Receptor de Androstano Constitutivo , Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Hipertrofia , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Fosfoproteínas/genética , Cultivo Primario de Células , Piridinas/farmacología , Receptores Citoplasmáticos y Nucleares/genética , Verteporfina/farmacología , Proteínas Señalizadoras YAPRESUMEN
The pregnane X receptor (PXR) is well-known as a key regulator of drug/xenobiotic clearance. Upon activation by ligand, PXR transcriptionally upregulates the expression of drug-metabolizing enzymes and drug transporters. Recent studies have revealed that PXR also plays a role in regulating immune/inflammatory responses. Specific PXR activators, including synthetic ligands and phytochemicals, have been shown to ameliorate chemically induced colitis in mice. In this study, we investigated an anti-inflammatory effect of pregnenolone 16α-carbonitrile (PCN), a prototypical activator for rodent PXR, in concanavalin A (Con A)-induced liver injury, a model of immune-mediated liver injury, using wild-type and Pxr-/- mice. Unexpectedly, pretreatment with PCN significantly ameliorated Con A-induced liver injury in not only wild-type but Pxr-/- mice as well, accompanied with lowered plasma ALT levels and histological improvements. Pretreatment with PCN was found to significantly repress the induction of Cxcl2 and Ccl2 mRNA expression and neutrophil infiltration into the liver of both wild-type and Pxr-/- mice at the early time point of Con A-induced liver injury. Our results indicate that PCN has unexpected immunosuppressive activity independent of PXR activation to protect mice from immune-mediated liver injury induced by Con A.