Related Experiment Video
Updated: May 24, 2026
LPS and ATP-induced Death of PMA-differentiated THP-1 Macrophages and its Validation
Published on: May 3, 2024
Involvement of P2X4 receptor in P2X7 receptor-dependent cell death of mouse macrophages
Ayumi Kawano1, Mitsutoshi Tsukimoto, Taisei Noguchi
1Department of Radiation Biosciences, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi Chiba 278-8510, Japan.
Abstract:
Interaction of P2X7 receptor with P2X4 receptor has recently been suggested, but it remains unclear whether P2X4 receptor is involved in P2X7 receptor-mediated events, such as cell death of macrophages induced by high concentrations of extracellular ATP. Here, we present evidence that P2X4 receptor does play a role in P2X7 receptor-dependent cell death. Treatment of mouse macrophage RAW264.7 cells with 1mM ATP induced Ca(2+) influx, non-selective large pore formation, activation of extracellular signal-regulated protein kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK), and cell death via activation of P2X7 receptor. P2X4-knockdown cells, established by transfecting RAW264.7 cells with two short hairpin RNAs (shRNAs) targeting P2X4 receptor, showed a decrease of the initial peak of intracellular Ca(2+) after treatment with ATP, though pore formation and the P2X7-mediated activation of ERK1/2 and p38 MAPK were not affected. Intriguingly, P2X4 knockdown resulted in significant suppression of cell death induced by ATP or P2X7 agonist BzATP. In conclusion, our results suggest that P2X4 receptor is involved in P2X7 receptor-mediated cell death, but not pore formation or MAPK signaling.
Insights
The P2X4 receptor plays a role in P2X7 receptor-dependent macrophage cell death, but not in pore formation or MAPK signaling. This finding clarifies the P2X4 receptor's involvement in P2X7 receptor-mediated events.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- The P2X7 receptor mediates macrophage cell death upon stimulation with high extracellular ATP concentrations.
- The precise role of P2X4 receptor interaction in P2X7 receptor-mediated events, particularly cell death, remains largely undetermined.
Purpose of the Study:
- To investigate the involvement of P2X4 receptor in P2X7 receptor-dependent macrophage cell death.
- To elucidate the specific mechanisms by which P2X4 receptor influences P2X7 receptor signaling pathways.
Main Methods:
- Utilized mouse macrophage RAW264.7 cells treated with 1mM ATP or P2X7 agonist BzATP.
- Established P2X4-knockdown cell lines using short hairpin RNAs (shRNAs) targeting P2X4 receptor.
- Assessed calcium (Ca2+) influx, pore formation, extracellular signal-regulated protein kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK) activation, and cell death.
Main Results:
- P2X4 receptor knockdown decreased the initial peak of intracellular Ca2+ influx induced by ATP.
- Pore formation and P2X7-mediated activation of ERK1/2 and p38 MAPK remained unaffected by P2X4 knockdown.
- Significant suppression of ATP- or BzATP-induced cell death was observed in P2X4-knockdown cells.
Conclusions:
- P2X4 receptor is implicated in P2X7 receptor-mediated macrophage cell death.
- P2X4 receptor does not appear to be essential for P2X7 receptor-induced pore formation or MAPK signaling.
- These findings highlight a specific role for P2X4 receptor in the downstream events of P2X7 receptor activation leading to cell death.
