Abstract
Purpose:
We have reported that knockdown by p120 siRNA selectively activates p120-catenin/Kaiso signaling and successfully expands contact-inhibited HCEC monolayers to an average size of 2.1 ± 0.3 mm in diameter from 1/8 of the descemet membrane stripped from the corneoscleral rim. Herein, we would like to show how incorporation of other means might further expand the monolayer size.
Methods:
HCEC monolayers derived from 1/8 stripped descemet membrane and cultured to 7 days in SHEM were treated with different concentrations of p120 siRNA weekly with or without 100 nM Kaiso siRNA or 5 µg/ml nocodazole, a microtubule disrupting agent, for up to 5 weeks. Before termination, cells were further treated with 10 µM BrdU for 4 h. Immunostaining was performed to monitor cytolocalization of F-actin, α-catenin, β-catenin, p120 catenin, N-cadherin, ZO-1, Na+/K+-ATPase, and BrdU labeling.
Results:
Consistent with our recent report, p120 siRNA knockdown uniquely promoted proliferation of contact-inhibited HCEC by nuclear translocation of p120 catenin to relieve repression by nuclear Kaiso. Such proliferation was further enhanced to achieve an average monolayer size of 4.1 ± 0.3 mm in diameter (n=3, p<0.05) when the concentration of p120 siRNA was increased from 40 nM to 100 nM and the duration was prolonged from 2 to 5 weeks. Additional knockdown by 100 nM Kaiso siRNA, but not treatment of 5 µg/ml norcodazole, further synergistically promoted the monolayer size to 5.0 ± 0.4 mm in diameter (n=3, p<0.05). Proliferating HCEC still maintained a hexagonal shape, an comparable in vivo density, and a normal expression pattern of F-actin, α-catenin, β-catenin, N-cadherin, ZO-1, and Na+/K+-ATPase. Furthermore, downregulation of p120 catenin at the cell junction was completely reversed after withdrawal of p120 siRNA for 1 week.
Conclusions:
This new strategy of perturbing contact inhibition by selective activation of p120 catenin-Kaiso signaling without disrupting adherent junction can be exploited to engineer surgical grafts containing normal human corneal endothelial cells to meet a global corneal shortage and for endothelial keratoplasties.
Keywords: 481 cornea: endothelium •
654 proliferation