April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
The Phenotype Is Preserved by p120-catenin Knockdown but Lost by EDTA Treatment to Perturb Adherent Junction to Promote Proliferation in Contact-Inhibited Human Corneal Endothelial Monolayers
Author Affiliations & Notes
  • Yingting Zhu
    Research, Ocular Surface Ctr and Tissue Tech, Miami, Florida
  • Hung-Chi J. Chen
    Ophthalmology, Chang Gung Memorial Hospital, Taoyuan County, Taiwan
  • Szu-Yu Chen
    Ocular Surface Research & Edu Foundation, Tissue Tech Inc, Miami, Florida
  • Scheffer C. Tseng
    Ocular Surface Center, Ocular Surface Res & Educ Fndtn, Miami, Florida
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 6441. doi:
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      Yingting Zhu, Hung-Chi J. Chen, Szu-Yu Chen, Scheffer C. Tseng; The Phenotype Is Preserved by p120-catenin Knockdown but Lost by EDTA Treatment to Perturb Adherent Junction to Promote Proliferation in Contact-Inhibited Human Corneal Endothelial Monolayers. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6441.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Contact inhibition of post-confluent human corneal endothelial cells (HCEC) can be released by EDTA. However, we speculate such an approach runs the risk of losing the cell phenotype to EMT. Thus, we sought a different approach to unlock such mitotic block without altering the cell phenotype.

Methods: : HCEC monolayers isolated by collagenase directly from stripped Descemet membrane were cultured to Day 19, and treated by 5 mM EDTA for 1 h with or without 20 ng/ml bFGF for 2 days followed by with or without 10 ng/ml TGF-β for 3 days. The parallel cultures were transfected by 100 nM siRNA to p120-catenin (p120), β-catenin, N-cadherin, or ZO-1. Before termination, the cells were labeled with 10 µM BrdU for 24 h. Cytolocalization and quantitation of p120, Kaiso, N-cadherin, ZO-1, β-catenin, LEF1, S-100A4, α-SMA and BrdU were assessed by immunostaining and Western or dot blotting.

Results: : EDTA promoted BrdU labeling in HCEC monolayers only when bFGF was added, but also changed from a hexagonal to fibroblastic-like shape expressing α-SMA in the cytoplasm and S-100A4 in the nucleus with additional TGFβ. Such an EMT phenotype was correlated with nuclear translocalization of β-catenin and LEF1, suggestive of activation of the Wnt signaling. In contrast, knockdown with siRNA to p120, but not to β-catenin, N-cadherin, or ZO-1, also promoted BrdU labeling; such promotion was not affected by bFGF. Furthermore, HCEC remained hexagonal, retained junctional expression of β-catenin, and did not activate the Wnt signaling and EMT despite addition of bFGF/TGF-β. Intriguingly, membranous p120 was reduced while nuclear p120 increased accompanied with downregulation of nuclear Kaiso, suggestive of activation of the p120/Kaiso signaling. Moreover, the normal HCEC phenotype was retained with a higher cell density after withdrawal of p120 siRNA.

Conclusions: : These results collectively indicate that the mitotic block of contact-inhibited HCEC can be unlocked by downregulating junctional p120 without affecting the normal phenotype. This novel strategy can be deployed to engineer HCEC for endothelial keratoplastiies.

Keywords: cornea: endothelium • proliferation • cell adhesions/cell junctions 
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