Abstract:
Mechanical stress is an important factor in maintaining periodontium homeostasis. We previously reported that mechanical stress could activate ATP release in human periodontal ligament (HPDL) cells. In this study, we found that ATP mediated mechanical stress-induced mRNA expression and release of the pro-inflammatory cytokine IL-1β. The released ATP later activates P2 receptor. By using inhibitors and siRNA experiments, we found that P2X7 receptor was the main P2 subtype responsible for ATP-induced upregulation of IL-1β in HPDL cells. In macrophages, Pannexin-1 (Panx1) hemichannel has been proposed to combine with P2X7 receptor, forming a mechanosensitive complex involving in the regulation of IL-1β. In this work, we found that, in the absence of Panx1, upregulation of IL-1β upon both mechanical and ATP stimulation was drastically diminished. Additionally, immunocytochemistry and co-immunoprecipitation revealed the co-localization of P2X7 receptor and Panx1 hemichannel in HPDL cells, which found to be increased after stress application. Results also showed that Panx1 hemichannel might serve as one of the ATP release pathway in HPDL cells. The IL-1β releasing mechanism was also investigated. Pretreatment with vesicular trafficking inhibitors significantly reduced IL-1β release from HPDL cells, while mRNA induction remained unaffected. Membrane co-localization of SNAP-25, a core protein of vesicular-membrane fusion SNARE complex, with P2X7 receptor/Panx1 was observed. Collectively, these data indicated vesicular release of IL-1β. In conclusion, mechanical stress could directly regulate IL-1β expression and vesicular release in HPDL cells through ATP-gated P2X7 receptor/Panx1-dependent pathway. Function of Panx1/P2X7 receptor might be required in the IL-1β induction mechanism, and their possible novel function as docking sites for IL-1β vesicular release was also indicated.
