BCL-2 protects peroxynitrite-treated thymocytes from poly(ADP-ribose) synthase (PARS)-independent apoptotic but not from PARS-mediated necrotic cell death

Abstract

In thymocytes, peroxynitrite induces poly(ADP-ribose) synthetase (PARS) activation, which results in necrotic cell death. In the absence of PARS, however, peroxynitrite-treated thymocytes die by apoptosis. Because Bcl-2 has been reported to inhibit not only apoptotic but also some forms of necrotic cell death, here we have investigated how Bcl-2 regulates the peroxynitrite-induced apoptotic and necrotic cell death. We have found that Bcl-2 did not provide protection against peroxynitrite-induced necrotic death, as characterized by propidium iodide uptake, mitochondrial membrane potential decrease, secondary superoxide production, and cardiolipin loss. In the presence of a PARS inhibitor, peroxynitrite-treated thymocytes from Bcl-2 transgenic mice showed no caspase activation or DNA fragmentation and displayed smaller mitochondrial membrane potential decrease. These data show that Bcl-2 protects thymocytes from peroxynitrite-induced apoptosis at a step proximal to mitochondrial alterations but fails to prevent PARS-mediated necrotic cell death. Activation of tissue transglutaminase (tTG) occurs in various forms of apoptosis. Peroxynitrite did not induce transglutaminase activity in thymocytes and did not have a direct inhibitory effect on the purified tTG. Basal tTG was not different in Bcl-2 transgenic and wild type cells.

Introduction

Peroxynitrite, a potent oxidant formed in the reaction of nitric oxide and superoxide [1] is a mediator of various forms of inflammation, shock, and reperfusion injury [2], [3]. Our previous work has demonstrated that in thymocytes, low concentrations (10–15 μM) of peroxynitrite cause apoptosis, whereas higher doses (20–40 μM) induce necrosis [4]. In the thymocyte model, the necrotic cell death induced by peroxynitrite, hydrogen peroxide, or superoxide is mediated by poly(ADP-ribose) synthase (PARS) (also called poly(ADP-ribose) polymerase, PARP) activation [4], [5]. PARS is a nuclear nick sensor enzyme, which becomes activated by DNA single strand breaks [6]. Upon activation, PARS cleaves NAD to nicotinamide and ADP-ribose and catalyses the addition of (ADP-ribose)_n adducts to proteins, including PARS itself. Excessive PARS activation depletes cellular NAD and ATP pools [7], [8] and causes necrotic cell death characterized by the breakdown of plasma membrane integrity and deterioration of mitochondrial function and structure in the absence of DNA fragmentation [4]. Inhibition of the enzyme preserves the cellular energy stores and allows the oxidatively injured cells to undergo the energy-demanding apoptotic process [4]. Inhibition of the cytotoxic PARS activation pathway proved useful in various disease models [9], [10].

The regulatory mechanisms of peroxynitrite-induced cytotoxicity, however, remain undefined. Possible candidates for such a regulatory role may be members of the Bcl-2 family, which recently emerged as important regulators of various forms of both apoptotic and necrotic death [11], [12], [13]. The Bcl-2 family consists of pro-apoptotic (bax, bak, bad, bik) and antiapoptotic (Bcl-2, Bcl-x_L, mcl-1) molecules each of which are capable of forming homo- or heterodimers with some members of the antagonist group [14]. The ratio of pro- and anti-apoptotic Bcl proteins is thought to determine the fate of stimulated cells, death vs. survival. The Bcl proteins are predominantly located in the mitochondria but can also be found in the nuclear membrane and in the endoplasmic reticulum [14]. In the mitochondria, Bcl-2 blocks the opening of the mitochondrial permeability pore [15], [16] thereby preventing the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm and caspase activation [17], [18], [19], [20].

The unique feature of the anti-apoptotic Bcl proteins is their ability to protect not only from apoptotic but also from necrotic death [21], [22], [23], [24]. We have previously characterized the cytotoxic effect of peroxynitrite on mouse thymocytes and found that peroxynitrite-induced PARS activation results in necrotic cell death [4]. However, in the absence of PARS, peroxynitrite-treated cells die by apoptosis characterized by phosphatidylserine exposure, caspase activation, and DNA fragmentation [4]. Thus, this model allows us to examine how the peroxynitrite-induced PARS-mediated necrotic and PARS-independent apoptotic death pathways are regulated by Bcl-2.

To further characterize the peroxynitrite-induced apoptotic process, we have also investigated the possible involvement of the tissue transglutaminase, a common mediator of various apoptosis processes, in the peroxynitrite-induced apoptosis.