Masayuki Takigawa

Effect of transforming growth factor-beta1 on expression of the connective tissue growth factor (CCN2/CTGF) gene in normal human gingival fibroblasts and periodontal ligament cells

BACKGROUND AND OBJECTIVE Connective tissue growth factor (CCN2/CTGF) plays an important role in wound healing and regulation of the extracellular matrix in periodontal tissue. However, the functional relationship between altered transforming growth factor-beta1 levels and CCN2/CTGF has not been extensively investigated in human gingival fibroblasts and periodontal ligament cells. This study investigated the effects of transforming growth factor-beta1 on the expression of the CCN2/CTGF gene in human gingival fibroblasts and periodontal ligament cells in vitro. MATERIAL AND METHODS Cells were isolated from normal periodontal tissues and cultured in Dulbeccos modified Eagles minimal essential medium/F12 containing 10% fetal bovine serum. Subconfluent cells were maintained under serum deprivation for 24 h then treated with Dulbeccos modified Eagles minimal essential medium/F12 containing 0.5% fetal bovine serum (control) and 0.1, 1, 5 or 10 ng/mL of transforming growth factor-beta1 for 24, 48 or 72 h. The effects of transforming growth factor-beta1 on CCN2/CTGF mRNA expression were measured by reverse transcription-polymerase chain reaction. CCN2/CTGF protein was quantitatively analyzed using enzyme-liked immunosorbent assay. Subcellular distribution of CCN2/CTGF protein in both human gingival fibroblasts and periodontal ligament cells was observed using immunofluorescence microscopy. RESULTS In both human gingival fibroblasts and periodontal ligament cells, the expression of CCN2/CTGF mRNA and CCN2/CTGF protein was significantly increased, in a dose- and time-dependent manner, in the presence of transforming growth factor-beta1. Moreover, immunofluorescence analysis indicated that immunoreactivity to CCN2/CTGF showed a granular pattern of protein localization. CONCLUSION The expression of CCN2/CTGF mRNA and protein was induced by transforming growth factor-beta1 in human gingival fibroblasts and periodontal ligament cells. These results suggest that CCN2/CTGF plays an important role in wound healing and in the regeneration of periodontal tissue.

The Inhibition of Interferon-γ-Induced Upregulation of HLA-DR Expression on Cultured Human Gingival Fibroblasts by Interleukin-1β or Tumor Necrosis Factor-α*

The purpose of this study was to examine the effect of inflammatory cytokines on IFN-γ-induced HLA-DR expression on cultured human gingival fibroblasts by flow cytometry. Natural human IFN-γ, recombinant human interleukin-1β (rhIL-1β), and rh tumor necrosis factor-α (rhTNF-α) were used. IFN-γ-induced upregulation of HLA-DR expression was inhibited by simultaneously adding rhIL-1β or rhTNF-α (65.9% and 31.4% inhibition, respectively). Both rhIL-1β and rhTNF-α induced endogenous Prostaglandin E2 (PGE2 ) from gingival fibroblasts, while IFN-γ did not. The inhibitory effect of rhIL-1β or rhTNF-α on IFN-γ-induced upregulation of HLA-DR expression was partially abated in the presence of indomethacin (reductions of 65.9% and 41.7%, respectively). Both rhIL-1β- and rhTNF-α-induced endogenous PGE2 synthesis were completely inhibited by adding indomethacin (P < 0.001). The addition of exogenous PGE2 inhibited the IFN-γ-induced HLA-DR expression (P < 0.001). These observations suggest that the MCH class II expression on human gingival fibroblasts are influenced by the cytokine network and indirectly by the cytokine-mediated fibroblast PGE2 . J Periodontol 1994;65:336-341.

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 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 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. 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.

Effect of TNF-.ALPHA. on DNA Synthesis and Extracellular Matrix Protein Synthesis in Human Gingival Fibroblasts.

腫瘍壊死因子 (TNF) -αは, 初期の炎症反応として産生される炎症性サイトカインのひとつで, 炎症病巣の成立に関与していると考えられている。我々は, 歯肉結合組織におけるTNF-αの役割を理解するために, ヒト歯肉線維芽細胞のDNA合成, コラーゲン合成, および非コラーゲン蛋白合成に対するTNF-αの作用を, 線維芽細胞自らが産生するプロスタグランジン (PG) E2, および血小板成長因子 (PDGF) のオートクラインな影響を考慮して調べた。結果は以下の通りである。1) TNF-αはヒト歯肉線維芽細胞におけるDNA合成, コラーゲン合成, および非コラーゲン蛋白合成を促進した。2) TNF-αはヒト歯肉線維芽細胞におけるPGE2産生を著明に促進した。3) インドメタシンを用いてPGE2産生を阻害することにより, TNF-αがヒト歯肉線維芽細胞におけるDNA合成, コラーゲン合成, および非コラーゲン蛋白合成を促進する効果を増強した。4) TNF-αはヒト歯肉線維芽細胞におけるPDGF-A鎖mRNAの発現を促進した。以上の結果から, TNF-αはヒト歯肉線維芽細胞のDNA合成および細胞外基質蛋白合成に対し促進的に作用することが明らかとなった。また, これらの作用は, TNF-αが産生を誘導するPGE2によって抑制を受けていた。さらにTNF-αがDNA合成を促進する作用には, 内因性PDGFが関与している可能性が示唆された。