title: N-sulfation of heparan sulfate is critical for syndecan-4-mediated podocyte cell-matrix interactions.
abstract: Previous research has shown that podocytes unable to assemble heparan sulfate on cell surface proteoglycan core proteins have compromised cell-matrix interactions. This report further explores the role of N-sulfation of intact heparan chains in podocyte-matrix interactions. For the purposes of this study, a murine model in which the enzyme N-deacetylase/N-sulfotransferase 1 (NDST1) was specifically deleted in podocytes and immortalized podocyte cell lines lacking NDST1 were developed and used to explore the effects of such a mutation on podocyte behavior in vitro. NDST1 is a bifunctional enzyme, ultimately responsible for N-sulfation of heparan glycosaminoglycans produced by cells. Immunostaining of glomeruli from mice whose podocytes were null for Ndst1 (Ndst1(-/-)) showed a disrupted pattern of localization for the cell surface proteoglycan, syndecan-4, and for α-actinin-4 compared with controls. The pattern of immunostaining for synaptopodin and nephrin did not show as significant alterations. In vitro studies showed that Ndst1(-/-) podocytes attached, spread, and migrated less efficiently than Ndst1(+/+) podocytes. Immunostaining in vitro for several markers for molecules involved in cell-matrix interactions showed that Ndst1(-/-) cells had decreased clustering of syndecan-4 and decreased recruitment of protein kinase-Cα, α-actinin-4, vinculin, and phospho-focal adhesion kinase to focal adhesions. Total intracellular phospho-focal adhesion kinase was decreased in Ndst1(-/-) compared with Ndst1(+/+) cells. A significant decrease in the abundance of activated integrin α5β1 on the cell surface of Ndst1(-/-) cells compared with Ndst1(+/+) cells was observed. These results serve to highlight the critical role of heparan sulfate N-sulfation in facilitating normal podocyte-matrix interactions.
authors.affiliation: Department of Cell Biology and Anatomy, LSU Health Sciences Center, Shreveport, Louisiana;
authors.forename: Terrel
authors.initials: T
authors.lastname: Sugar
authors.affiliation: Department of Pathology, LSU Health Sciences Center, Shreveport, Louisiana; and.
authors.forename: Deborah J
authors.initials: DJ
authors.lastname: Wassenhove-McCarthy
authors.affiliation: Department of Pathology, LSU Health Sciences Center, Shreveport, Louisiana; and.
authors.forename: A Wayne
authors.initials: AW
authors.lastname: Orr
authors.affiliation: Department of Pathology, LSU Health Sciences Center, Shreveport, Louisiana; and.
authors.forename: Jonette
authors.initials: J
authors.lastname: Green
authors.affiliation: Department of Biochemistry, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
authors.forename: Toin H
authors.initials: TH
authors.lastname: van Kuppevelt
authors.affiliation: Department of Cell Biology and Anatomy, LSU Health Sciences Center, Shreveport, Louisiana; Department of Pathology, LSU Health Sciences Center, Shreveport, Louisiana; and kmccar2@lsuhsc.edu.
authors.forename: Kevin J
authors.identifier: 0000-0001-9317-5624
authors.initials: KJ
authors.lastname: McCarthy
mesh_terms: D000185:Actinin / Q000378:metabolism; D000818:Animals; D002448:Cell Adhesion; D002465:Cell Movement; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D004195:Disease Models, Animal; D005109:Extracellular Matrix / Q000378:metabolism*; D022001:Focal Adhesions / Q000378:metabolism; D050533:Glomerular Basement Membrane / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism*; D039081:Integrin alpha5beta1 / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D050199:Podocytes / Q000378:metabolism*; D015238:Sulfotransferases / Q000235:genetics*; D053671:Syndecan-4 / Q000378:metabolism*
chemical_list: D000185:Actinin; D006497:Heparan Sulfate; D039081:Integrin alpha5beta1; D015238:Sulfotransferases; D053671:Syndecan-4; C491752:Actn4 protein, mouse; C508306:Sdc4 protein, mouse; C021350:heparitin sulfotransferase
keywords: glycosaminoglycans; heparan sulfate; podocyte; proteoglycans; syndecan