Poster Sessions

DEV-12

Vaishali Patel

V. Patel, K. Lassiter, S. Cowherd, Y. Xu, J. Liu, M. Hoffman

A Novel Role of 3-O-Sulfation during Submandibular Gland Epithelial Branching Morphogenesis

Heparan sulfates (HS), which are glycosaminoglycan chains attached to the proteoglycan protein core, mediate high affinity binding of FGFs to their receptors. During their synthesis, HS is modified by a series of sulfotransferase enzymes. Although 3-O-sulfotransferases (3-OST) are the largest sulfotransferases family, with seven isoforms, they act late in HS biosynthesis forming the rarest HS modification. The 3-O-sulfated HS is known to form binding epitopes for antithrombin and herpes simplex virus. Here we identify a novel role for 3-O-sulfated HS, which binds FGFR2b and increases FGF10-dependent submandibular gland epithelial proliferation and morphogenesis. FGF10-mediated proliferation occurs only at the tips of branching epithelium in culture, whereas its receptor, FGFR2b, is localized throughout the epithelium, suggesting an HSPG co-receptor is located on the tip cells. We therefore profiled gene expression of the HS biosynthetic enzymes in the proliferating tip of the epithelium, and identified a striking increase in 3-OST isoforms. In situ analysis of intact SMGs confirmed that the Hs3st isoforms were localized in the peripheral epithelial end bud cells. To study the function of 3-O-sulfation, under-sulfated kidney HS was modified with 3-OST enzymes, adding two 3-O-sulfates/HS chain. Over 3-O-sulfated HS increased branching and proliferation of FGF10-cultured epithelia. Gene expression of end bud markers downstream of FGF10 signaling, Etv4 and Etv5, increased as did Aqp5, an acinar cell differentiation marker. In loss of function experiments, a reduction in Hs3st expression inhibited morphogenesis, which was restored by adding exogenous over-3-O-sulfated HS. Further, over 3-O-sulfated HS increased the amount of FGF10 bound to FGFR2b in pull-down assays, suggesting that 3-O-sulfation helps stabilize FGF10-FGFR2b complex. Interestingly, we found FGFR2b but not FGF10 specifically bound immobilized Hs3st3-modified kidney HS. Collectively, we identify a novel and important role for 3-O-sulfated HS during SMG development. 3-O-sulfated HSPGs localized at the peripheral end buds in SMG epithelium specifically bind FGFR2b. This increases FGF10/FGFR2b signaling resulting in end bud proliferation, differentiation and branching morphogenesis. 3-O sulfated HS may be useful for targeting FGFR2b-mediated regeneration of salivary gland tissue.