PURPOSE: Although the cornea expresses high levels of clusterin (CLU), the role of CLU in the cornea is poorly understood. This study was performed to investigate the possible role of CLU in corneal epithelial homeostasis. METHODS: CLU was overexpressed in 3T3 cells by transfection of a vector encoding full-length CLU (Clu-3T3). Colony-forming efficacy (CFE) was compared in mouse corneal cell line (TKE2) and human primary corneal/limbal epithelial cells that were cocultured with Clu-3T3 and mock-3T3. To determine whether feeder cells have a contact effect, cocultures without feeder-epithelium contact were also performed. Neutralizing antibody against CLU was used to assess the effects of secretory CLU in TKE2 cells cocultured with Clu-3T3 cells. The expression of growth factors associated with limbal stem/progenitor cell maintenance and growth were analyzed by RT-PCR and Western blot analysis. RESULTS: TKE2 cells cocultured with Clu-3T3 feeders showed higher CFE and were larger in colony size than TKE2 cells cocultured with mock-3T3 feeders. Increased CFE of TKE2 was observed without direct contact with Clu-3T3 cells, which was significantly blocked by treatment with CLU neutralizing antibody. Clu-3T3 cells expressed higher levels of HGF than mock-3T3 cells, which were significantly suppressed with anti-HGF neutralizing antibodies. Collectively, the promotion of colony-forming and cell proliferation by Clu-3T3 cells was partially mediated by the induction of HGF. CONCLUSIONS: Clusterin indirectly enhances the CFE of corneal/limbal epithelial cells by inducing the production of HGF by feeder cells, suggesting a role in epithelial-mesenchymal interaction.
PURPOSE: Although the cornea expresses high levels of clusterin (CLU), the role of CLU in the cornea is poorly understood. This study was performed to investigate the possible role of CLU in corneal epithelial homeostasis. METHODS:CLU was overexpressed in 3T3 cells by transfection of a vector encoding full-length CLU (Clu-3T3). Colony-forming efficacy (CFE) was compared in mousecorneal cell line (TKE2) and human primary corneal/limbal epithelial cells that were cocultured with Clu-3T3 and mock-3T3. To determine whether feeder cells have a contact effect, cocultures without feeder-epithelium contact were also performed. Neutralizing antibody against CLU was used to assess the effects of secretory CLU in TKE2 cells cocultured with Clu-3T3 cells. The expression of growth factors associated with limbal stem/progenitor cell maintenance and growth were analyzed by RT-PCR and Western blot analysis. RESULTS: TKE2 cells cocultured with Clu-3T3 feeders showed higher CFE and were larger in colony size than TKE2 cells cocultured with mock-3T3 feeders. Increased CFE of TKE2 was observed without direct contact with Clu-3T3 cells, which was significantly blocked by treatment with CLU neutralizing antibody. Clu-3T3 cells expressed higher levels of HGF than mock-3T3 cells, which were significantly suppressed with anti-HGF neutralizing antibodies. Collectively, the promotion of colony-forming and cell proliferation by Clu-3T3 cells was partially mediated by the induction of HGF. CONCLUSIONS:Clusterin indirectly enhances the CFE of corneal/limbal epithelial cells by inducing the production of HGF by feeder cells, suggesting a role in epithelial-mesenchymal interaction.