Nuclear Translocation of Procaspase-3 by TRB3 by densitometric image analysis. (B) CASP3/7 activity in HeLa cells was measured as described in the legend of Figure 2C, except that here apoptosis was induced by incubation with 5 mM tunicamycin for the indicated times. Each data point represents mean 6SD of three independent experiments. (C) Twenty-four hours after transfection with the indicated V5-TRB3 expression plasmid or control vector, HeLa cells were treated with tunicamycin for 48 hr. The resulting dead cells were counted by trypan blue staining. Error bars indicate mean 6SD of four independent experiments. *P,0.001. (D) Twenty-four hours after transfection with the given V5-TRB3 expression plasmid, HeLa cells were treated with TNFa/CHX or tunicamycin for the indicated times. The cell lysates were subjected to immunoblot analysis using the anti-V5 antibody. a-Tubulin was used as an internal control. (E) HeLa cells plated in 96-well plates (1.06104 cells/well) were treated with TNFa/CHX or tunicamycin for the indicated times. CASP3/7 activity was measured using the luminometric Caspase-GloH 3/7 Assay Kit. Each data point represents mean 6SD of three independent experiments. (F-H) HeLa cells were transfected with negative control siRNA (siNC) or TRB3 siRNA (siTRB3). Twenty-four hours after, the cells were treated with tunicamycin for the indicated time points (F). The cell lysates were subjected to immunoblot analysis using the anti-TRB3 antibody (G). Cell lysates were also used for the Caspase-GloH 3/7 assay to measure the CASP3/7 activity (H). The relative CASP3/7 activity was then determined after normalizing each value with respect to the relative amount of expressed a-Tubulin as estimated by densitometric image analysis. Each data point represents mean 6SD of three independent experiments. doi:10.1371/journal.pone.0042721.g003 nuclear DsRed-NLS (Figure 5D and see Movie S1). These results strongly suggest that nuclear distribution of proCASP3 is not critical for the execution of apoptosis. Taken together with the previous section, our results suggest that TRB3 expression prevents CASP3 activation by nuclear translocation of proCASP3, and thereby inhibits apoptosis. Discussion TRB3 expression is up-regulated in a variety of cell types under various stress conditions including ER stress, nutrient deprivation, hypoxia and oxidative stress [1,6,17,26]. However, the exact role of TRB3 expression in stress response is controversial because it has been reported that the stress-induced expression of TRB3 exhibited both pro-apoptotic [6,16,27,28] and anti-apoptotic [17,26] effects. Several studies have led to the suggestion that the opposite behavior of TRB3 was due to its putative role as a stress sensor [6,13,29]. The exact molecular mechanism, however, remains unclear. Recently, we found that TRB3 was cleaved by CASP3, and this observation could easily be correlated to the previous reports by suggesting that TRB3 is involved in the regulation of apoptosis. In this study, we further found that the C-terminal region of TRB3 was a target for most caspases (Figure 1C). In fact, the expressed TRB3 was cleaved within a few hours of inducing apoptosis using TNFa/CHX or anti-Fas antibody (Figure 1D and 3D). In this case, CASP3/7 activation and subsequent apoptosis were pro- moted in WT-TRB3 expressing cells, but the pro-apoptotic effect of TRB3 was not observed in cells expressing the non-cleavable D338A-TRB3 (Figure 2). Additionally, we have found that DC20- TRB3 (see Figure 1A), which could be produced by multiple caspases during the apoptotic process (Figure 1C and D), did not have any pro-apoptotic effect (Figure S9), suggesting that the pro- apoptotic effect is not mediated by the cleaved TRB3 (DC20) but likely requires the C-terminal region of TRB3 and/or the cleavage event to function. Indeed, a peptide, around 10 residues long, such as amyloid b-peptide, has been shown to induce apoptosis [30,31]. In contrast to the above case, TRB3 cleavage was hardly observed even if the cells were exposed to ER stress by tunicamycin for as long as 24 hours (Figure 3D). CASP2 has been shown to be involved in ER stress with treatment of high tunicamycin condition (.20 mg/mL = 24 mM) [32,33] and has TRB3 cleavage ability (Figure 1C). However CASP2 activation may be not found in low tunicamycin condition (,5 mg/mL = 6 mM) [32]. Actually, in our condition (5 mM) it was not detected (data not shown), and TRB3 cleavage was also hardly observed (Figure S10A), like Figure 3D. In addition, treatment of CASP2 inhibitor (z-VDVAD- FMK) rescued only small number of the dead cells (Figure S10B). Therefore, at least in our condition, CASP2 seems to have only limited impact on apoptosis by tunicamycin. Interestingly, in these cases, CASP3/7 activation and subsequent apoptosis were slightly inhibited in WT-TRB3 expressing cells (Figure 3B, C and S10B), and the results shown in Figure 4 and 5 suggested that the anti- apoptotic effect was due to the TRB3-mediated nuclear trans- location of proCASP3. The non-cleavable D338A-TRB3 was also localized in the nucleus (Figure S6A), and its translocation efficiency of proCASP3 was almost same as that by WT-TRB3 (Figure S6B). But, in the case of chronic stress, the anti-apoptotic effect of WT-TRB3 was lower than that of D338A-TRB3 (Figure 3B and C), likely as a result of the combined pro-apoptotic effect of the cleavable TRB3 (i.e. WT-TRB3). To investigate how TRB3 affects apoptosis induced by intrinsic stimulus independent of ER stress, we further used staurosporine (STS), a wide spectrum inhibitor of protein kinase. As well as the case of ER stress condition, TRB3 exerted the anti-apoptotic effect on STS-treated cells at least in our condition (Figure S11A), and in which TRB3 cleavage was also hardly observed (Figure S11B and C), even though STS-induced apoptosis is essentially mediated by CASP9 [34] that has TRB3 cleavage ability (Figure 1C). These results support our hypothesis that the anti-apoptotic effect of TRB3 is exerted during adaptive phase of stress as long as TRB3 is not cleaved. Taken together, our findings indicate that TRB3 has dual function – both in cell survival and also in apoptosis. This dual function of TRB3 might have contributed to the previously reported controversial role of TRB3 in apoptosis. Initiator caspases, especially CASP8, CASP9, and CASP10, are known to play important roles in the beginning of apoptotic signal transduction [35]. Activated initiator caspases could then lead to the activation of downstream, effector caspases (i.e. CASP3, CASP6, and CASP7). Most caspases, including CASP8, CASP9, and CASP10 are predominantly located in the cytoplasm, and not in the nucleus [36,37]. Using fluorescence resonance energy transfer (FRET) technique it was shown that during the apoptotic process CASP3 activation is first initiated in the cytoplasm and then in the nucleus [25]. These results suggest that the cytoplasmic initiator caspases cannot easily activate the nuclear proCASP3. In fact, our results showed that expression of the nuclear proCASP3 (proCASP3-NLS-EmGFP) did not enhance cell death under apoptotic conditions compared with control (DsRed-NLS), although expression of the cytoplasmic proCASP3 (proCASP3- EmGFP) enhanced it markedly (Figure 5). We found that TRB3 expression can induce cytoplasmic-to-nuclear translocation of proCASP3 under ER stress condition (Figure 4). TRB3 is a stress- inducible protein, and stress response is a mechanism by which cells adapt to stressful situations. Thus, TRB3 might insulate proCASP3 by promoting its entry into the nucleus to prevent CASP3 activation in the cytoplasm under TRB3-inducing stress conditions. Taken together, these results suggest that the overall CASP3 activity is regulated not only by proteolytic activation of proCASP3 but also by its subcellular distribution. To the best of our knowledge, TRB3-mediated nuclear translocation of pro- PLoS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e42721