title: Localization of the laminin alpha4 chain in the skin and identification of a heparin-dependent cell adhesion site within the laminin alpha4 chain C-terminal LG4 module.
abstract: The laminin alpha4 chain, a component of laminin-8/9, is expressed in basement membranes of endothelial cells, the peripheral nerves, and muscle fibers. The localization and functions of laminin alpha4 chain in the skin have not been elucidated. By immunostaining with specific antibodies, we demonstrate here that the alpha4 chain is located in the basement membrane zones of blood vessels and is also associated with fibroblast-like cells in the dermis. Western blot showed that cultured fibroblasts secreted a laminin trimer containing the alpha4 chain. We have also focused on the cell adhesion activities of the human laminin alpha4 LG4 module since the corresponding LG4 module of laminin alpha3 was previously identified as active for cell adhesion. Recombinant human alpha4 LG4 was active for heparin-dependent fibroblast adhesion. Screening assays with 19 synthetic peptides covering the entire alpha4 LG4 module identified three peptides (HA4G82: TLFLAHGRLVYM; HA4G83: LVYMFNVGHKKL; and HA4G90: TEATWKIKGPIYL) as active sites for heparin- and heparan sulfate-dependent cell adhesion. Serine-substituted peptides demonstrated that two basic residues, His and Arg, within HA4G82 were essential for cell adhesion activity. The cell surface heparan sulfate proteoglycans (HSPGs), syndecan-2, -4, and glypican-1, were stably expressed in 293T cells to estimate whether they function as cell adhesion receptors. 293T cells overexpressing syndecan-2 or -4 bound to recombinant alpha4 LG4 and to HA4G82, but parental or glypican-1-overexpressing 293T cells did not. Therefore, syndecan-2 and -4 could mediate cell adhesion to the laminin alpha4 LG4 module. Our study suggests that the laminin alpha4 LG4 module may play an important role in cell adhesion and/or vessel wall formation in the skin by interacting with syndecan-2 and/or -4.
authors.affiliation: Department of Dermatology, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Matsuura
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Momota
authors.forename: Kaoru
authors.initials: K
authors.lastname: Murata
authors.forename: Hironori
authors.initials: H
authors.lastname: Matsushima
authors.forename: Nobuharu
authors.initials: N
authors.lastname: Suzuki
authors.forename: Motoyoshi
authors.initials: M
authors.lastname: Nomizu
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Shinkai
authors.forename: Atsushi
authors.initials: A
authors.lastname: Utani
mesh_terms: D001665:Binding Sites; D002448:Cell Adhesion / Q000187:drug effects*; D002478:Cells, Cultured; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D007797:Laminin / Q000032:analysis* / Q000737:chemistry / Q000502:physiology; D008562:Membrane Glycoproteins / Q000502:physiology; D011509:Proteoglycans / Q000502:physiology; D012867:Skin / Q000737:chemistry*; D053669:Syndecan-2; D053671:Syndecan-4
chemical_list: C501369:LAMA4 protein, human; D007797:Laminin; D008562:Membrane Glycoproteins; D011509:Proteoglycans; C508245:SDC2 protein, human; C508304:SDC4 protein, human; D053671:Syndecan-4; D053669:Syndecan-2; D006493:Heparin