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Identification of transactivation‐responsive DNA ‐binding protein 43 ( TARDBP 43; TDP ‐43) as a novel factor for TNF ‐α expression upon lipopolysaccharide stimulation in human monocytes
Author(s) -
Murata H.,
Hattori T.,
Maeda H.,
Takashiba S.,
Takigawa M.,
Kido J.,
Nagata T.
Publication year - 2015
Publication title -
journal of periodontal research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.31
H-Index - 83
eISSN - 1600-0765
pISSN - 0022-3484
DOI - 10.1111/jre.12227
Subject(s) - transactivation , microbiology and biotechnology , electrophoretic mobility shift assay , tumor necrosis factor alpha , western blot , biology , nuclear protein , gene knockdown , gene expression , transcription factor , binding protein , gene , biochemistry , immunology
Background and 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. Material and Methods To identify DNA ‐binding proteins that bound to the target region of TNF ‐α promoter, a c DNA 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 m RNA 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 c DNA library and transactivation‐responsive DNA ‐binding protein 43 ( TARDBP 43; 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 m RNA 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. Conclusion 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.