April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
Activation of TLR3 by 21nt-siRNA and Intracellular Trafficking of the Human Toll-like Receptor 3 Upon 21nt-siRNA Stimulation
Author Affiliations & Notes
  • W. G. Cho
    Ophthal & Visual Science, University of Kentucky, Lexington, Kentucky
  • M. E. Kleinman
    Ophthal & Visual Science, University of Kentucky, Lexington, Kentucky
  • R. J. Albuquergue
    Ophthal & Visual Science, University of Kentucky, Lexington, Kentucky
  • J. Z. Baffi
    Ophthal & Visual Science, University of Kentucky, Lexington, Kentucky
  • H. Kaneko
    Ophthal & Visual Science, University of Kentucky, Lexington, Kentucky
  • K. Saito
    Ophthal & Visual Science, University of Kentucky, Lexington, Kentucky
  • M. G. Rich
    Ophthal & Visual Science, University of Kentucky, Lexington, Kentucky
  • J. Ambati
    Ophthal & Visual Science, University of Kentucky, Lexington, Kentucky
  • Footnotes
    Commercial Relationships  W.G. Cho, None; M.E. Kleinman, None; R.J. Albuquergue, None; J.Z. Baffi, None; H. Kaneko, None; K. Saito, None; M.G. Rich, None; J. Ambati, Allergen, C; Novatis, C; Quark, C; University of Kentucky, P.
  • Footnotes
    Support  NEI/NIH, Doris Duke Distinguished Clinical Scientist Award, Burroughs Wellcome Fund Clinical Scientist Award in Translational Research, Dr. E. Vernon Smith and Eloise C. Smith Macular Degeneration End
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 1189. doi:
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      W. G. Cho, M. E. Kleinman, R. J. Albuquergue, J. Z. Baffi, H. Kaneko, K. Saito, M. G. Rich, J. Ambati; Activation of TLR3 by 21nt-siRNA and Intracellular Trafficking of the Human Toll-like Receptor 3 Upon 21nt-siRNA Stimulation. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1189.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Toll-like receptor 3 (TLR3) is a double-stranded RNA (dsRNA) sensor that mediates an anti-viral innate immune response. Recently, 21nt-siRNA has been reported as a TLR3 agonist (Kleinman et al. Nature 2008), but details of how 21nt-siRNA binds or activates TLR3 remains to be elucidated. How 21nt-siRNA differentially engages extracellular and intracellular TLR3, what mechanisms drive internalization of 21nt-siRNA conjugated to cholesterol (a cell-permeating moiety), and how TLR3 traffics in the cell after siRNA exposure are equally unclear. We sought to mechanistically dissect these questions.

Methods: : Primary human RPE cells were treated with naked or cholesterol-conjugated 21nt-siRNA.TLR3 activation was monitored by western blotting using phospho-specific antibody. Cellular uptake of fluorescein-conjugated siRNA (Fl-siRNA) with or without cholesterol conjugation was visualized by time lapse confocal live imaging. Intracellular trafficking and subcellular localization of Fl-21nt-siRNA and GFP-tagged TLR3 were visualized by immunofluorescent confocal microscopy.

Results: : Fl-21nt-siRNA was internalization within 30 minutes if it was conjugated to cholesterol but not if it was unmodified. Yet naked 21-nt siRNA induced cell surface TLR3 phosphorylation. Fl-siRNA-chol internalization requires scavenger receptors and involves vesicular transport. GFP-tagged TLR3 trafficked to different subcellular locations depending on whether it ligated extracellular or internalized 21-nt siRNA.

Keywords: retinal pigment epithelium • RNAi • receptors 
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