Study of Alveolar Bone Remodeling Using Deciduous Tooth Stem Cells and Hydroxyapatite by Vascular Endothelial Growth Factor Enhancement and Inhibition of Matrix Metalloproteinase-8 Expression in vivo.

Background: Periodontitis progression is characterized by alveolar bone loss, and its prevention is a major clinical problem in periodontal disease management. Matrix metalloproteinase-8 (MMP-8) has been shown to adequately monitor the treatment of chronic periodontitis patients as gingival crevicular fluid MMP-8s were positively associated with the severity of periodontal disease. Moreover, modulating the vascular endothelial growth factor (VEGF) levels in bones could be a good way to improve bone regeneration and cure periodontitis as VEGF promotes endothelial cell proliferation, proteolytic enzyme release, chemotaxis, and migration; all of which are required for angiogenesis. Purpose: The aim of this study was to determine the effect of hydroxyapatite incorporated with stem cells from exfoliated deciduous teeth (SHED) in Wistar rats' initial alveolar bone remodeling based on the findings of MMP-8 and VEGF expressions. Methods: A hydroxyapatite scaffold (HAS) in conjunction with SHED was transplanted into animal models with alveolar mandibular defects. A total of 10 Wistar rats (Rattus norvegicus) were divided into two groups: HAS and HAS + SHED. Immunohistochemistry staining was performed after 7 days to facilitate the examination of MMP-8 and VEGF expressions. Results: The independent t-test found significant downregulation of MMP-8 and upregulation VEGF expressions in groups transplanted with HAS in conjunction with SHED compared with the HAS group (p < 0.05). Conclusion: The combination of SHED with HAS on alveolar bone defects may contribute to initial alveolar bone remodeling as evident through the assessments of MMP-8 and VEGF expressions.

The Identification of Marker Genes for Predicting the Osteogenic Differentiation Potential of Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) have the potential to differentiate into a variety of mature cell types and are a promising source of regenerative medicine. The success of regenerative medicine using MSCs strongly depends on their differentiation potential. In this study, we sought to identify marker genes for predicting the osteogenic differentiation potential by comparing ilium MSC and fibroblast samples. We measured the mRNA levels of 95 candidate genes in nine ilium MSC and four fibroblast samples before osteogenic induction, and compared them with alkaline phosphatase (ALP) activity as a marker of osteogenic differentiation after induction. We identified 17 genes whose mRNA expression levels positively correlated with ALP activity. The chondrogenic and adipogenic differentiation potentials of jaw MSCs are much lower than those of ilium MSCs, although the osteogenic differentiation potential of jaw MSCs is comparable with that of ilium MSCs. To select markers suitable for predicting the osteogenic differentiation potential, we compared the mRNA levels of the 17 genes in ilium MSCs with those in jaw MSCs. The levels of 7 out of the 17 genes were not substantially different between the jaw and ilium MSCs, while the remaining 10 genes were expressed at significantly lower levels in jaw MSCs than in ilium MSCs. The mRNA levels of the seven similarly expressed genes were also compared with those in fibroblasts, which have little or no osteogenic differentiation potential. Among the seven genes, the mRNA levels of IGF1 and SRGN in all MSCs examined were higher than those in any of the fibroblasts. These results suggest that measuring the mRNA levels of IGF1 and SRGN before osteogenic induction will provide useful information for selecting competent MSCs for regenerative medicine, although the effectiveness of the markers is needed to be confirmed using a large number of MSCs, which have various levels of osteogenic differentiation potential.

BHLHE41/DEC2 Expression Induces Autophagic Cell Death in Lung Cancer Cells and Is Associated with Favorable Prognosis for Patients with Lung Adenocarcinoma.

Lung cancer constitutes a threat to human health. BHLHE41 plays important roles in circadian rhythm and cell differentiation as a negative regulatory transcription factor. This study investigates the role of BHLHE41 in lung cancer progression. We analyzed BHLHE41 function via in silico and immunohistochemical studies of 177 surgically resected non-small cell lung cancer (NSCLC) samples and 18 early lung squamous cell carcinoma (LUSC) cases. We also examined doxycycline (DOX)-inducible BHLHE41-expressing A549 and H2030 adenocarcinoma cells. BHLHE41 expression was higher in normal lung than in lung adenocarcinoma (LUAD) tissues and was associated with better prognosis for the overall survival (OS) of patients. In total, 15 of 132 LUAD tissues expressed BHLHE41 in normal lung epithelial cells. Staining was mainly observed in adenocarcinoma in situ and the lepidic growth part of invasive cancer tissue. BHLHE41 expression constituted a favorable prognostic factor for OS (p = 0.049) and cause-specific survival (p = 0.042) in patients with LUAD. During early LUSC, 7 of 18 cases expressed BHLHE41, and this expression was inversely correlated with the depth of invasion. DOX suppressed cell proliferation and increased the autophagy protein LC3, while chloroquine enhanced LC3 accumulation and suppressed cell death. In a xenograft model, DOX suppressed tumor growth. Our results indicate that BHLHE41 expression prevents early lung tumor malignant progression by inducing autophagic cell death in NSCLC.

CHRONO and DEC1/DEC2 compensate for lack of CRY1/CRY2 in expression of coherent circadian rhythm but not in generation of circadian oscillation in the neonatal mouse SCN.

Clock genes Cry1 and Cry2, inhibitory components of core molecular feedback loop, are regarded as critical molecules for the circadian rhythm generation in mammals. A double knockout of Cry1 and Cry2 abolishes the circadian behavioral rhythm in adult mice under constant darkness. However, robust circadian rhythms in PER2::LUC expression are detected in the cultured suprachiasmatic nucleus (SCN) of Cry1/Cry2 deficient neonatal mice and restored in adult SCN by co-culture with wild-type neonatal SCN. These findings led us to postulate the compensatory molecule(s) for Cry1/Cry2 deficiency in circadian rhythm generation. We examined the roles of Chrono and Dec1/Dec2 proteins, the suppressors of Per(s) transcription similar to CRY(s). Unexpectedly, knockout of Chrono or Dec1/Dec2 in the Cry1/Cry2 deficient mice did not abolish but decoupled the coherent circadian rhythm into three different periodicities or significantly shortened the circadian period in neonatal SCN. DNA microarray analysis for the SCN of Cry1/Cry2 deficient mice revealed substantial increases in Per(s), Chrono and Dec(s) expression, indicating disinhibition of the transactivation by BMAL1/CLOCK. Here, we conclude that Chrono and Dec1/Dec2 do not compensate for absence of CRY1/CRY2 in the circadian rhythm generation but contribute to the coherent circadian rhythm expression in the neonatal mouse SCN most likely through integration of cellular circadian rhythms.

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.

Genetic Markers Can Predict Chondrogenic Differentiation Potential in Bone Marrow-Derived Mesenchymal Stromal Cells.

The precise predictions of the differentiation direction and potential of mesenchymal stromal cells (MSCs) are an important key to the success of regenerative medicine. The expression levels of fate-determining genes may provide tools for predicting differentiation potential. The expression levels of 95 candidate marker genes and glycosaminoglycan (GAG) contents after chondrogenic induction in 10 undifferentiated ilium and 5 jaw MSC cultures were determined, and their correlations were analyzed. The expression levels of eight genes before the induction of chondrogenic MSC differentiation were significantly correlated with the GAG levels after induction. Based on correlation patterns, the eight genes were classified into two groups: group 1 genes (AURKB, E2F1, CDKN2D, LIF, and ACLY), related to cell cycle regulation, and group 2 genes (CD74, EFEMP1, and TGM2), involved in chondrogenesis. The expression levels of the group 2 genes were significantly correlated with the ages of the cell donors. The expression levels of CDKN2D, CD74, and TGM2 were >10-fold higher in highly potent MSCs (ilium MSCs) than in MSCs with limited potential (jaw MSCs). Three-dimensional (3D) scatter plot analyses of the expression levels of these genes showed reduced variability between donors and confirmed predictive potential. These data suggest that group 2 genes are involved in age-dependent decreases in the chondrogenic differentiation potential of MSCs, and combined 3D analyses of the expression profiles of three genes, including two group 2 genes, were predictive of MSC differentiation potential.

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.

Characterization of human dental pulp cells grown in chemically defined serum-free medium.

Dental pulp cells (DPCs) are promising candidates for use as transplantable cells in regenerative medicine. However, ex vivo expansion of these cells typically requires culture media containing fetal bovine serum, which may cause infection and immunological reaction following transplantation. In addition, the proliferation and differentiation of DPCs markedly depend upon serum batches. Therefore, the present study examined whether DPCs could be expanded under serum-free conditions. DPCs obtained from four donors were identified to proliferate actively in the serum-free medium, STK2, when compared with those cells in control medium (Dulbecco's modified Eagle's medium containing 10% serum). The high proliferative potential with STK2 was maintained through multiple successive culture passages. DNA microarray analyses demonstrated that the gene expression profile of DPCs grown in STK2 was similar to that of cells grown in the control medium; however, a number of genes related to cell proliferation, including placental growth factor and inhibin-ßE, were upregulated in the STK2 cultures. Following induction of osteogenesis, DPCs grown in STK2 induced alkaline phosphatase activity and calcification at higher levels compared with the control medium cultures, indicating maintenance of differentiation potential in STK2. This serum-free culture system with DPCs may have applications in further experimental studies and as a clinical strategy in regenerative medicine.

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.

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.

Rhythmic expression of DEC2 protein in vitro and in vivo.

Basic helix-loop-helix (bHLH) transcription factor DEC2 (bHLHE41/Sharp1) is one of the clock genes that show a circadian rhythm in various tissues. DEC2 regulates differentiation, sleep length, tumor cell invasion and apoptosis. Although studies have been conducted on the rhythmic expression of DEC2 mRNA in various tissues, the precise molecular mechanism of DEC2 expression is poorly understood. In the present study, we examined whether DEC2 protein had a rhythmic expression. Western blot analysis for DEC2 protein revealed a rhythmic expression in mouse liver, lung and muscle and in MCF-7 and U2OS cells. In addition, AMP-activated protein kinase (AMPK) activity (phosphorylation of AMPK) in mouse embryonic fibroblasts (MEFs) exhibited a rhythmic expression under the condition of medium change or glucose-depleted medium. However, the rhythmic expression of DEC2 in MEF gradually decreased in time under these conditions. The medium change affected the levels of DEC2 protein and phosphorylation of AMPK. In addition, the levels of DEC2 protein showed a rhythmic expression in vivo and in MCF-7 and U2OS cells. The results showed that the phosphorylation of AMPK immunoreactivity was strongly detected in the liver and lung of DEC2 knockout mice compared with that of wild-type mice. These results may provide new insights into rhythmic expression and the regulation between DEC2 protein and AMPK activity.

DEC2-E4BP4 Heterodimer Represses the Transcriptional Enhancer Activity of the EE Element in the Per2 Promoter.

The circadian oscillation of clock gene expression in mammals is based on the interconnected transcriptional/translational feedback loops of Period (Per) and Bmal1. The Per feedback loop initiates transcription through direct binding of the BMAL1-CLOCK (NPAS2) heterodimer to the E-box of the Per2 promoter region. Negative feedback of PER protein on this promoter subsequently represses transcription. Other circadian transcription regulators, particularly E4BP4 and DEC2, regulate the amplitude and phase of Per2 expression rhythms. Moreover, a direct repeat of E-box-like (EE) elements in the Per2 promoter is required for its cell-autonomous circadian rhythm. However, the detailed mechanism for repression of the two core sequences of the EE element in the Per2 promoter region is unknown. Here, we show that E4BP4 binds to the Per2 EE element with DEC2 to repress transcription and identify the DEC2-E4BP4 heterodimer as a key repressor of the tightly interlocked Per2 feedback loop in the mammalian circadian oscillator. Our results suggest an additional modulatory mechanism for tuning of the phase of cell-autonomous Per2 gene expression cycling.

Characteristic expression of MSX1, MSX2, TBX2 and ENTPD1 in dental pulp cells.

Dental pulp cells (DPCs) are a promising source of transplantable cells in regenerative medicine. However, DPCs have not been fully characterized at the molecular level. The aim of the present study was to distinguish DPCs from various source-derived mesenchymal stem cells (MSCs), fibroblasts (FBs) and other cells by the expression of several DPC-characteristic genes. DPCs were isolated from human pulp tissues by the explant method or the enzyme digestion method, and maintained with media containing 10% serum or 7.5% platelet-rich plasma. RNA was isolated from the cells and from dental pulp tissue specimens. The mRNA levels were determined by DNA microarray and quantitative polymerase chain reaction analyses. The msh homeobox 1, msh homeobox 2, T-box 2 and ectonucleoside triphosphate diphosphohydrolase 1 mRNA levels in DPCs were higher than that of the levels identified in the following cell types: MSCs derived from bone marrow, synovium and adipose tissue; and in cells such as FBs, osteoblasts, adipocytes and chondrocytes. The enhanced expression in DPCs was consistently observed irrespective of donor age, tooth type and culture medium. In addition, these genes were expressed at high levels in dental pulp tissue in vivo. In conclusion, this gene set may be useful in the identification and characterization of DPCs in basic studies and pulp cell-based regeneration therapy.

Basic helix-loop-helix transcription factor DEC1 regulates the cisplatin-induced apoptotic pathway of human esophageal cancer cells.

DEC1 [basic helix-loop-helix (BHLH) E40/Stra13/Sharp2] and DEC2 (BHLHE41/Sharp1) are BHLH transcription factors that are associated with the regulation of apoptosis, cell proliferation, and circadian rhythms, as well as malignancy in various cancers. However, the roles of DEC1 and DEC2 expression in esophageal cancer are poorly understood. In this study, we examined the roles of DEC1 and DEC2 in human esophageal cancer TE 5 and TE 10 cells that had been treated with cis-diamminedichloroplatinum (II) (cisplatin: CDDP). Expression of DEC1 and DEC2 was decreased with CDDP treatment in TE 5 cells; however, knockdown or overexpression of DEC1/DEC2 had little effects on CDDP-induced apoptosis in TE 5 cells. DEC1 expression was up-regulated in CDDP-treated TE 10 cells, whereas DEC2 expression was unchanged. DEC1 knockdown by siRNA in TE 10 decreased the amount of cleaved poly (ADP-ribose) polymerase (PARP) after treatment with CDDP, whereas DEC2 knockdown had no effects on the amount of cleaved PARP in both the presence and absence of CDDP. We also demonstrated that DEC1 overexpression promoted cleaved PARP expression, whereas DEC2 overexpression had no effects on the amount of cleaved PARP in TE 10 cells. These results suggested that DEC1 has pro-apoptotic effects on human esophageal cancer TE 10 cells of well-differentiated type.

Involvement of c-Myc in the proliferation of MCF-7 human breast cancer cells induced by bHLH transcription factor DEC2.

Differentiated embryonic chondrocyte expressed gene 1 (DEC1; BHLHE40/Stra13/Sharp2) and differentiated embryonic chondrocyte expressed gene 2 (DEC2; BHLHE41/Sharp1) are basic helix-loop-helix (bHLH) transcriptional factors that are involved in the regulation of cell differentiation, circadian rhythms, response to hypoxia and carcinogenesis. Previous studies have demonstrated that the expression of DECs is induced under hypoxic conditions in various normal and cancer cell lines. In the present study, using RT-qPCR and western blot analysis, we demonstrated that hypoxia induced the expression of DEC1 and DEC2 in MCF-7 human breast cancer cells; their expression levels reached a peak at different time points. In particular, we found that the expression pattern of the hypoxia-inducible factor (HIF)-1α protein was similar to DEC1, and that of the HIF-2α protein was identical to that of DEC2. The knockdown of HIF-2α using siRNA suppressed the phosphorylation of Akt, as well as the expression of DEC2 and c-Myc. Hypoxia failed to affect the expression of DEC2 and c-Myc when the PI3K/Akt signaling pathway was blocked. In addition, the overexpression of DEC1 and DEC2 was induced by transfecting the cells with a pcDNA vector. The overexpression of DEC2, but not that of DEC1, increased the proliferation of the MCF-7 cells under both normoxic and hypoxic conditions. Concomitantly, the expression of c-Myc was upregulated by exposure to hypoxia and by the overexpression of DEC2. In conclusion, DEC2 participates in hypoxia-induced cell proliferation by functioning as a target gene of the PI3K/Akt signaling pathway and regulating the expression of c-Myc.

DEC1/STRA13/SHARP2 and DEC2/SHARP1 coordinate physiological processes, including circadian rhythms in response to environmental stimuli.

Daily physiological and behavioral rhythms are regulated by endogenous circadian molecular clocks. Clock proteins DEC1 (BHLHe40) and DEC2 (BHLHe41) belong to the basic helix-loop-helix protein superfamily, which contains other clock proteins CLOCK and BMAL1. DEC1 and DEC2 are induced by CLOCK:BMAL1 heterodimer via the CACGTG E-box in the promoter and, thereafter, suppress their own expression by competing with CLOCK:BMAL1 for the DNA binding. This negative feedback DEC loop together with the PER loop involving PER and CRY, the other negative clock regulators, maintains the circadian rhythm of Dec1 and Dec2 expression. DEC1 is induced by light pulse and adjusts the circadian phase of the central clock in the suprachiasmatic nucleus, whereas DEC1 upregulation by TGF-ß resets the circadian phase of the peripheral clocks in tissues. Furthermore, DEC1 and DEC2 modulate the clock output signals to control circadian rhythms in behavior and metabolism. In addition to the functions in the clocks, DEC1 and DEC2 are involved in hypoxia responses, immunological reactions, and carcinogenesis. These DEC actions are mediated by the direct binding to the E-box elements in target genes or by protein-protein interactions with transcription factors such as HIF-1α, RXRα, MyoD, and STAT. Notably, numerous growth factors, hormones, and cytokines, along with ionizing radiation and DNA-damaging agents, induce Dec1 and/or Dec2 in a tissue-specific manner. These findings suggest that DEC1 and DEC2 play a critical role in animal adaptation to various environmental stimuli.

A novel protein, CHRONO, functions as a core component of the mammalian circadian clock.

Circadian rhythms are controlled by a system of negative and positive genetic feedback loops composed of clock genes. Although many genes have been implicated in these feedback loops, it is unclear whether our current list of clock genes is exhaustive. We have recently identified Chrono as a robustly cycling transcript through genome-wide profiling of BMAL1 binding on the E-box. Here, we explore the role of Chrono in cellular timekeeping. Remarkably, endogenous CHRONO occupancy around E-boxes shows a circadian oscillation antiphasic to BMAL1. Overexpression of Chrono leads to suppression of BMAL1-CLOCK activity in a histone deacetylase (HDAC) -dependent manner. In vivo loss-of-function studies of Chrono including Avp neuron-specific knockout (KO) mice display a longer circadian period of locomotor activity. Chrono KO also alters the expression of core clock genes and impairs the response of the circadian clock to stress. CHRONO forms a complex with the glucocorticoid receptor and mediates glucocorticoid response. Our comprehensive study spotlights a previously unrecognized clock component of an unsuspected negative circadian feedback loop that is independent of another negative regulator, Cry2, and that integrates behavioral stress and epigenetic control for efficient metabolic integration of the clock.

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.

The BHLH transcription factor DEC1 plays an important role in the epithelial-mesenchymal transition of pancreatic cancer.

DEC1 (BHLHE40/Stra13/Sharp2) is a basic helix-loop-helix (bHLH) transcription factor that is involved in the regulation of apoptosis and cell proliferation and the response to hypoxia. Epithelial-mesenchymal transition (EMT) is an important step leading to invasion and migration of various tumor cells, and TGF-ß treatment has been shown to induce cancer cells to undergo EMT. However, the significance of DEC1 in TGF-ß-induced EMT remains unknown. We examined the role of DEC1 in EMT of PANC-1 cells, a human pancreatic cancer cell line. As a result, we found that DEC1 was upregulated by TGF-ß in PANC-1 cells, and regulated the expression and the levels of nuclear, cytoplasmic or membrane localization of EMT-related factors, including phosphorylated Smad3 (pSmad3), snail, claudin-4 and N-cadherin. In the presence of TGF-ß, DEC1 knockdown by siRNA inhibited morphological changes during EMT processes, while TGF-ß induced PANC-1 cells to taken on a spindle-shaped morphology. Furthermore, a combination treatment of DEC1 expression with TGF-ß was closely linked to the migration and invasion of PANC-1 cells. Immunohistochemically, DEC1 and pSmad3 were detected within pancreatic cancer tissues, whereas claudin-4 expression was weaker in the cancer tissues compared with the adjacent non-cancer pancreatic tissues. These findings suggest that DEC1 plays an important role in the regulation of these EMT-related factors in pancreatic cancer.