Molecular interaction between glucocorticoid and circadian clock genes

Abstract

The circadian clock is an internal timekeeping system that regulates physiology, metabolism and behavior with about 24-hr period. In mammals, it has hierarchical structure: the master clock in the brain regulates the peripheral clocks. The master clock, which is located in the suprachiasmatic nucleus of the hypothalamus, synchronizes peripheral clocks through neural and humoral factors. Among many synchronizing signals, an attractive one is glucocorticoid (GC) hormone, of which the plasma level exhibits the circadian pattern. The hormone can entrain most of peripheral tissues without the interference of the master clock, because the GC receptor (GR) is ubiquitously expressed in peripheral tissues, not in the master clock. GC directly regulates circadian clock genes, including Period1 (Per1) and Period2 (Per2). The previous study revealed that there is a functional glucocorticoid response element (GRE) in Per1 promoter, however, the GRE had not been found in Per2 promoter. Therefore, I attempted to explore the molecular mechanism of GC-mediated Per2 expression. I observed that GC induced a prominent Per2 induction and delayed circadian phase compared to other synchronizing signals. Using the promoter prediction tool, I found that the GRE in the highly conserved region of Per2 promoter. The GRE was overlapped with E-box by 1-bp. Interestingly, the mutation of E-box inhibited GC responsiveness of Per2, which was also abolished in Bmal1-/- MEFs. The involvement of Bmal1 in GC responsiveness might be a special regulatory mechanism because other GRE-containing genes, such as Per1 and mouse mammary tumor virus (MMTV) still exhibited GC responsiveness in Bmal1-/- MEFs. The chromatin immunoprecipitation assay using the GR antibody showed that Bmal1 was required for the binding of GR to the GRE of Per2 promoter. To identify the functional importance of GC-mediated Per2 induction, I generated the adenovirus that expresses the fusion protein of PER2 and LUCIFERASE under Per2 promoter. When Per2-/- MEFs were transduced with either GRE or E-box mutant adenovirus, GC-mediated Per2 induction was decreased and the circadian phase was advanced. Taken together, this study reveals that Bmal1-dependent Per2 induction by GC signaling rendered the circadian phase delay. Based on a novel GC regulation mechanism, which requires Bmal1, I investigated global regulatory patterns of Bmal1 on GC-responsive genes to discover other Bmal1-modulated GC-responsive genes. I performed the microarray experiment using WT and Bmal1-/- MEFs after the treatment of vehicle or dexamethasone and analyzed the data by 2-way ANOVA. More than half of GC-responsive genes exhibited altered GC responsiveness in Bmal1-/- MEFs. To explore the biological meaning of this regulation mechanism, I analyzed the enrichment of gene ontology terms with the bioinformatics tool to find out Bmal1-modulated GC-responsive genes. Interestingly, Bmal1-modulated GC-responsive genes were highly related to the terms, glycoprotein, oxidation-reduction and response to stimulus, while non-Bmal1-modulated GC-responsive genes were highly related to the terms, intracellular, protein binding and intracellular signal transduction. Then, results suggest that Bmal1 is widely involved in GC-regulation mechanism and Bmal1-modulated GC-responsive genes are involved in some biological processes, including glycosylation, oxidation-reduction and the response to stimulus. These finding may contribute to future understanding for GC action mechanisms and the interaction between GC and circadian clock genes.