Purchase this article with an account.
O. Nichini, S. Cottet, T. Favez, N. Voirol, C. Ruegg, D.F. Schorderet; Impaired Viability, Morphology and Cell–Surface Expression of Integrins in the Retinal Pigment Epithelial Cell Line ARPE–19 Showing siRNA–Targeted Silencing of Rpe65 Protein . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2321.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
RPE65 protein, preferentially expressed in RPE cells, is required to maintain physiological functions of the RPE and retina. It is notable that mutations in RPE–expressed genes of the visual cycle can cause diverse forms of retinal dystrophies. However, the molecular mechanisms by which mutations in RPE65 contribute to retinal diseases remain largely unresolved, affected patients showing marked RPE damage and photoreceptor degeneration. Photoreceptor cell death can be caused by any changes that alter the signal transduction cascade, influence the energy metabolism or disturb the structural and functional interactions between the RPE and the photoreceptors.
The human RPE cell line, ARPE–19, has proven to be an advantageous cell model to study retinal pigment epithelium physiology because it retains many RPE–specific morphologic and biochemical markers. To evaluate RPE65–dependent cellular defects in the RPE, siRNA–targeted RPE65 expression was knockdowned in ARPE–19 cells. Cells were transduced with recombinant lentiviruses containing control– and RPE65 siRNA– constructs and the decrease in RPE65 level was assessed by real–time RT–PCR. Modulation of the expression of integrins at the cell surface was assessed by FACS analysis. Cell viability was assessed in control– and siRNA–expressing RPE cells.
Quantitative RT–PCR experiments showed siRNA–mediated reduction of RPE65 gene expression by 70 %. As observed by FACS analysis, cell–surface expression of integrins, among them αvß5, was significantly reduced in siRNA–expressing cells as compared to control ARPE–19 cells. A significant decrease in cell viability was also specifically observed in retinal epithelium cells expressing RPE65 siRNA.
Lentiviral–mediated expression of target siRNA leads to efficient knockdown of human RPE65. Downregulation of RPE65 is correlated with impaired cell–surface expression of integrins and decreased RPE cell viability. ARPE–19 cells stably expressing RPE65 siRNA represent a valuable cellular model for detailed studies of putative unraveled functions of RPE65 protein besides its intrinsic isomerase activity and point out intracellular pathways that may impact on RPE function in the absence of RPE65.
This PDF is available to Subscribers Only