GENE SILENCING PART II 

Minerva Biotecnologica 2008 June;20(2):59-67

Copyright © 2008 EDIZIONI MINERVA MEDICA

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Non-redundant roles of cathepsins L, B and S in CD1a+ dendritic cells knocked-down for cathepsin S by RNA interference

Tiribuzi R., Martino S., Ciraci E., D’Angelo F., Di Girolamo I., Datti A., Bottazzo G. F., Berardi A. C., Orlacchio A.

¹ Section of Molecular Biology and Biochemistry Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy 2 Stem Cells Research Laboratory, IRCCS Bambino Gesù Pediatric Hospital, Rome, Italy

Aim. The ability to modulate the functional properties of dendritic cells (DCs) with chemical drugs or via RNA-based technologies may lead to significant therapeutic applications. In light of their relevance to the biology of DCs, particularly within the antigen-presentation pathway, cysteine cathepsins L, B and S were investigated with the long-term objective of assessing their value as molecular target.
Methods. Cathepsin expression was monitored via a cell-based model in which human, CD34+hematopoietic stem cells (HSCs) were induced to differentiate into CD1a+DCs. Time-course analyses were performed via Real time RT-PCR and Western blotting. The same experiments were conducted, in parallel, using HSCs subjected to cathepsin S knockdown by RNA interference.
Results. Processing of cathepsins L, B and S is subjected to temporal patterns of expression throughout DC differentiation (a 14 day process). The mature form of cathepsin S appeared in the lysosomal fraction on day 7, while mature cathepsin L and B proteins displayed such localization only upon completion of differentiation (day 14). The non-redundant roles of these cathepsins were evident on day 7, as CatS-RNAi-mediated knockdown cells were found to show a marked decrease of HLA-DR expression.
Conclusion. Cathepsins L, B and S are subjected to a temporal regulation of expression that is apparently associated with the progress of the differentiation process. CD1a+DCs knocked-down for cathepsin S show the non-redundant roles of cathepsin L, B and S within the DC maturation pathway and highlight the potential of cathepsin S as a molecular target for drug discovery research.