MG-132 inhibited TSP-1 mRNA expression in THP-1 cells.

<p>THP-1 cells were pretreated with 5.0 ng/ml of MG-132 (inhibitor of NF-κB) for 1 h, followed by the addition of 1.0 µg/ml of <i>P. gingivalis</i> LPS and 10 ng/ml of IL-17F. <i>P. gingivalis</i> LPS-induced TSP-1 mRNA was significantly reduced by MG-132. MG-132 also significantly reduced TSP-1 mRNA expression induced by <i>P. gingivalis</i> LPS together with IL-17F.</p

Upregulation of TSP-1 in THP-1 cells by <i>P. gingivalis</i> LPS stimulation.

<p>(A) THP-1 cells were stimulated by <i>P. gingivalis</i> LPS at concentrations of 0, 0.0001, 0.001, 0.01, 0.1, or 1.0 µg/ml for 4 h. <i>P. gingivalis</i> LPS increased TSP-1 mRNA expression in a dose-dependent manner in THP-1 cells. (B) THP-1 cells were stimulated by 1.0 µg/ml of <i>P. gingivalis</i> LPS for 0, 1, 2, 4, 12, 24, 48, or 72 h. <i>P. gingivalis</i> LPS increased TSP-1 mRNA expression in a time-dependent manner in THP-1 cells. (C) THP-1 cells were stimulated with <i>P. gingivalis</i> LPS at concentrations of 0, 0.01, 0.1, or 1.0 µg/ml for 72 h. <i>P. gingivalis</i> LPS increased TSP-1 protein production in a dose-dependent manner in THP-1 cells. (D) THP-1 cells were stimulated with 1.0 µg/ml of <i>P. gingivalis</i> LPS for 0, 1, 4, 6, 12, 24, 48, or 72 h. <i>P. gingivalis</i> LPS increased TSP-1 protein production in a time-dependent manner in THP-1 cells. (E) PMA-treated THP-1 cells were stimulated with <i>P. gingivalis</i> LPS at concentrations of 0, 0.01, 0.1, or 1.0 µg/ml for 72 h. <i>P. gingivalis</i> LPS increased TSP-1 protein production in a dose-dependent manner in PMA-treated THP-1 cells.</p

The level of TSP-1 mRNA expression was significantly upregulated in periodontitis gingival tissues.

<p>Human gingival tissues were obtained from periodontal surgical operations. TSP-1 mRNA expression was enhanced in inflammatory gingival tissues. Its expression was significantly higher in regions of deep periodontal pockets than in shallow periodontal pockets and healthy sites (<i>P</i><0.001).</p

Thrombospondin-1 Production Is Enhanced by <i>Porphyromonas gingivalis</i> Lipopolysaccharide in THP-1 Cells

<div><p>Periodontitis is a chronic inflammatory disease caused by gram-negative anaerobic bacteria. Monocytes and macrophages stimulated by periodontopathic bacteria induce inflammatory mediators that cause tooth-supporting structure destruction and alveolar bone resorption. In this study, using a DNA microarray, we identified the enhanced gene expression of thrombospondin-1 (TSP-1) in human monocytic cells stimulated by <i>Porphyromonas gingivalis</i> lipopolysaccharide (LPS). TSP-1 is a multifunctional extracellular matrix protein that is upregulated during the inflammatory process. Recent studies have suggested that TSP-1 is associated with rheumatoid arthritis, diabetes mellitus, and osteoclastogenesis. TSP-1 is secreted from neutrophils, monocytes, and macrophages, which mediate immune responses at inflammatory regions. However, TSP-1 expression in periodontitis and the mechanisms underlying TSP-1 expression in human monocytic cells remain unknown. Here using real-time RT-PCR, we demonstrated that TSP-1 mRNA expression level was significantly upregulated in inflamed periodontitis gingival tissues and in <i>P. gingivalis</i> LPS-stimulated human monocytic cell line THP-1 cells. TSP-1 was expressed via Toll-like receptor (TLR) 2 and TLR4 pathways. In <i>P. gingivalis</i> LPS stimulation, TSP-1 expression was dependent upon TLR2 through the activation of NF-κB signaling. Furthermore, IL-17F synergistically enhanced <i>P. gingivalis</i> LPS-induced TSP-1 production. These results suggest that modulation of TSP-1 expression by <i>P. gingivalis</i> plays an important role in the progression and chronicity of periodontitis. It may also contribute a new target molecule for periodontal therapy.</p></div

Effect of various cytokines on TSP-1 expression in THP-1 cells.

<p>THP-1 cells were co-stimulated with 1.0 µg/ml of <i>P. gingivalis</i> LPS and 10 ng/ml of each of IL-4, IL-17A, IL-17F, or IFN-γ for 4 h. TSP-1 mRNA expression was significantly enhanced by all co-stimulatory factors (<i>P</i><0.001). In particular, TSP-1 mRNA expression by <i>P. gingivalis</i> LPS together with IL-17F was high.</p

<i>P. gingivalis</i> LPS-induced TSP-1 mRNA expression was inhibited by TLR2-neutralizing antibody in THP-1 cells.

<p>THP-1 cells were pretreated with 10 ng/ml of IgG2a isotype control, TLR2-neutralizing antibody, and TLR4-neutralizing antibody for 30 min, followed by the addition of 1.0 µg/ml of <i>P. gingivalis</i> LPS, Pam2CSK4, and <i>E. coli</i> LPS for 4 h. (A) TLR2-neutralizing antibody and TLR4-neutralizing antibody neutralized the activity of Pam2CSK4 and <i>E. coli</i> LPS, respectively. (B) <i>P. gingivalis</i> LPS with TLR2-neutralizing antibody significantly reduced TSP-1 mRNA expression compared with <i>P. gingivalis</i> LPS alone in THP-1 cells (<i>P</i><0.001).</p

Induction of Wnt5a expression is partly JAK/STAT dependent.

<p>(A–D) The expression of Wnt5a mRNA was detected by real-time PCR. Relative expression levels of Wnt5a are presented. (A, B) THP-1 cells were pre-treated with AG490 (A) or fludarabine (B) for 1 hr and then stimulated with <i>P. gingivalis</i> LPS for 4 hrs. DMSO is a solvent control for both inhibitors. (C) THP-1 cells were stimulated with <i>P. gingivalis</i> LPS for 4 hrs after transfection with STAT1 siRNA or control siRNA for 18 hrs. (D) THP-1 cells were pre-treated with STA21 for 1 hr and stimulated with <i>P. gingivalis</i> LPS for 4 hrs. *p<0.05.</p

Characteristics of the Study Subjects.

*<p>Mann-Whitney U test for continuous data, and Fisher's exact test for binary data.</p>**<p>Measured at the sampling sites.</p

<i>P. gingivalis</i> LPS-induced Wnt5a expression was inhibited by wedelolactone or dnIκBα.

<p>(A, B) THP-1 cells were pre-treated with the IKK inhibitor wedelolactone or the IKK inhibitor VII for 1 hr. The expression of Wnt5a mRNA was detected by real-time PCR. Relative expression levels of Wnt5a are presented. DMSO is a solvent control for both inhibitors. (C) THP-1 cells were transfected with a dominant-negative form of IκBα (dnIκBα) or the parent plasmid (pUSE) for 12 hrs. Fold induction of Wnt5a mRNA expression is shown. *p<0.05.</p

Induction of Wnt5a expression is NF-κB dependent.

<p>(A) THP-1 cells were stimulated with <i>E. coli</i> LPS or <i>P. gingivalis</i> LPS for 30 mins or 4 hrs. Whole cell extracts were prepared and analyzed by Western blot by using antibodies against IκBα. β-actin served as the protein loading control. (B) THP-1 cells were stimulated with <i>P. gingivalis</i> LPS for 4 hrs after being transfected with NF-κB reporter plasmid for 12 hrs, and the transcription activity was assessed by luminometer. The activity is represented by the relative luciferase activity. (C) Nuclear extracts were prepared and EMSA was performed with γ³²P-labeled oligonucleotides representing the NF-κB consensus sequence as a probe. Anti-p65 antibody was used for supershift assays. The lower arrow shows the DNA-protein complex, and the upper arrow shows the supershifted band. *p<0.05.</p