Rhythmic expression of DEC2 protein in vitro and in vivo

Abstract

Basic helix-loop-helix (bHLH) transcription factor DEC2 (bHLHE41/Sharp1) is one of the clock genes that show a circadian rhythm in various tissues. DEC2 regulates differentiation, sleep length, tumor cell invasion and apoptosis. Although studies have been conducted on the rhythmic expression of DEC2 mRNA in various tissues, the precise molecular mechanism of DEC2 expression is poorly understood. In the present study, we examined whether DEC2 protein had a rhythmic expression. Western blot analysis for DEC2 protein revealed a rhythmic expression in mouse liver, lung and muscle and in MCF-7 and U2OS cells. In addition, AMP-activated protein kinase (AMPK) activity (phosphorylation of AMPK) in mouse embryonic fibroblasts (MEFs) exhibited a rhythmic expression under the condition of medium change or glucose-depleted medium. However, the rhythmic expression of DEC2 in MEF gradually decreased in time under these conditions. The medium change affected the levels of DEC2 protein and phosphorylation of AMPK. In addition, the levels of DEC2 protein showed a rhythmic expression in vivo and in MCF-7 and U2OS cells. The results showed that the phosphorylation of AMPK immunoreactivity was strongly detected in the liver and lung of DEC2 knockout mice compared with that of wild-type mice. These results may provide new insights into rhythmic expression and the regulation between DEC2 protein and AMPK activity.

Keywords: DEC2; circadian rhythm; clock gene; phosphorylation of AMP-activated protein kinase.

Figure 1.

Rhythmic expression of DEC2 protein in mice. (A) Mice were housed under light-dark conditions as described in Materials and methods. Liver, lung and muscle samples were collected from the mice and subjected to western blotting for DEC2, pAMPK, AMPK and actin levels. One representative sample from two independent experiments with similar results is shown. (B) The intensities of DEC2, pAMPK and AMPK bands were quantified and divided by that of actin. AMPK, AMP-activated protein kinase.

Figure 2.

Rhythmic expression of DEC2 protein in U2OS and MCF-7 cells by the medium change. (A and B) U2OS and (C and D) MCF-7 cells were cultured and subjected to a medium change. The cells were sampled at 0 and 3 h and then every 6 h until 60 h. Samples were subjected to western blotting for DEC2, pAMPK, AMPK and actin levels. One representative sample from two independent experiments with similar results is shown. The intensities of DEC2, pAMPK and AMPK bands were quantified as described above. AMPK, AMP-activated protein kinase.

Figure 3.

Rhythmic expression of DEC2 protein in MEF is decreased by medium change. (A) MEF were cultured and subjected to medium change or glucose-depleted medium. The cells were sampled at 0 and 6 h and then every 6 h until 48 h from the time of the medium change (0 h) or glucose-depleted medium. Samples were subjected to western blotting for DEC1, DEC2, pAMPK, AMPK and actin levels. One representative sample from two independent experiments with similar results is shown. (B) The intensities of DEC1, DEC2, pAMPK and AMPK bands were quantified as described above. MEF, mouse embryonic fibroblast; AMPK, AMP-activated protein kinase.

Figure 4.

pAMPK immunoreactivity was strongly detected in DEC2 knockout mice tissues. Strong pAMPK immunoreactivity was detected in the liver and lung of DEC2 KO mice compared with WT mice. AMPK, AMP-activated protein kinase; KO, knockout; WT, wild-type.