April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Expression Profiling of Nuclear Receptors in Human Retinal Pigment Epithelial Cells: Relevance to Age-Related Macular Degeneration
Author Affiliations & Notes
  • G. Malek
    Ophthalmology and Pathology,
    Duke University, Durham, North Carolina
  • P. Hu
    Ophthalmology, Duke University Eye Center, Durham, North Carolina
  • M. Dwyer
    Pharmacology and Cancer Biology,
    Duke University, Durham, North Carolina
  • S. W. Cousins
    Ophthalmology,
    Duke University, Durham, North Carolina
  • D. P. McDonnell
    Pharmacology and Cancer Biology,
    Duke University, Durham, North Carolina
  • Footnotes
    Commercial Relationships  G. Malek, None; P. Hu, None; M. Dwyer, None; S.W. Cousins, None; D.P. McDonnell, None.
  • Footnotes
    Support  International Retinal Research Foundation and Research to Prevent Blindness
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 5966. doi:
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      G. Malek, P. Hu, M. Dwyer, S. W. Cousins, D. P. McDonnell; Expression Profiling of Nuclear Receptors in Human Retinal Pigment Epithelial Cells: Relevance to Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5966.

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

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Abstract

Purpose: : Retinal pigment epithelial cells (RPE) play a central role in retinal physiology, forming the outer blood retinal barrier and supporting the photoreceptors. As RPE cells are affected in all forms of age-related macular degeneration (AMD), it is clear that a better understanding of the cell biology of these cells is necessary to tease out potential pathogenic pathways. Nuclear receptors (NRs) are a superfamily of ligand-dependent transcription factors, critical in regulation of differentiation, development, and many physiological processes. Currently, synthetic agonist/antagonists for NRs are used to treat asthma, diabetes, atherosclerosis and cancer. We report the systematic profiling of NRs in 3 model systems of human RPE cells and identify receptors that may be relevant to the biology of AMD.

Methods: : We used three human RPE samples: an immortalized cell line (ARPE19), primary cell lines derived from adult donors without known eye disorders, and RPE cells isolated from freshly obtained tissue. Three biological replicates were used for all experiments. In our cell culture systems, cells were grown for over 3 weeks prior to RNA isolation. Expression of the 48 human NRs was assessed by quantitative real-time PCR. The primer/probe sets for the NRs were validated in ARPE19 and primary cells based on a single peak in the dissociation curve, slope and R2 value of the standard curve plot of CT value versus cDNA quantity. Protein expression and activity of select NRs were evaluated by Western blot and luciferase based assays.

Results: : NR expression was divided into four groups: high, medium, low, or absent (CT value: less than 23, 23.1-28, 28.1-31 and greater that 31.1). In general, expression of NRs between immortalized and primary RPE cell lines were highly concordant. Expression of NRs that play a role in different aspects of AMD biology including lipid metabolism and cholesterol homeostasis (PPAR, LXR, FXR, HNF4), inflammation and complement regulation (LXR, FXR, TLX), extracellular matrix regulation (PPAR, FXR, RXR and PR) and apoptosis (TR3, TR4) were identified as medium, high or when low, protein expression was confirmed by Western blot. Evaluation of freshly isolated RPE cells from donor eyes is ongoing.

Conclusions: : Given the degree of cross-talk between some nuclear receptors and the complexity of AMD, these data should provide a unique resource to further explore the role of receptors relevant to the initiation and progression of this disease.

Keywords: transcription factors • retinal pigment epithelium • age-related macular degeneration 
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