Abstract
Death-associated protein 3 (DAP3) is crucial for promoting apoptosis induced by various stimulations. This report demonstrates that DAP3 is also important for T cell receptor (TCR)-mediated apoptosis induction in immature thymocytes. Enforced expression of DAP3 accelerated the negative selection in developing thymocytes, using the reaggregate thymus organ culture system. In addition, expression of DAP3 accelerated TCR-mediated apoptosis induction in DO11.10 cells. We also demonstrated that DAP3 translocates into the nucleus during TCR-mediated apoptosis in a Nur77 dependent manner. It is concluded that DAP3 is critical for TCR-mediated induction of apoptosis at the downstream of Nur77.
Introduction
Negative selection is a quite important event for acquisition of self-tolerance by elimination of self-reactive thymocytes [1], [2]. During T cell development, immature CD4+CD8+ thymocytes undergo negative selection events based on the specificities of the αβT cell receptor (TCR) complexes.
Death-associated protein 3 (DAP3) is ubiquitously expressed in various tissue including the immune system, such as in the thymus of human and mice [3], [4]. DAP3 is a GTP-binding protein that has been identified as a positive mediator in interferon (IFN)-γ-induced cell death [5]. The gene of DAP3 encodes for a 46 kDa protein with a potential P-loop motif, a potential nuclear receptor-interacting domain (NR box), and a putative cleavage site for the N-terminal mitochondrial targeting sequence [5], [6], [7]. It has been reported that mitochondrial DAP3 regulates cellular senescence though an oxidative stress response [8]. Moreover, DAP3 is reportedly phosphorylated in an Akt-dependent manner, correlating with the suppression of DAP3-facilitated apoptosis in anoikis [9], and IFN-β promoter stimulator 1 (IPS-1) binds DAP3, resulting in induced anoikis by caspase activation [10]. Although the accumulating evidence shows that DAP3 plays roles in apoptosis induction, the role of DAP3 in thymocyte development is still unknown.
An orphan nuclear receptor, Nur77, belonging to the steroid/thyroid hormone receptor superfamily, is a transcription factor responsible for inducing apoptosis [11]. Nur77 is activated by various kinds of stimulation for apoptosis induction, and TCR stimulation is known to be a potent activator of Nur77 transcription [12], [13]. A previous report indicated that thymocytes from transgenic mice that express a dominant-negative form of Nur77 (DN-Nur77) are protected from negative selection, and conversely transgenic mice that express wild-type Nur77 exhibit promoted negative selection [14]. Therefore, Nur77 is assumed to be an important factor in the development of thymocytes.
This study demonstrates the physiological importance of DAP3 on negative selection of immature thymocytes. The data indicate that DAP3 is a critical factor for induction of apoptosis induced by TCR stimulation at the downstream of Nur77.
Section snippets
Antibodies
The specific antibodies used in this study, anti-DAP3 (Clone 10; BD Biosciences, San Jose, CA), anti-Crk (clone 22; BD Biosciences), anti-Hsp60 (Clone LK-1; StressGen, Victoria BC, Canada), anti-nucleoporin p62 (clone 53; BD Biosciences), anti-Nur77 (clone 12.14; BD Biosciences), anti-mouse CD3 antibody (clone 145-2C11; BD Biosciences) and anti-mouse CD28 antibody (BD Biosciences) were purchased from commercially available products.
Cell culture
The T cell hybridoma, DO11.10 cells were kindly provided by Dr.
DAP3. is critical for negative selection
To understand DAP3 functioning on T cell development in the thymus, the DAP3 expression in each population of thymocytes was initially analyzed by RT-PCR analysis (Fig. 1A). Here, DAP3 was shown to be expressed in CD4+CD8+ double positive cells. CD4+ single positive cells, and in CD8+ single positive cells, but not in CD4−CD8− double negative cells. CD4+CD8+ double positive cells are known to be screened against autoreactivity during development in the thymus. The results suggest that DAP3 is
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