Lacrimal Gland Development and Fgf10-Fgfr2b Signaling Are Controlled by 2-O- and 6-O-sulfated Heparan Sulfate

Abstract

Heparan sulfate, an extensively sulfated glycosaminoglycan abundant on cell surface proteoglycans, regulates intercellular signaling through its binding to various growth factors and receptors. In the lacrimal gland, branching morphogenesis depends on the interaction of heparan sulfate with Fgf10-Fgfr2b. To address if lacrimal gland development and FGF signaling depends on 2-O-sulfation of uronic acids and 6-O-sulfation of glucosamine residues, we genetically ablated heparan sulfate 2-O and 6-O sulfotransferases (Hs2st, Hs6st1, and Hs6st2) in developing lacrimal gland. Using a panel of phage display antibodies, we confirmed that these mutations disrupted 2-O and/or 6-O but not N-sulfation of heparan sulfate. The Hs6st mutants exhibited significant lacrimal gland hypoplasia and a strong genetic interaction with Fgf10, demonstrating the importance of heparan sulfate 6-O sulfation in lacrimal gland FGF signaling. Altering Hs2st caused a much less severe phenotype, but the Hs2st;Hs6st double mutants completely abolished lacrimal gland development, suggesting that both 2-O and 6-O sulfation of heparan sulfate contribute to FGF signaling. Combined Hs2st;Hs6st deficiency synergistically disrupted the formation of Fgf10-Fgfr2b-heparan sulfate complex on the cell surface and prevented lacrimal gland induction by Fgf10 in explant cultures. Importantly, the Hs2st;Hs6st double mutants abrogated FGF downstream ERK signaling. Therefore, Fgf10-Fgfr2b signaling during lacrimal gland development is sensitive to the content or arrangement of O-sulfate groups in heparan sulfate. To our knowledge, this is the first study to show that simultaneous deletion of Hs2st and Hs6st exhibits profound FGF signaling defects in mammalian development.Heparan sulfate is a cell-surface glycosaminoglycan playing important roles in the transport and signaling of multiple growth factors, including Hedgehog, Wnt, bone morphogenic protein (BMP), and fibroblast growth factor (FGF) (1-3). Heparan sulfate is first synthesized from the activated monosaccharides, UDP-glucuronic acid and UDP-N-acetylglucosamine, by an Ext copolymerase complex to form a copolymer of glucuronic acid and N-acetylglucosamine. Polymerization is followed by N-deacetylation/N-sulfation of subsets of N-acetylglucosamine residues by N-deacetylase-N-sulfotransferase (Ndst) 2 enzymes (4). Because of the incomplete processing by the Ndst enzymes, the polysaccharide backbone is divided into stretches of variable length of N-sulfated disaccharides (NS domains) and N-acetylated disaccharides (NA domains). A portion of the D-glucuronic acid residues in the NS domains is next converted by glucuronyl C5-epimerase (Hsepi) into l-iduronic acids. A 2-O-sulfotransferase (Hs2st) transfers a sulfate group to the C-2 carbon of the iduronic acids and less frequently to glucuronic acid. Finally, 6-O-sulfotransferases (Hs6st) and more rarely 3-O-sulfotransferases (Hs3st) add sulfate groups to the C-6 and C-3 carbon of the glucosamine residues, respectively. These reactions do not go to completion, lea...

Heparan sulfation is essential for the prevention of cellular senescence

“…54,64,65 However, the exact role of HS in FGF signaling is still a matter of considerable debate. 33 In the present study, we found that FGF1 superfamily, FGF1/2 readily binds to FGFR1 and leads the hyper-activation of FGFR1 signaling in the absence of sulfated HSPG. In contrast, HGF/Met signaling, another well-known ligand/ receptor signaling regulated by HS, is not affected by HS sulfation status.…”

“…[30][31][32] However, the exact role of HS in FGF signaling is still a matter of considerable debate. 33 In addition, how FGF signaling is tumor promoting in some contexts, but tumor-suppressive in others remains unclear. 34 In this study, we investigate the role of HS sulfation status and FGF signaling in cellular senescence.…”

Attachment ofChlamydia trachomatisL2 to host cells requires sulfation

“…Lacrimal gland development (17), viability and neural development in C. elegans (18), FGF-induced signaling (19), and cartilage homeostasis (20) all depend on sulfation. Sulfation of tyrosine residues is important for the interaction of chemokines and their receptors (21)(22)(23) and for binding and entry of HIV (24).…”

Overexpression of heparan sulfate 6-O-sulfotransferase-2 enhances fibroblast growth factor-mediated chondrocyte growth and differentiation

“…It has also been reported that HS6ST2 plays a critical role in mediating energy metabolism by controlling signals from the FGF-19 subfamily proteins, and that HS6ST2 dysregulation is associated with glucose and insulin intolerance (34). The deletion of HS6ST1 and HS6ST2 has been shown to disrupt the FGF-10-FGFR2b-heparan sulfate complex on the cell surface, thus demonstrating the profound effect of FGF signaling on mammalian development (35). In the present study, we demonstrated that the FGF-2-mediated chondrocyte growth and differentiation was regulated by HS6ST2, that the overexpression of HS6ST2 significantly enhanced FGF-2-mediated chondrocyte growth and differentiation, while the silencing of HSTST2 abrogated the FGF-2-mediated chondrocyte growth and differentiation.…”