TRPM2 is an ion channel that modulates hematopoietic cell death through activation of caspases and PARP cleavage.
TRPM2 is a Ca(2+)-permeable channel activated by oxidative stress or TNF-alpha, and TRPM2 activation confers susceptibility to cell death. The mechanisms were examined here in human monocytic U937-ecoR cells. This cell line expresses full-length TRPM2 (TRPM2-L) and several isoforms including a short splice variant lacking the Ca(2+)-permeable pore region (TRPM2-S), which functions as a dominant negative. Treatment with H(2)O(2), a model of oxidative stress, or TNF-alpha results in reduced cell viability. Expression of TRPM2-L and TRPM2-S was modulated by retroviral infection. U937-ecoR cells expressing increased levels of TRPM2-L were treated with H(2)O(2) or TNF-alpha, and these cells exhibited significantly increased intracellular calcium concentration ([Ca(2+)](i)), decreased viability, and increased apoptosis. A dramatic increase in cleavage of caspases-8, -9, -3, and -7 and poly(ADP-ribose)polymerase (PARP) was observed, demonstrating a downstream mechanism through which cell death is mediated. Bcl-2 levels were unchanged. Inhibition of the [Ca(2+)](i) rise with the intracellular Ca(2+) chelator BAPTA blocked caspase/PARP cleavage and cell death induced after activation of TRPM2-L, demonstrating the critical role of [Ca(2+)](i) in mediating these effects. Downregulation of endogenous TRPM2 by RNA interference or increased expression of TRPM2-S inhibited the rise in [Ca(2+)](i), enhanced cell viability, and reduced numbers of apoptotic cells after exposure to oxidative stress or TNF-alpha, demonstrating the physiological importance of TRPM2. Our data show that one mechanism through which oxidative stress or TNF-alpha mediates cell death is activation of TRPM2, resulting in increased [Ca(2+)](i), followed by caspase activation and PARP cleavage. Inhibition of TRPM2-L function by reduction in TRPM2 levels, interaction with TRPM2-S, or Ca(2+) chelation antagonizes this important cell death pathway.
Zhang
Wenyi
W
Department of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, USA
Hirschler-Laszkiewicz
Iwona
I
Tong
Qin
Q
Conrad
Kathleen
K
Sun
Shao-Cong
SC
Penn
Linda
L
Barber
Dwayne L
DL
Stahl
Richard
R
Carey
David J
DJ
Cheung
Joseph Y
JY
Miller
Barbara A
BA
D017398:Alternative Splicing; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death / Q000502:physiology*; D002460:Cell Line; D024161:Collagen Type XI / Q000378:metabolism*; D004789:Enzyme Activation; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000502:physiology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism; D018384:Oxidative Stress; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050053:TRPM Cation Channels / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
C499319:COL11A2 protein, human; D024161:Collagen Type XI; D016877:Oxidants; D020033:Protein Isoforms; D011498:Protein Precursors; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D050053:TRPM Cation Channels; C121934:TRPM2 protein, human; D014409:Tumor Necrosis Factor-alpha; D006861:Hydrogen Peroxide; D020169:Caspases; D002118:Calcium