Methods: HGF were separated into Thy-1High and Thy-1Low subpopulations using a magnetic bead selection. Following incubation of each subset 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.
Results: Thy-1High HGF were >90% positive for Thy-1, and Thy-1Low HGF were <10% positive. Thy-1High HGF showed higher levels of bead binding and its internalization than Thy-1Low HGF. AS02 treatment stimulates actin reorganization, Akt phosphorylation and subsequent bead phagocytic route. Pretreatment with PI3K inhibitors or Cytochalasin D inhibited the AS02 actions.
Conclusion: Thy-1 positively regulates collagen phagocytosis by HGF. Further, the PI3K/Akt signaling pathway is thought to partially mediate the Thy-1-activated collagen phagocytic pathway, which may be dependent on actin cytoskeletal rearrangement.