Zhang Wenyi, Hirschler-Laszkiewicz Iwona, Tong Qin, Conrad Kathleen, Sun Shao-Cong, Penn Linda, Barber Dwayne L, Stahl Richard, Carey David J, Cheung Joseph Y, Miller Barbara A
Department of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, USA
Am J Physiol Cell Physiol. 2006 Apr;290(4):C1146-59. doi: 10.1152/ajpcell.00205.2005. Epub 2005 Nov 23.
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+)), 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+) 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+) in mediating these effects. Downregulation of endogenous TRPM2 by RNA interference or increased expression of TRPM2-S inhibited the rise in Ca(2+), 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+), 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.
瞬时受体电位 melastatin 2型（TRPM2）是一种由氧化应激或肿瘤坏死因子-α（TNF-α）激活的钙离子通透通道，TRPM2的激活会导致细胞死亡易感性增加。本文在人单核细胞U937-ecoR细胞中研究了其机制。该细胞系表达全长TRPM2（TRPM2-L）和几种异构体，包括一种缺少钙离子通透孔区域的短剪接变体（TRPM2-S），其作为显性负性起作用。用氧化应激模型过氧化氢（H₂O₂）或TNF-α处理会导致细胞活力降低。通过逆转录病毒感染调节TRPM2-L和TRPM2-S的表达。用H₂O₂或TNF-α处理表达水平升高的TRPM2-L的U93七千-ecoR细胞，这些细胞表现出细胞内钙浓度（[Ca²⁺]i）显著升高、活力降低和凋亡增加。观察到半胱天冬酶-8、-9、-3和-7以及聚（ADP-核糖）聚合酶（PARP）的切割显著增加，证明了介导细胞死亡的下游机制。Bcl-2水平未改变。用细胞内钙离子螯合剂1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA）抑制[Ca²⁺]i升高可阻断TRPM2-L激活后诱导的半胱天冬酶/PARP切割和细胞死亡，证明[Ca²⁺]i在介导这些效应中的关键作用。通过RNA干扰下调内源性TRPM2或增加TRPM2-S的表达可抑制[Ca²⁺]i升高，增强细胞活力，并减少暴露于氧化应激或TNF-α后的凋亡细胞数量，证明了TRPM2的生理重要性。我们的数据表明，氧化应激或TNF-α介导细胞死亡的一种机制是TRPM2的激活，导致[Ca²⁺]i增加，随后半胱天冬酶激活和PARP切割。通过降低TRPM2水平、与TRPM2-S相互作用或钙离子螯合来抑制TRPM2-L功能可拮抗这一重要的细胞死亡途径。
