Purinergic receptors responsible for ATP-dependent ROS production and activation of inflammasomes in gingival epithelial cells
- Author(s): Ho, Marcus
- Advisor(s): Ojcius, David M.
- et al.
P2X4 and P2X7 are purinergic receptors prevalent in human immune and epithelial cells. Some of their functions in the cell include mediating cell death and the inflammatory response. The multiprotein inflammasome complex in the innate immune system helps process and release cytokines such as interleukins 1β and 8. Pannexin-1 is a membrane channel suggested to play a role in inflammasome activation by interacting with purinergic receptors. This thesis evaluates whether extracellular ATP activates the inflammasome in a P2X7, P2X4 or pannexin-1-dependent manner. The characterization of this mechanism is to be elucidated in gingival epithelial cells (GECs), which are part of the oral cavity’s first line of defense in the innate immune system. GECs express P2X4 and P2X7 receptors as well as pannexin-1, and are sensitive to ATP-induced apoptosis. However, the mechanisms by which the inflammasomes are activated are uncertain. In this study, we compared reactive oxygen species (ROS) production levels between GECs treated with ATP and those without ATP. ROS production has been shown to promote the assembly of inflammasomes, leading to caspase-1 activation. We also did treatments with various receptor and channel inhibitors, and generated GEC P2X4, P2X7 and pannexin-1 knockdown cell lines to compare the amount of ROS and caspase-1 activation due to ATP stimulation. Our results show that depletion of P2X4, P2X7 and pannexin-1 by either specific antagonists or shRNA diminished the ability of ATP to induce ROS production and to a lesser extent, caspase-1 activation in GECs. P2X4, P2X7 and pannexin-1 are therefore likely to be important mediators of the inflammatory response in GECs. Lastly, we sought to find protein-protein interactions between the three proteins, and how they may affect inflammasome activation. So far, this study shows that P2X4 and P2X7 are able to interact, and are enhanced upon ATP stimulation.