DEC1 regulates the rhythmic expression of PPARγ target genes involved in lipid metabolism in white adipose tissue.

Previously, we found that the basic helix-loop-helix transcriptional repressor DEC1 interacts with the PPARγ:RXRα heterodimer, a master transcription factor for adipogenesis and lipogenesis, to suppress transcription from PPARγ target genes (Noshiro et al., Genes to Cells, 2018, 23:658-669). Because the expression of PPARγ and several of its target genes exhibits circadian rhythmicity in white adipose tissue (WAT), we examined the expression profiles of PPARγ target genes in wild-type and Dec1-/- mice. We found that the expression of PPARγ target genes responsible for lipid metabolism, including the synthesis of triacylglycerol from free fatty acids (FFAs), lipid storage and the lipolysis of triacylglycerol to FFAs, oscillates in a circadian manner in WAT. Moreover, DEC1 deficiency led to a marked increase in the expression of these genes at night (Zeitgeber times 16 and 22), resulting in disruption of circadian rhythms. Serum FFA levels in wild-type mice also showed circadian oscillations, but these were disrupted by DEC1 deficiency, leading to reduced FFA levels. These results suggest that PPARγ:RXRα and DEC1 cooperatively generate the circadian expression of PPARγ target genes through PPAR-responsive elements in WAT.

Deficiency of the basic helix-loop-helix transcription factor DEC1 prevents obesity induced by a high-fat diet in mice.

Obesity is a major public health problem in developed countries resulting from increased food intake and decreased energy consumption and usually associated with abnormal lipid metabolism. Here, we show that DEC1, a basic helix-loop-helix transcription factor, plays an important role in the regulation of lipid consumption in mouse brown adipose tissue (BAT), which is the major site of thermogenesis. Homozygous Dec1 deletion attenuated high-fat-diet-induced obesity, adipocyte hypertrophy, fat volume and hepatic steatosis. Furthermore, DEC1 deficiency increased body temperature during daytime and enhanced the expression of uncoupler protein 1, a key factor of thermogenesis, and various lipolysis-related genes in interscapular BAT. In vitro experiments suggested that DEC1 suppresses the expression of various lipolysis-related genes induced by the heterodimer of peroxisome proliferator-activated receptor γ and retinoid X receptor α (RXRα) through direct binding to RXRα. These observations suggest that enhanced lipolysis in BAT caused by DEC1 deficiency leads to an increase in lipid consumption, thereby decreasing lipid accumulation in adipose tissues and the liver. Thus, DEC1 may serve as an energy-saving factor that suppresses lipid consumption, which may be relevant to managing obesity.

Regulation of basic helix-loop-helix transcription factors Dec1 and Dec2 by RORα and their roles in adipogenesis.

DEC1 and DEC2, members of the basic helix-loop-helix superfamily, are involved in various biological phenomena including clock systems, cell differentiation and metabolism. In clock systems, Dec1 and Dec2 expression are up-regulated by the CLOCK:BMAL1 heterodimer via E-box (CACGTG), exhibiting a circadian rhythm in the suprachiasmatic nucleus (SCN), the central circadian pacemaker and other peripheral tissues. In this study, using assays of luciferase reporters, electrophoretic mobility shift and chromatin immunoprecipitation, we identified novel nuclear receptor response elements, ROR response elements (RORE), in Dec1 and Dec2 promoters. These ROREs responded to the transcriptional activator RORα, but not to the repressor REVERBα, although the Bmal1 promoter responded to both RORα and REVERBα. Therefore, RORα, but not REVERBα, is involved in the regulation of Dec1 and Dec2 expression without significantly affecting their rhythmicity. Since RORα, DEC1 and DEC2 reportedly suppressed adipogenic differentiation, we examined expression of Rorα, Dec1, Dec2 and other clock-controlled genes in differentiating 3T3-L1 adipocytes. The results suggested that RORα suppresses adipogenic differentiation at a later stage of differentiation by RORE-mediated stimulation of Dec1 and Dec2 expression.

Smad3 and Snail show circadian expression in human gingival fibroblasts, human mesenchymal stem cell, and in mouse liver.

Smads are intracellular signaling mediators. Complexes of Smad2 and Smad3 with Smad4 transmit transforming growth factor-beta (TGF-ß) receptor-induced signaling. Snail plays important roles in mesoderm formation, gastrulation, neural crest development, and epithelial mesenchymal transition. However, it remains unknown whether Smad3 and Snail expression is circadian rhythm-dependent. Here, we showed for the first time that Smad3 and Snail show circadian expression in human gingival fibroblasts (HGF-1) and human mesenchymal stem cells (MSC) after serum shock. They also showed circadian expression in the mouse liver. We confirmed that BMAL1/2, DEC1/2, VEGF, and PER1/2/3 also show circadian expression in both HGF-1 and MSC. The mRNA peaks and phases in circadian expression of these genes differed between HGF-1 and MSC. In a luciferase assay, Smad3 promoter activity was upregulated by CLOCK/BMAL1. These findings suggest that Smad3 and Snail have circadian rhythm in vitro and vivo, and that circadian expression of Smad3 depends on CLOCK/BMAL1.

Anti-apoptotic effect of the basic helix-loop-helix (bHLH) transcription factor DEC2 in human breast cancer cells.

DEC1 (BHLHB2/Stra13/Sharp2) and DEC2 (BHLHB3/Sharp1) are basic helix-loop-helix (bHLH) transcription factors that are involved in circadian rhythms, differentiation and the responses to hypoxia. We examined whether DEC1 and DEC2 are involved in apoptosis regulation, in human breast cancer MCF-7 cells. We found that siRNA-mediated knockdown of DEC2 resulted in marked enhancement of apoptosis compared with that in control cells transfected with nonspecific siRNA. However, knockdown of DEC1 by siRNA did not affect cell survival. Knockdown of DEC2 affected the expression of mRNA or proteins related to apoptosis, such as Fas, c-Myc, caspase-8, poly (ADP-ribose) polymerase (PARP) and Bax. We also showed that tumor necrosis factor-alpha (TNF-alpha) up-regulates the expression of DEC1 and DEC2. DEC2 over-expression caused by the transfection of an expression vector reduced the amounts of cleaved PARP and caspase-8 induced by TNF-alpha treatment, whereas DEC1 over-expression increased it. Finally, we revealed that treatment with double knockdown against both DEC1 and DEC2 decreased the amounts of cleaved PARP and caspase-8 induced by DEC2 siRNA with or without TNF-alpha. These data indicate that DEC2 has an anti-apoptotic effect, whereas DEC1 has a pro-apoptotic effect, which are involved in the balance of survival of human breast cancer MCF-7 cells.

Liver X receptors (LXRalpha and LXRbeta) are potent regulators for hepatic Dec1 expression.

DEC1 (BHLHB2/Stra13/Sharp2)-a basic helix-loop-helix transcription factor-is known to be involved in various biological phenomena including clock systems and metabolism. In the clock systems, Dec1 expression is dominantly up-regulated by CLOCK : BMAL1 heterodimer, and it exhibits circadian rhythm in the suprachiasmatic nucleus (SCN)-the central circadian pacemaker-and other peripheral tissues. Recent studies have shown that the strong circadian rhythmicity of Dec1 in the SCN was abolished by Clock mutation, whereas that in the liver was affected, but not abolished, by Clock mutation. Moreover, feeding conditions affected hepatic Dec1 expression, which indicates that Dec1 expression is closely linked with the metabolic functions of the liver. Among ligand-activated nuclear receptors examined, LXRalpha and LXRbeta with T0901317-agonist for LXR-were found to be potent enhancers for Dec1 promoter activity, and a higher expression level of LXRalpha protein was detected in the liver than in the kidney and heart. T0901317 increased the levels of endogenous Dec1 transcript in hepatoma cells. Chromatin immunoprecipitation assay indicated that LXRalpha bound to the Dec1 promoter, and an LXRalpha-binding site was identified. These observations indicate that hepatic DEC1 mediates the ligand-dependent LXR signal to regulate the expression of genes involved in the hepatic clock system and metabolism.

The basic helix-loop-helix proteins differentiated embryo chondrocyte (DEC) 1 and DEC2 function as corepressors of retinoid X receptors.

The basic helix-loop-helix proteins differentiated embryo chondrocyte 1 (DEC1) and DEC2 are involved in circadian rhythm control. Because the metabolism of dietary nutrients has been linked to circadian regulation, we examined the effect of DEC1 and DEC2 on the function of the metabolite-sensing nuclear receptors, ligand-dependent transcription factors, including retinoid X receptor (RXR) and liver X receptor (LXR). Transfection assays showed that DEC1 and DEC2 repressed ligand-dependent transactivation by RXR. Knockdown of endogenous DEC1 and DEC2 expression with small interfering RNAs augmented ligand-dependent RXRalpha transactivation. DEC1 and DEC2 interacted directly with RXRalpha, and ligand addition enhanced their association. DEC1 and DEC2 modified interaction of RXRalpha with cofactor proteins. Transfection assays using DEC1 and DEC2 mutants revealed that the C-terminal region of DEC2 is required for repression and that an LXXLL motif in DEC1 and DEC2 is necessary for RXRalpha repression. DEC1 and DEC2 repressed the induction of LXR target genes, associated with the promoter of an LXR target gene, and dissociated from the promoter with ligand treatment. Knockdown of endogenous DEC1 and DEC2 enhanced the LXR target gene expression in hepatocytes. Expression of Dec1, Dec2, and Srebp-1c showed a circadian rhythm in the liver of mice, whereas that of Lxralpha, Lxrbeta, and Rxralpha was not rhythmic. DEC1 and DEC2 also repressed the transactivation of other RXR heterodimers, such as farnesoid X receptor, vitamin D receptor, and retinoic acid receptor. Thus, the repressor function of DEC1 and DEC2 may be extended to other RXR heterodimer nuclear receptors.

Basic-helix-loop-helix (bHLH) transcription factor DEC2 negatively regulates vascular endothelial growth factor expression.

DEC1 (BHLHB2/Sharp2/Stra13) and DEC2 (BHLHB3/Sharp1) are basic-helix-loop-helix (bHLH) transcription factors, involved in cellular differentiation, responses to hypoxia and circadian rhythms. We recently showed that the expression of DEC1 and DEC2 was up-regulated by hypoxia; however, the functions of these two factors under hypoxic conditions have not been elucidated in detail. It is well established that the expression of vascular endothelial growth factor (VEGF) is up-regulated by hypoxia, and the expression of VEGF in response to hypoxia depends on transcriptional activation by a heterodimer comprising hypoxia-inducible factor 1alpha (HIF-1alpha) and arylhydrocarbon receptor nuclear translocator 1 (ARNT1). In the present study, we showed that DEC2, but not DEC1, suppressed VEGF gene expression under hypoxic conditions. DEC2 protein was co-immunoprecipitated with HIF-1alpha but not with ARNT1. The binding of HIF-1alpha to the hypoxia response element (HRE) in the VEGF promoter was decreased by DEC2 over-expression, and increased by DEC2 knockdown. We also showed that the circadian expression of VEGF showed a reciprocal pattern to that of DEC2 in cartilage. DEC2 had a circadian oscillation in implanted Sarcoma 180 cells. We conclude that DEC2 negatively regulates VEGF expression and plays an important role in the pathological conditions in which VEGF is involved.

Multiple mechanisms regulate circadian expression of the gene for cholesterol 7alpha-hydroxylase (Cyp7a), a key enzyme in hepatic bile acid biosynthesis.

Cholesterol 7alpha-hydroxylase (CYP7A) and sterol 12alpha-hydroxylase (CYP8B) in bile acid biosynthesis and 3-hydroxyl-3-methylglutaryl CoA reductase (HMGCR) in cholesterol biosynthesis are the key enzymes in hepatic metabolic pathways, and their transcripts exhibit circadian expression profiles in rodent liver. The authors determined transcript levels of these enzymes and the regulatory factors for Cyp7a--including Dbp, Dec2, E4bp4, Hnf4alpha, Pparalpha, Lxralpha, Rev-erbalpha, and Rev-erbbeta--in the liver of wild-type and homozygous Clock mutant mice (Clock/Clock) and examined the effects of these transcription factors on the transcription activities of Cyp7a. The expression profile of the Cyp7a transcript in wild-type mice showed a strong circadian rhythm in both the 12L:12D light-dark cycle and constant darkness, and that in Clock/Clock also exhibited a circadian rhythm at an enhanced level with a lower amplitude, although its protein level became arrhythmic at a high level. The expression profile of Cyp8b mRNA in wild-type mice showed a shifted circadian rhythm from that of Cyp7a, becoming arrhythmic in Clock/Clock at an expression level comparable to that of wild-type mice. The expression profile of Hmgcr mRNA also lost its strong circadian rhythm in Clock/Clock , showing an expression level comparable to that of wild-type mice. The expressions of Dbp, Dec2, Rev-erbalpha, and Rev-erb beta--potent regulators for Cyp7a expression--were abolished or became arrhythmic in Clock/Clock, while other regulators for Cyp7a-Lxralpha, Hnf4alpha, Pparalpha, and E4bp4--had either less affected or enhanced expression in Clock/Clock. In luciferase reporter assays, REV-ERBalpha/beta, DBP, LXRalpha, and HNF4alpha increased the promoter activity of Cyp7a, whereas DEC2 abolished the transcription from the Cyp7a promoter: E4BP4 and PPARalpha were moderate negative regulators. Furthermore, knockdown of REV-ERBalpha/beta with siRNA suppressed Cyp7a transcript levels, and in the electrophoretic mobility shift assay, REV-ERBalpha/beta bound to the promoter of Cyp7a . These observations suggest that (1) active CLOCK is essential for the robust circadian expression of hepatic metabolic enzymes (Cyp7a, Cyp8b, and Hmgcr); (2) clock-controlled genes--DBP, DEC2, and REV-ERBalpha/beta--are direct regulators required for the robust circadian rhythm of Cyp7a; and (3) the circadian rhythm of Cyp7a is regulated by multiple transcription factors, including DBP, REV-ERBalpha/beta, LXRalpha, HNF4alpha DEC2, E4BP4, and PPARalpha.

Dec1 and CLOCK Regulate Na+/K+-ATPase ß1 Subunit Expression and Blood Pressure.

Blood pressure shows a circadian rhythm, and recent studies have suggested the involvement of a molecular clock system in its control. In the clock system, the CLOCK (circadian locomotor output cycles kaput):BMAL1 (brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein-1) heterodimer enhances promoter activity of clock genes, and DEC1 (BHLHE40/STRA13/SHARP-2) represses CLOCK/BMAL1-enhanced promoter activity through competition for binding to the clock element, CACGTG E-box. However, the molecular mechanisms by which this system regulates blood pressure remain unclear. Here, we show that DEC1 suppressed the expression of ATP1B1, which encodes the ß1 subunit of the Na+/K+-ATPase and elevated blood pressure. Using chromatin immunoprecipitation and chromatin immunoprecipitation-on-chip analyses, we found that DEC1 and CLOCK bound to E-boxes in the ATP1B1 promoter. Luciferase assays revealed that CLOCK:BMAL1 heterodimer enhanced transcription from the ATP1B1 promoter, whereas DEC1 suppressed this transactivation. Accordingly, Atp1b1 mRNA and protein levels in mouse kidney, aorta, and heart showed a circadian rhythm that was antiphasic to the blood pressure rhythm. Furthermore, Dec1-deficient mice showed enhanced Atp1b1 expression in these tissues and reduced blood pressure. In contrast, Clock-mutant mice showed reduced Atp1b1 expression and elevated blood pressure. Our results raise the possibility that transcriptional regulation of Atp1b1 by DEC1 and CLOCK:BMAL1 contributes to blood pressure.

Transcriptional repression by the basic helix-loop-helix protein Dec2: multiple mechanisms through E-box elements.

Dec2, a member of the basic helix-loop-helix (bHLH) superfamily, has been shown to function as a transcriptional repressor and is implicated in cell proliferation and differentiation. In addition, Dec2 transcripts exhibit a striking circadian oscillation in the suprachiasmatic nucleus. To identify the molecular mechanisms by which Dec2 regulates gene expression, we carried out structure-function analyses. Gel retardation and luciferase assays showed that Dec2, as well as its related protein Dec1, preferentially binds to class B E-box elements (CACGTG) as a homodimer and represses the transcription of target genes in a histone deacetylase (HDAC)-dependent manner. Functional studies with the GAL4-DNA binding domain fusion proteins identified the domain responsible for the repression activity of Dec2 in its C-terminal region, which is also necessary to recruit HDAC1. In addition, the basic and HLH domains of Dec2 were required for DNA binding and homodimerization, respectively. In contrast, Dec proteins repressed a MyoD-activated promoter activity of muscle creatine kinase gene through class A E-box in an HDAC1-independent manner. Dec2 formed a heterodimer with MyoD through the basic and HLH domains. Consistent with this, both the basic and HLH domains were required for the ability of Dec2 to inhibit the transcriptional activity of MyoD. These findings indicate that Dec2 employs multiple mechanisms, including DNA-binding and protein-protein interactions, to achieve E-box-dependent transcriptional repressions.

Effects of overexpression of basic helix-loop-helix transcription factor Dec1 on osteogenic and adipogenic differentiation of mesenchymal stem cells.

We recently reported that forced expression of basic helix-loop-helix transcription factor Dec1 accelerated chondrogenic differentiation of mesenchymal stem cells (MSC) in pellet cultures (Shen, M., Yoshida, E., Yan, W., Kawamoto, T., Suardita, K., Koyano, Y., Fujimoto, K., Noshiro, M., Kato, Y., 2002. Basic helix-loop-helix protein DEC1 promotes chondrocyte differentiation at the early and terminal stages. J. Biol. Chem. 277, 50112-50120). Since MSC have multilineage differentiation potential, we investigated the roles of Dec1 in osteogenic and adipogenic differentiation of human bone marrow-derived MSC. After osteogenic induction of MSC in medium containing dexamethasone, beta-glycerophosphate, and ascorbic acid, Dec1 expression gradually increased from day 5 to day 14, while expression levels of Dec1 mRNA markedly decreased on days 3 and 7 after adipogenic induction. Infection with adenovirus expressing Dec1 raised mRNA levels of several bone characteristic molecules such as osteopontin, PTH receptor, and alkaline phosphatase, even in the absence of the osteogenic induction medium, although it had little effect on Runx2 expression or calcification. In the osteogenic induction medium, Dec1 overexpression enhanced the expression of osteopontin and alkaline phosphatase and induced matrix calcification. Knockdown of Dec1 with siRNA suppressed the expression of osteoblastic phenotype by the induced MSC. Using MSC cultures, we also confirmed that forced expression of Dec1 suppressed adipogenic differentiation. These findings suggest that Dec1 modulates osteogenic differentiation of MSC by inducing the expression of several, but not all, bone-related genes.

Effects of fasting and re-feeding on the expression of Dec1, Per1, and other clock-related genes.

To elucidate the food-entrainable oscillatory mechanism of peripheral clock systems, we examined the effect of fasting on circadian expression of clock genes including Dec1 and Dec2 in mice. Withholding of food for 2 days had these effects: the expression level of Dec1 mRNA decreased in all tissues examined, although Per1 mRNA level markedly increased; Per2 expression was reduced in the liver and heart only 42-46 h after the start of fasting; and expression profiles of Dec2 and Bmal1 were altered only in the heart and in the liver, respectively, whereas Rev-erbalpha mRNA levels did not change significantly. Re-feeding after 36-h starvation erased, at least in part, the effect of fasting on Dec1, Dec2, Per1, Per2, and Bmal1 within several hours, and restriction feeding shifted the phase of expression profiles of all examined clock genes including Dec1 and Dec2. These findings indicate that short-term fasting and re-feeding modulate the circadian rhythms of clock genes to different extents in peripheral tissues, and suggest that the expression of Dec1, Per1, and some other clock genes was closely linked with the metabolic activity of these tissues.

57Arg in the bHLH transcription factor DEC2 is essential for the suppression of CLOCK/BMAL2-mediated transactivation.

The basic helix-loop-helix (bHLH) transcription factors, DEC2 and DEC1, play critical roles in the circadian rhythm of the suprachiasmatic nucleus (SCN). It is known that mammalian circadian rhythms are regulated by molecular clockwork systems based on a negative-feedback loop, and CLOCK/BMAL1 and CLOCK/BMAL2 enhance DEC2 transcription via CACGTG E-boxes. To understand the role of arginine 57 ((57)Arg) within the basic region of DEC2, we examined the effect of substituting this residue into DEC2 on CLOCK/BMAL2-mediated transactivation. A luciferase assay showed that substituting (57)Arg for Ala or Lys in DEC2 diminished the suppressive activity of wild-type DEC2 on CLOCK/ BMAL2-mediated transactivation, while substituting (48)Pro for Ala in DEC2 did not alter it, and the same was true for wild-type DEC2. We also showed that proteins which were wild-type and substitution mutants of DEC2 were expressed at nearly equivalent levels by Western blotting. These findings demonstrate that (57)Arg in the basic region of DEC2 is essential for its activity in suppressing CLOCK/BMAL2-mediated transactivation.

Tissue-specific disruption of rhythmic expression of Dec1 and Dec2 in clock mutant mice.

DEC1 and DEC2-basic helix-loop-helix transcription factors-exhibit a circadian expression in the suprachiasmatic nucleus and other peripheral tissues and seem to play roles in regulating the mammalian circadian rhythm by suppressing the CLOCK/BMAL1-activated promoters of Per1, Dec1, and Dec2. The authors present data on the expression patterns of mRNA for Dec1, Dec2, Per2, Dbp, and Npas2 in various tissues of wild-type and homozygous Clock mutant mice (Clock/Clock). The Clock mutation resulted in extreme reduction of Dec1 expression in kidney, heart, and skeletal muscle but not in liver, whereas it strongly repressed Dec2 expression in liver, kidney, and heart, while Dec2 expression in skeletal muscle remained rhythmic. Per2 also showed the tissue-dependent disruption of the rhythmicity by Clock mutation, whereas rhythmic expression of Dbp in Clock mutant mice disappeared in all tissues examined. Npas2, a structurally and functionally related gene to Clock, showed significant levels of expression in the liver and kidney with a robust rhythmicity, which was also affected by Clock mutation. These marked changes in the Dec1 and Dec2 expression, as well as in the Per2, Dbp, and Npas2 expression in the periphery by Clock mutation, indicated that CLOCK plays a major role in the expression of these genes in most tissues. However, circadian expression of Dec1 in liver and kidney and that of Dec2 in skeletal muscle of Clock mutant mice suggested that CLOCK-independent circadian regulation operates in some tissues.

DEC2 is a negative regulator for the proliferation and differentiation of chondrocyte lineage-committed mesenchymal stem cells.

Differentiated embryo chondrocyte 2 (DEC2) is a basic helix-loop-helix-Orange transcription factor that regulates cell differentiation in various mammalian tissues. DEC2 has been shown to suppress the differentiation of mesenchymal stem cells (MSCs) into myocytes and adipocytes. In the present study, we examined the role of DEC2 in the chondrogenic differentiation of human MSCs. The overexpression of DEC2 exerted minimal effects on the proliferation of MSCs in monolayer cultures with the growth medium under undifferentiating conditions, whereas it suppressed increases in DNA content, glycosaminoglycan content, and the expression of several chondrocyte-related genes, including aggrecan and type X collagen alpha 1, in MSC pellets in centrifuge tubes under chondrogenic conditions. In the pellets exposed to chondrogenesis induction medium, DEC2 overexpression downregulated the mRNA expression of fibroblast growth factor 18, which is involved in the proliferation and differentiation of chondrocytes, and upregulated the expression of p16INK4, which is a cell cycle inhibitor. These findings suggest that DEC2 is a negative regulator of the proliferation and differentiation of chondrocyte lineage-committed mesenchymal cells.

Role of MSX1 in Osteogenic Differentiation of Human Dental Pulp Stem Cells.

Msh homeobox 1 (MSX1) encodes a transcription factor implicated in embryonic development of limbs and craniofacial tissues including bone and teeth. Although MSX1 regulates osteoblast differentiation in the cranial bone of young animal, little is known about the contribution of MSX1 to the osteogenic potential of human cells. In the present study, we investigate the role of MSX1 in osteogenic differentiation of human dental pulp stem cells isolated from deciduous teeth. When these cells were exposed to osteogenesis-induction medium, runt-related transcription factor-2 (RUNX2), bone morphogenetic protein-2 (BMP2), alkaline phosphatase (ALPL), and osteocalcin (OCN) mRNA levels, as well as alkaline phosphatase activity, increased on days 4-12, and thereafter the matrix was calcified on day 14. However, knockdown of MSX1 with small interfering RNA abolished the induction of the osteoblast-related gene expression, alkaline phosphatase activity, and calcification. Interestingly, DNA microarray and PCR analyses revealed that MSX1 knockdown induced the sterol regulatory element-binding protein 2 (SREBP2) transcriptional factor and its downstream target genes in the cholesterol synthesis pathway. Inhibition of cholesterol synthesis enhances osteoblast differentiation of various mesenchymal cells. Thus, MSX1 may downregulate the cholesterol synthesis-related genes to ensure osteoblast differentiation of human dental pulp stem cells.

Expression of the gene for Dec2, a basic helix-loop-helix transcription factor, is regulated by a molecular clock system.

Dec2, a member of the basic helix-loop-helix superfamily, is a recently confirmed regulatory protein for the clockwork system. Transcripts of Dec2, as well as those of its related gene Dec1, exhibit a striking circadian oscillation in the suprachiasmatic nucleus, and Dec2 inhibits transcription from the Per1 promoter induced by Clock/Bmal1 [Honma, Kawamoto, Takagi, Fujimoto, Sato, Noshiro, Kato and Honma (2002) Nature (London) 419, 841-844]. It is known that mammalian circadian rhythms are controlled by molecular clockwork systems based on negative-feedback loop(s), but the molecular mechanisms for the circadian regulation of Dec2 gene expression have not been clarified. We show here that transcription of the Dec2 gene is regulated by several clock molecules and a negative-feedback loop. Luciferase and gel retardation assays showed that expression of Dec2 was negatively regulated by binding of Dec2 or Dec1 to two CACGTG E-boxes in the Dec2 promoter. Forced expression of Clock/Bmal1 and Clock/Bmal2 markedly increased Dec2 mRNA levels, and up-regulated the transcription of the Dec2 gene through the CACGTG E-boxes. Like Dec, Cry and Per also suppressed Clock/Bmal-induced transcription from the Dec2 promoter. Moreover, the circadian expression of Dec2 transcripts was abolished in the kidney of Clock/Clock mutant mice. These findings suggest that the Clock/Bmal heterodimer enhances Dec2 transcription via the CACGTG E-boxes, whereas the induced transcription is suppressed by Dec2, which therefore must contribute to its own rhythmic expression. In addition, Cry and Per may also modulate Dec2 transcription.

Dec1 and Dec2 expression is disrupted in the suprachiasmatic nuclei of Clock mutant mice.

DEC1 and DEC2 are basic helix-loop-helix transcription factors that functionally resemble negative feedback components of the mammalian circadian clock. The genes Dec1 and Dec2 are expressed rhythmically in the rat suprachiasmatic nuclei, and Dec1 expression is stimulated by light in a time-dependent manner with the kinetics of an immediate early gene. DEC1 and DEC2 can inhibit CLOCK:BMAL1 transactivation of the clock gene Per1, suggesting that these transcription factors may help regulate circadian timing. The authors present data on the expression pattern of Dec1 and Dec2 in wild-type and homozygous Clock mutant mice. In the suprachiasmatic nuclei, the Clock mutation significantly reduces the expression of Dec1 and Dec2. Dec1 becomes arrhythmic; Dec2 remains weakly rhythmic in a 12L:12D light-dark cycle but is arrhythmic in constant darkness. A robust attenuation of the Dec1 and Dec2 signals in Clock mutant mice was detected in all brain areas examined. These data point to up-regulation of Dec1 and Dec2 by Clock in vivo.

Regulation of bile acid synthesis under reconstructed enterohepatic circulation in rats.

Cholesterol 7alpha-hydroxylase (CYP7A1) is regulated by bile acids through the farnesoid X receptor (FXR) mechanism in a negative feedback fashion. However, the fact that CYP7A1 is down-regulated by intraduodenal administration of bile acid, but not by intravenous administration may not be explained only by this mechanism. The aim of this study was to establish a new rat model with reconstructed or simulated enterohepatic circulation to examine if intravenous or portal administration of bile acid can regulate CYP7A1. Under biliary drainage, taurocholate (0 or 6 micromol/h/100g body weight) was administered continuously for 48h into the duodenum (ID-0/ID-6), femoral vein (IV-0/IV-6), or portal vein (IP-0/IP-6) to create a condition in which biliary bile acids were continuously lost, and a similar dose of taurocholate was supplied to the liver simultaneously. CYP7A1 activity and mRNA expression of the ID-0 group were significantly increased compared with the no treatment (NT) group. CYP7A1 activity and mRNA expression of the ID-6 group were suppressed significantly to 41 and 46% of those of the ID-0 group, respectively. In the IV-6 and IP-6 groups, however, enzyme activity and mRNA expression were decreased slightly, but the suppression was not statistically significant. The results suggested that portal as well as intravenous administration of bile acids cannot suppress bile acid synthesis as effectively as intraduodenal administration. It was concluded that an unidentified regulatory factor other than the nuclear receptors may be involved in bile acid synthesis in vivo.