Epigenetic and Glucocorticoid Receptor-Mediated Regulation of Glutathione Peroxidase 3 in Lung Cancer Cells

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.

Keywords: dexamethasone; gene regulation; glucocorticoid receptor; glucocorticoid response element; glucocorticoids; glutathione peroxidase-3.

Fig. 1.

Methylation of the GPx3 promoter suppresses its expression in lung cancer cells. GPx3, GR, and actin mRNA expression levels were analyzed by RT-PCR in the H157, H460, A549, H1299, H1650, and H1975 lung cancer cell lines. Actin was used as an internal control for normalization. (A) Lung cancer cells treated with DMSO. (B) Lung cancer cells treated with 5-Aza-CdR (10 μM) for 24 h.

Fig. 2.

GPx3 is down-regulated by hyper-methylation of its promoter CpG in lung cancer cells. Methylation-specific PCR of the GPx3 gene in lung cancer cells. In the gel panels on the left, levels of unmethylated DNA were higher in cells with high levels of GPx3 expression under normal conditions (H157, H460, A549), whereas methylated DNA was only weakly detected. In the panels on the right, cells with low levels of GPx3 expression under normal conditions exhibited low levels of unmethylated DNA and higher levels of methylated DNA.

Fig. 3.

GPx3 is up-regulated by Dex treatment in lung cancer cells. GPx3, GR, and actin mRNA expression levels were analyzed by RT-PCR in the H157, H460, A549, H1299, H1650, and H1975 lung cancer cell lines. Actin was used as an internal control for normalization. (A) Lung cancer cells treated with DMSO. (B) Lung cancer cells treated with Dex (100 nM) for 24 h. (C) To examine the time course of the effect of Dex, GPx3 expression was assessed in H1299 and H1975 at the indicated time points after treatment.

Fig. 4.

hGR protein binds specifically to GREs on the GPx3 promoter in lung cancer cells. ChIP was performed using A549 cells. ChIP-PCR was then performed using a primer pair [GPH1010877(+)02A] specific for GRE6 and GRE7 in intron 1 of the GPx3 gene. As negative controls, primers for GAPDH, which does not possess a GRE, and an exon-specific GPx3 primer pair were used.

Fig. 5.

Functional analysis of GR and GREs in lung cancer cells. The effect of dual mutation of GRE6 and GRE7 on the GPx3 promoter was determined using a luciferase reporter system in lung cancer cells. A reporter plasmid with nano luciferase [pNL1.1::GPx3 promoter-GRE (WT) or (MT)] and a control plasmid with firefly luciferase (pGL4.54) were co-transfected into lung cancer cells. Nano luminescence was determined and normalized to the firefly luciferase signal. The fold-inductions were then calculated by normalization to the pNL1.1 control.

Fig. 6.

Suppression of GR mRNA reduces GPx3 expression levels in lung cancer cells. Effect of GR knockdown on GPx3 expression in lung cancer cells. (A) A549 and H1975 cells were transfected with siRNA against GR or a control siRNA, and expression of GPx3 was evaluated 48 h later by RT-PCR. As shown, the levels of GPx3 were significantly decreased in the presence of GR siRNA, but not in the presence of control siRNA. (B) Western blot analysis of total cell lysates of A549 and H1975 cells transfected with GR siRNA. As shown, transfection with GR siRNA decreased the levels of GPx3 expression in lung cancer cells.