To study the effect of mechanical stress on the expression of toll-like receptors in human PDL cells.
Human periodontal ligament cell line #2630 (ScienCell Research Laboratories) cells were cultured in alpha-MEM with 10% FBS and 1% Pen/Strep at 37 °C and 5% CO2. The cells (1-10 passages) were seeded at a density of 2000 cells/cm2 on 75X38 mm2 glass slides coated with type I collagen. After 2 days of growth, the cells were starved for 24 hours then subjected to 12 dynes/cm2 fluid shear stress (FSS) for 1 hour. After FSS, the cells were post incubated for 6 hours and then lysed to test the expression of toll-like receptor (TLR) -2 and -4. To explore the possible involvement of MAPK signaling pathway, a specific MEK1 inhibitor - PD98059 was added during the flow and post incubation period. To quantify the protein production, 50 micrograms of whole cell lysate from each group were separated by 10% SDS-PAGE and immunoblotted with anti-TLR2 and anti-TLR4 antibodies, respectively. The signals of interest were determined using the ECL method. For quantification, densitometries of gel bands of interest were normalized to that of vinculin by using Fuji-1000 Imaging system. One-way ANOVA was used to compare the results among the experimental groups, with p value being set at 0.05.
TLR4 but not TLR2 were strongly expressed in human PDL cells. Compared to the basal level of TLR4 expression in the static controls, FSS significantly reduced the expression of TLR4 by about 55% (p<0.001, n=3, One-way ANOVA, Minitab, version 18.104.22.168). When PD98059 was added during FSS and post incubation, the FSS-induced reduction of TLR4 was significantly recovered back to the basal level as seen in the static controls.
Conclusion: Mechanical stress down-regulates the expression of TLR4 but not TLR2, which is medicated by MAPK signaling pathway.
Keywords: Biophysics, Bone, Cell biology, Orthodontics and Periodontal disease
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