May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
The Effect of Chemical Chaperone on ARPE-19 Under Oxidative Stress
Author Affiliations & Notes
  • T. C. Sauer, III
    National Eye Institute, Bethesda, Maryland
  • X. Ding
    National Eye Institute, Bethesda, Maryland
  • D. Shen
    National Eye Institute, Bethesda, Maryland
  • A. Maminishkis
    National Eye Institute, Bethesda, Maryland
  • J. Tuo
    National Eye Institute, Bethesda, Maryland
  • C.-C. Chan
    National Eye Institute, Bethesda, Maryland
  • Footnotes
    Commercial Relationships  T.C. Sauer, None; X. Ding, None; D. Shen, None; A. Maminishkis, None; J. Tuo, None; C. Chan, None.
  • Footnotes
    Support  NEI Intramural Research Program
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 5919. doi:https://doi.org/
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      T. C. Sauer, III, X. Ding, D. Shen, A. Maminishkis, J. Tuo, C.-C. Chan; The Effect of Chemical Chaperone on ARPE-19 Under Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5919. doi: https://doi.org/.

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

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Abstract

Purpose: : Endoplasmic reticulum (ER) stress, protein misfolding and subsequent aggregation have been implicated in a variety of neurodegenerative diseases including age-related macular degeneration (AMD). Oxidative stress is a potential cause of AMD and a factor in ER stress that may lead to retinal pigment epithelium (RPE) cell death and neurodegeneration. Chemical chaperones are known to promote proper protein folding and ameliorate ER stress. We assessed the effect of chemical chaperone, 4-phenyl butyric acid (PBA) on cell proliferation and ER and chaperone protein expression in ARPE-19 under oxidative stress.

Methods: : ARPE-19 cells were grown in MEME growth media to 90% confluence. Cells were treated for 24hrs with 10mM PBA, followed by 2hrs of 2mM H2O2 treatment. Controls included untreated ARPE-19 cells and cells treated with either PBA or H2O2. Cell proliferation was assessed by the reduction of yellow MTT tetrazole to purple formazan by the mitocondria of active cells as observed by spectrophotometer at 570nm. Protein and mRNA expression were assessed for the chaperone protein ERp29 and the ER stress responsive proteins GRp78(BiP) and C/EBP Homologous Protein (CHOP) by western blot and real time RT-PCR, respectively.

Results: : Cell proliferation was unaffected by PBA treatment. However, H2O2 treatment suppressed proliferation 34% whether or not combined with PBA treatment. Protein levels of GRp78, CHOP, and ERp29 increased 1.0 to 2.5-fold after both PBA and H2O2 exposure. Additionally, repeated analysis found mRNA expression to rise 1.5 to 3.0-fold after the individual treatments. But, the most significant increase in protein expression resulted from H2O2 exposure following PBA treatment. Interestingly, transcription levels did not mirror the changes in protein expression after the combined treatment.

Conclusions: : The stress response of ARPE-19 to H2O2 involves upregulation of ER-stress induced chaperone proteins. The chemical chaperone PBA can effectively increase expression of chaperones and other ER-stress responsive proteins. Although the potential role for PBA to ameliorate the long-term effects of oxidative stress in RPE is unclear, alleviating ER stress by chemical chaperone treatment may be a potential pharmacological therapy for retinal degenerative disorders, such as AMD.

Keywords: retinal pigment epithelium • chaperones • oxidation/oxidative or free radical damage 
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