title: Identification of transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) as a novel factor for TNF-α expression upon lipopolysaccharide stimulation in human monocytes.
abstract: OBJECTIVE
  Tumor necrosis factor alpha (TNF-α) is a major cytokine implicated in various inflammatory diseases. The nature of the nuclear factors associated with human TNF-α gene regulation is not well elucidated. We previously identified a novel region located from -550 to -487 in human TNF-α promoter that did not contain the reported binding sites for nuclear factor kappa B (NF-κB) but showed lipopolysaccharide (LPS)-induced transcriptional activity. The purpose of this study is to identify novel factors that bind to the promoter region and regulate TNF-α expression.
  
  
  METHODS
  To identify DNA-binding proteins that bound to the target region of TNF-α promoter, a cDNA library from LPS-stimulated human monocytic cell line THP-1 was screened using a yeast one-hybrid system. Cellular localizations of the DNA-binding protein in the cells were examined by subcellular immunocytochemistry. Nuclear amounts of the protein in LPS-stimulated THP-1 cells were identified by western blot analysis. Expression of mRNA of the protein in the cells was quantified by real-time polymerase chain reaction. Electrophoretic mobility shift assays were performed to confirm the DNA-binding profile. Overexpression of the protein and knockdown of the gene were also performed to investigate the role for TNF-α expression.
  
  
  RESULTS
  Several candidates were identified from the cDNA library and transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) was focused on. Western blot analysis revealed that nuclear TDP-43 protein was increased in the LPS-stimulated THP-1 cells. Expression of TDP-43 mRNA was already enhanced before TNF-α induction by LPS. Electrophoretic mobility shift assay analysis showed that nuclear extracts obtained by overexpressing FLAG-tagged TDP-43 bound to the -550 to -487 TNF-α promoter fragments. Overexpression of TDP-43 in THP-1 cells resulted in an increase of TNF-α expression. Knockdown of TDP-43 in THP-1 cells downregulated TNF-α expression.
  
  
  CONCLUSIONS
  We identified TDP-43 as one of the novel TNF-α factors and found that it bound to the LPS-responsive element in the TNF-α promoter to increase TNF-α expression.
authors.affiliation: Department of Periodontology and Endodontology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Murata
authors.affiliation: Department of Biochemistry & Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Hattori
authors.affiliation: Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Maeda
authors.affiliation: Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Takashiba
authors.affiliation: Department of Biochemistry & Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Takigawa
authors.affiliation: Department of Periodontology and Endodontology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan.
authors.forename: J
authors.initials: J
authors.lastname: Kido
authors.affiliation: Department of Periodontology and Endodontology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Nagata
mesh_terms: D002460:Cell Line; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics; D055785:Gene Knockdown Techniques; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D009000:Monocytes / Q000187:drug effects*; D010957:Plasmids / Q000235:genetics; D011110:Polymorphism, Genetic / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000187:drug effects / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D008070:Lipopolysaccharides; D034741:RNA, Small Interfering; C000598624:TARDBP protein, human; D014409:Tumor Necrosis Factor-alpha
keywords: TDP-43; lipopolysaccharide; monocyte; tumor necrosis factor alpha