HS4C3-Binding Epitope Contributes to Fas Signaling with primary antibody. In the right panel, the values shown are the mean fluorescence intensity 6 SD after normalization against mESCs cultured in the presence of LIF (arbitrary value = 1). *, P,0.01; **, P,0.05. Three independent experiments were performed in each case. doi:10.1371/journal.pone.0043440.g003 indicate that overexpression of the HS4C3-binding epitope induced both differentiation and apoptosis of mESCs in a similar manner to LIF withdrawal. Caspase-3 is activated during mESC differentiation (Figure 2D, [37]). Therefore, we investigated whether caspase-3 is activated in cells overexpressing 3OST-5 and found that its level of activation was indeed higher in these cells than in control cells (Figure 2D). Given that activated caspase-3 degrades Nanog, which results in mESC differentiation [38], we compared the amounts of Nanog in cells overexpressing 3OST-5 and control cells. The amount of Nanog protein was decreased in cells overexpressing 3OST-5 (Figure 2E), whereas the level of Nanog mRNA was not decreased (Figure 2F). These results suggest that induction of differentiation in cells overexpressing 3OST-5 depends on the activation of caspase-3 followed by the degradation of Nanog. The Expression of Fas Increased on the Surface of Cells Overexpressing 3OST-5 To clarify the mechanism by which caspase is activated during mESC differentiation, we investigated the contribution of upstream components of the caspase cascade. Fas (CD95 or Apo-1) is a type I membrane protein and a member of the tumor necrosis factor receptor family. Fas signaling, which is activated by interaction between Fas and the Fas ligand (FasL) or simply by the redistribution of Fas into lipid rafts, comprises part of the caspase activation cascade [39,40]. First, we determined the distribution of Fas on cells overexpressing 3OST-5. Fas was present at a very low level on the surface of control cells but this level was clearly higher on cells overexpressing 3OST-5 (Figure 3A left histogram and 3B). However, the total level of Fas did not change between the two cell types (Figure 3A right histogram). The increase in the level of Fas on the surface of cells overexpressing 3OST-5 was inhibited by treating the cells with brefeldin A, which inhibits the transport of proteins from the ER to the Golgi and leads to the accumulation of proteins inside the ER (Figure 3B). Furthermore, the level of Fas on the surface of mESCs differentiated by culture in the absence of LIF for 8 days was increased (Figure 3C). Overexpression of the HS4C3-Binding Epitope Recruits Fas into Lipid Rafts, which Activates Fas Signaling Next, we analyzed biochemically whether Fas was localized in lipid rafts in differentiated cells. The separation of lipid rafts and non-rafts was confirmed by western blotting using Flotillin-1 as a marker of the raft fraction (fractions 3–5) and the transferrin receptor as a marker of the non-raft fraction (fractions 7–9) (Figure 4A). Fas was located in the raft fraction in cells overexpressing 3OST-5 in the presence of LIF (+LIF) and in mESCs differentiated by culture in the absence of LIF for 6 days (2LIF) (Figure 4A), which indicated that the activation of Fas signaling in these cells was mediated by the redistribution of Fas into lipid rafts. In addition, the HS4C3-binding epitope was localized in lipid rafts in cells overexpressing 3OST-5 (Figure S2). Caspase-8 is a downstream component of the Fas signaling pathway. We also found that the level of activated caspase-8 was increased, and the level of unactivated caspase-8 decreased, in cells overexpressing 3OST-5 and in mESCs after the induction of differentiation by LIF withdrawal for 8 days (Figure 4B). These data indicated that Fas signaling had been activated. To determine whether Fas signaling induced the differentiation of mESCs, we performed a self-renewal assay with cells overexpressing Fas. FACS analysis showed that the amount of Fas on the cell surface was increased in cells overexpressing Fas (Figure 4C). The number of AP-positive colonies that were derived from cells overexpressing Fas was reduced significantly to the same number as those derived from cells overexpressing 3OST-5, even in the presence of LIF and serum in clonal density culture (Figure 4D). This indicated that up-regulation of Fas reduced the self-renewal of mESCs. We examined the putative interaction of Fas and the HS4C3- binding epitope by using an overlay assay. Given that we expected the heparin-binding domain (KLRRRVH) in the Fas ectodomain to bind in the Golgi to sulfated regions of HS chains, including the HS4C3-binding epitope, we used several types of the recombinant Fas ectodomain for the assay (Figure 4E). As shown in Figure 4F (F-2), overlaid Fas (indicated by ** in the figure) increased in parallel with the HS4C3-binding epitope (indicated by * in Figure 4F (F-1)) in cells overexpressing 3OST-5, which indicated that the Fas ectodomain bound specifically to the HS4C3-binding epitope. Addition of HS4C3 blocked Fas binding completely, which confirmed the interaction was specific (Figure 4F (F-3)). Furthermore, point mutations in the heparin-binding domain (ALAAAVA) of Fas abolished the binding (Figure 4F (F-4)). In addition, we prepared two separate fragments of the Fas ectodomain, amino acids 19–38 and 39–168, (Figure 4E) and performed the overlay assay with these fragments. Binding of the fragment that comprised amino acids 19-38, which included the heparin-binding domain, was increased in cells overexpressing 3OST-5 (Figure 4G (G-2)). In addition, the fragment that comprised amino acids 39-168 did not bind to samples from control cells and cells overexpressing 3OST-5 (Figure 4G (G-4)). Addition of HS4C3 also blocked fragments binding (Figure 4G (G- 3, G-5)). Therefore, we concluded that amino acids 19–38 of Fas, which include the heparin-binding domain, are necessary for interaction with the HS4C3-binding epitope. These findings demonstrated for the first time that Fas binds to HS chains, including the HS4C3-binding epitope, through the region that contains the heparin-binding domain (KLRRRVH). Another important caspase cascade is mediated by changes in mitochondrial permeability, which are regulated by proapoptotic Bcl-2 proteins such as Bad. Bad is phosphorylated as a result of the activation of signal transduction by several survival factors, including growth factors. We examined the phosphorylation of Akt, which is upstream of Bad, after serum stimulation and observed a similar increase in the level of phosphorylated Akt in control cells and cells overexpressing 3OST-5 (Figure S3A). Interestingly, the expression of Bad was markedly decreased in cells overexpressing 3OST-5, which indicated a reduction in the Bad-mediated proapoptotic state (Figure S3B). These results demonstrate that activation of caspase-3 in cells overexpressing 3OST-5 was not due to a defect in the mitochondrial pathway. Taken together, our findings provide the first demonstration that redistribution of Fas from intracellular pools to lipid rafts on the cell surface depends on an interaction between Fas and the HS4C3-binding epitope, and is followed by the activation of Fas signaling and caspase-3. PLOS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e43440