3-O -Sulfated Heparan Sulfate Recognized by the Antibody HS4C3 Contribute to the Differentiation of Mouse Embryonic Stem Cells via Fas Signaling Kazumi Hirano 1 , Norihiko Sasaki1, Tomomi Ichimiya1, Taichi Miura1, Toin H. Van Kuppevelt2, Shoko Nishihara1* 1 Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, Hachioji, Tokyo, Japan, 2 Nijmegen Centre for Molecular Life Sciences, Department of Biochemistry, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands Abstract Maintenance of self-renewal and pluripotency in mouse embryonic stem cells (mESCs) is regulated by the balance between several extrinsic signaling pathways. Recently, we demonstrated that heparan sulfate (HS) chains play important roles in the maintenance and differentiation of mESCs by regulating extrinsic signaling. Sulfated HS structures are modified by various sulfotransferases during development. However, the significance of specific HS structures during development remains unclear. Here, we show that 3-O-sulfated HS structures synthesized by HS 3-O-sulfotransferases (3OSTs) and recognized by the antibody HS4C3 increase during differentiation of mESCs. Furthermore, expression of Fas on the cell surface of the differentiated cells also increased. Overexpression of the HS4C3-binding epitope in mESCs induced apoptosis and spontaneous differentiation even in the presence of LIF and serum. These data showed that the HS4C3-binding epitope was required for differentiation of mESCs. Up-regulation of the HS4C3-binding epitope resulted in the recruitment of Fas from the cytoplasm to lipid rafts on the cell surface followed by activation of Fas signaling. Indeed, the HS4C3-binding epitope interacted with a region that included the heparin-binding domain (KLRRRVH) of Fas. Reduced self-renewal capability in cells overexpressing 3OST resulted from the degradation of Nanog by activated caspase-3, which is downstream of Fas signaling, and was rescued by the inhibition of Fas signaling. We also found that knockdown of 3OST and inhibition of Fas signaling reduced the potential for differentiation into the three germ layers during embryoid body formation. This is the first demonstration that activation of Fas signaling is mediated by an increase in the HS4C3-binding epitope and indicates a novel signaling pathway for differentiation in mESCs. Citation: Hirano K, Sasaki N, Ichimiya T, Miura T, Van Kuppevelt TH, et al. (2012) 3-O-Sulfated Heparan Sulfate Recognized by the Antibody HS4C3 Contribute to the Differentiation of Mouse Embryonic Stem Cells via Fas Signaling. PLoS ONE 7(8): e43440. doi:10.1371/journal.pone.0043440 Editor: Austin John Cooney, Baylor College of Medicine, United States of America Received December 6, 2011; Accepted July 23, 2012; Published August 16, 2012 Copyright: 2012 Hirano et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was partially supported by funds from Ministry of Education, Culture, Sports, Science and Technology, the Grant-in-Aid for Scientific Research (B) to SN, 20370051, 2008–2010, and from Ministry of Education, Culture, Sports, Science and Technology, the Matching Fund for Private Universities, S0901015, 2009–2014. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: shoko@soka.ac.jp Introduction Heparan sulfate (HS) is a ubiquitous component of proteogly- cans in the extracellular matrix and on the cell surface. In proteoglycans, the HS polysaccharide chains are attached covalently to Ser residues in the core proteins through the linkage region GlcAb1-3Galb1-3Galb1-4Xylb1-O-Ser [1]. HS chains are synthesized in the Golgi by several enzymes, including members of the EXT protein family, which elongate the HS chain by adding repeating disaccharide units of D-glucuronic acid-N-acetyl-D- glucosamine (-4GlcAb1-4GlcNAca1-)n. These repeating units are then modified differentially by epimerization and sulfation to produce a wide range of structurally and functionally diverse compounds [1]. Many molecules that are important for develop- ment, including members of the fibroblast growth factor (Fgf) family, Wnt, and bone morphogenic protein (BMP), can bind to specific sulfated regions of HS chains, which regulate signaling by these molecules [2,3]. The control of sulfated HS structures is considered essential for the spatiotemporal regulation of cellular differentiation and growth throughout development. Embryonic stem cells (ESCs) [4,5] are promising tools for biotechnology and possess key features that should allow their exploitation in cell replacement therapies [6]. To exploit this potential, a better understanding of the molecular mechanisms that control ESC pluripotency is required. Pluripotency is regulated by a combination of intrinsic and extrinsic factors [7]. A number of these intrinsic factors have been identified, including Oct3/4 and Nanog [8]. Leukemia inhibitory factor (LIF) [9,10], a known extrinsic factor, plays an important role in maintaining the self-renewal of mouse ESCs (mESCs) via signal transduction and activator of transcription 3 (STAT3) activation [11]. It has been reported that mESCs differentiate into primitive endoderm upon the blockage of LIF/STAT3 signaling, and Nanog inhibits this differentiation process [12,13]. Another extrinsic factor, BMP4, acts in synergy with LIF to maintain self-renewal by modulating the Smad-mediated induction of the Id (inhibitor of differentiation) gene [14]. Wnt/b-catenin signaling also plays a role in the regulation of self-renewal that is independent of LIF/STAT3 signaling but involves Nanog expression [15–17]. Fgf4 is produced PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e43440