AIRE-induced apoptosis is associated with nuclear translocation of stress sensor protein GAPDH Ingrid Liiv ⇑ , Uku Haljasorg, Kai Kisand, Julia Maslovskaja, Martti Laan, Pärt Peterson Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, Tartu, Estonia article info Article history: Received 10 May 2012 Available online 18 May 2012 Keywords: AIRE CARD domain Apoptosis GAPDH abstract AIRE (Autoimmune Regulator) has a central role in the transcriptional regulation of self-antigens in med- ullary thymic epithelial cells, which is necessary for negative selection of autoreactive T cells. Recent data have shown that AIRE can also induce apoptosis, which may be linked to cross-presentation of these self- antigens. Here we studied AIRE-induced apoptosis using AIRE over-expression in a thymic epithelial cell line as well as doxycycline-inducible HEK293 cells. We show that the HSR/CARD domain in AIRE together with a nuclear localization signal is sufficient to induce apoptosis. In the nuclei of AIRE-positive cells, we also found an increased accumulation of a glycolytic enzyme, glyceraldehyde-3-phosphate (GAPDH) reflecting cellular stress and apoptosis. Additionally, AIRE-induced apoptosis was inhibited with an anti-apoptotic agent deprenyl that blocks GAPDH nitrosylation and nuclear translocation. We propose that the AIRE-induced apoptosis pathway is associated with GAPDH nuclear translocation and induction of NO-induced cellular stress in AIRE-expressing cells. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction AIRE (Autoimmune Regulator) is expressed in medullary thymic epithelial cells [1], where it controls the ectopic expression of peripheral tissue-specific self-antigens [2]. The presentation of self-antigen proteins to developing T-cells is required for the neg- ative selection of developing thymocytes [3]. Mutations in the AIRE gene cause autoimmune polyendocrinopathy–candidiasis–ecto- dermal dystrophy or APECED (OMIM 240300), a monogenic disease where patients develop multiple autoimmune disorders [4]. At the subcellular level, AIRE is located in nuclear structures that resem- ble but are distinct from PML (promyelocytic leukemia) bodies [1,5]. AIRE interaction with many proteins involved in transcrip- tional regulation, pre-mRNA processing, nuclear transport, and chromatin binding have been found [6,7]. For example, these in- clude DNA-PK and topoisomerase 2, which are involved in repara- tion of DNA double strand breaks [6,8]. AIRE has been shown to act as a transcriptional regulator via binding of its PHD-type zinc finger to histone H3 [9,10], which is a mechanism currently thought to be central in AIRE-induced acti- vation of tissue-specific antigens. The AIRE N-terminal region also contains a homogenously staining region or caspase recruitment domain (HSR/CARD). As this domain is characteristic of several pro-apoptotic proteins, such as APAF-1, caspase-1, -2, -4 and -9 [11], a number of recent studies have focused on the role of AIRE in apoptosis induction. Accordingly, AIRE has been shown to cause apoptosis in mouse thymic epithelial cell line 1C6 [6,12,13] as well as in mouse primary medullary epithelium [13]. The apoptotic activity of AIRE was also reported in a recent study that analyzed its effect on the cellular proteome using quantitative proteomics [14]. In addition, the transcriptional regulator DAXX has been shown to repress the transcriptional activity of AIRE [15], while DAXX itself is involved in Fas-induced apoptosis [16] and colocal- izes with Sp100 protein in PML bodies [17]. AIRE shares a similar HSR/CARD domain with the Sp100 protein, which is involved in the DNA damage-induced apoptosis pathway [18]. Collectively, there is increasing evidence linking AIRE with thymic medullary epithelial cell apoptosis. As a result, it has been suggested that this mechanism may in fact promote cross-presentation of tissue-spe- cific antigens to the developing thymocytes [12]. Thus, the path- ways behind AIRE-induced apoptosis may be directly linked to the induction of central tolerance in thymus. Glyceraldehyde-3-phosphate (GAPDH), a common housekeep- ing gene, is known for its role in glycolysis. Recently, GAPDH has also been implicated in cellular stress and apoptosis [19–23]. Apoptotic stimuli, such as oxidative or genotoxic stress, results in GAPDH translocation and accumulation into nuclei [23,24]. For example, cell stressors activate nitric oxide synthase (NOS) which increases generation of nitric oxide (NO), leading to S-nitrosylation of GAPDH [19]. This event abolishes GAPDH glycolytic activity and elicits nuclear translocation of GAPDH [19]. The nuclear transloca- tion depends on interaction with E3-ubiquitin ligase Siah-1, which has a nuclear localization signal and specifically binds to GAPDH 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2012.05.057 ⇑ Corresponding author. E-mail address: ingrid.liiv@ut.ee (I. Liiv). Biochemical and Biophysical Research Communications 423 (2012) 32–37 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc