Figure 6. Decrease in the HS4C3-binding epitope inhibits differentiation and apoptosis. (A) and (J) Real time PCR analysis of 3OST-5 in stable (A) and transient (J) 3OST-5 KD cells. The values shown are means 6 SD after normalization against control cells (arbitrary value = 1). (B) FACS analysis using the anti-HS antibody HS4C3 (black line, control ESCs; blue line, stable 3OST-5-1 KD ESCs; red line, control EBs; green line, stable 3OST-5-1 KD EBs). The control cells were transfected with the construct that targeted EGFP. The gray line shows the result obtained for cells not treated with primary antibody. (C) Self- renewal assay with stable 3OST-5 KD cells. Left panels show photographs of representative colonies. Scale bars, 200 mm. The right panel shows the proportion of AP-positive colonies. The values shown are the mean 6 SD. (D) and (E) Real time PCR analysis of marker genes in stable 3OST-5 KD cells at 6 days after LIF withdrawal (D) and at 3 (Fgf5, Goosecoid, and Sox17), 4 (Nanog and Oct3/4), or 12 (Pax6) days after EB formation (E). The values shown are means 6 SD after normalization against control cells (arbitrary value = 1). (F) Western blot HS4C3-Binding Epitope Contributes to Fas Signaling analysis, using anti-pErk1/2 and Erk1/2 antibodies, of cells stimulated with Fgf4. The histograms show mean densitometric readings 6 SD expressed as the ratio p-Erk1/2/Erk1/2. Representative results are shown. (G) Real time PCR analysis of marker genes, Fgf5 and Goosecoid, in cells treated with IETD at 3 days after EB formation. The values shown are means 6 SD after normalization against nontreated cells (arbitrary value = 1). (H) FACS analysis using the anti-HS antibody HS4C3 in cells overexpressing 3OST-5 at 2 days after EB formation. The values shown are mean fluorescence intensity 6 SD. (I) Real time PCR analysis of Fgf5 and Goosecoid in cells overexpressing 3OST-5 at 0–4 days after EB formation (black line, control cells; red line, cells overexpressing 3OST-5). The values shown are means 6 SD from duplicate measurements from one representative experiment. (K) FACS analysis using the anti-HS antibody HS4C3 (black line, control cells; red line, transient 3OST-5-2 KD cells). The gray line shows the result obtained for cells not treated with primary antibody. (L) Measurement of apoptosis in transient 3OST-5 KD cells using an annexin V-FITC kit at 2 days after transfection. The values shown are the means 6 SD after normalization against control cells in the absence of LIF (arbitrary value = 1). KD, knockdown; RNAi, RNA interference; EB, embryoid body. *, P,0.01; **, P,0.05. Three independent experiments were performed in each case. doi:10.1371/journal.pone.0043440.g006 Discussion Herein we report for the first time that activation of Fas signaling via the HS4C3-binding epitope induced the differenti- ation into primitive endoderm and primitive ectoderm from mESCs. From our results, we propose the following scheme (Figure 7B). The level of the HS4C3-binding epitope in mESCs increased during differentiation (Figure 1A, 1B, and Figure 6B). The HS4C3-binding epitope interacted with the heparin-binding domain (KLRRRVH) of Fas in the Golgi, which resulted in the redistribution of Fas to lipid rafts and the activation of Fas signaling. Activation of Fas caused degradation of Nanog via activated caspase-3, which induced differentiation (Figure 2D and 2E). The proposed scheme was supported by rescue experiments in which Fas signaling was blocked (Figure 5) and by confocal slices (Figure 7A). Therefore, we propose that the activation of Fas signaling mediated by the HS4C3-binding epitope is a novel mechanism for the regulation of differentiation in mESCs. It has been reported that Fas is transported from intracellular pools to the cell surface in response to various stimuli [32,41]. However, the identities of the molecules that contribute to this transport of Fas have yet to be determined. Two possible transport pathways have been implicated. 1) The intracellular domain of secreted proteins might interact with Fas in the cytoskeletal network and recruit Fas from the cytoskeletal network to the cell surface. Ivanov et al. reported that Fas-associated phosphatase-1 (FAP-1) contributes to the localization of Fas by binding via the intracellular domain of Fas within the cytoskeletal network [41]. 2) The extracellular domain of secreted proteins might interact with Fas in the Golgi lumen and recruit Fas from the Golgi to the cell surface. Our proposed scheme corresponds to this second possible pathway. As shown in Figure 3B, the effect of brefeldin A on the translocation of Fas was probably due to the inhibition of secretion of HS proteoglycans (HSPGs) that contained the HS4C3-binding epitope. Recently, it has been demonstrated that Fas is redistrib- uted from intracellular pools to lipid rafts on the cell surface in Swiss 3T3 cells overexpressing syndecan-2 [42]. However, the mechanism of this redistribution has not yet been clarified. We obtained data that demonstrated that syndecan-2 was localized to lipid rafts in mESCs (data not shown). Considering our results and the above reported findings, we propose that HS chains on syndecan-2 interact with Fas in the Golgi lumen and mediate the transport of Fas from the Golgi to the cell surface, that is, via pathway 2) described above. PLOS ONE | www.plosone.org 12 August 2012 | Volume 7 | Issue 8 | e43440