N-(3-oxododecanoyl)-homoserine lactone regulates intracellular calcium of MC3T3 osteoblasts

Published: 26-08-2020| Version 1 | DOI: 10.17632/2hxv9cr73m.1
Contributors:
jiajie guo,
Ziyi Wang,
Lihong Qiu

Description

Pseudomonas aeruginosa (P. aeruginosa) is associated with periapical periodontitis. The lesions are characterized by a disorder in osteoblast metabolism. Quorum sensing molecular N-(3-oxododecanoyl)-homoserine lactone (AHL) is secreted by P. aeruginosa and governs the expression of numerous virulence factors. AHL can trigger intracellular calcium ([Ca²⁺]i) fluctuations in many host cells. However, it is unclear whether AHL can regulate osteoblast metabolism by affecting [Ca²⁺]i changes or its spatial correlation. We explored AHL-induced apoptosis and differentiation in pre-osteoblastic MC3T3-E1 cells and evaluated [Ca²⁺]i mobilization using several extraction methods. The spatial distribution pattern of [Ca²⁺]i among cells was investigated by Moran's I, an index of spatial autocorrelation. We found that 30 μM and 50 μM AHL triggered opposing osteoblast fates. At 50 μM, AHL inhibited osteoblast differentiation by promoting mitochondrial-dependent apoptosis and negatively regulating osteogenic marker genes, including Runx2, Osterix, bone sialoprotein (Bsp), and osteocalcin (OCN). In contrast, prolonged treatment with 30 μM AHL promoted osteoblast differentiation concomitantly with cell apoptosis. The elevation of [Ca²⁺]i levels in osteoblasts treated with 50 μM AHL was spatially autocorrelated, while no such phenomenon was observed in 30 μM AHL-treated osteoblasts. The blocking of cell-to-cell spatial autocorrelation in the osteoblasts provoked by 50 μM AHL significantly inhibited apoptosis and partially restored differentiation. Our observations suggest that AHL affects the fate of osteoblasts (apoptosis and differentiation) by affecting the spatial correlation of [Ca²⁺]i changes. Thus, AHL acts as a double-edged sword for osteoblast function. Please cite this article below if using any data from this dataset. Guo J, Wang Z, Weng Y, Yuan H, Yoshida K, Ikegame M, Uchibe K, Kamioka H, Ochiai K, Okamura H, Qiu L. N-(3-oxododecanoyl)-homoserine lactone regulates osteoblast apoptosis and differentiation by mediating intracellular calcium. Cellular Signalling. 2020 Aug 18:109740. https://doi.org/10.1016/j.cellsig.2020.109740

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Cells were cultured in µ-Dishes (Ibidi, Grafelfing, Germany) and loaded with 5 µM Calbryte™ 520 AM (AAT Bioquest, Sunnyvale, CA) and 5 µM Fura Red AM (AAT Bioquest) in the culture medium for 1 hr at 37°C. The cells were washed with HBSS three times, and then a fresh culture medium was added to the dishes. Dye-loaded cells were monitored using an LSM780 confocal laser scanning microscope. The cells were treated with AHL and continuously monitored over time. The scanning rate was 3.13 seconds/scan, and time-lapse images were collected from a single Z plane and recorded at 5-sec intervals. The fluorescence of Calbryte™ 520 AM and Fura Red AM was measured using 488/525 nm (excitation/emission) and 488/647 nm (excitation/emission), respectively.