Thomas Jefferson University Jefferson Digital Commons Department of Pathology, Anatomy, and Cell Department of Pathology, Anatomy, and Cell Biology Faculty Papers Biology 9-26-2005 Selective role for superoxide in InsP3 receptor-mediated mitochondrial dysfunction and endothelial apoptosis. Muniswamy Madesh University of Pennsylvania Brian J Hawkins University of Pennsylvania Tatyana Milovanova University of Pennsylvania CunnigaiperFollow this and D additionalBhanumathy works at: https://jdc.jefferson.edu/pacbfp Thomas Part ofJeff theerson Medical Univ Anatersityomy Commons, Medical Cell Biology Commons, and the Medical Pathology SurCommonsesh K Joseph LetThomas us Jeff knowerson Univ howersity access to this document benefits ouy SeeRecommended next page for Citation additional authors Madesh, Muniswamy; Hawkins, Brian J; Milovanova, Tatyana; Bhanumathy, Cunnigaiper D; Joseph, Suresh K; Ramachandrarao, Satish P; Sharma, Kumar; Kurosaki, Tomohiro; and Fisher, Aron B, "Selective role for superoxide in InsP3 receptor-mediated mitochondrial dysfunction and endothelial apoptosis." (2005). Department of Pathology, Anatomy, and Cell Biology Faculty Papers. 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Authors Muniswamy Madesh, Brian J Hawkins, Tatyana Milovanova, Cunnigaiper D Bhanumathy, Suresh K Joseph, Satish P Ramachandrarao, Kumar Sharma, Tomohiro Kurosaki, and Aron B Fisher This article is available at Jefferson Digital Commons: https://jdc.jefferson.edu/pacbfp/115 JCB: ARTICLE Selective role for superoxide in InsP3 receptor–mediated mitochondrial dysfunction and endothelial apoptosis Muniswamy Madesh,1,2 Brian J. Hawkins,1 Tatyana Milovanova,1 Cunnigaiper D. Bhanumathy,3 Suresh K. Joseph,3 Satish P. RamachandraRao,4 Kumar Sharma,4 Tomohiro Kurosaki,5 and Aron B. Fisher1 1Institute for Environmental Medicine and 2Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA 19104 3Department of Pathology, Anatomy, and Cell Biology and 4Dorrance Hamilton Research Laboratories, Thomas Jefferson University, Philadelphia, PA 19107 5Laboratory for Lymphocyte Differentiation, Institute of Physical and Chemical Research, Research Center for Allergy and Immunology, Turumi-ku, Yokohama 230-0045, Japan eactive oxygen species (ROS) play a divergent pendent, and cells lacking the three InsP3 receptor 2ϩ role in both cell survival and cell death during is- (InsP3R) isoforms failed to display the [Ca ]i transient. .Ϫ 2ϩ R chemia/reperfusion (I/R) injury and associated Importantly, the O2 -triggered Ca mobilization pre- inflammation. In this study, ROS generation by activated ceded a loss in mitochondrial membrane potential that 2ϩ 2ϩ macrophages evoked an intracellular Ca ([Ca ]i) was independent of other oxidants and mitochondrially transient in endothelial cells that was ablated by a com- derived ROS. Activation of apoptosis occurred selec- .Ϫ bination of superoxide dismutase and an anion channel tively in response to O2 and could be prevented by 2ϩ 2ϩ .Ϫ blocker. [Ca ]i store depletion, but not extracellular [Ca ]i buffering. This study provides evidence that O2 2ϩ 2ϩ Ca chelation, prevented [Ca ]i elevation in response facilitates an InsP3R-linked apoptotic cascade and may Ϫ . to O2 that was inositol 1,4,5-trisphosphate (InsP3) de- serve a critical function in I/R injury and inflammation. Introduction Receptor-mediated generation of reactive oxygen species iron leads to hydroxyl radical formation via Fenton chemistry. .Ϫ (ROS) is necessary for signal transduction, gene expression, During I/R injury, O2 production in the vasculature is sub- and cell proliferation in smooth muscle cells, T and B lympho- stantially increased (Wei et al., 1999) and is accompanied by cytes, and fibroblasts (Devadas et al., 2002). Conversely, ROS endothelial cytotoxicity (for review see Li and Shah, 2004). produced under pathological conditions such as ischemia/re- However, the molecular mechanisms by which ROS lead to perfusion (I/R) or inflammation are associated with cellular organ damage are poorly understood. dysfunction and apoptosis (Davies, 1995). Endothelial cells In pathological conditions, cell death is facilitated by an THE JOURNAL OF CELL BIOLOGY 2ϩ respond to numerous external stimuli by producing the super- elevation in intracellular calcium ([Ca ]i; Hajnoczky et al., .Ϫ oxide anion (O2 ). In physiological conditions, mitochondrial 2003; Orrenius et al., 2003) via inositol 1,4,5-trisphosphate .Ϫ respiratory chain proteins produce O2 , which can be dismu- (InsP3). InsP3 is a second messenger produced by the hydrolysis tated into hydrogen peroxide (H2O2) or react with nitric oxide of phosphatidylinositol-4,5-bisphosphate by PLC. InsP3 receptor 2ϩ to produce peroxynitrite. In addition, reaction of H2O2 with (InsP3R)–mediated [Ca ]i changes are associated with a rapid, transient Ca2ϩ release from Ca2ϩ stores in the ER followed by Ca2ϩ entry through slow-activating plasma membrane store- Correspondence to Muniswamy Madesh: [email protected] operated channels (Putney and Bird, 1993; Parekh and Penner, 2ϩ Abbreviations used in this paper: 2-APB, 2-aminoethoxydiphenyl borate; BAPTA, 1997; Berridge et al., 1998). InsP3 [Ca ]i signals control a Ј Ј 2ϩ 1,2-bis(2-aminophenoxy)ethane-N,N,N ,N -tetracetate; [Ca ]i, intracellular ⌬⌿ wide range of cellular functions, including cell proliferation calcium; m, mitochondrial membrane potential; DCF, dichlorofluorescein; DPI, diphenyleneiodonium; ECM, extracellular medium; GPCR, G protein–coupled re- and apoptosis (Berridge et al., 2000; Orrenius et al., 2003). ceptor; InsP3, inositol 1,4,5-trisphosphate; InsP3R, InsP3 receptor; I/R, ischemia/ Apoptosis is reduced in cells lacking all three InsP R isoforms reperfusion; KO, knockout; LPS, lipopolysaccharide; MPTP, mitochondrial perme- 3 ability transition pore; PI, propidium iodide; PMVEC, pulmonary microvascular (DT40 avian B cells) and after selective suppression of InsP3R-3 endothelial cell; ROS, reactive oxygen species; SOD, superoxide dismutase; (Jayaraman and Marks, 1997; Sugawara et al., 1997), indicat- t-BuOOH, tert-butyl hydroperoxide; Tg, thapsigargin; TKO, triple KO; TMRE, tetramethylrhodamine, ethyl ester, perchlorate. ing the important role of InsP3 in cell death mechanisms (Pan et 2ϩ The online version of this article contains supplemental material. al., 2001). Alterations in [Ca ]i after oxidative stress facilitate © The Rockefeller University Press $8.00 The Journal of Cell Biology, Vol. 170, No. 7, September 26, 2005 1079–1090 http://www.jcb.org/cgi/doi/10.1083/jcb.200505022 JCB 1079 activation of the mitochondrial permeability transition pore (MPTP), which releases cytochrome c from the mitochondrial intermembrane space, leading to mitochondrial membrane po- tential (⌬⌿m) loss, assembly of the apoptosome, and activation of downstream caspases (Crompton, 1999). Recent evidence suggested that cytochrome c transiently released from mito- 2ϩ chondria interacts with InsP3R and amplifies Ca -mediated apoptosis (Boehning et al., 2003). Endothelial cells subjected to oxidative stress undergo apoptosis (Warren et al., 2000). Although there is evidence that 2ϩ 2ϩ perturbations of cellular Ca homeostasis (including [Ca ]i elevation, ER Ca2ϩ depletion, and mitochondrial Ca2ϩ in- creases) occur, the mechanisms by which oxidative stress me- diates endothelial apoptosis remain unclear. Events in the early 2ϩ stages of stress signaling include the mobilization of [Ca ]i (Patterson et al., 2004), the generation of ROS, and the forma- tion of lipid peroxides. However, it is unclear whether radical formation is a consequence of Ca2ϩ mobilization or a parallel event in early stress signaling. The proximity between mito- chondria and the ER facilitates a higher Ca2ϩ exposure in mito- chondria relative to the cytosol when released from the ER (Rizzuto et al., 1998). During pathological situations, excess ER-released Ca2ϩ may be detrimental to mitochondrial func- tion and may trigger mitochondrial fragmentation and apopto- sis. Previously, Bcl-2 family proteins have been implicated in apoptosis by affecting cellular Ca2ϩ homeostasis (Pinton et al., 2000; Pan et al., 2001; Li et al., 2002). A recent study reported 2؉ that a functional interaction of Bcl-2 with InsP3R attenuated Figure 1. Endothelial cell Ca mobilization in response to activated 2ϩ macrophage-derived ROS. (A) J774A.1 murine macrophages were acti- InsP3R activation, which in turn controlled InsP3-evoked Ca vated by 1 ␮g/ml LPS for 3 h in the presence or absence of the flavoprotein release (Chen et al., 2004), in contrast to our findings that Bcl-XL inhibitor DPI for 1 h. Cells were incubated with 10 ␮M of the ROS-sensitive activates InsP3R (White et al., 2005). In addition, ER-localized dye H2DCF-DA