May 2006
Volume 47, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2006
Silencing of Sigma–1 Receptor Induces Cell Death in Human Lens Cells
Author Affiliations & Notes
  • L. Wang
    School of Biological Sciences, University of East Anglia, Norwich, United Kingdom
  • G. Duncan
    School of Biological Sciences, University of East Anglia, Norwich, United Kingdom
  • M. Handsley
    School of Biological Sciences, University of East Anglia, Norwich, United Kingdom
  • J.R. Reddan
    Biological Sciences, Oakland University, Rochester, MI
  • Footnotes
    Commercial Relationships  L. Wang, None; G. Duncan, None; M. Handsley, None; J.R. Reddan, None.
  • Footnotes
    Support  The Humane Research Trust
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 3517. doi:
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      L. Wang, G. Duncan, M. Handsley, J.R. Reddan; Silencing of Sigma–1 Receptor Induces Cell Death in Human Lens Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3517.

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

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Abstract

Purpose: : Sigma receptors have no known homology with other receptor systems, have no known natural ligands, but appear to play a critical role in a large diversity of cell functions. In the absence of a conventional pharmacology, siRNA technology provides a direct means of elucidating the major cell signalling pathways influenced by this receptor system.

Methods: : The non–transformed human lens cell line FHL124 expresses the sigma–1 receptor (Sig–1R) and was employed for these studies. FHL124 cells were maintained in serum–free Eagle’s minimum essential medium (EMEM) or EMEM supplemented with 5% fetal calf serum (FCS) at 350C in 5% CO2. Two independent Sig–1R siRNAs and universal scrambled control siRNA were designed and generated by Ambion and cells were transfected in standard Oligofectamine medium. Cell growth was assessed by measuring total protein and cell death by quantifying LDH leakage. Expression of the Sig–1R was assayed by RT–PCR. Western immunoblot techniques were employed to measure levels of Sig–1R protein, pERK/ERK, pAkt/Akt and caspase–3.

Results: : 72 hours of transfection with either of the two siRNAs directed against Sig–1R reduced messenger RNA and protein levels by over 70 and 60% respectively. Subsequent incubation for 96 hours in culture medium (EMEM) supplemented with 5% serum gave a partial recovery of message, but there was no significant increase in protein expression. LDH leakage assays showed that significant cell death occurred during this time accompanied by an increased expression of caspase–3. Thrombin (10 nM) stimulated cell growth with an accompanying increase in the level of ERK and Akt phosphorylation. These increases were inhibited in the cells where knock down had occurred, but not in cells exposed to scrambled control siRNA.

Conclusions: : This study establishes a central role for the Sig–1R in lens cell death and survival through modulating the activity of the ERK, Akt and caspase pathways.

Keywords: apoptosis/cell death • cell survival • signal transduction 
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