Original contributionMicroarray analysis of H2O2-, HNE-, or tBH-treated ARPE-19 cells
Introduction
Oxidative stress has been shown to play a role in normal aging of the posterior pole of the human eye as well as the pathogenesis of age-related macular degeneration (AMD). The proposed role of oxidative stress in AMD is similar to the role proposed in several neurodegenerative diseases, such as amyotrophic lateral sclerosis, Alzheimer’s disease, and Parkinson’s disease.
The oxidative stress response of individual cell types has been studied in vitro. The oxidative stress response can be induced in vitro using a variety of oxidants, including hydrogen peroxide (H2O2), tert-butylhydroperoxide (tBH), 4-hydroxynonenal (HNE), sodium arsenite, and various oxygen tensions, as well as UV irradiation (reviewed in [1]). H2O2, HNE, and tBH, in particular, are metabolized by overlapping as well as distinct enzymatic pathways, and as such are useful for investigating the complex and common transcriptional response to oxidative stress in vitro. In the cell, catalase is the predominant enzyme involved in H2O2 metabolism, although both glutathione peroxidase and molecular iron can eliminate H2O2 as well [2]. Inactivation of tBH is predominantly accomplished by glutathione peroxidase [3]. In contrast, HNE can be metabolized by aldehyde dehydrogenases, alcohol dehydrogenases, aldose reductase, or through conjugation with glutathione by glutathione transferases 4, 5.
The transcriptional response to oxidative stress has been studied extensively in a variety of cell types in vitro. Several genes involved have been identified, including members of the DNA repair, apoptosis, transcriptional regulation, and signal transduction pathways (for review, 1, 6, 7, 8, 9, 10). Some previous studies in RPE cells in vitro have focused on the metallothioneins, heme oxygenase-1, thioredoxin, catalase, superoxide dismutase, gluthatione peroxidase, and reductase, vascular endothelial growth factor, pigment epithelium-derived factor, lens epithelium-derived growth factor, and FGFs and their receptors 11, 12, 13, 14, 15, 16, 17. These previous studies have been limited in that they generally examine the response of a small number of genes to a single oxidant rather than defining a comprehensive response. In this study we sought to use a single well-defined cell line exposed to three different oxidants with thorough evaluation of a large number of genes by microarray analysis. The human retinal pigmented epithelium cell line ARPE-19 was chosen for this study because it is well characterized and has many differentiated properties [18]. In addition, the RPE may play a primary role in the pathogenesis of AMD.
Section snippets
Cell culture
The human retinal pigmented epithelium (RPE) cell line ARPE-19 was maintained in DMEM/F12 (BioWhittaker, Walkersville, MD, USA) with 15 mM HEPES buffer, 2 mM L-glutamine, 0.348% sodium bicarbonate, and 10% FBS (BioWhittaker) [18]. Cells were used between passages 16 and 18. The cells were plated onto 150 cm2 tissue culture flasks at confluence (100,000 cells/cm2). After 3 d, the cells were placed in medium without FBS to achieve quiescence [19]. On day two of serum withdrawal, the medium was
Determination of optimum doses for microarray analysis
Experiments were designed to determine the oxidant dose that preserved maximal cell survival while simultaneously producing the highest induction of heme oxygenase 1 (HO-1), a well-known indicator of cellular oxidative stress 27, 28. Cells were treated with increasing doses of each of the oxidants for 4 h and viability determined. Northern analysis was used to determine the maximal induction of the HO-1 gene at increasing doses of the three separate oxidants after 4 h of treatment [29].
Discussion
This study’s hypothesis stated that when exposed to different oxidants the human retinal pigmented epithelium cell line ARPE-19 would show a common transcriptional response even though each oxidant has its own specific degradation pathway. To address this question, microarray analysis examined nearly 1200 genes after cells were exposed to H2O2, HNE, or tBH. Control and treated replicates for each oxidant were compared in a pairwise analysis using the recently described Significance Analysis of
Abbreviations
AMD—age-related macular degeneration
ARE—antioxidant response element
Cu,Zn-SOD—copper, zinc superoxide dismutase (cytosolic SOD)
FDR—false discovery rate
FGF—fibroblast growth factor
GST—glutathione S-transferase
H2O2—hydrogen peroxide
HNE—4-hydroxynonenal
HO-1—heme oxygenase 1
IAP3—inhibitor of apoptosis protein 3
IGFBP2—insulin-like growth factor binding protein 2
MnSOD—manganese superoxide dismutase (mitochondrial SOD)
MPTP—N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
ROI—reactive oxygen intermediate
Acknowledgements
This work was supported by: NIH/EY06473 (L.M.H.), NIH/EY14005 (J.T.H.), Research to Prevent Blindness Manpower Award (J.T.H.), and an unrestricted grant from Research to Prevent Blindness (Department of Ophthalmology, University of California, Davis). Supplementary data not published here will be available at the authors’ website, http://www.mcb.ucdavis.edu/faculty-labs/hjelmeland/. We would like to thank Dr. Sharon Boylan for helpful discussions and critical reading of the manuscript.
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