Clusterin promotes corneal epithelial cell growth through upregulation of hepatocyte growth factor by mesenchymal cells in vitro

Naoko Okada, Tetsuya Kawakita, Kenji Mishima, Ichiro Saito, Hideyuki Miyashita, Satoru Yoshida, Shigeto Shimmura, Kazuo Tsubota

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

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 feederepithelium 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 colonyforming 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.

Original languageEnglish
Pages (from-to)2905-2910
Number of pages6
JournalInvestigative Ophthalmology and Visual Science
Volume52
Issue number6
DOIs
Publication statusPublished - 2011 May

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Clusterin
Hepatocyte Growth Factor
3T3 Cells
Up-Regulation
Epithelial Cells
Growth
Neutralizing Antibodies
Feeder Cells
Cornea
Stem Cells
In Vitro Techniques
Coculture Techniques
Cell Size
Transfection
Intercellular Signaling Peptides and Proteins
Homeostasis
Western Blotting
Maintenance
Cell Proliferation
Cell Line

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Clusterin promotes corneal epithelial cell growth through upregulation of hepatocyte growth factor by mesenchymal cells in vitro. / Okada, Naoko; Kawakita, Tetsuya; Mishima, Kenji; Saito, Ichiro; Miyashita, Hideyuki; Yoshida, Satoru; Shimmura, Shigeto; Tsubota, Kazuo.

In: Investigative Ophthalmology and Visual Science, Vol. 52, No. 6, 05.2011, p. 2905-2910.

Research output: Contribution to journalArticle

Okada, Naoko ; Kawakita, Tetsuya ; Mishima, Kenji ; Saito, Ichiro ; Miyashita, Hideyuki ; Yoshida, Satoru ; Shimmura, Shigeto ; Tsubota, Kazuo. / Clusterin promotes corneal epithelial cell growth through upregulation of hepatocyte growth factor by mesenchymal cells in vitro. In: Investigative Ophthalmology and Visual Science. 2011 ; Vol. 52, No. 6. pp. 2905-2910.
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AU - Okada, Naoko

AU - Kawakita, Tetsuya

AU - Mishima, Kenji

AU - Saito, Ichiro

AU - Miyashita, Hideyuki

AU - Yoshida, Satoru

AU - Shimmura, Shigeto

AU - Tsubota, Kazuo

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N2 - 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 feederepithelium 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 colonyforming 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.

AB - 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 feederepithelium 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 colonyforming 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.

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