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

Background and ObjectiveTumor necrosis factor alpha (TNF-) is a major cytokine implicated in various inflammatory diseases. The nature of the nuclear factors associated with human TNF- gene regulation is not well elucidated. We previously identified a novel region located from -550 to -487 in human TNF- promoter that did not contain the reported binding sites for nuclear factor kappa B (NF-B) 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- expression. Material and MethodsTo identify DNA-binding proteins that bound to the target region of TNF- 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- expression. ResultsSeveral 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- 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- promoter fragments. Overexpression of TDP-43 in THP-1 cells resulted in an increase of TNF- expression. Knockdown of TDP-43 in THP-1 cells downregulated TNF- expression. ConclusionWe identified TDP-43 as one of the novel TNF- factors and found that it bound to the LPS-responsive element in the TNF- promoter to increase TNF- expression.