3-O-Sulfated Heparan Sulfate Recognized by the Antibody HS4C3 Contribute to the Differentiation of Mouse Embryonic Stem Cells via Fas Signaling

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.

Changes in glycosaminoglycan structure on differentiation of human embryonic stem cells towards mesoderm and endoderm lineages

“…HS3ST1 is found in kidney, brain and heart, whereas HS3ST2 was reported only in brain, which coincides with the increase in HS3ST1 transcripts in the cardiac progenitor Isl1 + line. These enzymes are responsible for introducing 3- O -sulfo groups into the HS/HP chain and these are important for interaction with proteins, such as antithrombin [37] and are also critical for differentiation of mouse ESCs through Fas signaling [49]. Due to the lack of available enzymes that can cut the HS/HP chains to yield 3- O -sulfo group containing disaccharides, we were unable to use LC-MS to verify whether the change in HS3ST transcript levels leads to a change in HS/HP structure.…”

Heparan sulfate 3-O-sulfation: A rare modification in search of a function

“…In tissue culture, withdrawal of LIF results in differentiation of mouse embryonic stem cells and a concurrent upregulation of Hs3st-5 (Hirano et al, 2012). That cell surface 3- O -sulfation increased was also suggested by cell surface binding of a single chain antibody (HS4C3) with propensity to bind 3- O -sulfated heparan sulfate (Ten Dam et al, 2006).…”

Heparan sulfate: a key regulator of embryonic stem cell fate

“…The possible function of this low-sulfated form of HS is unclear, but may serve to shield ESCs from differentiation-induction signaling, as will be discussed in the next section of this review. As ESCs transition into committed cell types, their HS chains become increasingly sulfated (Johnson et al, 2007; Nairn et al, 2007; Hirano et al, 2012). Changes in sulfation upon lineage commitment arise primarily as a result of increased N-, 3-O-, and 6-O-sulfation.…”

“…At least in the context of glypican 4, an abundant glypican expressed by ESCs, HS may be a positive regulator of self-renewal, although it has not been demonstrated whether HS chains or core proteins are the actual mediators of Wnt signaling (Fico et al, 2012). Recently, Fas signaling was reported to be implicated in promoting ESC self-renewal and was shown to depend on 3-O-sulfation of HS for the activation of this pathway (Hirano et al, 2012). Although the role of Fas signaling in ESC self-renewal needs further characterization, this and other studies have merged the strict structure-function relations of HS into the modulation of ESC self-renewal.…”