EXPRESSION OF CATHEPSIN S ANTISENSE TRANSCRIPTS BY ADENOVIRUS IN RETINAL-PIGMENT EPITHELIAL-CELLS

Citation
Pe. Rakoczy et al., EXPRESSION OF CATHEPSIN S ANTISENSE TRANSCRIPTS BY ADENOVIRUS IN RETINAL-PIGMENT EPITHELIAL-CELLS, Investigative ophthalmology & visual science, 39(11), 1998, pp. 2095-2104
Citations number
44
Categorie Soggetti
Ophthalmology
ISSN journal
01460404
Volume
39
Issue
11
Year of publication
1998
Pages
2095 - 2104
Database
ISI
SICI code
0146-0404(1998)39:11<2095:EOCSAT>2.0.ZU;2-0
Abstract
PURPOSE. TO show the production of sense or antisense transcripts by r ecombinant adenoviruses, to investigate whether the transcripts produc ed were suitable for downregulating the expression of the targeted gen e, cathepsin S (CatS), and to examine the effect of antisense transcri pt production on the biologic function of retinal pigment epithelial ( RPE) cells, including the regulation of endogenous aspartic protease e xpression. METHODS. Ad.MLP.CatSAS, Ad.RSV.CatSAS, and Ad.MLP.CatSS rec ombinant viruses were produced by homologous recombination. The recomb inant viruses were tested by restriction enzyme digestion to confirm t he orientation of the inserts. The expression of antisense transcripts was tested by not-them blot analysis. Western blot analysis was used to study the regulation of the endogenous CatS protein in ARPE19 cells . The biologic effect of CatS downregulation in ARPE19 cells was teste d by proliferation and phagocytosis assays, de novo cathepsin D (CatD) synthesis, and measurement of aspartic protease activity. RESULTS. Af ter characterization of the recombinant adenovirus constructs, the pro duction of antisense and sense CatS transcripts was shown in ARPE19 ce lls. The transcripts appeared at approximately 1.9 kb 48 hours after t ransduction, and the expression of the antisense transcripts was simil ar in constructs carrying either the MLP or the RSV promoter. Western blot analysis showed that ARPE19 cells transduced with Ad.MLP.CatSAS a nd Ad.RSV.CatSAS had no detectable CatS. In contrast, there was a stro ng signal appearing at 24 kDa in ARPE19 cells transduced with Ad.MLP.C atSS. ARPE19 cells were transduced to a high level. The transduction o f ARPE19 cells with the recombinant adenoviruses did not affect the mo rphologic appearance of the cells, their proliferation, or their phago cytosing ability. However, ARPE19 cells transduced by Ad.MLP.CatSAS re combinant adenoviruses showed a significant downregulation of de novo CatD synthesis and a twofold decrease in aspartic protease activity. C ONCLUSIONS. Recombinant adenoviruses were shown to be suitable for pro ducing antisense CatS transcripts to modulate endogenous CatS expressi on in RPE cells. It is proposed that CatS may play an important role, directly or indirectly, in the lysosomal digestion of outer segments t hrough the regulation of other lysosomal enzyme activity, such as the expression of CatD.