HS4C3-Binding Epitope Contributes to Fas Signaling Figure 7. Subcellular localization of HS4C3-binding epitope and Fas, and scheme for Fas-mediated differentiation via HS4C3- binding epitope. (A) Immunostaining, using HS4C3 and anti-Fas antibodies, of permeabilized mESCs in the presence of LIF (+LIF) or mESCs cultured for 5 or 7 days in the absence of LIF (2LIF day 5 or day 7). Scale bar, 5 mm. Representative confocal images are shown. (B) Scheme for the induction of differentiation by the activation of Fas signaling via the HS4C3-binding epitope. (1) The HS4C3-binding epitope is synthesized on HSPGs in the Golgi. (2) The HS4C3-binding epitope on HSPGs interacts with Fas in the Golgi. (3) Fas then translocates into lipid rafts on the cell surface through interaction between the HS4C3-binding epitope and Fas. Fas signaling is activated by this redistribution of Fas from the Golgi into the lipid rafts. (4) Fas signaling activates caspase-3, and then activated caspase-3 degrades Nanog. (5) mESCs differentiate due to the degradation of Nanog. HSPGs, heparan sulfate proteoglycans. doi:10.1371/journal.pone.0043440.g007 Gabriella et al. have reported that FasL is not expressed in undifferentiated mESCs [43]. Our preliminary studies showed that expression of FasL was not increased in mESCs during differen- tiation induced by LIF withdrawal or in cells overexpressing 3OST-5 (Figure S5). In tumor cells, Fas signaling is amplified by the redistribution of Fas into lipid rafts alone, even if FasL does not bind to Fas [39]. Therefore, the data suggest that, in cells overexpressing 3OST-5, Fas signaling can be activated indepen- dently of Fas/FasL interaction simply by redistributing Fas into lipid rafts. The in vitro activities of mouse 3OSTs have not been reported yet. However, it has been reported that the human 3OST-2, 3, 4, and 5 isoforms synthesize the HS4C3-binding epitope [25]. Furthermore, among human 3OST-2, 3, 4, and 5, 3OST-2 and -5 correspond clearly to mouse 3OST-2 and 3OST-5, respectively. We performed FACS analysis using the HS4C3 antibody and confirmed that the HS4C3-binding epitope increased in cells overexpressing 3OST-2 or -5 (Figure S6A). In 3OST-2-transfected cells, we obtained the same results as in 3OST-5-transfected cells (Figure S6A–C). These data indicated that 3OST-2 also activated Fas signaling and induced the differentiation. As shown in Figure 1G, cells overexpressing 3OST-5 had the potential to differentiate not only into primitive endoderm but also into primitive ectoderm. On the other hand, stable 3OST-5 KD cells lost the potential to differentiate into primitive endoderm and primitive ectoderm (Figure 6A–E). Furthermore, treatment with IETD, an inhibitor of caspase-8, interfered with normal differen- tiation into primitive endoderm and primitive ectoderm (Figure 5F and 6G). Therefore, the activation of Fas signaling via the HS4C3- binding epitope, which was indicated in this study, is involved in the differentiation of mESCs into both primitive endoderm by LIF withdrawal and primitive ectoderm by EB formation. We showed clearly that Fas signaling functioned during these differentiation processes. Interestingly, although Fgf4/Erk signaling was func- tioning, differentiation into primitive endoderm, primitive ecto- derm, and subsequently all three germ layers was inhibited in the stable 3OST-5 KD cells. Therefore, a signal other than Fgf4/Erk signaling must function to induce differentiation. Hence, in addition to Fgf4/Erk signaling, we propose that the activation of Fas signaling via the HS4C3-binding epitope plays an important role in the early stages of all types of differentiation in mESCs. Supporting Information Figure S1 The expression of HS in cells overexpressing 3OST-5. FACS analysis using the anti-HS antibody 10E4 (black line, control cells; red line, cells overexpressing 3OST-5). The gray line shows the result obtained for cells not treated with primary antibody. (TIF) Figure S2 HS4C3-binding epitope localized in lipid rafts in cells overexpressing 3OST-5. Western blot analysis of raft and non-raft fractions using anti-Flotillin-1 (raft), anti-transferrin receptor (non-raft), and HS4C3 antibodies. Representative results are shown. (TIF) Figure S3 Mitochondrial pathways mediated by surviv- al factors are not affected in cells overexpressing 3OST- 5. (A) and (B) Western blot analysis, using antibodies against p- Akt, Akt, and Bad, of cells stimulated with FBS. The histograms show mean densitometric readings 6 SD for the ratio p-Akt/Akt or Bad/b-actin. Three independent experiments were performed, and representative results are shown. (TIF) Figure S4 Pluripotency was maintained in stable 3OST- 5 knockdown cells. Real time PCR analysis of markers of the undifferentiated and differentiated states in stable 3OST-5 knockdown cells. The values shown are means 6 SD after normalization against control cells (arbitrary value = 1). Three independent experiments were performed. *, P,0.01. (TIF) Figure S5 The expression of Fas ligand was not increased in cells overexpressing 3OST-5. RT-PCR analysis of the expression of Fas ligand in cells overexpressing 3OST-5. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (TIF) Figure S6 The overexpression of 3OST-2 also activated Fas signaling and induced the differentiation of mESCs. (A) FACS analysis, using the HS4C3 antibody, of mESCs at 2 days after transfection with the 3OST-2 expression construct (black line, control cells; green line, cells overexpressing 3OST-2). The gray line shows the result obtained from cells not treated with primary antibody. (B) Real time PCR analysis of markers of the undifferentiated state in cells overexpressing 3OST-2. The values were normalized against control cells (arbitrary value = 1). (C) western blot analysis using an antibody against uncleaved caspase- 8, cleaved caspase-3, or Nanog. The histograms show mean densitometric readings 6 SD after normalization against control cells (arbitrary value = 1). **, P,0.05. Three independent experiments were performed. (TIF) List of gene specific primers for real time List of gene specific probes for real time PCR. Table S1 PCR. (TIF) Table S2 (TIF) Acknowledgments We thank Dr. Seiji Hitoshi and Prof. Kumiko Ui-Tei for the kind gift of experimental material. Author Contributions Conceived and designed the experiments: SN. Performed the experiments: KH NS TI TM. Analyzed the data: KH NS. Contributed reagents/ materials/analysis tools: THVK. Wrote the paper: KH NS SN. PLOS ONE | www.plosone.org 14 August 2012 | Volume 7 | Issue 8 | e43440