Abstract
Purpose: :
The limited proliferative capability of human corneal endothelial cells (HCEC) is due to an arrest in G1 phase. A brief EDTA treatment is necessary to unlock the mitotic block of confluent HCEC derived from EDTA/trypsin dissociated single cells. We have developed a new method of generating HCEC monolayers without dissociating them into single cells. Because such monolayers expressing all adherent junction components also exhibit the mitotic block, we wonder whether EDTA can similarly unlock this mitotic block.
Methods: :
Both post-confluent ARPE-19 and HCEC monolayers cultured up to Day 21 were treated with 2 mg/ml EDTA for one hour and cultured for two days, or by 100 nM p120 siRNA3 for 2 days, and compared to their respective controls similarly treated with PBS or scrambled RNA. Before termination, cells were labeled with 10 µM BrdU for 24h. Real-time PCR, western blot and immunostaining to p120 catenin, β-catenin, N-cadherin, VE-cadherin and ZO-1 with double labeling to BrdU were compared.
Results: :
In ARPE-19 cells, EDTA treatment markedly downregulated β-catenin, but not p120, N-cadherin and ZO-1 transcripts and proteins when compared to the control of PBS. In contrast, p120 siRNA transfection significantly downregulated p120, N-cadherin and ZO-1, but not β-catenin trasnscripts and proteins when compared to the control of scrambled RNA. Similarly in HCEC monolayers, immunostaining showed that EDTA treatment still largely preserved the intercellular staining of p120, N-cadherin, and VE-cadherin, but abolished that of β-catenin when compared to the control. In contrast, siRNA3 knockdown completely dissolved the intercellular and cytoplasmic staining of p120, N-cadherin, and VE-cadheri, but not that of β-catenin. Double immunostaining showed intercellular, cytoplasmic and nuclear staining of p120 without BrdU-labeled nuclei in cells at the periphery and the center of the control HCEC treated with PBS or scrambled siRNAi. Similarly, EDTA-treated monolayers did not reveal BrdU but showed a slight decrease in the intercellular location of p120, total disappearance at cytoplasmic and more prominent nuclear staining at the periphery and the center. Although p120 staining pattern was similar to that of EDTA treatment, BrdU-labeled nuclei were abundant in the periphery of HCEC monolayer treated by siRNA3.
Conclusions: :
These results collectively indicate that disruption of adherent junction mediated by β-catenin via EDTA cannot, but that by p120 catenin via siRNA can unlock the mitotic block of HCEC monolayers when all adherent junction components are maintained after isolation from stripped Descemet membrane.
Keywords: cornea: endothelium • proliferation • retinal pigment epithelium