Regulation of P2X7-dependent inflammatory functions by P2X4 receptor in mouse macrophages
Ayumi Kawano1, Mitsutoshi Tsukimoto, Daisuke Mori
1Department of Radiation Biosciences, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba, Japan.
Abstract:
Activation of the P2X7 receptor of macrophages plays an important role in inflammation. We recently reported that co-expression of P2X4 receptor with P2X7 receptor facilitates P2X7 receptor-mediated cell death via Ca(2+) influx. However, it remained unclear whether P2X4 receptor is involved in P2X7 receptor-mediated inflammatory responses, such as cytokine production. Here, we present evidence that P2X4 receptor modulates P2X7 receptor-dependent inflammatory functions. Treatment of mouse macrophage RAW264.7 cells with 1mM ATP induced high mobility group box 1 (HMGB1) release and IL-1β production via activation of P2X7 receptor. Knockdown of P2X4 receptor or removal of extracellular Ca(2+) suppressed ATP-induced release of both HMGB1 and IL-1β. On the other hand, knockdown of P2X4 receptor or removal of extracellular Ca(2+) enhanced P2X7-dependent LC3-II expression (an index of autophagy), suggesting that P2X4 receptor suppresses P2X7-mediated autophagy. Since LC3-II expression was inhibited by pretreatment with antioxidant and NADPH oxidase inhibitor, we examined P2X7-mediated production of reactive oxygen species (ROS). We found that activation of P2X7 receptor-mediated production of ROS was significantly facilitated in P2X4-knockdown cells, suggesting that co-expression of P2X4 receptor with P2X7 receptor may suppress anti-inflammatory function-related autophagy via suppression of ROS production. We conclude that co-expression of P2X4 receptor with P2X7 receptor enhances P2X7-mediated inflammation through both facilitation of release of cytokines and suppression of autophagy.
Insights
The P2X4 receptor influences inflammation by modulating P2X7 receptor activity. Co-expression of P2X4 and P2X7 receptors enhances inflammation by increasing cytokine release and reducing autophagy.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- The P2X7 receptor (P2X7R) on macrophages is crucial for inflammation.
- P2X4 receptor (P2X4R) co-expression with P2X7R facilitates P2X7R-mediated cell death.
- The role of P2X4R in P2X7R-mediated inflammatory responses, like cytokine production, was unclear.
Purpose of the Study:
- To investigate the role of P2X4R in P2X7R-dependent inflammatory functions.
- To determine if P2X4R modulates P2X7R-mediated cytokine production and autophagy.
Main Methods:
- Utilized mouse macrophage RAW264.7 cells.
- Stimulated cells with ATP to activate P2X7R.
- Employed P2X4R knockdown and assessed levels of high mobility group box 1 (HMGB1), IL-1β, LC3-II, and reactive oxygen species (ROS).
Main Results:
- ATP-induced HMGB1 release and IL-1β production via P2X7R were suppressed by P2X4R knockdown.
- P2X4R knockdown enhanced P2X7R-dependent LC3-II expression (autophagy).
- P2X4R knockdown increased P2X7R-mediated ROS production, suggesting P2X4R suppresses ROS and subsequent autophagy.
Conclusions:
- Co-expression of P2X4R with P2X7R enhances P2X7R-mediated inflammation.
- This enhancement occurs through increased cytokine release (HMGB1, IL-1β) and suppressed autophagy.
- P2X4R may suppress autophagy via inhibition of ROS production, indicating a complex role in regulating inflammatory processes.
