DNA Methylation Analysis of Human Trabecular Meshwork Cells During Dexamethasone Stimulation

Abstract

Purpose.: We investigated the changes in the DNA methylation status of cultured human trabecular meshwork (TM) cells during glucocorticoid exposure, and evaluated the effect of epigenetic modification on the gene expression of TM cells.

Methods.: Three batches of primary culture TM cells were treated with and without 100 nM dexamethasone (DEX) for 14 days. Genome-wide methylation analysis was done using Illumina 450 K methylation chips. The gene expression profile of the TM cells also was examined. The epigenetic effect of DEX stimulation on gene expression in TM cells was further verified by treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-dC) and subsequent real-time PCR analysis.

Results.: After DEX stimulation, we found demethylated cytosine-phosphate-guanine (CpG) sites within the FKBP5, ZBTB16, and SCNN1A gene promoter regions with increases of corresponding gene expression for all three TM batches, and methylated CpG sites within the ARSI, HIC1, GREM2, and MATN2 gene promoter regions with decreases of corresponding of gene expression for all three batches. Inhibition of DNA methylation by 5-aza-dC treatment induced a further increase of FKBP5 and SCNN1A mRNA expression under DEX stimulation.

Conclusions.: The DEX stimulation induces alteration of the DNA methylation status in human TM cells. Epigenetic modification could affect the TM gene expression profile.

Glucocorticoid-induced glaucoma is an adverse effect of glucocorticoid therapy. Secondary open-angle glaucoma with elevated IOP, optic nerve head damage, and visual field defects are the clinical features of this condition. Elevation of IOP usually occurs weeks to months after glucocorticoid administration, and the degree of the elevation depends on the potency and dose of the glucocorticoid.^1,2 The IOP elevation may persist even after cessation of glucocorticoid treatment, and sometimes glaucoma surgery is needed to control the IOP.³ 

Previous studies showed increased deposition of extracellular matrix in the trabecular meshwork (TM),⁴ and decreased phagocytosis of TM cells in an organ culture model supplemented with dexamethasone (DEX).⁵ Other lines of investigation showed the glucocorticoid-induced gene expression profiles in TM cells, elucidating several important molecular pathways relevant for the disease.^6–8 

The effects of glucocorticoids are mediated by the glucocorticoid receptor (GR), which serves as a ligand-activated nuclear transcription factor. Epigenetic mechanisms have some roles in glucocorticoid-induced signal transduction. For example, glucocorticoid-induced loss of DNA methylation occurs at FK506 binding protein 5 (FKBP5), and, thus, increases FKPB5 transcription.⁹ The initial idea for investigating the roles of epigenetic regulation in TM cells during DEX exposure came from these relationships between glucocorticoid signaling pathways and epigenetic regulation. 

On the other hand, previous studies have suggested that responsiveness to glucocorticoids is affected by genetic predisposition,¹⁰ but not simply determined by genetics.¹¹ Becker et al.¹⁰ showed that patients with primary open angle glaucoma (POAG) and those having a first-degree relative with POAG were more likely to respond to steroids. Another study by Schwartz et al.¹¹ observed no intrapair differences for steroid responses between monozygotic and dizygotic twin pairs, indicating that the responsiveness to glucocorticoids was not simply determined by genetics. The contribution of genetic factor(s) to the pathophysiology of glucocorticoid-induced glaucoma is complex and might be affected by epigenetic factors. 

Recently, the roles of epigenetics in the pathophysiology of many noncancerous diseases, such as metabolic,¹² collagen,¹³ fibrotic,¹⁴ and allergic¹⁵ diseases, are beginning to be explored. To investigate the roles of epigenetic modification in the pathophysiology of glucocorticoid-induced glaucoma, we performed genome-wide DNA methylation analysis of primary culture TM cells subjected to DEX exposure. 

Materials and Methods

Cell Culture

Three lots (lot Nos. 2584, 3423, and 4973) of primary culture human TM cells were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). All TM cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Tokyo, Japan) with 10% fetal calf serum (FCS; Life Technologies), using 6-well culture dishes (Iwaki, Tokyo, Japan). The DEX was obtained from Sigma Japan (Tokyo, Japan). The DEX-stimulated TM cells were prepared by adding 10⁻⁷ M (100 nM) DEX to the culture medium and changing the medium every 2 days for 14 days. Extraction of DNA and RNA from the cultured TM cells was done using a NucleoSpin Tissue DNA purification kit and NucleoSpin RNA II kit (Macherey-Nagel, Duren, Germany), respectively. This study was conducted in accordance with the tenets of the Declaration of Helsinki. 

Genome-Wide DNA Methylation Analysis

Illumina human methylation 450K chips (Illumina, San Diego, CA, USA) were used according to the manufacturer's protocol to examine the DNA methylation status of untreated and DEX-treated TM cells. Genomic DNA samples were subjected to bisulfite conversion using an EZ DNA methylation kit (Zymo Research, Irvine, CA, USA). The bisulfite-converted DNA was used in the whole-genome amplification reaction, and then the DNA was fragmented and resuspended in hybridization buffer. The fragmented DNA was reacted with the methylation 450 K chip and hybridized for 20 hours. After a primer extension process, the chips were imaged on an Illumina iScan. Methylation levels in Illumina assays are quantified by the β-value, Display Formula , the ratio of signal intensities of methylated (M) and unmethylated (U) alleles. The signal intensities of M alleles and U alleles are normalized with normalization control probes using GenomeStudio Methylation Module software (Illumina). The β-value is continuous and ranges from 0 (unmethylated) to 1 (completely methylated).¹⁶ We excluded the probes with low detection P values (P > 0.01). We performed Illumina human methylation 450 K chip analysis using the same lots of TM cells and DEX stimulations two times to verify the reproducibility of the whole-genome DNA methylation analysis. Statistical analysis was done to detect significant alteration of β-value in response to DEX treatment using the Wilcoxon signed-rank test. A value of P < 0.05 was considered statistically significant.  

Genome-Wide Expression Profile of DEX-Treated TM Cells

The whole-genome expression profiles of untreated and DEX-treated TM cells were examined using SurePrint G3 Human Gene Expression 8 × 60 K DNA chips (Agilent Japan, Tokyo, Japan), according to the manufacturer's protocols. The data were analyzed using GeneSpring GX version 12.6.1 (Agilent), and excluded probes by using the default settings of “Expression Filtering (exclude the probes with very low expression [less than 20 percentile of the expression data] in both conditions)” and “Flag Filtering (exclude the probes with compromised hybridization).” 

DNA Methyltransferase Inhibitor Treatment and Subsequent Real-Time PCR Analysis

Three batches of TM cells were incubated with various concentrations of the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-dC; Sigma Japan) for 4 days, with and without 100 nM DEX treatment. Mock-treated control samples also were prepared. After total RNA extraction, reverse transcription was done using RevaTra Ace reverse transcriptase and random hexamers (both from Toyobo, Osaka, Japan) according to the manufacturer's protocol. Real-time PCR analysis was done using FAST SYBR master mix (Life Technologies). The primers used for this PCR analysis were designed by QuantPrime software (available in the public domain at http://www.quantprime.de) and are listed in Supplementary Table S1. All the real-time PCR analysis data are a representative data of at least three independent cell stimulation experiments, and real-time PCR analysis was done using triplicate samples. 

Validation of Typical TM Cell Gene Expression in Response to DEX

To validate the characteristics of the three batches of primary cultured TM cells used in this study, we performed real-time PCR analysis of the response to DEX treatment. Three batches of TM cells were incubated with and without 100 nM DEX for 4 days. Reverse transcription and real-time PCR analysis were performed as described above. 

Results

Differentially Methylated DNA Regions in TM Cells After DEX Exposure

We found 30 significantly demethylated cytosine-phosphate-guanine (CpG) sites (P < 0.05 by the Wilcoxon signed-rank test), which also showing average Δβ = (β-value of unstimulated condition) – (β-value of the DEX-stimulated condition) > 0.10, and 49 significantly methylated CpG sites (P < 0.05 by the Wilcoxon signed-rank test), which showing average Δβ < −0.10 at 14 days after DEX treatment (Supplementary Tables S2, S3). The lists of differentially methylated CpG sites in each lot are shown in Supplementary Tables S4 through S9. The reproducibility of the whole-genome DNA methylation analysis was verified by repeating the analysis two times using the same lots of cells and stimuli. The Pearson's correlation coefficients between the same experiments were 0.987/0.987 (naive/DEX stimulated) for lot 2584 TM cells, 0.961/0.961 for lot 3423, and 0.991/0.990 for lot 4973. 

Genome-wide Expression Analysis of DEX-Treated TM Cells and Selection of Genes Based on the Results of DNA Methylation

To select biologically meaningful changes of DNA methylation in response to DEX stimulation, we performed genome-wide gene expression analysis. The genes showing changes of mRNA expression (increases or decreases of more than 2-fold) in all three TM cell batches after 14 days of DEX exposure are listed in Supplementary Tables S10 and S11. The gene expression data were deposited in Gene Expression Omnibus (accession No. GSE65240). We then integrated the lists of DNA methylation analysis and gene expression analysis, and selected the differentially methylated genes from among those with corresponding changes of gene expression in all three batches of TM cells (summarized in Tables 1, 2). We found three genes (FKBP5, ZBTB16, SCNN1A) that showed statistically significant DNA demethylation in at least one CpG site, differential demethylation (Δβ > 0.1) in all three TM batches, and increased mRNA expression in all three TM batches after DEX exposure; and one gene (GATA6) with significant DNA demethylation in one CpG site, differential demethylation (Δβ > 0.1) in all three TM batches, and decreased mRNA expression in all three TM batches after DEX exposure (Table 1). We also found four genes (ARSI, HIC1, GREM2, MATN2) showing statistically significant DNA methylation in one CpG site, differential demethylation (Δβ < −0.1) in all three TM batches, and decreased mRNA expression after DEX exposure in all three TM batches (Table 2). 

Table 1

 

DNA Demethylated Genes With the Corresponding Changes of Gene Expression

 

Table 2

 

DNA Methylated Genes With the Corresponding Changes of Gene Expression

 

Upregulation of Myocilin Gene Expression and Constant α-B-Crystallin Expression in Response to 4-Day DEX Treatment

To validate typical gene expression of TM cells in response to DEX treatment, expression of myocilin (MYOC) mRNA and α-B-crystallin (CRYAB) mRNA was quantified by real-time PCR. The MYOC mRNA expression was upregulated in all three TM batches (Fig. 1A), whereas relatively constant CRYAB mRNA expression was observed (Fig. 1B). The increases in MYOC mRNA induced by DEX treatment were 148.9-fold (in lot 2584), 37.5-fold (in lot 3423), and 8.8-fold (in lot 4973) greater than those of CRYAB. 

Figure 1

 

Upregulation of myocilin gene expression in response to DEX treatment. Expression of MYOC mRNA (A) and CRYAB mRNA (B) with and without 100 nM DEX treatment (4 days) was quantified by real-time PCR. Expression of MYOC mRNA was upregulated in all three TM batches (A), whereas relatively constant CRYAB mRNA expression was observed (B). Error bars: mean ± SD.

DNA Demethylation in the Promoter Region of the FKBP5 Gene and Increased Gene Expression Induced by 5-aza-dC Treatment Under DEX Stimulation

The DNA demethylation was observed in the promoter region of the FKBP5 gene (Fig. 2, arrows for left lane) in all three batches of TM cells. Stimulation with 100 nM DEX for 4 days and simultaneous DNA methyltransferase inhibitor (5-aza-dC) treatment induced further upregulation of FKBP5 mRNA expression (Fig. 2, right lane). 

Figure 2

 

DEX-induced reductions of β-values in the FKBP5 gene region and alteration of mRNA expression by 5-aza-dC treatment. Left lane: Alteration of β-values after DEX treatment in the FKBP5 gene regions of the three batches of TM cells. The β-values ranged from 0 (unmethylated) to 1 (completely methylated). TSS1500, within 1500 base pairs (bp) upstream from the TSS; TSS 200, within 200 bp upstream from TSS; 5′UTR, 5′-side of the untranslated region (from TSS to upstream of the initiation codon); Body, CpG site within the gene body (the gene region including exon[s]) and intron[s]); 3′UTR, 3′-side of the untranslated region (downstream of the termination codon). The FKBP5 gene has two TSSs, shown as TSS1 and TSS2. Arrows indicate CpG sites showing demethylation after DEX treatment. Right lane: 100 nM DEX treatment upregulated FKBP5 mRNA expression and 5-aza-dC treatment further increased FKBP5 mRNA expression. Error bars: mean ± SD.

DNA Demethylation in the Promoter Region of the SCNN1A Gene and Increased Gene Expression Induced by 5-aza-dC Treatment Under DEX Stimulation

The DNA demethylation was observed in the promoter region of SCNN1A (Fig. 3, left lane, arrows). Increased SCNN1A mRNA expression was observed with 5-aza-dC treatment only under DEX stimulation (Fig. 3, right lane). 

Figure 3

 

DEX-induced decreases of β-values in the SCNN1A gene region and alteration of mRNA expression by 5-aza-dC treatment. Left lane: Alteration of β-values after DEX treatment in the SCNN1A gene regions of the three batches of TM cells. Arrows indicate CpG sites showing demethylation after DEX treatment. Right lane: 100 nM DEX treatment upregulated SCNN1A mRNA expression and 5-aza-dC treatment further increased SCNN1A mRNA expression. Error bars: mean ± SD.

DNA Demethylation in the Promoter Region of the SAA1 Gene and Increased Gene Expression Induced by 5-aza-dC Treatment Under DEX Stimulation

The SAA1 and SAA2 gene promoters are located in tandem and the transcription occurs in opposite directions (indicated by arrows with transcription start sites [TSS]). The CpG sites within the promoter regions of the SAA1 gene were demethylated in lot 2584 TM cells (Fig. 4, top left arrow). Treatment with 5-Aza-dC induced further upregulation of SAA1 mRNA expression upon DEX stimulation (Fig. 4, middle lane). We also found increased SAA1 mRNA expression induced by 5-aza-dC treatment even without DEX treatment (Fig. 4, right). 

Figure 4

 

DEX-induced decreases of β-values in the SAA1 gene region and alteration of mRNA expression by 5-aza-dC treatment. Left lane: Alteration of β-values after DEX treatment in SAA1 and SAA2 gene regions of TM cells. Genes SAA1 and SAA2 have their own TSS and the transcription starts in opposite directions (horizontal arrows). Note the decreased β-values in the promoter regions of the SAA1 gene after DEX treatment (arrow and arrowheads). Middle and right lanes: SAA1 mRNA expression was increased by DEX treatment and was further increased by 5-aza-dC in TM cells (middle lane), and SAA1 mRNA expression also was increased by 5-aza-dC treatment without DEX treatment (right lane). Error bars: mean ± SD.

DNA Methylation in the Promoter Region of the ARSI Gene and Increased Gene Expression Induced by 5-aza-dC Treatment Under DEX Stimulation

Methylation of DNA was observed in the promoter region of the ARSI gene (Fig. 5, arrows, left lane). Increased ARSI mRNA expression was observed after 5-aza-dC treatment without DEX stimulation in all lots of TM cells, and DEX-induced downregulation of ARSI mRNA expression was partially abolished in lot 3423 TM cells (Fig. 5, right lane). 

Figure 5

 

DEX-induced increases of β-values in the ARSI gene region and alteration of mRNA expression by 5-aza-dC treatment. Left lane: Alterations of β-values in the ARSI gene regions after DEX treatment. Note the increased β-values at the first intron of the ARSI genes (arrows). Horizontal arrow indicates the direction of transcription. Right lane: 100 nM DEX treatment reduced ARSI mRNA expression and 5-aza-dC treatment increased ARSI mRNA expression without DEX treatment. Error bars: mean ± SD.

Discussion

To our knowledge, this is the first genome-wide analysis of DEX-induced differential DNA methylation using primary cultured TM cells. The Illumina human methylation 450 K chip covers 96% of the CpG islands within the human genome, and the reproducibility of our studies was verified by good correlations of β-values between duplicated studies (r = 0.96–0.99, Pearson's correlation coefficients). We performed statistical analysis of DNA methylation data using the Wilcoxon signed-rank test, and selected the CpG sites showing statistically significant alteration between the pairs of control and simultaneously-DEX–stimulated samples of the same lots (Supplementary Tables S2, S3). Some genome-wide DNA methylation studies have processed their data with stringent multitesting corrections, like Bonferroni correction, which multiplies each probability by the total number of tests performed.¹⁷ To obtain significant results after Bonferroni correction, the P values should be less than 1 × 10⁷, and our results did not reach such significance. In this study, we tried to detect the alterations of DNA methylation in response to DEX stimulation using the same cell batches. The DEX stimulation could affect DNA methylation status through glucocorticoid receptor binding sites in DNA,¹⁸ so we tried to detect focal alteration of DNA methylation occurring in quite limited DNA regions as shown in Figures 2 through 5. Therefore, we performed additional experiments (expression analysis and 5-aza-dC treatment analysis) instead of stringent genome-wide multitesting corrections to avoid false positive results. 

To validate the characteristics of the primary cultured TM cells used in this study, we evaluated MYOC mRNA expression in response to DEX treatment, because induction of the MYOC gene by DEX is reported to be specific for human TM cells.¹⁹ As a control, we also analyzed CRYAB mRNA expression, which is relatively constant in response to DEX treatment.²⁰ The three lots of primary cultured TM cells showed prominent increases of MYOC mRNA expression in response to 4-day DEX treatment, and relatively constant CRYAB mRNA expression (Fig. 1). These results were consistent with the previous report on the characteristics of TM cells. In addition, we compared the results of our genome-wide expression analysis of DEX-treated TM cells with previous reports. Our data showed upregulation of ANGPTL7, FKBP5, MAOA, MYOC, SAA1, SERPINA3, SLPI, TSC22D3, and ZBTB16 in response to 14-day DEX treatment, which was commonly observed in the two genome-wide expression analyses.^7,8 Downregulation of IL33 (c9orf26), IL6, GRP, NET1, BMP2, and TNFRSF11B also was observed in the study of Rozsa et al.⁷ Taken together, we considered that the TM cells used in this study showed typical gene expression profiles of human TM cells as reported previously. 

Among the differentially methylated gene regions, we selected genes with the corresponding changes of expression in all three batches of TM cells (Tables 1, 2). Integrating genome-wide DNA methylation data with gene expression analysis can be a useful method to elucidate important biological pathways under epigenetic control.^21,22 We found demethylated CpG sites near the promoter region of the FKBP5 gene after DEX stimulation (Fig. 2, arrows). This result was consistent with a previous report that showing ligand-binding GR could trigger DNA demethylation via DNA strand breaks at the 3′ side of the glucocorticoid-responsive unit,¹⁸ and it is known that DNA strand breaks induce the base excision repair process and subsequent restoration of unmodified cytosines (the DNA demethylation process).²³ 

Zhang et al.²⁴ reported that FKBP5 was involved in nuclear transport of the dominant negative receptor for glucocorticoid (GRβ), suggesting protective effects of FKBP5 against glucocorticoid-induced glaucoma. In our results, the magnitudes of DNA demethylation in the FKBP5 gene upon DEX stimulation were variable among the three TM batches (Fig. 2), which might affect the ability of negative feedback against glucocorticoid-induced signals. Further studies using TM cells established from steroid-induced glaucoma patients will be useful to draw conclusions about the roles of FKBP5 gene regulations in the pathophysiology of glucocorticoid-induced glaucoma. 

The finding of DNA demethylation and prominent upregulation (50–60-fold) of SCNN1A mRNA by 5-aza-dC treatment under DEX stimulation (Fig. 3) suggested that DNA demethylation in the SCNN1A gene promoter could enhance transcription under DEX exposure. Our result was consistent with a previous report showing that SCNN1A gene expression was induced by glucocorticoid stimulation²⁵ and reduction of DNA methylation in the SCNN1A gene after systemic steroid exposure in chronic obstructive pulmonary disease.²⁶ The expression of the α subunit of the epithelial sodium channel, encoded by the SCNN1A gene, was reported in human TM cells,²⁷ and this molecule has essential roles in aqueous humor secretion and reabsorption.²⁸ 

We also analyzed the SAA1 gene region because of the prominent upregulation of SAA1 mRNA induced by DEX treatment (Table 1), and a previous report showing recombinant SAA protein-induced elevation of IOP in human ocular perfusion organ culture.²⁹ We found that CpG sites within the promoter regions of the SAA1 gene were demethylated in lot 2584 TM cells (Fig. 4, top left arrow, the β-value was decreased 18% by DEX treatment) and also were demethylated in lot 3423 (Fig. 4, middle left arrowhead, the β-value was decreased 8% by DEX treatment), although they did not reach to the cutoff value of our study (10% alteration of the β-value during DEX treatment). Prominent upregulation of SAA1 mRNA expression was observed with 5-aza-dC treatment under DEX stimulation (Fig. 4, middle lane), and even without DEX stimulation (Fig. 4, right lane). These results suggested strong dependency of the SAA1 transcription machinery on the demethylated status of DNA. The insufficient numbers of CpG probes within the SAA1 promoter region hindered the detection of significant DNA demethylation in lot 4973, so we are now investigating the DNA methylation status of the SAA1 promoter region more precisely by analyzing bisulfite sequences data. Our results were consistent with a report showing increased SAA1 mRNA expression and a decrease of DNA methylation in the SAA1 promoter region in colonic biopsy samples obtained from ulcerative colitis patients compared to control samples.³⁰ 

We also found some gene regions showing DNA methylation in response to DEX treatment with corresponding changes of expression (Table 2). For example, significant DNA methylation was induced by DEX treatment at the first intron of the arylsulfatase I (ARSI) gene (Fig. 5, arrows). Treatment with 5-Aza-dC increased ARSI mRNA expression without DEX stimulation and reversed DEX-induced suppression of ARSI expression in lot 3423 TM cells (Fig. 5, right lane), suggesting a role of DNA methylation in DEX-induced suppression of the ARSI gene. The CpG site within the ARSI gene shows DEX-induced DNA methylation (position 149680926 at chromosome 5) located within DNaseI hypersensitivity clusters (data from thw University of California, Santa Cruz [UCSC; Santa Cruz, CA, USA] genome browser, available in the public domain at http://genome.ucsc.edu/index.html). The location suggested that DEX-induced DNA methylation inhibited the transcription machinery of the ARSI gene. Preferential ARSI mRNA expression is observed in human retinal pigment epithelial cells,³¹ and we also confirmed ARSI mRNA expression in TM cells. 

We also found alteration of the DNA methylation status in the gene regions of ZBTB16 (DNA demethylation, increased mRNA expression induced by DEX treatment), GATA6 (DNA demethylation, decreased mRNA expression), HIC1/GREM2/MATN2 (DNA methylation, decreased mRNA expression), and confirmed alteration of their mRNA expression by DEX treatment using real-time PCR analysis (Supplementary Figures S1, S2). However, additional detailed studies are required to investigate the roles of these molecules in the pathophysiology of glucocorticoid-induced glaucoma. 

Previous studies showed the roles of the integrin³² and cytoskeletal signaling pathways³³ in the outflow resistance of the TM. Therefore, we searched for genes related to the integrin and cytoskeletal pathways within our data. We found some integrin genes (ITGA1, ITGA10, ITGB4, and ITGBL1) showing upregulation of gene expression in all three TM cell batches after DEX treatment (Supplementary Table S10); however, we could find only inconsistent alteration of the DNA methylation status within the ITGBL1 gene region in lot 4973 TM cells. For the cytoskeletal signaling pathway, we found significant differential DNA methylation (methylation and demethylation) in the gene body region of ARHGAP26 (Rho GTPase activating protein 26) gene (Supplementary Tables S2, S3). However, gene array expression analysis showed that there was no change in the mRNA expression level of the ARHGA26 gene (data not shown). Therefore, we could not find any integrin/cytoskeleton-related genes showing differential DNA methylation with corresponding change of gene expression in our study. 

The main limitation of our present study is that, since we used primary cultured human TM cells, the results may not reflect the in vivo DNA methylation status in glucocorticoid-induced glaucoma. Theoretically it is possible to analyze the DNA methylation status in small amounts of tissue, so we will perform DNA methylation analysis of surgical samples obtained from glucocorticoid-induced glaucoma patients in the near future. Ewald et al.³⁴ found a correlation of the FKBP5 DNA methylation status in response to glucocorticoid exposure between peripheral blood and the brain, so if we are able to find any correlation of the FKBP5 DNA methylation status between TM tissue and peripheral blood cells, the FKBP5 gene's DNA methylation status in peripheral blood could be a useful biomarker for glucocorticoid-induced glaucoma. 

In conclusion, we found DEX-induced alteration of DNA methylation and subsequent changes of gene expression. Detailed functional analysis of the effects of DEX-induced alterations of epigenetic status, including other types of epigenetic changes, such as histone modifications, which may further affect the transcriptional machinery, are ongoing. 

Acknowledgments

Supported by Grants-in-Aid from the Japanese Society for the Promotion of Science (No. 24592652 and No. 21592239 to AMa), and by the Institute for Environmental and Gender-Specific Medicine, and by the Institute for Diseases of Old Age, Juntendo University. 

Disclosure: A. Matsuda, None; Y. Asada, None; K. Takakuwa, None; J. Sugita, None; A. Murakami, None; N. Ebihara, None