Nuclear Translocation of Procaspase-3 by TRB3 arrowhead indicated V5-WT-TRB3 positive and negative cells respectively. Scale bars = 10 mm. (B) Twenty-four hours after transfection with the C163S-proCASP3-EmGFP expression plasmid, HeLa cells were incubated with or without tunicamycin for 8 hr. Arrowheads showed representative phenotypes of C163S-proCASP3-EmGFP localization in each condition. Scale bars = 20 mm. (C) Twenty-four hours after transfection with the C163S- proCASP3-EmGFP expression plasmid, HeLa cells were treated with tunicamycin, and localization of C163S-proCASP3-EmGFP was simultaneously monitored for 24 hr by live imaging. Representative frames displaying scenes of nuclear translocation (arrows) are shown. Scale bars = 10 mm. (D) Localization of C163S-proCASP3-EmGFP was quantified as cytoplasmic, mainly cytoplasmic or cytoplasmic equal to nuclear (C, C.N, C = N), or as nuclear or mainly nuclear (N, N.C) under conditions described above (Figure 4A, V5-WT-TRB3; Figure 4B, ctrl or Tunicamycin). This localization was also assessed in cells coexpressing V5-WT-TRB3 (right bar graph). Over 30 cells were assessed in three independent experiments. Error bar: mean 6SD. *P,0.01, **P,0.005. (E) HeLa cells were cotransfected with the C163S-proCASP3-HA and indicated artificial miRNA expression plasmids. After 24 hr, the cells were incubated with tunicamycin for 8 hr. The localization of C163S-proCASP3-HA was observed by immunofluorescence staining with an anti-HA antibody (red). The miRNA expressing cells of inner panel were visualized by cocistronic expression of EmGFP. Arrowheads showed the C163S-proCASP3-HA expressing cells. Scale bars = 20 mm. (F) The cells positive for nuclear C163S-proCASP3-HA (N, N.C, N = C) were quantified by immunofluorescence staining with an anti-HA antibody. Over 30 cells positive for EmGFP were assessed in six independent experiments. Error bar: mean 6SD. *P,0.005, **P,0.001. miR-NC denotes negative control miRNA. doi:10.1371/journal.pone.0042721.g004 CASP3 is the first evidence supporting the above idea. However, we could not confirm the direct interaction between TRB3 and proCASP3 either by in vitro binding assay or by immunoprecip- itation using the overexpressing cells. Further studies are required for better understanding of the mechanism. Our results indicated that TRB3 behaved as both an anti- apoptotic and a pro-apoptotic factor by whether or not TRB3 is cleaved by caspases under stressful situations. We propose the following model for the stress response mechanism of TRB3 (Figure S12). During the adaptive phase of stress, expressed TRB3 prevents CASP3 activation by nuclear translocation of proCASP3, which in turn leads to an extended adaptive phase and cell survival (Figure 3 and 4). However, when the stress becomes over- whelming, such as under prolonged stress, TRB3 is cleaved by caspases. According to indirect evidence, cleavage of TRB3 could be a trigger for further activation of caspases and thereby induces apoptosis (Figure 2), even if the condition that a part of proCASP3 had been localized to the nucleus by TRB3 (Figure 4A). Therefore, it seems like the anti-apoptotic effect of TRB3 had been abrogated when caspase activity is beyond tolerance level by lethal apoptotic stimuli such as TNFa/CHX unlike tunicamycin. During ER stress, ‘‘because the UPR controls cell fate by switching between pro-survival and pro-apoptotic signaling, it is crucial to understand the parameters or events defining this transition, as well as gain insights on the components involved in its regulation’’ [12]. The exact mechanism controlling the transition between the adaptive and cell death programs is not well understood. In this study, we propose that TRB3 functions as a stress sensor that detects caspase activity and also as a switch molecule that assists cells to commit to either survival or apoptotic pathway, and these functions of TRB3 are exerted through its own cleavage by caspases depending on the cellular context. Thus, TRB3 may play an important role in this strategic commitment mechanism. Recently, it has been reported that CASP3 have cellular functions other than apoptosis, such as in cell development and differentiation [35]. In fact, during the development of iPS cells, CASP3/8 were activated and cleaved retinoblastoma (Rb), a known transcription coactivator [38]. The cleavage of Rb by CASP3/8 enhanced iPS formation. Among the initiator caspases, proCASP2 has been identified in the cell nucleus [39,40]. This raises a possibility that proCASP3 is actually activated in the nucleus via the mediation of TRB3, and that transcriptional regulators, such as Rb, could be targets of the nuclear form of active CASP3 under nonlethal condition (i.e. TRB3 non-cleavable conditions for nonapoptotic function). Alternatively, apoptosis accompanied by iPS formation is also suggestive of pro-apoptotic effect of TRB3. In another report, designed ER stress enhanced differentiation-associated apoptosis of myoblasts [41]. It is likely that ER stress-induced apoptosis selectively eliminates vulnerable cells. Interestingly, surviving myoblasts were resistant to apoptosis and they differentiated well. This result could be related to our finding that ER stress-induced TRB3 expression exerted anti- apoptotic effect by translocating proCASP3 into the nucleus. Because TRB3 expression is induced by various types of stresses, these results suggest that proCASP3 localizes in the nucleus under a variety of stress conditions. That proCASP3 was found to be localized in the nucleus, could now open avenues for finding new insights on biological roles of CASP3. Materials and Methods Plasmid Constructions Full-length human TRB3 cDNA and cDNA coding for amino acids 1–338 of human TRB3 (DC20-TRB3) were amplified by PCR. To construct the expression plasmids pcDNA3.1-V5-WT- TRB3 and pcDNA3.1-V5-DC20-TRB3, the above cDNAs were individually subcloned into the pcDNA3.1nV5-DEST vector (Invitrogen, Carlsbad, CA, USA) via the donor vector pDONR221 of the Gateway Cloning Technology kit (Invitrogen). An expression plasmid containing a cDNA of a caspase non- cleavable D338A mutant of TRB3 (pcDNA3.1-V5-D338A-TRB3) was generated from the pcDNA3.1-V5-WT-TRB3 plasmid by using the PrimeSTAR Mutagenesis Basal kit (TakaraBio, Otsu, Japan) and utilizing the mutagenic primers (59- GTCCCTGCGGGACTGGGGCTGGACGAA-39 and 59- CAGTCCCGCAGGGACCACCTGGGCAGC-39) as described in the manufacturer’s instructions. In the same way, cDNAs of human caspase-3 and C163S mutant of caspase-3 (kindly provided by Dr. K. Sakamaki, Kyoto Univ.) were subcloned into the pcDNA6.2/C-EmGFP-DEST vector (Invitrogen) to construct the expression plasmids pcDNA6.2-proCASP3-EmGFP and pcDNA6.2-C163S-proCASP3-EmGFP, respectively. Three copies of the SV40 T-antigen nuclear localization signal (NLS) and HA tag sequences were fused by PCR to construct pcDNA6.2- proCASP3-NLS-EmGFP and pcDNA6.2-C163S-proCASP3-HA plasmids, respectively. The DsRed-NLS expression plasmid (pDsRed2-Nuc) was purchased from Clontech (Palo Alto, CA, USA). In vitro Cleavage Assay Construction of DNA templates and cell-free protein synthesis for N-terminal biotinylated-TRB3 were performed as described previously [21]. For the cleavage reaction, 3 ml of translation mixture was added to 7 ml of reaction mixture (20 mM HEPES, pH 7.8, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 10% sucrose), with 0.3 (CASP3, CASP7, CASP8), 10 (CASP9) or 20 (CASP2, CASP6, CASP10) units of each active caspase (1 unit is defined as the amount that will hydrolyze 1 nmole of the caspase substrate per minute or hour) (Sigma-Aldrich, St. Louis, MO), and PLoS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e42721