RESUMO
The proteins of homeobox (HOX) genes are transcription regulators involved in cell type-specific differentiation and patterning of the body plan in vertebrates. Particularly, the HOXD family is involved in limb formation in mice and chicks. There is also some evidence that the HOXD9 gene, a member of the HOXD family, is involved in the pathology of rheumatoid arthritis (RA). The purpose of the present study was to determine if the HOXD9 protein was expressed in RA synovium and then to characterize the HOXD9-expressing cell. Western blotting and immunohistochemical analysis showed that the HOXD9 protein was expressed in the synovium from patients with RA, but not in those from patients with osteoarthritis or healthy individuals. The HOXD9-positive cells were localized in both the lining and sublining areas of the synovium. Furthermore, fluorescent double-staining showed that the HOXD9 protein was expressed in fibroblast-like synoviocytes (FLS). These findings not only indicate that the HOXD9 gene is exclusively expressed in the RA synovium but also suggest that the HOXD9 gene contributes to the pathology of rheumatoid arthritis through the FLS.
Assuntos
Artrite Reumatoide/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Neoplasias/biossíntese , Membrana Sinovial/metabolismo , Western Blotting , Células Cultivadas , Imunofluorescência , Proteínas de Homeodomínio , Humanos , Imuno-Histoquímica , Membrana Sinovial/citologiaRESUMO
CREB binding protein (CBP) plays a central role in cell differentiation and proliferation, interacting with a large number of nuclear factors. To find novel nuclear factors associating with CBP, we have carried out yeast two-hybrid screening of human chondrocyte cDNA library using the C/H3 region of CBP as a bait and cloned CDK4 binding protein p34SEI-1, the recently found cell cycle regulator. The association of p34SEI-1 with CBP was confirmed in vitro by GST pull-down assay and in vivo by coimmunoprecipitation. Results of the immunofluorescence assay also supported the association of p34SEI-1 and CBP. In reporter assay using CRE promoter, p34SEI-1 strongly suppressed CREB-mediated transcription, and this suppression was overcome by excess amount of CBP, but not by CBPDeltaCH3. It is suggested that the association of p34SEI-1 and CBP is not only involved in cell cycle regulation by CBP, but also have some effect on other CBP-dependent transcription.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Proteína de Ligação a CREB , Ciclo Celular , Linhagem Celular , Condrócitos/metabolismo , Clonagem Molecular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , DNA Complementar/genética , Humanos , Técnicas In Vitro , Substâncias Macromoleculares , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/química , Transativadores/genética , Fatores de Transcrição , Transcrição Gênica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Synovial hyperplasia is an important feature of rheumatoid arthritis (RA) and we have reported that several transcription factors were highly activated in rheumatoid synoviocytes. The purpose of this study was to examine nuclear acetylation in synoviocytes as an activation marker and determine its role in cell activation. Autonomous acetylation of approximately 53 and 62 kDa nuclear proteins was detected in rheumatoid synoviocytes by anti-acetylated lysine specific antibody. Furthermore, tumor necrosis factor alpha (TNFalpha), a potent mitogen for synoviocytes, dose-dependently increased their state of acetylation. Immunoprecipitation analysis revealed that 53 kDa acetylated protein (ap53) was identical with p53, a tumor suppressor gene product. Since enhanced p53 binding to the promoter by TNFalpha treatment was detected by gel shift assay, we analyzed p53 promoter activity by reporter assay system. Contrary to enhanced binding activity, the transcriptional activity was attenuated in a TNFalpha concentration-dependent manner. Since p53 activation requires recruitment of CREB binding protein (CBP) as a coactivator, we also examined the effect of CBP on TNFalpha-induced attenuation of p53 promoter activation. Overexpression of CBP induced p53 transcriptional activity and recovery of TNFalpha-induced inhibition. Our results clearly indicate that autonomous nuclear acetylation is characteristically enhanced in rheumatoid synoviocytes and that p53 is one of acetylated protein. Our results also demonstrate that TNFalpha-induced acetylation of p53 attenuated its transcriptional activation via CBP depletion, and that overexpression of CBP enhanced TNFalpha-induced cell death in rheumatoid synoviocytes, suggesting that regulation of transcriptional coactivator become a novel strategy for RA therapy.
Assuntos
Proteínas Nucleares/metabolismo , Líquido Sinovial/citologia , Transativadores/metabolismo , Ativação Transcricional , Fator de Necrose Tumoral alfa/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Apoptose , Artrite Reumatoide/metabolismo , Western Blotting , Proteína de Ligação a CREB , Núcleo Celular/metabolismo , Relação Dose-Resposta a Droga , Humanos , Imunoglobulina G/metabolismo , Microscopia de Fluorescência , Plasmídeos/metabolismo , Testes de Precipitina , Transfecção , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND AND AIM: Chronic hepatic damage leads to liver fibrosis, which is characterized by the accumulation of collagen-rich extracellular matrix. However, the mechanism by which E3 ubiquitin ligase is involved in collagen synthesis in liver fibrosis is incompletely understood. This study aimed to explore the involvement of the E3 ubiquitin ligase synoviolin (Syno) in liver fibrosis. METHODS: The expression and localization of synoviolin in the liver were analyzed in CCl(4)-induced hepatic injury models and human cirrhosis tissues. The degree of liver fibrosis and the number of activated hepatic stellate cells (HSCs) was compared between wild type (wt) and Syno(+/-) mice in the chronic hepatic injury model. We compared the ratio of apoptosis in activated HSCs between wt and Syno(+/-) mice. We also analyzed the effect of synoviolin on collagen synthesis in the cell line from HSCs (LX-2) using siRNA-synoviolin and a mutant synoviolin in which E3 ligase activity was abolished. Furthermore, we compared collagen synthesis between wt and Syno(-/-) mice embryonic fibroblasts (MEF) using quantitative RT-PCR, western blotting, and collagen assay; then, we immunohistochemically analyzed the localization of collagen in Syno(-/-) MEF cells. RESULTS: In the hepatic injury model as well as in cirrhosis, synoviolin was upregulated in the activated HSCs, while Syno(+/-) mice developed significantly less liver fibrosis than in wt mice. The number of activated HSCs was decreased in Syno(+/-) mice, and some of these cells showed apoptosis. Furthermore, collagen expression in LX-2 cells was upregulated by synoviolin overexpression, while synoviolin knockdown led to reduced collagen expression. Moreover, in Syno(-/-) MEF cells, the amounts of intracellular and secreted mature collagen were significantly decreased, and procollagen was abnormally accumulated in the endoplasmic reticulum. CONCLUSION: Our findings demonstrate the importance of the E3 ubiquitin ligase synoviolin in liver fibrosis.
Assuntos
Cirrose Hepática/fisiopatologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase , Interferência de RNA , Ubiquitina-Proteína Ligases/genéticaRESUMO
OBJECTIVE: Synoviolin is an E3 ubiquitin ligase, and its overexpression is implicated in the pathogenesis of rheumatoid arthritis (RA). We reported previously that Ets binding site 1 (EBS-1) within the synoviolin promoter is crucial for the expression of synoviolin, and GA binding protein (GABP) binds to this site. This study was undertaken to elucidate the precise mechanisms of transcriptional regulation via EBS-1. METHODS: We performed purification and identification of complex components that bind to EBS-1 and inspected their contributions to the transcriptional regulation of synoviolin in rheumatoid synovial cells. We biochemically purified proteins that had EBS-1 binding activity and identified the proteins using liquid chromatography tandem mass spectrometry analysis. The identified proteins were verified to recruit and form the complex on EBS-1 using electrophoretic mobility shift assay and coimmunoprecipitation assay. Furthermore, their transcription activities were tested by reporter assays and RNA interference experiments. RESULTS: We identified interleukin enhancer binding factor 3 (ILF-3) as a novel factor in the complex. ILF-3 was demonstrated to activate the synoviolin promoter via association with GABPalpha in rheumatoid synovial cells. In addition, further activation was observed with ILF-2 and GABPbeta, previously reported interactants of ILF-3 and GABPalpha, respectively. Moreover, ILF-3-knockdown experiments showed reduced expression of the synoviolin gene. CONCLUSION: Our findings indicate that ILF-3, which has been known to regulate IL-2 expression in T cells, up-regulates synoviolin expression with GABPalpha in rheumatoid synovial cells. ILF-3 might be a target for RA treatment through its effect on IL-2 in T cells and synoviolin in rheumatoid synovial cells.
Assuntos
Artrite Reumatoide/fisiopatologia , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Proteínas do Fator Nuclear 90/metabolismo , Membrana Sinovial/fisiologia , Ubiquitina-Proteína Ligases/genética , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Regulação da Expressão Gênica/fisiologia , Células HeLa , Humanos , Células Jurkat , Camundongos , Células NIH 3T3 , Proteínas Nucleares/metabolismo , Osteoartrite/metabolismo , Osteoartrite/fisiopatologia , Regiões Promotoras Genéticas/fisiologia , Interferência de RNA , Membrana Sinovial/citologiaRESUMO
Synoviolin, also called HRD1, is an E3 ubiquitin ligase and is implicated in endoplasmic reticulum -associated degradation. In mammals, Synoviolin plays crucial roles in various physiological and pathological processes, including embryogenesis and the pathogenesis of arthropathy. However, little is known about the molecular mechanisms of Synoviolin in these actions. To clarify these issues, we analyzed the profile of protein expression in synoviolin-null cells. Here, we report that Synoviolin targets tumor suppressor gene p53 for ubiquitination. Synoviolin sequestrated and metabolized p53 in the cytoplasm and negatively regulated its cellular level and biological functions, including transcription, cell cycle regulation and apoptosis. Furthermore, these p53 regulatory functions of Synoviolin were irrelevant to other E3 ubiquitin ligases for p53, such as MDM2, Pirh2 and Cop1, which form autoregulatory feedback loops. Our results provide novel insights into p53 signaling mediated by Synoviolin.
Assuntos
Citoplasma/metabolismo , Proteína Supressora de Tumor p53/química , Ubiquitina-Proteína Ligases/fisiologia , Animais , Linhagem Celular Tumoral , Drosophila melanogaster , Retículo Endoplasmático/metabolismo , Humanos , Plasmídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Transdução de Sinais , Transfecção , Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina-Proteína Ligases/químicaRESUMO
RNA helicase A (RHA), an ATPase/helicase, regulates the gene expression at various steps including transcriptional activation and RNA processing. RHA is known to shuttle between the nucleus and cytoplasm. We identified the nuclear localization signal (NLS) of RHA and analyzed the nuclear import mechanisms. The NLS of RHA (RHA-NLS) consisting of 19 amino acid residues is highly conserved through species and does not have the consensus classical NLS. In vitro nuclear import assays revealed that the nuclear import of RHA was Ran-dependent and mediated with the classical importin-alpha/beta-dependent pathway. The binding assay indicated that the basic residues in RHA-NLS were used for interaction with importin-alpha. Furthermore, the nuclear import of RHA-NLS was supported by importin-alpha1 and preferentially importin-alpha3. Our results indicate that the nuclear import of RHA is mediated by the importin-alpha3/importin-beta-dependent pathway and suggest that the specificity for importin may regulate the functions of cargo proteins.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Sinais de Localização Nuclear/metabolismo , alfa Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , RNA Helicases DEAD-box , Células HeLa , HumanosRESUMO
Transcription factor GATA-1 and its cofactor FOG-1 coordinate erythroid cell maturation by activating erythroid-specific genes and repressing genes associated with the undifferentiated state. Here we show that FOG-1 binds to the NuRD corepressor complex in vitro and in vivo. The interaction is mediated by a small conserved domain at the extreme N-terminus of FOG-1 that is necessary and sufficient for NuRD binding. This domain defines a novel repression module found in diverse transcriptional repressors. NuRD is present at GATA-1/FOG-1-repressed genes in erythroid cells in vivo. Point mutations near the N-terminus of FOG-1 that abrogate NuRD binding block gene repression by FOG-1. Finally, the ability of GATA-1 to repress transcription was impaired in erythroid cells expressing mutant forms of FOG-1 that are defective for NuRD binding. Together, these studies show that FOG-1 and likely other FOG-like proteins are corepressors that link GATA factors to histone deacetylation and nucleosome remodeling.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histona Desacetilases/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Células Cultivadas , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Inativação Gênica , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Camundongos , Dados de Sequência Molecular , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Proteínas Nucleares/genética , Mutação Puntual , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Repressoras/genética , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genéticaRESUMO
Transcriptional coactivators have crucial roles in eukaryotic transcription. It has been suggested that one of the coactivators, cAMP response element binding protein (CREB) binding protein (CBP), regulates gene expression with a number of transcription factors via two mechanisms. One is the recruitment of general transcriptional machinery to the promoters. The other is its intrinsic and associated histone acetyltransferase (HAT) activity, which increases the accessibility of the activator to DNA, and the acetylation of nonhistone proteins. Rheumatoid arthritis (RA) is characterized by the inflammation and proliferation of synovium, leading to the destruction of articular cartilage and bone. To understand the pathogenesis of RA, we focused the transcription mechanism through CBP in synoviocytes and chondrocytes. We identified Notch-1 in synoviocytes and p34(SEI-1) in chondrocytes as CBP binding proteins by yeast two-hybrid screening. It was also suggested that the acetylation of p53 could repress transactivation in RA synoviocytes. These associations may regulate proliferation and apoptosis. This study suggests that regulation of the coactivator could become a novel strategy for RA therapy.
RESUMO
The transcription factor GATA-1 and its cofactor FOG-1 are essential for the normal development of erythroid cells and megakaryocytes. FOG-1 can stimulate or inhibit GATA-1 activity depending on cell and promoter context. How the GATA-1-FOG-1 complex controls the expression of distinct sets of gene in megakaryocytes and erythroid cells is not understood. Here, we examine the molecular basis for the megakaryocyte-restricted activation of the alphaIIb gene. FOG-1 stimulates GATA-1-dependent alphaIIb gene expression in a manner that requires their direct physical interaction. Transcriptional output by the GATA-1-FOG-1 complex is determined by the hematopoietic Ets protein Fli-1 that binds to an adjacent Ets element. Chromatin immunoprecipitation experiments show that GATA-1, FOG-1 and Fli-1 co-occupy the alphaIIb promoter in vivo. Expression of several additional megakaryocyte-specific genes that bear tandem GATA and Ets elements in their promoters also depends on the physical interaction between GATA-1 and FOG-1. Our studies define a molecular context for transcriptional activation by GATA-1 and FOG-1, and may explain the occurrence of tandem GATA and Ets elements in the promoters of numerous megakaryocyte-expressed genes.