Makoto Akashi

BACKGROUND: The circadian rhythm of about 24 hours is a fundamental physiological function observed in almost all organisms from prokaryotes to humans. Identification of clock genes has allowed us to study the molecular bases for circadian behaviors and temporal physiological processes such as hormonal secretion, and has prompted the idea that molecular clocks reside not only in a central pacemaker, the suprachiasmatic nuclei (SCN) of hypothalamus in mammals, but also in peripheral tissues, even in immortalized cells. Furthermore, previous molecular dissection revealed that the mechanism of circadian oscillation at a molecular level is based on transcriptional regulation of clock and clock-controlled genes. RESULTS: We systematically analyzed the mRNA expression of clock and clock-controlled genes in mouse peripheral tissues. Eight genes (mBmal1, mNpas2, mRev-erbalpha, mDbp, mRev-erbbeta, mPer3, mPer1 and mPer2; given in the temporal order of the rhythm peak) showed robust circadian expressions of mRNAs in all tissues except testis, suggesting that these genes are core molecules of the molecular biological clock. The bioinformatics analysis revealed that these genes have one or a combination of 3 transcriptional elements (RORE, DBPE, and E-box), which are conserved among human, mouse, and rat genome sequences, and indicated that these 3 elements may be responsible for the biological timing of expression of canonical clock genes. CONCLUSIONS: The observation of oscillatory profiles of canonical clock genes is not only useful for physiological and pathological examination of the circadian clock in various organs but also important for systematic understanding of transcriptional regulation on a genome-wide basis. Our finding of the oscillatory expression of canonical clock genes with a temporal order provides us an interesting hypothesis, that cyclic timing of all clock and clock-controlled genes may be dependent on several transcriptional elements including 3 known elements, E-box, RORE, and DBPE.

Recent studies have shown that casein kinase I epsilon (CKIepsilon) is an essential regulator of the mammalian circadian clock. However, the detailed mechanisms by which CKIepsilon regulates each component of the circadian negative-feedback loop have not been fully defined. We show here that mPer proteins, negative limbs of the autoregulatory loop, are specific substrates for CKIepsilon and CKIdelta. The CKI phosphorylation of mPer1 and mPer3 proteins results in their rapid degradation, which is dependent on the ubiquitin-proteasome pathway. Moreover, CKIepsilon and CKIdelta are able to induce nuclear translocation of mPer3, which requires its nuclear localization signal. The mutation in potential phosphorylation sites on mPer3 decreased the extent of both nuclear translocation and degradation of mPer3 that are stimulated by CKIepsilon. CKIepsilon and CKIdelta affected the inhibitory effect of mPer proteins on the transcriptional activity of BMAL1-CLOCK, but the inhibitory effect of mCry proteins on the activity of BMAL1-CLOCK was unaffected. These results suggest that CKIepsilon and CKIdelta regulate the mammalian circadian autoregulatory loop by controlling both protein turnover and subcellular localization of mPer proteins.

Although the suprachiasmatic nucleus (SCN) is the major pacemaker in mammals, the peripheral cells or immortalized cells also contain a circadian clock. The SCN and the periphery may use different entraining signals-light and some humoral factors, respectively. We show that induction of the circadian oscillation of gene expression is triggered by TPA treatment of NIH-3T3 fibroblasts, which is inhibited by a MEK inhibitor, and that prolonged activation of the MAPK cascade is sufficient to trigger circadian gene expression. Therefore, such prolonged activation of MAPK by entraining cues may be involved in the resetting of the circadian clock.

A thorough understanding of the circadian clock requires qualitative evaluation of circadian clock gene expression. Thus far, no simple and effective method for detecting human clock gene expression has become available. This limitation has greatly hampered our understanding of human circadian rhythm. Here we report a convenient, reliable, and less invasive method for detecting human clock gene expression using biopsy samples of hair follicle cells from the head or chin. We show that the circadian phase of clock gene expression in hair follicle cells accurately reflects that of individual behavioral rhythms, demonstrating that this strategy is appropriate for evaluating the human peripheral circadian clock. Furthermore, using this method, we indicate that rotating shift workers suffer from a serious time lag between circadian gene expression rhythms and lifestyle. Qualitative evaluation of clock gene expression in hair follicle cells, therefore, may be an effective approach for studying the human circadian clock in the clinical setting.