Controlled production of active cathepsin D in retinal pigment epithelial cells following adenovirus-mediated gene delivery.
The transduction of a low cathepsin D-producing retinal pigment epithelial cell line with a recombinant adenovirus, Ad.proCatD, carrying a viral promoter and the precursor form of the lysosomal enzyme cathepsin D, procathepsin D, led to the upregulation of proCatD expression. However, the resultant aspartic protease activity did not exceed that observed in normal primary human retinal pigment epithelial cells. Following the injection of Ad. proCatD into rat eyes, immunohistochemistry and Western blot analysis localized the expression of procathepsin D to the retinal pigment epithelial cell layer and to the sclera/choroid/retinal epithelial cell layers, respectively. This upregulation of procathepsin D expression was accompanied by a limited increase in aspartic protease activity. The injected eyes did not demonstrate any of the retinal changes that have been associated with the overproduction and secretion of active cathepsin D. Immunoelectronmicroscopy of Ad.proCatD-transduced retinal pigment epithelial cells demonstrated the presence of cathepsin D not only in cytoplasmic vesicles and lysosomes but also in the nucleoli and, less strongly, elsewhere in euchromatic regions of some 10% of cells. In spite of the upregulated expression of procathepsin D, the production of active cathepsin D in Ad.proCatD-transduced retinal pigment epithelial cells was strictly controlled. It is proposed that active cathepsin D production is controlled at the point of posttranslational modification by an intranuclear feedback mechanism initiated by the relative excess of procathepsin D in Ad. proCatD-transduced retinal pigment epithelial cells.
Lai
C M
CM
Center for Ophthalmology and Visual Science, Lions Eye Institute, Nedlands, 6009, Australia.
Robertson
T
T
Papadimitriou
J
J
Shen
W Y
WY
Daw
N
N
Constable
I J
IJ
Rakoczy
P E
PE
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002402:Cathepsin D / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D004792:Enzyme Precursors / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D005822:Genetic Vectors; D006801:Humans; D007267:Injections; D016253:Microscopy, Immunoelectron; D010587:Phagocytosis; D010857:Pigment Epithelium of Eye / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D011499:Protein Processing, Post-Translational; D051381:Rats; D014161:Transduction, Genetic; D019076:Transgenes
D004792:Enzyme Precursors; D002402:Cathepsin D