The circadian clock is reset by external time cues for synchronization to environmental changes. In mammals, the light-input signalling pathway mediated by Per gene induction has been extensively studied. On the other hand, little is known about resetting mechanisms that are independent of Per induction. Here we show that activation of activin receptor-like kinase (ALK), triggered by TGF-beta, activin or alkali signals, evoked resetting of the cellular clock independently of Per induction. The resetting was mediated by an immediate-early induction of Dec1, a gene whose physiological role in the function of the circadian clock has been unclear. Acute Dec1 induction was a prerequisite for ALK-mediated resetting and upregulation was dependent on SMAD3, which was phosphorylated for activation in response to the resetting stimuli. Intraperitoneal injection of TGF-beta into wild-type or Dec1-deficient mice demonstrated that Dec1 has an essential role in phase-shift of clock gene expression in the kidney and adrenal gland. These results indicate that ALK-SMAD3-Dec1 signalling provides an input pathway in the mammalian molecular clock.

Daily behavioral rhythms in mammals are governed by the central circadian clock, located in the suprachiasmatic nucleus (SCN). The behavioral rhythms persist even in constant darkness, with a stable activity time due to coupling between two oscillators that determine the morning and evening activities. Accumulating evidence supports a prerequisite role for Ca 2+ in the robust oscillation of the SCN, yet the underlying molecular mechanism remains elusive. Here, we show that Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) activity is essential for not only the cellular oscillation but also synchronization among oscillators in the SCN. A kinase-dead mutation in mouse CaMKIIa weakened the behavioral rhythmicity and elicited decoupling between the morning and evening activity rhythms, sometimes causing arrhythmicity. In the mutant SCN, the right and left nuclei showed uncoupled oscillations. Cellular and biochemical analyses revealed that Ca 2+ -calmodulin-CaMKII signaling contributes to activation of E-box-dependent gene expression through promoting dimerization of circadian locomotor output cycles kaput (CLOCK) and brain and muscle Arnt-like protein 1 (BMAL1). These results demonstrate a dual role of CaMKII as a component of cell-autonomous clockwork and as a synchronizer integrating circadian behavioral activities.

In cardiac myocytes, regulation of mitochondrial Ca2+ is important for cellular signaling and cardiac contraction. Ca2+ entry into the mitochondria is mediated by a highly selective Ca2+ channel called the mitochondrial calcium uniporter, which consists of a pore-forming subunit MCU and regulatory subunits such as MICU1. Although pharmacological regulation of the mitochondrial Ca2+ influx is a promising approach to controlling the cellular functions, a cell-permeable and specific inhibitor of the mitochondrial calcium uniporter has not yet been developed. Here, we identify a novel cell-permeable inhibitor of the uniporter by a high-throughput screening of 120 000 small-molecule compounds. In our study, DS16570511 dose-dependently inhibited serum-induced mitochondrial Ca2+ influx in HEK293A cells with an IC50 of 7 μM. DS16570511 inhibited Ca2+ uptake of isolated mitochondria from human cells, rat heart and pig heart. Overexpression of hMCU or hMICU1 in HEK293A cells increased mitochondrial Ca2+ influx, and the increases were completely suppressed by the pretreatment with DS16570511. DS16570511 also blocks mitochondrial Ca2+ overload in a Langendorff perfused beating rat heart. Interestingly, DS16570511 increased cardiac contractility without affecting heart rate in the perfused heart. These results show that DS16570511 is a novel cell-permeable inhibitor of the mitochondrial calcium uniporter and applicable for control of the cardiac functions.

The circadian clock controls daily rhythms in many physiologic processes, and the clock oscillation is regulated by external time cues such as light, temperature, and feeding. In mammals, the transcriptional regulation of clock genes underlies the clock oscillatory mechanism, which is operative even in cultured fibroblasts. We previously demonstrated that glucose treatment of rat-1 fibroblasts evokes circadian expression of clock genes with a rapid induction of Tieg1 transcript encoding a transcriptional repressor. Here, we found diurnal variation of both Tieg1 mRNA and nuclear TIEG1 protein levels in the mouse liver with their peaks at day ⁄ night transition and midnight, respectively. In vitro experiments showed that TIEG1 bound to Bmal1 gene promoter and repressed its transcriptional activity through two juxtaposed GC boxes near the transcription initiation site. The GC box ⁄ TIEG1-mediated repression of Bmal1 promoter was additive to RORE-dependent repression by REV-ERBa, a well-known repressor of Bmal1 gene. In cell-based real-time assay, siRNA-mediated knock-down of TIEG1 caused period shortening of cellular bioluminescence rhythms driven by Bmal1-luciferase and Per2-luciferase reporters. These findings highlight an active role of TIEG1 in the normal clock oscillation and GC box-mediated regulation of Bmal1 transcription.

Circadian rhythms are based on biochemical oscillations generated by clock genes/proteins, which independently evolved in animals, fungi, plants, and cyanobacteria. Temperature compensation of the oscillation speed is a common feature of the circadian clocks, but the evolutionary-conserved mechanism has been unclear. Here, we show that Na+/Ca2+ exchanger (NCX) mediates cold-responsive Ca2+ signaling important for the temperature-compensated oscillation in mammalian cells. In response to temperature decrease, NCX elevates intracellular Ca2+, which activates Ca2+/calmodulin-dependent protein kinase II and accelerates transcriptional oscillations of clock genes. The cold-responsive Ca2+ signaling is conserved among mice, Drosophila, and Arabidopsis. The mammalian cellular rhythms and Drosophila behavioral rhythms were severely attenuated by NCX inhibition, indicating essential roles of NCX in both temperature compensation and autonomous oscillation. NCX also contributes to the temperature-compensated transcriptional rhythms in cyanobacterial clock. Our results suggest that NCX-mediated Ca2+ signaling is a common mechanism underlying temperature-compensated circadian rhythms both in eukaryotes and prokaryotes.