Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 11 de 11
Filtrar
1.
Stem Cells ; 36(2): 218-229, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29143419

RESUMEN

Early lung development is a tightly orchestrated process encompassing (a) formation of definitive endoderm, (b) anteriorization of definitive endoderm, followed by (c) specification and maturation of both proximal and distal lung precursors. Several reports detailing the interaction of genes and proteins during lung development are available; however, studies reporting the role(s) of long noncoding RNAs (lncRNA) in lung morphogenesis are limited. To investigate this, we tailored a protocol for differentiation of human-induced pluripotent stem cells into distal and proximal lung progenitors to mimic in vivo lung development. The authenticity of differentiated cells was confirmed by expression of key lung markers such as FoxA2, Sox-17, Nkx2.1, Pitx2, FoxJ1, CC10, SPC, and via scanning as well as transmission electron microscopy. We employed next generation sequencing to identify lncRNAs and categorized them based on their proximity to genes essential for lung morphogenesis. In-depth bioinformatical analysis of the sequencing data enabled identification of a novel lncRNA, RP11-380D23.2, which is located upstream of PITX2 and includes a binding site for PARP1. Chromatin immunoprecipitation and other relevant studies revealed that PARP1 is a repressor for PITX2. Whole genome microarray analysis of RP11-380D23.2/PITX2 knockdown populations of progenitors demonstrated enrichment in proximal progenitors and indicated altered distal-proximal patterning. Dysregulation of WNT effectors in both knockdowns highlighted direct modulation of PITX2 by RP11-380D23.2. Most of these results were validated in four independent hiPSC lines (including a patient-specific CFTR mutant line). Taken together, these findings offer a mechanistic explanation underpinning the role of RP11-380D23.2 during lung morphogenesis via WNT signaling. Stem Cells 2018;36:218-229.


Asunto(s)
Proteínas de Homeodominio/metabolismo , Pulmón/metabolismo , ARN Largo no Codificante/metabolismo , Factores de Transcripción/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Inmunoprecipitación de Cromatina , Proteínas de Homeodominio/genética , Humanos , Células Madre Pluripotentes/metabolismo , Interferencia de ARN , ARN Largo no Codificante/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Factores de Transcripción/genética , Proteína del Homeodomínio PITX2
2.
Biol Pharm Bull ; 37(1): 87-95, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24389485

RESUMEN

Inhibitor of kappaB (IκB) kinase beta (IKKß) plays a critical role in nuclear factor-kappaB (NF-κB) activation and production of proinflammatory cytokines in various inflammatory diseases including rheumatoid arthritis. We previously reported a novel IKKß inhibitor Compound D, 4-[6-(cyclobutylamino)imidazo[1,2-b]pyridazin-3-yl]-2-fluoro-N-{[(2S,4R)-4-fluoropyrrolidin-2-yl]methyl}benzamide, which is efficacious in experimental arthritis models. In the present study, we characterized the pharmacological properties of Compound D and investigated the mechanisms of the anti-arthritic effect. Compound D inhibited IKKß kinase activity with 160-fold selectivity against IKKα. The cellular analyses revealed that Compound D selectively blocked NF-κB promoter activity among major cellular signaling pathways, such as the activator protein-1 pathway, consistent with inhibition of the NF-κB signaling pathway including phosphorylation of IκBα. In addition, Compound D inhibited NF-κB-driven production of tumor necrosis factor alpha (TNFα) and interleukin-6 comparably. The correlation between inhibitory effect on TNFα production and plasma concentration of the compound was observed in vivo. Consecutive administration of Compound D decreased gene expression of proinflammatory cytokines and inflammatory mediators in the paws of arthritic mice with attenuation of paw swelling. Notably, Compound D was rapidly distributed to the arthritic paws, rather than healthy paws, and where it decreased the gene expression of proinflammatory cytokines by a single oral administration. Furthermore, Compound D completely inhibited arthritis progression even when treatment occurred after disease development. These data suggest that the downregulation of proinflammatory cytokines in local inflamed joints is one of the mechanisms underlying the anti-arthritic effect of the IKKß inhibitor, Compound D.


Asunto(s)
Artritis Experimental/tratamiento farmacológico , Artritis Reumatoide/metabolismo , Benzamidas/farmacología , Citocinas/metabolismo , Compuestos Heterocíclicos con 2 Anillos/farmacología , Quinasa I-kappa B/antagonistas & inhibidores , Mediadores de Inflamación/metabolismo , Articulaciones/efectos de los fármacos , Animales , Antirreumáticos/farmacología , Antirreumáticos/uso terapéutico , Artritis Experimental/metabolismo , Artritis Experimental/patología , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/patología , Benzamidas/uso terapéutico , Regulación hacia Abajo , Femenino , Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos con 2 Anillos/uso terapéutico , Proteínas I-kappa B/metabolismo , Interleucina-6/metabolismo , Articulaciones/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Inhibidor NF-kappaB alfa , FN-kappa B/antagonistas & inhibidores , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Transducción de Señal , Distribución Tisular , Factor de Necrosis Tumoral alfa/biosíntesis
3.
Microbiol Immunol ; 57(6): 445-54, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23773023

RESUMEN

Collagen-induced arthritis (CIA) is an animal model for rheumatoid arthritis (RA). Lipopolysaccharide (LPS) is known to accelerate CIA; however, the pathogenetic mechanisms are not yet fully understood. In this study, type II collagen (CII)-immunized mice were found to have marked increases in degree of expression of mRNA of inflammatory mediators such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-1ß, and macrophage inflammatory protein-2 (MIP-2) in their arthritic paws and of serum anti-CII antibody concentration before the onset of arthritis induced by LPS injection. The gene expression was rapid and continuous after direct activation of nuclear factor κB. The amounts of mRNA of TNF-α, IL-1ß, and MIP-2, as well as of matrix metalloproteinases and the receptor activator of nuclear factor κB ligand, increased with the development of arthritis, correlated positively with clinical severity and corresponded with histopathological changes. Moreover, anti-TNF-α neutralizing antibody inhibited the development of LPS-accelerated CIA and a single injection of recombinant mouse TNF-α induced increases in anti-CII antibody concentrations, suggesting TNF-α may contribute to the development of arthritis by both initiation of inflammation and production of autoantibodies. These data suggest that exacerbation of RA by LPS is associated with rapid and continuous production of inflammatory mediators and autoantibodies.


Asunto(s)
Artritis Experimental/inmunología , Autoanticuerpos/inmunología , Quimiocina CXCL2/biosíntesis , Colágeno Tipo II/inmunología , Interleucina-1beta/biosíntesis , Lipopolisacáridos/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Artritis Experimental/patología , Quimiocina CXCL2/genética , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Histocitoquímica , Interleucina-1beta/genética , Ratones , Factor de Necrosis Tumoral alfa/genética
4.
J Biol Chem ; 286(36): 31409-17, 2011 Sep 09.
Artículo en Inglés | MEDLINE | ID: mdl-21733845

RESUMEN

The retinoic acid-related orphan nuclear receptor γt (RORγt)/RORγ2 is well known as a master regulator of interleukin 17 (IL-17)-producing helper T (Th17) cell development. To develop a therapeutic agent against Th17-mediated autoimmune diseases, we screened chemical compounds and successfully found that digoxin inhibited IL-17 production. Further studies revealed that digoxin bound to the ligand binding domain of RORγt and suppressed Th17 differentiation without affecting Th1 differentiation. To better understand the structural basis for the inhibitory activity of digoxin, we determined the crystal structure of the RORγt ligand-binding domain in complex with digoxin at 2.2 Å resolution. The structure reveals that digoxin binds to the ligand-binding pocket protruding between helices H3 and H11 from the pocket. In addition, digoxin disrupts the key interaction important for the agonistic activity, resulting in preventing the positioning of helix H12 in the active conformation, thus antagonizing coactivator interaction. Functional studies demonstrated that digoxin inhibited RORγt activity and decreased IL-17 production but not RORα activity. Digoxin inhibited IL-17 production in CD4(+) T cells from experimental autoimmune encephalomyelitis mice. Our data indicates that RORγt is a promising therapeutic target for Th17-derived autoimmune diseases and our structural data will help to design novel RORγt antagonists.


Asunto(s)
Digoxina/química , Interleucina-17/biosíntesis , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Células Th17/efectos de los fármacos , Animales , Enfermedades Autoinmunes/tratamiento farmacológico , Linfocitos T CD4-Positivos , Diferenciación Celular/efectos de los fármacos , Cristalografía por Rayos X , Digoxina/farmacología , Encefalomielitis Autoinmune Experimental , Interleucina-17/antagonistas & inhibidores , Ratones , Estructura Molecular , Células Th17/citología
5.
Bioorg Med Chem Lett ; 21(3): 904-8, 2011 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-21232950

RESUMEN

We have increased the potency of imidazo[1,2-b]pyridazine derivatives as IKKß inhibitors with two strategies. One is to enhance the activity in cell-based assay by adjusting the polarity of molecules to improve permeability. Another is to increase the affinity for IKKß by the introduction of additional substituents based on the hypothesis derived from an interaction model study. These improved compounds showed inhibitory activity of TNFα production in mice.


Asunto(s)
Quinasa I-kappa B/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Piridazinas/química , Animales , Sitios de Unión , Simulación por Computador , Evaluación Preclínica de Medicamentos , Quinasa I-kappa B/metabolismo , Masculino , Ratones , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Piridazinas/síntesis química , Piridazinas/farmacología , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/metabolismo
6.
Bioorg Med Chem Lett ; 21(15): 4550-5, 2011 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-21705219

RESUMEN

We have discovered imidazo[1,2-b]pyridazine derivatives that show suppressive activity of inflammation in arthritis models. We optimized the substructures of imidazo[1,2-b]pyridazine derivatives to combine potent IKKß inhibitory activity, TNFα inhibitory activity in vivo and excellent pharmacokinetics. The compound we have acquired, which had both potent activities and good pharmacokinetic profiles based on improved physicochemical properties, demonstrated efficacy on collagen-induced arthritis models in mice and rats.


Asunto(s)
Artritis Experimental/tratamiento farmacológico , Quinasa I-kappa B/antagonistas & inhibidores , Imidazoles/química , Inhibidores de Proteínas Quinasas/química , Piridazinas/química , Administración Oral , Animales , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Quinasa I-kappa B/metabolismo , Ratones , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridazinas/farmacocinética , Piridazinas/uso terapéutico , Ratas , Factor de Necrosis Tumoral alfa/metabolismo
7.
Bioorg Med Chem Lett ; 20(17): 5113-8, 2010 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-20675134

RESUMEN

Imidazo[1,2-b]pyridazine derivatives from high-throughput screening were developed as IKKbeta inhibitors. By the optimization of the 3- and 6-position of imidazo[1,2-b]pyridazine scaffold, cell-free IKKbeta inhibitory activity and TNFalpha inhibitory activity in THP-1 cell increased. Also, these compounds showed high kinase selectivity. The structure-activity relationship was revealed and the interaction model of imidazo[1,2-b]pyridazine compounds with IKKbeta was constructed.


Asunto(s)
Quinasa I-kappa B/antagonistas & inhibidores , Imidazoles/química , Imidazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridazinas/química , Piridazinas/farmacología , Descubrimiento de Drogas , Modelos Moleculares , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad
8.
J Immunol Methods ; 303(1-2): 105-21, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16048727

RESUMEN

Novel, cell-based assays, based on bioluminescence resonance energy transfer, have been developed for FcepsilonRI- and GPVI-FcRgamma complex-mediated signaling at receptor-proximal steps. In a stable transfectant of the HEK-293 cell line expressing human FcepsilonRIalpha, FcepsilonRIbeta, and FcRgamma-GFP2 and Syk(1-265)-Rluc fusion proteins, FcepsilonRI cross-linking markedly increased BRET2 ratios, which are the ratios of GFP2 emission to Rluc emission. These ratios reflect the FcRgamma-GFP2-Syk(1-265)-Rluc interaction in living cells. The signals are specifically inhibited by the Src-family kinase inhibitor PP2. Separately, in transient transfectants expressing GPVI, FcRgamma-GFP2, and Syk(1-265)-Rluc, the GPVI-specific ligand convulxin induced a two-fold increase in the BRET2 ratio and this increase was also inhibited by PP2. Finally, a differential assay was developed which permits the measurement of FcepsilonRI- and GPVI-FcRgamma complex-mediated signaling in the same cell. These assays provide useful methods for monitoring FcRgamma-Syk interaction in real time in living cells and may contribute to the understanding of signal regulation through FcRgamma-containing receptors.


Asunto(s)
Mediciones Luminiscentes/métodos , Receptores Fc/análisis , Receptores Fc/fisiología , Transducción de Señal/inmunología , Western Blotting , Línea Celular , Precursores Enzimáticos/metabolismo , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana Plaquetaria/análisis , Proteínas Tirosina Quinasas/metabolismo , Receptores Fc/biosíntesis , Quinasa Syk , Transfección
9.
Endocr J ; 50(6): 681-7, 2003 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-14709838

RESUMEN

The aim of this study was to investigate the effect of daily oral administration of calcitriol on calcium metabolism in Japanese postmenopausal women. For this purpose, we administered 0.5 microg of daily calcitriol to 18 Japanese postmenopausal women for up to 24 weeks. During the first 28 days, daily administration of 0.5 microg of oral calcitriol increased fasting serum 1,25(OH) 2D levels significantly in 9 women (Group B) (p<0.005), while no significant change was seen in another 9 women without calcitriol administration (Group A). The first 28-day calcitriol supplement increased fasting urinary calcium excretion (urinary Ca/Cr) from 0.133 +/- 0.072 to 0.171 +/- 0.089 (p<0.05) and fractional excretion of calcium (FECa) without changing serum Ca2+. Urinary NTx/Cr excretion, an index of bone resorption, decreased significantly from 64.8 +/- 24.5 to 50.3 +/- 27.2 nMBCE/mMCr in Group B. Following the 28-day control period, 0.5 microg of oral calcitriol was also administered to women in Group A for another 20 weeks. At the end of the 24-week investigation period, the effects of oral calcitriol on urinary calcium excretion and bone resorption were still significant in both Group A and B. A positive correlation was found between urinary Ca/Cr and NTx/Cr excretion at the start (r = 0.657, p<0.05), but this correlation was lost by calcitriol treatment (r = 0.135). These results indicated that calcitriol supplement was effective in suppressing bone resorption in postmenopausal women, and that an increased fasting urinary calcium excretion due to calcitriol supplement was predominantly caused by increased intestinal calcium absorption in these women.


Asunto(s)
Pueblo Asiatico , Calcitriol/administración & dosificación , Agonistas de los Canales de Calcio/administración & dosificación , Calcio/metabolismo , Posmenopausia/sangre , Vitamina D/análogos & derivados , Vitamina D/sangre , Administración Oral , Anciano , Densidad Ósea/efectos de los fármacos , Resorción Ósea/prevención & control , Calcio/sangre , Calcio/orina , Ayuno/sangre , Femenino , Humanos , Persona de Mediana Edad , Nitratos/orina , Nitritos/orina , Posmenopausia/orina
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA