Inhibitory role of REV-ERBα in the expression of bone morphogenetic protein gene family in rat uterus endometrium stromal cells.

Uterus circadian rhythms have been implicated in the gestation processes of mammals through entraining of the clock proteins to numerous downstream genes. Bone morphogenetic proteins (BMPs), having clock-controlled regulatory sites in their gene promoters, are expressed in the uterus during decidualization, but the regulation of the Bmp gene expression is poorly understood. The present study was designed to dissect the physiological roles of the uterus oscillators in the Bmp expression using the uterus endometrial stromal cells (UESCs) isolated from Per2-dLuc transgenic rats on day 4.5 of gestation. The in vitro decidualization of UESCs was induced by medroxyprogesterone acetate and 2-O-dibutyryl cAMP. A significant decline of Per2-dLuc bioluminescence activity was induced in decidual cells, and concomitantly, the expression of canonical clock genes was downregulated. Conversely, the expression of the core Bmp genes Bmp2, Bmp4, Bmp6, and Bmp7 was upregulated. In UESCs transfected with Bmal1-specific siRNA, in which Rev-erbα expression was downregulated, Bmp genes, such as Bmp2, Bmp4, and Bmp6 were upregulated. However, Bmp1, Bmp7, and Bmp8a were not significantly affected by Bmal1 silencing. The expression of all Bmp genes was enhanced after treatment with the REV-ERBα antagonist (SR8278), although their rhythmic profiles were differed from each other. The binding of REV-ERBα to the proximal regions of the Bmp2 and Bmp4 promoters was revealed by chromatin immunoprecipitation-PCR analysis. Collectively, these results indicate that the Bmp genes are upregulated by the attenuation of the cellular circadian clock; in particular, its core component REV-ERBα functions as a transcriptional silencer in the Bmp gene family.

Removal of Rev-erbα inhibition contributes to the prostaglandin G/H synthase 2 expression in rat endometrial stromal cells.

The rhythmic expression of clock genes in the uterus is attenuated during decidualization. This study focused on Ptgs2, which is essential for decidualization, as a putative clock-controlled gene, and aimed to reveal the functions of clock genes in relation to Ptgs2 during decidualization. We compared the transcript levels of clock genes in the rat uterus on days 4.5 (D4.5) and 6.5 of pregnancy. The transcript levels of clock genes (Per2, Bmal1, Rorα, and Rev-erbα) had decreased at implantation sites on day 6.5 (D6.5e) compared with those on D4.5, whereas Ptgs2 transcripts had increased on D6.5e. Similar observations of Rev-erbα and Ptgs2 were also obtained in the endometrium on D6.5e by immunohistochemistry. In the decidual cells induced by medroxyprogesterone and 2-O-dibutyryl-cAMP, the rhythmic expression levels of clock genes were attenuated, whereas Ptgs2 transcription was induced. These results indicate that decidualization causes the attenuation of clock genes and the induction of Ptgs2. Furthermore, in the experiment of Bmal1 siRNA, the rhythmic expression of clock genes and Ptgs2 was attenuated by the siRNA. Transcript levels of Ptgs2 and prostaglandin (PG)E2 production were increased by treatment with the Rev-erbα antagonist, suggesting the contribution of the nuclear receptor Rev-erbα to Ptgs2 expression. Moreover, Rev-erbα knockdown enhanced the induction of Ptgs2 transcription and PGE2 production by forskolin. Chromatin immunoprecipitation-PCR analysis revealed that Rev-erbα could directly bind to a proximal RORE site of Ptgs2. Collectively, this study demonstrates that the attenuation of the circadian clock, especially its core component Rev-erbα, contributes to the induction of Ptgs2 during decidualization.

System-level identification of transcriptional circuits underlying mammalian circadian clocks.

Mammalian circadian clocks consist of complexly integrated regulatory loops, making it difficult to elucidate them without both the accurate measurement of system dynamics and the comprehensive identification of network circuits. Toward a system-level understanding of this transcriptional circuitry, we identified clock-controlled elements on 16 clock and clock-controlled genes in a comprehensive surveillance of evolutionarily conserved cis elements and measurement of their transcriptional dynamics. Here we report the roles of E/E' boxes, DBP/E4BP4 binding elements and RevErbA/ROR binding elements in nine, seven and six genes, respectively. Our results indicate that circadian transcriptional circuits are governed by two design principles: regulation of E/E' boxes and RevErbA/ROR binding elements follows a repressor-precedes-activator pattern, resulting in delayed transcriptional activity, whereas regulation of DBP/E4BP4 binding elements follows a repressor-antiphasic-to-activator mechanism, which generates high-amplitude transcriptional activity. Our analysis further suggests that regulation of E/E' boxes is a topological vulnerability in mammalian circadian clocks, a concept that has been functionally verified using in vitro phenotype assay systems.

Downregulation of core clock gene Bmal1 attenuates expression of progesterone and prostaglandin biosynthesis-related genes in rat luteinizing granulosa cells.

Ovarian circadian oscillators have been implicated in the reproductive processes of mammals. However, there are few reports regarding the detection of ovarian clock-controlled genes (CCGs). The present study was designed to unravel the mechanisms through which CCG ovarian circadian oscillators regulate fertility, primarily using quantitative RT-PCR and RNA interference against Bmal1 in rat granulosa cells. Mature granulosa cells were prepared from mouse Per2-destabilized luciferase (dLuc) reporter gene transgenic rats. A real-time monitoring system of Per2 promoter activity was employed to detect Per2-dLuc oscillations. The cells exposed to luteinizing hormone (LH) displayed clear Per2-dLuc oscillations and a rhythmic expression of clock genes (Bmal1, Per1, Per2, Rev-erbα, and Dbp). Meanwhile, the examined ovarian genes (Star, Cyp19a1, Cyp11a1, Ptgs2, Lhcgr, and p53) showed rhythmic transcript profiles except for Hsd3b2, indicating that these rhythmic expression genes may be CCGs. Notably, Bmal1 small interfering (si)RNA treatment significantly decreased both the amplitude of Per2-dLuc oscillations and Bmal1 mRNA levels compared with nonsilencing RNA treatment in luteinizing granulosa cells. Depletion of Bmal1 by siRNA decreased the transcript levels of clock genes (Per1, Per2, Rev-erbα, and Dbp) and examined ovarian genes (Star, Cyp19a1, Cyp11a1, Ptgs2, Hsd3b2, and Lhcgr). Accordingly, knockdown of Bmal1 also inhibited the synthesis of progesterone and prostaglandin E2, which are associated with crucial reproductive processes. Collectively, these data suggest that ovarian circadian oscillators regulate the synthesis of steroid hormones and prostaglandins through ovarian-specific CCGs in response to LH stimuli. The present study provides new insights into the physiologic significance of Bmal1 related to fertility in ovarian circadian oscillators.

FSH induces the development of circadian clockwork in rat granulosa cells via a gap junction protein Cx43-dependent pathway.

The present study was designed to assess the relationship between gap junctions and the maturation of a clock system in rat granulosa cells stimulated by follicle-stimulating hormone (FSH). Immature and mature granulosa cells were prepared by puncturing the ovaries of diethylstilbestrol- and equine chorionic gonadotropin (eCG)-treated mouse Period2 (Per2)-dLuc reporter gene transgenic rats, respectively. Mature granulosa cells exposed to dexamethasone (DXM) synchronization displayed several Per2-dLuc oscillations and a rhythmic expression of clock genes. Intriguingly, we observed clear evidence that the FSH stimulation significantly increased the amplitude of Per2 oscillations in the granulosa cells, which was confirmed by the elevation of the Per2 and Rev-erbα (Nr1d1) mRNA levels. FSH also induced a major phase-advance shift of Per2 oscillations. The mature granulosa cells cultured for 2 days with FSH expressed higher mRNA levels of Per2, Rev-erbα, Bmal1 (Arnt1), Lhcgr, and connexin (Cx) 43 (Gja1) compared with the immature granulosa cells. Consistently, our immunofluorescence results revealed abundant Cx43 protein in antral follicles stimulated with eCG and weak or no fluorescence signal of Cx43 in primary and preantral follicles. Similar results were confirmed by Western blotting analysis. Two gap junction blockers, lindane and carbenoxolone (CBX), significantly decreased the amplitude of Per2 oscillations, which further adhered significant decreases in Per2 and Rev-erbα transcript levels. In addition, both lindane and CBX induced a clear phase-delay shift of Per2 oscillations. These findings suggest that FSH induces the development of the clock system by increasing the expression of Cx43.

Regional circadian period difference in the suprachiasmatic nucleus of the mammalian circadian center.

The suprachiasmatic nucleus (SCN) is the mammalian circadian rhythm center. Individual oscillating neurons have different endogenous circadian periods, but they are usually synchronized by an intercellular coupling mechanism. The differences in the period of each oscillating neuron have been extensively studied; however, the clustering of oscillators with similar periods has not been reported. In the present study, we artificially disrupted the intercellular coupling among oscillating neurons in the SCN and observed regional differences in the periods of the oscillating small-latticed regions of the SCN using a transgenic rat carrying a luciferase reporter gene driven by regulatory elements from a per2 clock gene (Per2::dluc rat). The analysis divided the SCN into two regions--aregion with periods shorter than 24 h (short-period region, SPR) and another with periods longer than 24 h (long-period region, LPR). The SPR was located in the smaller medial region of the dorsal SCN, whereas the LPR occupied the remaining larger region. We also found that slices containing the medial region of the SCN generated shorter circadian periods than slices that contained the lateral region of the SCN. Interestingly, the SPR corresponded well with the region where the SCN phase wave is generated. We numerically simulated the relationship between the SPR and a large LPR. A mathematical model of the SCN based on our findings faithfully reproduced the kinetics of the oscillators in the SCN in synchronized conditions, assuming the existence of clustered short-period oscillators.

Contribution of FSH and triiodothyronine to the development of circadian clocks during granulosa cell maturation.

The involvement of FSH and triiodothyronine (T(3)) in circadian clocks was investigated using immature granulosa cells of ovaries during the progress of cell maturation. Granulosa cells were prepared from preantral follicles of mouse Period2 (Per2)-dLuc reporter gene transgenic rats injected subcutaneously with the synthetic nonsteroidal estrogen diethylstilbestrol. Analysis of the cellular clock of the immature granulosa cells was performed partly using a serum-free culture system. Several bioluminescence oscillations of Per2-dLuc promoter activity were generated in the presence of FSH + fetal bovine serum, but not in the presence of either FSH or serum. As revealed by bioluminescence recording and analysis of clock gene expression, the granulosa cells lack the functional cellular clock at the immature stage, although Lhr was greatly expressed during the period of cell maturation. The granulosa cells gained a strong circadian rhythm of bioluminescence during stimulation with FSH, whereas LH reset the cellular clock of matured granulosa cells. During strong circadian rhythms of clock genes, the Star gene showed significant expression in matured granulosa cells. In contrast, T(3) showed an inhibitory effect on the development of the functional cellular clock during the period of cell maturation. These results indicate that FSH provides a cue for the development of the functional cellular clock of the immature granulosa cells, and T(3) blocks the development of the cellular clock.

Rev-erbα regulates circadian rhythms and StAR expression in rat granulosa cells as identified by the agonist GSK4112.

The Rev-erbα gene is regarded as a circadian clock gene and clock-regulated gene which regulates the circadian transcriptional/translational loop in a subtle way. Here, we first detected the circadian oscillation in mature granulosa cells from antral follicles using a real-time monitoring system of Per2 promoter activity with the addition of FSH. Then we used GSK4112, an agonist ligand of Rev-erbα, to investigate the function of Rev-erbα. GSK4112 treatment significantly reduced the Per2-dLuc amplitude and induced the Per2 oscillation phase advance shift. GSK4112 significantly inhibited Bmal1 mRNA expression, whereas it did clearly stimulate expression of StAR mRNA in a dose-dependent manner. Our data are the first to show the Rev-erbα function in the steroid biosynthesis of rat granulosa cells, and to suggest that Rev-erbα may coordinate circadian rhythm and metabolism in rat ovaries.

Identification of functional clock-controlled elements involved in differential timing of Per1 and Per2 transcription.

It has been proposed that robust rhythmic gene expression requires clock-controlled elements (CCEs). Transcription of Per1 was reported to be regulated by the E-box and D-box in conventional reporter assays. However, such experiments are inconclusive in terms of how the CCEs and their combinations determine the phase of the Per1 gene. Whereas the phase of Per2 oscillation was found to be the most delayed among the three Period genes, the phase-delaying regions of the Per2 promoter remain to be determined. We therefore investigated the regulatory mechanism of circadian Per1 and Per2 transcription using an in vitro rhythm oscillation-monitoring system. We found that the copy number of the E-box might play an important role in determining the phase of Per1 oscillation. Based on real-time bioluminescence assays with various promoter constructs, we provide evidence that the non-canonical E-box is involved in the phase delay of Per2 oscillation. Transfection experiments confirmed that the non-canonical E-box could be activated by CLOCK/BMAL1. We also show that the D-box in the third conserved segment of the Per2 promoter generated high amplitude. Our experiments demonstrate that the copy number and various combinations of functional CCEs ultimately led to different circadian phases and amplitudes.

Regular feeding plays an important role in cholesterol homeostasis through the liver circadian clock.

RATIONALE: Peripheral clock control and the relevance of the circadian rhythm to physiology and disease are major questions in mammalian circadian biology. OBJECTIVE: We examined the physiological functions of the liver clock. METHODS AND RESULTS: We established a suppressed feeding schedule regimen constituting a high-cholesterol diet delivered every 6 hours without changes in energy and cholesterol intake. We found that rats exposed to this regimen developed hypercholesteremia. In the liver, the rhythmicity of expression of several clock genes was disrupted. Furthermore, the nocturnal expression of the CYP7A1 gene, which encodes the rate-limiting enzyme for the conversion of cholesterol to bile acids, was shifted to a diurnal pattern. Indeed, suppression of a regular feeding rhythm increased the secretion rate of very-low-density lipoprotein cholesterol from the liver and decreased the excretion of fecal bile acids. CONCLUSIONS: Our results demonstrated that not only the amount and quality of food but also the timing of meals has crucial health implications.

Up-regulation of circadian clock gene Period 2 in the prostate mesenchymal cells during flutamide-induced apoptosis.

Androgen regulates the proper development and physiological function of the prostate. Here, we investigated the modulation of androgen and androgen receptor (AR) antagonist on circadian oscillations of a clock core gene Period 2 (Per2) in rat prostate mesenchymal cells (PMCs). Circadian oscillations were analyzed with the real-time monitoring system of gene expression using transgenic rats introduced with mouse Per2 promoter fused to a destabilized luciferase (Per2-dLuc) reporter gene. Analyses of circadian oscillations, immunofluorescence, and androgen response element (ARE)-luciferase reporter assay revealed that circadian clocks are operative and the AR protein is functional in PMCs in vitro. Androgen such as testosterone (T) and dihydrotestosterone (DHT) did not cause any changes in circadian Per2-dLuc oscillations of confluent cells. Conversely, flutamide (FL) up-regulated the amplitude of circadian Per2-dLuc oscillations in a dose-dependent manner, whereas T antagonized the action of FL. The PER2 protein was markedly accumulated by FL treatment and localized in both the nucleus and cytoplasm during the first peak period of circadian Per2-dLuc oscillations. Simultaneously, FL treatment increased apoptotic cell death. Collectively, the present study demonstrates that a clock gene Per2 is up-regulated in PMCs during FL-induced apoptotic cell death. Thus, circadian oscillations of Per2 gene expression may be closely linked to the cellular states of PMCs such as apoptotic cell death.

Progesterone, but not estradiol, synchronizes circadian oscillator in the uterus endometrial stromal cells.

The circadian oscillator is generated within the suprachiasmatic nuclei and synchronizes circadian clocks in numerous peripheral tissues. The molecular basis is composed of a number of genes and proteins that form transcriptional and translational feedback loops. Such molecular oscillators are also operative in peripheral tissues, including in the uterus. Although ovarian steroids regulate the function of uterine endometrial stromal cells, the modulation of ovarian steroids on the circadian rhythms remains unknown. Here we investigate the possibility that estradiol (E2) and progesterone (P4) modulate the circadian oscillator of the stromal cells. The study using transgenic rats constructed with Period 2 (Per2) promoter-destabilized luciferase (Per2-dLuc) gene, with the real-time monitoring system of Per2-dLuc oscillation. The stromal cells displayed constant Per2-dLuc oscillation after treatment with dexamethasone, suggesting that the circadian oscillator is operative. However, the circadian oscillator was disrupted by in vivo administration of human chorionic gonadotropin (hCG) following equine chorionic gonadotropin (eCG), although it was altered into a rhythmic pattern 4 days later following hCG. Chronic treatment with P4 induced constant Per2-dLuc oscillation in the stromal cells from eCG-treated immature and pregnant rats, whereas E2 did not promote such a rhythmic Per2-dLuc oscillation. Collectively, P4 synchronizes the circadian oscillator of the uterus endometrial stromal cells through transcriptional and translational feedback loops of the clockwork system.

Angiopoietin-related growth factor suppresses gluconeogenesis through the Akt/forkhead box class O1-dependent pathway in hepatocytes.

Angiopoietin-related growth factor (AGF; or Angptl6) is a liver-derived, circulating factor and is considered to be a regulator of metabolic homeostasis. AGF is capable of counteracting both obesity and obesity-related insulin resistance. However, the target tissues and the molecular mechanisms underlying the antiobesity and antidiabetic actions of AGF have not been completely defined. Using rat hepatoma H4IIEc3 cells or primary hepatocytes, we demonstrate that AGF suppresses glucose production in a concentration-dependent manner through reduced expression of a key gluconeogenic enzyme, glucose-6-phosphatase (G6Pase), at both transcriptional and translational levels. The action of AGF on glucose production was inhibited by pretreatment of the cells with LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], a phosphoinositide 3-kinase (PI3K) inhibitor, and Akt (protein kinase B) inhibitors. AGF increased the phosphorylation of Akt and its substrates, glycogen synthase kinase 3beta and forkhead box class O1 (FoxO1), a key transcription factor for G6Pase expression. Furthermore, an immunohistochemical approach with anti-FoxO1 antibody demonstrated that AGF stimulation promoted translocation of FoxO1 from the nucleus to the cytoplasm in the cells. These results suggest that in hepatocytes, AGF suppresses gluconeogenesis via reduced transcriptional activity of FoxO1 resulting from the activation of PI3K/Akt signaling cascades.

The disruption of circadian clockwork in differentiating cells from rat reproductive tissues as identified by in vitro real-time monitoring system.

The circadian clock, regulating hormonal secretion and metabolisms in accordance with the environmental light-dark cycle, resides in almost all peripheral tissues as well as in the superchiasmatic nucleus. Clock gene expression has been found to be noncyclic during spermatogenesis and the differentiation of thymocytes. However, currently little is known about how cell differentiation could affect circadian clockwork. We performed this study using the in vitro real-time oscillation monitoring system to examine the clockwork in several types of differentiating cells originated from reproductive tissues of transgenic rats (constructed with Period gene 2 (Per2) promoter-destabilized luciferase reporter gene). After treatment with dexamethasone (DXM), persistent oscillation of Per2 expression was observed in both gonadotropin-induced and pregnant ovarian luteal cells, proliferative uterine stromal cells (USCs), and nondifferentiating testicular interstitial cells, with a cyclic period of ~24 h. In contrast to these cell types, only one cycle of oscillation was sustained in granulosa cells undergoing differentiation. Additionally, Per2 oscillation was irregular in USCs undergoing decidualization induced by medroxyprogesterone acetate plus N6, 2-O-dibutyryl adenosine 3':5'-cyclic monophosphate. Furthermore, no oscillation of Per2 expression was evoked by DXM in Leydig cells and thymocytes. In conclusion, the present study characterized the oscillation of Per2 gene expression in several types of ovarian, uterine, and testicular cells, and it is strongly suggested that circadian clockwork is affected during cellular differentiation.

Proinsulin C-peptide stimulates a PKC/IkappaB/NF-kappaB signaling pathway to activate COX-2 gene transcription in Swiss 3T3 fibroblasts.

Proinsulin C-peptide causes multiple molecular and physiological effects, and improves renal and neuronal dysfunction in patients with diabetes. However, whether C-peptide controls the inhibitor kappaB (IkappaB)/NF-kappaB-dependent transcription of genes, including inflammatory genes is unknown. Here we showed that 1 nM C-peptide increased the expression of cyclooxygenase-2 (COX-2) mRNA and its protein in Swiss 3T3 fibroblasts. Consistently, C-peptide enhanced COX-2 gene promoter-activity, which was inhibited by GF109203X and Go6976, specific PKC inhibitors, and BAY11-7082, a specific nuclear factor-kappaB (NF-kappaB) inhibitor, accompanied by increased phosphorylation and degradation of IkappaB. These results suggest that C-peptide stimulates the transcription of inflammatory genes via activation of a PKC/IkappaB/NF-kappaB signaling pathway.

The nuclear receptor REV-ERBα represses the transcription of growth/differentiation factor 10 and 15 genes in rat endometrium stromal cells.

Cellular oscillators in the uterus play critical roles in the gestation processes of mammals through entraining of the clock proteins to numerous downstream genes, including growth/differentiation factor (Gdf)10 and Gdf15. The expression of Gdf10 and Gdf15 is significantly increased in the uterus during decidualization, but the mechanism underlying the regulation of Gdf gene expression in the uterus is poorly understood. Here, we focused on the function of the cellular oscillators in the expression of Gdf family by using uterine endometrial stromal cells (UESCs) isolated from pregnant Per2-dLuc transgenic rats. A significant decline of Per2-dLuc bioluminescence activity was induced in in vitro decidualized UESCs, and concomitantly the expression of canonical clock genes was downregulated. Conversely, the expression of Gdf10 and Gdf15 of the Gdf was upregulated. In UESCs transfected with Bmal1-specific siRNA, in which Rev-erbα expression was downregulated, Gdf10 and Gdf15 were upregulated. However, Gdf5, Gdf7, and Gdf11 were not significantly affected by Bmal1 silencing. The expression of Gdf10 and Gdf15 was enhanced after treatment with a REV-ERBα antagonist in the presence or absence of progesterone. Chromatin immunoprecipitation-PCR analysis revealed the inhibitory effect of REV-ERBα on the expression of Gdf10 and Gdf15 in UESCs by recognizing their gene promoters. Collectively, our findings indicate that the attenuation of REV-ERBα leads to an upregulation of Gdf10 and Gdf15 in decidual cells, in which cellular oscillators are impaired. Our results provide novel evidence regarding the functions of cellular oscillators regulating the expression of downstream genes during the differentiation of UESCs.

Integration of the nuclear receptor REV-ERBα linked with circadian oscillators in the expressions of Alas1, Ppargc1a, and Il6 genes in rat granulosa cells.

The nuclear receptor REV-ERBα links circadian rhythms and numerous physiological processes, but its physiological role in ovaries remains largely unknown. The aim of this study was to determine the potential role of REV-ERBα in the regulation of the transcription of its putative target genes in granulosa cells (GCs) prepared from Per2-destablized luciferase (dLuc) reporter gene transgenic rats. Alas1, Ppargc1a, and Il6 were chosen as representatives for genes analysis. A real-time monitoring system of Per2 promoter activity was performed to detect Per2-dLuc circadian oscillations. Two agonists (GSK4112, heme) and an antagonist (SR8278) of REV-ERBα as well as Rev-erbα siRNA knockdown were used to identify its target genes. Clear Per2-dLuc circadian oscillations were generated in matured GCs after synchronization with GSK4112 or SR8278. GSK4112 treatment lengthened and SR8278 treatment shortened the period of circadian oscillations in matured GCs stimulated with or without luteinizing hormone (LH). GSK4112 showed an inhibitory effect on the amplitude of circadian oscillations and caused an arrhythmic expression of canonical clock genes. SR8278 also had a subtle effect on their daily expression profiles, but the treatment resulted only in the arrhythmic expression of Rev-erbα. These findings indicate the functional biological activity of REV-ERBα in response to its ligands. Its natural ligand heme further elongated the period of circadian oscillations and alleviated their amplitudes in GCs cultured with LH. Heme treatment also repressed the expressions of clock genes, Alas1, Il6, and Ppargc1a. Rev-erbα knockdown up-regulated these transcript levels. Collectively, these data extend the recent finding to rat GCs and demonstrate that REV-ERBα represses the expressions of Alas1, Ppargc1a, and Il6, providing novel insights into the physiological significance of REV-ERBα in ovarian circadian oscillators.

Profiling of circadian genes expressed in the uterus endometrial stromal cells of pregnant rats as revealed by DNA microarray coupled with RNA interference.

The peripheral circadian oscillator plays an essential role in synchronizing local physiology to operate in a circadian manner via regulation of the expression of clock-controlled genes. The present study aimed to evaluate the circadian rhythms of clock genes and clock-controlled genes expressed in the rat uterus endometrial stromal cells (UESCs) during the stage of implantation by a DNA microarray. Of 12,252 genes showing significantly expression, 7,235 genes displayed significant alterations. As revealed by the biological pathway analysis using the database for annotation, visualization, and integrated discovery online annotation software, genes were involved in cell cycle, glutathione metabolism, MAPK signaling pathway, fatty acid metabolism, ubiquitin mediated proteolysis, focal adhesion, and PPAR signaling pathway. The clustering of clock genes were mainly divided into four groups: the first group was Rorα, Timeless, Npas2, Bmal1, Id2, and Cry2; the second group Per1, Per2, Per3, Dec1, Tef, and Dbp; the third group Bmal2, Cry1, E4bp4, Rorß, and Clock; the fourth group Rev-erbα. Eleven implantation-related genes and 24 placenta formation-related genes displayed significant alterations, suggesting that these genes involved in implantation and placenta formation are controlled under circadian clock. Some candidates as clock-controlled genes were evaluated by using RNA interference to Bmal1 mRNA. Down-regulation of Igf1 gene expression was observed by Bmal1 silencing, whereas the expression of Inhßa was significantly increased. During active oscillation of circadian clock, the apoptosis-related genes Fas and Caspase3 remained no significant changes, but they were significantly increased by knockdown of Bmal1 mRNA. These results indicate that clock-controlled genes are up- or down-regulated in rat UESCs during the stage of decidualization. DNA microarray analysis coupled with RNA interference will be helpful to understand the physiological roles of some oscillating genes in blastocyst implantation and placenta formation.

Different circadian expression of major matrix-related genes in various types of cartilage: modulation by light-dark conditions.

We screened circadian-regulated genes in rat cartilage by using a DNA microarray analysis. In rib growth-plate cartilage, numerous genes showed statistically significant circadian mRNA expression under both 12:12 h light-dark and constant darkness conditions. Type II collagen and aggrecan genes--along with several genes essential for post-translational modifications of collagen and aggrecan, including prolyl 4-hydroxylase 1, lysyl oxidase, lysyl oxidase-like 2 and 3'-phosphoadenosine 5'-phosphosulphate synthase 2--showed the same circadian phase. In addition, the mRNA level of SOX9, a master transcription factor for the synthesis of type II collagen and aggrecan, has a similar phase of circadian rhythms. The circadian expression of the matrix-related genes may be critical in the development and the growth of various cartilages, because similar circadian expression of the matrix-related genes was observed in hip joint cartilage. However, the circadian phase of the major matrix-related genes in the rib permanent cartilage was almost the converse of that in the rib growth-plate cartilage under light-dark conditions. We also found that half of the oscillating genes had conserved clock-regulatory elements, indicating contribution of the elements to the clock outputs. These findings suggest that the synthesis of the cartilage matrix macromolecules is controlled by cell-autonomous clocks depending upon the in vivo location of cartilage.

Identification of a new clock-related element EL-box involved in circadian regulation by BMAL1/CLOCK and HES1.

Several cis-acting elements play critical roles in maintaining circadian expression of clock and clock-controlled genes. Using in silico analysis, we identified 10 sequence motifs that are correlated with the circadian phases of gene expression in the cartilage. One of these motifs, an E-box-like clock-related element (EL-box; GGCACGAGGC), can mediate BMAL1/CLOCK-induced transcription, which is typically regulated through an E-box or E'-box. Expression of EL-box-containing genes, including Ank, Dbp, and Nr1d1 (Rev-erbα), was induced by BMAL1/CLOCK or BMAL1/NPAS2. Compared with the E-box, the EL-box elements had distinct responsiveness to DEC1, DEC2, and HES1: suppressive actions of DEC1 and DEC2 on the EL-box were less potent than those on the E-box. HES1, which is known to bind to the N-box (CACNAG), suppressed enhancer activity of the EL-box, but not the E-box. In the Dbp promoter, an EL-box worked cooperatively with a noncanonical (NC) E-box to mediate BMAL1/CLOCK actions. These findings suggest that in addition to known clock elements, the EL-box element may contribute to circadian regulation of clock and clock-controlled genes.