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. 2016 Jan;58(1):34-9.
doi: 10.3164/jcbn.15-16. Epub 2015 Nov 20.

Exendin-4 promotes extracellular-superoxide dismutase expression in A549 cells through DNA demethylation

Hiroyuki Yasuda  1 , Koji Mizukami  1 , Mutsuna Hayashi  1 , Tetsuro Kamiya  1 , Hirokazu Hara  1 , Tetsuo Adachi  1
Affiliations

Exendin-4 promotes extracellular-superoxide dismutase expression in A549 cells through DNA demethylation

Hiroyuki Yasuda et al. J Clin Biochem Nutr. 2016 Jan.

Abstract

Exendin-4 is an agonist of the glucagon-like peptide 1 receptor (GLP-1R) and is used in the treatment of type 2 diabetes. Since human GLP-1R has been identified in various cells besides pancreatic cells, exendin-4 is expected to exert extrapancreatic actions. It has also been suggested to affect gene expression through epigenetic regulation, such as DNA methylation and/or histone modifications. Furthermore, the expression of extracellular-superoxide dismutase (EC-SOD), a major SOD isozyme that is crucially involved in redox homeostasis, is regulated by epigenetic factors. In the present study, we demonstrated that exendin-4 induced the demethylation of DNA in A549 cells, which, in turn, affected the expression of EC-SOD. Our results showed that the treatment with exendin-4 up-regulated the expression of EC-SOD through GLP-1R and demethylated some methyl-CpG sites (methylated cytosine at 5'-CG-3') in the EC-SOD gene. Moreover, the treatment with exendin-4 inactivated DNA methyltransferases (DNMTs), but did not change their expression levels. In conclusion, the results of the present study demonstrated for the first time that exendin-4 regulated the expression of EC-SOD by reducing the activity of DNMTs and demethylation of DNA within the EC-SOD promoter region in A549 cells.

Keywords: DNA demethylation; epigenetics; exendin-4; extracellular-superoxide dismutase.

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Figures

Fig. 1
Fig. 1
Effects of exendin-4 on EC-SOD expression in A549 cells. (A) A549 cells were treated with the indicated concentrations of exendin-4 or 1 µM Aza for 24 h, followed by the measurement of SODs mRNA levels. (B) A549 cells were pretreated with (+) or without (–) 200 nM exendin-(9-39) for 1 h, and then treated with (+) or without (–) 100 nM exendin-4 for 24 h. Real-time RT-PCR data were normalized using 18S rRNA levels. Data are shown as the mean ± SD (n = 3). *p<0.01 vs vehicle, #p<0.05 vs exendin-4-treated cells.
Fig. 2
Fig. 2
Involvement of DNA demethylation in exendin-4-inducible EC-SOD expression in A549 cells. A549 cells were treated with 100 nM exendin-4 or 1 µM Aza for 24 h. (A) After these treatments, genomic DNA from A549 cells was purified, and followed by McrBC digestion-real-time RT-PCR. Data are shown as the mean ± SD (n = 3). *p<0.05, **p<0.01 vs vehicle. (B) The genomic DNA of A549 cells was purified and treated with bisulfite, followed by methylation-specific PCR (MSP) amplification with methylation (M) and non-methylation (U) site primers. Data are shown as the mean ± SD (n = 3). *p<0.05, **p<0.01 vs control (U). #p<0.01 vs control (M). (C) Amplified sequences were subjected to a DNA sequence analysis. At least 10 individual clones were sequenced for both cell types.
Fig. 3
Fig. 3
Effects of exendin-4 on DNMTs in A549 cells. A549 cells were treated with 100 nM exendin-4 or 1 µM Aza for 24 h. mRNA expression levels of DNMTs (A) and DNMT activity (B) were then assayed. (A) Real-time RT-PCR data were normalized using 18S rRNA levels. (B) The activity of DNMT in nuclear extracts was determined by the method described in the Materials and Methods. Data are shown as the mean ± SD (n = 3). *p<0.01 vs vehicle.
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