RESUMEN
BACKGROUND: Early recurrence (ER) within a 90-day blanking period (BP) in catheter ablation (CA) for atrial fibrillation (AF) is a risk factor for late recurrence (LR) after 90 days postoperatively. However, few reports have examined them in the second CA and compared them to the first CA. Moreover, in recent years, there have been reports suggesting that BP should be reduced from 90 to 30 days. Therefore, the association between ER and LR in the first and the second CA was examined, and the validity of a 30-day BP was evaluated. METHODS: A total of 511 consecutive patients undergoing the first CA and 116 of these patients undergoing the second CA for AF at a single institution from November 2016 to December 2020 were analyzed retrospectively. RESULTS: When ER within a 90-day BP was divided into 0-30 days and 31-90 days according to the timing of the last ER episode, the hazard ratios on LR of them relative to no ER were 2.7 {95% confidence interval (CI) 1.7-4.2} and 9.7 (95% CI 6.6-14.3), respectively, for the first CA and 15.3 (95% CI 4.7-50.1) and 44.1 (95% CI 14.0-139.4), respectively, for the second CA. CONCLUSIONS: ER was strongly associated with LR, especially in patients with the last episode of ER more than 30 days after CA. This was pronounced in cases after the second CA, when PVI appeared to be completed. With the current improvement in PVI durability, BP may be acceptable for 30 days.
Asunto(s)
Fibrilación Atrial , Ablación por Catéter , Recurrencia , Fibrilación Atrial/cirugía , Humanos , Ablación por Catéter/métodos , Masculino , Femenino , Estudios Retrospectivos , Persona de Mediana Edad , Factores de Tiempo , Factores de Riesgo , AncianoRESUMEN
A 71-year-old man was transferred urgently to our hospital after collapsing near his home post the first shot of the BNT162b2 coronavirus disease 2019 vaccine (Pfizer-BioNTech, Comirnaty®). Immediately after arrival at our hospital, cardiac arrest due to complete atrioventricular block with no ventricular escaped beats was observed on electrocardiogram. Echocardiography showed preserved left ventricular ejection fraction, however, diffuse severe hypokinesia was revealed after 3â¯weeks, and he died 3â¯months after admission because of worsening heart failure. An autopsy examination revealed eosinophilic myocarditis or hypersensitivity myocarditis with extensive fibrosis and widespread myocardial dropout throughout the heart. Learning objective: 1. Severe myocarditis occurs extremely rarely after mRNA coronavirus disease 2019 (COVID-19) vaccination. 2. Myocarditis after mRNA COVID-19 vaccination might cause complete atrioventricular block, followed by a course of decreased left ventricular ejection fraction. 3. Histologically, severe myocarditis after mRNA COVID-19 vaccination seems to present as fulminant necrotizing eosinophilic myocarditis or hypersensitivity myocarditis.
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Glycosaminoglycan polysaccharides are components of animal extracellular matrices and regulate cell functions based on their various sulfation and epimerization pattern structures. The present study aimed to find glycosaminoglycan structures to promote neural differentiation. We investigated the effect of exogenous glycosaminoglycans with well-defined structures on the all-trans-retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells, which is an ideal model culture system for studying neural differentiation. We found that chondroitin sulfate E and heparin, but not any other glycosaminoglycans, upregulated the expressions of neural specific markers but not a grail specific marker. Chondroitin sulfate E was suggested to function during spheroid formation, however, equimolar concentration of its oligosaccharide did not show promotive effect on the neural differentiation. Another finding was that hyaluronan oligosaccharide mixture markedly downregulated the expressions of a myelin specific marker. These findings suggested that the specific sulfation pattern and/or chain length of exogenous added glycosaminoglycan is important to regulate neural differentiation and myelination.
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Diferenciación Celular/efectos de los fármacos , Células Madre de Carcinoma Embrionario/patología , Glicosaminoglicanos/química , Glicosaminoglicanos/farmacología , Neuronas/patología , Tretinoina/farmacología , Animales , Biomarcadores/metabolismo , Bovinos , Ratones , Neuronas/efectos de los fármacos , Oligosacáridos/metabolismo , PorcinosRESUMEN
BACKGROUND: Timely differentiation of monocytes into M2-like macrophages is important in the cardiac healing process after myocardial infarction (MI), but molecular mechanisms governing M2-like macrophage differentiation at the transcriptional level after MI have not been fully understood.MethodsâandâResults:A time-series microarray analysis of mRNAs and microRNAs in macrophages isolated from the infarcted myocardium was performed to identify the microRNAs involved in regulating the process of differentiation to M2-like macrophages. Correlation analysis revealed 7 microRNAs showing negative correlations with the progression of polarity changes towards M2-like subsets. Next, correlation coefficients for the changes in expression of mRNAs and miRNAs over time were calculated for all combinations. As a result, miR-27a-5p was extracted as a possible regulator of the largest number of genes in the pathway for the M2-like polarization. By selecting mouse mRNAs and human mRNAs possessing target sequences of miR-27a-5p and showing expression patterns inversely correlated with that of miR-27a-5p, 8 potential targets of miR-27a-5p were identified, includingPpm1l. Using the mouse bone marrow-derived macrophages undergoing differentiation into M2-like subsets by interleukin 4 stimulation, we confirmed that miR-27a-5p suppressed M2-related genes by negatively regulatingPpm1lexpression. CONCLUSIONS: Ppm1land miR-27a-5p may be the key molecules regulating M2-like polarization, with miR-27a-5p inhibiting the M2-like polarization through downregulation ofPpm1lexpression.
Asunto(s)
MicroARNs , Infarto del Miocardio , Animales , Perfilación de la Expresión Génica , Macrófagos , Ratones , MicroARNs/genética , Monocitos , Infarto del Miocardio/genética , ARN MensajeroRESUMEN
A 77-year-old woman with symptomatic paroxysmal atrial fibrillation (PAF) underwent pulmonary vein isolation (PVI), but subsequently experienced recurrence. In the second session, unidirectional left atrium (LA)-left superior pulmonary vein (LSPV) conduction was revealed to exist at the carina of the LSPV. Left pulmonary vein (LPV) pacing performed in a cycle between 300 and 260 ms revealed rate-dependent pulmonary vein (PV)-LA conduction, and the location was estimated to be in the roof of the LSPV. PV isolation was achieved after ablation of two gaps. Consideration of the presence of rate-dependent gaps may be useful to confirm bidirectional block lines after ablation.
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Ppm1b, a metaldependent serine/threonine protein phosphatase, catalyzes the dephosphorylation of a variety of phosphorylated proteins. Ppm1b/ mouse embryos die at the fertilized oocyte stage, whereas Ppm1b+/ mice with a C57BL/6 background exhibit no phenotypic abnormalities. Because the C57BL/6 strain produces a limited number of pups, in an attempt to produce Ppm1b/ mice, congenic Ppm1b+/ mice with an ICR background were established, which are more fertile and gave birth to more pups. As a result, however, no Ppm1b/ offspring were obtained when pairs of Ppm1b+/ ICR mice were bred again. Ppm1b+/ male and female ICR mice were analyzed from the viewpoint of fecundity. The Ppm1b haploinsufficiency had no effect on testicular weight or the number of sperm in male mice. Despite the fact that the levels of Ppm1b protein in the ovaries of sexually mature Ppm1b+/ mice were decreased compared with those of Ppm1b+/+ mice, there appeared to be no significant difference in the histological appearance of the ovaries, litter sizes or plasma progesterone levels at the estrous stage. When superovulation was induced by stimulation using a hormone treatment, the number of ovulated oocytes were the same for Ppm1b+/ and Ppm1b+/+ mice at 4 weeks of age when the estrous cycle did not proceed, however, the number of ovulated oocytes was lower in sexually mature Ppm1b+/ mice at 11 weeks of age compared with Ppm1b+/+ mice in the first and the second superovulation cycles. These collective results suggest that follicle development is excessive in Ppm1b+/ mice, and that this leads to a partial depletion of matured follicles and a corresponding decrease in the number of ovulated oocytes.
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Proteína Fosfatasa 2C/genética , Superovulación , Animales , Gonadotropina Coriónica/farmacología , Femenino , Heterocigoto , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Oocitos/citología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Ovario/metabolismo , Embarazo , Progesterona/sangre , Proteína Fosfatasa 2C/metabolismo , Superovulación/efectos de los fármacosRESUMEN
Renal fibrosis is closely related to chronic inflammation and is under the control of epigenetic regulations. Because the signaling of transforming growth factor-ß1 (TGF-ß1) and tumor necrosis factor-α (TNF-α) play key roles in progression of renal fibrosis, dual blockade of TGF-ß1 and TNF-α is desired as its therapeutic approach. Here we screened small molecules showing anti-TNF-α activity in the compound library of indole derivatives. 11 out of 41 indole derivatives inhibited the TNF-α effect. Among them, Mitochonic Acid 35 (MA-35), 5-(3, 5-dimethoxybenzyloxy)-3-indoleacetic acid, showed the potent effect. The anti-TNF-α activity was mediated by inhibiting IκB kinase phosphorylation, which attenuated the LPS/GaIN-induced hepatic inflammation in the mice. Additionally, MA-35 concurrently showed an anti-TGF-ß1 effect by inhibiting Smad3 phosphorylation, resulting in the downregulation of TGF-ß1-induced fibrotic gene expression. In unilateral ureter obstructed mouse kidney, which is a renal fibrosis model, MA-35 attenuated renal inflammation and fibrosis with the downregulation of inflammatory cytokines and fibrotic gene expressions. Furthermore, MA-35 inhibited TGF-ß1-induced H3K4me1 histone modification of the fibrotic gene promoter, leading to a decrease in the fibrotic gene expression. MA-35 affects multiple signaling pathways involved in the fibrosis and may recover epigenetic modification; therefore, it could possibly be a novel therapeutic drug for fibrosis.
Asunto(s)
Indoles/farmacología , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Línea Celular , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Fibrosis , Hepatitis/tratamiento farmacológico , Hepatitis/etiología , Hepatitis/metabolismo , Hepatitis/patología , Histonas/metabolismo , Humanos , Quinasa I-kappa B/metabolismo , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/etiología , Lipopolisacáridos/efectos adversos , Masculino , Metilación , Ratones , Modelos Biológicos , Fosforilación/efectos de los fármacos , Proteína smad3/metabolismoRESUMEN
PPM1L, a member of the metal-dependent protein phosphatase (PPM) family, is involved in regulating the stress-activated protein kinase pathway and ceramide trafficking. However, the physiological function of PPM1L in the brain is unclear. In this study, we generated and analyzed ppm1l-deficient mice in order to investigate PPM1L functions in the brain. Our results indicate that ppm1l is highly expressed in the central nervous system during mouse development and that ppm1lΔ/Δ mice display impaired motor performance and morphological abnormalities in the forebrain. Electron microscopic and immunohistochemical analyses suggest that these abnormalities are due to impaired axonal tract formation. Our novel findings suggest an important role for PPM1L in brain development.
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Encéfalo/anomalías , Fosfoproteínas Fosfatasas/deficiencia , Animales , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Sistema de Señalización de MAP Quinasas , RatonesRESUMEN
The BCNT (Bucentaur) superfamily is classified by an uncharacteristic conserved sequence of â¼80 amino acids (aa) at the C-terminus, BCNT-C (the conserved C-terminal region of Bcnt/Cfdp1). Whereas the yeast Swc5 and Drosophila Yeti homologues play crucial roles in chromatin remodelling organization, mammalian Bcnt/Cfdp1 (craniofacial developmental protein 1) remains poorly understood. The protein, which lacks cysteine, is largely disordered and comprises an acidic N-terminal region, a lysine/glutamic acid/proline-rich 40 aa sequence and BCNT-C. It shows complex mobility on SDS/PAGE at â¼50 kDa, whereas its calculated molecular mass is â¼33 kDa. To characterize this mobility discrepancy and the effects of post-translational modifications (PTMs), we expressed various deleted His-Bcnt in E. coli and HEK cells and found that an acidic stretch in the N-terminal region is a main cause of the gel shift. Exogenous BCNT/CFDP1 constitutively expressed in HEK clones appears as a doublet at 49 and 47 kDa, slower than the protein expressed in Escherichia coli but faster than the endogenous protein on SDS/PAGE. Among seven in vivo phosphorylation sites, Ser(250), which resides in a region between disordered and ordered regions in BCNT-C, is heavily phosphorylated and detected predominantly in the 49 kDa band. Together with experiments involving treatment with phosphatases and Ser(250) substitutions, the results indicate that the complex behaviour of Bcnt/Cfdp1 on SDS/PAGE is caused mainly by an acidic stretch in the N-terminal region and Ser(250) phosphorylation in BCNT-C. Furthermore, Bcnt/Cfdp1 is acetylated in vitro by CREB-binding protein (CBP) and four lysine residues including Lys(268) in BCNT-C are also acetylated in vivo, revealing a protein regulated at multiple levels.
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Fosfoproteínas/química , Fosfoproteínas/metabolismo , Acetilación , Sustitución de Aminoácidos , Proteína de Unión a CREB/química , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Epigénesis Genética , Células HEK293 , Humanos , Mutación Missense , Proteínas Nucleares , Fosfoproteínas/genética , Fosforilación/fisiología , Estructura Terciaria de ProteínaRESUMEN
A variety of genetic backgrounds cause the loss of function of thiazide-sensitive sodium chloride cotransporter, encoded by SLC12A3, responsible for the phenotypes in Gitelman syndrome. Recently, the phenomenon of exon skipping, in which exonic mutations result in abnormal splicing, has been associated with various diseases. Specifically, mutations in exonic splicing enhancer (ESE) sequences can promote exon skipping. Here, we used a bioinformatics program to analyze 88 missense mutations in the SLC12A3 gene and identify candidate mutations that may induce exon skipping. The three candidate mutations that reduced ESE scores the most were further investigated by minigene assay, and two (p.A356V and p.M672I) caused abnormal splicing in vitro. Furthermore, we identified the p.M672I (c.2016G>A) mutation in a patient with Gitelman syndrome and found that this single nucleotide mutation causes exclusion of exon 16 in the SLC12A3 mRNA transcript. Functional analyses revealed that the protein encoded by the aberrant SLC12A3 transcript does not transport sodium. These results suggest that aberrant exon skipping is one previously unrecognized mechanism by which missense mutations in SLC12A3 can lead to Gitelman syndrome.
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Exones , Síndrome de Gitelman/genética , Mutación Missense , Terminación de la Transcripción Genética , Adulto , Línea Celular , Femenino , Síndrome de Gitelman/fisiopatología , Humanos , Linaje , Polimorfismo de Nucleótido Simple/genética , ARN Mensajero/genética , Análisis de Secuencia de ADN , Miembro 3 de la Familia de Transportadores de Soluto 12/genéticaRESUMEN
Bone mass is maintained by the balance between the activities of bone-forming osteoblasts and bone-resorbing osteoclasts. It is well known that adequate mechanical stress is essential for the maintenance of bone mass, whereas excess mechanical stress induces bone resorption. However, it has not been clarified how osteoblasts respond to different magnitudes of mechanical stress. Here we report that large-magnitude (12%) cyclic stretch induced Ca(2+) influx, which activated reactive oxygen species generation in MC3T3-E1 osteoblasts. Reactive oxygen species then activated the ASK1-JNK/p38 pathways. The activated JNK led to transiently enhanced expression of FGF-inducible 14 (Fn14, a member of the TNF receptor superfamily) gene. Cells with enhanced expression of Fn14 subsequently acquired sensitivity to the ligand of Fn14, TNF-related weak inducer of apoptosis, and underwent apoptosis. On the other hand, the ASK1-p38 pathway induced expression of the monocyte chemoattractant protein 3 (MCP-3) gene, which promoted chemotaxis of preosteoclasts. In contrast, the ERK pathway was activated by small-magnitude stretching (1%) and induced expression of two osteogenic genes, collagen Ia (Col1a) and osteopontin (OPN). Moreover, activated JNK suppressed Col1a and OPN induction in large-magnitude mechanical stretch-loaded cells. The enhanced expression of Fn14 and MCP-3 by 12% stretch and the enhanced expression of Col1a and OPN by 1% stretch were also observed in mouse primary osteoblasts. These results suggest that differences in the response of osteoblasts to varying magnitudes of mechanical stress play a key role in switching the mode of bone metabolism between formation and resorption.
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Apoptosis , Regulación de la Expresión Génica , MAP Quinasa Quinasa Quinasa 5/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Osteoblastos/fisiología , Receptores del Factor de Necrosis Tumoral/genética , Estrés Mecánico , Células 3T3 , Animales , Sistema de Señalización de MAP Quinasas , Ratones , Osteoblastos/metabolismo , Receptor de TWEAKRESUMEN
PPM [metal-dependent protein phosphatase, formerly called PP2C (protein phosphatase 2C)] family members play essential roles in regulating a variety of signalling pathways. While searching for protein phosphatase(s) that act on AMPK (AMP-activated protein kinase), we found that PPM1A and PPM1B are N-myristoylated and that this modification is essential for their ability to dephosphorylate the α subunit of AMPK (AMPKα) in cells. N-Myristoylation was also required for two other functions of PPM1A and PPM1B in cells. Although a non-myristoylated mutation (G2A) of PPM1A and PPM1B prevented membrane association, this relocalization did not likely cause the decreased activity towards AMPKα. In in vitro experiments, the G2A mutants exhibited reduced activities towards AMPKα, but much higher specific activity against an artificial substrate, PNPP (p-nitrophenyl phosphate), compared with the wild-type counterparts. Taken together, the results of the present study suggest that N-myristoylation of PPM1A and PPM1B plays a key role in recognition of their physiological substrates in cells.
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Fosfoproteínas Fosfatasas/metabolismo , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Dominio Catalítico/genética , Células HEK293 , Células HeLa , Humanos , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Ácido Mirístico/metabolismo , Nitrofenoles/metabolismo , Compuestos Organofosforados/metabolismo , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Fosforilación , Proteína Fosfatasa 2C , Procesamiento Proteico-Postraduccional , ARN Interferente Pequeño/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
The metal-dependent protein phosphatase family (PPM) governs a number of signaling pathways. PPM1L, originally identified as a negative regulator of stress-activated protein kinase signaling, was recently shown to be involved in the regulation of ceramide trafficking at ER-Golgi membrane contact sites. Here, we identified acyl-CoA binding domain containing 3 (ACBD3) as an interacting partner of PPM1L. We showed that this association, which recruits PPM1L to ER-Golgi membrane contact sites, is mediated by a GOLD (Golgi dynamics) domain in ACBD3. These results suggested that ACBD3 plays a pivotal role in ceramide transport regulation at the ER-Golgi interface.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Sitios de Unión , Células HEK293 , Células HeLa , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Modelos Biológicos , Modelos Moleculares , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Técnicas del Sistema de Dos Híbridos , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Mechanical stress plays a key role in bone remodeling. Previous studies showed that loading of mechanical stretch induces a rapid Ca(2+) influx and subsequent activation of stress-activated protein kinase pathways in osteoblasts. However, the activation mechanism and its significance in bone remodeling have not been fully elucidated. Here we show that TAK1 MAPKKK was activated by cyclic stretch loading of MC3T3-E1 cells. Knockdown of TAK1 attenuated the stretch-induced activation of JNK, p38, and NF-κB. Extracellular (EGTA) or intracellular (BAPTA/AM) Ca(2+) chelator prevented the stretch-induced activation of TAK1. Activation of TAK1 and its associated downstream signaling pathways were also suppressed by CaMKII inhibitors (KN-93 and KN-62). Furthermore, TAK1-mediated downstream pathways cooperatively induced the expression of IL-6 mRNA in the stretched MC3T3-E1 cells. We also confirmed that TAK1 mediates cyclic stretch-induced IL-6 protein synthesis in the cells using immunoblotting and ELISA. Finally, stretch loading of murine primary osteoblasts induced the expression of IL-6 mRNA via TAK1. Collectively, these data suggest that stretch-dependent Ca(2+) influx activates TAK1 via CaMKII, leading to the enhanced expression of IL-6 through JNK, p38, and NF-κB pathways in osteoblasts.
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Interleucina-6/biosíntesis , Quinasas Quinasa Quinasa PAM/fisiología , Osteoclastos/fisiología , Estrés Mecánico , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Bencilaminas/farmacología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Línea Celular , Técnicas de Silenciamiento del Gen , MAP Quinasa Quinasa 4/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Ratones , FN-kappa B/metabolismo , Osteoclastos/enzimología , Sulfonamidas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
IL-1 (interleukin-1) is a pro-inflammatory cytokine that has a variety of effects during the process of inflammation. Stimulating cells with IL-1 initiates a signalling cascade that includes the activation of NF-kappaB (nuclear factor kappaB), and subsequently induces a variety of inflammatory genes. Although the molecular mechanism for the IL-1-induced activation of NF-kappaB has been well documented, much less is known about the mechanism by which protein phosphatases down-regulate this pathway. Here we show that mouse PP2Ceta-2 (protein serine/threonine phosphatase 2Ceta-2), a novel member of the protein serine/threonine phosphatase 2C family, inhibits the IL-1-NF-kappaB signalling pathway. Ectopic expression of PP2Ceta-2 in human embryonic kidney HEK293IL-1RI cells inhibited the IL-1-induced activation of NF-kappaB. TAK1 (transforming-growth-factor-beta-activated kinase 1) mediates the IL-1 signalling pathway to NF-kappaB, and we observed that the TAK1-induced activation of NF-kappaB was suppressed by PP2Ceta-2 expression. Expression of IKKbeta [IkappaB (inhibitory kappaB) kinase beta], which lies downstream of TAK1, activates NF-kappaB, and this activation was also readily reversed by PP2Ceta-2 co-expression. Additionally, PP2Ceta-2 knockdown with small interfering RNA further stimulated the IL-1-enhanced phosphorylation of IKKbeta and destabilization of IkappaBalpha in HeLa cells. PP2Ceta-2 knockdown also increased the IL-1-induced expression of IL-6 mRNA. Furthermore, IKKbeta was readily dephosphorylated by PP2Ceta-2 in vitro. These results suggest that PP2Ceta-2 inhibits the IL-1-NF-kappaB signalling pathway by selectively dephosphorylating IKKbeta.
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Interleucina-1/farmacología , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Fosfoproteínas Fosfatasas/fisiología , Animales , Núcleo Celular/metabolismo , Células Cultivadas , Regulación hacia Abajo , Células HeLa , Humanos , Quinasa I-kappa B/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Isoenzimas/metabolismo , Isoenzimas/fisiología , Ratones , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteína Fosfatasa 2C , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
The protein phosphatase 2C (PP2C) family represents one of the four major protein Ser/Thr phosphatase activities in mammalian cells and contains at least 13 distinct gene products. Although PP2C family members regulate a variety of cellular functions, mechanisms of regulation of their activities are largely unknown. Here, we show that PP2Czeta, a PP2C family member that is enriched in testicular germ cells, is phosphorylated by c-Jun NH 2-terminal kinase (JNK) but not by p38 in vitro. Mass spectrometry and mutational analyses demonstrated that phosphorylation occurs at Ser (92), Thr (202), and Thr (205) of PP2Czeta. Phosphorylation of these Ser and Thr residues of PP2Czeta ectopically expressed in 293 cells was enhanced by osmotic stress and was attenuated by a JNK inhibitor but not by p38 or MEK inhibitors. Phosphorylation of PP2Czeta by TAK1-activated JNK repressed its phosphatase activity in cells, and alanine mutation at Ser (92) but not at Thr (202) or Thr (205) suppressed this inhibition. Taken together, these results suggest that specific phosphorylation of PP2Czeta at Ser (92) by stress-activated JNK attenuates its phosphatase activity in cells.
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Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/metabolismo , Fosfoserina/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Ratones , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosforilación , Proteína Fosfatasa 2CRESUMEN
Protein phosphatase 2Cepsilon (PP2Cepsilon), a mammalian PP2C family member, is expressed in various tissues and is implicated in the negative regulation of stress-activated protein kinase pathways. We show that PP2Cepsilon is an endoplasmic reticulum (ER) transmembrane protein with a transmembrane domain at the amino terminus and the catalytic domain facing the cytoplasm. Yeast two-hybrid screening of a human brain library using PP2Cepsilon as bait resulted in the isolation of a cDNA that encoded vesicle-associated membrane protein-associated protein A (VAPA). VAPA is an ER resident integral membrane protein involved in recruiting lipid-binding proteins such as the ceramide transport protein CERT to the ER membrane. Expression of PP2Cepsilon resulted in dephosphorylation of CERT in a VAPA expression-dependent manner, which was accompanied by redistribution of CERT from the cytoplasm to the Golgi apparatus. The expression of PP2Cepsilon also enhanced the association between CERT and VAPA. In addition, knockdown of PP2Cepsilon expression by short interference RNA attenuated the interaction between CERT and VAPA and the sphingomyelin synthesis. These results suggest that CERT is a physiological substrate of PP2Cepsilon and that dephosphorylation of CERT by PP2Cepsilon may play an important role in the regulation of ceramide trafficking from the ER to the Golgi apparatus.
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Encéfalo/enzimología , Ceramidas/metabolismo , Retículo Endoplásmico/enzimología , Aparato de Golgi/enzimología , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Transporte Biológico/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Ceramidas/genética , Retículo Endoplásmico/genética , Aparato de Golgi/genética , Células HeLa , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/genética , Proteína Fosfatasa 2C , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína/fisiología , ARN Interferente Pequeño/genética , Ratas , Esfingomielinas/biosíntesis , Esfingomielinas/genética , Técnicas del Sistema de Dos Híbridos , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Protein phosphatase 2Cbeta (PP2Cbeta) is a member of a family of protein Ser/Thr phosphatases (PP2C) that is composed of at least twelve different gene products. Recent studies have revealed that PP2Cbeta mRNA accumulates in mature sperm, unfertilized metaphase II-arrested oocytes and zygotes, but that the mRNA level then decreases sharply between the early two-cell and eight-cell stages, remaining at low levels during the 16-cell to blastocyst stages of mice. These observations raised the possibility that PP2Cbeta plays a crucial role during gametogenesis, fertilization, and/or early stages of embryonic development. In this study, we employed a gene knockout technique in mice to test this possibility. We found that PP2Cbeta(Delta/wt) mice generate normal mature gametes. However, PP2Cbeta(Delta/Delta) embryos die between the two-cell and eight-cell stages. To our interest, PP2Cbeta(Delta/Delta) ES cells which had been generated by transfecting PP2Cbeta(3lox/3lox) ES cells with Cre-expressing plasmid were viable. In addition, knockdown of PP2Cbeta using siRNA did not affect the proliferation of wild-type ES cells. These observations suggest that relatively high PP2Cbeta expression is specifically required during the early stages of pre-implantation development. The possible mechanisms for the early pre-implantation lethality of PP2Cbeta(Delta/Delta) mice are discussed.
Asunto(s)
Implantación del Embrión/genética , Desarrollo Embrionario/genética , Células Madre Embrionarias/enzimología , Fosfoproteínas Fosfatasas/fisiología , Animales , Secuencia de Bases , Supervivencia Celular , Células Madre Embrionarias/fisiología , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/genética , Proteína Fosfatasa 2C , ARN Interferente Pequeño/farmacologíaRESUMEN
ASK1 (apoptosis signal-regulating kinase 1), a MKKK (mitogen-activated protein kinase kinase kinase), is activated in response to cytotoxic stresses, such as H2O2 and TNFalpha (tumour necrosis factor alpha). ASK1 induction initiates a signalling cascade leading to apoptosis. After exposure of cells to H2O2, ASK1 is transiently activated by autophosphorylation at Thr845. The protein then associates with PP5 (protein serine/threonine phosphatase 5), which inactivates ASK1 by dephosphorylation of Thr845. Although this feedback regulation mechanism has been elucidated, it remains unclear how ASK1 is maintained in the dephosphorylated state under non-stressed conditions. In the present study, we have examined the possible role of PP2Cepsilon (protein phosphatase 2Cepsilon), a member of PP2C family, in the regulation of ASK1 signalling. Following expression in HEK-293 cells (human embryonic kidney cells), wild-type PP2Cepsilon inhibited ASK1-induced activation of an AP-1 (activator protein 1) reporter gene. Conversely, a dominant-negative PP2Cepsilon mutant enhanced AP-1 activity. Exogenous PP2Cepsilon associated with exogenous ASK1 in HEK-293 cells under non-stressed conditions, inactivating ASK1 by decreasing Thr845 phosphorylation. The association of endogenous PP2Cepsilon and ASK1 was also observed in mouse brain extracts. PP2Cepsilon directly dephosphorylated ASK1 at Thr845 in vitro. In contrast with PP5, PP2Cepsilon transiently dissociated from ASK1 within cells upon H2O2 treatment. These results suggest that PP2Cepsilon maintains ASK1 in an inactive state by dephosphorylation in quiescent cells, supporting the possibility that PP2Cepsilon and PP5 play different roles in H2O2-induced regulation of ASK1 activity.