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1.
Int Immunol ; 29(2): 87-94, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28338898

RESUMO

PU.1 is a hematopoietic cell-specific transcription factor belonging to the Ets family, which plays an important role in the development of dendritic cells (DCs). CD11c (encoded by Itgax) is well established as a characteristic marker of hematopoietic lineages including DCs. In the present study, we analyzed the role of PU.1 (encoded by Spi-1) in the expression of CD11c. When small interfering RNA (siRNA) for Spi-1 was introduced into bone marrow-derived DCs (BMDCs), the mRNA level and cell surface expression of CD11c were dramatically reduced. Using reporter assays, the TTCC sequence at -56/-53 was identified to be critical for PU.1-mediated activation of the promoter. An EMSA showed that PU.1 directly bound to this region. ChIP assays demonstrated that a significant amount of PU.1 bound to this region on chromosomal DNA in BMDCs, which was decreased in LPS-stimulated BMDCs in accordance with the reduced levels of mRNAs of Itgax and Spi-1, and the histone acetylation degree. Enforced expression of exogenous PU.1 induced the expression of the CD11c protein on the cell surface of mast cells, whereas control transfectants rarely expressed CD11c. Quantitative RT-PCR also showed that the expression of a transcription factor Irf4, which is a partner molecule of PU.1, was reduced in PU.1-knocked down BMDCs. IRF4 transactivated the Itgax gene in a synergistic manner with PU.1. Taken together, these results indicate that PU.1 functions as a positive regulator of CD11c gene expression by directly binding to the Itgax promoter and through transactivation of the Irf4 gene.


Assuntos
Antígeno CD11c/metabolismo , Células Dendríticas/fisiologia , Hematopoese , Fatores Reguladores de Interferon/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Acetilação , Animais , Antígeno CD11c/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Hematopoese/genética , Histonas/metabolismo , Fatores Reguladores de Interferon/genética , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/genética , Transativadores/genética , Ativação Transcricional
2.
Mol Immunol ; 53(3): 295-301, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22990073

RESUMO

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.


Assuntos
Antígeno CD11c/genética , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Antígeno 2 Relacionado a Fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Mapeamento Cromossômico , Primers do DNA/genética , Elementos Facilitadores Genéticos , Antígeno 2 Relacionado a Fos/antagonistas & inibidores , Antígeno 2 Relacionado a Fos/química , Antígeno 2 Relacionado a Fos/genética , Regulação da Expressão Gênica , Camundongos , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-jun/química , Proteínas Proto-Oncogênicas c-jun/genética , RNA Interferente Pequeno/genética , Fator de Transcrição AP-1/química
3.
Int Immunol ; 21(7): 803-16, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19502584

RESUMO

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.


Assuntos
Interferon gama/imunologia , Mastócitos/imunologia , Monócitos/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Animais , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Antígeno CD11b/imunologia , Antígeno CD11b/metabolismo , Antígeno CD11c/imunologia , Antígeno CD11c/metabolismo , Meios de Cultivo Condicionados/farmacologia , Genes MHC da Classe II , Humanos , Interferon gama/farmacologia , Interleucina-3/farmacologia , Interleucina-4/farmacologia , Interleucina-6/biossíntese , Interleucina-6/imunologia , Lipopolissacarídeos/farmacologia , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Monócitos/efeitos dos fármacos , Proteínas Nucleares/genética , Proteínas/farmacologia , Proteínas Proto-Oncogênicas/genética , Receptores de Interferon/genética , Receptores de Interferon/imunologia , Receptores de Interferon/metabolismo , Fator de Células-Tronco/farmacologia , Transativadores/genética , Fator de Necrose Tumoral alfa/farmacologia , Tirosina Quinase 3 Semelhante a fms/farmacologia , Receptor de Interferon gama
4.
Biochem Biophys Res Commun ; 347(2): 388-93, 2006 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-16836979

RESUMO

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.


Assuntos
Antígenos HLA-DR/biossíntese , Queratinócitos/metabolismo , Proteínas Nucleares/genética , Transativadores/genética , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Citometria de Fluxo , Humanos , Interferon gama/farmacologia , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Cinética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética
5.
Int Immunol ; 17(7): 847-56, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15967781

RESUMO

The high-affinity receptor for IgE (FcepsilonRI) that is expressed on the surface of mast cells plays an important role in antigen/IgE-mediated allergic reactions. We have previously found that critical elements in the promoter of the FcepsilonRI alpha- and beta-chain genes are recognized by the transcription factor GATA-1 in electrophoretic mobility shift assays coupled with a transient expression system for the alpha- and beta-chain promoters. To confirm that GATA-1 is involved in the expression of FcepsilonRI definitively, we generated bone marrow-derived mast cells from GATA-1 knockdown (KD) heterozygous mice. FACS analysis showed that the frequency of FcepsilonRI-positive cells was significantly decreased in mast cells derived from bone marrow of GATA-1 KD mice. Reverse transcription-PCR analysis showed that the level of transcripts not only for GATA-1 but also for both the alpha- and beta-chains was significantly lower in KD mast cells, whereas that of the FcepsilonRI gamma-chain was not affected. Degranulation caused by cross-linking of FcepsilonRI on mast cells prepared from KD mice was markedly repressed in comparison with that of wild-type mast cells. We concluded that the transcription factor GATA-1 positively regulates FcepsilonRI alpha- and beta-chain expression and therefore is involved in mast cell development.


Assuntos
Células da Medula Óssea/imunologia , Degranulação Celular/imunologia , Mastócitos/imunologia , Receptores de IgE/imunologia , Animais , Degranulação Celular/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Camundongos , Camundongos Knockout , Receptores de IgE/genética
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