in an autocrine fashion in mESCs, and Fgf4/extracellular signal- regulated kinase (Erk) signaling contributes to the differentiation of mESCs into neural and mesodermal lineages [18]. Recently, we demonstrated that HS chains, including sulfated regions, contribute to the self-renewal and pluripotency of mESCs through Wnt/b-catenin and BMP/Smad signaling in culture media containing serum and LIF [16,19]. In contrast, other groups have reported that HS chains contribute to the initiation of differentiation via Fgf4 signaling following LIF withdrawal [20,21]. Therefore, HS chains contribute to both the maintenance of the undifferentiated state and induction of differentiation. It has also been reported that the pattern of HS chain sulfation changes during the differentiation of mESCs into mesodermal and neuroectodermal lineages [22,23]. Specific sulfated structures in HS chains might contribute to these differentiation processes by regulating several signal transduction pathways, although the significance of the changes in the sulfation pattern of HS chains remains unclear. We suggest that alteration of HS chain sulfation patterning regulates differentiation in mESCs by controlling several signaling cascades. 3-O-sulfated HS structures that are recognized by the anti-HS antibody HS4C3 [24], which include GlcA/IdoA2S-GlcNS3S6S, are synthesized by HS 3-O-sulfotransferase (3OST), which transfers sulfate from PAPS (adenosine 39-phosphate 59-phos- phosulphate) to the 3-OH position of a glucosamine residue to form 3-O-sulfated HS [25]. Six different isoforms of 3OST have been identified so far in mouse (3OST-1, -2, -3A, -3B, -5, and - 6). 3-O-sulfated HS serves as an entry receptor for herpes simplex virus 1 (HSV-1) and binds to glycoprotein D of HSV-1 and antithrombin [26]. However, it remains unclear whether 3-O- sulfated HS structures, including the HS4C3-binding epitope, have other functional roles, for example within signal transduc- tion. In this study, we investigated the contribution of the HS4C3- binding epitope to the regulation of mESC differentiation. We found that HS4C3-binding epitope on mESCs increased after the induction of differentiation into primitive endoderm and primitive ectoderm. Up-regulation of the HS4C3-binding epitope by overexpression of 3OST induced mESC differentia- tion even in the presence of LIF and serum, and demonstrated that this differentiation resulted from the redistribution of Fas to lipid rafts. In contrast, knockdown of 3OST reduced the potential for differentiation into primitive endoderm and primitive ectoderm. The results showed that Fas signaling via the HS4C3-binding epitope contributes to general differentiation in mESCs. Materials and Methods Construction of Expression Vectors The 3OST-2, -5, and Fas expression vectors for transfection into mESCs were constructed using the vector pCAGIPuro (a kind gift of Prof. Kumiko Ui-Tei). The Fas ectodomain expression vectors, for the production of recombinant proteins, were constructed using the vector pGEX-6P-1 (GE Healthcare). These constructs were produced by using the GATEWAYTM cloning system (Invitrogen) as described previously [27]. Each construct contained the appropriate full-length coding sequence (3OST-2, amino acids 1–367; 3OST-5, amino acids 1–346; Fas, amino acids 1–328) or the sequence for the putative extracellular domain of Fas, or fragments of this domain (amino acids 19–168, 19–38, and 39– 168). HS4C3-Binding Epitope Contributes to Fas Signaling Cell Culture and Transfection R1 [28] and E14TG2a [29] mESC lines were maintained on mouse embryonic fibroblasts (MEFs) inactivated with 10 mg/ml mitomycin C (Sigma) in ESC medium (DMEM supplemented with 15% FBS {Hyclone}, 1% penicillin/streptomycin {Gibco}, 0.1 mM 2-mercaptoethanol {Gibco}, and 0.1 mM non-essential amino acids {Gibco}) with 1000 U/ml LIF (Chemicon). R1 and E14TG2a lines were gifts from Dr. Seiji Hitoshi (National Institute for Physiological Sciences, Japan) and Prof. Kumiko Ui-Tei (Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Japan), respectively. MEFs were prepared from embryos at embryonic day 14.5 (E14.5; ICR). All experi- ments were performed in the R1 line and most were repeated using the E14TG2a line to confirm that the results were consistent. Prior to transfection, the mESCs were harvested and 16106 cells were replated in gelatin-coated feeder-free 60-mm culture dishes (Iwaki) in ESC medium with LIF, and incubated for 16 h. Subsequently (day 1), the cells were transfected with 4 mg of pCAGI containing 3OST-2, 3OST-5, Fas or no insert (control) using Lipofectamine 2000 (Invitrogen). On day 2, the cells were subjected to selection with 2 mg/ml puromycin (Sigma) for 24 h. The transfection efficiency was approximately 60%, but only transfected cells survived after puromycin selection. On day 3 (2 days after transfection), the transfected cells were harvested and used in the various experiments as described below. To induce primitive endoderm, mESCs were harvested at the first and second passages and 26105 cells were replated in gelatin-coated feeder-free 60-mm culture dishes in ESC medium without LIF. At the third and fourth passages, the cells were harvested and 56105 cells were replated in gelatin-coated feeder-free 60-mm culture dishes in ESC medium without LIF. To induce embryoid body (EB) formation, the transfected cells were transferred to 60-mm Low Cell Binding dishes (Nunc) and cultured in ESC medium without LIF. To analyze the inhibition of Fas signaling, the cells were cultured in medium that contained 10 mM Ac-IETD-CHO or 20 mM Ac-DEVD-CHO (Peptide Institute Inc) dissolved in DMSO. Ac-IETD-CHO and Ac-DEVD-CHO are inhibitors of caspase-8 and caspase-3, respectively. We generated siRNA expression plasmids that targeted 3OST-5 or EGFP, as a negative control, by inserting the appropriate dsDNAs between the BamHI and HindIII sites of pSilencer 3.1- H1 (Ambion) or pSUPER.retro.puro (OligoEngine). The siRNA sequences used for RNA interference (RNAi) were designed as described previously [19] using ‘‘siDirect’’: EGFP, 59- GATCCCGCCACAACGTCTATATCATGGGGAAAATC- CATGATATAGACGTTGTGGCTTTTTTGGAAA-39; 3OST- 5-1, 59-GATCCCGTAGACCCCTCCGTCAT- TACCGCTTCCTGTCACGGTAATGACGGAGGGGTC- TACTTTTTTA-39; 3OST-5-2, 59-GATCCCCGGTTAGGAC- CAGCATATACAGCTTCCTGTCACTGTA- TATGCTGGTCCTAACCGTTTTTTA-39. Stable knockdown of 3OST-5 mRNA was carried out as follows. To produce retrovirus, the pSUPER.retro.puro constructs were transfected into ecotropic virus-packaging (PLAT-E) cells. Super- natants that contained virus and were derived from these PLAT-E cultures were mixed with 8 mg/ml polybrene (Sigma) and the virus/polybrene mixtures were incubated with mESCs for 24 h. After infection, the cells were replated with ESC medium containing LIF and 2 mg/ml puromycin and cultured for 5–7 days. For transient knockdown of 3OST-5 mRNA by RNAi, 4 mg of the pSilencer 3.1-H1 construct for 3OST-5 were transfected into mESCs by the method described above. PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e43440