Cementoblast Gene Expression is Regulated by Porphyromonas gingivalis Lipopolysaccharide Partially via Toll-like Receptor-4/MD-2
F.H. Nociti, Jr.1,2,
B.L. Foster2,
S.P. Barros3,
R.P. Darveau2, and
M.J. Somerman2,*
1 Department of Prosthodontics/Periodontics, Division of Periodontics, School of Dentistry at Piracicaba, University of Campinas, Brazil;
2 Department of Periodontics, School of Dentistry, 1959 NE Pacific, D322-Health Science Center, University of Washington, Seattle, WA 98195-7444, USA; and
3 Department of Morphology, Division of Histology, School of Dentistry at Piracicaba, University of Campinas, Brazil;
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Figure 1. Characterization of cementoblasts: mRNA expression of (A) Toll-like receptors 2 and 4 (TLR-2/TLR-4), and (B) MD-2 and CD-14. Bars represent mean ± SD from two separate experiments, with similar results (n = 2). Cementoblasts (OCCM-30) were seeded, and upon reaching confluence (4 days), mRNA was extracted and analyzed by real-time RT-PCR; the results were normalized to GAPDH and to calibrator (OCCM-30) values. (C) Proliferation assay: Cementoblasts (OCCM-30) were seeded and, after 24 hrs, were treated with P. gingivalis lipopolysaccharide at 100 ng/mL. Cells were counted before treatment (D0), and after 2, 4, and 6 days. Graph illustrates the proportion of viable (open bar) vs. non-viable cells (dark bar) (Trypan blue staining) for a representative experiment with similar results seen in two separate experiments (n = 2). *P < 0.001 and **P < 0.05 vs. control within the same period of time. CN = control group and P-LPS = Porphyromonas gingivalis-treated group.
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Figure 2. Effect of P. gingivalis lipopolysaccharide (P-LPS) on gene expression: (A) Cementoblasts (OCCM-30) were cultured in DMEM (5% FBS) with ascorbic acid (AA) (50 µg/mL) (control - CN) or plus P-LPS (1–1000 ng/mL). mRNA was extracted after 24 hrs, and expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen I (Col I), and osteopontin (OPN) was analyzed by Northern blot. Results from two separate experiments (n = 2) showed that P-LPS regulates expression of mineral-associated genes in a dose-dependent manner, with a marked decrease in OCN and an increase in OPN and Col I transcripts. Effect of P-LPS on genes/proteins associated with bone resorption: (B) Cementoblasts (OCCM-30) were cultured as described above (n = 2). RNA was extracted after 24 hrs and analyzed by real-time PCR, and values were normalized to GAPDH and calibrator values. P-LPS significantly decreased RANKL and increased OPG expression in a dose-dependent manner. (C) Protein levels evaluated by ELISA. Cell lysate and medium were used to obtain RANKL and OPG levels, respectively. Note that the impact of P-LPS on protein levels parallels findings on gene expression. Bars represent mean ± SD from two separate experiments, with similar results (n = 2). *P < 0.001 and **P < 0.05 vs. control.
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Figure 3. Effect of P. gingivalis lipopolysaccharide (P-LPS) on gene expression: Time-course assay: Cementoblasts (OCCM-30) were cultured in DMEM (5% FBS) with ascorbic acid (AA) (50 µg/mL) (control-CN) or plus P-LPS (100 ng/mL). RNA was extracted after days 2, 4, and 6, and expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen I (Col I), and osteopontin (OPN) was analyzed by Northern blot. Values were normalized to 18S, and bars represent mean ± SD from two separate experiments, with similar results (n = 2). Results indicated that the effect of P-LPS on gene expression was observed as early as 6 hrs. OCN mRNA levels were markedly decreased at 12 and 24 hrs, whereas OPN levels were significantly noted by 6 hrs. Col I mRNA levels were slightly and consistently up-regulated by P-LPS at 24 hrs, whereas minimal changes were observed in BSP expression when the control group was compared with the P-LPS group. *P < 0.001 and **P < 0.05 vs. control within the same period of time.
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Figure 4. Lipopolysaccharide-receptor interactions: Cementoblasts (OCCM-30) were cultured in DMEM (5% FBS) with ascorbic acid (AA) (50 µg/mL) (control-CN), plus P-LPS (100 ng/mL), or P-LPS plus Antibody (against TLR-4/MD-2 complex-MTS510) (20 µg/mL). (A) RNA was extracted after 24 hrs, and expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen I (Col I), and osteopontin (OPN) was assessed. Values were normalized to 18S, and bars represent mean ± SD from two separate experiments, with similar results (n = 2). Note that antibody treatment partially blocked the effect of P-LPS on OCN/OPN/Col I mRNA levels. (B) Real-time PCR for RANKL/OPG mRNA expressions. Results illustrate that antibody also partially blocked the effect of P-LPS on RANKL/OPG. Antibody treatment resulted in a significant reduction of the effect of P-LPS on OPG, whereas its effect on RANKL expression was not statistically significant. Bars represent mean ± SD from two separate experiments (n = 2). *P < 0.001 and **P < 0.05 vs. control.
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