title: Heparanase cleavage of perlecan heparan sulfate modulates FGF10 activity during ex vivo submandibular gland branching morphogenesis.
abstract: Heparan sulfate proteoglycans are essential for biological processes regulated by fibroblast growth factors (FGFs). Heparan sulfate (HS) regulates the activity of FGFs by acting as a coreceptor at the cell surface, enhancing FGF-FGFR affinity, and being a storage reservoir for FGFs in the extracellular matrix (ECM). Here we demonstrate a critical role for heparanase during mouse submandibular gland (SMG) branching morphogenesis. Heparanase, an endoglycosidase, colocalized with perlecan in the basement membrane and in epithelial clefts of SMGs. Inhibition of heparanase activity in organ culture decreased branching morphogenesis, and this inhibition was rescued specifically by FGF10 and not by other FGFs. By contrast, exogenous heparanase increased SMG branching and MAPK signaling and, surprisingly, when isolated epithelia were cultured in a three-dimensional ECM with FGF10, it increased the number of lateral branches and end buds. In a solid-phase binding assay, an FGF10-FGFR2b complex was released from the ECM by heparanase. In addition, surface plasmon resonance (SPR) analysis showed that FGF10 and the FGF10-FGFR2b complex bound to purified perlecan HS and could be released by heparanase. We used the FGF10-FGFR2b complex as a probe for HS in SMGs, and it colocalized with perlecan in the basement membrane and partly colocalized with syndecan 1 in the epithelium, and binding was reduced by treatment with heparanase. In summary, our results show heparanase releases FGF10 from perlecan HS in the basement membrane, increasing MAPK signaling, epithelial clefting, and lateral branch formation, which results in increased branching morphogenesis.
authors.affiliation: Matrix and Morphogenesis Unit, Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, Bethesda, MD, USA.
authors.forename: Vaishali N
authors.initials: VN
authors.lastname: Patel
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Knox
authors.forename: Karen M
authors.initials: KM
authors.lastname: Likar
authors.forename: Colin A
authors.initials: CA
authors.lastname: Lathrop
authors.forename: Rydhwana
authors.initials: R
authors.lastname: Hossain
authors.forename: Siavash
authors.initials: S
authors.lastname: Eftekhari
authors.forename: John M
authors.initials: JM
authors.lastname: Whitelock
authors.forename: Michael
authors.initials: M
authors.lastname: Elkin
authors.forename: Israel
authors.initials: I
authors.lastname: Vlodavsky
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Hoffman
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000166:cytology; D005109:Extracellular Matrix / Q000502:physiology / Q000648:ultrastructure; D051526:Fibroblast Growth Factor 10 / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism*; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics; D008648:Mesoderm / Q000166:cytology; D051379:Mice; D008813:Mice, Inbred ICR; D008822:Mice, Transgenic / Q000196:embryology / Q000235:genetics; D009024:Morphogenesis / Q000502:physiology*; D016133:Polymerase Chain Reaction; D051497:Receptor, Fibroblast Growth Factor, Type 2 / Q000378:metabolism; D013363:Submandibular Gland / Q000196:embryology*; D020349:Surface Plasmon Resonance; D000099175:Heparanase
chemical_list: D051526:Fibroblast Growth Factor 10; D005966:Glucuronidase; D019812:Heparan Sulfate Proteoglycans; D007141:Immunoglobulin Fc Fragments; D051497:Receptor, Fibroblast Growth Factor, Type 2; D000099175:Heparanase; C496464:Fgf10 protein, mouse; C071927:perlecan