P2X7 receptor-Pannexin1 interaction mediates stress-induced interleukin-1 beta expression in human periodontal ligament cells

Background and Objective: Pannexin 1 (Panx1) has been found to form nonjunctional hemichannels. It is also proposed to combine with the P2X7 receptor, forming a complex involved in adenosine triphosphate (ATP)-induced interleukin-1beta (IL-1 beta) release in macrophages. Previously, we reported that mechanical stress induced IL-1 beta expression via the ATP/P2X7 receptor-dependent pathway in human periodontal ligament (HPDL) cells and that ATP was released through the connexin 43 (Cx43) hemichannel. In the present work, we examined the role of Panx1 in stress-induced IL-1 beta induction in HPDL cells. Material and Methods: Cultured HPDL cells were treated with compressive loading or ATP to stimulate IL-1 beta expression. Inhibitors, antagonists and the small interfering RNA technique were used to investigate the involvement of Panx1 in IL-1 beta induction. Co-immunoprecipitation (Co-IP) and immunostaining were used to determine the association of Panx1 with the P2X7 receptor. The IL-1 beta release mechanism was analyzed using inhibitors. Results: Blocking Panx1 significantly decreased ATP release, as well as IL-1 beta up-regulation, upon stimulation with stress or ATP. Co-IP revealed the association of Panx1 and the P2X7 receptor in HPDL cells, which was increased in response to mechanical loading. Pretreatment with vesicular trafficking inhibitors significantly reduced the amount of IL-1 beta released from stimulated cells, suggesting that IL-1 beta might be released through vesicles. Conclusion: We clearly illustrated the contribution of Panx1 in ATP release, as well as in IL-1 beta induction in HPDL cells. The association of Panx1 and the P2X7 receptor might be required for IL-1 beta induction, and their possible novel role in IL-1 beta vesicular release was indicated.