Role of TRPM2 in H(2)O(2)-induced cell apoptosis in endothelial cells.
Melastatin-like transient receptor potential channel 2 (TRPM2) is an oxidant-sensitive and cationic non-selective channel that is expressed in mammalian vascular endothelium. Here we investigated the functional role of TRPM2 channels in hydrogen peroxide (H(2)O(2))-induced cytosolic Ca(2+) ([Ca(2+)](i)) elavation, whole-cell current increase, and apoptotic cell death in murine heart microvessel endothelial cell line H5V. A TRPM2 blocking antibody (TM2E3), which targets the E3 region near the ion permeation pore of TRPM2, was developed. Treatment of H5V cells with TM2E3 reduced the [Ca(2+)](i) rise and whole-cell current change in response to H(2)O(2). Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA) had similar inhibitory effect. H(2)O(2)-induced apoptotic cell death in H5V cells was examined using MTT assay, DNA ladder formation analysis, and DAPI-based nuclear DNA condensation assay. Based on these assays, TM2E3 and TRPM2-specific shRNA both showed protective effect against H(2)O(2)-induced apoptotic cell death. TM2E3 and TRPM2-specific shRNA also protect the cells from tumor necrosis factor (TNF)-α-induced cell death in MTT assay. In contrast, overexpression of TRPM2 in H5V cells resulted in an increased response in [Ca(2+)](i) and whole-cell currents to H(2)O(2). TRPM2 overexpression also aggravated the H(2)O(2)-induced apoptotic cell death. Downstream pathways following TRPM2 activation was examined. Results showed that TRPM2 activity stimulated caspase-8, caspase-9 and caspase-3. These findings strongly suggest that TRPM2 channel mediates cellular Ca(2+) overload in response to H(2)O(2) and contribute to oxidant-induced apoptotic cell death in vascular endothelial cells. Down-regulating endogenous TRPM2 could be a means to protect the vascular endothelial cells from apoptotic cell death.
Li Ka Shing Institute of Health Sciences and School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China.
Lei
L
Sun
Ho-Yan
HY
Yau
Wei-Yan
WY
Wong
Ronald A
RA
Li
Yu
Y
Huang
Xiaoqiang
X
Yao
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002460:Cell Line; D053938:DNA Fragmentation / Q000187:drug effects; D004594:Electrophysiology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D051379:Mice; D050053:TRPM Cation Channels / Q000235:genetics / Q000378:metabolism*
D050053:TRPM Cation Channels; C544443:TRPM2 protein, mouse; D006861:Hydrogen Peroxide