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

Thy-1是一种25-37 kDa的糖基磷脂酰肌醇（GPI）锚定糖蛋白，在许多细胞类型中表达。由于Thy-1影响广泛的非免疫功能，如细胞粘附、迁移和增殖，该分子可能是细胞-细胞和细胞-基质相互作用的重要调节因子。我们研究了Thy-1在人牙龈成纤维细胞（HGF）吞噬胶原中的作用及其机制。采用磁珠分选法将HGF分为Thy-1High亚群和Thy-1Low亚群。用胶原包被的荧光珠孵育每个亚群后，计数与细胞层结合的珠数，并用流式细胞仪分析珠的内化情况。在PI3K抑制剂或Cytochalasin D存在或不存在的情况下，用抗thy -1抗体（AS02）预处理HGF，分别用罗丹明-phalloidin染色和Western blotting检测肌动蛋白重组和Akt磷酸化，并进一步检测蛋白的结合和内化。thy -1阳性细胞的平均百分比在thy -1高HGF组为约90%，在thy -1低HGF组<10%。Thy-1High HGF比Thy-1Low HGF表现出更高的头结合水平和内化水平。AS02处理诱导肌动蛋白重组、Akt磷酸化并刺激随后的头细胞吞噬途径。PI3K抑制剂或细胞松弛素D预处理可抑制AS02的作用。这些结果表明，Thy-1的表达参与了HGF中肌动蛋白细胞骨架组织和组成型胶原吞噬的维持。在抗Thy-1抗体（或Thy-1配体）刺激的HGF中，Thy-1信号可能部分通过PI3K/Akt刺激肌动蛋白细胞骨架变化和胶原吞噬通路。

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.