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Regulation of Cementoblast Function by P. gingivalis Lipopolysaccharide via TLR2

¹ Department of Periodontics, School of Dentistry, University of Washington, D322-Health Science Center Box 356365, Seattle, WA 98195-6365, USA;
² Division of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575, Japan; and
³ FBDC-Curso de Odontologia, Brazil

View larger version (16K): [in a new window]   Figure 1. Effects of P. gingivalis and E. coli LPS on selected mRNA levels. Confluent OCCM-30 cells in 60-mm-diameter culture dishes was cultured in DMEM (5% FBS) with AA (50 µg/mL) in the presence or absence of P. gingivalis LPS (1 µg/mL) for 1, 3, 6, 12, and 24 hrs (A) or E. coli LPS (10 ng/mL) for 3 hrs (B). Total cellular RNA was extracted, and the mRNA expressions were analyzed by real-time quantitative RT-PCR. Data in (A) represent mean ± SD from 3 separate experiments. Data in (B) are representative of 2 independent experiments. Statistical significance is shown (*P < 0.05 vs. control).

View larger version (19K): [in a new window]   Figure 2. Effect of P. gingivalis LPS on selected protein levels. Confluent OCCM-30 in 35-mm-diameter culture dishes was cultured in DMEM (5% FBS) with AA (50 µg/mL) in the presence or absence of LPS (1 µg/mL) for 3, 6, 12, and 24 hrs. The amounts of OPG, MCP-1, IL-6 RANTES, and MIP-1 in the supernatant and RANKL in the cell lysate were analyzed by ELISA. Data represent mean ± SD from 2 separate experiments done by triplicate assay. Statistical significance is shown (*P < 0.05 vs. control).

View larger version (16K): [in a new window]   Figure 3. Effects of anti-TLR-2 and TLR-4 mAbs on NF-B activation. OCCM-30 cells were co-transfected with pNK-B-TA-Luc and pß-actin Renilla luciferase, as described in MATERIALS & METHODS. Eighteen hours later, transfectants were pre-treated with 20 µg/mL of anti-TLR-2 and -4 mAbs, as well as with 20 µg/mL of control Abs, for 30 min, followed by stimulation with E. coli LPS (10 ng/mL), peptidoglycan (1 µg/mL), PgLPS1690 (1 µg/mL), or PgLPS1435/1449 (1 µg/mL) for 5 hrs. Cells were harvested, and luciferase activity was measured. Values were calculated as fold increase of relative luciferase units (firefly luciferase/Renilla luciferase) compared with the non-stimulated control response, which was set at 1. Representative data of 3 separate experiments are shown as means ± SD of triplicate assays. Statistical significance is shown (*P < 0.05 vs. control).

View larger version (20K): [in a new window]   Figure 4. Effects of anti-TLR-2 and TLR-4 mAbs on gene expression. Confluent OCCM-30 cells in 35-mm-diameter culture dishes were pre-treated with 20 µg/mL of anti-TLR-2 and -4 mAbs, as well as with 20 µg/mL of control Abs, for 30 min, followed by stimulation with PgLPS1690 (1 µg/mL) or PgLPS1435/1449 (1 µg/mL) in DMEM (5% FBS) with AA (50 µg/mL) for 3 hrs. Total cellular RNA was extracted, and mRNA expression of RANKL, IL-6, and RANTES was analyzed by real-time quantitative RT-PCR. Values were calculated as fold increase compared with calibrator cDNA, which was generated from OCCM-30 stimulated with 50 µg/mL AA for 5 days. Representative data of 3 separate experiments are shown as means ± SD of triplicate assays. Statistical significance is shown (*P < 0.05 vs. control).