MODULATION OF CATHEPSIN-D ACTIVITY IN RETINAL-PIGMENT EPITHELIAL-CELLS

This project used retinal pigment epithelial (RPE) cells to investigat e the effects of up- and down-regulation of cathepsin D expression on the processing of cathepsin D and on the normal phagocytic and digesti ve function of these cells. RPE cells were transfected with a pH beta Apr-1-neo vector construct carrying the full-length sequence of the tr anslated region of human cathepsin D in sense and antisense directions . Transfected cells were characterized for the presence and expression of the transgene by PCR amplification using transgene-specific primer s. Total aspartic proteinase activity present in transformed RPE cells was measured by an enzyme assay using haemoglobin as substrate. Flow cytometry was used to quantify phagocytosis of fluorescein isothiocyan ate-labelled rod outer segments (ROS), and lysosomal digestion of ROS was monitored by immunofluorescence. A 435 bp fragment was present in RPE cells carrying the cathepsin D transgene in sense and antisense or ientations after PCR amplification. Expression of both 52 kDa procathe psin D and 34 kDa active cathepsin D was significantly up-regulated in sense cathepsin D-transfected RPE cells and down-regulated in RPE cel ls transfected with antisense cathepsin D. No other forms of cathepsin D were detected in the transfected cells, suggesting that, if pseudo- cathepsin D exists in RPE cells in vivo, it requires the presence of u nknown specific regulatory elements. The up- and down-regulation of ca thepsin D expression was further confirmed by enzyme assay. Transfecte d cells retained their phagocytosing ability after ROS challenge and m aintained their ability to process ROS. The processing of ROS was sign ificantly slower in RPE cells transfected with antisense than control vector or in sense-cathepsin D-transfected cells. These results demons trate that cathepsin D is a major proteolytic enzyme participating in the lysosomal digestion of photoreceptor outer segments.