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Hongyu Ying, Xiang Shen, Beatrice Y. Yue; Alterations in the Barrier Function and Cell Migration in stable RGC5 Cell Lines Induced by Expression of Wild-type and Mutated Myocilin. Invest. Ophthalmol. Vis. Sci. 2012;53(14):785.
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Myocilin is a gene linked directly to juvenile- and adult-onset open angle glaucoma. Mutations including Pro370Leu (P370L) and Gln368stop (Q368X) have been identified in patients. The purpose of the study is to examine the effects of wild type and mutant myocilin expression in tetracycline inducible (Tet-on) stable retinal ganglion RGC5 cell lines.
RGC5 cell lines plated on 8W10E+ arrays were induced by treatment of doxycycline (Dox, 1 μg/ml) for 48 h to express wild type or mutated (P370L or Q368X) myocilin-GFP. The barrier function (represented by the total electrical resistance) in both non-induced and induced cells was measured using an electrical cell-substrate impedance sensing system (ECIS). The expression levels of ZO-1 and occludin in total cell lysates was assessed by immunoblotting. For migratory activity, confluent non-induced and induced RGC5 cells in 6 well plates were scratched with a pipet tip. The ability of cells to migrate into the scratch wound was examined using a Zeiss inverted microscope 6- 8 h later. The percentage of scratched areas covered by migratory cells in each sample was determined. For actin staining, non-induced and induced cells were fixed and stained with rhodamine-phalloidin. The rounding up of cells following addition of trypsin to the cultures was monitored by a Zeiss live cell imaging system.
The ECIS readings from cells induced to express wild type myocilin were decreased. Similar results were also obtained from cells expressing P370L and Q368X myocilin. The levels of junction proteins ZO-1 or occludin were lower in all induced cells compared with non-induced controls. In addition, cell migration was markedly inhibited, and actin stress fibers were fewer and shorter. The trypsinization time needed to round up cells was also reduced in all cell lines following Dox induction.
The barrier function of RGC5 cells was impaired and their migration was hindered upon induced expression of wild type and mutated myocilin. The reduced barrier function might be related to the declined levels of ZO-1 or occludin. The retarded cell migration was consistent with the loss of actin fibers and cell-matrix adhesiveness.
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