Early changes in gene expression induced by blue light irradiation of A2E-laden retinal pigment epithelial cells

Purpose: Accumulation of bisretinoids as lipofuscin in retinal pigment epithelial (RPE) cells is implicated in the pathogenesis of some blinding diseases including age-related macular degeneration (AMD). To identify genes whose expression may change under conditions of bisretinoid accumulation, we investigated the differential gene expression in RPE cells that had accumulated the lipofuscin fluorophore A2E and were exposed to blue light (430 nm). Methods: A2E-laden RPE cells were exposed to blue light (A2E/430 nm) at various time intervals. Cell death was quantified using Dead Red staining, and RNA levels for the entire genome was determined using DNA microarrays (Affymetrix GeneChip Human Genome 2.0 Plus). Array results for selected genes were confirmed by real-time reverse-transcriptase polymerase chain reaction. Results: Principal component analysis revealed that the A2E-laden RPE cells irradiated with blue light were clearly distinguishable from the control samples. We found differential regulation of genes belonging to the following functional groups: transcription factors, stress response, apoptosis and immune response. Among the last mentioned were downregulation of four genes that coded for proteins that have an inhibitory effect on the complement cascade: (complement factor H, complement factor H-related 1, complement factor I and vitronectin) and of two belonging to the classical pathway (complement component 1, s subcomponent and complement component 1, r subcomponent). Conclusion: This study demonstrates that blue light irradiation of A2E-laden RPE cells can alter the transcription of genes belonging to different functional pathways including stress response, apoptosis and the immune response. We suggest that these molecules may be associated to the pathogenesis of AMD and can potentially serve as future therapeutic targets. Bisretinoid adducts accumulate in retinal pigm'ent epithelial (RPE) cells with age and constitute the autofluorescent lipofuscin of the cells. The lipofuscin deposits of RPE are primarily derivatives of vitamin A that originate in photoreceptor cells and are deposited in RPE as components of phagocytized photoreceptor outer segments. RPE lipofuscin can be monitored in vivo as fundus autofluorescence and may be associated with retinal degenerative diseases such as best macular dystrophy, recessive Stargardt disease and age-related macular degeneration (AMD; Sparrow & Boulton 2005; Sparrow 2007). Numerous fluorophores have been detected in RPE lipofuscin such as the pyridinium bis-retinoid A2E and isoA2E (Eldred & Lasky 1993; Parish et al. 1998), oxidized derivatives of A2E, conjugates of the all-trans-retinal dimer and trans-retinal dimer-phosphatidylethanolamine (atRAL dimer-PE; Kim et al. 2007; Sparrow 2007) and A-aldehyde dihydropyridine-phosphatidylethanolamine (A2-DHP-PE; Wu et al. 2009). Of these, the best characterized is A2E, an amphiphilic molecule that contains a quaternary amine nitrogen and that forms from two molecules of all-trans-retinal and one of phosphatidylethanolamine; the latter is a component of photoreceptor outer segment membranes (Liu et al. 2000). A2E is photoreactive, and blue light exposure of intracellular accumulated A2E leads to singlet oxygen production. The formed singlet oxygen adds to the carbon–carbon-double bonds of the retinoid derived side-arms of the A2E molecule resulting in formation of oxygen-containing moieties (Ben-Shabat et al. 2002; Sparrow et al. 2002; Jang et al. 2005). The oxygen-containing groups in photooxidized A2E include epoxides that rearrange to furanoid structures and cyclic peroxides, which are highly reactive and therefore suspected to be cytotoxic (Ben-Shabat et al. 2002; Dillon et al. 2004; Jang et al. 2005). It is well established that exposure of cultured A2E-laden RPE cells to blue light (430 nm) results in cell death (Schutt et al. 2000; Sparrow et al. 2000; Davies et al. 2001; Sparrow & Cai 2001; Westlund et al. 2009). This process has been shown to be dependent on the formation of reactive oxygen species (Sparrow et al. 2002). Of particular importance is the finding that A2E-laden RPE cells irradiated at 430 nm as well as photooxidized forms of A2E tested in a noncellular assay can activate the complement system (Zhou et al. 2006, 2009). Studies indicate that complement activation by this mechanism may be dependent on the alternative pathway (Zhou et al. 2009). Age-related macular degeneration is considered to be a multifactorial disease with many contributing factors and mechanisms working together in a complicated interplay. Different elements, for instance, complement dysregulation and oxidative mechanisms are suspected to play a role in the pathogenesis (Beatty et al. 2000; Holz et al. 2001; Anderson et al. 2002; Hageman et al. 2005). The role of photooxidized A2E in retinal disorders is far from fully elucidated. Using microarrays, we sought to clarify how intracellular A2E-accumulation and exposure to blue light affects gene expression. Studying changes in gene expression can provide important information about pathways that might be implicated in RPE cell death and provide clues to other cellular processes that might be influenced by photooxidative process in RPE. Cells were treated as previously described (Sparrow et al. 1999, 2000, 2002). In short, A2E was synthesized from all-trans-retinal and ethanolamine (2:1; Parish et al. 1998). A human adult RPE cell line (ARPE-19; American Type Culture Collection, Manassas, VA, USA) lacking endogenous A2E (Sparrow et al. 1999) was grown to confluence in 35-mm dishes in cell culture medium consisting of Dulbecco’s Modified Eagle Medium (DME; Gibco, Gaithersburg, MD, USA) with 10% heat-inactivated fetal calf serum (Hyclone, Logan, UT, USA), 2 mm glutamine (Gibco), 0.1 mm minimum essential medium nonessential amino acids solution (Gibco) and gentamicin sulphate (10 μg/ml). Subsequently, cells were incubated with synthesized A2E in cell culture media at a concentration of 10 μm to allow A2E accumulation; the latter was confirmed by light microscopy. Culture medium was replaced with Dulbecco’s phosphate-buffered saline containing calcium, magnesium (Gibco) and 5.5 mm glucose, and cells grown for a minimum of 4 weeks were irradiated with blue light (430 ± 30 nm 1 mW/cm2, 6, 10, 15 or 20 min exposure) followed by 6 hr incubation. These wavelengths are consistent with the excitation spectrum of A2E. (Parish et al. 1998; Sparrow et al. 2000) The time intervals were chosen to ensure both sublethal and lethal exposure to blue light of the A2E-laden RPE cells. Controls included untreated, A2E-free RPE cells, A2E-free RPE cells irradiated for 20 min and RPE cells that had accumulated A2E but were not exposed to blue light. The experiment was repeated in triplicates. The three experiments were performed on subsequent passages of the ARPE-19 cells and therefore in the same passage intervals. Cell viability was quantified 8 hr after light exposure by labelling nuclei of nonviable cells with the membrane impermeant dye Dead Red (Molecular Probes, Eugene, OR, USA) and the nuclei of all cells with 4′,6′-diamino-2-phenylindole (DAPI), as previously described (Sparrow et al. 2002). Total RNA was extracted using NucleoSpin® RNA II (Macherey-Nagel GmbH, Düren, Germany) according to the manufacturer’s protocol. The genome-wide Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA: http://www.affymetrix.com) representing more than 54 000 probe sets translating into approximately 47 000 transcripts and 38 500 well-characterized genes were used. Processing of the Affymetrix GeneChips® were performed by the RH Microarray Centre at Rigshospitalet (Copenhagen, Denmark), following the guidelines from Affymetrix. Summation and normalization of Probe set expression measures were carried out using the Robust Multichip Average (RMA) method of background correction, quantile normalization and summarization of signal intensity (Irizarry et al. 2003). Calculations were made using the statistical software environment r (http://www.R-project.org). Principal component analysis (PCA), functional annotation for biological processes and identification of overrepresentation of potential transcription factor binding sites were calculated using r functions developed by the authors as previously described (Hansen et al. 2012). In brief, the 2.5% of genes contributing the most to the first principal component were identified and overrepresentation of gene ontology (GO) terms for biological processes and of predicted transcription factor binding sites were calculated using Fisher’s exact test for proportions followed by correction for multiple testing using the Bonferroni procedure. Student’s t-test was used to compare RPE cell cultures analysed at different time points. Data were pooled for groups where no statistically significance was observed. Microarray data are reported according to the MIAME statement (Brazma et al. 2001). Several differentially regulated genes with relation to the immune system or cell death were selected and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was performed for these candidate genes to validate the results obtained from the microarrays. cDNA was produced from 2 μg of each original RNA sample using the ‘Revertaid First Strand Synthesis’ kit (Fermentas, Burlington, ON, Canada). For the qPCR reaction, a Brilliant SYBR Green QPCR Mastermix was used according to the manufacturer’s instructions (Stratagene; AH Diagnostics, Aarhus, Denmark). The reactions were run on the Stratagene Mx3000P. Reactions were performed in triplicates plus a control without reverse-transcriptase and a control without template. Table 1 shows primer sequences used. To account for differences in the amount of total RNA added to each reaction, the housekeeping gene GAPDH was used as internal amplification control. Melting curves were routinely checked to rule out the amplification of unrelated fragments during qRT-PCR. Pfaffl’s method (Pfaffl 2001) was used to calculate the relative gene expression, and mean and Standard Error of Mean (SEM) of the relative quantity of target mRNA were calculated for all groups (n = 3). Means for all experimental groups were compared with untreated controls by one-way anova and the Newman–Keul Multiple Comparison test (Prism; GraphPad Software, San Diego, CA, USA). Values were considered statistical significantly different when p < 0.05. Retinal pigment epithelial cells that had accumulated A2E were exposed to blue light (A2E/430 nm) for 6, 10, 15 and 20 min. Cell death was measured after 8 hr by colabelling all cells with DAPI and nuclei of dead cells with a membrane impermeant dye. In A2E-containing RPE cells irradiated for 6 or 10 min, <1% of the cells were nonviable. The rate of cell death increased to 5.1% (±3.4%, p > 0.05) and 16.3% (±3.6%, p < 0.001) for A2E-laden RPE cells irradiated for 15 and 20 min, respectively. Cell death did not occur in any of the control groups (Fig. 1A). Cell death and changes in the gene expression profile induced by blue light exposure of A2E-laden retinal pigment epithelial (RPE) cells were more pronounced with longer exposure time. A2E-laden RPE cells were exposed to blue light for 6, 10, 15 and 20 min. Untreated A2E-free RPE cells, A2E-free RPE cells irradiated for 20 min and A2E-laden RPE cells that had not been exposed to blue light served as controls. The experiment was repeated three times. Cell death was quantified, and RNA was extracted for microarray analysis in each experiment. (A) Per cent of nonviable cells was determined by labelling nuclei in death cells with death red staining and all nuclei with DAPI. Mean ± SEM of three experiments, *p < 0.001, + presence of condition + presence of The gene expression data from the microarrays were analysed by principal component analysis principal components were and the of the were in a using the first and principal component The first most of the in the (RPE) to A2E-free RPE cells and + to A2E-laden RPE cells. The of blue light exposure is in 10, RPE + A2E RPE RPE + A2E 6, RPE + A2E 10 RPE + A2E RPE + A2E It is the that many genes change expression in a in the A2E-laden RPE cells. The of the analysis is to this of the many genes to identify in the data and to the in a to this is to principal component analysis The genes that are and into a a principal which the gene expression of such The genes are analysed for other gene expression that be by which constitute a principal This the most important of the gene expression to be in a where each component a of genes with expression In this that are to each other are more with to gene expression than that are carried out on the gene expression data revealed that the first principal component and the component 10% of the A using these first two components as differential of the (Fig. from the irradiated A2E-laden RPE cells were in the on the for the first principal component (Fig. The longer of blue light exposure the more the from untreated cells, with the A2E-laden RPE cells irradiated for 20 min the In irradiation of A2E-free RPE cells for 20 min and A2E did not changes in gene expression, and the from these two groups and the from the untreated RPE cells were to each other and the on the of the first principal component (Fig. Subsequently, the genes contributing the most to the of the first principal component were identified and to functional annotation analysis for overrepresentation of terms for biological processes by the This functional analysis that the of the first processes to cell p = p = to p = and to p = expression is by transcription factors that to transcription factor binding which are in the of the The of gene expression can of different pathways that on transcription factors that of genes same for transcription factor binding analysis of can be to how sets of genes can be by a of To identify mechanisms and we analysed the genes the first of the for overrepresentation of potential transcription factor binding The transcription factors are shown in Table Among these where transcription factors belonging to the This of transcription factors has been implicated in the regulation of a of biological functions including regulation of cell apoptosis and immune system & Green et al. 2009). from the three control groups RPE cells, A2E-free RPE cells irradiated for 20 min and A2E-laden RPE cells that had not been exposed to blue were compared using This analysis revealed that blue light exposure of the A2E-free RPE cells and A2E did not a statistical effect gene expression. We compared the A2E-laden cells irradiated for 6 and 10 min with a t-test and found no statistically The same were when the A2E-laden cells irradiated for 15 and 20 min. the data from A2E-free RPE cells, A2E-free RPE cells irradiated for 20 min and the A2E-laden RPE cells were pooled as a control The A2E-laden RPE cells irradiated for 6 and 10 min were pooled to a nm (A2E/430 and A2E-laden RPE cells irradiated for 15 and 20 min were pooled as a to as (A2E/430 the control and the and groups were analysed with a and the set of was used to calculate the rate & which was chosen to be This revealed that probe sets to genes were differentially regulated in the sublethal irradiated A2E-laden RPE cells as compared to controls change or = minimum expression were set to The were used to differentially regulated genes in stress and in the immune 3). In differentially regulated genes with biological functions that might have in the pathogenesis of AMD are shown in Table some of the genes not significantly change in expression levels in both and the change for are shown in Table The genes to and immune and the gene and to and were found to be In that is in and processes was found to be the control with the that probe sets to genes were differentially regulated in the lethal irradiated A2E-laden RPE cells as compared to controls change or = minimum expression were set to were used to for genes in stress and in the immune results for are included in to a for an expression of a when the expression is significantly different in the We found that genes were and 20 were in the We found of genes and downregulation of three proteins are that of synthesized proteins and the of the for can be in to or and are to and both and on the cell and cellular of the were in the 1, 2 and 4 and and 1 We found that various genes to the immune system were in the Among these were 8 6 and the for of both and are regulated by the transcription factor that were found to be as has been shown to in the We found an of in to Studies the complement as an important role in of AMD et al. and we have previously that irradiated A2E-laden RPE cells and photooxidized of A2E can activate the complement (Zhou et al. 2006, 2009). In the we found statistically downregulation of genes in A2E-laden RPE cells irradiated with blue light. of these genes proteins that have an inhibitory effect on the complement cascade: complement factor complement factor H-related 1 complement factor I and and two belonging to the classical complement component 1, s subcomponent and complement component 1, r subcomponent genes found by t-test to be significantly or were investigated by qRT-PCR. The results can be in genes were found to be regulated in the same with both microarray and and changes were found to be is that the longer irradiation of the A2E-laden RPE cells, the more pronounced is the change in mRNA levels for the differential regulated genes we tested with qRT-PCR. reverse-transcriptase analysis of selected genes confirmed the expression in the Affymetrix microarrays. Retinal pigment epithelial (RPE) cells that had accumulated A2E were irradiated at 430 nm for 6, 10, 15 and 20 min. RNA was extracted after and expression of selected genes was measured by quantitative to validate the microarray Controls were A2E-free RPE cells that were not irradiated or were irradiated for 20 min and cells that had accumulated A2E but were not irradiated The RNA of GAPDH was used as internal amplification and method was used to calculate the relative gene expression. (A) and apoptosis Mean ± SEM of three *p < < and < Using microarrays, we investigated the changes in gene expression induced by blue light exposure of A2E-laden RPE cells. We used the well-characterized human RPE cell line is of and endogenous A2E (Sparrow et al. The of these is essential to to in the of these which on light we are that one be about from the in to the in vivo The study that were no differences in gene expression untreated RPE cells and A2E-free RPE cells irradiated for 20 min and A2E-laden RPE cells that had not been exposed to blue light. were no to blue light or A2E were when the of blue light and A2E were The analysis of the microarray data clearly that the from A2E-laden RPE cells exposed to blue light were from the controls. The longer the were exposed to blue light the more from the controls. is that some of are the same experimental The are three for each experimental and the is to the in biological to the that microarrays are to to the functional analysis performed with the were no pathways or functional that account for the This that A2E and blue light might changes in various cellular processes than cell death was detected in the and RNA expression was for This might be an important that changes with exposure to a amount of blue light and RPE cell death the differentially genes according to we found that of these genes and transcription factors were in to cell death and immune response. and genes and transcription factors were in to and This might but indicate that a and is of importance for the cell in to and In the were from to cell The that a of the cells were dead and that changes in RNA levels were detected in the that of the changes in gene expression originate from but not dead cells. in genes for complement factors have been shown to the of AMD et al. et al. has been considered to be a and the of the alternative pathway in the complement and has been that of of the complement system might in of the RPE cells. The microarray analysis demonstrates that the gene expression of genes belonging to the complement system was in A2E-laden RPE cells irradiated with lethal of blue light. performed with the more method that a sublethal of blue light is to downregulation of and We that a of these of the alternative pathway the cells to be to complement This might the to AMD to in genes for In a of recessive Stargardt macular the RPE lipofuscin formation is et al. et al. It has been reported that the mRNA the complement proteins and are in RPE cells of when compared to in the are increased and a of genes to oxidative stress and are et al. These are in with of downregulation of and of in RPE cells that have accumulated A2E and are exposed to blue light. et al. reported of complement genes in human RPE cells after these cells with photoreceptor outer from in The the in vivo and in is not in data did not any differential gene regulation of ARPE-19 cells in to of A2E but after exposure to blue light of A2E RPE cells. stress has been suspected to play an important role in retinal degenerative diseases such as and have been performed to clarify the mechanisms of oxidative stress to the RPE cells. et al. differential gene expression of RPE cells exposed to a sublethal of oxidative stress and found of genes in and Of these we found that and were in the In were differentially regulated in to oxidative but no to or RNA levels were increased for the genes and in cells with oxidative stress as well as in treated cells. the other et al. found of the and results a downregulation of and are to play a role in of might that the downregulation of the in RPE cells with the to This to of The differences in gene expression of in RPE cells with oxidative stress as compared to photooxidative stress that the of the two forms of stress might be of lipofuscin to light reactive oxygen This process not leads to the of singlet oxygen with the A2E molecule with highly reactive moieties resulting from this The with the study of et al. suggest that might be some in the cellular to and to are differences on the gene expression and is that the of by for some of the and that the and of A2E et al. for a as In the data that intracellular A2E with exposure to blue light in a sublethal as well as a lethal differential expression of genes belonging to various functional including to cell death and the immune response. These data the that nm conditions can RPE cells and changes in gene expression in the of cell These have to be investigated gene expression and presence of for transcription factors may in for RPE cells and in vivo as compared to the ARPE-19 cell gene in RPE cells from the serve as an in vivo to and in in This was by and The to and by of to regulated genes in 430 The is not for the or of any information by the than be to the for the

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