Natural Compounds that Modulate Circadian Rhythms.

Circadian rhythms of innate 24 h cycles comprise well-conserved biological phenomena from cyanobacteria to mammalian. They are driven by light and regulated by clock genes that work as transcription factors and control the expression of many other genes and physiological functions in the cells. The expression of ~ 40% of protein-coding genes shows 24 h oscillation patterns in mice, implying their importance in normal body functions. Indeed, the physiological and behavioural rhythmicity generated through clock genes-mediated multiple mechanisms affects the quality of life at large. Disrupted circadian rhythmicity is associated with several kinds of diseases. For example, cancer cells show abnormal expression patterns for circadian rhythm genes that have been shown to regulate oncogenesis, drug responses, and disease prognosis. Furthermore, the modern globalisation of human lifestyle and business and social activities have disrupted innate circadian rhythm, resulting in a variety of diseases through disrupted humoral, immunological, and neuronal pathways. Safe and sustainable modulation of circadian rhythm has become a prevalent need that warrants basic and interventional research, as well as clinical investigations. Although traditional systems of medicine suggest some natural compounds with circadian rhythmmodulating potential, most of these have not been validated in laboratory or clinical studies. Reliable read-outs of the effects of test compounds on circadian rhythmicity have been limited by the availability of live cell assays. We have, herein, provided an overview of living cell-embedded real- time reporter gene assays designed for screening compounds that modulate circadian rhythm, and discussed the potential of some natural compounds for circadian rhythm modulation as validated by cell-based assay systems, and their role in disease therapeutics.

Withanolide Derivative 2,3-Dihydro-3ß-methoxy Withaferin-A Modulates the Circadian Clock via Interaction with RAR-Related Orphan Receptor α (RORa).

Withanolide derivatives have anticancer, anti-inflammatory, and other functions and are components of Indian traditional Ayurvedic medicine. Here, we found that 2,3-dihydro-3ß-methoxy withaferin-A (3ßmWi-A), a derivative of withaferin-A (Wi-A) belonging to a class of withanolides that are abundant in Ashwagandha (Withania somnifera), lengthened the period of the circadian clock. This compound dose-dependently elongated circadian rhythms in Sarcoma 180 cancer cells and in normal fibroblasts including NIH3T3 and spontaneously immortalized mouse embryonic fibroblasts (MEF). Furthermore, 3ßmWi-A dose-dependently upregulated the mRNA expression and promoter activities of Bmal1 after dexamethasone stimulation and of the nuclear orphan receptors, Rora and Nr1d1, that comprise the stabilization loop for Bmal1 oscillatory expression. We showed that 3ßmWi-A functions as an inverse agonist for RORa with an IC50 of 11.3 µM and that 3ßmWi-A directly, but weakly, interacts with RORa (estimated dissociation constant [Kd], 5.9 µM). We propose that 3ßmWi-A is a novel modulator of circadian rhythms.

ß-Arrestin 1 (ARRB1) serves as a molecular marker of the peripheral circadian rhythm.

The control of the circadian rhythm is important for health because it regulates physiological functions and is associated with health hazards. We aimed to identify a circadian biomarker of health status in human saliva, since collecting saliva is non-invasive, straightforward, and cost-effective. Among 500 genes potentially controlled by the salivary clock identified using chromatin immunoprecipitation (ChIP) assays, 22 of them showed reasonable transcriptional responses according to a DNA array in a salivary model system. Among these 22 genes, ARRB1, which is expressed in human salivary glands, was also expressed in model HSG cells at the transcriptional and translational levels. The profile of ARRB1 expression in human saliva was circadian, suggesting that ARRB1 could serve as a candidate circadian biomarker in saliva. We compared ARRB1 with other biomarkers in salivary samples from jet-lagged individuals. The circadian profile of ARRB1 reflected the time lag more than the profile of melatonin, whereas the profiles of cortisol and α-amylase did not reflect the time lag. Overall, these findings suggest that salivary ARRB1 could serve as a candidate biomarker that could be used to monitor the internal body clock.

Epigenetic regulation of the circadian clock: role of 5-aza-2'-deoxycytidine.

We have been investigating transcriptional regulation of the BMAL1 gene, a critical component of the mammalian clock system including DNA methylation. Here, a more detailed analysis of the regulation of DNA methylation of BMAL1 proceeded in RPMI8402 lymphoma cells. We found that CpG islands in the BMAL1 and the PER2 promoters were hyper- and hypomethylated, respectively and that 5-aza-2'-deoxycytidine (aza-dC) not only enhanced PER2 gene expression but also PER2 oscillation within 24 h in RPMI8402 cells. That is, such hypermethylation of CpG islands in the BMAL1 promoter restricted PER2 expression which was recovered by aza-dC within 1 day in these cells. These results suggest that the circadian clock system can be recovered through BMAL1 expression induced by aza-dC within a day. The RPIB9 promoter of RPMI8402 cells, which is a methylation hotspot in lymphoblastic leukemia, was also hypermethylated and aza-dC gradually recovered RPIB9 expression in 3 days. In addition, methylation-specific PCR revealed a different degree of aza-dC-induced methylation release between BMAL1 and RPIB9 These results suggest that the aza-dC-induced recovery of gene expression from DNA methylation is dependent on a gene, for example the rapid response to demethylation by the circadian system, and thus, is of importance to clinical strategies for treating cancer.

Shikonin shortens the circadian period: possible involvement of Top2 inhibition.

The naphthoquinone pigment, shikonin, is a natural product derived from Lithospermum erythrorhizon and an active component of a Chinese traditional herbal therapeutic. We identified shikonin as a candidate for shortening the circadian period using real-time reporter gene assays based on NIH3T3-derived stable reporter cells. Period length that became shortened in cells incubated with shikonin or etoposide reverted to that of control cells after continued incubation without these compounds. These findings indicated that shikonin and etoposide shorten the circadian period reversibly and through similar mechanisms. Topoisomerase II (Top2)-specific decatenation assays confirmed that shikonin, liker etoposide, is a Top2 inhibitor. Shikonin was incorporated into the nucleus and Top2 was located in the Bmal1 promoter, suggesting the relationship between Bmal1 transcription and Top2 inhibition. Top2a siRNA also shortened period length, suggesting that Top2 is involved in this process. Promoter assays showed that Top2a siRNA, etoposide and shikonin reduce Bmal1 promoter activity. These findings indicated that Top2 is involved in Bmal1 transcription and influences the circadian period, and that shikonin is a novel contributor to the control of period length in mammalian cells.

Rhythmic binding of Topoisomerase I impacts on the transcription of Bmal1 and circadian period.

The Bmal1 gene is essential for the circadian system, and its promoter has a unique open chromatin structure. We examined the mechanism of topoisomerase I (Top1) to understand the role of the unique chromatin structure in Bmal1 gene regulation. Camptothecin, a Top1 inhibitor, and Top1 small interfering RNA (siRNA) enhanced Baml1 transcription and lengthened its circadian period. Top1 is located at an intermediate region between two ROREs that are critical cis-elements of circadian transcription and the profile of Top1 binding indicated anti-phase circadian oscillation of Bmal1 transcription. Promoter assays showed that the Top1-binding site is required for transcriptional suppression and that it functions cooperatively with the distal RORE, supporting that Bmal1 transcription is upregulated by Top1 inhibition. A DNA fragment between the ROREs, where the Top1-binding site is located, behaved like a right-handed superhelical twist, and modulation of Top1 activity by camptothecin and Top1 siRNA altered the footprint profile, indicating modulation of the chromatin structure. These data indicate that Top1 modulates the chromatin structure of the Bmal1 promoter, regulates Bmal1 transcription and influences the circadian period.

Lycorine, a candidate for the control of period length in mammalian cells.

Plants of the Amaryllidaceae family have been used as therapeutic agents against CNS related maladies such as Alzheimer's disease. The known primary alkaloid constituents have significant biological activity. We identified the Lycoris alkaloids lycorine and lycoricidinol from Amaryllidaceae using a real-time reporter gene assay system based on NIH3T3 cells. These alkaloids have a wide spectrum of pharmacological actions and dose-dependently lengthen the circadian period. When cells that had been incubated with lycorine or lycoricidinol were washed and then incubated without these alkaloids, period length reverted to that of control cells, suggesting that elongation of the circadian period induced by lycorine and lycoricidinol is reversible. Although one of its major activities is the inhibition of protein synthesis, lycorine induced dose-dependent period elongation regardless of the presence of cycloheximide and moreover, cycloheximide, itself did not affect period length, suggesting that lycorine dose-dependently extends the circadian period by a mechanism other than translational inhibition. Real-time RT-PCR showed that lycorine enhanced RORα and Bmal1 transcription, and exogenous expression and knockdown of Bmal1 also caused long and short periods, respectively, thus confirming the phenotype indicated by lycorine. These data indicate that lycorine and lycoricidinol modulate Bmal1 transcription and the circadian period, and also suggest that Lycoris alkaloids are novel contributors to the control of period length in mammalian cells.

The harmala alkaloid harmine is a modulator of circadian Bmal1 transcription.

Biological rhythms are orchestrated by a cell-autonomous clock system that drives the rhythmic cascade of clock genes. We established an assay system using NIH 3T3 cells stably expressing the Bmal1 promoter-driven luciferase reporter gene and used it to analyse circadian oscillation of the gene. Modulators of PKC (protein kinase C) revealed that an activator and an inhibitor represented short- and long-period phenotypes respectively which were consistent with reported effects of PKC on the circadian clock and validated the assay system. We examined the effects of the alkaloid harmine, contained in Hoasca, which has a wide spectrum of pharmacological actions, on circadian rhythms using the validated assay system. Harmine dose dependently elongated the period. Furthermore, EMSA (electrophoretic mobility-shift assay) and Western-blot analysis showed that harmine enhanced the transactivating function of RORα (retinoid-related orphan receptor α), probably by increasing its nuclear translocation. Exogenous expression of RORα also caused a long period, confirming the phenotype indicated by harmine. These results suggest that harmine extends the circadian period by enhancing RORα function and that harmine is a new candidate that contributes to the control of period length in mammalian cells.

HSG cells, a model in the submandibular clock.

Circadian rhythm of vital processes is essential to health, and various tissues show unique peripheral rhythms. HSG is the human submandibular gland cell line that has been used for analysing the effects of steroids and growth factors. In the present study, we analysed the transcriptional regulation of the BMAL1 gene, a critical component of the mammalian clock system to investigate the possibility of using HSG cells as a model system of the submandibular clock. The BMAL1 gene was expressed with circadian oscillation after stimulation with dexamethasone, and its regulatory region contained two recognition motifs for ROR (retinoic acid-receptor-related orphan receptor) and ROREs [RORα (ROR α-subunit)-binding elements] in hypomethylated CpG islands with an open chromatin structure. REV-ERBα was expressed with circadian oscillation, and knockdown experiments suggested that REV-ERBα is involved in circadian transcription of the BMAL1 gene in HSG cells. These results are similar to those in NIH 3T3 cells, a standard model for the circadian system, whereas RORα required for REV-ERBα antagonism was expressed very little in HSG cells. These findings show that in the salivary gland cell line HSG there is a rhythm in the core oscillator components BMAL1 and REV-ERBα, indicating that circadian-based transcriptional regulation can be modelled in this peripheral cell type.

Conserved amino acid residues in C-terminus of PERIOD 2 are involved in interaction with CRYPTOCHROME 1.

We investigated the amino acid sequences of rat PERIOD2 (rPER2) that are required for interaction with CRYPTOCHROME1 (CRY1) to understand the molecular mechanism of the circadian clock. Co-immunoprecipitation assays using various C-terminal fragments of rPER2 with internal deletions revealed that amino acid residues 1179-1198 are necessary for interaction with CRY1. To identify precisely which amino acid residues are responsible for the interaction, we substituted alanine for residues conserved among PER isoforms and species. We found that more than three mutations of conserved PER2 residues impaired not only binding to CRY1 but also subsequent nuclear translocation, although mutations of non-conserved residues did not affect interaction with CRY1. Thus, the conserved amino acid residues of 1179-1198 in PER2 are apparently responsible for binding to CRY1.

Humidification during high-frequency oscillation ventilation is affected by ventilator circuit and ventilatory setting.

BACKGROUND: High-frequency oscillation ventilation (HFOV) is an accepted ventilatory mode for acute respiratory failure in neonates. As conventional mechanical ventilation, inspiratory gas humidification is essential. However, humidification during HFOV has not been clarified. In this bench study, we evaluated humidification during HFOV in the open circumstance of ICU. Our hypothesis is that humidification during HFOV is affected by circuit design and ventilatory settings. METHODS/MATERIALS: We connected a ventilator with HFOV mode to a neonatal lung model that was placed in an infant incubator set at 37 degrees C. We set a heated humidifier (Fisher & Paykel) to obtain 37 degrees C at the chamber outlet and 40 degrees C at the distal temperature probe. We measured absolute humidity and temperature at the Y-piece using a rapid-response hygrometer. We evaluated two types of ventilator circuit: a circuit with inner heating wire and another with embedded heating element. In addition, we evaluated three lengths of the inspiratory limb, three stroke volumes, three frequencies, and three mean airway pressures. RESULTS: The circuit with embedded heating element provided significantly higher absolute humidity and temperature than one with inner heating wire. As an extended tube lacking a heating wire was shorter, absolute humidity and temperature became higher. In the circuit with inner heating wire, absolute humidity and temperature increased as stroke volume increased. CONCLUSION: Humidification during HFOV is affected by circuit design and ventilatory settings.

Molecular characterization of Mybbp1a as a co-repressor on the Period2 promoter.

The circadian clock comprises transcriptional feedback loops of clock genes. Cryptochromes are essential components of the negative feedback loop in mammals as they inhibit CLOCK-BMAL1-mediated transcription. We purified mouse CRY1 (mCRY1) protein complexes from Sarcoma 180 cells to determine their roles in circadian gene expression and discovered that Myb-binding protein 1a (Mybbp1a) interacts with mCRY1. Mybbp1a regulates various transcription factors, but its role in circadian gene expression is unknown. We found that Mybbp1a functions as a co-repressor of Per2 expression and repressed Per2 promoter activity in reporter assays. Chromatin immunoprecipitation (ChIP) assays revealed endogenous Mybbp1a binding to the Per2 promoter that temporally matched that of mCRY1. Furthermore, Mybbp1a binding to the Per2 promoter correlated with the start of the down-regulation of Per2 expression and with the dimethylation of histone H3 Lys9, to which it could also bind. These findings suggest that Mybbp1a and mCRY1 can form complexes on the Per2 promoter that function as negative regulators of Per2 expression.

Rhythmic SAF-A binding underlies circadian transcription of the Bmal1 gene.

Although Bmal1 is a key component of the mammalian clock system, little is understood about the actual mechanism of circadian Bmal1 gene transcription, particularly at the chromatin level. Here we discovered a unique chromatin structure within the Bmal1 promoter. The RORE region, which is a critical cis element for the circadian regulation of the Bmal1 gene, is comprised of GC-rich open chromatin. The 3'-flanking region of the promoter inhibited rhythmic transcription in the reporter gene assay in vitro even in the presence of RORalpha and REV-ERBalpha. We also found that the nuclear matrix protein SAF-A binds to the 3'-flanking region with circadian timing, which was correlated with Bmal1 expression by footprinting in vivo. These results suggest that the unique chromatin structure containing SAF-A is required for the circadian transcriptional regulation of the Bmal1 gene in cells.

Identification of negative transcriptional factor E4BP4-binding site in the mouse circadian-regulated gene Mdr2.

The hepatic transporter Mdr2 is an ATP-binding cassette transporter which excretes phosphatidylcholine into the bile. We showed that the level of Mdr2 mRNA oscillated in circadian fashion in mouse liver whereas such oscillation was dampened in the liver of Clock mutants. To examine transcriptional regulation of the Mdr2 gene we performed luciferase reporter assays using plasmid constructs containing the 5'-flanking region of the Mdr2 gene. Reporter assays using deletion constructs demonstrated that E4BP4 represses the transcriptional activity of the promoter including the D1 and D2 sites within four putative E4BP4-binding sites. Chromatin immunoprecipitation and gel shift assays showed that E4BP4 binds to the D2 site, but not to the D1 site. These data suggested that E4BP4 is a negative transcription factor for circadian Mdr2 mRNA expression.

The negative transcription factor E4BP4 is associated with circadian clock protein PERIOD2.

The bZIP transcription factor E4BP4, is a mammalian homologue of vrille that functions as a key negative component of the circadian clock. We have shown that the E4BP4-binding site (B-site) is required in addition to a non-canonical E-box (E2 enhancer) for robust circadian Period2 (Per2) expression in the cell-autonomous clock. While the E2 enhancer and the B-site are closely situated, correlations between each component bound to the E2 enhancer and the B-site remain obscure. Here, we show that E4BP4 interacts with PER2, which represses transcriptional activity via the E-box enhancer. Interaction with PER2 required the carboxyl-terminal region that contains the repression domain of E4BP4. We also found that E4BP4 interacts with CRYPTOCHROME2 (CRY2), a key negative regulator in the mammalian circadian clock. These results suggest that E4BP4 is a component of the negative regulator complex of mammalian circadian clocks.

A novel E4BP4 element drives circadian expression of mPeriod2.

Period2 (Per2) is an essential component of the mammalian clock mechanism and robust circadian expression of Per2 is essential for the maintenance of circadian rhythms. Although recent studies have shown that the circadian E2 enhancer (a non-canonical E-box) accounts for most of the circadian transcriptional drive of mPer2, little is known about the other cis-elements of mPer2 oscillatory transcription. Here, we examined the contribution of E4BP4 to Per2 mRNA oscillation in the cell-autonomous clock. Knockdown experiments of E4BP4 in both Northern blots and real-time luciferase assays suggested that endogenous E4BP4 negatively regulates Per2 mRNA oscillation. Sequence analysis revealed two putative E4BP4-binding sites (termed A-site and B-site) on mammalian Per2 promoter regions. Luciferase assays with mutant constructs showed that a novel E4BP4-binding site (B-site) is responsible for E4BP4-mediated transcriptional repression of Per2. Furthermore, chromatin immunoprecipitation assays in vivo showed that the peak of E4BP4 binding to the B-site on the Per2 promoter almost matched the trough of Per2 mRNA expression. Importantly, real-time luciferase assays showed that the B-site in addition to the E2 enhancer is required for robust circadian expression of Per2 in the cell-autonomous clock. These findings indicated that E4BP4 is required for the negative regulation of mammalian circadian clocks.

Biochemical screening of stable dinucleosomes using DNA fragments from a dinucleosome DNA library.

The dinucleosome is an informative unit for analysis of the higher-order chromatin structure. DNA fragments forming stable dinucleosomes were screened from a dinucleosome DNA library after the reconstitution of nucleosomes in vitro and digestion with micrococcal nuclease. Reconstituted dinucleosomes showed a diversity of sensitivity to micrococcal nuclease, suggesting that the biochemical stability of a dinucleosome depends, in part, on the DNA fragments. The DNA fragments after the screening were classified into three groups represented by clones bf10, af14 and af32 according to the sensitivity to micrococcal nuclease. Mapping of the nucleosome boundaries by Southern blotting of the DNA after restriction digestion and by primer extension analysis showed that each nucleosome position of clone af32 was fixed. Analysis of reconstituted dinucleosomes using mutant DNA fragments of clone af32 revealed a unique property characteristic of a key nucleosome, given that the replacement of a DNA fragment corresponding to the right nucleosome position resulted in marked sensitivity to micrococcal nuclease, whereas the replacement of the other nucleosome fragment had almost no effect on sensitivity as compared to the original af32 construct. The mutant construct in which the right nucleosome was removed showed multiple nucleosome phases, suggesting that the right nucleosome stabilized first each mononucleosome and then the dinucleosome. An oligonucleotide bending assay revealed that the DNA fragment in the right nucleosome included curved DNA, suggesting that the positioning activity of the nucleosome was attributed to its DNA structure. These results suggest that information for forming stable dinucleosome is embedded in the genomic DNA and that a further characterization of the key nucleosome is useful for understanding the building up of the chromatin structure.

Alternative splicing of the human Kank gene produces two types of Kank protein.

The human Kank gene encodes an ankyrin repeat domain-containing protein which regulates actin polymerization. There are at least two types of Kank protein depending on cell type, likely due to differences in transcription. Here, to examine the transcriptional initiation and genomic organization of the human Kank gene, we performed 5'-RACE (rapid amplification of cDNA ends) using total RNA from normal kidney and a human kidney cancer cell line, VMRC-RCW cells. The results suggest that the human Kank gene has several alternative first exons. While mRNA from VMRC-RCW cells encoded Kank protein (referred to as Kank-S) as reported previously, mRNA from the normal kidney tissue encoded a novel type of Kank protein (referred to as Kank-L), which contained an additional N-terminal sequence 158 amino acids long. Promoter activity and the expression of the Kank variants in normal and cancer tissues were examined.

Expressional regulation of neuronal and cancer-related genes by estrogen in adult female rats.

Adult female rats were ovariectomized and treated with or without estrogen for two weeks. mRNA was obtained from the hypothalamus, uterus, liver, kidney and skeletal muscle and analyzed by Northern blotting and/or RT-PCR. We examined two types of estrogen-responsive genes from rats, neuronal system-related genes (Amphiregulin, AR; Neuropeptide Y-Y1 receptor, NPY-Y1R; Bassoon, BSN; N-Cadherin, N-CADH) and estrogen-susceptible cancer-related genes (C-terminal binding protein interacting protein, CtIP), based on the results of a cDNA microarray analysis which was carried out to profile estrogen-responsive genes in the human breast cancer cell line MCF-7. The N-CADH gene showed identical response to that in MCF-7 cells. In the hypothalamus, all except the AR gene were down-regulated in their expression. In other tissues, the expression showed marked differences: expression of the BSN gene was not detected by either method, and the NPY-Y1R gene showed down-regulation in most tissues except for skeletal muscle. We then analyzed the time course of the estrogen-responsiveness of these genes in several tissues, finding changes in expression patterns especially in skeletal muscle but not in the hypothalamus. Our results show that the estrogen-responsive genes, which were demonstrated simply as either up- or down-regulated in their expression by estrogen in a human cell line using cDNA microarrays, exhibit tissue and temporal-specific expression patterns in adult female rats.

Preparation and characterization of an anti-DNA monoclonal antibody showing size selectivity toward DNA fragments.

Anti-DNA monoclonal antibodies were prepared using an in vitro immunization method. Balb/c mouse splenocytes were immunized with HeLa cell nuclear extract in the presence of N-acetylmuramyl-L-alanyl-D-isoglutamine and fused with P3U1 myeloma cells using PEG 4000. After HAT selection and ELISA using fragmented HeLa genomic DNA, an anti-DNA monoclonal antibody was obtained. The monoclonal antibody D-1-1, whose isotype was IgM, interacted with a variety of double-stranded DNA. The antibody reacted only with DNA fragments longer than 0.8 kbp, and its apparent dissociation constant for a 1.0-kbp DNA fragment was 34 nM. This antibody will be a helpful tool for the detection of DNA structures.