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1.
FASEB J ; 32(9): 5119-5131, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29913557

RESUMEN

Hepatocyte growth factor (HGF) is a multifunctional protein that contains angiogenic and neurotrophic properties. In the current study, we investigated the analgesic effects of HGF by using a plasmid DNA that was designed to express 2 isoforms of human HGF-pCK-HGF-X7 (or VM202)-in a chronic constriction injury (CCI) -induced mouse neuropathic pain model. Intramuscular injection of pCK-HGF-X7 into proximal thigh muscle induced the expression of HGF in the muscle, sciatic nerve, and dorsal root ganglia (DRG). This gene transfer procedure significantly attenuated mechanical allodynia and thermal hyperalgesia after CCI. Injury-induced expression of activating transcription factor 3, calcium channel subunit α2δ1, and CSF1 in the ipsilateral DRG neurons was markedly down-regulated in the pCK-HGF-X7-treated group, which suggested that HGF might exert its analgesic effects by inhibiting pain-mediating genes in the sensory neurons. In addition, suppressed CSF1 expression in DRG neurons by pCK-HGF-X7 treatment was accompanied by a noticeable suppression of the nerve injury-induced glial cell activation in the spinal cord dorsal horn. Taken together, our data show that pCK-HGF-X7 attenuates nerve injury-induced neuropathic pain by inhibiting pain-related factors in DRG neurons and subsequent spinal cord glial activation, which suggests its therapeutic efficacy in the treatment of neuropathic pain.-Nho, B., Lee, J., Lee, J., Ko, K. R., Lee, S. J., Kim, S. Effective control of neuropathic pain by transient expression of hepatocyte growth factor in a mouse chronic constriction injury model.


Asunto(s)
Factor de Crecimiento de Hepatocito/genética , Neuralgia/genética , Neuralgia/terapia , Traumatismos de la Médula Espinal/genética , Animales , Canales de Calcio/genética , Constricción , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Ganglios Espinales/metabolismo , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Humanos , Hiperalgesia/genética , Factor Estimulante de Colonias de Macrófagos/genética , Masculino , Ratones , Ratones Endogámicos ICR , Músculo Esquelético/metabolismo , Neuroglía/metabolismo , Nervio Ciático/metabolismo , Células Receptoras Sensoriales/metabolismo , Asta Dorsal de la Médula Espinal/metabolismo , Factor de Transcripción 3/genética
2.
Arch Virol ; 161(5): 1151-8, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-26831934

RESUMEN

CCAAT/enhancer-binding protein (C/EBP) α, a member of the C/EBP family of transcription factors, is known to be involved in gene expression and DNA replication of human cytomegalovirus (HCMV). This study aimed to understand the regulation of endogenous C/EBPα during HCMV infection using an in vitro infection model. The expression and localization of C/EBPα were investigated in fibroblasts infected with HCMV. The overexpression of C/EBP homologous protein (CHOP), the endogenous inhibitor of C/EBP, was also employed to test the involvement of C/EBPα during HCMV infection. Our data showed that HCMV infection increases the expression of the full-length C/EBPα isoform (p42) especially during the late stage of infection at the transcriptional and post-translational levels. The increased p42 accumulated in the viral DNA replication compartment. p42 expression was not induced in cells treated with UV-irradiated virus or in cells infected with normal virus in the presence of ganciclovir. CHOP-mediated inhibition of C/EBP activity suppressed viral gene expression and DNA replication, which lowered the level of viral production. Together, our data suggest that HCMV-mediated C/EBPα regulation might play a beneficial role in the lytic cycle of HCMV.


Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/fisiología , Citomegalovirus/fisiología , Proteína alfa Potenciadora de Unión a CCAAT/genética , Citomegalovirus/genética , Infecciones por Citomegalovirus/virología , Fibroblastos/virología , Técnica del Anticuerpo Fluorescente , Regulación Viral de la Expresión Génica/genética , Regulación Viral de la Expresión Génica/fisiología , Células HEK293/virología , Humanos , Immunoblotting , Reacción en Cadena en Tiempo Real de la Polimerasa , Replicación Viral/fisiología
3.
Biochem Biophys Res Commun ; 466(1): 52-9, 2015 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-26325468

RESUMEN

Interferon-gamma (IFN-γ) is one of the critical cytokines released by host immune cells upon infection. Despite the important role(s) of IFN-γ in host immune responses, there has been no in vivo study regarding the effects of IFN-γ on brain development, and the results from many in vitro studies are controversial. In this study, the effects of IFN-γ on embryonic neurogenesis were investigated. Treatment of E14.5 mouse neural progenitor cells (NPCs) with IFN-γ resulted in a decrease in the percentage of TuJ1-positive immature neurons but an increase in the percentage of Nestin-positive NPCs. Similar results were obtained in vivo. Treatment of NPCs with a JAK inhibitor or the knockdown of STAT1 expression abrogated the IFN-γ-mediated inhibition of neurogenesis. Interestingly, the expression of one of proneural genes, Neurogenin2 (Neurog2) was dramatically inhibited upon IFN-γ treatment, and cells overexpressing Neurog2 did not respond to IFN-γ. Taken together, our results demonstrate that IFN-γ inhibits neuronal differentiation of NPCs by negatively regulating the expression of Neurog2 via the JAK/STAT1 pathway. Our findings may provide an insight into the role of IFN-γ in the development of embryonic brain.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Interferón gamma/inmunología , Quinasas Janus/inmunología , Proteínas del Tejido Nervioso/genética , Células-Madre Neurales/citología , Neurogénesis , Factor de Transcripción STAT1/inmunología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Células Cultivadas , Regulación hacia Abajo , Ratones , Proteínas del Tejido Nervioso/inmunología , Células-Madre Neurales/inmunología , Células-Madre Neurales/metabolismo , Transducción de Señal
4.
J Gen Virol ; 94(Pt 7): 1658-1668, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23580430

RESUMEN

NF-E2 related factor 2 (Nrf2) is a transcription factor that plays a key role(s) in cellular defence against oxidative stress. In this study, we showed that the expression of Nrf2 was upregulated in primary human foreskin fibroblasts (HFFs), following human cytomegalovirus (HCMV/HHV-5) infection. The expression of haem oxygenase-1, a downstream target of Nrf2, was also increased by HCMV infection, and this induction was suppressed in HFFs expressing a small hairpin RNA (shRNA) against Nrf2. The HCMV-mediated increase in Nrf2 expression was abolished when UV-irradiated virus was used or when the activity of casein kinase 2 was inhibited. Host cells infected by HCMV had higher survival rates following oxidative stress induced by buthionine sulfoximine compared with uninfected control cells, but this cell-protective effect was abolished by the use of Nrf2 shRNA. Our results suggest that HCMV-mediated activation of Nrf2 might be beneficial to the virus by increasing the host cell's ability to cope with oxidative stress resulting from viral infection and/or inflammation.


Asunto(s)
Citomegalovirus/patogenicidad , Fibroblastos/fisiología , Fibroblastos/virología , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , Regulación hacia Arriba , Butionina Sulfoximina/farmacología , Supervivencia Celular , Infecciones por Citomegalovirus/virología , Citoprotección , Fibroblastos/metabolismo , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Humanos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/farmacología , Estrés Oxidativo/efectos de los fármacos
5.
Nitric Oxide ; 33: 42-55, 2013 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-23747519

RESUMEN

PG201 is an ethanol extract prepared from a specially designed botanical formulation and has previously been shown to contain strong anti-arthritic activities by controlling inflammation and cartilage destruction in two animal models [1,2]. In the present study, we evaluated the effects of PG201 on the expression of heme oxygenase-1 (HO-1). The treatment of Raw264.7 cells (a murine macrophage cell line) and bone marrow-derived macrophages (BMDMs) with PG201 increased the protein and RNA levels of HO-1. The results from a reporter plasmid assay indicated that PG201 induced HO-1 promoter activity through the stress response element present in the two enhancers of the HO-1 promoter. The treatment of cells with PG201 increased the total amount and the nuclear level of NF-E2-related factor 2 (Nrf2). Protein analysis using BMDMs from Nrf2 knockout mice showed that Nrf2 was necessary for the PG201-mediated induction of HO-1 expression. The PG201-mediated induction of these anti-oxidative stress factors was inhibited by a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), but not by inhibitors of p38, ERK and JNK mitogen-activated protein kinases. Furthermore, the results from an experiment involving a specific siRNA and chemical inhibitors for HO-1 showed that the PG201-mediated increase of the HO-1 protein contributed to the suppression of inducible nitric oxide synthase (iNOS) and nitrite production stimulated by lipopolysaccharide. Taken together, these results suggest that PG201 activates Nrf2 through the PI3K signal transduction pathway, increases the expression of HO-1, and subsequently decreases the production of iNOS and nitrite, eventually exerting anti-inflammatory activities.


Asunto(s)
Hemo-Oxigenasa 1/biosíntesis , Factor 2 Relacionado con NF-E2/metabolismo , Nitritos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Extractos Vegetales/farmacología , Análisis de Varianza , Animales , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Ratones , Ratones Noqueados , Factor 2 Relacionado con NF-E2/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos
6.
Exp Biol Med (Maywood) ; 240(9): 1136-46, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25605059

RESUMEN

Heme oxygenase-1 (HO-1) has been suggested to be a key neuroprotective enzyme because of its widespread distribution in the brain as well as its strong antioxidative effects. HX106N, a water-soluble botanical formulation, has previously been demonstrated to prevent amyloid ß-induced memory impairment and oxidative stress in mice by upregulating HO-1 levels. In this study, the underlying molecular mechanisms of HX106N-induced HO-1 expression were investigated using BV-2 cells, a murine microglial cell line, and primary microglia. Treatment with HX106N induced the expression of HO-1 at the transcriptional level through the stress-responsive element-containing enhancer present in the ho-1 promoter. Nuclear factor E2-related factor 2 (Nrf2) was activated in cells treated with HX106N. The results from knockdown assay showed that small interfering RNA of Nrf2 attenuated HX106N-mediated HO-1 expression. Pharmacological inhibitors of p38 and JNK mitogen-activated protein kinases suppressed the HX106N-mediated induction of HO-1. The NF-κB signaling pathway was activated by HX106N and played a role in HX106N-induced HO-1 expression. Furthermore, HO-1 and one of its by-products during the enzymatic degradation of heme, CO, were found to be involved in HX106N-mediated suppression of NO production. Taken together, these data indicate that HX106N exerts potent antioxidative effects by increasing the expression of HO-1 through multiple signaling pathways, leading to the suppression of NO production.


Asunto(s)
Antioxidantes/farmacología , Hemo-Oxigenasa 1/genética , Óxido Nítrico/biosíntesis , Extractos Vegetales/farmacología , Animales , Monóxido de Carbono/metabolismo , Línea Celular , Técnicas de Silenciamiento del Gen , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Microglía/efectos de los fármacos , Microglía/metabolismo , Modelos Biológicos , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Factor de Transcripción AP-1/metabolismo , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos
7.
Stem Cells Dev ; 23(10): 1121-33, 2014 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-24397546

RESUMEN

Glycogen synthase kinase 3 (GSK3) acts as an important regulator during the proliferation and differentiation of neural progenitor cells (NPCs), but the roles of the isoforms of this molecule (GSK3α and GSK3ß) have not been clearly defined. In this study, we investigated the functions of GSK3α and GSK3ß in the context of neuronal differentiation of murine NPCs. Treatment of primary NPCs with a GSK3 inhibitor (SB216763) resulted in an increase in the percentage of TuJ1-positive immature neurons, suggesting an inhibitory role of GSK3 in embryonic neurogenesis. Downregulation of GSK3ß expression increased the percentage of TuJ1-positive cells, while knock-down of GSK3α seemed to have no effect. When primary NPCs were engineered to stably express either isoform of GSK3 using retroviral vectors, GSK3ß, but not GSK3α, inhibited neuronal differentiation and helped the cells to maintain the characteristics of NPCs. Mutant GSK3ß (Y216F) failed to suppress neuronal differentiation, indicating that the kinase activity of GSK3ß is important for this regulatory function. Similar results were obtained in vivo when a retroviral vector expressing GSK3ß was delivered to E9.5 mouse brains using the ultrasound image-guided gene delivery technique. In addition, SB216763 was found to block the rapamycin-mediated inhibition of neuronal differentiation of NPCs. Taken together, our results demonstrate that GSK3ß, but not GSK3α, negatively controls the neuronal differentiation of progenitor cells and that GSK3ß may act downstream of the mammalian target of rapamycin complex1 signaling pathway.


Asunto(s)
Diferenciación Celular/fisiología , Glucógeno Sintasa Quinasa 3/metabolismo , Complejos Multiproteicos/inmunología , Células-Madre Neurales/metabolismo , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/inmunología , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Humanos , Indoles/farmacología , Maleimidas/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Complejos Multiproteicos/genética , Células-Madre Neurales/citología , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
8.
Stem Cells Dev ; 23(23): 2841-50, 2014 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-25003789

RESUMEN

Notch has a broad range of regulatory functions in many developmental processes, including hematopoiesis, neurogenesis, and angiogenesis. Notch has several key functional regions such as the RBP-Jκ/CBF1 association module (RAM) domain, nuclear localization signals (NLS), and ankyrin (ANK) repeats. However, previous reports assessing the level of importance of these domains in the Notch signaling pathway are controversial. In this study, we have assessed the level of contribution of each Notch domain to the regulation of mammalian neural stem cells in vivo as well as in vitro. Reporter assays and real-time polymerase chain reactions show that the ANK repeats and RAM domain are indispensable to the transactivation of Notch target genes, whereas a nuclear export signal (NES)-fused Notch intracellular domain (NICD) mutant defective in nuclear localization exerts a level of activity comparable to unmodified NICD. Transactivational ability appears to be tightly coupled to Notch functions during brain development. Unlike ANK repeats and RAM domain deletion mutants, NES-NICD recapitulates NICD features such as promotion of astrogenesis at the expense of neurogenesis in vitro and enhancement of neural stem cell character in vivo. Our data support the previous observation that intranuclear localization is not essential to the oncogenesis of Notch1 in certain types of cells and imply the importance of the noncanonical Notch signaling pathway in the regulation of mammalian neural stem cells.


Asunto(s)
Encéfalo/embriología , Núcleo Celular/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis/fisiología , Señales de Localización Nuclear/metabolismo , Receptor Notch1/metabolismo , Animales , Repetición de Anquirina , Encéfalo/metabolismo , Núcleo Celular/genética , Ratones , Células 3T3 NIH , Señales de Localización Nuclear/genética , Estructura Terciaria de Proteína , Receptor Notch1/genética , Transducción de Señal/fisiología
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