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1.
Mol Immunol ; 53(3): 295-301, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22990073

RESUMEN

CD11c, a member of the ß(2) integrin family of adhesion molecule, is expressed on the surface of myeloid lineages and activated lymphoid cells and forms a heterodimeric receptor with CD18. We analyzed the mouse CD11c promoter structure to elucidate the transcriptional regulation in dendritic cells (DCs). By reporter assay, the -84/-65 region was identified to be essential for activity of the mouse CD11c promoter in the mouse bone marrow-derived (BM) DCs and monocyte cell line RAW264.7. An electrophoretic mobility shift assay using a number of antibodies against transcription factors revealed that the target region was recognized by a complex including JunD and Fra2, which are transcription factors belonging to the AP-1 family. The direct interaction of JunD and Fra2 with the CD11c promoter was further confirmed by a chromatin immunoprecipitation assay using CD11c-positive cells purified from BMDCs. Finally, mouse JunD and/or Fra2 siRNA was introduced into BMDCs to evaluate the involvement of these factors against CD11c transcription and found that Fra2 siRNA reduced cell surface expression level of CD11c. These results indicate that AP-1 composed with JunD and Fra2 protein plays a primary role in enhancing the transcription level of the CD11c gene in DC.


Asunto(s)
Antígeno CD11c/genética , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Antígeno 2 Relacionado con Fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Transcripción AP-1/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Mapeo Cromosómico , Cartilla de ADN/genética , Elementos de Facilitación Genéticos , Antígeno 2 Relacionado con Fos/antagonistas & inhibidores , Antígeno 2 Relacionado con Fos/química , Antígeno 2 Relacionado con Fos/genética , Regulación de la Expresión Génica , Ratones , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-jun/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-jun/química , Proteínas Proto-Oncogénicas c-jun/genética , ARN Interferente Pequeño/genética , Factor de Transcripción AP-1/química
2.
J Allergy Clin Immunol ; 129(3): 814-824.e6, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22112519

RESUMEN

BACKGROUND: PU.1 is a hematopoietic cell-specific transcription factor belonging to the Ets family. We hypothesized that PU.1 is involved in MHC class II expression in dendritic cells (DCs). OBJECTIVE: The role of PU.1 in MHC class II expression in DCs was analyzed. METHODS: Transcriptional regulation of the DC-specific pI promoter of the class II transactivator (CIITA) gene and subsequent MHC class II expression was investigated by using PU.1 small interfering RNA (siRNA) and reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays. RESULTS: PU.1 siRNA introduction suppressed MHC class II expression, allogeneic and syngeneic T-cell activation activities of bone marrow-derived DCs (BMDCs) with reduction of CIITA mRNA driven by the DC-specific promoter pI, and MHC class II mRNA. The chromatin immunoprecipitation assay showed constitutive binding of PU.1 to the pI region in BMDCs, whereas acetylation of histone H3 on pI was suppressed by LPS stimulation in parallel with shutdown of CIITA transcription. PU.1 transactivated the pI promoter through cis-elements at -47/-44 and -30/-27 in a reporter assay and to which PU.1 directly bound in an electrophoretic mobility shift assay. Acetylation of histones H3 and H4 on pI was reduced in PU.1 siRNA-introduced BMDCs. Knockdown of interferon regulatory factor 4 or 8, which is a heterodimer partner of PU.1, by siRNA did not affect pI-driven CIITA transcription or MHC class II expression. CONCLUSION: PU.1 basally transactivates the CIITA pI promoter in DCs by functioning as a monomeric transcription factor and by affecting histone modification, resulting in the subsequent expression and function of MHC class II.


Asunto(s)
Células de la Médula Ósea/metabolismo , Células Dendríticas/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Acetilación , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/inmunología , Regulación de la Expresión Génica/genética , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Histonas/metabolismo , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/inmunología , ARN Interferente Pequeño/genética , Linfocitos T/inmunología , Transactivadores/genética , Transactivadores/inmunología , Activación Transcripcional/genética
3.
Blood ; 117(7): 2211-22, 2011 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-21119111

RESUMEN

In this study, we investigated the role of a transcription factor, PU.1, in the regulation of CD80 and CD86 expression in dendritic cells (DCs). A chromatin immunoprecipitation assay revealed that PU.1 is constitutively bound to the CD80 and CD86 promoters in bone marrow-derived DCs. In addition, co-expression of PU.1 resulted in the transactivation of the CD80 and CD86 promoters in a reporter assay. The binding of PU.1 to cis-enhancing regions was confirmed by electromobility gel-shift assay. As expected, inhibition of PU.1 expression by short interfering RNA (siRNA) in bone marrow-derived DCs resulted in marked down-regulation of CD80 and CD86 expression. Moreover, overexpression of PU.1 in murine bone marrow-derived lineage-negative cells induced the expression of CD80 and CD86 in the absence of monocyte/DC-related growth factors and/or cytokines. Based on these results, we conclude that PU.1 is a critical factor for the expression of CD80 and CD86. We also found that subcutaneous injection of PU.1 siRNA or topical application of a cream-emulsified PU.1 siRNA efficiently inhibited murine contact hypersensitivity. Our results suggest that PU.1 is a potential target for the treatment of immune-related diseases.


Asunto(s)
Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Administración Tópica , Animales , Antígeno B7-1/genética , Antígeno B7-2/genética , Secuencia de Bases , Sitios de Unión/genética , ADN Complementario/genética , Dermatitis por Contacto/genética , Dermatitis por Contacto/inmunología , Dermatitis por Contacto/prevención & control , Regulación hacia Abajo , Femenino , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Transactivadores/antagonistas & inhibidores , Transactivadores/genética , Sitio de Iniciación de la Transcripción , Activación Transcripcional
4.
Trends Pharmacol Sci ; 31(8): 345-50, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20591510

RESUMEN

Cannabinoids have emerged as powerful drug candidates for the treatment of inflammatory and autoimmune diseases due to their immunosuppressive properties. Significant clinical and experimental data on the use of cannabinoids as anti-inflammatory agents exist in many autoimmune disease settings, but virtually no studies have been undertaken on their potential role in transplant rejection. Here we suggest a theoretical role for the use of cannabinoids in preventing allograft rejection. The psychotropic properties of CB1 agonists limit their clinical use, but CB2 agonists may offer a new avenue to selectively target immune cells involved in allograft rejection. Moreover, development of mixed CB1/CB2 agonists that cannot cross the blood-brain barrier may help prevent their undesired psychotropic properties. In addition, manipulation of endocannabinoids in vivo by activating their biosynthesis and inhibiting cellular uptake and metabolism may offer another pathway to regulate immune response during allograft rejection.


Asunto(s)
Cannabinoides/farmacología , Rechazo de Injerto/prevención & control , Trasplante de Órganos/métodos , Animales , Barrera Hematoencefálica/metabolismo , Moduladores de Receptores de Cannabinoides/biosíntesis , Moduladores de Receptores de Cannabinoides/metabolismo , Cannabinoides/efectos adversos , Cannabinoides/farmacocinética , Rechazo de Injerto/inmunología , Humanos , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB2/agonistas
5.
FEBS Lett ; 584(11): 2315-20, 2010 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-20371366

RESUMEN

Mast cells (MCs) are activated upon stimulation via TLRs or FcepsilonRI, contributing to immune protection and/or leading to allergic diseases. In the present study, the effects of Trichostatin A (TSA) on the activation of MCs were analyzed with bone marrow-derived (BM) MCs. TSA increased the transcription and protein secretion of IL-6 in case of LPS-stimulation, in contrast to the suppressive effect on IgE-mediated activation of BMMCs. Chromatin immunoprecipitation assay showed IgE-mediated signaling-specific suppression of transcription factors recruitment to the IL-6 promoter. TSA-treatment inhibited nuclear translocation of NF-kappaB following IgE-mediated, but not LPS-induced activation in MCs.


Asunto(s)
Ácidos Hidroxámicos/farmacología , Interleucina-6/genética , Interleucina-6/metabolismo , Mastocitos/metabolismo , FN-kappa B/metabolismo , Lipopolisacáridos/farmacología , Mastocitos/efectos de los fármacos , FN-kappa B/genética , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
6.
Immunogenetics ; 61(9): 649-55, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19685047

RESUMEN

Three frequent genetic polymorphisms in the human high-affinity IgE receptor alpha-subunit (FcepsilonRIalpha) were shown to be associated with allergic disorders and/or total serum IgE levels in allergic patients. Two of these were previously demonstrated to affect FcepsilonRIalpha expression while the third -18483A>C (rs2494262) has not yet been subjected to functional studies. We hypothesized that the -18483A>C variant affects transcriptional activity of the FcepsilonRIalpha distal promoter in monocytes in which FcepsilonRIalpha transcription is driven through that regulatory region. Indeed, we confirmed preferential binding of the YY1 transcription factor to the -18483C allele, resulting in lower transcriptional activity when compared with the -18483A allele.


Asunto(s)
Polimorfismo de Nucleótido Simple , Receptores de IgE/genética , Transcripción Genética , Factor de Transcripción YY1/metabolismo , Pueblo Asiatico/genética , Ensayo de Cambio de Movilidad Electroforética , Humanos , Población Blanca/genética
7.
Biochem Biophys Res Commun ; 388(1): 102-6, 2009 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-19646961

RESUMEN

PU.1 is a myeloid- and lymphoid-specific transcription factor that serves many important roles in the development and specific gene regulation of hematopoietic lineages. Mast cells (MC) and dendritic cells (DC) express PU.1 at low and high levels, respectively. Previously, we found that enforced expression of PU.1 in MC resulted in acquisition of DC-like characteristics, including repression of several IgE-mediated responses due to reduced expression of IgE-signaling related molecules. In contrast, PU.1 overexpression in MC up-regulated TNF-alpha production in response to IgE- and LPS-stimulation suggesting that PU.1 positively regulates TNF-alpha expression. However, the role of PU.1 in the expression of TNF-alpha is largely unknown. In the present study, the effects of PU.1 on the TNF-alpha promoter in mouse bone marrow-derived (BM) MC and DC were studied. Real-time PCR, ELISA, and chromatin immunoprecipitation assays indicated that the kinetics and magnitude of TNF-alpha expression levels following LPS- or IgE-stimulation are related to the amount of PU.1 binding to the promoter. In brief, higher and delayed up-regulation of TNF-alpha promoter function was observed in DC, whereas there were lower and rapid responses in MC. When PU.1-overexpressing retrovirus vector was introduced into MC, the amount of PU.1 recruited to the TNF-alpha promoter markedly increased. The knockdown of PU.1 in BMDC by siRNA resulted in a reduction of TNF-alpha protein produced from LPS-stimulated BMDC. These observations indicate that PU.1 transactivates the TNF-alpha promoter and that the amount of PU.1 binding on the promoter is associated with promoter activity.


Asunto(s)
Células de la Médula Ósea/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Activación Transcripcional , Factor de Necrosis Tumoral alfa/genética , Animales , Secuencia de Bases , Técnicas de Silenciamiento del Gen , Interleucina-6/genética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/genética , Transactivadores/genética , Transcripción Genética
8.
Int Immunol ; 21(7): 803-16, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19502584

RESUMEN

Over-expression of PU.1, a myeloid- and lymphoid-specific transcription factor belonging to the Ets family, induces monocyte-specific gene expression in mast cells. However, the effects of PU.1 on each target gene and the involvement of cytokine signaling in PU.1-mediated gene expression are largely unknown. In the present study, PU.1 was over-expressed in two different types of bone marrow-derived cultured mast cells (BMMCs): BMMCs cultured with IL-3 plus stem cell factor (SCF) and BMMCs cultured with pokeweed mitogen-stimulated spleen-conditioned medium (PWM-SCM). PU.1 over-expression induced expression of MHC class II, CD11b, CD11c and F4/80 on PWM-SCM-cultured BMMCs, whereas IL-3/SCF-cultured BMMCs expressed CD11b and F4/80, but not MHC class II or CD11c. When IFN-gamma was added to the IL-3/SCF-based medium, PU.1 transfectant acquired MHC class II expression, which was abolished by antibody neutralization or in Ifngr(-/-) BMMCs, through the induction of expression of the MHC class II transactivator, CIITA. Real-time PCR detected CIITA mRNA driven by the fourth promoter, pIV, and chromatin immunoprecipitation indicated direct binding of PU.1 to pIV in PU.1-over-expressing BMMCs. PU.1-over-expressing cells showed a marked increase in IL-6 production in response to LPS stimulation in both IL-3/SCF and PWM-SCM cultures. These results suggest that PU.1 overproduction alone is sufficient for both expression of CD11b and F4/80 and for amplification of LPS-induced IL-6 production. However, IFN-gamma stimulation is essential for PU.1-mediated transactivation of CIITA pIV. Reduced expression of mast cell-related molecules and transcription factors GATA-1/2 and up-regulation of C/EBPalpha in PU.1 transfectants indicate that enforced PU.1 suppresses mast cell-specific gene expression through these transcription factors.


Asunto(s)
Interferón gamma/inmunología , Mastocitos/inmunología , Monocitos/inmunología , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Activación Transcripcional , Animales , Antígenos de Diferenciación/inmunología , Antígenos de Diferenciación/metabolismo , Antígeno CD11b/inmunología , Antígeno CD11b/metabolismo , Antígeno CD11c/inmunología , Antígeno CD11c/metabolismo , Medios de Cultivo Condicionados/farmacología , Genes MHC Clase II , Humanos , Interferón gamma/farmacología , Interleucina-3/farmacología , Interleucina-4/farmacología , Interleucina-6/biosíntesis , Interleucina-6/inmunología , Lipopolisacáridos/farmacología , Mastocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Monocitos/efectos de los fármacos , Proteínas Nucleares/genética , Proteínas/farmacología , Proteínas Proto-Oncogénicas/genética , Receptores de Interferón/genética , Receptores de Interferón/inmunología , Receptores de Interferón/metabolismo , Factor de Células Madre/farmacología , Transactivadores/genética , Factor de Necrosis Tumoral alfa/farmacología , Tirosina Quinasa 3 Similar a fms/farmacología , Receptor de Interferón gamma
9.
Biochem Biophys Res Commun ; 375(1): 95-100, 2008 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-18680724

RESUMEN

An Ets-family transcription factor PU.1 is involved in the development and specific gene regulation of hematopoietic cells. PU.1 also determines the commitment between several lineages via its expression level. Although enforced expression of PU.1 in mast cells (MC) induced expression of monocyte-specific markers and morphological change from MC to monocytes, especially dendritic cells (DC), in the previous report, intracellular events caused by PU.1 are largely unknown. In the present study, effect of PU.1 on IgE- and LPS-mediated stimulation degrees was analyzed. The amounts of IL-6, IL-13, and TNF-alpha produced from LPS-stimulated MC were markedly increased by overexpression of PU.1. In contrast, IL-6 and IL-13 production levels in response to IgE were reduced by PU.1, whereas that of TNF-alpha was up-regulated. beta-Hexosaminidase release as a means of degranulation was decreased in PU.1 transfectants. When eicosanoid generation in response to IgE-stimulation was analyzed, overexpression of PU.1 reduced leukotriene C(4) (LTC(4)) release, but enhanced PGD(2) production. Microarray analysis suggested that expression of FcepsilonRI signal pathway related molecules were suppressed in PU.1 overexpressing MC as well as DC. These observations indicate that up-regulation of PU.1 suppresses expression of FcepsilonRI signal transduction-related intracellular molecules, but increases the potential of transcription activity of monocyte characters.


Asunto(s)
Mastocitos/inmunología , Proteínas Proto-Oncogénicas/fisiología , Receptores de IgE/metabolismo , Transactivadores/fisiología , Animales , Degranulación de la Célula/genética , Eicosanoides/biosíntesis , Regulación de la Expresión Génica , Inmunoglobulina E/inmunología , Interleucina-13/biosíntesis , Interleucina-6/biosíntesis , Leucotrieno C4/biosíntesis , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Mastocitos/efectos de los fármacos , Ratones , Prostaglandina D2/biosíntesis , Proteínas Proto-Oncogénicas/genética , Retroviridae , Transducción de Señal , Transactivadores/genética , Transfección , Factor de Necrosis Tumoral alfa/biosíntesis
10.
Immunogenetics ; 60(10): 557-63, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18629488

RESUMEN

The high-affinity receptor for immunoglobulin E (IgE), FcepsilonRI, is specifically expressed in mast cells and basophils and plays a key role in IgE-mediated allergic reactions. The transcription factor Elf-1 has been previously identified to bind to the promoter of the human FcepsilonRI alpha-chain, which is essential for the function and expression of FcepsilonRI. In the present study, Elf-1 siRNA was conducted to evaluate the effects of Elf-1 on FcepsilonRI alpha-chain expression in the primary mouse mast cells, bone marrow-derived mast cells (BMMC). Introduction of Elf-1 siRNA effectively reduced expression levels of Elf-1 mRNA and protein in BMMC. Transient reporter assay showed that the knockdown of Elf-1 by siRNA resulted in increased FcepsilonRI alpha-chain promoter activity, while overexpression of Elf-1 suppressed alpha-chain promoter activity in BMMC. Elf-1 siRNA-treated BMMC exhibited marked upregulation of FcepsilonRI alpha-chain transcription, whereas beta-chain mRNA was not affected by Elf-1 siRNA. Chromatin immunoprecipitation assay showed that the amount of transcription factor PU.1, recognizing the cis-element close to the Elf-1-site on the FcepsilonRI alpha-chain promoter, was significantly increased by introduction of Elf-1 siRNA. These results indicate that Elf-1 negatively regulates FcepsilonRI alpha-chain expression by suppressing PU.1-mediated transcription of the alpha-chain in BMMC.


Asunto(s)
Efrina-A2/antagonistas & inhibidores , Regulación de la Expresión Génica , Mastocitos/metabolismo , ARN Interferente Pequeño/farmacología , Receptores de IgE/genética , Animales , Western Blotting , Médula Ósea/metabolismo , Inmunoprecipitación de Cromatina , Humanos , Luciferasas/metabolismo , Ratones , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de IgE/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/genética , Transactivadores/metabolismo , Transcripción Genética , Regulación hacia Arriba
11.
J Immunol ; 180(12): 8204-10, 2008 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-18523286

RESUMEN

The alpha-chain is a specific component of FcepsilonRI, which is essential for the cell surface expression of FcepsilonRI and the binding of IgE. Recently, two single nucleotide polymorphisms (SNPs) in the alpha-chain promoter, -315C>T and -66T>C, have been shown by statistic studies to associate with allergic diseases. The effect of -66 SNP on GATA-1-mediated promoter activity has been already indicated. In the present study, to investigate roles of the -315 SNP on the alpha-chain promoter functions, the transcription activity was evaluated by reporter assay. The alpha-chain promoter carrying -315T (minor allele) possessed significantly higher transcriptional activity than that of -315C (major allele). EMSA indicated that the transcription factor Sp1, but not Myc-associated zinc finger protein (MAZ), was bound to the -315C allele probe and that a transcription factor belonging to a high mobility group-family bound to the -315T allele probe. The chromatin immunoprecipitation assay suggested that high mobility group 1, 2, and Sp1 bound around -315 of FcepsilonRIalpha genomic DNA in vivo in the human basophil cell line KU812 with -315C/T and in human peripheral blood basophils with -315C/C, respectively. When cell surface expression level of FcepsilonRI on basophils was analyzed by flow cytometry, basophils from individuals carrying -315T allele expressed significantly higher amount of FcepsilonRI compared with those of -315C/C. The findings demonstrate that a -315 SNP significantly affects human FcepsilonRI alpha-chain promoter activity and expression level of FcepsilonRI on basophils by binding different transcription factors to the SNP site.


Asunto(s)
Proteínas del Grupo de Alta Movilidad/metabolismo , Polimorfismo de Nucleótido Simple/inmunología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores de IgE/genética , Receptores de IgE/metabolismo , Factor de Transcripción Sp1/metabolismo , Alelos , Animales , Basófilos/inmunología , Basófilos/metabolismo , Línea Celular Tumoral , Membrana Celular/genética , Membrana Celular/inmunología , Membrana Celular/metabolismo , Nucleótidos de Citosina/genética , Nucleótidos de Citosina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/genética , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/inmunología , Unión Proteica/genética , Unión Proteica/inmunología , Subunidades de Proteína/biosíntesis , Ratas , Receptores de IgE/biosíntesis , Factor de Transcripción Sp1/genética , Nucleótidos de Timina/genética , Nucleótidos de Timina/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
12.
J Invest Dermatol ; 128(1): 96-103, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17597815

RESUMEN

The ATP2A2 gene encodes Ca2+-dependent ATPase, the dysfunction of which causes Darier disease. In this study, we analyzed the promoter structure of the human ATP2A2 gene using primary normal human keratinocytes (NHK). Reporter assays showed that deletion of -550/-529, -488/-472, -390/-362, or -42/-21 resulted in a significant decrease in human ATP2A2 promoter activity. Electrophoretic mobility shift assay (EMSA) showed that Sp1 is a transcription factor that binds to the -550/-529 and -488/-472 regions of the promoter. Chromatin immunoprecipitation (ChIP) assay demonstrated that Sp1, but not Sp3, binds to the promoter region of the ATP2A2 gene in NHK cells in vivo. Knockdown of Sp1 expression by small interfering RNA resulted in a marked reduction in ATP2A2 promoter activity and ATP2A2 mRNA levels in NHK, suggesting that Sp1 positively transactivates the ATP2A2 promoter in NHK. This is early evidence demonstrating that Sp1 plays an important and positive role in ATP2A2 gene expression in NHK in vivo and in vitro.


Asunto(s)
Queratinocitos/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Factor de Transcripción Sp1/fisiología , Transcripción Genética , Calcio/metabolismo , Células Cultivadas , Inmunoprecipitación de Cromatina , Enfermedad de Darier/etiología , Enfermedad de Darier/terapia , Ensayo de Cambio de Movilidad Electroforética , Humanos , Pénfigo Familiar Benigno/terapia , Regiones Promotoras Genéticas , ARN Interferente Pequeño/farmacología , Factor de Transcripción Sp1/antagonistas & inhibidores , Factor de Transcripción Sp1/genética
13.
Blood ; 109(11): 4846-55, 2007 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-17289816

RESUMEN

Interleukin-12 (IL-12), a heterodimeric cytokine (p35/p40) produced mainly from macrophages and dendritic cells, is an important regulator of T-helper 1 cell responses and for host defense. We found that interferon (IFN) consensus sequence binding protein (ICSBP), which is a transcription factor essential for the expression of p40, was expressed in mouse bone marrow-derived mast cells (BMMCs). The transcription levels of p35 and p40 were increased by stimulation of BMMCs with IFN-gamma/lipopolysaccharide (LPS). IL-12 was secreted from BMMCs in response to LPS but not by FcepsilonRI cross-linking. The p40 levels in the peritoneal cavity of mast cell-deficient W/W(v) and W/W(v) reconstituted with p40(-/-) BMMCs were significantly lower than those of WBB6F(1)(+/+) and wild-type (WT) BMMC-reconstituted W/W(v) in the acute septic peritonitis model. The survival rate of W/W(v) reconstituted with p40(-/-) BMMCs was significantly decreased compared to those of WBB6F(1)(+/+) and WT-BMMC-reconstituted W/W(v), which was due to reduced production of IFN-gamma and subsequent impaired activation of neutrophils in the peritoneal cavity. Survival rate of p40(-/-) mice was also restored by adoptive transfer of WT-BMMCs. These results demonstrate that mast cells play a significant role in the production of IL-12 required for host defense. This is the first report to demonstrate that mast cells are a crucial source of functional IL-12.


Asunto(s)
Infecciones Bacterianas/metabolismo , Células Dendríticas/inmunología , Subunidad p40 de la Interleucina-12/biosíntesis , Interleucina-12/biosíntesis , Mastocitos/metabolismo , Neutrófilos/microbiología , Peritonitis/microbiología , Sepsis/sangre , Animales , Infecciones Bacterianas/patología , Interferón gamma/metabolismo , Interleucina-12/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Neutrófilos/metabolismo
14.
Biochem Biophys Res Commun ; 347(2): 388-93, 2006 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-16836979

RESUMEN

A transcriptional cofactor, the MHC class II transactivator (CIITA), is a master regulator of MHC class II gene expression. CIITA expression is restricted in MHC class II-positive cells and is regulated by 4 (human) or 3 (mouse) promoters. To clarify the usage of human CIITA promoters in keratinocytes, we analyzed CIITA mRNA levels in IFN-gamma-stimulated normal human keratinocytes (NHK) by real-time PCR using promoter-specific primers. When the amount of total CIITA mRNA in NHK was quantified at 6h after IFN-gamma-stimulation, the amount of CIITA mRNA detected in NHK did not differ from that seen in the B cell line Raji or the IFN-gamma-stimulated macrophage cell line THP-1. Quantitative real-time PCR using promoter-specific primers showed that type IV CIITA mRNA was strongly transcribed and that type III CIITA mRNA was weakly transcribed in stimulated NHK, while no transcripts from pI and pII were detected. Although type IV mRNA in THP-1 was transiently transcribed by IFN-gamma-stimulation, transcription of type IV in IFN-gamma-stimulated keratinocytes was prolonged. This difference subsequently caused significantly higher expression at 72 h of MHC class II on NHK, compared with THP-1. This is the first report to quantitatively analyze each type of CIITA transcript in NHK.


Asunto(s)
Antígenos HLA-DR/biosíntesis , Queratinocitos/metabolismo , Proteínas Nucleares/genética , Transactivadores/genética , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Citometría de Flujo , Humanos , Interferón gamma/farmacología , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Cinética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética
15.
Blood ; 108(1): 262-9, 2006 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-16522818

RESUMEN

Cell-type-specific transcription of mouse high-affinity IgE receptor (FcepsilonRI) beta-chain is positively regulated by the transcription factor GATA-1. Although GATA-1 is expressed in erythroid cells, megakaryocytes, and mast cells, the expression of mouse FcepsilonRI beta-chain is restricted to mast cells. In the present study, we characterized the role of GATA-associated cofactor FOG-1 in the regulation of the FcepsilonRI beta-chain promoter. The expression levels of FOG-1, GATA-1, and beta-chain in each hematopoietic cell line were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. FOG-1 expression was higher in the beta-chain-negative hematopoietic progenitor cell line Ba/F3 than in the beta-chain-positive mast cell line PT18. By contrast, GATA-1 expression was similar when comparing the 2 cell lines. A transient reporter assay demonstrated that the beta-chain promoter functioned in PT18 but not in Ba/F3 and that the transcription activity of the beta-chain promoter in PT18 was markedly suppressed by overexpression of FOG-1. Although the activity of the beta-chain promoter, which was upregulated by coexpression of GATA-1, was significantly suppressed by coexpression of FOG-1 in the simian kidney CV-1 cells (beta-chain(-), GATA-1(-), and FOG-1(-)), the transactivation of the beta-chain promoter by the GATA-1 mutant V205G, which cannot bind FOG-1, was not affected by coexpression of FOG-1. Further, overexpression of FOG-1 in PT18 resulted in decreases in cell surface expression of FcepsilonRI and beta-chain transcription. Finally, suppression of FOG-1 expression using an siRNA approach resulted in increased beta-chain promoter activity in Ba/F3. These results suggest that FOG-1 expression level regulates the GATA-1-dependent FcepsilonRI beta-chain promoter.


Asunto(s)
Factor de Transcripción GATA1/antagonistas & inhibidores , Regulación de la Expresión Génica/genética , Mastocitos/inmunología , Proteínas Nucleares/farmacología , Receptores de IgE/antagonistas & inhibidores , Receptores de IgE/genética , Factores de Transcripción/farmacología , Transcripción Genética , Animales , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Factor de Transcripción GATA1/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/inmunología , Receptores de IgE/inmunología , Relación Estructura-Actividad , Factores de Transcripción/biosíntesis , Factores de Transcripción/inmunología
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