Investigation of the role of Thy-1 in collagen phagocytosis by gingival fibroblasts and its mechanisms

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Thy-1, a 25-37 kDa glycosylphosphatidylinositol (GPI)-anchored glycoprotein, is expressed on many cell types. Since Thy-1 affects widespread nonimmunologic functions such as cellular adhesion, migration and proliferation, this molecule may be an important regulator of cell-cell and cell-matrix interactions. We investigated here a role of Thy-1 in collagen phagocytosis by human gingival fibroblasts (HGF) and its mechanisms. HGF were separated into Thy-1High and Thy-1Low subpopulations using the magnetic bead sorting. Following incubation of each subpopulation with collagen-coated fluorescent beads, number of beads bound to cell layer was counted, and internalization of beads was analyzed using a flow cytometer. After HGF were pretreated with anti-Thy-1 antibody (AS02) in the presence or absence of PI3K inhibitors or Cytochalasin D, actin reorganization and phosphorylation of Akt were assessed by rhodamine-phalloidin staining and Western blotting, respectively, and further the binding and internalization of bead were determined. Mean % of Thy-1-positive cells were >90% in Thy-1High HGF and <10% in Thy-1Low HGF. Thy-1High HGF showed higher levels of bead binding as well as its internalization than Thy-1Low HGF. AS02 treatment induces actin reorganization, Akt phosphorylation and stimulates subsequent bead phagocytic route. Pretreatment with PI3K inhibitors or Cytochalasin D inhibited the AS02 actions. These results suggest that Thy-1 expression participates with maintenance of actin cytoskeletal organization and constitutive collagen phagocytosis in HGF. In HGF stimulated with anti-Thy-1 antibody (or Thy-1 ligand), Thy-1 signal may stimulate actin cytoskeletal changes and collagen phagocytic pathway via PI3K/Akt, in part.