Elsevier

Free Radical Biology and Medicine

Volume 33, Issue 10, 15 November 2002, Pages 1419-1432
Free Radical Biology and Medicine

Original contribution
Microarray analysis of H2O2-, HNE-, or tBH-treated ARPE-19 cells

https://doi.org/10.1016/S0891-5849(02)01082-1Get rights and content

Abstract

Oxidative stress plays a key role in aging diseases of the posterior pole of the eye such as age-related macular degeneration. The oxidative stress response of in vitro RPE cells has been studied for a small number of genes. However, a comprehensive transcriptional response has yet to be elucidated. The purpose of this study was to determine if the transcription of a common set of genes is altered by exposure of ARPE-19 cells to three major generators of oxidative stress, hydrogen peroxide (H2O2), 4-hydroxynonenal (HNE), and tert-butylhydroperoxide (tBH). As expected, a common response was observed that included 35 genes differentially regulated by all three treatments. Of these, only one gene was upregulated, and only by one oxidant, while all other responses were downregulation. The majority of these genes fell into five functional categories: apoptosis, cell cycle regulation, cell-cell communication, signal transduction, and transcriptional regulation. Additionally, a large number of genes were differentially regulated by one oxidant only, including the majority of the conventional oxidative stress response genes present on the Clontech Human 1.2 microarray. This study raises questions regarding the generality of results that involve the use of a single oxidant and a single cell culture condition.

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.

References (47)

  • J. Li et al.

    Microarray analysis reveals an antioxidant responsive element-driven gene set involved in conferring protection from an oxidative stress-induced apoptosis in IMR-32 cells

    J. Biol. Chem.

    (2002)
  • Z. Li et al.

    Genes regulated in human breast cancer cells overexpressing manganese-containing superoxide dismutase

    Free Radic. Biol. Med.

    (2001)
  • J.D. Moehlenkamp et al.

    Activation of antioxidant/electrophile-responsive elements in IMR-32 human neuroblastoma cells

    Arch. Biochem. Biophys.

    (1999)
  • W.R. Pearson et al.

    Increased synthesis of glutathione S-transferases in response to anticarcinogenic antioxidants. Cloning and measurement of messenger RNA

    J. Biol. Chem.

    (1983)
  • M.B. Yim et al.

    Enzyme function of copper, zinc superoxide dismutase as a free radical generator

    J. Biol. Chem.

    (1993)
  • B. Chance et al.

    Hydroperoxide metabolism in mammalian organs

    Physiol. Rev.

    (1979)
  • C.Y. Chu et al.

    Protective effects of capillarisin on tert-butylhydroperoxide-induced oxidative damage in rat primary hepatocytes

    Arch. Toxicol.

    (1999)
  • K. Irani et al.

    Ras, superoxide and signal transduction

    Biochem. Pharmacol.

    (1998)
  • M. Wada et al.

    Density-dependent expression of FGF-2 in response to oxidative stress in RPE cells in vitro

    Curr. Eye Res.

    (2001)
  • K. Ohno-Matsui et al.

    Novel mechanism for age-related macular degenerationan equilibrium shift between the angiogenesis factors VEGF and PEDF

    J. Cell. Physiol.

    (2001)
  • H. Matsui et al.

    Lens epithelium-derived growth factorincreased survival and decreased DNA breakage of human RPE cells induced by oxidative stress

    Invest. Ophthalmol. Vis. Sci.

    (2001)
  • M. Alizadeh et al.

    Expression and splicing of FGF receptor mRNAs during APRE-19 cell differentiation in vitro

    Invest. Ophthalmol. Vis. Sci.

    (2000)
  • S.S. Singhal et al.

    Induction of glutathione S-transferase hGST 5.8 is an early response to oxidative stress in RPE cells

    Invest. Ophthalmol. Vis. Sci.

    (1999)
  • Cited by (90)

    View all citing articles on Scopus
    View full text