Signalling mechanisms mediating Zn2+-induced TRPM2 channel activation and cell death in microglial cells.
Excessive Zn2+causes brain damage via promoting ROS generation. Here we investigated the role of ROS-sensitive TRPM2 channel in H2O2/Zn2+-induced Ca2+signalling and cell death in microglial cells. H2O2/Zn2+induced concentration-dependent increases in cytosolic Ca2+concentration ([Ca2+]c), which was inhibited by PJ34, a PARP inhibitor, and abolished by TRPM2 knockout (TRPM2-KO). Pathological concentrations of H2O2/Zn2+induced substantial cell death that was inhibited by PJ34 and DPQ, PARP inhibitors, 2-APB, a TRPM2 channel inhibitor, and prevented by TRPM2-KO. Further analysis indicate that Zn2+induced ROS production, PARP-1 stimulation, increase in the [Ca2+]cand cell death, all of which were suppressed by chelerythrine, a protein kinase C inhibitor, DPI, a NADPH-dependent oxidase (NOX) inhibitor, GKT137831, a NOX1/4 inhibitor, and Phox-I2, a NOX2 inhibitor. Furthermore, Zn2+-induced PARP-1 stimulation, increase in the [Ca2+]cand cell death were inhibited by PF431396, a Ca2+-sensitive PYK2 inhibitor, and U0126, a MEK/ERK inhibitor. Taken together, our study shows PKC/NOX-mediated ROS generation and PARP-1 activation as an important mechanism in Zn2+-induced TRPM2 channel activation and, TRPM2-mediated increase in the [Ca2+]cto trigger the PYK2/MEK/ERK signalling pathway as a positive feedback mechanism that amplifies the TRPM2 channel activation. Activation of these TRPM2-depenent signalling mechanisms ultimately drives Zn2+-induced Ca2+overloading and cell death.
School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, United Kingdom.
Sharifah Syed
SS
Mortadza
School of Life Sciences, University of Manchester, United Kingdom.
Joan A
JA
Sim
School of Molecular and Cell Biology, Faculty of Biological Sciences, University of Leeds, United Kingdom.
Martin
M
Stacey
School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, United Kingdom.|Sino-UK Joint Laboratory of Brain Function and Injury, and Department of Physiology and Neurobiology, Xinxiang Medical University, PR China.
Lin-Hua
LH
Jiang
D000818:Animals; D002118:Calcium / Q000378:metabolism; D016923:Cell Death; D002478:Cells, Cultured; D051418:Focal Adhesion Kinase 2 / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D020935:MAP Kinase Signaling System; D051379:Mice; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D008954:Models, Biological; D019255:NADPH Oxidases / Q000378:metabolism; D000071137:Poly (ADP-Ribose) Polymerase-1 / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D050053:TRPM Cation Channels / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D017382:Reactive Oxygen Species; D050053:TRPM Cation Channels; C544443:TRPM2 protein, mouse; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; D000071137:Poly (ADP-Ribose) Polymerase-1; D051418:Focal Adhesion Kinase 2; C496053:Ptk2b protein, mouse; D011493:Protein Kinase C; D015032:Zinc; D002118:Calcium