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. (9th September 2014)

• Record Type: Journal Article
• Authors: Murata, H.; Hattori, T.; Maeda, H.; Takashiba, S.; Takigawa, M.; Kido, J.; Nagata, T.
• Is Part Of: Journal of periodontal research. Volume 50:Number 4(2015:Aug.)
• Page Start: 452
• Page End: 460
• Publication Date: 2014-09-09
• Subjects: Periodontics -- Periodicals; 617.632
• DOI: 10.1111/jre.12227
• Languages: English
• ISSNs: 0022-3484

Abstract

<abstract abstract-type="main" id="jre12227-abs-0001"> <title> <x xml:space="preserve">Abstract</x> </title> <sec id="jre12227-sec-0001" sec-type="section"> <title>Background and Objective</title> <p>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 <italic>TNF‐α</italic> 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 <italic>TNF‐α</italic> expression.</p> </sec> <sec id="jre12227-sec-0002" sec-type="section"> <title>Material and Methods</title> <p>To identify DNA‐binding proteins that bound to the target region of <italic>TNF‐α</italic> 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 <italic>TNF‐α</italic> expression.</p> </sec> <sec id="jre12227-sec-0003" sec-type="section"> <title>Results</title> <p>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 <italic>TDP‐43</italic> mRNA was already enhanced before <italic>TNF‐α</italic> 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 <italic>TNF‐α</italic> promoter fragments. Overexpression of TDP‐43 in THP‐1 cells resulted in an increase of <italic>TNF‐α</italic> expression. Knockdown of TDP‐43 in THP‐1 cells downregulated <italic>TNF‐α</italic> expression.</p> </sec> <sec id="jre12227-sec-0004" sec-type="section"> <title>Conclusion</title> <p>We identified TDP‐43 as one of the novel <italic>TNF‐α</italic> factors and found that it bound to the LPS‐responsive element in the <italic>TNF‐α</italic> promoter to increase <italic>TNF‐α</italic> expression.</p> </sec> </abstract>