Epigenetic and Glucocorticoid Receptor-Mediated Regulation of Glutathione Peroxidase 3 in Lung Cancer Cells
Byung Chull An, Nak-Kyun Jung, Chun Young Park, In-Jae Oh, Yoo-Duk Choi, Jae-Il Park, and Seung-won Lee
Abstract
Glutathione peroxidase 3 (GPx3), an antioxidant enzyme, acts as a modulator of redox signaling, has immunomodulatory function, and catalyzes the detoxification of reactive oxygen species (ROS). GPx3 has been identified as a tumor suppressor in many cancers. Although hyper-methylation of the GPx3 promoter has been shown to down-regulate its expression, other mechanisms by which GPx3 expression is regulated have not been reported. The aim of this study was to further elucidate the mechanisms of GPx3 regulation. GPx3 gene analysis predicted the presence of ten glucocorticoid response elements (GREs) on the GPx3 gene. This result prompted us to investigate whether GPx3 expression is regulated by the glucocorticoid receptor (GR), which is implicated in tumor response to chemotherapy. The corticosteroid dexamethasone (Dex) was used to examine the possible relationship between GR and GPx3 expression. Dex significantly induced GPx3 expression in H1299, H1650, and H1975 cell lines, which exhibit low levels of GPx3 expression under normal conditions. The results of EMSA and ChIP-PCR suggest that GR binds directly to GRE 6 and 7, both of which are located near the GPx3 promoter. Assessment of GPx3 transcription efficiency using a luciferase reporter system showed that blocking formation of the GR-GRE complexes reduced luciferase activity by 7–8-fold. Suppression of GR expression by siRNA transfection also induced down-regulation of GPx3. These data indicate that GPx3 expression can be regulated independently via epigenetic or GR-mediated mechanisms in lung cancer cells, and suggest that GPx3 could potentiate glucocorticoid (GC)-mediated anti-inflammatory signaling in lung cancer cells.
INTRODUCTION
Lung cancer is a malignant lung tumor characterized by uncontrolled cell growth in
tissues of the lung. If not treated, lung cancer metastasizes beyond the lung into
nearby tissue or other parts of the body. This consequence is the leading cause of
cancer-related deaths. To improve the survival rate in patients with lung cancer,
many studies have sought to identify reliable biological markers of lung cancer (Supplementary Fig. S1). GRE 6 and 7 are of particular interest, as they are located in the downstream regulatory
region while the other GREs are located more than 5 kb (up- or downstream) from the
transcription start site (TSS). Glucocorticoids (GCs) can manipulate GR expression
and signaling in cells. Moreover, GCs are essential for survival and are involved
in a variety of physiological processes, including regulation of metabolism, immunity,
and apoptosis (
MATERIALS AND METHODS
Reagents and antibodies
Human recombinant GR was purchased from Calbiochem (USA). GPx3, GR, and actin antibodies were purchased from Santa Cruz Biotechnology (USA). Anti-mouse antibody conjugated to horseradish peroxidase was obtained from Santa Cruz Biotechnology. For drug treatment, cell lines were treated with 10 μM 5-aza-2′-deoxycytidine (5-Aza-CdR) (Sigma, USA) and 100 nM dexamethasone (Dex) (Sigma). Control cells were incubated with DMSO in culture medium.
Cell culture
H157, H460, A549, H1299, H1650, and H1975 lung cancer cell lines (ATCC, USA) were
cultured in appropriate media (RPMI 1640) (Gibco, USA) with 10% fetal bovine serum
(FBS) (Gibco) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml
streptomycin (Invitrogen, USA) at 37°C in a 5% CO2 incubator (
MSP
Genomic DNA was prepared from the six lung cancer cell lines using the QIAamp® DNA Mini Kit (Qiagen, USA). Extracted gDNA was bisulfate-modified using the MethylEasy™ Xceed kit (Human Genetic Signatures, Australia), as described previously (
DNA methylation arrays
Samples were bisulfite-converted using the EZ DNA Methylation Kit (Zymo Research,
USA) according to the manufacturer’s protocol. Controls on the array were used to
track the bisulfite conversion efficiency. The Infinium HumanMethylation450 BeadChip
array (Illumina Inc., USA) was used to measure genome-wide CpG methylation using beads
with target-specific probes designed to interrogate individual CpG sites on bisulfite-converted
genomic DNA (
Construction of plasmids
A GPx3 promoter fragment spanning the −881/+1546 region was amplified by PCR from
lung cancer cell genomic DNA using the following primers: −881 (
Reporter assays
Lung cancer cells were transfected with 200 ng of nanoluciferase reporter construct
(pNL1.1::GPx3 promoter) and 200 ng of firefly luciferase construct encoded by the
pGL 4.54 plasmid using Lipofectamine 3000 (Invitrogen). After 2 days of transfection,
cells were analyzed using the Nano-Glo Luciferase Assay according to the manufacturer’s
instructions (Promega) and the Infinite PRO 2000 multimode reader (Tecan, Germany).
Measured luciferase values were normalized to the internal firefly luciferase control
(
Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA (1 μg) from lung cancer cells was reverse transcribed to complementary DNA (cDNA) using Hyperscript™ RT premix (with oligo dT) (GeneAll) in a final volume of 20 μl. This mixture was incubated for 1 h at 55°C and then heated for 10 min at 95°C to inactivate the reverse transcriptase. The resulting cDNAs were used for PCR amplification of the following specific targets: GPx3, GR, and the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin. Primers were designed using Primer3 so that any genomic DNA product could be distinguished from the target cDNA based on size difference (Table 1). For PCR, 2 μl of cDNA and 20 pmol of each primer were amplified in a total volume of 20 μl using AmpONE™ Taq premix (GeneAll). PCR conditions were 95°C for 10 min, 37 cycles of 95°C for 1 min, annealing at 58°C for 1 min and 72°C for 1 min, followed by a final extension step at 72°C for 10 min.
Site-directed mutagenesis of GREs by PCR
Double mutations in GRE6 and GRE7 of the GPx3 promoter were generated by PCR-mediated
site-directed mutagenesis. For single mutation of GRE6, the complementary primers
contained a triple-base mismatch in GRE6 that converts TGT to CAG using the pNL1.1::GPx3
promoter-GRE (WT) as the template. For double mutations of GRE6 and GRE7, the complementary
primers contained a triple-base mismatch in GRE7 that converts GTCC to ATAA using
the pNL1.1::GPx3 promoter-GRE6 mutant as the template. Briefly, specific PCR was carried
out in 20 μl mixtures containing 10 ng of plasmid DNA and 20 pmol of each primer using
AmpONE™ Taq premix (GeneAll). PCR conditions were 95°C for 10 min, 30 cycles of 95°C for
1 min, annealing at 45°C for 1 min and 72°C for 5 min, followed by a final extension
step at 72°C for 10 min. The PCR products of the single and double mutations were
collected and purified, treated with DpnI (Enzynomics, Korea) to remove the original
DNA, and then transformed into DH5α cells. The single and double mutations were confirmed
by nucleotide sequencing after plasmid isolation (
Gel mobility shift assays
DNA-protein binding was assayed by gel mobility shift EMSA as described previously
(
ChIP
ChIP experiments were performed as described previously (
Western blot analysis
Following the indicated treatments, cells were washed in phosphate-buffered saline
(PBS) and lysed in ice-cold RIPA buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA,
0.5% sodium deoxycholate, 1% IGEPAL CA-630, and 0.1% sodium dodecyl sulfate) supplemented
with protease inhibitor cocktail (Sigma). The cell extracts were cleared by centrifugation
at 13,000 rpm for 30 min at 4°C, and the protein concentration was quantified using
a BCA Bio-Rad protein assay kit (Bio-Rad, USA). The cell lysates were resolved on
4% to 20% Tris-glycine Ready gels (Bio-Rad) and transferred onto PVDF membranes. Subsequently,
the membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (20 mM
Tris base, pH 7.5, and 150 mM NaCl) containing 0.05% Tween 20 (TBS-T) and then probed
with primary antibodies (GPx3, GR, and actin) diluted in 1% nonfat milk in TBS-T.
After incubation with primary antibody in TBS-T, the membranes were incubated with
the appropriate horseradish peroxidase-conjugated secondary antibodies (Santa Cruz
Biotechnology), and the blots were visualized by chemiluminescence (GE Healthcare,
USA) (
RESULTS
GPx3 expression is suppressed by an epigenetic mechanism in lung cancer cells
Suppression of GPx3 expression via an epigenetic mechanism has been reported in many
types of human cancer and is proposed to play an important role in tumorigenesis (

GPx3 expression is up-regulated by Dex treatment in lung cancer cells
GCs are one of the steroid hormones that function to maintain homeostasis. The effects
of GCs are mediated by ubiquitously expressed GR. The GR is an intracellular ligand-activated
transcription factor and member of the nuclear receptor superfamily. The GR is expressed
in almost every cell in the body and regulates genes controlling development, metabolism,
and the immune response (Supplementary Fig. S1). To determine the role of GRs in mediating GPx3 expression, Dex was used to examine
the possible relationship between GR and GPx3 expression. Dex treatment of the lung
cancer cell lines showed that GPx3 expression levels significantly increased in the
low GPx3 expression level group, while the high GPx3 expression level group was not
affected and GR expression was slightly decreased (Fig. 3B). In agreement with this finding, a reduction in GR mRNA expression by Dex treatment
in lung cancer was reported previously (
Recombinant hGR protein binds specifically to GREs of the GPx3 promoter
Binding of recombinant hGR protein to GREs in the GPx3 promoter was studied by EMSA using 3′-biotinylated GRE6 (34 bp: sequence +2194 to +2329) or 3′-biotinylated GRE7 (31 bp: +2233 to +2298) DNA probes. As shown in Supplementary Fig. S2, both the GRE6 and GRE7 DNA probes shifted significantly in the presence of Dex due to the formation of a complex between the GREs and hGR protein. The ChIP-PCR revealed that the GR protein bound specifically to the GREs of the GPx3 promoter region in lung cancer cells (Fig. 4). To verify the specificity of the GR-GRE complex on the GPx3 promoter region, we employed primer pairs [Qiagen; GPH1010877(+)02A; Table 1] specific for the GPx3 promoter region (1 kb). A primer pair for GAPDH was used as a negative control, as this gene does not contain a GRE. In addition, a GPx3 exon-specific primer pair was used as a second negative control. Together, these results indicate that GR binds specifically to GREs in the GPx3 promoter in lung cancer cells.
Enhanced transcription of GPx3 is associated with GR binding
The functional importance of the GREs was then tested by mutational analysis. Mutations targeted to GR-binding motifs on both GRE6 and GRE7 are described in Table 2. The promoter activities of the wild-type GRE (WT) and double-mutated GRE (MT) constructs were tested in lung cancer cells. Interestingly, Dex-induced transcription of the GPx3 gene was accompanied by decreased transcription of GR mRNA, as assessed by RT-PCR (Fig. 3B). The GR-mediated increase in GPx3 expression shown in Fig. 3 was then confirmed by a cell-based luciferase reporter assay, which showed that the double mutation of GRE6 and GRE7 led to significantly reduced GR-mediated transactivation of the GPx3 promoter (Fig. 5). The GRE (WT) construct exhibited approximately 7–8-fold higher luciferase activity than the GRE (MT) construct (Fig. 5). Together, the EMSA, ChIP, and luciferase reporter assay results suggest that the GR binds specifically to GREs in the GPx3 promoter, leading to induction of GPx3 expression.
Silencing of GR reduces GPx3 expression in lung cancer cells
To confirm this conclusion, we also performed transient siRNA transfection to specifically suppress GR expression. In both groups of lung cancer cells, especially the A549 and in H1975 lines, transfection of siRNA against GR resulted in decreased GR expression and decreased transcription of the GPx3 gene as assessed by RT-PCR and Western blot (Fig. 6).
DISCUSSION
GPx3 is the only extracellular glutathione peroxidase to play a critical role in detoxifying
ROS and protecting DNA from damage caused by ROS in mammalian cells (
Many studies have reported silencing of GPx3 caused by hyper-methylation of the promoter
in human prostate cancer, breast cancer, lung cancer, ovarian clear cell adenocarcinoma,
gastric carcinoma, and Barrett’s disease (
Despite the growing interest in GPx3 as a molecule functionally involved in tumor suppression, information about GPx3 gene regulation other than the epigenetic control remains limited. Here, we demonstrated for the first time that GR-mediated up-regulation of GPx3 is independent of the epigenetic regulation of GPx3 in lung cancer cells.
In conclusion, our demonstration that GR mediates upregulation of the GPx3 gene in
lung cancer cells provides important insights into cancer biology. GPx3 is well known
to act as an antioxidant and tumor suppressor in cancer cells (
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