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Mechanical Stress Induces Osteopontin via ATP/P2Y1 in Periodontal Cells

Department of Anatomy and Graduate School of Oral Biology, Faculty of Dentistry, Chulalongkorn University, Henri Dunant Road, Pathumwan, Bangkok 10330, Thailand

View larger version (34K): [in this window] [in a new window]   Figure 1. Stress-induced OPN expression was inhibited by suramin and NF449. HPDL cells were stimulated with 1.5 g/cm2 of force for 24 hrs. Increased OPN mRNA (a) and protein (b) expression was observed. The stress-induced OPN expression was significantly abolished by both suramin (A) and NF449 (B). The graph represents the band density from Western blot analysis. The results are expressed as mean ± SD from 3 different experiments. *p < 0.05.

View larger version (34K): [in this window] [in a new window]   Figure 2. The conditioned medium (CM-S) induced the expression of OPN. (A) Cells were incubated for 24 hrs with the medium transferred from the stress-stimulated (CM-S) and non-stimulated (CM-C) cultures. CM-S induced OPN expression when compared with control (CM-C). The inductive effect exerted by CM-S was similar to that resulting from stress (S) and could be inhibited by NF449. (B) The Luciferin-Luciferase bioluminescence assay revealed that the amount of ATP in the medium collected from stress-stimulated cultures (1, 1.5, 2 g/cm2) was increased. (C) Apyrase partially inhibited the CM-S-induced OPN expression. The graph shows the band density from Western blot analysis. The results are expressed as mean ± SD from 3 separate experiments. *p < 0.05; a, mRNA expression; b, protein expression.

View larger version (41K): [in this window] [in a new window]   Figure 3. HPDL cells expressed P2Y1 and P2Y2 ATP receptors. (A) HPDL cells expressed P2Y1 and P2Y2 receptors detectable at both mRNA and protein levels. (B) MRS2179, a specific P2Y1 antagonist, exerted an inhibitory effect on the stress-induced OPN expression in a dose-dependent manner. The graph represents the mean ± SD of the band density from Western blot analysis. The data are from 3 separate experiments. *p < 0.05; a, mRNA expression; b, protein expression.

View larger version (35K): [in this window] [in a new window]   Figure 4. ATP induced the up-regulation of OPN via Rho kinase. (A) HPDL cells were incubated with exogenous ATP (0.1, 1, and 10 µM) for 24 hrs. The results showed that ATP stimulated the expression of OPN in a dose-dependent manner. Cells were incubated with Rho kinase inhibitor (Rhoi) for 30 min before the application of ATP (1 µM) or stress (1.5 g/cm2) for 24 hrs. The results demonstrated that the increase in OPN expression (B), but not that of ATP (C), induced by stress was abolished by Rhoi. The graph in (B) represents the band density from Western blot analysis. The results are expressed as mean ± SD from 3 separate experiments. *p < 0.05; a, mRNA expression; b, protein expression.

Journal of Dental Research, Vol. 87, No. 6, 564-568 (2008)
DOI: 10.1177/154405910808700601


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