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Expression of cathepsin S antisense transcripts by adenovirus in retinal pigment epithelial cells
P E Rakoczy1, M C Lai, M G Baines
1Department of Molecular Biology, Centre for Ophthalmology and Visual Science, University of Western Australia, Nedlands, Australia.
Purpose:
To show the production of sense or antisense transcripts by recombinant adenoviruses, to investigate whether the transcripts produced were suitable for downregulating the expression of the targeted gene, cathepsin S (CatS), and to examine the effect of antisense transcript production on the biologic function of retinal pigment epithelial (RPE) cells, including the regulation of endogenous aspartic protease expression.
Methods:
Ad.MLP.CatSAS, Ad.RSV.CatSAS, and Ad.MLP.CatSS recombinant viruses were produced by homologous recombination. The recombinant viruses were tested by restriction enzyme digestion to confirm the orientation of the inserts. The expression of antisense transcripts was tested by northern 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 tested by proliferation and phagocytosis assays, de novo cathepsin D (CatD) synthesis, and measurement of aspartic protease activity.
Results:
After characterization of the recombinant adenovirus constructs, the production of antisense and sense CatS transcripts was shown in ARPE19 cells. The transcripts appeared at approximately 1.9 kb 48 hours after transduction, and the expression of the antisense transcripts was similar in constructs carrying either the MLP or the RSV promoter. Western blot analysis showed that ARPE19 cells transduced with Ad.MLP.CatSAS and Ad.RSV.CatSAS had no detectable CatS. In contrast, there was a strong signal appearing at 24 kDa in ARPE19 cells transduced with Ad.MLP.CatSS. ARPE19 cells were transduced to a high level. The transduction of ARPE19 cells with the recombinant adenoviruses did not affect the morphologic appearance of the cells, their proliferation, or their phagocytosing ability. However, ARPE19 cells transduced by Ad.MLP.CatSAS recombinant adenovirus showed a significant downregulation of de novo CatD synthesis and a twofold decrease in aspartic protease activity.
Conclusions:
Recombinant adenoviruses were shown to be suitable for producing antisense CatS transcripts to modulate endogenous CatS expression in RPE cells. It is proposed that CatS may play an important role, directly or indirectly, in the lysosomal digestion of outer segments through the regulation of other lysosomal enzyme activity, such as the expression of CatD.
Insights
Recombinant adenoviruses effectively produced antisense transcripts to downregulate cathepsin S (CatS) in retinal pigment epithelial cells. This approach modulated CatS expression and affected other lysosomal enzymes like cathepsin D (CatD).
Area of Science:
- Molecular Biology
- Gene Expression Regulation
- Cell Biology
Background:
- Retinal Pigment Epithelial (RPE) cells are crucial for retinal health.
- Cathepsin S (CatS) is an aspartic protease implicated in cellular processes.
- Understanding CatS regulation in RPE cells is important for ocular health research.
Purpose of the Study:
- To demonstrate recombinant adenovirus-mediated production of sense and antisense transcripts.
- To assess the efficacy of these transcripts in downregulating CatS expression.
- To investigate the impact of CatS downregulation on RPE cell function and other protease activities.
Main Methods:
- Production of recombinant adenoviruses (Ad.MLP.CatSAS, Ad.RSV.CatSAS, Ad.MLP.CatSS).
- Confirmation of viral constructs via restriction enzyme digestion.
- Analysis of antisense transcript expression using Northern blot.
- Assessment of CatS protein levels via Western blot in ARPE19 cells.
- Evaluation of RPE cell biologic functions (proliferation, phagocytosis) and aspartic protease activity.
Main Results:
- Successful production of antisense and sense CatS transcripts in ARPE19 cells.
- Significant reduction of detectable CatS protein in cells transduced with antisense constructs.
- No adverse effects on RPE cell morphology, proliferation, or phagocytosis.
- Downregulation of de novo CatD synthesis and a decrease in overall aspartic protease activity.
Conclusions:
- Recombinant adenoviruses are effective tools for generating antisense CatS transcripts to modulate gene expression in RPE cells.
- CatS may play a regulatory role in lysosomal digestion, potentially influencing other lysosomal enzymes like CatD.