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D. Simic, D. Baltrukonis, S. Sokolowski, C. Somps; Effect of Reduced Cathepsin D on Phagocytosis of Rat RPE Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5535.
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© ARVO (1962-2015); The Authors (2016-present)
The aspartyl protease, Cathepsin–D, is a lysosomal enzyme expressed in the retinal pigmented epithelium (RPE) and is believed to play a role in the digestion of outer segments shed from retinal rods and cones. The purpose of the present study was to determine if a reduction in Cathepsin D (Cat–D) activity causes a change in the phagocytosis of rod outer segments (ROS) by rat RPE cells.
Cells from the RPE–J cell line were plated in 12–well dishes (3.5cm2), incubated in normal media (high glucose DMEM w/10% FBS and 1% L–Glutamine) until ∼90% confluent. Cells were then treated with either a non–cytotoxic dose (30 uM) of Pepstatin A (a non–specific aspartyl protease inhibitor) for 24, 48 or 72h, or with Cat–D specific siRNA (25nM) for 6 hrs followed by normal media for total incubation times of 24, 48, or 72h. RNAi–transfected cells were analyzed for Cat–D knockdown by RT–PCR and Western blot. Treated and control cells were assessed for their ability to phagocytose FITC–labeled, bovine rod outer segments (ROS) during a 6h challenge using flow cytometry at 24h, 48h and 72h.
Under normal conditions 50–70% of RPE–J cells phagocytose the FITC–labeled ROS. Treatment with 30 uM of the nonspecific Cat–D inhibitor, Pepstatin A, had no effect on phagocytosis of ROS by RPE–J cells. Cells transfected with Cat–D specific siRNA showed a 70–80% decrease in the amount of Cathepsin D message 24 and 48h post transfection, with some recovery by 72h. Reductions in Cat–D expression with RNAi were confirmed with Western blot. Consistent with the pepstatin A results, specific reduction in CAT–D by RNAi did not result in a change in the ability of RPE–J cells to phagocytose ROS.
Significant reduction in Cathepsin D activity, sustained over 3 days using either non–specific pharmacologic inhibition or specific RNAi knockdown of the enzyme, does not affect RPE–J phagocytosis of ROS. This finding suggests that a large, but incomplete, inhibition of cathepsin D activity will not cause changes in RPE phagocytosis in vivo.
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