Modulation of cathepsin D activity in retinal pigment epithelial cells.
This project used retinal pigment epithelial (RPE) cells to investigate the effects of up- and down-regulation of cathepsin D expression on the processing of cathepsin D and on the normal phagocytic and digestive function of these cells. RPE cells were transfected with a pHbetaApr-1-neo vector construct carrying the full-length sequence of the translated 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 primers. 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 isothiocyanate-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 orientations after PCR amplification. Expression of both 52 kDa procathepsin D and 34 kDa active cathepsin D was significantly up-regulated in sense cathepsin D-transfected RPE cells and down-regulated in RPE cells 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 unknown specific regulatory elements. The up- and down-regulation of cathepsin D expression was further confirmed by enzyme assay. Transfected cells retained their phagocytosing ability after ROS challenge and maintained their ability to process ROS. The processing of ROS was significantly slower in RPE cells transfected with antisense than control vector or in sense-cathepsin D-transfected cells. These results demonstrate that cathepsin D is a major proteolytic enzyme participating in the lysosomal digestion of photoreceptor outer segments.
Rakoczy
P E
PE
Molecular Biology, Centre for Ophthalmology and Visual Science, Lions Eye Institute, University of Western Australia, 2 Verdun St., Nedlands 6009, WA, Australia.
Lai
C M
CM
Baines
M
M
Di Grandi
S
S
Fitton
J H
JH
Constable
I J
IJ
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D015153:Blotting, Western; D002402:Cathepsin D / Q000378:metabolism*; D002417:Cattle; D002648:Child; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006801:Humans; D010587:Phagocytosis; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000201:enzymology* / Q000276:immunology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism; D014162:Transfection
D012333:RNA, Messenger; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; D002402:Cathepsin D