Localization of the Laminin α4 Chain in the Skin and Identification of a Heparin-Dependent Cell Adhesion Site Within the Laminin α4 Chain C-Terminal LG4 Module.

The laminin α4 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 α4 chain in the skin have not been elucidated. By immunostaining with specific antibodies, we demonstrate here that the α4 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 α4 chain. We have also focused on the cell adhesion activities of the human laminin α4 LG4 module since the corresponding LG4 module of laminin α3 was previously identified as active for cell adhesion. Recombinant human α4 LG4 was active for heparin-dependent fibroblast adhesion. Screening assays with 19 synthetic peptides covering the entire α4 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 α4 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 α4 LG4 module. Our study suggests that the laminin α4 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. [ABSTRACT FROM AUTHOR]